| Hypoxia is involved in the processes of many diseases. No adequate oxygen supplying the tissue cells had been reported to induce the disturbance of metabolism and dysfunction, even necrosis and apoptosis of the cells. Many studies have shown that noise and local ischemia/hypoxia may be responsible for the cochlear diseases. Furthermore hypoxia could aggravate the cochlear damage caused by other factors, even induce the tissue necrosis. It is of great significance to study the injury induced by hypoxia in cochlea and explore the mechanism of the death of the cochlear cells for the further understanding about the pathogenesis, pathological changes and treatments of the cochlear diseases and tumors. In this study we establish a practical model for cochlear organ cultivated in vitro model and observe the morphological changes of cochlear cells during hypoxia by the cytochemical methods, immunofluorescence and molecular biological techniques such as RT-PCR etc.; the expression of BNIP3 (Bcl-2 apoptotic protein family) and anti-apoptotic protein Bcl-2 in the cochlear tissue; the effect of hypoxia on the activity of caspase-3 and the protective effects of iNOS (inducible nitric oxide synthase) on the damage induced by hypoxia in the hair cells (HCs) of cochlea. From the following levels, that's of cell, of protein and of gene level, to explore the apoptosis of hypoxic cochlear cells; the rule of dynamic expression of various apoptotic factors in the process and discuss the relevance between the hypoxic condition and the expression of BNIP3 gene in the cochlear dells cultured in vitro during hypoxia, which would be the cornerstone of the research about the etiology, pathology and treatment of hypoxia-related ear diseases.Methods and materialsThe cochlear basal membrane from 3-day-old Wistar rats (whatever gender) were used. After the basal menbranes were isolated, they were layed in the medium containing DMEM (6BMs/dish), which were put in the incubator (37℃, 5% CO2) for overnight. The better basal membranes were randomized assigned into groups, each two dishes as a group. The control group was cultured in incubator (37℃, 5% CO2), however the experimental groups were put in Thermo multi- airway incubator (37℃, 90% N2, 5% CO2 and 5% O2) and cultured in hypoxia respectively for 0.5h, 1h, 2h, 6h, 12h, 24h and 48h according to various methods. To observe the deletion of cochlear HCs and the effect of iNOS (SMT) on hypoxic HCs by cytochemical methods; to observe the impact of hypoxia on spiral ganglion cells (SGC) and neurofiber, the expression of BNIP3 and the changes of mitochondrial membrane potential (MMP) during hypoxia by immunofluore- scence; to measure the changes of the activity of caspase-3 in the cochlear tissue under hypoxia by Western Blot method; to test the expression of Bcl-2 family apoptotic protein and anti-apoptotic protein and the effect of iNOS inhibitor (SMT) on the apoptosis of cochlear cells by RT-PCR technique. Eventually we would make a corresponding statistical analysis. Results1. The effect of hypoxia on the cochlear cultured in vitro with mmunofluor- escence staining showed that in the early stage of hypoxia, the HCs had no obvious changes; at 2h, the punctiform deletion of IHCs (inner hair cells of cochlea) occurred and the numbers of the cells decreased. The density of the HCs had remarkable difference compared with control (P<0.01). With the prolonged time of hypoxia, the amount of HCs apparently reduced and the HCs density descended continuously and the deletion level of HCs increased, esp. at 48h, at which point few IHCs could survive and the HCs density had remarkable difference compared with the control (P<0.01). At 6h of hypoxia, the deletion of OHCs (outer hair cells of cochlea) occurred and the density also had remarkable difference compared with the control (P<0.01). And the injury showed time-dependence. The result exhibited that hypoxia could induce the damage of the hair cells cultured in vitro, especially IHCs.Immunofluorescence staining showed that there were no obvious changes in morphous and density at 6h of hypoxia, but at 12h, the border of the SGCs became obscure and the amounts of cells in unit area decreased (the density of cells had remarkable difference, P<0.01). With the prolonged time of hypoxia, the degree of cells deletion exacerbated; the neurofiber appeared swollen and obscure at 6h of hypoxia, and we may