| Background and purpose:According to the Global burden of disease(GBD)study,stroke is the leading cause of death in China,with approximately 2.4 million new cases and 1.1 million strokerelated deaths each year.Stroke is the most common cause of disability and the second most common cause of dementia worldwide.There are two types of stroke:ischemic and haemorrhagic,with ischemic strokes accounting for up to 87%of strokes.The most effective treatment for ischemic strokes is currently considered to be thrombolysis,but the short time window for treatment means that not all patients have access to thrombolysis,and the limited efficacy of commonly used drugs makes it urgent to explore new stroke treatments.Stroke brings with it a range of functional impairments due to neuronal death in the primary focus area and possible secondary lesions in the surrounding or distant area.There is growing clinical evidence that delayed,secondary white matter degeneration can occur in remote,non-ischemic cortical areas.Axonal connectivity is thought to be key in linking remote neuronal injury to the site of the primary lesion,suggesting that underlying white matter degeneration also occurs after cerebral ischemia.The use of light and electron microscopic methods in early animal studies showed that focal cortical ischemia damages white matter.Secondary degeneration of cerebral white matter following ischemia has been observed in several studies using non-invasive neuroimaging tools in a clinical setting.Although the role of secondary degeneration in stroke recovery has not yet been fully investigated,recent clinical studies suggest that secondary white matter degeneration is associated with neurological deficits and can predict functional outcome after stroke.An important pathological feature of cerebral ischemia is the disruption of energy supply,which usually results in mitochondrial dysfunction.Defective bioenergetics,abnormal mitochondrial morphology and structure and aberrant mitochondrial dynamics play a crucial role in triggering cell death.Mitochondria can be involved in the pathological mechanisms of ischemic stroke through increased facilitated ROS formation,oxidative stress,calcium overload,MPTP induction,control of cell death and inflammation.Mitochondrial dysfunction associated with the pathology of cerebral ischemia and targeting of mitochondria through novel therapeutic strategies can be used to mitigate the devastating consequences of cerebral ischemia.This provides direction for the use of novel mitochondrial-targeted therapies for the treatment of cerebral ischemia.Mitochondrial damage is recognised as a major cause of cell death and oxidative stress.Storing or promoting mitochondrial function is thought to hold promise for reducing secondary damage following cerebral ischemia.Studies have shown that in neurons the half-life of mitochondria is typically 18 to 24 days.Studies of mitochondrial transfer from astrocytes to ischemic neurons have provided the basis for the use of exogenous sources of mitochondria in the treatment of cerebral ischemia.There is now a series of studies that support that replacement of dysfunctional mitochondria would be an effective way to restore mitochondrial function and rescue cellular damage after stroke.These studies have focused on mitochondrial transplantation to improve symptoms of cerebral ischemia through the protection of neurons,astrocytes and other pathways.The brain is composed of two parts:grey matter and white matter.The grey matter consists mainly of neuronal cytosol and unmyelinated axons,which are responsible for information processing in the central nervous system.The white matter,on the other hand,consists mainly of myelinated axons in bundles,myelin-producing oligodendrocytes and other glial cells.White matter plays a vital role in signal transmission and communication between different areas of the brain.White matter damage is usually present in at least half of the lesion volume in stroke and traumatic brain injury.Oligodendrocytes(OLs)are responsible for the production and maintenance of myelin sheaths and for remyelination of myelin after axonal injury,OPCs are extremely sensitive to ischemia.Myelinated oligodendrocytes are very vulnerable to ischemic or traumatic injury and the loss of oligodendrocytes is thought to be an important factor in postinjury demyelination.During severe periods of ischemic white matter injury,oligodendrocyte structure is altered and the number of OPCs increases during the acute phase of ischaemia,a phenomenon that is more pronounced in the ischemic semidark zone and is considered a compensatory response.In fact,during the stage of cerebral white matter lesions,OPCs tend to proliferate and migrate to the injury zone,where they then differentiate into mature oligodendrocytes that repair damaged axons.Ischemia leads to progressive white matter damage accompanied by the loss of myelin and oligodendrocytes.There is a great lack of effective methods to protect oligodendrocytes and demyelinating cells from white matter lesions,both clinically and in basic research.The loss of oligodendrocytes is thought to be an important factor in demyelination after nerve injury.Previous studies of transplanted mitochondria after spinal cord injury have shown that green fluorescent protein(GFP)-labelled mitochondria can enter brain macrophages,endothelial cells,astrocytes,pericytes and oligodendrocytes.The study showed that transplanted allogeneic mitochondria survived for at least 28 d in the injured region of the traumatic spinal cord injury model,and that mitochondrial fragmentation,apoptosis,neuroinflammation and oxidative stress were significantly reduced after transplantation.Exogenous mitochondrial transplantation improved the recovery of somatosensory and motor functions after spinal cord injury and promoted the survival of white matter in the spinal cord of rats with