c mice caused by Angiostrongylus cantonensis

Veterinary Parasitology 171 (2010) 74–80

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Alterations of myelin proteins in inflammatory demyelination of BALB/c mice caused by Angiostrongylus cantonensis K.Y. Lin a , K.M. Chen b , K.P. Lan c , H.H. Lee b , S.C. Lai b,d,∗ a b c d

Institute of Medicine, Chung Shan Medical University, Taichung 402, Taiwan Department of Parasitology, Chung Shan Medical University, Taichung 402, Taiwan Department of Laboratory, CiShan Hospital, Department of Health, 60 Chung-Hsueh Road, CiShan Chen, Kaohsiung County 842, Taiwan Clinical Laboratory, Chung Shan University Hospital, Taichung 402, Taiwan

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Article history: Received 17 November 2009 Received in revised form 8 March 2010 Accepted 8 March 2010 Keywords: Angiostrongylus cantonensis Demyelination MAG MBP MOBP PLP

a b s t r a c t Angiostrongylus cantonensis causes eosinophilic meningitis or meningoencephalitis, yet little is known about demyelination caused by this parasite. To define the course of demyelination caused by A. cantonensis, we analyzed the expression of myelin proteins including myelin-associated glycoprotein (MAG), myelin basic protein (MBP), myelinassociated oligodendrocytic basic protein (MOBP), and proteolipid protein (PLP) in brain and cerebrospinal fluid (CSF)-like fluid of infected and uninfected BALB/c mice. In A. cantonensis-infected mice, the expression of MAG, MBP, MOBP, and PLP mRNAs in brain tissue was decreased, while expression of the corresponding proteins was significantly increased in CSF-like fluid. Light microscopy revealed perivascular infiltrates in the brain during meningoencephalitis, suggesting that the cause of demyelination in angiostrongyliasis was immune system attack on the oligodendrocytic myelin sheath and subsequent release of myelin proteins into the CSF. Thus, intracerebral myelin breakdown in angiostrongyliasis may be a response to inflammatory mediators and the cause of increased myelin proteins in the CSF-like fluid. © 2010 Elsevier B.V. All rights reserved.

1. Introduction The rat lung worm Angiostrongylus cantonensis undergoes an obligatory migration into the brain of its hosts (Mackerras and Sandars, 1954; Jindrak, 1968). This parasite causes severe central nervous system (CNS) infection and produces eosinophilic meningitis (Hsu et al., 1990), eosinophilic meningoencephalitis (Gardiner et al., 1990), Purkinje cell degeneration (Chen et al., 2004), or demyelination (Lin and Lai, 2009). Neurological disorders in A. cantonensis-infected nonpermissive hosts may be ascribed not only to the mechanical damage caused by worm migra-

∗ Corresponding author at: Department of Parasitology, Chung Shan Medical University, 110, Section 1, Chien-Kuo North Road, Taichung 402, Taiwan. Tel.: +886 4 24730022x1641; fax: +886 4 23823381. E-mail address: [email protected] (S.C. Lai). 0304-4017/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2010.03.019

tion into the brain (Yoshimura et al., 1994) but also to the neurotoxicity of eosinophil-derived basic proteins (Durack et al., 1979; Fredens et al., 1982). Most common inflammatory demyelinating disorders of the CNS result from T-lymphocyte driven aberrant immune responses to a number of myelin antigens such as myelin-associated glycoprotein (MAG), myelin basic protein (MBP), myelin-associated oligodendrocytic basic protein (MOBP), and proteolipid protein (PLP) (Hartung and Rieckmann, 1997). Myelin is composed of lipid (70–75% dry weight) and protein (25–30%)(Compston et al., 1997). PLP (approximately 50% of total myelin protein) and MBP (30–40%) are the main myelin proteins. MAG and MOBP constitute less than 1% myelin proteins (Compston et al., 1997; Montague et al., 2006). MAG is a transmembrane structural protein that is thought to be required for the formation and/or maintenance of the myelin sheath (Stebbins et al., 1997). MBP contains clusters of positively

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charged amino acid residues that facilitate myelin sheath compaction (Richter-Landsberg, 2000). MOBP is a soluble cytoplasmic protein required for maintenance of compact myelin (Yamamoto et al., 1994). PLP may modify myelin structure but is not essential for its formation (Boison and Stoffel, 1994; Klugmann et al., 1997). In A. cantonensis-infected mice showing demyelination (Lin and Lai, 2009), no analysis of the expression of myelin proteins has been performed. The aim of this study was to determine whether myelin proteins were differentially expressed in BALB/c mice infected with A. cantonensis.

