The Pathophysiology of Chronic Relapsing Experimental Allergic Encephalomyelitis in the Lewis Rat

Electrophysiological studies were performed in Lewis rats with chronic relapsing experimental allergic encephalomyelitis (EAE) induced by inoculation with guinea-pig spinal cord and adjuvants and treatment with low dose cyclosporin A. During clinical episodes there was conduction failure in the central nervous system (CNS), namely the spinal cord dorsal columns, and in the afferent fibres in the peripheral nervous system (PNS). The following observations indicated that the conduction failure was mainly due to demyelination-induced conduction block: (1) rate-dependent conduction block in the CNS and PNS; (2) temporal dispersion due to slowing of PNS conduction; (3) restoration of PNS conduction by cooling; (4) restoration of CNS conduction by ouabain; (5) previously demonstrated histological evidence of primary demyelination in the dorsal columns, dorsal root ganglia and dorsal roots; and (6) the temporal association of restoration of conduction with remyelination. However, it is likely that CNS and PNS axonal degeneration, which occurs in this disease, also contributed to the conduction failure. In clinical remissions there was restoration of conduction in the CNS and PNS which can be explained by ensheathment/remyelination by oligodendrocytes and Schwann cells, respectively. In most rats during clinical episodes the cerebral somatosensory evoked potential was reduced in amplitude and prolonged in latency, which can be accounted for by demyelination and axonal degeneration in the CNS and PNS components of the afferent pathway. In 2 rats with episodes of EAE, however, this potential was markedly increased in amplitude, which might have been due to demyelination-induced conduction block of descending pathways that normally inhibit synaptic transmission in the afferent pathway. In well-established remission there was residual conduction failure in the CNS and PNS which can be mainly accounted for by axonal degeneration

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

Cortical Plasticity and Behavioral Recovery Following Focal Lesion to Primary Motor Cortex in Adult Rats

Acquired brain injuries, such as ischemic stroke and traumatic brain injury, are the leading causes of physical disabilities. Previously, scientists have shown that damage of the primary motor cortex induced neural plasticity in the premotor area in human and non-human primate studies. Neural plasticity, particularly within the same hemisphere of the lesion (ipsilesional), is thought to contribute to and account for functional recovery. It is not yet known to what extent plasticity mediates recovery and how to take advantage of neural plasticity to maximize the functional outcome. Rodent models are most often used not only for studying the role of motor cortex in motor skill learning but also in neurodegenerative research. To further elucidate the role of adaptive plasticity in the ipsilesional hemisphere during the recovery of upper limb function, we aimed to establish the baseline neural changes after a focal cortical injury. Therefore, we took advantage of two separate cortical motor areas, in the Rattus norvegicus, from which the corticospinal tracts terminate in the motor nuclei of the cervical level spinal cord, controlling upper extremity musculature--the first, a more caudally located subregion of M1, often referred to as the caudal forelimb area (CFA), and the second, a more rostrally located non-primary area, referred to as the rostral forelimb area (RFA). The objective of this dissertation work was to characterize physiological changes in RFA during the complex and lengthy process of recovery using rat models of focal cortical trauma and cortical ischemia restricted to CFA. The results demonstrated that the post-injury cortical plasticity in RFA may play a role in functional recovery. Further, we showed differential effects of rehabilitative training on ipsilesional RFA plasticity after CFA ischemic injury. Extensive physiological changes were evident past rehabilitative training. Thus, neural plasticity in RFA appeared to be dependent both on post-lesion motor experience and time. The dissertation work supports the hypothesis that cortical plasticity within the spared RFA after restrictive damage to CFA mediates use-dependent physiological reorganization, which provides a substrate for sustaining rehabilitation-aided motor functional recovery

Afferent information modulates spinal network activity in vitro and in preclinical animal models

