Novel Surgical Approaches for Transplanting Three-Dimensional Constructs of Olfactory Ensheathing Cells to Repair the Injured Spinal Cord

Abstract

Neurological injuries are very difficult for the human body to heal, as neurons are highly specialized cells which do not undergo cell division in adulthood. Central nervous system (CNS) neurons exhibit particularly low capacity for regeneration, due to both intrinsic cellular and environmental factors. For this reason, spinal cord injuries are usually irreversible in nature and they usually result in at least some motor paralysis and/or loss of sensory function. Higher level (cervical) injuries typically lead to widespread paralysis and sometimes death. Spinal cord injuries are a serious health problem and a very large burden on health care system. Therefore, therapies that repair the injured spinal cord will bring considerable benefits to patients as well as large socioeconomic benefits to the society. One particular region of the nervous system that naturally constantly undergoes regeneration is the primary olfactory nervous system, which is comprised of the olfactory nerve and the outer layer of the olfactory bulb (the nerve fibre layer). Olfactory neurons continuously degenerate, and new neurons originate from progenitor cells in the olfactory neuroepithelium of the nasal cavity. The glial cells of this system, olfactory ensheathing cells (OECs), are thought crucial for this process and are considered to have unique growth-promoting properties. For these reasons, transplantation of OECs into damaged nervous system regions is emerging as a promising therapy. OECs can be cultured from biopsies from either the olfactory mucosa (which contains olfactory nerve fascicles) or from the olfactory bulb. To date, numerous studies of OEC transplantation into rodent models of spinal cord injury as well as human clinical trials have been conducted. OEC transplantation has proven to be safe and feasible for spinal cord injury repair in humans, but outcomes in both animal studies and human clinical trials are highly variable and the method needs considerable improvement and standardization. One of the major limiting factors for the efficacy of this therapy is cell survival and integration following transplantation. One interesting avenue for improving both cell survival and integration is to transplant the cells as a three-dimensional construct rather than as cells suspended in liquid. The mechanisms by which OECs can induce and sustain neural regeneration are becoming well characterised in the literature, however, little in vivo evidence exists regarding the interrelationship between (1) cell survival and structural repair, (2) structural repair and functional improvement and (3) different injury conditions and cell survival/integration. Moreover, over two thirds of studies to date have used OECs derived from the olfactory bulb (OB-OECs) rather than mucosa-derived OECs (OMOECs). From a clinical viewpoint, OM-OECs are highly favourable since bulbar biopsies induce damage to the CNS and require intracranial brain surgery, whilst OMOECs can be isolated from a peripheral biopsy of the olfactory mucosa at the roof of the nasal cavity. Thus, more studies need to focus on transplantation of OM-OECs rather than OB-OECs. The primary focus of this thesis was to improve the therapeutic potential of OMOEC transplantation for spinal cord injury repair, with particular focus on enhancing cell survival and integration, by improving the surgical transplantation approach. A key component of this strategy was to transplant the cells in a 3D conformation (spheroids). This work also evaluated the effects of different injury conditions on the structural repair and functional regain following treatments. The aims of this Thesis included (1) the establishment of a robust murine spinal cord transection-type injury model and, later, adaptation to contusion-type injury, (2) testing of different methods for delivering OECs to the spinal cord injury site (different configurations of cell suspension, cell spheroids), (3) determination of how the chronicity of injury (time between injury and OEC transplantation) affects cell survival and integration, (4) assessment of how cell transplantation affects structural repair and (5) determination of the link between structural repair and functional outcomes. Throughout these aims, the aspect of OEC purity was focused on. OEC cultures from the olfactory mucosa not only contain OECs but also other cell types, in particular olfactory nerve fibroblasts, which can affect both the transplanted cells and cells endogenous to the injury site. Therefore, OECs were transplanted at different purities and resultant structural/functional outcomes were assessed. A minor aspect of this Thesis also focused on optimisation of a treatment delivery matrix. A set of in vitro experiments were conducted to determine the best composition of the matrix which could not only augment and contain the OECs in a 3D form while transplanting, but also enable them to migrate and form bridges across the injury site. This project was designed to ultimately provide insight for the clinical translation of this therapy with focus on improving cell survival and integration at the injury site, leading to enhanced structural repair and functional regain, paving the way for clinical trials in the coming future. As a result of this undertaking, the following key outcomes have been achieved: Development of a murine transection-type spinal cord injury model: A novel