Interferon-γ plus lipopolysaccharide induction of delayed neuronal apoptosis in rat hippocampus

NEUROCHEMISTRY International Neurochemistry International 23 "0888# 80Ð88

Interferon!g plus lipopolysaccharide induction of delayed neuronal apoptosis in rat hippocampus Yasuji Matsuokaa\\ Yoshihisa Kitamuraa\ Hideaki Takahashia\ Ikuo Tooyamab\ Hiroshi Kimurac\ Peter J[ Gebicke!Haerterd\ Yasuyuki Nomurae\ Takashi Taniguchia a

Department of Neurobiolo`y\ Kyoto Pharmaceutical University\ Yamashina\ Kyoto 596!7301\ Japan b Division of Neurochemistry\ Shi`a University of Medical Science\ Otsu\ Shi`a 419!1081\ Japan c Division of Neuroanatomy\ Shi`a University of Medical Science\ Otsu\ Shi`a 419!1081\ Japan d Department of Psychiatry and Psychotherapy\ Albert!Ludwi`s!University of Freibur`\ D!68093\ Germany e Department of Pharmacolo`y\ Faculty of Pharmaceutical Sciences\ Hokkaido University\ Sapporo 959!9701\ Japan Received 08 June 0887^ accepted 19 July 0887

Abstract Interferon!g and lipopolysaccharide "IFN!g:LPS# induce expression of inducible nitric oxide synthase "iNOS# protein both in cells in vitro and in the brain in vivo[ In cultured cells\ excessive production of nitric oxide "NO# induces neuronal cell death[ However\ it is still unclear whether IFN!g and LPS might induce neuronal cell death in vivo[ In this study\ we examined the neuronal cell death and induction of major histocompatibility complex "MHC# antigens after microinjection of IFN!g:LPS into the rat hippocampus[ Although microglia appeared morphologically rami_ed in the normal and vehicle!injected hippocampus\ microinjection of IFN!g: LPS immediately induced the ameboid type[ From days 0Ð6\ iNOS was expressed in ameboid microglia surrounding the site of the microinjection[ Terminal deoxynucleotidyl transferase!mediated dUTP nick!end labeling "TUNEL#!positive cells appeared among the granular neurons of the dentate gyrus on day 2 and peaked about 6 days after microinjection[ When the NOS inhibitor NG!nitro! L!arginine "L!NA# was intraperitoneally administered prior to the microinjection\ the number of TUNEL!positive neurons decreased in a L!NA dose!dependent manner[ These results suggest that IFN!g:LPS induces delayed neuronal apoptosis in the hippocampus in vivo\ and it possibly involves excessive NO production by iNOS[ Thus\ this animal model may be one of neurodegenerative with extensive in~ammatory activation in the hippocampus[ Þ 0888 Elsevier Science Ltd[ All rights reserved[

0[ Introduction In the central nervous system "CNS#\ the concentration of several cytokines\ such as interleukin!0 or tumor necrosis factor!a\ is increased with mechanical stress[ Microglial cells secrete these cytokines in response to endotoxin stimulation "Banati et al[\ 0882^ Benveniste\ 0882#[ Acti! vated microglia and astrocytes accumulate in the neigh! borhood of neurodegenerative sites in the brains of patients with Alzheimer|s disease\ Parkinson|s disease\ and acquired immunode_ciency syndrome "AIDS# "Dickson et al[\ 0882^ McGeer et al[\ 0882#[ Nitric oxide "NO# is synthesized in several mammalian systems such as the immune\ cardiovascular\ and neu! ronal systems\ where NO acts as a signaling and:or cyto! toxic molecule "Dawson et al[\ 0881^ Nathan\ 0881#[ Three isomers of NO synthase "NOS# have been ident!