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Food and water were withheld for 12 h before infection. The mice of experimental groups (D5 , D10 , D15 , D20 , and D25 ) were inoculated with 50 A. cantonensis larvae by oral inoculation and sacrificed on days 5, 10, 15, 20, and 25 postinoculation (PI), respectively. The control mice received only water and sacrificed on day 25 PI. The mice were sacrificed by cervical dislocation at different time points, and the brain was rapidly removed and frozen in liquid nitrogen. 2.5. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

2. Materials and methods 2.1. Experimental animals Five-week-old male mice, BALB/c strain, were purchased from the National Laboratory Animal Center, Taipei, Taiwan. Mice were maintained at a 12 h light/dark cycle photoperiod, provided with Purina Laboratory Chow and water ad libitum, and kept in our laboratory for more than one week before the experimental infection. All of the procedures involving animals and their care in this study were approved by the Institutional Animal Care and Use Committee of Chung-Shan Medical University in accordance with institutional guidelines for animal experiments. 2.2. Antibodies Monoclonal rat anti-MBP IgG was purchased from Sigma (St Louis, MO, USA). Polyclonal goat anti-mouse PLP IgG and polyclonal goat anti-mouse MAG IgG were purchased from Santa Cruz (California, USA). Monoclonal mouse anti-MOBP IgG was purchased from Lab Vision (Fremont, CA, USA). HRP-conjugated anti-mouse IgG, HRPconjugated anti-rat IgG and HRP-conjugated anti-goat IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). 2.3. Larval preparation The infective larvae (L3 ) of A. cantonensis originally obtained from wild giant African snails (Achatina fulica) that were propagated for several months in our experimental farm of Wufeng (Taichung, Taiwan) by cycling through rats and A. fulica. The larvae within tissues were recovered using a modification of the method of Parsons and Grieve (1990). Briefly, the shells were crushed, the tissues were homogenized and digested in a pepsin–HCl solution (pH 1–2, 500 I.U. pepsin/g tissue), and incubated with agitation in a 37 ◦ C waterbath for 2 h. Host cellular debris was removed from the digest by centrifugation at 1400 × g for 10 min. The larvae in the sediment were collected by serial washing in double-distilled water and counted under a microscope. The morphological criteria for identification of L3 of A. cantonensis was described previously (Hou et al., 2004).

Total brain RNA was isolated using the TRIZOL reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. RNA quality was analyzed on the 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA). RNA samples were reverse transcribed for 30 min at 42 ◦ C with the MultiScribe Reverse Transcriptase and the oligo d(T)16 primer according to the standard protocol of the supplier (Applied Biosystems, Foster City, CA). Primer sequences were designed by the Mission Biotech Company (Taipei, Taiwan) using the Primer Express Software (Applied Biosystems). MAG forward primer: 5 -CGCAACGTGACTGTGAATGAG3 ; reverse primer: 5 -CCGGATCGTGAGGATGCT-3 . MBP forward primer: 5 -TGTCCCTCTGGCCACTTCTC-3 ; reverse primer: 5 -CCGAGGTTAGTGTGTACCAATGG-3 . MOBP forward primer: 5 -TGCAGCACAGACTGAGAGGAA-3 ; reverse primer: 5 -GCACCCTCAGGAAGTGAGGAT-3 . PLP forward primer: 5 -CAATGGACTATTTAAGCCCTAACTCA-3 ; reverse primer: 5 -CGCCCCGCTTTGTCTATACA-3 . Quantitative PCR was performed with the SYBR Green master mix (Applied Biosystems) and analyzed on an ABI PRISM 7300 sequencedetection system (Applied Biosystems) according to the manufacturer’s instructions. PCR was performed in MicroAmp® optical 96-well reaction plates (Applied Biosystems). Each well contained 12.5 ␮L 2X SYBR Green PCR master mix, 0.5 ␮L 5 ␮M forward and reverse primers for each specific gene, 40 ng cDNA, and water to a final 25 ␮L volume. PCR was performed as follows: incubation at 50 ◦ C for 2 min, initial denaturation at 95 ◦ C for 10 min to activate the AmliTaq Gold DNA polymerase followed by 40 cycles of 95 ◦ C, 15 s and 60 ◦ C, 1 min. Melting curve analysis was performed after the final cycle to examine the specificity in each reaction well for all tested samples in a given run. The dissociation step thermal cycles included a 95 ◦ C for 15 s step, a 60 ◦ C for 30 s step, and a 95 ◦ C for 15 s step. A single melting peak for each reaction confirmed a single PCR product without non-specific amplification or primer-dimers. Quantitative values were obtained from the threshold cycle (CT ) number, and target gene expression levels were normalized to ␤-actin expression in each sample. The relative mRNA level of the target gene was determined by relative quantitation CT method. 2.6. Collection of CSF-like fluid

