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(Stroke. 1995;26:1415-1422.)
© 1995 American Heart Association, Inc.

Articles

Protective Effect of Cyclosporin A on White Matter Changes in the Rat Brain After Chronic Cerebral Hypoperfusion

Hideaki Wakita, MD; Hidekazu Tomimoto, MD; Ichiro Akiguchi, MD Jun Kimura, MD

From the Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto, Japan.

Correspondence to Hideaki Wakita, Department of Neurology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.

	Abstract

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Background and Purpose Activation of glial cells and rarefaction of the white matter have been reported in rat brain after bilateral permanent occlusion of the common carotid arteries. Using this model, we investigated the effects of the immunosuppressant cyclosporin A on the activation of glial cells and the white matter rarefaction.

Methods Both common carotid arteries were ligated bilaterally in 40 male Wistar rats. Twenty-two of these rats received an intraperitoneal injection of cyclosporin A, and the remaining 18 received a vehicle-solution injection. Microglia/macrophages were investigated with immunohistochemistry for the major histocompatibility complex class I and II antigens as well as for leukocyte common antigen. Astroglia were examined with glial fibrillary acidic protein as a marker. Activation of glial cells and white matter rarefaction were then investigated from 7 to 30 days after the ligation.

Results In vehicle-treated animals, there was a persistent and extensive activation of both microglia/macrophages and astroglia in the white matter, including the optic nerve, optic tract, corpus callosum, internal capsule, and traversing fiber bundles of the caudoputamen. In cyclosporin A–treated rats, the number of activated microglia/macrophages was significantly reduced (P<.01) to approximately one fifth of that in vehicle-treated animals. Similarly, rarefaction of the white matter was much less intense in cyclosporin A–treated rats (P<.01).

Conclusions Cyclosporin A suppressed both glial activation and white matter changes after chronic cerebral hypoperfusion. These results suggest that immunologic reaction may play a role in the pathogenesis of the white matter changes and that the present model may be useful in investigating the pathophysiology of white matter changes induced by chronic cerebral hypoperfusion.

Key Words: cyclosporin • hypoperfusion • white matter • rats

	Introduction

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Subcortical hyperintensity lesions are frequent findings on T₂-weighted MR images of the elderly.¹ Although some of these lesions may correspond to a known pathological process, most are incidental and poorly correlated with previous neurological symptoms.¹ The pathological correlates of these lesions are usually white matter pallor with gliosis, a dilated perivascular space, and lacunar infarcts,^{1 2 3} although they may also correspond to the normal white matter.⁴ The pathophysiological mechanism responsible for these lesions is generally believed to be chronic cerebral hypoperfusion.^{5 6} However, a pathogenetic heterogeneity may exist, since these lesions are observed in various pathological conditions such as Binswanger's disease, Alzheimer's disease,⁷ amyloid angiopathy,⁸ cerebral tumors,⁹ and head trauma.¹⁰

We have previously reported T-cell infiltration and activation of microglia in the white matter of a heterogeneous group in leukoencephalopathy.¹¹ Similarly, in rat brain, chronic cerebral hypoperfusion induces activation of microglia in the white matter and subsequently rarefaction of the corresponding region.¹² Recent evidence indicates that a variety of inflammatory responses ensue after the cessation of cerebral blood flow. Neutrophils infiltrate the cerebral infarct loci and release various chemical mediators such as proteases, lipid-derived prostanoids, and oxygen radicals during the reperfusion process.¹³ Microglia, the immune effector cells of the central nervous system, are activated in acute cerebral ischemia and express MHC antigens.^{14 15} This is also true of chronic cerebral ischemia, in which the expression of MHC antigens in the microglia/macrophages is preferentially observed in the white matter.¹² Since microglia induce neuronal death in culture^{16 17} and the inhibition of mononuclear phagocytes reduces ischemic injury,¹⁸ microglia may be involved in the pathogenesis of white matter changes after chronic cerebral hypoperfusion.

