Combination Of Lenti-ChABC And Schwann Cell Transplantation In The Treatment Of Spinal Cord Injury In Rats

Objective: Spinal cord injury(SCI) is devastating, affecting hundreds of thousands of patients alone, with thousands more diagnosed each year. Although much progress has been made in the scientific community to understand the pathology and molecular mechanisms of the injury, and to develop new repair strategies for it, effective treatments for significant recovery remain to be developed. Functional loss following SCI results primarily from the severing of long descending and ascending axons within the spinal cord by direct trauma and by complex secondary injury mechanisms. In both human and animal models of SCI, the formation of gaps or cavities inevitably occurs. The failure of axonal regeneration must therefore be caused, at least in part, by these anatomical spaces across which axons are unable to grow. Cell-based strategies are thus essential to provide a physical substrate upon which axons can grow across the lesion site. Additionally, formidable impediments formed by reactive astrocytes, oligodendrocytes and extracellular matrix-associated inhibitory molecules may further inhibit axonal regeneration. Consequently, additional treatments aimed at modifying the host-graft interfaces or the host cord proper would promote further growth of regenerating axons back into the host spinal cord. Furthermore, regenerating axons need to innervate appropriate targets, form new synapses, and be myelinated to achieve optimal functional recovery. Clearly, a combinatorial strategy aimed at addressing each of the aforementioned obstacles is essential. Schwann cells(SCs) are considered to be one of the most attractive cell types for transplantation after SCI because they express a variety of trophic factors, extracellular matrix molecules, and adhesion molecules that are strong promoters of axonal growth. The major challenge exist with SC-based therapy for SCI is the migration of SCs into the host spinal cord. When placed into the astrocyte-rich CNS environment, the SCs and astrocytes do not intermingle. The SCs are walled off by astrocytes and migrate poorly from the site of transplantation into the host. Astrocytes in contact with SC grafts become hypertrophied and upregulate chondroitin sulfate proteoglycans(CSPGs). The sharp boundaries that form between SCs and astrocytes are a barrier to regenerating axons, particularly axons attempting to leave the growth-promoting SC environment and to reenter the astrocyte territory of the host spinal cord. The molecular events that prevent SCs and astrocytes intermingling are only partially understood. There is clearly evidence, however, that astrocyte-produced CSPGs block SC migration. Application of Ch ABC which degrades the inhibitory CSPGs, was shown to increase the intermingling of SCs with astrocytes in vitro.If SCs can migrate out from the graft into the host spinal cord, axons within the graft may elongate, along with the migratory SCs, into the host spinal cord where they may establish new synapses on target neurons. Here the lentiCh ABC was applied to digest away inhibitory CSPGs at the glial border that allows the migration of SCs into the host spinal cord.Methods: 1. SCs were obtained from the sciatic nerve(GFP-rat). The explants were kept for 6 weeks, then under culturing, purifying and proliferation. 2. In vitro, T293 cells transfected by lenti-Ch ABC were supposed to secrete chondroitinase, which can digest the CSPGs. In vivo, lenti-GFP was injected into the spinal cord. 3. SD rats were divided by five groups: sham(n=10), control group(n=12), SCs group(n=12), lenti-Ch ABC group(n=12) and lenti-Ch ABC+SCs group(n=12). All animals(except sham group) were under contusion injury through NYU. 7-10 days after injury, SCs and/or lenti-Ch ABc were injected into the spinal cord. SCs were injected into the epicenter of the lesion cavity, and lenit-Ch ABC was injected into the caudal and rostral of the lesion cavity. Control group rats were injected with DMEM. 4. BBB open field testing was performed every weeks after SCI for up to 8 weeks to evaluate functional recovery. 5. Grid walk: the rats were examined in a clear grid field. The number of foot fall errors where the hind limb failed between the bars was recorded. Generally the animals were allowed to walk over the grid three times, and each trial was videotaped. The foot falls were counted by the blind observer from the videos and the average of foot fall errors was calculated. 