2.4. Animal infection A total of 120 male mice were randomly allocated to six groups (control, D5 , D10 , D15 , D20 , and D25 ) of 20 mice each.

The mice were sacrificed and their brains removed into a 35 mm dish. The cranial cavity and cerebral ventricles (lateral, third and fourth ventricles) were rinsed with 1 ml

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0.15 M phosphate buffered saline (PBS) and CSF was thus harvested with PBS, the washing solution being the socalled “CSF-like fluid”. 2.7. Western blot analysis The CSF-like fluid of mice was centrifuged at 12,000 × g at 4 ◦ C for 10 min, and the protein contents of the supernatants were determined with protein assay kits (Bio-Rad, Hercules, CA, USA) using BSA as the standard. An equal volume of loading buffer (62.5 mM Tris–HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol, 0.05% bromophenol blue) was added to the samples. The mixture was boiled for 5 min prior to electrophoresis on SDS-polyacrylamide gel and electrotransferred to nitrocellulose membrane at a constant current of 190 mA for 90 min. Afterward, the membrane was saturated with PBS containing 0.1% Tween20 for 30 min at room temperature. The membrane was allowed to react with primary antibodies (anti-MAG, MBP, MOBP, and PLP antibodies) at 37 ◦ C for 1 h. Then the membrane was washed three times with PBS containing 0.1% Tween-20 (PBS-T), followed by incubation with HRPconjugated secondary antibody (1:5000 dilution) at 37 ◦ C for 1 h to detect the bound primary antibody. The reactive protein was detected by enhanced chemiluminescence (Amersham Pharmacia, Amersham, UK). The bands were quantitated using a computer assisted imaging densitometer system. 2.8. Histopathological examinations The mouse brains were fixed separately in 10% neutral buffered formalin for 24 h. The fixed specimens were dehydrated in a graded ethanol series (50%, 75%, and 100%) and xylene, and then embedded in paraffin at 55 ◦ C for 24 h. Serial sections were cut at 5 ␮m thickness for each brain from each mouse. Paraffin was removed by heating the sections for 5 min at 65 ◦ C. These sections were dewaxed by washing three times for 5 min each in xylene; and then rehydrated through 100%, 95%, and 75% ethanol for 5 min each, and finally rinsed with distilled water. After staining with haematoxylin (Muto, Tokyo, Japan) and eosin (Muto, Tokyo, Japan), pathological changes were examined under a light microscope.

Fig. 1. Time course studies for MAG. (a) MAG mRNA level was decreased (*P < 0.05) in the brain of mice infected with Angiostrongylus cantonensis on day 15 post-inoculation (PI) than control. (b) Two MAG protein bands of CSF-like fluid were revealed. (c) Both the 100- and 92-kDa MAGs were significantly elevated (*P < 0.05) in the CSF-like fluid on days 15, 20, and 25 PI than controls. *P < 0.05 indicates a significant difference in A. cantonensis-infected-mice versus controls.

