Monocyte chemoattractant protein-1 is a mediator of acute excitotoxic injury in neonatal rat brain.

Monocyte chemoattractant protein-1 is a chemokine with potent monocyte activating and chemotactic effects. Monocyte chemoattractant protein-1 gene and protein expression is rapidly up-regulated in response to a variety of acute and chronic central nervous system disorders. The activation and recruitment of microglia and monocytes into areas of inflammation may play a critical role in the pathogenesis of acute brain injury. Monocyte chemoattractant protein-1 could be a pathophysiologically important mediator of the microglial and monocyte responses in the brain. Using a well-characterized model of acute excitotoxic brain injury in neonatal rats, experiments were designed to evaluate whether monocyte chemoattractant protein-1 plays a role in the progression of tissue damage. Direct co-administration of recombinant monocyte chemoattractant protein-1 with the excitotoxin N-methyl-D-aspartate exacerbated injury, both in the striatum and in the hippocampus, by 55% and 167%, respectively. Complementary experiments to determine the effect of functional inhibition of monocyte chemoattractant protein-1, using an anti-monocyte chemoattractant protein-1-neutralizing antibody, revealed that co-administration of the antibody with N-methyl-D-aspartate attenuated tissue injury in the striatum and hippocampus by 57% and 39%, respectively.Together, these data suggest that monocyte chemoattractant protein-1 is a mediator of acute excitotoxic brain injury in neonatal rats and that inflammatory mechanisms contribute significantly to the pathogenesis of acute neonatal brain injury. Whether chemokines are pathophysiologically relevant mediators of neuronal injury in human neonates remains to be determined.

Acute excitotoxic injury induces expression of monocyte chemoattractant protein-1 and its receptor, CCR2, in neonatal rat brain.

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.

Inhibition of TNF-alpha can attenuate or exacerbate excitotoxic injury in neonatal rat brain.

Tumor necrosis factor (TNF)-alpha, a multifunctional pro-inflammatory cytokine, has been implicated in the pathogenesis of acute ischemic brain injury. Recent data also suggest that TNF-alpha is a clinically relevant mediator of neonatal brain injury. We hypothesized that inhibition of TNF-alpha activity would reduce excitotoxic brain injury in neonatal rats. To test this hypothesis, we evaluated the efficacy of a TNF binding protein (bp) in attenuating NMDA-induced injury in 7 day old rats. Intrastriatal co-injection of TNFbp (3.75 microg) with NMDA (10 nmol) reduced striatal injury by 26%; in contrast, intra-hippocampal co-injection of TNFbp (3.75 microg) with NMDA (10 nmol) increased hippocampal damage by 68%. These findings indicate that TNF-alpha may have both beneficial and deleterious effects in the injured neonatal brain.

Experimental gliosarcoma induces chemokine receptor expression in rat brain.

Macrophage/microglial infiltration is a characteristic feature of brain tumors. The functional role(s) of these cells is complex and could include both trophic and suppressive effects on tumors. Information has recently emerged about the molecular signals that regulate the accumulation and function of monocytes in pathological disorders. Recent data indicate that the chemokine, monocyte chemoattractant protein-1 (MCP-1), a potent monocyte activating and chemotactic factor, is a primary regulator of the macrophage response in brain tumors. We hypothesized that if MCP-1 regulates macrophage/microglial infiltration, then expression of the specific MCP-1 receptor, CCR2, will be induced in peritumoral tissue and/or within brain tumors. Identification of a specific receptor that is preferentially expressed in brain tumors could be important both in terms of tumor biology and as a potential therapeutic target. We used an established experimental gliosarcoma model, induced by intracranial transplantation of cultured 9L cells into adult rat brain, to test this hypothesis. RT-PCR analysis showed high levels of both MCP-1 and CCR2 mRNA and Western blot analysis demonstrated increased CCR2 protein in tumor extracts. Immunocytochemistry showed CCR2 immunoreactive microglia in peritumoral tissue and, unexpectedly, that intrinsic tumor cells, rather than monocytes, were the predominant source of CCR2. These results demonstrate that CCR2 expression is markedly upregulated in this brain tumor model.

Excitotoxic brain injury stimulates expression of the chemokine receptor CCR5 in neonatal rats.

Chemokines interact with specific G-protein-coupled receptors to activate and direct recruitment of immune cells. Some chemokines are up-regulated in pathological conditions of the central nervous system, and recently several chemokine receptors, including CCR5, were identified in the brain. However, little is known about the regulation of expression of chemokine receptors in the brain. Direct intracerebral injection of N-methyl-D-aspartate (NMDA), an excitatory amino acid agonist, elicits reproducible focal excitotoxic brain injury; in neonatal rats, intrahippocampal NMDA injection stimulates expression of pro-inflammatory cytokines and elicits a robust microglia/monocyte response. We hypothesized that NMDA-induced neurotoxicity would also stimulate expression of CCR5 in the neonatal rat brain. We evaluated the impact of intrahippocampal injections of NMDA on CCR5 expression in postnatal day 7 rats. Reverse transcription polymerase chain reaction revealed an increase in hippocampal CCR5 mRNA expression 24 hours after lesioning, and in situ hybridization analysis demonstrated that CCR5 mRNA was expressed in the lesioned hippocampus and adjacent regions. Western blot analysis demonstrated increased CCR5 protein in hippocampal tissue extracts 32 hours after lesioning. Complementary immunocytochemistry studies identified both infiltrating microglia/monocytes and injured neurons as the principal CCR5-immunoreactive cells. These results provide the first evidence that acute excitotoxic injury regulates CCR5 expression in the developing rat brain.

Synergistic effect of intrastriatal co-administration of L-NAME and quinolinic acid.

Chronic dialytic intrastriatal co-administration of quinolinic acid (QUIN) and four concentrations of the nitric oxide synthase (NOS) inhibitor NG nitro-L-arginine methyl ester (L-NAME) produced variable results. Low concentrations of L-NAME (1 microM and 50 microM) co-administered with 15 mM QUIN produced lesions not significantly different from those produced by 15 mM QUIN alone. In contrast, higher concentrations of L-NAME (1 mM and 100 mM) co-administered with 15 mM QUIN produced striatal lesions significantly larger than those produced by 15 mM QUIN alone. Administered by itself, 100 mM L-NAME produced little striatal damage. These findings suggest that low levels of NOS inhibition have little or no effect on NMDA neurotoxicity in the striatum, whereas high levels of NOS inhibition increase NMDA-induced striatal lesion volume.