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Neuronal precursor cell proliferation in the hippocampus after transient cerebral ischemia: a comparative study of two rat strains using stereological tools.

ABSTRACT: BACKGROUND: We are currently investigating microglial activation and neuronal precursor cell (NPC) proliferation after transient middle cerebral artery occlusion (tMCAo) in rats. This study aimed: (1) to investigate differences in hippocampal NPC proliferation in outbred male spontaneously hypertensive rats (SHRs) and Sprague-Dawley rats (SDs) one week after tMCAo; (2) to present the practical use of the optical fractionator and 2D nucleator in stereological brain tissue analyses; and (3) to report our experiences with an intraluminal tMCAo model where the occluding filament is advanced 22 mm beyond the carotid bifurcation and the common carotid artery is clamped during tMCAo. METHODS: Twenty-three SDs and twenty SHRs were randomized into four groups subjected to 90 minutes tMCAo or sham. BrdU (50 mg/kg) was administered intraperitoneally twice daily on Day 4 to 7 after surgery. On Day 8 all animals were euthanized. NeuN-stained tissue sections were used for brain and infarct volume estimation with the 2D nucleator and Cavalieri principle. Brains were studied for the presence of activated microglia (ED-1) and hippocampal BrdU incorporation using the optical fractionator. RESULTS: We found no significant difference or increase in post-ischemic NPC proliferation between the two strains. However, the response to remote ischemia may differ between SDs and SHRs. In three animals increased post-stroke NPC proliferation was associated with hippocampal ischemic injury. The mean infarct volume was 89.2 +/- 76.1 mm3 in SHRs and 16.9 +/- 22.7 mm3 in SDs (p < 0.005). Eight out of eleven SHRs had ischemic neocortical damage in contrast to only one out of 12 SDs. We observed involvement of the anterior choroidal and hypothalamic arteries in several animals from both strains and the anterior cerebral artery in two SHRs. CONCLUSIONS: We found no evidence of an early hippocampal NPC proliferation one week after tMCAo in both strains. Infarction within the anterior choroidal artery could induce hippocampal ischemia and increase NPC proliferation profoundly. NPC proliferation was not aggravated by the presence of activated microglia. Intraluminal tMCAo in SHRs gave a more reliable infarct with neocortical involvement, but affected territories supplied by the anterior cerebral, anterior choroidal and hypothalamic arteries.

SUBMITTER: Kelsen J

PROVIDER: S-EPMC2868803 | biostudies-other | 2010

REPOSITORIES: biostudies-other

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Neuronal precursor cell proliferation in the hippocampus after transient cerebral ischemia: a comparative study of two rat strains using stereological tools.

Kelsen Jesper J Larsen Marianne H MH Sørensen Jens Christian JC Møller Arne A Frøkiaer Jørgen J Nielsen Søren S Nyengaard Jens R JR Mikkelsen Jens D JD Rønn Lars Christian B LC

Experimental & translational stroke medicine 20100406

<h4>Background</h4>We are currently investigating microglial activation and neuronal precursor cell (NPC) proliferation after transient middle cerebral artery occlusion (tMCAo) in rats. This study aimed: (1) to investigate differences in hippocampal NPC proliferation in outbred male spontaneously hypertensive rats (SHRs) and Sprague-Dawley rats (SDs) one week after tMCAo; (2) to present the practical use of the optical fractionator and 2D nucleator in stereological brain tissue analyses; and (3) ...[more]

PMID: 20370927

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Project description:Purpose:To evaluate the effect of transient Global Cerebral Ischemia(tGCI) with Hypoxic Postconditioning (HPC) on piRNA expression profiles, piRNAs sequencing was performed. Methods: Total RNA of each sample (n=3 in each group) was prepared using Trizol reagent (Invitrogen), and the RNA quality and integrity were confirmed. 5’- and 3’-Adaptors were ligated to the obtained small RNA. Reverse transcription followed by PCR was used to create cDNA constructs. Subsequently, an about 150 bp fraction corresponding to approximate the adaptor-ligated constructs derived from the 30 nts piRNA fragment was excised and purified. The purified libraries were sequenced on Illumina HiSeq 2000 apparatus. The raw data were refined using cutadapt software (v1.9.1). After filtering out low quality reads and short reads (<15 nts), trimmed reads from all samples were pooled, and piano software was used to predict novel piRNAs. The trimmed reads were aligned to the merged rat piRNA databases (known piRNA from piRNABank plus the newly predicted piRNAs) using Novoalign software (v3.02.12) with at most one mismatch. PiRNA-binding transposon and targets were predicted by miranda software (v3.3a). piRNA-targets networks were plotted by cytoscape software (v2.8.0). Results: After tGCI, a total of 5574 piRNAs in CA1 was detected after 26 h of reperfusion. Of those, 12 were significantly upregulated compared with Sham rats (≥1.5-fold change, p<0.05), including 6 novel piRNAs. Of the 12 piRNAs, 4 showed ≥2-fold change. In comparison to tGCI group, 6 piRNAs in CA1 were significantly downregulated after HPC. Interestingly, the piRNAs rno_piR_000618, rno_piR_017990 and rno_piR_014971 were significantly changed in both tGCI and HPC groups Conclusions: In our study, we identified a cohort of piRNAs in CA1 of rats. Furthermore, we demonstrated directly contrast changes of piRNAs interacted with Piwil2 in CA1 after tGCI, as compared to those in HPC rats. Therefore, this study revealed that PIWI/piRNAs respond to ischemic brain damage and neuroprotection mediated by HPC. To the best of our knowledge, this is the first study ever on piRNAs associated with Piwil2 in cerebral ischemic tolerance.

Dataset Information

Neuronal precursor cell proliferation in the hippocampus after transient cerebral ischemia: a comparative study of two rat strains using stereological tools.

Publications

Neuronal precursor cell proliferation in the hippocampus after transient cerebral ischemia: a comparative study of two rat strains using stereological tools.

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