| With the development of the society, Spinal Cord Injury(SCI), havingbecome a disease with a high incidence, tends to cause very serious disability.The spinal nerve regeneration and the functional recovery of limbs are still themedical difficulties in the world. Recent years witness the possibility ofolfactory ensheathing cells(OECs), among the transplanted cells in the treatmentof SCI, to become one of the cells with a brighter propect due to its uniquebiological characteristic and the ability to regenerate injured axons. Currently, agreat controversy has arisen about the time of using the method of OECstransplantation for SCI. There are few related studies on this subject both athome and abroad with a lack of convincing report about it.ObjectivesTo guide clinically the choice of transplanting time in the treatment ofspinal cord injury, and to promote the repair and regeneration of the injuriedspinal cord by studying the influence of the transplanting time of OECs on thefunctional recovery of injured spinal nerve and the possible mechanisms.Materials and methods1. The culturing, purifying, evaluating, marking and calculating of OECsPick healthy Wistar rats of two months old, either male or female, with aweight of200-250g. After narcotizing the Wistar rats, get the olfactory bulbswith a craniotomy. Use the cell culturing method combining the improvedNASH differential time attachment and Ara-c excitation to carry out the primaryculture, purification and evaluation of olfactory ensheathing cells, then digest,collect and caculate the olfactory ensheathing cells, using Hoechst to mark thenucleus. 2. Establishment, grouping and intramedullary transplantation of spinalcord injuried animal model:Use80healthy grown-up Wistar rats to establish the left side of the T10spinal cord hemisection injury model by surgical operation. Divide the animalmodels into8groups at random: the group with an immediate transplantationafter the spinal cord being injuried (Group A1): the group with a one-week latertransplantation after the spinal cord being injuried (Group B1): the group with afour-week-later transplantation after the spinal cord being injuried (Group C1):the group with eight-week-later transplantation after the spinal cord beinginjuried (Group D1): Meanwhile, a matched blank control is set up for everygroup (respectively named Group A2, Group B2, Group C2and Group D2,)3. Assessment on the neural repair of injuried spinal cord:Select the Wistar rats which respectively have had the transplantation for1,2,4,6and8weeks and evaluate their limb fuctions by using the combinedbehavioral scoring method(the modified CBS grading system), and observe theSCI model motor function; prepare the spinal cord specimen after the rats havethe transplantation for4and8weeks. Observe the general morphologicalchange first, and then have the specimen HE and argyrophil stained to check theregeneration of spinal cord nerve axon on the injured part, and finally give theimmumohistochemical staining to observe and mark the survival anddistribution of olfactory ensheathing cells on the spinal cord injuried part with afluorescence microscope.Results1. It gets olfactory ensheathing cells of higher purity by using thepurification culturing method combining differential attachment and Ara-cexcitation, with the quantity of these olfactory ensheathing cells standing at1×108/ml and the purity being above70%, which meet the experimentalrequirements. Through a seven-day culture and purification, the cell densityincreases, the cell protrusions becoming slender, bipolar and tripolar cellstaking up a higher proportion, the cell bodies taking on the shape of fusiform,disciform and multiple protrusion. The staining reaction of both NGFRp75andGFAP is positive.2. The Wistar rat’s motor function after the transplantation graduallyrecovers with the passage of time. It gets as follows after having had the transplantation for four weeks through CBS grading system: the differencesamong group A1, B1, C1and D1have distinctive significance (P<0.05);however, the differences between A1and B1have no statistical significance(P>0.05). While the curative effects are similar, group A1is superior to groupB1slightly, with the curative effect of group C1medium and group D1theworst. Blank control group has no obvious improvement.3. The CBS grades of group A1, B1, C1, D1, A2, B2, C2and D2arerespectively10.60±0.96、12.10±2.91、20.20±1.98、17.10±1.19、35.10±2.28、36.80±2.04、30.10±1.66、29.50±0.52after having had the transplantation for8weeks. Through observation, HE and argyrophil staining in group A1, B1andC1all have more regeneration axon, with partial regeneration nerve fibrespassing the injuried part. There is porosis on the injuried spinal cord in groupD1with a small amount of regeneration axons, distributed in the injuried placeswhich are passed by uncontinuous nerve fibres. After the operation of fourweeks, surviving OECs are found on the injuried places by observing under theimmunohistochemical staining and fluorescence microscope, of which thequantity of the cells is as follows: group A1is greater than group B1, group B1greater than C1and group C1greater than group D1.Conclusions1. This experiment can get cells of higher purity by using the methodcombining differential attachment and Ara-c excitation to purify and cultureolfactory ensheathing cells, which has better promotional value because of itspracticability and economical efficiency.2. The transplantation of olfactory ensheathing cells is the effective way tocure spinal cord injury.3. The transplantation with the best curative effect should be doneimmediately or one week later when having had the spinal cord injuried with nobig differences between the two cases. With the time for treatment beingpostponed after injury, the curative effect of transplantation reduces gradually. |
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