Study On Time Sequence Of Spiral Ganglion Cells And Dorsal Cochlear Nucleus Degeneration Of Sprague-Dawley Rat Following Chronic Kanamycin-induced Deafness

Part IStudy on time course and mechanism of spiral ganglion cell degeneration of Sprague-Dawley Rat following chronic kanamycin-induced deafnessObjective:Establish the model of chronic kanamycin-induced deafness in adult rats. We investigated the dynamic morphological changes and degeneration process of the spiral ganglion cells (SGCs), and detected whether the endoplasmic reticulum (ER) stress was involved in the degeneration of spiral ganglion neurons (SGNs) following chronic kanamycin-induced deafness.MethodsRats were treated with kanamycin by subcutaneous injection at500mg/kg per day for10days. The morphological and density changes of SGCs were investigated at1,7,14,28,56and70days after kanamycin treatment. The expression of Bip, IRE1α, caspase-12, Phospho-eIF2-alpha (Ser51), Phospho-PERK (Thr980), GADD153and ATF-6a at each time point after kanamycin treatment were detected. Meanwhile, TUNEL staining was also performed.Result:1.Evaluation of auditory function:In control group, the tone burst ABR average thresholds at4K,8K,16K,24K and32K Hz frequency was24.00±3.16,20.50±4.38,25.00±5.77,30.50±5.50and34.00±3.94dB SPL, respectively. At1,7,14,28,56,70and140days after kanamycin treatment, the tone burst ABR at4K Hz frequency average thresholds was37.00±10.33,37.50±13.79,57.00±8.24,60.00±11.06,72.00±8.56,48.00±9.78and50.00±11.55dB SPL, respectively. At each time point after kanamycin treatment as above, the tone burst ABR at8K Hz frequency average thresholds was42.00±12.51,40.50±11.17,52.00±11.35,63.00±9.19,81.00±8.43,56.50±5.80and57.00±14.18dB SPL, respectively. At16K Hz frequency average thresholds was59.0±11.50,58.00±11.83,64.50±6.85,67.00±11.35,83.50±5.30,66.00±12.43and61.50±19.59dB SPL, respectively. At24K Hz frequency average thresholds was61.50±12.26,61.00±9.66,73.50±9.14,80.50±5.99,86.00±5.68,76.50±11.80and68.50±15.64dB SPL, respectively. At32K Hz frequency average thresholds was71.50±6.69,67.50±11.37,80.50±8.96,81.00±7.75,86.50±6.26,75.50±9.56and75.00±13.33dB SPL, respectively. There was a significant increase in the tone burst ABR average thresholds of each frequency at each time point after kanamycin treatment compared with control group, excepting for4K Hz at1and7days (P<0.001).2. SGN density measurements:After kanamycin administration, the number of SGCs in basal turn was (/mm2)2334.06±169.62,2100.00±237.01,1664.47±139.45,1400.03±279.79,1334.66±85.96,1314.85±164.18,1166.43±41.69and1140.27±96.09in the control and experimental groups (1,7,14,28,56,70and140days), respectively. There were significant differences in SGC counts between control and each experimental group except the one at1day after kanamycin treatment (P<0.001).3. SGC ultrastructural morphology: The amount of (type-Ⅰ) SGCs had dramatically diminished, resulting in great enlargement of free space between the individual cells. Most of the SGCs exhibited a more compact distribution of organelles and intracellular content, resulting in an electron-dense appearance. The endoplasmic reticula were dilated obviously and the mitochondria were progressively swelling at an early phase, which gradually recovered from28days to140days after kanamycin treatment. Meanwhile, focal vacuoles in mitochondria occurred at1,7,14,28and56days after kanamycin administration.4. TUNEL staining:SGCs positive to both TUNEL and caspase-12were increased at7,14and28days after kanamycin treatment, and at7days were much higher than at other time points after kanamycin treatment.5. Western blot analysis:IREla level was significantly upregulated at1(P<0.001),7(P<0.001),14(P<0.05) and56days (P<0.001). In comparison with control group, Bip level was upregulated from1day to56days (P<0.001), while downregulated at140days (P<0.05) after kanamycin treatment. GADD153protein level was significantly upregulated at1day (P<0.001), persisted until70days (P<0.001) and reached highest level at70days (P<0.001) after kanamycin treatment. Second, in comparison with control group, p-PERK level was upregulated from7days to140days (P<0.001), and p-eIF2a protein level was upregulated from1day to56days (P<0.001) after kanamycin treatment. Third, it was hard to detect the expression of ATF-6a in control and experimental groups. Finally, Caspase-12protein level was significantly upregulated at1(P<0.05),7(P<0.05),14(P<0.001) and56days (P<0.05), while downregulated at140days (P<0.001).Conclusion:1,The degeneration of SGNs induced by Kanamycin was sequential.2,ER stress pathway was involved in kanamycin induced apoptosis of SGNs.3,Kanamycin treatment-induced apoptosis is mediated, at