The Study Of Partition Type Tissue-engineered Spinal Cord In Repairing Rat Spinal Cord Injury And Inhibiting Neural Apoptosis

PartⅠThe Study of Partition Type Tissue-engineered Spinal Cordon Repairing T8Complete Transaction Injury in Rat Spinal CordObjectiveTo investigate the combinational therapeutic strategy using the partition-type tubescaffold (PtTS) and bone marrow stromal cells (BMSCs) in treating the T85mmtransaction injury in rat spinal cord.MethodsExperiment in vitro:1. Cell culture and passage BMSCs labeled by green fluorescent protein (GFP)derived from rats.2. Generate PtTS and hollow tube scaffold (HTS) using chitosan and chitin.3. Prepare leach liquor of the tissue-engineered spinal cord scaffold for culturingBMSCs. Test the compatibility of BMSCs and the tissue-engineered spinal cord scaffoldusing CCK8.4. Monitor the growing condition of BMSCs on PtTS in by light microscopy.Experiment in vivo:1. Animal groups: SD rats were randomized into6groups. Within the6ones, fourof them, the HTS, PtTS, HTS+BMSCs and PtTS+BMSCs groups, were used to repairthe the T85mm transaction injury in rat spinal cord, while the other two groups, injurywithout repair (unbridged) and non-injury (sham) control groups were used as thecontrols. The animals were kept in separate cages for up to12months.2. Behavior test: BBB test and CatWalk-automated quantitative gait analysis wereperformed on all the animals before the surgical injury and1w,2w,4w,6w,8w,10w, 6M,9M and12M after it.3. Motor evoked potentials (MEPs) anlyses: Positioned the stimulating electrodesin the cerebral cortex and epidermis below the injured spinal cord (around T10), andpositioned the recordingelectrode in the gastrocnemius muscle.The MEPs from all the ratswere measured and the latencies and amplitudes were statistically analyzed.4. Anterograde tracing of rubrospinal tract (RST) axons: Fluoro ruby were injectedinto the center of the red nuclei using the stereotaxic apparatus2weeks beforeharvesting the tissues. Record the RST growing condition in the injury region of thespinal cord.5. Immunohistochemistry: Spinal cord tissues from C4to L4were collected andstained for5-HT1Aand NF to evaluate the axonal regeneration and BMSCdifferentiation. Nerve fiber numbers were quantified in different spinal cord sections,and compared with control groups. All the data were processed with statistics.6. Western Blot:3mm spinal cord tissues from the injury center to both rostral andcaudal ends were collected and processed for Western blots using antibodies against5-HT1Aand NF-200.7. Electron microscopy (EM) analyses: Regenerated tissues from the injury centerwere obtained and prepared for EM studies. Myelinated nerve fibers and the thicknessof the myelin were quantified.ResultsExperiment in vitro:1. After6passages of being cultured in vitro, the efficiency of GFP labeling inBMSCs was about90%.2. PtTS and HTS were successfully generated.3. Evaluation of compatibility of BMSCs and the tissue-engineered spinal cordscaffold: Cell proliferation curve obtained by CCK8showed the continuous increasednumbers of BMSCs when they were cultured in vitro. It reached the platform at day3. Itwas detected that BMSCs adhesively grew on the luminal side of the tube with goodviability by light microscopy.Experiment in vivo:1. Post surgery general condition:12months after surgery, the rats from all the groups except the sham group suffered from the muscle dystrophy of the hind limbs, aswell as vertebral deviation to a certain extent. However, the rats from the PtTS+BMSCsand HTS+BMSCs groups were better than the other lesioned groups.2. Behavior test:(1) BBB score: BBB score decreased to0after the surgical injury, and slowlyincreased thereafter. The scores of the PtTS+BMSCs and HTS+BMSCs groups weresignificantly higher than those of the other three lesioned groups between week10andmonth12post surgery. No significant difference was detected among those three otherlesioned groups.