Neuropathic pain is a tremendous medical problem that afflicts millions. It is also distinct from other pain conditions. It persists in the absence of non-noxious stimuli or in response to formerly innocuous stimuli due to unique neurochemical and neurophysiological changes. These changes include acute excitation of peripheral neurons associated with regeneration of axons near the injury site. The causes of injuries leading to neuropathic pain include viral infection as well as trauma. Recently, a highly specific angiotensin II type 2 receptor (AGTR2) antagonist known as EMA401 showed efficacy as a treatment for postherpetic neuralgia in clinical trials. Together with previous immunohistochemical studies of the effects of EMA401 on in vitro neurite outgrowth and the presence of AGTR2 in rodent and human dorsal root ganglion, it ignited interest in AGTR2 as a pharmacological target for neuropathic pain. However, the role of AGTR2 in the modulation of neuropathic pain is not well understood. Despite previous studies, its anatomical expression in dorsal root ganglion and trigeminal ganglion remains uncertain. Additionally, few mechanisms for its modulation of nociceptive transmission have been extensively elucidated. Finally, differential expression of AGTR2 between mouse and human dorsal root ganglion has not been fully explored, especially given the availability of high-throughput expression data. Therefore, this study attempts to develop understanding of the role of AGTR2 in neuropathic pain through bioinformatic analysis of Gene Expression Omnibus (GEO) microarray data for multiple tissues and RNA Seq data acquired for human DRG.

Cerebral oximetry has been shown to effectively identify declining regional cerebral oxygen saturations (rSO2) in coronary artery bypass graft (CABG) surgery. Prolonged intraoperative cerebral desaturations have been significantly associated with an increased risk of cognitive decline after CABG surgery. We compared conventional CABG to minimally invasive robotic coronary artery bypass surgery (r-CABG) using cerebral oximetry to determine the beneficial effects of the less invasive procedure. A retrospective study of 32 isolated CABG patients were treated for coronary artery disease (CAD) via conventional CABG (n=20) or r-CABG (n=12) with analysis of cerebral oximetry tracings and intraoperative data. Parameters, such as, blood loss, mean arterial pressure (MAP), partial pressure of carbon dioxide (PaCO2), cardiopulmonary bypass (CPB), and diabetes mellitus (DM) were analyzed against the area under the curve (AUC) from the cerebral oximetry tracing, an indicator of rSO2 desaturations. Many of these parameters showed statistical significance (p<0.05) between conventional CABG and r-CABG including a decreased mean AUC in the latter. In conclusion, minimally invasive r-CABG tends to show beneficial effects for patients by reducing the total mean AUC in comparison to conventional CABG, especially in the DM patient.

The most common cancer types have a high likelihood of metastasizing to the bone and can cause cancer-induced bone pain (CIBP). Current therapeutic options do not offer proper management and thus CIBP can severely affect a patient's quality of life. Dysregulation of the excitatory neurotransmitter, glutamate, may be involved in the complex and multifaceted mechanisms of CIBP. Because glutamatergic signaling promotes pain, a local rise in glutamate in the bone-tumor microenvironment may contribute to CIBP. Glutamate levels are regulated in part by the cystine/glutamate antiporter, system xc⁻. System xc⁻ is known to be expressed by many different cancer cell types. It functions by transporting cystine into cells and in return releasing glutamate into the extracellular space. Elevated glutamate levels driven by the upregulated expression of this antiporter may contribute to CIBP. Here we demonstrate that system xc⁻ is expressed on a spontaneously occurring murine mammary tumor cell line (66.1) and that treatment of these cells with the established inhibitor and anti-inflammatory agent, sulfasalazine, decreases glutamate secretion in a time and dose-dependent manner. Furthermore, in a novel model of breast CIBP, systemic sulfasalazine treatment not only reduces glutamate levels within the femur, but also significantly attenuates CIBP behaviors. Studies utilized 66.1 cells implanted into the femur intramedullary space of immunocompetent mice. Measurements of spontaneous and evoked pain were made 7 and 10 days post cancer cell inoculation. Systemic administration of sulfasalazine for 4 days (on days 7-10) significantly reduced spontaneous pain-related behaviors and glutamate in femur extrudate as compared to vehicle treated controls. In summary, we demonstrate that pharmacological inhibition of the system xc⁻ transporter attenuates CIBP related behaviors in mice. These data support a role for system xc⁻ in CIBP and validate it as an analgesic target. Further research is warranted to evaluate the potential repurposing of sulfasalazine as an antinociceptive agent for patients with CIBP.

