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Cajal’s Contributions to the Study of Alzheimer’s Disease
Virginia Garc´a-Mar´n1,∗ , Pablo Garc´a-L´ pez1 and Miguel Freire ı ı ı o
´ Museum Ramon y Cajal, Instituto Cajal, CSIC, Avda. Doctor Arce, 37; Madrid – 28002, Spain
Abstract. Last year 2006, we commemorated two important events in the history of Neuroscience. One hundred years ago, on November 3, Alois Alzheimer (1864–1915) presented the ﬁrst case of a patient with symptoms of a disease that later would be called Alzheimer’s disease. One month later, on December 10, Santiago Ram´ n y Cajal (1852–1934) and Camilo Golgi o (1843–1926) received the Nobel Prize “in recognition of their work on the structure of the Nervous System”. These facts seem not to be related, but working in the Museum Cajal we found 37 histological preparations of material from patients suffering from Alzheimer’s disease, revealing that Cajal also studied this disease. This paper deals with Cajal’s contribution to the study of Alzheimer’s disease and it is fully illustrated by original pictures of Cajal’s slides preserved in the Cajal Museum, Madrid. Keywords: Cajal, Alzheimer’s disease, senile plaques, neuroﬁbrillary tangles
INTRODUCTION Ram´ n y Cajal is broadly known by his studies of o the anatomy, functional organization and development of the nervous system. However, his observations and theories on pathology of the nervous systems are little known, with the exception of his experimental studies about degeneration and regeneration of the nervous system . Cajal made important contributions to pathological anatomy including his studies on cancer , Alzheimer’s disease (AD), neurosyphilis, Korsakoff’s syndrome, and rabies. AD is one of the most important neurological disorders. Since it was discovered by Alzheimer in 1906, much research has been done in order to clarify the mechanism that underlies the disease. Cajal also paid attention to this disorder and following his own research about degeneration and regeneration of the nervous system gave some ideas about the degenerative and regenerative processes that occur in AD. Our aim is to study the original histological preparations conserved in the Museum Cajal from patients sufauthors contributed equally to this work. ∗ Address for correspondence. Tel.: +34 91 585 47 43; Fax: +34 91 585 47 53; E-mail: firstname.lastname@example.org.
fering from AD. We will show original pictures of the main pathological hallmarks of AD taken from these slides, and review some of Cajal’s ideas about AD from a historical and present-day perspective. Cajal’s main contributions to AD are summarized in his Span´ ´ ish books Degeneraci on y Regeneraci on del sistema nervioso2  and Manual General de Anatomia patol´ gica y bacteriolog´a 3 . o ı
CAJAL’S HISTOLOGICAL PREPARATIONS In the Museum Cajal, 4,529 histological preparations of Cajal are conserved, 690 are related to pathology: cancer (92 slides), degeneration and regeneration of the nervous system (391), AD (37), neurosyphilis (135), Korsakoff syndrome (17) and rage (18). Among Cajal’s histological preparations of AD (Fig. 1), we have found different histological methods. Table 1 summarizes the different components of each method and their main staining targets. Cajal used to write on the labels of his
book has been translated into English in 1928 (translation by May)  and 1991 (translation by translation by May, with introduction and additional translation by DeFelipe and Jones) . 3 Not English version of this book.
