CONTEXT: Nitric Oxide (NO) is an endothelial-derived vasoactive molecule that causes an increase in blood flow and oxygen delivery to tissue. A reduction in NO bioavailability has been found to occur in adults over the age of 60 and can be reversed pharmacologically by improving NO synthase (NOS) activity. Reversing these age-related changes with alternative interventions, such as aerobic exercise, has shown some promising results. OBJECTIVE: To quantify blood NO-bioavailability (as measured by blood nitrite levels) in a population of aerobically trained elderly men and compare these data to a group of age-matched, inactive individuals. In addition, we measured the cutaneous vasodilator response to local skin heating as a bioassay for NO-mediated cutaneous dilation. SETTING: BYU Human Performance Research Center (HPRC). PARTICIPANTS: 16 healthy elderly men (age = 66 ± 7.07 years) were divided into two groups based on physical fitness levels and estimated VO2max in ml O2•kg-1•min-1 (Trained = 39.1 ± 1.21, Untrained = 29.0 ± 2.70). INTERVENTIONS: A blood sample was collected and analyzed for NO. A microdialysis study was performed and dialysate was collected at 32°C and at 42°C. During the heating process, skin blood flow (skin vasomotor activity) was monitored and reported as cutaneous vascular conductance (CVC). MAIN OUTCOME MEASURES: Whole blood nitrite concentrations, pre- and post-heat nitrite concentrations, and CVCmax were compared between trained and untrained groups. RESULTS: Whole blood nitrite concentration was similar in trained subjects and untrained subjects averaging 25.77 ± 6.75 and 21.43 ± 7.20 µM, respectively (F1,13 = 0.19; P = 0.6671]. Local skin heating had no impact on the concentration of nitrite in dialysate samples ([NOx]dialysate F1,26 = 0.01; P = 0.7567). In addition, the plateau in % CVCmax following 30 minutes of local heating was similar for trained and untrained subjects averaging 67.7 ± 5.8 and 68.0 ± 6.2 % CVCmax, respectively (F1,13 = 0.00; P = 0.9673). CONCLUSIONS: The results of this study indicate that age-mediated reductions in whole blood NO-bioavailability and decrements in NO-mediated cutaneous vasodilation during local heating were similar in aerobically fit and sedentary adults 60 years old or older. We conclude that a commitment to aerobic fitness was unable to overcome the age-related dysfunction of the NOS system.

<p>Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives & Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury.</p><p>PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.</p>

A model with human melanoma xenografts transplanted to athymic mice has been adopted for in vivo studies of 5-S-cysteinyldopa (an intermediate pigment metabolite), glutathione, and cysteine. L-Dopa is an intermediate metabolite in pigment formation and is also important in the treatment of Parkinson's disease, and therefore 1 have also studied the pharmacokinetics of this compound. We were first to describe in vivo microdialysis in melanoma tissue and showed that dialysis membranes of cuprophane or polyamide are suitable for studies of interstitial 5-S-cysteinyldopa and selected thiols. Analytical procedures were also improved for quantitation of 5-S-cysteinyldopa, L-dopa, glutathione, cysteine, and N-acetylcysteine (NAC). In the melanoma xenografts the interstitial concentration of 5-S-cysteinyldopa reflected the high intracellular production of this intermediate metabolite. For in vivo manipulation of glutathione in the melanoma tissue we gave intraperitoneal injection of buthionine sulphoximine to the animals and thus reduced the glutathione concentrations substantially. We showed that restitution of glutathione in melanoma tissue occurs spontaneously and is not much improved by treatment with the cysteine deliverers NAC and L-2-oxothiazolidine-4-carboxylate (OTC). 5-S-Cysteinyldopa was not substantially affected by great variations in glutathione concentrations. Transport of NAC from intraperitoneal injection to melanoma tissue occurred rapidly and deacetylation to cysteine in vivo could be detected soon after NAC injection. In vivo formation of cysteine was slower from OTC than from NAC. Pharmacokinetic studies of L-dopa in human subjects indicated a slight to moderate protein binding. Plasma free L-dopa had similar elimination T½ as interstitial L-dopa, but in some cases the elimination of total L-dopa was slower. Difficulties in intestinal absorption of L-dopa were revealed by microdialysis in blood and subcutaneous tissue. Studies showed that this was due to delayed emptying of the stomach. L-Dopa intake increased 5-S-cysteinyldopa concentrations in blood within 30 min in patients with Parkinson's disease and a history of melanoma. No melanoma activation occurred during long-term treatment with L-dopa. Microdialysis is thus a safe and easily applied method for in vivo studies of both pigment metabolites from human melanoma tissue transplanted to nude mice and for pharmacokinetic studies of L-dopa. / On the day of the public defence the status of the articles IV, V and VI was: Submitted.

