ELSEVIER

BRAIN RESEARCH

Brain Research 664 (1994) 69,76

Research report

Astroglial and microglial reactions in the gerbil hippocampus with induced ischemic tolerance

Hiroyuki Kato a,*, Kyuya Kogure b, Tsutomu Araki a, Yasuto Itoyama a

a Department of Neurology, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980, Japan b Foundation for Brain and Nerve Diseases and the Institute ofNeuropathology, Kumagaya, Japan

Accepted 23 August 1994

Abstract

Preconditioning of the brain with sublethal ischemia protects against neuronal damage following subsequent longer periods of ischemia (ischemic tolerance). In this study, we investigated astroglial and microglial reactions in the hippocampus following ischemia in a gerbil model of ischemic tolerance. Two minutes of forebrain ischemia (preconditioning ischemia) or sham operation was followed by 3 min of ischemia (second ischemia) 3 days later. The brains were perfusion-fixed after 4 h, 1 day, 2 days, and 7 days. Paraffin sections were used for the visualization of astrocytes by immunostaining against glia ! fibrillary acidic protein (GFAP) and for the visualization of microglia by histochemical staining with isolectin-B4 from Griffonia simplicifolia. The preconditioning ischemia induced a moderate increase in astroglial and microglial staining. Two days after the second ischemia, GFAP staining further increased in astrocytes in the hippocampus with ischemic tolerance. In the CA1 region of the hippocampus without ischemic tolerance, in contrast, microglial activation with increased staining and morphological changes was pronounced. After 7 days, neuronal destruction resulted in the CA1 region without tolerance, where hypertrophic reactive astroglia and reactive microglia with phagocytic transformation accumulated intensely. However, the ischemic preconditioning prevented the CA1 neuronal damage and the activation of microglia was subsiding after 7 days. Thus, activation of glial cells occurred in a graded fashion in response to different degrees of neuronal injury. Astroglial but not microglial activation may have implications in neuronal survival in ischemic tolerance. The findings also suggest that neuron-glial and glia-glial interactions are under strict control and play a critical role in neuronal survival and death after ischemia.

Keywords: Cerebral ischemia; Ischemic tolerance; Astroglia; Microglia; Immunohistochemistry; Neuron-glia interaction; Hip- pocampus; Gerbil

I. Introduct ion

Neurons in specific brain regions are selectively susceptible to ischemic insults [2,20,39]. A brief period of cerebral ischemia, as short as 3 min in gerbils, destroys neurons in specific neuronal populations, such as pyramidal neurons in the CA1 subfield of the hip- pocampus [19]. The neuronal destruction occurs after 3 - 4 days of reperfusion and has been known as de- layed neuronal death [20]. However, this selective vul- nerability can be modified by preconditioning of the brain with a sublethal period of ischemia [19,21,22]. A

* Corresponding author. Fax: (81) (22) 272-5818.

0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006 -8993 (94)01047-1

2-min period of forebrain ischemia in gerbils produces no appreciable neuronal damage in the brain. How- ever, preconditioning of the brain with this period of ischemia followed by 1-7 days of reperfusion protects against neuronal damage following a longer period of ischemia which normally damages the CA1 neurons [19]. This phenomenon has been termed ischemic tol- erance and has received attention because the elucida- tion of its mechanism may afford a clue for protection against ischemic brain damage [19,21,22,23]. However, the mechanism of ischemic tolerance remains elusive although the role of altered gene expression in neu- rons, such as that of stress proteins, has been suggested [21,23].

On the other hand, glial cells in the brain greatly influence the maintenance and survival of neurons

7{I H. Kato et aL /Brain Research 664 (1994) 69-76

[3,9,12,14,15,36]. Astroglia and microglia are rapidly activated after a wide range of brain injury, including cerebral ischemia [10,11,13,17,26,32]. Earlier studies, especially those in vitro, have shown that these two types of glial cells have opposing actions in regulating neuronal survival [12,14]. These studies have shown that astroglia support neuronal growth and survival. In contrast, microglia, when stimulated, release a variety of cytotoxic agents which may be important mediators of neuronal injury [3,12,14,34,36]. It is very important to clarify how these glial cells influence neuronal sur- vival and death following cerebral ischemia in vivo. The purpose of this study was therefore to investigate the activation of astroglia and microglia in the hippocam- pus following ischemia in a gerbil model of ischemic tolerance.

