| At the current stage of the domestic animal husbandry breeding system,intensive breeding has gradually replaced the traditional breeding model,which has largely avoided the occurrence of cold stress reactions.However,the temperatures required for each growth stage of livestock and poultry are not the same,and discontinuous cold exposure during the ventilation of the livestock house and the long-distance transportation of the animal is still unavoidable.Therefore,the chronic cold stress response still occurs.As the primary target for the role of various cytokines,stress hormones,and neurotransmitters,what effect will the hippocampus suffer when a chronic cold stress reaction occurs? What kind of complicated regulation mechanism exists?In-depth research on the effects of chronic cold stress on the structure and function of the hippocampus in mice is still necessary to more effectively and proactively avoid the risks caused by cold stress.Farming welfare therefore remains important.To investigate the impact on the function and anatomical structure of the hippocampus in mice,a chronic cold stress model of C57BL/6 mice of different sexes was used with the conditions of 4°C for 3 h per day for 7 days.During this process,the serum glucocorticoid levels in each group of mice were continuously monitored,and blood gas analysis of mice was detected immediately after the end of the cold exposure process to assess whether the chronic cold stress model was successfully developed.The results showed that compared with the control group,the levels of glucocorticoids(GCs)in the serum of the model group mice were significantly increased(P < 0.001)at 1,3,5,and 7 days of cold exposure.The blood gas results also showed significant changes in pH and other factors after the cold exposure cycle(P < 0.01).The combination of two experimental results confirmed that the chronic cold stress mouse model was successfully developed.Samples of the hippocampus and whole brain tissue of each group of mice were then collected,and western blotting,mass spectrometry,immunohistochemistry,immunofluorescence,and Nissl staining were used to measure the activation markers of microglia integrin alpha M(CD11B)and hippocampal neuron markers.Microtubule-associated protein 2(MAP2),heat shock protein 70(Hsp70),brain-derived neurotrophic factor(BDNF),inflammatory factor interleukin-1(IL-1?),and neurotransmitter metabolites were used to assess the homeostasis of the CNS of the hippocampus to determine the effects on hippocampalstructure and neuron function following chronic cold exposure.The results showed that compared with the control group,the expression of BDNF in hippocampal tissues of mice in the chronic cold stress model group was downregulated(P < 0.05)and the expression of Hsp70 and a number of cytokine proteins including IL-1? were significantly increased(P < 0.01).In addition,the results of the immunofluorescence assay of mouse brain sections showed that the numbers of CD11 B positive cells in the hippocampus CA1 and CA3 functional regions were remarkably increased(P < 0.001).In addition,the expression of the neuronal marker,MAP2,was decreased(P < 0.05)and the number of Nissl positive bodies was significantly decreased(P< 0.001).There were also significant differences in the metabolites of various neurotransmitters(P < 0.05).Moreover,the results in the cold stress model group not only showed differences in statistical analysis with the control,but also in the model group,where statistical analysis of the data between different mouse sexes also showed significant changes.The above results showed that the chronic cold stress response disrupted the homeostasis of the CNS in the hippocampus of mice,mediating the activation of microglia in the CA1 and CA3 regions of the hippocampus,and reducing the number of neurons,resulting in changes in neurotransmitter metabolism.Sex differences were also found.In addition,the content of glucocorticoids(GCs)in hippocampal tissues,the expression of the GC metabolic enzyme,corticosteroid 11? dehydrogenase isozyme 1(HSD11B1),and the glucocorticoid receptor(GR)all showed significant upregulation(P <0.001).Overall,the results indicated that GCs may play an important regulatory role in the imbalance process of homeostasis induced by cold exposure.It was speculated that GCs excessively induced the occurrence and development of various processes in hippocampus tissues,and because GCs have different metabolic efficiencies in male and female mice,they also produced sex differences in the occurrence of various phenomena.To