Monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 are involved in both excitotoxin-induced neurodegeneration and regeneration.

Intrahippocamal injections of kainic acid (KA) significantly increase the expression of monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) in the ipsilateral hippocampus at 2-4 h and 21-45 days post-administration, suggesting the possible involvement of these chemokines in both neurodegenerative and regenerative processes. To examine the possible role of these chemokines on neuronal cell death, hippocampal neurons were incubated with either MCP-1 or MIP-2 in vitro and examined to assess the effects on neuronal cell viability. These treatments resulted in significant neuronal apoptosis that could be abrogated by prior treatment with the caspase-1 inhibitor, Z-VAD-FMK, the caspase-3 inhibitor, Z-DEVD-FMK, the Galphai inhibitor, pertussis toxin, or the MAO-B inhibitor, (-)deprenyl. Furthermore, this chemokine apoptotic effect could also be observed in vivo as intrahippocampal injections of MCP-1 or MIP-2 resulted in the apoptosis of hippocampal neurons, thus supporting a direct role of these chemokines in neuronal death. In contrast, immunohistological analysis of kainic acid lesions on days 21-45 revealed significant expression of MCP-1 and MIP-2 associated with reactive astrocytes and macrophages, respectively, with no apoptotic populations being observed. These results suggested that these chemokines might also mediate distinct biological effects on local microenvironmental cell populations at various stages post truama and during cellular repair. To address this possibility, astrocyte were cultured in the presence or absence of these chemokines and examined by microarray analysis for effects on astrocytes gene expression. A number of genes encoding proteins associated with inflammation, cellular signaling, differentiation, and repair were directly modulated by chemokine treatment. More specifically, the RNA and protein expression of the neurotrophic factor, basic fibroblast growth factor (bFGF), was found to be significantly increased upon culture with MCP-1 and MIP-2. Conditioned media derived from chemokine-stimulated astrocytes also facilitated bFGF-dependent neuronal cell differentiation and promoted survival of H19-7 neurons in vitro, suggesting a possible role for chemokine-activated astrocytes as a source of trophic support. Taken together, these data support possible autocrine and paracrine roles for MCP-1 and MIP-2 in both the "death and life" of hippocampal neurons following CNS injury.

Differential transcriptional regulation by human immunodeficiency virus type 1 and gp120 in human astrocytes.

Astrocytes may be infected with the human immunodeficiency virus type 1 (HIV-1) or exposed to the HIV protein gp120, yet their role in the pathogenesis of HIV dementia is largely unknown. To characterize the effects of HIV on astrocytic transcription, microarray analysis and ribonuclease protection assays (RPA) were performed. Infection of astrocytes by HIV or treatment with gp120 had differential and profound effects on gene transcription. Of the 1153 oligonucleotides on the immune-based array, the expression of 108 genes (53 up; 55 down) and 82 genes (32 up; 50 down) were significantly modulated by gp120 and HIV infection respectively. Of the 1153 oligonucleotides on the neuro-based array, 58 genes (25 up; 33 down) and 47 genes (17 up; 30 down) were significantly modulated by gp120 and HIV infection respectively. Chemokine and cytokine induction occurred predominantly by HIV infection, whereas gp120 had no significant effect. These results were confirmed by RPA. The authors conclude that profound alterations of astrocytic function occur in response to HIV infection or interaction with viral proteins, suggesting that astrocytes may play an important role in the pathogenesis of HIV dementia.

Aged mice exhibit greater mortality concomitant to increased brain and plasma TNF-alpha levels following intracerebroventricular injection of lipopolysaccharide.

BACKGROUND: Age-related defects in the development of peripheral inflammatory responses have been observed in rodents and humans. OBJECTIVE: We examined the effects of age on a centrally injected endotoxin-induced cytokine production and cellular activation in mice. METHODS: Male C57BL/6J (B6) mice, C3H/HeN mice, and C3H/HeJ mice received an intracerebroventricular injection of lipopolysaccharide (LPS) and were sacrificed at various times (2, 4, 8 h) thereafter. ELISA for IL-1beta, IL-6, IL-12, and TNF-alpha were conducted on forebrain tissue homogenates as well as plasma samples, and lectin staining to detect activated microglia was prepared for selected brain slices. RESULTS: Intracerebroventricular injection of LPS in B6 mice produced an age-associated increase in mortality which was paralleled with a significant increase in brain and plasma levels of TNF-alpha. AntiTNF-alpha- and IL-6-immunoreactive cells possessed macrophagelike morphologies and were observed along the LPS injection tract and scattered throughout the hilus of the dorsal hippocampus and cerebral cortices. This LPS-mediated response was found to be specific in that the LPS-hyporesponsive mouse strain (C3H/HeJ) failed to demonstrate significant brain or plasma levels of TNF-alpha after LPS administration compared to C3H/HeN mice. CONCLUSION: These results suggest that the age-related increases in TNF-alpha production and mortality following the intracerebroventricular administration of LPS may be due to an increased endotoxin hypersensitivity of brain microglia/macrophages within aged animals.

