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Amoeboid microglial response following X-ray-induced apoptosis in the neonatal rat brain
ELSEVIER
Neuroscience Letters 193 (1995) 109-112
HEUROSCIEHC[ LETTERS
Amoeboid microglial response following X-ray-induced apoptosis in the neonatal rat brain I. Ferrer a,*, M. Oliv6 a, R. Blanco a, J. Ballabriga a, C. Cin6s b, A.M. P l a n a s c aunitat de Neuropatologia, Servei d'Anatomia Patolbgica, Hospital Princeps d'Espanya, Universitat de Barcelona, 08907 Hospitalet de Llobregat, Barcelona, Spain bServei de Neuroprotecci6, Hospital Princeps d'Espanya, Universitat de Barcelona, Barcelona, Spain CDepartament de Farmacologia i Toxicologia CID, CSIC, Barcelona, Spain Received 15 May 1995; revised version received 29 May 1995; accepted 29 May 1995
Abstract
The phagocytic response following X-ray-induced apoptosis in the neonatal rat brain was examined by immunohistochemistry with the antibodies OX-6 and OX-42 which recognize MHC class II antigens and the CR3 complement receptor, respectively. Few OX-6immunoreactive cells were observed in control rats, and in rats irradiated at postnatal day 2 and examined during the first 2 postnatal weeks. However, a transient increase in the number of OX-42-immunoreactive amoeboid microglia, containing large numbers of apoptotic cells, occurred at 6, 24 and 48 h after irradiation when compared with age-matched controls. These results show that X-rayinduced apoptosis promotes a short-lasting phagocytic response.
Keywords: Irradiation; Apoptosis; Cell death; Microglia; Developing brain
Apoptosis is a particular form of cell death characterized morphologically by early chromatin condensation, extreme nuclear shrinkage and formation of apoptotic bodies, and biochemically by double-strand cleavage of DNA to produce fragments which are multiples of about 180-200 bp (endonuclease-mediated internucleosomal DNA fragmentation) [1,6,10,17]. In contrast to necrosis, apoptosis affects isolated cells which are eventually removed by neighbouring cells and local phagocytes [10]. X-ray-induced cell death in the developing nervous system has the morphological features of apoptosis and is associated with internucleosomal DNA fragmentation [2,3,7-9]. Yet the phagocytic response involved in the shedding of dead cells in the irradiated developing brain has not been examined in detail. In the present study we have used immunohistochemistry with the antibodies OX-42 and OX-6 to learn about the phagocytic response following X-ray-induced apoptosis. The antibody OX-42 recognizes the CR3 complement receptor and is a specific marker for resting and amoeboid microglia [13,16]. The antibody OX-6 is di* Corresponding author, Fax: +34 3 2045065.
0304-3940/95/$09.50
rected against MHC class II antigens and has been used as a marker of perivascular macrophages [5]. Sprague-Dawley rats were irradiated at postnatal day 2 (P2) with a single dose of 2 Gy using a 300 kVp Stabilipan with a half-value layer of 3.3 mm Cu. Pups were anaesthetized with diethyl-ether at 6, 24 and 48 h, and 4 and 13 days, and perfused through the heart with saline followed by 4% paraformaldehyde in phosphate buffer saline (PBS). Once removed, the brain was immersed in 4% paraformaldehyde in PBS for 24 h, washed overnight in PBS, and cut with a vibratome. Serial 50 m thick sections were processed free-floating for immunohistochemistry using the avidin-biotin-peroxidase (ABC kit, Vector Vectastain) method. Monoclonal antibodies OX-42 and OX-6 (Serotec) were used at dilutions of 1:500 and 1:1000, respectively. Sections were then incubated with rat-adsorbed biotinylated IgG antibody diluted 1:100 for 1 h, and finally with ABC at a dilution of 1:100 for 1 h. The antibody reaction was visualized with 0.05% diaminobenzidine and 0.01% hydrogen peroxide. Some sections were incubated without the primary antibody to rule out false positive results. Some sections were counterstained with haematoxylin. Control animals aged P2, P3,
© 1995 Elsevier Science Ireland Ltd. All rights reserved
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110
L Ferrer et al. /Neuroscience Letters 193 (1995) 109-112
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Fig. I. OX-42-immunoreactive cells in the cingulum of rats irradiated at postnatal day 2 (P2) and examined at 6 h (A), 24 h (C), 48 h (E) and 4 days (G), compared with normal controls at ages P2 (B), P3 (D), P4 (F) and P6 (H). The number of immunoreactive cells is higher in irradiated animals at 6, 24 and 48 h than in age-matched controls. Bar = 25urn.
