| Hypercapnia is usually encountered in some clinical conditions,such as acute exacer'oation of chronic obstructive pulmonary disease(AECOPD),severe pneumonia,and severe acute asthma in the intensive care unit(ICU).The patients had been found to complicate with different degrees of cognitive dysfunction after being discharged in the clinic.Also,the lung protective ventilation strategies of low tidal volume has been used as a ventilation strategy to reduce the mortality of ARDS,but such ventilation strategies often cause hypercapnia.Previous epidemiological surveys have shown that:70%-100%of the survived ARDS patients have been found to complicate with different degrees of cognitive dysfunction after being discharged,46%-80%after 1 year,and 20%after 5 years.The various cognitive dysfunctions such as attention disorder,memory impairment,and executive dysfunction after being discharged of critically ill patients can lead to a serious decline in the self-care ability.After being returned to the society,the declining ability of self-care brought great economic burden to the patients themselves,families and society.The disruption of blood-brain barrier(BBB)might contribute to the exacerbation of the cognitive impairment.Under normal circumstances,BBB provides a safe environment for the central nervous system to protect it from external pathogens and toxins.The increasing of BBB permeability is likely to induce the more peripheral inflammatory factors and neurotoxic substances entering into the central nervous system and can lead to cognitive dysfunction.BBB is a cell complex composed of pore-free capillary endothelial cells and intercellular tight junctions(TJs),basement membrane,pericytes,neurons,the feet processes of astrocytes and extremely narrow extracellular space of the cells.The astrocytes in BBB are closely linked to the surrounding pericytes,neurons,and endothelial cells through their owner end-feet.Astrocytes participate in the maintenance and formation of the BBB mainly through the synergistic action of secretory actives,gene transcription and protein synthesis.Astrocytes regulate the BBB permeability primarily by regulating the expression of aquaporin 4(AQP-4)and matrix metalloproteinases(MMPs).Under hypoxic conditions,upregulation of the expression of AQP-4 in the astrocytes may lead to an increment of BBB permeability and aggravate the brain edema.In a rat model of ischemia and hypoxia,astrocytes mainly secrete MMP-9,which leads to the degradation of tight junction protein(Tjps)between the brain microvascular endothelial cells and enhances the BBB permeability.Therefore,the expression of AQP4 and MMP-9 protein is closely related to the BBB permeability.Hypoxia-inducible Factor-1(HIF-1)is a transcriptionally active protein which produced under hypoxic conditions and can bind to hypoxia response elements(HRE),where it modulates the transcription of a host of targets against hypoxic conditions.HIF-1 is a heterodimer composed mainly of 120kD HIF-1? and 91?94kD HIF-1?subunits.The HIF-1? subunit is stably expressed in the cytoplasm and plays an important role in maintaining the cell structure.HIF-1? is the active subunit of HIF-1,which is regulated by hypoxia signal.The HIF-1? subunit is degraded by ubiquitin-protease hydrolysis complex after translation,so it is barely detected in cells under normal oxygen saturation.In hypoxia,the degradation of HIF-1? subunit is inhibited.HIF-1? firstly stabilizes in the cytoplasm and then translocates from cytoplasm into the nucleus,where it dimerizes with HIF-1?.The HIF-1 heterodimer binds to hypoxia response elements(HRE),where it modulates the transcription of a host of targets against hypoxic conditions.In hypoxic ischemic injury,it participates in repairing processes in various organs and tissues.In the procession of hypoxemia,ischemia-reperfusion and inflammation,the activation of HIF-1 signaling pathway can cause an increment of BBB permeability.In the mouse model of brain trauma,it was also found that the activation of HIF-1 signal transduction pathway can increase the BBB permeability and cerebral edema by up-regulating the expression of AQP-4 and MMP9.However,at present,there is no relevant literature reported that under the condition of hypoxemia,whether the HIF-1 signal pathway is further regulated by hypercapnia,and then the upregulation of the AQP-4 and MMP9 in astrocytes of the central nervous system,eventually