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Progress in Neurobiology 69 (2003) 229285

Diabetic neuropathy and nerve regeneration


Hitoshi Yasuda , Masahiko Terada, Kengo Maeda, Shuro Kogawa, Mitsuru Sanada, Masakazu Haneda, Atsunori Kashiwagi, Ryuichi Kikkawa
Division of Neurology, Department of Medicine, Shiga University of Medical Science, Seta, Otsu, Shiga 520-2192, Japan Received 20 June 2001; accepted 20 February 2003

Abstract Diabetic neuropathy is the most common peripheral neuropathy in western countries. Although every effort has been made to clarify the pathogenic mechanism of diabetic neuropathy, thereby devising its ideal therapeutic drugs, neither convinced hypotheses nor unequivocally effective drugs have been established. In view of the pathologic basis for the treatment of diabetic neuropathy, it is important to enhance nerve regeneration as well as prevent nerve degeneration. Nerve regeneration or sprouting in diabetes may occur not only in the nerve trunk but also in the dermis and around dorsal root ganglion neurons, thereby being implicated in the generation of pain sensation. Thus, inadequate nerve regeneration unequivocally contributes to the pathophysiologic mechanism of diabetic neuropathy. In this context, the research on nerve regeneration in diabetes should be more accelerated. Indeed, nerve regenerative capacity has been shown to be decreased in diabetic patients as well as in diabetic animals. Disturbed nerve regeneration in diabetes has been ascribed at least in part to all or some of decreased levels of neurotrophic factors, decreased expression of their receptors, altered cellular signal pathways and/or abnormal expression of cell adhesion molecules, although the mechanisms of their changes remain almost unclear. In addition to their steady-state changes in diabetes, nerve injury induces injury-specic changes in individual neurotrophic factors, their receptors and their intracellular signal pathways, which are closely linked with altered neuronal function, varying from neuronal survival and neurite extension/nerve regeneration to apoptosis. Although it is essential to clarify those changes for understanding the mechanism of disturbed nerve regeneration in diabetes, very few data are now available. Rationally accepted replacement therapy with neurotrophic factors has not provided any success in treating diabetic neuropathy. Aside from adverse effects of those factors, more rigorous consideration for their delivery system may be needed for any possible success. Although conventional therapeutic drugs like aldose reductase (AR) inhibitors and vasodilators have been shown to enhance nerve regeneration, their efcacy should be strictly evaluated with respect to nerve regenerative capacity. For this purpose, especially clinically, skin biopsy, by which cutaneous nerve pathology including nerve regeneration can be morphometrically evaluated, might be a safe and useful examination. 2003 Elsevier Science Ltd. All rights reserved.

Abbreviations: ABC, avidin-biotinylated enzyme complex; ALS, amyotrophic lateral sclerosis; AP-1, activator protein-1; AR, aldose reductase; ARI, aldose reductase inhibitor; ATF-2, activating transcription factor-2; BDNF, brain-derived neurotrophic factor; bFGF, basic broblast growth factor; Cdk5, cyclin-dependent kinase 5; CGRP, calcitonin gene-related protein; CMAP, compound muscle action potential; CNS, central nervous system; CNTF, ciliary neurotrophic factor; CRE, cAMP responsive element; CSF, cerebrospinal uid; DRG, dorsal root ganglion; ELISA, enzyme-linked immunosorbent assay; ERK1, extracellular signal-related kinase 1 (42 kDa); ERK2, extracellular signal-related kinase 2 (44 kDa); GAP-43, growth-associated protein-43; GDC, granular disintegration of the cytoskeleton; GDNF, glial cell-derived neurotrophic factor; GFR, GDNF family receptor component; GPI, glycosylphosphatidylinositol; GSK-3, glycogen synthase-3; ICAM, intercellular cell adhesion molecule; IDDM, insulin-dependent diabetes mellitus; IGF, insulin-like growth factor; IGFBP, insulin-like growth factor binding protein; JNK, c-Jun N-terminal kinase; L1, L1-CAM (L1 cell adhesion molecule); LIF, leukemia inhibitory factor; MAG, myelin-associated glycoprotein; MAP, mitogen-activated protein; MAPK, MAP kinase; MAPKK, MAP kinase kinase; MMPs, matrix metalloproteinases; MNF, myelinated nerve ber; NCAM, neural cell adhesion molecule; NF, neurolament; NF-H, high molecular weight mass (200 kDa) neurolament; NF-L, light molecular weight mass (68 kDa) neurolament; NF-M, medium molecular weight mass (145 kDa) neurolament; NGF, nerve growth factor; NIDDM, non-insulin-dependent diabetes mellitus; NPY, neuropeptide Y; NT-3, neurotrophin-3; NT-4/5, neurotrophin-4/5; p75NTR, p75 neurotrophin receptor; pak, p21-activated kinase; PGE1, prostaglandin E1; PGI2, prostaglandin I2; PGP 9.5, protein gene-product 9.5; PKC, protein kinase C; PNS, peripheral nervous system; RET, glial cell-derived neurotrophic factor receptor tyrosine kinase; rhNGF, recombinant human nerve growth factor; SAPK, stress-activated protein kinase; SCa, slow component a; SCb, slow component b; STZ, streptozocin; TNF-, tumor necrosis factor-; TrkA, tyrosine-receptor kinase A; TUNNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling; UMNF, unmyelinated nerve ber; VEGF, vascular endothelial growth factor; VIP, vosoactive intestinal polypeptide Corresponding author. Tel.: +81-77-548-2222; fax: +81-77-543-3858. E-mail address: hyasuda@belle.shiga-med.ac.jp (H. Yasuda). 0301-0082/03/$ see front matter 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0301-0082(03)00034-0

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Contents
1. Introduction: aims and scope of review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Intrinsic and extrinsic factors associated with nerve regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Intrinsic neuronal regenerating activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. Growth-associated protein-43 (GAP-43) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Tubulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Neurotrophins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1.1. Nerve growth factor (NGF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1.2. Brain-derived neurotrophic factor (BDNF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1.3. Neurotrophin-3 (NT-3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1.4. Neurotrophin-4/5 (NT-4/5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Insulin-like growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.1. Insulin-like growth factor-I and -II (IGF-I/II) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2.2. Insulin-like growth factor binding protein (IGFBP) . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Hematopoietic cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3.1. Ciliary neurotrophic factor (CNTF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3.2. Tumor necrosis factor- (TNF-) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3.3. Interleukin-6 (IL-6) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Glial cell line-derived neurotrophic factor (GDNF) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Extracellular matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. Laminin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Fibronectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3. Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4. Matrix metalloproteinases (MMPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Cell adhesion molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.1. Neural cell adhesion molecule (NCAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.2. L1 cell adhesion molecule (L1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.3. Myelin-associated glycoprotein (MAG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4.4. Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Cell signal messengers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.1. cAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.2. Protein kinase C (PKC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.3. Mitogen-activated protein kinases (MAPKs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.3.1. c-Jun N-terminal protein kinase (JNK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.3.2. Extracellular signal-related kinase 1/2 (ERK1/2) . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5.4. Cyclin-dependent kinases and small GTPases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Immediate early genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1. c-Jun . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.2. c-Fos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.3. Activating transcription factor-2 (ATF-2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Endoneurial microenvironment including vascularization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Experimental studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Relevance of examining nerve regeneration in experimental diabetic models . . . . . . . . . . . . . . . 3.2. Evaluation of nerve regeneration in animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Degeneration and regeneration of the peripheral nerve . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Cellular events in Wallerian degeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Early axonal changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Schwann cell responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.4. Macrophage responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.5. Nerve regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.6. Axonal sprouting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.7. Growth cone and axonal elongation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.8. Cell body reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.9. Maturation of regenerating nerve bers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Wallerian degeneration in experimental diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1. Axonal degeneration and pathway clearance in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2. Degradation of cytoskeletal proteins in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3. Macrophage responses in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.4. Schwann cell response in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 232 232 232 233 234 234 234 237 237 238 238 238 238 239 239 239 239 240 240 240 241 241 241 241 242 242 242 243 243 243 245 245 246 247 247 248 248 249 249 249 250 250 250 250 250 251 251 252 252 252 252 253 253 253 253 253 255 256

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3.5. Nerve regeneration in experimental diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.1. Axonal sprouting, elongation and maturation in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.2. Cell body reaction in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5.3. Partial denervation in diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Clinical observation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Pathological ndings suggesting nerve regeneration in diabetic nerves . . . . . . . . . . . . . . . . . . . . 4.2. Rationale for using neurotrophic factors for diabetic patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Clinical trials: current state . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1. Lessens from other trials: amyotrophic lateral sclerosis and toxic neuropathies . . . . . . 4.3.2. Nerve growth factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3. Other neurotrophic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.4. Aldose reductase inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. Specic problems for the use of neurotrophic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Regenerating nerve bers with special reference to pain generation . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Evaluation of nerve pathology including regeneration by skin biopsy . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Morphometric analysis of cutaneous nerves by immunohistochemistry . . . . . . . . . . . . . . . . . . . . 6.2. Morphometric analysis of cutaneous nerves by ultrastructural examination . . . . . . . . . . . . . . . . 6.3. Neurotrophins and cutaneous innervation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Evaluation of therapeutic compounds for diabetic neuropathy by skin biopsy . . . . . . . . . . . . . . 7. Neuronal cell death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.1. Apoptosis induced by the sera from diabetic patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Apoptosis is induced under high glucose or hyperglycemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Apoptosis and neurotrophins/MAP kinase/cAMP in hyperglycemia . . . . . . . . . . . . . . . . . . . . . . . 7.3.1. Neurotrophins and their receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.2. MAP kinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3.3. cAMP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction: aims and scope of review Diabetic polyneuropathy, the most common of the peripheral neuropathies, occurs widely in western countries. It most often develops in the midst of complications observed in diabetes. The putative pathogenesis of diabetic neuropathy includes increased polyol pathway activity leading to the accumulation of sorbitol and fructose (Gabby et al., 1966; Gabby and OSullivan, 1968) and imbalances in nicotinamide adenine dinucleotide phosphate/nicotinamide adenine dinucleotide, reduced form (Williamson et al., 1993); auto-oxidation of glucose leading to the formation of reactive oxygen species (Low et al., 1997); advanced glycation end-products produced by non-enzymatic glycation of proteins (Brownlee et al., 1988); inappropriate activation of protein kinase C (PKC) (Greene et al., 1985, 1999a,b; Koya and King, 1998); and a decit of neurotrophic supports (Tomlinson et al., 1997) (Fig. 1). Based on these observations, several therapeutic drugs including aldose reductase inhibitors (ARIs) (Kikkawa et al., 1984; Sima et al., 1988a,b), anti-oxidant drugs (Low et al., 1997), aminoguanidine (Yagihashi et al., 1992), a selective PKC inhibitor (Nakamura et al., 1999), and neurotrophic factors (Tomlinson et al., 1996, 1997) have been used to treat diabetic neuropathy and have been reported to ameliorate nerve dysfunction and/or morphologic abnormalities in diabetic animals and/or patients.

Although some of these compounds have had signicant effects on morphological abnormalities of nerve bers as well as nerve dysfunction, usually the magnitude of the effects has been smaller in clinical trials than in experimental studies. The discrepancy may be due in part to the difference in the amount of nerve ber lesions: patients with diabetic neuropathy usually show nerve ber loss to some degree, whereas diabetic animals, especially streptozocin (STZ)-induced ones, do not have any signicant degree of nerve ber loss (Yasuda et al., 1989a,b; Zochodne et al., 2001). Moreover, pancreas transplantation has not produced the expected benecial effects on nerve dysfunction; complete normalization of blood glucose has resulted in a notable but still mild degree of improvement in nerve function even 10 years after transplantation (Navarro et al., 1997). Patients who received pancreas transplantation usually had moderate to severe neuropathy; thus, it may follow that the ineffectiveness of glycemic control for diabetic neuropathy may be due to the difculty for nerve bers to regenerate once degenerated. This tendency toward irreversibility in the peripheral nerve in diabetes has also been supported in many clinical trials on the therapeutic drugs for diabetic neuropathy in which nerve function has been improved only by a few meter/s in nerve conduction velocity during 1 year (Boulton et al., 1990; Goto et al., 1995; Greene et al., 1999a,b). These results may suggest that peripheral nerve tissue damage cannot be measurably reversed once pathological

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Fig. 1. Schematic hypothetical mechanisms of diabetic neuropathy. In this gure, possible causative factors are simply presented, although each factor is closely associated with other factors. In addition, there are other putative factors which may be involved in its pathogenesis. One of the points which should be emphasized in this schema is that nerve regeneration is closely involved in nerve degeneration such that the balance between nerve degeneration and regeneration may be directed toward nerve degeneration in diabetic condition. PKC, protein kinase C.

changes even mildly occur. Thus, early diagnosis of diabetic neuropathy followed by drug therapy combined with glycemic control may be warranted to prevent the progression of diabetic neuropathy, i.e. arrest the degenerative changes of nerve ber pathology. However, even with these early efforts, it may be nearly impossible to normalize glycemic level. The pathology of diabetic polyneuropathy includes axonal atrophy, demyelination, loss of nerve bers, and the blunted regeneration of nerve bers (Sima et al., 1988a,b; Dyck and Giannini, 1996). Thus, nerve regeneration coexists with degeneration and contributes to nerve function as well as nerve pathology especially at the later stage of diabetic neuropathy. The goal in treating diabetic neuropathy is not only to prevent the progression of neuropathic symptoms and nerve dysfunction and degeneration (Kennedy et al., 1990; The Diabetes Control and Complications Trial Research Group, 1993) but also to promote regeneration of degenerated nerve bers. Thus, in addition to prevention of nerve degenerative process, augmentation of nerve ber regenerative capacity may be important and useful for the treatment of diabetic neuropathy (Fig. 1). For this purpose, the fundamental understanding about nerve regenerative mechanisms especially with respect to the pathogenesis of diabetic neuropathy should be rigorously studied. The decreased nerve regenerative capacity in diabetes has been associated with impaired neurotrophic tone, which could reect diminished synthesis, secretion or responsiveness of neurotrophic factors such as nerve growth factor (NGF) in sensory and autonomic nerve bers. Although neurotrophic factors including NGF are required not only for the development but also for the maintenance of NGF-sensitive sensory and autonomic neurons and their axons, most of their serum and nerve levels have been reported to be decreased in the diabetic condition. Retrograde axonal transport of

peripherally synthesized neurotrophic factors including NGF from target organs to neuronal cell bodies, which is required for normal maintenance and regeneration of the peripheral nervous system (PNS), is also disturbed in the diabetic state (Schmidt et al., 1985; Fernyhough et al., 1998a,b). In addition, novel neurotrophic factors are still being discovered and some of these may be decient in the diabetic condition. Furthermore, the signal transduction of neurotrophic factors has not been fully uncovered although it is considerably changed in diabetes, and its abnormality is closely implicated in either disrupted neuronal survival or disturbed nerve regeneration. Most diabetic animals have not shown nerve ber loss in steady state, and it is not satisfactory to use their uninjured nerves for nerve regeneration research in diabetic condition. However, examining nerve ber regeneration in animal models of nerve injuries, e.g. crush, freezing, or transection, may be a useful alternative method. In this manuscript, we focus on the pathophysiology of nerve regeneration with special reference to diabetic sensory polyneuropathy and the roles of its associated neurotrophic factors, their receptors, extracellular matrices, etc. in the pathogenesis and treatment of diabetic polyneuropathy. Our work with respect to nerve regeneration in diabetic state is described.

2. Intrinsic and extrinsic factors associated with nerve regeneration 2.1. Intrinsic neuronal regenerating activity 2.1.1. Growth-associated protein-43 (GAP-43) The synthesis and axonal transport of GAP-43/B-50 are induced in the process of axonal elongation. GAP-43 is a

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Fig. 2. Molecular biological changes during nerve regeneration after nerve injury. See Abbreviations for details.

major constituent of the axonal growth cone (Fig. 2), where it is localized exclusively in the membrane skeleton. GAP-43 is never induced in injured neurons of the central nervous system (CNS), where nerve regeneration does not occur under physiological conditions. By contrast, the protein is dramatically induced after nerve injury in the PNS (Vanselow et al., 1994). In steady state, GAP-43 is expressed only in small dorsal root ganglion (DRG) neurons under normal condition, whereas it is expressed by all sizes of DRG neurons after nerve injury. However, the induction depends on the level of the axotomized site along the entire length of axons; central axotomy does not induce GAP-43 mRNA in DRG (Chong et al., 1994). This observation suggests that unknown substrates from the periphery might regulate GAP-43 expression. The substrates may include NGF, which increases GAP-43 mRNA by increasing its half-life. This effect of NGF was mimicked by phorbol ester, PKC agonist, and was inhibited by PKC inhibitor or down-regulation of PKC in PC12 cells, suggesting the possible PKC-dependent stabilization of mRNA (Perrone-Bizzozero et al., 1993). Sensory neurons from mice lacking GAP-43, however, can extend neurites and form lopodia in culture condition, suggesting that GAP-43 is not required to create growth cones. In certain decision points, such as optic chiasm, GAP-43 is necessary for pathnding (Strittmatter et al., 1995). Apart from neuronal cells of the PNS, unmyelinating Schwann cells also express GAP-43, although its function is unknown. In diabetic rats, the mRNA level of GAP-43 has been reported to be reduced in uninjured DRGs. After nerve

crush, the level is consistently up-regulated in DRG neurons, although the magnitude of its increase by quantitative analysis was different between reports; Maeda et al. (1996) reported a lower increase of GAP-43 mRNA in DRGs of diabetic rats than in those of control rats, whereas Pekiner et al. (1996) reported its unchanged expression in DRGs of diabetic rats compared with those of non-diabetic rats. A decrease in GAP-43 mRNA after nerve crush in diabetic rats was reported by others (Mohiuddin and Tomlinson, 1997). The protein level of GAP-43 in injured DRG neurons was reduced in the sciatic nerve of diabetic rats (Pekiner et al., 1996). In the autonomic nervous system, there was no difference in immunohistochemical localization of GAP-43 in sympathetic ganglions between control and long-term diabetic rats (Schmidt et al., 1991). 2.1.2. Tubulin Tubulin protein is a cytoskeleton that is up-regulated after nerve injury (Hoffman and Cleveland, 1988; Miller et al., 1989; Oblinger et al., 1989; Moskowitz et al., 1993). Microtubules which are the major cytoskeletal component of regenerating axons are formed by heterodimers of - and -tubulin. In uninjured diabetic DRG, T1-tubulin mRNA is decreased (Mohiuddin et al., 1995a; Scott et al., 1999). Recently, class III -tubulin mRNA has been shown to be increased in diabetic DRG by in situ hybridization (Liuzzi et al., 1998). It is difcult to explain the discrepancy

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H. Yasuda et al. / Progress in Neurobiology 69 (2003) 229285 Table 1 List of neurotrophic factors Neurotrophins (NT) Nerve growth factor (NGF) Brain-derived neurotrophic factor (BDNF) NT-3 NT-4/5 NT-6 Hematopoietic cytokines Ciliary neurotrophic factor (CNTF) Leukemia inhibitory factor (LIF) Oncogene M Interleukin (IL)-1 IL-3 IL-6 IL-7 IL-9 IL-11 Granulocyte colony-stimulating factor Insulin-like growth factors (IGF) Insulin IGF-I IGF-II Heparin-binding family Acidic-broblast growth factor (FGF) Basic FGF int-2 onc hst/k-fgf onc FGF-4 FGF-5 FGF-6 Keratinocyte growth factor Epidermal growth factor (EGF) family EGF Transforming growth factor (TGF)- TGF- family TGF-1 TGF-2 TGF-3 Glial-derived neurotrophic factor (GDNF) Neurturin Persephin Activin A Bone morphogenetic proteins Tyrosine kinase-associated cytokines Platelet-derived growth factor (PDGF) Colony-stimulating factor-1 Stem cell factor Others ACTH analogues Gangliosides Amyloid precursor protein -Interferon Galectin-1

between -and -tubulin mRNA in diabetic DRG. In addition, it remains to be claried whether these alterations of tubulin synthesis are associated with regeneration failure in diabetic nerves. Tubulin protein is modied posttranslationally. Non-enzymatic glycation of tubulin has been observed as early as 2 weeks after induction of diabetes (Cullum et al., 1991; McLean et al., 1992). Posttranslational modication might have some effects on tubulin assembly. However, if any, its direct association with regeneration failure in diabetes is also uncertain. 2.2. Growth factors Although many neurotrophic factors are known (Table 1), few are studied in the eld of the pathogenesis and treatment of diabetic neuropathy; most of them are associated with nerve growth factor (NGF). Although the mechanism of action is as yet unknown, the current knowledge of growth factors and their relationship to diabetic neuropathy suggest a pathophysiological role of reduced levels of growth factors in the development of diabetic neuropathy; neuronal function may be compromised by their decits (Table 2). Furthermore, atrophy of neurons or nerves and even neuronal death may be induced due to growth factor reduction in diabetic neuropathy. It also remains to be established whether growth factor deciency is due to a decrease in its synthesis, an inability of the factor to bind to its receptor, or disturbances in retrograde axonal transport or intraneuronal processing. Further studies aimed at understanding the disturbances in expression of the genes and proteins involved in diabetic neuropathy, as well as their receptor binding and subsequent transport from sites of synthesis to sites of action, may clarify the relationship between growth factors and diabetic neuropathy. 2.2.1. Neurotrophins 2.2.1.1. Nerve growth factor (NGF). NGF shows trophic effects on a subpopulation of sensory neurons in DRG and sympathetic postganglionic neurons (Smeyne et al., 1994; Crowley et al., 1994). NGF is produced by the target tissues including skeletal muscle (Amano et al., 1991) and skin. NGF released by the target tissue is incorporated with its high afnity receptor, TrkA (Figs. 24) at the nerve ending. NGFphosphorylated TrkA complex is retrogradely transported to the neuronal body and transduces its signal to the nucleus. The function of another NGF receptor, p75NTR, which is also bound to other neurotrophins with low afnity, is controversial. The DRG neurons supported by NGF are small in size and mediate nociception. During development, NGF is essential for the survival of DRG small neurons. However, in adult, NGF is not necessary for the survival but maintains neuropeptide levels such as substance P. When the nerve is injured, the delivery of NGF from the target is decreased. Interleukin (IL)-1 released by macrophages inltrating the endoneurium increases NGF expression in

broblasts in the distal stump (Heumann et al., 1987a,b; Lindholm et al., 1987, 1988; Brown et al., 1991; Rotshenker et al., 1992). Schwann cells, which do not respond to IL-, also increase NGF expression (Matsuoka et al., 1991). NGF mRNA is also increased in the denervated skin (Mearow

H. Yasuda et al. / Progress in Neurobiology 69 (2003) 229285 Table 2 Neurotrophins and their receptors in experimental diabetes Neurotrophins Nerve growth factor (NGF) Serum Skin Nerve Heart Brain-derived neurotrophic factor (BDNF) Muscle Nerve DRG Neurotrophin-3 Muscle Skin Nerve Neurotrophin-4/5 Nerve Receptors TrkA DRG

235

TrkB Not reported

TrkC DRG

TrkB Not reported

et al., 1993). The expression of both trkA and p75NTR mRNA in DRGs is decreased after nerve injury. What effect NGF has on nerve regeneration is a controversial issue. Although the early studies showed that NGF improved nerve regeneration (Chen et al., 1989; Rich et al., 1989; Gold et al., 1991), it has been clearly shown that NGF stimulates collateral sprouting from uninjured neurons but not regeneration from injured neurons (Diamond et al., 1992; Mearow et al., 1994). This fact may not be disappointing in terms of the treatment of diabetic peripheral neuropathy,

since the goal of treatment is reinnervation of the target tissues. NGF increases tubulin mRNA in sympathetic neurons (Mathew and Miller, 1990) and DRG neurite outgrowth in three-dimensional extracellular matrix via the up-regulation of matrix metalloproteinase (MMP)-2 (Muir, 1994). In diabetic patients, serum (Faradji and Soleto, 1990) and skin NGF levels were reported to be decreased (Anand et al., 1996), comparable to the data from experimental diabetes studies (Table 2). However, discrepant results have been reported; skin NGF mRNA was increased in diabetic patients (Diemel et al., 1999), and the serum NGF level in IDDM patients was increased compared with age-matched control subjects and NIDDM patients (Azar et al., 1999). In experimental models, tissue NGF levels were decreased in diabetic mice, and this decrease was ameliorated by transplantation of islet cells (Hellweg et al., 1991) (Table 2). Others also found a decrease in NGF content in the sciatic nerves of diabetic rats (Hanaoka et al., 1992). Insulin treatment was shown to improve decreased NGF mRNA in the skin of diabetic rats (Fernyhough et al., 1994). These reports suggest that hyperglycemia and/or hypoinsulinemia induce a decit of NGF synthesis. This decit might also be caused by altered corticosterone and 1,25-(OH)2 D3 (Neveu et al., 1992). Corticosterone decreases NGF, whereas 25-(OH)2 D3 increases NGF; in diabetes, serum concentration of corticosterone is increased, whereas that of 1,25-(OH)2 D3 is decreased. A Vitamin D3 derivative not only induces NGF but also improves neuropeptide content (Riaz et al., 1999). Accumulation of polyol could decrease NGF synthesis; ARI

Fig. 3. Distribution of neurotrophic factors in the peripheral nerve under normal and injured conditions. Solid circles imply neurotrophic factors which are secreted from various cells including Schwann cells, captured and taken up from the axons and transported to the neuronal body. The mode of production and secretion are extraordinarily different either among involved factors or among involved conditions.

