Early response of brain resident microglia to kainic acid-induced hippocampal lesions

BRAIN RESEARCH ELSEVIER

Brain Research 635 (1994) 257-268

Research report

Early response of brain resident microglia to kainic acid-induced hippocampal lesions Haruhiko Akiyama a,,, Ikuo Tooyama b, Hiromi Kondo a, Kenji Ikeda a, Hiroshi Kimura b, Edith G. McGeer c Patrick L. McGeer c a Tokyo Institute of Psychiatry (Formerly, Psychiatry Research Institute of Tokyo), 2-1-8, Kamtkttazawa, Setagaya-ku, Tokyo 156, Japan, b Institute of Molecular Neuroblology, Shtga Unwerstty of Medical Science, Otsu, Japan, c Kmsmen Laboratory of Neurological Research, Unwerstty of Brmsh Columbia, 2255 Wesbrook Mall, Vancouver, BC V6T 1W5, Canada (Accepted 21 September 1993)

Abstract

We investigated the early response of microglia with complement and other proteins in well controlled rat central nervous system lesions. A selective neuronal degeneration in the hippocampal CA3 region was induced without direct tissue damage by an intraventricular injection of a small amount of kainic acid. As early as 1 h post injection, complement proteins Clq, C4, and C3 and immunoglobulin(Ig)G were found in the lesioned area. After 2 h, non-specific leakage of other plasma proteins occurred. By 3 h, reactive microglia gathered around the injured pyramidal neurons. Areas surrounding the lesions were depleted, on the other hand, indicating that these reactive microglia had originally resided in and migrated from such vacant areas. Upregulation of ICAM-1 expression by vascular endothelial cells commenced after 6 h. LFA-l-positive leucocytes were, then, accumulated in the vasculature, which was followed by an infiltration of leucocytes into the lesioned brain parenchyma. These results indicate that, following an acute neuronal injury, the response of the humoral factors such as complement proteins and IgG precedes the microglial reaction. Activation of vascular endothehal cells and subsequent infiltration of blood leucocytes occurs much later than the activation and migration of brain resident microglia. The origin of complement proteins and IgG in the lesioned brain parenchyma remains to be determined, although the production of complement proteins by microglia is suggested. Key words: Microglia; Complement receptor; Complement; ICAM-1; LFA-1; Blood-brain barrier

1. Introduction

Brain microglia share a number of cell surface molecules with monocyte-macrophages. These include receptors for Fc portion of immunoglobulins(Ig)Gs [22,32] and fl2-integrins [1,10,32]. fl2-integrins are a family of cell adhesion molecules that comprise leucocyte function associated antigen (LFA)-I, complement receptors CR3 and CR4. Expression of fl2-integrins by microglia is upregulated in the affected areas of a number of degenerative neurological diseases [1,17,37]. Complement receptors on microglia interact with complement fragments generated by activation of either the classical or alternative pathway. When either of these pathways is activated, controlled proteolysis of

* Corresponding author Fax. (81) 3-3329-8035. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 2 9 3 - C

complement components commences. Multiple biologically active fragments are generated, which are involved in phagocytosis, chemotaxis and cellular interactions [7,18]. Either pathway leads to the cleavage of C3 to produce C3a and C3b, the latter of which is further processed via proteolytic breakdown to iC3b, C3c and C3d,g, to C3d [19]. Complement receptors CR3 and CR4, which are the fl2-integrins C D 1 1 b / C D 1 8 and C D 1 1 c / C D 1 8 , both bind to iC3b. Activation of the classical but not the alternative, complement pathway is known to occur in association with such central nervous system (CNS) lesions as senile plaques and neurofibrillary tangles in Alzheimer disease [8,14,21,22]. Complement-activated oligodendroglia are seen in various neurodegenerative diseases [46,47]. In these lesions, reactive microglia with highly upregulated levels of CR3 and CR4 appear to attack complement coated structures [2,22,47].