 Corresponding author[ Tel[] ¦70!64!484!3695^ Fax] ¦70!64!484! 3685^ E!mail]myasuji!indÝumin[ac[jp

i_ed] neuronal type "nNOS#\ inducible type "iNOS#\ and endothelial type "eNOS#\ and iNOS is induced upon exposure to cytokines and:or bacterial endotoxin such as interferon!g "IFN!g# and:or lipopolysaccharide "LPS#\ in macrophages and hepatocytes in vitro "Nathan\ 0881#[ LPS and cytokines also induce iNOS in glial cells in vitro "Kitumura et al[\ 0885a\b^ Murphy et al[\ 0882#[ Recently\ we examined the e}ect of microinjection of IFN!g plus LPS "IFN!g:LPS# into rat brain in vivo[ Microinjection of IFN!g:LPS induces expression of iNOS in ameboid microglia accompanied by expression of major his! tocompatibility complex "MHC# class II antigen which is a glycoprotein on the cell surface that plays a central role in self recognition in the immune system "Kitamura et al[\ 0885c#[ In this model\ NO production was strongly enhanced "Kitamura et al[\ 0885c#[ Recently\ we also found that NO!donor induces apoptotic cell death in human neuroblastoma SH!SY4Y cells "Kitamura et al[\ 0887#[ Apoptosis "programmed cell death#\ may be involved in neuronal degenerative diseases of the CNS "Dickson\

9086!9075:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[ PII ] S 9 0 8 6 ! 9 0 7 5 " 8 7 # 9 9 4 2 ! 8

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0884#[ In fact\ terminal deoxynucleotidyl transferase "TdT#!mediated dUTP nick!end labeling "TUNEL#!posi! tive "a marker for apoptotic cell death# cells are located around neuronal degeneration sites in the brains of pat! ients with Alzheimer|s disease "Smale et al[\ 0884#\ Par! kinson|s disease "Mochizuki et al[\ 0885# and AIDS "Petito and Roberts\ 0884#[ In addition\ excessive NO production induces neuronal death "Dawson et al[\ 0880#[ However\ the causal relationship between neuronal apop! tosis and cytokines and:or endotoxins which possibly act in neurodegenerative sites\ was still unclear\ especially in vivo[ In this study\ we examined the delayed changes after microinjection with IFN!g:LPS in the rat brain\ with special focus on neuronal apoptosis[

1[ Experimental procedures 1[0[ Animals and materials Male Wistar rats "Crj] Wistar\ Charles River\ Atsugi\ Japan# weighing approximately 399 g were adapted to and maintained at 12>C under a 01!h light:dark cycle "lights on 7]99Ð19]99#[ All animals were housed in stan! dard laboratory cages and had free access to food and water throughout the period of the study[ Institutional guidelines for experimental animal care were strictly observed\ and the experimental protocol was approved by the Committee for Animal Research at Kyoto Phar! maceutical University[ We used the following reagents] recombinant rat IFN!g from Life Technologies "Grand Island\ NY#^ LPS "E[ coli Serotype 944]B4#\ DNase I and NG!nitro!L!arginine "L! A# from Sigma Chemical Company "St Louis\ MO#^ mouse and anti!glial _brillary acidic protein "GFAP# monoclonal antibody from Chemicon International "Temecula\ CA#^ mouse monoclonal antibodies against rat CD00b "MRC OX!31#\ rat MHC class I "MRC OX07# and rat MHC class II "MRC OX5# from Harlan Sera! Lab "Loughborough\ U[K[#^ Vectastain ABC Elite kits from Vector Laboratories "Burlingame\ CA#^ and in situ cell death detection kits from Boehringer Mannheim "Mannheim\ Germany#[ Rabbit anti!iNOS polyclonal antibody "Ohshima et al[\ 0881# was a gift from Dr H[ Esumi "National Cancer Research Institute\ Japan#[ 1[1[ Animal model and experimental protocol The rats were fasted overnight with free access to water[ For stereotaxic injection\ rats were anesthetized "sodium pentobarbital\ 49 mg:kg\ i[p[# and immobilized in a Kopf stereotaxic frame[ The Paxinos and Watson "Paxinos and Watson\ 0875# coordinates relative to the bregma were 2[9 mm caudal\ 1[9 mm right lateral\ 2[4 mm ventral[ A mixture of 0 unit of IFN!g and 0 mg LPS dissolved in 1 ml sterilized physiological saline was infused via an injection