2. Materials and methods

2.1. Induction of ischemia

We used male Mongolian gerbils, 12-13 weeks old and weighing 60-80 g (Seiwa Experimental Animals, Fukuoka, Japan). Forebrain ischemia and ischemic tolerance were induced as reported previously [19]. Briefly, the gerbils were anesthetized with 2% halothane in a mixture of 30% oxygen and 70% nitrous oxide and both common carotid arteries were gently exposed. One minute after discontinua- tion of anesthesia, the carotid arteries were occluded with aneurysm clips. This procedure produces a severe reduction of blood flow in the forebrain [18]. A 2-min period of occlusion (ischemic precondi- tioning) or sham operation was followed by 3 days of reperfusion. Then 3 min of occlusion (the second ischemia) was again induced. Rectal temperatures of the animals were maintained at 37°C during surgery and ischemia with a heating pad and a lamp, and were also monitored after ischemia for 2 h to confirm lhe occurrence of

Fig. 1. Immunostaining against glial fibrillary acidic protein (GFAP) for the visualization of astrocytes (a,b,c) and isolectin staining for the visualization of microglia (d,e,f) in the hippocampus (CA1 subfield and dentate gyrus), a,d: sham-operated. Normal appearance, b,e: 3 days after 2-min ischemia. GFAP-sta ined astroglia and iectin-stained microglia increased in number , especially in the dentate hilus, c,f: 7 days after 3-min ischemia without ischemic preconditioning. Note accumulation of reactive astroglia and microglia in the CA1 subfield. Bar = 0.2 mm.

H. Kato et al. / Brain Research 664 (1994) 69-76 71

postischemic hyperthermia [19]. The animals were sacrificed 3 days after the preconditioning ischemia or sham operation, and at 4 h, 1 day, 2 days, and 7 days after the second ischemia. Under anesthesia

with pentobarbital (50 mg/kg, i.p.), the brains were perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. The brains were removed 2 h later and postfixed in the same fixative overnight at 4°C. The brains were then routinely embedded in paraffin. Sec- tions at a thickness of 5/xm were prepared. Each group contained 5 animals.

2.2. Visualization of glial cells

The sections were immunohistochemically stained with an anti- body against glial fibrillary acidic protein (GFAP; Chemicon) for the visualization of astrocytes. The paraffin sections were deparaffinized and preincubated with 10% normal serum. The sections were then incubated with the anti-GFAP antibody (1:200) overnight at 4°C. Then the sections were incubated with biotinylated second antibody for 1 h at room temperature, followed by incubation with avidin-bio- tin-peroxidase complex for 30 min at room temperature according to the supplier's recommendations (Vectastain elite ABC kit, Vector

Laboratories). Lastly, the sections were reacted with diaminobenzi- dine and H20 2 for color development.

The sections were also histochemically stained with peroxidase-

labeled isolectin-B4 from Griffonia simplicifolia seeds (Sigma) for the visualization of microglia as described previously [35]. Briefly, the sections were incubated with the isolectin (20 p,g/ml) in phosphate buffered saline with cations overnight at 4°C. Then, the sections were reacted with diaminobenzidine and H20 2 for color development. The sections were counterstained with hematoxylin.

3. Results

3.1. Astroglia

In s h a m - o p e r a t e d gerbi ls , a s t rog l i a w e r e on ly weak ly

i m m u n o s t a i n e d fo r G F A P (Fig. l a , Fig. 2a). T h e y w e r e

re la t ive ly a b u n d a n t in s t r a t u m l a c u n o s u m - m o l e c u l a r e

o f t h e C A 1 subf ie ld . T h e y w e r e a lso s e e n in s t r a t a

r a d i a t u m and o r i e n s o f t h e C A 1 , bu t w e r e on ly ra re ly

Fig. 2. Immunostaining against glial fibrillary acidic protein for the visualization of astrocytes in the CA1 subfield of the hippocampus, a: sham-operated. Normal appearance, b,c: 2 days and 7 days after 3-min ischemia without ischemic preconditioning. Note hypertrophic reactive astrocytes in c. d: 3 days after 2-rain ischemia, e,f: 2 days and 7 days after 3-min ischemia with ischemic preconditioning. Increased astroglial staining in d, e and f. Bar = 0.1 mm.