1. Ferrer et al. / Neuroscience Letters 193 (1995) 109-112 Table 1 Controls P2 P3 P4 P6 P15
X-Irradiated rats 488.4 552.2 469.3 468.6 52.8
_+ 57.2 +_29.4 +_44.3 _+33.0 + 17.4
P2 P2 P2 P2 P2
+6h + 24 h + 48 h + 4 days + 13 days
675.3 1069.2 741.4 510.4 46.3
_+45.7* +__90.1 * -+ 85.6 * + 68.6 _+ 17.4
Density of OX-42-immunoreactive amoeboid microglial cells in the cingulum of control rats and animals irradiated at P2 and examined at different postnatal ages. Five sections per rat and three animals per age were analyzed. Results are expressed as mean values _+ SD per mm 2. *P < 0.05 (Mann-Whitney U-test)
P4, P6 and P15 were processed in the same manner. Animal welfare was safeguarded according to the regulations of the European Community. The amount and distribution of apoptotic cells in irradiated animals was similar to that already observed using an identical radiation protocol [2,3]. Briefly, apoptotic cells occurred in the upper layers of the neocortex, piriform and entorhinal cortex, stratum oriens and pyramidal cell layer of the hippocampus, hilus of the dentate gyrus, striatum, thalamus, subcortical white matter, periventricular germinal zone, cerebellar cortex and cerebellar white matter. In control rats, few weakly stained cells were labelled with the antibody OX-6 during the first 2 postnatal weeks,
III
b u t iarge numbers o f amoeboid microglial cells were stained with the antibody OX-42. Immunoreactive amoeboid cells were localized in the periventricular germinal zone, septum, subcortical white matter, hippocampus, fornix and lateral thalamus. A few immunoreactive amoeboid microglial cells were observed in the upper regions of the cerebral cortex and meninges. Amoeboid microglial cells were abundant during the first week, but their numbers dwindled in the following days so that a few OX-42-immunoreactive amoeboid cells were found in the brain of rats aged 2 weeks. These results are in accordance with observations of pioneering studies using the same antibodies to examine the development of amoeboid microglia in the rat brain [11,12], Few OX-6-immunoreactive cells were found in rats irradiated at P2 and examined up to 13 days after exposure to X-rays. OX-42-immunoreactive cells in irradiated animals had the same distribution as in control rats. However, the number of immunoreactive amoeboid microglial cells was transiently higher in irradiated animals when compared with controls in all regions with increased numbers of dead cells (Fig. 1). For practical reasons, this transient increase was quantified in the cingular region because it is a representative zone in which dead cells and microglial cells are abundant. The number of immunoreactive amoeboid cells was counted in the cingulum at the level of the anterior hippocampus in controls and irradiated rats at different postnatal ages. Counts were made
Fig. 2. OX-42-immunoreactive cells in the periventricular germinal layer (A), subcortieal white matter (B) and layer VI of the cerebral cortex (C) in rats treated with 2 Gy X-rays at P2 and examined 6 h later. Many pyknotic dying cells are being engulfed by immunoreactive cells (arrow). Sections counterstained with haematoxylin. Bar = 10/~m.