traumatic spinal cord injury.Whether exogenous mitochondrial transplantation can exert a protective effect on postischemic white matter lesions and oligodendrocyte spectrum cells deserves further investigation.ObjectiveThis project intends to observe the protective effect of exogenous mitochondrial transplantation on myelin degeneration after photochemical brain ischemic injury in mice.Methods1.A mouse cerebral ischemia model was established by tail vein injection of Rose Bengal photochemical method,and the success of the model was verified by TTC staining 24h after ischemic injury,and the infarct volume was measured.2.Liver-derived mitochondria from homozygous mice were extracted by the kit,and the activity and ATP content of the extracted mitochondria were measured.The extracted fresh exogenous mitochondria were then injected by brain stereotaxic injection into the damaged area of cerebral ischemic mice,and the endocytosis of oligodendrocyte spectrum cells was assessed by immunofluorescence double staining using Mito-Tracker,and the effects of NG2 and CNPase distribution and expression were observed by immunofluorescence staining at 24h and 72h after perfusion of the sampled material.The endocytosis of exogenous mitochondria in oligodendrocyte lineage was observed in the damaged area after 24h and 72h of perfusion,and the mitochondrial activity and ATP content in the damaged area at 72h were also measured.3.The mice were randomly divided into 2 groups:ischemic control group and mitochondria treatment group.The survival and proliferation of OPCs after mitochondrial transplantation were observed by TUNEL/NG2 and Cleaved Caspase3/Sox10 immunofluorescence double staining at 3 d and Ki67/Sox10 and Ki67/PDGFRα double immunostaining at 5 d.4.Changes in motor function were assessed in both groups of mice at 1d,3d,7d and 21 d postoperatively(day post injury,dpi)by performing forelimb mobility tests,Slinding score and Rotarod test.MBP was detected by Western Blot and immunofluorescence staining,and demyelinating lesions in the brain tissue of both groups of mice were observed using electron microscopy.5.Functional clustering of genes after exogenous mitochondrial treatment was studied by RNA-seq sequencing and qPCR to investigate the relevant pathways that promote myelin formation.Result:1.A brain ischemia model was successfully prepared using photochemical methods.The activity and ATP content of the extracted liver-derived mitochondria were measured,suggesting high activity of liver-derived mitochondria.The activity and ATP content of mitochondrial respiratory chain complexes(Ⅰ,Ⅱ and Ⅲ)in the ischemic region were significantly enhanced after transplantation.Mito-Trackerlabelled exogenous mitochondrial endocytosis was assessed at 24 h and 3 d posttransplantation.Approximately 58%of NG2-positive cells and 40%of CNPasepositive cells were found to be Mito-Tracker-positive in a region 300μ m from the edge of the lesion border at 24 h post-transplantation.At 3 d post-transplantation,approximately 52%of NG2-positive cells and 43%of CNPase-positive cells in this region were Mito-Tracker-labelled.Exogenous mitochondria could be effectively endocytosed by oligodendrocytes of ischemic tissue after transplantation.2.we investigated the effect of exogenous mitochondria on the survival and proliferation of OPCs.double staining with TUNEL and NG2 immunostaining showed that TUNEL/NG2-positive cells in the ischemic zone were significantly reduced in the cortex of the mitochondrial transplantation group at 3 d posttransplantation(p<0.01).In addition,double staining with immunofluorescence of Sox 10 and activated Caspase-3(CC-3)showed a significant decrease in CC-3/Sox10 positive cells at 3 d post-transplantation(P<0.001).This indicates that exogenous mitochondrial transplantation promotes the survival of OPCs in the ischemic cortex.immunofluorescence double staining of Ki67 with two OPC markers,PDGFRa and Sox10,showed a significant increase in Ki67/PDGFRα positive and Ki67/Sox10 positive cells in the mitochondria-treated cortex at 5d compared to controls(P<0.001).It is suggested that exogenous mitochondrial transplantation can effectively promote the survival and proliferation of oligodendrocyte precursor cells around the ischemic area.3.Immunofluorescence staining and Western blot showed significantly higher levels of MBP in the damaged area in the mitochondria-treated group.In addition,demyelinated axons with thin,loosely myelinated sheaths were normally detected in the ischaemic control group under electron microscopy,whereas more axons with thick,intact myelin sheaths were observed in the damaged areas of the mitochondrial transplantation group.The G-ratio of myelin sheaths in the damaged areas of the mitochondria-treated group was significantly lower than that of the ischemic group.This suggests that exogenous mitochondrial transplantation can effectively promote remyelination in the ischemic cortex.4.The motor function was assessed using the stick-turning test,forelimb mobility test,and Sliding scores.Starting from 1 d after transplantation,forelimb mobility function was significantly improved in the mitochondria-treated group compared with the ischaemic group.Mice in the mitochondria-treated group showed significantly longer dwell time on the pole.It is suggested that exogenous mitochondrial transplantation helps to promote the recovery of neurological function in mice with cerebral ischemia.5.RNA-seq sequencing and qPCR revealed that the mitochondrial transplantation group had a large amount of metabolism-related gene expression and enrichment of pathways related to myelin formation such as lipid metabolism after exogenous mitochondrial transplantation compared with the ischemic control group,which may be one of the mechanisms of neuroprotection against cerebral ischemia by exogenous mitochondria. |
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