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Fig. 4. The receptors for neurotrophins. TrkA, TrkB and TrkC are high afnity receptors specic for NGF, BDNF and NT-4/5 and NT-3, respectively, all of which have intracellular kinase domain. Both TrkA and TrkB also show an afnity for other neurotrophins, although relatively weak. All neurotrophins are able to bind to low afnity receptor p75 which does not have intracellular kinase domain.

epalrestat corrects decreases in both NGF content in diabetic nerves and NGF mRNA in cultured Schwann cells under high glucose condition (Ohi et al., 1998). The anti-oxidant -lipoic acid, which is known to be effective for diabetic neuropathy, was also reported to reverse nerve NGF content in diabetic rats (Garrett et al., 1997; Hounsom et al., 1998). The uptake of exogenous NGF into DRG or superior mesenteric ganglion and retrograde axonal transport of NGF have been reported to be decreased in diabetic rats (Jakobsen et al., 1981; Schmidt et al., 1983; Hellweg et al., 1994). NGF ameliorates decreases in mRNAs and proteins of substance P and CGRP in peripheral nerves, DRGs or dorsal horns of the spinal cord (Diemel et al., 1994; Fernyhough et al., 1995a,b; Schmidt et al., 1995; Unger et al., 1998), nociceptive threshold (Apfel et al., 1994) and amplitude of electrically evoked C-ber response (Elias et al., 1998). In addition, neurogenic cutaneous vasodilatation and plasma extravasation (Bennett et al., 1998a,b) and myelinated nerve ber (MNF) morphology in the sural nerve (Unger et al., 1998) are also restored by NGF, whereas it does not improve nerve blood ow or motor nerve conduction velocity in diabetic rats (Maeda et al., 1997). Decreased NGF pro-

duction may lead to up-regulation of trkA mRNA in the keratinocytes (Terenghi et al., 1997). In the autonomic nervous system, NGF mRNA (Kanki et al., 1999) and protein (Hellweg and Hartung, 1990) were increased in cardiac muscle at the early stage of diabetes and decreased in long-term diabetes (Schmid et al., 1999). In the iris, NGF mRNA was also increased in untreated diabetic rats (Brewster et al., 1995). Although the regeneration of postganglionic noradrenergic nerves after chemical denervation by 6-hydroxydopamine was well preserved in diabetic rats, a response to exogenous NGF was defective when assessed by the content of noradrenaline, suggesting loss of TrkA function (Vo and Tomlinson, 1999). It was reported that the expression of p75NTR was decreased in DRGs of STZ-induced diabetic rats compared with those of control rats (Delcroix et al., 1997), whereas that of TrkA was unchanged (Delcroix et al., 1997) or decreased in DRGs of diabetic rats (Mohiuddin and Tomlinson, 1997). In addition, anterograde and retrograde axonal transport of p75NTR was also decreased, while that of TrkA was unaffected in diabetic rats (Delcroix et al., 1998). NGF treatment reversed changes in transcripts and protein of p75NTR but

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did not reverse those of TrkA (Delcroix et al., 1998). Thus, the ability to capture and retrogradely transport NGF may be impaired in diabetic state because of suboptimal production of p75NTR, and NGF therapy may overcome this deciency. It was reported that extracellular cleavage product of NGF receptor (p75NTR) was increased in the urine of diabetic patients with neuropathy (Hurska et al., 1993), although the signicance is not clear. Decreased NGF effects in diabetes were also observed after nerve injury. The expression of NGF mRNA in the distal stump was unexpectedly increased in diabetic rats compared with non-diabetic rats. By contrast, the expression of trkA mRNA in DRG was decreased in diabetic rats. Messenger RNAs of substance P and GAP-43, which are regulated by NGF, were also decreased in diabetes (Maeda et al., 1996). Combined with the early observation that the uptake of exogenous NGF into DRG was decreased in diabetic rats (Jakobsen et al., 1981), this experiment suggests that both decits in the synthesis of NGF and in the coupling of NGF and trkA may contribute to a decrease in the effects of NGF on diabetic nerves. NGF that was administered at the proximal stumps of transected nerves corrected the molecular changes in DRGs caused by axotomy; both an increase in GAP-43 mRNA in DRG and a decrease in -preprotachykinin mRNA were reversed by NGF therapy (Mohiuddin et al., 1999). In this situation, interestingly, NGF supernormalized decreased p75NTR mRNA but did not affect decreased trkA mRNA in DRGs. The decreased ratio of TrkA/p75NTR was observed in axotomized DRG in diabetes. It was reported that excessive NGF might cause apoptosis via p75NTR (Frade et al., 1996). In the neuroblastoma cell line, expressing p75NTR, NGF, but neither BDNF nor NT-3, induced apoptosis (Kuner and Hertel, 1998). Thus, DRG neurons may undergo apoptotic changes when nerves are injured in diabetic condition. This phenomenon is discussed in Section 7. 2.2.1.2. Brain-derived neurotrophic factor (BDNF). BDNF has trophic effects on DRG sensory neurons (Ernfors et al., 1994a,b; Klein et al., 1993). Although BDNF is a member of NGF family, it has a distinct aspect different from NGF. BDNF is produced not only by target tissues but also by the neuron itself and transported anterogradely (Zhou et al., 1996) (Figs. 2 and 3). This neuronal production suggests its local neurotrophic effect via paracrine or autocrine action. BDNF supports medium-sized DRG neurons and motoneurons at the spinal anterior horn via its high afnity receptor TrkB (Fig. 4). Peripheral axotomy increases BDNF mRNA linearly in the distal stump of axotomized nerves (Meyer et al., 1992) and denervated muscles (Funakoshi et al., 1993). The transient increase in NGF synthesis in the distal stump of injured nerves may serve to stimulate the expression of BDNF (Apfel et al., 1996). BDNF mRNA is also increased in axotomized DRG neurons (Ernfors et al., 1993). Its receptor trkB mRNA was reported to be increased (Ernfors et al.,

1993) or unchanged in rat DRGs after nerve injury (Sebert and Shooter, 1993). Reports on BDNF in diabetes are less available than those on NGF. Messenger RNAs of BDNF in soleus muscle and DRG were increased in diabetic rats, and this increase was reversed with insulin treatment (Fernyhough et al., 1995a,b). However, endogenous BDNF protein in the sciatic nerve and antero- and retrograde axonal transport of BDNF are decreased in STZ-induced diabetic rats. The observation that transport of radio-labeled BDNF is not affected by diabetes suggests that reduced BDNF transport in diabetes is not a result of impaired capacity for receptor-mediated transport (Mizisin et al., 1999). The trkB mRNA in both full-length and truncated forms was also decreased in the sciatic nerve of 6-week diabetic rats but returned to the control level at 12-week diabetes (Rodiguez-Pena et al., 1995). Decreased protein and increased mRNA levels of BDNF may indicate compensatory up-regulation of BDNF production. These alterations may be due, in part, to the osmotic effect of hyperglycemia since similar results are reported in galactose-fed rats (Mizisin et al., 1999). BDNF did not ameliorate a decrease of substance P or CGRP in the sciatic nerve of diabetic rats (Diemel et al., 1994). In galactose-fed rats, which develop a neuropathy characterized by nerve conduction decits and axonal atrophy, BDNF improved motor nerve conduction velocity decit in the sciatic nerve and ameliorated the diminution of the caliber of dorsal root sensory axons but did not improve sensory nerve conduction velocity decit (Mizisin et al., 1997a). During nerve regeneration, muscular BDNF mRNA is regulated differently in soleus and gastrocnemius muscles in diabetic rats (Fernyhough et al., 1996). 2.2.1.3. Neurotrophin-3 (NT-3). NT-3 is the third member of the NGF family. This neurotrophin supports large neurons of DRGs that mediate proprioception (Klein et al., 1994; Ernfors et al., 1994a,b; Tessarollo et al., 1994). NT-3 binds to its high afnity receptor TrkC (Fig. 4) and promotes peripheral nerve regeneration (Sterne et al., 1997). After peripheral axotomy, NT-3 mRNA was decreased in the distal stump of the transected nerve and spinal cord but was unchanged in the gastrocnemius (Funakoshi et al., 1993) (Fig. 2). NT-3 enhances neurite outgrowth and up-regulates mRNAs for GAP-43 and T1-tubulin in culture condition (Mohiuddin et al., 1995a,b). In uninjured diabetic rats, NT-3 mRNA in the hindlimb skeletal muscle and sciatic nerve was decreased compared with control rats (Rodiguez-Pena et al., 1995; Ihara et al., 1996; Fernyhough et al., 1998a,b), while that in the dorsal root and sural nerve was increased (Cai et al., 1999) (Table 2). The expression of trkC mRNA in the sciatic nerve was also decreased in diabetic rats (Rodiguez-Pena et al., 1995). NT-3 mRNA in the skin was not altered in diabetic rats (Cai et al., 1999). NT-3 protein (Kennedy et al., 1998) and trkC mRNA (Terenghi et al., 1997) were increased in human diabetic skin. Together with the results of decreased

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axonal transport of NT-3 and decreased trkC mRNA expression in DRGs in diabetes (Fernyhough et al., 1998a,b), this suggests that diabetic DRG neurons receive less support by NT-3, which can be ameliorated with insulin treatment (Fernyhough et al., 1998a,b). 2.2.1.4. Neurotrophin-4/5 (NT-4/5). NT-4 and NT-5 were separately discovered, but it became apparent that these two factors were likely to be inter-species variants of the same protein. That is why these proteins are commonly referred to as NT4/5. NT-4/5 shows trophic effects via binding to trkB. Thus, this protein has a spectrum of activity similar to that of BDNF with respect to neuronal populations. In the diabetic uninjured nerve, NT-4/5 mRNA is decreased at 12-week diabetes (Rodiguez-Pena et al., 1995). 2.2.2. Insulin-like growth factors 2.2.2.1. Insulin-like growth factor-I and -II (IGF-I/II). Insulin-like growth factors (IGFs) are a family of structurally and functionally related proteins that include insulin, IGF-I and IGF-II. IGFs have been implicated in the growth and differentiation of neurons; both IGFs promote neurite outgrowth of neuroblastoma cells (Recio-Pinto et al., 1984a,b). IGFs bind to their own receptors, type-I and type-II IGF receptors, although there is considerable cross reactivity. The liver is the main source of serum IGFs in rats, although neural tissue also produces IGFs. The major sites of production and secretion of circulating IGF-II in the postnatal animal are the choroid plexus and leptomeninges, and its mRNA also exists in the nervous tissue in the adult. IGFs also accelerate regeneration of sensory (Fernyhough et al., 1993) and motor nerves (Near et al., 1992). Following nerve injury, the expression of IGFs is increased in Schwann cells in the distal stump (Glazner et al., 1994) (Fig. 2). IGF receptors exist in neuronal cell bodies, axons and nerve terminals. Serum IGFs are able to gain access to neurons through fenestrated capillaries in DRG. Serum IGF-I level is lower in diabetic patients than in non-diabetic subjects, while serum IGF-II level is not different between the two groups. In comparing STZ-induced diabetic rats with control rats, there is a decrease of mRNAs of both IGF-I and IGF-II in the nerve of the STZ-induced diabetic rats. The mRNA and protein expression of IGF-I receptor is also decreased in the superior cervical ganglia of STZ-diabetic rats (Bitar et al., 1997). This is also the case with the model of NIDDM, obese Zucker rats, in which IGF-II mRNA was decreased in the sciatic nerve, spinal cord, and brain, while IGF-I mRNA was decreased in the liver but not in the nervous system (Zhuang et al., 1997). Insulin treatment partially improves nerve IGFs mRNA (Wuarin et al., 1994). Systemic IGF-I or IGF-II treatment and local application of IGF-I improved the regenerating rate of sensory nerves in diabetic rats without affecting plasma glucose levels (Ishii, 1995).

2.2.2.2. Insulin-like growth factor binding protein (IGFBP). IGFBPs comprise a family of several proteins that bind IGFs with high afnity and specicity and thereby regulate IGF-dependent actions. Thus, IGFs exist not as free peptides but bound to one of their binding proteins; six IGF-binding proteins (IGFBPs) have been cloned, sequenced and characterized (Shimasaki et al., 1991). IGFBP-3 is the major form in plasma and is responsible for regulating the half-life of IGFs. In circulation, most IGFs are found in a ternary complex with IGFBP-3 and an acid-labile subunit that does not bind IGFs (Baxter, 1988). The ternary complex (150 kDa) is unable to leave circulation because of its size, leading to a prolonged half-life in IGFs within the complex (Binoux and Hossenlopp, 1988). Therefore, this complex acts as a circulating reservoir for IGFs. Small complexes of IGFs and other IGFBPs (50 kDa) than IGFBP-3 are able to cross the capillary wall and serve to transport IGFs to the tissues. Thus, IGFBPs are closely implicated in the turnover of IGFs. The binary complexes, including IGFs and the binding protein, have increased afnity for the acid-labile unit, thereby forming the ternary complex. In this state, IGF-I has reduced bioavailability and prolonged half-life. By contrast, when IGF-I is bound to any of the binding proteins without the acid-labile subunit, IGF-I has a shorter turnover and increased bioavailability. IGFBPs are synthesized in all tissues, including the nervous system, and act as local regulators of IGF actions. IGFBPs have several functions including transporting the IGFs in the circulation, mediating IGF transport out of the vascular compartment, localizing the IGFs to specic cell types and modulating both IGF binding to receptors and growth-promoting actions. The functions of IGFBPs appear to be altered through the reduction of the binding afnity of IGFBPs for IGFs due to proteolysis or posttranslational modications including glycosylation and phosphorylation. Proteolysis of the IGFBP decreases its afnity for IGF, promoting its release to the receptor. Phosphorylation alters the binding afnity of IGFBP-1 for IGFs and also may alter its ability to bind to cell surfaces. The role of the oligosaccharide chains of IGFBPs is unknown. Circulating levels of IGFBP-1 are increased in IDDM patients (Suikkari et al., 1988) and IDDM patients with neuropathy (Crosby et al., 1992). An inverse relationship between IGFBP-1 and insulin level was demonstrated in adolescents with IDDM (Batch et al., 1991). In addition, IGFBP-1 levels are correlated with mean 12-month HbA1c levels, and improvement of glycemic control with continuous subcutaneous insulin infusion for 2 months results in normalization of IGFBP-1 levels in adolescents with IDDM (Batch et al., 1991). These ndings may well explain the regulation of IGFBP-1 by insulin. However, it was also reported that diabetic patients had markedly elevated plasma IGFBP-1 levels and lower plasma IGF-I levels even though these patients were hyperinsulinemic. Thus, overall, poor glycemic control in type 1 diabetes is associated with

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elevated serum IGFBP-1 levels and reduced IGF-I levels. Increasing age is accompanied by a further decrease in serum IGF-I levels as well as an increase in IGFBP-1 levels in adult diabetic type 1 and type 2 subjects. IGFBP-3 levels are decreased in poorly controlled IDDM subjects (Baxter and Martin, 1986). The puberty-related rise in IGFBP-3 levels is also blunted by diabetes (Batch et al., 1991). These observations may be explained by the decreased levels of IGF-I in patients with IDDM (Bach and Rechler, 1992). As in IDDM patients, STZ-diabetic rats showed increased IGFBP-1 (Unterman et al., 1990) and decreased IGFBP-3 serum level (Zapf et al., 1989). 2.2.3. Hematopoietic cytokines Peripheral nerve production of cytokines originates from resident and recruited macrophages, lymphocytes, mastocytes, Schwann cells and probably neurons. Cytokines are involved in nerve lesions and repair. Tumor necrosis factor- (TNF-) injected into nerve induces Wallerian degeneration, whereas interleukin-1 (IL-1) production promotes detersion by scavenger macrophages and synthesis of neurotrophic factors (NGF and leukemia inhibitory factor (LIF)). After experimental axotomy, other neurotrophic factors, including IL-6, LIF and transforming growth factor-1 (TGF-1), are overexpressed in the nerve and promote axonal growth until axonSchwann cell contact. 2.2.3.1. Ciliary neurotrophic factor (CNTF). CNTF is produced by myelinating Schwann cells in the intact nerve, although it is not released under normal condition. Once the nerve is injured, CNTF is released and incorporated into DRG neurons (Sendtner et al., 1992). Although DRG neurons express CNTF receptor complex consisting of CNTFR-, gp130 and LIFR-, they do not increase the expression of CNTF receptor complex after axotomy (Curtis et al., 1993). An increase in the uptake of CNTF after axotomy might be associated with increased release of CNTFR- from the denervated muscles (Davis et al., 1993). In the spinal cord, CNTFR mRNA is increased after nerve injury (Mata et al., 1993). The treatment with CNTF after axotomy resulted in an increase in the number of MNFs and myelination in rats. CNTF receptor is more important for the nervous system than CNTF itself; mice lacking CNTFR- showed signicant motor neuron loss unlike CNTF knockout mice (De Chiara et al., 1995). Immunohistochemical staining showed a decrease in CNTF expression in the sciatic nerve of patients with motor neuron disease but not in patients with diabetic motor neuropathy (Lee et al., 1996). In diabetic rats, nerve CNTF mRNA and bioactivity (Calcutt et al., 1992) are reported to be decreased or to be unchanged (Ohi et al., 1998). The production failure seen in experimental models might be due to metabolic changes caused by an accelerated polyol pathway, since ARI treatment increased CNTF in the galactose-fed rat, which is the model of the accumulation of polyol (Mizisin et al., 1997b).

2.2.3.2. Tumor necrosis factor- (TNF-). TNF- is one of the major cytokines and is implicated in a variety of actions, including regulation of immune response and control of cell growth and differentiation through paracrine and autocrine networks in a variety of tissues, including the nervous system (Vassalli, 1992). There is growing circumstantial evidence that TNF- plays a role in the pathogenesis of inammatory demyelinating disorders, including Gullain-Barre syndrome (Tsukada et al., 1991) and multiple sclerosis (Hofman et al., 1989). TNF- is expressed in macrophages, Schwann cells or broblasts within the endoneurium in healthy subjects, and its immunoreactivity has been reported to be enhanced in neuropathies of various etiologies (Deprez et al., 2001). Indeed, TNF- exerts deleterious effects including demyelination on nerve bers either in vitro (Selmaj and Raine, 1988) or in vivo (Redford et al., 1995). Although by immunohistochemistry the expression of TNF- has not been shown to be increased in the PNS in diabetic condition, its serum level has been reported to be elevated in diabetic patients (Katsuki et al., 1998; Lechleitner et al., 2000) as well as in diabetic animals. Interestingly, inhibitors of TNF- including N-acetylcysteine (Sagara et al., 1996), troglitazone (Qiang et al., 1998a,b) and gliclazide (Qiang et al., 1998a,b) have been shown to inhibit the development of peripheral neuropathy in STZ-induced diabetic rats. Since these compounds also have the feature of free radical scavenger, it is unclear whether the effect of those compounds on nerve dysfunction is mediated through inhibiting TNF- activity and whether the increased level of serum TNF- contributes to nerve dysfunction or disturbed nerve regeneration in diabetes. 2.2.3.3. Interleukin-6 (IL-6). IL-6 belongs to the neuropoietic cytokine superfamily, which includes various cytokines presented in Table 1. All of these cytokines use the common signal-transducing receptor compound gp130 (Ip et al., 1992). The activation of this receptor is triggered by several types of receptorligand interactions. IL-6 is synthesized in a subpopulation of developing peripheral sensory and sympathetic neurons (Murphy et al., 1995; Gadient and Otton, 1996). In the adult nervous system, IL-6 level is hardly detectable, but IL-6 synthesis appears to be strongly increased during pathological situations. The level of IL-6 mRNA was elevated in the non-neuronal cells surrounding the motor bers of the facial nucleus after motoneuron axotomy (Kiefer et al., 1993). An increase in IL-6 synthesis was found either in the sciatic nerve at sites undergoing Wallerian degeneration (Bolin et al., 1995; Bourde et al., 1996) or in the DRG neurons within 1 day after sciatic nerve injury (Murphy et al., 1995). After sciatic nerve crush, its functional recovery was delayed in IL-6 gene knockout mice as analyzed from a behavioral footprint assay. Compound action potentials after crush lesion showed that there was a very low level of recovery of the sensory but not of the motor branch of the mice. Thus, sensory functions were

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impaired in the intact adult animals and regeneration of the lesioned sensory axons was delayed in IL-6 gene knockout mice (Zhong et al., 1999). The magnitude of increased IL-6 level after sciatic nerve transection was smaller in diabetic nerves than in control nerves, although its signicance remains unclear (Takagi et al., 2001). 2.2.4. Glial cell line-derived neurotrophic factor (GDNF) GDNF is a neurotrophic polypeptide, distantly related to transforming growth factor- (TGF-). Although GDNF was originally reported to support dopaminergic neurons (Lin et al., 1993, 1994), it has more recently been shown to be a potent neurotrophic factor for peripheral neurons, including enteric, sympathetic, motor and sensory ones. GDNF is produced in the skin, kidney, stomach and testis. Low levels of GDNF mRNAs have also been found in the skeletal muscle, ovary, lung, adrenal gland, spinal cord, superior cervical ganglion and DRG (Trupp et al., 1995). The receptor tyrosine kinase RET is the signaling receptor (Trupp et al., 1996) and acts in concert with one or several glycosylphosphatidylinositol (GPI)-linked proteins: GDNF family receptor components (GFR) 1-4 (Airaksinen et al., 1999). In DRG, non-peptidergic small neurons postnatally switch their dependency from NGF to GDNF (Silos-Santiago et al., 1995; Bennett et al., 1996; Molliver and Snider, 1997). These neurons bind the isolectin IB4 and express thiamine monophosphate (TMP). IB4-positive central axons terminate in the inner layer of lamina II in the dorsal horn (Averill et al., 1995). IB4-positive neurons synthesize RET, GFR 1 and GFR 2 (Molliver et al., 1997; Bennett et al., 1998a,b). The function of these neurons is unclear. Although IB4-positive neurons have a high density of voltage-gated tetrodotoxin-resistant Na+ channels compared to peptidergic neurons (Stucky and Lewin, 1999), noxious heat causes a great currents in peptidergic neurons, but much smaller currents in IB4 neurons (Stucky et al., 1999). IB4 neurons might be associated with neuropathic pain but not inammatory pain (Snider and McMahon, 1998). Two days after transection of the sciatic nerve, GDNF mRNA is increased dramatically (400500 times) (Trupp et al., 1995). In DRG, RET mRNA is increased only slightly. However, GFR1 and 2 are highly increased after axotomy (Kashiba et al., 1998; Bennett et al., 2000). The treatment with GDNF restores IB4 binding/TMP expression (Bennett et al., 2000) and stimulates the regeneration of P2X3-positive axons (Ramer et al., 2000). In addition, GDNF treatment improved the recovery of the sensitivity to noxious heat and pressure. Although any change in either GDNF synthesis or its receptor expression has not been reported in diabetes, intrathecal GDNF treatment restored TMP labeling in the inner layer of lamina II in STZ-induced diabetic mice (Akkina et al., 2001).

