CADASIL mutations sensitize the brain to ischemia via spreading depolarizations and abnormal extracellular potassium homeostasis

Abstract

Cerebral autosomal dominant arteriopathy, subcortical infarcts, and leukoencephalopathy (CADASIL) is the most common monogenic form of small vessel disease characterized by migraine with aura, leukoaraiosis, strokes, and dementia. CADASIL mutations cause cerebrovascular dysfunction in both animal models and humans. Here, we showed that 2 different human CADASIL mutations (Notch3 R90C or R169C) worsen ischemic stroke outcomes in transgenic mice; this was explained by the higher blood flow threshold to maintain tissue viability compared with that in wild type (WT) mice. Both mutants developed larger infarcts and worse neurological deficits compared with WT mice, regardless of age or sex after filament middle cerebral artery occlusion. However, full-field laser speckle flowmetry during distal middle cerebral artery occlusion showed comparable perfusion deficits in mutants and their respective WT controls. Circle of Willis anatomy and pial collateralization also did not differ among the genotypes. In contrast, mutants had a higher cerebral blood flow threshold, below which infarction ensued, suggesting increased sensitivity of brain tissue to ischemia. Electrophysiological recordings revealed a 1.5- to 2-fold higher frequency of peri-infarct spreading depolarizations in CADASIL mutants. Higher extracellular K+ elevations during spreading depolarizations in the mutants implicated a defect in extracellular K+ clearance. Altogether, these data reveal a mechanism of enhanced vulnerability to ischemic injury linked to abnormal extracellular ion homeostasis and susceptibility to ischemic depolarizations in CADASIL.

Keywords: Microcirculation; Neuroscience; Potassium channels; Stroke.

Figure 1. Filament middle cerebral artery occlusion in TgNotch3^R90C and TgNotch3^R169C cohorts.

(A) Left: A representative image of 2,3,5-triphenyltetrazolium chloride–stained (TTC-stained) coronal sections 24 hours after transient filament middle cerebral artery occlusion (fMCAO). Infarct can be seen as nonstained tissue involving MCA territory. Right: Representative laser Doppler flowmetry (LDF) tracing shows decrease in regional cerebral blood flow (CBF) after common carotid artery occlusion (CCAO) followed by MCAO and reperfusion. Shaded segments indicate where CBF was measured relative to baseline. (B) Infarct volume (indirect method), neurological deficit score, and CBF after CCAO, during MCAO, and after reperfusion in the entire TgNotch3^R90C cohort (all ages pooled). In addition to nontransgenic WT mice, transgenic mice overexpressing the human WT Notch 3 (TgNotch3^WT mice) were used to control for overexpression in TgNotch3^R90C mice. One-way ANOVA followed by Tukey’s multiple comparisons for infarct volume and neurological deficit score (*P = 0.0035 vs. WT, *P = 0.0074 vs. TgNotch3^WT; †P = 0.0039 vs. WT, †P = 0.0372 vs. TgNotch3^WT) and 2-way ANOVA for repeated measures followed by Šidák’s multiple comparisons for CBF. ANOVA P values are also shown on each panel. Sample sizes are provided in Table 4. (C) Infarct volume (indirect method), neurological deficit score, and CBF after CCAO, during MCAO, and after reperfusion in the entire TgNotch3^R169C cohort (all ages pooled, Table 4). Unpaired t test for infarct volume and neurological deficit score, and two-way ANOVA for repeated measures followed by Šidák’s multiple comparisons for CBF (*P = 0.0326, TgNotch3^R169C vs. WT). ANOVA P values are also shown on each panel. Mean ± SD. Sample sizes are provided in Table 4.

Figure 2. Distal middle cerebral artery occlusion in TgNotch3^R90C and TgNotch3^R169C cohorts.