Brief episodes of ischemia and reperfusion render the myocardium more resistant to necrosis from a subsequent, otherwise lethal ischemic insult. This phenomenon is called ischemic preconditioning(IP). Today, much is known about the signalling pathways involved in IP; however, the details of the final steps leading to cardioprotection, remain elusive. Adenosine (a catabolite of ATP) plays a major role in the signalling pathways of IP. Following IP there is an unexplained discrepancy between an increased adenosine production (evidenced by increased 5’-nucleotidase activity) and the successively lower adenosine levels observed in the interstitial space. We propose that this discrepancy in adenosine production vs. availability may be due to an increased metabolic utilisation of adenosine by the IP myocardium. According to our hypothesis, IP induces/activates a metabolic pathway involving deamination of adenosine to inosine. Inosine is further catalysed (in presence of Pi) to hypoxanthine and ribose-1-phosphate. Ribose-1-phosphate can be converted to ribose-5-phosphate in a phosphoribomutase reaction. Ribose-5-phosphate is an intermediate of the hexose monophosphate pathway also operative under anaerobic conditions. Hence the ribose moiety of adenosine can be utilised to generate pyruvate and ultimately ATP (via lactate formation) n.b. without any initial ATP investment. Such cost-effective adenosine utilisation may at least partly explain the cardioprotective effect of IP. Objectives &amp; Methods: In the current studies we investigated the role of adenosine metabolism according to the suggested metabolic pathway by addition of adenosine and inhibition of its metabolism during IP as well as by comparing tissue and interstitial levels of key energy-metabolites following different protocols of IP. Furthermore, we studied the importance of the IP protocol with regard to the number of ischemia and reperfusion cycles for the cardioprotective effect of IP. In addition, the validity of the microdialysis technique for experimental in vivo studies of myocardial energy metabolism was evaluated. For these purposes the microdialysis technique, tissue biopsies, and planimetric infarct size estimation in an open chest porcine heart-model was used. Results: Addition of adenosine via microdialysis probes enhanced the interstitial release of inosine, hypoxanthine and lactate in the myocardium of IP-subjects during prolonged ischemia. This finding did not occur in non-preconditioned subjects. Similar addition of deoxyadenosine a non-metabolizable adenosine receptor-agonist, did not evoke the same metabolic response. Purine nucleoside phosphorylase (PNP) is responsible for the conversion of inosine to hypoxanthine being a key enzyme in the above mentioned metabolic pathway. Inclusion of 8' aminoguanosine (a competitive inhibitor of PNP) decreased interstitial hypoxanthine release (as a token of PNP inhibition) and increased the release of taurine (marker of cellular injury) in the ischemic IP myocardium. Addition of inosine (a natural substrate of PNP) reverted these changes. Four IP cycles protected the heart more than one IP cycle as evidenced by morphometric and energy-metabolic data.Proportionally more hypoxanthine was found in the myocardium of IP subjects during prolonged ischemia. The ratio of tissue levels of inosine/hypoxanthine (used as an indicator of PNP activity) was significantly smaller in the IP groups. In addition, myocardial interstitial levels of energy-related metabolites (lactate, adenosine, inosine, and hypoxanthine) obtained by the microdialysis technique correlated with tissue biopsy levels of corresponding metabolites. Conclusions: IP activated a metabolic pathway favouring metabolism of exogenous adenosine to inosine, hypoxanthine and eventually lactate. Inhibition of adenosine metabolism following IP (via inhibition of PNP-activity resulted in enhanced cellular injury. PNP-activity is proportionally higher in IP-myocardium. Metabolic utilisation of adenosine in IP-myocardium (as outlined above) may represent a costeffective way to produce ATP and at least partly explain the cardioprotective effect of IP. IP protects the myocardium in a graded fashion. Furthermore, we confirmed the validity of the microdialysis technique (in the current setting) for studying dynamic changes of myocardial energy metabolism.

