Rationale. For centuries, medications derived from the marijuana plant have been used for therapeutic purposes across numerous cultures. In 1964, the primary psychoactive ingredient in cannabis, delta-9-tetrahydrocannabinol (-9-THC) was defined. This, followed by the discovery of the endocannabinoid system, marked the beginning of comprehensive research into the beneficial exploitation of this system. The cannabis plant contains various other cannabinoids besides -9-THC. Most of the effects of cannabinoid-based therapies are based on the agonistic action of -9-THC through cannabinoid receptors. Alternatively, some of these effects are caused by the actions of other cannabinoids, like cannabidiol, which does not have high affinity for cannabinoid receptors. Like -9-THC, cannabidiol (CBD), the non-psychoactive phytocannabinoid in Cannabis sativa, has been hypothesized to ameliorate adverse effects of -9-THC. Cannabidiol possesses neuroprotective, antiemetic, and anti-inflammatory properties. Sativex, a 1:1 ratio of CBD and -9-THC, is currently an approved medication in Europe for the treatment of conditions such as neuropathic pain, and has been fast tracked by the USFDA for late stage clinical trials for a host of disorders, ranging from epilepsy to irritable bowel disease. Additionally, increasing preclinical evidence demonstrates that treatment with Cannabidiol alone produces efficacy on a variety of nervous system injuries, including neuropathic pain, schizophrenia and anxiety disorders. Furthermore, there is mounting evidence of an “entourage effect” in cannabinoid-based pharmacotherapies. This effect occurs when treatment with a combination of cannabinoids derived from the plant produce more efficacy than treatment with a single cannabinoid (1). As cannabinoid-based treatments continue to develop and clinical data increases, further investigation of the entourage effect is necessary to facilitate the appropriate future treatment regimens for nervous system disorders. Hypotheses. We hypothesized that treatment with the non-psychoactive cannabis compound cannabidiol would be as effective as the psychoactive cannabis compound -9-THC, or a combination of the two, in mitigating neuropathic pain in a mouse model of chemotherapy-induced peripheral neuropathy. We additionally hypothesized that cannabidiol would not affect classic cannabinoid-agonist induced cognitive impairment in rodent models of learning and memory. Methodology. Neuropathic pain was induced by repeated injections of the chemotherapeutic agent Paclitaxel. Mechanical hypersensitivity to Paclitaxel was assessed using the Von Frey assay. Cognition was assessed using three rodent models of learning and memory: 1) Conditional Discrimination, 2) Conditional Discrimination with a reversal component, and 3) Barnes Maze. Results. Cannabidiol was found to be more potent and more effective than -9-THC in attenuating neuropathic pain in a dose dependent manner. Combinations of CBD+-9-THC revealed that lower, ineffective doses of CBD and -9-THC display supra-additive effects when given in combination while higher, individually effective doses exhibit sub-additive effects in combination. Cognitively, no deficits were observed over a range of doses of any cannabinoid tested in the conditional discrimination tasks, although a slight trend was observed in animals administered the synthetic mixed CB1/CB2 agonist WIN55,212-2. In the Barnes Maze task, treatment with -9-THC alone dose-dependently decreased number of entries and total time spent in the target zone. Cannabidiol did not produce any effects in the Barnes Maze alone, nor did it attenuate the effects seen in animals treated with -9-THC alone. Lastly, -9-THC did not affect total distance traveled or average speed, whereas combination treatment increased both locomotor measurements at all but the highest combination dose. Conclusions. The results of these studies indicate that cannabidiol is more potent than -9-THC in attenuating neuropathic pain. Results of cognitive testing indicate subtle impairment in animals treated with -9-THC and WIN55,212-2 that were not reversed by CBD. / Biomedical Sciences

