| The morbidity of human spinal cord injury presented the trend of progressive increase in recent years with self-evident disabling nature.Spinal cord injury mainly contains two categories of primary injury and secondary injury,among which the latter means that,after the exogenic action,complicated physiological and biochemical reaction generated in injured area that damages normal spinal cord tissues around and enlarges injury area.At present,most researches on spinal cord injury focus on secondary injury.Meanwhile,spinal cord injury involves in many pathology mechanisms with poor therapeutic effect;so current treatment of spinal cord injury mainly starts with anti-inflammatory reaction,anti-oxidative stress and anti-apoptosis.Oxidative stress is the significant factor of nerve cell necrosis,apoptosis and neurolo gical dysfunction in secondary spinal cord injury.The research made by Japanese scholar Ohsawa in 2007 indicated that,hydrogen can selectively remove hydroxyl and peroxynitrite anion,lower cerebral infarction size and improve brain function,which triggered people’s extensive research on hydrogen.For the past few years,more and more experimental researches discovered that,hydrogen molecule plays role in oxidative stress injury.As a new antioxidant,hydrogen carries the characteristics of excellent permeability and extensive source.Therefore,by cultivating spinal cord neuronal cells and making neurone mechanical damage model,our research found that,hydrogen in high concentration can alleviate oxidative stress injury,protect mitochondrial function and inhibit neuronal cell apoptosis,which is hopeful to provide selection of treating spinal cord injury.Part 1: Separation,culture and identification of spinal cord neuronal cellsIn this experiment,the female mice just four days after birth were selected as raw material.The primary neuronal cells were purified and cultured based on differential attachment;neurone identification was implemented through neurone specificity dyeing(enolase dyeing);immunofluorescence microscope,in full view,saw yellow and green neurone with clear neurite.The result indicated successfully separated and cultured mice spinal cord neuronal cells.Part 2: Analysis of function of hydrogen in different concentrations on morphology of mechanical injured neuroneThis experiment researched the influence of hydrogen in three different concentrations(50%,65% and 75%)on mechanical injured neurone morphology.By means of neurone mechanical injury model,the light microscope saw that,in 75% hydrogen group,a few spinal cord neuronal cells in injured area migrated after hydrogen therapy;the differences of nerve specificity enolase(NSE)immunofluorescence dyeing result in different groups were not obvious.Microfilament microtubule immunofluorescence dyeing result saw that,in 75% hydrogen group,cell morphology basically recovered uniformly;cytoskeleton stretched and indicated that,compared with hydrogen in concentrations of 50% and 65%,the hydrogen in concentration of 75% was more beneficial to the transfer of neurone in neurone mechanical injury model,which maintained the morphology of neurone.Part 3: Analysis of protective function of high concentration hydrogen on mechanical injured neuroneThis experiment researched the protective function of hydrogen in concentration of 75% on mechanical injured neurone.Superoxide anion fluorescent probe was used to detect reactive oxide species of neurone in different groups after operation;Flow cytometry and Tunel dyeing were applied to detect neuronal apoptosis;Enzyme linked immunosorbent assay was conducted to detect MDA and 8-OhdG contents;Western blot was carried out to detect contents of SOD1,SOD2,Nox2 and Nox4.The result showed that,the contents of ROS,MDA,8-OhdG,Nox2 and Nox4 increased after spinal cord neuron injury;the contents of SOD1 and SOD2 decreased;after hydrogen therapy in high concentration,the contents of ROS,MDA,8-OhdG,Nox2 and Nox4 decreased;the contents of SOD1 and SOD2 increased.Flow cytometry and Tunel dyeing result showed that,neuronal apoptosis increased after spinal cord neuron injury;the number of neuronal apoptosis decreased after hydrogen therapy in high concentration.The above results indicated that,hydrogen in concentration of 75% inhibited the expressions of Nox2 and Nox4,enhanced the expressions of SOD1 and SOD2,controlled ROS generation,alleviated lipid peroxidation and restricted neuronal apoptosis.Part 4: Research on function of high concentration hydrogen on spinal cord neurone mitochondriaThis experiment researched the influence of hydrogen in concentration of 75% on spinal cord neurone mitochondria,observed the ultrastructure,detected mitochondrial membrane potential change,neurone ATP content,antioxidant enzyme and apoptin content.The result showed that,after nurone mechanical injury,mitochondria was injured,membrane potential was lowered,ATP content was lowered,Bax and caspase 3 expressions up regulated,Bcl-2 expression down regulated,and antioxidant enzyme PrxIII and Trx2 contents were lowered.After rendering hydrogen therapy,injured mitochondria recovered to some extent,membrane potential and ATP contents enhanced,Bax and caspase 3 expressions down regulated,Bcl-2 expression up regulated,and antioxidant enzyme Prx III and Trx2 contents were enhanced.The above results indicated that,hydrogen in concentration of 75 resisted oxidative damage,inhibits mitochondria apoptosis pathway activation,protected mitochondria function,and controled neuronal apoptosis.To sum up,hydrogen in high concentration can up regulate the expressions of SOD1,SOD2,PrxIII and Trx2 while inhibiting expressions of Nox2 and Nox4 proteins so as to reduce generation of reactive oxygen species ROS,inhibit expressions of Caspase3 and Box,control activation of mitochondria apoptosis pathway,and restrain neuronal apoptosis.This research is hopeful to provide another feasible selection of treating spinal cord injury. |
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