Investigating local circuit function following spinal cord injury and cell transplant therapy

Spinal cord injury (SCI) leads to severe functional deficits and currently there are no effective treatments. In addition to the initial mechanical damage to axons, neurons and glia, traumatic injury also triggers a complex array of cellular and molecular events. Some of these processes are thought to contribute to mechanisms of progressive damage (secondary mechanisms) whilst others concurrently promote limited repair. Functional outcome depends on the interaction of these two processes. However, the interacting dynamics and time course with which they impact the functional outcome is not fully understood. Secondary processes are studied with molecular and anatomical approaches and loss of axons or neurons are used to infer reduced function. Similarly, recovery of functional outcome is mostly assessed using behavioural approaches. However, these are unable to differentiate recovery due to compensatory plastic changes in the brain which might contribute to recovery. Therefore, the first aim of the thesis was to develop an electrophysiological approach to directly assess functional changes in spinal cord circuits following a contusion SCI. The second aim was to use this protocol combined with behavioural testing in order to investigate the use of human embryonic stem cell derived mesenchymal stem cells (hESC-MSCs) as a potential therapy for spinal cord injury. An electrophysiological approach was used to assess spinal cord function after contusion injuries at the cervical (C6) level of the rat spinal cord. Contusion injuries due to high velocity impact were made using an Infinite Horizon Impactor device. Cord dorsum potentials (CDPs) evoked by supramaximal electrical stimulation of a radial nerve (to activate sensory fibres) or within the pyramids (to activate corticospinal fibres) were used to measure changes in the function of these fibre systems in the vicinity of the injury. Animals were investigated at different time points from acute up to 6 months post injury to characterise the temporal progression of changes in spinal cord function. Contusion injury produced an immediate substantial reduction in sensory circuit function which was highly localized to the site of the impact. However, CDPs continued to be evoked both above and below the injury site. This suggests that the main sensory axons in the dorsal column which carry impulses past the level of the injury are not substantially affected and deterioration of function at the injury centre is a result of damage to the neurons and axon collaterals located at the site of impact. Further worsening occurred over the following 3 days but was more marked above the level of the injury possibly because it is due to demyelination of the main sensory axons. However, CDP mapping at 2 weeks showed almost complete recovery of the potentials above this compared to 3 days reflecting a repair mechanism which is possibly remyelination. The fact that CDP amplitudes at 2 weeks are very close to those recorded immediately after injury suggests that most of the loss of function produced by the contusion is due to primary mechanical trauma and that secondary mechanisms contribute relatively little to the loss of function. CDP amplitudes recorded 4 weeks, 7 weeks and 3 months after injury remained closely similar to 2 weeks suggesting a prolonged period of stability in the sensory system. However, a later phase of deterioration is observed at 6 months below the injury possibly due to extension of the injury cavity causing further neuronal loss. Histology also revealed significantly larger cavities in 6 month survival animals. CDPs evoked by stimulation of the corticospinal tract below the injury level were profoundly depressed immediately following a contusion reflecting extensive damage to the main component of the CST in the dorsal columns while potentials above the injury were not acutely affected. There was a further reduction in CDP amplitudes rostral to the injury at 3 days due to demyelination or die back of CST fibres in the dorsal columns. Substantially this improved at 2 weeks and then remained stable up to 6 months. Below the injury there was a gradual increase in the strength of corticospinal connections evident by an increase in CDP amplitudes at 7 weeks which was maximal by 3 months. This reflects plasticity in the spared corticospinal projection probably those in the lateral component. Mesenchymal stem cells (MSCs) from bone marrow (BM) have been reported to promote repair and some functional improvement in animal models. However, autologous transplantation of BM-MSCs has several disadvantages such as the need for invasive harvesting, variable cell quality and slow proliferation. We have successfully derived MSC-like cells from human embryonic stem cells (hESCs) and have found that these cells show anatomical evidence of repair after transplantation in an animal model of SCI (Mohamad, 2014). Delayed transplants of hESC derived MSCs (hESC-MSCs) were performed in a cervical (C6) contusion (175 kdyn) injury model. These cells were transplanted 3 weeks after injury and immunosuppression was begun two days prior to this and continued for the remainder of the study. Functional outcomes were evaluated behaviourally using grip strength and horizontal ladder walking to assess improvement in forelimb function. In electrophysiology experiments cord dorsum potentials (CDPs) evoked by supra-maximal electrical stimulation of a radial nerve or within the pyramids were used to measure changes in the function of these fibre systems in the vicinity of a spinal cord injury at 7 weeks post-transplant. There was a substantial reduction in the gripping ability immediately after the contusion injury followed by a modest spontaneous recovery. Weekly assessment of the grip strength score before and after transplantation revealed no differences in hESC-MSC transplanted and control groups of animals. Similarly, ladder walk analysis revealed loss of co-ordinated forelimb-hindlimb stepping and increased errors of forepaw placement on the ladder rungs after injury. There was no difference in the recovery of co-ordination or improvement of forelimb function after hESC-MSC transplants compared to control animals. Similarly, terminal electrophysiological experiments showed that the amplitudes of CDPs evoked by radial nerve stimulation were closely similar at all recording locations in both groups of animals. However, corticospinal evoked CDPs showed deterioration of function in the transplanted animals compared to the control group of animals. Histology indicated that hESC-MSCs survived in considerable numbers and were present in all transplanted animals at 7 weeks leading to solid infilling of the injury site and a reduction in injury site extent compared to the medium injected control group of animals. In conclusion, the results in the first part of the thesis suggest that maximum damage after contusion injury occurs at the time of injury and spontaneous recovery of function largely mitigates the small amount of the secondary damage. Therefore, injury due to high velocity impact might not be amenable to neuroprotective therapies. Furthermore, evidence of modest spontaneous plasticity in the spared corticospinal system suggests some compensatory plasticity after the injuries.