Effects Of Mild Hypothermia On Tunneling Nanotubes Formation And Astrocytic Mitochondria Transfer To Damaged Neurons Subjected To Oxygen-glucose Deprivation/reoxygenation

Objective:Astrocytes and neurons were cultured and neurons OGD/R injury model was established.To investigate the effect of the mild hypothermia（33℃）on astrocytes mitochondrial transfer to OGD/R injured neurons.Methods:1.Primary astrocytes and neurons were cultured from cerebral cortex of newborn SD rats within 24h.A three-gas(94%+5%CO₂₊1%O₂)incubator combined with sugar-free DMEM were used for 1h and reoxygenated for 24h to manipulate the mature neurons,which was to establish the neurons OGD/R injury model.The OGD/R injury model was evaluated by visual observation under light microscope,CCK-8 kit,thionin-staining and Hoechst-PI staining.2.Mixed culture of astrocytes and neurons.The purified astrocytes were transferred to a six-well plate,and when70%～80%of the cells were covered,the neurons culture medium was added to the second generation of astrocytes after digesting and centrifuging.Then all of these were transferred to the OGD/R damaged neuron culture plate.3.Formation of TNTs between various cells at normal temperature.（1）Normal astrocytes（A）group:Mature astrocytes after passage were cultured at normal temperature for 24h.（2）Normal（N）group:normal cultured neurons were cultured for 24h at normal temperature.（3）Injury（OGD/R）group:Neurons were cultured at normal temperature for 24h after OGD injury for 1h.（4）Normal（A+N）group:normal cultured neurons and astrocytes were cultured 1:1 at normal temperature for 24h.（5）Normal injury（A+OGD/R）group:after 1h OGD injury,neurons were reoxygenated and reoxygenated with astrocytes 1:1 at normal temperature for24h.Fluorescence microscope was used to observe whether TNTs formed between cells in each group and the number of them.4.Effects of neuronal oxygen glucose deprivation on the number of TNTs formation and the number of mitochondrial transfer from astrocytes to neurons.（1）Normal（A+N）group:normal cultured neurons and astrocytes were cultured 1:1 at normal temperature for 24h.（2）Normal temperature injury（A+OGD/R）group:After 1h OGD injury,neurons were cultured 1:1 with astrocytes for 24h at normal temperature.Fluorescence microscope was used to observe mitochondria transfer between cells and the number of TNTs in each group.5.Effects of oxygen-glucose deprivation on mitochondrial membrane potential（MMP）,mortality and damage of neurons after mixed culture of astrocytes and neurons.（1）Control（Ctrl）group:The neurons were cultured normally without any treatment.（2）Injury（OGD/R）group:The Neurons were cultured at normal temperature（37℃）for 24h after 1h OGD injury.（3）Normal（A+N）group:After 1h OGD injury,the neurons were reoxygenated with astrocytes in a ratio of one to one at normal temperature for24h.（4）Normal injury（A+OGD/R）group:Normal neurons were cultured with astrocytes in a ratio of one to one for 24h at normal temperature.JC-1 kit was used to detect MMP of neurons.Hoechst-PI staining was used to detect the mortality of neurons.The damage of neurons was detected by thionin-staining.6.Effects of TNTs on the number of mitochondria transferred from astrocytes to OGD/R injured neurons.（1）Normal injury（A+OGD/R）group:After 1h OGD injury,neurons were cultured 1:1 with astrocytes for 24h at normal temperature.（2）Normal Cyto D（A+N+Cyto D）group:Neurons were cultured 1:1 with astrocytes and added cytochalasin D at normal temperature for 24h after OGD injury for 1h.Fluorescence and light microscope were used to observe the morphology of astrocytes after adding cytochalasin D.Fluorescence microscope was used to observe the number of mitochondria transferred from astrocytes to OGD/R injured neurons.7.Effects of mild hypothermia on the transfer of astrocytes mitochondria to OGD/R injured neurons via TNTs.7.1 Effects of mild hypothermia on TNTs formation between astrocytes and OGD/R damaged neurons and the number of astrocytic mitochondria transferred to OGD/R injured neurons.（1）Normal temperature injury（A+OGD/R）group:After 1h OGD injury,neurons were cultured 1:1 with astrocytes for 24h at normal temperature.（2）Mild hypothermia injury（HA+OGD/R）group:After OGD injury for1h,the neurons were cultured with astrocytes at mild hypothermia（33℃）for24h.The number of TNTs formed between astrocytes and neurons in each group was observed by fluorescence microscope.7.2 Effects of mild hypothermia on the number of astrocytic mitochondria transferred to OGD/R injured neurons and MMP of OGD/R damaged neurons.