No description available.

No description available.

No description available.

No description available.

This thesis studies sensory innervation in mammals using an organotypic co-culture of spinal cord-dorsal root ganglion and skeletal muscle of embryonic rat, the histological changes of reinnervated muscle spindles after nerve section and the localisation of the calcium-binding protein calretinin in cat mechanoreceptor organs. The immediate importance of this project concerns the better understanding of how the normal process of development differs from reinnervation following nerve lesion or section. A range of classical and well defined materials and methods as been used in the work described. The thesis Is divided into Ove chapters: Chapter 1 reviews aspects of the mechanosensory organs which have been studied experimentally in relation to their sensory innervation, including proprioceptive muscle spindle development, reinnervation, and finally, the presence of the calcium-binding protein, calretinin in the mechanoreceptor organs. This provides an introduction and background to the work. Chapter 2 describes the organotypic organisation of spinal-cord, dorsal-root ganglia and skeletal muscle co-culture in vitro. Results show that slices of the spinal-cord, dorsal- root ganglia survive well under experimental conditions and can live for several weeks with feeding every 1-3 days. Sensory neurons can develop and grow in a medium without any additional promoting factor. The presence of structurally identifiable synapses indicates that other neurons are also maintained in culture and have functional connections. In the organotypic culture new muscle fibres can form either from the original explant or from the additional explant. In chapter 3 I describe two abnormal endings present in spindles of the tenuissimus of the cat that had been reinnervated following section of the nerve more than one year previously. The reconstruction of the endings of these two spindles supports the hypothesis of modulation of the primary-ending response by the mechanical properties of the intrafusal muscle fibres, rather than by intrinsic properties of the la afferent itself. They further indicate that, in the absence of a la afferent, intrafusal-fibre differentiation can be maintained by a group II afferent. Chapter 4 concerns the localisation of the calcium-binding protein calretinin, which was studied immunohistochemically in the abductor digiti quinti medius muscle of the cat hind limb. The calretinin immunoreactivity was found in some intrafusal fibres, the primary endings and the cqjsule of the muscle spindles and the sensory terminals of tendon organs and Paciniform corpuscles. The present findings contradict a recent hypothesis that calretinin is associated with rapid adaptation, but suggest that calretinin has a specific function in muscle proprioceptors. Finally, Chapter 5 outlines the conclusions of this study and gives some suggestions for continuation of the work in the future.

No description available.

The olfactory system has the remarkable capacity for neurogenesis following injury. However, the molecular mechanisms important for reinnervation of the olfactory bulb (OB) remain unknown. The matrix metalloproteinases (MMPs) are important components in many central nervous system (CNS) injury paradigms, yet remain unexplored in olfactory injury and recovery. To address the role of MMPs, the temporal expressions of MMP-9 and MMP-2 were examined in 3 olfactory injury models: nerve transection (NTx), methyl bromide gas (MeBr) exposure, and nerve transection with Teflon barrier (NTx-TB). Each injury model represents a different degree of olfactory injury and neuronal recovery. In NTx, sensory axons are lesioned, leading to neuronal degeneration and subsequent reinnervation of the OB. MeBr exposure damages the cell bodies of sensory neurons in the peripheral olfactory epithelium (OE), leading to degeneration and reinnervation of the OB without direct trauma to the OB. In NTx-TB, sensory axons are lesioned and a barrier is inserted following injury that blocks regenerated neurons from reinnervation of the OB. Following NTx, MMP-9 increased immediately in the OB and was localized to neutrophils, an inflammatory leukocyte. The elevated levels of MMP-9 corresponded to neuronal degeneration. To confirm this relationship, MMP-9 expression was measured following MeBr injury. MMP-9 increased during neuronal degeneration in the OB and was localized to neutrophils in the area of sensory axon degradation. These experiments demonstrated that MMP-9 is important for both neuronal degeneration and the acute inflammatory response following olfactory injury. In NTx injury, MMP-2 expression peaked at day 7 and corresponded to the transition between degeneration and reinnervation of the OB. MMP-2 was localized to the granule cell and external plexiform layers in control and day 7 bulbs. Following NTx-TB, MMP-2 remained low and was not expressed by regenerated axons. The absence of a MMP-2 peak in the NTx-TB injury suggests that this peak depends on reinnervation of the OB. This study demonstrates a temporal correlation between MMP-9 and degeneration and MMP-2 and reinnervation following olfactory injury. These findings provide new insight into the molecular mechanisms underlying olfactory nerve injury. Modulation of MMPs could provide novel therapeutic interventions to improve neuronal recovery following injury.