H Aktyama et al /Bram Research 635 (1994) 257-268

258

In this study, we investigated the early response of CR3-positive microglia with complement and other proteins in well controlled rat CNS lesions. We employed injections of a small amount of kainic acid into the lateral ventricle [24,25] to produce hippocampal CA3 lesions. Complement components Clq, C3 and C4 were localized immunohistochemically in these lesions. In this model system, acute selective degeneration of CA3 pyramidal neurons was produced without direct tissue damage caused by needle insertion into the target area [25]. We also report the expression of LFA-1, another member of /32-integrins and its preferred ligand, intercellular adhesion molecule (ICAM)1 in the early stage of this model lesion. 2. Materials and methods Male W~star rats weighing approximately 200 g were used in thns study Test rats were rejected wnth 1 4 nmol of kalmc acid dmsolved m 0 3 tzl of physiological sahne by stereotaxlc rejection into the left lateral ventricle. Sham-operated rats were rejected w~th the same amount of physnologncal sahne The Paxmos and Watson [30] coordnnates relatwe to the bregma were 0 2 m m caudal, 1 2 m m left lateral, and 3 8 m m ventral• Rats were sacrificed after periods of 1, 2, 3, 6, 9, 12, 24, and 48 h following the rejection. At least three experiment rats and one sham-operated rat were used for each time period Rats were kdled by intrapentoneal m lect~on of an excess amount of s o d m m pentobarbltal (200 m g / k g b.wt ). The brains were ~mmed]ately removed, cut into 5 m m coronal slabs, and fixed m 2% paraformaldehyde, 1% p~cnc acnd in 0 1 M phosphate buffer, p H 7 4, for 24 h at 4°C S~x add~honal rats were perfus~on-ftxed at 1 or 2 h after kamic acnd mjectnon These rats were rejected with 20 mg horse radish perox~dase (HRP, Wako) m 1 ml of physiological saline 10 m m prior to sacrifice U n d e r deep anesthesm, HRP-mjected rats were perfused transcardmlly wnth 100 ml of 0 01 M phosphatebuffered physiological sahne (PBS), pH 7 4 and then with 400 ml of the same fixative as that used for ~mmers~on fixahon The brains

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were postfixed m the same fixatwe for 24 h After cryoprotectton wnth PBS containing 15% sucrose, 30 # m sechons were cut on a freezing m~crotome A few sections containing h~ppocampal CA3 area from each rat were Nissl stained wnth Cresyl V~olet for evaluahon of neuronal damage For ~mmunohnstochemlstry, free-floating sections were hrst pretreated w~th 0 2% hydrogen peroxide solution m PBS for 30 mln to ehmlnate endogenous perox~dase actw]ty Sections were then incubated w~th primary antibody for 72 h m the cold The primary antibody was diluted m PBS containing 0.3% Triton X-100 (PBS-Tx) and 0 5% bovine serum albumin (BSA, Sngma) The sections were next treated w~th b~otmylated secondary antibody (Vector Lab) d~luted 1.1000 for 2 h at room temperature followed by incubation m the aVldln-biotmylated H R P complex (ABC Ehte, Vector Lab) for 1 h at room temperature Peroxldase labelhng was wsuahzed by incubating with a solution containing 0 01% 3,3'-diamlnobenz]dme (DAB, Sngma), 0.6% mckel a m m o m u m sulfate, 005 M ;m]dazole and 0 00015% hydrogen peroxide A dark purple react;on product appeared after about 15-45 ram, at which tnme the react;on was terminated by transferring the sechons to PBS-Tx For double ]mm u n o s t a m m g , sechons were treated for 30 m m w~th 0.5% hydrogen peroxide m PBS alter the D A B reaction of the first cycle. The second ~mmunohnstochemncal cycle was carried out s;mfiarly to the first, except that mckel a m m o n i u m sulfate was e h m m a t e d from the D A B solut;on, yielding a brown precipitate m the second cycle Sections were mounted on glass shdes, dried, dehydrated and covershpped w~th Entellan (Merck) As controls for nmmunohnstochemnstry, the primary anhbody was subshtuted with enther a mouse monoclonal antnbody indifferent to rat brain antigens, non-;mm u m z e d rabb~t serum or goat serum. No posntwe staining was seen m such control staining The primary ant]bodnes employed m this study, as well as the source, type and dilution are gwen in Table 1 [36,41,42] To compare the relative senslt]whes of detection system for plasma proteins, spot test was performed [43] Rat plasma was spotted onto gelatin coated mtrocellulose m e m b r a n e (Amersham) F~ve spots were made at ddutnons of 1.5, 1'25, 1 125, 1 625, and 1 3,125 from the original plasma w~th PBS. M e m b r a n e s were fixed with formaldehyde vapor at 80°C for 1 h, and washed m 0 05 M T n s buffered saline pH 7 4 containing 0 1% Twin-20 (TBS-Tw) Followmg the p r e m c u b a h o n m TBS-Tw contalmng 1% BSA, m e m b r a n e s

