Anat Cell Biol.  2013 Jun;46(2):131-140. 10.5115/acb.2013.46.2.131.

Characterization of nestin expression in astrocytes in the rat hippocampal CA1 region following transient forebrain ischemia

• Cho JM
• Shin YJ
• Park JM
• Kim J
• Lee MY

• KMID: 2263105
• DOI: http://doi.org/10.5115/acb.2013.46.2.131

Abstract

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 S100beta, 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.

Keyword

Nestin; Transient forebrain ischemia; Reactive astrocytes; Hippocampus

MeSH Terms

Animals
Astrocytes
CA1 Region, Hippocampal
Hippocampus
Intermediate Filament Proteins
Ischemia
Nerve Tissue Proteins
Neurons
Prosencephalon
Pyramidal Cells
Rats
Transcription Factors
Intermediate Filament Proteins
Nerve Tissue Proteins
Transcription Factors

Figure

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  Fig. 1 Spatiotemporal relationship between nestin and glial fibrillary acidic protein (GFAP) in the hippocampal CA1 region in the early phase following transient forebrain ischemia. (A-C) No specific staining for nestin was detected in the hippocampal CA1 region of sham-operated rats, while GFAP-positive astrocytes showed thin glial processes. pcl, pyramidal cell layer; slm, stratum lacunosum moleculare; so, stratum oriens; sr, stratum radiatum. (D-F) At day 3 after ischemia, significant induction of nestin expression was detected in the CA1 pyramidal cell layer, while a rather uniform upregulation of GFAP immunoreactivity was observed in all laminae of the CA1 region. (G-I) At 7 days, GFAP immunoreactivity was markedly increased in all strata of the CA1 region, while nestin expression was more pronounced in the CA1 dendritic layers and absent in the stratum lacunosum moleculare. Scale bars in (C, F, I)=200 µm (A-I).

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  Fig. 2 Spatiotemporal relationship between nestin and glial fibrillary acidic protein (GFAP) in the hippocampal CA1 region during the later phase following transient forebrain ischemia. (A-C) At 14 days after ischemia, GFAP-positive astrocytes were distributed in all strata of the hippocampal CA1 region, while nestin expression was localized in the CA1 dendritic layers including the strata radiatum (sr) and oriens (so), but was rare in the pyramidal cell layer (pcl) and in the stratum lacunosum moleculare (slm). Over the chronic interval of 21 days (D-F) and 28 days (G-I), the labeling intensity and patterns of GFAP staining were similar to those at day 14. Nestin expression was still observed in the CA1 dendritic layers at 21 days after ischemia, while it remained only in the stratum radiatum at 28 days. Note the absence of nestin expression in the stratum lacunosum moleculare. Scale bars in (C, F, I)=200 µm (A-I).

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  Fig. 3 Spatiotemporal distribution of nestin-positive astrocytes in the CA1 hippocampus of control (A) and ischemic rats (B-F). Nestin expression appeared in the pyramidal cell layer (pcl) by 3 days (B), and extended into the stratum radiatum (sr) by 7 days (C). By 14 days after ischemia (D), nestin expression was more pronounced in the stratum radiatum, and further increased in this stratum at 21 days (E) and 28 days (F). Note that nestin-positive astrocytes were rarely observed in the pyramidal cell layer by 14 days, despite the evident astroglial reaction. (G, H) Temporal profile of glial fibrillary acidic protein (GFAP) single-labeled cells and nestin/GFAP double-labeled cells in the pyramidal cell layer (G) and stratum radiatum (H) of the CA1 region after transient forebrain ischemia. The data are expressed as the mean±SD; *P<0.01 from the Bonferroni test with each time point compared to sham-operated controls. Scale bars in (C, F)=100 µm (A-F).

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  Fig. 4 Characterization of nestin-positive cells in the CA1 hippocampal region after transient forebrain ischemia. (A-F) Double-labeling with nestin and Ki-67 showed that some nestin-positive cells exhibited Ki-67-labeled nuclei (arrowheads) on days 3 (A-C) and 14 (D-F) after ischemia. pcl, pyramidal cell layer; sr, stratum radiatum. (G-L) Double-labeling with nestin and the transcription factors Sox-2 (G-I) or Sox-9 (J-L). Note that almost all nestin-positive cells expressed Sox-2- or Sox-9-labeled nuclei at days 3 (H, K) and 14 (I, L) after ischemia. Also note that Sox-2- or Sox-9-positive nuclei were also observed in the control hippocampus (G, J), where no nestin expression was observed. Scale bars in (C, F, I, L)=100 µm (A-L).

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  Fig. 5 Relationship between S100β and nestin (A, B, D, E) or glial fibrillary acidic protein (GFAP) (C, F) in the CA1 hippocampal region after transient forebrain ischemia. All of the nestin-positive cells expressed S100β, corresponding to only a small fraction of all of the S100β-positive cells at all time points examined. Note that virtually all of the S100β-positive cells in the ischemic CA1 region were GFAP-positive astrocytes. Scale bars in (C, F)=100 µm (A-F).

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