2.3. Extracellular matrix The extracellular matrix of the peripheral nerve mechanically supports the cells that it surrounds, but it also regulates their behavior through specic interactions mediated via molecules on the cell surface, such as integrin receptors and cell surface proteoglycans. Thus, changes in the structure and composition of the extracellular matrix may alter cellular functions in multiple ways. At the ultrastructural level, these changes include thickening of vascular, perineurial and Schwann cell associated basement membranes; accumulation of microbrillar material in the vicinity of perineurial cells; and increased diameter of endoneurial collagen brils. At the molecular level, the changes may be associated with altered metabolism of various collagen types, such as type IVI collagens, laminin and bronectin. 2.3.1. Laminin Laminin is an 800 kDa heterotrimeric glycoprotein consisting of three subunits, a large A chain and two smaller chains, B1 and B2, and constitutes one of the major extracellular matrices consisting of basement membrane (Lander, 1987). Laminin readily binds to itself, type VI collagen, proteoglycans, entactin and perhaps other extracellular matrix constituents. In the PNS, the A chain is replaced by the merosin M chain, a major component of basal lamina. Nerve elongation is modied by neurite outgrowth domain peptide (p20) of B2 chain (Liesi et al., 1989) via 31 integrin receptor (Tomaselli et al., 1993). Antibodies against laminin (Wang et al., 1992) or its receptor (Toyota et al., 1990) inhibit peripheral nerve regeneration. Although the signicance is unknown, neuronal cells, as well as non-neuronal cells, produce laminin B2 chain (Le Beau et al., 1994) and up-regulate its mRNA during nerve regeneration (Le Beau et al., 1995). Attachment of neurons to extracellular substrate is an important phase for neurite outgrowth in culture. Diabetic DRG neurons are impaired to adhere to laminin, type I and IV collagens, and bronectin (Sango et al., 1995). This defect was prevented by treatment with aldose reductase inhibitor (ARI) ONO-2235 (Sango et al., 1999). The mechanism of this therapeutic effect of ARI remains unclear, although the function of receptors for extracellular matrix might be ameliorated. Extracellular matrix proteins are glycated under long-term hyperglycemia. Indeed, the presence of advanced glycosylation end products was shown in the nerve of STZ-induced diabetic rats (Yagihashi et al., 1992) and diabetic patients (Sugimoto et al., 1997). Glycation may prevent binding of laminin to members of the integrin family and thus compromise its metabolic activity. It was reported that nonenzymatic glycosylation of laminin and the laminin peptide IKVAV inhibited neurite outgrowth by cultured neuroblastoma cells (Federoff et al., 1993). Immunohistochemical study showed the labeling for laminin in the basal laminae of blood vessels and Schwann cells. In the perineurium, it was restricted to the innermost layer, where the amount

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of laminin appeared to be increased in the diabetic nerve compared with the control nerve (Bradley et al., 2000). 2.3.2. Fibronectin Fibronectin, a dimer of 440 kDa, is a major constituent of extracellular matrix in the developing PNS and contributes to nerve regeneration. It is also found in soluble form in plasma, which leaks into the endoneurium from endoneurial microvessels immediately after nerve injury (Salonen, 1987). There are various related isoforms because of RNA splicing and posttranslational modications. Its neuronal receptors are molecules in the integrin family (Reichardt et al., 1989). During nerve development and regeneration, axonal integrin 51, which is localized in the lopodia and central region of growth cones (Yanagida et al., 1999), is thought to bind to bronectin and interact with actin lament (Lefcort et al., 1992). Non-neuronal cells from the sciatic nerve express increased levels of mRNAs for bronectin and type I and IV collagens under high glucose concentration (Muona et al., 1991). By imunohistochemistry, the labeling for bronectin was noted in the basal laminae of blood vessels and Schwann cells and in those of the perineurial cells throughout all layers in the sural nerve. The distribution and staining of bronectin was almost similar among normal nerves, the nerves from diabetic neuropathy and those from other neuropathies (Bradley et al., 2000). 2.3.3. Collagen Type IV collagen is another major constituent of the basement membrane and also supports nerve regeneration. Under high glucose condition, mRNAs of pro-1 and pro-2 type IV collagen and pro-1 type I collagen chains have been reported to be increased in Schwann cells and perineurial cells (Muona et al., 1991). Indeed, increased synthesis of type IV collagen might make a massive deposit of microbrils between perineurial cell layers (Muona et al., 1993; Bradley et al., 2000). Circular basal laminal tubes after axonal degeneration are characteristically observed in diabetic polyneuropathy (King et al., 1989). In normal condition, these Schwann cell basal laminal tubes collapse following the removal of the axonal and myelin debris. By contrast, these tubes are lled with densely packed collagen brils in diabetes, forming circular basal laminal tubes. These brils may impede nerve ber regeneration. The diameter of endoneurial collagen brils is increased in diabetic BB Wistar rats (Muona et al., 1989). Although the signicance of this nding remains unclear, this is also found in patients with hereditary motor and sensory neuropathy as well as in diabetic patients, suggesting that the increase in the diameter of collagen brils is not specic for diabetic neuropathy but merely a part of nerve degeneration (Bradley et al., 2000). Among various types of collagen, type IV collagen was increased in the endoneurium in diabetic patients (Muona et al., 1993; Bradley et al., 2000). The deposit of collagen brils per se might prevent nerve regeneration. In addition, glycated collagen, which is expected

to be increased in diabetic condition, is resistant to protease digestion, which could be enhanced in regenerating axons (Lubec and Pollak, 1980), possibly leading to impaired nerve regeneration in diabetic neuropathy. 2.3.4. Matrix metalloproteinases (MMPs) Both tissue repair and elongation of axons play important roles in the process of nerve regeneration. Proteases participate in the former event; matrix metalloproteinases (MMPs) are one group of proteases involved in wound healing. MMPs are expressed within various cells in inammatory lesions. The substrates for MMPs include extracellular matrixes, including type IV collagen and bronectin. In the PNS, both MMP-2 and MMP-9 are known to be expressed during nerve regeneration after crush injury (La Fleur et al., 1996). Sensory neurons responsive to NGF in DRG express MMP-2 (Muir, 1994). Schwann cells and perineurial cells also express MMP-2 (Kherif et al., 1998). Remodeling of the extracellular matrix by proteolytic activity is known to be crucial for growth-cone motility. Neurite outgrowth of DRG neurons was reported to be suppressed by MMP inhibitor and inversely activated by NGF treatment accompanied with increased expression of MMP-2 (Muir, 1994). However, in vivo study showed the discrepant results that MMP inhibitor BB-1101 had no effects on compound muscle action potential recorded from denervated extensor digitorum or morphometric results on the diameter of regenerating axons (Demestre et al., 1999). Extracellular matrix is glycated in diabetic condition. Glycated type IV collagen is resistant to proteolysis by MMPs (Mott et al., 1997). This may be true for the basement membrane of the epidermis. Our skin biopsy studies suggest that either epidermal nerve ber number or length, which are decreased in diabetic patients, is unable to be elongated by treatment with aldose reductase inhibitor, whereas dermal nerve ber length is able to be elongated (Hirai et al., 2000; Yasuda et al., 2000). Since epidermal reinnervation is obtained mainly by the reentry of dermal nerves into the epidermis through the basement membrane of epidermal basal cells, it is likely that glycated extracellular matrix prevents the penetration of regenerating or sprouting nerve bers into the epidermis, although there is no clear evidence. 2.4. Cell adhesion molecules Interactions of cell membranes with adhesion molecules expressed either on other cell membranes or on extracellular matrices may play a potentially important role in the morphogenesis of the nervous tissue. Among cell adhesion molecules (CAMs), neural cell adhesion molecule (NCAM), L1 cell adhesion molecule (L1) and myelin-associated glycoprotein (MAG) all belong to the immunoglobulin superfamily and are best characterized. The former two molecules and N-cadherin are expressed on axons and Schwann cells. These molecules are involved in the axon-to-axon and axon-to-Schwann cell attachments using

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homophilic interactions. Axonal integrins such as 11 and 61, which function as receptors, mediate the attachment between axons (integrin) and Schwann cell basal laminae (laminin) using heterophilic interactions. Growing cultured neurites are slowed by the presence of antibodies against these proteins. The level of CAMs in Schwann cells are decreased with myelination and re-expressed at high levels during Wallerian degeneration after nerve transection. These observations suggest that CAMs contribute to membrane interactions with other membranes or matrix during development and axonal regeneration after injury. 2.4.1. Neural cell adhesion molecule (NCAM) NCAM is the rst adhesion molecule that was identied in the nervous system, and it is known that NCAM has an important role in axon guidance and projection to targets. Therefore, changes in its expression or nature may provide a signicant inuence on the function of nerve bers. It was already reported that NCAM, tenascin and N-cadherin were signicantly up-regulated, whereas polysialic acid was signicantly decreased with direct and indirect enzyme-linked immunosorbent assays (ELISAs) in diabetic rats, although no differences were detected by immunohistochemistry between diabetic (6-month BB/W) and control rats. In view of the fact that impaired nodal Na+ currents are associated with displacement of nodal Na+ channels across the damaged paranodal barrier, which is made up of adhesion molecules, these data may suggest that imbalances between highly interactive molecules responsible for the adhesiveness between the terminal Schwann cell loop and paranodal axolemma in diabetes may underlie the critical paranodal barrier defect in diabetic neuropathy (Merry et al., 1998). In addition, since glycating hexoses, including glucose and fructose, bind to the lysine residue of peptides, which is the amino acid residue to which polysialic acid associates with NCAM, it may be possible that increasing glycation of NCAM may prevent the interaction between polysialic acid and NCAM, thereby diminishing the adhesiveness of junctional complexes. 2.4.2. L1 cell adhesion molecule (L1) L1 is a cell adhesion molecule that is expressed on the surface of developing axons, growth cones, and Schwann cells of unmyelinated bers (Martini and Schachner, 1986) and is involved in neurite outgrowth, migration and fasciculation. These actions are elicited by its homophilic and heterophilic interactions (Lagenaur and Lemmon, 1987; Lemmon et al., 1989). The homophilic interactions on growth cones activate the broblast growth factor (FGF) receptor, which initiates arachidonic acid production, Ca2+ inux, cytoskeletal rearrangements and neurite outgrowth (Williams et al., 1994). The cytoplasmic domain of L1 can bind ankyrin (Davis, 1994) and may provide a mechanism for transducing extracellular signals and dynamic cytoskeletal rearrangements required for cell migration (Burden-Gulley et al., 1997). L1 heterophilic interactions with TAG-1/axonin-1,

DM1-GRASP and 13 integrin have also been implicated in neurite outgrowth (Kuhn et al., 1991; DeBernardo and Chang, 1996), although the downstream mechanisms are not well understood. An analysis of L1-decient mice showed that axonal-L1 maintains Schwann cell ensheathment of adult sensory unmyelinated axons by heterophilic binding mechanisms and that loss of axonal-L1 resulted in axonal degeneration (Haney et al., 1999). Although L1 is a membrane glycoprotein expressed on neural cells, the soluble form of L1 is generated in vivo by proteolysis. The soluble form of L1 without cytoplasmic and membrane spanning domains, which is secreted from a stable transfectant of CHO cells, induced neurite outgrowth of explants from embryonic chick brain stem comparable with that with substrate-bound L1 (Sugawa et al., 1997). It was also reported that cerebellar neurons responded to a soluble recombinant L1-Fc chimera by extending longer neurites than controls. The response was inhibited by pretreating neurons with antibodies to L1 (Doherty et al., 1995). These data suggest that the ability of CAMs to stimulate neurite outgrowth can be dissociated from their ability as substrate-associated adhesion molecules and point to the potential of using the soluble form of L1 to promote nerve regeneration. In the PNS, in uninjured animals, L1 and its close homologue CHL1 mRNAs were expressed at moderate levels by small- to medium-sized sensory neurons, and L1 mRNA was expressed at moderate levels by motor neurons (Zhang et al., 2000). Many large sensory neurons expressed neither L1 nor CHL1 mRNAs, and motor neurons expressed little or no CHL1 mRNA. Neither up-regulation of L1 mRNA in all neurons nor that of CHL1 mRNA was found after axotomy. CHL1 mRNA was transiently increased following sciatic nerve crush and declined to control levels. CHL1 mRNA was also up-regulated by many presumptive Schwann cells in injured sciatic nerves. There has been neither data on the expression of L1 nor reports on its contribution to impaired nerve regeneration in diabetes. 2.4.3. Myelin-associated glycoprotein (MAG) MAG, a well-characterized myelin protein, is a bifunctional molecule: (1) inhibiting neurite outgrowth from both developing cerebellar and adult DRGs in vitro (Mukhopadhyay et al., 1994) and axonal regeneration (Tang et al., 1997) and sprouting (Shen et al., 1998); or (2) promoting neurite outgrowth from newborn DRG (Mukhopadhyay et al., 1994). MAG knockout mice reveal that MAG is not essential for the initiation of myelination, although it plays an important role in maintaining a stable interaction between axon and myelin (Bartsch et al., 1997). The distal segment of the crush-injured sciatic nerve showed a decrease in the level of MAG mRNA 2 days after crush injury, which was followed by its progressive increase between 7 and 21 days after injury. By Western blot, the level of MAG protein was shown to be substantially decreased between 7 and 21 days

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after injury (Gupta et al., 1990). By contrast, both mRNA and protein of MAG were undetectable in the distal segment of the sciatic nerve 35 days after permanent transection, suggesting distinct down-regulation of MAG gene expression after permanent transection of the peripheral nerve (Gupta et al., 1990). Thus, the expression of MAG may play a signicant role in the process of nerve regeneration. Myelin is synthesized about the time of birth. The Src-family tyrosine kinase Fyn, which is present in myelin-forming cells, is activated through stimulation of cell surface receptors such as large myelin-associated glycoprotein (L-MAG) at the initial events of myelination (Umenomori et al., 1994). Myelin basic protein (MBP), which is a major myelin-specic protein and plays special roles in the initial stages of myelinogenesis, is signicantly reduced in Fyn-decient mice. In addition, Fyn has been shown to stimulate the promoter activity of the MBP gene, suggesting an important role of Fyn in myelination through transactivation of the MBP gene (Umenomori et al., 1999); although, the possibility that MAG and Fyn act independently to initiate myelination has been proposed in that possible compensatory mechanisms other than MAGFyn signaling pathway may well explain a slight hypomyelination in MAG-decient mice (Bifger et al., 2000). Although the expression of MAG in diabetic nerves does not differ from that in control nerves, the expression of MAG during Wallerian degeneration after nerve crush is larger in diabetic than control nerves (unpublished observations). This increase may contribute to impaired nerve regeneration in diabetic state. 2.4.4. Others Na+ /K+ -ATPase plays an important role in peripheral nerve function. Three isoforms of catalytic subunits and two isoforms of glycosylated subunits of the enzyme have been identied (Shull et al., 1986), and all distribute in a cell- and tissue-specic manner. Nerve injury experiments using immunohistochemical and Western blot analysis have revealed that 3 and 1 isoforms are exclusive for axons and 2 and 2 isoforms are exclusive for Schwann cells, although axonal contact regulates 2 and 2 isoform expressions; after sciatic nerve injury, 3 and 1 isoforms completely disappeared from the distal segment, whereas 2 and 2 isoform expressions are markedly increased in Schwann cells in the distal segment of the injured sciatic nerve, followed by a return to the baseline with nerve regeneration (Kawai et al., 1997). Because the 2 isoform is known as an adhesion molecule on glia (AMOG) (Gloor et al., 1990), increased expression of AMOG/2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/2 may act as an adhesion molecule in peripheral nerve regeneration. Although possibly altered AMOG/2 expression in diabetes may contribute to disturbed nerve regeneration, there have been no data on its expression in diabetic nerves.

The serum levels of soluble forms of CAMs including intercellular adhesion molecule-1 (sICAM-1) have been reported to be elevated under poor glycemic control and to be reversed by intensive insulin treatment. It was reported that plasma CAMs might be a predictor of the development of diabetic neuropathy (Jude et al., 1998): in a 5-year follow-up study of 28 diabetic patients, they found that both P-selectin and ICAM-1 were increased at baseline in patients with neuropathy compared to non-neuropathic patients. P-selectin and E-selectin were also found to be signicantly higher at baseline in patients who at follow-up showed deterioration in peroneal nerve conduction velocity of more than 3 m/s. P-selectin and ICAM strongly correlated with the velocity. Univariate and multivariate regression analyses showed a signicant inverse association between increasing log P-selectin, log E-selectin and log ICAM-1 with decreasing velocity. This was true even after adjustment for glycemic control. P-selectin and E-selectin were signicantly associated with the risk of deterioration of the conduction velocity after 5 years. These results suggest an important role of CAMs in the development and progression of peripheral neuropathy in diabetes mellitus. However, it remains unclear how important they are and whether they are associated with the process of nerve regeneration in diabetic condition. 2.5. Cell signal messengers 2.5.1. cAMP Cyclic AMP, synthesized from ATP by adenylate cyclase, is a second messenger of various cellular signals. In neuronal cells, adenylate cyclase is associated with receptors of various molecules, such as neurotransmitters, neurotrophic factors, prostaglandins, and others. The activated adenylate cyclase promotes synthesis and accumulation of cAMP in neuronal cells, and the increased cAMP activates protein kinase A. There is increasing evidence that the activation of cAMP-signal promotes neuronal survival and axonal elongation in neuronal cells. In a series of experiments, Roisen et al. (1972) reported that dibutyryl cAMP, a cAMP analogue, promoted the in vitro elongation of neurites from chick sensory ganglia. In addition to the in vitro effect, intramuscular injections of this compound enhanced signicantly the regenerative rate of sensorimotor nerves in either crushed or hemisected rat sciatic nerves (Pichichero et al., 1973). Furthermore, the compound accelerated the initial process of Wallerian degeneration and enhanced the rate of increase in number and diameter of myelinated nerve bers (Gershenbaum and Roisen, 1980). However, discrepant results also have been reported; McQuarrie and Partlow (1976) found no effect of dibutyryl cAMP on the rate of axonal growth of crushed rat sciatic nerve and Black and Lasek (1979) did not observe enhanced axonal regeneration in crushed rat sciatic nerve with dibutyryl cAMP using axonal transport as an index of nerve elongation.

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Fig. 5. The effects of increasing intracellular cAMP content by administration with prostaglandin E1 (PGE1), dibutyryl cAMP etc. Various insults including high glucose and hypoxia exert deleterious effects including arrest of axonal extension and apoptosis on neurons through activation of JNKc-Jun pathway. Increase of intracellular cAMP content has the preventive effects through activating protein kinase A (PKA).

Similar to cAMP, forskolin, adenylate cyclase activator, was also reported to increase cAMP concentration in either proximal or distal stump of freeze-lesioned sciatic nerves and stimulate peripheral nerve regeneration in Rana pipiens (Kilner and Carlsen, 1984). Prostaglandin E1 (PGE1) increased cAMP content in neuroblastoma cells and enhanced neurite outgrowth (Lando et al., 1990) (Fig. 5). These in vitro and in vivo observations reveal that accumulation of cAMP in neurons has an important role in axonal elongation. Although the mechanism of axonal elongation remains unclear, cAMP-induced neurite outgrowth may be independent of NGF-stimulated neurite outgrowth, and both cAMP and NGF have complementary effects but function through different mechanisms to stimulate neurite outgrowth (Simeone et al., 1994). The induction of microtubule-associated proteins (MAPs) (Sano et al., 1990), increased production of mRNA for -tubulin and MAP2 (Simeone et al., 1994) or reorganization of cytoskeletal proteins including F-actin (Sano and Iwanaga, 1992; Bolsover and Gilbert, 1992) are elicited by activation of cAMP-dependent signaling pathways and may contribute to neurite outgrowth. Cyclic AMP content has been reported to be reduced in the sciatic nerve of diabetic animals (Shindo et al., 1992). The signicance of decreased cAMP content in the pathogenesis of diabetic neuropathy remains unclear, although an amelioration of nerve cAMP content restores a decrease in nerve Na+ /K+ -ATPase activity (Shindo et al., 1993a,b), which is thought to be essential for maintaining nerve function. The mechanism by which cAMP content is

decreased in diabetic nerves is controversial; adenylate cyclase activity was decreased (Shindo et al., 1993a,b) or the activity of both adenylate cyclase and phosphodiesterase was not changed with a signicant lower response of cAMP production to isopreterenol, suggesting that the transduction from receptor to adenylate cyclase, which regulates cAMP production, may be disturbed in diabetic nerves (Maeda et al., 1994). Based on experimental results that cAMP ameliorates decreases in Na+ /K+ -ATPase activity and nerve conduction velocity in diabetic animals, PGE1 and PGI2 have been tried for the treatment of human diabetic neuropathy with successful effects, although the clinical efcacy has not been substantiated in controlled randomized trials. On the other hand, the effect of cAMP on nerve regeneration has not been conrmed in diabetic nerves. However, our recent experiment on the effect of PGE1, which increases cAMP content in DRG neurons, on nerve regeneration after nerve crush injury may provide promising information (Kogawa et al., 2000). The nerve regeneration distance was reduced in diabetic rats at 7 days after crush injury of sciatic nerve. The PGE1 treatment ameliorated its impairment and a decrease in cAMP content of DRG. This result may indicate that the restoration of cAMP content may contribute to an amelioration of nerve regeneration after nerve crush injury in diabetic rats. However, in view of the observations that compounds, including cilostazol (Yamamoto et al., 1998) and angiotensin II AT1 receptor antagonist (Love et al., 1995), which are able to ameliorate nerve blood ow, restore a defect

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of nerve regenerative capacity in diabetic rats, an amelioration of nerve blood ow with PGE1 treatment may also contribute to an improvement of disturbed nerve regeneration in diabetes. Thus, cAMP may be one of potential target second messengers for the treatment of diabetic neuropathy. Aside from nerve regeneration, cAMP plays a role in preventing apoptosis under several conditions as described in other sections. 2.5.2. Protein kinase C (PKC) PKC family of enzymes is activated by the diacylglycerol resulting from receptor-mediated hydrolysis of inositol phospholipids. PKC participates in a variety of functions, including signal transduction, regulation of ion channels and neurotransmitter release, control of cell growth and differentiation, and changes in cell morphology and gene expression. PKC immunoreactivity has been demonstrated in the afferent and efferent nerve endings of the PNS. PKC has several isoforms, and the existence of some of these in the peripheral nerve was conrmed by immunoblotting (, I, II, , , , , , , , ) (Khasar et al., 1999; Roberts and McLean, 1997) or immunohistochemistry (, , , , , ) (Okajima et al., 1995; Kawano et al., 1997). The expression and distribution of PKC isoforms are changed under pathological conditions (Roberts and McLean, 1997; Okajima et al., 1995). For example, the immunoreactivities of these isoforms were patchy in the axolemma and subaxolemmal peripheral zones of myelinated nerves, whereas they distributed diffusely throughout the axoplasm of axonal sprouts and growth cones after nerve crush (Okajima et al., 1995; Kawano et al., 1997). Several substrates that possibly are responsible for some of PKC-specic biological effects have been identied. GAP-43, one of the substrates, is strongly expressed in developing and regenerating axons, especially in their growth cones, and preferentially phosphorylated by the II isozyme (Sheu et al., 1990). Thus, each PKC isozyme phosphorylate its specic target protein. PKC may also promote neurite outgrowth on the extracellular matrix by affecting integrin interactions with laminin. Indeed, PKC inhibitor chelerythrine reduced anterograde and retrograde axonal transport and prevented neurite growth in cultured isolated mouse DRG neurons, suggesting that PKC activity is required to maintain axonal transport and thereby neurite growth (Hiruma et al., 1999). Thus, PKC may play an important role in phosphorylation necessary for the elongation of growth cones. However, the observation that PKC inhibition with chelerythrine arrested only short-term axon growth in adult mice sensory ganglia cultured in an extracellular matrix gel which was subsequently recovered, whereas a combination of chelerythrine and HA-1004, protein kinase A/G inhibitor, induced the permanent inhibition of axonal growth, suggests that compensatory mechanisms associated with other kinases than PKC function in the regulation of axonal growth by protein kinases (Wiklund and Ekstrom, 1999).