Recent approaches in depression therapeutics include triple reuptake inhibitors, drugs that target three monoamine systems. Using in vivo electrophysiological and microdialysis techniques, the effects of 2- and 14-day treatments of escitalopram, nomifensine and the co-administration of these two drugs (TRI) were examined in male Sprague-Dawley rats. Short- and long-term TRI administration decreased NE firing and had no effect on DA neurons. Normal 5-HT firing rates were maintained after 2-day TRI administration compared to the robust inhibitory action of selective serotonin reuptake inhibitors (SSRIs). Escitalopram treatment enhanced the tonic activation of the 5-HT1A receptors given the increase in firing observed following WAY100635 administration. Nomifensine treatment enhanced tonic activation of the α2–adrenoceptors following idazoxan administration. TRI treatment caused a robust increase in extracellular DA levels that was in part mediated by a serotonergic contribution. Therapeutic effects of the drugs examined in this study may be due to the enhancement of 5-HT, NE and/or DA neurotransmission.

This thesis tested the hypothesis that ethanol suppresses respiratory muscle activity by effects at the central motor pool and/or by state-dependent regulation of motor activity via influences on sleep/arousal processes. Ten rats were implanted with electroencephalogram and neck electrodes to record sleep-wake states, and genioglossus and diaphragm electrodes for respiratory recordings. Studies were performed following intraperitoneal injection of ethanol (1.25g/kg) or vehicle. The effects on genioglossus activity of ethanol (0.025-1M) or vehicle applied directly to the hypoglossal motor nucleus were also determined in sixteen isoflurane-anaesthetized rats. The results of these studies suggest that ethanol at physiologically relevant concentrations promoted sleep, and altered electroencephalogram and postural motor activities indicative of a sedating effect. The lack of effect on genioglossus activity with ethanol applied directly to the hypoglossal motor pool suggests that the suppression observed with systemic administration may be mediated via effects on state-dependent processes rather than direct effects at the motor pool per se.

This thesis tested the hypothesis that ethanol suppresses respiratory muscle activity by effects at the central motor pool and/or by state-dependent regulation of motor activity via influences on sleep/arousal processes. Ten rats were implanted with electroencephalogram and neck electrodes to record sleep-wake states, and genioglossus and diaphragm electrodes for respiratory recordings. Studies were performed following intraperitoneal injection of ethanol (1.25g/kg) or vehicle. The effects on genioglossus activity of ethanol (0.025-1M) or vehicle applied directly to the hypoglossal motor nucleus were also determined in sixteen isoflurane-anaesthetized rats. The results of these studies suggest that ethanol at physiologically relevant concentrations promoted sleep, and altered electroencephalogram and postural motor activities indicative of a sedating effect. The lack of effect on genioglossus activity with ethanol applied directly to the hypoglossal motor pool suggests that the suppression observed with systemic administration may be mediated via effects on state-dependent processes rather than direct effects at the motor pool per se.

<p>The success of implanted medical devices, such as biosensors, is dependent on the immune reaction to the surface of the implanted material. This immune reaction, termed the foreign body reaction, is potentially affected by the physical and chemical properties of the implanted material. Macrophages interact with the surface of the implanted material and secrete intercellular signals, including cytokines and growth factors, which direct the actions of immune cells in the surrounding tissue. The type and quantity of cytokines and growth factors produced by macrophages at an implant surface could be an indicator of the outcome of the foreign body reaction. </p><p>This study investigated the effect of the surface chemistry of an implanted device on the production of cytokines and growth factors. First, microdialysis sampling was characterized as a technique for collecting cytokines and growth factors from the tissue surrounding an implant. Based on this characterization, it was determined that a direct sampling method would be more suitable than microdialysis sampling for determining accurate tissue concentrations of cytokines and growth factors. Second, an in vitro model was developed and utilized to assess cytokine and growth factor production from monocyte/macrophage cultures seeded onto commonly implanted polymeric biomaterials with varying surface chemistries. The materials included in this study were polyethylene (PE), polyurethane (PU), polymethyl methacrylate (PMMA), expanded polytetrafluoroethylene (ePTFE), and a cytotoxic organo-tin polyvinyl chloride (ot-PVC) as a positive control. From this in vitro model, it was determined that the varying surface chemistries of these non-toxic materials, excluding ot-PVC, did not significantly affect the types and quantities of cytokines and growth factors produced. Finally, an in vivo model for evaluating the cytokine and growth factor response to an implanted biomaterial was utilized for comparison with the in vitro findings. In this model, biomaterials were implanted subcutaneously within the lumen of a stainless steel mesh cage. The mesh cage served to create a "pocket" where wound exudate fluid collected within the cage, surrounding the implanted biomaterial. The materials included in this study were PE, PU, and ot-PVC. Cytokines and growth factors produced at the material surface were sampled directly from the exudate fluid. The results from this in vivo study indicate that cytokine and growth factor production were not significantly impacted by the varying surface chemistries of the implanted biomaterials. The in vivo data support the findings from the in vitro model, suggesting that the foreign body reaction proceeds in a similar fashion for each of these non-cytotoxic, polymeric biomaterials with varying surface chemistries.</p> / Dissertation

