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Cerebral Palsy Article 2

Cerebral Palsy Article 2

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Special Article

Maturational Effects of Lipopolysaccharide on White-Matter Injury in Fetal Sheep
Pernilla Svedin, MSc; Ingmar Kjellmer, MD, PhD; Anna-Karin Welin, MD; Sofia Blad, BSc; Carina Mallard, PhD

ABSTRACT White-matter damage has been associated with the development of cerebral palsy in children born both prematurely and at term, and it has been suggested that intrauterine infection can contribute to the brain injury. However, the relative importance of age on white-matter injury following infectious exposure in utero remains unclear. In this study, fetal sheep were exposed to systemic endotoxemia by administration of Escherichia coli lipopolysaccharide (88.7 ± 7.7 ng/kg) at 65% or 85% of gestation. These gestational ages approximately correspond to human brain development in preterm and near-term infants respectively. White-matter injury was evaluated 3 days after lipopolysaccharide exposure with regard to microglia activation and loss of neurofilament and myelin basic protein. The expression of oligodendrocytes at different maturational stages was demonstrated in preterm and near-term fetuses with the oligodendroglial markers O4 and 2´,3´-cyclic nucleotide 3´-phospodiesterase. Forty percent of the fetuses in the preterm group and 22% in the near-term group died within 8 hours of the endotoxin exposure. Three of six preterm and two of seven near-term surviving fetuses demonstrated pathologic changes in the brain with regard to increased microglia activation and loss of neurofilament staining. The number of activated microglia was enhanced in the subcortical white matter in both the preterm lipopolysaccharideexposed fetuses (lipopolysaccharide: 235 ± 64 cells/mm2; control: 72 ± 28 cells/mm2; P = .0374) and the near-term fetuses ( lipopolysaccharide: 180 ± 40 cells/mm2; control 23 ± 16 cells/mm2; P = .0152). There was a loss of neurofilament staining in both preterm fetuses (lipopolysaccharide: 2.20 ± 0.77 pixel units; control: 0.20 ± 0.10 pixel units; P = .0306) and nearterm fetuses (lipopolysaccharide: 1.15 ± 0.48 pixel units; control: 0.06 ± 0.06 pixel units; P = .0285). O4-positive cells were detected at both gestational ages, whereas 2,3-cyclic nucleotide 3-phospodiesterase-positive cells and myelin basic protein staining were mainly detected in the near-term fetuses. In summary, we found white-matter injury in a proportion of both preterm and near-term fetuses after administration of lipopolysaccharide. These results are in agreement with clinical evidence suggesting that both preterm and term infants are at risk of periventricular leukomalacia in association with intrauterine infection. (J Child Neurol 2005;20:960–964).

Received August 10, 2005. Accepted for publication August 10, 2005. From the Departments of Physiology and Pharmacology (Ms Svedin, Dr Mallard, and Ms Blad), Pediatrics (Dr Kjellmer), and Obstetrics and Gynecology (Drs Welin and Mallard), Perinatal Center, Sahlgrenska Academy, Göteborg University, Göteborg, Sweden. This work was supported by the Swedish Medical Research Council (K200433X-14185-03A), the Åhlen Foundation, the Sven Jerring Foundation, the Magnus Bergvall Foundation, the Wilhelm and Martina Lundgren Foundation, the Linnéa and Josef Carlsson Foundation, the Frimurare Barnhus Foundation, the Göteborg Medical Society, and the Åke Wibergs Foundation. Presented in part at the United Cerebral Palsy Workshop on Experimental Models of Cerebral Palsy, San Francisco, CA, June 13–14, 2004. Address correspondence to Ms Pernilla Svedin, Department of Physiology, Sahlgrenska Academy, Göteborg University, 405 30 Göteborg, Sweden. Tel: +46-31-7733539; fax: +46-31-7733512; e-mail: pernilla.svedin@fysiologi.gu.se.

The risk of cerebral palsy increases dramatically with decreasing gestational age at birth and is particularly high among extremely premature infants (< 34 weeks’ gestation).1 The prevalence of cerebral palsy in more mature infants is much lower, however, because the majority of births occur close to term; half of all cases of cerebral palsy occur in term or near-term infants.2 A majority of infants with cerebral palsy demonstrate injury to the cerebral white matter, characterized by focal necrosis in the periventricular region, so-called periventricular leukomalacia, or diffuse injury.3,4 Although less frequent, white-matter injury is also observed in full-term infants and is then more often of the focal than the diffuse type.5 The etiology of white- matter damage in cerebral palsy is not fully understood, but it has been suggested that intrauterine infection plays a role6,7 and chorioamnionitis has been identified as a risk factor for cerebral palsy and cystic periventricular leukoma-


