| Neuroprotection treatment is applied before or after nerve injury by means of medicine or some special procedure to minimize the degree of nerve injury.There are many aspects of prevention and cure for spinal cord injury(SCI),including prevention for secondary lesion,neuroprotection for subacute and chronic stage,neuraxis and neuron regeneration and reinnervation obtaining etc.,and there are many issues remained unclear in each aspect.In the past two decades, researchers in and abroad did a lot experimental and clinical studies to investigate the biology mechanism of SCI,which led to a more profound understanding of SCI on cellular and molecular level.Based on these researches,this study is to explore new neuroprotection treatment for improving the prognosis of SCI. Preconditioning is an important prevention method which is different from traditional neuroprotection treatments,and researchers has been paying more and more attention on this new subject.Preconditioning could mobilize the defence system in corpore so as to produce powful protection for the sequentia major injury.Hypoxia preconditioning has been studied a lot in the protection for ischemical reperfusion injury in central nervous system and made significant progress,but the study of its role in SCI protection is just started.This study utilized SD rat as a SCI model, divided them into different groups applying hyperbaric oxygen(HBO) and hypobaric hypoxia preconditioning respectively, observed the neuroprotective effect for SCI,investigated its possible mechanism in each group,and also evalutaed the curative effect of HBO treatment for SCI.There are three parts in this study as follows:1.neuroprotective effect of HBO and hypobaric hypoxia preconditioning on SCI in ratsObject:To establish an animal model for HBO and hypobaric hypoxia preconditioning, investi- gate the impact of such preconditioning on the adult rat in different preriod after acute SCI,and evaluate the neuroprotective effect of such preconditioning in SCI.Method:1) Animal model establishment for HBO and hypobaric hypoxia preconditioning:SD rats were divided into two groups randomly,and preconditioned for 5 days respectively in HBO or hypobaric hypoxia chambers.2) SCI animal model establishment: Micrurgy were applied on the rats under anaesthesia to expose canalis spinalis and dural sac,utilizing a 30g lash stick to strike on the spinal cord from a height of 5 cm.The inferior extremity of the lash stick is 3 mm in diameter,with a shock energy of 150 gcf.3) Observation items:(1) Behavioral assessment:We applied BBB motor function score system in this study, and assessed the score at 1d,1w,2w,4w,and 8w after SCI.(2) Evoked potential examination: Somatosensory evoked potentia(SEP) and motor evoked potential(MEP)were detected at different time after SCI in each group.(3) Transmission electron microscope(TEM) observation:We removed a volume of 1 mm3 tissue right in the central of the injured region after being reperfused and fixed,placed the tissue into 10% glutaric dialdehyde fluid,made TEM slices and did the observation.(4) HE dyeing and observation under light microscope: the samples of spinal cord were obtained at different time after SCI, made paraffin section in anteroposterior axes,performed HE dyeing and observed the slices under light microscope.(5) Nerve-tract tracing: Anterogradely and retrogradely traceing were performed using biotin dextran amine(BDA) and horseradish peroxidase(HRP) respectively,observed the regenerated neuraxis ascendingly and descendingly,so as to identify the effect of such preconditioning on neuraxis regeneration.4) Data were analyzed by using SPSS12.0 statistics software.Result:1)The experimental animal model established is reliable and stable,presenting a good repeatability.2) The BBB score was dramatically dropped right after SCI,and then gradually increased afterward.The score of preconditioning groups was higher than the control group,especially in the hypobaric hypoxia group of which the difference was significant;the score in all groups entered a platform 4w after the SCI.3) SEP and MEP showed a prolonged lantency after SCI in all groups,the lantency in preconditioning groups was shorter compared with control group, and there was significant difference in hypobaric hypoxia preconditioning group.The SEP and MEP was relatively stable 4w after SCI.4) light microscope and TEM observation: In control group, the central gray of injured region appeared hemorrhage,cellular swelling,tigroid body abolition,and a great quantity of mononuclear or polynuclear inflammatory cells infiltration at 1d after SCI;Necrosis focuses appeared in injured region