- Email: [email protected]
Cyclosporin A prevents ischemia-induced reduction of muscarinic acetylcholine receptors with suppression of microglial activation in gerbil hippocampus
ESEARCH ELSEVIER
Neuroseience Research 22 (1995) 123-127
Rapid communication
Cyclosporin A prevents ischemia-induced reduction of muscarinic acetylcholine receptors with suppression Of microglial activation in gerbil hippocampus Yoichi Kondo a'b, Norio Ogawa *a, Masato Asanuma a, Sakiko Nishibayashi a, Emi Iwata a, Akitane Mori a aDepartment of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan bThird Department of Internal Medicine, Okayama University Medical School, 2-5-1 Shikata-cho, Okayarna 700, Japan Received 7 November 1994; accepted 26 December 1994
Abstract
We previously reported the late onset reduction of muscarinic acetylcholinereceptors (LORMAR) which begins 7 days after a 5-min period of experimentallyinduced forebrain ischemia in the gerbil hippocampus. This study demonstratedthat post-ischemic administration of cyclosporin A (CsA) reduced LORMAR 10 days after 5 min of forebrain ischemia in the gerbil hippocampus, suggesting that immunosuppression by CsA may reduce damage to the cholinergic system after ischemia. Microglia positive for HLA-DR class II antigen which presented in the hippocampal CA 1 area, the region most vulnerable to ischemia,were also reduced by CsA. CsA may suppress microglial activation especially with regard to the antigen-presenting function, and LORMAR may be attenuated by this modulation of microgliai function.
Keywords:CyclosporinA; Muscarinicacetylcholinereceptor; Late onset damage; Microglia;HLA-DR class 1I; Transient forebrain ischemia; Hippocampus; Gerbil
In the 5-min bilateral common carotid artery occlusion model in gerbils, which produces transient forebrain ischemia, pyramidal cell death is seen in the selectively vulnerable CAI area of the hippocampus 2-3 days after the ischemic period. This delayed neuronal death has been described in both gerbils (Ito et al., 1975; Kirino, 1982; Kirino and Sano, 1984) and in the fourvessel occlusion model in rats (Kirino et al., 1984; Petito and Pulsinelli, 1984), and is known to accompany microglial activation with several immunoregulatory surface antigens, recognized immunohistochemically, such as major histocompatibility complex (MHC) class I and
* Corresponding author. Tel.: +81 86 223 7151, ext. 2643; Fax: +81 86 234 2426.
II, CD4 and complement receptor type 3 (Gehrmann et al., 1992; Morioka et al., 1992). Microglia are known as scavenger cells which phagocytose cellular debris in the developing central nervous system (CNS) and in the adult CNS with inflammation or injury. Recent studies disclose, however, that microglia are involved in some immune responses in the CNS, as evidenced by its antigen-presenting activities and by the fact that microglia both secrete some immunoregulatory cytokines and have receptors for them (Perry and Gordon, 1988). We previously reported that 5 min of transient forebrain ischemia provokes the late onset reduction of muscarinic acetylcholine receptors (LORMAR) which begins 7 days after ischemic insult in the gerbil hippocampus (Haba et al., 1991; Ogawa et al., 1991). To
0168-0102/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-0102(95)00878-W
124
Y. Kondo et al./ Neuroscience Research 22 (1995) 123-127
elucidate whether the LORMAR has some relationship to immunoregulatory events in the CNS, we investigated post-ischemic hippocampal changes in the muscarinic acetylcholine receptor binding capacity and activation of microglia as a member of immunoregulatory networks in the CNS and the effect of an immunosupressant, cyclosporin A (CsA), on these changes. Gerbils weighing 60-80 g were used for this study. Under ketamine (100 mg/kg body weight, i.p.) anesthesia, both common carotid arteries were exposed and a silk thread was loosely placed around them without interrupting blood flow. On the next day, under light ether anesthesia, both common carotid arteries were reexposed and occluded with aneurysmal clips to induce transient forebrain ischemia. At the end of 5 min of bilateral carotid occlusion, blood flow was restored by releasing the clips. Body temperature was maintained at 37°C using a heating lamp with thermostat. Shamoperated controls were treated similarly to the ischemic group, but neither of the common carotid arteries was occluded. Until the animals were sacrificed, CsA (4 mg/kg per day, s,c.; Sandoz, Basel, Switzerland) or the same volume of vehicle (2% castor oil) was given once a day beginning just after