leading to cognitive dysfunction.In this study,a rat model of hypoxia/hypercapnia was established through mechanical ventilation.The neurocognitive function of SD rats was evaluated by Morris water maze(MWM)test;The extravasation of Evans Blue(EB)and brain water content wered used to assess the BBB permeability;Western Blot and immunofluorescence assay were employed to detect the protein expression of total and nuclear HIF-1a in the astrocytes of SD rats in hippocampus.Furthermore,the expression of AQP-4,MMP9,occluding and claudin-5 proteins were further examined.And then,methoxyestradiol(2-Methoxyestradiol:2ME2,an inhibitor of nuclear translocation of HIF-1?)was administered 1h before the ventilation in the 2ME2 group to explore the effect of hypercapnia on nuclear translocation of HIF-1?protein and upregulation of AQP-4 and MMP9.Finally,Tris-base was used to neutralize the acidosis induced by the high concentration of CO2.After the acidosis was neutralized,we observed the protein expressions of HIF-1?,AQP-4,MMP9 occluding and claudin-5 in the astrocytes.The study is divided into six chapters to explore the mechanisms:Chapter 1 The effect of hypoxia in combination with hypercapnia on neurocognitive function in experimental ratsObjectiveThe SD rat model of hypoxia/hypercapnia was established through mechanical ventilation.Rat arterial blood samples were drawn from the left femoral artery.Blood gas analysis was performed to determine the optimal O2 concentration and CO2 concentration to establish the hypoxic/hypercapnia rat model.MWM experiments were performed to evaluate the effect of hypoxia/hypercapnia on the spatial and temporal memory ability of SD rats.Method:Pentobarbital sodium(30 mg/kg)was used to anesthetize the rats through the intra peritoneal injection.Through oral intubation,the rats were connected to an animal-specific ventilator and ventilated for 3 h.By adjusting the proportion of gas in the ventilator cylinder,rats were randomly divided into 4 groups according to the randomization principle:sham operation group(Sham operation/S group:normal air condition),hypoxemia group(Hypoxemia/H group:84%N2+16%O2),hypercapnia group(Hypercapnia/HC group:74%N2+21%O2+5%CO2),hypoxic hypercapnia group(Hypoxemia and Hypercapnia group/HH group:79%N2+16%O2+5%CO2).Blood gas samples were drawn from each group during mechanical ventilation at the time of 30 min,60 min,120 min,and 180 min.And then,the O2 partial pressure(PaO2),CO2 partial pressure(PaCO2),and pH values were analyzed.Through adjusting the proportion of gas O2,CO2 and N2 in the ventilator cylinder,the clinical scenario of hypoxia in combination with hypercapnia was simulated.In the MWM experiment,the escape latency time was used to evaluate the spatiotemporal memory ability of rats.Repeated measures analysis of variance was used to analyze repeated measures data;factorial design analysis of variance was used to assess whether there was an interaction effect between hypoxia treatment and hypercapnia treatment;Simple-effect analysis were further peformed if an interaction effect was found.The difference was considered statistically significance when P<0.05.Results:In S and H group,PaCO2 was maintained between 35-45 mmHg;The PaCO2 values of HC and HH group were maintained between 60-69 mmHg,the corresponding pH value was maintained between 7.20-7.25.The 5%concentration of CO2 intervention in the HC and HH group significantly increased the PaCO2 value(30 min,60 min,120 min,180 min:all P<0.01),while the PH values were decreased(30 min,60 min,120 min,180 min:all P<0.01).The PO2 values of the H and HH group were hovered around 60 mmHg.The PaO2 value was significantly reduced by 16%O2 intervention(30 min,60 min,120 min,180 min:all P<0.01);In the MWM experiment,significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(48 h,72 h:all P<0.05).Further simple effects analysis showed that there was no significant difference in escape latency between the S group and the HC group(48 h,72 h:all P>0.05),while group escape latency in the H group was significantly longer than the S group(48 h,72 h:All P<0.05),the escape latency of the HH group was significantly longer than that of the H group(48 h,72 h:all P<0.05)and the HC group(48 h,72 h:all P<0.05).Conclusions:1.The experimental rat model of hypoxia in combination with hypercapnia was successfully established through the mechanical ventilation method.2.Hypercapnia alone does not