| BackgroundZebrafish has emerged as an ideal vertebrate animal model, and it possesses a host of advantages such as small individuals, simple farming, in vitro fertilization, in vitro development, short reproductive cycle,strong fertility, transparent embryos, besides, it has about 87% genes as similar as human. Although there is no outer and middle ear, but it has vertebrate typical inner ear structures. In addition to inner ear, zebrafish has another sensory organs-lateral line, comprised by a series of individual neuromasts, which are containing with cell clusters of mechanosensory hair cells.In view of this, a large number of international researchers choose larval zebrafish to study the damage and regeneration of lateral line hair cells, but some researchers use adult zebrafish to study the damage and regeneration of inner hair cells.Cisplatin is a commonly used anticancer drugs in clinical, with the treatment of cancer is also associated with varying degrees of side effects, one of the most serious side effects is damaging human inner ear hair cell and leading to permanent hearing loss. Unfortunately, the accurate mechanisms of cisplatin injury are not fully clear. As in mammal, the damage of inner ear hair cells is irreversible, but in Lower vertebrates such as birds, fish and amphibians, it has varying degrees capability of regeneration after hair cell damage. In recent years, studies have shown that hair cells in mammalian inner ear and vestibular retain certain ability of regeneration, as kindled hope of regeneration in human inner hair cell. Hence, it is necessary to focus on the study of the hair cell damage, the mechanism of hair cell regeneration and gene therapy for hair cell.As in the past study, there have research reports about cisplatin toxicity to inner ear hair cells of mammals and birds, but there is still no cisplatin damage model of adult fish inner ear. In this study, through the establishment of adult zebrafish model of cisplatin ototoxicity will help to further reveal the mechanism of hair cell injury, may provide new clues for the hair cell damage protection. Study also showed that cisplatin ototoxicity blocks sensory hair cell regeneration in the avian inner ear in vitro. Considering the advantages of zebrafish model and its powerful hair cell regenerative capacity, we use adult and larval zebrafish to study cisplatin ototoxicity, discussing the effect of cisplatin injury on the regeneration of hair cells, uncovering the similarities and differences of hair cells regeneration after cisplatin ototoxicity between adult and larval zebrafish, which may lay a foundation of genetic studies for hair cell regeneration in zebrafish.ObjectiveThis research first established cisplatin ototoxicity model of adult zebrafish which will make up for gaps in the relevant research area. Through discuss toxic damage of cisplatin on adult zebrafish inner ear hair cells and larval zebrafish lateral line hair cells, and the effect on hair cells regeneration after ciplatin damage, will lay the foundation for revealing gene regulation associated with hair cell regeneration.Methods1. Cisplatin damage zebrafish inner ear hair cells1.1 The intraperitoneal injection of adult zebrafishAdult zebrafish (aged range from 4-5 months, weight range from 0.38-0.55g) were randomly divided into cisplatin group, gentamycin group and control group (N =6).Cisplatin group were given two consecutive intraperitoneal injection of 24 mg/kg cisplatin solution, interval of 12h. Gentamycin group were firstly given the same doses normal saline as cisplatin group,12h later was given 250 mg/kg gentamycin sulfate solution. The control group received the same doses of normal saline as cisplatin group.1.2 Dyeing the stereocilium of adult zebrafish inner ear hair cellsAt the first day and tenth day after the last intraperitoneal injection, part of zebrafish in cisplatin group and control group were killed after anesthesia. The heads were removed and fixed in 4% paraformaldehyde at 4℃ overnight. Then the fixed tissues were cleaned by phosphate buffered saline (PBS)and dissected under tereoscopic microscope to separate the inner ear structures(saccules, lagenae, and utricles).Finally the ear tissues were incubated with 1:100 fluorescein phalloidin in dark box,cleaned again by PBS, placed on glass slides, cover-slipped, then photographed and observed under fluorescence microscope.1.3 Determination of cell proliferation in adult zebrafish inner ear24h、48h、72h after the last injection of cisplatin, the fish were injected intraperitoneally with 15ul BrdU(5mg/ml) in all group.After 10h recovery, fish were euthanized, fixed, and the inner ear maculae were excised. All tissue was incubated overnight at 4℃ temperature in 1:100 mouse monoclonal anti-BrdU antibody. After washing in PBS, tissues were incubated with 1:200 Alexa Fluor 488-conjugated goat anti-mouse IgG for 1 h at room temperature in a dark box. The tissues were washed with PBS again, placed on glass slides, cover-slipped, then photographed and observed under fluorescence microscope.2. The discussion of cisplatin damage larval zebrafish lateral line hair cells and dexamethasone promote the regeneration of lateral line hair cellsFive day post fertilization (dpf) larval zebrafish were selected and randomized into different groups with 10-15 zebrafish each.2μM fluorescent dye activity Yo-Pro-1 was selected to mark neuromast hair cells. Choose P1、P7、P8 three neuromasts to count up the hair cell numbers.2.1 Cisplatin damage lateral line hair cells of larval zebrafish5dpf larval zebrafish which were incubated with 2μm fluorescent dye Yo-Pro-1 in dark for 30min, were randomly divided into control group and experiment group. The experiment group were added into 1nM cisplatin solution with the control group added into the same amount of system water, both group were cultivate in dark room for 3h,6h. After the treatment of cisplatin and allow for 0h,3 h and 6 h recovery,took photos and counted the number of hair cells in neuromast