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Fxg. 1. Cresyl Vnolet staining of the hnppocampal CA3 pyramidal layer A control (unlesloned) rat. B 3 h after lesionlng Many pyramidal neurons shrank and were stained darkly A and B are the same magmflcatlon Bar = 100/~m m B

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Fig. 2. OX42 staining of hippocampal CA3 area A: unlesloned rat hippocampus Many resting mlcroglia with n u m e r o u s thin, branched processes are distributed evenly in hlppocampal gray matter. Inset (upper left) is a higher power photomicrograph of resting microgha, p, pyramidal cell layer; alv, alveus Bar = 200 ~cm, and 25/.tm in inset B. 1 h after kalmc acid Injection Processes of microglia In the pyramidal layer have started to become thicker and shorter compared with those in the insert of A Bar = 100 p.m. C' 3 h after lesionlng. A n u m b e r of microglia have gathered around the CA3 pyramidal cells Microgha extending their processes toward the pyramidal cell layer are also seen. Inset (upper left) shows a high power photomicrograph of such a cell, which is indicated at low power by the arrow head. Bar = 200/.tm. Inset in C is the same magnification as the insert in A. D: at 12 h, microgha in the pyramidal cell layer have become more reactive with shorter processes and larger cell bodies• Notice the area vacated of microgha which appear to have migrated towards the pyramidal cells. The same magnification as C. E: at 24 h, amoeboid cells predominate in the lesioned area and are intermingled with a few round cells. Bar = 100 p.m. F' n u m b e r s of reachve microgha, both process bearing and amoeboid, as well as round cells are dramatically increased at 48 h The same magnification as E.

H Aktyama et al / Bram Research 635 (1994) 257-268

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were incubated with rabbit antibodies to Clq, C4c, C3c, a-1 antltrypsln or fibrlnogen in TBS-Tw containing 1% BSA overnight Membranes were treated with alkahne phosphatase-labelled secondary antibody (Dakopatts) and then colored In a solution containing 5-bromo-4-chloro-3-1ndolylphosphate p-tolmdme salt and Nitroblue Tetrazolmm chloride (BCIP and NBT k~t, Bethesda Research Lab.) at the recommended concentration by the company

3. Results

In Nissl preparations, degeneration of pyramidal neurons in the hippocampal CA3 area became evident 3 h after the intraventricular kainic acid injection (Fig. 1A,B). The cell bodies shrank and the cytoplasm was stained more darkly. Twelve hours after lesioning, most damaged neurons were not stained or stained only lightly with Cresyl Violet. Fig. 2 illustrates the time course of microglial response to the degeneration of CA3 pyramidal neurons. In control rats, monoclonal antibody OX42, which rec-

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ognizes complement receptor CR3 [36], stained numerous microglia distributed evenly throughout the gray matter of the hippocampus (Fig. 2A). The morphology of these cells was consistent with that of resting microglia [6,31] having slender cell bodies and many long, thin, branched processes (Fig. 2A, inset). As early as 1 h after kainic acid administration, a small number of microglia in the CA3 pyramidal layer had become hypertrophic with shorter, thicker and less branched processes (Fig. 2B). By 3 h, numerous microglia had gathered around the degenerated pyramidal neurons (Fig. 2C). The stratum radiatum, on the other hand, was depleted compared with that of control rats. Other microglia in the stratum radiatum and oriens of CA3 extended their processes towards the pyramidal layer (Fig. 2C, inset). At 12 h, the number of microglia gathering around the pyramidal neurons increased, and the area vacated by resident microglia became more evident (Fig. 2D). Reactive microglia in the pyramidal layer had even shorter, thicker processes and swollen

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Fig. 3. G F A P staimng of h~ppocampal CA3 area Numbers in the upper right indicate t~me periods (h) after lessoning. All photographs are at the same magnification. Bar = 1 0 0 / z m m A A 1 h after kalmc acid mject~on. Astrocytes w~th thin processes are dmtributed evenly in the brain parenchyma. No s~gnlficant change ~s discerned compared w~th control rats. B: at 3 h, apparent fragmentanon of some GFAP-positwe astrocytes is seen (arrowheads) C' at 12 h, the number of GFAP-posltwe astrocytes in the pyramidal layer is reduced D: at 48 h Astrocytes in the stratum orlens and stratum radmtum have become reactwe with thicker processes, swollen cell bodies and more intense G F A P labelling. They surround the pyramidal layer, which has been vacated of GFAP-pos~twe astrocytes

