Investigations into long tract function following spinal cord injury and cell transplant therapy

Traumatic spinal cord injury (SCI) leads to severe functional deficits for which there are currently no effective treatments. About 50% of SCIs are incomplete leaving varying numbers of spared axons intact whilst damaging the cells which ensheathe them. These spared fibres provide targets for therapeutic interventions which aim to maximise their potential for supporting residual functions. In preclinical studies, functional outcomes are most commonly assessed using behavioural approaches. However they are unable to provide information on the mechanisms of recovery or differentiate between mechanisms occurring in the spinal cord and compensatory mechanisms occurring in the brain. This study had two main aims: firstly to develop an electrophysiological protocol for assessing transmission in the ascending dorsal column pathway, to use this protocol to characterise the effects of contusion injuries of different severities and to investigate the time course of changes to long tract function following SCI. The second aim of this project was to use this protocol combined with behavioural testing to investigate the use of human lamina-propria mesenchymal stem cells (hLP-MSCs) as a potential therapy for spinal cord injury. An electrophysiological approach was used to investigate function in rats subjected to T9 contusion injuries of the dorsal columns. Changes in the function of this pathway were assessed by recording sensory evoked potentials (SEPs) from the surface of the exposed somatosensory cortex, following stimulation of the contralateral sciatic nerve. Functional effects of increasing injury severities were investigated in normal animals and animals 6 weeks after receiving contusion injuries of increasing severity. Maximum SEP amplitudes and isopotential plot areas were reduced with injury severity, and latency to sciatic SEP onset was seen to increase in a graded fashion with increasing injury severity. SEP mapping revealed that the region of cortex from which SEPs could be recorded at or greater than certain amplitudes remained focused in the same location with increasing injury severity. Animals were investigated at different time points from acute up to 6 months post injury. Acute investigation revealed that sciatic SEPs are ablated immediately following injury and after incomplete recovery stabilise within hours of injury. Maximum sciatic SEP amplitudes and cortical areas both show 2 phases of recovery: One at 2 weeks post injury and one at 6 months. Onset latencies are seen to increase initially before gradually returning nearer to normal levels by 6 months. SEP mapping revealed that the region of cortex from which SEPs could be recorded at or greater than certain amplitudes remained focused in the same location with increasing post injury survival time. Histological observations confirmed that the injury causes substantial damage to the dorsal columns. To assess the effects of potential therapeutic hLP-MSC transplants, the functional effects of T9 150 Kdyn contusion injuries were investigated in medium injected controls and 3 week delayed hLP-MSC transplanted Sprague Dawley rats, at 10 weeks post injury. Behavioural testing was performed throughout, with terminal electrophysiological and immunohistological investigations performed at the end of the study. Animals were behaviourally tested at pre- and post-operative time points for the duration of the experiment. Electrophysiological recordings suggest some recovery of function with time after injury. Two phases of recovery are seen, one at about 2 weeks after injury and the other at about 6 months after injury; however other measurements suggest hLP-MSC transplants had little or no effect on the functional integrity of the dorsal column pathway. Open field locomotor testing using the Basso, Beattie and Bresnahan (BBB) locomotor scale revealed no differences between the recoveries of cell transplanted and control groups. Gait analysis was performed using the Digigait™ Imaging System revealing a trend for earlier recovery of co-ordination between forelimbs and hindlimbs in hLP-MSC transplanted animals compared to control animals. Moreover the step sequence data also suggested a better recovery of co-ordinated stepping in transplanted compared to medium injected animals. Dynamic weight bearing apparatus (BIOSEB) was used to measure the percentage of body weight borne on the forepaws and hindpaws, this demonstrated no effect of transplanted cells on postural changes. hLP-MSC transplants did not increase indicators of neuropathic pain in our model suggesting they are unlikely to exacerbate neuropathic pain following spinal cord injury. At present there are no immunohistochemical (IHC) markers that can be used to differentiate axons which have been remyelinated with central-type myelination from those which survived the injury. Thus, the degree of peripheral-type myelination was investigated as a simple way of assessing remyelination. This suggested that there was a greater degree of remyelination in transplanted animals, and that this was specifically in areas where transplanted cells were located.