Acute Excitotoxic Injury Induces Expression of Monocyte Chemoattractant Protein-1 and Its Receptor, CCR2, in Neonatal Rat Brain

Experimental Neurology 165, 295–305 (2000) doi:10.1006/exnr.2000.7466, available online at http://www.idealibrary.com on

Acute Excitotoxic Injury Induces Expression of Monocyte Chemoattractant Protein-1 and Its Receptor, CCR2, in Neonatal Rat Brain 1 John M. Galasso,* Mark J. Miller,† Rita M. Cowell,* Jeffrey K. Harrison,‡ Jeffrey S. Warren,† and Faye S. Silverstein* ,§ *Neuroscience Program, †Department of Pathology and §Department of Pediatrics and Neurology, University of Michigan, Ann Arbor, Michigan 48109-0646; and ‡Department of Pharmacology and Therapeutics, University of Florida, Gainesville, Florida 32610-0267 Received January 10, 2000; accepted April 12, 2000

Chemokines are a family of structurally related cytokines that activate and recruit leukocytes into areas of inflammation. The “CC” chemokine, monocyte chemoattractant protein (MCP)-1 may regulate the microglia/monocyte response to acute brain injury. Recent studies have documented increased expression of MCP-1 in diverse acute and chronic experimental brain injury models; in contrast, there is little information regarding expression of the MCP-1 receptor, CCR2, in the brain. In the neonatal rat brain, acute excitotoxic injury elicits a rapid and intense microglial response. To determine if MCP-1 could be a regulator of this response, we evaluated the impact of excitotoxic injury on MCP-1 and CCR2 expression in the neonatal rat brain. We used a reproducible model of focal excitotoxic brain injury elicited by intrahippocampal injection of NMDA (10 nmol) in 7-day-old rats, to examine injury-induced alterations in MCP-1 and CCR2 expression. RT-PCR assays demonstrated rapid stimulation of both MCP-1 and CCR2 mRNA expression. MCP-1 protein content, measured by ELISA in tissue extracts, increased >30-fold in lesioned tissue 8 –12 h after lesioning. CCR2 protein was also detectable in tissue extracts. Double-immunofluorescent labeling enabled localization of CCR2 both to activated microglia/monocytes in the corpus callosum adjacent to the lesioned hippocampus and subsequently in microglia/monocytes infiltrating the pyramidal cell layer of the lesioned hippocampus. These results demonstrate that in the neonatal brain, acute excitotoxic injury stimulates expression of both MCP-1 and its receptor, CCR2, and suggests that MCP-1 regulates the microglial/monocyte response to acute brain injury. ©

2000 Academic Press

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This research was supported by USPHS Grants NS35059 and NS31504 (to F.S.S.), HL 48287 (to J.S.W.), and UM-MAC (NIH P60-AR20557).

Key Words: chemokine; chemokine receptor; inflammation; excitotoxicity.

INTRODUCTION

It is increasingly evident that inflammatory mechanisms contribute to the pathogenesis of brain injury in a variety of acute and chronic neurodegenerative disorders. Activation and recruitment of leukocytes into areas of inflammation constitute critical processes during host-mediated immune responses to injury. Microglia and blood-derived monocytes may contribute to the progression of neuronal injury through the release of inflammatory mediators or other soluble toxins (1, 2). There is growing interest in identifying the molecular signals that regulate the inflammatory response in the brain. Mediators of the inflammatory response to brain injury may include chemokines, a family of structurally related cytokines that regulate the activation and directed migration of leukocytes (for review see 3). Monocyte chemoattractant protein (MCP)-1 is one of the best characterized CC chemokines. MCP-1 has powerful activating and recruiting effects on mononuclear phagocytes, activated T cells, and B cells (4 – 6). Increased MCP-1 expression has been reported in several experimental models of acute and chronic CNS disorders including cerebral ischemia (7, 8), experimental autoimmune encephalitis (EAE) (9 –11), mechanical injury (10, 12), and brain tumors (13, 14). In a well-characterized model of acute excitotoxic brain injury elicited by direct intracerebral administration of the selective glutamate agonist, N-methyl-D-aspartate (NMDA), into postnatal day (P) 7 rats, MCP-1 mRNA is rapidly upregulated in areas where activated microglia/monocytes subsequently accumulate and where irreversible neuronal injury evolves (15). However, the precise role of MCP-1 in the progression of neuronal injury is uncertain.

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0014-4886/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

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Most chemokines elicit their effects through interactions with seven-transmembrane domain, G-proteincoupled receptors. MCP-1 receptors have been identified in monocytes (16) and lymphocytes (17), as well as in nonhematopoietic cells (18). Knowledge of the structure, function, and regulation of chemokine receptors is rapidly expanding; interest in these receptors stems in part from evidence that several chemokine receptors, including CCR2 and the closely related chemokine receptor, CCR5, play pivotal roles in the pathogenesis of HIV-1 infection (19). Several chemokine receptors have been identified in human postmortem brain tissue and in experimental animals (20). In an experimental gliosarcoma model in adult rats, we recently reported that expression of the MCP-1 receptor, CCR2, is markedly upregulated within and around the brain tumors, both in infiltrating microglia/monocytes and in tumor cells (14). In order to evaluate whether MCP-1 could be an important mediator of the inflammatory response initiated by acute excitotoxic brain injury in the neonatal brain, we performed three related groups of experiments in P7 rats. We evaluated the impact of direct intrahippocampal NMDA injection on MCP-1 and CCR2 mRNA expression; we examined NMDA-induced changes in MCP-1 and CCR2 protein content in the acutely injured brain; and we used immunofluorescence assays and confocal microscopy to localize and identify CCR2-immunoreactive cells in the acutely injured hippocampus. MATERIALS AND METHODS

