Mitochondria dysfunction, a potential cytoprotection target against ischemia-reperfusion injury in a mouse stroke model

Abstract

More than 50 ​% of patients undergoing mechanical thrombectomy (MT) for ischemic stroke have a poor functional outcome despite timely and successful angiographic reperfusion, highlighting the need for adjunctive treatments to reperfusion therapy. Mitochondria are key regulators of cell fate, by controlling cell bioenergetics via oxidative phosphorylation (OXPHOS) and cell death through the mitochondrial permeability transition pore (mPTP). Whether these two main mitochondrial functions are altered by reperfusion and could represent a new cytoprotective approach remains to be elucidated in mice. Swiss male mice underwent either permanent or transient middle cerebral artery occlusion (pMCAO or tMCAO), with neuroscore evaluation and multimodal imaging. The area at risk of necrosis was evaluated by per-occlusion dynamic contrast-enhanced ultrasound. Final infarct size was assessed at day 1 by MRI. Cortical mitochondrial isolation was subsequently performed to assess mPTP sensitivity by calcium retention capacity (CRC) and OXPHOS. A cytoprotective treatment targeting mitochondria, ciclosporine A (CsA), was tested in tMCAO, to mimick the clinical situation of patients treated with MT. Reperfusion after 60 ​min of ischemia improves neuroscores but does not significantly reduce infarct size or mitochondrial dysfunction compared to permanent ischemia. CsA treatment at reperfusion mitigates stroke outcome, decreases final infarct size and improves mitochondrial CRC and OXPHOS. Mitochondrial dysfunctions, i.e. reduced mPTP sensitivity and decreased oxygen consumption rates, were observed in pMCAO and tMCAO regardless of the reperfusion status. CsA improved mitochondrial functions when injected at reperfusion. These suggest that both mPTP opening and OXPHOS alterations are thus early but reversible hallmarks of cerebral ischemia/reperfusion.

Keywords: Cerebral ischemia-reperfusion; Mitochondrial respiration; PTP; Therapeutic.

Graphical abstract

Fig. 1

Description of the study. A. Study design. Ischemic stroke was induced by middle cerebral artery occlusion (MCAO) using the monofilament model. Mice underwent permanent or transient MCAO (60 ​min) or Sham surgery. Ciclosporin A (CsA) was given in a subgroup of mice with tMCAO. Per-occlusion ultrasound imaging was performed to verify: (i) arterial occlusion using color Doppler mode and (ii) hypoperfusion of the MCA territory using contrast (microbubbles)-enhanced ultrasound. Neuroscores and MRI were obtained 24 ​h post-surgery. Brains were sampled and mitochondria extracted for functional analysis (CRC and OXPHOS). B. Study Flow Chart. A total of 52 animals were randomized. Inclusion/exclusion criteria were defined a priori. MCAO mice that did not have occlusion and/or perfusion defect on per-occlusion ultrasound were excluded. C. Imaging protocol: the yellow arrow shows the right MCA occlusion on Doppler and the perfusion defect appears in blue on CEUS. The yellow dotted line on the MRI delineates the infarct.

Fig. 2

Comparison between transient middle cerebral artery (tMCAO) and permanent MCAO (pMCAO) mice. A. Neuroscores in the two groups, tMCAO and pMCAO. Three outcome categories were defined based on the Bederson score (0–5): mild (4–5), moderate (2–3) and severe (0–1). One mouse from the tMCAO group and 2 mice from the Sham group did not get a neuroscore because of neurobehavior experimenter unexpected absence. Neuroscores were significantly improved in tMCAO compared to pMCAO. Chi-square test. B. Quantification of the area at risk (AAR) in tMCAO and pMCAO groups. The AAR was defined as the hypoperfused region on per-occlusion contrast-enhanced ultrasound. As expected, AAR were not statistically different between tMCAO and pMCAO. Mann-Whitney test, Median [interquartile range 25 ​%; 75 ​%]. C. Quantification of the infarct size calculated as the ratio of lesion volume measured on T2-weitghed MRI over AAR (%HLVc/%AAR) in tMCAO and pMCAO groups. Infarct sizes were not different between groups. Mann-Whitney test, Median [interquartile range 25 ​%; 75 ​%]. D. Lesion volume presented as percentage of the hemisphere (%HVLc) relative to the AAR, showing that for a given AAR, infarct size was lower for tMCAO mice, although this was not significant (p ​= ​0.11).

Fig. 3

Comparison of mitochondrial functions between sham, transient middle cerebral artery (tMCAO) and permanent MCAO (pMCAO) mice. Upper figures: right ischemic hemisphere. A. Calcium Retention Capacity (CRC), B. OXPHOS after stimulation of complex I, II or IV. Bottom figures: left non-ischemic hemisphere. C. CRC, D. OXPHOS after stimulation of complex I, II or IV. Data are expressed as Median [interquartile range 25 ​%; 75 ​%]; Kruskal-Wallis test. As expected, no statistical difference was found between groups in the non-ischemic hemisphere. In contrast, CRC and OXPHOS were decreased in the ischemic hemisphere in MCAO groups vs Sham, with no statistically significant difference between permanent and transient MCAO.

Fig. 4

Effect of Cyclosporine (CsA) treatment in mice with transient middle cerebral artery (tMCAO). A. Neuroscores in tMCAO and tMCAO+CsA groups. Neurofunctional outcome was improved in CsA-treated mice although statistical significance was not reached. B. Representative infarct lesions in tMCAO and tMCAO ​+ ​CsA groups (4 central slices on T2-weitghed MRI). C. Quantification of the area et risk (AAR) in tMCAO and tMCAO ​+ ​CsA groups. As expected, AAR were not statistically different between tMCAO and pMCAO. Mann-Whitney test, Median [interquartile range 25 ​%; 75 ​%]. D. Quantification of the infarct size calculated as the ratio of lesion volume over AAR (%HVLc/%AAR) in tMCAO and tMCAO ​+ ​CsA groups. The primary endpoint was statistically lower in CsA-treated animals. Mann-Whitney test, Median [interquartile range 25 ​%; 75 ​%]. E. Lesion volume presented as percentage of the hemisphere (%HVLc) relative to the area at risk (%AAR). For a given AAR, infarct size was significantly lower for CsA-treated mice (p ​= ​0.0494).

Fig. 5

Effect of CsA treatment on mitochondrial function in mice with transient middle cerebral artery (tMCAO). Upper figures: right ischemic hemisphere. A. Calcium Retention Capacity (CRC), B. OXPHOS after stimulation of complex of I, II or IV. Bottom figures: left non-ischemic hemisphere. C. CRC, D. OXPHOS after stimulation of complex I, II or IV. Data are expressed as Median [interquartile range 25 ​%; 75 ​%]; Mann-Whitney test. As expected, no statistical difference was found between groups in the non-ischemic hemisphere. In contrast, CRC and OXPHOS were improved in the ischemic hemisphere of CsA-treated mice.

Fig. 6

Correlation between CRC and %HVLc in the left non-ischemic hemisphere (A) and right ischemic hemisphere (B). There was no relationship between CRC and infarct size in the non-ischemic hemisphere. In contrast, CRC was negatively correlated to infarct size in the ischemic hemisphere.