The neurochemical problem (1) and the directive of the neuroanalytical chemist (2) can be stated as follows: (1) The chemical space of the nervous system is populated by hundreds of neuroactive species linked through extensive biological circuits which are dynamically changing in time and space in response to myriad inputs. (2) Neurochemical analysis techniques should therefore have the appropriate temporal, spatial, and chemical resolution to study these systems, while perturbing them so minimally as to allow unfettered in vivo measurements. New tools and concepts for analytical measurements of neurotransmitters, neuropeptides, and synaptic vesicles are developed and presented in this dissertation on analytical measurements for addressing the neurochemical problem. The introduction gives a broad overview of chemical neuroscience and introduces quantitative visualization of the multidimensional resolution paradigm for analytical chemists seeking to design effective experiments. Chapters two through four detail advancements in data processing and instrument design which decrease detection limits and allow for improved spatial, temporal, chemical resolution in capillary electrophoresis measurements of neurotransmitters, metabolites, and synaptic vesicles. Chapter five discusses the development of fast-scan controlled-adsorption voltammetry which has dramatically increases the spatial and temporal resolution of basal dopamine measurement in vivo. Chapter six introduces online-preservation microdialysis as a way to overcome enzymatic degradation of endogenous opioid neuropeptides during in vivo sample collection. Because of this discovery of the secretory behavior these neuropeptides is reported in the anterior cingulate cortex (ACC), a region of the brain deeply associated with pain signaling. The advancement of peptide drugs particularly glycosylated neuropeptide analogs through new methods of mass spectrometry analysis for rapid feedback in drug development are presented in chapter 7. Chapter eight concludes this work with future directions pointing towards single-cell electrochemical and mass spectrometry measurements, shotgun-microdialysis for high-throughput screening of neurotherapeutics, preliminary data on the effect of chronic pain of endogenous opioids in the ACC, and the beginnings of in vivo neuroproteomics analysis in rodent pain models.

Sustained administration of morphine in humans and in animals induces a state of abnormal pain (i.e., hyperalgesia) which may be associated with the development of reduced analgesic efficacy (i.e., tolerance). Evidence suggests that opiate treatment may upregulate cholecystokinin (CCK), a pronociceptive peptide, in the brain and spinal cord. Therefore, we hypothesized that CCK may be upregulated by opiate treatment in the rostral ventromedial medulla (RVM) and to subsequently drive descending facilitation mechanisms to elicit hyperalgesia and antinociceptive tolerance in rats.CCK administered into the RVM of naive rats elicited hyperalgesia which was blocked by either RVM CCK2 receptor antagonist L365,260; or by bilateral lesion of dorsolateral funiculus, a major bulbospinal descending pain modulation pathway from the RVM to spinal cord.Sustained subcutaneous morphine induced hyperalgesia and spinal antinociceptive tolerance. Both effects were reversed by RVM CCK2 antagonist, suggesting that the up-regulation of the endogenous RVM CCK system played a critical role in the expression of these phenomena.Lesion of cells in the RVM which selectively express CCK2 receptors with a saporin construct (CCK-SAP) to inhibit ribosome activity, prevented morphine-induced hyperalgesia and spinal antinociceptive tolerance. These findings suggest that the integrity of the RVM CCK system is required for the development of hyperalgesia and antinociceptive tolerance induced by sustained morphine.The CCK system does not seem to play a role in setting the baseline sensory thresholds in normal rats because neither RVM L365,260 nor CCK-SAP treatment altered baseline sensory thresholds in naive rats.CCK appears to be present exclusively in nerve terminals of RVM neurons in naive rats. There was no obvious change in the levels of CCK-LI, CCK2 receptor, or CCK2 receptor mRNA in the RVM after sustained morphine treatment. However, microdialysis studies showed an approximately 5-fold increase in basal CCK levels in the RVM after sustained morphine treatment.Taken together, our results support the hypothesis that increased release of CCK in the RVM is induced by sustained morphine and drives descending facilitation to mediate morphine-induced paradoxical "pain" and spinal antinociceptive tolerance.