(2) Regularity index (RI) of the pattern regularity: All the lesioned groups hadsignificant decrease in their RI, while they started to increase after2weeks. RI of thePtTS+BMSCs group was higher than the other4lesioned groups after8weeks. RI wassignificantly higher in the HTS+BMSCs group than that in the unbridged group. Nosignificant difference was detected among the HTS, PtTS and unbridged groups.(3) Base of support: The values were increased to more than50mm in all thelesioned groups, as they started to decrease after2weeks. The values in thePtTS+BMSCs and HTS+BMSCs groups were significantly smaller than the other threelesioned goups.(4) Hindpaw pressure: The values were significantly decreased after the surgeries.However, the value in the PtTS+BMSCs group started to increase after4weeks andhigher than the other4lesioned groups. The value of the HTS+BMSCs group started todecrease after12weeks. However, the values of the HTS, PtTS and unbridged groupskept on low level.(5) Swing time: At all the checking time points, the values of swing timein in allthe surgery groups fluctuated.3. MEPs testing: MEPs were obtained from stimulation of both the cerebral cortexand the lower SCI site in all of the4bridged groups. When the animals were stimulatedin the cerebral cortex, the latency periods in the PtTS+BMSCs and HTS+BMSCsgroups were significantly shorter than those in the PtTS and HTS groups. In addition,the amplitudes in the PtTS+BMSCs group were significantly higher than those in theother3bridged groups. When stimulated at the lower SCI site, there was no significantdifference between all groups in the latency period; however, the amplitudes in thePtTS+BMSCs and HTS+BMSCs groups were significantly higher than those in the PtTS, HTS and unbridged groups.4. General observation: Samples from the unbridged group showed noregenerated tissue between the two stumps of residual dura mater. In the4implantedgroups, newly-regenerated tissue connected the stumps across the gap in the spinal cord,accompanied with debris from degraded chitosan, showing anatomical completeness tovarying degrees.5. Anterograde tracing of RST axons: That red fluorescence-labeled rubrospinalaxons were found to be present in the vicinity of the rostral edge of the spinal cordlesioned gaps in the PtTS+BMSCs group. The axons arranged regularly and most ofthem distributed in the partition of the RST.6. The regeneration of the5-HT1A+nerve fibers: In both the PtTS+BMSCs andPtTS groups,5-HT1A+nerve fibers were found to form bundles in an orderly fashion.And more positive fibers were observed in the PtTS+BMSCs group. A number oftransplanted BMSCs in the PtTS+BMSCs group showed5-HT1A+/GFP+. In theHTS+BMSCs and HTS groups, a small number of5-HT1A+nerve fibers were arrangedirregularly. Compared with the other4lesioned groups, the PtTS+BMSCs groupexhibited a greater number of regenerated5-HT1A+nerve fibers from3mm rostral to3mm caudal within the transected lesion site. The HTS+BMSCs group exhibited agreater number of5-HT1A+nerve fibers from2mm rostral to the epicenter and in the3mm caudal to the epicenter areas compared to the HTS, PtTS and unbridged groups. Inaddition, the results of Western blotting showed that the expression level of5-HT1Aprotein in the groups was in-line with the results of the immunohistochemical analyses.7. The regeneration of the NF+nerve fibers: NF+nerve fibers were detected toform bundles in an orderly fashion and across the entire defect gap in the PtTS+BMSCsgroup. Many BMSCs which were positive both for GFP and NF were detected amongthe nerve fibers. The PtTS+BMSCs group exhibited greater regeneration of NF+nervefibers from3mm rostral to3mm caudal within the transected lesion site compared tothe other4lesioned groups. And the HTS+BMSCs group exhibited more NF+nervefibers compared with the HTS, PtTS and unbridged groups. The results of Westernblotting showed that the expression level of NF protein in the groups was in-line withthe results of the immunohistochemical analyses.8. Myelin formation observation by EM: Nothing but the glia scar tissue wasfound in the center of the injured spinal cord in the unbridged group. There was no sign of axonal regeneration. In the HTS and PtTS groups, very few axon fibers were detectedsporadically, some