| Background and objectiveBrain hypoxia, a central pathogenetic syndrome causing various structural changes of the brain and its malformations, frequently occurs during early development of variable pathological states. Cerebral edema is a clinical patho-condition when excess water accumulates in the intra- and/or extracellular spaces of the brain. As the most frequent and serious complication of brain hypoxia, the developing molecular pathological mechanism of cerebral edema remains unclear. According to different pathogenesis, cerebral edema can be classified as vasogenic and cytotoxic edema. Vasogenic edema occurs when the blood-brain barrier becomes leaky, permitting the entry of plasma fluid into the brain parenchyma; whereas cytotoxic edema consists mainly of intracellular fluid accumulation that occurs during water intoxication and anoxia-generating conditions such as stroke, trauma and hypoxia. It is believed that astrocyte is the major cell type showing swelling in the later conditions.A new family of related water-transporting proteins, called aquaporins(AQP), has been identified over the past decade. Widely scattering in mammals, amphibians, plants, yeast, bacteria and many other lower organisms, it has been confirmed that AQP plays an imperative role in the pathophysiology of brain edema. At least 12 AQP water channels have been cloned (AQPO, AQP1~11) from mammals, among which AQP4 is expressed most strongly in the brain, mainly around the brain parenchyma microvessels, including astrocyte foot processes near capillaries and in ependymal cells lining the ventricles-key sites for water movement between the cellular, vascular, and ventricular compartments. Recently, the hypothesis was tested that AQP4 was likely to be of the prime importance in the brain's handling of water. However, most researches on the relationship between AQP4 and brain edema were performed in vivo, which is easily influenced by uncertain reasons. This study was aimed to further explore the molecular pathological mechanism of cerebral edema developing using astrocytes in vitro after hypoxia and reoxygenation.Nowaday, Researches on relation between AQP4 and cerebal edema under ischemiaand hypoxia condition almost focus on vivo level. But it is difficult to come to a reliable conclusion in vivo experiment for complex of inner-environment and individual difference. And reseach about AQP4 expression of brain cell under hypoxia condition in vitro is still untouched. Therefore, we intend to investrgate the changes of AQP4 expression under different degree of hypoxia and reoxgenation consequently in vitro by building up hypoxia and reoxgenation astrocyte model and then discuss the molecular pathological mechanism of ischemia and hypoxia brain edema. So as to providly experiment evidence for clinical treatment of ischemia and hypoxia brain edema.MethodsAstrocytes were isolated from cerebral cortices of SD rats. After incubation and subculturing, homogeneity of astrocytes was confirmed by immunocytochemical examinations with anti-glial fibrillary acidic protein(GFAP) to be over 95%. Hypoxia culture chamber was prepared with inflow and outflow valves, into which a mixture of 95% N2 and 5%CO2 was flushed for 15 minutes at a flow rate. During the exposure to hypoxia aliquots of the culture medium were collected at the indicated times from 0 to 12 hours for monitoring the partial pressures of oxygen (PO2), concentrations of glucose and lactate and lactate dehydrogenase (LDH) activity to ensure the best hypoxia time point for cultural astrocytes to study further. On this basis, 8 hours was selected as the best time point for cultural astrocytes for study in vitro.After exposure to hypoxia for 8 hours, the culture dishes were transferred to an ordinary incubator with 95% air and 5% CO2 from the cells for reoxygenation for indicated periods (2 hours, 4 hours, 6 hours, 8 hours and 10 hours specifically). At each indicated time point, total RNA was extracted from the cells with TRIZOL reagent to examine the expression of AQP4 on mRNA level by semi-quantified RT-PCR. Meanwhile, cells at different reoxygenation time points were fixed with acetic acid and polyformaldehyde for immunocytochemistry in conjunction with confocal microscopy to investigate expression spectrum and expression level of AQP4 in cell.ResultsThe astrocytes became to swell, float and die with the prolongation of the time of hypoxia. Compared with the control group, changes of number of dead cells, concentration of glucose and lactate, and activity of LDH of the hypoxia group were significant (PO.05) after hypoxia lOh. However, the flat and polygonal morphology of astrocytes almost remained unchanged after hypoxia 8h, although whose changes in media were also significant. Therefore, 8 hours was selected for the time of hypoxia to study AQP4 expression in astrocytes in vitro.The results of semi-quantified RT-PCR showed that expression of AQP4 in astrocytes in vitro on mRNA level was reduced most significantly (p<0.01) comparing with the normal control. However, if reoxygenation occurred after hypoxia for 8 hours, the expression of AQP4 was up back gradually as the reoxygenation time extended. When reoxygenating for 6 hours, the level of AQP4 mRNA in astrocytes in vitro almost reached the normal level (P>0.05). If reoxygenation continued, mRNA of AQP4 mRNA would be higher than normal control. Furthermore, the sequences of our PCR products were identical with the sequences of AQP4 mRNA of rat in Genbank with the exact sites from bp to bp.It was indicated by immunocytochemistry combining confocal microscopy that expression of AQP4 in astrocytes in vitro on protein level was reduced very obviously, and reoxygenation after hypoxia for 8 hours could increase the expression of AQP4 till up to the normal level when reoxygenation continued for 10 hours. Meanwhile, it was also found that in cultured astrocytes, unlike in astrocytes in vivo, expression of AQP4 was not only limited in astrocytes foot process any more. Instead, AQP4 was scattered evenly through the whole plasma membrane.ConclusionIn summary, the newly prepared hypoxia chamber is effective and 8 hours of hypoxia is the best time for astrocytes when further study on their biological properity is needed; expression of AQP4 in astrocytes after hypoxia for 8 hours in vitro reduces significantly may prevent astrocyte from swelling in the early stage of hypoxia damage. However, reoxygenation after hypoxia can increase the expression level of AQP4 even beyond that of the normal control, which cause the astrocyte swelling to balance the osmotic pressure and the irons level for better inner environment of neuron. This phenomena is consistant with the study in vivo that AQP4 in area surround the infarct center will highly express. This finding implies the inhibitor of AQP4 maybe prevent or postpone the procession brain edema.
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