cause neurocognitive dysfunction in the rats.3.Hypercapnia can further aggravate neurocognitive dysfunction in rats with hypoxemia.Chapter 2 The effect of hypoxia in combination with hypercapnia on the blood-brain barrier function in experimental ratsObjectiveThe brain tissue water content and extravasation of Evans blue were used to evaluate the effects of hypoxia in combination with hypercapnia on the BBB function in SD rats.Methods:SD rats were randomly divided into four groups:sham operation group(Sham operation/S group:normal air condition),hypoxemia group(Hypoxemia/H group:84%N2+16%O2),hypercapnia group(Hypercapnia/HC group:74%N2+21%O2+5%CO2),hypoxemia and hypercapnia group(Hypoxemia and Hypercapnia group/HH group:79%N2+16%O2+5%CO2).Brain tissue was immediately obtained after 3 hours mechanical ventilation in each of the above groups.The brain tissue water content of SD rats was calculated as follows:%H2O=(wet weight-dry weight)/wet weight ×100%.The extravasation of Evans blue was measured by spectrophotometer at a wavelength of 620 nm.The total EB content in the brain was quantified in reference to a linear standard curve derived from known amounts of the dye and was expressed as micrograms per gram of tissue.Repeated measures analysis of variance was used to analyze repeated measures data;factorial design analysis of variance was used to assess whether there was an interaction effect between hypoxia treatment and hypercapnia treatment;Simple-effect analysis were further peformed if an interaction effect was found.The difference was considered statistically significance when P<0.05.Results:1.The BWC experiment:significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(BWC:p<0.05).Further simple effect analysis showed that there was no significant difference in the BWC between the S group and HC group(P>0.05),while the BWC of SD rats in the group was significantly longer than that in S group.The BWC in HH group was significantly longer than that in H group(P<0.05)and HC group(P<0.05).2.The EB leakage experiment:significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(EB:p<0.05).Further simple effect analysis showed that there was no significant difference in the extravasation of Evans blue in SD rats compared with HC group(P>0.05),while the extravasation of Evans blue in H group was significantly more than that in S group(P<0.05).the extravasation of Evans blue in HH group was significantly more than that in H group(P<0.05)and HC group(P<0.05).Conclusions:1.Hypercapnia alone does not disrupt the BBB in the experimental rats.2.Hypercapnia can further aggravate the disruption of the BBB in rats with hypoxemia.Chapter 3 The effects of hypoxemia in combination with hypercapnia on HIF-1a-AQP-4/MMP-9 signaling pathway-associated proteins and tight junction proteins in BBB of hippocampus in experimental ratsObjective:To explore the effects of hypoxemia in combination with hypercapnia on the expressions of HIF-la total and nuclear protein,AQP-4 protein and MMP-9 protein in the astrocytes of hippocampus in SD rats and tight junction proteins which include occludin and claudi-5 protein.Methods:SD rats were randomly divided into four groups:sham operation group(Sham operation/S group:normal air condition),hypoxemia group(Hypoxemia/H group:84%N2+16%O2),hypercapnia group(Hypercapnia/HC group:74%N2+21%O2+5%CO2),hypoxemia and Hypercapnia group(Hypoxemia and Hypercapnia group/HH group:79%N2+16%O2+5%CO2).The brain tissue from hippocampus in the above groups were obtained after 3 hours of mechanically ventilation.The Western Blot was used to detected the total and nuclear protein of HIF-1a,AQP-4 protein,MMP-9 protein,occludin protein and claudin-5 protein in the hippocampus of SD rats.The immunofluorescence assay was used to detected the expression of HIF-1a protein,AQP-4 protein,MMP-9 protein in the astrocytes(GFAP as marker)in the hippocampus of SD rats.Repeated measures analysis of variance was used to analyze repeated measures data;factorial design analysis of variance was used to assess whether there was an interaction effect between hypoxia treatment and hypercapnia treatment;Simple-effect analysis were further peformed if an interaction effect was found.The difference was considered statistically significance when P<0.05.Results:1.Western Blot experiment:1.1 HIF-1a total protein:no significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(p>0.05);HIF-1a nuclear