The Research Of Damage On Primary Cultured Neurons Suffered From High Temperature

Heatstroke is a seriously disease which threatens human health, character as high morbidity, high morality, high disability rate. As global warming, its morbidity raises year by year. Heatstroke should cause injury involving multiple organs and systems, especially in nervous system. Heatstroke patients should express some nervous symptom, such as headache, convulsion, loss of consciousness, mania, haziness of spirit-mind, cognitive impairment, and so on.There are two problems. The first problem is that some research on heatstroke mostly focus on people or animals, nevertheless the disadvantage is that the models for animals are not uniform, which should influence the research results more or less. The second problem is that we didn’t obtain the general data about neurons suffered from different high temperature. If directly heating the primary cultured cortical neurons bypass animals model, it should resolve the contradiction well. We investigated the effects of the different combinations of high temperature and exposure time on the neurons, and observe their pathological change, which had simulated the pathological traits well, and laid the foundation for preventing and treating heatstroke.We explored and built a new method for culture high quality neurons with high purity, high survival rate and high yield by newborn rat’s cortex, through lots of researches. We heated neurons in different high temperature for different exposure time, and observe the morality rate in different situation, in order to explore the pathological feature of neuronal death due to heat. In the same time, we further did some researches on apoptosis and necrosis rate in common high temperature result to heatstroke. Moreover, detected some related protein(Cas-3 and HSP70). For preventing and treating heatstroke, performed some initiatory researches.Part Ⅰ Build a modified model for culturing primary cortical neurons on ratsObjective: To build a stable method culturing rats cortical neurons with high purity and low morality.Methods: We referred Beaudoin’s method and improved it by exploration. The procedure as below : use newborn Wistar rats within 12 h old. After anesthesia, the rats were dissected under cold temperatures, the bilateral cerebral cortex was separated and digested by trypsin, and then gently blow it. After that, the cortical tissues were screened and centrifuged, the supernatant was abandoned, and the resultant was added with PM. Briefly blow and centrifuge it again, count neurons under microscope, plant neurons with suitable concentration. Change full PM in 4h, 48 h and 96 h after plantation.Results: At Day 5 of the culture, the neurons show plumpness with clear background and few pyknosis dead cells. The positive rate of immunocytochemical staining of specific marker for neuron, neuron-specific enolase(NSE), was above 95%. N-methyl-D-aspartate receptor 1(NMDAR1) staining showed that the cells presented plenty of axons and dendrites, effective crosslinking. The positive rate of trypan blue staining was lower than 5%, indicating low mortality of cultured neurons.Conclusions: Build a stable and convenient method culturing rats cortical neurons with high purity, low morality, high yield and low background. Part II The research on neurons injury suffered from different high temperature and exposure timeObjective: To explore the pathological feature and dead traits of neurons suffered from different high temperature and exposure time.Methods: At Day 5 of the culture, the neurons were suffered from high temperature(37°C, 39°C, 41°C,43°C, 45°C and 47°C) and exposure time(45 min and 1h) in the 5% CO2 incubator under 37°C. Observed the change of neuronal morphology 24 h later after heating. Moreover, observed the neurons after trypan blue staining and counted the neuronal morality in different model. At last, observed the neurons after Annexin/PI staining and view the apoptosis and necrosis neurons.Results: Under the treatment of 45 min-heat, the neurons were not sensitive and could resist high temperature up to 45℃, and under the treatment of 1 h-heat, the mortality of neurons increased as the temperature rising. Below 43℃ heating for 1h, dead neurons mainly were apoptosis. Beyond 45℃ for 1h heat, most of neurons were necrosis.Conclusions: Different high temperature and exposure time were two important factors affecting the neuronal morality. Neurons could resist high temperature up to 45℃ for 45 min. Heat injury below 43℃ mainly result some neurons to apoptosis. Up to 45℃, most of neurons occurred to necrosis.Part III The further research of neuronal injury due to common high temperatureObjective: To explore the dead traits of neuronal apoptosis and necrosis, the changing feature of related protein, under common high temperature.Methods: At Day 5 of the culture, the neurons were suffered from high temperature(41°C and 43°C) for 1h in the 5% CO2 incubator under 37°C. Observed the change of neuronal morphology 24 h later after heating. After Annexin V/PI immunofluorescence staining, the neurons were deteced by flow cytometry and counted the number of neuronal apoptosis and necrosis cells. Moreover, perform Caspase-3 and HSP-70 immunocytochemical staining, The results were analyzed using Image-Pro Plus 6.0 to perform IOD scanning..Results: Under the treatment of 41℃、43℃ for 45 min, the neurons were partially dead, which the apoptosis neurons were in the majority, and the rate of apoptosis neurons increased as the temperature rising. The survival neurons showed no obvious changes. Caspase-3 expression intensity : Group a(normal control cells)< Group b(41°C for 1 h)< Group c(43°C for 1 h). HSP70 expression intensity : Group c(43°C for 1 h)<Group a(normal control cells)< Group b(41°C for 1 h).Conclusions: Under the common heat injury, only a small ratio of the neurons were dead, which the apoptosis neurons were in the majority. The apoptosis neurons were less in 41℃for 1 h than 41℃for 1 h,and the expression of Cas-3 was lower, the expression of HSP70 was higher, which should be related with the protection of HSP70.