6. Hargreaves test: for thermal allodynia testing, a rat was placed in a Plexiglas enclouse, and the rat wa s tested by the lab’s protocol. The test was done at 4 and 8 weeks postoperatively. 7. The metabolic cage were used to test the bladder function at 6 weeks postoperatively. Before the start of the experiment, each rat was placed in an individual metabolic cage for 3 consecutive days with free access to food and water. Urine output was monitored every 2 minutes for 12 hours. The balance was connected to a personal computer data acquisition. The parameters were analyzed through 12-hour urine production, 12-hour voiding frequency and mean voided volume. 8. The rats were sacrificed on the 8th weeks. The tissue was cut into 25μm slides longitudinally. P0, ED-1, Reca-1 and Tunnel were used to test the function and condition of the SCs. CS56, GFAP and 2B6 were used to test the CSPGs. GFP-SCs were used to test the migration of transplanted cells. TH and 5-HT were marked axons. All the images were analyzed by image J. All the statistics were performed by Graph pad 6.0.Results: 1. SCs can be obtained through the sciatic nerve. It was easy to get the SCs from this method and easy to passage. The purity can reach to 98%. It was good choice to select cell before passage 4. 2. The T293 cell transfected with lenti-Ch ABC can continuously secrete chondroitinase, and can digest the CS which can be proved through DMB assay. T293, transfected by lenti-GFP, like the commercial chondroitinase, can effectively digest the CSPGs. The lenit-GFP can be successfully applied in the spinal cord. 3. For the behavioral assessments, after SCI, all of the rats can reach to 0. 1 and 2 weeks after SCI, the rats recovered well. BBB in the lenti-Ch ABC+SCs were higher than the rest of the groups after the 4th weeks(p<0.05). On the 8th weeks, the BBB between these groups showed significant difference, through there were no difference in weights. 4. In the grid walk test, lenti-Ch ABC+SCs rats showed lower drop percent than the rest of the groups on the 4th and 8th weeks(p<0.05). 5. For the Hargreaves test, no difference was found during these groups, indicating that lenti-Ch ABC or SCs did not cause any sensory loss.6. After SCI, control rats showed lower frequency and higher void per frequency.Interestingly, lenti-Ch ABC and SCs group showed better recovery(p<0.05).7. Grafted SCs-GFP myelinated regenerated axons and expressed protein zero. ED-1staining showed that macrophages rarely engulfed degenerated SCs-GFP. Within the SCs-GFP grafts. New blood vessels were formed. Tunnel showed that very few grafted SCs-GFP had underwent apoptosis. Immunofluorescence staining of CS56 and 2B6 showed CSPGs were digested and the lesion cavity was reduced. GFAP staining showed the astrocytes were less reactive. Immunofluorescence staining of TH and 5-HT showed SCs-GFP were in close association with axons growing into the graft, and promoted the axon regeneration in the lesion cavity. 5-HT and TH axons in the epicenter of graft area were correlated with BBB. The numbers of 5-HT and TH axons in the combination group were significantly higher in the caudal spinal cord.Conclusion: 1. SCs culture in this project was excellent for the high purity, proliferation and passage, indicating that the SCs could be used for cell transplantation.2. Lenti-Ch ABC can effectively secrete chondroitinase after transfecting the T293 cells. In vivo, the lenti-GFP successfully transfected the spinal cord.3. SCs can secrete neurotrophic factors and extracellular matrix, which can promote axon regeneration. SCs supported axonal growth and myelinated regenerated axons within the lesion site, providing a unique opportunity for axon regeneration. Grafted SCs-GFP myelinated regenerated axons and expressed protein zero. Macrophages rarely engulfed degenerated SCs-GFP. Within the SCs-GFP grafts, new blood vessels were formed. Few grafted SCs-GFP had underwent apoptosis.4. Combination of lenti-Ch ABC and Schwann cell can promote functional recovery,including BBB and grid walk. What else, lenti-Ch ABC and Schwann cell did not cause sensory damage.5. Lenti-Ch ABC and Schwann cell can improve the frequency and reduce the voids per frequency.6. Combination of lenti-Ch ABC and Schwann cell can effectively secrete chondroitinase, which can digest CSPGs. The CS56 showed lower expression. 2B6 was showed in the lenti-Ch ABC group. Astrocytes were less reactive. 7. Combination of lenti-Ch ABC and Schwann cell can promote the migration of Schwann cells.8. Combination of lenti-Ch ABC and Schwann cell can promote axon regeneration of TH and 5-HT.