Dysregulation of RNF213 promotes cerebral hypoperfusion

Abstract

RNF213 is a susceptibility gene for moyamoya disease, yet its exact functions remain unclear. To evaluate the role of RNF213 in adaptation of cerebral blood flow (CBF) under cerebral hypoperfusion, we performed bilateral common carotid artery stenosis surgery using external microcoils on Rnf213 knockout (KO) and vascular endothelial cell-specific Rnf213 mutant (human p.R4810K orthologue) transgenic (EC-Tg) mice. Temporal CBF changes were measured by arterial spin-labelling magnetic resonance imaging. In the cortical area, no significant difference in CBF was found before surgery between the genotypes. Three of eight (37.5%) KO mice died after surgery but all wild-type and EC-Tg mice survived hypoperfusion. KO mice had a significantly more severe reduction in CBF on day 7 than wild-type mice (KO, 29.7% of baseline level; wild-type, 49.3%; p = 0.038), while CBF restoration on day 28 was significantly impaired in both KO (50.0%) and EC-Tg (56.1%) mice compared with wild-type mice (69.5%; p = 0.031 and 0.037, respectively). Changes in the subcortical area also showed the same tendency as the cortical area. Additionally, histological analysis demonstrated that angiogenesis was impaired in both EC-Tg and KO mice. These results are indicative of the essential role of RNF213 in the maintenance of CBF.

Figure 1

Brain magnetic resonance (MR) imaging. MR images on day 7 of the KO mouse (Animal Code: KO-5 in Supplementary Table 1) that died on day 11 after surgery are shown. Cerebral infarction is seen in the caudate nucleus (red arrowheads), and pre-Wallerian degeneration is seen in the cerebral peduncle (yellow arrowheads). T2WI, T2 weighted image; DWI, diffusion weighted image; ADC, apparent diffusion coefficient.

Figure 2

Temporal profiles of cerebral blood flow (CBF) of mice with bilateral common carotid artery stenosis (BCAS). (a) Regions of interest (ROIs) used in the analyses of CBF images obtained from arterial spin-labelling magnetic resonance imaging. The CBF values in the cerebral cortex and the subcortical area were calculated from the six blue and two red ROIs, respectively. (b) Representative coronal CBF images obtained from arterial spin labelling in WT, KO and EC-Tg mice. (c) Temporal profiles of CBF presented as absolute values (mL/100 g/min) in the cortical and subcortical parenchymal areas. A column with a bar represents mean ± SD of CBF. Two-way repeated measures ANOVA was conducted for CBF values between genotypes and time interaction terms. In the cortical area, genotypes significantly affected the CBF values (p = 0.012) and the interaction was marginally significant (p = 0.066). In the subcortical area, both genotypes (p = 0.049) and interaction (p = 0.014) were significant. Additionally, in both the cortical and the subcortical areas, there were significant differences in the CBF values between the three genotypes using one-way ANOVA on day 7 (Cortical, p = 0.045; Subcortical, p = 0.046 (WT, n = 15; KO, n = 7; EC-Tg, n = 8)) and day 28 (Cortical, p = 0.035; Subcortical, p = 0.048 (WT, n = 14; KO, n = 5; EC-Tg, n = 8), but not pre-surgery (pre) (Cortical, p = 0.17; Subcortical, p = 0.61 (WT, n = 15; KO, n = 8; EC-Tg, n = 8)). On day 7, CBF in KO mice was significantly decreased compared with the other genotypes according to Tukey’s test. On day 28, CBF in KO and EC-Tg mice was significantly decreased compared with WT mice; *p < 0.05.

Figure 3

Intracranial arterial flow after BCAS assessed by magnetic resonance angiography (MRA). Representative images of intracranial arterial flow in WT, KO, EC-Tg mice are shown. Images were obtained by a 7 Tesla brain MRA before (pre) and at 7 and 28 days after BCAS.

Figure 4

Cerebral cortex stained for glucose transporter 1 (Glut1) of surviving mice. (a) Representative images of Glut1-immunostained sections of cerebral cortex of WT, KO and EC-Tg mice at 28 days after BCAS. Scale bar represents 100 μm. (b) Quantified results of cerebral microvessels. A column with a bar represents mean ± SD of the number of cerebral microvessels/mm². There was a significant difference in the number of cerebral microvessels between the three genotypes using one-way ANOVA (WT, n = 6; KO, n = 5; EC-Tg, n = 8; p = 0.002). There were significant differences between KO and WT (p = 0.034) and between EC-Tg and WT (p = 0.001) using Tukey’s test; *p < 0.05 vs WT.