Animals and reagents. P7 Sprague–Dawley rats were obtained from Charles River (Wilmington, MA). Reagents were purchased from the following sources: NMDA, protease inhibitors (aprotinin, phenylmethylsulfonyl fluoride (PMSF), and sodium orthovanadate), BSA (Cat. No. 9430), and teleostean (cold water fish skin) gelatin from Sigma (St. Louis, MO); 3-((RS)-2carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) from RBI (Natick, MA); Tri-Reagent from Molecular Research Center (Cincinnati, OH); MuLV RT, random hexamers, dNTP’s, and RNase inhibitor from Perkin Elmer (Foster City, CA); and DNase from Ambion (Austin, TX). Antibodies and related reagents. 96-well microtiter plates were purchased from NUNC (Denmark). Reagents were obtained from the following sources: purified and biotinylated mouse anti-MCP-1 antibodies from PharMingen (San Diego, CA); recombinant rat MCP-1 from PeproTech (Rocky Hill, NJ); horseradish peroxidase (HRP)-conjugated neutralite-avidin from Southern Biotechnology Associates (Birmingham, AL); and goat anti-CCR2 and normal goat IgG from Santa Cruz Biotechnology (Santa Cruz, CA). HRP-conjugated anti-goat IgG, BCA protein assay kit, and enhanced

luminol were purchased from Pierce (Rockford, IL). ED-1 mAb was obtained from Serotec (Oxford, England) and NeuN mAb was purchased from Chemicon (Temecula, CA). Fluorescent secondary Ab’s Alexa 488 donkey anti-goat IgG, Alexa 568 rabbit anti-mouse IgG, and ProLong anti-fade mounting medium were obtained from Molecular Probes (Eugene, OR); ABTS was purchased from Boehringer Mannheim (Indianapolis, IN). Animal methods. Surgical protocols were approved by the University of Michigan Committee on the Care and Use of Animals; lesioning was performed in P7 Sprague–Dawley rats of both genders, using previously reported methods (21) with minor modifications. Animals were deeply anesthetized by methoxyflurane inhalation and NMDA (10 nmol/0.5 ␮l) was microinjected into the right dorsolateral hippocampus (stereotaxic coordinates relative to bregma: AP 2.0 mm; L 2.5 mm; D 4.0 mm). All experiments included controls that underwent the same procedures, in which an equal volume of PBS was substituted for NMDA, as well as unlesioned littermate controls. Animals were killed either by decapitation or by administration of a lethal dosage of pentobarbital followed by perfusion–fixation. Treatment with NMDA antagonist. To evaluate the effects of an NMDA antagonist, selected animals received intraperitoneal (ip) injections of the competitive NMDA receptor antagonist, CPP (20 mg/kg), 15 min after the injection of NMDA (22); controls for these experiments received ip injections of equal volumes of PBS. RNA isolation. RNA samples were prepared from the left and right hippocampus of animals that received right intrahippocampal injections of 10 nmol NMDA 2, 4, 8, 16, 24, 48, or 72 h earlier and from animals that had received PBS injections (8 and 16 h earlier). Normal P8 hippocampus was also collected. Brains were divided along the midline and left and right hippocampus were microdissected on ice. Four hippocampi were pooled/sample. Total RNA was isolated using Tri-Reagent according to manufacturer’s directions and stored at ⫺70°C. Concentration and purity of RNA samples were determined by spectrophotometric analysis. Reverse transcription-polymerase chain reaction (RT-PCR). Total RNA (1 ␮g) was pretreated with DNase (15 min at room temperature); the reaction was stopped with EDTA (final concentration 2.5 mM), and DNase was heat-inactivated (65°C, 15 min). RT was performed as previously described (23) with minor modifications. DNase-treated RNA (1 ␮g) was incubated with 50 U MuLV RT, 2 ␮M random hexamers, and 0.5 mM each dNTP (reaction volume 25 ␮l) under the following conditions: 10 min at room temperature, 15 min at 42°C, 5 min at 99°C. The RT product was diluted to a final volume of 100 ␮l in sterile water.

NMDA INDUCES MCP-1 AND CCR2 EXPRESSION

TABLE 1 Primer Sequences Used in RT-PCR Assays Target

Size (bp)

Primers

MCP-1

396

CCR2

409

GAPDH

298

5⬘-TCTACAGAAGTGCTTGAGGTGGTTG-3⬘ 5⬘-CCTGTTGTTCACAGTTGCTGCC-3⬘ 5⬘-GGAATCCTCCACACCCTGTTTC-3⬘ 5⬘-ACCCAACTGAGACTTCTTGCTCCC-3⬘ 5⬘-CAGATCCACAACGGATACATTGG-3⬘ 5⬘-TCCTGCACCACCAACTGCTTAG-3⬘

Note. The left column indicates the target mRNA. The middle column shows the predicted nucleotide base pair (bp) size of the amplified cDNA based on the respective rat cDNA sequence. The right column shows the respective primer sequences used: the top primer denotes the sense sequence and the bottom primer shows the antisense sequence.

