Hypoxic preconditioning: effect, mechanism and clinical implication (Part 1) / 中国应用生理学杂志

Hypoxic preconditioning (HPC) refers to exposure of organisms, systems, organs, tissues or cells to moderate hypoxia/ischemia that is able to result in a resistance to subsequent severe hypoxia/ischemia in tissues and cells. The effects exerted by HPC are well documented. The original local in situ (LiHPC) is now broadened to remote ectopic organs-tissues (ReHPC) and extended crossly to cross pluripotential HPC(CpHPC) induced by a variety of stresses other than hypoxia/ischemia, including cancer, for example. We developed a unique animal model of repetitive autohypoxia in adult mice, and studied systematically on the effects and mechanisms of HPC on the model in our laboratory since the early 1960s. The tolerances to hypoxia and protection from injury increased significantly in this model. The adult mice behave like hypoxia-intolerant mammalian newborns and hypoxia-tolerant adult animals during their exposure to repetitive autohypoxia. The overall energy supply and demand decreased, the microorganization of the brain maintained and the spacial learning and memory ability improved but not impaired, the detrimental neurochemicals such as free radicals down-regulated and the beneficial neurochemicals such as adenosine(ADO) and antihypoxic gene(s)/factor(s) (AHGs/AHFs) up-regulated. Accordingly, we hypothesize that mechanisms for the tolerance/protective effects of HPC are fundamentally depending on energy saving and brain plasticity in particular. It is thought that these two major mechanisms are triggered by exposure to hypoxia/ischemia via oxygen sensing-transduction pathways and HIF-1 initiation cascades. We suggest that HPC is an intrinsic mechanism developed in biological evolution and is a novel potential strategy for fighting against hypoxia-ischemia and other stresses. Motivation of endogenous antihypoxic potential, activation of oxygen sensing--signal transduction systems and supplement of exogenous antihypoxic substances as well as development of HPC appliances and HPC medicines such as AHFs are encouraged based on our basic research on HPC. HPC may result in therapeutic augmentation of the endogenous cytoprotection in hypoxic-ischemic or suffering from other diseases' patients. Evolutionary consideration of HPC and clinical implications of HPC are both discussed to guide future research. The product of AHF is expected to be one of the most effective first aid medicines to rescue patients in critical condition. HPC is beginning to be used in surgery and is expected to be developed into a feasible adaptive medicine in the near future.

Effect of brain homogenate from hypoxia-preconditioned mice on rat embryonic hippocampal neurons with hypoxia/reoxygenation injury / 中华神经医学杂志

Objective To observe the effect of brain homogenate (BH) extracted from hypoxia-preconditioned mice on the viability and apoptosis of rat embryonic hippocampal neurons with hypoxia/reoxygenation-induced injury. Methods Rat embryonic hippocampal neurons primarily cultured for 8 days in 96 well tissue culture plate were divided into 5 groups, namely the normal control group (treated with PBS), H<,4>R<,48> group (with hypoxia for 4 h followed by reoxygenation for 48 h and PBS treatment), H0 group (with BH from normal mice prior to hypoxia/reoxygenation), H1 group (with BH from acute hypoxia-preconditioned mice and hypoxia/reoxygenation), and H4 group (with BH from hypoxia-preconditioned mice and hypoxia/reoxygenation). The viability and apoptosis of the cells in the 5 groups were observed by MTT assay and flow cytomertry, respectively. Results The cell viability was significantly higher in the normal control group than in H<,4>R<,48> group. In H0, H1, and H4 groups, the cell viability increased significantly as compared with that in H<,4>R<,48> group, and the cells in H4 group showed the highest viability. Apoptotie cells were scarcely observed in the normal control group, but were numerous in H<,4>R<,48> group. Compared with H<,4>R<,48> group, H0, H1, and H4 groups showed obviously reduced apoptotic cells, and the reduction was the most conspicuous in H4 group. Conclusion BH extracted from hypoxia-preconditioned mice may offer protection against hypoxic injury of rat embryonic hippocampal neurons challenged with hypoxia-reoxygenation by promoting the cell viability and decreasing cell apoptosis.

Changes in Bcl-2 and Caspase-3 expressions in cortex of hypoxic preconditioning mice / 生理学报

The purpose of the present study was to explore the roles of Bcl-2 and Caspase-3 in mouse cortex in hypoxic preconditioning. Blb/c mice were randomly divided into three groups: control group, hypoxic group and hypoxic preconditioning group. Fluorescence intensity of Bcl-2 and Caspase-3 was observed and number of positive cells was counted in parietal cortex by immunofluorescence and confocal laser scanning microscope. Fluorescence intensity of Bcl-2 in the normal group, hypoxic group and hypoxic preconditioning group was 6.2±1.7, 68.5±13.1, 180.6±34.8, respectively, and number of Bcl-2-positive cells was 18.5±4.9, 52.3±10.5, 150.8±24.7, respectively. Fluorescence intensity of Caspase-3 in the control group, hypoxic group and hypoxic preconditioning group was 8.6±2.0, 40.2±8.2, 26.4±6.1, respectively, and number of Caspase-3-positive cells of was 4.3±1.2, 63.6±12.5, 45.7±9.8, respectively. The results showed that the expressions of Bcl-2 in both hypoxic group and hypoxic preconditioning group were significantly higher than that in the control group; and the expression of Bcl-2 in hypoxic preconditioning group was even higher than that in hypoxic group. The expressions of Caspase-3 in hypoxic group and hypoxic preconditioning group were also significantly higher than that in the control group; whereas the expression of Caspase-3 in hypoxic preconditioning group was significantly lower than that in hypoxic group. These results suggest that cortex cells are resistant to apoptosis via increased expression of Bcl-2 and lowered expression of Caspase-3 in the cortex and brain cells are thereby protected during hypoxic preconditioning.