cannula connected by polyethylene tubing to a motor! driven 09!ml Hamilton syringe\ at a rate of 0 ml:min[ The injection cannula was retained for a further 4 min after injection[ Control rats were injected with the same amount "1 ml# of sterile physiological saline alone[ Five rats in each group were anesthetized with sodium pento! barbital "099 mg:kg\ i[p[#\ 0\ 2\ and 6 days following intrahippocampal injection[ L!NA "9[0\ 0[9\ and 09 mg:kg# was dissolved in sterile physiological saline and intraperitoneally administered 29 min before intrahippocampal injection with the IFN!g: LPS mixture[ The same amount of sterile physiological saline was administered as the vehicle[

1[2[ Tissue preparation\ TUNEL stainin`

immunohistochemistry

and

Rats were perfused through the ascending aorta with 049 ml of 09 mM phosphate bu}ered saline "PBS\ pH 6[3#\ followed by 299 ml of a cold _xative consisting of 3) paraformaldehyde in 099 mM phosphate bu}er "PB\ pH 6[3# under deep anesthesia with sodium pentobarbital "099 mg:kg\ i[p[#[ After perfusion\ brains were quickly removed and post _xed for 0 day with 3) par! aformaldehyde in 099 mM PB[ After cryoprotection with 04) sucrose in 099 mM PB\ 19 mm!sections were cut in a cryostat and collected into 099 mM PBS[ The sections were incubated with primary antibodies against CD00b "0 ] 09\999#\ GFAP "0 ] 3\999#\ MHC class I "0 ] 1\999#\ MHC class II "0 ] 1\999#\ and iNOS "0 ] 1\999# for 3 days at 3>C\ followed by biotinylated secondary antibody and avidin!biotin!peroxidase "ABC elite\ 0 ] 3\999# for 0 h at room temperature[ Peroxidase labeling was visualized after incubation with 49 mM Tris!HCl bu}er\ pH 6[5\ containing 9[91) 2\2?!diaminobenzidine\ 9[94) hydrogen peroxide and 9[2) nickel ammonium sulfate[ The TUNEL reaction proceeded according to the manufacturer|s protocol supplied with the in situ cell death detection kit with slight modi_cations[ In brief\ sections prepared as described above\ were mounted on glass slides[ Sections were incubated with TdT and ~u! orescein "FITC#!labeled 05!1?!dUTP for 89 min at 26>C\ then incubated with the horseradish peroxide!conjugated Fab fragment of anti!FITC antibody for 0 h at room temperature[ Finally\ peroxidase labeling was visualized as described above[ Some of the sections were incubated without TdT to determine non!speci_c staining[ For posi! tive controls\ sections were pre!incubated with 0 unit of DNase I in 09 mM PBS for 19 min[ All reagents were diluted as described in the manufacturer|s protocol[ After each incubation\ all sections were washed in PBS several times[ The immunostained samples were scanned using a high resolution camera "ProgRes 2997\ Zeiss\ Jena\ Germany#[