of which do not have myelin. In the HTS+PtTS and PtTS+BMSCsgroups, more axonal fibers with myelin and capillaries were detected. In thePtTS+BMSCs group, the axons were more organized and the structure of myelin sheetswas clear with obvious synapses. The amount of myelin coated axon fibers in thePtTS+BMSCs group was bigger than those in the HTS, PtTS and HTS+BMSCs groups;whereas the amount of axon fibers in the HTS+BMSCs group was more than that in thePtTS and HTS groups. No significant difference has been detected between the PtTSand HTS groups. The thickness of the myelin sheets from all surgery groups had nosignificant difference.Conclusion：Tissue-engineered spinal cord scaffold had a good compatibility with BMSCs.Compared to the HTS+BMSCs, PtTS and HTS groups, the combination of the PtTS andBMSCs had a better motor functional recovery in treating the spinal cord injury. All ofthe electrophysiological properties in the descending motor nerve pathway of all thebridged groups were partially recovered. Compared to the HTS, the PtTS could inducethe regenerated axons to grow in a more organized fashion. The transplanted BMSCscould differentiate into neurons and assit the axonal regeneration. The combination ofthe PtTS and BMSCs could promote the myelinated nerve fiber regeneration after spinalcord injury and guide the organized outgrowth so as to restore the correspondingfunctional reconnection with the injuried caudal end. Our findings may provide apotential combinational therapeutic strategy to treat spinal cord injuries. Part Ⅱ Effect ofThe Partition Type Tissue-engineered Spinal Cordon Neural Apoptosis after Spinal Cord Injury in RatObjective：To investigate the effect of BMSCs in the partition type tissue-engineered spinalcord on nerve cell apoptosis after spinal cord injury and explore the mechanism of itsrepairing nerve injury and promoting functional recovery.Methods：1. The SD rats were divided into4groups at random: in the BMSCs group(PtTS+BMSCs), PBS group (PtTS+PBS) and z-VAD-fmk group (PtTS+z-VAD-fmk),the5mm spine cord gap at T8of rats were bridged by different transplants, respectively.Sham group was set as control. All animals were maintained for3weeks followingsurgery.2. Behavioral testing: BBB behavioral test and CatWalk-automated quantitativegait analysis were conducted on each group of rats before surgery and at3d,7d,14d and21d post surgery.3. TUNEL staining: The spinal cord at T7of each group’s rats were collected toconduct TUNEL staining at1w,2w,3w after surgery. The number of positive cells wereanalyzed and the nerve cell apoptosis was observed.4. Electron microscopy analyses: The spinal cord at T7of each group’s rats werecollected for electron microscopy observation.5. Immunohistochemical analyses: The spinal cord at T7of each group’s rats werecollected to cut into sections on a cryostat.(1) Double-labeled staining of TUNEL/NeuN was used to detect the neuronapoptosis of the anterior horn in the gray matter and the number of TUNEL+neuronswas analyzed.(2) Double-labeled staining of TUNEL/CNPase and TUNEL/GFAP were used todetect the neuroglial cell apoptosis in the white matter and the number of TUNEL+oligodendrocytes and TUNEL+astrocytes were analyzed, respectively.(3) Double-labeled staining of NeuN/caspase-3was used to detect the expressionof caspase-3in the neurons of the anterior horn in the gray matter.(4) Double-labeled staining of CNPase/caspase-3and GFAP/caspase-3were used to detect the expression of caspase-3in the oligodendrocytes and astrocytes of the whitematter, respectively.6. Western blot analyses: Spinal cord tissues located3mm rostral and3mmcaudal to the epicenter were collected to detect the expression of caspase-3p17, PARP,and cleave PARP.Results：1. TUNEL staining: No significant cell apoptosis was observed in the sham group.Many TUNEL+cells were found in3other groups1w after surgery, while there was nosignificant difference among them. The numbers of the TUNEL+cells in the BMSCsand z-VAD-fmk groups were less than that of the PBS group since the2ndweek postsurgery. And the numbers of the TUNEL+cells in the BMSCs