protein:significant interaction effects were observed between low O2(16%)intervention and high CO2(5%)intervention(p<0.05);Further simple effect analysis shown that:there was no significant difference between the S group and HC group in the expression of HIF-la nuclear protein in the hippocampus of SD rats(p>0.05),while the expression in H group was significantly increased in comparison with S group(P<0.05).The expression of HIF-la nuclear protein in HH group was significantly increased in comparison with the H group(P<0.05)and the HC group(P<0.05).1.2 AQP-4 protein,MMP-9 protein:significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(AQP-4 protein:p<0.05;MMP-9 protein:p<0.05).Further simple effect analysis showed that:there was no significant difference in the expressions of AQP-4 protein and MMP-9 protein in the hippocampus of SD rats in comparison with HC group(AQP-4 protein:P>0.05;MMP-9 protein:P>0.05),while the expression of AQP-4 protein and MMP-9 protein in hippocampus of SD rats was significantly increased in comparison with the S group(AQP-4 protein:p<0.05;MMP-9 protein:p<0.05).The expression of AQP-4 protein and MMP-9 protein in hippocampus of SD rats in HH group was significantly increased in comparison with the H group(AQP-4 protein:p<0.05;MMP-9 protein:p<0.05)and the HC group(AQP-4 protein:p<0.05;MMP-9 protein:p<0.05).1.3 Tight junction proteins:significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(occludin protein:p<0.05;claudin-5 protein:p<0.05).Further simple effect analysis shown that:compared with the HC group,there was no significant difference in the expressions of occludin protein and claudin-5 protein in the hippocampus of SD rats in the S group(occludin protein:P>0.05;claudin-5 protein:P>0.05).The expression s of occludin protein and claudin-5 protein in the hippocampus of SD rats were significantly decreased in comparison with the S group(occludin protein:p<0.05;claudin-5 protein:p<0.05).The expressions of occludin protein and claudin-5 protein in HH group were significantly decreased in comparison with the H group(occludin protein:p<0.05;claudin-5 protein:p<0.05)and the HC group(occludin protein:p<0.05;claudin-5 protein:p<0.05).2 Double immunofluorescence experiment:2.1 HIF-1a nuclear protein:hypercapnia alone can not increase the expression of HIF-la nuclear protein in the astrocytes of the hippocampus in SD rats;The intervention of low concentration of O2(16%)can significantly up-regulate the expression of HIF-1a nuclear protein in the astrocytes of the hippocampus in the H group.High concentration of CO2(5%)intervention can further increase the expression of HIF-1a nuclear protein in the astrocytes of the hippocampus in the rats with hypoxemia.2.2 AQP-4 protein,MMP-9 protein:hypercapnia alone can not increase the expression of AQP-4 protein and MMP-9 protein in the astrocytes of the hippocampus in SD rats;low concentration of O2(16%)intervention can significantly up-regulate the expression of AQP-4 protein and MMP-9 protein in the astrocytes of hippocampus in the H group.Low concentration of O2(16%)and high concentration of CO2(5%)intervention can further increase the expression of AQP-4 protein and MMP-9 protein in the astrocytes of hippocampus in the rats with hypoxemia.Conclusions:1.Hypercapnia alone does not induce any change in the expression of HIF-1a total and nuclear protein,AQP-4 protein and MMP-9 protein in the astrocytes of hippocampus,in the rats.It will not induce the degradation of the blood-brain barrier tight junction proteins which include occludin and claudin-5 protein in the hippocampus.2.Hypercapnia can not increase the expression of HIF-1a total protein in astrocytes of hippocampus in the rats with hypoxemia,but can further increase the expression of HIF-1a nuclear protein.3.Hypercapnia can further increase the expression of AQP-4 protein and MMP-9 protein in the astrocytes of hippocampus in hypoxemia rats,and also further increase the degradation of occludin and claudin proteins.Chapter4 The effect of hypercapnia on the translocation of HIF-1a nuclear protein and the protein expression of AQP-4,MMP-9,occludin and claudin-5 in the hippocampus of hypoxic rats.ObjectiveTo explore the role of hypoxemia in combination with hypercapnia in the translocation of HIF-1a protein into the nuclear of astrocyte in hypoxic rats,as well as the effets on the expressions of AQP-4,MMP-9,occludin and claudin-5 protein.Methods:Drug