Establishment of U6 promoter-based Nox1 shRNA/AAV vector and identification of the transduction in cells of the peri-infarct regions.

(a) Nox1 shRNA sequence. Nox1 shRNA was designed based on the siRNA sequence (boxed red nucleotides) (b) Establishment of the Nox1 shRNA/AAV construct. U6 promoter-driven shRNA expression system was established in the AAV2 vector. EGFP expression is separately controlled by a CMV promoter as a marker for the transduction efficiency. (c) Schematic diagrams of the infarct core and the peri-infarct region delineated by Nissl staining at 2 weeks after MCAO—reperfusion. Black: infarct core; black dotted line: peri-infarct region; gray colored boxes: EGFP expression observed areas; A: peri-infarct area in the cortex; B: peri-infarct area in the striatum. (d) Representative photographs of tissue sections expressed EGFP (green) and stained with the NeuN (red) antibody from rat cortex and striatum tissue taken from rats at 6 weeks after Nox1 shRNA/AAV2 injection. shRNA expression in NeuN⁺ neurons is demonstrated as yellow staining after merging green (EGFP) and red (NeuN) images. (e) EGFP expression levels were measured as cell counts of EGFP expression in NeuN⁺ neurons in the cortex and striatum n = 6/group. (f, g, h, and i) Nox1 knockdown efficiency (f and g) and specificity (h and i) in the cortex and striatum was verified by western blot analysis for Nox1, Nox2, and β-actin performed at 8 weeks after AAV injection. n = 4/group, **p<0.01, ***p < 0.001 vs sham control with scb shRNA, ###p < 0.001 versus MCAO control with scb shRNA. Scb, scramble.