(A) Resting CBF calculated using laser speckle contrast inverse correlation time values before distal middle cerebral artery occlusion (dMCAO) did not differ between CADASIL mutant mice and controls. Sample sizes are provided in Table 4. Student’s t test. (B) A representative laser speckle contrast image taken 60 minutes after dMCAO shows regions with severe (residual CBF <20%), moderate (21%–30%), and mild (31%–40%) CBF deficit. Composite bar graphs show the areas of severe, moderate, and mild CBF deficit in TgNotch3^R90C and TgNotch3^R169C mice and their respective controls (TgNotch3^WT and WT) 60 minutes after dMCAO (all ages pooled). Two-way ANOVA for repeated measures. P values on each panel are those of main ANOVA. CBF deficit area is shown as mean ± SEM. (C) A representative laser speckle contrast image showing the perfusion defect during dMCAO (left) and 2,3,5-triphenyltetrazolium chloride–stained (TTC-stained) brain showing the infarct in the same brain 48 hours later (right). Images were spatially coregistered using surface landmarks. A line profile was drawn between lambda and the clip occluding the middle cerebral artery (yellow arrowhead). For each animal, CBF was plotted along the line profile as a function of distance from lambda using laser speckle images (bottom). The CBF at the infarct edge was determined (blue dotted lines), representing the CBF threshold for viability, below which tissue infarcted in each mouse. The average viability threshold was significantly higher in TgNotch3^R90C vs. TgNotch3^WT mice (all ages pooled). Unpaired t test.

Figure 3. Cerebrovascular anatomy in Notch3^R90C and Notch3^R169C cohorts.

Representative (A) ventral and (B) dorsal views show the circle of Wills anatomy and pial arterial anastomoses between middle and anterior cerebral arteries. Circles on the dorsal surface in B indicate the pial anastomoses analyzed for their number and distance to midline. ACA, anterior cerebral artery; BA, basilar artery; ICA, internal carotid artery; MCA, middle cerebral artery; PCA, posterior cerebral artery; PComA, posterior communicating artery. The sample size is 39 in total and details are provided in Table 4. One-way ANOVA, 2-way ANOVA, or unpaired t test. Panel A shows diameters of major arteries in the circle of Willis. Panel B shows the distance of pial collaterals from midline. Mean ± SD.

Figure 4. Peri-infarct spreading depolarization during filament middle cerebral artery occlusion in Notch3^R90C and Notch3^R169C cohorts.

(A) Representative extracellular DC potential recordings from peri-infarct cortex showing higher frequency of peri-infarct spreading depolarizations (SDs) in TgNotch3^R169C mice compared with WT mice after filament middle cerebral artery occlusion (fMCAO). Experimental setup shows intracortical glass micropipettes placed outside the ischemic core (purple area) to detect SDs. (B) Left: Experimental timelines showing the time of onset and end of recordings in each mouse, and time of occurrence of SDs (round symbols) in WT and TgNotch3^R90C or TgNotch3^R169C mice. Middle: Pooled cumulative SD numbers per animal over time after fMCAO. Right: The frequency of SDs and cumulative SD duration in WT and TgNotch3^R90C mice or TgNotch3^R169C mice. Unpaired t test. Sample sizes are provided in Table 4.

Figure 5. Extracellular K⁺ rise during spreading depolarizations in nonischemic cortex.

Representative extracellular DC potential and K⁺-sensitive electrode tracings show the measurement of the amplitude (a), area under curve (auc), onset slope (s), and duration (d) of the K⁺ surge during an SD. Graphs show these measurements. Unpaired t test for repeated measures. Sample sizes are provided in Table 4. Mean ± standard error.

Figure 6. Proposed gliovascular mechanism of extracellular K⁺ regulation when local buffering mechanisms are exceeded during spreading depolarizations.

(i) Upon intense depolarization states, such as anoxic or spreading depolarization, extracellular K⁺ concentration ([K⁺]_e) can rise above the 10–12 mM ceiling. (b) Astrocytes play a major role in regulating [K⁺]_e via rapid uptake and spatial buffering through the astrocytic syncytium. (c) Astrocytes send their end feet, almost completely encasing the cerebral vasculature, including the capillary bed, providing a route for gliovascular K⁺ siphoning. (d) The massive rise in [K⁺]_e during an SD might also facilitate direct entry of K⁺ into the perivascular space to reach the capillary endothelium. (e) Astrocyte end feet have high K⁺ conductance, in part, due to BK channels activated by intracellular Ca²⁺ elevations, such as those observed during SD, and release large amounts of K⁺ into the tight perivascular space. (f) This perivascular K⁺ is then taken up by the endothelial Na⁺/K⁺-ATPase, which is densely — and asymmetrically — localized on the abluminal membranes. (g) Endothelial cells then release the K⁺ into the blood stream via channels and/or pumps on the luminal membrane, including K_ir2.1, which is known to be activated by elevated perivascular [K⁺]_e. (h) Notch3^R169C mutation is associated with impaired endothelial K_ir2.1 channel function, linking CADASIL to impaired vascular K⁺ clearance.