CsA, a widely used immunosuppressant, binds cyclophillin and inhibits calcineurin activity, thereby suppressing cytokine production^{19 20} and NMDA-triggered NO-mediated neurotoxity.^{21 22} In the present study, we demonstrate that CsA has a protective effect on glial activation and white matter rarefaction in a rat model of chronic cerebral hypoperfusion.¹²

	Materials and Methods

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Male Wistar rats (Shimizu Experimental Supply Co) weighing 150 to 200 g were used in all experiments. The protocol for the chronic cerebral hypoperfusion has been described previously.¹² Briefly, the animals were anesthetized with sodium pentobarbital (25 mg/kg IP) and were allowed spontaneous respiration throughout the surgical procedure. Through a midline cervical incision, both common carotid arteries were exposed and double-ligated with silk sutures. The rectal temperature was monitored and maintained between 36.5°C and 37.5°C during the surgical procedure. After the operation, the rats were kept in animal quarters with food and water ad libitum. The experimental rats received a daily intraperitoneal injection of CsA (10 mg/kg) diluted in vehicle solution from 1 day before the operation to 14 days after and thereafter on every third day beyond the 14 days.

At 7, 14, and 30 days after the ligation of the carotid arteries, the animals were deeply anesthetized with sodium pentobarbital, perfused transcardially with 0.01 mol/L phosphate-buffered saline, and then perfused with a fixative containing 4% paraformaldehyde and 0.2% picric acid in 0.1 mol/L phosphate buffer (pH 7.4). Leukocyte count, the ratio of T cells to leukocytes, serum glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, blood urea nitrogen, and creatinine levels were measured 7 and 30 days after the ligation. Six to 9 animals were used at each postischemic period in the CsA-treated group. As a control, 6 animals underwent the same surgery but received an intraperitoneal administration of vehicle only (130 mg/kg polyoxyethylated castor oil and 6.6% ethanol in saline) in the same manner at each postischemic period. Another 5 animals were subjected to a sham operation (the same surgery except for occlusion) but were killed immediately.

For investigating dose-response relations, 8 rats underwent an injection of 5 mg/kg CsA and 9 rats received 15 mg/kg CsA in the same manner as the group treated with 10 mg/kg CsA. These animals were killed 14 days after the ligation of the carotid arteries.

The brains and optic nerves were immersed for 12 hours in 4% paraformaldehyde in 0.1 mol/L phosphate buffer (pH 7.4), and coronal brain blocks including the caudoputamen and hippocampus were embedded in paraffin for histological examination. Paraffin sections 2 µm thick were then cut on a microtome and stained with Klüver-Barrera and Bielschowsky's staining. The severity of the white matter changes was graded as normal (grade 0), disarrangement of the nerve fibers (grade 1), formation of marked vacuoles (grade 2), and disappearance of myelinated fibers (grade 3) by two independent investigators (Fig 1). Four animals were used for grading the white matter changes, whereas the immunohistochemical examination was performed on 4 animals or more.

View larger version (144K): [in this window] [in a new window]   Figure 1. Photomicrographs of Klüver-Barrera–stained optic nerves after bilateral occlusion of the common carotid arteries. The degree of white matter change was graded as follows: normal, grade 0 (A); disarrangement of nerve fibers, grade 1 (B); formation of marked vacuoles, grade 2 (C); and disappearance of myelinated fibers, grade 3 (D). Bars indicate 30 µm.

For immunohistochemistry, the rest of the coronal blocks were stored in 15% sucrose in 0.1 mol/L phosphate buffer (pH 7.4) until used. Serial sections (20 µm thick) were cut in a cryostat and were incubated overnight with the following monoclonal antibodies (dilutions in parentheses): OX18 (Sera Lab, 1:400) against MHC class I antigen, OX6 (Sera Lab, 1:100) against MHC class II (Ia) antigen, OX1 (Sera Lab, 1:100) against LCA, W3/25 (Biotrin International, 1:200) against CD4 antigen, and OX8 (Chemicon, 1:400) against CD8 antigen. A polyclonal antiserum against GFAP (Dako, 1:20 000) was used to identify astroglia. The sections were subsequently incubated with either biotinylated anti-mouse or biotinylated anti-rabbit IgG (Vector Laboratories, 1:200) for 1 hour and then incubated with an avidin-biotin peroxidase complex solution (Vector Laboratories, 1:100) for 1 hour. After each incubation, the sections were rinsed for 15 minutes with 0.01 mol/L phosphate-buffered saline containing 0.3% Triton X-100. Finally, the immunoreaction products were visualized with a solution of 0.02% 3,3'-diaminobenzidine tetrahydrochloride and 0.005% H₂O₂ in 0.05 mol/L Tris buffer (pH 7.6), and each section was counterstained with hematoxylin. The number of nuclei with immunoreactive perikarya and processes were then counted. The numerical density of the immunoreactive cells was expressed as the number of cells per 0.3 mm² in the region of interest. Values are expressed as mean±SD. Differences between groups of the laboratory data were determined with Student's t test, and those of mortality rates between each group were determined by Fisher's exact probability test. Differences of the numerical densities of the immunoreactive cells and the grading scores between each group were determined by two-factor factorial ANOVA followed by Fisher's protected least-significant difference procedure. A value of P<.05 was considered statistically significant. Kidney tissues were examined with hematoxylin and eosin staining in the animals that received either 10 mg/kg CsA or vehicle for 7 days.