The effect of high glucose or hyperglycemia on PKC may be tissue and isoform specic. PKC activity was increased in the retina, aorta, heart, and glomeruli, but not in the brain and peripheral nerves. Total and II isozyme immunoreactivity were decreased, whereas total I and isozyme immunoreactivity were unchanged in diabetic nerves (Roberts and McLean, 1997), although no difference in their immunoreactivities among isozymes was reported in diabetic nerves (Borghini et al., 1994). Although there has been controversy regarding the activity of PKC in the peripheral nerve in diabetes, the recent report that PKC inhibitor ameliorates decreases in both nerve conduction velocity and nerve blood ow in STZ-induced diabetic rats suggests that this amelioration may be obtained through improving nerve blood ow possibly via inhibiting PKC activity of endoneurial microvasculature by this inhibitor (Nakamura et al., 1999). In addition, although Nakamura and colleagues could not show any difference in the activities of each PKC isozyme among the groups treated with or without PKC inhibitors, the in vitro assay of PKC activity might not precisely reect its in vivo activity. Another concern is that each isoform may show different activity, expression and/or distribution in diabetic state; it was reported that total PKC immunoreactivity was unchanged with its signicantly reduced cytosolic fraction, whereas total and cytosolic PKCII immunoreactivity were reduced in diabetes, and nerve crush increased only PKC immunoreactivity (Roberts and McLean, 1997). 2.5.3. Mitogen-activated protein kinases (MAPKs) MAPKs play an instrumental role in the transmission of signals from cell surface receptors and of environmental cues to the transcriptional machinery (Cobb and Goldsmith, 1995; Karin and Hunter, 1995). MAPKs are activated in response to extracellular stimuli through dual phosphorylation at conserved threonine and tyrosine residues (serine/threonine specic kinases). In vertebrae, at least three such pathways have been identied; these activate different MAP kinase classes known as extracellular signal-related kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 (Treisman, 1996; Robinson and Cobb, 1997). MAPKs have key roles in cellular proliferation, differentiation, and apoptosis. Previous studies indicated that ERK1/2 had an opposite role to JNK and p38 in neuronal survival; ERK1/2 promotes neuronal survival, whereas JNK/p38 brings the neuronal cell to apoptosis (Xia et al., 1995). However, it is controversial whether even JNK/p38 induces neuronal cells to apoptosis or to survival. Three major hypothetic pathways in the pathogenesis of diabetic complications including enhanced sorbitol pathway, non-enzymatic glycation of proteins and increased oxidative stress may contribute to the activation of MAPK, leading to tissue dysfunction through the phosphorylation of transcription factors. In addition, PKC, which has recently been shown to be a potent candidate for diabetic complications (Haneda et al., 1997), is able to activate MAPK (Haneda et al., 1995, 1997). Thus, MAPK may act as a signal

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Fig. 6. Schematic representation of neurotrophin signal transduction. Neurotrophins use two types of receptors, TrkA and p75NTR, to regulate the growth, development, survival and repair of the nervous system. These receptors collaborate with or inhibit each others actions to mediate neurotrophin effects. The signaling pathways include Akt and MAPK-induced signaling via TrkA and JNK and p53 via p75NTR. Neurotrophin binding to Trk stimulates receptor transphosphorylation, resulting in the recruitment of a series of signaling proteins to docking sites on the receptor. These proteins participate in activating MAPK through Raf. The pathways including MEK and ERK regulate neurite outgrowth. The pathways using RSK through ERK and using Akt through PI3 kinase are involved in cell survival, respectively, via subsequent activation of Bcl-2 and via inhibition of p53. Unlike Trk, our understanding of p75NTR is considerably lagged behind. However, there is growing evidence that several signal cascades implicated with p75NTR contribute to apoptotic changes in cells including neurons. It is known that p75NTR signals apoptosis in a Trk-independent fashion. TrkA activation silences p75NTR apoptotic signaling. p75 NTR signals apoptosis in a few ways. Either p75NTR activation or NGF withdrawal activates JNKp53Bax pathway. Elevation of ceramide is reported to inhibit NGF-dependent growth of distal sympathetic neurites. p75 is likely to regulate axonal growth either positively or negatively, depending on the proportion of unliganded to liganded p75NTR present in the local microenvironment. In addition, p75NTR binds a number of interacting proteins which may be involved in cell apoptosis.

transducer and trigger all the cellular events in the development of diabetic complications, including neuropathy, possibly in part by affecting nerve regenerative capacity (Tomlinson, 1999) (Figs. 6 and 7). 2.5.3.1. c-Jun N-terminal protein kinase (JNK). There have been few studies that examine the role of MAPKs in peripheral nerve regeneration in vivo. Kenny and Kocsis (1998) reported that c-Jun was activated in rat DRG after axon injury. They revealed that c-Jun activation lasted for over 30 days in the axotomized DRG, whereas the activation diminished gradually in regenerating DRG after crush injury. Some animal studies indicated that axotomy, which mimics withdrawal of neurotrophic factor in vitro, induced JNK activation in DRG (Herdegen et al., 1998; Kenny and Kocsis, 1998). In fact, the recent studies showed that inhibition of JNK by PGE1 or JNK inhibitor prevented apoptosis of neurons with removal of neurotrophic factor (Fig. 5) (Kawamura et al., 1999; Maloney et al., 1998; Glicksman et al., 1998). In addition, it may be possible that inhibition of JNK itself may play a role in nerve regeneration.

Indeed, our recent study showed that inhibition of persistent JNK activation in DRG neurons after sciatic nerve crush injury was correlated with improvement of nerve regeneration in diabetic rats (Kogawa et al., 2000). PGE1 analogue prevented long lasting JNK signal activation in DRG of diabetic rats. JNK in DRG was activated at 1 day after crush injury in all groups. At 7 days after crush injury, JNK activity was reversed to the basal level in control and PGE1 treated diabetic rats. However, in untreated diabetic rats, the activation of JNK was observed even at 7 days after crush injury. These observations suggest that long lasting JNK activation in DRG of diabetic rats may impede nerve regeneration, and PGE1 analogue may promote nerve regeneration of diabetic rats through suppressing the long lasting JNK activation. Recently, Fernyhough et al. (1999) showed that neurolament (NF) was aberrantly phosphorylated by activated JNK in DRG of diabetic rats. It is known that hyperphosphorylation of NF is implicated in human neurodegenerative disorders including Alzheimers disease, Parkinsons disease, and amyotrophic lateral sclerosis. Thus, long-lasting

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In diabetes, ERK phosphorylation was increased in DRG but not in the sural nerve (Fernyhough et al., 1999), implying an absence of any direct association of ERK with NF phosphorylation. The relationship between impaired nerve regeneration and ERK1/2 activity in diabetic rats remains to be studied. 2.5.4. Cyclin-dependent kinases and small GTPases The cell division cycle gene cdc2 in ssion yeast plays an essential role in both G1/S and G2/M transitions (Murray and Kirschner, 1989). The protein product of the gene is a catalytic subunit of serine/threonine protein kinase and associates with specic cyclins to form functional protein kinases. Mammalian cells contain a large number of cdc2 homologous proteins called cyclin-dependent kinases (Cdks), as well as an extended family of cyclins (Sherr, 1993). The cdc2-like protein kinases have functions other than cell division; a novel cdc2-like kinase is a heterodimer of a cdc2-related protein cyclin-dependent kinase-5 (Cdk5) and 35 kDa regulatory subunit (p35) (Lew et al., 1994; Tsai et al., 1994). Cdk5 has been shown to phosphorylate NFs (Hellmich et al., 1992), thereby being implicated in neurodegenerative changes, and is likely to play a role in neurite outgrowth and neuronal differentiation. Temporal and spatial patterns of expression of Cdk5 and p35 have been demonstrated in the CNS (Tsai et al., 1993). They may play a pivotal role in cytoskeletal dynamics and nerve regeneration in the PNS (Nikolic et al., 1996; Ohshima et al., 1996). Although their expression pattern and kinase activity in the PNS have been claried (Terada et al., 1998a,b), the possible changes and their signicance have not been elucidated in diseased conditions, including diabetes. Small GTPases have been described as molecular switches, which play a role in signaling of the extracellular stimulation to intracellular downstream effectors. In particular, the Rho family of small GTPases, such as Rho, Rac and cdc42, is implicated in a variety of cellular functions, including cell migration (Zipkin et al., 1997), cytoskeletal actin organization (Ridley and Hall, 1992) and cell adhesion, in response to extracellular stimulation (Ridley and Hall, 1992). In neuroblastoma cells, neurites are promoted to be extended by activated Rac/cdc42, whereas they are induced to be retracted by activated Rho (Kozma et al., 1997; Leeuwen et al., 1997). Thus, the Rho-family GTPases play an important role in neurite formation. The p21-activated kinase (pak) families of serine/threonine kinases are targets for active Rac and cdc42 in rat tissues, including brain (Manser et al., 1994). Phosphorylation of pak induces its conformational change leading to an increase in its kinase activity (Manser et al., 1994). In PC12 cells, pak was demonstrated to regulate neurite outgrowth and to be necessary for nerve growth factor-induced neurite formation (Daniels et al., 1998). It was also demonstrated that both Rho-family GTPases and pak existed in the PNS and that Rac/cdc42pak signaling pathway functions through mediating some intracellular signals (Terashima et al., 2001).

Fig. 7. Hypothetical mechanism of diabetic complications suggesting the possible roles of mitogen-activated protein kinases (MAPKs) in the development of the complications including diabetic neuropathy (modied synopsis from the reference by Tomlinson, 1999). Hyperglycemia exerts several metabolic effects on cells, leading to activation of MAPKs, which may be involved in cell functional or degenerative changes through activation of transcription factors.

activation of JNK pathway in DRG of diabetic rats after crush injury may be responsible for the degenerating process, like other neurodegenerative diseases. 2.5.3.2. Extracellular signal-related kinase 1/2 (ERK1/2). In various mammalian cells, ERK1 (42 kDa) and ERK2 (44 kDa) are activated by growth factors. In neuronal cells, neurotrophic factors such as NGF and BDNF activate ERK1/2 and induce the cells to proliferation or differentiation. In fact, in the rat pheochromocytoma PC12 cell line, ERK activation is linked to induction of neuronal differentiation, which converts these cells to post mitotic cells similar to sympathetic neurons (Traverse et al., 1992). It is well-known that ERK1/2 is activated by means of its phosphorylation by MAPKK and that ERK1/2 is translocated to the nucleus. These processes are believed to be necessary for the functioning of ERK1/2 in cells. However, our immunohistochemical study reveals that phosphorylated ERK1/2 exists not only in the nucleus but also in the cytoplasm and distal site of axons (unpublished data). Even after sciatic nerve crush injury, the localization of ERK1/2 does not change in DRG neurons. This observation indicates that ERK1/2 works not only in the nucleus but also in the cytoplasm in vivo. It has been shown that ERK1/2 phosphorylates NF. After crush injury, ERK1/2 is activated in DRG neurons as well as distal to the crushed site (Svensson et al., 1995), where NF is hyperphosphorylated. Therefore, it is possible that ERK1/2 may phosphorylate NF and regulate its axonal transport in regenerating axons in addition to transcriptional control.

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In addition, the activated pak was reported to be able to activate the c-Jun N-terminal kinase (JNK) leading to transcriptional and cytoskeletal events in COS-1 cells (Bagrodia et al., 1995). It was also reported that the Rac/cdc42pak cascade contributed to the activation of the MAPK including ERK and JNK. Furthermore, the Racpak complex was shown to colocalize and associate with Cdk5/p35 at neuronal growth cone (Nikolic et al., 1998). Thus, Rho-family GTPasespak cascade may be acting in up-streams of both MAPK and Cdk5/p35 and, through affecting cytoskeletal dynamics, may be involved in morphological changes under pathological as well as physiological conditions in the PNS. Although these enzymes may be closely associated with the pathophysiology including nerve regenerative capacity in diabetic neuropathy, we do not have any data on these enzymes in diabetic state at the present time. 2.6. Immediate early genes 2.6.1. c-Jun The transcription factor c-Jun is one of the earliest and most consistent markers for neurons that respond to nerve

ber transection, and its expression can be related to both degeneration and survival including target reinnervation, known as bipotential mediator of neuronal death and regeneration (Herdegen et al., 1997a,b) (Fig. 8). Among a variety of inducible transcription factors that mediate cellular responses to environmental change, the c-Jun is the predominant transcription factor that is expressed following various neurodegenerative disorders, including axotomy, ischemic tolerance hypoglycemia, Alzheimers disease, NGF withdrawal and X-ray irradiation. c-Jun can dimerize with Jun, Fos, activating transcription factor (ATF) and other transcription factors to control numerous target genes and is known to participate in regulation of the cell cycle, cellular differentiation, organogenesis, tumor transformation and apoptosis. The level of c-Jun expression is closely linked to the intensity of the cell body reaction, which depends on the injured level, proximal or distal to cell body. The expression of GAP-43 and cytoskeleton proteins is associated with the response of the cell body of DRG neurons following axotomy (Jenkins et al., 1993; Chong et al., 1994). There is also evidence that c-Jun and GAP-43 are regulated in parallel following neuronal damage (Tetzlaff et al., 1994; Schreyer

Fig. 8. Neuronal response after nerve transection, which may depend on the expression of c-Jun, followed by that of other transcription factors and regeneration-associated protein like GAP-43. Proximal transection of nerve bers increases both the potency for regeneration and the risk for cell death. It is not clearly understood whether these two neuronal responses are in parallel but independently activated or are caused by a common signal cascade linked with certain transcription factors that decide upon survival or death.

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and Skene, 1993; Bisby et al., 1995). GAP-43 is a major growth-cone component, which unequivocally contributes to axon regeneration. Its gene has a highly conserved AP-1 site (Eggen et al., 1994), which can strongly modulate transcription of GAP-43 (Weber and Skene, 1998). Here, c-Jun homo- or heterodimers can directly regulate expression of axonal growth-cone components. c-Jun coexpresses with other effector proteins, including -amyloid precursor protein, tyrosine hydroxylase and galanin, which can be driven by AP-1 promoter sites and possibly play a role in neuronal survival or degeneration. Furthermore, c-Jun is also involved in the transcription of the large pool of genes regulated by CRE consensus sites and nuclear receptors (Kamei et al., 1996). We found that long lasting phosphorylation of c-Jun was observed in parallel with JNK activation in DRG and may contribute to decreased regenerative capacity of sciatic nerves of diabetic rats after crush injury (Kogawa et al., 2000). Although it is controversial whether c-Jun phosphorylation induces the neuron into degeneration or regeneration, our data might support that dephosphorylation of c-Jun is an important process in nerve regeneration. 2.6.2. c-Fos Unlike c-Jun, c-Fos is not clearly expressed after nerve injuries, including axotomy, ischemia and hypoglycemia. NGF withdrawal is an exclusive condition that enhances the expression of c-Fos. Facial motoneurons respond to peripheral transection of the facial nerve with a number of molecular changes. Northern blot analysis of RNA extracted from the facial nucleus revealed that axotomy resulted in a unique pattern of immediate early gene (c-Jun, Jun B, 12-O-tetradecanoylphorbol-13-acetate-induced sequence (TIS) 11 sequence mRNA) induction in the facial nucleus (Haas et al., 1993). Increased levels of these mRNAs were strongly induced in a long-term fashion after nerve injury. By contrast, c-Fos mRNA was neither expressed in the unoperated nucleus nor induced by axotomy (Haas et al., 1993). These data suggest that c-Fos may not play a role in triggering the regeneration program of motoneurons. No induction of c-Jun and marked induction of c-Fos were also reported in the rabbit retinal ganglion cells after optic nerve crush (Koistinaho et al., 1993). c-Fos may contribute to the nerve regenerative process under diabetic condition, although such studies have not been reported. 2.6.3. Activating transcription factor-2 (ATF-2) ATF-2 is a component of the transcriptional network that interacts with c-Jun activity and is involved with the fate of injured neurons. ATF-2 is known to play a role in neuronal protection because the targeted disruption of the ATF-2 locus results in the degeneration and disturbed migration of neurons in the CNS (Reimold et al., 1996). ATF-2, as well as c-Jun, is the major substrate in neuronal cells that are known to be phosphorylated by JNKs (Gupta et al., 1995). ATF-2 controls the transcription of c-Jun following stimula-

tion by cellular stressors, such as UV irradiation (Herr et al., 1994; van Dam et al., 1995a,b), and also forms active heterodimers with c-Jun, which enhance the activation of the c-Jun promoter (van Dam et al., 1995a,b). On the other hand, ATF-2 is down-regulated in damaged adult neurons following axotomy, ischemia and UV irradiation, and the suppression of ATF-2 occurs in parallel with the up-regulation of c-Jun (Herdegen et al., 1997a,b). Because of ATF-2 decay, c-Jun cannot form heterodimers with ATF-2 or compete with ATF-2 for formation of other heterodimers, thereby being unable to act as a substrate for JNK. Thus, ATF-2 might be involved in neuronal cell death by modulating c-Jun function. We found that the expression of ATF-2 was increased in DRG neurons of STZ-induced diabetic rats, although its signicance remains unclear (unpublished data). 2.7. Endoneurial microenvironment including vascularization Axons apart from neuronal cell bodies locally receive oxygen and nutrition. Thus, axons are much inuenced by local vascularization and oxygenation. Vasculitis either in the epineurium or in the endoneurium causes nerve ischemia followed by axonal breakdown. Similarly, nerve ischemia and/or decreased endoneurial oxygen tension are believed to be the major putative mechanisms in the pathogenesis of diabetic neuropathy (Low, 1987; Yasuda and Dyck, 1987; Dyck, 1989). In fact, endoneurial microenvironment, including nerve blood ow, has to be strictly regulated to maintain normal nerve function and promote the recovery process after nerve injury. Otherwise, nerve regenerative capacity as well as nerve function might be deteriorated. The observation that regenerating myelinating nerve bers (MNF) were less frequent in type 2 diabetes than in type 1 diabetes suggests that endoneurial vascularization may be one of the important determinant factors for endoneurial nerve regenerative capacity (Bradley et al., 2000). This has also been supported by the experimental observations. The rate of nerve regeneration after nerve infarction is slower than that following mechanical injury (Korthals et al., 1991). In addition, a vascularized nerve graft results in a better outcome than a non-vascularized graft (Shupeck et al., 1989). Vasotrophic drugs have improved the rate of nerve regeneration as well as nerve blood ow in diabetes (Maxeld et al., 1995; Kogawa et al., 2000), suggesting possible implication either of decreased hypoperfusion in disturbed nerve regeneration or of ameliorated nerve blood ow in restorated nerve regenerative capacity. On the other hand, the microvasculature is inuenced by the endoneurial milieu. The microvasculature is altered in the distal stump of axotomized nerves; increased permeability of blood nerve barrier (Weerasuriya, 1988) and dilatation of the vascular lumen have been reported (Podhajsky and Myers, 1993). In our morphological study, increases in vascular and fascicular area and arteriolization of posttransperineurial capillaries were observed in the endoneurium of

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STZ-induced diabetic rats at 3 months after transection of the sciatic nerve (Maeda et al., 1999). These observations suggest that unknown growth factor may induce angiogenesis in the injured endoneurium in diabetic condition. In fact, it was reported that arterio-venous shunting and proliferating new vessels were seen in the epineurium of the sural nerve in diabetic patients with acute painful neuropathy of rapid glycemia control (Tesfaye et al., 1996). A ne network of the latter vessels resembles the new vessels of the retina, which is believed to be caused by growth factors including vascular endothelial growth factor (VEGF), suggesting that the same event may develop in both tissues possibly under hypoxic condition associated with diabetes. Although endoneurial vascularization has not been accepted to be impaired in patients and animals with diabetes, some reports have shown that increased endoneurial angiogenesis is induced by vasodilators and is able to ameliorate nerve conduction velocity as well as nerve blood ow (Cameron et al., 1991, 1992). Four weeks after intramuscular gene transfer of plasmid DNA encoding VEGF-1 or VEGF-2, both vascularity and blood ow in the nerves of diabetic rats were recently reported to be signicantly improved to the level similar to those of non-diabetic control animals. Constitutive overexpression transgenes resulted in restoration of large and small ber peripheral nerve function (Schratzberger et al., 2001). These ndings raise the possibility of a novel treatment strategy for patients with diabetic neuropathy for whom conventional therapy cannot relieve disabling neuropathic symptoms. 3. Experimental studies 3.1. Relevance of examining nerve regeneration in experimental diabetic models From the morphological standpoint of view, pathological ndings reported in diabetic patients include axonal atrophy, demyelination, loss of nerve bers, and blunted regeneration of nerve bers (Sima et al., 1988a,b; Dyck and Giannini, 1996). The progressive nerve ber loss found in human diabetic neuropathy may be due, in part, to an impaired ability of the diabetic nerve to regenerate in response to the degenerative process. Number of nerve bers of the peripheral nerve is balanced between nerve degeneration and regeneration under non-neuropathic conditions. If nerve degeneration exceeds nerve regeneration in pathological conditions like diabetic neuropathy, nerve ber loss may progress. However, if the specic therapy decreases nerve degeneration and/or increases nerve regeneration, pathological ndings, including nerve ber degeneration and loss, could be improved. The goal in treating diabetic neuropathy is not only to prevent the progression of neuropathic symptoms and nerve dysfunction and degeneration (Kennedy et al., 1990; The Diabetes Control and Complications Trial Research Group, 1993) but also to promote regeneration of degenerated nerve

bers. Since the recovery of diseased nerve function depends on both regeneration of degenerated nerve bers and reestablishment of their functional connections with the target tissue, it is important to examine how diabetic condition inuences nerve regeneration and whether potential therapeutic agents can promote satisfactory regeneration of functioning nerve bers. 3.2. Evaluation of nerve regeneration in animal models Recently, ARIs, sorbinil and tolrestat, have been reported not only to improve clinical symptoms and neurophysiologic function, but also to increase nerve regeneration in human diabetic neuropathy (Sima et al., 1988a,b, 1993). Although these reports provide strong evidence that ARIs may be potent in promoting nerve regeneration in diabetic neuropathy, an animal model is necessary to precisely and objectively evaluate the ability of potential therapeutic agents to promote nerve regeneration. However, most animal models, including STZ-induced diabetic animals, have not been demonstrated to show nerve ber loss in the peripheral nerve. In addition, although some animal models including BB Wistar rats have been reported to show considerable nerve ber loss, the magnitude of their ber loss does not mimic human diabetic neuropathy (Sima et al., 1983; Sima and Sugimoto, 1999). Furthermore, it takes longer for nerve ber loss to occur in these animals and thus to evaluate the specic effects of test compounds on nerve regeneration. In this sense, nerve crush and cut model may be useful to study nerve regeneration, since nerve degeneration and regeneration after axotomy occurs in a predictable manner (Asbury and Thomas, 1978) (Fig. 9). 3.3. Degeneration and regeneration of the peripheral nerve In axotomy models, nerve bers regenerate after degenerated bers are cleared. Thus, successful nerve regeneration may be closely linked with the process of Wallerian degeneration (Fig. 10). 3.3.1. Cellular events in Wallerian degeneration The structural changes in Wallerian degeneration are divided into several stages: early axonal changes, macrophage recruitment, clearance of myelin and axon debris, Schwann cell responses, and behavior of the other neural cells. The changes begin with the enzymatic proteolysis of axonal cytoskeleton. In short, the cytoskeleton of transected axons undergoes granular disintegration within 2448 h. The Schwann cells stop making myelin proteins and discard their myelin sheaths. Next, circulating macrophages promptly enter the endoneurium. Here, they phagocytize myelin and axon debris together with Schwann cells, initiate mitosis and differentiation of Schwann cells, induce the production of several neurotrophic factors and extracellular matrixes via the release of cytokines, assist in recycling neural lipids, and set the stage for regeneration of nerve bers.

