| Stroke is a disease that causes high mortality and disability worldwide,and it is a serious threat to human health.Mitochondria dysfunction is now considered an important marker of neuronal death after stroke.Studies have shown that mitochondria transfer from astrocytes to injured neurons after stroke,which triggers endogenous neuroprotective mechanisms.Basic and clinical studies indicate that mild hypothermia has a definite protective effect on neurons after cerebral ischemic injury,but the effect of mild hypothermia on mitochondria transfer from astrocytes to injured neurons remains unclear.Therefore,this project aims to investigate whether mild hypothermia can promote the transfer of mitochondria from astrocytes to ischemic injured neurons and strengthen the endogenous neuroprotective effect.Part 1 Primary culture of rat astrocytes and neurons and establishment of a neuron oxygen glucose deprivation/reoxygenation injury modelObjevtive: Primary SD rat astrocytes and neurons were cultured and neuron OGD/R injury model was established.Methods:The astrocytes and neurons of the neonatal SD rat(<24 h)cerebral cortex were cultured.The astrocytes were cultured with 10%FBS+DMEM/F12+1% PS medium,and the cells were purified and passaged when the cells reached 80% or more of the bottom of the culture flask at about 7 days.When the cells cover more than 90% of the bottom of the culture plate,the purity is identified by cell immunofluorescence.Neuronal cells were cultured in 97% Neurobasal +2%B27+1%PS serum-free medium.When cultured to about 7-11 days,the cell was identified by cell immunofluorescence method.A three-gas incubator combined with sugar-free DMEM was used to establish a neuronal cell OGD/R injury model.Neuronal cells were deprived of oxygen and glucose for 1 hour and reoxygenated for 24 hours.The OGD/R injury model was evaluated by observation under a microscope,Hoechst-PI staining and CCK-8 kit.Results:1.morphology of astrocytes and neuronal cells is typical.They were identified with GFAP and MAP2,respectively,and the results showed that the astrocytes had a purity of more than 95% and neurons had a purity of more than 90%.2.Microscopic results showed that the neuron cells in the Ctrl group had typical morphology and were intertwined to form a neural network;the neuron cells in the OGD group were oedematous and the neural network-like structure was damaged.3.CCK8 results showed that compared with the Ctrl group,the neuron activity in the OGD/R group was significantly reduced,and the difference was statistically significant(P<0.01).4.Hoechst-PI staining showed that the nuclei of neurons in the Ctrl group showed uniform blue fluorescence and regular morphology.Compared with the control group,the number of dead cells in the OGD/R group was significantly increased,the nuclei were red,and chromatin was condensed.Conclusions:1.The cortical astrocytes and neurons of neonatal SD rats cultured in vitro have typical morphology,which can meet the further experimental requirements.2.The neuron OGD/R injury model was successfully established.Part2 Mild hypothermia promotes mitochondria transfer from astrocytes to oxygen glucose deprivation/reoxygenation injured neuronsObjevtive: To study the effect of mild hypothermia(33?)on the transfer of mitochondria from astrocytes to OGD/R injured neurons.Methods:The culture of astrocytes and neurons and OGD/R injury model is the same as the first part.1 To observe the effect of mild hypothermia on astrocyte mitochondria release.(1)Normal temperature group: Astrocytes were cultured in a normal incubator(37?)for 24 hours.(2)Mild hypothermia group: Astrocytes were cultured in a mild hypothermia incubator(33?)for 24 h.Two groups of extracellular medium were collected to determine their extracellular ATP content.2 To observe the effect of mild hypothermia on the transfer of mitochondria from astrocytes to OGD/R injured neurons.2.1 Laser confocal microscopy to observe the effect of mild hypothermia on the number of mitochondria transferred from astrocytes to OGD/R injured neurons.(1)Normal temperature group: After OGD injury for 1 h,the neuron cell medium was replaced with the astrocyte normal temperature medium and incubated for 24 h.(2)Mild hypothermia group: After OGD injury for 1 h,the neuron cell medium was replaced with the astrocyte mild hypothermia medium and incubated for 24 h.Transfer of mitochondria was observed by laser confocal microscopy after immunofluorescence staining.2.2 To observe the effect of mild hypothermia on mitochondria function.(1)Control(Ctrl)group: The neuronal cells were cultured normally without any treatment.(2)OGD/R group: After OGD injury for 1 h,the neuron cell medium was replaced with the normal medium and incubated for 24 h.(3)Normal temperature(ACM+OGD/R)group: After OGD injury for 1h,the neuron cell medium was replaced with the astrocyte normal temperature medium and incubated for 24 h.(4)Mild hypothermia(HACM+OGD/R)group: After OGD injury for 1h,the neuron cell medium was replaced with the astrocyte mild hypothermia medium and incubated for 24 h.The Cell-titer-Glo kit was used to detect the content of ATP in neurons in each group,and the JC-1 staining kit was used to detect the changes of mitochondria membrane potential in neurons in each group.2.3 To observe the effect of mild hypothermia on the survival rate of neurons.(1)Control(Ctrl)group: The neuronal cells were cultured normally without any treatment.(2)OGD/R group: After OGD injury for 1 h,the neuron cell medium was replaced with the normal medium and incubated for 24 h.(3)Mild hypothermia(HACM+OGD/R)group: After OGD injury for 1h,the neuron cell medium was replaced with the astrocyte mild hypothermia medium and incubated for 24 h.(4)Mild hypothermia normal(HACM+N)group: The neuron cell medium cultured normally was replaced with the astrocyte mild hypothermia medium and incubated for 24 h.(5)Normal temperature(ACM+OGD/R)group: After OGD injury for 1h,the neuron cell medium was replaced with the astrocyte normal temperature medium and incubated for 24 h.(6)Normal temperature normal(ACM+N)group: The neuron cell medium cultured normally was replaced with the astrocyte normal temperature medium and incubated for 24 h.CCK8 kit was used to detect the neuron activity in each group.Hoechst-PI staining was used to detect the mortality of neurons in each group.Results:1.Compared with the normal temperature group,the extracellular ATP content in the mild hypothermia group was significantly increased,and the difference was statistically significant(P <0.01).2.Compared with the normal temperature group,the number of mitochondria transferred from astrocytes to OGD-injured neurons was significantly increased in the mild hypothermia group.3.Compared with the Ctrl group,the ATP content in the neurons was significantly reduced in the OGD/R group(P<0.01),and the mitochondria membrane potential was significantly reduced;compared with the OGD/R group,the ATP content in neurons was significantly increased in HACM+OGD/R group and ACM+OGD/R group(P<0.01),and the mitochondria membrane potential increased significantly;compared with ACM+OGD/R group,the ATP content in the neurons was increased in the HACM+OGD/R group(P<0.01),and the mitochondria membrane potential was increased.4.Compared with the Ctrl group,the activity of neurons decreased significantly in OGD/R group(P<0.01),and the mortality rate increased significantly(P<0.01).Compared with the Ctrl group,the neuron activity of the HACM+N group and the ACM+N group increased slightly,and the mortality rate decreased slightly,but there was no significant difference(P>0.05).Compared with the OGD/R group,the neuron activity of the HACM+OGD/R group and the ACM+OGD/R group increased significantly(P<0.01),and the mortality rate decreased significantly(P<0.01).Compared with the ACM+OGD/R group,the neuron activity of the HACM+OGD/R group increased significantly(P<0.01),and the mortality rate decreased significantly(P<0.01).Conclusions:Mild hypothermia promoted the transfer of mitochondria from astrocytes to OGD/R neurons and strengthened the endogenous neuroprotective effect. |
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