Acute Neuroinflammation Exacerbates Excitotoxicity in Rat Hippocampus in Vivo

Experimental Neurology 177, 95–104 (2002) doi:10.1006/exnr.2002.7991

Acute Neuroinflammation Exacerbates Excitotoxicity in Rat Hippocampus in Vivo Kiyoshi Morimoto, 1 Takako Murasugi, and Tomiichiro Oda Neuroscience and Immunology Research Laboratories, Sankyo Co., Ltd., 2-58, Hiromachi 1-chome, Shinagawa-ku, Tokyo 140-8710, Japan Received March 22, 2002; accepted June 13, 2002

cells, which release proinflammatory and cytotoxic factors including interleukin-1 (IL-1) 2, tumor necrosis factor-␣ (TNF-␣), and nitric oxide (NO) (4, 6, 24). However, it is little known about whether activation of microglia is capable of causing neuronal damage in vivo (22). Lipopolysaccharide (LPS), the major glycolipid component of gram-negative bacterial outer membranes, is a potent proinflammagen and is frequently used to stimulate the immune system. In vitro, activation of microglia and subsequent production of cytotoxic mediators such as NO and TNF-␣ have been attributed to increased neurotoxicity in neuron-glia coculture (4, 5, 13). In vivo, there are some reports showing that acute injection of LPS into rat brain causes microglial activation (2, 17, 33), but does not cause neurodegeneration in the hippocampus (19). Recently, it has been reported that chronic LPS infusion into rat basal forebrain decreases cortical choline-acetyltransferase activity, whose toxicity is suppressed by the glutamate receptor antagonist (12, 40, 41). Excitotoxicity also contributes to some neurodegenerative disorders (7). As an in vitro study revealed potentiation of N-methyl-D-aspartate (NMDA)-mediated neurotoxicity by stimulated microglia (19), microglial activation by LPS might affect the glutamate toxicity of rat hippocampus in vivo. It is also shown that intrastriatal coinfusion of IL-1 with the NMDA or ␣-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid (AMPA) receptor agonist caused extensive cell death in the cortex. Microglia are the major early source of IL-1 in response to NMDA agonists (1, 23, 34).

Accumulating evidence suggests that inflammation may play an important part in neurodegenerative diseases such as Alzheimer’s disease. Inflammation itself, however, is insufficient to produce acute neurodegeneration in vivo. In this report, we determined whether inflammation increases excitotoxicity in hippocampal neurons. A proinflammagen, bacterial endotoxin lipopolysaccharide, was coinjected with ibotenate, an Nmethyl-D-aspartate receptor agonist, into rat hippocampus. One week after coinjection, significant neuronal degeneration and severe tissue collapse were observed in the hippocampus. Astroglial and microglial infiltration were also detected. The neurodegeneration was suppressed by dizocilpine maleate, an N-methyl-D-aspartate receptor antagonist. We then examined whether microglial activation takes part in synergistic neuronal loss. One day after the lipopolysaccharide injection into the rat hippocampus, substantial microglial activation and induction of inducible nitric oxide synthase were observed, while neither neuronal nor astrocytic changes were detected. On the other hand, ibotenate injection at the same place 1 day after lipopolysaccharide injection in the hippocampus produced significant neuronal degeneration and gross microglial activation. These results suggest that inflammation by lipopolysaccharide might play an important role in ibotenate/lipopolysaccharide neurotoxicity. © 2002 Elsevier Science (USA) Key Words: glutamate; inflammation; LPS; microglia; neurodegeneration; toxicity.

INTRODUCTION

Recent analyses suggest that inflammatory processes are involved in a lot of neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, acquired immunodeficiency syndrome (AIDS) dementia, and multiple sclerosis (6, 26, 27, 35). The hallmark of neuroinflammation is the activation of microglial

2 Abbreviations used: AIDS, acquired immunodeficiency syndrome; AMPA, ␣-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazolepropanoic acid; GFAP, glial fibrillary acidic protein; IBO, ibotenate; iNOS, inducible nitric oxide synthase; IFN, interferon; IL, interleukin; LPS, lipopolysaccharide; MAP2, microtubule- associated protein 2; MK-801, dizocilpine maleate; NMDA, N-methyl-D-aspartate; NeuN, neuron-specific nuclear protein; NO, nitric oxide; PBS, phosphate-buffered saline; SEM, standard error of mean; TNF, tumor necrosis factor.