MCP-1 and CCR2 mRNA were assayed in aliquots from the same batches of reverse-transcribed RNA. Oligonucleotide primer sets were generated (Table 1) to amplify fragments of the rat MCP-1 and CCR2 cDNA sequences. GAPDH mRNA was assayed concurrently to evaluate the equivalence of RNA content among samples. RT-PCR reaction products were visualized in ethidium bromide-stained 2% agarose gels. Results were quantified by fluorometric scanning of the gels; measurement of arbitrary optical density units (counts/mm) of each band was performed using the Molecular Analyst imaging system (Bio-Rad, Hercules, CA). Preliminary experiments established conditions within the linear range of coamplification for MCP-1/ GAPDH (12 ␮l RT product; 0.2 ␮mol/L MCP-1 primers, 0.075 ␮mol/L GAPDH primers; 94°C, 90 s; 60°C, 30 s; 72°C, 30 s; 30 cycles) and for CCR2/GAPDH [25 ␮l RT product; 0.4 ␮mol/L CCR2 primers, 0.075 ␮mol/L GAPDH primers; 94°C, 90 s; 55°C, 60 s; 72°C, 120 s; 38 cycles (with addition of GAPDH primers after first eight cycles)]; their sensitivity was confirmed by assays of serial dilutions of cDNA’s. To verify the identity of the amplified products, restriction digests were performed (with BglII and EcoRI, for MCP-1 and CCR2 cDNA’s, respectively) which yielded products of the predicted size (data not shown). To evaluate the efficacy of the DNase treatment, selected RNA samples (from right hippocampus at 8 and 16 h post-NMDA injection for MCP-1 and CCR2, respectively) were DNase treated and amplified under the conditions described above without preceding RT reaction. Enzyme-linked immunosorbent assay (ELISA) for MCP-1. We developed an MCP-1 ELISA to measure concentrations of MCP-1 in tissue extracts. Animals were killed, brains were removed, and left and right hemispheres were separated on ice. Based on an approach that had been used successfully to measure tissue content of interleukin (IL)-1␤ (24), each hemi-

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sphere was rapidly homogenized in 500 ␮l PBS (pH 7.2) and centrifuged (12,000 rpm, 15 min, 4°C); supernatants were collected and stored at ⫺70°C. Protein content of the supernatant was determined using a BCA protein assay kit, according to manufacturer’s directions. Samples were collected from animals that received NMDA 2 h (n ⫽ 3), 4 h (n ⫽ 3), 8 h (n ⫽ 5), 12 h (n ⫽ 4), 16 h (n ⫽ 3), 24 h (n ⫽ 3), 48 h (n ⫽ 3), and 96 h (n ⫽ 2) earlier. Samples were also collected from PBS-injected (8 h earlier; n ⫽ 3), from normal P8 (n ⫽ 5) animals, and from NMDA-lesioned animals (12 h earlier) that were treated with an ip injection of the NMDA antagonist CPP (n ⫽ 3) or an ip injection of PBS (n ⫽ 3). Ninety-six-well microtiter plates were coated with 4 ␮g/ml (50 ␮l/well) of affinity-purified anti-MCP-1 Ab diluted in 0.1 M Na 2HPO 4 (pH 9.0; 24 h at 4°C). Plates were washed (PBS/0.05% Tween 20; 200 ␮l/well) and incubated in blocking buffer [PBS/0.05% Tween-20/1% BSA (200 ␮l/well; 2 h, room temperature)]. Plates were washed twice and samples were loaded in triplicate (100 ␮l/well; 24 h, 4°C). Recombinant rat MCP-1 diluted in blocking buffer was used to generate a standard curve. After three washes, plates were incubated with 1 ␮g/ml biotinylated anti-MCP-1 Ab (100 ␮l/well; 30 min, room temperature) and then were washed and incubated with HRP-conjugated neutralite–avidin diluted 1:4000 (100 ␮l/well; 30 min, room temperature). Chromogenic detection was performed using ABTS. An automated plate reader was used to measure optical density at 405 nm and MCP-1 content was derived from a standard curve. This assay consistently detected concentrations of recombinant MCP-1 as low as 10 pg/ml. The rectilinear relation between log concentration and log OD extended up to 100 ng/ml. MCP-1 tissue content was expressed as pg MCP1/mg protein. Left and right hemisphere values were compared using ANOVA; Fisher’s LSD post hoc tests were performed to assess differences from normal values. Preliminary experiments were also performed in pooled hippocampal samples, and concentrations were in the same range as in the cerebral hemisphere extracts (data not shown). Western blotting. A Western blot assay was developed to detect CCR2 in brain tissue extracts. Goat anti-human CCR2 Ab directed against a sequence of the human CCR2B protein known to be conserved in rat CCR2 was used; according to the manufacturer’s specifications, this Ab does not cross-react with other known gene encoded CC chemokine receptors. Preliminary immunocytochemistry data (see below) suggested that CCR2 was expressed in the lesioned dorsal hippocampus and adjacent corpus callosum; thus, an approach for tissue dissection was devised to obtain tissue samples that incorporated both regions. Brains were rapidly removed and microdissected on ice; coro-