	Results

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Of the 22 animals that received 10 mg/kg CsA, 1 (4.5%) died within 7 days after the operation and 3 more (13.6%) died after 7 days. Of the 8 animals that received 5 mg/kg CsA, 2 (25%) died within 7 days after the operation and none died after 7 days. Of the 9 animals that received 15 mg/kg CsA, 2 (22.2%) died within 7 days after the operation and 1 more (11.1%) died after 7 days. Of the 18 animals that received the vehicle, 4 (22.2%) died within 7 days and none died after 7 days. There were no significant differences between the mortality rates of the CsA-treated groups versus those of the vehicle-treated group. In each group, the surviving animals occasionally showed ptosis and transient difficulties in feeding. Leukocyte count, the ratio of T cells to leukocytes, serum glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, blood urea nitrogen, and creatinine levels were not significantly different between the vehicle-treated versus the CsA-treated animals. There were no obvious histological changes in the kidneys of the animals that received 10 mg/kg CsA. No large infarcts were observed in vehicle-treated animals. In the 10-mg/kg CsA–treated group, 2 animals showed a large infarct in the gray matter and were excluded from the statistical analysis.

In the white matter of the sham-operated animals, there was positive immunostaining for MHC class I and II (Ia) antigens and LCA in only a few glial cells. GFAP-positive astroglia were distributed predominantly in the white matter. From 7 to 30 days after the ligation, the brains of vehicle-treated animals showed an increase in the number of microglia/macrophages, which were immunoreactive for MHC class I and II antigens and LCA, in various white matter regions such as the optic nerve, optic tract, corpus callosum, internal capsule, anterior commissure, and traversing fiber bundles of the caudoputamen (Fig 2A and 2C). Microglia/macrophages immunoreactive for MHC antigens and LCA were most numerous at 14 days in these regions (Fig 5). The number of astroglia immunoreactive for GFAP increased from 7 to 30 days after the ligation (Fig 3A and 3C). CD4- and CD8-positive lymphocytes were scattered sparsely throughout the white matter of these regions from 7 to 30 days after the ligation (Fig 4A and 4C).

View larger version (102K): [in this window] [in a new window]   Figure 2. Photomicrographs of the immunohistochemical staining for MHC class I (A, B) and class II (C, D) antigens in the internal capsule. The animals received an intraperitoneal administration of vehicle (A, C) or CsA (B, D) for 7 days. In the CsA-treated animals, microglia/macrophages immunoreactive for MHC class I and II antigens were much less prominent when compared with vehicle-treated animals. Bars indicate 30 µm.

View larger version (23K): [in this window] [in a new window]   Figure 5. Histograms show the numerical densities of MHC class II immunopositive microglia/macrophages in the white matter of rats receiving either vehicle or CsA for 7 (A), 14 (B), and 30 (C) days. D, Diagram shows the regions examined, indicated by number: , ON (optical nerve); , OT (optical tract); , IC (internal capsule); , CP (caudoputamen); and , CC (corpus callosum). In the CsA-treated animals, the number of MHC class II immunopositive microglia/macrophages was significantly reduced at P<.01 by two-factor factorial ANOVA from 7 to 30 days compared with the vehicle-treated animals. Solid bars indicate vehicle; shaded bars, CsA. *Significant at P<.01 by Fisher's protected least-significant difference procedure when compared with vehicle-treated animals; significant at P<.05 by Fisher's protected least-significant difference procedure when compared with vehicle-treated animals.

View larger version (116K): [in this window] [in a new window]   Figure 3. Photomicrographs of the immunohistochemical staining for GFAP in the optic tract. The animals received an intraperitoneal administration of vehicle (A, C) or CsA (B, D) for 7 (A, B) or 30 days (C, D). In the CsA-treated animals, astroglia immunoreactive for GFAP were much less prominent when compared with vehicle-treated animals. Bars indicate 30 µm.

View larger version (101K): [in this window] [in a new window]   Figure 4. Photomicrographs of the immunohistochemical staining for CD4 (A, B) and CD8 (C, D) in the internal capsule. The animals received an intraperitoneal administration of vehicle (A, C) or CsA (B, D) for 7 days. The infiltration of CD4- or CD8-immunopositive lymphocytes was reduced in number for the CsA-treated rats. Bars indicate 30 µm.

