Focal Ischemic Injury with Complex Middle Cerebral Artery in Stroke-Prone Spontaneously Hypertensive Rats with Loss-Of-Function in NADPH Oxidases

Abstract

By means of introgressing a loss-of-function mutation in the p22phox gene from the Matsumoto Eosinophilia Shinshu (MES) rat to stroke-prone spontaneously hypertensive rats (SHRSP), we constructed the SHRSP-based congenic strain lacking the P22PHOX expression (i.e., lacking NADPH oxidases [NOX] activities) (SHRSP.MES-Cyba mes/Izm; hereafter referred to as SP.MES). To examine the effects of Nox activities on the focal ischemic injury or stroke, we performed middle cerebral artery (MCA) occlusion in this new congenic strain; the distal MCA was occluded by 561-nm laser-driven photothrombosis. Resting mean arterial blood pressure was significantly lower in SP.MES when compared with the control PM0/SHRSP (150±11 mmHg vs. 166±11 mmHg). Cerebral blood flow decreased to 37±13% in SP.MES and 35±17% in PM0/SHRSP at 10 min after MCA occlusion (not significant). Infarct volume determined at 24 h after MCA occlusion in SP.MES was 89±39 mm3, which was not significantly different from 83±35 mm3 in PM0/SHRSP. The distal MCA pattern was more complex in SP.MES (median 3, IQR 3–5) than PM0/SHRSP (median 2, IQR 1–3) (p = 0.001). Because more complex distal MCA is known to produce larger infarction after distal MCA occlusion in SHR, we adjusted for the branching pattern in an ANCOVA. The adjusted mean of infarct volume was significantly smaller in SP.MES compared with that in PM0/SHRSP (67 [95% CI 46 to 87] mm3 vs. 100 [95% CI 82 to 118] mm3, p = 0.032). Elimination of the P22PHOX expression induced complex distal MCA, which would suggest the presence of ‘loss of complexity’ induced by enhanced oxidative stress in SHRSP; infarct size in SP.MES—when adjusted for distal MCA complexity—was significantly attenuated compared with that in PM0/SHRSP. Therefore, the present results suggest that Nox is harmful for ischemic brain tissue.

Effect of p22phox depletion on sympathetic regulation of blood pressure in SHRSP: evaluation in a new congenic strain

“…S3b and ref. 7). Since P22PHOX is an essential subunit of the active NOX, the ROS level was expected to be low in SP.MES, and indeed, DHE staining (see below for the method) showed a lower level of superoxide in RVLM in SP.MES (Fig.…”

“…To examine this hypothesis, we introduced a P22PHOX-depleted congenic SHRSP, which was recently developed in our laboratory7. This congenic strain (called as SP.MES) was established to harbor the null mutation in the P22phox gene of the Matsumoto Eosinophilic Shinshu rat (MES)8.…”

NADPH Oxidase-Related Pathophysiology in Experimental Models of Stroke

“…Because Nox1-4 need to form heterodimers with the membrane-bound p22 phox subunit [ 1 ], SP.MES lacks Nox activities on the background of the SHRSP. Although we have reported the results of photothrombotic distal MCAO in this new congenic strain [ 73 ], the findings are summarized below.…”

“…Physiological variables such as blood pressure are critically important in experimental stroke; for example, congenic removal of a blood pressure quantitative trait locus (decreased blood pressure by 12% or −29 mmHg) attenuated infarct size produced by MCAO [ 70 ]. Our previous study mentioned above showed slightly but significantly decreased blood pressure in SP.MES (i.e., Nox dysfunction due to the absence of p22 phox protein) when compared with SHRSP/PM0, suggesting that decreased levels of resting or pre-stroke blood pressure in hypertensive rats would attenuate infarct size produced by MCAO [ 73 ]. Therefore, decreases in blood pressure due to the absence of p22 phox may explain at least in part the protective effects against focal ischemic injury observed in SP.MES.…”