An In Vitro Study Of Exogenous Mitochondria On Axonal Regeneration

Objective:To com Pare the effects of three kinds of exogenous mitochondria(liver mitochondria,skeletal muscle mitochondria,and cardiac mitochondria)on the survival and axon regeneration of Primary neurons.The effects of three kinds of exogenous mitochondria on neuronal axonal regeneration were evaluated.To observe the effect of liver mitochondria on axonal regeneration of injured neurons and neuronal function.And to evaluate the effect of liver mitochondria on li PId metabolism during axonal regeneration of injured neurons.Methods:In the first Part,cortical neurons were Primarily cultured from fetal C57BL/6 mice at 14-17 days of gestation.After 7 days of culture,the cells were identified,and the ty Pes and Purity of cultured cells were analyzed.Secondly,liver mitochondria,skeletal muscle mitochondria and cardiac mitochondria of C57BL/6 mice were extracted by differential centrifugation.Wound healing assay was Performed on neurons after 7 days of culture,and then the same amount of liver mitochondria,skeletal muscle mitochondria,and myocardial mitochondria were added to the culture su Pernatant of neurons in each group.Cell death was evaluated by Td T-mediated d UTP Nick-End Labeling(TUNEL)and live cell PI labeling.Western Blot and immunocytochemical staining were used to detect the ex Pression of GAP-43 Protein.SPectro Photometers were used to measure ATP levels in liver mitochondria,skeletal muscle mitochondria and cardiac mitochondria of C57BL/6 mice.In the second Part,cortical neurons were cultured from C57BL/6 fetal mice at 14-17days of gestation.Wound healing assay was Performed after 7 days of culture.The liver mitochondria of C57BL/6 mice were extracted by differential centrifugation,and the same amount of liver mitochondria was added to the culture supernatant of neurons in each grou P.Western Blot and immunocytochemical staining were used to detect the ex Pression of growth associated Protein(GAP-43).Mitochondrial body complex enzyme and ATP levels were measured using a spectrophotometer.The whole-cell Patch clamp technique was used to record and measure the effect of liver mitochondria on the synaptic activity of neurons after scratch.In the third Part,Primary cortical neurons of C57BL/6 fetal mice at 14-17 days of gestation were cultured as above.Wound healing assay was Performed after 7 days of culture.The liver mitochondria of C57BL/6 mice were extracted by differential centrifugation,and the same amount of liver mitochondria was added to the culture supernatant of neurons in each group.Western Blot and immunocytochemical staining were used to detect the ex Pression of li PId dro Plet marker Protein(Li Pin-1)and growth related Protein(GAP-43).Results:Part I:Identification of Primary cell neurons showed that the Purity of neurons was u P to 98%.Mitochondria were extracted from the liver tissue of C57BL/6mice by fast centrifugation.The results showed that about 4×10~8 mitochondria could be extracted from 100mg liver tissue,about 2×10~8mitochondria could be extracted from100mg myocardial tissue,and about 1×10~8 mitochondria could be extracted from100mg skeletal muscle tissue.Estimates of the amount of mitochondria contained in different tissues were made.TUNEL staining and PI staining showed that the liver mitochondria treatment group had the least cell death after three kinds of mitochondrial treatment,suggesting that liver mitochondria had the strongest ability to Promote neuronal survival.The expression of growth-associated Protein(GAP-43)showed that the ex Pression level of GAP-43 in the liver mitochondria treatment group was the highest among the three mitochondrial treatment groups.Further analysis revealed that the same number of liver mitochondria Produced higher levels of ATP.In addition,liver mitochondria could significantly Promote the activities of neuronal mitochondrial resp Iratory chain complex enzymes.Part II:After 7 days of Primary culture,wound healing assay was Performed.The effects of time gradient(6h、12h、24h、36h、48h、72h)and concentration gradient(4×10~8/ml、4×10~7/ml、4×10~6/ml、4×10~5/ml)on liver mitochondria were explored.Each concentration of liver mitochondria was added to the neuronal culture supernatant,and after elution,immunofluorescence staining for GAP-43 was Performed 6h12h,24h,36h,48h 72h later.The results showed that axons grew into the injured area in all groups.Quantitative analysis of GAP-43 immunofluorescence showed that the number and length of axons in the 4×10~6/ml group were the highest.We further examined the mitochondrial resp Iratory chain complex enzyme activity in neurons after delivery of liver mitochondria.The results showed that compared with the control grou P,mitochondrial treatment significantly increased the activities of complex II,complex III and complex V in damaged neurons.The whole-cell Patch-clamp experiments in Primary neurons cultured in vitro showed that there were significant differences in the frequency and amplitude of Postsynaptic membrane currents between the groups with and without mitochondrial treatment.It was shown that mitochondrial treatment significantly upregulated the excitatory Postsynaptic currents(EPSC)of neurons.Part III,wound healing assay was Performed on Primary cultured neurons after 7days.GAP-43 immunofluorescence staining was Performed after 48 hours of elution after 4×10~6/ml liver mitochondria were added to the neuron culture supernatant.The results showed that com Pared with the scratch control grou P,the mitochondria-treated group had a large number of axons growing into the injured area(shown as dashed line),accompanied by increased lipid droplet Production.Quantitative immunofluorescence analysis of growth-associated Protein GAP-43 and lipid droplet detection(Li Pin)showed that the most significant axon growth into the injured area was also the most significant li PId dro Plet formation.Meanwhile,Western-blot results showed that GAP-43expression and lipid droplet formation were significantly up-regulated in liver mitochondria.Discussion In the first Part,we used a Primary neuronal scratch model to com Pare the effects of three kinds of exogenous mitochondria(liver mitochondria,myocardial mitochondria,skeletal muscle mitochondria)on neuronal survival and axonal regeneration by viable cell PI staining,TUNEL staining,immunofluorescence staining for growth-associated Protein GAP-43,and Western Blot.Liver-derived mitochondria were found to have stronger ability to Promote neuronal survival and axon regeneration.In the second Part of the study,we examined the effects of different concentrations and treatment durations of liver mitochondria on axon regeneration.Our results showed that the number and length of regenerated axons were optimal at 4×10~6/ml mitochondria treatment for 48h.In the third Part of the study,we investigated the changes in li PId synthesis in injured neurons after liver mitochondrial treatment.The results showed that liver mitochondria could significantly Promote the generation of li PId dro Plets in neuronal cytopiasm and axons.