see the sporadic intense fluorescence body; at 12h the swollen degeneration became more obvious, the intense F body increased, and the neurofiber fragmentation/ disintegration occurred in the local region. If the time of hypoxia prolonged, there would be larger area of fragmentation/ disinteg- ration, which could indicate neurofiber was more sensitive to hypoxia.To further observe whether the cell apoptosis was involved in the deletion of cochlear cells during hypoxia, we recorded the changes of cochlear cells at various time points. Stained with Hoechst, the nuclei of the control HCs were blue, round, uniform in size and dyeing and regular in array;at 6h of hypoxia the nuclei of HCs swelled and distorted, fluorescence weakened but a part of nuclei with fluorescence enhancement accompanied with subtle pyknosis;at 12h, the pykno- sis of the nuclei became more obviously, which induced the single nucleus fluore- scence enhancement, but the whole fluorescence intensity weakened and the nucl- ear fragmentation appeared; at 24h, pyknosis became more obviously, array disordered and in local region there was serious nuclear fragmentation and lamellar deletion of the nuclei of HCs. After the nuclear fragmentation, the cells would form many apoptotic bodies by budding. This experiment indicated that the apoptosis of cells also take part in the injury of cochlear cells induced by hypoxia.2. The apoptotic mechanism of cells. During the last 10 years the molecular mechanisms of programmed cell death had rapidly been clarified. Among these, caspase-3 is the most widely investigated member of the caspase family involved in the execution of cell death. The activation of caspase-3 is an essential step where a protease cascade pathway converges. Once the caspase-3 was activated, the process of apoptosis was irreversible. To evaluate indirectly the changes of the activity of caspase-3 protein in cochlear HCs at various periods during hypoxia, we measure the degradation degree of spectrin, which is the special substrate of caspase-3 by Western Blot and observe whether caspase-3 was evolved in the apoptotic process of HCs during hypoxia. In control group, there was the low level of degradation for caspase-3 substrate; while at 6h and 12h, no obvious change occurred, but at 24h spectrin degradated into 120KDa and 150KDa fragments, and the grey scale increased obviously. The mean grey scale had remarkable difference compared with the control (P<0.01). The result showed that caspase-3 was involved in the apoptotic process during hypoxia and long-term hypoxia could activate the caspase-3 induced signal transduction pathway, thus induce the apoptosis of cochlear HCs.MMP is a sensitive index to estimate the function of mitochondria and the value could directly reflect the function and number of mitochondria. If the MMP stepped down, the dysfunction of oxidative phosphorylation occurred and ATP decreased, which induced the apoptosis and necrosis. Rhodamine123 was a lipophilic cationic fluorescent dye which could penetrate into the cells and had the permeability of the membrane, thereby its fluorescence signals concentrated at mitochondria. The changes of fluorescence intensity could reflect those of MMP. It not only could show the morphous of mitochondria, but also reflect the permeability of membrane by the changes of the fluorescence intensity and calculation of the changes of MMP, so it is a fine reagent to study the morphous and function of mitochondria. By Laser scanning confocal microscopy we use Rhodamine123 as an indicator and observe the changes of MMP during hypoxia.After dyeing with Rhodamine123, the green fluorescence light can be observed in the mitochondria of living cells immediately by the laser scanning confocal microscopy. The array of HCs in control was regular and the cells could present the green fluorescence; at 6h the array became irregular, and the deletion of HCs occurred, meanwhile the fluorescence intensity in the cells decreased; at 12h the deletion of HCs became more obviously; at 24h the loss of HCs appeared in a larger area and the green fluorescence still can be seen in the residual. With the prolonged time of hypoxia, the intensity of fluorescence decreased gradually, which indicated that the MMP decreaed. The experiment demonstrated that hypoxia can induce the dysfunction of mitochondria in the process of the injury of cochlear cells. 