Spatial transcriptomics approach to hypercontractile parenchymal arteries associated with ischemia-reperfusion

Abstract

Background: Despite vessel recanalization, up to 67% of ischemic-stroke patients experience futile reperfusion increasing the risk of neurological morbidity and death. Arterial hypercontractility has been suggested to underlie the no-flow phenomenon, however, this remains to be fully elucidated. Parenchymal arteries in the area surrounding the ischemic core (penumbra) display an elevated vascular tone due to increased intracellular Ca 2+ sensitivity. Ischemia-reperfusion (IR) reduces Na + , K + -ATPase activity and is inversely proportional to Src activation, a tyrosine kinase, which is known to sensitize the contractile machinery to Ca 2+ via myosin light chain kinase (MLCK) activation. We hypothesize that IR reduces Na + , K + -ATPase activity and increases Src with consequent Ca 2+ sensitization and hypercontractility of parenchymal arteries. Materials and Methods: Five 3-month-old C57BL/6J male mice were exposed to one-hour transient middle cerebral artery occlusion by a filament followed by 24-hour reperfusion. Mice were anesthetized with isoflurane (3% induction, 1.5–2% maintenance) and intraperitoneally injected with Temgesic (0.1 mg/kg) 0.5- and 6 hours post-operative. Nissl staining was used to detect infarct areas. Spatial transcriptomics (ST) of coronal slices was performed. Infarct size was corrected for edema and calculated as the percentage of the whole hemisphere. ST was cell-predicted, and the cortex region was specified using gene markers from a single-cell RNA sequencing dataset. Pearson’s correlation coefficient was used to predict cell-cell correlation in the ST spots. Cells of interest were used for average gene expression analysis of scaled data. The expression levels were compared between the two hemispheres using t-tests. Data are presented as mean ± SD. Results: The ischemic core area was 0.67 ± 0.34 cm 2 corresponding to 18.11 ± 8.28% of the whole hemisphere. An interrelationship between cell types from the cerebral blood vessels and astrocytes was found, and these cell types were pooled. When comparing the average gene expressions between the contralateral and ipsilateral hemisphere, the Na + , K + -ATPase α 1 and α 2 isoforms were downregulated in the ipsilateral cortex area (p=2.7e -6 and p=3.3e -6 , respectively). Src (p=2.3e -4 ) and MLCK were downregulated in the ipsilateral cortex (4.8e -2 ). All genes for the myosin light chain phosphatase (MLCP) were downregulated. Conclusion: The data suggest that potentiated hypercontractility of parenchymal arteries is not due to Na + , K + -ATPase, and Src kinase-associated activation of the Ca 2+ -dependent MLCK. However, hypercontractility of parenchymal arteries may be due to a downregulated MLCP. Funding: This study was funded by the Lundbeck Foundation (R344-2020-952). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.