at 1w after SCI,and the inflammatory reaction was stronger;the inflammatory cell infiltration began to abate at 2w after SCI in injured and peri-injured region;capsular space-like change occurred in injured myeloid tissue at 4w after SCI, and at 8w after SCI glial scar became the major component.Apoptotic cells showed cytoplasm depletion,karyo-anachromasis and karyopyknosis under electron microscope.In HBO and hypobaric hypoxia preconditioning groups,the pathological change were less severe within 2w after SCI compared with control group,especially the surrounding area of injured region,and no significant difference were found after 4w after SCI.5) Nerve tract tracing revealed that the number of regeneration neuraxis ascending and descending increased in preconditioning groups,of which the hypobaric hypoxia group was much more obvious.Conclusion:1)the rat SCI model is resemblant with human SCI ,which is reliable with an insignificant individual difference and a good reproducibility,and a simple preparation process.2) Compared with the control group,rats in the preconditioning groups present a higher BBB score,a shorter lantency of SEP and MEP,a less severe pathological change,an increased regeneration neuraxis etc.,which indicate a neuroprotective effect of such preconditioning on rat SCI.3) Our study indicates that HBO and hypobaric hypoxia preconditioning have an effect of neuroprotection at certain degree.4) Hypobaric hypoxia preconditioning is more effective in neuroprotection for SCI than HBO preconditioning.2.Mechanism of the neuroprotective effect of HBO and hypobaric hypoxia preconditioning in rat SCI.Object:To investigate the mechanism of the neuroprotective effect of HBO and hypobaric hypoxia preconditioning in rat SCI,so as to provide rationale for its clinical application.Method:After divided into different groups for distinct preconditioning situation and preparation for SCI model, samples were collected at different time after SCI,then immunohistochemistry,RT-PCR and Westernblot were performed to identify neurocyte apoptosis,nerve stem cells,neuroglia cells,vascular endothelial growth factor,axon growth inhibitor and purinoceptor etc. Data were analyzed by using SPSS12.0 statistics software.Observation items:1) neurocyte apoptosis observation by means of TUNEL.2) immunohistochemistry for GFAP observation.3) immunohistochemistry for Nestin observation.4) immunohistochemistry for VEGF observation.5) immunohistochemistry for Nogo observation.6) quantitative observation for P2X7 purinoceptor.Result:1)In control group,apoptotic-positive cells increased from 1d after SCI,with a maldistribution in both gray and white matter, then gradually deceased from 1w after SCI with a mainly distribution in white matter surrounding the injured area,and only few apoptic-positive cells were found 2w after SCI.In preconditioning groups apoptic-positive cells appeared simultaneously with control group,but mainly concentrated in marginal part of injured region,and at 1w after SCI the apoptic-positive cells in preconditioning groups are much more less than that in control group.2) GFAP expresses in both gray matter and white matter in normal spinal cord.In the control group,GFAP expression began to increase surrounding the injured region at 1d till 2w after SCI,especially in the ependyma of spinal medullary canal.Capsular space began to form 4w after SCI,and GFAP expression decreased and mainly centered in the glia cell body and process surrouding the capsular space.GFAP expression in HBO preconditioning group is similar with control group;while in the hypobaric hypoxia preconditioning group,GFAP-positive cells were notablely increased at 2w after SCI and significantly decreased at 4w after SCI compared with control group.3) In control group,a few nestin immunostaining positive cells were observed at 1w after SCI in spinal cord parenchyma,and at 2w after SCI,a large amount of nestin-positive cells were seen.Most nestin-positive cells were polygon or orbicular-ovate in cell body with a lot of processes, which extended coincidently with the cell long axis;a minority of nestin-positive neurons were found in the vicinal gray matter of injured region.Nestin- positive cells were obviously decreased at 4w after SCI.In the preconditioning groups, nestin expression was stronger than that in control group after SCI,and hypobaric hypoxia preconditioning group is much more significant.4) VEGF expression increased in the injured and perimeter region at 1d after SCI,mainly in the cytoplasm of vascular endothelia cell(VEC) of the spinal pia mater and gray and white matter of spinal cord, as well as glial cell and macrophage of