Satellite glial cells (SGC) are laminar cells that wrap completely around the sensory neuron and are responsible for buffering extracellular K+ after neuronal excitation. A decrease in the potassium buffering capacity of SGC has been associated with neuropathic pain (NP) behavior and apoptosis. This dissertation investigated the role of the potassium buffering capacity and apoptosis of trigeminal satellite glial cells (SGC) in the maintenance and development of orofacial NP in rats using in vivo and in vitro methodologies. In vivo endpoints were evaluated after performing chronic constriction injury (CCI) of the infraorbital nerve (IoN). NP signs and behavior were evaluated at 5, 10, 20 40 and 80 hours after injury. We evaluated the potassium buffering capacity of SGC by measuring the intracellular potassium concentration and protein levels and gene expression of the Kir4.1 and the SK3 potassium channels and gap junction protein connexin 43 (Cx43). We evaluated apoptosis endpoints including protein levels and gene expression of apoptotic related proteins bcl-2, caspase 9, caspase 3 and p53. Results indicate that NP signs developed as early as 5 hours after injury. After PNI, SGC responded by increasing their intracellular potassium concentration and by increasing protein levels of Kir4.1, SK3 and Cx43. Nonetheless, this increase in protein levels was not accompanied by an increase in gene expression. Apoptosis results revealed that SGC decreased protein levels and gene expression of anti-apoptotic protein Bcl-2. Using in vitro methodologies, we developed primary trigeminal SGC cultures and evaluated how a decrease in the intracellular potassium concentration modulates apoptosis induced by the mitochondrial and death receptor pathways. SGC depleted of potassium after hypoosmotic shock showed a significant increase in early apoptosis after incubation with mitochondrial pathway apoptotic inducer staurosporine when compared to SGC with normal intracellular concentration. This research has revealed that SGC respond early to PNI by increasing their potassium buffering capacity. We also determined that the mitochondrial apoptotic pathway might be involved in the trigeminal SGC response to PNI. From our in vitro experiments we have revealed that potassium is an important modulator of apoptosis induced by the mitochondrial pathway in cultured trigeminal SGC. / Ph. D.

The aim of this project was to get insight into mass spectrometry based proteomics, get familiarized with novel techniques, and obtain the operating skills with modern Orbitrap mass spectrometers. In order to achieve this, the proteome changes in neuropathic pain responses corresponding to nerve injury side in individual rat’s spinal cord were explored. We focused in protein identification and quantification of the expressed proteins in 3 different set of samples, SNL, Sham and Naive rats.

Aim of Investigation: The neurological manifestations of Fabry’s disease, a rare, X-linked, multisystem disorder caused by alpha-galactosidase A deficiency and globotriosylceramide (Gb3) accumulation, include both peripheral and central nervous system symptoms. Here we evaluated a prospectively recruited cohort of patients with Fabry’s disease for pain, small nerve fiber function, and skin innervation. Methods: 66 patients (31 male and 35 female) were enrolled&#65292;31 patients were on ERT. All patients underwent quantitative sensory testing (QST), electrophysiological examination, and extra- and transcranial Doppler sonography. For pain and mood assessment standardized questionnaires were used. Skin biopsies were performed at the left distal leg in 38 patients for intraepidermal nerve fiber density (IENFD) assessment. Results: Age at examination did not differ significantly between women (40.2+/-16.2 years) and men (38.9+/-13.8; n.s.). 29/31 male and 19/35 female patients complained of acroparesthesias or neuropathic pain. QST abnormalities indicative of small fiber impairment were found in 26/31 male and 28/35 female patients. Electrophysiological examination of large fibers and autonomic fibers revealed pathological findings in 11/31 male and 3/35 female patients. All patients had normal Doppler sonography results. Indicators for depression were present in 14/31 male and 10/35 female patients. 20/31 male and 18/35 female patients had a skin biopsy, the IENFD was significantly reduced in male (2.0+/-2.8 fibers/mm) compared with female patients (6.7 +/- 4.4 fibers/mm). In 10 patients free from neurological symptoms, QST and IENFD abnormalities were still detected. Follow up examination after one year in 12 patients under ERT (2.1+/-1.7 years) showed improvement in some symptoms and in QST and neurophysiology in six patients with normal renal function. 20/35 female patients older than 40 y had concomitant diseases, while none of the 18 younger female patients did. The corresponding radio in male patients was 5/19 (>=40y) and 2/13 (<40y) respectively. Conclusions: Neuropathic pain and sensory deficits of the distal extremities are common in patients with Fabry’s disease. QST and IENFD analysis are important for early diagnosis of nerve involvement in Fabry’s disease. Small fiber function may improve under ERT in patients without severe renal impairment.

