Preliminary Studies On The Mechanism Of Oxidizing Damage Induced By Oxygen Free Radicals To The Auditory Neurons And Its Protection

Part I Isolation and culture of the spiral ganglion cellsObjective Damage of the auditory neurons is one of the major causes of sensorineural deafness. Some previous studies have indicated that the auditory neuron damage was closely related to oxidant stress. This study aim to establish a method of isolation and culture of the spiral ganglion cells(SGCs) of the mouse cochlea in vitro, so as to provide a new approach for investigating oxidizing damage of the cochlear neurons.Methods Newborn to 1~3-day postnatal Kunming mouse pups were killed by decapitation , the cochleae were removed from the temporal bone and placed in Hank1 balanced salt solution (HBSS) by an aseptic technique. The bony cochlear capsule and the spiral ligament as well as the organ of Corti were removed, and the spiral ganglion neurons (SGNs) within the modiolus were remained. Then the surrounding connective tissue was removed. The spiral ganglia were collected in HBSS. Enzymatic dissociation was then performed in Ca2\/Mg2\-free HBSS with 0.125% collagenase at 37℃ for 20 minutes, sequentially 0.125% trypsin at 37℃ for 15 minutes. The digestive process was terminated by adding DMEM/F-12 (containing 10% fetal calf serum), followed by there times washes in serum-free DMEM and one wash in the culture medium. After centrifuging at 1000rpm for 7~10 minutes, the supernatants were replaced with high-glucose DMEM/F-12 culture medium (4.5 mg/mL). The SGCs were inoculated to 96-well culture plates treated by poly-lysine with the cell density of 1.0 × 105/mL added with penicillin (100U/mL), fresh insulin (10 ug/mL) and Cytosine arabinoside(5umol/L) to suppress the growth of microbial and non-nerve cells. Half culture medium was changed daily. Twenty-four to forty-eight hours after culture, the neurons were identified by immunocytochemistry using rabbit anti-neuron-specific enolase(anti-NSE) based on SP program.Results The SGCs were successfully cultured in vitro. The SGCs began to paste the wall at 30 minutes after inoculation, and the fibroblast cells began to paste wall earlier. Fibroblasts were significantly reduced by using cytosine arabinoside. After culture for 24 hours, the typic bipolar neurons and occasionally tri-pole neurons could be observed. The SGCs were identified using specific rabbit anti-NSE antibody according to streptavidin-peroxidase(SP) procedures. The SGCs were stained by 3,3'-diaminobenzidine (DAB). The whole cell bodies including cell body and terminates of some SGCs were stained as brown-yellow by DAB, but only the cell body in some cells were stained.Conclusions The SGCs of the cochlea could be successfully cultured in vitro. Ara-C could suppress the growth of non-nerve cells and the quantity and activity of cultured SGCs could meet requirement of experimented researches in cellular, sub-cellular and molecular levels. The culture of the SGCs in vitro was an effective method for the study of physiology, pathophysiology and pharmacology of the auditory neurons.