| Objective: To study the expression localizations and changes with time of hypoxia-inducible factor-1α(HIF-1α) and cytoglobin (Cygb),and make approach to the relationship between them. RT-PCR and immunofluorescin histochemistry were applied after hypobaric hypoxia brain injury in the hippocampus of rats.Methods: Male Sprague-Dawley rats were randomly divided into control group and injury groups. The rats of injury groups were subdivided into 6h,12h,18h and 24h groups according to the duration in hypobaric hypoxia. The morphological changes of hippocampus neurons after hypobaric hypoxia injury were studied by H.E and toluidine blue stain. Water concentration in brain and a series of biochemical indicators (SOD, CAT, MDA, NOS, NO) were determinated with physical and biochemistry method individually. The expression levels of HIF-1αand Cygb on mRNA were studied by reverse transcription polymerase chain reaction (RT-PCR). The expression localizations and changes with time of HIF-1αand Cygb in the hippocampus of rats were investigated by double immunofluorescence staining.Results: 1 The Morphological changes in the hippocampus of rats after hypobaric hypoxia: Neurocytes in hippocampal CA1 were more sensitive to hypobaric hypoxia than in hippocampal CA2, CA3 and CA4, and the morphology of many injured cells appeared significant changes that neurite and dendrite obviously shortened, cyton deflated, nucleus condensed and cytoplasm stained with homogeneous oxyphilic red colour (red neurocyte). Meanwhile, the main character of neurocytes in hippocampal CA2 to CA4 was cellular swelling that cyton swelled, nucleus inflated or disappeared, cytoplasm stained light. The main character of neurocytes in 12h, 18h and 24h injury groups was cellular necrosis and apoptosis. With increasing duration of hypoxia, the incidence of shrinkage, karyorrhexis and karyolysis became serious. A lot of dead cells, cell debris and apoptotic bodies were observed densely in intercellular substance.By toluidine blue stain, the normal neurocyte nucleus was blue and cytoplasm was light blue with lots of dark blue Nissl bodies. The red neurocytes stained by H.E in hypobaric hypoxia groups were dark blue, the Nissl bodies were lost. The neurocytes with swollen cyton and light stained cytoplasm stained with H.E stain were light blue, the Nissl bodies were decreased in number or lost.2 The detection of water concentration in brain: Compared to control group, the water concentration in brain was remarkably increased in hypobaric hypoxia groups (P<0.01). Following the injure duration increase, the water concentration was increased remarkably.3 The detection of biochemical indicators: Compared to control group, the activities of SOD and CAT were remarkably depressed in hypobaric hypoxia groups (P<0.01). Compared to control group, the activities of NOS were raised (P<0.01). Compared to control group, the contents of MDA were increased in 12h group (P<0.05), and remarkably increased in 18h and 24h groups (P<0.01). The contents of NO were increased in 12h, 18h and 24h groups (P<0.01).4 The results of RT-PCR:(1) HIF-1α: the expressions of HIF-1αwere negative in control group, but observed after hypobaric hypoxia brain injury. Following the injure persteturing, the expressions of HIF-1αwere increased remarkably. Compared to control group, the expressions of HIF-1αwere increased in hypobaric hypoxia groups (P<0.01).(2) Cygb: the expressions of Cygb were small in control group. Compared to control group, the expressions of Cygb were increased in 6h group (p<0.05), 12h group, 18h group and 24h group (P<0.01), and had character of time dependent.5 The results of immunofluorescence staining: HIF-1αpositive cells showed green fluorescence; Cygb positive cells showed red fluorescence; both of them showed yellow fluorescence. There is no expression of HIF-1αin control group. But the positive signals of Cygb appeared in both cytoplasm and nucleus, especially high in nuclear membranes and nucleolus. The expressions of HIF-1αappeared in 6h group, but were mainly located in cytoplasm. The expressions of Cygb were increased, and the positive signals also appeared at neurite and dendrite. The expressions of HIF-1αappeared to both cytoplasm and nucleus in 12h group. The expressions of Cygb were increased obviously. The expressions of HIF-1αand Cygb were both increased remarkably. Although lots of conoid cells emerged karyorrhexis and karyolysis, the expressions of HIF-1αwere concentrated in nucleus region in 18h and 24h groups. The expressions of Cygb were increased in both cytoplasm and nucleus.Conclusion: 1 The morphological changes in the hippocampus of rats after hypobaric hypoxia was cellularoedema, cellular necrosis and apoptosis. With increase of the injure duration, the injury was character as time-dependent. Neurocytes in hippocampal CA1 were more sensitive to hypobaric hypoxia than in hippocampal CA2, CA3 and CA4. 2 The water concentration in brain was increased after hypobaric hypoxia injury. The degree of cerebral edema was aggravated with the hypobaric hypoxia injury duration increase. 3 The hypobaric hypoxia injury lead to the neurocytes in brain subjected oxidative stress injury. The activities of SOD and CAT were remarkably depressed, the activities of NOS were raised, and the contents of MDA and NO were increased after hypobaric hypoxia injury. 4 The expressions of HIF-1αcan not be found in normal hippocampal neurocytes. After hypobaric hypoxia injury, the expressions of HIF-1αappeared in cytoplasm firstly. With the hypobaric hypoxia injury duration increase, the expression of HIF-1αincreased progressively, and concentrated into cytoplasm. This hints that HIF-1αexpressed in cytoplasm, but took action by combination with DNA in nucleus as a transcription factor. 5 The expressions of Cygb appeared in both cytoplasm and nucleus at small amount, and were mainly located in nuclear membranes and nucleolus. Following hypobaric hypoxia injury, the expressions of Cygb were increased in both cytoplasm and nucleus. This hints that Cygb possible took action by combination with DNA in nucleus as a transcription factor, besides as a globin in cell. 6 The different temporal patterns and localization of HIF-1αand Cygb expressions following injury were observed in hippocampus neurons. It was suggested that there was a close relationship between HIF-1αand Cygb during hypobaric hypoxia injury. The expressions of HIF-1αand Cygb had important roles in the progression of hypobaric hypoxia brain injury. The study of expressions of HIF-1αand Cygb would be helpful for revealing the mechanisms of hypobaric hypoxia brain injury.
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