Neuroscience. 2016 Apr 21;320:69-82. doi: 10.1016/j.neuroscience.2016.01.066. Epub 2016 Feb 4.
Transplantation of neural progenitor cells in chronic spinal cord injury
Jin Y*, Bouyer J, Shumsky JS, Haas C, Fischer I*.
Department of Neurobiology and Anatomy, Drexel University, College of Medicine, Philadelphia PA 19129, United States
Previous studies demonstrated that neural progenitor cells (NPCs) transplanted into a subacute contusion injury improve motor, sensory, and bladder function. In this study we tested whether transplanted NPCs can also improve functional recovery after chronic spinal cord injury (SCI) alone or in combination with the reduction of glial scar and neurotrophic support. Adult rats received a T10 moderate contusion. Thirteen weeks after the injury they were divided into four groups and received either: 1. Medium (control), 2. NPC transplants, 3. NPC+lentivirus vector expressing chondroitinase, or 4. NPC+lentivirus vectors expressing chondroitinase and neurotrophic factors. During the 8 weeks post-transplantation the animals were tested for functional recovery and eventually analyzed by anatomical and immunohistochemical assays. The behavioral tests for motor and sensory function were performed before and after injury, and weekly after transplantation, with some animals also tested for bladder function at the end of the experiment. Transplant survival in the chronic injury model was variable and showed NPCs at the injury site in 60% of the animals in all transplantation groups. The NPC transplants comprised less than 40% of the injury site, without significant anatomical or histological differences among the groups. All groups also showed similar patterns of functional deficits and recovery in the 12 weeks after injury and in the 8 weeks after transplantation using the Basso, Beattie, and Bresnahan rating score, the grid test, and the Von Frey test for mechanical allodynia. A notable exception was group 4 (NPC together with chondroitinase and neurotrophins), which showed a significant improvement in bladder function. This study underscores the therapeutic challenges facing transplantation strategies in a chronic SCI in which even the inclusion of treatments designed to reduce scarring and increase neurotrophic support produce only modest functional improvements. Further studies will have to identify the combination of acute and chronic interventions that will augment the survival and efficacy of neural cell transplants.
Supplement: What we learned, What is next:
According to National Spinal Cord Injury Statistical Center (NSCISC) there are approximately 17,000 people in US experiencing a spinal cord injury (SCI) each year with an estimated 282,000 people living with SCI at the chronic stage1. Despite the impressive progress in elucidating the mechanisms of the injury, the development of promising preclinical therapeutic strategies and the beginning of approved clinical trials, there is no effective treatment for patients who suffer spinal cord injury. Furthermore, most of the research in SCI is focused on acute and sub-acute injury models where the potential for improvement is relatively high. In contrast, studies of chronic SCI are more challenging and complex as a cavity is formed at the injury center due to the loss of host cells and a dense scar surrounds the injury site with reactive astrocytes present at the scar producing inhibitory molecules. Together they form a physical and chemical barrier to axon growth and regeneration2, 3. The lack of spontaneous repair and continued inflammation at the chronic stages of SCI results in not only motor and sensory deficits but often also neuropathic pain and autonomic dysreflexia. Indeed, interventions that have demonstrated beneficial effects and recovery following acute and sub-acute SCI in animal models are not effective in chronic injury. Our recent research interest has been focused on cell transplantation as a promising strategy for SCI treatment with emphasis on the use of neural stem cells and neural progenitor cells (NPCs) derived from the embryonic spinal cord4-6. The present study has examined the efficacy of this strategy in chronic SCI.
Our previous studies showed that transplanted NPCs, composed of glial and neuronal restricted progenitors, into the sub-acute spinal thoracic contusion injury site can improve motor, sensory and autonomic function7 and in an acute model of cervical hemisection can form a functional relay across the injury4. Using similar strategies, we transplanted NPCs into a chronic injury site with additional steps to reduce scar formation and increase growth factors support (divided into 3 groups). We found that consistent survival of the grafted cells presents a major challenge for chronic cell transplantation. There were large variations in survival of the transplants in all 3 groups ranging from no survival, isolated pockets of cells around the lesion cavity, and excellent survival with cells filling the lesion cavity (Fig.1 A-C, respectively). Two important findings, which were reported in the present study include 1) The survived NPCs in the chronic lesion area differentiated into neurons and astrocytes (Fig. 1 D and E); 2) The NPCs transplant reduced scar formation if the cells were able to integrate within lesion area.