Maturational Effects of Lipopolysaccharide on White-Matter Injury / Svedin et al


lacia in both term and preterm infants.7–9 Elevated levels of cytokines in the amniotic fluid and/or fetal blood increase the risk of periventricular leukomalacia and cerebral palsy.10,11 It has also been established that the number of tumor necrosis factor – and interleukin-6–positive cells is significantly higher in the brains of infants who die with periventricular leukomalacia, suggesting that inflammatory cytokines can play a role in the neuropathology of periventricular leukomalacia.12,13 The age at which premature infants are at greatest risk of periventricular leukomalacia (24–32 gestational weeks) coincides with the developmental stage, when there is a high prevalence of premyelinating oligodendrocytes in the white matter.14,15 In vitro studies have shown that these cells, but not mature myelin basic protein–expressing oligodendrocytes, are particularly sensitive to free radical injury and death.16 The susceptibility of oligodendrocyte progenitor cells to nitric oxide involves mitochondrial dysfunction and activation of the caspase-independent apoptosis-inducing factor pathway.17 Oligodendroglia progenitor cells are also very sensitive to -amino-3-hydroxy-5-methyl-4isoxazoleproprionic acid (AMPA) receptor–mediated excitotoxicity,18 which appears to be associated with transiently enhanced AMPA glutamate receptor–mediated calcium signaling.19 Several studies in fetal sheep have shown that experimentally induced intrauterine infection at 65% to 70% of gestation is associated with cerebral white-matter lesions similar to those seen in children with cerebral palsy.20–22 However, it is not known whether infectious exposure to the more mature fetus is associated with periventricular leukomalacia–like lesions. In this study, we exposed fetal sheep at different gestational ages to in utero infection by intravenous lipopolysaccharide. The sheep fetuses were exposed at either 65% or 85% of pregnancy, which approximately corresponds to human brain development at the second and third trimesters, respectively.23 The fetal brains were evaluated with regard to axonal injury, myelination, microglia activation, and neuronal injury. MATERIAL AND METHODS Surgery
Twenty-six pregnant sheep were subjected to aseptic surgery (isoflurane, 1.5% in O2) at either 89.1 ± 0.2 (n = 14) or 121.3 ± 0.8 (n = 12) days of gestation (term = 147 days).20 Polyvinyl catheters were inserted into each fetal axillary artery and one vein for blood sampling and lipopolysaccharide administration. After the surgical procedure, the ewes were lodged in individual cages with free access to food and water.

evenly spaced, through the forebrain, and adjacent sections were stained with acid fuchsin and thionin24 for morphologic analysis or immunohistochemically for the identification of oligodendrocytes, myelin, neurofilament, and microglia as previously described.25 The following primary antibodies and dilutions were used from Sternberger Monoclonal Incorporated (Lutherville, MD): anti–myelin basic protein (SMI 94, 1:10,000, overnight, 4°C) and anti–phosphorylated neurofilament (SMI 312, 1:2000, 1 hour, room temperature, room temperature); and from Lab Vision Corporation (Fremont, CA): anti–2´,3´-cyclic nucleotide 3´-phospodiesterase (CNPase, MS-349-P, 1:100, 1 hour, RT), followed by secondary antibody incubation (horse-antimouse biotinylated, 1:250, Vector Laboratories, Burlingame, CA; 1 hour, room temperature). Immunoreactivity was visualized by 3,3-diaminobenzidine. To identify microglia, sections were incubated with 10 g/mL Griffonia simplicifolia isolectin B4, which was horseradish peroxidase–labeled (Sigma, L5391) overnight (4°C) and visualized using 3,3-diaminobenzidine. For the detection of oligodendroglia progenitor cells, free floating sections (40 m) were incubated with 10 g/mL antioligodendrocyte marker O4 (MAB345, Chemicon International, Temecula, CA), 2% bovine serum albumin, and 0.3% triton X-100 for 48 hours at 4°C. The staining procedure was continued as described above.

Analysis of Brain Injury
Histopathologic analysis was performed on coded slides, with the observer unaware of the experimental grouping. Evenly spaced sections throughout the brain were used for the acid fuchsin and thionin staining to make a gross morphologic examination. Control fetuses at both 65% and 85% of gestation were examined with regard to white-matter maturation (O4, 2,3-cyclic nucleotide 3-phospodiesterase, myelin basic protein). Microglia activation was examined in the white matter at three different brain levels according to the stereotaxic atlas of the fetal sheep brain26: level 1: A26.5 at the level of the anterior commissure; level 2: A22.0 at the level of the anterior thalamus; and level 3: A18.0 at the level of the anterior hippocampus. Activated microglia were identified as those cells with an ameboid appearance. The number of activated microglial cells was counted in each section in three different areas within the subcortical and periventricular white matter using a 40 objective. Neurofilament and myelin basic protein staining was analyzed in sections adjacent to those stained for microglia. The staining was converted into a black and white image and isolated from the background image using segmentation processing using Micro Image, version 4.0 (MicroMacro AB, Göteborg, Sweden). This allows measurements of areas where there is a loss of staining (pixel sum).

