Objective The purpose of this study was to explore a suitable method to model no-reflow phenomenon following ischemic stroke and to evaluate perfusion decrease from multiple perspectives.Methods Laser scatter contrast imaging and two-photon live imaging were used to compare transient middle cerebral artery occlusion in C57BL/6 and BALB/c mice and perfusion alterations in BALB/c mice with 1 or 1.5 h of ischemia.Several imaging techniques including laser scatter contrast imaging,low and higher magnification images of perfused brain slices and two-photon microscopy to monitor erythrocyte flow rate and flux were used to assess in vivo dynamics as well as whole brain sections and microvasculature for decreased cerebral perfusion after transient middle cerebral artery occlusion.Infarct size and behavioral deficits were assessed with microtubule-associated protein 2 staining and behavioral scoring.Results In C57BL/6 mice,most capillaries in the middle cerebral artery region remained flowing during ischemia,whereas most capillaries were blocked in BALB/c mice.In addition,cortical perfusion at 24 h of recanalization was significantly reduced to 76.1%of baseline following 1.5 h of ischemia in BALB/c mice(P=0.046 compared with the sham group),whereas for it was reduced to 79.9%following 1h of ischemia which was not significantly different from the sham group(P=0.299).Transient middle cerebral artery occlusion in BALB/c mice for 1.5 h resulted in a reduction in whole-brain perfusion to 75.1%(P＜0.001 compared with the sham group),and erythrocyte flow rate assessed by two-photon live-imaging of erythrocyte flow on the cortical surface of the middle cerebral artery basin was reduced to 50.3%of baseline levels at 24 h of recanalization(P=0.010 compared with the sham group),and erythrocyte flux decreased to 38.9%of baseline levels(P= 0.010 compared with the sham group);high-magnification imaging of sections assessed an approximately 76%reduction in the length of capillaries with perfusion(P=0.0001 compared with the sham group),and a reduction in the fraction of the total volume occupied by perfused capillaries by an approximately 76%reduction(P＜0.001 compared with the sham-operated group).Microtubule-associated protein 2 staining suggested that transient middle cerebral artery occlusion for 1.5 h in BALB/c mice resulted in infarcts that accounted for approximately 36%of the total cerebral area and behavioral scores elevated to 9,suggesting behavioral deficits.Conclusion Transient ischemia in BALB/c mice for 1.5 h resulted in a significant decrease in cerebral perfusion as well as capillary no-reflow and thus can model the no-reflow phenomenon following ischemic stroke.The combination of laser scatter contrast imaging,low magnification and higher magnification images of perfused brain slices,and two-photon microscopy live imaging allows for a multifaceted assessment of perfusion changes.

Transcriptional regulation is crucial for regulated gene expression. Due to the complexity, it has been difficult to engineer eukaryotic transcription factor (TF) and promoter pairs. The few availabilities of eukaryotic TF and promotor pairs limit their practical use for clinical or industrial applications. Here, we report a de novo construction of synthetic inhibitory transcription factor and promoter pairs for mammalian transcriptional regulation. The design of synthetic TF was based on the fusion of DNA binding domain and Kruppel associated box transcription regulating domain (KRAB). The synthetic promoter was constructed by inserting the corresponding TF response element after SV40 promoter. We constructed and tested five synthetic inhibitory transcription factor and promoter pairs in cultured mammalian cells. The inhibition capability and orthogonality were verified by flow cytometry. In summary, we demonstrate the feasibility of constructing mammalian inhibitory TF and promoter pairs, which could be standardized for advanced gene-circuit design and various applications in the mammalian synthetic biology.
Animals ; Gene Expression Regulation ; Gene Regulatory Networks ; Mammals ; Promoter Regions, Genetic ; Transcription Factors ; Transcription, Genetic