Rho-kinase inhibition improves ischemic perfusion deficit in hyperlipidemic mice

Abstract

Hyperlipidemia is a major cardiovascular risk factor associated with progressive cerebrovascular dysfunction and diminished collateral perfusion in stroke. Rho-associated kinase (ROCK) may be an important mediator of hyperlipidemic vascular dysfunction. We tested the efficacy of acute or chronic ROCK inhibition on the size of dynamic perfusion defect using laser speckle flowmetry in hyperlipidemic apolipoprotein E knockout mice fed on a high-fat diet for 8 weeks. Mice were studied at an age before the development of flow-limiting atherosclerotic stenoses in aorta and major cervical arteries. Focal ischemia was induced by distal middle cerebral artery occlusion (dMCAO) during optical imaging. The ROCK inhibitor fasudil (10 mg/kg) was administered either as a single dose 1 hour before ischemia onset, or daily for 4 weeks. Fasudil decreased both baseline arterial blood pressure and cerebrovascular resistance (CVR) by ∼15%, and significantly improved tissue perfusion during dMCAO. Interestingly, peri-infarct depolarizations were also reduced. Chronic treatment did not further enhance these benefits compared with acute treatment with a single dose. These data show that ROCK inhibition improves CVR and ischemic tissue perfusion in hyperlipidemic mice.

Figure 1

Effects of fasudil on ischemic tissue perfusion after distal middle cerebral artery occlusion (dMCAO) in hyperlipidemic apolipoprotein E (ApoE) knockout mice. (A) Left panel: Laser speckle imaging field (gray-shaded area) was positioned over the right hemisphere. Right panel: A representative laser speckle flow map shows the dorsolateral location of the perfusion defect after dMCAO. The area of perfusion defect was calculated by using a thresholding paradigm, where pixels with ⩽20% (light blue), ⩽30% (dark blue), and ⩽40% (not shown) residual cerebral blood flow (CBF) compared with preischemic baseline were identified, integrated and their area calculated based on the known scale factor. The optical artifact caused by the microvascular clip placed on the middle cerebral artery through a temporal craniotomy is also seen on the lower right of the image. (B) The area of perfusion defect is shown at different thresholding levels in vehicle or fasudil-treated mice. Whiskers, minimum and maximum values; boxes, 25% to 75% range; horizontal line, median; ‘+', mean. Individual data points are also shown. *P<0.05 versus vehicle. Two-way ANOVA followed by Tukey's test to correct for multiple comparisons.

Figure 2

Effects of single dose or chronic fasudil treatment on resting cortical blood flow (CBF), mean arterial blood pressure (MABP), and cerebrovascular resistance (CVR) in hyperlipidemic apolipoprotein E (ApoE) knockout mice. Data are from preischemic baseline obtained at the beginning of experiments before distal middle cerebral artery occlusion (dMCAO). CBF was measured by calculating the τ_c⁻¹ values using laser speckle contrast images, which were then normalized to the vehicle group average and expressed as %. CVR was calculated using the MABP and CBF, and expressed as % of vehicle group average. Please see Materials and Methods for details. Whiskers, minimum and maximum values; boxes, 25% to 75% range; horizontal line, median; ‘+', mean. Individual data points are also shown. *P<0.05 versus vehicle.

Figure 3

Effects of Rho-associated kinase (ROCK) inhibition on peri-infarct depolarization (PID) occurrence and its impact on the size of ischemic cerebral blood flow (CBF) deficit. (A) Representative CBF tracing obtained by laser speckle flowmetry during distal middle cerebral artery occlusion (dMCAO). On dMCAO, CBF abruptly decreased to 80% (moderate ischemia, upper tracing) or 35% of baseline (severe ischemia, lower tracing) as measured in two representative regions of interest (ROIs; circles in inset). Anoxic depolarization (AD) caused a second stepwise reduction in perfusion in both ROIs. Two PIDs occurred at 21 and 32 minutes, associated with typical transient hyperemia and hypoperfusion in moderately and severely ischemic ROIs, respectively. These CBF transients were used as surrogates to identify PID occurrence. (B) The number of PIDs detected during 1 hour imaging is shown from vehicle and pooled fasudil- or hydroxyfasudil-treated groups (ROCKi). Each symbol represents one animal. Mann–Whitney U-test. *P<0.05 versus vehicle. (C) The effect of PIDs on the area of perfusion defect (residual CBF⩽30%) is shown from vehicle and pooled fasudil- or hydroxyfasudil-treated groups (ROCKi). Data are expressed as % change in CBF deficit area induced by each PID. Whiskers, minimum and maximum values; boxes, 25% to 75% range; horizontal line, median; ‘+', mean.