| Objective: To develop a spinal cord injury device usable in animal studies for spinal cord injury.Methods:⑴The production of a rats spinal cord injury device: the device is consistedof an impacting equipment and a fixed supporting structure. The impacting equipment isconsisted of an impacting head and casing pipes. Production of the impacting head: use a5ml injector and cut all of its bottom part off; connect it to the bottom of a10g weight andtie a fine line to the upper end of the weight. Production of the casing pipes: use a10mlinjector and cut off its front end; trim the front end of the injector until it can hold thenozzle of a5ml injector. The fixed support structure is made of wood sticks and consistedof a supporting structure and a mobile structure; the supporting structure supports themobile structure and the casing pipes are fastened upon the mobile structure.⑵Preparation of a rats spinal cord impacting injury model: use100SD rats with weightsbetween200and250g; divide them randomly into three model groups (group A1,group A2and group A3) and one sham operation group (group B); select25rats for each group.Establishment of a rat spinal cord injury model with a simple impacting spinal cordimpactor: remove T9-11spinal vertebral arches of the rat; use a10g weight and impactspinal cord of group A1at a distance of2cm, group A2at a height of3cm and group A3from a height of4cm by having the weight fall freely; to the sham operation group (groupB), remove T9-11spinal vertebral arches of the rat and do not practice any operation on thespinal cord.⑶Behavioral assessment after the spinal cord of the rat is injured：1）obliqueboard test: randomly select five rat from every model group1d before the operation and3d,7d,14d and28d after the operation for undertaking the oblique board operation; place therat in a way that their body long axis is vertical to the longitudinal axis of the oblique boardand record the maximal angles the rat for a duration of5seconds as the caption values; measure3times and use the average value.2)BBB score: practice BBB motor functionassessments on rat immediately after they have completed the oblique board test.⑷Observation of histomorphology of the rat after their spinal cord injury: practiceparaformaldehyde heart perfusion on rat after the behavioral assessments have beencompleted; use fixed spinal cord and cut at both ends from the injury center; observe underLM with HE staining and an electron microscope respectively.Results:⑴Behavioral assessments on the rat after their spinal cord was injured: thesham operation group was observed at different time and the assessment on the obliqueexperience was identical with the BBB score; the result of oblique board test on group A13d after the operation by falling the weight from a height of2cm was （26.3±1.75）o andthe BBB score is0.40±0.55; as the time was prolonged to28d after the operation, the resultof oblique board test was（56.20±1.30）o and the BBB score was15.40±1.82, so the resultsof assessment was typically increase (P＜0.05); the result of oblique board test on groupA23d after the operation by falling the weight from a height of3cm was （28.00±1.58）and the BBB score was0.20±0.45; the result of oblique board test was7d after theoperation was（37.80±1.79）o; when the time was prolonged to28d, no obvious differenceamong the results was identified (P＞0.05); the BBB score28d after the operation was5.40±0.55, which was obviously higher than that3d after the operation (P＜0.05). Theresult of oblique board test on group A33d after the operation by falling the weight from aheight of4cm was （26.80±0.84）o and the BBB score was0.20±0.45; the result of obliqueboard test7d after the operation was （34.20±0.84）o, which was not obviously differentfrom that28d after the operation (P＞0.05), and the BBB score28d after the operation was5.20±0.84, which was obviously higher than that3d after the operation(P＜0.05).⑵Observation on histomorphology of the rat spinal cord injury:1)HE staining observation:①as indicated by the HE staining observation, the internal spinal cord structure of thesham operation group was complete at different time; morphologies of neurons wereregular, tissue spaces were normal and nerve fibers were lined up tightly;②3d after theoperation, the HE staining indicated that the spinal cord tissues of group A1were bleedingslightly, their white matter nerve fibers were lined up disorderly and there were cavities in them; as the time was prolonged, the bleeding in the spinal cord tissues disappearedgradually; the destruction of gray and white matter structures were most severe7d afteroperation; from14d to28d after the operation, the gray and white matter structures wererecovered to certain extent and the nerve fibers were lined up orderly.③3d afer theoperation, the HE staining result indicated that the spinal cord tissues of group A2wasbleeding severely, the cell spaces in white matters were increased, there were cavities inthe white matters and the nerve fibers were lined up loosely; as time was prolonged, thebleeding in spinal cord tissues disappeared gradually, the missing tissues increasedgradually, the number of neurons decreased and structure of the white matters weredisorderly;14d after the operation, there were less residual tissues of gray maters and thewhite matters were lined up disorderly;28d after the operation, some neurons in the graymatters were recovered and the white matters were lined up in a way more orderly thanthat14d after the operation.④3d afer the operation, the HE staining result indicated thatthere were a great deal of bleeds in the spinal cord tissues of group A3after being impactedby the weight fallen from a height of4cm; moreover, the tissue structure in the whitematters was lined up sparsely and there were cavities; the bounder between gray and whitematters was vague; as the time was prolonged to28d after the operation, the bleeding inspinal cord tissues disappeared gradually, the area of gray matter necrosis became severer,the structure of white matters became more disorder and only a small quantity of residualtissues were identified.2) results of observation under an electron microscope:①atdifferent time when observing with an electron microscope, rat of the sham operationgroup were tight in myelin sheath lamellar structure; moreover, no abnormal axonalspheroid was identified and the mitochondria were clear enough to be seen.②the resultfrom observing an electron microscope on group A13d after the operation indicated thatsome myelin sheath lamellar became loose, the axonal spheroids were comparativelycomplete and the mitochondria were clear enough to be seen; as time went by, the myelinsheath lamellar became looser, the axonal spheroids began to depauperate and themitochondria began to disappear gradually.③the result from observing with an electronmicroscope on group A23d after the operation indicated that the myelin sheath lamellar was loose and the axonal spheroids and mitochondria were vague; as time went by, themyelin sheath lamellar became looser, the axonal spheroids began to depauperate todisappear and the myelin figures disappeared completely.④The result from observingwith an electron microscope on group A33d after the operation indicated that the myelinsheath lamellar was severely loose and there was folding phenomenon; moreover, theaxonal spheroids began to depauperate and no any mitochondrion was identified; as timewent by, the myelin sheath lamerllar became looser and the axonal spheroids weredisappeared;28d after the operation, the myelin figures disappeared completely.Conclusion:⑴Injuries resulted from impacting spinal cord with this equipment becomemore severe with the rising of impacting heights.⑵Complete injuries can be resultedfrom impacting spinal cord with this equipment from a height of more than3cm whileinjuries resulted from impacting with this equipment from a height of2cm wereincomplete.⑶The self-prepared spinal cord impactor used in this experiment is aneconomical and practical equipment that is easy to operate with high repeatability forinjuring spinal cord of animals; the equipment can provide a reliable method for studyingspinal cord injuries and treatment through stem cell transplantations. |
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