Combined transplantation of human fetal olfactory ensheathing cells and rat embryonic spinal cord tissues in the treatment of spinal cord injury in rats

Weijiang WU; Guozhen HUI; Ranbo Lü; Zongning MIAO

ABSTRACT

BACKGROUND:

Spinal cord injury occurs frequently and its consequence is very severe. There is no effective method to rebuild the function of demylinated nerves. Transplantation of a kind of special glial cells in olfactory system of mammal attracts more attention.

OBJECTIVE:

To observe whether combined transplantation of human fetal olfactory ensheathing cells (human OECs) and rat embryonic spinal cord tissues (rat ECS) possesses synergistic effect in promoting axonal regeneration in the rats following spinal cord transection.transection.

DESIGN:

Open experiment.

SETTING:

Cell Room, Third People's Hospital of Wuxi City; Department of Neurology, First Hospital Affiliated to Soochow University; Department of Neurosurgery, Zhongda Hospital Affiliated to Central South University MATERIALS This experiment was carried out in the cell laboratory of the Third People's Hospital of Wuxi from September 2002 to October 2004. ① Totally 36 adult female SD rats, of clean grade, were selected and randomly divided into 4 groups human OECs group (n=10), rat ECS group (n=10), combined transplantation group (n=10) and sham-operation group (n=6). ② Fresh 12-week aborted human embryo was used for culture and purification of human OECs (Informed consent was obtained from the parturient). ③One SD rat at embryonic 14 days underwent caesarean operation, and fetal rat and fetal membrane were taken out together and used for preparing new embryonic spinal cord.

METHODS:

①Rats of 4 groups were all created into hemisection cavity models. Gelatin sponge and complete culture medium of 8 μL were packed into the injured cavity of rats in the model group, and the same culture medium of 2 μL was injected at 1 mm above or below injure; Human OECs suspension of 8 μL was added to gelatin sponge in human fetal Human OECs group, and human OECs suspension of 2 μL was injected at 1 mm above and below injure; rat embryonic spinal cord tissue of rat ECS group was chipped into pieces, which were packed into the cavity,and gelatin sponge was spread on the injury part. Embryonic spinal cord with the same size was packed into the cavity of combined transplantation group, then 8 μL human OECs suspension was injected into cavity with micro sample injector, and gelatin sponge was spread on the injury part, and then cellular suspension of 2 μL was injected at 1 mm above and below the cavity, and muscular layer skin was sutured layer by layer. ②The rats of each group were performed ethological evaluation periodically. Combined with pathological observation, effect of human OECS and rat ECS on neuronal survival and regeneration was evaluated by performing horseradish peroxidase-tetramethyl benzidine tracer technique.MAIN OUTCOME

MEASURES:

①In vitro culture and purification of human fetal human OECs. ② In vitro immunocytochemistrical analysis. ③BBB scoring of motor function of hindlimb of rats. ④ Immunohistochemical detection of implants and injured spinal cord repair⑤ Quantitative analysis on labeled neurons at the cortex and mesencephalic red nucleus ineach group with horseradish peroxidase-tetramethyl benzidine tracer technique.

RESULTS:

① Most of human fetal OSCs presented double-polar spindle.Five to seven days after culture, OSCs weaved into net and a lot of mitosis phases were found. The cellular purity was 85%. ② The rate of P75 positive cells was (83±7)%. Glial fibrillary acidic protein was found in about (81±6)% of cells and Vimentin in (91±9)% of cells and the rate of Nestin positive was (77±5)%. ③Three to five days after operation, affected limb of rats of sham-operation group began to contract, the activity of hindlimb of intact side was limited a little. Fewer obvious contraction symptoms were found in the other 3 groups. From 2 weeks after operation,behavioral function recovered significantly fast in each group. BBB scores of combined transplanted group were significantly high than those of human OECs group, rat ECS group and sham-operation group [(6.2±1.13) vs.(5.0±1.15)vs.(3.9±0.88)vs.(3.3±1.03)scores,P ＜ 0.05]. ④In bipolar or multipolar cells, in which basic protein(+)granules were found, P75 and glial fibrillary acidic protein positive were found at the implanted part in the range of 2.0 to 5.0 mm of transplanted region in the human OECs group and combined transplantation group. A great many of small MAP2 positive neurons were found in the spinal defected focus in the rat ECS group and combined transplantation group. Nerve plexus positive fibers were observed in spinal defected region of human OECs group, rat ECS group and combined transplantation group to different extents, especially significantly in the combined transplantation group, but they were not found in the model group. ⑤ Horse radish peroxidase labeling was hardly found in neurons at the injured side of sham-operation group, while the number of labeled neurons at the cortex and nesencephalic red nucleus was significantly higher in the combined transplantation group than in the human OECs group and rat ECS group (P ＜ 0.05).

CONCLUSION:

Combined transplantation of OECs and ESC can obviously protect injured spinal cord, promote host spinal axonal regeneration and play s a complementary and synergetic effect in speeding up the functional recovery of rats.