recirculation. For M1-R binding assays, the animals were killed 10 days after recirculation and the brains were immediately removed (n = 6 in each group). The isolated hippocampi were dissected by the method of Glowinski and Iversen (1966). The M r R binding assays were done using [3H]pirenzepine as a specific ligand for the MFR. The dissected hippocampal tissues were homogenized in 10 volumes of ice-cold sodium potassium phosphate buffer (50 mM, pH 7.4) and centrifuged at 12 000 x g for 20 min at 4°C. The pellets were washed twice with the same buffer by resuspension and recentrifugation before final suspension in 10 volumes of the buffer. An aliquot of the membrane fraction, containing about 0.1 mg protein in 0.25 ml of buffer, was incubated for 120 min at 25°C with 0.25 ml of the same buffer containing 0.125-10.0 nM [3H]pirenzepine (72.9 Ci/mmol; New England Nuclear, Boston, MA) with or without atropine. The reaction was stopped by filtration through GF/B glass fiber filters (Whatman, Maidstone, UK). Non-specific binding was determined in the presence of 1 #M atropine. Scatchard plots of the data were computer analyzed by the method of Marquardt (1963). At 3 and 10 days following transient ischemia, the animals (n = 4 in each group) were deeply anesthetized with sodium pentobarbital (60 mg/kg body weight, i.p.) and then perfusion fixed transcardially with ice-cold 4% paraformaldehyde/0.1 M phosphate buffer (PB, pH 7.4). The brains removed from the skulls were postfixed for 24 h in the same perfusion medium at 4°C, immersed in 20% sucrose for 24 h in 0.1 M PB at 4°C, and then 20/~m sections were cut coronally on a microslicer (Dohan E.M., Osaka, Japan). Sections including the hippocam-
pus were stocked in 0.1 M PB and subjected to immunohistochemical staining by the free floating method. Neuronal death was confirmed by hematoxylin-eosin staining. Sections were processed with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) for immunohistochemical analysis. To identify microglial activation, we used anti-HLA-DR class II mouse monoclonal antibody diluted at 1:50 (Bekton Dickinson, Mountain View, CA), recognizing DR class II antigen of human MHC that is the counterpart of MHC class II antigen in rodents. The peroxidation activity was visualized with 0.05% 3,3'-diaminobenzidine, 50 mM imidazole, 0.006% H20 2 and 1% nickel ammonium sulfate in 50 mM Tris buffer (pH 7.6). For immunohistoehemical controls, primary antibodies were replaced by mouse IgG at the same dilution. No positive staining was seen in any of the control sections. The data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan's post hoc test, and P < 0.01 was used as the level of significance. Fig. 1 shows the binding capacity Bmax for M1-R in the hippocampus on the 10th day after 5 min transient forebrain ischemia. There was no significant change in affinity in any of the group (Kd ranged from 10.06 + 5.05 to 12.27 ± 3.22 mM, data not shown). In the sham-operated gerbils, there was no difference in the Bmax between the CsA- and vehicle-treated groups. Although the Bmax in the ischemic group treated with vehicle was significantly decreased compared with the sham-operated group treated with vehicle, the Bmax in the ischemic group treated with CsA did not significantly differ from the sham-operated groups. In the ischemic group, moreover, the Bmax was significantly higher (about two-fold) in the CsA group than in the vehicle group. CsA administration induced no pathological change in sham-operated controls (Fig. 2a,d). Delayed neuronal death was seen in the pyramidal neurons in the hippocampal CA1 region by 3 days after transient ischemia both in the CsA- and in the vehicle-treated groups (Fig.
._~" 400 = ~. 300" U ~-- 200' ',.z.,
"t-~
~--~ IO0E ,.I-i 0
i
]
sham-operated + vehicle
~2~
sham-operated+ CsA ischemia + vehicle
]
ischemia+ CsA .
p
Fig. 1. Effects of CsA administration on the reduction of muscarinic M I receptor binding in the hippocampus following transient forebrain isehemia. Values are means 4- S.E.M. for 6 animals in each group.
Y. Kondo et al. / Neuroscience Research 22 (1995) 123-127
~i~
125
! ~ilii !L ¸ ~i•
~ii ~!!!i!ii~ij~•ili ~i¸
Fig. 2. Photomicrographs of the gerbil hippocampus at 3 and 10 days after 5-min transient forebrain ischemia. Hematoxylin-eosin staining at day 10 (a,d,g,j). Scale bars, 200 ~m. lmmunohistochemical staining for HLA-DR class 11 at 3 days (b,e,h,k) and l0 days (c,f,i,1) after transient ischemia. Scale bars, 100 ~m. Sham-operated vehicle (a,b,c), sham-operated CsA (d,e,f), ischemia vehicle (g,h,i), ischemia CsA (j,k,l).
