Attenuation of oxidative damage by targeting mitochondrial complex I in neonatal hypoxic-ischemic brain injury

Abstract

Background: Establishing sustained reoxygenation/reperfusion ensures not only the recovery, but may initiate a reperfusion injury in which oxidative stress plays a major role. This study offers the mechanism and this mechanism-specific therapeutic strategy against excessive release of reactive oxygen species (ROS) associated with reperfusion-driven recovery of mitochondrial metabolism.

Aims and methods: In neonatal mice subjected to cerebral hypoxia-ischaemia (HI) and reperfusion, we examined conformational changes and activity of mitochondrial complex I with and without post-HI administration of S-nitrosating agent, MitoSNO. Assessment of mitochondrial ROS production, oxidative brain damage, neuropathological and neurofunctional outcomes were used to define neuroprotective strength of MitoSNO. A specificity of reperfusion-driven mitochondrial ROS production to conformational changes in complex I was examined in-vitro.

Results: HI deactivated complex I, changing its conformation from active form (A) into the catalytically dormant, de-active form (D). Reperfusion rapidly converted the D-form into the A-form and increased ROS generation. Administration of MitoSNO at the onset of reperfusion, decelerated D→A transition of complex I, attenuated oxidative stress, and significantly improved neurological recovery. In cultured neurons, after simulated ischaemia-reperfusion injury, MitoSNO significantly reduced ROS generation and neuronal mortality. In isolated mitochondria subjected to anoxia-reoxygenation, MitoSNO restricted ROS release during D→A transitions.

Conclusion: Rapid D→A conformation in response to reperfusion reactivates complex I. This is essential not only for metabolic recovery, but also contributes to excessive release of mitochondrial ROS and reperfusion injury. We propose that the initiation of reperfusion should be followed by pharmacologically-controlled gradual reactivation of complex I.

Keywords: Hypoxia/ischaemia; Ischaemia/reperfusion damage; Mitochondrial complex I; Nitrosation.

Fig. 1.

A – Illustration of the study design. B, C - C-I activity and the D-form content in naïve (n = 6) and HI-brains at the end of HI-insult (0 min of reperfusion, n = 4), at 15 (n = 4) and 30 min of reperfusion (n = 6). D – The D-form content in naïve (n = 4) and HI-mice treated with vehicle (veh, n = 6) or with MitoSNO (n = 6). E - MitoSNO inactive metabolite (MitoNAP) in different regions of the brain following intranasal administration of MitoSNO to HI-mice (n = 5). F and G – Activities of C-II and C-IV in naïve (n = 6) and HI-mouse brains (n = 4–6).

Fig. 2.

A – Immunostaining and statistical analysis for 4-HNE positive cells (red) in the HI-cortex of mice treated with vehicle (n = 6) or MitoSNO (n = 6) and image of the cortex of naïve mouse. Counterstaining, MAP-2 (green), Dapi (blue). B – Immunoblot and statistical analysis of 3-NT expression in the HI-brains of vehicle-treated and MitoSNO-treated (n = 6) and naive mice (n = 6). C – left and middle; MitoSOX fluorescence (red) at 60 min of reperfusion following OGD-insult in hippocampal murine neurons in the presence (n = 5) or absence (n = 6) of 0.5 μM of MitoSNO, compared to naïve cells (n = 7). Right; Cellular viability of OGD-cells treated with MitoSNO or vehicle for the initial 60 min of reperfusion (n = 5).

Fig. 3.

A – Actual and mean values of cerebral infarct volumes at 24 h of reperfusion and representative images of TTC-stained brains from HI-mice treated with vehicle or MitoSNO. B – Sensorimotor performance tested at ten days after HI-insult in mice treated with vehicle (Veh, n = 17) or MitoSNO (n = 18) compared to Naives (n = 8). C – Representative images of Nissl-stained coronal sections obtained at ten days following HI-insult and D – Actual and mean values of residual volume of the ipsilateral hemisphere (% of the contralateral hemisphere).

Fig. 4.

A – Changes in the A/D ratio in intact mitochondria during anoxia and reoxygenation (n = 3 for each time point). B – The rate of succinate-supported H₂O₂ production by control (black) mitochondria and mitochondria pretreated with MitoSNO (red, MitoSNO) during reoxygenation. Four traces were analyzed to obtain mean ± SEM. C – Representative tracing of superoxide generation by succinate-supported SMP with the A-form of C-I (Black) or the D-form of C-I (Red) with statistical analysis. D – Phosphorylating respiration rate (state 3) at 60 min of reperfusion in mitochondria isolated from HI-mice treated with vehicle (n = 4) or MitoSNO (n = 4). Substrate, Malate-Glutamate. E – Succinate-supported H₂O₂ emission rate in the brain mitochondria isolated at 60 min of reperfusion from the ipsilateral hemisphere of HI-mice treated with vehicle (n = 8) or MitoSNO (n = 10).

Fig. 5.

Schematic presentation of the proposed mechanism and therapeutic strategy. During circulatory arrest (ischaemia) accumulated succinate does not generate ATP (lack of O₂) and ROS (C-I in the D form). Upon ROSC (reperfusion), succinate metabolism produces ATP (FET) and contributes to ROS burst as C-I transitions to the A-form which activates RET. Administration of MitoSNO by preservation of C-I in the D-form restricts RET and limits ROS release.