Peripheral sensitisation and central sensitisation are implicated in the development of neuropathic pain with neuroplasticity occurring at multiple levels of the pain pathway. Hypersensitivity of the spinothalamic tract has been described in neuropathic animal models of diabetes. Spinal dorsal horn neurones of diabetic rats exhibit abnormally high spontaneous firing, suggesting an imbalance between excitatory and inhibitory signals converging within this structure. GABAergic neurones within the spinal cord and thalamus are crucial for the transmission of painful stimuli to higher centres of the brain that are involved in pain perception. GABAA receptors (GABAARs) are an important target for many clinical drugs, and certain endogenous neurosteroids act as potent allosteric modulators of these receptors. A developmental change in the rate of exponential decay of GABAergic synaptic events has been observed in other types of neurones and this may be related in part to fluctuations in endogenous neurosteroid tone. The objective of this study was to investigate changes to inhibitory neurotransmission with development in three levels of the pain pathway and to explore potential mechanisms underlying diabetic neuropathy. The whole-cell patch-clamp technique was used on slices of neural tissue. Electrophysiological recordings were obtained from wild type mice between the ages of 6 and 80 days in lamina II of the spinal cord, the nucleus reticularis (nRT) of the thalamus and the cerebral cortex. Recordings were also obtained from mice with diabetic neuropathy (ob/ob and db/db) between the ages of 60 and 80 days. Neurosteroids and their precursors were employed along with compounds that prevented their activity at the GABAAR such as ?-cyclodextrin, which is a barrel-shaped cyclic oligosaccharide with a lipophilic interior that sequesters neurosteroids. Behavioural experiments were also performed using von Frey filaments and the tail flick test to examine mechanical and thermal nociception. Recordings from the spinal cord, the thalamus and the cerebral cortex revealed that the decay time of miniature inhibitory postsynaptic currents are significantly reduced with development. The neurosteroids allopregnanolone and ganaxolone were significantly more effective in neurones from the older mice. In contrast, ?-cyclodextrin had significantly less effect in neurones from the older mice. In mature diabetic mice (ob/ob mice), the endogenous neurosteroid tone is reduced compared to control mice, but certain neurosteroid compounds have a greater effect on the GABAARs of these diabetic mice. In addition, the diabetic mice exhibit mechanical allodynia and hyperalgesia, which is responsive to exogenously applied neurosteroids. These results are consistent with the hypothesis that a dramatic reduction in endogenous neurosteroid tone occurs as development progresses and that this impacts on the exponential decay time of GABAergic mIPSCs within neurones of the pain pathway. The higher neurosteroid tone in the youngest mice may confer a degree of neural protection over the nervous system as it develops. The reduction of endogenous neurosteroid tone in diabetic mice may be associated with their hypersensitivity. It is possible that pregnane-derived neurosteroids may exert analgesic effects in pathological pain states by attempting to restore the physiological GABAergic inhibitory tone that is observed in immature animals.

A Bell in the Storm - Persistent unexplained pain and the language of the uncanny in the creative neurophenomenal reference is a doctoral work comprised of three parts. Part 1 is an exegesis Persistent unexplained pain and the language of the uncanny in the creative neurophenomenal reference; Part 2 is The Plays, A Bell in the Storm (produced by deckchair theatre in May, 2005) and the radio play To Fall Without Landing (produced by the Australian Broadcasting Commission for Radio National in October 2005); and, Part 3 the book of monochord poems, Secrets of the Driftwood.