verify the scientific hypothesis proposed in vivo,different concentrations of GCs(CORT)were used to stimulate the microglial cell BV2,and appropriate concentrations were selected to develop the model of BV2 cell GC excess in vitro.Immunofluorescence,western blotting,dual luciferase reporter assays,co-immunoprecipitation,and other methods were used to detect the relevant mechanisms of microglial activation during GC exposure.The results showed that 200?M CORT significantly inhibited the expression and enzyme activity of Siltuin1(SIRT1)in BV2cells(P < 0.05).Moreover,the acetylation of histone H3 promoted the translocation of p65 subunits into the nucleus,enhancing the interaction between NF-?B and histones,accelerating the transcription of IL-1? downstream target gene,mediating microglial activation,and actively releasing acetylated high-mobility group box-1(HMGB1)of damage-associated molecular patterns.According to the results of the BV2 cell activation process in the presence of GCs,200?M CORT could mediate microglial activation and actively release acetylated HMGB1 by upregulating intracellular acetylation.Hypothesizing that GCs induce microglial activation,to further clarify the effect of excessive GCs on hippocampal neurons,different concentrations of GCs were used to treat hippocampal neuronal HT22 cells to develop the GC excess model in HT22 cells in vitro.The mitochondrial function and structural integrity in HT22 cells exposed to GCs were then determined using immunoblotting,immunofluorescence,a dual luciferase reporter assay,co-immunoprecipitation,and transmission electron microscopy.Moreover,an inhibitor of GR,Mifepristone(RU486)was used for the pre-processing of HT22 cells,and was used to characterize the programmed cell death processes and related mechanisms such as autophagy,mitochondrial autophagy,and apoptosis,to verify the scientific hypothesis proposed for in vivo effects.The results showed that when HT22 cells were treated with 400 ?M CORT for 3 h,the mitochondrial membrane potential decreased significantly with the extension of treatment time in HT22 cells(P < 0.05).The reactive oxygen species(ROS)level was also significantly increased(P < 0.001)and the mitochondrial structural integrity disappeared.At the same time,the signaling pathway of Nrf2/Keap1,an intracellular antioxidant system,was activated,and Nrf2 was transported into the nucleus and showed strong binding with acetylated H3 to promote target gene transcription,including downstream SOD1 and HO-1,mediating the programmed cell death progress of autophagy,mitochondrial autophagy,and endogenous apoptosis.However,after pretreatment with RU486,400 ?M CORT was used to treat HT22 cells,and the processes of autophagy,mitochondrial autophagy,and endogenous apoptosis were all increased.It was concluded that excessive CORT directly damaged mitochondria without inducing interaction with their receptors,to induce endogenous apoptosis and mediate autophagy and mitochondrial autophagy via the AMPK/mTOR and PINK1/Parkin signaling pathways.The occurrence of phagocytosis promoted the programmed death process of HT22 cells.It was confirmed that GCs induced BV2 cell activation and mediated the programmed cell death of HT22 cells in vitro.The mechanism of microglia activation and hippocampal neuron death in the hippocampus tissue of the cold-stressed mouse model was investigated again to verify and supplement the conclusions obtained from in vitro experiments.The results showed that cold stress mediated microglial activation by upregulating the acetylation level of the NF-?B signaling pathway in the mouse hippocampus,and affected hippocampal neurons through autophagy and apoptosis by affecting mitochondrial functioning to cause programed cell death.The in vitro results showed the processes of activation of BV2 cells and the death of HT22 cells mediated by GCs.The mechanism of microglia activation and hippocampal neuron death in a cold stress mouse model was tested again to verify the results in vivo.The results showed that cold stress mediated microglial activation by upregulating the acetylation level of the NF-?B signaling pathway in the hippocampus of cold stressed mice.It also induced hippocampal neurons through autophagy and apoptotic pathways by affecting the death of mitochondria.In addition,through comprehensive analyses of the in vivo and in vitro results,it was concluded that the activation process of microglial cells induced by cold stress preceded the occurrence of hippocampal neuron death. |
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