Effect Of Olfactory Ensheathing Cell On Growth Of Spinal Cord Neurons And Its Protective Effect On Neurons After Injury In Vitro

OBJECTIVE To investigate the effect of olfactory ensheathing cell culture medium(OECCM) on the growth of spinal cord neurons and its protective effect on the injuried neurons by H₂O₂,and to discuss the probable protective mechanisms of olfactory ensheathing cells(OECs).METHODS The primary OECs were isolated and cultured from olfactory bulb of adult SD rat,and OECCM were prepared.The morphology of OECs were observed with inverted phase contrast microscope,then the OECs were identified by rabbit-anti-rat low-affinity nerve growth factor receptor p75(NGFRp75) and its purity were calculated.Primary SD rat embryonic spinal cord neurons were cultured from 15-17-day pregnant SD rat,and injury model of neurons were prepared by H₂O₂.OECCM and control culture medium were added into the normal spinal cord neurons(groups A,B).OECCM and control culture medium were added into the injuried spinal cord neurons by H₂O₂ (groups C,D).In groups A and C,200μL of control culture medium were used;in groups B and D,100μL of control culture medium and 100μL of OECCM were used.Then The growth index such as average diameter of neuron body,the number and length of neuron axons were measured with inverted phase contrast microscope.The viability of normal and injuried neurons were assessed with MTT.RESULTS OECs showed bipolar or tripolar after 6-9 days of culture. Primary SD rat embryonic spinal cord neurons were round and bigger,and neuron axons grew significantly and showed bipolar after 5-7 days of culture.The immunocytochemistry of OECs by NGFRp75 showed that membrane were stained.The degree of purity >80%. (1)Primary spinal cord neurons grew well after 6-9 days of culture,and compared with group A,neurons of group B grew strong,whose cell density and diameter were bigger.The average diameter of neuron body,number and length of neuron axons were(33.38±6.80)D/μm,(1.67±0.80),and(91.19±62.64)L/μm in group A,and(37.39±7.28)D/μm(,1.76±0.82),and(121.33±81.13)L/μm in group B,showing statistically significant differences(P<0.05).The viability of neurons was 0.402 0±0.586 9 in group A and 0.466 0±0.479 0 in group B,showing statistically significant differences(P<0.01).(2)The neurons were injuried by H2O2,.after 2 hours of injury ,the cell density of spinal cord neurons decreased,and neuron axons shortened. The viability of injuried neurons was 0.149 0±0.030 0 in group C and 0.184 0±0.052 0 in group D,showing statistically significant difference(P<0.01).CONCLUSION The results above suggest that OECs could improve the growth of spinal cord neurons and protect the injuried neurons.The neurotrophic factors that OECs secrete play a important role in the treatment of spinal cord injury.