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Fig. 9. Evaluation of regenerating nerve bers after nerve injuries. (a) Motor nerve regeneration after sciatic nerve crush is evaluated using electromyography; the sciatic nerve is stimulated at the sciatic notch (proximal to the crush site) and tibial nerve is stimulated posterior to the ankle with steel needle electrode using supramaximal stimulation. Motor nerve conduction velocity and the peak-to-peak amplitude of the compound muscle action potentials (CMAPs) are measured. CMAPs are recorded from the plantar muscle with a unipolar needle electrode. (b) Sensory nerve regeneration after sciatic nerve crush is evaluated using pinch-reex test, in which beginning 30 mm distal to the crush site, nerves were lightly pinched with forceps at successive proximal 0.5 mm intervals until a reex contraction of the upper leg occurs (the regeneration front). (c) Nerve sprouting from denervated motor nerve terminals within tensor fascie latae (TNL) muscle after cutting L4 ventral root is morphologically evaluated. Motor end-plates are visualized by means of a silver-cholinesterase stain; sprouting from L5 ventral root is quantitatively assessed by the percentage of end-plate with terminal sprouts and the length of these terminal sprouts with a computer-assisted digitizer.

Fig. 10. Schematic representation of nerve regenerating process after nerve injury like transection. Axons at the distal stump of the transected nerve undergo degenerative change of granular appearance, followed by recruitment of circulating macrophages into the endoneurium. Axons start to regenerate in several ways; axonal sprouting from the transected end of axons, collateral sprouting from nodes of Ranvier and terminal sprouting from synaptic endings. The former pattern is the main process; regenerating axons grow along the Bungner band and increase in diameter, followed by remyelination after they connect with the target. Eventually, partial or complete functional recovery is induced.

3.3.2. Early axonal changes The pivotal event in Wallerian degeneration is granular disintegration of the cytoskeleton (GDC). It begins near the site of axotomy and spreads down the axon with time. The axoplasm composed of NFs and microtubules is converted into granular and amorphous debris several hours to a few days after axotomy. GDC correlates with development of the degradation of NF protein to lower molecular weight products (Schlaepfer and Micko, 1978, 1979). GDC is triggered by an increase in axoplasmic calcium concentration and mediated by a neutral calcium-sensitive protease (Nixon and Logvinenko, 1986; Zimmerman and Schlaepfer, 1984), calpain II, which is activated by millimolar calcium concentrations (Nixon and Logvinenko, 1986). In normal nerves, intra-axonal calcium concentration is kept at a very low level (Baker et al., 1971). The calcium concentration gradient between intra- and extra-axonal uid is maintained by an active calcium pump and intra-axonal particulate organelles that act as calcium sink. Nerve transection results in a rise in intra-axonal calcium throughout the distal stump (LoPachin Jr. et al., 1990). Chelation of extracellular calcium delays the time of GDC (Schlaepfer, 1971, 1977). George et al. have shown that GDC is retarded by a variety of agents that inhibit extracellular calcium entry in vitro (George et al., 1995). These studies indicate that entry of extracellular calcium is critical to the process. 3.3.3. Schwann cell responses Schwann cell responses include entry into cell cycle, alteration in protein synthesis, and formation of Bungner bands (Grifn and Hoffman, 1992). In Wallerian degeneration,

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myelin sheaths are discarded by Schwann cells and separate into short segments within the internodes. These segments form ovoid and collapse and fold around the cavity occupied by the original axon. Here each myelin internode separates into 610 ovoids, which become shorter and are separated by intervening Schwann cell cytoplasm with time. By 3 weeks, most myelins in rodent nerves have been cleared. Next, Schwann cells divide and form long chains of cells called Bungner bands, which provide a substrate for axonal regeneration. Changes in protein synthesis of Schwann cells correspond to the start of cell division. They include down-regulation of most myelin and myelin-related proteins, including P0, P2, MBP, MAG, and peripheral myelin protein 22 and up-regulation of CAMs, including L1 and NCAM (Grifn, 1992; Poduslo, 1992). Synthesis of neurotrophins is increased; NGF production has been reported to be stimulated by activated macrophages via IL-1 (Lindholm et al., 1987). 3.3.4. Macrophage responses Normal peripheral nerves contain a few resident macrophages. In the distal stump of transected nerves, circulating macrophages promptly recruit into the endoneurium and migrate specically to degenerating bers (Stoll et al., 1989). Recruited macrophages initially appear around day 2 after nerve transection and its number increases sharply on day 4 in the rat sciatic nerve. They pass through the basal lamina of degenerating bers to become intratubal phagocytes and play a central role for pathway clearance together with Schwann cells. At this stage, they acquire the characteristic foamy appearance of postphagocytic macrophages. 3.3.5. Nerve regeneration There are several kinds of regeneration patterns, including axonal sprouting from the transected ends of axons, collateral sprouting from nodes of Ranvier and other sites along uninterrupted bers, and terminal sprouting from synaptic endings. Here, we focus on regenerative axonal sprouting from the transected ends of axons, but the principles of regeneration apply to the other forms of regeneration as well. Following axonal injury, the injured axons sprout growth cones mainly from proximal nodes within 324 h. Each axon generates multiple sprouts, but most of these disappear as axons connect with targets. The regenerating axons penetrate into the distal stump with a delay of 12 days under the proper condition. At the cell body of the injured neurons, many metabolic and structural changes take place. Some of these changes observed during the embryonic period seem to be necessary for regeneration. The regenerating axons grow along the Bungner band at rates of up to 4 mm per day and could grow back to the original target if their original basal lamina tubes have not been disrupted. The regenerating axons increase in diameter and remyelination occurs.

3.3.6. Axonal sprouting Shortly after nerve injury, regenerating axons sprout at nodes proximal to the injury site. When the regenerating distance of sciatic nerve axons were plotted against lapse of time after nerve injury, a best straight line tting indicated that axon outgrowth after nerve injury began after 3060 h, suggesting that there was an initial delay between the injury and start of regeneration (Forman and Bereberg, 1978; Bisby and Pollock, 1983). However, recent observations show that this long latent period following nerve injury is an exploration artifact. It can be explained by an initial lower rate of elongation and its acceleration 3 days after injury. Therefore, actual delay for sprouting is less than 3 h (Sjoberg and Kanje, 1990). 3.3.7. Growth cone and axonal elongation The growth cone is the extending terminal of the axonal sprout. The most favorable local environment is the denervated distal stump prepared for regeneration by the process of Wallerian degeneration. The various factors on growth cone behavior (growth factors, substrate, membrane contact) have been postulated to be integrated by means of growth cone calcium concentration (Rehder et al., 1992). The calcium concentration is regulated by both environmental and intra-axonal factors. Optimal outgrowth occurs within a small range of calcium concentrations, and it is likely that the various extrinsic inuences affect both rate of extension and intra-axonal calcium concentration (Kater and Mills, 1991; Al-Mohanna et al., 1992). The growth cone contains the special axonal cytoskeleton and the smooth membrane system. The lopodia and lamellipodia extend on a meshwork of actin microlaments. The microtubule cytoskeleton is the predominant cytoskeletal element in the preterminal axonal sprout. The GAP-43 is one of proteins enriched in growth cones. It is dramatically induced in nerve cell body by axotomy and transported to the growth cone by fast axonal transport. Skene et al. have reported that it is an essential constituent for growth cone extension (Skene and Willard, 1981a,b). There is extensive endocytosis and exocytosis within growing tips (Hughes, 1953; Tessler et al., 1980; Grifn et al., 1981). Many of these endocytotic materials are taken into larger vesicles, some of which return to the cell body via fast retrograde transport. These materials such as NGF can affect the protein synthesis in the cell body. On the other hand, some of these exocytotic materials may be a new membrane, which is carried by fast axonal transport and added at the growth cones through the calcium-mediated mechanism. In the regenerating sprout, cytoskeletal proteins including actin and tubulin are transported in slow components a and b (SCa and SCb), while NFs are carried by SCa. The rate of SCb is equal to that of axonal elongation (Wujek and Lasek, 1983). This observation suggests that transport velocity of these cytoskeletal proteins in SCb is a limiting factor in the rate of neurite extension.

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3.3.8. Cell body reaction Following axotomy, most surviving cell bodies show a variety of morphological changes and modications in gene expression and cellular metabolism. The main morphological change is central chromatolysis. The onset of regeneration is associated with expression of new genes and proteins. Synthesis of several growth-associated proteins is markedly increased as described above (Skene and Willard, 1981a,b). Synthesis of cytoskeletal proteins changes within the rst few days after axotomy. Nerve cell bodies show a substantial decrease in expression of mRNA and in synthesis for NF proteins (Goldstein et al., 1988). This is associated with a decrease in content of NF and a decrease in diameter of regenerating nerve bers. On the other hand, several other proteins are increased in synthesis. mRNA content for tubulin and actin is increased after axotomy (Hoffman and Cleveland, 1988). 3.3.9. Maturation of regenerating nerve bers Axonal maturation insufciently occurs before the growing axon reaches an appropriate target. Maturation includes an increase in axonal diameter, formation of the axonSchwann cell unit, and myelination in appropriate bers. In the maturation process, NF synthesis, which is reduced after axotomy, recovers and correlates with a slowly sustained increase in axon diameter (Bisby and Tetzlaff, 1992). Recovery of NF synthesis does not occur in the neurons that do not connect with their targets. Because axonal caliber is proportional to the number of NFs, nal axonal diameter could be determined by the amount of NF synthesis and its transport (Muma et al., 1991). However, axonal diameter is also determined by a local interaction between axon and Schwann cells, as shown by local changes in axonal diameter between nodes and internodes. Recent study has indicated that myelinating Schwann cells interact with the axon to modulate a balance of axonal NF kinase and/or phosphatase activities (de Waegh et al., 1992). Changes in the phosphorylation state of C-terminal Lys-Ser-Pro (KSP) region in NFs might affect the conformation of their side arms and NF packing densities, and thus axonal diameter. Associated with an increase in axonal diameter, the ensheathing Schwann cells show reburst of proliferation, resulting in the relationship of one axon per Schwann cell. This process is a necessary prelude for myelination. Although the axonal signal that induces remyelination is not claried, there is a critical relationship between axonal diameter and myelination (Voyvodic, 1989), and interactions between CAMs (L1, NCAM, MAG) may be involved, since antibodies to neural adhesion molecules abolish Schwann cell differentiation of Schwann cell processes into neurite bundles (Seilheimer et al., 1989). As remyelination advances, regenerating sprouts increase in axonal caliber. The thickness of their myelin sheaths cannot reach that of the original axons, even though axonal caliber may reach that of the parent.

3.4. Wallerian degeneration in experimental diabetes 3.4.1. Axonal degeneration and pathway clearance in diabetes It is well-known that Wallerian degeneration is a prerequisite for normal regeneration following injury to the peripheral nerve. For example, the C57BL/Wld mouse is a mutant substrain that has the distinctive phenotype of prolonged axonal survival. In this mouse, the decreased Schwann cell and macrophage responses after nerve transection lead to a marked retardation in regeneration of the sensory and motor axons (Bisby and Chen, 1990; Glass et al., 1993). Thus, a slow Wallerian degeneration could impair nerve regeneration. There have been a few reports about Wallerian degeneration after nerve injury in experimental diabetes. We examined the time course of axonal degeneration during Wallerian degeneration in the sciatic nerve of rats with STZ-induced diabetes (Terada et al., 1998a,b). Morphometric analysis on the distal stump of the transected sciatic nerve was performed at intervals after axotomy; following nerve transection, swollen axons rounded by thin myelin gradually disappeared, and other changes, which included myelin sheath degeneration and macrophage invasion and phagocytosis, developed later. We considered these morphologically characteristic axons to be the surviving axons and counted them. By quantitative analysis, the number and density of surviving axons were signicantly greater in diabetes than in controls. These results indicate that axonal degeneration during Wallerian degeneration is delayed in diabetes. 3.4.2. Degradation of cytoskeletal proteins in diabetes It has been reported that early morphological changes in axons and their clearance during Wallerian degeneration correlates with the development of cleavage and degradation of NF proteins. NF is a class of intermediate lament and is a major component of axonal cytoskeleton (Fig. 11). NFs are composed of three subunits: low (NF-L), medium (NF-M), and high (NF-H) molecular-weight NF proteins. NFs have been reported to be highly phosphorylated in vivo and occur primarily in the axon. The site of phosphorylation is located on serine residues in KSP motifs within the carboxyl-terminal tail domains of the NF-M and NF-H subunits (Elhanany et al., 1994). Although the specic function of NF phosphorylation is unknown, the incorporation of phosphate may regulate NF interactions (Hisanaga and Hirokawa, 1988), NF subunit assembly (Lee et al., 1993), NF calcium binding (Lafebre and Mushynski, 1987), and NF susceptibility to degradation by proteases (Pant, 1988). Axon contains calcium-activated neutral proteases called calpains, which rapidly degrade NFs, thereby playing a pivotal role in axonal degeneration during Wallerian degeneration (Kamakura et al., 1983). It has been reported that phosphorylated NF is resistant to degradation by calpains (Pant, 1988). Namely, the time course of axonal degeneration may depend on the amount of phosphorylated NFs.

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Fig. 11. Schematic representation of the rat neurolament molecules. A unique feature of NF-M and NF-H is the presence of extended carboxyl-terminal tails containing multiple repeats of the lysine-serine-proline (KSP) sequence (05 for NF-M and approximately 50 for NF-H) indicated by vertical bars. The KSP serine residues are NF-specic phosphorylation sites. The phosphorylation provides NFs with several functions indicated.

We have examined whether the phosphorylation state of NFs is altered in diabetic nerves and whether the rate of NF degradation is inuenced by phosphorylation of NFs in diabetic nerves (Terada et al., 1998a,b). The immunoreactivity of phosphorylated NF-H and NF-M was about two to three times greater in diabetic than in control nerves, whereas the immunoreactivity of unphosphorylated NF-H was lower in diabetic nerves (50%). At 5 and 7 days after nerve transection, the immunoreactivity of NF-M was higher in diabetic than in control nerves, suggesting that degradation of NF-M is delayed in diabetes. Simultaneously, the degradation of phosphorylated NF was also delayed in diabetic compared with control nerves, whereas unphosphorylated NF did not differ between the two groups. Increased NF phosphorylation was also reported in the spinal cord of STZ-diabetic rats (Pekiner and McLean, 1991). In addition, Fernyhough et al. (1999) have indicated that aberrant NF phosphorylation occurs in sensory neurons of diabetic rats using the same antibodies as we used. Axons contain calcium-activated neutral proteases called calpains that are implicated in axonal degeneration (Kamakura et al., 1983). NFs are rapidly degraded by calpains in the presence of sufcient levels of axonal calcium, leading to loss of the immunoreactivity for NFs (Schlaepfer and Micko, 1978). Thus, prolonged survival of transected axons in diabetic nerves may variously involve the alterations in calcium entry, calcium buffering, calpain activity, and/or stability of NF response to calpain, although it is not known whether each of these putative factors is altered in diabetes during axonal degeneration.

Immunoblot analysis showed a delay in the degradation process of NFs during Wallerian degeneration in diabetic condition. Phosphorylated NFs detected were more recognized in diabetes, while the opposite was true for unphosphorylated NFs. Since it is known that the sensitivity of NF to calpain-mediated proteolysis is modulated by its carboxyl-terminal phosphorylation state and phosphorylated NFs are resistant to calpains (Pant, 1988), we concluded that abnormal NF phosphorylation state in diabetes could be one of the mechanisms by which axonal degeneration was delayed. Several possible mechanisms may explain the increase in phosphorylated NFs. If content of NFs was simply increased in the sciatic nerve of diabetic rats, the immunoreactivity of phosphorylated NFs would be increased, resulting in a delay in their degradation. However, Medori et al. (1985) showed that an axonal cross-sectional area of the spinal nerve did not differ between control and diabetic rats 46 weeks after STZ injection. They also showed that diabetic axons maintained the number of NFs per cross-sectional area at the control level (Medori et al., 1988). Because the caliber of axons in large MNFs is determined by the number of NFs (Hoffman et al., 1984), it is unlikely that the content of NF is increased in diabetes. The observation that the levels of mRNA that encode NF-M and NF-H in DRG are reduced in rats with STZ-induced diabetes (Mohiuddin et al., 1995a,b) is also against an increase of NFs in diabetes. The excessive phosphorylation of NFs observed in STZ-diabetic rats could be due to an increase in the kinase activity of NFs, a decrease in the phosphatase activity of

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them, or both. Proline-directed protein kinases, such as Cdk5 (Shetty et al., 1993), glycogen synthase kinase-3 (GSK-3), GSK-3 (Yang et al., 1995), and MAPKs, including ERK (Veeranna et al., 1998), JNK (Giasson and Mushynski, 1996, 1997) and p38 kinases (Giasson and Mushynski, 1997), are potential candidates for NF kinases. Recently, Fernyhough et al. (1999) reported that DRG and sural nerve from rats with STZ-induced diabetes showed strong steady-state activation of both ERK and JNK. However, other candidate kinases are not known to be activated in diabetes. While the kinases that phosphorylate NF proteins have been characterized, less is known about protein phosphatases (PPs) in the nervous system. Veeranna et al. (1995) reported that the KSPXKX motifs of NF-H were selectively phosphorylated by Cdk5 and dephosphorylated by protein phosphatase 2A (PP2A). A decreased PP2A activity may induce hyperphosphorylation of KSP motifs of NF-H. Shea et al. (1993) reported that NF-H subunits from cytoskeletons of NB2a/d1 cells treated with the PP inhibitor okadaic acid were less susceptible to the degradation by calpain. However, it is not known whether the activity of PPs is altered in diabetes. A series of our experiments may be summarized as follows (Fig. 12). Axonal degeneration following injury to the peripheral nerve is delayed in STZ-induced diabetic rats. This delay may have originated from an increase in the resistance of NF to calpain induced by its hyperphosphorylation and may lead to impaired nerve regeneration. Although the mechanism of the increase in NF phosphorylation is un-

clear, we suggest that this excessive phosphorylation of NF may be due to an increase in its kinase activity, a decrease in its phosphatase activity, or both, either of which may be induced by metabolic abnormality in diabetic nerves. 3.4.3. Macrophage responses in diabetes The recruited macrophages phagocytize axonal and myelin debris together with Schwann cells, contributing to pathway clearance for nerve regeneration. Beuche and Friede (1984) investigated the extent to which non-resident macrophages participate in Wallerian degeneration and concluded that invading macrophages played a critical role in pathway clearance. To know the implication of macrophages in the delayed process of Wallerian degeneration in diabetic condition, the time course of macrophage recruitment during Wallerian degeneration in the sciatic nerves was examined and compared between control and diabetic rats (Terada et al., 1998a,b). By immunohistochemical analysis using avidin-biotinylated enzyme complex (ABC) method with monoclonal antibody against ED1, which was a marker for activated macrophages, the number of macrophages per fascicle was counted in the distal stump of the transected sciatic nerve at intervals up to 24 days after axotomy. Although the extent of macrophage recruitment was decreased at the late stage (15 and 24 days after nerve crush) of Wallerian degeneration in the diabetic nerve, the timing and extent of macrophage recruitment did not differ between the two groups at its early stage (9 days after crush). Because axonal degeneration had been already delayed as

Fig. 12. Suggested pathogenesis of decreased nerve regenerative capacity in diabetes. Phosphorylation of neurolaments (NF) which may be closely linked with increased activity of several kinases including cyclin-dependent kinase 5 (Cdk5), extracellular signal-related kinase (ERK) and c-Jun N-terminal kinase (JNK) is increased in diabetes. Since phosphorylated NF is resistant to digestion by proteases activated due to axonal injury, axonal degenerative process may be delayed. Recruitment of macrophages into the endoneurium is disturbed after early stage of Wallerian degeneration, possibly resulting in disturbed clearance process of axon myelin debris. The following processes including axonal regeneration and maturation of axons and remyelination may be impaired, leading to possible disruption of functional recovery in diabetes.

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early as 3 days after crush (Terada et al., 1998a,b), the delay in axonal breakdown rather than the decrease in the invasion of macrophages may be the primary cause for the observed delay in axonal degeneration in diabetes. Thus, the macrophage-associated process may not profoundly contribute to delayed Wallerian degeneration in diabetic condition. In STZ-diabetic mice, Kennedy and Zochodne (2000) showed that the macrophages, which recruited into the tibial fascicle, were fewer in diabetic than control mice at 2 weeks after crush, whereas their numbers and densities increased later and persisted at least up to 8 weeks. Simultaneously, although the regenerative delay of MNFs was not apparent at 2 weeks after crush, ber number and density were decreased and ber and axon diameter were smaller in diabetics at 8 weeks following crush, suggesting the possible role of abnormalites in macrophage participation in either nerve repair or nerve regeneration. 3.4.4. Schwann cell response in diabetes Schwann cell responses in Wallerian degeneration start from their proliferation, followed by changes in protein synthesis and formation of Bungner bands. Our experiments showed that the number of the nuclei of Schwann cells in the distal stump of transected sciatic nerves was not different between control and diabetic groups throughout the experimental period up to 24 days after axotomy, indicating that the mitotic ability of Schwann cells during Wallerian degeneration may not be disturbed in diabetic condition. 3.5. Nerve regeneration in experimental diabetes 3.5.1. Axonal sprouting, elongation and maturation in diabetes The regeneration of the peripheral nerve after nerve injury has been reported to be impaired in STZ-induced diabetic rats (Longo et al., 1986; Ekstrom and Tomlinson, 1989; Triban et al., 1989; Terada et al., 1996). This impairment is thought to be due to defects in one or more steps in the series of processes that include a delay in the start of regeneration, a reduction in the rate of regeneration, and an impairment of the maturation of regenerated nerve bers. Bisby showed that nerves from diabetic animals took signicantly longer to commence regeneration after nerve crush (Bisby, 1980). This nding indicates that there is a longer delay of duration between the injury and start of regeneration in diabetes. However, Ekstrom et al. (1989) showed an opposite result using a different method from Bisby. More rened methods may conrm a longer initial delay in diabetes. There are several reports as to the elongation rate of regenerating nerves after crush injury in diabetic animals. In general, rats with diabetes have a decreased rate of nerve regeneration. This nding is conrmed by several authors using different methods. Some authors employed a pinch-reex test which assessed both small myelinated and unmyelinated sensory bers (Ekstrom and Tomlinson,