In the CsA-treated animals, the microglia/macrophages labeled for MHC antigens as well as for LCA were much fewer in number in all parts of the white matter from 7 to 30 days compared with the vehicle-treated animals (Fig 2B and 2D; Fig 5). The number of GFAP-positive astroglia was also reduced in the CsA-treated animals in the corresponding regions (Fig 3B and 3D). The infiltration of CD4- and CD8-positive lymphocytes was also much less in the white matter regions after the administration of CsA (Fig 4B and 4D).

The results of the grading scores from the white matter are summarized in the Table. In the sham-operated animals, there was no detectable rarefaction in the white matter (Table, Sham). After 7 days of ligation, the most severe rarefaction was observed in the optic nerve and optic tract of vehicle-treated animals. Less intense changes were observed in the medial part of the corpus callosum adjoining the lateral ventricle, the anterior commissure, the internal capsule, and the fiber bundles of the caudoputamen. In the 10-mg/kg CsA–treated animals, these changes were significantly reduced by two-factor factorial ANOVA (Table, 7 days). After either 14 or 30 days, the grading scores remained at the same level as at 7 days of postligation in the vehicle-treated animals by two-factor factorial ANOVA. The severity of the rarefaction was attenuated in the animals treated with 10 mg/kg CsA (Table, 14 and 30 days; Fig 6).

View this table: [in this window] [in a new window]   Table 1. Summary of the White Matter Rarefaction Gradings in Sham-Operated Rats and in Rats Receiving Cyclosporin A or Vehicle for 7, 14, and 30 Days

View larger version (138K): [in this window] [in a new window]   Figure 6. Photomicrographs of Klüver-Barrera–stained optic nerve (A, B) and medial part of the corpus callosum (C, D). The animals received an intraperitoneal administration of vehicle (A, C) or CsA (B, D) for 30 days. In the CsA-treated rats, the extent of white matter rarefaction was reduced. Bars indicate 30 µm.

After 14 days of ligation, every dose of CsA significantly reduced the grade of the rarefaction by two-factor factorial ANOVA. The grades were 2.0±0.82 (optic nerve), 1.75±0.5 (optic tract), 1.0±0.82 (corpus callosum), 0.5±0.58 (anterior commissure), 0.5±0.58 (internal capsule), and 0.75±0.5 (caudoputamen) in the 5-mg/kg CsA–treated animals and 1.5±1.0 (optic nerve), 1.0±0.82 (optic tract), 0.5±0.58 (corpus callosum), 0.25±0.5 (anterior commissure), 0 (internal capsule), and 0.25±0.5 (caudoputamen) in the 15-mg/kg CsA–treated animals. The grades were significantly reduced in the 10- and 15-mg/kg CsA–treated animals compared with the 5-mg/kg–treated ones by two-factor factorial ANOVA. However, there were no differences between 10- and 15-mg/kg–treated ones by two-factor factorial ANOVA.

In the vehicle-treated animals, Bielschowsky's staining revealed a decrease in the number of axons in the above regions from 7 to 30 days after ligation. With administration of 10 mg/kg CsA, the decrease in the number of axons was less severe in all white matter regions.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
After the bilateral occlusion of the common carotid arteries, regional cerebral blood flow was reduced significantly compared with sham-operated animals. This reduction rate is reported to be 47% in the parietal cortex from 0 to 21 days after surgery²³ and between 39% to 51% immediately after ligation and 18% to 28% after 7 days in the white matter.²⁴ The reduction of cerebral blood flow to the same extent also produces white matter lesions in the gerbil.²⁵ The mortality rate in the present study (22.2%) is comparable with those reported in previous studies: 1/10 (10%, Payan et al²⁶ ), 4/25 (16%, Ogata et al²⁷ ), 17/102 (17%, Iwasaki et al²⁸ ), and 8/38 (21%, Tsuchiya et al²⁴ ).

In vivo, CsA forms a complex with cyclophillin, a binding protein specific for CsA. This complex then interacts with calcineurin/calmodulin and thereby inhibits the phosphatase activity of calcineurin and interferes with Ca²⁺-regulated signal transduction and gene expression in T cells.²⁹ CsA does not influence brain perfusion,³⁰ but it does reduce the size of focal ischemic injury and prevents the late-onset decrease of muscarinic acetylcholine receptors after transient global ischemia.^{31 32} In the present study, white matter changes induced by chronic cerebral hypoperfusion were attenuated by CsA. Although the mechanism underlying these effects on ischemic damage remains uncertain, some aspects of the above-mentioned actions of CsA may be involved.

