| Hypoxia occurs under a large number of physiological and pathophysiological processes, such as embryonic development, adaptation to high altitudes, wound healing, in?ammation, cancer, and ischemic diseases. To study the human diseases associated with hypoxia, a variety of hypoxia models have been developed, such as physical and chemical hypoxia models. Although physical hypoxia models mimic the hypoxic environment and cause hypoxia-related insults to animals and cells, the establishment of these models requires a protocol that is not easy to control. Gas cylinders, which usually contain a desired gas mixture of 95% nitrogen and 5% carbon dioxide, and hypoxia chambers are generally needed to produce and maintain the conditions of physical hypoxia. Oxygen analyzers are also needed to precisely control the oxygen concentration. For the establishment of chemical hypoxia models, hypoxia-mimicking agents, such as cobalt chloride, deferoxamine, and sodium hyposulfite, are usually used. Although the mechanism by which cobalt and the iron chelator deferoxamine mimic the hypoxic response is based on competition with iron (a key factor of the oxygen sensing mechanism), cobalt is a heavy metal that leads to tissue and cellular toxicity during exposure. Despite all of these facts, cobalt only mimics some of the hypoxic insults induced by physical hypoxia. Another hypoxia model uses sodium hyposulfite to simulate the hypoxic response by depriving aqueous solutions of oxygen, similar to physical hypoxia. However, sodium hyposulfite spontaneously combusts, and its reaction with water is exothermic and accompanied by the generation of sodium sulfite (SS; Na2SO3) and sulfur dioxide (SO2). Therefore, it is important to develop new hypoxia models to illustrate the underlying mechanism of hypoxic insults.Analysis of the literature indicates that SS has been widely used as an oxygen scavenger to remove the last trace of oxygen from boiler feedwater in industrial practices. In addition, SS has also been used as a leading food preservative because of its ability to induce anaerobic conditions that effectively inhibit the growth of aerobic microorganisms. Taken together, these findings indicate that, as an oxygen scavenger used in biological research, SS has more advantages than sodium hyposulfite. The reaction of SS with water does not result in spontaneous combustion and does not generate sulfur dioxide.First of all, we examined the possibility of establishing a hypoxia model using SS in aqueous solutions. The result shows that DO (dissolved oxygen) could be suppressed by SS to create hypoxic conditions for a period of time. Furthermore, the levels and duration of the hypoxic conditions were dependent on the concentration of SS in the solutions. These data strongly suggest that SS is a potential and valuable chemical agent that can be used to mimic hypoxia in aqueous solutions. Secondly, we established the hypoxia model in C. elegans using SS. Comparing with physical hypoxic model, we evaluated the mortality, pharyngeal injury, neuronal axonal breakage and expression levels of hypoxia inducible factor 1 in C. elegans after SS treatment.We chose three different strains, wild-type N2 strain, hypoxia-resistent strain of daf-2(e1370) and hypoxia-sensitive strain of daf-16(mu86) in our study. The mortality patterns of all these three different strains are consistent with what the document have demonstrated. Additionally, thorough comparing the death rate of C. elegans after treatment of SS with different concentration and physical hypoxia, we found that there are not statistically significant between 2.0 g/L of SS solution and physical hypoxia groups. So we propose that SS lead to worm death by the way of removing off oxygen and 2.0 g/L of SS solution could replace physical hypoxia model in hypoxia-related studies.Necrotic-looking pharyngeal cells cannot be clearly observed after short period of hypoxic treatment. Based on the results of preliminary experiments, we observed the pharynx of C. elegans by DIC after 16 h hypoxia treatment. From the results, we could also make the conclusion that 2.0 g/L of sodium sulfite can replace physical hypoxia in C. elegans. Secondly, hypoxia could also affect neurons and result in the specific performance of axonal fragmentation. The results also show that, 2.0 g/L of sodium sulfite have the same function with physical hypoxic model. In protein expression level, both CeHIF-1(F38A6.3a) and CeHIF-1(F38A6.3c) are also stablized after SS treatment, which demonstrated that SS could mimic the hypoxia conditon and active the HIF-1 signaling pathway.Hypoxia is an important contributing factor for nervous system damage. Cerebral vascular disease, the third most common cause of death and the most common disabling neurologic disorder, have brought a great burden on patients and society. Therefore, studing the key molecular mechanisms of hypoxic injury to clinical prevention and treatment of such diseases has practical significance. Accordingly, clinical drug development on cerebral vascular disease, including stroke, has been the focus of medical research.Therefore, we also developed the mouse middle cerebral artery occlusion (MCAO) model to evaluate the neuroprotection of Neuroglobin (Ngb) with TAT tag. Neuroglobin, the newly discovered third globin, was reported to be expressed mainly in nervous system and retina. It has been reported that Ngb can protect neurons against ischemic/hypoxic insults. Several studies also suggested that Ngb may protect neuronal cells against oxidative damage by scavenging ROS, but there is still no clear idea for the underlying mechanism.We developed the mouse middle cerebral artery occlusion (MCAO) model by nylon line. Then, the triphenyltetrazolium chloride (TTC) staining method was optimized. The optimized'sandwich TTC staining'approach is a valuable method to determine the location and area of focal infarction in the ischemic brain, and will promote the detection efficiency of focal infarction after cerebral ischemia. Based on the MCAO model, we analyzed the change of infarction area after the mice were given different concentrations of TAT-Ngb protein by intravenous injection. The results show that low concentration of TAT-Ngb protein could significantly reduce the brain infarct size caused by ischemia/reperfusion insults. This may be related to the TAT tag can promote Ngb protein across the blood-brain barrier.In conclusion, our data clearly show that SS is a candidate hypoxia inducer that mimics hypoxic stress in C. elegans and can also be used in the study of hypoxic preconditioning and in the development of antihypoxic agents. In addition, throughing the mouse MCAO model we concluded that the TAT-Ngb protein has neuroprotective effect. Our study was important to the research of hypoxic preconditioning and in the development of antihypoxic agents and neuroprotective agents. |
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