2g,j). The extent of neuronal damage in these two groups, as confirmed by hematoxylin-eosin staining, was the same. Morphologically, HLA-DR class II immunoreactive cell types appeared to include resting and reactive microglia. In both sham-operated groups, resting microglia positive for HLA-DR class II were sporadically seen in all brain regions examined (Fig. 2b,c,e,f). In both ischemic groups, HLA-DR class IIpositive microglia (both resting and reactive) were increased in number in the hippocampal CAI area at 3 days after transient ischemia (Fig. 2h,k). At 10 days
after transient ischemia, HLA-DR class II immunoreactivity in the hippocampal CAI area remained intense in the vehicle-treated ischemic group (Fig. 2i). The CsAtreated ischemic group exhibited fewer HLA-DR class II-positive microglia in the hippocampal CA1 area than the vehicle-treated ischemic group at 10 days after transient ischemia (Fig. 21). Other regions except the hippocampal CA I area showed only sporadic HLA-DR class II-positive microglia (resting form) in all groups. The decrease in the Bmax seen in the ischemic group treated with vehicle (Fig. 1) is in keeping with our pre-
126
E Kondo et al./ Neuroscience Research 22 (1995) 123-127
vious finding of LORMAR in the gerbil hippocampus following transient forebrain ischemia (Haba et al., 1991; Ogawa et al., 1991), and in this study, LORMAR was prevented by CsA administration. CsA is a cyclic polypeptide of 11 amino acids which exerts its immunosuppressive effect by inhibiting transcription of interleukin (IL)-2 and several other cytokines, mainly in helper T cells (Faulds et al., 1993). IL-2 in the CNS has been identified in some neurological disorders such as Alzheimer's disease (Luber-Narod and Rogers, 1988) and multiple sclerosis (Woodroofe et al., 1986). In addition, IL-2 has some neuromodulatory effect on the CNS, as evidenced by the fact that IL-2 administration in cancer therapy produces some CNS side-effects such as somnolence, depression and confusion (Nisticb and De Sarro, 1991), Thus, it might be possible that CsA influences the CNS by inhibiting IL-2 production. We identified microglia immunohistochemically using anti- HLA-DR class II antibody following transient forebrain ischemia in the gerbil hippocampus. HLA-DR class II is a surface antigen on immune cells involved in their antigen-presenting ability and is known to be expressed on microglia in some CNS diseases (McGeer et al., 1988). Some studies have reported reduced expression of MHC class II antigen on T cells, monocytes and endothelial cells by CsA, and others have not confirmed any effect (Di Padova, 1989). Moreover, the effects of CsA on MHC class II expression on microglia have never been reported. Although the role of HLA-DR class II antigen on microglia is not fully understood, we confirmed that CsA reduced its expression and thus CsA can be assumed to alter microglial function. Processes of ischemic neuronal injuries depend on cellular interactions and the integrated effects of cytokines that are produced by CNS and immune system cells. The contribution of microglia to cell death in the CNS is substantial because of its phagocytotic function (Lees, 1993). Moreover, microglia are supposed to cause death of damaged but still viable neurons as a result of ischemia by releasing cytotoxic agents such as glutamate, tumor necrosis factor a, nitric oxide, hydrogen peroxides and oxygen containing free radicals (Lees, 1993). Thus, suppression of microglial activation may preserve some of these neurons to some extent and subsequently minimize LORMAR. It is reported that IL-2 has no effect on cultured microglia (Giulian and Ingeman, 1988). Microglia, however, express IL-2 receptor, suggesting that IL-2 does have some modulatory effects on them (Mouzaki and Diamantstein, 1987). Our results suggest that CsA suppresses microglial activation via suppression of IL-2 production in vivo. This may minimize LORMAR and decrease the number of damaged neurons that are subsequently phagocytosed by microglia. Another possible explanation for the protective effect
of CsA on LORMAR is a direct influence on the interaction between IL-2 and the cholinergic system. IL-2 reduces the amount of acetylcholine released from rat hippocampal slices (Araujo et al., 1989) and enhances amnesia and hyperactivity induced by the anticholinergic drug scopolamine in mice (Bianchi and Panerai, 1993), suggesting that IL-2 has an anticholinergic effect on the CNS. Thus, inhibition of IL-2 production by CsA administration could have a protective effect on the cholinergic system after the ischemic insult. Considering that the cholinergic system is essential for the higher functions of the CNS, CsA administration may be of therapeutic benefit to attenuate LORMAR following ischemic insult. The immunosupressive pharmacology of CsA is highly complex (Di Padova, 1989), and additional studies on the target(s) of CsA in the CNS are required.
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas and Scientific Research from the Japanese Ministry of Education, Science and Culture, and by Grants from the Japan Research Foundation for Clinical Pharmacology.
