Hyperlipidemia disrupts cerebrovascular reflexes and worsens ischemic perfusion defect

Abstract

Hyperlipidemia is a highly prevalent risk factor for coronary and cervical atherosclerosis and stroke. However, even in the absence of overt atherosclerosis, hyperlipidemia disrupts endothelial and smooth muscle function. We investigated the impact of hyperlipidemia on resting-brain perfusion, fundamental cerebrovascular reflexes, and dynamic perfusion defect during acute focal ischemia in hyperlipidemic apolipoprotein E knockout mice before the development of flow-limiting atherosclerotic stenoses. Despite elevated blood pressures, absolute resting cerebral blood flow was reduced by 20% in apolipoprotein E knockout compared with wild type when measured by [(14)C]-iodoamphetamine technique. Noninvasive, high spatiotemporal resolution laser speckle flow imaging revealed that the lower autoregulatory limit was elevated in apolipoprotein E knockout mice (60 vs. 40 mm Hg), and cortical hyperemic responses to hypercapnia and functional activation were attenuated by 30% and 64%, respectively. Distal middle cerebral artery occlusion caused significantly larger perfusion defects and infarct volumes in apolipoprotein E knockout compared with wild type. Cerebrovascular dysfunction showed a direct relationship to the duration of high-fat diet. These data suggest that hyperlipidemia disrupts cerebral blood flow regulation and diminishes collateral perfusion in acute stroke in the absence of hemodynamically significant atherosclerosis.

Figure 1

Resting cerebral perfusion. Laser speckle flowmetry provides a two-dimensional perfusion map of the entire dorsal cortex. Imaging field position over the right hemisphere (left) and a representative speckle contrast perfusion image (middle) are shown. Dotted lines show the region of interest for resting perfusion measurements in arbitrary units. Resting cerebral blood flow (CBF) progressively decreased after high-fat diet (HFD) in ApoE^−/− mice, but not in the wild type. n=9, 5, 8, 5 WT, and 6, 4, 9, 6 ApoE^−/− at 0, 4, 8 and 10 week time points, respectively. *P<0.05 vs. wild type (WT), ^†P<0.05 vs. 0 weeks; two-way analysis of variance (ANOVA).

Figure 2

Hypercapnic hyperemia. Representative laser speckle perfusion images through intact skull show hypercapnic hyperemia (5% CO₂ inhalation) throughout the dorsal cortex in wild type and ApoE^−/− mice (upper row). Color bar indicates cerebral blood flow (CBF) relative to baseline. Imaging field position is shown on the lower left. Dotted outline indicates the cortical region of interest to quantify the time course of hypercapnic CBF changes (lower right). Both the rate of CBF increase and the peak hyperemia were attenuated in ApoE^−/− mice during hypercapnia (n=11 and 7, wild type and ApoE^−/− mice, respectively; *P<0.05, two-way analysis of variance (ANOVA) for repeated measures).

Figure 3

Autoregulation of cerebral blood flow. The lower limit of cerebral blood flow (CBF) autoregulation was tested by stepwise reduction in blood pressure by controlled exsanguination. Both CBF and cerebrovascular resistance (CVR) autoregulation curves significantly differed between wild type and ApoE^−/− mice (n=11 and 7, respectively; *P<0.05, two-way analysis of variance (ANOVA) for repeated measures). The lower limit of autoregulation was 40 mm Hg in wild type compared with 60 mm Hg in ApoE^−/− mice (arrowheads), defined as the blood pressure level below which CBF was statistically significantly lower than the baseline CBF at 80 mm Hg blood pressure (see Methods). Moreover, both the lowest level of CVR attained during hypotension (i.e., maximal vasodilation), and the associated blood pressure level were higher in ApoE^−/− mice compared with wild type. CVR change was calculated as 100 × (BP/BP₀)/(CBF/CBF₀), where BP₀ and CBF₀ are baseline values (i.e., at 80 mm Hg blood pressure).

