Bone Marrow Mesenchymal Stem Cells Derived Mitochondria Promote Survival Of Injured Neurons After Spinal Cord Injury

BackgroundSpinal cord injury(SCI)refers to the obvious somatic dysfunction caused by exogenous injury on the spinal cord,and even the irreversible delayed death of spinal cord neurons.Spinal cord injury includes primary injury and secondary injury,of which secondary injury is more serious.Pathological changes of secondary injury include production of oxygen free radicals(ROS),inflammation and necrosis,and release of cytokines,which increase the permeability of blood-brain barrier.Chronic injury leads to endoplasmic reticulum stress response,incorrect folding and unfolded protein aggregation in the endoplasmic reticulum,Ca balance disorder,induced CHOP/GAD153 expression,resulting in neuron apoptosis and limit the recovery of motor function.Bone marrow mesenchymal stem cells(BMSCs)have been widely reported in the treatment of spinal cord injury.Its protective effects include:paracrine exocrine,brain-derived growth factor(BDNF),vascular endothelial growth factor(VEGF),neurotrophic factor(NT-3,NT-4/5),nerve growth factor(NGF)and so on;regulating macrophages,reducing the expression of inflammatory factors to improve the microenvironment;regulating the activation of protease,protecting nerves,promoting the synthesis of vascular endothelial growth factor;stimulating new blood vessels.Formation;Reduction of endoplasmic reticulum and oxidative stress stimulate myelin sheath formation;Inhibition of glial scar formation.In addition,BMSCs also contain abundant mitochondria,which are the organelles of cell energy production and the main place for cell aerobic respiration.Some studies have shown that BMSCs can transport mitochondria to injured cells,which may play an important protective role by increasing ATP production and reducing oxidative stress damage.We hypothesize that BMSCs-derived mitochondria can be transferred to damaged neurons and may play a protective role by alleviating endoplasmic reticulum stress injury.ObjectiveTo explore whether BMSCs-derived mitochondria can transfer to injured neurons after spinal cord injury(SCI),alleviate endoplasmic reticulum stress injury and promote motor function recovery,and to explore its possible protective mechanism in order to provide new ideas for clinical treatment.Method(1)In vivo experiment:SCI model was established in SD rat.BMSCs were cultured and mitochondria were isolated.Mitochondria were labeled by MitoTracker Red before injection.The experimental animals were divided into three groups(n=11/group):Control group(Sham),SCI+vehicle group(SCI+Vehicle),SCI+mitochondrial injection group(SCI+Mito).SCI rats were perfused and fixed by 4%PFA at 48 h after spinal cord injury and the spinal cord sections were observed under laser confocal microscope.The number of apoptotic cells after SCI was detected by TUNEL staining;The lesion cavity of spinal cord was observed by H&E staining after 6 weeks of SCI;The expression of neurofilament-H(NF)was detected by immunohistochemistry;The expression of growth associated protein-43(GAP43)was detected by western blot.BBB scores were used to evaluate the recovery of motor function after injury.(2)In vitro experiment:Mitochondria in BMSCs were labelled by MitoTracker Red before co-culture.VSC4.1 motor neurons were labelled by CFSE and oxygen-glucose deprivation(OGD)model was established.Neurons were divided into three groups:Control group,OGD+Vehicle group,OGD+mitochondria group(OGD+Mito).BMSCs were directly or indirectly co-cultured with post-OGD neurons to observe the transfer of mitochondria.Meanwhile,primary cortical neurons were co-cultured with BMSCs to confirm the transfer between primary cultured neurons and BMSCs.Next,BMSCs were cultured in serum-free high glucose medium for 24 hours,then the supernatant was collected to obtain conditioned medium.Mitochondria in conditioned medium were examined by transmission electron microscopy.ATP content in neurons was detected by ATP kit.Neuron death was detected by LDH kit.The apoptotic rate was detected by AnnexinV-PI flow cytometry.The expression of endoplasmic reticulum stress apoptotic protein CHOP was detected by western blot.Result(1)In vivo experiment:Laser confocal microscopy confirmed that mitochondria could be uptaken by neurons;TUNEL staining showed that the number of apoptotic cells in SCI+Mito group was less than that in SCI+Vehicle group(25.67±5.51 vs 52±6.08);H&E staining showed that the area of lesion cavity in SCI+Mito group was less than that in SCI+Vehicle group(0.88±0.01 fold of SCI+Vehicle group).Immunohistochemical staining showed that the positive staining area of NF in SCI+Mito group was increased than that in SCI+Vehicle group(1.52±0.05 fold of SCI+Vehicle);Western blot showed that the expression level of GAP43 in SCI+Mito group was higher than that in SCI+Vehicle group(0.75±0.09 fold of Sham 0.50±0.07 fold of Sham);BBB score showed that the locomotor function of the SCI+Mito group was better than that in SCI+Vehicle group(9.8±0.80 vs 5.5±0.50).(2)In vitro experiment:OGD model of VSC4.1 motor neurons was successfully established.In direct co-culture condition,BMSCs derived mitochondria was transferred to injured neurons through tunnel nanotubes(TNTs),which was observed by Laser confocal microscopy.In transwell co-culture condition,BMSCs derived mitochondria was paracrinely secreted into the culture medium,which was uptaken by injured neurons.The single and intact mitochondria were observed under transmission electron microscopy.The transfer of mitochondria between primary cultured neurons and BMSCs was also confirmed.It was found that all injured neurons internalized MitoTracker red labeled mitochondria within 30 min.The content of ATP in the OGD+Mito group(1.48±0.01 nmol/mg)was higher than that in the SCI+Vehicle group(1.13±0.06 nmol/mg).LDH in the OGD+Mito group was less than that in the OGD+Vehicle group(63.40±0.05 fold of OGD+Vehicle).The apoptotic rate in the OGD+Mito group was lower than that in OGD+Vehicle group(1.49±0.09 fold of control vs 2.50±0.17 fold of control).Western blot showed that the expression level of CHOP in the OGD+Mito group was significantly lower than that in the OGD+Vehicle group(3.80±0.65 fold of control vs 9.31±1.72 fold of control).ConclusionBMSCs derived mitochondria could be transferred to injured neurons through TNTs or paracrine mechanisms,which reduced CHOP expression and cell apoptosis,promoted the expression of GAP43 and NF,lessened the area of lesion cavity,and significantly promoted the recovery of motor function in SCI rats.