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This thesis sought to investigate the role of orexin receptors in cardiovascular control, and to determine the role ofthe orexin system in essential hypertension.Immunolabelling revealed that most orexin neurons express orexin receptors (OxR1 and OxR2) themselves, with65% co-expressing both receptors, whereas sympathetic preganglionic neurons of the intermediolateral columnwere only seen to express OxR1. This suggests that much of the actions of orexin receptor antagonists may be on theorexin neurons themselves, which would potentially reduce all the orexin mediated components of a response atthe same time.Orexin levels in the CSF were shown to be tonically upregulated in the adult spontaneously hypertensive rat (SHR)model of essential hypertension, as compared to normotensive Wistar Kyoto (WKY) and Wistar rats. Using orexinimmunolabelling, we determined that the previously reported increased expression of orexin neurons in the medialhypothalamus is present in pre-hypertensive juvenile SHRs, and therefore precedes the hypertensive state in theseanimals. We also discovered a significantly increased orexin neuron population in brains of the BPH/2J Schlagermouse, another animal model of essential hypertension. This suggests that orexin upregulation may be an indicator,if not a precursor to essential hypertension across multiple mammalian species.Both destruction of medial hypothalamic neurons through vivo-morpholinos and targeted rAAV-delivered shRNAknockdown of orexin within this region were successful in significantly and permanently attenuating hypertensionin the adult SHR. Similar injections had no effect on resting blood pressure in normotensive WKY rats. The consistencyof the reduction seen in both experiments suggests that orexin is the primary source of the hypertensive toneproduced from this region in the SHR.Thus, this thesis has identified potential pathways for the cardiovascular actions of orexin receptor antagonists,characterised an overactive and upregul

Neuropathic pain occurring as a consequence of a disease or lesion in the somatosensory nervous system is devastating and refractory to current treatments. Neuropathic pain negatively affects the quality of life of patients and causes an economic burden to the sufferers and the global economy. Thus, there is an overwhelming need to investigate the underlying mechanisms and develop novel treatment approaches. Over the past decade, it has become clear that neuroinflammation plays a critical role in the pathogenesis of neuropathic pain. Indeed, the contribution of leukocytes such as T cells and macrophages, immune-like glial cells such as microglia and astrocytes, and inflammatory cytokines have been implicated in neuropathic pain caused by nervous system injury. Modification of such neuroinflammatory responses via immunomodulation is a potential novel therapeutic approach for the treatment of neuropathic pain.Myelin-derived altered peptide ligands (APLs) generated by transforming the amino acid sequence of native myelin peptides are known to modify immune and inflammatory responses, and are able to inhibit antigen-specific T cell function. APLs have demonstrated promising therapeutic effects in several in vivo and in vitro assays by changing the activation and functions of immune cells, and altering their cytokine profiles. In this thesis, we found that immunisation with the native myelin basic protein (MBP) peptide produced pain hypersensitivity and neuroinflammation in uninjured animals and that co-immunisation with the relevant APL reduced these effects. Next, we showed that immunisation with MBP-derived APL in animals with peripheral nerve injury significantly attenuated pain hypersensitivity and suppressed neuroinflammation, as well as altered some systemic immune responses. However, the same immunisation strategy failed to exert an analgesic effect following spinal cord injury. Lastly, the effects of gene therapy to introduce the anti-inflammatory cytokine inter

In recent times, oxidative stress has been identified as a major instigator of numerous conditions of aging and inflammation. The retina is subject to high levels of oxidative stress due to its high oxygen per weight requirements and perpetual exposure to light stress. Several diseases, such as macular degeneration and diabetic retinopathy are positively identified as having an oxidative stress pathophysiological component. These conditions are the leading causes of blindness in the first world adult population, thus representing significant morbidity to both economy and society. The retina has endogenous protection mechanisms that provide a degree of protection against common insults. One such mechanism involves induction of changes to the trans-retinal Müller cell. In response to certain physical or biological stimuli, the Müller cell becomes reactive, resulting in phenotypic changes such as increased release of growth factors, changes in extracellular fluid and ion uptake or proliferation that in certain contexts may limit retinal damage. However, in other contexts, such a response may be inappropriate, exacerbating retinal damage or disease. In order to understand and perhaps control Müller cell reactive expression, an unprecedented look at the early signalling cascades in response to prototypical oxidative stress agent, hydrogen peroxide, was undertaken. Conditions whereby hydrogen peroxide induced reactive Müller cell migration, proliferation and hypertrophy were optimized. Using these conditions, assay of the expression of select major signal transducing entities was performed in the presence and absence of inhibitors. This method identified hydrogen peroxide as enhancing ADAM-17 activity and tyrosine kinase receptors such as the epidermal growth factor receptor. Transduction of the hydrogen peroxide stimulated signal caused activation of ERK/MAPK and PI3K/Akt signalling pathways with subsequent induction of Egr1 expression and activity. Concomitantly, the ER