Asthma is a lung disease which features chronic inflammation. Multiple genetic and environmental factors increase susceptibility and provoke episodes of asthma. However, the mechanisms responsible for asthma development are not well characterized. Although allergy is associated with asthma, it has not been shown to precede or predict asthma. To date, there are no clearly established biomarkers of asthma, reflecting our less adequate understanding of asthma pathobiology. In order to identify a plasma proteomic biomarker as an indicator that plasma constituents are altered early in childhood asthma, this study employed a high-throughput antibody array technique which simultaneously profiled relative expression of 507 proteins in human plasma samples from asthma and non-asthma groups. It was hypothesized that alterations of proteomic profiles are accompanied with asthma development. Out of 444 proteins, 4 proteins (erythropoietin, sGP130, galectin-3, and eotaxin-3) were identified with differential expression between asthma and non-asthma groups. Erythropoietin and sGP130 were validated with quantitative differences, which were consistent in direction with the findings from the antibody array, between two groups after having all 4 proteins assessed by ELISAs. Erythropoietin then was assessed for its biological effects in in vivo and in vitro models. It was hypothesized that EPO has influences on acetylcholine-induced airway resistance in animals and on cytokine production from peripheral blood mononuclear cells. EPO's inhibitory effect on IL-2 production and its excitatory effect on IL-6 production were demonstrated; however, the inhibitory effect of EPO on increases in airway resistance in animals was not evident. The results here suggested that asthma has identifiable components in the circulation; these plasma biomarkers may develop via distinct pathways. The demonstrated EPO's capacity of influencing on cytokine production from human immune cells, together with its systemic involvement in asthma, may reveal new opportunities for therapeutics and insights into pathogenesis of asthma.

Chronic pain has been estimated to impact the economy of the United States by an annual cost of $635 billion per year and to affect approximately 100 million Americans (1). Pain is the primary reason patients seek medical attention yet physicians have few options for therapies and there remains a vast unmet medical need for effective and safe analgesics. Most of the drugs clinically available today either have limited efficacy or a variety of unwanted side effects. Discovery of novel therapeutics has been challenging with scientists struggling to find ways to better translate research from the bench-top to the bedside. One impediment in this process has been differences in preclinical and clinical assessment of pain. Preclinical models have historically relied heavily on evoked or reflexive endpoints in non-verbal animals while clinical measures of pain have the advantage of assessing changes in self-reported pain ratings. It is likely, and data from the studies reported in this dissertation show, that mechanisms that underlie threshold responses to evoked stimuli differ from those mediating affective (i.e., aversive) qualities of pain. A further confound is that many effective analgesics are narcotics that carry risk of addiction. Fear of addiction and possibly misuse for chronic treatment of pain may result in undertreatment in many patients. The most clinically relevant question in the management of pain is whether or not a treatment improves the patient's quality of life. Here, we demonstrate that the aversiveness of ongoing pain can be assessed using motivated behavior (conditioned place preference; CPP) and neurochemical output (in vivo NAc microdialysis). Additionally, we assessed the mechanistic effects of three clinically relevant analgesics. Our results show that: (1) pain relief is rewarding and activates reward circuitry that differs from circuits mediating addictive qualities of opiates, and (2) that drugs that mimic the consequences of engagement of descending inhibitory systems act by increasing spinal norepinephrine (NE) levels. These studies provide much needed information that helps build a platform from which more effective analgesics can be discovered and characterized in the preclinical setting and that may help in the introduction of new therapies for patients.

Pathogenesis of pain in pancreatitis is multifactorial, however little is known about the mechanisms by which inflammation in the pancreas causes pain. Here, we hypothesized that pancreatitis-induced pain is dependent upon sensitization of primary afferents by inflammatory mediators such as nerve growth factor (NGF) and interleukin-6(IL-6) and that such a pain is mediated through ascending pathways via the nucleus gracilis. Inflammation in the pancreas resulted in a significant increase in the levels of NGF in the pancreas. Pre-treatment with an anti-NGF peptibody delayed the development of pancreatitis-induced pain. Double injection of anti-NGF peptibody completely prevented the development of pancreatitis-induced pain. Post-treatment with anti-NGF peptibody significantly decreased the number of abdominal withdrawals, compared to the placebo group. Treatment with TRPV1 antagonists reversed the referred abdominal hypersensitivity. Intrathecal administration of p38 inhibitor blocked the pancreatitis-induced pain within minutes after administration. The levels of the TRPV1 in the pancreas, DRG and the spinal cord were not significantly different in the animals with pancreatitis compared to the controls. Pancreatic inflammation resulted in a significant increase in the levels of IL-6 in the pancreas. Treatment with an IL-6 receptor antagonist blocked the referred abdominal hypersensitivity in animals with pancreatitis after systemic and oral administration. Intrathecal administration of the antagonist did not reduce the number of abdominal withdrawals. The possibility that IL-6 might sensitize TRPV1 channels was tested. Cultured DRG neurons were stimulated with IL-6, IL-6 antagonist or a combination of IL-6 with an antagonist, followed by stimulation with capsaicin. Stimulation of DRG with IL-6 produced a marked increase in the levels of CGRP release; combination of IL-6 with an antagonist returned the levels of CGRP to the control levels. To determine the role of the nucleus gracilis in the pancreatitis-induced pain bilateral cannulation of the nucleus gracilis was performed. Microinjection of CNQX into the nucleus gracilis produced a reversal of pancreatitis-induced pain, suggesting an important role of ascending pathway to the n.gracilis in pancreatic pain transmission.