Primary afferents are responsible for the transmission of peripheral sensory information to the spinal cord. Spinal circuits involved in sensory processing and in motor activity are directly modulated by incoming input conveyed by afferent fibres. Current neurorehabilitation exploits primary afferent information to induce plastic changes within lesioned spinal circuitries. Plasticity and neuromodulation promoted by activity-based interventions are suggested to support both the functional recovery of locomotion and pain relief in subjects with sensorimotor disorders. The present study was aimed at assessing spinal modifications mediated by afferent information. At the beginning of my PhD project, I adopted a simplified in vitro model of isolated spinal cord from the newborn rat. In this preparation, dorsal root (DR) fibres were repetitively activated by delivering trains of electrical stimuli. Responses of dorsal sensory-related and ventral motor-related circuits were assessed by extracellular recordings. I demonstrated that electrostimulation protocols able to activate the spinal CPG for locomotion, induced primary afferent hyperexcitability, as well. Thus, evidence of incoming signals in modulating spinal circuits was provided. Furthermore, a robust sensorimotor interplay was reported to take place within the spinal cord. I further investigated hyperexcitability conditions in a new in vivo model of peripheral neuropathic pain. Adult rats underwent a surgical procedure where the common peroneal nerve was crushed using a calibrated nerve clamp (modified spared nerve injury, mSNI). Thus, primary afferents of the common peroneal nerve were activated through the application of a noxious compression, which presumably elicited ectopic activity constitutively generated in the periphery. One week after surgery, animals were classified into two groups, with (mSNI+) and without (mSNI-) tactile hypersensitivity, based on behavioral tests assessing paw withdrawal threshold. Interestingly, the efficiency of the mSNI in inducing tactile hypersensitivity was halved with respect to the classical SNI model. Moreover, mSNI animals with tactile hypersensitivity (mSNI+) showed an extensive neuroinflammation within the dorsal horn, with activated microglia and astrocytes being significantly increased with respect to mSNI animals without tactile hypersensitivity (mSNI-) and to sham-operated animals. Lastly, RGS4 (regulator of G protein signaling 4) was reported to be enhanced in lumbar dorsal root ganglia (DRGs) and dorsal horn ipsilaterally to the lesion in mSNI+ animals. Thus, a new molecular marker was demonstrated to be involved in tactile hypersensitivity in our preclinical model of mSNI. Lastly, we developed a novel in vitro model of newborn rat, where hindlimbs were functionally connected to a partially dissected spinal cord and passively-driven by a robotic device (Bipedal Induced Kinetic Exercise, BIKE). I aimed at studying whether spinal activity was influenced by afferent signals evoked during passive cycling. I first demonstrated that BIKE could actually evoke an afferent feedback from the periphery. Then, I determined that spinal circuitries were differentially affected by training sessions of different duration. On one side, a short exercise session could not directly activate the locomotor CPG, but was able to transiently facilitate an electrically-induced locomotor-like activity. Moreover, no changes in reflex or spontaneous activity of dorsal and ventral networks were promoted by a short training. On the other side, a long BIKE session caused a loss in facilitation of spinal locomotor networks and a depression in the area of motor reflexes. Furthermore, activity in dorsal circuits was long-term enhanced, with a significant increase in both electrically-evoked and spontaneous antidromic discharges. Thus, the persistence of training-mediated effects was different, with spinal locomotor circuits being only transiently modulated, whereas dorsal activity being strongly and stably enhanced. Motoneurons were also affected by a prolonged training, showing a reduction in membrane resistance and an increase in the frequency of post-synaptic currents (PSCs), with both fast- and slow-decaying synaptic inputs being augmented. Changes in synaptic transmission onto the motoneuron were suggested to be responsible for network effects mediated by passive training. In conclusion, I demonstrated that afferent information might induce changes within the spinal cord, involving both neuronal and glial cells. In particular, spinal networks are affected by incoming peripheral signals, which mediate synaptic, cellular and molecular modifications. Moreover, a strong interplay between dorsal and ventral spinal circuits was also reported. A full comprehension of basic mechanisms underlying sensory-mediated spinal plasticity and bidirectional interactions between functionally different spinal networks might lead to the development of neurorehabilitation strategies which simultaneously promote locomotor recovery and pain relief