2.9. Statistical analysis Results in the different groups of animals were compared using the nonparametric Kruskal–Wallis test followed by post hoc using Dunn’s multiple comparison of means. All results were presented as mean ± standard deviation (S.D.). P values of <0.05 were considered statistically significant.

compared with that of the housekeeping gene (␤-actin). In the brains of mice infected with A. cantonensis, the expression of MAG mRNA was decreased (P < 0.05) on day 15 PI than control (Fig. 1), and Western blot analysis of the CSFlike fluid revealed both 100- and 92-kDa MAGs (Fig. 1b) and found significant elevation of MAG protein (P < 0.05) on days 15, 20, and 25 PI than control (Fig.1c).

3. Results

3.2. Time course studies for MBP

3.1. Time course studies for MAG

The expression for MBP mRNA was decreased (P < 0.05) in the brain on days 15, 20, and 25 PI than control (Fig. 2a). Western blot analysis of CSF-like fluid detected both 21and 14-kDa MBP (Fig. 2b), and significantly elevated 21kDa MBP (P < 0.05) on days 20 and 25 PI and, significantly

RT-qPCR was used to quantify MAG gene expression at six different time points (n = 5 per time point). Relative changes in MAG gene expression were calculated, as

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Fig. 2. Time course studies for MBP. (a) MBP mRNA level was decreased (*P < 0.05) in the brain of mice infected with Angiostrongylus cantonensis on days 15, 20, and 25 post-inoculation (PI) than control. (b) Two MBP protein bands of CSF-like fluid were revealed. (c) The 21-kDa protein was significantly elevated (*P < 0.05) on days 20 and 25 PI, whereas the 14-kDa protein was significantly elevated (*P < 0.05) on days 10, 15, 20, and 25 PI than controls. *P < 0.05 indicates a significant difference in A. cantonensisinfected-mice versus controls.

elevated 14-kDa MBP (P < 0.05) on days 10, 15, 20, and 25 PI than control (Fig. 2c). 3.3. Time course studies for MOBP The expression of MOBP mRNA was decreased (P < 0.05) in the brain on days 15, 20, and 25 PI than control (Fig. 3a). Western blot analysis of CSF-like fluid detected 48- and 46kDa MOBPs, two smaller bands (27- and 22-kDa) (Fig. 3b), significantly elevated 48- and 46-kDa MOBP (P < 0.05) on days 15, 20, and 25 PI, and significantly elevated 27- and 22-kDa MOBP (P < 0.05) on days 20 and 25 PI than control (Fig. 3c).

Fig. 3. Time course studies for MOBP. (a) MOBP mRNA level was decreased (*P < 0.05) in the brain of mice infected with Angiostrongylus cantonensis on days 15, 20, and 25 post-inoculation (PI) than control. (b) Four MOBP bands of the CSF-like fluid were revealed. (c) The 48- and 46-kDa MOBPs were significantly elevated (*P < 0.05) on days 15, 20, and 25 PI. Both 27and 22-kDa proteins were significantly elevated (*P < 0.05) on days 20 and 25 PI than controls. *P < 0.05 indicates a significant difference in A. cantonensis-infected-mice versus controls.

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3.5. Histological analysis of mouse brains Fig. 5 compares the perivascular areas of the cerebrum of uninfected control mice (a) with those of infected mice. Perivascular infiltration was observed on days 5 (Fig. 5b), 10 (c), 15 (d), 20 (e), and 25 (f) PI. Inflammation was first seen on day 10 PI. The peak period of infection (15–25 days PI) was characterized by perivascular cuffing and infiltrating leukocytes. 4. Discussion

Fig. 4. Time course studies for PLP. (a) PLP mRNA level was decreased (*P < 0.05) in the brain of mice infected with Angiostrongylus cantonensis on days 15, 20, and 25 post-inoculation (PI) than control. (b) Two PLP bands of CSF-like fluid were revealed. (c) The 28- and 26-kDa PLPs were significantly elevated (*P < 0.05) on days 20 and 25 PI than controls. *P < 0.05 indicates a significant difference in A. cantonensis-infected-mice versus controls.

3.4. Time course studies for PLP The expression of PLP mRNA was decreased (P < 0.05) in the brain on days 15, 20, and 25 PI than control (Fig. 4a). Western blotting analysis of the CSF-like fluid detected both 28- and 26-kDa PLP bands (Fig. 4b) and significantly elevated 28- and 26-kDa PLP (P < 0.05) on days 20 and 25 PI than control (Fig. 4c).