1 To whom correspondence should be addressed. Fax: ⫹81-3-54368566. E-mail: [email protected].

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0014-4886/02 $35.00 2002 Elsevier Science (USA) All rights reserved.

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FIG. 1. Sections of rat hippocampus 1 week after injection. Nissl-stained (A–D) and MAP2-immunostained (E–H) sections after injection of LPS (4 ␮g) (A, E), ibotenate (1 ␮g) (B, F), LPS (4 ␮g) ⫹ ibotenate (1 ␮g) (C, G), and LPS (4 ␮g) ⫹ ibotenate (1 ␮g) ⫹ MK-801 (1 ␮g) (D, H). The insets represent a higher magnification of each section of the CAl cell layer. Bars ⫽ 100 ␮m.

In this report, we injected LPS and/or ibotenate, an NMDA receptor agonist, into rat hippocampus, and investigated the morphological changes of neurons and glial cells, and also the putative role of activated microglia in neuronal death.

EXPERIMENTAL PROCEDURES

Materials LPS and ibotenate were purchased from Sigma (St. Louis, MO). Dizocilpine maleate (MK-801) was

NEURONAL LOSS BY LPS/IBOTENATE COINJECTION

TABLE 1 Degenerated Areas of Rat Hippocampal Neuronal Cell Layers 1 Week after Injection Damaged volume (mm 3) LPS (4 ␮g) IBO (1 ␮g) IBO (1 ␮g) ⫹ IBO (1 ␮g) ⫹ IBO (1 ␮g) ⫹ IBO (1 ␮g) ⫹

LPS LPS LPS LPS

(1 (2 (4 (4

␮g) ␮g) ␮g) ␮g) ⫹ MK (1 ␮g)

0.00043 ⫾ 0.00043 0.062 ⫾ 0.016 0.22 ⫾ 0.08* 0.27 ⫾ 0.08* 0.56 ⫾ 0.04** 0.023 ⫾ 0.021#

Note. Data shown are means ⫾ SEM of n ⫽ 7. LPS, IBO, and MK indicate lipopolysaccharide, ibotenate, and MK-801, respectively. * P ⬍ 0.05 and **P ⬍ 0.001 vs IBO (1 ␮g) as determined by Williams’ test; #P ⬍ 0.001 vs IBO (1 ␮g) ⫹ LPS (4 ␮g), as determined by Student’s t test.

from RBI (Natick, MA). Dulbecco’s phosphate-buffered saline (PBS) tablets (Dainippon Pharmaceutical., Japan) were dissolved in double-distilled water and sterilized. LPS (10 mg/ml), ibotenate (5 mg/ml), and MK801 (5 mg/ml) were dissolved in the PBS and stored at 4°C. Animals Adult male Sprague-Dawley rats (Japan SLC, Japan) weighing 250 –300 g at the beginning of the study were used. They were kept on a 12-h light/dark cycle

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with ad libitum access to food and water. Surgical and animal care procedures were carried out with strict adherence to the guidelines of Sankyo Ethical Committee for Animal Experiments. The number of animals used was 6 to 8 per group. Surgical Procedures After anesthetization with sodium pentobarbital (50 mg/kg, Abbott Laboratories, North Chicago, IL), the rats were positioned in a stereotaxic instrument. Injection materials in the PBS were mixed in a microtube. One microliter of solution was injected over 5 min through a 26-gauge Hamilton syringe into the rat left hippocampus (coordinates: anterior-posterior ⫽ ⫺3.5, medial-lateral ⫽ 2.0, dorsal-ventral ⫽ 2.7 mm from bregma, determined according to the atlas of Paxinos and Watson (1986)). Injection materials (doses) were PBS, LPS (0, 1, 2, 4 ␮g), LPS (0, 1, 2, 4 ␮g) ⫹ ibotenate (1 ␮g), and LPS (4 ␮g) ⫹ ibotenate (1 ␮g) ⫹ MK-801 (1 ␮g). After injection, the needle of the syringe was left in place for 5 min and then slowly withdrawn. One day or 1 week after the injection, the rats were deeply anesthetized and transcardially perfused with approximately 50 ml of saline, followed by about 200 ml of 10% formalin neutral buffer solution (Wako Pure Chemical Industries, Osaka, Japan). The brains were removed and postfixed in the same fixative.