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Photographs were printed with a full color digital photo printer "Pictrography 2999\ Fuji Film\ Tokyo\ Japan#[ 1[3[ Ima`e analysis and statistical analysis Sections were scanned using a high resolution camera "ProgRes 2997# and images were analyzed using the pub! lic domain NIH Image 0[45 program "written by Wayne Rasband at the U[S[ National Institute of Health and available from the Internet by anonymous ftp from zippy[nimh[nih[gov[#[ TUNEL!positive and iNOS! immunopositive cells in the hippocampal formation were counted\ and the data were presented number of cells in each square millimeter "cells mm1#[ Statistics were analyzed using one!or two!way analysis of variance "ANOVA#[ Groups were compared using the Bonferroni:Dunn posthoc test "Stat View\ Abacus Con! cepts\ Berkeley\ CA#[ Data are presented as means2stan! dard deviation "S[D[#[ 2[ Results 2[0[ Immunohistochemical chan`es in CD00b\ GRAP\ and MHC Class I and II anti`ens CD00b!immunoreactive cells in the proximity of the vehicle injection site consisted of highly branched\ rami! _ed microglia "Fig[ 0A#\ similar to resting resident mic! roglia in the normal hippocampus[ However\ in the hippocampus surrounding the site of an injection with IFN!g:LPS\ numerous CD00b!immunoreactive mic! roglia appeared with shorter\ thicker processes and swol! len cell bodies\ some of which appeared to assume an ameboid shape "Fig[ 0C and E# from 0 to 6 days after[ GFAP!immunoreactive astrocytes were slightly activated by injection with IFN!g:LPS "Fig[ 0B\ D and F#\ and were not changed between 0 and 6 days after[ MHC class I! and class II!immunoreactivities were not detectable in normal and vehicle!microinjected rat hippocampi "Fig[ 1A and B#[ MHC class II!immu! noreactivity appeared 0 day after microinjection with IFN!g:LPS "Fig[ 1D#[ MHC class II!immunoreactivity appeared in the ameboid microglia[ Between 2 and 6 days later\ the number of MHC class II!immunoreactive ameboid microglia gradually decreased "Fig[ 1F#[ In con! trast\ MHC class I!immunoreactivity was induced 2 days after and increased for up to 6 days "Fig[ 1E# after injec! tion\ although it was not evident 0 day after injection "Fig[ 1C#[ MHC class I!immunoreactivity also appeared in the ameboid microglia[ 2[1[ Induction of iNOS protein and TUNEL!positive apoptotic cells The rabbit anti!iNOS polyclonal antibody used in this study was originally raised against rat iNOS which was

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puri_ed from LPS!treated rat liver "Ohshima et al[\ 0881#[ This anti!iNOS antibody was immunoreacted with 029 kDa iNOS protein in the LPS!treated cultured glial^ how! ever^ it did not react with 059 kDa of puri_ed cerebellar nNOS "Kitamura et al[\ 0885a# and 039 kDa of puri_ed aortic eNOS "unpublished observation#[ In addition\ this anti!iNOS antibody "the 029 kDa iNOS# was detected in the rat hippocampus after microinjection with IFN!g: LPS\ although it was not detected in the vehicle!injected rat hippocampus "Kitamura et al[\ 0885c#[ In the same experimental system\ NO− 1 accumulation\ an indicator for NO production\ was observed even in the untreated rat brain which may be produced from constitutive NOSs^ however\ NO− 1 accumulation was signi_cantly enhanced by injection with IFN!g:LPS "Kitamura et al[\ 0885c#[ Thus\ iNOS protein which was detected by anti! iNOS antibody functioned to produce marked NO in the hippocampus[ Although iNOS!immunoreactivity was not detected in the normal and vehicle injected hippocampi "Fig[ 2A#\ iNOS!immunoreactivity was induced by microinjection of IFN!g:LPS "Fig[ 2B#[ iNOS!immunoreactivity was observed after days 0Ð6\ and then declined[ Immu! noreactivity to iNOS appeared in CD00b!immu! noreactive ameboid microglia but not GFAP! immunoreactive astrocytes\ as our previous _ndings "Kit! amura et al[\ 0885c#[ In the preliminary experiments\ we examined the e}ect of LPS or IFN!g alone\ and its con! centration[ iNOS!immunoreactivity and TUNEL!posi! tive cells were not induced by treatment with either LPS or IFN!g alone\ and lower concentration "data not shown#[ TUNEL!positive cells were observed in the granular neurons of the dentate gyrus[ TUNEL!positive cells were not observed when TdT enzyme was omitted from reac! tion mixture "Fig[ 3A#[ Cells were not TUNEL!positive even 6 days after vehicle!microinjection "Fig[ 3B#[ Although a few TUNEL!positive cells were detected 0 day after injection with IFN!g:LPS\ more appeared after 2 days\ which became marked after 6 days "Fig[ 3C and D#[ Therefore\ we quanti_ed the TUNEL!positive neu! rons after intrahippocampal injection with IFN!g:LPS[ TUNEL!positive neurons in the hippocampus were increased at day 2 "4026 cells:mm1#\ peaked about day 6 "013217 cells:mm1#\ and then slowly decreased "Fig[ 4#[ The number of TUNEL!positive neurons after mic! roinjection with IFN!g:LPS was signi_cantly increased vs vehicle injection "P ³ 9[90 using ANOVA followed by Bonferroni:Dunn posthoc test#[ 2[2[ L!NA affects upon apoptotic neuronal death caused by IFN!g:LPS The NOS inhibitor\ L!NA "9[0\ 0[9 and 09 mg:kg# was intraperitoneally administered 29 min before an intrah! ippocampal injection of IFN!g:LPS[ The number of

