Mitochondrial crisis in cerebrovascular endothelial cells opens the blood-brain barrier

Abstract

Background and purpose: The blood-brain barrier (BBB) is a selectively permeable cerebrovascular endothelial barrier that maintains homeostasis between the periphery and the central nervous system. BBB disruption is a consequence of ischemic stroke and BBB permeability can be altered by infection/inflammation, but the complex cellular and molecular changes that result in this BBB alteration need to be elucidated to determine mechanisms.

Methods: Infection mimic (lipopolysaccharide) challenge on infarct volume, BBB permeability, infiltrated neutrophils, and functional outcomes after murine transient middle cerebral artery occlusion in vivo; mitochondrial evaluation of cerebrovascular endothelial cells challenged by lipopolysaccharide in vitro; pharmacological inhibition of mitochondria on BBB permeability in vitro and in vivo; the effects of mitochondrial inhibitor on BBB permeability, infarct volume, and functional outcomes after transient middle cerebral artery occlusion.

Results: We report here that lipopolysaccharide worsens ischemic stroke outcome and increases BBB permeability after transient middle cerebral artery occlusion in mice. Furthermore, we elucidate a novel mechanism that compromised mitochondrial function accounts for increased BBB permeability as evidenced by: lipopolysaccharide-induced reductions in oxidative phosphorylation and subunit expression of respiratory chain complexes in cerebrovascular endothelial cells, a compromised BBB permeability induced by pharmacological inhibition of mitochondrial function in cerebrovascular endothelial cells in vitro and in an in vivo animal model, and worsened stroke outcomes in transient middle cerebral artery occlusion mice after inhibition of mitochondrial function.

Conclusions: We concluded that mitochondria are key players in BBB permeability. These novel findings suggest a potential new therapeutic strategy for ischemic stroke by endothelial cell mitochondrial regulation.

Keywords: blood-brain barrier; mitochondria; stroke.

Fig. 1. LPS exacerbates stroke infarct volume in mice

(A) Scheme depicting the experimental design. LPS (100 μg/kg, i.p.) or vehicle (saline, i.p.) was administered 30 min prior to right transient middle cerebral artery occlusion (tMCAO; 30 min) and 48 hour reperfusion was performed. (B) Infarct volumes were measured at 48 hours after ischemia induction. Representative TTC-stained coronal sections used to analyze infarction of mice treated with vehicle vs. LPS. (C) Mice treated with LPS had significantly larger infarct volume than vehicle group in cortex, striatum and total hemisphere. Mean ± S.D.; n = 7 per group; Student's t test. *, P < 0.05; **, P < 0.01.

Fig. 2. LPS increases early BBB leakage and infiltration of neutrophils into the brain of stroke mice

(A) Scheme of experimental design and workflow. LPS (100 μg/kg, i.p.) or vehicle (saline, i.p.) was administered 30 min prior to right tMCAO (30 min occlusion) and 6 hour reperfusion was performed. (B) Evans blue accumulation was visible in the brain of LPS pre-treated stroke mice (Red arrows) and quantification of Evans Blue extravasation (μg/g brain tissue) in contralateral (left) and ipsilateral (right) hemispheres. Vehicle, n = 6; LPS, n = 7. (C) Representative FACS data for individual hemispheres from mice treated with vehicle vs. LPS after 30 min tMCAO plus 6 hours reperfusion, and statistical analysis of infiltrated PMNs in the contralateral (left) and ipsilateral (right) hemispheres measured by FACS. N = 5 per group. (D) Neurological score at combined two end points (6 hours and 48 hours) after tMCAO. Vehicle, n = 18, LPS, n = 23. Data are expressed as mean ± S.D.; One-way ANOVA followed by post-hoc Tukey's test was used for multiple group comparison and Student's t test was used for two group statistical analysis. (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001.)