Myelin deficiency can result from myelin breakdown (demyelination) or from failure to produce the normal amount of myelin during development (hypomyelination) (Vela et al., 1998). Likewise, diseases causing both hypomyelination and demyelination can be classified as either acquired disorders having an allergic, infectious, toxic, or nutritional basis, or genetically determined (hereditary) disorders (Raine, 1984; Hauw et al., 1992; Quarles et al., 1994). An immune system attack on the oligodendrocytic myelin sheath is one of the most frequent causes for myelin loss in the CNS, leading to demyelinating diseases (Lucchinetti et al., 2000). In this study, light microscopy revealed perivascular inflammatory infiltrates in the brain during meningoencephalitis in mice infected with A. cantonensis. These inflammatory cells directed against myelin proteins may cause destabilization and destruction of myelin. Thus, an inflammatory cell attack on the oligodendrocytic myelin sheath can cause demyelination in A. cantonensis infection. A number of complex alterations in myelin protein expression at the transcriptional and post-transcriptional level have been described in oligodendrocytes of the jimpy mutant mouse (Vela et al., 1998). The number of oligodendrocytes expressing PLP mRNA was drastically reduced in jimpy mutant mice (Gardinier and Macklin, 1988; Macklin et al., 1991) and the surviving oligodendrocytes expressing the PLP gene expressed fewer PLP transcripts per cell than normal oligodendrocytes (Bongarzone et al., 1997). In addition, the transcriptional activity of the MBP gene was also reduced in jimpy mutant mice (Shiota et al., 1991). In this study, the expression of four myelin proteins, MAG, MBP, MOBP, and PLP was assessed in the brain of A. cantonensisinfected mice by RT-qPCR. The mRNA expression of these proteins was significantly lower in brains of A. cantonensisinfected mice during meningoencephalitis than in brains of the controls, probably reflecting oligodendrocyte damage. The enzyme 2 ,3 -cyclic nucleotide 3 phosphohydrolase (CNP) is involved in A. cantonensisinduced demyelination (Lin and Lai, 2009) and its activity appears to reflect the rate of breakdown of the myelin sheath (Banik et al., 1979). Further, in the present study, MAG, MBP, MOBP, and PLP were significantly increased in CSF-like fluid of A. cantonensis-infected mice. How does myelin pass from the brain into the CSF ? Cserr and Ostrach (1974) showed that interstitial fluid from the white matter of the brain drains directly through the ependyma into the ventricular CSF and is thus able to reach the subarachnoid space. Also, Kooi et al. (2009) indicated that myelin debris might drain from the brain parenchyma via this route in

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Fig. 5. Histological analysis of mouse brains. (a) Perivascular area of the cerebrum in uninfected control mice. Perivascular cellular infiltrate in the cerebrum of BALB/c mice infected with Angiostrongylus cantonensis on days 5 (b), 10 (c), 15 (d), 20 (e), and 25 (f) post-inoculation. Arrows indicate perivascular areas of infiltration. Arrowheads indicate infiltrating polymorphonuclear cells, the possible cell types including neutrophils and eosinophils. Bar scales = 80 ␮m.

MS patients. They also postulate that following myelin (or oligodendrocyte) damage, myelin debris passes successively from cortical lesions to the perivascular space to the meninges. The debris either accumulates in the meninges or is further drained to the CSF. This pattern might account for our findings of significantly increased myelin protein levels in CSF-like fluid during meningoencephalitis of A. cantonensis-infected mice. The release of myelin proteins into CSF may be a sequela of brain inflammation and tissue injury. Degraded CSF myelin proteins can also be indicators of demyelination in angiostrongyliasis. Thus, measurement of myelin protein levels in the CSF can help determine whether there is myelin breakdown or inflammation in CNS. Plasminogen activators (PAs) and matrix metalloproteases (MMPs) are mediators of extracellular proteolysis during inflammatory demyelination of the CNS (Cuzner and

