In Vitro Effects Of Bilirubin On Rat Cochlear Tissue

PART1In vitro effects of bilirubin in different concentration on the cultured cochlear tissueObject To investigate the in vitro toxic effects of bilirubin in different concentration on the cultured P3rat cochlear tissue.Method Fifty cochlear basilar membranes (CBMs) containing the organ of Corti and spiral ganglion neurons (SGNs) were gently isolated from twenty-five healthy P3Sprague-Dawley (SD) rats of both genders. After12hour’s primary culture, the culture medium was replaced with fresh prepared DMEM/F12medium containing5%FBS.40CBMs in good survival shape were selected for treatment and were randomly divided into four groups (10each),3experimental group treated with bilirubin (3experiment group:randomly be10μmol/L,50μmol/L,100μmol/L) respectively and one control group treated with normal saline. After24h’s culture, all the samples in the four groups were fixed and underwent immunofluorescence staining. The morphology of the SGNs or HCs were observed under laser confocal microscope. The numbers of the nerve fibers and hair cells were counted and underwent statistical analysis.Result After24h’s bilirubin treatment, the in vitro cultured cochlear tissue in the10μmol/L group didn’t show detectable toxic effects compared with control group, while in the50μmol/L group,the number of neuronal cell bodies and dendrites fiber reduced significantly, with the well kept hair cells morphology and quantity. The neurons almost disappeared on the100μmol/L experiment group, at the same time, some of the hair cells were also damaged.Conclusion SGNs injury by bilirubin in a concentration-dependent manner. That is, the number of the nerve fibers reduced apparently with the increasing bilirubin concentration. The SGNs were damaged firstly, and the hair cells were also damaged with the increased bilirubin concentration at last.PART2In vitro effects of bilirubin in different time on the cultured cochlear basilar membraneObjective To investigate the in vitro toxic effects of bilirubin in different time on the cultured P3rat cochlear tissue.Method Seventy pieces of CBMs containing the organ of Corti and spiral ganglion neurons were gently isolated from thirty-five healthy P3Sprague-Dawley (SD) rats of both genders. After12hour’s primary culture, the culture medium was replaced with freshly prepared DMEM/F12medium containing5%FBS.On the2nd day,60basilar membranes in good survival shape were selected for treatment and were randomly divided into six groups (10each) to receive treatment with normal saline (3control group:randomly be24h,48h,72h normal saline treatment) or treatment with bilirubin (3experimental group:randomly be24h,48h,72h bilirubin treatment) respectively. Bilirubin was added to the three experiment groups with the final concentration was50μmol/L in each culture dish, while the same volume saline was added into the control groups. We fixed the experiment group and the control group in the corresponding time point (24h,48h,72h). All specimens were fixed and immunofluorescence dyed. The morphology of the SGNs or HCs were observed under confocal microscope. At the same time, we count the number of the neurofilaments and hair cells in each group. The statistical methods were used to analysis the data of the six groups.Result After24h’s bilirubin treatment, the SGNs and its dendritic fibers reduced significantly on the in vitro cultured cochlea, while the morphology and quantity of the hair cells were good. With the processing time prolonging, the neurons decreased more apparent on48h treatment group, and the outer hair cells of the basal turn began to missing. The neurons almost disappeared on the72h experiment group, at the same time, the outer hair cells of the basic turn and middle turn decreased significantly, what’s more, the number of inner hair cells of the basal turn also showed the sign of reduction.Conclusion1. SGNs injury by bilirubin in a time-dependent manner, that is, the number of the nerve fibers reduced gradually with the prolonging bilirubin processing time.2. Bilirubin injury to neurons and hair cells with orderliness. That is, in the same time period (24h), the neurons have been injured apparently while the morphology and the quantity of the hair cells were good in the treatment group. However, with the treatment time prolonging (48h and72h), Hair cells were also damaged and the outer hair cells were injured originally. We find the inner hair cells were relatively more resistant to bilirubin toxicity. Therefore, we conclude that the bilirubin toxicity on the structure of in vitro cultured cochlear basilar membrane is orderly with the SGNs were damaged first, followed by the OHCs, and the last is the IHCs.PART3Caspase-3expression and apoptosis process in spiral ganglion neuron under the condition of bilirubinObject To investigate the in vitro toxic manner of bilirubin on the cultured P3rat cochlear basilar membrane.Method forty-six of CBMs containing the SGNs and Corti were gently isolated from twenty-three healthy P3Sprague-Dawley (SD) rats of both genders. After12hour’s primary culturing, the culture medium was removed and replaced with5%FBS fresh medium. Then the40CBMs in good survival shape were randomly divided into four groups (10each) and treated with normal saline (12h control group) or bilirubin (3experiment group:randomly be12h,18h,36h bilirubin treatment) respectively. For the experimental group, bilirubin was added to the three experiment groups with the final concentration was50μmol/L in each culture dish, while the same volume saline was added into the control groups. We fixed samples in the corresponding time point (12h,18h,36h). Then all the samples in the four groups were underwent immunofluorescence staining. The protein distribution of caspase-3were observed under confocal microscope.Result The apoptosis protein caspase-3high expressed in SGNs on12h experiment group. On the18h experiment group, We can find the crumbled SGNs or much of cellular debris. With the prolonged duration of bilirubin on36h, the number of the cell body and its dendrite fibers reduced significantly, in which the type I neurons reduced more obvious than Type Ⅱ.Conclusion Bilirubin is able to active the cell apoptosis program on the SGNs with the protein caspase-3expresssion. the Type I neurons reduced more obvious than TypeⅡ.