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Fig 4 Staining of the hippocampal CA3 area for complement and other plasma proteins• N u m b e r s In the upper right of C, E and F indicate time periods (h) after lesioning. A. C l q staining of a control rat hlppocampus A few microgha are stained positively. Bar = 50/.tm. B: low power photomicrograph of C3 staining of a control rat hlppocampus. Only residual blood plasma is stained posmvely. B, E and F are the same magnification Bar = 200/.Lm In B C' C l q staining at 1 h post kainic acid injection. C l q is diffusely distributed in the parenchyma of the stratum lucldum of hlppocampal CA3 area It is also found in blood vessels. Bar = 200/.~m D" high-power magnification of Clq-posltlVe microgha in the alveus Enlarged from the area indicated by an arrow head in 4C. Same magnification as A. E C3 staining of a nearby section to C. A simdar distribution is obtained. F: al-antitrypsin staining of another nearby section to C Only residual blood plasma is stained positively

262

H Aktyama et al /Brain Research 635 (1994) 257-268

cell bodies, some of which appeared to be amoeboid in shape. At 24 h, most microglia were of amoeboid morphology. In addition, a few round OX42-positive cells were seen (Fig. 2E). After 48 h, the number of OX42-positive cells of both amoeboid and round shape had dramatically increased (Fig. 2F). By this time period, the stratum radiatum, which was once devoid of microglia, had been replenished. Fig. 3 shows comparative changes in GFAP-posttive astrocytes. At 1 h, no significant change was observed in the astrocytic network in the hippocampal CA3 region (Fig. 3A). After 3 h, a few fragmented astrocytes were seen in the CA3 pyramidal layer (Fig. 3B, arrowheads). At 12 h, the number of GFAP-posttive astrocytes in the pyramidal layer was reduced (Fig. 3C). By 48 h, however, astrocytes around the lesion showed hypertrophy and looked reactive, while the pyramidal layer appeared to be devoid of GFAP-positive astrocytes (Fig. 3D).

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Staining for complement and other plasma proteins at early stages of the leston is illustrated in Figs. 4 and 5. Complement proteins C3c and C4c are activation fragments of larger proteins C3 and C4, respecttvely. Therefore, polyclonal antibodies to these fragments also recognize their larger precursors. Antibodies to C3 and C3c as well as those to C4 and C4c yielded identical staining results. In control rats, staining for immunoglobulin G (IgG), fibrinogen, al-antitrypsin, antithrombin III and prothrombin were confined to residual blood plasma in the vasculature. Antibodies to complement proteins C l q and C3, on the other hand, stained a number of microglial cells (Fig. 4A) in addition to residual blood plasma (Fig. 4B). Microglia positive for C4 were also found but were very rare. Such complement positive mtcroglia were more frequent in white matter. As early as 1 h after a kainic acid injection, Clq (Fig. 4C,D), C3 (Fig. 4E), C4 and IgG were detected in the brain parenchyma of the hip-

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Fig 5 Staining of the hlppocampal CA3 area for complement and other plasma proteins Numbers in the upper right of each photograph indicate time periods (h) after leslonlng A C3 staining at 2 h post kalnlC acid injection Parenchymalstaining has spread over the entire CA3 area A-D are the same magmficalton Bar = 200 p.m B fibrlnogen staining at 2 h A HRP-mjected and perfuslon fixed rat Diffuse parenchymal staining in the CA3 area is seen C AntI-HRP staining of a nearby section to B Diffuse parenchymal staining as well as some neuronal staining is seen. D C3 staining at 48 h In addition to the diffuse parenchymal labelling, intense granular staining ISdiscerned Some of the granular staining displays a neuronal contour (insert) Bar = 25 p.m in insert

H. Aklyama et aL / Bram Research 635 (1994) 257-268

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Table 2

Table 1 Antibody

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Molecular weight

Plasma concentration

OX42 [36] (CR3) WT.1 [42] (LFA-1) 1A29 [41] (ICAM-1) complement proteins Clq * C3 C3c * C4 * C4c * IgG IgG al-antitrypsln * antlthrombln III * fibrlnogen * prothrombln * GFAP collagen (IV) * HRP

mouse monoclonal (ascltes) mouse monoclonal (diluted ascites) mouse monoclonal (diluted ascites)