least in part, by ER stress-induced upregulation of caspase-12and GADD153. Part II Study on time sequence and mechanism of dorsal cochlear nucleus degeneration following chronic kanamycin-induced deafness in the Sprague-Dawley RatObjective:Examine dynamic pathological changes of the fusiform cell (FC) of the dorsal cochlear nucleus (DCN) and investigated whether apoptosis or autophagy was involved in the neurotoxic course of kanamycin on DCN after kanamycin treatment.Methods:Rats were treated with kanamycin sulfate/kg/day at dose of500mg by subcutaneous injection for10days. Dynamic pathological changes, neuron density and neuron apoptosis of the dorsal cochlear nucleus were examined at1,7,14,28,56,70and140days after kanamycin treatment. The expression of JNK1, DAPK2, Bcl-2, p-Bcl-2, Caspase-3, LC3B and Beclin-1were also detected at each time point after kanamycin treatment.Result:1. Evaluation of auditory function:In control group, the tone burst ABR average thresholds at4K,8K,16K,24K and32K Hz frequency was24.00±3.16,20.50±4.38,25.00±5.77,30.50±5.50and34.00±3.94dB SPL, respectively. At1,7,14,28,56,70and140days after kanamycin treatment, the tone burst ABR at4K Hz frequency average thresholds was37.00±10.33,37.50±13.79,57.00±8.24,60.00±11.06,72.00±8.56,48.00±9.78and50.00±11.55dB SPL, respectively. At each time point after kanamycin treatment as above, the tone burst ABR at8K Hz frequency average thresholds was42.00±12.51,40.50±11.17,52.00±11.35,63.00±9.19,81.00±8.43,56.50±5.80and57.00±14.18dB SPL, respectively. At16K Hz frequency average thresholds was59.00±11.50,58.00±11.83,64.50±6.85,67.00±11.35,83.50±5.30,66.00±12.43and61.50±19.59dB SPL, respectively. At24K Hz frequency average thresholds was61.50±12.26,61.00±9.66,73.50±9.14,80.50±5.99,86.00±5.68,76.50±11.80and68.50±15.64dB SPL, respectively. At32K Hz frequency average thresholds was71.50±6.69,67.50±11.37,80.50±8.96,81.00±7.75,86.50±6.26,75.50±9.56and75.00±13.33dB SPL, respectively. There was a significant increase in the tone burst ABR average thresholds of each frequency at each time point after kanamycin treatment compared with control group, excepting for4K Hz at1and7days (P<0.001).2. The ultrastructural changes of the fusiform cell (FC) of the DCN:The mitochondria were progressively swelling and the endoplasmic reticulum were dilated initially, and gradually recovered from28days to140days after the treatment of kanamycin. At lday after drug treatment, focal vacuoles in mitochondria were occurred. Meanwhile, experiment group showed a remarkable increase in the density of APs or ALs at1,7,14,28and56days compared to control group, followed by a progressive recovery. Partially degraded cargo contents within ALs were then manifested as unevenly distributed dense masses and eventually as evenly distributed dense materials.3. Counting and calculation of total neurons and LC3positive cells:The number of LC3positive cells on the experiment group was significantly higher than those on the control group at1(P<0.01),7(P<0.01) and14days (P<0.05), while that of the total neurons did not change obviously after drug treatment in comparison with control group.4. Western blot analysis:First, JNK1level did not distinctly change at1day after kanamycin treatment, but significantly upregulated at7days (P<0.05), followed by gradually downregulation after7days. Second, in comparison with control group, DAPK2level did not change evidently, nor did caspase-3. Third, Bcl-2protein level did not change clearly initially, but was downregulated at140days (P<0.05). P-Bcl-2protein level was significantly upregulated at1day (P<0.01), highest at7days (P<0.01) and persisted until28days (P<0.05) after kanamycin treatment. Finally, relative to control group, the Beclin-1conjugate was significantly increased in tissue homogenate at1day after drug treatment (P<0.05), highest at7days (P<0.01) and tended to decrease from14days to140days, but the difference did not reach statistical significance after kanamycin treatment during14days to140days. In comparison with the control group, LC3was drastically upregulated at7days (P<0.01) and then fell back to the normal level from14 days to140days after kanamycin treatment.5. TUNEL staining:In comparison with control group, the number of TUNEL positive cells did not increase obviously, in any experiment group.Conclusion:1. Kanamycin have a direct neurotoxic effect on the the fusiform cell (FC) of the dorsal cochlear nucleus (DCN).2. The neurotoxic effect of kanamycin on the neurons was reversible.3. Autophagy might be involved in the neurotoxic course of kanamycin on DCN.4. Increase of autophagy might be mediated by JNK1-mediated phosphorylation of Bcl-2pathway after kanamycin treatment.