Statistics Experimental Protocol
Systemic endotoxemia was induced at 91.0 ± 0.3 (preterm, n = 10) or 124.2 ± 1.1 (near term, n = 9) days of gestation by fetal administration of Escherichia coli lipopolysaccharide (Sigma, Saint Louis, MO; 055:B4, 88.7 ± 7.7 ng/kg based on their postmortem weight, intravenously). Control fetuses underwent sham experimental endotoxemia at 92.3 ± 0.3 (n = 6) or 125.3 ± 0.6 (n = 6) days of gestation. This study was approved by the Animal Ethical Committee of the University of Göteborg. The Mann-Whitney nonparametric ranking test was used for the histochemical statistical comparisons between the lipopolysaccharide-exposed animals and the control animals. Values were considered significant at P < .05, and data are presented as mean ± standard error of measurement. All statistical analyses were performed using StatView for Windows (SAS Institute Inc., Cary, NC), version 5.0.1.

RESULTS Ten preterm fetuses received lipopolysaccharide, of which four fetuses (40%) died within 8 hours. Three of the surviving fetuses (30%) demonstrated pathologic changes in the brain with regard to increased microglial activation and loss of neurofilament staining in the subcortical white matter (Figure 1). In three fetuses, there

Tissue Preparation and Histochemical Procedures
After a maternal overdose of sodium pentobarbital (130 mg/kg, intravenously), fetal brains were perfused in situ through the carotid arteries with saline (0.9% NaCl), followed by 5% paraformaldehyde in 0.1 M phosphate buffer. Paraffin-embedded brains were cut in coronal sections (8 m),


Journal of Child Neurology / Volume 20, Number 12, December 2005

The number of activated microglia was enhanced in the subcortical white matter at level 3 in the preterm lipopolysaccharideexposed fetuses (235 ± 64 cells/mm2) compared with the control group (72 ± 28 cells/mm2; P = .0374) and the near-term fetuses (180 ± 40 cells/mm2) compared with the control group (23 ± 16 cells/mm2; P = .0152) (Figure 2A). The loss of neurofilament staining at level 2 was decreased in the near-term lipopolysaccharideexposed fetuses (1.15 ± 0.48 pixel units) compared with control fetuses (0.06 ± 0.06 pixel units; P = .0285), and the lipopolysaccharide-exposed preterm fetuses showed a significant reduction at level 3 (2.20 ± 0.77 pixel units) compared with the control animals (0.20 ± 0.10 pixel units; P = .0306) (Figure 2B). No myelin basic protein staining was detected in the forebrain of preterm fetuses. Patches of myelin basic protein loss were observed in two lipopolysaccharide-exposed near-term fetuses; however, there was no significant difference in the near-term lipopolysaccharideexposed group compared with control fetuses (Figure 2C). Positive O4 cells were observed in both preterm and near-term control fetuses. There was very little 2,3-cyclic nucleotide 3phospodiesterase and myelin basic protein staining in preterm fetuses, whereas the near-term fetuses showed high expression of both 2,3-cyclic nucleotide 3-phosphodiesterase and myelin basic protein staining (Figure 3). DISCUSSION The relative importance of age on white-matter injury following infectious exposure in utero remains unclear. In this study, we compared the brain injury after systemic administration of bacterial endotoxin in fetal sheep at 65% (preterm) and 85% (near term) of gestation, respectively. These gestational ages are believed to approximately correspond to preterm respectively term infants, with respect to brain development. In agreement with our previous study in fetal sheep20 and other studies,21,22 we found that 50% of the surviving preterm fetuses developed pathologic changes in the brain in response to endotoxin exposure. We also observed injury in 28% of the surviving near-term fetuses. The pathologic changes were characterized by an increased number of activated microglia and loss of neurofilament protein in the periventricular and subcortical white matter. There was also a trend, although not significant, for a loss of myelin basic protein in the near-term group. We observed a significant number of microglia with ameboid appearance in preterm control fetuses, particularly in the white matter of the internal and external capsule, whereas this was rarely seen in near-term control fetuses. These findings are in agreement with previous studies demonstrating numerous ameboid isolectin B4–positive cells in white-matter tracts in fetal sheep before 96 days of gestation.27 There have been similar observations in the human brain showing colonization of ameboid-like microglia cells during the second trimester, which has been suggested to predispose these areas to injury.28 There was an increase in the number of activated microglia cells in the lipopolysaccharide-exposed preterm and near-term fetuses. Systemic administration of lipopolysaccharide is believed to trigger microglia activation at the level of the blood–brain barrier, with the subsequent release of proinflammatory cytokines such as tumor necrosis factor , via the activation of the lipopolysaccharide receptor Toll-like receptor 4.29 Several studies have shown that the cerebral white matter appears to be vulnerable to cytokines and oligodendrocyte progenitor cells appear to be particularly