Figure 1. Variability of transplant cell survival following chronic SCI. NPCs were derived from AP transgenic rats and identified by AP histology. A: no transplanted cells survived at lesion area. B: Cells survived at both stumps of the lesion but not connected the lesion. C: transplanted cells filled almost lesion area. Scale bar = 1mm. Transplanted cells differentiated in the lesion area. Confocal images show transplant cells (green) differentiate into neurons (D, red) as well as astrocytes (E, red). Scale bar =20um.
In analyzing the implications of these results, it is important to keep in mind that the primary goals of cell transplantation in SCI are to replace lost cells at the injury area, to provide a cellular matrix at the lesion cavity forming a bridge or relay across the injury, and to promote axons growth into and through the transplant to reconnect the damage the pathways. Without transplant survival within the lesion cavity and cell integration with host tissue to bridge the injury, damaged host axons will not be able to grow into and through the transplant/lesion site to reconnect with potential targets. However, if the goals of the transplants are to promote endogenous axon remyelination (e.g., using oligodendrocyte progenitors, OPC) or provide neurotrophic support (e.g., using glial restricted progenitors, GRPs), it may not be critical for the transplants to be localize at the injury site and cells can be injected rostral and caudal to the lesion site, or around the lesion area8, 9. In our present study of chronic SCI, about 60% of the animals in all groups showed some cell survival at either rostral or caudal stump of the spinal cord, but only few examples where the lesion cavity has been bridged. Without a neural bridge filling the cavities, damaged host axons could not grow into or through the transplant/lesion site. Even in acute or sub-acute SCI, where transplanted cells survive and bridge the lesion cavity, damaged axons show modest growth into the transplant with very few axons ever growing through the transplant5, underscoring that long distance regeneration of damaged CNS axons is limited and therefore need additional strategies to increase their regenerative capacity (e.g., modulating the mTOR signaling). An alternative strategy to reconnect the damaged pathway can be achieved by the formation of a functional relay, which requires that the transplants will generate neurons to form the relay and astrocytes to produce a permissive environment to promote growth of host axons into the transplant to make connection with graft-derived neurons. To complete the relay graft-derived neurons have to grow their axons out of the transplant and connect with the host target (which may require the formation of guidance cues along a directional pathway). Indeed, recent studies have demonstrated that transplanted NPCs at acute or sub-acute stage can form relay in both sensory and motor system4, 10. It is therefore important to note that in our study the transplanted cells differentiated into both neurons and astrocytes inside the lesion/transplant area (Fig. 1 D and E), which indicates that even at the chronic state (13 weeks after initial injury) this phenotypic requirement can be achieved using NPCs. Future studies will be focused on improving cell survival at the chronic stage and promote either long distance axon regeneration or formation of a relay to reconnect the damaged pathways.
Another finding from our present study relates to the changes in locomotor function after transplantation in the chronic stage. With the standard open field locomotion test, the functional changes, which are expressed by BBB scores, showed no significant changes comparing all experimental groups with the control group. To analyze the locomotion test in more details we used the BBB sub-score scale, showing that although there were no significant differences among all groups, all treated groups showed a tendency to increase the BBB sub-score (Fig. 2). We concluded that to further improve functional recovery after chronic injury, cell transplantation needs to be combine additional intervention, such as exercise. Exercise as a non-invasive treatment has shown many benefits for SCI: promoting synaptic plasticity in the spinal cord interneurons11, restoring motor and sensory function12, increasing neurotrophic factors in the spinal cord and muscles13, 14, and reducing inflammation around the lesion site12. In future studies we will examine whether combined cell transplant with exercise can improve functional recovery after chronic SCI.
In summary, our study addressed some of the challenges of cell transplantation in chronic SCI, and revealed the importance that needs to improve cell survival at lesion site if the goal is reconnection across the injury by host axon regeneration or through relays formed with graft-derived neurons. With successful restoration of connectivity together with exercise and training, which facilitates and stabilizes these connections it may possible to achieve functional improvement and eventually to translate the finding to treat patients with chronic SCI.
Figure 2. BBB sub-score shows locomotion changes 8 weeks after transplantation (21 weeks after SCI). The black line indicates the level of control group. There is no significant difference compared all groups, but treated groups show a little tendency to increase the sub-score.
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Authors would like to thank Dr. Vladimir Zhukarev for assistance of the confocal images.
This work was founded by grants from The Craig H. Neilsen Foundation (#160746) and NIH (SP01NS055976).