References Araujo, D.M., Lapchak, P.A., Collier, B. and Quirion, R. (1989) Localization of interleukin-2 immunoreactivity and interleukin-2 receptors in the rat brain: interaction with the cholinergic system. Brain Res., 498: 257-266. Bianchi, M. and Panerai, A.E. (1993) Interleukin-2 enhances scopolamine-induced amnesia and hyperactivity in the mouse. NeuroReport, 4: 1046-1048. Di Padova, F.E. (1989) Pharmacology of cyclosporine (Sandimune) V. Pharmacological effects on immune function: in vitro studies. Pharmacol. Rev., 41: 373-405. Faulds, D., Goa, K.L. and Benfield, P. (1993) Cyclosporin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders. Drugs, 45: 953-1040. Gehrmann, J., Bonnekoh, P., Miyazawa, T., Hossmann, K.A. and Kreutzburg, G.W. (1992) lmmunocytochemical study of an early microglial activation in ischemia. J. Cereb. Blood Flow Metab., 12: 257-269. Giulian, D. and Ingeman, J.E. (1988) Colony-stimulating factors as promoters of ameboid microglia. J. Neurosci., 8: 4707-4717. Glowinski, J. and lversen, L.L. (1966) Regional studies of catecholamine in the brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3HIDOPA in various regions of the brain. J. Neurochem., 13: 655-669. Haba, K., Ogawa, N., Mizukawa, K. and Mori, A. (1991) Time course of changes in lipid peroxidation, pre- and postsynaptic cholinergic indices, NMDA receptor binding and neuronal death in the gerbil hippocampus following transient ischemia. Brain Res., 540: 116-122. Ito, U., Spatz, M., Walker, J.T. and Klatzo, I. (1975) Experimental cerebral ischemia in Mongolian gerbils. I. Light microscopic observations. Acta Neuropathol. (Bed.), 32: 209-223. Kirino, T. (1982) Delayed neuronal death in the gerbil hippocampus following transient ischemia. Brain Res., 239: 57-69.
Y. Kondo et al./Neuroscience Research 22 (1995) 123-127
Kirino, T. and Sano, K. (1984) Selective vulnerability in the gerbil hippocampus following transient ischemia. Acta Neuropathol. (Berl.), 62: 201-208. Kirino, T., Tamura, A. and Sano, K. 0984) Delayed neuronal death in the rat hippocampus following transient forebrain ischemia. Acta Neuropathol. (Bed.), 64- 139-147. Lees, G.J. (1993) The possible contribution of microglia and macrophages to delayed neuronal death after ischemia. J. Neurol. Sci., ll4: 119-122. Luber-Narod, J. and Rogers, J. 0988) Immune system associated antigens expressed by cells of the human central nervous system. Neurosci. Lett., 94: 17-22. Marquardt, D W. (1963) An algorithm for least-squares estimation of nonlinear parameters. J. Soc. Ind. Appl. Math., ll: 431-441. McGeer, P.L., Itagaki, S. and McGeer, E.G. (1988) Expression of the histocompatibility glycoprotein HLA-DR in neurological disease. Acta Neuropatol., 76" 550-557. Morioka, T., Kalehua, A.N. and Streit, W.J. (1992) Progressive expression of immunomolecules on microglial cells in rat dorsal hippocampus following transient forebrain ischemia. Acta Neuropathol. (Berl.), 83: 149-157.
127
Mouzaki, A. and Diamantstein, T. (1987) Four epitopes on rat 55-kDa subunit of the interleukin 2 receptor as defined by newly developed mouse anti-rat interleukin 2 receptor monoclonal antibodies. Eur. J. Immunol., 17: 1661-1664. Nistic6, G. and De Sarro, G.B. (1991) Is interleukin 2 a neuromodulator in the brain? Trends Neurosci., 14: 146-150. Ogawa, N., Haba, K., Asanuma, M., Mizukawa, K. and Mori, A. (1991) Super-delayed changes of muscarinic acetylcholine receptor in the gerbil hippocampus following transient ischemia. Adv. Exp. Med. Biol., 287: 343-347. Perry, V.H. and Gordon, S. 0988) Macrophages and microglia in the nervous system. Trends Neurosci., l l: 273-277. Petito, C.K. and Pulsinelli, W.A. 0984) Delayed neuronal recovery and neuronal death in rat hippocampus following severe cerebral ischemia: possible relationship to abnormalities in neuronal processes. J. Cereb. Blood Flow Metab., 4: 194-205. Woodroofe, M.N., Bellamy, A.S., Feldmann, M., Davison, A.N. and Cuzner, M.L. 0986) Immunocytochemical characterisation of the immune reaction in the central nervous system in multiple sclerosis. Possible role for microglia in lesion growth. J. Neurol. Sci., 74: 135-152.