1989, 1990; Ekstrom et al., 1989) and others morphologically evaluated nerve regeneration through silicone chambers (Longo et al., 1986; Tantuwaya et al., 1997) or used in vitro electrophysiological techniques, which assessed large myelinated motor bers (Terada et al., 1996; Love et al., 1995, 1996). Instead of nerve crush, the freeze-injury was also applied to sciatic nerves, and nerve regeneration could be evaluated along the sciatic/tibial nerves (Bondoux-Jahan and Sebille, 1989); this method may produce a more punctual lesion, which causes all bers of the nerve to degenerate, than nerve crush with forceps, providing a more standardized lesion. Impaired nerve regeneration in diabetic rats was also shown using this method (Love et al., 1995; Maxeld et al., 1995). Transected and reapposed nerves in spontaneously diabetic rats have also been reported to show a delayed regeneration (Kamijo et al., 1996). Moreover, the effects of several therapeutic drugs on reduced regeneration rate after axotomy in diabetes was evaluated; gangliosides (Triban et al., 1989; Ekstrom and Tomlinson, 1990), anti-oxidants (butylated hydroxytoluene) (Love et al., 1995), metal chelator (trientine) (Love et al., 1996), antiplatelet agent (cilostazol) (Yamamoto et al., 1998), ARI (ZD5522) (Love et al., 1995) and angiotensin II AT1 receptor antagonist partially or completely ameliorated a decit in nerve regeneration rate in diabetes, but ARI ponalrestat (Ekstrom and Tomlinson, 1989; Terada et al., 1996) did not. The number of regenerating nerve bers after axotomy in diabetes has been assessed by several researchers. Both immunohistochemical analysis with anti-NF antibody and morphometric study indicated that the number of regenerating bers was decreased in diabetic animals, and both gangliosides (Triban et al., 1989) and ARI WAY121-509 (Kamijo et al., 1996) restored this decrease. We also evaluated nerve regeneration, including axonal sprouting, elongation, and maturation, electrophysiologically and morphologically after nerve crush injury to delineate the ability of diabetic nerves to regenerate and to determine the effect of ARI tolrestat on nerve regeneration in diabetes (Terada et al., 1996). We performed nerve conduction and morphometric studies on three groups of experimental animals: control, untreated diabetic and tolrestat-treated diabetic rats. Motor nerve conduction study was done at intervals after sciatic nerve crush. This study showed that the reappearance of compound muscle action potentials (CMAPs) after crush injury was delayed by 1 week in untreated diabetic rats (5 weeks) as compared with control rats (4 weeks). CMAPs are thought to reappear when regenerating MNFs have reached their target organ. Therefore, its delay in reappearance may be attributed to the reduced rate of axonal growth and/or impaired reestablishment of the neuromuscular junction in diabetes. Furthermore, following the reappearance of CMAPs, persistent and signicant decreased motor nerve conduction velocity was observed in the distal stump of the crushed nerve in untreated diabetic rats as compared to control rats. These

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ndings suggest that the maturation process of regenerating MNFs is impaired in diabetes. Regenerating MNFs and unmyelinated nerve bers (UMNFs) were morphologically evaluated 5 and 24 weeks after crush injury. The density and number of MNFs per fascicle were signicantly decreased in untreated diabetic rats, but tolrestat signicantly prevented the former decrease at 5 weeks and the latter at 24 weeks. The mean diameter of large MNFs (>4 m) was smaller in the untreated diabetic group than in the control group, but this decrease was also signicantly prevented with tolrestat treatment. These morphometric studies indicated that both number and size of regenerating MNFs after crush were reduced in diabetes and that tolrestat partially ameliorated these defects. The potential mechanism by which tolrestat improves numbers and sizes of regenerating nerve bers may be explained as follows. Axon caliber is determined by a number of cytoskeletal proteins such as NF (Hoffman et al., 1984), which is regulated by slow axonal transport (Jacobsen et al., 1986). This component of axonal transport has been reported to be decreased in STZ-diabetic rats and to be restored with ARI. These results may be expected in the nerve regenerative process in diabetic condition. In addition, Schwann cells play an important role in nerve regeneration because they synthesize and secrete several neurotrophic factors, adhesion molecules, and extracellular matrixes (Snider and Johnson, 1989; Martini, 1994). Enhanced polyol metabolism,

which is exclusively found in Schwann cells (Gobi and OSullivan, 1968), might disturb this function of these cells and thereby impair nerve regeneration. The deleterious effect of increased polyol metabolism on nerve regeneration has been demonstrated in animal models with galactose intoxication in which nerve CNTF was found to be decreased (Calcutt et al., 1992), and nerve regeneration across a silicone tube gap was delayed (Powell et al., 1986). Regarding the regenerative ability of UMNFs, density and number of UMNFs were signicantly increased in both control and diabetic rats at 5 weeks after injury (Yasuda et al., 1999) (Fig. 13). This increase returned to the baseline level in control rats but not in diabetic rats at 24 weeks. Although the axon size showed a marked decrease at 5 weeks and an incomplete recovery at 24 weeks in both groups, the recovery was signicantly worse in diabetic than in control rats. Tolrestat did not have any effect on regeneration of UMNFs in diabetic rats. These features of regenerative UMNFs are in striking contrast with MNFs. UMNFs regenerate more than MNFs after crush injury. Bray and colleagues also reported the same results in the distal segment of crushed cervical sympathetic trunks of rats (Bray et al., 1973; Bray and Aguayo, 1976). It is unclear what causes the difference in the regenerative capacity between the two types of bers. UMNFs of diabetic nerves were shown to regenerate to the same extent in control nerves at 5 weeks after crush, although the regenerative capacity of MNFs is known to be signicantly

Fig. 13. Schematic drawing of regenerative capacity of MNFs and UMNFs after sciatic nerve crush in diabetic rats and the therapeutic effect of ARI. Principally, ARI is effective on impaired regenerative capacity of MNFs but not that of UMNFs. ARI ameliorates decreases in both nerve density and ber size of MNFs but does not improve elongation rate of regenerating MNFs. Regenerative capacity of UMNFs after nerve crush are in striking contrast with that of MNFs in that the magnitude of either an increase in their number or a decrease in their axon size is much larger in UMNFs that in MNFs. In addition, either nerve density or size tends to be restored to the basal level at least 24 weeks after nerve crush in MNFs but does not in UMNFs.

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disturbed in diabetes. This preserved regenerative capacity of UMNFs may be partly attributable to a high NGF level distal to the site of nerve injury in diabetes (Maeda et al., 1996). In addition, regeneration of neurites of UMNFs from transected vagus nerve terminals of nodal ganglia in vitro was also reported to be more enhanced in diabetic than in control mice (Saito et al., 1999). Although the increased number of UMNFs returned to the basal level in control rats at 24 weeks, it was signicantly delayed in diabetic rats. This delay may be due to disturbed or delayed pruning of unnecessary regenerating bers in diabetes. Excessive UMNFs may not connect with target organs or may show more connection than expected between nerve endings and targets. ARI torlestat did not have any effect on nerve regeneration of UMNFs in diabetes. This is in marked contrast with that on MNFs. It has been reported that ARI restores reduced number and size of regenerating MNFs in crushed or axotomized diabetic nerves (Pekiner and McLean, 1990). Since AR exclusively localized in Schwann cells of MNFs rather than in those of UMNFs (Gobi and OSullivan, 1968), ARI would not be expected to have much effect on regeneration of UMNFs. The observation that AR activity was reduced in the distal segment of crushed nerves (Wong et al., 1992) may also support the ineffectiveness of ARI on regeneration of UMNFs in diabetic nerves. 3.5.2. Cell body reaction in diabetes The synthesis of cytoskeletal proteins in the retina and the uptake of precursor amino acids in DRG neurons have been reported to be decreased in STZ-diabetic rats (Chihara et al., 1987; Thomas et al., 1984). These deleterious effects may impair nerve regeneration in diabetes, since the synthetic extent of cytoskeletal proteins is closely associated with nerve regeneration. Numerous metabolic and structural changes take place at the cell body of injured neurons. Some of these changes observed during the embryonic period seem to be necessary for regeneration, but others may induce the death of injured nerve cells. The onset of nerve regeneration is associated with the expression of new genes and proteins. Diabetes may induce impairment in nerve regeneration after nerve injury via reducing cell body reactions. There have been several reports about the changes of protein and gene expression in DRG after nerve crush in diabetic animals. Pekiner and McLean have shown that the induction of ornithine decarboxylase after crush injury was decreased in the DRG of STZ-diabetic rats, and this decrease was prevented by ARI ponalrestat (Pekiner and McLean, 1990). The conversion of ornithine to putrescine by ornithine decarboxylase is a rate-limiting step for the synthesis of polyamines, which have multiple roles in cell growth and proliferation. Mohiuddin and Tomlinson have also reported that both steady-state and post-lesion expression of GAP-43 and T1-tubulin mRNA in DRG are decreased in diabetic rats compared with control rats (Mohiuddin et al., 1995a,b). Moreover, they have shown down-regulation of steady-state expression of NFs and trkA mRNA in DRG of diabetic rats.

Taken together, they concluded that this reduced expression of mRNA coding for GAP-43 and cytoskeletal proteins could participate in the reduced ability of regenerative response to nerve crush in diabetes. 3.5.3. Partial denervation in diabetes There are several kinds of regeneration patterns in the PNS. As described above, many authors focused on regenerative axonal sprouting from the transected ends of axons and claried that nerve regeneration was impaired in diabetes. It has been shown that partial denervation of muscles is followed by intramuscular nerve sprouting from the remaining axons (terminal and nodal sprouting). Observation of intramuscular nerve sprouting after partial denervation is a useful model to investigate motor nerve regeneration in diabetic condition (Fig. 9). We examined the extent of intramuscular nerve sprouting in response to partial denervation in STZ-induced diabetic rats in order to elucidate the regenerative capacity of motor nerves in diabetes (Terada et al., 1995). Partial denervation in tensor fasciae latae muscle was produced by cutting the L4 spinal nerve. We evaluated motor nerve sprouting in tensor fasciae latae muscle 7 and 14 days after partial denervation in 1 and 12 weeks diabetic rats using a silver-cholinesterase stain method. The extent of sprouting was quantitatively assessed by both the percentage of end-plate with sprouts and length of terminal sprouts. The mean percentage of end-plates with sprouts and mean length of terminal sprouts at 2 weeks after denervation were signicantly increased in 1 week diabetic compared with control rats. In contrast, they were signicantly reduced in 12 weeks diabetic in comparison with control rats. These data demonstrated that motor nerve sprouting after partial denervation was decreased in long-term diabetic rats, whereas it was enhanced in short-term diabetic rats. It is postulated that motor nerve sprouting may be controlled by cytokines and/or neurotrophic factors from muscle bers, muscle satellite cells and cellular elements of intramuscular nerves. Candidate factors that may cause nerve sprouting include CNTF (Gurney et al., 1992), basic FGF (bFGF) (Gurney et al., 1992), and IGF-I and-II (Caroni and Grandes, 1990). In fact, muscle IGF-I and-II mRNA (Zhuang et al., 1997) and CNTF-like immunoreactivity in nerves were demonstrated to be reduced in diabetes (Calcutt et al., 1992). However, the precise mechanism that explains these discrepant results on motor nerve response after partial denervation between short- and long-term duration diabetic rats remains unclear. 4. Clinical observation 4.1. Pathological ndings suggesting nerve regeneration in diabetic nerves Diabetic polyneuropathy is characterized by a variety of neuropathologic ndings, including axonal degeneration and

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segmental demyelination (Dyck and Giannini, 1996; Gianni and Dyck, 1999). It has not been completely established whether the primary site of involvement is the axon or the Schwann cell. Although earlier investigators rather favored a hypothesis that Schwann cells or myelin are primarily involved in the pathogenesis of diabetic neuropathy (Bischoff, 1967; Ballin and Thomas, 1968), growing evidence appears to be more compatible with the notion that axonal degeneration is rather primary. Dyck et al. (1986) reported that the frequency of teased bers undergoing axonal degeneration and regeneration was more often abnormal than that of segmental demyelination and demyelination with remyelination in patients with diabetic polyneuropathy. These ndings, together with the prominent multifocality of pattern of MNF loss, may lead to the conclusion that axonal degeneration is primary in the pathogenesis of diabetic polyneuropathy (Dyck et al., 1986). However, teased nerve ber analysis demonstrated the existence of primary segmental demyelination (random occurrence of a demyelinated internode) suggesting that Schwann cells are primary in addition to secondary segmental demyelination (clustered occurrence of demyelinated internodes) due to axonal degeneration (Said et al., 1983). It is important to determine whether axonal atrophy occurs in distal axons of human diabetic nerves because axonal atrophy precedes demyelination and remyelination and axonal degeneration in other neuropathies (Dyck et al., 1971) and in a permanent axotomy model (Dyck et al., 1993) and is known to be characteristic of nerve bers in experimental diabetic neuropathy (Medori et al., 1985; Jakobsen, 1976). Axonal atrophy can be assessed as progressive attenuation of axon caliber that manifests as a decreased axon/myelin ratio. Although this has been extensively investigated in human diabetic polyneuropathy, the conclusion that axonal atrophy may develop in that condition has not been obtained (Sugimura and Dyck, 1981; Engelstad et al., 1997). Regenerative capacity including axonal regeneration and remyelination is also noted to be impaired in diabetic nerves. Electrophysiological examination can detect some evidence of muscle denervation and reinnervation. This is made possible by single-ber and macroelectromyography (Stalberg, 1982; Bril et al., 1996). However, evidence of nerve sprouting is more convincingly obtained by nerve biopsy specimens in diabetic neuropathy. Clusters of small myelinated bers closely applied to one another but without a common basement membrane as well as cytoplasmic processes of Schwann cells and individual bers or clusters of bers surrounded by a common basement membrane probably represent regenerated ber clusters (Dyck and Giannini, 1996). Although, to be strict, the detection of these ndings requires electron microscopic examination, careful light microscopical examination may wholly enable the evaluation of regenerating MNFs. The magnitude of regeneration has been reported to be profuse in milder cases of diabetic sensory polyneuropathy, but the distal nerve may be almost completely devoid of axons in severe cases, suggesting that

axonal regeneration could not compensate fully for axonal degeneration at the advanced stage of diabetic neuropathy. Bradley et al. (1995) reported that numbers of regenerating bers evaluated by regenerative cluster were found to decline in proportion to the reduction in total MNF density and suggested that axonal regeneration failed with advancing neuropathy. They also found that the relative number of regenerating bers was signicantly greater in patients with IDDM as compared with those with NIDDM after correction for age, leading to the notion that a superimposed ischemic process might explain the decreased regenerative capacity in NIDDM, since the magnitude of endoneurial vascular changes including basement membrane thickening is unequivocally greater in NIDDM than in IDDM patients The contribution of vascular changes to nerve ber degeneration has been reported to be unequivocally more important in the pathogenesis of diabetic polyneuropathy of the patients with NIDDM than of IDDM (Sima et al., 1988a,b). Nerve regeneration might contribute at least to a certain type of neuropathic pain in diabetic patients (Brown et al., 1976), although this is quite controversial. In addition, the treatment for painful state is very important for the management of diabetic patients with neuropathy. Therefore, it is useful to understand the pathogenesis of diabetic painful neuropathy with reference to nerve regeneration and neurotrophic factors. This specic topic will be described in Section 5. 4.2. Rationale for using neurotrophic factors for diabetic patients Distal symmetric polyneuropathy that predominantly affects sensory and autonomic function is the most common manifestation in the peripheral nerve disorders associated with diabetes (Thomas, 1997). Motor decit is usually less manifested than sensory and autonomic ones. In addition, the symptoms that disturb patients quality of life are usually associated with sensory and autonomic disorders especially at the middle to late stage of the diseases. Although the symptoms like pain and dysesthesia (positive symptoms) are becoming increasingly larger with duration of diabetes (Partanen et al., 1995), loss of all modalities of sensation (negative symptoms) may eventually develop at the late stage of diabetic neuropathy. However, patients may have disabling neuropathic pain even at this stage (stage 3: disabling neuropathy) (Dyck, 1988). On the other hand, autonomic neuropathy has recently been ascribed to sudden death in diabetic patients. The considerable percentage of death cause may be arrhythmia due to diabetic autonomic neuropathy (Bellavere et al., 1988; Ong et al., 1993). These facts may potentiate the clinical use of NGF that is selectively trophic for sympathetic ganglion neurons and small-size sensory neurons that are specically responsible for mediating pain and temperature sensation. In fact, diabetic patients were found to have early detectable abnormalities of temperature sensitivity and axon reex vasodilation, suggesting small-ber

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sensory and autonomic dysfunction even before the onset of symptomatic peripheral neuropathy (Anand et al., 1996); this dysfunction parallels with reducing levels of NGF in skin keratinocytes, which are target cells for NGF-responsive sensory neurons. The implication of this study is that abnormal production of NGF may contribute to the early small-ber dysfunction seen in diabetic patients, although the magnitude of this contribution to the pathogenesis of diabetic neuropathy is unknown. There is growing evidence suggesting that the defect of nerve regenerative capacity is due in part to the disturbed availability of neurotrophic factors through impaired retrograde axonal transport of NGF (Jakobsen et al., 1981; Schmidt et al., 1985; Hellweg and Hartung, 1990; Hellweg et al., 1994), and decreased production of NGF in various tissues (Kasayama and Oka, 1989; Fernyhough et al., 1994; Ordonez et al., 1994) is unequivocally implicated in the pathogenesis of diabetic polyneuropathy. The role of neurotrophic factors in the regulation of growth initiation, proliferation, and the apoptotic process by which the peripheral nerve is remodeled prompts us to use neurotrophic factors for treating diabetic neuropathy. In addition, the availability of large quantities of recombinant neurotrophic factors also makes it feasible to consider in the management of diabetic polyneuropathy. 4.3. Clinical trials: current state 4.3.1. Lessens from other trials: amyotrophic lateral sclerosis and toxic neuropathies Amyotrophic lateral sclerosis (ALS) and toxic neuropathies are potentially targeted by the treatment of neurotrophic factors since many experimental studies have shown signicant effects of these factors on animal models of such diseases. The results from clinical trials of these diseases may provide some clues for useful strategies to evaluate their potential effect on diabetic neuropathy. ALS was the rst target for the treatment with neurotrophic factors in major clinical trials. Three neurotrophic factors including BDNF and IGF-I, both made by muscles, and CNTF, made by Schwann cells, became early candidates. BDNF appeared to increase survival and retard loss of pulmonary function in ALS patients in a phase I through II study. The benecial effect of BDNF was examined in 1135 ALS patients in a multicenter phase III trial. Although the study failed to show the benet of BDNF treatment for the primary end points, post-hoc analyses showed that ALS patients with early respiratory impairment and those developing altered bowel function showed a statistically signicant benet (The BDNF Study Group, 1999). IGF-I has also been tested in clinical trials for treating ALS. A North American trial reported that a subcutaneous administration of 0.1 mg/kg per day of recombinant human IGF-I (rhIGF-I) signicantly delayed the progression of the disease (Lai et al., 1997). By contrast, a European study

failed to demonstrate an efcacy of IGF-I administration (Borasio et al., 1998). Recently, a phase III clinical trial of IGF-I failed to show the signicant effect on ALS. No major toxic side effects have been reported. Experimentally, CNTF proved to have undoubted effects on survival of motor neurons in vitro (Arakawa et al., 1990) and in vivo (Sendter et al., 1990). The substance was also shown to improve motor function in animal models of motor neuron disease (Mitsumoto et al., 1994). Thus, clinical trials were subsequently initiated, resulting in failure because of side effects, including anorexia nervosa, weight loss, fatigue and viral-like symptoms, as well as lack of efcacy (Cedarbaum et al., 1995; Miller et al., 1996). Since motor neurons are protected from systemic circulation by the bloodbrain barrier, CNTF, which was subcutaneously injected, would not gain ready access to the neurons. Then, an intrathecal approach has been tried, but only in a small-sized trial, but the relevance of such approach remains to be elucidated. Toxic neuropathies caused by a variety of therapeutic drugs, including anti-tumor agents (cisplatin, vincristine, paclitaxel, etc.), antibiotics (agyl, isoniazid, clioquinol, etc.), anti-viral agents, and cardiac drugs, and environmental toxins, including acrylamide and n-hexane, is becoming clinically important. Since the underlying pathology is usually well dened, susceptible neuronal populations can be inferred in each toxic polyneuropathy. Cisplatin, an anti-tumor agent, causes a predominantly large ber sensory neuropathy. Vincristine, another important chemotherapeutic agent, causes a mixed neuropathy affecting sensory, motor and autonomic bers (Caccia et al., 1977). Paclitaxel causes a predominantly sensory neuropathy (Lipton et al., 1989). These toxic neuropathies are induced as the result of dose limiting toxicity. Since the pathogenic mechanism is simple and may be similar between human and animals, the result can be extrapolated to humans. It has been well established that experimental toxic neuropathies can be abrogated by neurotrophic factors; NGF is effective for vinblastine- (Johnson, 1986) and taclitaxel-induced neuropathy (Hayakawa et al., 1994; Konings et al., 1994). NT-3 is effective for cisplatin neuropathy (Gao et al., 1995). In spite of very promising results from experimental studies, large scales of clinical trials have not been conducted. As in toxic neuropathy, sensory neurons are preferentially injured in diabetic neuropathy. Although diabetic neuropathy may show various clinical manifestations from hyperalgesic to ataxic forms, which may be associated with selective loss of a class of bers, a wide spectrum of nerve ber pathology from a tendency for small ber loss to that for large ber loss for each patient, a pattern which is similar to normal distribution but more variable, was found in diabetic neuropathy (Dyck et al., 1986). The information from the efcacy of each neurotrophic factor on corresponding toxic neuropathy may well be helpful for the treatment of diabetic neuropathy.

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4.3.2. Nerve growth factor NGF has been shown to prevent or delay the development of sensory neuropathy in STZ-induced diabetes (Apfel et al., 1994). Recombinant human NGF (rhNGF) is the only neurotrophic factor that has been tested in large-scale clinical trials for the treatment of diabetic polyneuropathy. In phase I of a double-blind, randomized, placebo-controlled study, the safety of single intravenous or subcutaneous doses of rhNGF was conrmed at doses ranging from 0.03 to 1 g/kg in healthy human volunteers. No serious adverse effects were seen at any dose. At doses above 0.1 g/kg, muscle pain was reported in the bulbar and truncal muscles. Although injection site hyperalgesia was induced in nearly all patients receiving subcutaneous injection of the compound, the drug was safe and well tolerated when administered subcutaneously at doses below 1 g/kg (Petty et al., 1994). Antibodies to NGF were not detected in any subject. A placebo-controlled, phase II clinical trial of rhNGF for the treatment of symptomatic diabetic neuropathy enrolled 250 patients from 15 centers across the United States (Apfel et al., 1998). Compared with placebo, rhNGF led to signicant improvement after 6 months of treatment. The global symptom assessment revealed a strong benecial effect of rhNGF treatment on the subjects overall perception of their neuropathic symptoms; 73% (0.1 g/kg) and 76% (0.3 g/kg) of the subjects in the rhNGF-treated groups felt improved, whereas only 46% of the subjects felt improved in the placebo group (P = 0.001). Both cooling detection threshold and perception of heat pain showed only a trend toward improvement in the rhNGF-treated group, and only pinprick sensation was signicantly improved in the treated groups. Prospectively designated multiple endpoint analyses (using OBriens rank-sum test) indicated signicant improvements in the rhNGF-treated groups over the placebo group. Similar to phase I study, the most common adverse event was injection site hyperalgesia, which was described by most subjects as mild to moderate discomfort to palpation at the injection site. Although the results appeared encouraging in spite of partial unbinding of the study due to the high incidence of injection hyperalgesia, the phase III clinical trials, which were carried out for 12 months in the United States and which recruited 1000 subjects with diabetic neuropathy, has failed (Apfel et al., 2000). The similar trials that were simultaneously under way in Europe and under consideration in Japan were both retracted in light of the failure of the trial in the United States. Although the reason for the discrepancy in the results between the phase II and III trials remains unclear, the negative result has led us to reconsider the best access of NGF to sensory neurons, or, furthermore, if NGF is really benecial to diabetic polyneuropathy. 4.3.3. Other neurotrophic factors In experimental diabetic neuropathy, in addition to NGF, both NT-3 (Tomlinson et al., 1997) and IGF-I/II (Ishii and

Lupien, 1995; Zhuang et al., 1996) have been shown to ameliorate decreased sensory nerve function and/or sensory nerve regeneration. However, clinical trials of these neurotrophic factors for diabetic neuropathy have not been conducted. 4.3.4. Aldose reductase inhibitors ARIs have provided the most convincing results, including experimental studies and human trials for the treatment of diabetic neuropathy. Although fundamentally ARIs do not have neurotrophic actions, either experimental or human study has shown that these compounds enhance regenerative capacity at least in part through ameliorating nerve concentration of neurotrophic factors. Experimental data are described in other sections. Only the results of clinical trials are presented here. Sima et al. (1988a,b) reported that number of regenerating nerve bers assessed by the percentage of regeneration/remyelination from teased bers as well as nerve ber density were signicantly larger in the group treated with ARI (sorbinil) than in the control group for 1-year observation period. However, this compound has been withdrawn from clinical testing because of frequent idiosyncratic adverse reactions due to its hydantoin-like structure. Amelioration of nerve regenerative capacity with ARI treatment was also suggested in the long-term open-label tolrestat study; the patients with diabetic neuropathy who had been elected for open-label treatment after completing clinical trials with tolrestat were enrolled in a randomized, double-blind continuation/withdrawal study for 12 months (Santiago et al., 1993). In this study, the percent of regenerating bers (remyelination/regeneration) was statistically higher in tolrestat-treated patients than in the reference population. In addition, the percent of regenerating bers correlated in all patients with the duration of tolrestat treatment when the tolrestat-continued and tolrestat-withdrawn groups were combined. It was estimated that 53% of 38 clinical trials on the efcacy of ARI were positive either for distal symmetrical polyneuropathy (59%) or autonomic neuropathy (40%) with the inclusion of ponalrestat trials in which the compound failed to penetrate the human nerve at doses sufcient to decrease nerve sorbitol levels (Greene and Sima, 1993), so that there is an argument that the data should not be included in any analysis of ARIs (Pfeifer et al., 1997). Thus, many clinical trials with ARIs have proved to be negative partly due to either low potency of ARIs or the inappropriateness of clinical trial design. In view of these disadvantages, a 52-week, randomized, placebo-controlled, double-blinded, multiple-dose, clinical trial with ARI zenarestat was conducted in patients with mild to moderate diabetic peripheral polyneuropathy (Greene et al., 1999a,b) (Fig. 14). Four treatment groups (placebo and ARI; 150, 300 and 600 mg) were assigned and highly sensitive objective and quantitative measures of nerve biochemistry, function and structure were used to assess the role of the AR pathway in the progression of diabetic

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Fig. 14. Ultrastructures and their schematic drawings of cutaneous UMNFs. Ultrastructural morphometric analysis is performed on electron micrographs of transverse proles of dermal UMNFs. This unit of UMNFs contains three axons, A1, A2 and A3. Both area and perimeter are obtained by digitizing outlines of axons and Schwann cells using image analyzer. Axon area (A1 + A2 + A3) is designated as axon area per cluster and median of area (A1, A2 and A3) is designated as axon area. Number of axon per cluster is three in this gure. The hatched area is Schwann cell area per cluster which is obtained by subtracting axon area per cluster from ber area per cluster.

polyneuropathy. In this study, the compound doses producing 80% sorbitol suppression were associated with a signicant increase in the density of small-diameter (<5 m) sural nerve MNFs. This increase of small MNFs may be attributable largely to regeneration of previously degenerated MNFs rather than better preservation of preexisting MNFs because of both the selective increase in MNF density of bers <5 m in diameter and a tendency for an increase in axon/myelin ratio in high dose ARI group, although the selective decrease of MNF density <5 m in the placebo treatment group would be consistent with the interpretation that some of the increase in MNFs <5 m in the high dose ARI group was due to the preservation of preexisting small MNFs. Accurate assessment of the relative contribution of regeneration versus preservation of preexisting bers would require ultrastructural examination to accurately identify regenerating clusters of MNFs. However, an increase of regenerative capacity per se would not imply clinical improvement, because regenerating bers do not necessarily accompany the reestablishment of nerve function. The possible mechanisms by which ARIs enhance the regenerative capacity are discussed in Section 3.5.1. With all mild but signicant effects of zenarestat on diabetic neuropathy that have been subsequently reproduced in the phase III clinical trial in the United States and Europe, the development of this drug has recently been suspended because of the possible induction of renal dysfunction depending on its dose.