112
1. Ferrer et al. / Neuroscience Letters 193 (1995) 109-112
directly by using a grid adapted to the ocular of the microscope at a magnification of x 160. Five sections per animal and 3 animals per age were analyzed. Results, expressed as mean values + SD per m m 2, are shown in Table 1. Statistical analysis was carried out with the M a n n - W h i t n e y U-test. Significant differences in the density of OX-42-immunoreactive amoeboid microglial cells were seen in irradiated rats at 6, 24 and 48 h after irradiation compared with the corresponding age-matched controls. However, differences were no longer observed from this time onwards. Immunostained sections counterstained with haematoxylin showed that OX-42-immunoreactive amoeboid microglial cells in irradiated rats were active phagocytes containing large numbers of apoptotic cells (Fig. 2). The present findings show that early recruitment of OX-42-immunoreactive amoeboid microglial cells occurs following X-ray exposure in the neonatal brain. However, the limited response of amoeboid microglia in irradiated rats contrasts with the massive entrance of macrophages following stab-wound-induced necrosis [4], and with the massive activation of OX-42- and OX-6-immunoreactive microglial cells after hypoxia/ischaemia-induced necrosis in developing rats [14,15]. Moreover, the present results show that the turnover of amoeboid microglia in irradiated neonatal animals is very rapid as no differences in the density of these cells were found 4 days after irradiation when compared with age-matched controls. We wish to thank T. Yohannan for editorial assistance. This work was supported by a grant FIS 95-1951. J. Ballabriga and M. Oliv6 are recipients of grants from the Fundacio Pi i Sunyer. R. Blanco has a grant from CIRIT. [1] Corcoran, G.B., Fix, L., Jones, D.P., Nicotera, P., Oberhammer, F.A. and Buttyan, R., Apoptosis: molecularcontrol point in toxicology, Toxieol. Appl. Pharmacol., 128 (1994) 161-181. [2] Ferrer, I., The effect of cycloheximide on natural and X-rayinduced cell death in the developing cerebral cortex, Brain Res., 588 (1992) 351-357.
[3] Ferrer, I., Macaya, A., Blanco, R., Olivr, M., Cin6s, C., Munell, F. and Planas, A.M., Evidence of internucleosomal DNA fragmentation and identificationof dying cells in X-ray-inducedcell death in the developing brain, Int. J. Dev. Neurosci., 13 (1995) 21-28. [4] Ferrer, 1. and Sarmiento, J., Reactive microglia in the developing brain, Acta Neuropathol., 50 (1980) 69-76. [5] Gehrmann, J., Bonnekoh, P., Miyazaka, T., Hossman, K.A. and Kreutzberg, G.W., Immunocytochemicalstudy of an early microglial activation in ischemia, J. Cereb. Blood Flow Metab., 12 (1992) 257-269. [6] Gerschenson,L.E. and Rotello, R.J., Apoptosis: a different type of cell death, FASEB J., 6 (1992) 2450--2455. [7] Harmon, B.V. and Allan, D.J., X-ray-induced cell death via apoptosis in the immature rat cerebellum, Scan. Microsc., 2 (1988) 561-568. [8] lnouye, M., Radiation-induced apoptosis and developmental disturbances of the brain, CongenitalAnomalies,35 (1995) 1-13. [9] Inouye, M, Tamaru, K. and Kameyama, Y., Effects of cycloheximide and actinomycin D on radiation-induced apoptotic cell death in the developingmouse cerebellum,Int. J. Radiat. Biol., 61 (1992) 669-674. [10] Kerr, J.F.R. and Harmon, B.V., Definition and incidence of apoptosis: a historical perspective, In L.D. Tomei and F.O. Cope (Eds.), Apoptosis: The molecular Basis of Cell Death, Cold Spring Harbor Laboratory Press, New York, 1991, pp. 5-29. [11] Ling, E.A., Kaur, C., Yick, T.Y. and Wong, W.C., Immunocytochemical localizationof CR complementreceptors with OX-42 in amoeboid microgliain postnatal rats, Anat. Embryol., 182 (1990) 481-486. [12] Ling, E.A., Kaur, C. and Wong, W.C., Expressionof major histocompatibility complex and leukocyte common antigens in amoeboid microgliain postnatal rats, J. Anat., 177 (1991) 117-126. [13] Ling, E.A. and Wong, W.C., The origin and nature of ramified and amoeboid microglia: a historical review and current concepts, Glia, 7 (1993) 9-18. [14] McRae, A., Gilland, E., Bona, E. and Hagberg, H., Microglia activation after neonatal hypoxia-ischemia,Dev. Brain Res., 84 (1995) 245-252. [15] Ohno, M., Aotani, H. and Shimada, M., Glial responses to hypoxic/ischemic encephalopathy in neonatal rat cerebrum, Dev. Brain Res., 84 (1995) 294-298. [16] Robinson, A.P., White, T.M. and Mason, D.W., Macrophage heterogeneity in the rat as delineated by two monoclonalantibodies, MRC OX-41 and MRC OX-42, the latter recognising complement receptor type 3, Immunology,57 (1986) 527-531. [17] Ueda, N. and Shah, S.V., Apoptosis, J. Lab. Clin. Med., 124 (1994) 169-177.