Neuroseience Research 22 (1995) 123-127
Rapid communication
Cyclosporin A prevents ischemia-induced reduction of muscarinic acetylcholine receptors with suppression Of microglial activation in gerbil hippocampus Yoichi Kondo a'b, Norio Ogawa *a, Masato Asanuma a, Sakiko Nishibayashi a, Emi Iwata a, Akitane Mori a aDepartment of Neuroscience, Institute of Molecular and Cellular Medicine, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700, Japan bThird Department of Internal Medicine, Okayama University Medical School, 2-5-1 Shikata-cho, Okayarna 700, Japan Received 7 November 1994; accepted 26 December 1994
Abstract
We previously reported the late onset reduction of muscarinic acetylcholinereceptors (LORMAR) which begins 7 days after a 5-min period of experimentallyinduced forebrain ischemia in the gerbil hippocampus. This study demonstratedthat post-ischemic administration of cyclosporin A (CsA) reduced LORMAR 10 days after 5 min of forebrain ischemia in the gerbil hippocampus, suggesting that immunosuppression by CsA may reduce damage to the cholinergic system after ischemia. Microglia positive for HLA-DR class II antigen which presented in the hippocampal CA 1 area, the region most vulnerable to ischemia,were also reduced by CsA. CsA may suppress microglial activation especially with regard to the antigen-presenting function, and LORMAR may be attenuated by this modulation of microgliai function.
Keywords:CyclosporinA; Muscarinicacetylcholinereceptor; Late onset damage; Microglia;HLA-DR class 1I; Transient forebrain ischemia; Hippocampus; Gerbil
In the 5-min bilateral common carotid artery occlusion model in gerbils, which produces transient forebrain ischemia, pyramidal cell death is seen in the selectively vulnerable CAI area of the hippocampus 2-3 days after the ischemic period. This delayed neuronal death has been described in both gerbils (Ito et al., 1975; Kirino, 1982; Kirino and Sano, 1984) and in the fourvessel occlusion model in rats (Kirino et al., 1984; Petito and Pulsinelli, 1984), and is known to accompany microglial activation with several immunoregulatory surface antigens, recognized immunohistochemically, such as major histocompatibility complex (MHC) class I and
* Corresponding author. Tel.: +81 86 223 7151, ext. 2643; Fax: +81 86 234 2426.
II, CD4 and complement receptor type 3 (Gehrmann et al., 1992; Morioka et al., 1992). Microglia are known as scavenger cells which phagocytose cellular debris in the developing central nervous system (CNS) and in the adult CNS with inflammation or injury. Recent studies disclose, however, that microglia are involved in some immune responses in the CNS, as evidenced by its antigen-presenting activities and by the fact that microglia both secrete some immunoregulatory cytokines and have receptors for them (Perry and Gordon, 1988). We previously reported that 5 min of transient forebrain ischemia provokes the late onset reduction of muscarinic acetylcholine receptors (LORMAR) which begins 7 days after ischemic insult in the gerbil hippocampus (Haba et al., 1991; Ogawa et al., 1991). To
0168-0102/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-0102(95)00878-W
124
Y. Kondo et al./ Neuroscience Research 22 (1995) 123-127
elucidate whether the LORMAR has some relationship to immunoregulatory events in the CNS, we investigated post-ischemic hippocampal changes in the muscarinic acetylcholine receptor binding capacity and activation of microglia as a member of immunoregulatory networks in the CNS and the effect of an immunosupressant, cyclosporin A (CsA), on these changes. Gerbils weighing 60-80 g were used for this study. Under ketamine (100 mg/kg body weight, i.p.) anesthesia, both common carotid arteries were exposed and a silk thread was loosely placed around them without interrupting blood flow. On the next day, under light ether anesthesia, both common carotid arteries were reexposed and occluded with aneurysmal clips to induce transient forebrain ischemia. At the end of 5 min of bilateral carotid occlusion, blood flow was restored by releasing the clips. Body temperature was maintained at 37°C using a heating lamp with thermostat. Shamoperated controls were treated similarly to the ischemic group, but neither of the common carotid arteries was occluded. Until the animals were sacrificed, CsA (4 mg/kg per day, s,c.; Sandoz, Basel, Switzerland) or the same volume of vehicle (2% castor oil) was given once a day beginning just after recirculation. For M1-R binding assays, the animals were killed 10 days after