First, CsA may have prevented the ischemic damage mediated by microglia. Microglia play a central role in the cytokine network by producing a variety of cytokines, such as IL-1, IL-6, and TNF-.^{33 34 35} They also release cytotoxins such as proteases, reactive oxygen radicals, and nitrogen intermediates.^{16 36} Since the mRNA for several cytokines such as IL-1ß^{37 38 39} and TNF-⁴⁰ and for transforming growth factor-ß³⁹ increases after cerebral ischemia, some of these cytokines may affect the development of inflammatory and reparative processes. Indeed, interferon- induces MHC antigen expression on microglia⁴¹ and potentiates demyelination.⁴² TNF- and lymphotoxin may mediate the degeneration of myelin and oligodendrocytes.^{43 44} CsA does not affect phagocytosis,⁴⁵ chemotaxis,⁴⁶ or cytotoxic activity⁴⁷ in macrophages, which are the systemic counterparts to microglia. However, CsA downregulates CD4 expression in microglia⁴⁸ and suppresses the production of several cytokines in macrophages and in cells of other lineage.^{19 20}

Second, CsA may have protected against white matter changes by acting on T cells. Under pathological conditions of the white matter such as multiple sclerosis⁴⁹ and in experimental allergic encephalomyelitis,⁵⁰ T-cell activation induced by the MHC-restricted antigen presentation of microglia/macrophages is a central pathological event. Obviously, it is highly unlikely that these processes play a major role in frank cerebral ischemia, which follows the complete cessation of cerebral blood flow. However, T-cell activation may be involved in chronic pathological conditions of the white matter such as Binswanger's disease and experimental white matter changes after chronic cerebral hypoperfusion, since these disease conditions are frequently accompanied by T-cell infiltration into the white matter.^{11 12} CsA suppresses not only IL-2 gene expression and IL-2 bioactivity in T cells by inhibiting the binding of lymphocyte-specific factors to the IL-2 enhancer but also the gene transcription of several members of the cytokine family.^{51 52 53 54}

Third, CsA may have protected against white matter changes by inhibiting NMDA-mediated neurotoxicity. CsA and FK506 inhibit calcineurin activity, enhance phosphorylation of NO synthase, and inhibit NO synthase catalytic activity. These actions may alleviate ischemic neuronal damages, since NMDA neurotoxicity is mediated at least in part by NO.⁵⁵

The present study demonstrated that CsA is beneficial for protecting the white matter after chronic cerebral hypoperfusion. This suggests an immunologic component to the white matter changes in chronic cerebral hypoperfusion and also indicates the possibility of the clinical use of immunosuppressive agents in vascular leukoencephalopathy. However, there are several problems to be resolved, such as the occasional side effects of CsA, such as nephrotoxity and hypertension. In addition, since patients with vascular leukoencephalopathy are generally of advanced age, they are particularly susceptible to these side effects and to CsA immunosuppression–mediated secondary infections. Another perplexing problem is the considerable neurotoxity of CsA, which may manifest as a cerebral white matter lesion in nonneurological patients undergoing an organ transplantation.⁵⁶ The model used in the present study may therefore be a useful tool to gain further insights into the pathophysiology of chronic cerebral hypoperfusion and may also be useful in screening alternative immunosuppressive or anti-inflammatory drugs that may prevent vascular leukoencephalopathy.

	Selected Abbreviations and Acronyms

 

CsA = cyclosporin A GFAP = glial fibrillary acidic protein IL = interleukin LCA = leukocyte common antigen MHC = major histocompatibility complex NMDA = N-methyl-D-aspartate NO = nitric oxide TNF = tumor necrosis factor

	Acknowledgments

 
This work was supported by a grant-in-aid from the Japanese Ministry of Education, Science, and Culture, by grants-in-aid for amyotrophic lateral sclerosis and peripheral neuropathy from the Japanese Ministry of Health and Welfare, and a grant from the Kanae Foundation. We are indebted to Dr M. Kameyama for helpful advice, Dr Y. Yamada (Department of Pathology, Kyoto University) for pathological examination of the kidney tissues, and Mrs Y. Takagi for excellent technical assistance.

Received October 28, 1994; revision received April 27, 1995; accepted May 4, 1995.

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