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nal cuts were made at the levels of the optic chiasm anteriorly and just anterior to the pons, posteriorly, and only tissue between these landmarks was retained; using the ventral tip of the lateral ventricles as a landmark, an axial cut was made to remove a substantial fraction of thalamus bilaterally; and then left and right hemispheres were separated. Tissue extracts were prepared from animals that had received right intrahippocampal injections of NMDA or PBS 24 h earlier and from unlesioned P8 animals. Adult rat spleen extracts (a rich tissue source of chemokine receptors) were prepared for use as positive controls. Samples were homogenized in 500 ␮l buffer (PBS/0.1% Nonidet P-40/0.1% SDS/0.5% deoxycholic acid) containing aprotinin (5.7 ␮g/ml) and sodium orthovanadate (1 mM); PMSF (100 ␮g/ml) was added after homogenization. Samples were centrifuged (15,000g, 30 min) and supernatants were recentrifuged (20,000g, 15 min), collected and stored at ⫺70°C. Sample protein content was measured using a BCA protein assay kit. Equal amounts of protein (10 ␮g) were resolved by SDS–10% polyacrylamide gel electrophoresis. Protein was electrotransferred to nitrocellulose in Tris– glycine buffer containing 20% methanol. Membranes were blocked overnight at 4°C in TBS containing 5% gelatin and 0.2% Tween-20, and then incubated with CCR2 Ab (1:5,000 in TBS/3% gelatin for 1 h at room temperature). Membranes were washed (TBS, 0.05% Tween20), incubated with HRP-conjugated rabbit anti-goat IgG (1:40,000 in 3% gelatin in TBS), and signal developed using enhanced luminol according to manufacturer’s directions. Immunofluorescence. Animals (euthanized with pentobarbital) were perfused transcardially with 10 ml of PBS, followed by 10 ml of 4% paraformaldehyde in PBS. Brains were removed and cryoprotected by sequential incubation in solutions containing increasing sucrose concentrations (5, 7.5, 10, 12.5, 15, and 20% in PBS, for at least 1 h at 4°C, or until the brains sank) (25). Brains were frozen and stored at ⫺70°C. Coronal brain sections (30 ␮m) were collected from animals that received right intra-hippocampal injections of 10 nmol NMDA 16 (n ⫽ 6) and 32 (n ⫽ 6) h earlier. ED-1 Ab was used to identify cells of the microglia/monocyte lineage (26) and NeuNAb, a neuronspecific marker, was used to identify hippocampal pyramidal cells. Sections were blocked in normal donkey and rabbit serum and incubated with either CCR2 and ED-1 primary Ab’s (CCR2, 1:25 dilution; ED-1, 1:50) or with CCR2 and NeuN primary Ab’s (CCR2, 1:25; NeuN, 1:50) in PBS/0.3% Triton X-100 for 72 h at 4°C. Sections were washed in PBS and then incubated for 2 h in a mixture of Alexa 568 rabbit anti-mouse Ab (1:200) and Alexa 488 donkey anti-goat IgG (1:200). Equal amounts of isotype-matched IgG were substituted for the primary Ab in control samples. Sections

FIG. 1. NMDA-induced hippocampal injury in neonatal rat brain. 20-␮m coronal frozen brain sections were prepared from animals that had received a right intrahippocampal injection of 10 nmol NMDA 8 or 24 h earlier; sections were stained with cresyl violet to evaluate tissue integrity. (A and C) The intact contralateral hippocampus at 8 and 24 h after lesioning, respectively. At 8 h after lesioning, no loss of Nissl staining is apparent in the right hippocampus (B, arrow). At 24 after lesioning, in the right hippocampus there is diffuse loss of Nissl staining in the pyramidal cell layer, which is maximal in the cornu ammon (CA)3 subfield (D, arrow). (CC, corpus callosum; DG, dentate gyrus) (bar, 0.5 mm).

were washed in PBS, briefly air-dried, and coverslipped with ProLong anti-fade mounting medium. Fluorescent sections were visualized on a Nikon Diaphot 200 microscope equipped with a Noran confocal laser scanning imaging system and Silicon Graphics Indy workstation. Images were processed using Photoshop 5.0 (Adobe Systems, Mountain View, CA). RESULTS

In P7 rats, intrahippocampal injection of 10 nmol NMDA elicits reproducible focal excitotoxic injury; the histopathological features of injury evolve predictably in the first 24 h after lesioning. In the lesioned hippocampus, at 8 h there are subtle histopathological changes in the CA3 subfield, but there is no loss of Nissl staining (Fig. 1B). By 24 h after lesioning, evidence of neuronal injury, e.g., loss of Nissl staining, is detectable in the lesioned hippocampus and is most pronounced within the CA3 region of the pyramidal cell layer (Fig. 1D); the contralateral hippocampus is intact (Fig. 1C). NMDA Differentially Regulates MCP-1 and CCR2 mRNA Expression NMDA-induced changes in MCP-1 and CCR2 mRNA expression were compared in aliquots prepared from the same batches of reverse-transcribed RNA. No MCP-1 mRNA was detected in normal hippocampus. A substantial increase in MCP-1 mRNA was seen as early as 2 h in lesioned hippocampus, and expression peaked at 8 h, when there was a greater than threefold increase in MCP-1 mRNA compared to the left hip-