recirculation and the brains were immediately removed (n = 6 in each group). The isolated hippocampi were dissected by the method of Glowinski and Iversen (1966). The M r R binding assays were done using [3H]pirenzepine as a specific ligand for the MFR. The dissected hippocampal tissues were homogenized in 10 volumes of ice-cold sodium potassium phosphate buffer (50 mM, pH 7.4) and centrifuged at 12 000 x g for 20 min at 4°C. The pellets were washed twice with the same buffer by resuspension and recentrifugation before final suspension in 10 volumes of the buffer. An aliquot of the membrane fraction, containing about 0.1 mg protein in 0.25 ml of buffer, was incubated for 120 min at 25°C with 0.25 ml of the same buffer containing 0.125-10.0 nM [3H]pirenzepine (72.9 Ci/mmol; New England Nuclear, Boston, MA) with or without atropine. The reaction was stopped by filtration through GF/B glass fiber filters (Whatman, Maidstone, UK). Non-specific binding was determined in the presence of 1 #M atropine. Scatchard plots of the data were computer analyzed by the method of Marquardt (1963). At 3 and 10 days following transient ischemia, the animals (n = 4 in each group) were deeply anesthetized with sodium pentobarbital (60 mg/kg body weight, i.p.) and then perfusion fixed transcardially with ice-cold 4% paraformaldehyde/0.1 M phosphate buffer (PB, pH 7.4). The brains removed from the skulls were postfixed for 24 h in the same perfusion medium at 4°C, immersed in 20% sucrose for 24 h in 0.1 M PB at 4°C, and then 20/~m sections were cut coronally on a microslicer (Dohan E.M., Osaka, Japan). Sections including the hippocam-
pus were stocked in 0.1 M PB and subjected to immunohistochemical staining by the free floating method. Neuronal death was confirmed by hematoxylin-eosin staining. Sections were processed with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) for immunohistochemical analysis. To identify microglial activation, we used anti-HLA-DR class II mouse monoclonal antibody diluted at 1:50 (Bekton Dickinson, Mountain View, CA), recognizing DR class II antigen of human MHC that is the counterpart of MHC class II antigen in rodents. The peroxidation activity was visualized with 0.05% 3,3'-diaminobenzidine, 50 mM imidazole, 0.006% H20 2 and 1% nickel ammonium sulfate in 50 mM Tris buffer (pH 7.6). For immunohistoehemical controls, primary antibodies were replaced by mouse IgG at the same dilution. No positive staining was seen in any of the control sections. The data were analyzed by one-way analysis of variance (ANOVA) followed by Duncan's post hoc test, and P < 0.01 was used as the level of significance. Fig. 1 shows the binding capacity Bmax for M1-R in the hippocampus on the 10th day after 5 min transient forebrain ischemia. There was no significant change in affinity in any of the group (Kd ranged from 10.06 + 5.05 to 12.27 ± 3.22 mM, data not shown). In the sham-operated gerbils, there was no difference in the Bmax between the CsA- and vehicle-treated groups. Although the Bmax in the ischemic group treated with vehicle was significantly decreased compared with the sham-operated group treated with vehicle, the Bmax in the ischemic group treated with CsA did not significantly differ from the sham-operated groups. In the ischemic group, moreover, the Bmax was significantly higher (about two-fold) in the CsA group than in the vehicle group. CsA administration induced no pathological change in sham-operated controls (Fig. 2a,d). Delayed neuronal death was seen in the pyramidal neurons in the hippocampal CA1 region by 3 days after transient ischemia both in the CsA- and in the vehicle-treated groups (Fig.
._~" 400 = ~. 300" U ~-- 200' ',.z.,
"t-~
~--~ IO0E ,.I-i 0
i
]
sham-operated + vehicle
~2~
sham-operated+ CsA ischemia + vehicle
]
ischemia+ CsA .
p
Fig. 1. Effects of CsA administration on the reduction of muscarinic M I receptor binding in the hippocampus following transient forebrain isehemia. Values are means 4- S.E.M. for 6 animals in each group.
Y. Kondo et al. / Neuroscience Research 22 (1995) 123-127
~i~
125
! ~ilii !L ¸ ~i•
~ii ~!!!i!ii~ij~•ili ~i¸
Fig. 2. Photomicrographs of the gerbil hippocampus at 3 and 10 days after 5-min transient forebrain ischemia. Hematoxylin-eosin staining at day 10 (a,d,g,j). Scale bars, 200 ~m. lmmunohistochemical staining for HLA-DR class 11 at 3 days (b,e,h,k) and l0 days (c,f,i,1) after transient ischemia. Scale bars, 100 ~m. Sham-operated vehicle (a,b,c), sham-operated CsA (d,e,f), ischemia vehicle (g,h,i), ischemia CsA (j,k,l).