and z-VAD-fmk groups at3thweek post surgery were less than that at1stweek post surgery. There was nosignificant change in the PBS group.2. Electromicroscope observation: Many myelinated nerve fibers were found inthe BMSCs group, along with minor apoptotic cells. The number of the myelinatednerve fibers decreased in the z-VAD-fmk group. Density of myelinated nerve fiberdecreased significantly in the PBS group, and more apoptotic and necrotic cells couldbe found, with demyelination on axons.3. Neuron apoptosis: No TUNEL-positive reaction was found in NeuN+neuronsof the sham group, and number of neurons was more than3other groups. The numberof NeuN+/TUNEL+cells in the BMSCs and z-VAD-fmk groups were less than that inthe PBS group.4. Oligodendrocyte apoptosis: No significant difference was found in thenumbers of the CNPase+oligodendrocytes among groups. No TUNEL-positive reactionwas found in the CNPase+cells of the sham group. The number of CNPase+/TUNEL+cells in the z-VAD-fmk group was less than that in the PBS group. There was nosignificant difference between the BMSCs and PBS group, and so was the same to theBMSCs and z-VAD-fmk group.5. Astrocyte apoptosis: No significant difference was found in the numbers of theGFAP+astrocytes among groups. No TUNEL-positive reaction was found in the GFAP+cells of the sham group and there was no significant difference in the numbers ofGFAP+/TUNEL+cells among the other3groups. 6. Caspase-3expression in neurons: The number of caspase-3+/NeuN+cells in theBMSCs group was less than that in the PBS group. There was no caspase-3-positivereaction found in the NeuN+neurons of the z-VAD-fmk and sham groups.7. Caspase-3expression in oligodendrocytes: The number ofcaspase-3+/CNPase+cells in the BMSCs group was less than that in the PBS group.There was no caspase-3-positive reaction found in the CNPase+oligodendrocytes of thez-VAD-fmk and sham groups.8. Caspase-3expression in astrocytes: There was no significant difference in thenumber of caspase-3+/GFAP+cells between the BMSCs and PBS group. Nocaspase-3-positive reaction was found in the GFAP+astrocytes of the z-VAD-fmk andsham groups.9. Expression of caspase pathway proteins: The level of the expression ofcaspase-3p17and cleave PARP in the BMSCs group was higher than that of thez-VAD-fmk and sham group, but lower than that of the PBS group.10. Behavioral observation:(1) BBB locomotor score: Except for the sham group, which scored stably21,scores of other3groups fall to0and rised gradually. Since the2ndweek after surgery,the scores of the BMSCs and z-VAD-fmk groups were higher than that of the PBSgroup, while thescore of the latter group remained constant.(2) RI of the pattern regularity: Except for sham group, whose RI was stably noless than95%, other3groups’ index fall to10%and rised gradually. There was nosignificant difference among the operated groups after surgery.(3) Base of support: Except for the sham group, which kept stable at30mm, other3groups increased to above50mm. Since the2ndweek after surgery the distances of theBMSCs and z-VAD-fmk groups were shorter than that of the PBS group.(4) Hindpaw pressure: Statistics of other3groups fall significantly, except thesham group which was stably above100a.u. Pressures of the BMSCs and z-VAD-fmkgroups was higher than that of the PBS group since the2ndweek after surgery.(5) Swing time: Except for no changes in the sham group, other3groupsexperienced the increase of the time. Time of the BMSCs and z-VAD-fmk groups wasshorter than that of the PBS group since the1stweek after surgery. No significantdifference was found among the BMSCs, z-VAD-fmk and sham group since the2ndweek after surgery. Conclusion：BMSCs of the partition type tissue-engineered spinal cord could inhibit the caspasemediated cell apoptosis pathway by prohibiting the expression of caspase-3, and furtherdecreased the apoptosis of neurons and oligodendrocytes after SCI, promoted thefunctional recovery of rat’s motion. Apoptosis of astrocytes after SCI had no directcorrelation with the caspase-mediated pathway, which may likely to occur via otherapoptotic pathway.