Stereological estimation of total microglia number in mouse hippocampus.

Microglia are brain cells of considerable interest because of their role in CNS inflammatory responses and strong association with neuritic plaques in Alzheimer's disease (AD). In the present study, immunocytochemistry was combined with unbiased stereology to estimate the mean total number of microglia in dentate gyrus and CA1 regions of the mouse hippocampus. Systematic-uniform-random (SUR) sections were cut through the hippocampal formation of male C57BL/6J mice (n = 7, 4-5 months) and immunostained with Mac-1, an antibody to the complement subunit 3 receptor (CR3). The total number of Mac-1 immunopositive cells was determined using the optical fractionator method. The mean total number of microglia in the mouse dentate gyrus was estimated to be 20,300 (CV = 0.21) with a mean coefficient of error (CE) = 0.09. The mean total number of microglia in the mouse CA1 was estimated to be 43,200 (CV = 0.24) with a CE = 0.11. Comparison of total number estimates, derived from fraction- or volume-based methods, supported stereological theory regarding the equivalence of the two techniques. The time required to determine total microglia number in both hippocampal sub-regions was approximately 6 h per mouse from stained sections. The combination of immunocytochemistry and stereology provides a reliable means to assess microglia number that can easily be adopted for studies of transgenic and lesion-based models of aging and neurodegenerative diseases.

Stereological analysis of astrocyte and microglia in aging mouse hippocampus.

Recent evidence suggests neuroglia-mediated inflammatory mechanisms may stimulate neurodegenerative processes in mammalian brain during aging. To test the hypothesis that the number of microglia and astrocytes increase in the hippocampus during normal aging, unbiased stereological techniques were used to estimate total cell number in hippocampal subregions (CA1, dentate gyrus and hilus) of male C57BL/6J mice of different ages: 4-5 months, 13-14 months and 27-28 months. Immunocytochemical visualization for microglia and astrocytes were via Mac-1 and GFAP antibody, respectively. Estimates of total microglia and astrocyte number were assessed using the optical fractionator. No statistically significant age differences were found in the numbers of microglia or astrocytes in the hippocampal regions sampled. These findings suggest that age-related increases in the total numbers of hippocampal microglia and astrocytes is not causal for observed age-related increases in cytokine response.

Loss of serotonin uptake sites and immunoreactivity in rat cortex after dexfenfluramine occur without parallel glial cell reactions.

The frontal cortices of rats which received either D,L- or D-fenfluramine (DFEN) for 4 days were examined 18 h to 2 weeks following treatment for changes in synaptosomal uptake of serotonin (5HT), paroxetine binding, 5HT-immunoreactivity (5HT-IR), and both astrocytic (GFAP) and microglial markers. Additional rats received intracerebroventricular injections of the neurotoxin 5,7-dihydroxytryptamine (DHT). Consistent with previous reports, D,L- and DFEN produced dose-dependent losses of both 5HT uptake and paroxetine binding, and loss of 5HT-IR which coincided with an abnormal or 'swollen' appearance of immunoreactive axon processes. Recovery of these serotonergic indices was greatest following the lowest doses of DFEN, but was absent after 5,7-DHT treatment. No evidence for an increase in GFAP synthesis or microglial activation was observed in frontal cortices of rats treated with either DFEN or 5,7-DHT. We conclude that the presence of swollen 5HT-IR axons in the cortices of both the 5,7-DHT and DFEN groups is insufficient to trigger the glial responses often associated with neuronal degeneration. Thus, it remains to be determined if swollen 5HT-IR axons are a prelude to neurodegeneration, or whether they represent reversible changes in axonal immunochemistry associated with decreases in 5HT levels. The implications of the data for the clinical safety of DFEN are briefly discussed.

Characterization of microglial reaction after middle cerebral artery occlusion in rat brain.