2g,j). The extent of neuronal damage in these two groups, as confirmed by hematoxylin-eosin staining, was the same. Morphologically, HLA-DR class II immunoreactive cell types appeared to include resting and reactive microglia. In both sham-operated groups, resting microglia positive for HLA-DR class II were sporadically seen in all brain regions examined (Fig. 2b,c,e,f). In both ischemic groups, HLA-DR class IIpositive microglia (both resting and reactive) were increased in number in the hippocampal CAI area at 3 days after transient ischemia (Fig. 2h,k). At 10 days
after transient ischemia, HLA-DR class II immunoreactivity in the hippocampal CAI area remained intense in the vehicle-treated ischemic group (Fig. 2i). The CsAtreated ischemic group exhibited fewer HLA-DR class II-positive microglia in the hippocampal CA1 area than the vehicle-treated ischemic group at 10 days after transient ischemia (Fig. 21). Other regions except the hippocampal CA I area showed only sporadic HLA-DR class II-positive microglia (resting form) in all groups. The decrease in the Bmax seen in the ischemic group treated with vehicle (Fig. 1) is in keeping with our pre-
126
E Kondo et al./ Neuroscience Research 22 (1995) 123-127
vious finding of LORMAR in the gerbil hippocampus following transient forebrain ischemia (Haba et al., 1991; Ogawa et al., 1991), and in this study, LORMAR was prevented by CsA administration. CsA is a cyclic polypeptide of 11 amino acids which exerts its immunosuppressive effect by inhibiting transcription of interleukin (IL)-2 and several other cytokines, mainly in helper T cells (Faulds et al., 1993). IL-2 in the CNS has been identified in some neurological disorders such as Alzheimer's disease (Luber-Narod and Rogers, 1988) and multiple sclerosis (Woodroofe et al., 1986). In addition, IL-2 has some neuromodulatory effect on the CNS, as evidenced by the fact that IL-2 administration in cancer therapy produces some CNS side-effects such as somnolence, depression and confusion (Nisticb and De Sarro, 1991), Thus, it might be possible that CsA influences the CNS by inhibiting IL-2 production. We identified microglia immunohistochemically using anti- HLA-DR class II antibody following transient forebrain ischemia in the gerbil hippocampus. HLA-DR class II is a surface antigen on immune cells involved in their antigen-presenting ability and is known to be expressed on microglia in some CNS diseases (McGeer et al., 1988). Some studies have reported reduced expression of MHC class II antigen on T cells, monocytes and endothelial cells by CsA, and others have not confirmed any effect (Di Padova, 1989). Moreover, the effects of CsA on MHC class II expression on microglia have never been reported. Although the role of HLA-DR class II antigen on microglia is not fully understood, we confirmed that CsA reduced its expression and thus CsA can be assumed to alter microglial function. Processes of ischemic neuronal injuries depend on cellular interactions and the integrated effects of cytokines that are produced by CNS and immune system cells. The contribution of microglia to cell death in the CNS is substantial because of its phagocytotic function (Lees, 1993). Moreover, microglia are supposed to cause death of damaged but still viable neurons as a result of ischemia by releasing cytotoxic agents such as glutamate, tumor necrosis factor a, nitric oxide, hydrogen peroxides and oxygen containing free radicals (Lees, 1993). Thus, suppression of microglial activation may preserve some of these neurons to some extent and subsequently minimize LORMAR. It is reported that IL-2 has no effect on cultured microglia (Giulian and Ingeman, 1988). Microglia, however, express IL-2 receptor, suggesting that IL-2 does have some modulatory effects on them (Mouzaki and Diamantstein, 1987). Our results suggest that CsA suppresses microglial activation via suppression of IL-2 production in vivo. This may minimize LORMAR and decrease the number of damaged neurons that are subsequently phagocytosed by microglia. Another possible explanation for the protective effect
of CsA on LORMAR is a direct influence on the interaction between IL-2 and the cholinergic system. IL-2 reduces the amount of acetylcholine released from rat hippocampal slices (Araujo et al., 1989) and enhances amnesia and hyperactivity induced by the anticholinergic drug scopolamine in mice (Bianchi and Panerai, 1993), suggesting that IL-2 has an anticholinergic effect on the CNS. Thus, inhibition of IL-2 production by CsA administration could have a protective effect on the cholinergic system after the ischemic insult. Considering that the cholinergic system is essential for the higher functions of the CNS, CsA administration may be of therapeutic benefit to attenuate LORMAR following ischemic insult. The immunosupressive pharmacology of CsA is highly complex (Di Padova, 1989), and additional studies on the target(s) of CsA in the CNS are required.
Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research on Priority Areas and Scientific Research from the Japanese Ministry of Education, Science and Culture, and by Grants from the Japan Research Foundation for Clinical Pharmacology.