Figure 1. Cross sections of acid fuchsin–stained periventricular white matter in preterm (A) and near-term (B) fetuses (original magnification, x10). Microglial activation (C–F) (original magnification, x400) and neurofilament staining (G–J) (original magnification, x100) in the periventricular white matter (indicated by boxes in A and B) after lipopolysaccharide exposure (E, F I, J) and control fetuses (C, D, G, , H), in preterm (A, C, E, G, I) and near-term fetuses (B, D, F H, J). ,

were no neuropathologic alterations compared with the control fetuses. In the near-term group, two of nine fetuses exposed to lipopolysaccharide (22%) died within 8 hours and another 2 fetuses showed an increased microglia activation and reduced expression of neurofilament and myelin basic protein compared with the control group (see Figure 1). There were no neuropathologic alterations in five lipopolysaccharide-exposed near-term fetuses. Some activated microglia was detected in most control animals, mainly in the internal and external capsules in preterm fetuses.

Maturational Effects of Lipopolysaccharide on White-Matter Injury / Svedin et al


Figure 3. Immunohistochemical staining for oligodendrocytes in preterm (A, C, E) and near-term (B, D, F) fetuses (original magnification; x 600). O4-positive cells were present in both preterm (A) and near-term (B) fetuses.There were more 2,3-cyclic nucleotide 3-phosphodiesterase-positive cells in near-term fetuses (D) compared with preterm fetuses (C). Almost no myelin was detected in preterm fetuses (E), whereas there was significant myelination in the near-term fetuses (F).

Figure 2. The number of activated microglia (A) and loss of neurofilament (B) and myelin basic protein (C) in the periventricular and subcortical white matter at brain levels 1, 2, and 3. A, At level 3, lipopolysaccharide-exposed groups showed an increased number of microglia in both preterm (lipopolysaccharide: 235 ± 64 cells/mm2; control: 72 ± 28 cells/mm2; P = .0374) and near-term (lipopolysaccharide: 180 ± 40 cells/mm2; control: 23 ± 16 cells/mm2; P = .0152) fetuses. B, The expression of neurofilament at level 2 was decreased in the nearterm lipopolysaccharide-exposed fetuses compared with controls and at level 3 in the preterm lipopolysaccharide-exposed group compared with controls. C, Myelin basic protein staining in the near-term group. Black bar = lipopolysaccharide preterm; gray bar = control preterm; striped bar = lipopolysaccharide near term; white bar = control near term. Data are presented as mean ± standard error of measurement, and values were considered significant at P < .05.

sensitive to tumor necrosis factor .30 Furthermore, we have shown that the proinflammatory cytokine interleukin-18 plays a role in immature white- matter injury because deletion of the gene attenuated the loss of neurofilament and myelin in neonatal rats following hypoxic-ischemic brain damage.31 Lipopolysaccharide is also known to increase the permeability of the blood–brain barrier, which can allow entry of leukocytes or cytokines into the brain.32,33 In the fetal sheep, a low dose of lipopolysaccharide induced increased expression of cyclooxygenase 2 in the brain, and at the same time, plasma albumin was observed in the parenchyma of the brain, indicating increased blood–brain barrier permeability.34 The effects of inflammation on the blood–brain barrier can be particularly severe in the immature brain because it has been shown that the increase in the permeability of the blood–brain barrier was much greater in juvenile than older rats following an injection of interleukin-1 .35 Lipopolysaccharide is a potent inducer of oxidative stress, and in vitro studies show that oligodendrocyte progenitors are particularly susceptible to depletion of antioxidants and exposure to free radicals, contributing to failure of myelination.16 In contrast, more mature oligodendrocytes that are myelin basic protein positive exhibit increased resistance.17 Our results demonstrate that the preterm fetuses are at an immature premyelinating stage (O4 positive and myelin basic protein negative), with very few 2,3-cyclic


Journal of Child Neurology / Volume 20, Number 12, December 2005

nucleotide 3-phosphodiesterase-positive cells, whereas the cerebral white matter of the forebrain in the near-term fetuses is populated by cells expressing myelin basic protein, 2,3-cyclic nucleotide 3-phosphodiesterase, and O4 antigen. These observations suggest that the near-term fetuses are at a transitional stage, which has also been associated with increased vulnerability.36 In summary, we found white-matter injury in both preterm and near-term fetuses after administration of lipopolysaccharide. These results are in agreement with clinical evidence suggesting that both preterm and term infants are at risk of periventricular leukomalacia in association with intrauterine infection. References
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