administrationa)methoxyestradiol(2-Methoxyestradiol:2ME2,an inhibitor of nuclear translocation of HIF-1?)2ME2 was first dissolved in dimethyl sulfoxide(DMSO)and further diluted in phosphate buffered saline(PBS)to a final dilution of 2 ml.In the 2ME2 group,the inhibitor was administered(5 mg/kg,intra peritoneal injection)1 h before mechanical ventilation1.2 control vector(Vehicle)DMSO was diluted in PBS and finally diluted into a control vehicle of 2 ml volume.2.Groups and drug intervention2.1 SD rats were randomly divided into five groups:sham operation group(S group:normal air condition),hypoxemia group(H group:84%N2+16%O2),hypercapnia group(HC group 74%N2+21%O2+5%CO2),hypoxemia and hypercapnia group(HH group:79%N2+16%O2+5%CO2),hypoxemia and hypercapnia+2ME2 group.The S group,H group,HC group and HH group were intraperitoneally injected with a control vehicle(Vehicle)1 hour before mechanical ventilation.In the 2ME2 group,the inhibitor was administered(5 mg/kg,intra peritoneal injection)1 h before mechanical ventilation.The brain tissues of the hippocampus were obtained after 3 hours mechanically ventilation in the above groups.2.2 Western blot was used to detect the total and nuclear protein,and AQP-4 protein,MMP-9 protein,occludin protein and claudin-5 protein in hippocampus of SD rats.The expressions of HIF-1a protein,AQP-4 protein,MMP-9 protein in the astrocytes of hippocampus were further verified by double immunofluorescence.Repeated measures analysis of variance was used to analyze repeated measures data;factorial design analysis of variance was used to assess whether there was an interaction effect between hypoxia treatment and hypercapnia treatment;Simple-effect analysis were further peformed if an interaction effect was found.The difference was considered statistically significance when P<0.05.Results:1.Western Blot experiment:1.1 HIF-la total protein:no significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(p>0.05);HIF-la nuclear protein,AQP-4 protein and MMP-9 protein:significant interaction effects were observed between low O2(16%)intervention and high CO2(5%)intervention(p<0.05);Further simple effect analysis shown that:there was no significant difference between the S group and HC group in the expression of HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein in the hippocampus of SD rats(p>0.05),while the expression in H group was significantly increased in comparison with S group(P<0.05).The expression of HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein in HH group was significantly increased in comparison with the H group(P<0.05)and the HC group(P<0.05).Pretreatment of 2ME2,the expressions of HIF-la nuclear protein,AQP-4 protein and MMP-9 protein in 2ME2 group were significantly deceased in comparison with the HH group.1.2 tight junction protein:significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(occludin protein:p<0.05;claudin-5 protein:p<0.05).Further simple effect analysis shown that:compared with the HC group,there was no significant difference in the expressions of occludin protein and claudin-5 protein in the hippocampus of SD rats in the S group(occludin protein:P>0.05;claudin-5 protein:P>0.05).The expression s of occludin protein and claudin-5 protein in the hippocampus of SD rats were significantly decreased in comparison with the S group(occludin protein:p<0.05;claudin-5 protein:p<0.05).The expressions of occludin protein and claudin-5 protein in HH group were significantly decreased in comparison with the H group(occludin protein:p<0.05;claudin-5 protein:p<0.05)and the HC group(occludin protein:p<0.05;claudin-5 protein:p<0.05).Pretreatment of 2ME2,the expressions of occludin protein and claudin-5 protein in 2ME2 group were significantly increased in comparison with the HH group.2 Double immunofluorescencehypercapnia alone can not increase the expression of HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein in the astrocytes of the hippocampus in SD rats;low concentration of O2(16%)intervention can significantly up-regulate the expression of HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein in the astrocytes of hippocampus in the H group.Low concentration of O2(16%)and high concentration of CO2(5%)intervention can further increase the expression of HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein in the astrocytes of hippocampus in the rats with hypoxemia.Pretreatment of 2ME2,the expression