Multimodal Evaluation of TMS - Induced Somatosensory Plasticity and Behavioral Recovery in Rats With Contusion Spinal Cord Injury

Introduction: Spinal cord injury (SCI) causes partial or complete damage to sensory and motor pathways and induces immediate changes in cortical function. Current rehabilitative strategies do not address this early alteration, therefore impacting the degree of neuroplasticity and subsequent recovery. The following study aims to test if a non-invasive brain stimulation technique such as repetitive transcranial magnetic stimulation (rTMS) is effective in promoting plasticity and rehabilitation, and can be used as an early intervention strategy in a rat model of SCI.Methods: A contusion SCI was induced at segment T9 in adult rats. An rTMS coil was positioned over the brain to deliver high frequency stimulation. Behavior, motor and sensory functions were tested in three groups: SCI rats that received high-frequency (20 Hz) rTMS within 10 min post-injury (acute-TMS; n = 7); SCI rats that received TMS starting 2 weeks post-injury (chronic-TMS; n = 5), and SCI rats that received sham TMS (no-TMS, n = 5). Locomotion was evaluated by the Basso, Beattie, and Bresnahan (BBB) and gridwalk tests. Motor evoked potentials (MEP) were recorded from the forepaw across all groups to measure integrity of motor pathways. Functional MRI (fMRI) responses to contralateral tactile hindlimb stimulation were measured in an 11.7T horizontal bore small-animal scanner.Results: The acute-TMS group demonstrated the fastest improvements in locomotor performance in both the BBB and gridwalk tests compared to chronic and no-TMS groups. MEP responses from forepaw showed significantly greater difference in the inter-peak latency between acute-TMS and no-TMS groups, suggesting increases in motor function. Finally, the acute-TMS group showed increased fMRI-evoked responses to hindlimb stimulation over the right and left hindlimb (LHL) primary somatosensory representations (S1), respectively; the chronic-TMS group showed moderate sensory responses in comparison, and the no-TMS group exhibited the lowest sensory responses to both hindlimbs.Conclusion: The results suggest that rTMS therapy beginning in the acute phase after SCI promotes neuroplasticity and is an effective rehabilitative approach in a rat model of SCI

Insular Cortex: Functional Mapping and Allodynic Behavior in the Rat

The insular cortex is the often forgotten 5th lobe of the brain, and examination of its functional anatomy as well as its role in behavior is still in its infancy. To elucidate these unknown details a complete mapping of the functional anatomy of the caudal granular insular cortex (CGIC), as well as its relative position to other somatosensory maps was undertaken revealing its mislabeling as the parietal ventral region (PV). Using this unprecedented localization of CGIC, targeted lesions revealed its role in the maintenance of long-term allodynia in the chronic constriction injury (CCI) model of neuropathic pain. In addition the efferent outputs were examined using neuroanatomical tract tracing techniques. Using this knowledge as an anatomical blueprint, a possible spinal-cortico-spinal loop was uncovered using laminar multi-unit analysis of the lumbar dorsal horn combined with evoked stimulation and inhibition of sciatic, CGIC, and primary somatosensory cortex (SI). Further, an electrophysiological signature of disinhibition was seen in CGIC two weeks post CCI, which was verified with laminar multi-unit analysis and protein analysis. Acute disinhibition of CGIC mimicked cold allodynia behavior in an operant two-plate temperature discrimination task. These data suggest that disinhibition of CGIC plays a critical role in the maintenance of allodynia following CCI in the rat

Recording of motor and somatosensory evoked potential in an anaesthetised Wistar rat using digital polyrite system