NMDA INDUCES MCP-1 AND CCR2 EXPRESSION

pocampus (Fig. 2A). MCP-1 mRNA declined to baseline levels at 24 h. MCP-1 mRNA expression also increased in the contralateral hippocampus, at 4 –16 h after NMDA lesioning. No MCP-1 mRNA was detected in control samples from animals that had received an intrahippocampal PBS injection 8 h earlier. CCR2 mRNA was detected in normal P7 hippocampus. In samples from the lesioned right hippocampus, a subtle increase in CCR2 expression was detected at 4 h after NMDA lesioning, and expression peaked at 16 h; CCR2 mRNA expression remained elevated, relative to both contralateral hippocampus and normal tissue up to 72 h after NMDA lesioning (Fig. 2B). Based on densitometric estimates, at 16 h there was a greater than eleven-fold increase in CCR2 mRNA compared to normal hippocampus, and expression remained elevated at 72 h. Smaller increases in CCR2 mRNA were also detected in the contralateral hippocampus 4 –16 h after lesioning. To evaluate temporal trends, densitometric estimates of the right hippocampus for MCP-1 and CCR2 mRNA signal intensity were expressed as percentage of peak signal (Fig. 2C). The patterns of MCP-1 and CCR2 mRNA expression in the NMDA-lesioned hippocampus were distinct. MCP-1 expression peaked (at 8 h) and declined (back to baseline at 24 h) much more rapidly than did CCR2 mRNA (peak at 16 h); increased CCR2 expression was sustained for up to 72 h after lesioning (Fig. 2C). NMDA Stimulates MCP-1 and CCR2 Protein Expression Total protein content in the lesioned hemisphere declined significantly (16 –29%, P ⬍ 0.02, compared to normal values) (Table 2) at 8, 12, 16, and 48 h after injection of NMDA. In samples obtained at 24 h after lesioning, protein content did not change; the explanation for the equivalence of protein content in the samples obtained at 24 h is uncertain. Reductions in total protein could result from injury-induced protease activity and/or suppression of protein synthesis. In Fig. 3, MCP-1 values are expressed as pg MCP-1/mg protein, and statistical analysis was performed, using these values. Since injury also influenced the total protein content of right hemisphere samples, we replicated the analysis using values for total MCP-1/cerebral hemisphere, and trends were similar (data not shown). MCP-1 was detected in all samples (Fig. 3A). Significant increases in MCP-1 were detected in the lesioned hemisphere 4 – 48 h (P ⬍ 0.03) after injection of NMDA compared to normal values. MCP-1 peaked at 8 –12 h after NMDA lesioning when values increased more than 30-fold, compared with normal P8 animals (P ⬍ 0.0001). Increases persisted in the lesioned hemisphere up to 48 h and declined to baseline levels at 96 h. MCP-1 content also rose contralaterally, at

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8 –24 h after lesioning (P ⬍ 0.003). Preliminary MCP-1 immunocytochemistry assays (27) indicated that at 8 h after NMDA injection, MCP-1-immunoreactive cells in the lesioned hippocampus included ependymal cells, injured neurons, and glia (data not shown). Treatment with the competitive NMDA antagonist CPP (15 min after lesioning) blocked NMDA-stimulated MCP-1 expression in tissue samples evaluated at 12 h later (Fig. 3B); comparison of MCP-1 content in concurrently lesioned animals that received ip injections of either CPP or PBS demonstrated a marked increase in MCP-1 ipsilaterally in the saline-treated group (P ⬍ 0.0001, compared with normal values), and concentrations did not differ from normal controls compared to the CPP-treated group. NMDA Stimulates MCP-1 Receptor (CCR2) Expression Preliminary immunocytochemistry experiments (see below) localized CCR2 protein expression to the ipsilateral corpus callosum, a region densely populated by physiologically activated microglia and to cells, with the morphology of activated microglia/monocytes, in the injured hippocampus (28). Using a Western blot assay, we confirmed that the anti-CCR2 Ab detected a single band of the appropriate size (42 kDa) in all samples tested (Fig. 4). CCR2 content was higher in extracts obtained from the lesioned hemisphere (at 24 h after the NMDA injection) than in samples from normal P8 and PBS-injected animals. To determine the identity of cells that expressed CCR2, immunocytochemistry assays were performed with fluorescence-labeled secondary antibodies, and sections were imaged with confocal microscopy. In normal P7 rat brain, physiologically activated microglia (ED-1 immunoreactive) are concentrated in the corpus callosum (26, 29). In a tissue sample from an animal evaluated at 16 h after NMDA, ED-1-immunoreactive activated microglia were identified within the corpus callosum bilaterally (Figs. 5A and 5B). Of note, their distributions differed; in the corpus callosum adjacent to the lesioned hippocampus, ED-1-immunoreactive cells were more widely dispersed (Fig 5B) than in the corresponding contralateral region (Fig. 5A). At 16 h after lesioning, no CCR2-immunoreactive cells were detected in the contralateral corpus callosum (Fig. 5C); in the ipsilateral corpus callosum, there were many CCR2-immunoreactive cells (Fig. 5D). CCR2 and ED-1 immunoreactivity were consistently colocalized in the corpus callosum (Figs. 5E–5G). At 24 – 48 h after NMDA lesioning, there is a prominent monocyte/microglial infiltrate into the pyramidal cell layer of the hippocampus (26). ED-1 immunostaining readily identified these cells within the pyramidal cell layer CA3 subfield at 32 h after injection of NMDA (Fig. 5H).