FIG. 2. Immunostained sections of rat hippocampus 1 week after injection. OX42 (A–C) and GFAP (D–F) immunostaining of LPS (4 ␮g) (A, D), ibotenate (1 ␮g) (B, E), and LPS (4 ␮g) ⫹ ibotenate (1 ␮g) (C, F) injection. The insets represent a higher magnification of each section. Bars ⫽ 100 ␮m.

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Ibotenate injection 1 day after LPS injection was performed as follows: On Day1, LPS was injected into the rat hippocampus as noted above. On Day 2, ibotenate was injected at the same place. One week after Day 2, the rat brains were fixed. PBS injection 1 day after LPS injection and ibotenate injection 1 day after LPS injection were done in the same manner. Histochemistry The dissected brains were cryoprotected in PBS containing 20% sucrose for 1 day and coronally cut into consecutive 50-␮m sections with a freezing microtome and stored in PBS containing 0.05% NaN 3. The sections were stained with cresyl violet, which stains Nissl bodies in the perikarya (Nissl staining). The other free-floating brain sections were treated with 3% H 2O 2 and blocked with 5% normal goat serum containing 0.3% Triton-X, followed by incubation with one of the primary antibodies. Then the sections were incubated with appropriate biotinylated secondary antibodies and visualized using the avidin-biotin-peroxidase complex method (Vector Labs, Burlingame, CA) with 3,3-diaminobenzidine as a chromagen. Neurons were stained with a monoclonal antibody to microtubule-associated protein 2 (MAP2, Roche, Switzerland, 1:5000) and a monoclonal antibody recognizing the neuron-specific nuclear protein (NeuN, Chemicon, Temecula, CA, 1:1000). Microglial cells were stained with monoclonal antibody OX42 (BMA, Switzerland, 1:200) and monoclonal antibody against phosphotyrosine (4G10, Upstate, Lake Placid, NY, 1:200). Inducible NO synthase (iNOS) was visualized with a polyclonal antibody against iNOS (Santa Cruz, CA, 1:200). Astrocytes were stained with anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (Roche, 1:200). Quantification of Degenerated Volume After the rat brains were cut into 50-␮m sections with a freezing microtome, all the sections containing hippocampal regions were collected, and every sixth section (at intervals of 0.3 mm) was extracted and stained with cresyl violet. Each section stained was photographed under a microscope and the area (S i) of depleted hippocampal cell layers was measured. The volume of depletion was calculated by quantifying the damaged area in the hippocampus as follows: V ⫽ a⌺S i, where V ⫽ damaged volume (mm 3), S i ⫽ area of depleted cell layer of CA1-4 and dentate gyrus (mm 2) of one section, and a ⫽ interval of sections (0.3 mm). Counting iNOS-Positive Cells After iNOS immunostaining, iNOS-positive cells in the rat hippocampal slices containing needle tracks were counted under a microscope. All such cells 10 ␮m to the left and right of the tip of the needle track, that

were in the field of view of the microscope (200X) were counted. The mean value of both the left and right side was taken as the number of iNOS-positive cells. Statistical Analysis All data are expressed as the means ⫾ standard error of the means (SEM). In Table 1, statistical analysis was assessed by Williams’ test (ibotenate [1 ␮g] vs ibotenate [1 ␮g] ⫹ LPS [1 ␮g], ibotenate [1 ␮g] ⫹ LPS [2 ␮g], and ibotenate [1 ␮g] ⫹ LPS [4 ␮g]). Williams’ test is a trend-sensitive test used to statistically compare parametric multiples. Only if ibotenate [1 ␮g] ⫹ LPS [4 ␮g] was significant compared with ibotenate [1 ␮g] was the next analysis done by Student’s t test (ibotenate [1 ␮g] ⫹ LPS [4 ␮g] vs ibotenate [1 ␮g] ⫹ LPS [4 ␮g] ⫹ MK-801 [1 ␮g]). The others were assessed by Student’s t test between two groups and by Tukey’s test among more than 3 groups. A value of P ⬍ 0.05 was considered statistically significant. RESULTS