We have studied the microglial reaction that accompanies cortical infarction induced by middle cerebral artery occlusion (MCAO). Lectin histochemistry with the B4-isolectin from Griffonia simplicifolia as well as immunocytochemistry with a panel of monoclonal antibodies directed against major histocompatibility complex (MHC) and lymphocytic antigens were performed. Principal attention was focused on neocortical and thalamic regions, representative of primary and secondary ischemic damage, respectively. With the lectin procedure, activated microglial cells were abundant in the neocortex 24 hours after MCAO. In contrast, microglial activation in the thalamus was not apparent until day 2 after MCAO. On day 5, MHC class II antigen was expressed by reactive microglia in fiber tracts traversing the striatum, but was absent from activated microglia in the primary cortical infarction area. MHC class I and lymphocytic antigens were expressed differentially on microglia with class I antigens appearing early and lymphocytic antigens appearing late in the time course after focal ischemia. The findings are compatible with previous studies during global ischemia and confirm the early activation and the progressive nature of immunomolecule expression on activated microglia after an ischemic insult. In addition to neocortical and thalamic sites, our results showed an early microglial activation to be present also in forebrain regions outside of the middle cerebral artery (MCA) territory, such as the contralateral cortex and hippocampus. A unilateral microglial reaction was also detectable after long-term survival (> or = 4 weeks) in the pyramidal tracts, as well as in the corticospinal tracts at cervical but not lumbar spinal cord levels. Ischemia-induced neuronal damage, as evaluated by Nissl staining, was found only in cortical and thalamic regions. We conclude that the demonstration of reactive microglia indicates not only imminent ischemic neuronal damage within MCA territory but can also delineate extra-focal disturbances, possibly reflecting subtle and transitory changes in neuronal activity.

MK-801 prevents microglial reaction in rat hippocampus after forebrain ischemia.

Delayed neuronal death induced by transient forebrain ischemia in the rat hippocampus is preceded by a prominent microglial reaction which begins within minutes after the ischemic injury. In the present study we have examined the effect of the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 on microglial activation and neuronal survival. Using lectin histochemistry to detect microglia, we show that the systemic administration of MK-801 prior to ischemia prevents microglial activation, as well as delayed death of CA1 pyramidal neurons. The results demonstrate that early blockage of the glutamate cascade prevents microglial activation, and could suggest a role for microglia in mediating ischemic injury.

Progressive expression of immunomolecules on microglial cells in rat dorsal hippocampus following transient forebrain ischemia.

We show a differential up-regulation of immunomolecules in the rat dorsal hippocampus accompanying neuronal cell death as a consequence of transient forebrain ischemia (four-vessel occlusion model). Using a panel of monoclonal antibodies (mAbs), we have examined the time course of expression of major histocompatibility complex (MHC) antigens class I (OX-18) and class II (OX-6), leukocyte common antigen (OX-1), CD4 (W3/25) and CD8 (OX-8) antigens, CR3 complement receptor (OX-42), as well as brain macrophage antigen (ED2). The study was performed at time intervals ranging from 1 to 28 days after reperfusion. Throughout all post-ischemic time periods, strongly enhanced immunoreactivity on microglial cells in the CA1 region and dentate hilus and, to a lesser extent, in CA3 was demonstrated with mAb OX-42. MHC class I-positive cells (OX-18) appeared on day 2, whereas cells immunoreactive with OX-1 and W3/25 became evident in the CA1 and hilar regions on post-ischemic day 6. In contrast, MHC class II (Ia) antigen was first detected on indigenous microglia by day 13. In some animals, the OX-8 antibody resulted in the labelling of scattered CD8-positive lymphocytes, but perivascular inflammatory infiltrates were absent. No changes in the expression of ED2 immunoreactivity on perivascular cells could be observed. The results show that following ischemic injury, microglial cells demonstrate a time-dependent up-regulation and de novo expression of certain immunomolecules, indicative of their immunocompetence. The findings are compared with those obtained in other models of brain injury.

The microglial reaction in the rat dorsal hippocampus following transient forebrain ischemia.

We have examined the distribution and time course of the microglial reaction in the rat dorsal hippocampus after 25-min transient forebrain ischemia (four-vessel occlusion model). Microglial cells were visualized in brain sections using lectin staining with the Griffonia simplicifolia B4-isolectin following intervals of reperfusion ranging from 20 min to 4 weeks. Increased staining of microglial cells was detected in the dentate hilus and area CA1 as early as 20 min after reperfusion. These same regions demonstrated more intense microglial staining after 24 h. The strongest microglial reaction was observed 4-6 days after reperfusion when reactive microglia were abundant throughout all laminae of CA1 and the dentate hilus. Following longer reperfusion intervals, the microglial reaction became less intense; however, it remained above normal levels until the end of the fourth week. At all time points examined, microglial reactivity in the CA3 pyramidal and dentate granule cell layers was considerably lower than that observed in CA1 and dentate hilus. Our results are consistent with the known serial pathological changes associated with cerebral ischemia, but, in addition, show that the examination of the microglial reaction provides an extremely sensitive indicator of subtle and morphologically nonapparent neuronal damage during the early stages of injury.