Browsing by Subject "Microdialysis"
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Item Collection of IL-1 [beta] in skeletal muscle using microdialysis(2021-05-03) Crownover, Erin Jane; Stone, Audrey J.Microdialysis is a technique that allows for the determination of interstitial concentrations of small molecules. Therefore, it can be used to quantify local levels of cytokines within skeletal muscle in both healthy and diseased states. The purpose of this project was to define a microdialysis method to collect interstitial interleukin-1β (IL-1β) in skeletal muscle of rats. Microdialysis probes were constructed with PE-10 tubing and a fiber membrane. The gastrocnemius muscle of a rat was isolated and four probes were inserted prior to attaching those probes to PE-50 tubing and a two-way splitter, that was then connected to a perfusion pump. The pump was set at a constant flow rate of 5 μl/min with saline solution running through the probes. An initial baseline sample of dialysate was taken after probe insertion and a second was taken following a 60 min recovery period. The cytokine of interest (IL-1β) was then injected directly into the skeletal muscle surrounding the probes. Samples were collected following injection and then every 10 min for an hour. For each time point, dialysate was collected for 4 min across all probes and then pooled together. Dialysate samples need to be analyzed to quantify IL-1β and this can be done using a Luminex 200 instrument (Luminex Corp, Austin TX). After establishment of this technique at rest, it can then be applied during muscle contraction. Microdialysis is a useful technique for quantifying local levels of cytokines within skeletal muscle. As such, this method will facilitate research into the concentrations of cytokines within skeletal muscle, both at rest and during contraction, in healthy and diseased statesItem Dopamine concentrations in the nucleus accumbens core-shell border during the early stages of operant ethanol self-administration(2010-12) Carrillo, Jennifer; Gonzales, Rueben Anthony; Harris, Adron; Duvauchelle, Christine L.; Morikawa, Hitoshi; Schallert, TimothyMesolimbic dopamine plays an important role in ethanol reinforcement, and studies have shown that accumbal dopamine increases during operant ethanol self-administration. However, no one has ever studied this dopaminergic response during the acquisition of ethanol self-administration. Furthermore, some studies have shown that the dopamine signal does not correlate with the pharmacological effects of ethanol, but with the time during which the animal consumes the majority of the ethanol solution and when the sensory stimuli of ethanol are strongest. However, there is currently no direct evidence showing that the sensory stimuli of ethanol is indeed what causes the brief increase in accumbal dopamine during ethanol self-administration. The studies in this dissertation attempted to elucidate these issues. We designed and tested a placebo spout, which was to be used to study the relationship between accumbal dopamine and the sensory stimuli of ethanol during self-administration. Unfortunately, the placebo designs were either not feasible for performing microdialysis or did not show promising behavioral data. We also developed and tested a self-administration protocol in which the concentrations of ethanol (10%) were kept constant throughout the study. The new protocol was successful in initiating and maintaining ethanol self-administration, and the animals doubled their intake from day 1 to day 2 of ethanol consumption. Using this protocol, we trained male Long Evans rats to self-administer ethanol and measured accumbal dopamine during the first two days of ethanol self-administration through microdialysis. The behavioral and neurochemical data matched. A single exposure to ethanol was sufficient for the animals to double their ethanol consumption by day 2 and to cause an increase in accumbal dopamine during the first 5 minutes of ethanol self-administration. The dopamine response was observed during the time when the sensory stimuli of ethanol were strongest, but before ethanol reached peak concentrations in the brain. Overall, these results suggest that the dopamine response to ethanol self-administration may not be solely pharmacological and that a single exposure to ethanol is sufficient to learn the association between ethanol and its cues. These findings give us greater insight into mesolimbic dopamine's role in the early stages of ethanol reinforcement.Item Effects of opioid antagonism on operant ethanol self-administration in adolescence and characterization of extracellular GABA in the ventral tegmental area(2016-12) Zandy, Shannon Laine; Gonzales, Rueben Anthony; Harris, Robert A; Marinelli, Michela; Morikawa, Hitoshi; Morrisett, Richard AThe endogenous opioid peptide system is hypothesized to be involved in ethanol self-administration and relapse