72 H. Kato et al. / Brain Research 004 (1994) 09-70

seen in other regions. GFAP immunostaining was seen in the cytoplasm and thin processes of astrocytes (Fig. 2a).

Three days after 2 min of preconditioning ischemia, GFAP immunostaining increased in astrocytes in the entire hippocampus as compared with those in sham- operated gerbils (Fig. lb). The increase was most pro- nounced in the dentate hilus, and was moderate in stratum radiatum of the CA1 (Fig. 2d). Astroglia in the dentate hilus were slightly hypertrophic (Fig. 4a).

Four hours and 1 day after 3-min ischemia without ischemic preconditioning, GFAP staining appeared similar to that of sham-operation. In animals with preconditioning, the GFAP staining was also appeared similar to that 3 days after 2 min of ischemia. Thus, GFAP-positive astroglia were seen in the entire hip- pocampus, especially in the dentate hilus and the CA1.

After 2 days, GFAP staining largely unchanged in animals without preconditioning (Fig. 2b). However, GFAP staining increased in the CA1 region of the animals with preconditioning (Fig. 2e) and the astroglia in the CA1 became slightly hypertrophic.

After 7 days, CA1 pyramidal neurons were de- stroyed in animals without preconditioning. Astroglia in the CA1 region and the dentate hilus became more immunoreactive to GFAP and became hypertrophic, showing a typical morphology of reactive astrocytes (Fig. lc). The accumulation of reactive astrocytes in the CA1 was seen predominantly in strata oriens, radia- turn, and lacunosum-moleculare, but was sparse in stratum pyramidale (Fig. 2c). GFAP staining also slightly increased in the CA3 and the dentate gyrus. In the hippocampus of animals with preconditioning where CA1 neurons survived, astroglia with increased GFAP

Fig. 3. Histochemical staining with isolectin-B4 from Griffonia simplicifolia for the visualization of microglia in the CAI subfield of the hippocampus, a: sham-operated. Quiescent microglia are only rarely seen (arrow). b,c: 2 days and 7 days after 3-min ischemia without ischemic preconditioning. Note an intense activation and accumulation of reactive microglia in b and c. d: 3 days after 2-min ischemia, e,f: 2 days and 7 days after 3-min ischemia with ischemic preconditioning. Microglia are moderately activated in d and e but are subsiding in f. Bar = 0.l mm.

H. Kato et al. / Brain Research 664 (1994) 69-76 73

T ~ ~ . ~

Fig. 4. Immunostaining against glial fibrillary acidic protein for the visualization of astrocytes (a) and isolectin staining for the visualiza- tion of microglia (b) in the dentate hilus 3 days after 2-min ischemia. Reactive astroglia (a) and microglia (b) are seen in the dentate hilus. Bar = 0.1 mm.

staining were seen but they were not so hypertrophic (Fig. 2D. GFAP staining in the CA3 and the dentate gyrus decreased but the reactive astrocytes in the den- tate hilus were still hypertrophic.

3.2. Microglia

In sham-operated animals, microglia were only rarely and weakly stained with the isolectin in the hippocam- pus (Fig. ld, Fig. 3a). When stained, the microglia had the morphology of quiescent microglia, i.e. small cell body and ramified thin processes (Fig. 3a).

As reported earlier, microglia stained intensely with this concentration of isolectin could be considered as activated microglia [24]. Three days after 2 rain of preconditioning ischemia, microglial cells increased throughout the hippocampus, particularly in the den- tate hilus and the CA1 subfield (Fig. le, Fig. 3d and Fig. 4b). The microglia had the morphology of acti- vated microglia with increased isolectin staining and ramified, thorny processes.

Four hours after 3-min ischemia in animals without preconditioning, some activated microglia were already

seen in the hipp0campus, especially in the CA1 sub- field. In animals with preconditioning, isolectin stain- ing was largely similar to that before the second is- chemia.

