Hyperbaric Oxygen Promotes Nerve Regeneration On Spinal Cord Injury Rats Via Mitochondrial GTPases-related Pathway

?Objective?Traumatic spinal cord injury(SCI)is an acute necrosis of spinal cord neurons suffering from external force.The secondary apoptosis will further aggravate the injury of the cells.It appears to dusfunctions in movement,sensation and many others,which seriously affects patients' life quality.As is known to all,oxidative stress after SCI is an important mechanism to activating Caspase apoptosis pathway,which is also the initial theoretical foundation for hyperbaric oxygen therapy(HBO).The latest research reveals that the regulation of mitochondrial GTPase structural protein Drp1 predates the classic apoptosis pathway activated.Therefore,we hypothesized that mitochondrial GTPase-related pathway may plays a key role in the treatment of HBO after SCI,which has' t been any discussion before.In this study,nerve motor function assessment,pathologcal section and Western Blot was detected to explore the regulation pattern of the key protein in GTPase-related pathway like Drp1,Fis1,Mfn1/Mfn2,Cyt C and Caspase-3,and the regulation mechanism of nerve regeneration on HBO in different time windows after SCI within the control of this pathway.Hoping to develop a new theoretical basis and technical choice for the clinical treatment of acute phase of SCI.?Methods? Part OneSD rats were randomly divided into Con group,which is for negative control,Sham group with claminectomy and SCI group with spinal cord injury.The BBB lower extremity motor function score,motor evoked potentials and tissue sections with HE staining is used to detect the state changes of nerve damage and repairment after SCI in rats.TUNEL positive cells was calculated as the symnol of apoptotic neurons after SCI.Detecting Drp1,Fis1,Mfn1/Mfn2 protein content at 6h,12 h,24h,48 h,72h,7d and 10 d after SCI by Western Blot so as to explore the temporal variation of mitochondrial GTPases related pathway.Part TwoOn the basis of the first part,the SD rats were randomly divided into sham group,SCI group and HBO group,while the HBO group is intervented with HBO in different time windows and is therefor divided into three sub-groups as HBO-24 h group,HBO-48 h group,and HBO-72 h group separately.During 3 days HBO course,the BBB lower extremity motor function score,motor evoked potentials is used to detect the state changes of apoptotic neurons after SCI.At the end of HBO,tissue sections with HE staining and TUNEL test is used to detect HBO's influence on neuronal apoptotic and nerve regeneration after SCI.Western Blot explores how the HBO regulates the protein expression of Drp1,Fis1,Mfn1/Mfn2,Cyt C and Caspase-3 in lesion area,which is related to mitochondrial GTPase pathway after spinal cord injury in rats.?Result? Part OneCompared with Con and Sham group,HE staining of SCI group showed spinal cord tissue severely damaged,blur boundaries between grey and white matter,white matter loose and irregular,following cell swelling,microcapsule cavity changes,glial scar and Nissl body formation in the damage zone.BBB score and MEP amplitude decreased demonstrably and then showed a spontaneous improvement trend,while the expression of TUNEL positive cells was abundant comparing with Con and Sham group,and these changes were significant different(P < 0.01).The expression of Drp1 and Fis1 decreased in 6h,increased after 12 h and beyond normal level,reached the peak at 24 h and then following a downward trend to normal level at 10 d.The expression of Mfn1/Mfn2 increased in 6h,decreased after 12 h and blow normal,reached the peak at 24 h and then following a upward trend to normal level at 10 d.The difference between the SCI group and the other groups were statistically significant(P < 0.05).Part TwoSeventh days after injury,the BBB score,amplitude of MEP and the HE staining indicated obvious improvement of nerve function and cellular structure on spinal cord tissue in three HBO sub-groups.The expression of TUNEL positive cells presented different degrees of reduction comparing with SCI group.In addition,the expression level of Drp1 and Fis1 increased,while that of Mfn1/Mfn2,Cyt C and Caspase-3 has a decline comparing with the SCI group,which have statistical significant(P < 0.05).Additionally,comparing with HBO-48 h group,HBO-72 h group,the diversities above were more obvious in HBO-24 h group(P < 0.05).?Conclusion?The disorder of motor function and the apoptosis of neurons were obvious in rats after SCI.And the protein expression of Drp1,Fis1 and Mfn1/Mfn2 present a phase variation within 10 days after SCI.Neuroprotective effect of early HBO intervention may be bound up with the regulation of Drp1,Fis1 and Mfn1/Mfn2,which leads to apoptosis attenuation.Furthermore,HBO at 24 h after SCI could have a more effective prevention through mitochondrial GTPase-related pathways.