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

Abstract

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

“…Based on our results, as well as those of previous studies [ 15 ], we successfully established the 0.16/0.18 mm BCAS hypoperfusion model for gray matter lesions, with microglia manifesting a state of neuroinflammatory activation in the cerebral cortex. We then utilized both RNA-seq and ATAC-seq to study the effect of chromatin accessibility on gene expression as a whole, in order to investigate the molecular mechanics of CCH.…”

“…Thus, it can be concluded that BCAS hypoperfusion resulting from 0.16 mm stenosis can cause clearly evident histological injury. In our previous work, we found activation of microglia occurred mostly in areas of neuronal injury, including the cerebral cortex on the 0.16 mm stenosis side of BCAS mice [ 15 ]. Microglia, as the primary innate immune cells and important cellular mediators in the brain, play an important role in neuroinflammation.…”

“…1 H). Chronic hypoperfusion has been reported to lead to microglia activation, often described as swollen cell bodies with thicker and shorter processes or amoeboid-like morphology [ 15 ]. In our current BCAS hypoperfusion mouse model, it was found that microglia on the 0.18 mm side of BCAS group exhibited hyper-ramification morphology characterized by an increased length of branches (Fig.…”

“…In our previous research, gene set enrichment analysis (GSEA) revealed that BCAS hypoperfusion-induced up-regulated genes were significantly enriched in the pathways of IFN-regulated signaling along with neuroinflammation signaling. Also, our work revealed elevated expression of IFN-inducible protein in the cerebral cortex following BCAS hypoperfusion [ 15 ]. Based on the integrative analysis of our RNA-seq data and the published scRNA-seq dataset, it can be seen that the change of gene expression caused by BCAS hypoperfusion is closely associated with the activation of microglia.…”

“…Several previous studies have reported the activation of the type I interferon (IFN-I) signaling pathway in rodent models of brain injury, where the interferon-beta (IFN-β) signaling and response can promote the release of pro-inflammatory cytokines and modulation of brain injury, involving interactions between different cell types in the brain [ 14 ]. In our previous work, RNA sequencing (RNA-seq) was performed to identify cortex-specific gene expression changes caused by BCAS hypoperfusion [ 15 ]. It was found that neuroimmune response mediated by IFN-β signaling plays an important role in the regulation of brain injury induced by CCH.…”