Figure 4

Functional neurovascular coupling in whisker barrel cortex. (A) The imaging field, approximate location of whisker barrel cortex (dotted area), and the region of interest (square) within which cerebral blood flow (CBF) changes were quantified are indicated. (B) Representative laser speckle perfusion images of peak CBF changes during 30 second mechanical whisker stimulation. Wild-type mice showed a marked regional hyperemia limited to the whisker barrel cortex during stimulation. Functional hyperemia was significantly diminished in ApoE^−/− mice (see text for data). Color bar indicates CBF (%) relative to baseline. (C) Representative somatosensory evoked potentials from whisker barrel cortex in response to electrical stimulation of the contralateral whisker pad in wild type and ApoE^−/− mice. The latency, onset slope, amplitude and area under curve did not differ between wild type and ApoE^−/− mice (Supplementary Table 4).

Figure 5

Perfusion defect during focal arterial occlusion. (A) Representative relative (cerebral blood flow) CBF image (left) after distal middle cerebral artery occlusion (dMCAO) shows the region of interest placed in the hemodynamic penumbra (square) to quantify CBF changes over time. Arrowhead shows the microvascular clip position. Imaging field is positioned as shown in Figure 1. dMCAO resulted in an abrupt CBF reduction to approximately 40% of baseline in penumbra (time 0). ApoE^−/− mice had lower residual perfusion compared with wild type (WT) after high-fat diet (HFD) (*P<0.05 vs. WT, two-way analysis of variance (ANOVA) for repeated measures); the difference persisted for the entire 60 minutes imaging period after dMCAO. (B) Representative relative CBF image (left) shows the line profile between lambda (0 mm) and the occluded middle cerebral artery branch (arrowhead), along which residual CBF was plotted as % of pre-ischemic baseline at 60 minutes after dMCAO. ApoE^−/− mice had lower residual perfusion compared to wild type after HFD (*P<0.05 vs. WT, two-way ANOVA for repeated measures). The difference was most prominent in the hemodynamic penumbra approximately 1–3 mm from lambda. (C) Representative speckle contrast images (upper row) 60 minutes after dMCAO show the area of cortex with residual CBF⩽30% (superimposed blue pixels) in wild type and ApoE^−/− mice on 8 or 10 weeks of HFD. The core ischemic area with severe CBF deficit (residual CBF⩽20%) was progressively larger in ApoE^−/−mice (*P<0.05 vs. WT, two-way ANOVA for repeated measures). N=9, 5, 8 and 5 wild type, and 6, 4, 9 and 6 ApoE^−/−, on regular diet, or 4, 8 or 10 weeks of HFD, respectively.

Figure 6

Infarct volume after transient focal arterial occlusion. Representative topical triphenyl-tetrazolium chloride (TTC) stained brains (upper row) from wild type and ApoE^−/− mice show cortical infarct 48 hours after 1 hour transient distal middle cerebral artery occlusion (arrowheads). Infarct volumes, calculated by integrating the infarct area in 1 mm thick coronal slices, were almost doubled in ApoE^−/− mice compared with wild type (WT) in a concentric fashion after 8 but not 4 weeks of high-fat diet (HFD) (lower row). Numbers of mice are shown on the graphs. *P<0.05 vs. WT.

Figure 7

Perfusion threshold for viability. The laser speckle contrast image of a representative ApoE^−/− mouse shows pixels with ≤35% residual cerebral blood flow (CBF) superimposed in blue at 60 minutes after distal middle cerebral artery occlusion (arrowhead) immediately before reperfusion (upper left). The triphenyl-tetrazolium chloride (TTC)-stained brain of the same animal shows the infarct 48 hours later (upper right). After spatially co-registering the two images using surface landmarks, a line profile (red dotted line) is drawn between lambda and the occluded middle cerebral artery branch, and CBF plotted along this profile as a function of distance from lambda using laser speckle images (lower left). The CBF level corresponding to the infarct edge (blue dotted line) is then located (green arrow) based on the distance of infarct edge from lambda on this profile. This value represents the CBF threshold for viability, below which the tissue infarcted (35% residual CBF in this representative mouse). When calculated in this way for each mouse, the viability threshold was significantly higher in ApoE^−/− mice after 8 but not 4 weeks of high-fat diet (HFD) compared with age and diet-matched controls (lower right), suggesting that, in addition to suffering worse perfusion defects after distal middle cerebral artery occlusion (dMCAO), ApoE^−/− brains require even higher CBF to survive. Numbers of mice in each group are shown on the bars. *P<0.05 vs. wild type (WT); ^†P<0.05 vs. 4 week HFD.