Neuroscience Letters 193 (1995) 109-112
HEUROSCIEHC[ LETTERS
Amoeboid microglial response following X-ray-induced apoptosis in the neonatal rat brain I. Ferrer a,*, M. Oliv6 a, R. Blanco a, J. Ballabriga a, C. Cin6s b, A.M. P l a n a s c aunitat de Neuropatologia, Servei d'Anatomia Patolbgica, Hospital Princeps d'Espanya, Universitat de Barcelona, 08907 Hospitalet de Llobregat, Barcelona, Spain bServei de Neuroprotecci6, Hospital Princeps d'Espanya, Universitat de Barcelona, Barcelona, Spain CDepartament de Farmacologia i Toxicologia CID, CSIC, Barcelona, Spain Received 15 May 1995; revised version received 29 May 1995; accepted 29 May 1995
Abstract
The phagocytic response following X-ray-induced apoptosis in the neonatal rat brain was examined by immunohistochemistry with the antibodies OX-6 and OX-42 which recognize MHC class II antigens and the CR3 complement receptor, respectively. Few OX-6immunoreactive cells were observed in control rats, and in rats irradiated at postnatal day 2 and examined during the first 2 postnatal weeks. However, a transient increase in the number of OX-42-immunoreactive amoeboid microglia, containing large numbers of apoptotic cells, occurred at 6, 24 and 48 h after irradiation when compared with age-matched controls. These results show that X-rayinduced apoptosis promotes a short-lasting phagocytic response.
Keywords: Irradiation; Apoptosis; Cell death; Microglia; Developing brain
Apoptosis is a particular form of cell death characterized morphologically by early chromatin condensation, extreme nuclear shrinkage and formation of apoptotic bodies, and biochemically by double-strand cleavage of DNA to produce fragments which are multiples of about 180-200 bp (endonuclease-mediated internucleosomal DNA fragmentation) [1,6,10,17]. In contrast to necrosis, apoptosis affects isolated cells which are eventually removed by neighbouring cells and local phagocytes [10]. X-ray-induced cell death in the developing nervous system has the morphological features of apoptosis and is associated with internucleosomal DNA fragmentation [2,3,7-9]. Yet the phagocytic response involved in the shedding of dead cells in the irradiated developing brain has not been examined in detail. In the present study we have used immunohistochemistry with the antibodies OX-42 and OX-6 to learn about the phagocytic response following X-ray-induced apoptosis. The antibody OX-42 recognizes the CR3 complement receptor and is a specific marker for resting and amoeboid microglia [13,16]. The antibody OX-6 is di* Corresponding author, Fax: +34 3 2045065.
0304-3940/95/$09.50
rected against MHC class II antigens and has been used as a marker of perivascular macrophages [5]. Sprague-Dawley rats were irradiated at postnatal day 2 (P2) with a single dose of 2 Gy using a 300 kVp Stabilipan with a half-value layer of 3.3 mm Cu. Pups were anaesthetized with diethyl-ether at 6, 24 and 48 h, and 4 and 13 days, and perfused through the heart with saline followed by 4% paraformaldehyde in phosphate buffer saline (PBS). Once removed, the brain was immersed in 4% paraformaldehyde in PBS for 24 h, washed overnight in PBS, and cut with a vibratome. Serial 50 m thick sections were processed free-floating for immunohistochemistry using the avidin-biotin-peroxidase (ABC kit, Vector Vectastain) method. Monoclonal antibodies OX-42 and OX-6 (Serotec) were used at dilutions of 1:500 and 1:1000, respectively. Sections were then incubated with rat-adsorbed biotinylated IgG antibody diluted 1:100 for 1 h, and finally with ABC at a dilution of 1:100 for 1 h. The antibody reaction was visualized with 0.05% diaminobenzidine and 0.01% hydrogen peroxide. Some sections were incubated without the primary antibody to rule out false positive results. Some sections were counterstained with haematoxylin. Control animals aged P2, P3,
© 1995 Elsevier Science Ireland Ltd. All rights reserved
SSDI 0 3 0 4 - 3 9 4 0 ( 9 5 ) 1 1679-N
110
L Ferrer et al. /Neuroscience Letters 193 (1995) 109-112
A IP
¢, Rtr
U & Q
e7
I!
g
19 Q
~
It"
C
"
[lg,, ~
~lp. o
t"1
qt
,P
Fig. I. OX-42-immunoreactive cells in the cingulum of rats irradiated at postnatal day 2 (P2) and examined at 6 h (A), 24 h (C), 48 h (E) and 4 days (G), compared with normal controls at ages P2 (B), P3 (D), P4 (F) and P6 (H). The number of immunoreactive cells is higher in irradiated animals at 6, 24 and 48 h than in age-matched controls. Bar = 25urn.
