Background and Purpose-The mechanisms of excitotoxic cell death in cerebral ischemia are poorly understood. In addition to necrosis, apoptotic cell death may occur. The purpose of this study was to determine whether an established model of cerebral hypoxia-ischemia in the neonatal rat demonstrates any features of apoptosis. Methods-Seven-day-old neonatal rats underwent bilateral, permanent carotid ligation followed by 1 hour of hypoxia, and their brains were examined 1, 3, and 4 days after hypoxia-ischemia. The severity of ischemic damage was assessed in the dentate gyrus and frontotemporal cortex by light microscopy. Immunocytochemistry was performed to detect the cleavage of actin by caspases, a family of enzymes activated in apoptosis. Terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling (TUNEL) reactivity was examined in the cortical infarction bed and dentate gyrus. Neonatal rat brain DNA was run on agarose gel electrophoresis to detect DNA fragmentation. Ethidium bromide-staining and electron microscopy were used to determine whether apoptotic bodies, 1 of the hallmarks of apoptosis, were present. Results-The frontotemporal cortex displayed evidence of infarction, and in most rats the dentate gyrus showed selective, delayed neuronal death. Immunocytochemistry demonstrated caspase-related cleavage of actin. TUNEL and DNA electrophoresis provided evidence of DNA fragmentation. Ethidium bromide-staining and electron microscopy confirmed the presence of chromatin condensation and apoptotic bodies. Conclusions-Features of apoptosis are present in the described model of cerebral hypoxia-ischemia. Apoptosis may represent a mode of ischemic cell death that could be the target of novel treatments that could potentially expand the therapeutic window for stroke. (Stroke. 1998;29:2622-2630.)

Surface levels of CCR5 on memory CD4+ T cells influence HIV-1/AIDS susceptibility. Alternative promoter usage results in the generation of CCR5 mRNA isoforms that differ based on whether they contain or lack the untranslated exon 1. The impact of exon 1-containing transcripts on CCR5 surface expression is unknown. In this study, we show that the increased cell surface expression of CCR5 on primary T cells is associated with selective enrichment of exon 1-containing transcripts. The promoter that drives exon 1-containing transcripts is highly active in primary human T cells but not in transformed T cell lines. The transcription factors Oct-1 and -2 inhibit and enhance, respectively, the expression of exon 1-containing transcripts and CCR5 surface levels. However, polymorphisms at homologous octamer-binding sites in the CCR5 promoter of nonhuman primates abrogate the binding of these transcription factors. These results identify exon 1-containing transcripts, and the cis-trans factors that regulate the expression levels of these mRNA isoforms as key parameters that affect CCR5 surface expression levels, and by extension, susceptibility to HIV/AIDS among humans, and possibly, the observed interspecies differences in susceptibility to lentiviral infection.

Chemotactic factors known as chemokines play an important role in the pathogenesis of Multiple Sclerosis (MS). Transgenic expression of TNFα in the central nervous system (CNS) leads to the development of a demyelinating phenotype (TNFα-induced demyelination; TID) that is highly reminiscent of MS. Little is known about the role of chemokines in TID but insights derived from studying this model might extend our current understanding of MS pathogenesis and complement data derived from the classic autoimmune encephalomyelitis (EAE) model system. Here we show that in TID, chemokines and their receptors were significantly increased during the acute phases of disease. Notably, the CCL2 (MCP-1)-CCR2 axis and the closely related ligand-receptor pair CCR1-CCL3 (MIP-1α) were among the most up-regulated during disease. On the other hand, receptors like CCR3 and CCR4 were not elevated. This significant increase in the levels of chemokines/receptors correlated with robust immune infiltration of the CNS by inflammatory cells, i.e., macrophages, and immune cells particularly T and B cells. Immunostaining and confocal microscopy, along with in vitro studies revealed that astrocytes were a major source of locally produced chemokines and expressed functional chemokine receptors such as CCR2. Using an in vitro system we demonstrate Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. that expression of CCR2 was functional in astrocytes and that signaling via this receptor lead to activation of NF-kB and Akt and was associated with increased astrocyte survival. Collectively, our data suggests that transgenic murine models of MS are useful to dissect mechanisms of disease and that in these models, up-regulation of chemokines and their receptors may be key determinants in TID. NIH Public Access