After 1 day, activated microglia were scattered in the hippocampus, especially in the CA1, in animals both with and without preconditioning.

After 2 days, isolectin staining appeared unchanged in animals with preconditioning (Fig. 3e). However, activated microglia increased strikingly in the CA1 of animals without preconditioning and the microglia be- came larger and stained stronger (Fig. 3b).

After 7 days, an intense accumulation of reactive microglia was seen in the CA1 of animals without preconditioning, especially in stratum pyramidale, where reactive microglia had the morphology of ame- boid-like appearance (Fig. If and Fig. 3c). In stratum radiatum of the CA1, many microglia had the morphol- ogy of rod cells. In the CA3, dentate gyrus, and the dentate hilus, the number of microglia decreased. The hippocampus of the animals with preconditioning re- turned to near control levels and only scattered mi- croglia with less activated morphology were seen in the CA1 (Fig. 3f).

4. Discussion

The present study showed that both astroglia and microglia were activated after ischemia in a graded fashion in response to different degrees of neuronal injury. The first step of glial activation, which occurred without neuronal destruction, was an increased GFAP staining in astrocytes and an increased isolectin stain- ing in microglia. This type of glial activation was gener- ally seen after ischemia including ischemia-resistant regions such as the CA3 and the dentate gyrus al- though stronger in the vulnerable CA1 region. Such glial activation was also seen in the hippocampus that acquired ischemic tolerance. This early activation of glial cells was more rapid and striking in microglia and was subsiding after 7 days when ischemia was sub- lethal. In contrast, astroglial activation occurred rela- tively slowly but lasted longer even when ischemia was sublethal.

Conspicuous differences in glial activation between animals with and without ischemic tolerance was ob- served 2 days after the second ischemia. In the hip- pocampus without ischemic tolerance, microglial acti- vation with increases in isolectin staining and cell size was seen in the CA1 subfield although neuronal de- struction was not yet evident. However, astroglial reac- tions were not obvious. In the CA1 subfield of the hippocampus with ischemic tolerance, in contrast, as-

74 H. Kato et al. / Brain Research 004 (1994) 09-70

troglial activation was evident but microglial activation was milder than that in animals without tolerance. These observations suggest that the balance between astroglial and microglial reactions may be related to the final outcome.

The astroglial and microglial reactions became far intense when neuronal destruction occurred as seen in the CA1 of animals without tolerance and in the den- tate hilus both with and without preconditioning. In the animal models used in this study, ischemia-induced neuronal damage is produced in these two regions. Even a 2-min period of preconditioning ischemia de- stroys a subpopulation of dentate hilar neurons [25]. Reactive astrocytes intensely immunoreactive to GFAP became hypertrophic. Many reactive microglia had the ameboid-like morphology, suggesting an increase in phagocytic activities. Thus, full-blown activation of glial cells was seen when neurons were destroyed.

The function of astrocytes is to maintain local mi- croenvironment under physiological conditions [1,6,29, 38]. The first is a rapid reuptake and inactivation of released neurotransmitters, such as excitatory amino acid glutamate, from synaptic spaces. Secondly, astro- cytes contribute to form blood-brain barrier (BBB), and thirdly they maintain water and ionic homeostasis.

It has been generally accepted that a massive re- lease of glutamate during ischemia triggers the chain reactions leading to ischemia-induced neuronal injury by excitotoxic mechanisms [4,8]. Ischemia also induces water and ionic imbalance and this derangement be- comes severe when BBB is broken down [16]. There- fore, if astroglial activation implies enhanced astroglial functions, reactive astroglia in the hippocampus with induced tolerance may have an increased capacity of glutamate reuptake and maintenance of water and ionic homeostasis. In fact, glutamine synthetase, which inactivates glutamate into glutamine and is present exclusively in astrocytes, increases after cerebral is- chemia [30]. Therefore, ischemic insult rendered in the presence of astroglial activation may facilitate recovery from ischemia-induced environmental derangements when blood flow is restored. Thus, an increased capac- ity of astroglial function may be one of the mechanisms of ischemic tolerance.