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Fig[ 0[ Photomicrographs of immunostained sections using antibodies against CD00b "A\ C\ and E# and GFAP "B\ D\ and F# in areas surrounding sites of microinjections[ Rat hippocampi were microinjected with sterile physiological saline as the vehicle "A and B#\ or a mixture of 0 unit of IFN!g plus 0 mg of LPS "CÐF#[ Rats were sacri_ced 0 "C and D# and 6 days after "A\ B\ E and F#[ Bar  099 mm[

TUNEL!positive neurons were decreased by L!NA[ We further quanti_ed the e}ect of L!NA upon the apoptotic neuronal death caused by IFN!g:LPS "Fig[ 5#[ iNOS! immunopositive cells were not signi_cantly changed by administration of L!NA\ i[e[\ 74220 cells:mm1 "vehicle#\

89215 cells:mm1 "9[0 mg:kg#\ 69216 cells:mm1 "0[9 mg:kg#\ and 64218 cells:mm1 "09 mg:kg#[ In contrast to the number of iNOS!immunopositive cells\ the number of TUNEL!positive cells was reduced by administration of L!NA\ i[e[\ 015225 cells:mm1 "in vehicle#\ 090221

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Fig[ 1[ Photomicrographs of the sections immunostained using antibodies against MHC class I "A\ C and E# and class II "B\ D and F# antigens in the areas surrounding microinjection sites[ Rat hippocampi were microinjected with sterile physiological saline as the vehicle "A and B#\ or a mixture of 0 unit of IFN!g and 0 mg of LPS "CÐF#[ Rats were sacri_ced 0 "C and D# and 6 days after "A\ B\ E and F#[ Bar  099 mm[

cells:mm1 "9[0 mg:kg#\ 77215 cells:mm1 "0[9 mg:kg#\ and 20204 cells:mm1 "09 mg:kg#[ The number of TUNEL! positive cells was signi_cantly reduced by L!NA "P ³ 9[90 using Bonferroni:Dunn posthoc test#\ whereas that of iNOS!immunoreactive cells was not signi_cantly changed[

3[ Discussion Endotoxins and cytokines induce expression of iNOS protein in macrophages "Hasko et al[\ 0885\ 0887#\ and also in brain glial cells such as microglia and astrocytes "Kitamura et al[\ 0885a\c^ Murphy et al[\ 0882#[ Recently\

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Fig[ 2[ Photomicrographs of the sections immunostained using antibodies against iNOS in the areas surrounding microinjection sites[ Rat hippocampi were microinjected with sterile physiological saline as a vehicle "A# or a mixture of 0 unit of IFN!g and 0 mg of LPS "B#[ Asterisks indicate the trace of the needle for microinjection[ Bar  099 mm[

Fig[ 3[ Photomicrographs of TUNEL!stained sections in the granular layer of the dentate gyrus[ Rat hippocampi were injected with sterile physiological saline as the vehicle "B#\ or a mixture of 0 unit of IFN!g and 0 mg of LPS "A\ C and D#[ Rats were sacri_ced 2 "C# and 6 days after "A\ B and D#[ Terminal deoxynucleotidyl transferase was omitted from the reaction to examine non!speci_c staining "A#[ Bar  49 mm[

