Expression Of Nf-κ Bp50, Caspase-3, Calbindind28k In Spiral Ganglion Of Aged Rats

Background and purpose:With the aging of the population,the number of presbycusis is more and more.But so far the pathogenesis of presbycusis is not entirely clear.In 1964,in accordance with changes of peripheral device of auditory organ,Schuknecht divided presbyeusis into four types,they are sensory presbycusis, nerve presbycusis,metabolism presbycusis and mechanical presbycusis.Above is the histopathological basis of studying age-related hearing loss.Decrease of Spiral ganglion cells is the most basic pathological changes of nerve presbycusis.Large studies show that age-related hearing loss have generally spiral ganglion cells decrease and usually accompanied pure tone hearing thresholds decreased,but the loss mechanism of spiral ganglion cells unclear.Since 1956,Harman,a proposed the free radical theory of aging,scholars from various fields of age-related diseases have done a large number of research and produced important results,the research suggests that free radicals can generate peroxidation to induce damage membrane,mitochondrial membrane and DNA,thus leading to cell death.In vivo,both produce free radicals system and free radical scavenging system, they must maintain balance,and many diseases are related with the imbalance.Superoxide dismutase(SOD) can eliminate free radicals and superoxide anion,play a role in the protection of cells,but Lipid peroxidation product malondialdehyde(MDA) can cause amino compounds as enzymes crosslinking, loss of activity,resulting in cell membrane structure and function damage.Recent studies suggest that in the occurrence process of age-related hearing loss,oxygen free radicals not only directly damage cochlear tissue,but also cause hair cells DNA damage and accelerate cell apoptosis,which led to decline in cochlear function.nuclear factor-κB(NF-κB)not only can mediate immune and inflammatory responses of many material,but also is an important factor of transcriptional regulation in the apoptosis signaling pathway.Recent research has shown that free radicals as a signaling molecule can activate NF-κB,activation of NF-κB can regulate gene transcription in order to control apoptosis.Study shows that many varieties of diseases of aging have NF-κB activation,but the age-related hearing loss in the cochlea of the expression of NF-κB have not yet reported at home and abroad.Research also showed that free radicals has led aging mitochondrial damage, caused obstacles of energy metabolism,increased release of calcium ions,induced in nerve cell structure and function injury.Calcium-binding protein D28k (CalbindinD-28k) is widely distributed in the nervous system,can maintain the level of intracellular calcium to maintain calcium homeostasis,play an important role. expression change of cochlear spiral ganglion CalbindinD-28k in age-related hearing loss is not been reported in domestic.In order to further explore the free radical injury cochlea mechanism of presbycusis,we subject the D-galactose aging rats to study object,by detecting the serum superoxide dismutase(SOD) vitality and malondialdehyd(MDA) concentration to understand the in vivo redox status,by immunohistochemical method to observe spiral ganglion NF-kBp50,caspase-3,calcium-binding protein D28k expression changes and analyze its significance.So that we provide a theoretical basis for clinical prevention and treatment of age-related hearing loss.Materials and Methods:1.Adult rats were used in this study.60 healthy wistar rats with sensitive auricular reflex were randomly divided into two groups:an experimental group and a control group,30 in each group.Experimental group was treated with intraperitoneal injection of D-galactose for four weeks in twice daily dosage of 300mg/kg,the control group was received the same amount of intraperitoneal injection of saline,for 4 weeks,twice daily.2.Before and after treatment,animals in each group were measured serum SOD vitality with xanthine oxidase and MDA concentration with thiobarbituric acid colorimetry.3.Before and after treatment,the animals in each group were examined the anditory brain response (ABR) thresholds,the latencies of waveⅠ,Ⅲ,Ⅴand the interwavel ateneies ofⅠ-Ⅲ,Ⅲ-Ⅴ,Ⅰ-Ⅴ.4.The animals in each group before and after treatment were used Maresch silver staining reaction to observe spiral ganglion neurons and their morphological changes.5.After treatment,rats were anesthetize with 10%ketamine hydrochloride(100mg/kg) by intraperitoneal injection,Just the final examination of ABR was completed,the chest was opened and 150-200ml normal saline was used to wash away the blood through the left ventricle rapidly.Soon after this,500ml 4% paraformaldehyde phosphate buffer(PH7.4) was used to perfused slowly.Then quickly decapitated and took out of acoustic capsule,under the microscope we used fine-needle to open round and oval window,and then 4%paraformaldehyde fixative PBS was reperfused from two windows and pointed cochlea.Finally,the specimens were placed in the same fixative at 4℃refrigerator overnight.The next day the specimen were placed into the 10%EDTA(pH 7.4) for 7-10 d.Finally,the specimen was embeded by paraffin and was sliced successively to be thickness of 4-5um.Five part of slices were taken.One part of slices were stained with Maresch silver staining.three parts of slices were studied by NF-κBp50,caspase-3, calcium-binding protein D28k immunohistochemical staining,the other part of slices was used to be negative control.6.Finally we use the German Leica image processing system to exam IOD(integrated optical density)of NF-κBp50,caspase-3, calcium-binding protein D28k positive reactivity.7.Statistical analysis was performed with SPSS10.0 software,using t-test and correlation.Statistically significant level was considered as "alpha equals 0.05".Results:1.In the control group,the thresholds of ABR,the latencies of waveⅠ,Ⅲ,Ⅴand the interwave latencies ofⅠ-Ⅲ,Ⅲ-Ⅴ,Ⅰ-Ⅴhad little change after the animals being administrated drugs.But in the experimental group,the ABR thresholds elevated,latencies of waveⅠ,Ⅲ,Ⅴand the interwave latencies ofⅠ-Ⅲ,Ⅲ-Ⅴ,Ⅰ-Ⅴdelayed significantly.2.The vitality of SOD andt hedensity of MDA in serum had little change after being administrated drugs in controlgroup.In experimental group,the vitality of SOD reduced and the density of MDA increased significantly after being administrated drugs.The means of the two groups are different obviously(P＜0.05).3.Maresch silver staining reaction under light microscope showed that the experimental group rat cochlear spiral ganglion cells decreased,cells can be seen a large number of lipofuscin deposition,in the control group spiral ganglion cells can beseen dense,uniform and rare lipofuscin deposition.4.Through Image Analysis System,we found IOD values of immunohistochemistry results of NF-κBp50,caspase-3,calcium-binding protein D28k in the experimental group were bigger than in the control group,and the difference was statistically significant(P＜0.05).Conclusion:1.Old rats cochlear spiral ganglion cells decreased,there is degeneration and apoptosis.2.The calcium-binding protein D28k in the old rats cochlear spiral ganglion cells increased,it suggest that calcium influx increased..3.NF-kB activity in the old rats cochlear spiral ganglion cells may be related to cell survival.