References Araujo, D.M., Lapchak, P.A., Collier, B. and Quirion, R. (1989) Localization of interleukin-2 immunoreactivity and interleukin-2 receptors in the rat brain: interaction with the cholinergic system. Brain Res., 498: 257-266. Bianchi, M. and Panerai, A.E. (1993) Interleukin-2 enhances scopolamine-induced amnesia and hyperactivity in the mouse. NeuroReport, 4: 1046-1048. Di Padova, F.E. (1989) Pharmacology of cyclosporine (Sandimune) V. Pharmacological effects on immune function: in vitro studies. Pharmacol. Rev., 41: 373-405. Faulds, D., Goa, K.L. and Benfield, P. (1993) Cyclosporin. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use in immunoregulatory disorders. Drugs, 45: 953-1040. Gehrmann, J., Bonnekoh, P., Miyazawa, T., Hossmann, K.A. and Kreutzburg, G.W. (1992) lmmunocytochemical study of an early microglial activation in ischemia. J. Cereb. Blood Flow Metab., 12: 257-269. Giulian, D. and Ingeman, J.E. (1988) Colony-stimulating factors as promoters of ameboid microglia. J. Neurosci., 8: 4707-4717. Glowinski, J. and lversen, L.L. (1966) Regional studies of catecholamine in the brain. I. The disposition of [3H]norepinephrine, [3H]dopamine and [3HIDOPA in various regions of the brain. J. Neurochem., 13: 655-669. Haba, K., Ogawa, N., Mizukawa, K. and Mori, A. (1991) Time course of changes in lipid peroxidation, pre- and postsynaptic cholinergic indices, NMDA receptor binding and neuronal death in the gerbil hippocampus following transient ischemia. Brain Res., 540: 116-122. Ito, U., Spatz, M., Walker, J.T. and Klatzo, I. (1975) Experimental cerebral ischemia in Mongolian gerbils. I. Light microscopic observations. Acta Neuropathol. (Bed.), 32: 209-223. Kirino, T. (1982) Delayed neuronal death in the gerbil hippocampus following transient ischemia. Brain Res., 239: 57-69.
Y. Kondo et al./Neuroscience Research 22 (1995) 123-127
Kirino, T. and Sano, K. (1984) Selective vulnerability in the gerbil hippocampus following transient ischemia. Acta Neuropathol. (Berl.), 62: 201-208. Kirino, T., Tamura, A. and Sano, K. 0984) Delayed neuronal death in the rat hippocampus following transient forebrain ischemia. Acta Neuropathol. (Bed.), 64- 139-147. Lees, G.J. (1993) The possible contribution of microglia and macrophages to delayed neuronal death after ischemia. J. Neurol. Sci., ll4: 119-122. Luber-Narod, J. and Rogers, J. 0988) Immune system associated antigens expressed by cells of the human central nervous system. Neurosci. Lett., 94: 17-22. Marquardt, D W. (1963) An algorithm for least-squares estimation of nonlinear parameters. J. Soc. Ind. Appl. Math., ll: 431-441. McGeer, P.L., Itagaki, S. and McGeer, E.G. (1988) Expression of the histocompatibility glycoprotein HLA-DR in neurological disease. Acta Neuropatol., 76" 550-557. Morioka, T., Kalehua, A.N. and Streit, W.J. (1992) Progressive expression of immunomolecules on microglial cells in rat dorsal hippocampus following transient forebrain ischemia. Acta Neuropathol. (Berl.), 83: 149-157.
127
Mouzaki, A. and Diamantstein, T. (1987) Four epitopes on rat 55-kDa subunit of the interleukin 2 receptor as defined by newly developed mouse anti-rat interleukin 2 receptor monoclonal antibodies. Eur. J. Immunol., 17: 1661-1664. Nistic6, G. and De Sarro, G.B. (1991) Is interleukin 2 a neuromodulator in the brain? Trends Neurosci., 14: 146-150. Ogawa, N., Haba, K., Asanuma, M., Mizukawa, K. and Mori, A. (1991) Super-delayed changes of muscarinic acetylcholine receptor in the gerbil hippocampus following transient ischemia. Adv. Exp. Med. Biol., 287: 343-347. Perry, V.H. and Gordon, S. 0988) Macrophages and microglia in the nervous system. Trends Neurosci., l l: 273-277. Petito, C.K. and Pulsinelli, W.A. 0984) Delayed neuronal recovery and neuronal death in rat hippocampus following severe cerebral ischemia: possible relationship to abnormalities in neuronal processes. J. Cereb. Blood Flow Metab., 4: 194-205. Woodroofe, M.N., Bellamy, A.S., Feldmann, M., Davison, A.N. and Cuzner, M.L. 0986) Immunocytochemical characterisation of the immune reaction in the central nervous system in multiple sclerosis. Possible role for microglia in lesion growth. J. Neurol. Sci., 74: 135-152.