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Fig[ 4[ The number of TUNEL!positive cells in the hippocampus 0\ 2 and 6 days after intrahippocampal injection with vehicle "V# or a mixture of 0 unit of IFN!g plus 0 mg of LPS "I¦L#[ TUNEL!positive cells in the hippocampal formation were counted\ and the data presented the number of the cells in each square millimeter "cells:mm1#[ Each point represents the mean2S[D[ of _ve rats[ Statistical signi_cance "by Bonferroni:Dun posthoc test#] P ³ 9[90 vs the value of vehicle!treated animals on equivalent days[ Values of vehicle! and IFN!g:LPS!microinjected groups are signi_cantly di}erent "P ³ 9[90\ two!way ANOVA#[

Fig[ 5[ E}ects of L!NA on the apoptotic cell death and iNOS induced by injections of IFN!g:LPS[ TUNEL!positive "open column# and iNOS! immunoreactive cells "closed column# in the hippocampus were counted\ and the data presented the number of the cells in each square millimeter "cells:mm1#[ L!NA "9[0\ 0[9\ and 09 mg:kg\ i[p[# was administered 29 min before intrahippocampal injection of a mixture of 0 unit of IFN!g and 0 mg of LPS[ Each point represents the mean2S[D[ of _ve rats[ The number of TUNEL!positive cells was signi_cantly reduced by L!NA "P ³ 9[90 using Bonferroni:Dunn posthoc test#\ whereas that of iNOS!immunoreactive cells was not signi_cantly changed[

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we found that microinjected IFN!g:LPS induced iNOS mRNA expression after 5 h\ iNOS protein in ameboid microglia after 01 h and NO production after 01 h in the rat hippocampus "Kitamura et al[\ 0885c#[ In the present study\ we examined the changes that occurred from 0 day after a single microinjection of IFN!g:LPS\ with par! ticular reference to neuronal cell death and induction of MHC antigens in the rat hippocampus[ Biochemically\ it is well known that cells undergoing apoptosis are associated with cleavage of genomic DNA\ and the fragmented genomic DNA can be histologically detected by the TUNEL!staining method "Gavrieli et al[\ 0881#[ Thus\ TUNEL!positivity indicates apoptotic cell death[ Cells in the normal and vehicle!injected hip! pocampus were not TUNEL positive\ even 6 days after injection[ However\ 2 and 6 days after injection into hip! pocampus with IFN!g:LPS\ granular neurons in the den! tate gyrus were markedly TUNEL!stained[ In addition\ the NOS inhibitor\ L!NA\ decreased the number of TUNEL!positive neurons dose!dependently[ Excessive NO production induces neuronal death "Dawson et al[\ 0880#[ Furthermore\ our recent results suggest that NO! donors induce apoptotic cell death in human neuro! blastoma SH!SY4Y cells "Kitamura et al[\ 0887# These _ndings clearly indicate that IFN!g:LPS induces apop! totic neuronal death through iNOS induction and excess! ive NO production in ameboid microglia in vivo brain[ Recently\ several papers indicated that marked iNOS induction occurred in the brain of patients with severe AIDS dementia "Adamson et al[\ 0885 Bukrinsky et al[\ 0884# and that slight iNOS protein was detected in Alzh! eimer|s disease brain "Dorheim et al[\ 0883^ Kitamura et al[\ 0886#[ In addition\ administration of anti!in~am! matory drugs to patients with Alzheimer|s disease appears to slow the course of the disease "Rogers et al[\ 0882#[ From these observations\ we consider that the present animal model microinjected with IFN!g:LPS into the hippocampus is one of dementia model by extensive in~ammatory activation[ Microglia were morphologically rami_ed in the normal and vehicle!treated hippocampus^ however\ mic! roinjection with IFN!g:LPS immediately changed them to the ameboid type[ MHC antigens are glycoproteins of the cell surface that play a central role in self recognition in the immune system\ and are required to present foreign antigens to CD3!positive lymphocytes "Widera\ 0875#[ The expression of MHC antigens has been observed in human diseases such as AIDS "Dickson et al[\ 0882#\ Alzheimer|s disease "Tooyama et al[\ 0889# and multiple sclerosis "Hayashi et al[\ 0877#\ and in animal models such as cerebral ischemia "Finsen et al[\ 0882#\ optic nerve degeneration "Rao and Lund\ 0882#\ experimental allergic encephalomyelitis "Traugott\ 0876# and kainic acid!injection "Akiyama et al[\ 0877#[ In these cases\ MHC antigens were observed in activated microglia phagocytose degenerate neuronal elements[ In this study\