4.4. Specic problems for the use of neurotrophic factors So far, the clinical effects of most neurotrophic factors on diabetic neuropathy do not appear much more than expected. In addition, there are considerable potential side effects for each compound. Even if any neurotrophic factor enhances growth action in patients nerves, nerve sprouting, which is subsequently induced, might establish aberrant connections. This may even disturb nerve function. The high level of serum neurotrophic factors that are exogenously provided may stimulate the production of neuropeptides, enzymes or other metabolic proteins, all of which may exert proliferative or even carcinogenic actions on a variety of tissues. Although a small number of clinical trials have not raised these problems, careful observation maybe necessary. NGF has been used in clinical trials for diabetes, dwarsm, and osteoporosis without showing serious side effects. However, injection site hyperalgesia was found in more than 90% of the patients who have received rhNGF in the phase II study. Other adverse events seen commonly in the patients treated with rhNGF include mildly painful conditions such as myalgia and arthralgia. However, myalgia was reported in some subjects as mild to moderate muscle ache typically after a single injection and not experienced with subsequent injections. It appears to occur in every case of accidental overdosing of rhNGF and did not recur when subjects were rechallenged with the intended dose of

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rhNGF. These observations suggest that rhNGF is largely safe for clinical use. Numerous mechanisms have been proposed to explain the NGF-induced hyperalgesia. NGF is trophic for nociceptive afferent and sympathetic neurons, which participate in mediating pain sensation. NGF also enhances the production of the nociceptive neuropeptides, such as substance P and CGRP, induces mast cell degranulation with the release of serotonin and histamine (Lewin et al., 1994) and potentiates the activities of bradykinin (Rueff et al., 1996) and prostaglandin E2. Together with the clinical observation that intradermally administered NGF induced local hyperalgesia within a few hours (Dyck et al., 1997), these results may suggest a peripheral action of NGF rather than a central one in which it is expected to take more time to retrogradely transport NGF to neuronal cell body. Careful attention should be placed on the possible value of the IGFs in the treatment of diabetic neuropathy. Although the experimental data on IGF-I/II for treating diabetic neuropathy are encouraging, there are many potential problems when they are systematically administered. IGF-I increases glomerular ltration and renal plasma ow acutely and has been associated with a number of undesirable side effects, including the development of entrapment neuropathies when administered for longer periods of time (Guler et al., 1989; Bondy et al., 1994). This is particularly problematic when considered in light of the increased predisposition of diabetic subjects to entrapment neuropathies. It remains unclear whether IGF-II does not have this side effect. Many tissue abnormalities have been reported in transgenic mice that overexpress IGF-I (Quaife et al., 1989). These include red blood cell hematopoiesis in spleen, aberrant collagen bundle formation in skin, and enlargement of glomeruli. IGF-I has also been reported to counteract the benecial effect of bFGF on neuronal survival (Blottner and Baumgarten, 1992). CNTF has been discovered to induce anorexia and marked weight loss in clinical trials. Similar effects were seen in the experimental studies using rodents as well (Henderson et al., 1994). CNTF induced cahexia in mice was associated with rapid catabolism of adipose tissue and muscle, decreased levels of serum glucose and triglycerides, and a loss of CD4+/CD8+ T cells. CNTF overproduction in transgenic mice was shown to increase the rate of onset of motor neuron disease symptoms (Winter et al., 1996), although most studies support a trophic role for CNTF in protecting motor neurons. Thus, many problems need to be solved before CNTF can be used clinically.

5. Regenerating nerve bers with special reference to pain generation Diabetic patients can experience a variety of different pain syndromes (Thomas, 1999). However, the mechanism by which such sensory disturbance develops remains unclear. Although neurophysiological abnormalities including

dysfunction of ion channels may contribute to the generation of pain in diabetic state (Hirade et al., 1999), it has been suggested that regenerating peripheral afferent neurons are implicated in painful symptoms (Asbury and Fields, 1984). Indeed, nerve biopsies from patients with painful diabetic neuropathy often reveal regenerative nerve sprouting (Brown et al., 1976); ectopic generation of impulses from regenerative sprouts might be responsible for the pain. This notion may be supported by the observation that ectopic impulse generates in sprouts from sensory axons in neuromas produced by sciatic nerve transection in rats (Wall and Gutnick, 1974). Regenerating bers are sensitive to acetylcholine (Diamond, 1959) and adrenaline (Scadding, 1981), which display ectopic impulse generation. Sprouts from regenerating neurons can also have ectopic pacemaker capacity (Calvin, 1980). Although pathological ndings on the peripheral nerve from diabetic patients with painful symptoms have conrmed the presence of regenerating bers (Archer et al., 1983; Said et al., 1983; Llewelyn et al., 1986), Llewelyn et al. (1991) noted that there was no correlation between number of regenerative clusters from MNFs and the occurrence of painful symptoms in diabetic patients. In addition, it was shown by electron microscopy that both degeneration and regeneration of MNFs and UMNFs were present in the nerves of diabetic patients irrespective of pain (Britland et al., 1992). Aside from ber regeneration, it was hypothesized that ber shrinkage could be implicated in the generation of pain in diabetic neuropathy; a correlation between the occurrence of pain in diabetic neuropathy and axonal atrophy was suggested (Britland et al., 1990). In fact, Dyck et al. (1976) reported that painfulness in 72 peripheral neuropathies of which only 1 diabetic neuropathy was included was found to be correlated with active nerve ber degeneration more frequently than with other types of pathologic change. However, others failed to detect a correlation between pain and active ber breakdown in nerve biopsies from patients with diabetic neuropathy (Llewelyn et al., 1991; Britland et al., 1992). Taken together, there seems to be little evidence for the concept that pain arises from regenerating axon sprouts (Thomas, 1999). On the other hand, it is known that emotional upset can either trigger or exacerbate severe pain in diabetic neuropathy (Archer et al., 1983). In addition, the treatment for pain with sympatholytic drugs is often successful. These facts suggest that some kinds of pain in diabetic patients may be attributed in part to sympathetic activity. Under normal condition, there is no communication between sympathetic postganglionic neurons and afferent neurons in the periphery; afferent neurons are not excited or sensitized by activation of sympathetic neurons. However, after nerve lesion and tissue trauma with inammation, excitation of sympathetic neurons may lead to sensitization or activation of afferent neurons, and terminals of sympathetic neurons may mediate sensitization of afferent receptors independent of excitation. During these processes, adrenoceptors may

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express or up-regulate. Alternatively, in view of the observation that adrenoceptor mRNAs are normally constitutively present in DRG cells (Nicholas et al., 1993; Pieribone et al., 1994), it is possible that non-functional receptors become uncovered and effective. Both lumbar sympathetic nerve stimulation and noradrenaline injection into femoral artery elicited adrenergic excitation of C-ber polymodal receptors recorded from saphenous nerve of STZ-induced diabetic rats. In addition, these responses were consistently blocked by 2-adrenoceptor antagonists, including yohinbin, but not by 1-adrenoceptor antagonist, prazosin. These results may suggest the up-regulation of 2-adrenoceptors on the polymodal receptors of somatic sensory nerves (Sato and Kumazawa, 1996). Morphological analysis has shown that within DRG postganglionic axons with noradrenergic varicosities are normally found along blood vessels but rarely amongst the cell bodies of afferent neurons (Kummer et al., 1990). Only a few DRG cells are surrounded or contained by axons. However, following transection and ligation of the sciatic nerve in rats, many ne catecholamine-containing axons start to penetrate the DRGs which contain somata with lesioned axons. After ligating and cutting the sciatic nerve, noradrenergic perivascular axons sprout in the DRG and form basket-like structures preferentially around cell bodies of large diameter axotomized sensory neurons but rarely around those of small diameter neurons, which contribute to nociception. Furthermore, only a few axotomized spontaneously active afferent neurons with C bers respond via their cell bodies in the DRG to sympathetic stimulation. Although these results do not appear to provide the positive data on the role of sympathetic nerve sprouting in the generation of pain, peripheral nerve lesions have also been shown to induce sprouting of large diameter afferent neurons into lamina II of the dorsal horn (Woolf et al., 1992; Shortland and Woolf, 1993), thereby having access to the nociceptive system by sympathetic activity. In fact, it is suggested that ectopic impulse activity in lesioned small-diameter afferents (Michaelis et al., 1995; Blenk et al., 1996) sensitizes dorsal horn neurons and that mechanical stimulation of cutaneous low-threshold afferents activates the sensitized dorsal horn neurons leading to the allodynic behavior. This behavior disappeared or did not develop when surgical lumbar sympathectomy has been performed either after or before sciatic nerve lesion (Kim and Chung, 1992; Kinnman and Levine, 1995). Although the mechanism leading to the sprouting in the DRG is unknown, some trophic signal may generate from afferent cells with lesioned axons in the DRG or their surrounding Schwann cells. This notion is compatible with the observation that satellite cells around axotomized large-diameter DRG cells express or up-regulate the low-afnity neurotrophin receptor p75NTR (Zhou et al., 1996). In spite of these interesting results, there have been no reports indicating the occurrence of sympathetic nerve sprouting into the DRG neurons in diabetic animals.

6. Evaluation of nerve pathology including regeneration by skin biopsy Since skin biopsy is a convenient and safe method, it is often used for the diagnosis and evaluation of certain diseases in which cutaneous vascular and nervous tissues are systematically involved. The diagnosis for certain neurological diseases by skin biopsy is usually made on the qualitative basis; lysosomal storage diseases and other degenerative neurological disorders are good candidates (Martin et al., 1977, 1979; Aresenio-Nunes et al., 1981). Specic ultrastructures within nerve bers, broblasts or other cells were diagnostic as benecial hallmarks for certain diseases. Aside from these qualitative ndings, quantitative measurements on tissue components of the dermis have proved to be useful for the evaluation of disease state. For instance, in diabetes mellitus basement membrane of capillary endothelial cells and other types of cells is thickened with the duration of hyperglycemia, thereby providing some information on the disease condition (Yasuda et al., 1990a,b). A highly signicant degree of perineurial cell basement membrane thickening in the dermis was also reported in diabetic patients (Johnson and Doll, 1984). Since cutaneous nerve bers are terminal parts of sensory nerves, the skin is the initial part from which nerve bers start to degenerate in dying-back type polyneuropathy. In addition, the skin biopsy procedure is safe and does not leave the sequel such as dysesthesia and hypesthesia which usually accompanies nerve biopsy. Therefore, if pathological ndings of the skin reect those of nerve biopsy, skin biopsy may be a potential method for the diagnosis of peripheral sensory neuropathy especially in the case without an indication of nerve biopsy. Although the degree of degeneration of cutaneous UMNFs does not necessarily reect UMNF counts in nerve biopsies (Periquest et al., 1999; Herrmann et al., 1999; Chung et al., 1990), possibly due to either distal branching of sensory nerve bers or dilution by seemingly remaining sympathetic nerves, a signicant correlation between MNF density of the sural nerve and the ratio of axon/Schwann cell area of cutaneous UMNFs obtained by ultrastructural morphometry may suggest potential usefulness of skin biopsy (Yasuda et al., 1990a,b). Although it remains unclear what kind of pathology in the nerve fascicle corresponds to each ultrastructural measurement of cutaneous nerves, including axon area per cluster (m2 ), Schwann cell area per cluster (m2 ), nerve area per cluster (m2 ), axon/Schwann cell area ratio, Schwann cell perimeter per cluster (m), Schwann cell perimeter/area, axon area (m2 ) and number of axons per cluster reect, each measurement might represent axonal or Schwann cell changes due to the neurodegenerative process (Fig. 15). Skin biopsy may be superior to nerve biopsy as well as electromyography for the evaluation of small-ber sensory neuropathies, since the latter conditions may include the patients with reduced intra-epidermal nerve ber density in spite of normal histology and/or normal electromyography (Holland et al., 1997, 1998). Aside from the potential

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Fig. 15. (a) Note ne tortuous bers that were running toward the epidermis (arrow). The nerve bers were visualized with anti-PGP 9.5 antibody labeled with diaminobenzidine (DAB) with nickel ammonium sulfate. The skin sample was taken from ARI-treated patient at the end of clinical trial. (b) Ultrastructure of an axonSchwann cell unit possessing dense deposits of immune complexes including PGP 9.5 corresponding to the ne bers indicated by arrows in this gure. (a) All axons had incomplete coverage of Schwann cell processes. (c) Ultrastructure of two axonSchwann cell units that were photographed in the section untreated with PGP 9.5 antibody. The samples were taken from the same patient as in (b). Both units had three to four axons, most of which were incompletely covered by Schwann cell processes.

substitution of nerve biopsy by skin biopsy, the best application of this technique may be to evaluate the therapeutic effect of given compounds because of repeatability. 6.1. Morphometric analysis of cutaneous nerves by immunohistochemistry There is increasing evidence suggesting that the assessment of cutaneous innervation by skin biopsy is useful

for evaluating the magnitude of peripheral neuropathies including diabetic neuropathy. Cutaneous innervation can be qualitatively and quantitatively evaluated on the nerves immunostained with specic antibodies such as protein gene-product 9.5 (PGP 9.5) (Wang et al., 1990; Dalsgaard et al., 1989) and neuropeptides, including substance P (Lindberger et al., 1989; Dalsgaard et al., 1989; Levy et al., 1989, 1992), CGRP (Gibbins et al., 1987; Lindberger et al., 1989; Dalsgaard et al., 1989; Levy et al., 1989, 1992; Karanth et al., 1990; Properzi et al., 1993), vasoactive intestinal polypeptide (VIP) (Levy et al., 1989, 1992; Karanth et al., 1990) and neuropeptide Y (NPY) (Karanth et al., 1990; Levy et al., 1992). Among these immunoreactive markers, PGP 9.5 is best established as a neuron-specic, pan-axonal marker (Thompson et al., 1983). In addition, the antisera against PGP 9.5 well demonstrates intra-epidermal innervation of cutaneous nerves. Therefore, PGP 9.5 may be the most convenient and reliable marker for human epidermal nerve bers. Overall, the antibody of PGP 9.5 can detect more sensitively nerve bers than can that of neuropeptides. Absence of PGP 9.5 is thought to signify axonal loss or degeneration rather than leakage of the antigen. If axonal degeneration shows weak or no immunoreactivity in spite of the presence of axons, the innervation might be underestimated so that the interpretation of decreased innervation in diabetic neuropathy should be balanced. PGP 9.5 is now known to be a neuronal-specic ubiquitin COOH-terminal hydrolase (Wilkinson et al., 1989). Since its antibody immunostains human and experimental dystrophic axons (Lowe et al., 1990) in which amyloid precursor protein and ubiquitin epitopes are expressed (Cochran et al., 1991) and the enzyme may play a role in the proteolytic process in the attempt to eliminate excessive or abnormal proteins (Parag et al., 1987), the possibility that the immunoreactivity of PGP 9.5 may be enhanced in the degenerative process of neurological diseases, including diabetic neuropathy, should be excluded. Our ultrastructural immunohistochemical observation using the antibody to PGP 9.5 showed no signicant difference in the degree of the immunoreactivity of PGP 9.5 between control and diabetic skins with preserved immunostain in almost all axons observed. However, further observation for degenerative axons and morphometric analysis on the density of the immunoreactivity is necessary and may provide denite answers to these questions. Both epidermal and dermal nerve bers may show qualitative structural abnormalities, including segmented degeneration and swelling in sensory neuropathies, including diabetic neuropathy (Holland et al., 1998) (Fig. 16). Since cutaneous nerve bundles run vertically and horizontally and nerve bers branch off the nerve bundle in all directions in the dermis, the precise quantitative analysis on nerve bers is not easy, especially in the dermis. In this aspect, semiquantitative analysis by counting the number of intra-epidermal bers per mm length of epidermis using immunostained sections may be useful.

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Fig. 16. The percent change of nerve ber length during ARI treatment for 13 months. Broken lines show the mean + 2S.D. of the percent change of nerve length in patients without ARI treatment (epidermis 619%, dermis 109%). Eight of 19 ARI-treated patients and only 1 of 12 untreated patients showed larger value than this level in the dermis (ARI-treated vs. untreated; P = 0.014 by Fishers direct probability test).

However, since intra-epidermal bers start to disappear at the early stage of neuropathy and dermal nerve bers appear to have difculty entering the dermis once they have retracted from the epidermis, it is hard to evaluate the moderate to severe degree of neuropathy in which intra-epidermal bers are almost gone or to evaluate the therapeutic effect of given compounds on neuropathy. In either normal or diseased conditions, ne tortuous bers were often seen along with normal appearing nerve bers. By electron microscopy using immunostain with PGP 9.5 antibody, some of the former bers were shown to be regenerating ones because of both increased number of axons and incomplete coverage of Schwann cells over axons. The quantitative measurement on these bers may provide a useful method to evaluate the potency of the therapeutic drugs for diabetic neuropathy. In spite of the difculty in the morphometric evaluation of cutaneous nerves, cutaneous innervation has been morphometrically evaluated in several ways: semiquantitative grading, measuring the nerve area that is visualized on sections and ultrastructural assessment. While all of these methods are used, each presents specic problems in the accurate quantitation of cutaneous nerves. Semiquantitative grading is not appropriate for precise quantitation (Levy et al., 1989, 1992). The measurement of the nerve area is qualied only when a bundle of nerve bers, especially in the dermis, is

evaluated because of the difculty in following each ber (Properzi et al., 1993). Also, there exists a technical difculty in measuring the nerve area because of irregularly tortured immunostained nerve bers and the requirement to subtract the background count. In spite of some disadvantages, these techniques all have demonstrated reduced cutaneous innervation in diabetic patients compared with control healthy subjects. In addition to methodological problems, the data from these methods have been obtained from two-dimensional planes, while cutaneous nerves are running in many directions. In this sense, the technique of measuring nerve length using confocal laser scanning microscopy provides three-dimensional information about nerve bundles or nerve bers that exist in a section (40 m thickness per section). The evaluation using confocal laser scanning immunohistochemistry using PGP 9.5 antibody is now the most appropriate for the accurate quantitation of cutaneous nerves, although the nerve length measured by this procedure does not completely trace the nerve bers, because the images which are projected into a single in-focus image are available for the measurement. Using this method, we (Hirai et al., 2000) and others (Kennedy et al., 1996) have reported the same results, that epidermal innervation is decreased in diabetic patients compared with controls. Unlike epidermal bers that run in a single ber, since they are a terminal part of bers, nerve length of nerve bundles within the dermis may be underestimated because a small nerve bundle that is composed of a few bers is assessed equally in dermal nerve length to a single nerve ber. Dermal nerve bers are known to be decreased in diabetic patients so that dermal nerve length in diabetes may be overestimated when compared with that of controls. However, even this evaluation uncovers a decrease in nerve length in diabetes; therefore, the interpretation of the ndings may not be altered or even emphasized. In evaluating cutaneous nerve innervation, it is technically important to minimize the variability and maximize the reproducibility in measuring cutaneous nerves. The density of epidermal innervation is known to vary among different body sites in patients with sensory neuropathy as well as in control subjects (Lauria et al., 1999); it was most severely reduced distally, with more normal intra-epidermal nerve ber densities in the skin from proximal sites in slowly progressive idiopathic small-ber sensory neuropathies (Holland et al., 1998). To lower this intrasite variability, skin samples should be biopsied from exactly the same area between subjects under comparison and between different occasions in the same subjects. We punch-biopsy a 5 mm diameter of calf skin, and six images showing the largest measurement of nerve length among acquired views in three sections are used in measuring nerve length with confocal laser scanning microscopy. Using this procedure, the intersection variability of epidermal nerve length is assessed; mean coefcients of error (S.E.M./mean) were 0.08 in control and 0.12 in diabetes, whereas coefcients of variance were 0.18 in control