OBJECTIVES: The aim of this article is to explain the detailed methodology to record Motor evoked potential (MEP) and somatosensory evoked potential (SSEP) in adult albino Wistar rat, male (200–250 g) which has not been defined previously. MATERIALS AND METHODS: We have standardised recording of both MEP and SSEP in these rats under anaesthesia on ADI digital polyrite system. RESULTS: Evoked potentials have been widely studied in spinal cord injured patients to estimate the degree of injury and to establish a predictive measure of functional recovery. MEPs and SSEPs, arising from the motor cortex or peripheral nerve and generated either by direct electrical stimulation or by transcranial magnetic stimulation, have been advocated as a reliable indicator of descending and ascending pathway integrity. In the rat brain, there is a physical overlap between the motor and somatosensory cortex. Hence, our objective was to identify the exact area for stimulation in the cortex where we could record maximum response with the application of minimum electrical stimulation. CONCLUSION: The recording of MEP and SSEP together provides a powerful neurological technique to monitor the tracts of the spinal cord

Sodium channel expression in the ventral posterolateral nucleus of the thalamus after peripheral nerve injury

Peripheral nerve injury is known to up-regulate the expression of rapidly-repriming Nav1.3 sodium channel within first-order dorsal root ganglion neurons and second-order dorsal horn nociceptive neurons, but it is not known if pain-processing neurons higher along the neuraxis also undergo changes in sodium channel expression. In this study, we hypothesized that after peripheral nerve injury, third-order neurons in the ventral posterolateral (VPL) nucleus of the thalamus undergo changes in expression of sodium channels. To test this hypothesis, adult male Sprague-Dawley rats underwent chronic constriction injury (CCI) of the sciatic nerve. Ten days after CCI, when allodynia and hyperalgesia were evident, in situ hybridization and immunocytochemical analysis revealed up-regulation of Nav1.3 mRNA, but no changes in expression of Nav1.1, Nav1.2, or Nav1.6 in VPL neurons, and unit recordings demonstrated increased background firing, which persisted after spinal cord transection, and evoked hyperresponsiveness to peripheral stimuli. These results demonstrate that injury to the peripheral nervous system induces alterations in sodium channel expression within higher-order VPL neurons, and suggest that misexpression of the Nav1.3 sodium channel increases the excitability of VPL neurons injury, contributing to neuropathic pain

Response of sensorimotor pathways of the spinal cord to injury and experimental treatments