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FIG. 2. NMDA-induced changes in MCP-1 and CCR2 gene expression. RT-PCR assays were performed to estimate changes in tissue content of MCP-1 and CCR2 mRNA, relative to GAPDH mRNA (see Materials and Methods). Samples from left (L) and right (R) hippocampus of normal P8 animals and from animals that received right intrahippocampal NMDA (10 nmol) injections (evaluated at 2, 4, 8, 16, 24, 48, and 72 h postinjection) were assayed concurrently; PBS-injected controls were included (obtained at 8 and 16 h postinjection for MCP-1 and CCR2, respectively, to coincide with the corresponding peaks of NMDA-induced mRNA expression, based on preliminary experiments). PCR products were electrophoresed through 2% ethidium bromide-stained agarose gels and scanned fluorometrically. In A and B, the histograms provide quantitative estimates of MCP-1 and CCR2 mRNA, respectively, based on normalization to GAPDH mRNA/sample (counts represent arbitrary optical density units). C compares the temporal patterns of NMDA-induced changes in MCP-1 and CCR2 mRNA in the lesioned hippocampus; values are expressed as percentages of peak expression. (A) In the NMDA-injected right hippocampus, increased MCP-1 expression was apparent at 2 h; a 3-fold increase in MCP-1 was detected in the lesioned hippocampus at 8 h, compared to the contralateral hippocampus. Contralateral increases, compared with normal tissue samples, were also detected 2–16 h after injection of NMDA. (B) CCR2 mRNA was detected in all samples. In the NMDA-injected right hippocampus, an increase in CCR2 mRNA was detected at 4 h; CCR2 gene expression peaked at 16 h (⬎11-fold increase compared to normal hippocampus). Smaller increases were detected in the contralateral hippocampus. (C) In the lesioned hippocampus, MCP-1 mRNA expression peaked at 8 h and returned to baseline levels at 24 h. CCR2 mRNA expression peaked later at 16 h and increased expression was sustained up until 72 h.

NMDA INDUCES MCP-1 AND CCR2 EXPRESSION

TABLE 2 NMDA Lesioning Reduces Tissue Protein Content Acutely Time

N

Left hemisphere

Right hemisphere

Normal 2h 8h 12 h 24 h 48 h

5 3 5 4 3 3

10.0 (⫾0.4) 9.7 (⫾0.6) 9.1 (⫾0.4) 10.2 (⫾0.5) 9.8 (⫾0.6) 10.3 (⫾0.6)

10.5 (⫾0.4) 8.8 (⫾0.6)* 7.5 (⫾0.4)** 8.7 (⫾0.5)** 9.8 (⫾0.6) 7.8 (⫾0.6)**

Note. Total protein was measured in tissue homogenates prepared from the left and right cerebral hemispheres of normal P8 animals and animals that received right intrahippocampal injections of 10 nmol NMDA (2, 8, 12, 24, or 48 h earlier) (see Materials and Methods), using a BCA protein assay kit. Values are expressed in mg protein (⫾SEM). Left and right hemisphere values were compared using ANOVA and Fisher’s LSD post hoc tests were performed to assess differences from normal values. ** P ⬍ 0.01. * P ⬍ 0.02.

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pression acutely, both in the hippocampal pyramidal cell layer and in adjacent injured structures, particularly in the adjacent ependyma (15, 27); these results suggest that, at this developmental stage, injured neurons and ependymal cells are important sources of MCP-1. Reactive astrocytes (11) and microglia are also potential sources of chemokine production in the acutely injured brain. The molecular mechanisms that regulate MCP-1 production in vivo are incompletely understood. Studies in experimental lung injury models indicate that MCP-1 expression can be upregulated by proinflammatory cytokines [IL-1␤ and tumor necro-

Many of these infiltrating cells expressed CCR2 (Figs. 5H–5J). NeuN immunocytochemistry identified pyramidal neurons in the injured hippocampus; however, there was no colocalization of CCR2 and NeuN immunoreactivity (data not shown). Control assays in which goat and mouse IgG were substituted for the primary antibodies showed no immunofluorescence (data not shown). DISCUSSION

