| ObjectiveCombination chondroitinase ABC (ChABC) and bone marrow mesenchymal stem cells (BMSCs) was delivered to the lateral ventricle after spinal cord hemisection injury in rats. To study the effect of strategy combining BMSCs and chondroitinase ABC on the regeneration and functional recovery of spinal cord injury and discuss its potential mechanisms. Analyse and evaculate the reliablity of the intracerebroventricular cannulation approach.MethodsBMSCs were separated with density gradient centrifugation and cell attachment DAPI was used for labeling before cell transplantation. A total of120adult male SD rats (280±10g) were used in these studies. To establish the right spinal cord hemisection injury model with SD rats.Rats randomly divided into four groups as follows (n=30):Group A (control, saline treatment), Group B (BMSCs treatment), Group C (ChABC treatment), Group D (BMSCs+ChABC treatment). A、B、C、D group was sub-divided for postinjury3-day,7-day,14-day,30-day and BDA inject goup,each subgroup has6rats. Observation index and collection data:Locomotor behavior was monitored and scored according to the Basso Beattie and Bresnahan (BBB) open field test. Locomotor behavior was tested at postinjury3days,7days,14days and30days. Then the spinal cord with spinal column (T9-T11) was cut and postfixed with4%PFA. Twelve hours later, only spinal cord tissue was drew out and fixedovernight and embedded in10%gelatin for histo morphology observation.Include HE. Immunochemistry analysis:GFAP, GAP-43, BDA tracing after spinal injury14days (n=5).Results1. BBB scores of preinjury were21’. After SCI, all rats were instantly observed typical paraplegia syndrome. Twelve hours later, rats could start to scramble with two hind legs towing and acrotarsium on the ground.3days later, BBB scores of rats with four groups were no different on statistics.7days,14days,21days and30days BBB scores of rats treated with BMSCs or ChABC or BMSCs+ChABC were much higher than that of rats treated with control. Compared with group B and C, group D was significantly different (P<0.05)2. HE staining:spinal cord extensive hemorrhage, red blood cells exosmotic, neuron nuclear condensed, necrosis and dissolved, microgliasis and inflammatory infiltration, capsular space formation. The pathologic appearance rats treated with individually BMSCs, ChABC or BMSCs+ChABC was better than that of control group.3. BMSCs with the DAPI labeled were delivered to the lateral ventricle after spinal cord hemisection injury in rats. Group D result showed:after21days, in the spinal cord injury site can still see a lot of positive labeled BMSCs cell survival.4. The positive GAP-43cells were appeared at postinjury1-day, which was mainly expressed in the grey matter, fewer in the white matter. The GAP-43expression reached the peak at postinjury21-day and then decreased. At postinjury14-day,21-day and30-day, the positive expression of GAP-43of spinal cord tissues in rats treated with group B, C and D were much more numbers and deeper staining than group A.5. In each group, GAFP expression gradually increased over time after spinal injury and reach the peak at14-day. At postinjury14-day,21-day and30day the positive expression of GAP-43of spinal cord tissues in rats treated with group B and D were much less area and lighter staining than group A, and there was statistic difference between group B and D.6. BDA result were observed after injection2weeks, quantitative analysis showed that group D showed significant difference compared with the other groups, which had much more BDA-positive nerve fibers below the level of injury.Conclusions1. Transplanted cells or drug through the intracerebroventricular catheter appoach was secure and stable. Cells and drug with the cerebrospinal fluid circulation can reach the damage area of spinal cord injury, also with high survival rate. This method is suitable for repeated use to delivery the drug in one experiment.2. Chondroitinase ABC can destroy chondroitin sulfate proteoglycans specificity, prevent scarring, profit axon growth, indirectly promote injuried spinal cord regeneration and recovery.3. BMSCs can regulate up the GAP-43expression, promote the impaired neuronal morphology and improve axonal regeneration after spinal cord injury.4. GFAP expression will increase after spinal cord injury, while the BMSCs combination with ChABC can mutual synergies, and inhibit the proliferation of reactive glial cells, they can promote the survival of spinal cord injury and regeneration, BMSCs transplantation can improve the rceovery of nervous function of rats with spinal cord injury. |
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