microinjection with IFN!g:LPS induced expression of MHC class II antigens at days 0 and 2\ then disappeared[ On the other hand\ MHC class I antigens were expressed after 2 days[ Both MHC class I and II antigens were expressed in ameboid microglia\ but not in astrocytes surrounding the area of neurodegeneration[ However\ MHC class I and II were expressed at di}erent times[ In addition\ IFN!g induces MHC class II antigens in cul! tured microglia "Frei et al[\ 0876^ Suzumura et al[\ 0876#[ We found iNOS!immunopositive cells in some MHC class II!immunoreactive ameboid microglia "Kitamura et al[\ 0885c#[ Our observations suggest that MHC class II! immunopositive ameboid microglia play a key role in neuronal degeneration[ On the other hand\ the expression of MHC class I antigens paralleled the appearance of TUNEL!positive neurons\ and were markedly expressed in ameboid microglia surrounding TUNEL!positive neu! rons[ These results may suggest that MHC class I antigens help scavenge dead neurons[ Further studies of detailed functions of MHC antigens are necessary before de_nite conclusions can be drawn[ In conclusion\ we have shown that an intrah! ippocampal microinjection of IFN!g:LPS induces iNOS expression in microglia and the appearance of TUNEL! positive neurons began from 0 and 2 days after\ respec! tively[ L!NA decreased the number of TUNEL!positive neurons\ but did not change the number of ameboid microglia expressing iNOS[ Although the microglia appeared morphologically rami_ed in the normal and vehicle!treated hippocampus\ microinjection with IFN!g: LPS immediately changed them to the ameboid type[ In addition\ MHC class II antigens were expressed at days 0 and 2\ and thereafter[ MHC class I and II antigens were expressed in ameboid microglia but not in astrocytes[ These results suggest that the delayed apototic neuronal death induced by an intrahippocampal microinjection of IFN!g:LPS is caused by iNOS induction in ameboid microglia[ Acknowledgements We thank Dr H[ Esumi "National Cancer Research Insti! tute\ Japan# for the gift of anti!rat iNOS antibody[ We thank Mr M[ Okazaki\ Ms T[ Ohnaka\ Ms M[ Matsutani and Ms K[ Imazu for technical assistance[ Y[M[ is the recipient of a Research Fellowship from the Japan Society for the Promotion of Science[ This paper was supported in part by Grants!in!Aid from the Ministry of Education\ Science\ Sports and Culture\ Japan "Y[M[\ Y[K[ and T[T[#[ References Adamson\ D[C[\ Wildemann\ B[\ Sasaki\ M[\ Glass\ J[D[\ McArthur\ J[C[\ Christov\ V[I[\ Dawson\ T[M[\ Dawson\ V[L[\ 0885[ Immu!