behaviors. Naltrexone, a nonselective opioid antagonist, is an approved medication for alcohol use disorder which has been shown to decrease drinking in adult animal models and select clinical populations, but little is known about the efficacy of naltrexone in animal models that begin drinking ethanol in adolescence. Therefore, we investigated the effects of systemic naltrexone administration in an adolescent rat model of operant ethanol self-administration. We found that naltrexone significantly reduced ethanol intake and motivation to obtain ethanol in adolescent and adult rats. Following a period of abstinence, naltrexone also significantly reduced “relapse” to alcohol seeking in both age groups. These results extend findings that naltrexone is effective at reducing ethanol intake to an adolescent animal model and support opioid antagonism as a treatment strategy for decreasing problem drinking in late adolescents and young adults. One potential mechanism underlying the effects of opioid receptor blockade on ethanol self-administration implicates γ-aminobutyric acid (GABA) neurons within the ventral tegmental area (VTA). Inhibitory signaling in the VTA is involved in the mechanism of action of many drugs of abuse yet there are few studies measuring extracellular GABA concentration in this region. Therefore, the remaining experiments focused on developing methods to quantify extracellular GABA in the VTA. We first describe a novel, sensitive fluorescence method to quantify GABA concentration using high performance liquid chromatography (HPLC) of an ophthalaldehyde/sulfite derivative, previously reported to produce low fluorescence not suitable for in vivo microdialysis applications. Next, we used quantitative microdialysis under transient conditions to characterize basal extracellular GABA concentration and the influence of uptake mechanisms in the VTA. Our results show that inhibition of GABA uptake significantly increased extracellular GABA concentration and reduced in vivo extraction fraction of the probe. Reduced in vivo extraction fraction caused significant underestimation of the increase in extracellular GABA by conventional microdialysis. Together, these results establish the foundation for future studies to investigate the regulation of extracellular GABA concentration and uptake mechanisms in the VTA in mediating the effects of ethanol, opioid antagonism and associated drug-related behaviors.Item Impaired endothelium-independent microvascular function in obese young adults(2014-08) Patik, Jordan Christopher; Brothers, Robert MatthewMicrovascular dysfunction is believed to precede the development and contribute to the progression of obesity related diseases such as insulin resistance, hypertension, and coronary artery disease. Multiple studies have found impaired microvascular endothelium-dependent vasodilation occurs prior to the onset of disease in middle aged adults. In order to test the hypothesis that the cutaneous microvasculature of young obese (BMI>30kg/m²), but otherwise healthy, adults would exhibit impaired microvascular response, we recruited 12 obese and 12 lean (BMI<25 kg/m²) individuals. Each group was age-matched and consisted of 5 females and 7 males. Each participant was instrumented with two microdialysis probes inserted in the dermis of the non-dominant forearm for a wide dose range of infusions of either the endothelium-dependent vasodilator methacholine (MCh) or the endothelium-independent vasodilator sodium nitroprusside (SNP). Each microdialysis site was clamped at 33°C with a local heater and affixed with a laser Doppler flux (LDF) probe for determination of local red blood cell flux, an index of blood flow. LDF was recorded continuously while 7 doses of each drug (MCh: 10⁻³-10³mM; SNP: 5x10⁻⁵-50mM) were infused at a rate of 2 [mu]l/min for 8 minutes per dose. Both sites finished with heating to 43°C and infusion of 50mM SNP to confirm site specific maximal vasodilation. Blood pressure was recorded in the last minute of each stage and the corresponding LDF was used to calculate cutaneous vascular conductance (CVC). Dose response curves for CVC at each dose, as well as maximal CVC were analyzed. MCh dose response showed a trend toward endothelium–dependent impairment in obese (p=0.06) and maximal absolute CVC at the MCh site was attenuated in obese versus lean (2.70 ± 0.73 vs 3.30 ± 0.81 LDF/mmHg, p=0.027). Endothelium-independent vasodilation with SNP was impaired at the 4 highest doses of SNP (all P<0.006) and maximal CVC was attenuated in obese compared to lean (2.44 ± 0.74 vs 3.31 ± 0.65 LDF/mmHg, p=0.004). These results support the hypothesis that microvascular function is impaired in young, healthy obese, individuals; however they suggest the impairment is partially endothelium-independent.Item Mechanisms of cutaneous microvascular endothelial dysfunction in young black Americans(2016-12) Kim, Kiyoung, active 