[Effect of olfactory ensheathing cells 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 injured neurons by H2O2, and to discuss the probable protective mechanisms of olfactory ensheathing cells (OECs).


METHODS
The primary olfactory ensheathing cells (OECs) were isolated from olfactory bulb of adult SD rat, and OECCM were prepared. The morphology of OECs was observed by inverted phase contrast microscope, identified by rabbit-antiratlow-affinity nerve growth factor p75 (NGFRp75), and its purity were calculated. Primary spinal cord neurons were cultured from 15 to 17 days pregnant SD rats, and injury model of neurons were prepared by H2O2. OECCM and control culture medium were added into the normal spinal neurons (groups A, B). OECCM and control culture medium were added into the injured spinal neurons by H2O2 (groups C, D). In groups A and C, 200 microL of control culture medium was used; in groups B and D, 100 microL of control culture medium and 100 microL of OECCM were used. Then the growth index such as average diameter of neuron body, the number and length of neuron axons were measured. The viabilities of normal and injured neurons were assessed by MTT.


RESULTS
OECs showed bipolar or tripolar after 6-9 days of culture. Primary 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 was more than 90%. 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, the number and length of neuron axons were (33.38 +/- 6.80) D/microm, (1.67 +/- 0.80), and (91.19 +/- 62.64) L/microm in group A, and (37.39 +/- 7.28) D/microm, (1.76 +/- 0.82), and (121.33 +/- 81.13) L/microm in group B; showing statistically significant differences (P < 0.05). The absorbency (A) value 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 difference (P < 0.01). After 2 hours of injury by H2O2, the cell density of spinal cord neurons decreased, and neuron axons shortened. The A value of injured neurons was 0.1490 +/- 0.0300 in group C and 0.1840 +/- 0.0520 in group D, showing statistically significant difference (P < 0.01).


CONCLUSIONS
The results above suggest that OECCM could improve the growth of spinal cord neurons and protect the injured neurons. The neurotrophic factors that OECs secrete play an important role in the treatment of spinal cord injury.
Ji
Zhenwei
Z
Orthopaedic Oncology Institute of Chinese PLA, Tangdu Hospital, Fourth Military Medical University, Xi'an Shaanxi, 710038, P.R. China.
Qian
Jixian
J
Han
Jianwei
J
Fan
Qingyu
Q
D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D008297:Male; D020932:Nerve Growth Factor / Q000096:biosynthesis; D009416:Nerve Regeneration; D009474:Neurons; D009830:Olfactory Bulb / Q000166:cytology* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013116:Spinal Cord; D013119:Spinal Cord Injuries
D020932:Nerve Growth Factor