Y[ Matsuoka et al[ : Neurochem[ Int[ 23 "0888# 80Ð88 nologic NO synthase] Elevation in severe AIDS dementia and induc! tion by HIV!0 gp30[ Science 163\ 0806Ð0812[ Akiyama\ H[\ Itagaki\ S[\ McGeer\ P[L[\ 0877[ Major histocompatibility complex antigen expression on rat microglia following epidural kainic acid lesions[ J[ Neurosci[ Res[ 19\ 036Ð046[ Banati\ R[B[\ Gehrmann\ J[\ Schubert\ P[\ Kreutzberg\ G[W[\ 0882[ Cytotoxicity of microglia[ Glia 6\ 000Ð007[ Benveniste\ E[N[\ 0882[ Astrocyte!microglia interactions[ In] Murphy\ S[\ "Ed[# Astrocytes] Pharmacology and Function[ Academic Press\ San Diego\ pp[ 244Ð271[ Bukrinsky\ M[I[\ Nottet\ H[S[L[M[\ Schmidtmayerova\ H[\ Dubrovsky\ L[\ Flanagan\ C[R[\ Mullins\ M[E[\ Lipton\ S[A[\ Gendelman\ H[E[\ 0884[ Regulation of nitric oxide synthase activity in human immu! node_ciency virus type 0 "HIV!0#!infected monocytes] Implications for HIV!associated neurological disease[ J[ Exp[ Med[ 070\ 624Ð 634[ Dawson\ T[M[\ Dawson\ V[L[\ Snyder\ S[H[\ 0881[ A novel neuronal messenger molecule in brain] the free radical\ nitric oxide[ Ann[ Neurol[ 21\ 186Ð200[ Dawson\ V[L[\ Dawson\ T[M[\ London\ E[D[\ Bredt\ D[S[\ Snyder\ S[H[\ 0880[ Nitric oxide mediates glutamates neurotoxicity on pri! mary cortical cultures[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 77\ 5257Ð5260[ Dickson\ D[W[\ Lee\ S[C[\ Mattiace\ L[A[\ Yen\ S[C[\ Brosnan\ C[\ 0882[ Microglia and cytokines in neurological disease\ with special reference to AIDS and Alzheimer|s disease[ Glia 6\ 64Ð72[ Dickson\ D[W[\ 0884[ Apoptosis in the Brain] Physiology and path! ology[ Am[ J[ Pathol[ 035\ 0939Ð0933[ Dorheim\ M[!A[\ Tracey\ W[R[\ Pollock\ J[S[\ Grammas\ P[\ 0883[ Nitric oxide synthase activity is elevated in brain microvessels in Alzheimer|s disease[ Biochem[ Biophys[ Res[ Commun[ 194\ 548Ð 554[ Finsen\ B[R[\  Jrgensen\ M[B[\ Diemer\ N[H[\ Zimmer\ J[ 0882[ Mic! roglial MHC antigen expression after ischemic and kainic acid lesions of the adult rat hippocampus[ Glia 6\ 30Ð38[ Frei\ K[\ Siepl\ C[\ Groscurth\ P[\ Bodmer\ S[\ Schwerdel\ C[\ Fontana\ A[\ 0876[ Antigen presentation and tumor cytotoxicity by inter! feron!gamma!treated microglia cells[ Eur[ J[ Immunol[ 06\ 0160Ð 0167[ Gavrieli\ Y[\ Sherman\ Y[\ Ben!Sasson\ S[A[\ 0881[ Identi_cation of programed cell death in situ via speci_c labeling of nuclear DNA fragmentation[ J[ Cell Biol[ 008\ 382Ð490[ Hayashi\ T[\ Morimoto\ C[\ Burks\ J[C[\ Kerr\ C[\ Hauser\ S[L[\ 0877[ Dual!labeled immunocytochemistry of the active multiple sclerosis lesion] Major histocompatibility complex and activation antigens[ Ann[ Neurol[ 13\ 412Ð420[ Hasko\ G[\ Nemeth\ Z[H[\ Szabo\ C[\ Zsilla\ G[\ Salzman\ A[L[\ Vizi\ E[S[\ 0887[ Isoproterenol inhibits II!09\ TNF!a\ and nitric oxide production in RAW 153[6 macrophages[ Brain Res[ Bull[ 34\ 072Ð 076[ Hasko\ G[\ Szabo\ C[\ Nemeth\ Z[H[\ Kvetan\ V[\ Pastores S[M[\ Vizi\ E[S[\ 0885[ Adenosine receptor agonists di}erentially regulate IL! 09\ TNF!a\ and nitric oxide production in RAW 153[6 macrophages and in endotoxemic mice[ J[ Immunol[ 046\ 3523Ð3539[ Kitamura\ Y[\ Imaizumi\ R[\ Kitayama\ Y[\ Esumi\ H[\ Nomura\ Y[\ 0885a[ Possible involvement of tyrosine kinase activation in lipo! polysaccharide!induced expression of Ca1¦!insensitive but cal! modulin!coupling nitric oxide synthase in rat glial cells[ J[ Neurosci[ Res[ 32\ 124Ð134[

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