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1.3000

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1' 200

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1. 200

rabbit goat rabbit goat rabbit rabbit rabbit rabbit rabbit rabbit rabbit rabbit goat rabbit

Dakopatts Cappel Dakopatts Chemlcon Dakopatts Vector Dakopans Dakopatts Dakopatts Dakopatts Dakopans Dakopatts Southern Cappel

1. 4000 1. 20000 1:5000 1 : 5000 1 : 2000 1 : 3000 1 : 15000 1- 5000 1:5000 1' 5000 1 5000 1' 10000 1.5000 1 10000

Clq * C3 * C4 * IgG al-antltrypsin * antlthrombln III * prothrombm * fibrmogen *

410 [kDa] 185 200 150 52 54-68 72 340

0.15 [/xM] 6 1.8-2.5 67 20-50 25 11 6-13

* These antibodies were onglnally raised against human plasma proteins but cross-reacted with rat proteins.

pocampal CA3 region. The parenchymal staining was limited to stratum lucidum of CA3, where mossy fibers terminate at the apical dendrites of CA3 pyramidal neurons. Fibrinogen, al-antitrypsin (Fig. 4F), antithrombin III and prothrombin were not detected in brain parenchyma of the hippocampus. All these proteins were present, however, in the brain parenchyma around the needle tract, which is approximately 3 mm away from the hippocampal region examined in this study. At 2 h after lesioning, complement proteins and IgG diffused out more widely (Fig. 5A). At this time period, fibrinogen (Fig. 5B), al-antitrypsin, antithrombin III and prothrombin appeared in the parenchyma, showing a similar staining pattern to complement proteins. At 3 h, staining for these plasma proteins spread over all layers of the hippocampal CA3 area. In HRP-injected and perfusion-fixed rats, HRP was first detected in the CA3 parenchymal area at 2 h (Fig. 5C), showing a similar time course of appearance to fibrinogen, al-antitrypsin, antithrombin III and prothrombin. Several neurons appeared to take up HRP that leaked out from blood vessels. At 24-48 h, complement proteins were still present in the brain parenchyma, showing somewhat granular staining (Fig. 5D), some of which displayed a distorted neuronal contour (Fig. 5D, insert). To avoid misinterpretation of this staining of plasma proteins, which could be caused by different sensitivities of detection, rat plasma was spotted at several concentrations onto a nitrocellulose membrane, fixed and stained with the same antibodies used for tissue sections. The sensitivity of an antibody system to whole

* Mechamsms m Blood Coagulation Ftbrmolysts and the Complement System, by Torben Halkler, translation by Palu Woolley, Cambridge University Press, Cambridge, 1991. Upon an estimation that the total blood volume of a rat is about 20 ml, the calculated plasma concentration of injected HRP (MW: 40 kDa) is 25 ~M

plasma depends mainly on the concentration of antigen in plasma, as well as on the affinity of antibodies employed. Table 2 summarizes the plasma concentration and the molecular weight of proteins investigated in this study. Values in Table 2 were obtained in human material but could not be far different from those in rat. Fig. 6 illustrates the results of the spot test, revealing that the negative staining of CA3 parenchyma for al-antitrypsin and fibrinogen at 1 h was not the result of the different sensitivity of the detection systems. Compared with the response of microglia and complement proteins, vascular expression of ICAM-1, as

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H Aktyama et al./Bram Research 635 (1994) 257-268

well as accumulation and infiltration of blood leucocytes into the brain parenchyma, occurred at later time periods of the lesion. In control rats, a very small number of blood vessels were positive for ICAM-1. Fig. 7A shows ICAM-1 staining of hippocampal CA3 region 3 h after a kainic acid injection, showing that the prevalence of ICAM-l-positive vessels is similar to that in control brain. After 6 h, the n u m b e r of ICAM-1 positive blood vessels began to increase gradually (Fig. 7B,C). At 48 h, numerous vessels were positive for ICAM-1 (Fig. 7D). A small number of ICAM-l-positive leucocytes were also seen (Fig. 7D, arrows). LFA-l-positive leucocytes were observed only occasionally in blood vessels of control rats. Prior to 6 h, the incidence of LFA-l-positive leucocytes in kainic acid-injected rats a p p e a r e d to be similar to control rats (Fig. 7E) but a shift became evident at 12 h (Fig. 7F). The number of LFA-l-positive leucocytes increased gradually up to 48 h (Fig. 7G,H). At 24 h, in addition to the accumulation in capillaries, LFA-l-positive leucocytes were detected in brain parenchyma when sections were counterstained with anticollagen IV to label blood vessels (data not shown). Such infiltrated leucocytes became much more numerous at 48 h.