Synergistic Neuronal Degeneration by Coinjection of Lipopolysaccharide and Ibotenate Injection materials were mixed in a microtube. One microliter of the solution was injected over 5 min into the rat left hippocampus as described in the experimental procedures. Nissl-stained and MAP2-immunostained sections of the rat brain 1 week after the injection of LPS (4 ␮g) alone or a small amount of ibotenate (1 ␮g) alone into the hippocampus showed only slight neuronal degeneration around the needle tracks of the injection site (Figs. 1A, B, E, and F). PBS injection also did not produce any morphological changes (data not shown). On the other hand, coinjection of LPS (4 ␮g) with ibotenate (1 ␮g) into the rat hippocampus caused synergistic neuronal loss and severe tissue collapse (Figs. 1C and G). The degeneration extended from the region of CA4 close to the injection site to the distal area. The volume of the neurodegeneration by LPS and ibotenate coinjection was significantly (P ⬍ 0.05) increased in a dose-dependent manner (Table 1). Furthermore, simultaneous injection of MK-801 (1 ␮g), an NMDA receptor antagonist, into rat hippocampus with LPS and ibotenate, significantly blocked the neurodegeneration caused by the coinjection of LPS and ibotenate. (Figs. 1D and H, and Table 1, P ⬍ 0.05). These results indicate that the neuronal loss is dependent on some factors induced by the LPS injection and also produced through NMDA receptor activation. GFAP and OX42 immunostaining revealed that astrocytes and microglia were activated by the coinjection of LPS/ ibotenate more than by the injection of LPS alone or ibotenate alone (Fig. 2).

NEURONAL LOSS BY LPS/IBOTENATE COINJECTION

Morphological Changes 1 Day after Lipopolysaccharide Injection To elucidate the factors associated with the neuronal death induced by coinjection of LPS and ibotenate, we examined the histological changes 1 day after LPS (4 ␮g) injection into the rat hippocampus. Nissl-stained sections showed only small morphological changes around the needle track when LPS was injected alone, compared with PBS (Figs. 3A and B). MAP2 and NeuN immunostaining, which are markers of neurons, also revealed slight changes between LPS and PBS injection (Figs. 3C–F). These suggest that 4 ␮g of LPS is not sufficient to cause neuronal death, although it might affect neuronal function and susceptibility to glutamate. We did not detect any changes by ibotenate injection (data not shown). On the other hand, activated microglial cells immunostained with OX-42 and anti-phosphotyrosine antibodies were observed after LPS injection (Figs. 4A–D). Immunostaining by anti-iNOS antibody revealed that iNOS was also highly expressed in the hippocampus by LPS injection (Figs. 4E and F). The number of iNOSpositive cells significantly (P ⬍ 0.005) increased 1 day after LPS injection compared with that after PBS injection (Table 2). We did not identify any iNOS-positive cells in the double-staining immunohistochemistry evaluation. However, considering the extent of immunostaining, iNOS-positive cells seem to be microglia. Furthermore, it has been reported recently that iNOS is induced in microglia but not in astrocytes by the injection of LPS and cytokines in in vivo brain (3, 11, 20, 32). Activation of astrocytes was, however, minimal (Figs, 4G and H). These results suggest that the microglial activation and iNOS induction is not sufficient to cause the neurodegeneration in vivo. Synergistic Neuronal Degeneration by Ibotenate Injection 1 Day after LPS Injection Although LPS injection alone induced microglial activation in the hippocampus, no neuronal loss was observed. To study the role of glutamate under these conditions, PBS, LPS (4 ␮g), and/or a small amount of ibotenate (1 ␮g) not sufficient to cause neuronal loss was injected into the hippocampus under three different conditions as follows: (1) injection of LPS followed by ibotenate, meaning injection of ibotenate 1 day after LPS injection; (2) injection of PBS followed by ibotenate, meaning injection of ibotenate 1 day after PBS injection; (3) injection of LPS followed by PBS, meaning injection of PBS 1 day after LPS injection. No neuronal loss was observed after injection of LPS followed by PBS (Figs. 5A and D), although microglia were activated by LPS injection at the time of PBS injection as shown in Fig. 4. Furthermore, injection of PBS followed by ibotenate caused trivial neuronal loss