Glial cell line derived neurotrophic factor (GDNF) and artemin maintain the structural and functional integrity of the adult nervous system and regulate the plasticity of the injured or diseased adult nervous system apparently by interacting with GFRalpha1/RET and GFRalpha3/RET systems.The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies provide limited efficacy due in part to dose-limiting and undesirable side effects. Here we show that chronic infusion of GDNF normalizes nerve injury-induced neurochemical changes and prevents the expression of neuropathic pain. Systemic artemin produces partial to complete normalization of multiple morphological and neurochemical features of the injury state.Damaged axons in the dorsal root of adult mammals rarely regrow into the spinal cord, leading to the permanent loss of sensory function. This continues to be a major unmet clinical challenge relevant to a host of disease and trauma-induced injuries to peripheral nerves. Here we show that systemic artemin restores sensory function, apparently permanently, in an experimental dorsal root injury model in rats, including responses to noxious heat, mechanical and chemical stimuli and sensory input-required proprioceptive responses of placement stabilization, targeting and grasping. These effects are likely to result from successful support of multiple classes of sensory afferents which cross the dorsal root entry zone into the spinal cord and make functional connections with spinal neurons. Delayed artemin treatment defines the "therapeutic window" for artemin application following injury to the nerve roots, indicating that this strategy may ultimately be of clinical benefit.Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes can be achieved with artemin. The damaged axons can re-grow perhaps into their original region of occupation, and make functional connections with spinal neurons, resulting in apparently permanent restoration of the lost sensory function following dorsal root injury. Our present studies provide experimental evidence that the neurotrophic factors GDNF and artemin may serve as clinically viable drugs in treating peripheral nerve injury or other neurodegenerative diseases.

Intrathecal injection of dynorphin or des-Tyr-dynorphin fragments, which do not bind to opioid receptors, produce tactile and thermal hypersensitivity in rodents. The maintenance, but not initiation, of experimental neuropathic pain depends upon pronociceptive effects of elevated levels of spinal dynorphin. Recent findings implicated a direct excitatory action of dynorphin A at bradykinin receptors in vitro. Here, the possibility that the pronociceptive actions of pharmacological dynorphin or of pathological levels of endogenous spinal dynorphin are mediated by interaction with bradykinin receptors was explored.While spinal administration of a wide range of bradykinin did not produce hyperalgesia in rats, intrathecal injection of non-opioid des-tyrosyl-dynorphin A(2-13) produced reversible tactile and thermal hypersensitivities that were reversed by bradykinin receptor antagonists. Dynorphin-induced behavioral hyperesthesias were observed in bradykinin B2 receptor wild-type but not in B2 receptor knockout mice. Spinal administration or infusion of B1, and especially B2, receptor antagonists reversed experimental neuropathic pain behaviors in rats with peripheral nerve injury but only when the antagonists were given at times at which dynorphin was upregulated. After nerve injury, both B1 and B2 receptor mRNA were increased in the dorsal root ganglion, but not in the spinal cord. While a marked increase in mRNA expression for prodynorphin in the lumbar spinal cord was found following nerve injury, expression of mRNA for kininogen was below detection levels. The possible interaction of spinal dynorphin with bradykinin receptors as a basis of the pronociceptive action of this peptide was further tested in the CFA-induced inflammatory pain and DBTC-induced pancreatitis pain. Intrathecal administration of bradykinin receptor antagonists or dynorphin antiserum reversed DBTC-induced abdominal hypersensitivity and CFA-induced hyperalgesia only when spinal dynorphin or prodynorphin is upregulated. The antihyperalgesic effect of the bradykinin receptor antagonists was not due to de novo production of bradykinin.Taken together, our results unravel a novel, non-opioid molecular target of dynorphin, and indicate that dynorphin acts at bradykinin receptors to produce persistent psin in the pathological pain states. This novel pronociceptive mechanism offers new approaches to the development of therapy for pathological pain states.

