Study On Time Course Of Cochlear Cell Degeneration And Dorsal Cochlear Nucleus Plasticity Of Guinea Pig Following Chronic Kanamycin-induced Deafness

PartⅠStudy on time sequence and mechanism of auditory nerve and spiral ganglion cell degeneration following chronic kanamycin-induced deafness in the guinea pigObjective:To construct a chronic kanamycin-induced deafness model, investigate the time sequence and mechanism of auditory nerve and spiral ganglion cell degeneration guinea pig following chronic kanamycin- induced deafness. Methods:Guinea pigs were treated with kanamycin by subcutaneous injection at 500 mg/kg per day for 7 days. Histological changes in hair cells, SGCs, Schwann cells and the area of the cross-sectional of the AN with vestibular ganglion (VG) in the internal acoustic meatus were quantified at 1,7,14,28, 56,70 and 140 days after kanamycin treatment. Results:1. The average threshold was 35.83±5.97,48.13±3.45,83.44±7.47,105.00±8.37,104.38±5.44,108.75±7.64,104.38±9.17 and 107.19±12.24 dB SPL respectively in the control group and, at 1,7,14, 28,56,70 and 140 days after kanamycin treatment, and the threshold was statistically unchanged at 28,56,70 and 140 days in comparison with 14 days group. There was a significant increase in ABR threshold at 1,7 and 14 days (p<0.001, compared with control group), and no statistic difference among 14,28,56,70 and 140 days groups (p>0.05).2. There was a similar time course of morphological changes in the overall cochlea and the basal turn. Outer hair cell (OHC) decreased at 7 and 14 days. Loss of inner hair cell (IHC) occurred at 14 and 28 days. The number of remaining OHCs was 98.56±1.26%, 27.56±2.94%, 20.81±2.76%, 19.19±3.15%, 19.25±2.24%, 19.56±3.35% and 18.60±2.73% at the seven locations, and 98.48±1.28%, 24.99±2.52%, 16.60±3.07%, 11.94±1.34%, 10.96±2.44%, 11.05±2.32% and 11.20±1.23% in basal turn respectively at 1, 7, 14, 28, 56, 70 and 140 days. Additional, the number of remaining IHCs was 99.00±0.97%, 97.63±1.20%, 67.56±4.60%, 59.19±5.05%, 56.81±3.97%, 56.87±3.69% and 57.20±3.45% at the seven locations, and 98.82±1.08%, 96.92±1.03%, 57.92±4.05%, 38.21±3.45%, 37.77±3.13%, 36.34±3.19% and 37.08±3.14% in basal turn respectively at 1, 7, 14, 28, 56, 70 and 140 days. There were significant decreases in the mean percentages of remaining OHCs in the seven locations and basal turn at 7, 14, 28, 56, 70 and 140 days (P <0.001) and IHCs at 14, 28, 56, 70 and 140 days after deafening (P<0.001, compared with normal controls). 3. Loss of SGCS occurred at 7, 14 and 28 days after deafening. The number of SGCs was 4105.17±82.96, 4008.00±94.19, 3130.69±127.67, 2858.38±153.48, 2633.00±151.95, 2618.06±129.76, 2624.38±142.59 and 2625.63±137.69 at the seven locations, and 2091.00±86.62, 2073.13±95.40, 1576.94±96.77, 1194.00±97.81, 859.31±64.92, 849.94±64.37, 852.50±52.08 and 840.19±53.55 in basal turn respectively in the normal and experimental groups (1, 7, 14, 28, 56, 70 and 140 days). There were significant differences in SGC counts between normal and each experimental group except for the day 1 group (P<0.001) for seven locations and the basal turn. 4. Loss of Schwann cells was present at 7, 14 and 28 days after deafening. The number of Schwann cell in the cross-section of AN with VG was 6167.17±80.81, 6082.88±121.00, 3946.00±128.67, 3004.31±167.45, 2742.44±90.61, 2747.63±98.69, 2730.38±78.08 and 2782.69±78.70 respectively in the normal and experimental groups (1, 7, 14, 28, 56, 70 and 140 days). There were significant differences in Schwann cell counts between the normal group and each experimental group except for the day 1 group (P<0.001). 5. The cross-sectional area of the AN with VG increased at 1 day and decreased shortly following loss of SGCs and Schwann cells at 7,14 and 28 days after deafening. The cross-sectional area of the AN with VG was 0.15±0.020,0.17±0.004,0.12±0.007,0.13±0.007,0.13±0.007,0.11±0.012,0.12±0.008 and 0.13±0.013mm2 respectively in the normal and experimental groups (1,7,14,28,56,70 and 140 days). The cross-sectional area of the AN with VG increased at day 1 (P<0.001) and decreased at days 7,14,28,56 and 70 (P< 0.001, compared with normal controls). Conclusions:1.The effects of kanamycin on hair cells, spiral ganglion and Schwann cells are progressive.2. Early degeneration of SGC and Schwann cell mainly results from the direct toxic effect of kanamycin. However, multiple factors such as loss of hair cell, degeneration of Schwann cell and the progressive damage of kanamycin, may participate in the late degeneration process of SGCs.3. There were some differences in the time course of cell degeneration between acute and chronic kanamycin deafening models.PartⅡStudy on time course and mechanism of neuronal and synaptic plasticity in dorsal cochlear nucleus of guinea pig following chronic kanamycin-induced deafnessObjective:To investigate the time sequence and mechanism of neuronal and synaptic plasticity in dorsal cochlear nucleus of guinea pig following chronic kanamycin- induced deafness. Methods:Guinea pigs were treated with kanamycin by subcutaneous injection at 500 mg/kg per day for 7 