Our data demonstrate that in neonatal rat brain excitotoxic injury rapidly induces a marked increase in MCP-1 accumulation and a temporally distinct increase in expression of the MCP-1 receptor, CCR2; CCR2 expression is localized to activated microglia in the corpus callosum adjacent to the injured hippocampus and also to ED-1-immunoreactive cells (microglia and/or monocytes) that infiltrate the injured pyramidal cell layer. Together, these findings provide strong support for the hypothesis that chemokines and their receptors are important mediators of the inflammatory response initiated by acute excitotoxic hippocampal injury. Our findings of injury-induced MCP-1 expression are congruent with results of several recent studies in adult and neonatal rodent brain. In the adult rodent brain, increased MCP-1 expression has been reported in models of temporary and permanent cerebral ischemia (8, 30), in EAE (9 –11), and in human and experimental brain tumors (13, 14). Compelling evidence that MCP-1 could contribute to CNS pathology was provided by the finding that treatment with antiMCP-1 neutralizing antibodies decreased the severity of clinical symptoms in a rodent EAE model (31). In P7 rats, intrahippocampal NMDA injection and hypoxic–ischemic injury induced MCP-1 mRNA ex-

FIG. 3. Quantification of MCP-1 protein content in brain by ELISA. P7 animals received right intrahippocampal injections of 10 nmol NMDA, and tissue extracts were prepared from the left and right cerebral hemispheres at 2, 4, 8, 12, 16, 24, 48, and 96 h postinjection. Control samples were collected from normal P8 and intrahippocampal PBS-injected (8 h postinjection) animals. Samples were also obtained (at 12 h postlesioning) from animals that had received intrahippocampal injections of NMDA, followed by ip injections of PBS or the competitive NMDA antagonist, CPP. Values, expressed as pg MCP-1/mg protein, are means of ⱖ3 samples, except at 96 h post-NMDA injection (n ⫽ 2) (see Materials and Methods); error bars represent SEM. (A) MCP-1 was detected in all samples. At 8 –12 h, there was a ⬎30-fold increase compared to normal P8 tissue; increases in MCP-1 content were detected in all lesioned hemisphere samples at 4 – 48 h, compared to values in normal P8 brain (P ⬍ 0.03; ANOVA). Contralaterally, at 8 –24 h, there were corresponding MCP-1 increases of lesser magnitude, compared to normal P8 brain (P ⬍ 0.003; ANOVA). No changes were detected in PBS-injected animals. (B) In PBS-treated controls, evaluated at 12 h after right intrahippocampal NMDA injections, there was a ⬎30-fold increase in MCP-1 ipsilaterally, compared to normal P7 brain; there was a ⬎8-fold increase in the MCP-1 content of the contralateral hemisphere. In NMDA-lesioned animals that received CPP, MCP-1 content did not differ from normal P8 rat brain.

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FIG. 4. Western blot analysis of CCR2 in neonatal rat brain. Tissue extracts were prepared from the left (L) and right (R) cerebral hemispheres (see Materials and Methods) of normal P8 animals [lanes 2 (L) and 3 (R)] and from animals that received right intrahippocampal injections of PBS [lanes 4 (L) and 5 (R)] or 10 nmol NMDA [lanes 6 (L) and 7 (R)] 24 h earlier. Adult rat spleen (lane 1) was included as a positive control. Equal amounts of protein (10 ␮g) were resolved by SDS–10% polyacrylamide gel electrophoresis. A single immunoreactive band of the expected size (approximately 42 kDa) was detected in all samples, and the signal was stronger in the sample from NMDA-lesioned tissue (lane 7), than in the other brain samples.

sis factor (TNF)-␣] (32). In P7 rats, NMDA rapidly stimulates IL-1␤ and TNF-␣ (23, 24), and these cytokines could be pathophysiologically significant regulators of MCP-1 expression in the injured brain. In this study, we focused on quantitative analysis of NMDA-induced MCP-1 mRNA and protein production. Although mechanical trauma (saline injection) elicited a minimal increase in MCP-1 expression [as was reported previously (10, 12)], the magnitude of stimulation of MCP-1 elicited by NMDA was substantially greater. Evidence that stimulation of MCP-1 expression was directly linked with NMDA receptor activation was provided by results of experiments in which the competitive antagonist CPP blocked NMDA-stimulated increases in MCP-1 production. Clinical and experimental data implicate MCP-1 in the pathogenesis of diverse CNS disorders. Increased MCP-1 expression has been reported in primary brain tumors (13) and in the cerebrospinal fluid and brains of individuals with AIDS-associated dementia (33, 34). In the context of clinical disorders of the developing nervous system, an intriguing link between MCP-1 and CNS pathology was provided by results of a recent epidemiological study in which it was found that in neonates who subsequently developed spastic diplegia, there were substantial increases in MCP-1 (along with other proinflammatory cytokines) in neonatal blood samples (35). With respect to the role of CCR2 and regulation of its expression in human neuropathology much less is known. Of interest, a recent report identified CCR2 expression in neuritic plaques of Alzheimer’s brains (36). The corpus callosum of the immature rat brain is densely populated with physiologically activated microglia (26); these microglia do not express CCR2. Acute excitotoxic hippocampal injury elicits a robust microglia/monocyte response characterized by activation of intrinsic microglia and recruitment of microglia