under a fluorescence microscope.2.2 The validation of dexamethasone promoting the hair cells proliferate in larval zebrafish lateral line5 dpf larval zebrafish were randomly divided into control group and experiment group. The experiment group were added into 10μM,25μM dexamethasone solution with the contol group into the same amount of system water, both of group were cultivate in dark room for 48h.Immediatly after remove the solution, dyed with Yo-Pro-1, then took photos and counted the number of hair cells in neuromast under a fluorescence microscope.2.3 The study of dexamethasone promoting regeneration of lateral line hair cells damaged by cisplatin or neomycin.5dpf larvae zebrafish were randomly divided into control group, cisplatin group and neomycin group. Zebrafish in the cisplatin group were incubated with 1nM cisplatin solution for 3h, with neomycin group incubated with 200μM neomycin sulfate solution for 1h. Three hour later the solution was removed, both groups were added into 25μM dexamethasone solution or system water, while the control group was added into equivalent system water. At the 24 hour and 48 hour after administration of dexamethasone, the three groups were respectively dyed with Yo-Pro-1, took photos and counted the number of hair cells in neuromast under a fluorescence microscope.3. Statistical analysis.Quantitative data were analyzed by SPSS 17.0 statistical software.Data was presented by mean±standard deviation (M±S.D.).Comparison between two groups used independent sample t-test, multiple comparision using one-way analysis of variance (one-way ANOVA), and the test of homogeneity of variance adopted Levene test. The result was statistically significant by P< 0.05.RESULT1. Effects of cisplatin injection on hair cell bundle of adult zebrafish inner earBy staining with FITC-phalloidin, we found that the patterns of auditory sensory epithelium (saccule, lagenae, and utricle) in zebrafish are different. As for saccule, hair cell bundle density was reduced in the cisplatin group 24 hours following last cisplatin injection compared to the contol group. Specifically, after cisplatin injection, loss of hair cell bundles was significant at locations of 5%,25%, 50% and 75% with each area of 1600um along the rostral-caudal axis of the saccules (P<0.05). As ten day after last cisplatin injection, compared to the control group, there was no significantly change in hair cell bundle density of 5%,50% and 75% along the rostral-caudal axis of the saccules (P>0.05), but significantly less hair cell bundle density of 25%(P<0.05)2. Cell proliferation in the adult zebrafish inner ear after cisplatin ototoxicityAfter staining with Brdu, we found that there were scattered proliferating cells in zebrafish sensory epithelium. As 24h,48h after consecutively intraperitoneal injection of 24mg/kg cisplatin solution, a significant decrease in cellular proliferation was observed in the saccules of the cisplatin-injected group compared to the buffer group (P<0.05) and the gentamicin group (P<0.001).But 72h after last intraperitoneal injection of cisplatin solution, a significant increase in cellular proliferation was observed in the saccules of the cisplatin-injected group compared to the buffer group (P<0.001). However, in the gentamicin group, when at 24h,48h and 72h after intraperitoneal injection, a significant decrease in cellular proliferation was observed in the saccules as time went on, but the number of proliferation cells was still significantly more than the control group.3. Cisplatin can damage the zebrafish lateral line hair cells6 hours after administration of 1 nM cisplatin with 0 hour,3 hours recovery, the respective average hair cell number in P1, p7 and P8 neuromast was 1.970±0.526(N=11),1.767±0.629(N=10).3 hours after administration of 1 nM cisplatin with 0 hour,3 hours,6 hours recovery, the respective average hair cell number in the three neuromast was 5.909±0.761(N=11),2.633±0.483(N=10), 2.528±0.643(N=12). Compared to the control group, there was significant less hair cells in each treatment group (P<0.05)4. Dexamethasone promoted the proliferation of normal lateral line hair cellsAfter 48 hours’ dexamethasone disposal, the respective average hair cell number of 10μM and 25μM dexamethasone group was 11.028±1.314 (N=12),11.364±1.545 (N=11).The hair cell number in 7pdf control group is 9.922±0.862 (N=13). Compared to the control group, there was significant more hair cells in 10μM and 25μM dexamethasone group (P<0.01).5.Dexamethasone promoted proliferation of hair cells after neomycin injury rather than cisplatinAfter counting lateral line hair cells and useing the one-way ANOVA for statistic analysis, the results showed that:there was no significant difference between neomycin+dexamethasone 24h group and neomycin group(P=0.178), but there was significant difference between neomycin+dexamethasone 48h group and neomycin 48h group(P=0.001). However, there was no significant difference between cisplatin +dexamethasone group and cisplatin group after 24 hour and 48 hour disposal of 25 μM dexamethasone (P>0.05)Conclusion1. Consecutive intraperitoneal injection of 24 mg/kg cisplatin solution, with interval of 12h, can damage the inner ear hair cells of adult zebrafish. Under normal circumstances, there is a phenomenon of spontaneous update in adult zebrafish inner ear hair cells, as damaged by gentamicin, the regeneration of hair cell is very quickly, however, when damaged by cisplatin, the process of regeneration is delayed.2. Cisplatin can damage hair cells of larvae zebrafish lateral line, but it dependent on the duration of cisplatin administration. As damaged by neomycin, the hair cell can quickly regenerate, however, when damaged by cisplatin, the regeneration speed of hair cell slow down.3. Dexamethasone can promote proliferation of zebrafish lateral line hair cells in normal and after injury of neomycin, but it can’t promote hair cell proliferation after cisplatin ototoxicity. |
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