days, ultrastructural changes in fusiform cell (FC) and at auditory nerve (AN) synapse on FC (AN/FC synapse) were observed, and local insulin-like growth factor 1 (IGF-1) mRNA were quantified using quantitative real time PCR at 1,7,14,28,56, 70 and 140 days after kanamycin treatment. Results:1. The average threshold was 36.00±4.76,46.46±3.45,80.63±5.95,103.95±6.59,106.25±5.16,108.75±8.40,106.67±9.17 and 106.88±7.91 dB SPL respectively in the control group and at 1,7,14,28,56,70 and 140 days after kanamycin treatment, and the threshold was statistically unchanged at 28,56,70 and 140 days in comparison with 14 days group. There was a significant increase in ABR threshold at 1,7 and 14 days (p<0.001, compared with control group), and no statistic difference among 14,28,56,70 and 140 days groups (p>0.05).2. Close-packed cytoplasm and shrink of nucleolemma were present with the progressive swelling of mitochondria and dilatation of endoplasmic reticulum, and focal vacuoles in some mitochondria and more lysosomes in FC also occurred at 7,14,28 and 56 days after kanamycin treatment. Moreover, the progressive swelling of mitochondria at the presynaptic ending of AN/FC synapse was observed at 7,14,28 and 56 days. These changes in the ultrastructure of AN/FC synapse and FC were no longer present at 70 and 140 days.3. The thickness of the postsynaptic densities (PSD) increased at 1,7 and 14 days. The average thickness was 72.54±1.98,67.56±1.58,66.49±2.24,47.78±2.16,49.42±1.60,47.32±4.45 and 49.48±2.47 nm respectively at 1,7,14,28,56,70 and 140 days after kanamycin treatment. In comparison with control group (47.91±4.56 nm), there was a significant increase in the thickness of the PSD at 1,7 and 14 days (p<0.001), and no statistic difference at 28,56,70 and 140 days (p>0.05).4. The fold change in local IGF-1 mRNA was 0.88±0.17,1.53±0.21,2.07±0.17,1.51±0.23,1.92±0.14,1.87±0.15 and 0.97±0.11 respectively at 1,7,14,28,56,70 and 140 days after kanamycin treatment. There was a significant upregulation of local IGF-1 mRNA at 7,14,28,56 and 70 days in comparison with the control group (P<0.001), and no statistic difference among 14,28,56 and 70 days groups (p>0.05). Conclusions:1.The effects of kanamycin on the ultrastructure of FC and AN/FC synapse are direct and progressive.2. FC and AN/FC synapse are capable of reviving and remodeling after kanamycin-induced lesion and incomplete deafferentation.3. Local IGF-1 might play a role in the lesion- and deafness-induced plasticity in FC and at AN/FC synapse following chronic kanamycin-induced deafness. PartⅢStudy on effects of excitory and inhibitory receptors on synaptic plasticity in dorsal cochlear nucleus of guinea pig following chronic kanamycin-induced deafnessObjective:To investigate the effects of excitory and inhibitory receptors on synaptic plasticity in dorsal cochlear nucleus of guinea pig following chronic kanamycin- induced deafness. Methods:Guinea pigs were treated with kanamycin by subcutaneous injection at 500 mg/kg per day for 7 days. Synaptophysin expression using immunohistochemistry and N-methyl-D-aspartate receptor 1(NR1) andγ-aminobutyric acid receptor type Aα1(GABRA1) mRNA using quantitative real time PCR were quantified at 1,7,14,28,56, 70 and 140 days after kanamycin treatment. Results:1. there were two categories of synaptic endings in the DCN. The punctate deposits were distributed throughout the neuropil and perisomatic profiles were observed surrounding cell bodies. The punctate deposits appeared to be more common than the perisomatic profiles in the DCN.2. There was an increase in the mean gray level of synaptophysin immunostaining (P< 0.05) and mean number of boutons (P<0.001, compared with normal controls) at 1,7,14,28 and 56 days after kanamycin treatment. There was a decrease in the synaptophysin-immunostained area at 7 and 14 days (P<0.01, compared with normal controls).3. The fold change in NR1 mRNA was 1.58±0.18,2.20±0.33,3.67±0.34,2.04±0.30,1.34±0.36,1.28±0.21 and 1.29±0.17 respectively at 1,7,14,28,56,70 and 140 days after kanamycin treatment. There was a significant upregulation of NR1 mRNA mRNA expression at 1,7, 14 and 28 days in comparison with the control group (P<0.001).4. The fold change in GABRA1 mRNA was 0.77±0.08,0.45±0.09,0.34±0.07,0.32±0.07,0.90±0.11,0.94±0.12 and 0.92±0.11 respectively at 1,7,14,28,56,70 and 140 days after kanamycin treatment. There was a significant downregulation of GABRA1 mRNA expression at 1,7,14 and 28 days in comparison with the control group (P<0.001). Conclusions:1.The effects of kanamycin on the number of synapse and distribution of the excitory and inhibitory recptors in DCN are progressive.2. The synapses in the DCN are capable of rearrangement and remodeling after kanamycin-induced lesion and incomplete deafferentation.3. There are significant upregulation in excitation of the glutamatergic auditory pathway, and downregulation in inhibition of the GABAergic auditory pathway.4. Regulation of excitory and inhibitory recptors expression might play a role in the lesion-and deafness- induced plasticity in the DCN following chronic kanamycin-induced deafness.