Transcriptome Profiling of Hippocampus After Cerebral Hypoperfusion in Mice.

Chronic cerebral hypoperfusion (CCH) is considered to be one of the major mechanism in the pathogenesis of vascular cognitive impairment (VCI). Increased inflammatory cells, particularly microglia, often parallel hypoperfusion-induced gray matter damage such as hippocampal lesions, but the exact mechanism remains largely unknown. To understand the pathological mechanisms, we analyzed hippocampus-specific transcriptome profiles after cerebral hypoperfusion. The mouse hypoperfusion model was induced by employing the 0.16/0.18 mm bilateral common carotid artery stenosis (BCAS) procedure. Cerebral blood flow (CBF) was assessed after 3-week hypoperfusion. Pathological changes were evaluated via hematoxylin staining and immunofluorescence staining. RNA-sequencing (RNA-seq) was performed using RNA samples of sham- or BCAS-operated mice, followed by quantitative real-time PCR (qRT-PCR) validation. We found that the 0.16/0.18 mm BCAS induced decreased CBF, hippocampal neuronal loss, and microglial activation. Furthermore, GSEA between sham and BCAS mice showed activation of interferon-beta signaling along with inflammatory immune responses. In addition, integrative analysis with published single-cell RNA-seq revealed that up-regulated differentially expressed genes (DEGs) were enriched in a distinct cell type of "microglia," and down-regulated DEGs were enriched in "CA1 pyramidal," not in "interneurons" or "S1 pyramidal." This database of transcriptomic profiles of BCAS-hypoperfusion will be useful for future studies to explore potential targets for vascular cognitive dysfunction.

Cortex-specific transcriptome profiling reveals upregulation of interferon-regulated genes after deeper cerebral hypoperfusion in mice.

Background: Chronic cerebral hypoperfusion (CCH) is commonly accompanied by brain injury and glial activation. In addition to white matter lesions, the intensity of CCH greatly affects the degree of gray matter damage. However, little is understood about the underlying molecular mechanisms related to cortical lesions and glial activation following hypoperfusion. Efforts to investigate the relationship between neuropathological alternations and gene expression changes support a role for identifying novel molecular pathways by transcriptomic mechanisms. Methods: Chronic cerebral ischemic injury model was induced by the bilateral carotid artery stenosis (BCAS) using 0.16/0.18 mm microcoils. Cerebral blood flow (CBF) was evaluated using laser speckle contrast imaging (LSCI) system. Spatial learning and memory were assessed by Morris water maze test. Histological changes were evaluated by Hematoxylin staining. Microglial activation and neuronal loss were further examined by immunofluorescence staining. Cortex-specific gene expression profiling analysis was performed in sham and BCAS mice, and then validated by quantitative RT-PCR and immunohistochemistry (IHC). Results: In our study, compared with the sham group, the right hemisphere CBF of BCAS mice decreased to 69% and the cognitive function became impaired at 4 weeks postoperation. Besides, the BCAS mice displayed profound gray matter damage, including atrophy and thinning of the cortex, accompanied by neuronal loss and increased activated microglia. Gene set enrichment analysis (GSEA) revealed that hypoperfusion-induced upregulated genes were significantly enriched in the pathways of interferon (IFN)-regulated signaling along with neuroinflammation signaling. Ingenuity pathway analysis (IPA) predicted the importance of type I IFN signaling in regulating the CCH gene network. The obtained RNA-seq data were validated by qRT-PCR in cerebral cortex, showing consistency with the RNA-seq results. Also, IHC staining revealed elevated expression of IFN-inducible protein in cerebral cortex following BCAS-hypoperfusion. Conclusion: Overall, the activation of IFN-mediated signaling enhanced our understanding of the neuroimmune responses induced by CCH. The upregulation of IFN-regulated genes (IRGs) might exert a critical impact on the progression of cerebral hypoperfusion. Our improved understanding of cortex-specific transcriptional profiles will be helpful to explore potential targets for CCH.

Integrated Analysis of Chromatin and Transcriptomic Profiling Identifies PU.1 as a Core Regulatory Factor in Microglial Activation Induced by Chronic Cerebral Hypoperfusion.

In addition to causing white matter lesions, chronic cerebral hypoperfusion (CCH) can also cause damage to gray matter, but the underlying molecular mechanisms remain largely unknown. In order to obtain a better understanding of the relationship between gene expression and transcriptional regulation alterations, novel upstream regulators could be identified using integration analysis of the transcriptome and epigenetic approaches. Here, a bilateral common carotid artery stenosis (BCAS) model was established for inducing CCH in mice. The spatial cognitive function of mice was evaluated, and changes in cortical microglia morphology were observed. RNA-sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were performed on isolated mouse cortical brain tissue. Then, a systematic joint analysis of BCAS hypoperfusion-induced cortex-specific RNA-seq and ATAC-seq was conducted in order to assess the extent of the correlation between the two, and PU.1 was found to be greatly enriched through motif analysis and transcription factor annotation. Also, the core regulatory factor PU.1 induced by BCAS hypoperfusion was shown to be colocalized with microglia. Based on the above analysis, PU.1 plays a key regulatory role in microglial activation induced by CCH. And the transcriptome and epigenomic data presented in this study can help identify potential targets for future research exploring chronic hypoperfusion-induced brain injury.