white matter;VEGF expression began to decrease at 1w after SCI.While in the preconditioning groups, VEGF expression was stronger than that of control group.5) In control group, no nogo-positive cells were found at 1d after SCI;2w later,a few nogo-positive cells were observed in the white matter nearby the injured region;8w after SCI,nogo were strongly expressed in the glial scar.No significant difference were seen in preconditioning groups.6) P2X7 receptor expression:In the HBO preconditioning and control group,strap for P2X7 receptor were obtained at 12h,3d and 5d after SCI,while in the hypobaric hypoxia preconditioning group nothing were found.No significant difference were found after between each group 7d after SCI.Conclusion:1)HBO and hypobaric hypoxia preconditioning has an effect of inhibiting nerve cell apoptosis;2) In the early state after SCI,HBO and hypobaric hypoxia preconditioning could both promote horizontal cell activation so as to protect neuron;while in the later stage, it may facilitate the hyperplasy of glial cell to form glial scar.3) Spinal cord injury could activate nerve stem or precursor cell,and HBO and hypobaric hypoxia preconditioning have an effect of promoting nerve stem or precursor cell proliferation and differentiation,while hypobaric hypoxia preconditioning is more effective.4) VEGF expression is stronger after SCI, and both preconditioning treatments could enhance its expression,thus to promote vascularizaiton and improve microcirculation to protect the nerve from hypoxia.5) In the later stage after SCI,the strong expression of axon growth inhibitor nogo may play an important role in hindering axon growth,and both HBO and hypobaric hypoxia preconditioning has no obvious impact on the nogo expression.6) After SCI,hypobaric hypoxia preconditioning could inhibit P2X7 receptor expression in a short term so as to lead a neuroprotective role,however,the HBO preconditioning is not involved in the same process.3.Evaluation of curative effect of HBO on experimental rat SCI Object:To evaluate the curative effect of HBO on rat SCI and provide reliable evidence for its clinical useage.Method:1)Rats were divided into 4 groups:SCI control group(group 1),SCI with HBO therapy group(group 2),SCI with HBO preconditioning and HBO therapy group(group 3) and SCI with hypobaric hypoxia preconditioning and HBO therapy group(group 4).The HBO therapy duration is 2w.2) Observation items:(1) behavioral assessment: BBB motor function score system was utilized in this study, and assessed the score at 1d,1w,2w,4w,and 8w after SCI.(2) Evoked potential examination: Somatosensory evoked potentia(SEP) and motor evoked potential(MEP)were detected at different time after SCI in each group.(3) TEM observation:Removed a volume of 1 mm3 tissue right in the central of the injured region after being perfused and fixed,placed the tissue into 10% glutaric dialdehyde fluid,made TEM slices and did the observation.(4) HE dyeing and observation: the samples of spinal cord were obtained at different time after SCI, made paraffin section in anteroposterior axes,performed HE dyeing and observed the slices under light microscope.(5) immunohistochemistry for nestin and GFAP expression.(6) Nerve-tract tracing: Anterogradely traceing were performed using biotin dextran amine(BDA) to see the regeneration of neuraxis descendingly,so as to identify the effect of HBO therapy on SCI.3) Data were analyzed by using SPSS12.0 statistics software.Result:1)BBB score in HBO therapy groups are higher than the control group,and the difference between group4 and group 1 is significant.(2) SEP and MEP showed a prolonged lantency after SCI in all groups,while after HBO therapy,the lantency in group2,3 and 4 is shortened compared with group1,and the difference in group 4 is significant.(3) The pathological change is lighter in group 2,3 and 4 compared with group 1,especially in the vicinity of the injured region.(4) GFAP and nestin expression increases in HBO therapy groups which is much more notable during 2w~4w after SCI,and group 4 shows a significant differnence.(5) Nerve-tract tracing indicates that the descending regeneration neuraxis increases in the distal end of injured region and peremeter of glial scar after HBO therapy.Conclusion:1)HBO therapy could relieve the pathological change after SCI,promote endogenous nerve stem or precusor cell proliferation,glial cell activation and neuraxis regeneration,thus to facilitate recovery of neurofuction after SCI.2) Combination of Hypobaric hypoxia preconditioning and immediately HBO treatment is effective in SCI prevention and cure.
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