approach for laminectomy at the T10 spinal level was developed that can be used to induce a complete transection-type injury (using a narrow blade). The advantage of this method is that it is precise, it minimises the collateral tissue damage and mitigates blood loss. The complete transection type injury results in complete loss of sensory-motor and autonomic function below the level of injury, which is why a manual bladder expression protocol was also established during the course of the post-surgical care. A subjective assessment system was also developed to study the sensory recovery and bladder function recovery along with standardised motor behavioural tests. Transplantation matrix can support OECs in vitro: The in vitro experiments show that the transplantation matrix can provide stable and long term support to the 3D cultured cells and allow them to freely migrate and interact with each other. The matrix provides a physiological scaffolding to the OECs that can hold the 3D constructs in place without hindering the movements of individual cells. Transplantation of OECs in 3D improves cell survival, integration and structural repair: To determine whether transplantation of cells in 3D rather than in suspension would improve spinal cord injury repair, a comparison was conducted between suspension injection (the literature gold-standard) and a number of different configurations of cell spheroid transplantations. The best results showed ~15-20% cell survival one week following the transplantation, which is a significant improvement upon that reported for cell suspension in the literature (~0.6% cell survival was reported in the previous study that most resembles the experimental design used in this Thesis). Spheroid transplantations also demonstrated relatively better cell morphology in vivo compared to the published reports and better structural repairs post transplantation compared to the untreated controls. Interestingly, contrary to the evidence presented throughout the literature, the chronicity of the injury appeared not to have any significant effects on the cell survival when cells were transplanted in spheroids. Transplantation of OECs spheroids leads to functional regain: The treatments with the best functional outcomes were the ones when OECs were delivered to the SCI site in the form of two medium-sized spheroids, as compared a single large spheroid or four smaller-sized spheroids. The best cell survival results were also observed with the same modality. Although all spheroid treatments resulted in functional recovery, best recovery was observed with large and midsized spheroids. This functional status of the animals was assessed by the conventionally used motor function measurements such as open field behavioural scores – Basso Mouse Scale and Toyama Mouse Scale, and once the animals showed consistent stepping – DigiGait (an automated gait analysis of ventral plane images of the mouse’s gait). As mentioned earlier, additional subjective assessments of reflex recovery, sensory functions and autonomic status (bladder function) were also employed for the same. Different cell purities show different recovery trends: Behavioural studies showed that significant differences in the functional recovery trends depending on the purity of transplanted cells. Unpurified transplants showed faster onset of recovery, however, the recovery plateaued soon after that. On the contrary, the purified transplants showed a slower onset of recovery, which continued throughout the follow up period. Unfortunately, due to the low purity of OECs in the unpurified transplants, a comparison between cell survival and integration could not be done. This was due to the fact that the treatment cells were sourced from a mouse line genetically modified to express DsRed reported protein driven by s100-β promoter. This means that the OECs (and some other cells such as Schwann cells) fluoresce red under the ultraviolet light, which makes it difficult to visualize the non-DsRed cells (or cells other than the OECs). Evidence of structural repair, which links to functional outcomes, and evidence of cell integration: Histological analysis of the treated spinal cords revealed structural repair in terms of injury gap size reduction, axonal sprouting and formation of cellular bridges across the injury site. High-magnification imaging revealed evidence of OEC integration with both astrocytes and the sprouting axons. The transplanted cells were observed to ensheathe the axons extending across the injury site. Overall, the animals for which considerable structural repair was observed also were the ones that showed functional improvement. Development of a contusion-type spinal cord injury model: In the final stages of this Thesis, the treatment was also optimised for the use in an incomplete – contusion type injury which is clinically more relevant. Here, a contusion-type injury using the Infinite Horizons Impactor was induced at T10 level (the same laminectomy approach as for transection-type injury was used). A longterm follow-up of the two main injury models – transection and contusion, was also conducted to establish and compare the two models. It was discovered that the incomplete contusion injury resulted in a degree of spontaneous functional recovery for a short period following the injury, while the complete transection type injury resulted in total and irreversible loss of function. Potential links of animal behaviour and physical condition to the different stages of injury were also observed.