levels of HIF-la nuclear protein,AQP-4 protein and MMP-9 protein in 2ME2 group were significantly significantly.increased in comparison with the HH group.Conclusions:1.Hypercapnia can further increase the expressions of HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein in the astrocytes of hippocampus,along with increased degradation of the occludin and claudin-5 proteins and eventually further increase the permeability of the BBB in hypoxic rats.2.Inhibition of nuclear translocation of HIF-1a protein can significantly reduce the expressions of HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein,along with decreased degradation of occludin and claudin-5 proteins.Chapter 5 The effect of HIF-la nuclear protein translocation in the astrocytes induced by hypercapnia on the cognitive dysfunction and BBB disruption in hypoxic rats.Objective:To explore the effect of HIF-1a nuclear protein translocation in the astrocytes induced by hypercapnia on the cognitive dysfunction and BBB disruption in hypoxic rats.Methods:Drug administrationb)methoxyestradiol(2-Methoxyestradiol:2ME2,an inhibitor of nuclear translocation of HIF-1?)2ME2 was first dissolved in dimethyl sulfoxide(DMSO)and further diluted in phosphate buffered saline(PBS)to a final dilution of 2 ml.In the 2ME2 group,the inhibitor was administered(5 mg/kg,intra peritoneal injection)1 h before mechanical ventilation1.2 control vector(Vehicle)DMSO was diluted in PBS and finally diluted into a control vehicle of 2 ml volume.2.Groups and drug intervention2.1 SD rats were randomly divided into five groups:sham operation group(S group:normal air condition),hypoxemia group(H group:84%N2+16%O2),hypercapnia group(HC group 74%N2+21%O2+5%CO2),hypoxemia and hypercapnia group(HH group:79%N2+16%O2+5%CO2),hypoxemia and hypercapnia+2ME2 group.The S group,H group,HC group and HH group were intraperitoneally injected with a control vehicle(Vehicle)1 hour before mechanical ventilation.In the 2ME2 group,the inhibitor was administered(5 mg/kg,intra peritoneal injection)1 h before mechanical ventilation.The experimental rats were subjected to MWM test,EB test and BWC test after 3 hours mechanical ventilation.2.2 The specific process of MWM experiment,EB experiment and BWC experiment(refer to the Method section of Chapter 2,Chapter 3).Repeated measures analysis of variance was used to analyze repeated measures data;Factorial design analysis of variance was used to assess whether there was an interaction effect between hypoxia treatment and hypercapnia treatment;Simple-effect analysis were further peformed if an interaction effect was found.The difference was considered statistically significance when P<0.05.Results:1.The BWC experiment:significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(BWC:p<0.05).Further simple effect analysis showed that there was no significant difference in the BWC between the S group and HC group(P>0.05),while the BWC of SD rats in the group was significantly longer than that in S group.The BWC in HH group was significantly longer than that in H group(P<0.05)and HC group(P<0.05).Pretreatment of 2ME2,the BWC of the 2ME2 group was significantly lower in comparison with the HH group(P<0.05).2.The EB leakage experiment:significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(EB:p<0.05).Further simple effect analysis showed that there was no significant difference in the extravasation of Evans blue in SD rats compared with HC group(P>0.05),while the extravasation of Evans blue in H group was significantly more than that in S group(P<0.05).the extravasation of Evans blue in HH group was significantly more than that in H group(P<0.05)and HC group(P<0.05).Pretreatment of 2ME2,the extravasation of Evans blue in the 2ME2 group was significantly lower in comparison with the HH group(P<0.05).3.MWM experiment:significant interaction effects were observed between low concentration of O2(16%)intervention and high concentration of CO2(5%)intervention(48 h,72 h:all P<0.05).Further simple effects analysis showed that there was no significant difference in escape latency between the S group and the HC group(48 h,72 h:all P>0.05),while group escape latency in the H group was significantly longer than the S group(48 h,72 h:All P<0.05),the escape latency of the HH group was significantly longer than that of the H group(48 h,72 h:all P<0.05)and the HC group(48 h,72 h:all P<0.05).Pretreatment of 2ME2,the escape latency