Unlike the peripheral nervous system (PNS), adult neurons in the central nervous system (CNS) have limited regenerative capacity after injury. One interesting phenomenon observed nearly four decades ago was that lesion of a peripheral nerve can significantly enhance the regenerative capacity of the central axons of the corresponding dorsal root ganglion (DRG) neurons, termed a ‘conditioning lesion’, but the underlying mechanism is still not fully understood. Since ATP is released after nerve injury and extracellular ATP has a broad range of biological activities, we postulated that ATP might be the injury signalling molecule that triggers the regenerative machinery in the injured neurons. If that were the case, injection of ATP into a peripheral nerve should be able to mimic the effect of a conditioning lesion. To test this theory, we injected ATP into a peripheral (sciatic) nerve after a dorsal column transection and found that ATP injection did promote the regeneration of injured axons into the lesion cavity. We also found that ATP injection activated transcription factor STAT3 and increased the expression of growth associated protein 43 (GAP43) in the corresponding DRG neurons. ATP injection increased the concentrations of ciliary neurotrophic factor and interleukin-6 in sciatic nerve and DRG. These results indicate that intraneural injection of ATP can mimic conditioning lesion to a certain degree. Most interestingly, we found that a second injection of ATP one week after the first one markedly boosted the effects of the first injection as many more axons grew into or across the lesion compared with double saline injection or ATP plus saline injection. Double ATP injection is also more effective in sustaining the expression of phospho- STAT3 and GAP43. Immunohistochemical analysis showed ATP injection caused little Wallerian degeneration at the injection site. Behavioural tests showed no long-term adverse effects to the injected sciatic nerve. In order to explore the underlying mechanism of ATP induced elevation of the regeneration state of DRG neurons and look for more potent purinoceptor agonists to stimulate axonal regeneration, we first tried to identify the expression of purinoceptor subtypes in sciatic nerves using quantitative PCR and immunohistochemistry. We found that mRNAs for all the four P1 and fourteen P2 purinoceptor subtypes were expressed in the sciatic nerve, DRG or dissociated Schwann cells at various levels. Immunohistochemical analysis showed that purinoceptor subtypes are expressed by different types of cells. Due to the expression of nearly all purinoceptor subtypes in the sciatic nerve, it will be a big challenge to identify the receptor subtype(s) responsible for ATP induced axonal regeneration. We have set up a compartmented co-culture system to test various agonists/antagonists of purinoceptors. Taken together, we have shown that intraneural ATP injection can mimic conditioning lesion in promoting sensory axonal regeneration. Identification of the receptor subtype(s) and other molecules involved in the enhanced regeneration capacity of injured neurons may lead to the development of therapeutic agents to effectively promote the axonal regeneration of both peripheral and central neurons.

No description available.

Spinal root avulsion leads to paralysis and loss of sensory function. Surgical methods can improve motor function and ameliorate pain but sensory recovery in adults is poor. Previous studies have shown that cell transplantation or treatment with trophic factors can improve functional outcome in rodents after dorsal root transection or crush. Here, a dorsal root injury model, more similar to human avulsion injuries, was used. The aims of this thesis were to investigate the behaviour of different stem cells following transplantation to avulsed dorsal roots and asses their potential to serve as possible regenerative therapy. In paper I, different murine stem cell types were transplanted to avulsed dorsal roots in rats. Murine embryonic stem cells remained outside the spinal cord and were surrounded by glutamatergic terminals. Boundary cap neural crest stem cells (bNCSC) formed elongated bands outside the spinal cord and migrated to the spinal cord as single cells. In paper II, transplanted bNCSC were further characterized. bNCSC remaining outside the spinal cord expressed glial markers and were associated with different types of sensory fibres. bNCSC that migrated into the injured spinal cord expressed different neuronal markers. In paper III, effects of bNCSC transplantation on local vasculature and glial scar formation were studied. bNCSC increase angiogenesis in a non dose response manner and participate in boundary glial scar formation. In paper IV, bNCSC spinal migration was analysed using two different injury models - dorsal root transection and dorsal root avulsion. In addition, bNCSC capacity to support sensory regeneration was assessed and the results suggest that bNCSC do not support robust regeneration of avulsed afferents. In paper V, an in vitro stem cell model system was used to assess the possibility of using artificial nanomaterials to deliver differentiation factors. Cells treated with either soluble factors or particle-delivered factors showed similar differentiation patterns. Stem cell transplantation offers several opportunities following dorsal root avulsion, including cell replacement and regenerative support. By elucidating the mechanisms by which stem cells can assist regeneration of avulsed afferents will allow for more targeted or combinatorial approaches, including growth factor treatment.