3. The expression of Bcl-2 gene family in the hypoxic cochlear tissues Bcl-2 family is closely related to the cell apoptosis. According to various structure and function, the family consists of two categories: anti-apoptotic gene and pro apopto- tic gene. The proportion of two kinds of gene decided the reaction of cells to the death signals and whether the apoptosis occurred. The expression of anti- and pro-apoptotic gene of Bcl-2 family in the HCs cultured in vitro during hypoxia was measured by RT-PCR technique. The results showed that in normal cochlear tissue there should be a low level of Bcl-2, Bcl-w, Bcl-x and BNIP3 mRNA. Hypoxia could induce the transcription enhancement of apoptotic gene, BNIP3 mRNA, but no obvious changes of Bcl-x, then the level of Bcl-2 and Bcl-w mRNA dropped down slightly. The transcription of BNIP3 mRNA at 6h of hypoxia enhanced obviously and the mean grey scale had remarkable difference compared with the control (P<0.01). The level of BNIP3 at 12h was almost same with that at 6h; at 24h the level began to decrease, but the mean grey scale had also statistical significance (P<0.05). The transcription of Bcl-x mRNA at various periods had not enhanced, and there was no statistical significance among the mean grey scales (P>0.05). The transcription of Bcl-2 and Bcl-w mRNA at 6h and 12h decreased slightly, and no remarkable difference existed between the groups (P>0.05). The results of experiment indicated that the apoptotic protein BNIP3 of Bcl-2 family was involved in the hypoxia-induced cochlear cells death, and when the transcription at 6h and 12h of hypoxia enhanced, the transcription of Bcl-2 and Bcl-w mRNA decreased.4. The correlation between hypoxia and expression/localization of BNIP3 protein in the tissue of cochlea. After immunofluorescence dyeing, laser scanning confocal microscopy observed the cochlear cells: the positive expression of BNIP3 was red fluorescence in the cytoplasm and blue fluorescence in the nuclei. In the normal control the nuclei of HCs were blue, inner hair cells for one row and outer hair cells for three rows were clear to count in a regular array and there was no red material produced by expression of BNIP3 in the cytoplasm. In sustentacular cells of OSC there was a little erythrous material around the blue nuclei. At 6h of hypoxia, there was a little sporadic red plaque in the cytoplasm, and no statistical significance existed between the groups (P>0.05). While at 12h, the nuclei of HCs partly were broken into pieces and vanished and there was few residual nuclei pyknosis. There were a number of sporadic red plaques with inequal sizes. Compared with the control group and 6h of hypoxia there was a remarkable difference (P<0.01). Hypoxia could induce the enhancement of expression of BNIP3 protein in the cochlear cells and the effect had time-dependence.The results of experiments indicated that in the normal cochlear cells there was no expression of BNIP3 by immunofluorescence, however the expression of BNIP3 could be observed in the sustentacular cells of OSC. Meanwhile the result of RT-PCR showed the control group had little BNIP3, which indicated there was a little BNIP3 in the normal cochlear cells, mainly in the Hensen cells and there was no correlation between hypoxia and apoptosis. In this study hypoxia could induce the expression of BNIP3 in the normal cochlear cells and continuous expression in a long term, which could promote the cell apoptosis. The results could further indicate that BNIP3 is susceptible to hypoxia.The experiment indicated that the apoptotic protein BNIP3 of Bcl-2 family was involved in the injury of cochlear cells induced by early hypoxia. At 24h of hypoxia, the transcription activity of BNIP3 mRNA deceased. Our experiment of early stage showed there were no obvious change between the activity of caspase-3 protein at 6h and 12h of hypoxia and the activity of caspase-3 enhanced obviously at 24h of hypoxia, which demonstrated at the early stage of hypoxia (6-12h) the apoptosis mainly induced by increasing BNIP3 and cutting MMP down, thus changing the structure of outer membrane of mitochondria instead of the effect of caspase-3. However, at advanced stage of hypoxia (24h) the apoptosis was chiefly by the pathway of activating caspase-3. The effect of BNIP3 in the process of cell death differed with other members in the Bcl-2 family.5. iNOS was invovled in the apoptosis of cochlear