Functional hyperemia is an endogenous regulatory process coupling synaptic activity and elevated neuronal energy demand with increased local blood flow. This involves signalling between neurons, astrocytes and blood vessels, comprising the neurovascular unit. Astrocyte processes ensheath both synapses and blood vessels, permitting multi-modal responses to synaptic activity, where astrocyte cytoplasmic Ca2+ is elevated, triggering endfeet processes to release vasoactive molecules, such as arachidonic acid (AA) metabolites and gliotransmitters, such as D-serine. D-Serine is a co-agonist of the glycine regulatory site at N-methyl-D-aspartate (NMDA)-type glutamate receptors, and NMDA receptors play a role in functional hyperemia in vivo. Thus, our aim was to examine the role of astrocyte D-serine in NMDA receptor-mediated vasodilation. Using isolated pressurized mouse middle cerebral arteries (MCAs), we determined that co-application of glutamate and D-serine induced dose-dependent dilation which was mediated by NMDA receptors and endothelial nitric oxide synthase (eNOS) in an endothelial-dependent mechanism. This is the first evidence of direct vascular effects of D-serine and glutamate and suggests a possible role for endothelial NMDA receptor activation. Several studies indicate vascular endothelial cells express NMDA receptor subunits. However, expression in mouse endothelial cells has not been well characterized, so we identified NR1 and NR2C/2D subunit expression in primary brain endothelial cultures by PCR and immunocytochemistry, and further confirmed endothelial NR2C/2D expression in situ by immunohistochemistry. To further investigate astrocyte D-serine release and NMDA receptor-mediated functional hyperemia within the neurovascular unit, we used an acute cortical brain slice model where stimulation of astrocyte cytoplasmic Ca2+ induced vasodilation of nearby arterioles. Pharmacologically, D-serine release and NMDA receptor activation were implicated in this vasodilation. Endothelial-derived nitric oxide was also determined to induce dilation by inhibiting the production of an AA metabolite, 20-hydroxyeicostetranoic acid (20-HETE), a vasoconstrictor. This suggests an interaction between astrocyte vasoactive molecules, nitric oxide and D-serine, which warrants further investigation. Overall, our results provide evidence of modulation of NMDA receptor-mediated neurovascular coupling by astrocytic D-serine.

Functional hyperemia is an endogenous regulatory process coupling synaptic activity and elevated neuronal energy demand with increased local blood flow. This involves signalling between neurons, astrocytes and blood vessels, comprising the neurovascular unit. Astrocyte processes ensheath both synapses and blood vessels, permitting multi-modal responses to synaptic activity, where astrocyte cytoplasmic Ca2+ is elevated, triggering endfeet processes to release vasoactive molecules, such as arachidonic acid (AA) metabolites and gliotransmitters, such as D-serine. D-Serine is a co-agonist of the glycine regulatory site at N-methyl-D-aspartate (NMDA)-type glutamate receptors, and NMDA receptors play a role in functional hyperemia in vivo. Thus, our aim was to examine the role of astrocyte D-serine in NMDA receptor-mediated vasodilation. Using isolated pressurized mouse middle cerebral arteries (MCAs), we determined that co-application of glutamate and D-serine induced dose-dependent dilation which was mediated by NMDA receptors and endothelial nitric oxide synthase (eNOS) in an endothelial-dependent mechanism. This is the first evidence of direct vascular effects of D-serine and glutamate and suggests a possible role for endothelial NMDA receptor activation. Several studies indicate vascular endothelial cells express NMDA receptor subunits. However, expression in mouse endothelial cells has not been well characterized, so we identified NR1 and NR2C/2D subunit expression in primary brain endothelial cultures by PCR and immunocytochemistry, and further confirmed endothelial NR2C/2D expression in situ by immunohistochemistry. To further investigate astrocyte D-serine release and NMDA receptor-mediated functional hyperemia within the neurovascular unit, we used an acute cortical brain slice model where stimulation of astrocyte cytoplasmic Ca2+ induced vasodilation of nearby arterioles. Pharmacologically, D-serine release and NMDA receptor activation were implicated in this vasodilation. Endothelial-derived nitric oxide was also determined to induce dilation by inhibiting the production of an AA metabolite, 20-hydroxyeicostetranoic acid (20-HETE), a vasoconstrictor. This suggests an interaction between astrocyte vasoactive molecules, nitric oxide and D-serine, which warrants further investigation. Overall, our results provide evidence of modulation of NMDA receptor-mediated neurovascular coupling by astrocytic D-serine.