1. Ferrer et al. / Neuroscience Letters 193 (1995) 109-112 Table 1 Controls P2 P3 P4 P6 P15
X-Irradiated rats 488.4 552.2 469.3 468.6 52.8
_+ 57.2 +_29.4 +_44.3 _+33.0 + 17.4
P2 P2 P2 P2 P2
+6h + 24 h + 48 h + 4 days + 13 days
675.3 1069.2 741.4 510.4 46.3
_+45.7* +__90.1 * -+ 85.6 * + 68.6 _+ 17.4
Density of OX-42-immunoreactive amoeboid microglial cells in the cingulum of control rats and animals irradiated at P2 and examined at different postnatal ages. Five sections per rat and three animals per age were analyzed. Results are expressed as mean values _+ SD per mm 2. *P < 0.05 (Mann-Whitney U-test)
P4, P6 and P15 were processed in the same manner. Animal welfare was safeguarded according to the regulations of the European Community. The amount and distribution of apoptotic cells in irradiated animals was similar to that already observed using an identical radiation protocol [2,3]. Briefly, apoptotic cells occurred in the upper layers of the neocortex, piriform and entorhinal cortex, stratum oriens and pyramidal cell layer of the hippocampus, hilus of the dentate gyrus, striatum, thalamus, subcortical white matter, periventricular germinal zone, cerebellar cortex and cerebellar white matter. In control rats, few weakly stained cells were labelled with the antibody OX-6 during the first 2 postnatal weeks,
III
b u t iarge numbers o f amoeboid microglial cells were stained with the antibody OX-42. Immunoreactive amoeboid cells were localized in the periventricular germinal zone, septum, subcortical white matter, hippocampus, fornix and lateral thalamus. A few immunoreactive amoeboid microglial cells were observed in the upper regions of the cerebral cortex and meninges. Amoeboid microglial cells were abundant during the first week, but their numbers dwindled in the following days so that a few OX-42-immunoreactive amoeboid cells were found in the brain of rats aged 2 weeks. These results are in accordance with observations of pioneering studies using the same antibodies to examine the development of amoeboid microglia in the rat brain [11,12], Few OX-6-immunoreactive cells were found in rats irradiated at P2 and examined up to 13 days after exposure to X-rays. OX-42-immunoreactive cells in irradiated animals had the same distribution as in control rats. However, the number of immunoreactive amoeboid microglial cells was transiently higher in irradiated animals when compared with controls in all regions with increased numbers of dead cells (Fig. 1). For practical reasons, this transient increase was quantified in the cingular region because it is a representative zone in which dead cells and microglial cells are abundant. The number of immunoreactive amoeboid cells was counted in the cingulum at the level of the anterior hippocampus in controls and irradiated rats at different postnatal ages. Counts were made
Fig. 2. OX-42-immunoreactive cells in the periventricular germinal layer (A), subcortieal white matter (B) and layer VI of the cerebral cortex (C) in rats treated with 2 Gy X-rays at P2 and examined 6 h later. Many pyknotic dying cells are being engulfed by immunoreactive cells (arrow). Sections counterstained with haematoxylin. Bar = 10/~m.