time of the 2ME2 group was significantly shorter than that of the HH group(48 h,72 h:all P<0.05).Conclusion:Inhibition of nuclear translocation of HIF-1? protein can significantly reduce the permeability of BBB,as result ameliorating neurocognitive dysfunctionChapter 6 The effects of acidosis induced by high concentration of CO2 on the proteins related with BBB permeability,BBB permeability and cognitive function in SD rats with hypoxemia.ObjectiveTo explore the effects of acidosis induced by high concentration of CO2 on the proteins related with BBB permeability,BBB permeability and cognitive function in SD rats with hypoxemia.1 Drug administration1.1 Tris-base:7.28%Tris-base(2-3ml/kg)was diluted with an equal amount of 5%-10%glucose solution1.2 Control vector(Vehicle):physiological saline(2-3ml/kg)was diluted with an equal amount of 5%-10%glucose solution,and then slowly intravenously drip into the experimental rats.2.Groups and Drug administration:SD rats were randomly divided into two groups:hypoxemia and hypercapnia group(HH group:79%N2+16%O2+5%CO2),hypoxemia and hypercapnia+Tris-base group(Tris-base group:79%N2+16%O2+5%CO2).In the HH group,a control vector(Vehicle)was intravenously administered through the tail vein in the experiment.In the Tris-base group,Tris-base was administered intravenously through the tail vein and at the same time,the pH value was neutralized between 7.35 and 7.45.2.1 The experimental rats were subjected to MWM test,EB test and BWC test after 3 hours mechanical ventilation..2.2 The brain tissues of the hippocampus were obtained after 3 hours mechanically ventilation in the above groups.Western blot was used to detect the total and nuclear protein,and AQP-4 protein,MMP-9 protein,occludin protein and claudin-5 protein in hippocampus of SD rats.The expressions of HIF-1a protein,AQP-4 protein,MMP-9 protein in the astrocytes of hippocampus were further verified by double immunofluorescence.Repeated measures analysis of variance was used to analyze repeated measures data;Factorial design analysis of variance was used to assess whether there was an interaction effect between hypoxia treatment and hypercapnia treatment;Simple-effect analysis were further peformed if an interaction effect was found.The difference was considered statistically significance when P<0.05.Results:1.Acidosis induced by high concentration of CO2 on the proteins related with BBB permeability in SD rats with hypoxemia.1.1 Western Blot experiment:Compared with the HH group,there were no significant differences in the expressions of HIF-1a total protein,HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein in the hippocampus of SD rats in the Tris-base group((P>0.05)Compared with the HH group,there were no significant differences in the expressions of occludin protein and claudin-5 protein in the hippocampus of SD rats in the Tris-base group(P>0.05)c)Double immunofluorescence:Compared with the HH group,there were no significant differences in the expressions of HIF-1a total protein,HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein in the the astrocytes of hippocampus in SD rats in the Tris-base group(P>0.05)2.Acidosis induced by high concentration of CO2 on BBB permeability in SD rats with hypoxemia:Compared with the HH group,there were no significant differences in the extravasation of EB and BWC in the Tris-base group(P>0.05).3.Acidosis induced by high concentration of CO2 on neurocognitive function in SD rats with hypoxemia:Compared with the HH group,there were no significant differences in escape latency in the Tris-base group(48 h,72 h:all P>0.05)Conclusions:1.The further upregulation of HIF-1a nuclear protein,AQP-4 protein and MMP-9 protein in the astrocytes of hippocampus in the hypoxic rats induced by hypercapnia was not related with the acidosis induced by high concentration of CO2.2.The further degradation of occludin protein and claudin-5 protein in the astrocytes of hippocampus in the hypoxic rats induced by hypercapnia was not related with the acidosis induced by high concentration of CO2.3.The further disruption of the BBB in the hypoxic rats induced by hypercapnia was not related with the acidosis induced by high concentration of CO2.4.The exacerbation of neurocognitive dysfunction in the hypoxic rats induced by hypercapnia was not related with the acidosis induced by high concentration of CO2. |
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