Neuropathic pain is defined as "pain initiated or caused by a primary lesion or dysfunction in the nervous system". In contrast to physiological pain that warns of noxious stimuli likely to result in tissue damage, neuropathic pain serves no protective function. Examples of neuropathic pain states include postherpetic neuralgia (shingles) and phantom limb / stump pain. This pain state also exists in the orofacial region, with the possibility of several variants including atypical odontalgia and burning mouth syndrome. There is a paucity of information on the prevalence of neuropathic pain in the orofacial region. One study assessed patients following endodontic treatment and found that approximately 3 to 6percent of patients reported persistent pain. Patients predisposed to the condition atypical odontalgia (phantom tooth pain) include those suffering from recurrent cluster or migraine headaches. Biochemical and neurobiological processes leading to a neuropathic pain state are complex and involve peripheral sensitisation, and neuronal plasticity of the central and peripheral nervous systems. Subsequent associated pathophysiology includes regional muscle spasm, sympathetic hyperfunction, and centralisation of pain. The relevant clinical features of neuropathic pain are: (i) precipitating factors such as trauma or disease (infection), (ii) pain that is frequently described as having burning, paroxysmal, and lancinating or sharp qualities, and (iii) physical examination may indicate hyperalgesia, allodynia and sympathetic hyperfunction. The typical patient complains of persistent, severe pain, yet there are no clearly identifiable clinical or radiographic abnormalities. Often, due to the chronicity of the problem, afflicted patients exhibit significant distress and are poor pain historians, thus complicating the clinician's task of obtaining a detailed and relevant clinical and psychosocial history. An appropriate analgetic blockade test for intraoral sites of neuropathic pain is mucosal application of topical anaesthetics. Other, more specific, tests include placebo controlled lignocaine infusions for assessing neuropathic pain, and placebo controlled phentolamine infusions for sympathetically maintained pain. The treatment and management of neuropathic pain is multidisciplinary. Medication rationalisation utilises first-line antineuropathic drugs including tricyclic antidepressants, and possibly an anticonvulsant. Topical applications of capsaicin to the gingivae and oral mucosa are a simple and effective treatment. Neuropathic pain responds poorly to opioid medication. Psychological assessment is often crucial in developing strategies for pain management. Psychological variables include distress, depression, expectations of treatment, motivation to improve, and background environmental factors. To enable a greater understanding of neuropathic pain, thereby leading to improved treatments, high-performance liquid chromatography-mass spectrometry is one analytical technique that has the potential to contribute to our knowledge base. This technique allows drugs and endogenous substances to be assayed from one sample in a relatively short time. The technique can identify, confirm, and measure the concentrations of multiple analytes from a single sample.

Neuronal cell death may be responsible for the pathogenesis of neuropathic pain. Glial cell line-derived neurotrophic factor (GDNF) protects sensory neurons after injury and offers a promising alternative for the management of intractable pain. However, continuous administration of trophic factors into the central nervous system is costly and difficult to maintain. Therefore, we evaluated the potential of intrathecal GDNF gene delivery for the treatment of neuropathic pain. Recombinant adenovirus encoding GDNF (Ad-GDNF) was characterized and shown to enhance viability of neuronal cultures. After intrathecal injection of Ad-GDNF, an elevated GDNF level was observed in spinal cord for four weeks. In rats with sciatic nerve axotomy,intrathecal injection of Ad-GDNF significantly ameliorated the duration of neuropathic pain. However, animals treated with Ad-GDNF developed hyperalgesia in the early stage of treatment. Immunofluorescence analysis indicated that intrathecal GDNF gene delivery prominently attenuated the neuronal loss due to nerve injury. Unexpectedly, varying degrees of hair loss was found in some rats receiving Ad-GDNF. Histological analysis revealed that hair loss resulted from severe degeneration of hair follicles in skin from Ad-GDNF-treated animals. In summary, the present study demonstrate the feasibility and limitations of GDNF gene delivery for the management of neuropathic pain.