Furthermore, reactive astrocytes upregulate the ex- pression of a large number of molecules that may benefit the injured neurons [9]. In vitro studies have shown that astrocytes actually produce a number of agents that support neuronal growth and survival [12,14]. In addition, astroglia-derived growth factors attenuate the toxic effects of microglia and may help to preserve neurons under attack by phagocytic cells [12,14]. Nerve growth factor and basic fibroblast growth factor, which are produced by astrocytes, have been shown to prevent ischemia-induced neuronal damage in vivo when administered exogenously before and

after the onset of ischemia [28,33]. Thus. it is assumed that the neuronal injury following the second ischemia is also reduced by the substances produced by the activated astrocytes in the hippocampus with ischemic tolerance. Growth factors released from astrocytes may also function at a later phase for wound repair.

In vitro studies have shown that microglial cells release toxic agents [12,14,34]. When stimulated, mi- croglia release a variety of cytotoxic agents which may be important mediators of neuronal injury, such as certain kinds of cytokines, reactive oxygen radicals, proteases, and glutamate. In this study, a strong activa- tion of microglia was seen in the CA I subfield 2 days after 3 min of ischemia without preconditioning before the CA1 neurons were destroyed. These activated cells may have a neuron-killing effect. The fate of ischemic neurons may therefore depend on the reactive astroglia and microglia as they compete to govern the survival of neurons.

However, considerable microglial activation, though not full-blown, was seen when ischemic insult was sublethal. Microglial ceils were frequently seen in close proximity to viable neurons. There was a rapid activa- tion of microglia next to neurons that do not die. Of particular interest in this regard is the phenomenon termed synaptic stripping. Blinzinger and Kreutzberg [5] reported that activated microglia after facial nerve lesioning displace afferent terminals from the facial nerve cell bodies causing deafferentation. Whether similar deafferentation occurs in the hippocampus fol- lowing ischemia is unknown and electron microscopic studies are necessary. However, if microglia cause simi- lar deafferentation after ischemic preconditioning, it may protect the CA1 neurons from ischemia because excitotoxicity caused by exposure to glutamate is blocked. Actually, deafferentation has been reported to protect CA1 neurons from ischemic injury [7,40]. Just the presence of microglia therefore does not mean neuronal death and possibly is neuroprotective. There are various levels of microglial activation and this graded activation may explain these conflicting obser- vations.

When neurons were destroyed after ischemia, far intense activation of astrocytes and microglia was seen. They also changed morphology, i.e. hypertrophy of astrocytes and macrophage-like transformation of mi- croglia. Elimination of damaged circuits would prevent continuing disruption and, perhaps, dysfunction of the entire neural system. Controlled cell death is a well-re- cognized process during wound healing and tissue re- newal and arises when inflammatory cells release cyto- toxins. Microglia may protect healthy neurons by re- moval of diseased cells because necrotic tissue elicits aggressive phagocytic microglia.

In conclusion, the findings of this study suggest that activation of astroglia and microglia following ischemia

H. Kato et al. / Brain Research 664 (1994) 69-76 75

is under strict control, and that they are activated in a graded fashion in response to different degrees of neuronal injury. The graded response of microglia have been shown by the differential expression of im- munomolecules and receptors on microglia [11,26], and therefore this kind of investigation may further clarify the differential activation of microglia in ischemic tol- erance. It is quite possible that neuron-glia interac- tions, especially astroglial activation, have implications in promoting neuronal survival in ischemic tolerance. The regulation of glial effects upon neurons is further complicated by microglia-astroglial interactions be- cause fully activated microglia may have neuron-killing effects while moderately activated microglia may not. Thus, the balance between neuron-glial and glia-glial interactions may play a critical role in neuronal survival and death after ischemia and in the manifestation of ischemic tolerance.

Acknowledgements

This study was supported in part by Grant-in-Aid for Scientific Research 03404028 and Grant-in-Aid for Scientific Research on priority areas from the Ministry of Education, Science, and Culture of Japan, and by Kanae Fund of Research for New Medicine.