Fig. 3. LPS reduces mitochondrial function in cultured and primary cerebrovascular endothelial cells

(A) Immunofluorescence staining of mitochondrial (MitoTracker, purple) co-stained with MyD88 (green), suggests that the MyD88 expresses on mitochondria of cultured cerebral vascular endothelial cells (cCVECs). Nuclei were stained with DAPI (blue). Results are representative from four independent experiments. Images were taken under 63× objective. Scale bars = 10 μm. (B) Bioenergetics functional assay in cCVECs exposed to various concentration of LPS compared to vehicle control for 24 hours. Basal respiration, ATP production, maximal respiration, and spare capacity were calculated from the assay presented in supplementary Figure III A. Results are representative from four independent experiments. N = 4 per group; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 vs. vehicle group. One way ANOVA followed by post-hoc Tukey's test was used for data analysis. (C) Bioenergetics functional assay in pCVECs exposed to various concentration of LPS compared to vehicle control for 24 hours. Basal respiration, ATP production, maximal respiration, and spare capacity were calculated from the assay presented in supplementary Figure III B. Results are representative from four independent experiments. N = 4 per group; *, P < 0.05 vs. vehicle group. One way ANOVA followed by post-hoc Tukey's test was used for data analysis. (D) Analysis of mitochondrial specific proteins for complex I proteins (NDUFAF1, NDUFS2, NDUFA2), complex II protein (succinate dehydrogenase, SDH), complex III protein (cytochrome c, Cyc), and complex IV protein (cytochrome c oxidase, cox IV) in cCVECs after a 1 μg/ml LPS challenge for 48 hours. Flow cytometry confirmed that LPS decreases the expression of complex I (NDUFS2 and NDUFA2), complex III (cytochrome c) and complex IV (cox IV) proteins. Results are representative from three independent experiments. N = 3 per group; *, P < 0.05; **, P < 0.01; ****, P < 0.0001 vs. vehicle group. Student's t test was used for data analysis.

Fig. 4. Pharmacological inhibition of mitochondria increases BBB permeability in vitro

FITC-Dextran-70 permeability after addition of 20 μM rotenone (A), 20 μM FCCP (B) and 20 μM oligomycin (C) vs. vehicle to cultured cerebrovascular endothelial cells (cCVECs). Data are presented as both real-time rate of permeability (Two-way ANOVA followed by post-hoc Dunnett's test) and calculated apparent permeability coefficient (Papp, Student t-test). N = 3 per group. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (D) Confocal fluorescence images of cCVEC confluent monolayers after treatment with 20 μM rotenone, 20 μM FCCP and 20 μM oligomycin vs. vehicle for 2 hours. The immunohistochemistry staining of ZO-1 (red) was performed for tight junctions. Nuclei were stained with DAPI (blue). Mitochondrial inhibitors apparently disrupted tight junctions and resulted in gaps between cells (white arrows).

Fig. 5. Pharmacological inhibition of mitochondria increases BBB permeability in vivo

(A) Graph depicting the epidural application (EA) model. (B) Exposure of epidural membrane: vessels were visible under surgical microscope. (C) TTC staining in the EA model showing no brain injury. (D) Evans blue accumulation was visible in the brain of rotenone applied mice (red arrows) in EA model and quantitative analysis of Evans blue extravasation (μg/g brain tissue) in individual brain. N = 5 per group; One-way ANOVA followed by post-hoc Tukey's test. Data are expressed as mean ± S.D.; **, P < 0.01.

Fig. 6. Impairment of mitochondria function exacerbates stroke outcomes

(A) Scheme of experimental design and workflow. Rotenone (1mg/kg, i.p.) or vehicle (saline, i.p.) was administered 30 min prior to right tMCAO (60 min occlusion), BBB permeability was evaluated at 6 hour reperfusion and infarct volume was measured at 24 hour reperfusion. (B) Representative brain coronal sections for Evans blue accumulation and quantitative analysis of Evans Blue extravasation (μg/g brain tissue) in contralateral (left) and ipsilateral (right) hemispheres. Vehicle, n = 4; Rotenone, n = 4. One-way Data are analyzed with ANOVA followed by post-hoc Tukey's test (*, P < 0.05). (C) Representative TTC-stained coronal sections used to analyze infarct of tMCAO mice and quantitative analysis of infarct volumes. Mice treated with rotenone had significant larger infarct volume than vehicle group, in cortex, striatum, and total hemisphere. Rotenone (1 mg/kg, i.p.) or vehicle (saline, i.p.) was administered 30 min prior to right tMCAO (1 hour occlusion) and 24 hour reperfusion was performed. Vehicle, n = 8; Rotenone, n = 8. Data are analyzed with Student's t tests (**, P < 0.01; ***, P <0.001; ****, P < 0.0001.). (D) Rotenone worsened neurological deficits at 6 and 24 hours after tMCAO. Vehicle, n = 12; Rotenone, n = 12. Data are analyzed with Student's t test (****, P < 0.0001).