The use of gene-based techniques to produce antinociceptive molecules has been actively investigated for treatment of neuropathic pain and trauma of central nervous system. Among the endogenous opioids, b-endorphin (b-EP) is the most potent one, which is derived from pro-opiomelanocortin (POMC). In addition to b-endorphin, POMC is also the precursor of many neuropeptides such as adrenocorticotropin hormone (ACTH), melanocyte-stimulating hormone (a-MSH), ¡Ketc. Appropriate administration of POMC gene is essential for the success of its clinical application. Thus, gene transfer approach seems to be suitable for continuous supply of b-endorphin to alleviate intractable pain. Recombinant adenovirus was used as gene delivery system for POMC because of its high titer, wide host range, and transduction efficiency. In the present study, we have generated and characterized the recombinant adenovirus encoding POMC (Ad-POMC) by PCR and western blot analysis, and detect the presence of opioid peptides including ACTH, a-MSH and b-EP by RIA and chemilluminiscent assay. GH3 cells infected with Ad-POMC showed significantly higher levels of ACTH, b-endorphin, and a¡VMSH comparing with cells of control groups. By using Ad-GFP, the optimal MOI for adenovirus vector to infect neuronal GH3 cells, glial C6 cells, hepatoma Hep3B cells, smooth muscle G8 cells, fibroblast CCD-965K cells, and endothelial EA.hy926 cells was determined at 50, 500, 50, 500, 500, and 200, respectively. The results of determining the efficiency of POMC processing in different types of cells after in vitro cell cultures gene delivery indicated that peripheral cells, though at a lower extent, are capable of cleaving POMC and releasing opioid peptides after POMC gene delivery like neuronal cells of central nervous system. In formalin test, the intrathecal POMC gene delivery significantly decreased the magnitude of the formalin-evoked flinching response phase 1 (P < 0.05) and phase 2 (P < 0.001) when compared with rats receiving saline or Ad-GFP. In conclusion, the intrathecal POMC gene delivery can produce effectively attenuation on the inflammatory pain response. So far, there have been various gene delivery studies confirming the potential role of POMC in antinociception. In the future, more experiments will be needed to characterize the effects of POMC expression on cellular lipid metabolism. This will enable us to evaluate the therapeutic potential of POMC on treatment of obesity.

Neuropathic pain is a prevalent disease with no effective, safe treatments and limited knowledge on the mechanisms involved. One target for neuropathic pain treatment may be the blockade of Dynorphin A (Dyn A). Dyn A is a unique endogenous ligand that possesses well-known neuroinhibitory effects via opioid receptors and neuroexcitatory effects that are mediated through the bradykinin 2 receptors (B2Rs). Extensive SAR was carried out to develop a ligand for the blockade of the excitatory actions of Dyn A at the B2R. A lead ligand was able to block Dyn A-induced hyperalgesia in naïve animals and was effective in a neuropathic pain model. However, the ligand was susceptible to enzymatic degradation. In an effort to increase the stability, modifications of the ligand using non-natural amino acids were performed. Analogues substituted at or near the N-terminus with a D-isomer retained binding at the receptor as well as provided a large increase in stability. These ligands were also found to be non-toxic in a cell toxicity assay. Dyn A has been found to not activate the classical signaling of the B2R, PI hydrolysis or Ca²⁺ mobilization. In an effort to determine Dyn A's signaling, a study was done examining up-regulation of phosphorylated proteins. It was found that Dyn A did not activate; pERK, 7 PKC isoforms or PKA. A well known B2R antagonist, HOE140, was found to have low affinity at rat and guinea pig brain B2Rs but high affinity in the guinea pig ileum. Further examination revealed that this discrepancy in binding may arise from a different isoform of the B2R that has not been previously examined. To date, we have discovered Dyn A analogues that have high affinity for the B2R, are very stable, and have low toxicity. The signaling pathway is still not fully understood, but further studies are underway. Also, there is evidence that the B2R in which the analogues are interacting at may be a different form than what has previously been described. Targeting this different isoform of the B2R with our current stable ligands may provide beneficial therapeutics for the treatment of neuropathic pain without the cardiovascular liabilities.