Many spinal cord injuries (SCI) are incomplete, variable numbers of spared fibres passing the lesion level and supporting some residual function below the injury. One approach to improving function following injury is to develop therapies that maximise the potential of these spared fibres. The aim of the work in this thesis was to investigate the spontaneous plasticity that occurs in spinal cord pathways following injury and then determine whether olfactory ensheathing cell (OEC) transplants or treatment with antibodies blocking the function of the myelin inhibitors Nogo and MAG could enhance this plasticity. An electrophysiological approach was used to investigate these questions in rats which were subjected to a lesion of the dorsal columns at the C4/5 segmental level. This lesion interrupts the main component of the corticospinal tract which descends in the ventromedial dorsal column and also interrupts the ascending collaterals of sensory fibres from forelimb nerves. In this study, changes in the function of both of these pathways were assessed by recording cord dorsum potentials (CDPs) after stimulation in the pyramids (corticospinal activation) or of the radial nerve (sensory fibre activation). To enable plasticity in the corticospinal system to be investigated a method was developed for maximally activating the corticospinal projection on one side of the pyramids, whilst avoiding activation of the opposite pyramid and structures surrounding the pyramids. It was found that this could be achieved by careful positioning of bipolar stimulating electrodes. Before investigating the effect of potential plasticity inducing agents, the degree to which plasticity occurs in the absence of treatments was first assessed in F344 rats. The function of corticospinal and sensory pathways was compared in normal animals, acutely lesioned animals, and at 1 week and 3 months after a dorsal column lesion. Corticospinally-evoked CDPs above the lesion were not altered following an acute lesion but were larger in 1 week and 3 month dorsal column lesioned animals than in normal animals. The increase in amplitude was similar in both lesioned animal groups. This suggests that plasticity occurs at the intact connections formed by corticospinal fibres axotomised more distally, that it occurs within a week of the lesion and persisits for at least 3 months. Corticospinally-evoked CDPs were almost abolished below an acute dorsal column lesion and remained of minimal amplitude 1 week after lesioning. However, there was some recovery of CDPs between 1 week and 3 months. This suggests plasticity either at the connections formed by spared fibres of the minor non-dorsal column components of the corticospinal tract or in propriospinal pathways originating above the lesion. This plasticity has a longer time-course than that at the connections of axotomised fibres above the lesion. Plasticity of the connections formed by larger diameter sensory fibres in the radial nerve was also seen below the level of the dorsal column lesion. This had a similar time course to the plasticity of corticospinal connections above the lesion CDPs being larger both 1 week and 3 months after injury compared to normal animals. A modest enhancement of transmission in both corticospinal and sensory systems therefore occurred following a dorsal column lesion. To investigate whether OEC transplants enhance plasticity after spinal cord injury, OECs were transplanted such that they became distributed within the spinal cord for several mm either above or below the lesion. Electrophysiological methods were then used, as above, to investigate whether transmission in the corticospinal and sensory fibre systems following a dorsal column lesion was improved in transplanted animals compared to 3 month survival animals. However, corticospinal actions rostral to the lesion were not enhanced by OEC transplants above the lesion and sensory transmission caudal to the lesion was not enhanced by cells below the lesion. OEC transplants are therefore unlikely to support recovery by promoting plasticity in the spinal cord after injury. To investigate whether antibodies designed to block the function of the myelin inhibitors Nogo and MAG would enhance plasticity following spinal cord injury, antibodies were delivered intrathecally via implanted osmotic minipumps over a period of six weeks following a dorsal column lesion. Vehicle treated and normal animals were investigated for comparison. Placement of the cannula and/or delivery of vehicle alone was found to have a detrimental effect on corticospinal actions above the lesion when compared to normal animals. Treatment with an anti-Nogo antibody (GSK577548) raised against a human Nogo-A fragment and targeting the amino-Nogo terminal was found to enhance transmission of corticospinal actions both above and below the dorsal column lesion. Corticospinal actions above the lesion were significantly greater than in the vehicle treated controls but did not exceed those in normal animals because of the detrimental effect of the intrathecal cannulae/vehicle treatment. Transmission at the terminals of sensory afferent fibres below the level of the lesion was also enhanced by anti-Nogo treatment. In this case the actions of sensory pathways were significantly greater than those in both vehicle treated and normal animals. The fact that enhanced transmission occurs on the ‘wrong side’ of the lesion to be explained by axonal regeneration and the sensory transmission is enhanced over normal, strongly suggests that anti-Nogo induces plasticity in spinal pathways. In contrast, treatment with the anti-MAG antibody (GSK249320A) had no effect on either corticospinal or sensory-evoked activity in the spinal cord above or below the lesion, CDPs evoked by these pathways being comparable to that in vehicle treated controls. Anti-MAG does not appear to induce plasticity but may have neuroprotective actions which cannot be adequately tested in this lesion model. The results show that both corticospinal and sensory fibre systems show modest spontaneous plasticity following a dorsal column lesion. Plasticity at the terminations of axotomised fibres occurs relatively rapidly (within one week) while plasticity in spared systems occurs more slowly. This spontaneous plasticity does not appear to be enhanced by transplants of OECs, so that improvements in spinal cord function previously demonstrated in transplanted animals are probably due to a neuroprotective mechanism. The results obtained using function blocking antibodies targeting myelin inhibitors suggest that anti-Nogo but not anti-MAG treatment may enhance plasticity in the spinal cord after injury. This observation adds to the accumulating evidence that interfering with Nogo-A signalling may be a useful approach for improving function after spinal cord injury