The effect of hypoxia preconditioning no binding activity of HIF-1 on the HRE with EPO in the hippocampus of mice / 中国应用生理学杂志

<p><b>AIM</b>To observe change of binding activity of HIF-1 with erythropoietin (EPO) hypoxia response element (HRE) in the hippocampus of mice preconditioned to hypoxia and explore relationship between the changes and the preconditioning.</p><p><b>METHODS</b>The hippocampus was removed from mice exposed to hypoxia for 0 run (control group), 1 run (H1 group) and 4 runs(H4 group). Electrophoretic mobility shift assays (EMSA), chromatin immunoprecipitation (ChIP)and real time PCR were used to detect the change of activity of HIF-1 on HRE of EPO.</p><p><b>RESULTS</b>Both in vitro and in vivo binding tests showed that the HIF-1 DNA-binding activities were increased in group H1 and markedly increased in group H4.</p><p><b>CONCLUSION</b>The increase of HIF-1 and HRE of EPO binding activities is thought be involved in hypoxic preconditioning.</p>

Hypoxic preconditioning increase nPKCepsilon membrane translocation in the brain of mice / 中国应用生理学杂志

<p><b>AIM</b>To explore the role of novel protein kinases C (nPKCs) in the development of cerebral hypoxic preconditioning.</p><p><b>METHODS</b>By using the mice model of hypoxic preconditioning, which was established before in our lab, the biochemistry techniques of SDS-PAGE and Western blot were applied to observe the effects of repetitive hypoxic exposure (H0-H4) on nPKCs (nPKCepsilon, delta, eta, mu and theta) membrane translocation in hippocampus and cortex.</p><p><b>RESULTS</b>nPKCepsilon membrane translocation was increased in response to the hypoxic exposure times in the hippocampus (H0: 41.6% +/- 1.4% vs. H1-H4: 46.9% +/- 4.5%, 52.7% +/- 3.9%, 58.8% +/- 2.7% and 61.3% +/- 3.7%) and cortex (H0: 38.4% +/- 4.5% vs. 42.4% +/- 5.0%, 48.7% +/- 6.5%, 55.3% +/- 8.9% and 61.2% +/- 10.2%) of mice, and there were statistic significances among H2, H3 and H4 in hippocampus, and H3 and H4 in cortex respectively (P < 0.01). But for nPKCdelta, eta, mu and theta membrane translocation, there were no any significant changes in hippocampus and cortex of hypoxic preconditioned mice.</p><p><b>CONCLUSION</b>nPKCepsilon may play an important role in the development of cerebral hypoxic preconditioning, but it need more evidence to prove.</p>

Brain mechanisms of hypoxic preconditioning / 中国应用生理学杂志

A Review: A concept of tissue adaptation to hypoxia (i. e. hypoxic preconditioning) was developed and its corresponding animal models were reproduced in 1966s. The methods of model reproduction in rat, rabbit, and mouse in particular and the main results are briefly introduced in this review. The tolerance to hypoxia of preconditioned animals is significantly increased. Regular changes in animals' behavior, neurophysiology, respiratory and circulatory physiology, neuron morphology in vivo and function of brain and spinal cord in vitro are briefly demonstrated. The protective effects in vivo and in vitro of homogenate extract taken from the brain of preconditioned animals, neurochemicals and molecular neurobiological alterations are briefly presented. The essence and significance of tissue adaptation to hypoxia/hypoxic preconditioning are discussed in the review in terms of evolution and practical implication.

Effect of repeated acute hypoxic treatment on the expression of alpha-synuclein in the mouse brain cortex / 生理学报

An anti-alpha-synuclein (alpha-SYN) monoclonal antibody produced in our laboratory was used to investigate the effect of repeated acute hypoxic treatments on the expression of alpha-SYN in the mouse cerebral cortex. Western blot analysis showed that the expression levels of alpha-SYN in the cortex changed accordingly upon hypoxic exposure times, as that the alpha-synuclein level significantly increased after the first hypoxic exposure and then dropped down to the background level after the fourth hypoxic exposure. Immunohistochemical staining revealed that the alpha-SYN-immunopositive substance was localized not only in the nerve endings, but also within the nuclei of some neurons. The cell density of the neurons with alpha-SYN immunopositive nuclei was increased significantly after the first hypoxic exposure but returned back to control levels after the fourth hypoxic exposure. Our results indicate that both of the alpha-SYN expression level in the brain and the number of the neurons with alpha-SYN positive nuclei are affected by the repeated acute hypoxic treatments and that this modification is hypoxic time-dependent. The mechanism and the physiological significance underlying these changes need to be further investigated.

Measurement of nitrate and nitrite concentrations in biological fluids using high performance liquid chromatography / 中国应用生理学杂志

<p><b>AIM AND METHODS</b>New method to analyse nitrate and nitrite concentrations in saliva, serum and urine was developed using high performance liquid chromatography.</p><p><b>RESULTS</b>The whole isolation process was completed in less than 7 minutes, the determination linearity of nitrate and nitrite were 0.7 ng-100 ng and 5 ng-100 ng, respectively. MINIMUM: Detectable limits of nitrate and nitrite were 0.3 ng and 2 ng, respectively. Nitrate recovery ratio was 99%-102% and nitrite recovery ratio was 99%-104%. The RSD of nitrate and nitrite was 0.8% and 1.7%, respectively.</p><p><b>CONCLUSION</b>In comparison with other methods available, the present method seems to be simpler, more sensitive and specific.</p>