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around the needle track (Figs. 5B and E). As shown in Fig. 4, few microglia were activated by PBS injection around the needle track at the time of ibotenate injection. On the other hand, a severe degeneration of hippocampal neurons was observed after injection of LPS followed by ibotenate (Figs. 5C and F). As shown in Fig. 4, microglia were activated by LPS injection at the time of ibotenate injection. These results suggest that some factors induced by activated microglia seem to be necessary to cause the neuronal loss by a small amount of ibotenate. The degeneration volume by injection of LPS followed by ibotenate was significantly increased compared with injection of PBS followed by ibotenate or LPS followed by PBS (Table 3, P ⬍ 0.001). Astrocytes and microglia were substantially activated by injection of LPS followed by ibotenate compared with injection of PBS followed by ibotenate or LPS followed by PBS (Figs. 5G–L). DISCUSSION

It has been reported in some in vitro studies that treatment with LPS alone or in combination with IL-1, TNF-␣, or interferon-␥ (IFN-␥) to neuron/microglial coculture shows neurotoxic effects by factors such as NO and TNF-␣ (4, 5, 16, 19). Flavin et al. (8) claimed that conditioned medium from microglial culture produces neuronal loss. These suggest that microglia-derived factors are important mediators of inflammation-mediated neurodegeneration. Furthermore, it has also been reported that LPS causes inappropriate excitation (38), and that microglia potentiates NMDA-mediated neurotoxicity through NO (19), suggesting that neuroinflammation might exacerbate excitotoxicity in neurons. In vivo, it has already been reported that LPS injected into rat brain activates IL-1- and OX-42-positive microglial cells considerably just after LPS injection (2, 17, 33). With respect to neuron loss, however, acute LPS injection into rat hippocampus does not degenerate rat hippocampal neurons (18). Consistent with these findings, we have shown in this report that even when a large number of OX-42-positive microglia are observed 1 day after LPS injection (Fig. 4), neuronal degeneration does not occur (Figs. 1A and E, and 2). Thus, microglial activation alone might be insufficient to produce neuronal degeneration. On the other hand, we have demonstrated here that coinjection of LPS with ibotenate, a glutamate agonist, could cause synergistic neuronal degeneration in the rat hippocampus. Besides, neurodegeneration was also observed by the injection of ibotenate 1 day after LPS injection. In some in vivo reports, it has been shown that in the basal forebrain the chronic LPS infusion reduces cholinergic neurons and that the degeneration was suppressed by NMDA receptor antagonists, cyclooxygenase-2 inhibitors, anti-inflammatory drugs, and

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FIG. 3. Immunostained sections of rat hippocampus 1 day after injection. Nissl staining (A, B) and MAP2 (C, D) and NeuN (E, F) immunostaining of PBS (A, C, and E) and LPS (4 ␮g) (B, D, and F) injection. The insets represent a higher magnification of each section of CA1 cell layer. Bars ⫽ 100 ␮m.

caspase inhibitors (39 – 41). They suggested that the cytotoxic effects of chronic neuroinflammation may involve prostanoid synthesis and may operate through NMDA receptors. In our study, LPS might also have

increased the glutamate concentration in the rat hippocampus by a similar mechanism, and exacerbated the excitotoxicity induced by the glutamate receptor

TABLE 3

TABLE 2 The Number of iNOS-Positive Cells within a Representative Section of the Rat Hippocampus 1 Day after Injection

PBS LPS

Numbers of iNOS-positive cells

SEM

18.4 582

10.4 122**

Note. Data shown are means ⫾ SEM of n ⫽ 8 and 7 of LPS (4 ␮g) and PBS injection, respectively. ** P ⬍ 0.005 vs PBS, as determined by Student’s t test.

Degenerated Areas of Rat Hippocampal Neuronal Cell Layers Damaged volume (mm 3) LPS3PBS PBS3IBO LPS3IBO

0.039 ⫾ 0.013 0.197 ⫾ 0.042 0.920 ⫾ 0.093*,#

Note. Data shown are means ⫾ SEM of n ⫽ 7 (mm 3). For abbreviations, see Fig. 5. * P ⬍ 0.001 vs LPS3 PBS, #P ⬍ 0.001 vs PBS3 IBO, as determined by Tukey’s test.