Opdenakker, 1999). Further, MMPs play a significant role in the fragmentation of MBP and in demyelination leading to multiple sclerosis and experimental autoimmune encephalomyelitis (Shiryaev et al., 2009). We present evidence that myelin proteins were significantly degraded during the inflammatory stage of A. cantonensis infection. The expression of myelin mRNA in brains was negatively correlated with myelin protein levels in CSF-like fluid, suggesting that the increased myelin protein level in CSF-like fluid is due to intracerebral myelin breakdown. The possible mediators of damage to oligodendrocytes and myelin sheaths may be proteolytic enzymes such as PAs or MMPs. To our knowledge, this is the first and most comprehensive study of myelin protein expression in CNS tissue and CSF-like fluid from mice with angiostrongyliasis. Taken together, our results suggest that inflammationinduced myelin protein breakdown occurs during the

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meningoencephalitis stage of angiostrongyliasis. The increase in CSF myelin protein level can be used as an indicator of demyelination in angiostrongyliasis. Acknowledgements We wish to thank Cheng-You Lu, Department of Parasitology, Chung Shan Medical University, for assistance in this study. This study was supported by a grant (NSC96-2320-B-040-007) from the National Science Council, Republic of China. References Banik, N.L., Mauldin, L.B., Hogan, E.L., 1979. Activity of 2 ,3 -cyclic nucleotide 3 -phosphohydrolase in human cerebrospinal fluid. Ann. Neurol. 5, 539–541. Boison, D., Stoffel, W., 1994. Disruption of the compacted myelin sheath of axons of the central nervous system in proteolipid protein-deficient mice. Proc. Natl. Acad. Sci. U. S. A. 91, 11709–11713. Bongarzone, E.R., Foster, L.M., Byravan, S., Schonmann, V., Campagnoni, A.T., 1997. Temperature-dependent regulation of PLP/DM20 and CNP gene expression in two conditionally-immortalized jimpy oligodendrocyte cell lines. Neurochem. Res. 22, 363–372. Chen, K.M., Lee, H.H., Lu, K.H., Tseng, Y.K., Hsu, L.S., Chou, H.L., Lai, S.C., 2004. Association of matrix metalloproteinase-9 and Purkinje cell degeneration in mouse cerebellum caused by Angiostrongylus cantonensis. Int. J. Parasitol. 34, 1147–1156. Compston, A., Zajicek, J., Sussman, J., Webb, A., Hall, G., Muir, D., Shaw, C., Wood, A., Scolding, N., 1997. Glial lineages and myelination in the central nervous system. J. Anat. 190, 161–200. Cserr, H.F., Ostrach, L.H., 1974. Bulk flow of interstitial fluid after intracranial injection of blue dextran 2000. Exp. Neurol. 45, 50–60. Cuzner, M.L., Opdenakker, G., 1999. Plasminogen activators and matrix metalloproteases, mediators of extracellular proteolysis in inflammatory demyelination of the central nervous system. J. Neuroimmunol. 94, 1–14. Durack, D.T., Sumi, S.M., Klebanoff, S.J., 1979. Neurotoxicity of human eosinophils. Proc. Natl. Acad. Sci. U. S. A. 76, 1443–1447. Fredens, K., Dahl, R., Venge, P., 1982. The Gordon phenomenon induced by the eosinophil cationic protein and eosinophil protein X. J. Allergy Clin. Immunol. 70, 361–366. Gardinier, M.V., Macklin, W.B., 1988. Myelin proteolipid protein gene expression in jimpy and jimpymsd mice. J. Neurochem. 51, 360–369. Gardiner, C.H., Wells, S., Gutter, A.E., Fitzgerald, L., Anderson, D.C., Harris, R.K., Nichols, D.K., 1990. Eosinophilic meningoencephalitis due to Angiostrongylus cantonensis as the cause of death in captive nonhuman primates. Am. J. Trop. Med. Hyg. 42, 70–74. Hauw, J.J., Delaere, P., Seilhean, D., Cornu, P., 1992. Morphology of demyelination in the human central nervous system. J. Neuroimmunol. 40, 139–152. Hartung, H.P., Rieckmann, P., 1997. Pathogenesis of immune-mediated demyelination in the CNS. J. Neural Transm. Suppl. 50, 173–181. Hou, R.F., Tua, W.T., Lee, H.H., Chen, K.M., Chou, H.L., Lai, S.C., 2004. Elevation of plasminogen activators in cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis. Int. J. Parasitol. 34, 1355–1364.

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