ISSN 1387-2877/07/$17.00 2007 – IOS Press and the authors. All rights reserved
V. Garc´a-Mar´n et al. / Cajal’s Contributions to the Study of Alzheimer’s Disease ı ı Box 1
Cajal proposed his neurotropic hypothesis, ﬁrstly named chymiotactic hypothesis in 1893 . He proposed the existence of neurotropic substances for explaining the guidance of axons to their targets during development. Cajal also admitted the secretion of neurotropic substances by intermediate targets. For instance, he explained the attraction of commissural axons to the ventral part of the epithelial barrel during the development of the spinal cord, as later was demonstrated discovering the secretion of netrins by the ﬂoor plate [64,126]. “It would be explained by the production of inducting substances of great force at the level of the ventral half of the epithelial barrel” . Furthermore Cajal admitted the possibility of negative neurotropism [102,116] although in some parts of his work he negated this possibility . However this Cajal’s concept of neurotropic substance evolved to a concept of neurotrophic agents especially under pathological conditions, in which the substances secreted by the Schwann cells would create the trophic ambient necessary for the growing of the axonal sprouts. “These substances have not only an orientating function, but they are also trophic in character, since the sprouts that have arrived at the peripheral stump are robust, show a great capacity for ramiﬁcation and grow straight to their target without vacillations, as though they were following an irresistible attraction” . Nowadays, this double concept of Cajal is still valuable for developing and regenerating nervous system, i) the growth and maturation of cells, under the inﬂuence of neurotrophic substances, and ii) the direction of neuronal processes, depending on neurotropic substances, mediated by guidance cues that can act attracting or repelling dendrites or axons. In this last group of neurotropic substances are also included surface-bound adhesive molecules. As examples of neurotrophic substances which allowed the growth and maturation are found neurotrophins such as; NGF [72,73], BDNF [13,29], NT-3, and NT4/5 [15,78] and their receptors (the trk receptor tyrosine kinases and the p75 neurotrophin receptors) [63,95]. On the other hand, neurotropic substances can act on axons such as: Netrins [127-64] /DCC-Unc [57,61], semaphorins [69,77] /plexinsNeuropilin [25,44,56,143], and Slits [22,65] /Robo [66,149]; on dendrites, for instance LNotch [14,127], Beta-catenin , Numb [121,127], Rho family of GTPases [52,76]; and on synapses for example: CAMs [16,135], integrins [93,36], cadherins [125,26], neurexinneuroligin [124,130] and syndecan 2 [38,58].
More studies focused on nerve growth factors are necessary to understand the mechanism of neurodegenerative disorders and their treatment. In addition growth factors constitute a therapeutic approach for the treatment of the disease. Neuroﬁbrillary pathology Neuroﬁbrillary tangles were described by Alzheimer . At Cajal’s time, scientists called them “Alzheimer lesions” as we can see in Cajal’s preparations (Fig. 1) and writings . Alzheimer wrote “Specimens which were prepared according to Bielschowsky’s silver method show very striking changes of the neuroﬁbrils. Inside of a cell which appears to be quite normal, one or several ﬁbrils can be distinguished by their unique thickness and capacity for impregnation. Further examination shows many ﬁbrils located next to each other which have been changed in the same way. Next, combined in thick bundles, they appear one by one at the surface of the cell. Finally, the nucleus and the cell itself disintegrate and only a tangle of ﬁbrils indicates the place where a neuron was previously located” (Fig. 5J) [3,53,133]. Interestingly, Cajal also observed neuroﬁbrillar alterations in physiological and pathological conditions such as hibernation, rage, or after a wound in the nervous system (Box 2). The different morphological variants of tangles found are in part due to the nature of the neurons in which they were formed. Flame-shaped NFT are characteristic of pyramidal neurons, and globose NFT are
found in large non-pyramidal neurons . The ﬂameshaped NFT sometimes have a great apical process that follows the direction of the apical dendrite (Fig. 5D–F). In addition, we have found other lesions related to neuroﬁbrillary degeneration, neuropil threads and dystrophic neurites. Neuropil threads are short and tortuous argyrophilic neurites dispersed in the neuropil that are immunoreactive for tau . Most neuropil threads are probably derived from dendritic processes , but up to 10% have been identiﬁed as myelinated axons with electron microscopy [94,100]. Dystrophic neurites appear surrounding the corona of neuritic senile plaques (Fig. 4). These dystrophic neurites could be the crown of nervous buds which Cajal refers to in 1918. Cajal specially proposed that these dystrophic neurites were axonal sprouts with terminal clubs directed to the plaque because the inﬂuence of some neurotropic substances. Interestingly these axonal sprouts were ﬁrstly seen by Cajal in regenerating axonal process after the section of the sciatic nerve (Fig. 7) [109,110] and also conﬁrmed by Perroncito . In addition to the axonal sprouting [45,46,59,84,85],dendritic sprouting [6–9,41,60,71,89,96,123] have been conﬁrmed in AD. This process of dendritic regeneration is mediated by structures that resemble dendritic growth cones Interestingly dendritic neoformation was observed by Cajal  in the sensitive ganglion of old humans and dogs and in the hippocampus of old dogs by Rodriguez Lafora , disciple of Cajal and Alzheimer. The regeneration studies of Cajal inﬂuenced the initial studies of AD. Fischer proposed that the senile
V. Garc´a-Mar´n et al. / Cajal’s Contributions to the Study of Alzheimer’s Disease ı ı
modify its own structural organization and functionings as an adaptative response to functional demands which is impaired in AD” . Coming back to Cajal and the loss of plasticity in the adult brain he stated: “Such plasticity of the cellular processes probably varies at different ages: greater in the young man, diminished in the adult and almost completely disappeared in the aged” [31,103].
