This thesis was an explorative study to evaluate potential possibilities for spinal cord regeneration. At present there is no medical treatment available to cure spinal cord injury and the person remain paralysed and incontinent for life. Based on the literature three major strategies were evaluated namely cell therapy, administration of growth factor and enzyme. All these studies were conducted in rat model of spinal cord injury.
Rat olfactory mucosa isolated from the posterior region of nasal septum, and lamina propria was enzymatically separated from the epithelium. Lamina propria was enzymatically dissociated to yield olfactory ensheathing cells (OEC) and olfactory nerve fibroblast (OEC). The cells were cultured, characterized by IHC and flow cytometry to test p75NTR (OEC), fibronectin (ONF). Fresh second passage cells were labeled with lentiviral-GFP and transplanted on the 9th day following spinal cord injury with different dosage/combination into injured cord.
Olfactory epithelium enzymatic digested to yield cells. These cells were cultured and Globose basal stem cells (GBC) were isolated by GBC III antibody and characterized immunohistochemically and flow cytometry methods for expression of neural stem cell marker (nestin, SOX2, NCAM), bone marrow MSC marker (CD90, CD54, CD29,CD105, CD73) and haematopoietic marker (CD45, CD34). The result showed that GBC has the properties of both neural stem cell and mesenchymal stem cell. In addition, GBC formed neurosphere in culture condition, which is the characteristic of neural stem cell in brain. These cells were neuronally induced and characterized for neuronal marker (βIII-tubulin, MAP2, NeuN, Neurofilament). The result showed invitro differentiation of GBC into neurons, and its multipotency. So this is considered as an alternative source of transplantation for spinal cord injury. Second passage GBCwere transplanted into spinal cord on day 9 following injury.
Bone marrow collected from rat femur and tibia, and then isolated MSC were cultured. Cultured MSC were characterized for MSC marker (CD90, CD54, CD29, CD105, CD73) and haematopoietic marker (CD45,CD14, CD34) by flow cytometry and IHC to prove purity of MSC, not contaminated with haematopoietic cells. These cells neuronally induced and characterized for positive marker of neurons and glia (MAP2, NF, NeuN,βIII tubulin, O4 and GFAP). Mature neuron express voltage-gated sodium channel, which is the hallmark of functional excitable cells. To address this issue, voltage-gated sodium channel (Nav1.1) expression was seen by IHC and patch-clamp studies was done to prove the existence of gated sodium channel; but only K+ channel was expressed. Multipotent bone marrow mesenchymal stem cells (MSC) were lentiviral GFP labeled and then transplanted on 9th day after spinal cord injury in rat.
Acidic fibroblast growth factor was administered to minimize the initial damage after spinal cord injury and inhibit secondary inflammatory cascade. The results show neuroprotective effects of aFGF.
Hence, it is concluded that the combinatorial treatment for CNS injury, taking into consideration of the therapeutic value of each stem cell type tried in this study, may not provide complete cure as expected. Nevertheless, cell therapy could give a definite relief to the patient suffering from spinal cord injury. Prolonged assessment of motor recovery for a year or so after cell transplantation is beyond the scope of this study.
Importantly; OEC, olfactory epithelial cells can provide an accessible source compared to intra-cranially located neural stem/progenitors for autologous neurotransplantation, eliminating the need for immunosuppression thus reducing GVHD problems. While olfactory bulb derived OECs shows remarkable regenerative potential, but practically olfactory bulb tissue harvesting is invasive for therapeutic autologous transplantation strategies. Bone marrow mesenchymal stem cell can also be isolated less invasively from iliac crest and could be a source of stem cell. Autologous neurotransplantation for spinal cord injury should be preceded by further studies in larger animals models for future clinical practice