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and 0.21 in diabetes. Similar values were reported by others (Stocks et al., 1996), although their method of obtaining epidermal nerve length was not exactly the same; the mean coefcient of error was 0.13 and the coefcient of variance was 0.29. The interobserver variability can be avoided by a single observer. As for the reproducibility, dermal nerve length obtained from 12 diabetic patients biopsied on two occasions at 1-year intervals showed highly signicant coefcient of correlation between two occasions (P < 0.005; n = 12, r = 0.796). 6.2. Morphometric analysis of cutaneous nerves by ultrastructural examination At the start of a series of our study about skin biopsy in diabetic patients, we rst chose ultrastrucural analysis, in which the density of UMNFs in the upper dermis was signicantly lower, and axonal vacuolation and Schwann cell clusters devoid of axons were more frequently found in diabetic patients than in age-matched control subjects (Yasuda et al., 1985). Although axonal vacuolation is sometimes indistinguishable from the artifact due to the preparation of skin samples, the observation that it is more frequently found in diabetes was thought to support that the nding may be attributable to diabetes. Axonal degeneration may result in loss or shrinkage of axons, leading to Schwann cell clusters devoid of axon and sometimes multiplying and tortuous basement membrane assembly without cellular component possibly due to rostral withdrawal of Schwann cells following axonal loss. In diabetic patients, the frequency of basement membrane multiplication of Schwann cells was higher in patients with abnormal sensation in their limbs than in patients without it. Patients with pain in their limbs showed a higher frequency of the basement membrane and Schwann cell clusters devoid of axons compared with patients without pain (Yasuda et al., 1992). MNFs were shown to infrequently exist in the upper dermis in the calf skin. Each ultrathin section used for ultrastructural analysis was cut vertically against the epidermal plane and was photographed and morphometrically assessed for cross-sectioned UMNFs, which exist up to 150 m in depth of the skin. The evaluation for nerve density may be better evaluated in the section parallel to the epidermal plane. However, the procedure to obtain such sections is technically complicated and not easy to be done precisely. Although this rather semiquantitative analysis provides important and useful information about pathology of Schwann cells and axons, it is unsatisfactory in terms of quantitation. Thus, morphometric measurements of dermal UMNFs were conducted to obtain more objective morphological indicators of diabetic neuropathy. Morphometric measurements were made using computer-assisted digitizer; cellular components of dermal UMNFs were digitized with a cursor to obtain the area and perimeter of axons and Schwann cells. Axon area per cluster (AA/C), axon/Schwann cell area ratio (A/S.R) and number of axons

per cluster (no.A/C) were signicantly lower in diabetic patients than in control subjects. These results may support that axonal degeneration or axonal atrophy, which is often observed in nerve bers within major nerve fascicles, takes place in cutaneous nerves. Among diabetic patients, those with pain in their extremities had signicantly lower values for AA/C, A/S.R and no.A/C than those with no pain. AA/C and no.A/C were signicantly correlated with both vibration sense evaluated by C128 tuning fork and the coefcient of variation of the RR-interval (Yasuda et al., 1992). The nding that axonal dwindling of cutaneous nerves is signicantly greater in diabetic patients with pain is compatible with the report from sural nerve biopsy that axonal atrophy may be involved in the generation of neuropathic pain (Britland et al., 1990). Although the signicance of these parameters remains unclear, A/S.R was shown to be signicantly correlated with MNF density of the sural nerve (Yasuda et al., 1990a,b), an observation which suggests that either a decreased number of axons or axonal atrophy of the dermal nerve may reect MNF density of the peripheral nerve in diabetic patients. Thus, ultrastructural morphometry of the dermal nerve may be useful to evaluate clinical or pathological features of diabetic neuropathy. 6.3. Neurotrophins and cutaneous innervation Neurotrophins may contribute to cutaneous nerve sprouting or reinnervation. Among neurotrophins, NGF, which is trophic to sensory and sympathetic bers, or its mRNA has been reported to be increased (Diemel et al., 1999) or decreased (Anand et al., 1996) in the skin of diabetic patients. The concentrations of endogenous NT-3, which may be necessary for the development of muscle spindle and Merkel cell afferent bers and was shown to be present in the suprabasal epidermis, where many unmyelinated sensory bers terminate, is higher in the skin in patients with diabetic polyneuropathy than in the age-matched control skin (Kennedy et al., 1998). The increase of neurotrophic factors may be caused by skin denervation through the up-regulation mechanism in neurotrophin-producing cells. By contrast, chronic denervation may cause its down-regulation. Simultaneously, length-dependent dysfunction of small-diameter bers may develop with depletion of NGF and the sensory neuropeptide substance P (Anand et al., 1996). The depletion of skin NGF was shown to be correlated with decreased skin-reex vasodilation. Thus, a decrease in endogenous skin-derived NGF inuences the presentation of diabetic polyneuropathy, although metabolic or vascular abnormalities may be primarily important as a cause of the neuropathy. Since a decreased nociception and axon-reex vasodilation contribute to diabetic foot ulceration, early and prolonged NGF treatment at an appropriate dose or the treatment with the compound that induces endogenous NGF production may well be rationalized. On the other hand, TrkA, high-afnity receptor of NGF, and TrkC, receptor of NT-3, both have been reported to be

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increased in the diabetic skin in patients with early subclinical diabetic neuropathy (Terenghi et al., 1997). The up-regulation of epidermal Trk receptors may result from decreased autocrine neurotrophin action and could represent a compensatory mechanism. 6.4. Evaluation of therapeutic compounds for diabetic neuropathy by skin biopsy In an attempt to obtain ideal therapeutic compounds for diabetic neuropathy, many clinical trials are continually in progress. It is extraordinarily important to accurately evaluate the efcacy of given compounds in each trial. Although electrophysiologiocal procedures, especially nerve conduction velocity, are considered practically most reliable for the evaluation of the effect of therapeutic drugs, morphological examination, like nerve biopsy, may be more accurate and objective. However, nerve biopsy is considerably aggressive and leaves some defect, including dysesthesia and pain, although not a serious one. In this context, together with the repeatability on occasions, the skin biopsy method may be convenient and advantageous. However, there have been no reports on skin biopsy with reference to the evaluation for the efcacy of given compounds on diabetic neuropathy except for ours. We evaluated the efcacy of 1 years administration of ARI epalrestat, whose efcacy was conrmed in a multicenter double-blind clinical trial (Goto et al., 1993, 1995) and which has been already launched into the market in Japan (Hotta et al., 1996), and found that the compound is able to elongate cutaneous nerve bers at least in some patients with diabetic polyneuropathy (Yasuda et al., 2000). In such patients, ne tortuous bers were more frequently shown to branch off the bundle that ran in the upper dermis. By ultrastructural immunohistochemistry, these ne bers proliferating in the upper dermis proved to be composed of an increased number of axons with incomplete Schwann cell coverage, a nding compatible with regenerating nerve bers. The effect of ARI on the regenerative capacity of MNFs has already been reported in clinical and experimental studies. Our study shows that small nerve bers, mainly composed of UMNFs of the sensory nerve terminal, could also regenerate or sprout with ARI (Yasuda et al., 2000). The possible mechanisms by which AR plays a role in the pathogenesis of diabetic neuropathy may also be relevant to the mechanisms by which ARI improves dermal nerve regeneration. In addition, ARI improves decreased content of NGF in the sciatic nerve of diabetic rats (Ohi et al., 1998). The collateral sprouting of intact cutaneous sensory neurons has been shown to be dependent upon the presence of NGF. In addition, the sprouting and associated increase in GAP-43 mRNA levels were blocked with anti-NGF treatment (Mearow and Kril, 1995). Therefore, NGF is essential for the development of collateral reinnervation from cutaneous C-bers but is depleted in the keratinocytes of the

skin in diabetic patients. ARI may reverse its depletion and induce sprouting of dermal nerves. Thus, it is possible that an increase in nerve ber length may be due to sprouting but not to regeneration. To be strict, it is not clear whether the ne bers that are proliferating in the upper dermis are regenerating or sprouting. The origin of reinnervated nerves into denervated adjacent skin was examined using the antiserum to NGF; all collateral sprouting was prevented and that in progress was halted, whereas sprouting resumed when the treatment was discontinued (Diamond et al., 1987). Interestingly, cutaneous nerve elds were reestablished or even enlarged by regenerating axons by discontinuation of anti-NGF treatment even after dorsal rhizotomy to eliminate any central trophic support. In addition to this systemic administration of anti-NGF antibodies, its local application can block collateral sprouting in rats with chronic constriction injury (Ro et al., 1996). These results suggest that reinnervation in the denervated skin is caused by collateral sprouting, which is dependent on the local availability of NGF by the nearby intact cutaneous nerve bers, whereas nerve regeneration by both nociceptive and non-nociceptive axons is not dependent on NGF. Aldose reductase (AR) is the rate-limiting enzyme of polyol pathway, which has been hypothesized as one of the major pathogenic factors of diabetic neuropathy (Gabby, 1973). Diabetic patients with a high level of tissue AR level expression may be subject to diabetic complications (Nishimura et al., 1994). The AR level in tissue can be assayed by ELISA (Nishimura et al., 1993). Although it is not known whether the erythrocyte AR level reects that of tissue AR level, a high erythrocyte AR level has been reported to be associated with diabetic neuropathy as well as diabetic retinopathy (Nishimura et al., 1994; Ito et al., 1997). In addition, dermal nerve length was signicantly shorter in the group with higher AR level than in the group with lower AR level in the setting of no difference between age, duration of diabetes and HbA1c between the two groups (P < 0.05) (Hirai et al., 2000). Thus, it is likely that erythrocyte AR level may reect cutaneous AR level, which is associated with cutaneous nerve degeneration assessed by nerve length. We found that AR protein was expressed in cutaneous tissue by ELISA (unpublished data). Unlike dermal nerves, neither number nor length of epidermal nerves correlate to erythrocyte AR. In addition, even when the reinnervation process was successfully achieved with ARI treatment, it was restricted within the dermis; epidermal innervation was not improved with ARI. This nding may suggest that regeneration or sprouting nerves of the dermis cannot readily enter the epidermis in diabetic state, rather than that epidermal nerves cannot regenerate or sprout, since epidermal innervation was very poor in most patients. Although the reason remains unclear, glycated basement membrane of the epidermis may prevent reinnervation of dermal nerve bers into the epidermis.

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7. Neuronal cell death The pathological characteristics of MNFs in diabetic patients include axonal degeneration and segmental demyelination. Although loss of neuronal cells including DRG neurons and anterior horn cells was previously described in diabetic patients (Dolman, 1963; Greenbaum et al., 1964), extensive morphometric analysis has not been done. Ohnishi et al. reported that there was no evidence of a decrease in the number of cell bodies in L5 DRG and in their diameters, although the results were obtained only in two diabetic patients with neuropathy; normal range of number of L5 DRG neurons were found in spite of decreased number of MNFs of the sural nerve (Ohnishi et al., 1983). Most diabetic animals manifest neither neuronal loss nor nerve ber loss (Zochodne et al., 2001; Yagihashi, 1995) except for BB/W rats (Sima and Sugimoto, 1999). This is typically true for STZ-induced diabetic animals, which are most frequently used for models for diabetes (Yasuda et al., 1989a,b). Thus, although the occurrence of neuronal death in the PNS in diabetes seems rather doubtful, there are some recent reports suggesting possible apoptotic changes of neuronal cells in the PNS in diabetic animals as described in Section 7.2. 7.1. Apoptosis induced by the sera from diabetic patients Immunoglobulins of the sera from type 1 diabetic patients with diabetic neuropathy have apoptotic effects on neuroblastoma cells possibly through Fas-related pathway (Pittenger et al., 1997). The sera from type 2 diabetic patients with neuropathy were also shown to induce complement-independent, calcium-dependent apoptosis in neuronal cells (Srinivasan et al., 1998). The sera may contain an autoimmune immunoglobulin causing Ca2+ inux, because serum-induced enhancement in cytosolic calcium and calcium current density was blocked by the treatment with trypsin and ltration of the serum using a 100,000 kDa molecular weight lter, and, furthermore, the treatment with an anti-human IgG antibody was associated with intense uorescence on the surface of neuronal cells exposed to the sera from type 2 diabetic patients with neuropathy. 7.2. Apoptosis is induced under high glucose or hyperglycemia It has been shown that neuroblastoma cells undergo programmed cell death under high glucose or hyperosmolar condition, conditions designed to mimic diabetic state (Matthews et al., 1997; Matthews and Feldman, 1996; Singleton et al., 1996a,b; Cheng and Feldman, 1998). Hyperglycemia also induces apoptotic changes in DRG neurons and Schwann cells including ballooning of mitochondria and disruption of the internal cristae that were observed in the presence of oxidative stress (Sasaki et al.,

1997; Kerezoudi et al., 1995) both in STZ-induced diabetic rats and in rats made acutely hyperglycemic with infused glucose (Russel et al., 1999). Hyperglycemia is believed to induce increased oxidative stress (Baynes, 1991; Cameron and Cotter, 1994; van Dam et al., 1995a,b), which, in turn, may activate death effector proteins that disrupt mitochondrial membrane potentials and trigger mitochondria to swell and become permeable (Susin et al., 1997). Changes in mitochondrial permeability lead to release of cytochrome c and other regulatory proteins into cytoplasm, promoting programmed cell death through activation of caspase-3-associated pathway. These results suggest that oxidative stress may promote the mitochondrial changes in diabetic animals, leading to activation of the caspase programmed cell death pathways, thereby causing apoptosis of Schwann cells as well as neuronal cells. Indeed, Russel et al. (1999) found that apoptotic changes occurred in parallel with activation of caspase-3, dependent on the concentration of glucose. In addition, Srinivasan et al. also observed that TUNNEL-positive apoptotic DRG neurons were increased in diabetic rats, and basal mitochondrial membrane potential was signicantly more positive in DRG neurons from diabetic rats and resulted in a signicantly more positive potential and a delay in its recovery with glutamate-induced depolarization in those neurons by evaluating confocal micrographs of mitochondria loaded with JC-1 dye (Srinivasan et al., 2000). Simultaneously, DRG neurons from diabetic rats demonstrated decreased Bcl-2 level and translocation of cytochrome c from the mitochondria to the cytoplasm, whereas the level of Bax or Bcl-XL was not altered in diabetic neuropathy. These abnormalities were corrected with insulin treatment. Thus, a selective decrease in Bcl-2 level in diabetic neuropathy results in loss of mitochondrial membrane potential, translocation of cytochrome c into the cytosol and activation of apoptosis. Since serum IGF level has been reported to be decreased with increased IGFBP-1 in type 1 diabetic patients (Janssen et al., 1997; Janssen and Lamberts, 2000), a hypothesis that defective bioactivity of neurotrophic factors like IGF-I due to chronic hyperglycemia induces apoptosis of Schwann cells as well as sensory and motor neurons may well be proposed. Indeed, IGF-I is able to prevent apoptosis of DRG neurons under high glucose in vitro (Russel et al., 1999). Although the mechanism by which IGF-I prevents glucose-mediated cell death in neurons remains unknown, it is likely that IGF-I exerts its neuroprotective effect through specic receptor signaling cascades that inhibit programmed cell death in DRG neurons. In fact, it has been reported that IGF-I prevents activation of caspase-3 (Russel et al., 1999). Although a series of observations provide important information for understanding the degenerative mechanism of neuronal cells in diabetes, the implication of apoptotic changes under hyperglycemia or high glucose condition in DRG neurons appears unclear. As described above, there have been no reports on loss of DRG neurons in both short- and long-term STZ-induced diabetic rats. Thus, the

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relationship of apoptotic changes of DRG neurons to neuronal loss in this animal model is not evident, because those neurons are likely to survive apoptotic changes. Similarly, TUNEL staining, frequently used as a marker of apoptosis, is often positive even in the absence of neuronal loss at the early stage of chronic neurodegenerative diseases, including Alzheimers disease (Stadelmann et al., 1999), amyotrophic lateral sclerosis (Martin, 1999), Parkinsons disease (Burke and Kholodilov, 1998) and other neurodegenerative diseases (Gray et al., 1999) in which the neurons with fragmented DNA would be expected to be rapidly phagocytosed, leading to neuronal loss. However, since we do not know the temporal sequence between fragmented DNA and DNA repair that allows neuronal cells to remain dysfunctional but resist nal dissolution and phagocytosis, the direct causal relationship between positive TUNEL staining and neuronal loss has to be examined. On the other hand, we found that, using TUNEL staining, no apoptotic changes developed in the DRG neurons in either control or STZ-induced diabetic rats, whereas sciatic nerve crush evoked the apoptotic change as early as 1 day after the crush only in diabetic rats, when cAMP content was more decreased and p-JNK/p-c-Jun was more increased in DRG neurons in diabetic than in control rats. Interestingly, since the treatment with PGE1analogue prevented the apoptotic change without affecting abnormalities in cAMP and p-JNK/p-c-Jun, the apoptotic change may be associated with the other factors. 7.3. Apoptosis and neurotrophins/MAP kinase/cAMP in hyperglycemia 7.3.1. Neurotrophins and their receptors NGF deprivation is fatal to neurons because NGF is a key factor in neuronal survival and maintenance of their normal functions. Indeed, NGF deprivation has been shown to induce neuronal apoptosis (Pittman et al., 1993). The activation of stress-activated protein kinase (SAPK)/JNK in target neurons is shown to be implicated in the suicide program (Xia et al., 1995). Thus, neuronal apoptosis may be blocked by inhibiting the activity of the intracellular messengers associated with SAPK/JNK pathway. Serum and tissue levels of many neurotrophins and the expression of their receptors are decreased in diabetic condition. In addition, the activity of JNK is increased in DRG neurons of STZ-induced diabetic rats (Fernyhough et al., 1999). This circumstance may contribute to neuronal degeneration or loss in diabetes, although it remains unclear whether neuronal loss primarily occurs in diabetic nerves. All family members of neurotrophins bind to neurotrophin receptor p75 (p75NTR), which belongs to the tumor necrosis factor superfamily. Although the biological function of p75NTR has been elusive, it has been suggested to mediate apoptosis of developing neurons in the absence of trk receptors. Thus, life-death decisions of cells are dependent on the expression and action of two different receptors

with distinctive signaling mechanisms. In the presence of TrkA receptors, p75NTR can participate in the formation of high afnity binding sites and enhanced NGF responsiveness leading to a survival signal. In the absence of TrkA receptors, p75NTR can generate a death signal: induced expression of the p75NTR intracellular domain within adult animals lead to increased motor neuron death after axotomy (Majdan et al., 1997). The mechanism of p75NTR-mediated neuronal or Schwann cell death is unclear, although Bcl-2 family members may be involved (Coulson et al., 1999; Soilu-Hanninen et al., 1999). These results may suggest that the balance in the expression between TrkA and p75NTR is involved in neuronal death or nerve regeneration. It was shown that, in diabetic rats, the expression of p75NTR was reduced, whereas that of TrkA was unchanged (Delcroix et al., 1997). This was true for anterograde and retrograde axonal transport of these receptors within sciatic nerves in diabetic rats (Delcroix et al., 1998). However, axotomy did not reduce the expressions of both p75NTR and TrkA in STZ-induced diabetic rats, while it reduced p75NTR but not TrkA in normal rats (Mohiuddin and Tomlinson, 1997; Delcroix et al., 1997). This situation may lead to a higher ratio of p75NTR/TrkA in diabetic than in normal rats under the circumstance of nerve lesions. This altered balance between the two receptors under hyperglycemic condition might underlie the susceptibility of neurons to nerve damage, although there is no clear evidence supporting this hypothesis. Furthermore, NGF mRNA in the distal stump of crushed nerves, which is the major source of NGF for axotomized DRG neurons, is increased in diabetic rats (Maeda et al., 1996). Thus, increased binding of NGF to p75NTR may also contribute to the vulnerability to apoptosis in axotomized DRG neurons in diabetes. The notion that the altered expression of NGF and p75NTR might induce apoptosis and regeneration failure (Carter and Lewin, 1997) was already proposed. 7.3.2. MAP kinases Among MAPKs, ERKs are most potently activated in response to signals originating from receptor and cytoplasmic protein tyrosine kinases. Unlike ERKs, JNK and p38 are most potently activated by environmental stresses such as UV radiation and osmotic shock, as well as by proinammatory cytokines such as TNF and IL-1 (Davis, 1994; Karin, 1995). However, there is little evidence of activation of p38 in neurons and no indication of stress-induced phosphorylation of this kinase. In addition to the contribution to Ras-mediated transformation of rat embryo broblasts and proliferation of mouse embryo broblasts, JNK was implicated in induction of apoptosis. By contrast, ERK activation may protect PC12 cells against apoptosis. This notion may suggest the possibility that the difference, if any, in the expression of the two kinds of MAP kinases contributes to neuronal death under pathologic conditions including diabetic neuropathy. Although Fernyhough et al. (1999) and we (unpublished data) found that phosphorylation of both

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MAP kinases were increased in DRG neurons of diabetic rats, Tomlinson (1999) pointed out that ERK was strikingly absent from the cell bodies of motor neurons. Tomlinson (1999) speculated that this nding might underlie the predisposition of diabetic neuropathy for sensory bers. 7.3.3. cAMP NGF regulates the developmental programmed cell death of certain neurons in the PNS. The survival and proper functioning of sympathetic neurons are dependent on NGF; when immature sympathetic neurons are deprived of NGF, they undergo an active dying process termed programmed cell death. This trophic factor dependence is age-related such that the cells become less dependent on NGF as they mature, which triggers the process of programmed cell death, resulting in a signicant decrease of intracellular cAMP levels. In contrast, when these cells mature in culture and become relatively NGF independent, NGF withdrawal does not lead to a drop of cAMP levels. Thus, cAMP content is closely linked with neuron survival. However, the role of cAMP in the induction of apoptosis remains controversial: stimulatory (Weiss et al., 1995) or inhibitory (Alok et al., 1994). Indeed, cAMP has divergent effects on MAPK activity through either Ras/Raf-1 or Rap1/B-raf as signal pathway. In neurons, cAMP activates MAPK in a Rap1/B-raf-dependent manner, while, in astrocytes, cAMP decreases MAPK activity. Inhibition of MAPK in neurons decreases neuronal growth factor-mediated survival, and activation of MAPK by cAMP analogues rescues neurons from death. Furthermore, constitutive expression of B-raf in astrocytoma cells increases MAPK activation, leading to enhancing proliferation. These data provide evidence that B-raf is the molecular switch that dominantly permits differential cAMP-dependent regulation of MAPK in neurons versus astrocytes (Dugan et al., 1999). The possibility that cAMP promotes neuronal survival by directly phosphorylating glycogen synthase kinase 3, which has been demonstrated to be associated with apoptosis of rat cerebellar granule neurons, is also suggested (Li et al., 2000). Since cAMP content is known to be decreased in DRG (Kogawa et al., 2000) and sciatic nerves (Shindo et al., 1992; Maeda et al., 1994) in diabetic rats, the contribution of decreased cAMP content to apoptotic change in DRG neurons may be considered. However, our data that TUNEL-positive apoptotic change was induced even 1 day after axotomy when cAMP content remains unchanged from before crush may suggest that this apoptotic change may not be associated with cAMP content. Peripheral nerve axotomy induces apoptosis in Schwann cell precursors. bFGF protects these cells from axotomyinduced apoptosis (Jessen et al., 1994; Gavrilovic et al., 1995). Cultured Schwann cells also undergo apoptotic change by treatment with 8-bromo-cAMP, a membranepermeable analogue of cAMP, which induces expression of galactocerebroside in the plasma membranes of Schwann cells. Treatment with bFGF reduced the percent-

age of galactocerebroside-bearing Schwann cells undergoing cAMP-induced DNA fragmentation. These ndings suggest that bFGF can enhance the survival of differentiated Schwann cells by preventing cAMP-induced apoptosis (Shaw et al., 1997). Although it is unclear whether apoptotic changes are induced in Schwann cells in human diabetic nerves, it was reported that dorsal root Schwann cells underwent apoptosis in STZ-induced diabetic rats and in acutely hyperglycemic rats, although the analysis was not precisely performed (Russel et al., 1999).

8. Conclusion In spite of much effort to introduce ideal therapeutic drugs for diabetic neuropathy, ARIs, whose effects have been best investigated both clinically and experimentally and have been shown to be mediated through multifactorial mechanisms including amelioration of neurotrophic support, are still the most established compounds among potent drugs. However, although experimental data on ARIs have been very promising, a reality is that their clinical efcacy seems limited even for mild degrees of diabetic neuropathy. That is the case with other compounds. Neurotrophic factors appear less effective than such conventional drugs; no extensive trials have shown their efcacy and a considerable number of adverse effects are also problematic. At the moment, early diagnosis and preventive therapy may be the best strategy for treating diabetic neuropathy. Nevertheless, in the near future, cell or tissue engineering technology combined with molecular biology may provide ideal therapies for nerve regeneration by enhancing axon extension and by modulating either axonSchwann cell or axonextracellular matrix interaction, although there are still many difculties to overcome. Furthermore, huge amount of unknown mechanisms about cell molecular biology and signal pathways may contribute to the process of nerve regeneration. Their exploration may provide us with benecial clues for the treatment of diabetic neuropathy with reference to nerve regenerative capacity as well as cell biology.

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