CONCLUSIONS Cajal described his ﬁndings about AD more than 10 years later than Alzheimer published his ﬁrst article in 1907. However, it is still useful to review his slides and writings in order to compare them with the actual knowledge about the disease and also to point out some of the ideas proposed by Cajal. The staining methods used by Cajal, such as reduced silver nitrate, sublimated gold chloride, ammoniacal silver oxide, and the Bielschowsky method, are very useful to observe the many alterations in AD. Ammoniacal silver oxide and the Bielschowsky methods give a general idea of the brain alterations because they stain senile plaques, neuroﬁbrillar lesions, reactive microglia, and active astrocytes. The reduced silver nitrate method is very useful to observe the evolution of the senile plaques, and ﬁnally, the sublimated gold chloride method allows a speciﬁc study of reactive astrocytes. These classic staining methods can still be useful in the research of this pathology, because today we can re-interpret them taking into account the knowledge acquired using speciﬁc antibodies. Furthermore, with the use of speciﬁc antibodies only we may loose and overlook other components of the senile plaques and neuroﬁbrillary tangles already shown by the classical staining methods. The original works of the pioneers and Cajal in the study of AD still propose hypotheses and questions that we have to corroborate and/or to answer to be able to advance the knowledge of AD. For instance, Cajal proposed in the evolution of senile plaques a degeneration of neurites in the surrounding area. Nowadays with modern technique it has been observed disruptions in neurites trajectories and reductions in dendritic spine density in the regions near the plaques have been observed. The dystrophic neurites (both axonal and dendritic origin) present swellings on their structure that was observed by Cajal with his silver nitrate method as we can see in his drawing. Moreover, Cajal suggested that axonal sprouts could be attracted towards the region of the plaque, under the inﬂuence of some special neurotropic substance. Future research should clarify these issues in order to advance in the knowledge of AD.
GLIAL ALTERATION Cajal observed that astrocytes in the senile plaques showed hypertrophy in their bodies and appendages, and that some of these processes ended in balls . This hypertrophy of astrocytes is clear in his histological preparations (Fig. 6), some of them are extremely big and show a great quantity of glioﬁlaments as was ﬁrstly noticed by Alzheimer in his famous report of Auguste, D.  “The glia have developed numerous ﬁbers and other glial cells have large lipid droplets”. Astrocytes acquire a ﬁbrous character like the astrocytes in white matter. The processes of the astrocytes surround the senile plaques. Mature senile plaques are not only wrapped by astrocytic processes, but they also penetrate the plaque core [79,92]. Astrocytes secrete growth factors such as nerve growth factor (NGF), basic ﬁbroblast growth factor (BFGF), etc. which are essential for survival and terminal remodeling of the surviving neurons because they act as a trophic stimulus inducing axonal growth and sprouting [74,137,140,142]. Furthermore, in AD there is an alteration in microglial cells, with hypertrophy and changes in their morphology towards round-shaped macrophage-like cells. Microglial cells were found surrounding senile plaques (Fig. 6B–C, E). Activated microglia are most evident in dense cored plaques [10,141]. These microglia have a very important role in the process of inﬂammation as Fischer point out in 1910. He thought that “the foreign substance” provoked the neuroregenerative response by inﬂammation, although he could not ﬁnd morphological signs of an inﬂammation process around the plaques. In the eighties of the last century using monoclonals antibodies directed against cells of the monocyte-macrophage cell lineage, senile plaques were shown to be associated with clusters of activated microglia [88,120,122]. The neuroinﬂammatory response includes a local upregulation of acutephase proteins, complement, cytokines and other inﬂammatory mediators . However the diffuse plaques are no associated with activated microglia
ACKNOWLEGDEMENTS ´ We acknowledge Santiago Ram on y Cajal Junquera for his help studying the histological preparations and
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