2013; Tanaka, Hirofumi, Ph. D.; Farrar, Roger P.; Castelli, Darla M.; Brothers, Robert Matthew; Davis, Scott L.Black Americans have an increased risk for developing a variety of cardiovascular disease (CVD) when compared to white Americans and other populations in the United States. It has also been demonstrated that the underlying impairments in black Americans manifest during early adulthood prior to any overt signs of risk, which leads to higher rates of CVD related morbidity and mortality in black Americans than other populations. Study 1 was designed to investigate the potential mechanisms of cutaneous microvascular dysfunction in young college-age black Americans. This was assessed by measuring the skin blood flow response to local heating while various vasoactive substances were delivered into the cutaneous interstitial space by intradermal microdialysis. We demonstrated that an attenuated nitric oxide (NO) mediated vasodilation due in part to a relative deficit of L-arginine in the endothelial cells is one mechanism by which microvascular dysfunction occurs in young black Americans. Study 2 conducted to investigate the effects of acute cocoa flavanol intake on cutaneous microvascular function in young black Americans. This was assessed by measuring the skin blood flow response to local heating and delivery of vasoactive substances (as described above) before and after consumption of a beverage high in flavanol content. Study 2 demonstrated that acute flavanol intake improved cutaneous microvascular function in response to local heating in young black Americans relative to young white Americans. Study 3 was designed to investigate the effects of acute flavonal intake on endothelium-dependent microvascular dilation in response to exogenous administration of methacholine (MCh) in young black Americans. This was assessed by skin blood flow responses to incremental dose of MCh, which was delivered by intradermal microdialysis, before and after consumption of a beverage high in polyphenol content. Study 3 identified that acute flavanol intake did not alter the dose-response curve of MCh-induced cutaneous vasodilation in either racial groups. Overall, the series of studies in this dissertation may provide evidence that young black Americans have attenuated microvascular function relative to young white Americans, and that a potential mechanism of decreased microvascular function is a decrease in NO bioavailability and/or NO mediated vasodilation, which is related to a deficit of L-arginine in the endothelial cells in young black Americans. Furthermore, our findings may provide evidence that the consumption of cocoa flavanols is an effective therapeutic strategy to prevent and/or delay the development of CVD at least in young black Americans.Item Novel findings to aid in alcohol use disorder research : ethanol vapor and the ventral tegmental area(2021-07-30) Ontiveros, Tiahna; Gonzales, Rueben AnthonyThe high prevalence of alcohol use disorder continues to impact individuals by heightening stress, anxiety and disrupting cognitive tasks (Morrow and Creese, 1986). Ethanol affects several neuromodulator systems, exerting its effects within the central nervous system (Deehan et al., 2013). One known effect of ethanol is its impact on the mesocorticolimbic system and related circuits, including the ventral tegmental area and its involvement in the regulation of motivation and goal-directed behavior (Doyon et al., 2020). The neurotransmitter norepinephrine has also been linked to mood stabilization, alertness, as well as the endocrine and autonomic nervous systems (Vazey et al., 2018). Additional issues remain to be explored, such as the interaction between norepinephrine signaling and other neuromodulators, and a better mechanistic understanding of ethanol withdrawal (Kushner et al., 2000; Skelly and Weiner 2014; Fredriksson et al., 2015; Becker and Koob, 2016; Petrakis et al., 2016). In order to increase our understanding of alcohol use disorder I carried out two experiments throughout the course of my thesis. We conducted an ethanol vapor study showing that we were able to successfully induce alcohol dependence and withdrawal through a short ten-day vapor exposure model in Long Evans male rats. Lastly, we created a time course of ethanol in the ventral tegmental area through gas chromatography analysis, also using microdialysis. In the appendix we also show pilot data from a study were we closely examined the estrous cycle in female rats that underwent surgery to place an intracranial guide cannula for microdialysis experiments. We found the possible surgical effects that can halt cycling and ultimately affect data looking at differences between males and females in alcohol dependance and withdrawal, however more experiments are necessary to make a precise conclusion. Findings from these three experiments can be used to continue the advancement of alcohol use disorder research in different ways.