（1）Normal temperature injury（A+OGD/R）group:After 1h OGD injury,the neuron were reoxygenated with astrocytes in a ratio of one to one at normal temperature for 24h.（2）Normal temperature injury with Cyto D（A+OGD/R+Cyto D）group:After 1h OGD injury,neurons were cultured with astrocytes in a ratio of one to one and the cytochalasin D was added to the media for 24h at normal temperature.（3）Mild hypothermia injury（HA+OGD/R）group:After 1h OGD injury,the neurons were cultured with astrocytes at the mild hypothermia（33℃）for24h.（4）Mild hypothermia injury with Cyto D（HA+OGD/R+Cyto D）group:After 1h OGD injury,neurons were cultured with astrocytes in a ratio of one to one and the cytochalasin D was added to the media for 24h under the mild hypothermia.Mitochondria of astrocytes were stained with Mito Tracker Red CMXRos,and the neurons were stained and labeled with CFSE.Then Mitochondria from astrocytes in each group and MMP of OGD/R injured neurons were observed by fluorescence microscope.7.3 Effects of mild hypothermia on damaged neurons mortality and damage after astrocytes mitochondrial transfer to damaged neurons via TNTs.（1）Normal temperature injury（A+OGD/R）group:After 1h OGD injury,the neuron were reoxygenated with astrocytes in a ratio of one to one at normal temperature for 24h.（2）Normal temperature injury with Cyto D（A+OGD/R+Cyto D）group:After 1h OGD injury,neurons were cultured with astrocytes in a ratio of one to one and the cytochalasin D was added to the media for 24h at normal temperature.（3）Mild hypothermia injury（HA+OGD/R）group:After 1h OGD injury,the neurons were cultured with astrocytes at the mild hypothermia（33℃）for24h.（4）Mild hypothermia injury with Cyto D（HA+OGD/R+Cyto D）group:After 1h OGD injury,neurons were cultured with astrocytes in a ratio of one to one and the cytochalasin D was added to the media for 24h under the mild hypothermia.Hoechst-PI staining was used to detect the mortality of neurons.Damage of neurons in each group was detected by thionin-staining.Results:1.Evaluation of the neuronal OGD/R injury model was successful.2.TNTs were found between astrocytes and normal neurons,between astrocytes and OGD/R damaged neurons,between astrocytes,between normal neurons and OGD/R damaged neurons.3.Compared with the A+N group,the number of astrocytes mitochondria in OGD/R injured neurons and TNTs formation in A+OGD/R group were increased（P<0.05）.4.It was observed under light and fluorescence microscope that astrocytes were round without protruding or forming TNTs to connect with neurons after adding cytocalasin D（TNTs inhibitor）.Compared with the A+OGD/R+Cyto D group,the number of mitochondria transferred from astrocytes to neurons in A+OGD/R group was increased（P<0.05）.5.Compared with the A+OGD/R group,the number of TNTs formed between astrocyte and neuron and the number of astrocytic mitochondria transferred to neurons in HA+OGD/R group were significantly increased（P<0.05）.6.Compared with the Ctrl group,MMP in OGD/R group and A+OGD/R group was decreased,and neuron death was increased（P<0.05）;The number of neuronal Nissl was decreased,staining became shallow,and damage increased significantly.There were no significant changes in mitochondrial membrane potential,number of deaths and apoptosis in A+N group.Compared with the OGD/R group,mitochondrial membrane potential was increased in A+OGD/R group,and neuron death was decreased（P<0.05）.The number of neuronal Nissl was increased,the staining was deeper,and the damage decreased.7.Compared with the normal temperature group,the number of astrocytic mitochondria transferred to neurons and neuronal MMP of the mild hypothermia group were increased（P<0.05）.Compared with the addition of cytochalasin D（TNTs inhibitor）group,the number of astrocytes mitochondria transferred to neurons and neuronal mitochondrial membrane potential in the group without addition of cytochalasin D were increased（P<0.05）.8.Compared with the normal temperature group,neuronal mortality and damage were decreased in mild hypothermia group.Compared with the addition of cytochalasin D group,neuronal mortality and damage were decreased in the group without addition of cytochalasin D（P<0.05）.Conclusions:1.There were TNTs between nerve cells,and the formation of TNTs between astrocytes and neurons were increased after oxygen-glucose deprivation/reoxygenation at normal temperature,initiating endogenous neuroprotective effect.2.Mild hypothermia promoted the formation of TNTs,increased the transfer of functional mitochondria from astrocytes to OGD damaged neurons and enhanced the endogenous neuroprotective effect.