4. Discussion The origin of brain macrophage has long been a matter of significant debate. The issue is further complicated by uncertainties concerning the origin, nature and function of brain resident microglia. Although the questions have yet to be fully answered, recent investigations have indicated that microglia belong to the mononuclear phagocytic system (MPS) [33]. Brain resident microglia share a n u m b e r of antigens with other tissue macrophages. These include leucocyte common antigen [1,15], receptors for Fc portion of I g G [1,22,32], receptor for M-CSF [35], and/32-integrins [1], of which LFA-1 and complement receptor CR3 are members. Availability of monoclonal antibodies to rodent CR3 has enabled investigators to follow the differentiation of monocytes to microglia in the developing CNS [20,32]. Radiation chimeras with different M H C antigens were used to show that in the adult rodent, the brain became endowed by cells originating from donor bone marrow. Some of these cells in the perivascular

265

parenchyma displayed the morphology of microglia [12]. Induction of major histocompatibility complex (MHC) class II antigen expression by intravenous [39] and intrathecal [44] injection of interferon y provided further evidence for the macrophage nature of brain resident microglia. In earlier works, the confusion about the nature of microglia was worsened by analyzing lesions where both brain resident microglia and blood borne monocytes responded to become phagocytes in varying proportions depending on the type of injury. More recently, investigators have carefully designed the type of lesion to avoid influx of blood leucocytes. Such lesions are degeneration of the facial nucleus following application of toxic ricirt to facial nerve [40], axonal degeneration induced by the destruction of neuronal cell bodies in remote area [9,23], and intraventricular administration of toxins that cause selective neuronal death [38]. In this study, we adopted selective degeneration of hippocampal CA3 pyramidal neurons induced by the intraventricular injection of a small amount of kainic acid [3,24,25]. In early time periods following the lesion, response of endogenous microglia was clearly differentiated from that of monocyte-derived macrophages. Andersson et al. induced the same type of lesion in mouse brain to observe the macrophage-microglial reaction [3]. The earliest time point of their experiment was 12 h postinjection, when they found morphological changes of resident microglia in the absence of leucocyte infiltration. Here, we investigated even earlier stages of such lesions. We demonstrated that activated microglia gathered around the degenerated hippocampal CA3 neurons as early as 1-3 h following lesioning. Areas surrounding the site of neuronal injury were depleted, on the other hand, indicating that the reactive microglia had originally resided in and migrated from such vacant areas. These microglia were then transformed to become amoeboid in shape before the c o m m e n c e m e n t of leucocyte infiltration from blood. By 48 h, the vacant areas were again occupied by microglia. Proliferation of microglia is known to occur in CNS lesions [10,40] and could be a source of newly resident microglia. Throughout the time course examined in this study, only round cells were stained positively for LFA-1. LFA-1 is a fl2-integrin, a m e m b e r of the family of cell

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Fig. 7. ICAM-1 staining (A-D) and LFA-1 staining (E-H) of the hlppocampal CA3 area. E-H are nearby sections to A-D respectively A to H are the same magnification. Bar = 100/,~m in A. A: 3 h after lesioning. A small number of blood vessels are positive for ICAM-1 (arrow) B: at 12 h, the number of ICAM-l-posttwe vessels has increased C: at 24 h. ICAM-1 staining of vessels has further increased. D at 48 h, intense vascular labelling, plus a few ICAM-l-posltive round cells are seen (arrows). E: LFA-1 staining at 3 h of a nearby section to A Occasional posmvely stained leukocytes are seen (arrows). F: at 12 h, a small number of LFA-1 positive leucocytes are margmating along some vessel walls. G: at 24 h, the number of margmatmg leucocytes has increased. A few leucocytes have entered the brain parenchyma but the majority remain within vessels. H at 48 h, a large number of leucocytes are seen in the brain parenchyma