References

[1] Abbott, N.J., Revest, P.A. and Romero, I.A., Astrocyte-endo- thelial interaction: physiology and pathology, Neuropathol. Appl. Neurobiol., 18 (1992) 424-433.

[2] Araki, T., Kato, H. and Kogure, K., Selective neuronal vulnera- bility following transient cerebral ischemia in the gerbil: distri- bution and time course, Acta Neurol. Scand., 80 (1989) 548-553.

[3] Banati, R.B., Gehrmann, J., Schubert, P. and Kreutzberg, G.W., Cytotoxicity of microglia, Glia, 7 (1993) 111-118.

[4] Benveniste, H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis, J. Neurochem., 43 (1984) 1369-1374.

[5] Blinzinger, K. and Kreutzberg, G.W., Displacement of synaptic terminals from regenerating motoneurons by microglial cells, Z. Zellforsch., 85 (1968) 145-157.

[6] Brightman, M., Implication of astroglia in the blood-brain bar- rier, Ann. NYAcad. Sci., 633 (1991) 343-347.

[7] Buchan, A.M. and Pulsinelli, W.A., Septo-hippocampal deaf- ferentation protects CA1 neurons against ischemic injury, Brain Res., 512 (1990) 7-14.

[8] Choi, D.W., Cerebral hypoxia: some new approaches and unan- swered questions, J. Neurosci., 10 (1990) 2493-2501.

[9] Eddleston, E. and Mucke, L., Molecular profile of reactive astrocytes. Implications for their role in neurologic disease, Neuroscience, 54 (1993) 15-36.

[10] Finsen, B.R., Jorgensen M.B., Diemer N.H. and Jimmer, J., Microglial MHC antigen expression after ischemic and kainic acid lesions of the adult rat hippocampus, Glia, 7 (1993) 41-49.

[11] Gehrmann, J., Bonnekoh, P., Miyazawa, T., Hossmann, K.-A.

and Kreutzberg, G.W., Immunocytochemical study of an early microglial activation in ischemia, J. Cereb. Blood Flow Metab., 12 (1992) 257-269.

[12] Giulian, D., Reactive glia as rivals in regulating neuronal sur- vival, Glia, 7 (1993) 102-110.

[13] Giulian, D. and Vaca, K., Inflammatory glia mediate delayed neuronal damage after ischemia in the central nervous system, Stroke, 24 (Suppl. I) (1993) 1-84-1-90.

[14] Giulian, D., Vaca, K. and Corpuz, M., Brain glia release factors with opposing actions upon neuronal survival, J. Neurosci., 13 (1993) 29-37.

[15] Hatten, M.E., Liem, R.K.H., Shelanski, M.L. and Mason, C.A., Astroglia in CNS injury, Glia, 4 (1991) 233-243.

[16] Hossmann, K.-A., Development and resolution of ischemic brain swelling. In Papius, H.M. and Feindel, W. (Eds.), Dynamics of Brain Edema, Springer, Berlin, 1976, pp. 219-227.

[17] J0rgensen, M.B., Finsen, B.R., Jensen, M.B., Castellano, B., Diemer, N.H. and Zimmer, J., Microglial and astroglial reac- tions to ischemic and kainic acid-induced lesions of the adult rat hippocampus, Exp. Neurol., 120 (1993) 70-88.

[18] Kato, H., Araki, T., Kogure, K., Murakami, M. and Uemura, K., Sequential cerebral blood flow changes in short-term cerebral ischemia in the gerbil, Stroke, 21 (1990) 1346-1349.

[19] Kato, H., Liu, Y., Araki, T. and Kogure K., Temporal profile of the effects of pretreatment with brief cerebral ischemia on the neuronal damage following secondary ischemic insult in the gerbil: cumulative damage and protective effects, Brain Res., 553 (1991) 238-242.

[20] Kirino, T., Delayed neuronal death in the gerbil hippocampus following ischemia, Brain Res., 239 (1982) 57-69.

[21] Kirino, T., Tsujita, Y. and Tamura, A., Induced tolerance to ischemia in gerbil hippocampal neurons. J. Cereb. Blood Flow Metab. 11 (1991) 299-307.