into injured areas (most evident in the CA3 pyramidal cell layer) (26, 29); this response peaks at 24 – 48 h after lesioning (26, 37). The timing of NMDA-induced MCP-1 expression and microglial activation/recruitment suggest that MCP-1 could be one of the molecular signals regulating this response. At 16 h after lesioning, CCR2-immunoreactive microglia were detected only in the corpus callosum ipsilateral to the injured hippocampus; the regional distribution of these cells had changed as well. They were more widely dispersed than in the corresponding contralateral region (a finding consistent with the hypothesis that these cells were being mobilized/recruited away). At 32 h after NMDA lesioning, ED-1/CCR2-immunoreactive cells infiltrated the injured hippocampal pyramidal cell layer; since fully activated microglia and monocytes are not distinguishable (38) we could not definitively establish the identity of these cells and it is certainly possible that both cell types are recruited in response to MCP-1. Several studies have reported constitutive and/or disease-related increases in expression of chemokine receptors in neurons and microglia in both human and animal brains (14, 29, 39 – 45). Constitutive expression of CCR2 mRNA has been reported in cultured rat microglia; upregulation of CCR2 mRNA expression was detected in an adult rat EAE model (20, 46). We recently reported that in an adult rodent brain tumor model (14), in which there is an intense microglial/ monocyte response to tumor implantation, CCR2 mRNA and protein expression are markedly upregulated within and around the brain tumors (both in infiltrating microglia/monocytes and in tumor cells) (14). The results of the present study provide the first evidence that brain injury upregulates expression of CCR2 protein acutely and also identified CCR2-immunoreactive cells as activated microglia/monocytes. Of note, no CCR2 immunoreactivity was detected in physiologically activated microglia in the contralateral corpus callosum or in unlesioned neonatal brain. The finding that only “pathologically” activated microglia, adjacent to the injured hippocampus, expressed CCR2 suggests that physiological and pathological activation of microglia involve independent and distinct regulatory mechanisms. Under the conditions of the immunocytochemistry assay of CCR2, no reactive cells were detected in normal neonatal brain or in the contralateral hippocampus of lesioned animals; yet, Western blot immunoassays of tissue homogenates indicated that CCR2 is also expressed (by as of yet unidentified cells) in normal neonatal brain. Of note, CCR2 knockout mice develop normally, but show deficiencies in peritoneal macrophage recruitment (47– 49) and are also highly susceptible to pathogenic infection. Whether these CCR2 knockout mice will have altered responses to brain injury is an intriguing question for future research.

FIG. 5. Immunofluorescence labeling to colocalize CCR2 and ED-1-immunoreactive cells. Coronal brain sections, prepared from animals that had received a right intrahippocampal injections of 10 nmol NMDA 16 h (A–G) or 32 h (H–J) earlier were double labeled to detect CCR2 and ED-1. Confocal microscopy was used to visualize the distributions of fluorescent-tagged secondary Ab’s (see Materials and Methods); an Alexa 488 donkey anti-goat secondary Ab was used to detect CCR2 Ab and an Alexa 568 rabbit anti-mouse secondary Ab was used to detect ED-1. A–G represent images of the left and right corpus callosum and H–J present images of the lesioned hippocampus. The distributions of ED-1- and CCR2-immunoreactive cells in the left (contralateral to the lesion) corpus callosum are illustrated in A and C, respectively; B and D show the distributions of ED-1- and CCR2-immunoreactive cells in the right corpus callosum. At 16 h after NMDA lesioning, ED-1-immunoreactive cells were detected in the corpus callosum bilaterally (A and B) but their distributions differed; ipsilaterally, these cells were more widely dispersed (B); CCR2-immunoreactive cells were evident only in the ipsilateral corpus callosum (D). E and F provide higher magnifications of ED-1- and CCR2-immunoreactive cells in the ipsilateral corpus callosum, and G is the composite showing colocalization of ED-1- and CCR2-immunoreactive cells. At 32 h after NMDA lesioning, ED-1- (H) and CCR2- (I) immunoreactive cells were identified in the lesioned hippocampus; J illustrates that CCR2 is colocalized to ED-1-immunoreactive cells infiltrating the CA3 pyramidal cell layer of the lesioned hippocampus. Bars, 100 ␮m (A–D) and 20 ␮m (E–J). 303

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Currently, there is no information about in vivo regulation of chemokine receptors in the CNS. The differences in the temporal patterns of MCP-1 and CCR2 expression suggest that distinct regulatory mechanisms are involved. MCP-1 expression preceded increases in CCR2 expression; whether MCP-1 itself could by a stimulus for CCR2 expression is unknown. In vitro studies indicate that inflammatory mediators downregulate CCR2 expression in monocytes (50, 51). We recently evaluated the impact of acute excitotoxic injury on expression of a closely related chemokine receptor, CCR5, in the same experimental model. In comparison with NMDA-stimulated CCR2 mRNA expression, peak CCR5 mRNA expression occurred later, at 24 – 48 h after lesioning and was less pronounced (twofold increase) (29). In contrast with CCR2 (which was not colocalized with NeuN, a neuron-specific marker), CCR5 was expressed by injured hippocampal neurons. These trends suggest that distinct signaling mechanisms regulate injury-induced chemokine receptor expression. In conclusion, our data demonstrate that acute excitotoxic injury induces MCP-1 and CCR2 expression in the neonatal rat brain. CCR2 was colocalized to infiltrating activated microglia, suggesting that MCP-1 may be an important regulator of the inflammatory response to acute brain injury.

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ACKNOWLEDGMENTS We thank Thomas Komorowski and the Michigan Diabetes Research and Training Center for assistance and training with confocal microscopy.

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