Evaluation of Mitochondrial Transplantation Therapy for Ischemia Reperfusion Injury

Abstract

Peripheral artery disease is a condition in which atherosclerotic obstruction of artery supplying blood to the lower limb produce hypoxia leading to loss of skeletal muscle function. Ischemia reperfusion injury (IRI) is the central manifestation of peripheral arterial disease that mainly compromises mitochondrial functions. IRI results in disturbance of mitochondrial calcium homeostasis, ROS scavenging system, and metabolic pathways of energy production which leads to mitochondrial Ca2+ and ROS accumulation triggering MPTP opening and cellular death. This urge to develop mitochondria-based therapeutic regimen. In IRI, cells that previously experienced ischemia due to atherosclerosis reestablishes the blood flow by reperfusion which causes further cellular damage. The present study was aimed to explore the effect of transplantation of allogenic mitochondria isolated from healthy mice liver into hind limb IRI model of mice. For this purpose, we first characterized two different populations of mitochondria for their integrity, viability, purity by performing MTT and MPTP assay. Acute hind limb IRI model was optimized by using grommets and orthodontic bands (ORB). Besides, the time-based model evaluation was also done using different ischemia time points. The IRI-elicited infarct size was observed by using TTC staining. The model development was then followed by in-vivo mitochondrial transplantation experiments accompanied with infarct size evaluation, histological and enzymatic (lactate dehydrogenase and Creatine kinase) level measurements. Results revealed that mitochondria isolated at 3000RPM were more viable and were used for mitochondrial transplantation. We proceeded with less invasive ORB for ischemia induction. Moreover, effect of ORB-induced ischemia at 45min and 120 min ischemia followed by 24 hours reperfusion yielded no difference. Histological as well as Lactate dehydrogenase assay (LDH) and Creatine kinase (CK) confirmed the IRI injury from 120 min ischemia with 24 hours reperfusion. Afterwards, viable and active mitochondria were injected at the onset of reperfusion into GS and TA muscle of IRI limb which resulted in significant reduction of LDH while non-significant reduction of infarct size and CK was observed. Our study confirmed the therapeutic benefit of mitochondrial transplantation in hind limb IRI injury.