112
1. Ferrer et al. / Neuroscience Letters 193 (1995) 109-112
directly by using a grid adapted to the ocular of the microscope at a magnification of x 160. Five sections per animal and 3 animals per age were analyzed. Results, expressed as mean values + SD per m m 2, are shown in Table 1. Statistical analysis was carried out with the M a n n - W h i t n e y U-test. Significant differences in the density of OX-42-immunoreactive amoeboid microglial cells were seen in irradiated rats at 6, 24 and 48 h after irradiation compared with the corresponding age-matched controls. However, differences were no longer observed from this time onwards. Immunostained sections counterstained with haematoxylin showed that OX-42-immunoreactive amoeboid microglial cells in irradiated rats were active phagocytes containing large numbers of apoptotic cells (Fig. 2). The present findings show that early recruitment of OX-42-immunoreactive amoeboid microglial cells occurs following X-ray exposure in the neonatal brain. However, the limited response of amoeboid microglia in irradiated rats contrasts with the massive entrance of macrophages following stab-wound-induced necrosis [4], and with the massive activation of OX-42- and OX-6-immunoreactive microglial cells after hypoxia/ischaemia-induced necrosis in developing rats [14,15]. Moreover, the present results show that the turnover of amoeboid microglia in irradiated neonatal animals is very rapid as no differences in the density of these cells were found 4 days after irradiation when compared with age-matched controls. We wish to thank T. Yohannan for editorial assistance. This work was supported by a grant FIS 95-1951. J. Ballabriga and M. Oliv6 are recipients of grants from the Fundacio Pi i Sunyer. R. Blanco has a grant from CIRIT. [1] Corcoran, G.B., Fix, L., Jones, D.P., Nicotera, P., Oberhammer, F.A. and Buttyan, R., Apoptosis: molecularcontrol point in toxicology, Toxieol. Appl. Pharmacol., 128 (1994) 161-181. [2] Ferrer, I., The effect of cycloheximide on natural and X-rayinduced cell death in the developing cerebral cortex, Brain Res., 588 (1992) 351-357.
[3] Ferrer, I., Macaya, A., Blanco, R., Olivr, M., Cin6s, C., Munell, F. and Planas, A.M., Evidence of internucleosomal DNA fragmentation and identificationof dying cells in X-ray-inducedcell death in the developing brain, Int. J. Dev. Neurosci., 13 (1995) 21-28. [4] Ferrer, 1. and Sarmiento, J., Reactive microglia in the developing brain, Acta Neuropathol., 50 (1980) 69-76. [5] Gehrmann, J., Bonnekoh, P., Miyazaka, T., Hossman, K.A. and Kreutzberg, G.W., Immunocytochemicalstudy of an early microglial activation in ischemia, J. Cereb. Blood Flow Metab., 12 (1992) 257-269. [6] Gerschenson,L.E. and Rotello, R.J., Apoptosis: a different type of cell death, FASEB J., 6 (1992) 2450--2455. [7] Harmon, B.V. and Allan, D.J., X-ray-induced cell death via apoptosis in the immature rat cerebellum, Scan. Microsc., 2 (1988) 561-568. [8] lnouye, M., Radiation-induced apoptosis and developmental disturbances of the brain, CongenitalAnomalies,35 (1995) 1-13. [9] Inouye, M, Tamaru, K. and Kameyama, Y., Effects of cycloheximide and actinomycin D on radiation-induced apoptotic cell death in the developingmouse cerebellum,Int. J. Radiat. Biol., 61 (1992) 669-674. [10] Kerr, J.F.R. and Harmon, B.V., Definition and incidence of apoptosis: a historical perspective, In L.D. Tomei and F.O. Cope (Eds.), Apoptosis: The molecular Basis of Cell Death, Cold Spring Harbor Laboratory Press, New York, 1991, pp. 5-29. [11] Ling, E.A., Kaur, C., Yick, T.Y. and Wong, W.C., Immunocytochemical localizationof CR complementreceptors with OX-42 in amoeboid microgliain postnatal rats, Anat. Embryol., 182 (1990) 481-486. [12] Ling, E.A., Kaur, C. and Wong, W.C., Expressionof major histocompatibility complex and leukocyte common antigens in amoeboid microgliain postnatal rats, J. Anat., 177 (1991) 117-126. [13] Ling, E.A. and Wong, W.C., The origin and nature of ramified and amoeboid microglia: a historical review and current concepts, Glia, 7 (1993) 9-18. [14] McRae, A., Gilland, E., Bona, E. and Hagberg, H., Microglia activation after neonatal hypoxia-ischemia,Dev. Brain Res., 84 (1995) 245-252. [15] Ohno, M., Aotani, H. and Shimada, M., Glial responses to hypoxic/ischemic encephalopathy in neonatal rat cerebrum, Dev. Brain Res., 84 (1995) 294-298. [16] Robinson, A.P., White, T.M. and Mason, D.W., Macrophage heterogeneity in the rat as delineated by two monoclonalantibodies, MRC OX-41 and MRC OX-42, the latter recognising complement receptor type 3, Immunology,57 (1986) 527-531. [17] Ueda, N. and Shah, S.V., Apoptosis, J. Lab. Clin. Med., 124 (1994) 169-177.