266

H. Aktyama et al / Bram Research 635 (1994) 257-268

adhesion molecules that are expressed exclusively by leucocytes and their kindred. In rat brain, reactive microglia also expressed LFA-1 but several days after lesioning (unpublished data). Interaction of LFA-1 with its ligand ICAM-1 is considered to be involved in leucocyte adhesion to vascular endothelial cells [4,28], which ts the first step in leucocyte infiltration into tissue [13]. In this study, the upregulation of vascular ICAM-1 expression preceded the gathering of leucocytes in the vasculature, which was then followed by the appearance of LFA-l-positive leucocytes in brain parenchyma. These events began much later than the response of brain resident microglia. Such a delay of leucocyte infiltration was also observed in rat brain lesions induced by an intraperitoneal 3-acetylpyridine injection, in which direct tissue damage was avoided [27]. ICAM-1 expression is upregulated by inflammatory cytokines such as IL-1 and TNF-a. It is reasonable to speculate, therefore, that in brain lesions, reactive microglia release some of these factors, which then activate vascular endothelial cells to initiate leucocyte infiltration. As early as 1 h after a kainic acid injection, complement proteins and IgG were detected m the brain parenchyma around degenerated neurons. Other plasma proteins as well as exogenously injected HRP were not then present in the brain parenchyma. One might speculate that the extremely high concentration of IgG in plasma could cause earlier detection of this protein during the time course of blood brain barrier opening. As for complement proteins, however, such a selective appearance in the early time point was proved to be unrelated to either plasma concentration of each I protein or sensitivity of the detection system. The initial distribution of complement proteins and IgG was sharply restricted to the stratum radiatum and pyramidale. Such a specific localization formed a striking contrast to the diffuse spreading of plasma proteins after 2 h. This suggests that the early presence of complement proteins and IgG is of some functional significances rather than being simply an initial phase of the non-specific blood-brain barrier (BBB) breakdown. Both IgG and activation fragments of complement C3 function as opsonins, which dramatically enhance phagocytosis. Microglia express receptors for IgG and iC3b [1,22] and may require these opsonins for effective phagocytosis in brain lesions. The origin of these complement proteins and IgG remains to be determined. Theoretically, they could be derived from either endogenous sources or blood plasma through selective permeability of BBB. Cytoplasmic staining of microglia for complement proteins in both normal and lesioned rats suggests that microglia are the source of these complement proteins. Such a notion is supported by the previous works that have demonstrated complement proteins mRNA in

brain tissue using polymerization chain reaction [45] or in situ hybridization [16,29]. In culture, microglia have been reported to upregulate C3 production upon activation [11]. Pasinetti, et al. detected a significant increase in Clq m R N A in rat brain lesions 48 h after a systemic injection of kainic acid [29]. We have observed much more rapid deposition of complement proteins in this study. This infers that, if endogenous, these complement proteins are released from intracellular storages at least in the initial stage of the lesions. On the other hand, a possible endogenous source of IgG is less certain. Very small number of B lymphocytes might be recruited into normal brain, be transformed into plasma cells and produce IgG following stimulation. Intrathecal production of antibodies has been reported in the presence of an intact BBB [5]. However, B lymphocytes in the brain parenchyma, if present, must be distributed only sparsely. It may be unlikely that such rare B lymphocytes release IgG that covers specifically and entirely the lesioned hippocampal CA3 area within an hour after an insult. Nitsch et al. examined by EM changes of the BBB in rat following kainic acid injection [26]. They suggested that the opening of BBB was a result of increased transendothelial pinocytosis w~th an intact tight junction. Such an observation might be supportive evidence for a selective uptake of some plasma proteins into brain at a site of injury. Resident microgha respond quickly to neuronal degeneration and become reactive microglia before monocytes enter the lesioned brain tissue. Some classical plasma proteins such as complement proteins and IgG appear to be involved in this early response of microglia. Perry et al. have reported that some yet undefined plasma protein regulates the expression of a macrophage siahc acid-binding receptor by microglia [34]. Further study is needed to clarify the entire picture of microglial activation as well as the involvement and source of these classical plasma proteins. This research was supported by grants from Sasakawa Research Foundauon, grants-m-aid from the Mlmstry of Education, Science and Culture of Japan (04770517), and the Alzhelmer Society of B C, as well as donations from mdwldual British Columbians The authors are grateful to Ms. Joane Sunahara for techmcal assistance and Mr Kazusuke Kato and Mr Hlroshl Shoda for photographic help

Acknowledgement

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