Conditioned fear to a tone is associated with disinhibition of brainstem-projecting neurons in the central nucleus of the amygdala (CeA). This is thought to trigger freezing via projections to the ventrolateral periaqueductal gray (VLPAG). However, the proportion of these projections containing GABA and glutamate is unknown. Using in situ hybridisation for glutamic acid decarboxylase (GAD67) and vesicular glutamate transporters (VGLUT1, VGLUT2) combined with retrograde labelling from the VLPAG, we showed that 90% of VLPAG-projecting CeA neurons contained GAD67, and none contained VGLUT1 or VGLUT2. Next, by combining Fos immunohistochemistry with retrograde labelling from the VLPAG, we found that sustained re-exposure to a feared context induced Fos expression in only 0.7% of VLPAG-projecting CeA neurons. We then attempted to selectively activate neurons in the medial CeA with orexin-A in freely moving rats, whilst recording cardiovascular and behavioural response. Orexin was associated with enhanced heart rate (HR), mean arterial pressure (MAP), activity, tail temperature and arousal. Next, we administered the same treatment to rats before sustained re-exposure to a fear-conditioned context. We showed that orexin prevented normal within-trial reductions in freezing, but had no effect on HR or MAP responses, activity or total time spent freezing. In a follow up re-exposure, rats previously injected with orexin were treated with saline. These rats did not show any sign of previous extinction learning, and spent more time freezing than rats receiving saline in the first re-exposure. Conversely, rats injected with orexin which were previously injected with saline displayed low levels of freezing. Finally, we sought to determine the behavioural and cardiovascular effects of microinjection of orexin-A in the VLPAG, an important structure in passive coping responses. We showed that orexin enhanced arousal, activity, HR, MAP and tail temperature. These findings support the

This thesis attempted to identify the site(s) of action of orexin and 5HT1A receptor agonists in the central network thatmediate/modulate the cardiovascular and behavioural changes evoked by psychological and physical stress.Central blockade of orexin receptors with Almorexant in the lateral ventricle significantly reduced the locomotor response toNovelty stress but did not significantly reduce the associated cardiovascular response. Comparison of the effect of orexin-A inthe lateral ventricle, fourth ventricle and thoracic cord showed that orexin injections in the upper thoracic cord could evokesubstantial cardiovascular effects, comparable to those evoked from the fourth and lateral ventricle, suggesting that orexin couldact at spinal level to mediate the cardiovascular effects of stress. This was confirmed by showing high levels of Fos expression inorexin neurons retrogradely labelled from the upper thoracic cord after Fear, Novelty and Exercise. Finally, orexin-A in themedullary raphe evoked clear cardiovascular effects, indicating that the medullary raphe could also be an important supraspinalsite of action of orexin in the cardiovascular response to stress.The 5HT1A receptor agonist 8-OH-DPAT evoked greater reduction in the cardiovascular response to Novelty, Restraint, Fear andCold after injection in the fourth ventricle than after injections in the lateral ventricle. This indicates that the lower brainstem islikely the main site of action of 8-OH-DPAT for the cardiovascular component of these stressors, suggesting a directsympathoinhibitory effect. However, the reduction of the freezing response evoked by Fear was greater after lateral ventricle thanfourth ventricle injections, suggesting an anxiolytic effect in the forebrain and/or midbrain region.Thus, this thesis has identified the most probable site(s) of action of orexin and 5HT1A agonists for the cardiovascular responseto stress. Orexin is likely to have a direct action in the upper thoracic cord toget

Disorders of the autonomic nervous system constitute serious complications of spinal cord injury (SCI) and their treatment is usually highly prioritised by spinal patients. Among these, autonomic dysreflexia and impaired thermoregulation are potentially life threatening conditions and require effective management. Olfactory ensheathing cells (OECs), progenitor cells and polymeric scaffolds have been tested in animal models of SCI and some of them have been considered for clinical trials. However, evaluation of the effect of such interventions on autonomic functions has received only rudimentary attention and would require a more thorough experimental assessment before the methods are utilised in human patients. This thesis tested two potential therapeutic strategies for autonomic dysreflexia and examined disorders of thermoregulatory functions in a rat model of spinal cord transection. Magnitude and duration of autonomic dysreflexia were evaluated with radio telemetry in spinalised animals treated with (i) implants of OECs and olfactory neurosphere-derived cells seeded in poly(lactic co glycolic) porous scaffolds or with (ii) transplants of OECs alone. (iii) Effects of SCI and of OECs on the morphology of sympathetic preganglionic neurons (SPNs; which are involved in pathogenesis of autonomic dysreflexia) stained for NADPH diaphorase were examined. (iv) Doppler ultrasonography and infrared thermography were used to assess responses of tail blood flow and surface temperature to cold.Transplants of OECs alone, but not in combination with olfactory neurosphere-derived cells and polymeric scaffolds, resulted in significantly shortened episodes of autonomic dysreflexia. This may be attributed to the alterations to the morphology of SPNs adjacent to the lesion: a transient increase in the morphometric features of the SPNs was evoked by spinal cord transection and this was further altered by transplantation of OECs. The thesis also showed that local responses of tail blood