cells induced by hypoxia Inducible nitric oxide synthase (iNOS) could be activated by some factors and produced a number of NO, which could induce body injury, but whether there was any changes in the cochlear tissues during hypoxia remained unknown. The early experiment showed that mainly increasing of BNIP3 produced the effect of hypoxia on apoptosis of cochlear cells. But the relationship between iNOS and BNIP3 was a puzzle. Thus by cytofluorescence chemistry and RT-PCR the study intended to explore the correlation of iNOS and BNIP3 and the effect of iNOS inhibitor (SMT) on apoptosis of cochlear cells induced by hypoxia.S-Methylisothiourea Sulfate (SMT) is a highly selective inhibitor of iNOS. The effect of hypoxia on the HCs was as follows: in the control group lack of oxygen for 24h, the array of inner/outer HCs'pilus disordered and there was lamellar deletion of HCs. In unit area, the cell densities of IHCs and OHCs were 8.325±0.104 and 47.512±0.125 respectively. In the experimental group with SMT, there was the deletion of HCs, but the array of the pilus of IHCs was very clear to see and there was a remarkable difference compared with the control cell density in unit area (Experimental group: 16.000±0.185, P<0.01). The experimental results showed SMT could inhibit the injury induced by hypoxia and protect the HCs, especially IHCs. The fact could be proved indirectly that iNOS was involved in the apoptosis induced by hypoxia. The results of RT-PCR indicated that in the control there was little transcription of BNIP3 and at 6h of hypoxia the transcription of BNIP3 mRNA enhanced obviously. The mean grey scales between groups had statistical significance (P<0.01). After adding SMT, the transcription activity of BNIP3 mRNA decreased greatly and compared with 6h of hypoxia there was a remarkable difference (P<0.01). We may draw a conclusion that SMT could inhibit the activity of iNOS, thus indirectly inhibit the transcription and protein expression of BNIP3 and lessen the injury of cochlear cells during hypoxia; what's more the protection of SMT was more obvious in IHCs. During hypoxia, activating iNOS to enhance the toxicity of NO and increasing the expression of pro-apoptotic protein BNIP3 induced the apoptosis of cochlear cells.Conclusion1. In this study we establish a practical model for cochlear organ cultured in vitro model and observe the toxic effects of hypoxia on the cochlear HCs and SGCs. It proved that the apoptosis of cells was the main pattern causing the injury of cochlear cells during hypoxia and the injury was of time-dependence. Meanwhile neurofiber is more susceptional to hypoxia and the injury induced by hypoxia varied from of ototoxicity by drug-induced.2. Long-term hypoxia could activate the signal transduction pathway of caspase-3, which induced apoptosis of cells, lowered MMP and deprived of the normal function of mitochondria. This experiment indicated that the mechanism of apoptosis induced by hypoxia was chiefly as follows: activating caspase-3, changing MMP and thus it deprived of the function of mitochondria. It proved indirectly that the apoptosis of cells induced by hypoxia was a complicated process in which many pathways and many factors were involved. 3. Hypoxia could induce the transcription enhancement of apoptotic gene BNIP3, but the levels of anti-apoptotic gene Bcl-2 and Bcl-w had no obvious changes. It indicated that the apoptotic protein BNIP3 of Bcl-2 family takes part in the process of cochlear cells apoptosis.4. The apoptotic protein BNIP3 of Bcl-2 family was involved in the injury induced by early hypoxia. Our experiment demonstrated at the early stage of hypoxia (6-12h) the apoptosis mainly induced by increasing BNIP3 and cutting MMP down instead of the effect of caspase-3. However, at advanced stage of hypoxia (24h) the apoptosis was chiefly by the pathway of activating caspase-3.5. iNOS was involved in and accelerated the apoptotic process induced by hypoxia and the effect of iNOS on apoptosis was produced by increasing the apoptotic protein BNIP3. It proved indirectly that during hypoxia the apoptosis of cochlear cells was induced by activating iNOS to enhance the toxicity of NO and increasing the expression of pro-apoptotic protein BNIP3. iNOS inhibitors (SMT) could lessen the injury of cochlear cells during hypoxia, what's more the protection of SMT was more obvious in IHCs.
|
PDF Full Text Request