The present study aimed to provide a detailed characterization of the cellular phenotypes of nestin-positive cells in a rat model of ischemic stroke. Nestin-positive cells included reactive astrocytes in the peri-infarct region. In the ischemic core, in which astrocytes had virtually disappeared, nestin expression was exclusively associated with the vasculature, including the microvasculature and larger caliber vessels. Induction of nestin expression in the ischemic core occurred by 3 days post-ischemia. Nestin expression continued through at least 28 days post-ischemia but the cellular profiles of nestin-positive cells changed over this period. In the ischemic core at day 3, nestin-positive cells frequently had long processes that ran parallel along the longitudinal axis of the vasculature. These cells were highly proliferative and expressed the transcription factor for neural/glial progenitors, Sox9. Based on their morphological characteristics and on a double-labeling study, most nestin-positive cells were clearly distinguishable from vasculature-associated cells including endothelial cells, smooth muscle cells and microglia/macrophages. Immunoelectron microscopic findings demonstrated that most nestin-positive cells lay in the perivascular space and had macrophage-like features, indicating morphological similarity to perivascular macrophages. Nestin expression was still associated with the vasculature 14 days after ischemia but appeared in fibroblast-like cells. Thus, our data indicated that, in the ischemic core, nestin expression was not limited to a progenitor/stem cell population but was induced in the vasculature-associated cells. These cell types included perivascular macrophages and fibroblast-like cells that appeared to undergo dynamic structural changes. These results suggest that nestin facilitates cellular structural remodeling in response to ischemic injury.

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Recent studies have suggested that nestin facilitates cellular structural remodeling in vasculature-associated cells in response to ischemic injury. The current study was designed to investigate the potential role of post-ischemic nestin expression in parenchymal astrocytes. With this aim, we characterized ischemia-induced nestin expression in the CA1 hippocampal region, an area that undergoes a delayed neuronal death, followed by a lack of neuronal generation after transient forebrain ischemia. Virtually all of the nestin-positive cells in the ischemic CA1 hippocampus were reactive astrocytes. However, induction of nestin expression did not correlate simply with astrogliosis, but rather showed characteristic time- and strata-dependent expression patterns. Nestin induction in astrocytes of the pyramidal cell layer was rapid and transient, while a long-lasting induction of nestin was observed in astrocytes located in the CA1 dendritic subfields, such as the stratum oriens and radiatum, until at least day 28 after ischemia. There was no detectable expression in the stratum lacunosum moleculare despite the evident astroglial reaction. Almost all of the nestin-positive cells also expressed a transcription factor for neural/glial progenitors, i.e., Sox-2 or Sox-9, and some cells were also positive for Ki-67. However, all of the nestin-positive astrocytes expressed the calcium-binding protein S100β, which is known to be expressed in a distinct, post-mitotic astrocyte population. Thus, our data indicate that in the ischemic CA1 hippocampus, nestin expression was induced in astroglia that were becoming reactive, but not in a progenitor/stem cell population, suggesting that nestin may allow for the structural remodeling of these cells in response to ischemic injury.

We investigated the spatiotemporal expression of vascular endothelial growth factor receptor–3 (VEGFR-3) in the spinal cord of Lewis rats with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. VEGFR-3 mRNA and protein were constitutively expressed in gray matter neurons and in a few white matter astrocytes. Induction of VEGFR-3 occurred predominantly in perivascular infiltrated macrophages in the spinal cord white matter during the inductive phase of EAE. VEGFR-3 expression was also induced in activated microglial cells in the gray and white matter, mainly in the peak phase. In addition, reactive astrocytes in the white matter, but not in the gray matter, expressed VEGFR-3 as disease severity increased. These data suggest that VEGFR-3 is involved in the recruitment of monocytic macrophages and in glial reactions during EAE.

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