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FIG. 4. Immunostained sections of rat hippocampus 1 day after injection. OX42 (A, B), phosphotyrosine (C, D), iNOS (E, F), and GFAP (G, H) immunostaining of PBS (A, C, E, and G) and LPS (4 ␮g) (B, D, F, and H) injection. The insets represent a higher magnification of each section. Bars ⫽ 100 ␮m.

agonist. Furthermore, it is reported that intrastriatal coinfusion of IL-1␤ with S-AMPA produced not only striatal damage but also extensive cortical damage (1, 23, 34). Infusion of IL-1␤ into the cortex, however, did not enhance the damage caused by the infusion of

glutamate agonist alone into the cortex. They concluded that the IL-1␤-dependent pathway from the striatum to the cortex leads to the cortical injury. On the other hand, coinjection of ibotenate with LPS in the hippocampus in the present study caused extensive

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FIG. 5. Sections of rat hippocampus of indicated treatments. LPS3 PBS (A, D, G, and J) indicates PBS injection 1 day after LPS (4 ␮g) injection. PBS3 IBO (B, E, H, and K) indicates ibotenate injection 1 day after PBS (1 ␮g) injection. LPS3 IBO (C, F, I, and L) indicates ibotenate (1 ␮g) injection 1 day after LPS (4 ␮g) injection. A–C, Nissl staining; D–F, MAP2 immunostaining; G–I, OX42 immunostaining; J–L, GFAP immunostaining. The insets represent a higher magnification of each section. Bars ⫽ 100 ␮m.

degeneration from the region of CA4 close to the injection site to the distal area in the hippocampus. This suggested that LPS with ibotenate seems to act di-

rectly in the hippocampus. Additionally, LPS causes neuronal degeneration only in the substantia nigra, but not in the hippocampus or cerebral cortex (18).

NEURONAL LOSS BY LPS/IBOTENATE COINJECTION

Analysis of the abundance of microglia reveals that the substantia nigra have the highest density of microglia. They concluded that the region-specific differential susceptibility of neurons to LPS is attributable to differences in the number of microglia present within the system. We could not see any neuronal degeneration in hippocampal neurons by injection of LPS, either. However, as coinjection of ibotenate and LPS caused synergistic neuronal degeneration, a low density of microglia might be sufficient to enhance the excitotoxicity. There is a possibility that neuronal degeneration could also be induced by the high degree of microglial activation itself, increasing the susceptibility of neurons to excitatory amino acids or by neuroinflammation over a long period of time. Although this could occur in the chronic phase, in the acute phase, microglial activation with excessive excitatory amino acids might be enough to produce neuronal degeneration. Inflammatory processes may be involved in a lot of neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, AIDS dementia, and multiple sclerosis (6, 26, 27, 35). ␤-Amyloid, which is thought to play a causal role in Alzheimer’s disease (21), is known to activate microglia in vitro (25, 36, 37, 42). In vivo, transgenic mice overexpressing ␤-amyloid show a lot of amyloid plaques and microglial activation in the brain (9, 14), but produce no neurodegeneration (15). This also suggests that the microglial activation alone is not sufficient to cause neurodegeneration. In fact, we have previously shown that coinjection of ␤-amyloid with ibotenate into rat hippocampus produces significant neuronal loss, whereas injection of ␤-amyloid alone does not (29, 30). Although activated microglia were observed under these conditions, the mechanism of the neuronal loss was unclear. This study suggests that microglial activation by ␤-amyloid might contribute to the neuronal degeneration. Harkany et al. (10) also reported that ␤-amyloid neurotoxicity is mediated by a glutamate-triggered excitotoxic cascade in rat nucleus basalis. In this report, we have shown that LPS as well as ␤-amyloid exacerbate ibotenate neurotoxicity. While the exact mechanism by which microglial activation increases the excitotoxicity remains unknown, degeneration by ␤-amyloid with ibotenate might be partially due to the microglial activation by ␤-amyloid. Thus, inhibition of microglial activation might be useful to prevent AD. In conclusion, injection of LPS with ibotenate into rat hippocampus synergistically produces neuronal degeneration and microglial activation might participate in this neuronal degeneration. REFERENCES 1.

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