[22] Kitagawa, K., Matsumoto, M., Tagaya, M., Hata, R., Ueda, H., Niinobe, M., Handa, N., Fukunaga, R., Kimura, K,. Mikoshiba, K. and Kamata, T., 'Ischemic tolerance' phenomenon found in the brain, Brain Res., 528 (1990) 21-24.

[23] Liu, Y., Kato, H., Nakata, N. and Kogure, K., Temporal profile of heat shock protein 70 synthesis in ischemic tolerance induced by preconditioning ischemia in rat hippocampus, Neuroscience, 56 (1993) 921-927.

[24] Marty, S., Dusart, I. and Peschanski, M., Glial changes following an excitotoxic lesion in the CNS. I. Microglia/macrophages, Neuroscience, 45 (1991) 529-539.

[25] Matsuyama, T., Tsuchiyama, M., Nakamura, H., Matsumoto, M. and Sugita M., Hilar somatostatin neurons are more vulnerable to an ischemic insult than CA1 pyramidal neurons, J. Cereb. Blood Flow Metab., 13 (1993) 229-234.

[26] Morioka, T., Kalehua, A.N. and Streit, W.J., The microglial reaction in the rat dorsal hippocampus following transient fore- brain ischemia, J. Cereb. Blood Flow Metab., 11 (1991)966-973.

[27] Morioka, T., Kalehua, A.N. and Streit, W.J., Progressive expres- sion of immunomolecules on microglial cells in rat dorsal hip- pocampus following transient forebrain ischemia, Acta Neu- ropathol., 83 (1992) 149-157.

[28] Nakata, N., Kato, H. and Kogure, K., Protective effects of basic fibroblast growth factor against hippocampal neuronal damage following cerebral ischemia in the gerbil, Brain Res., 605 (1993) 458-464.

[29] Nicholls, D. and Attwell, D., The release and uptake of excita- tory amino acids, Trends Pharmacol. Sci., 11 (1990) 462-468.

[30] Petito, C.K., Chung, M.C., Verkhovsky, L.M. and Coopwe, A.J.L., Brain glutamine synthetase increases following cerebral ischemia in the rat, Brain Res., 569 (1992) 275-280.

[31] Rothman, S.M. and Olney, J.W., Glutamate and the pathophysi- ology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111.

76 H. Kato et aL /Brain Research 604 (1994) 09-7(3

[32] Schmidt-Kastner, R., Szymas, J. and Hossmann, K.-A., lmmuno- histochemical study of glial reaction and serum-protein extrava- sation in relation to neuronal damage in rat hippocampus after ischemia, Neuroscience, 38 (1990) 527-540.

[33] Shigeno, T., Mima, T., Takakura, K., Graham, D.T., Kato, G., Hashimoto, Y. and Furukawa, S., Amelioration of delayed neu- ronal death in the hippocampus by nerve growth factor, J. Neurosci., 11 (1991) 2914-2919.

[34] Streit, W.J., Graeber, M.B. and Kreutzberg, G.W., Functional plasticity of microglia: a review, Glia, 1 (1988) 301-307.

[35] Streit, W.J., An improved staining method for rat microglial cells using the lectin from Griffonia simplicifolia (GSA I-B4), J. Histochem. Cytochem., 38 (1990) 1683-1686.

[36] Streit, W.J., Microglial-neuronal interactions, J. Chem. Neu- roanat., 6 (1993) 261-266.

[37] Vibulsreth, S., Hefti, F., Ginsberg, M.D., Dietrich, W.D. and Busto, R., Astrocytes protect cultured neurons from degenera- tion induced by anoxia, Brain Res., 422 (1987) 303-311.

[38] Walz, W., Role of glial cells in the regulation of the brain ion microenvironment, Prog. Neurobiol., 33 (1989) 309-333.

[39] Wieloch, T., Neurochemical correlates to selective neuronal vulnerability, Prog. Brain Res., 63 (1985) 69-85.

[40] Wieloch, T., Lindvall, O., Blomqvist, P. and Gage, F.H., Evi- dence for amelioration of ischaemic neuronal damage in the hippocampal formation by lesions of the perforant path, Neurot Res., 7 (1985) 24-26.