Cardiovascular

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/31252

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PYRUVATE DIMERIZATION BY DIVALENT METAL CATIONS
(2013-04-12) Barnes, David
Purpose: Pyruvate enriched cardioplegia has been shown to reduce cardiopulmonary bypass induced inflammation and oxidative stress. Cardioplegia solutions are stored in the hospital pharmacy at room temperature. It has been argued that pyruvate is unstable in aqueous solutions containing divalent metal cations and undergoes an aldol condensation reaction to form parapyruvate. This substance is a potential inhibitor of a critical step in the TCA cycle, oxoglutarate dehydrogenase, and therefore may be toxic. Our pilot study is testing whether pyruvate remains stable in solution in the presence of a metal catalyst. Methods: Aqueous solutions of 5 mM pyruvate were prepared in 0.5 M Tris and aliquoted into test tubes. Zn2+ (ZnCl2) was then added to the test tubes at 0, 0.1, 0.3 and 1.0 mM concentrations. The solution was buffered to pH 4 to prevent precipitation of the Zn2+ which occurred at neutral pH. The solutions were stored in the dark at 20 and 4 ℃ to assess the effect of temperature on the aldol condensation. Absorbance of ultraviolet light (315 nm wavelength) by the pyruvate-Zn solution was measured by spectrophotometry at 0 and 10 days to detect formation of parapyruvate, which absorbs light at that wavelength. Results: The changes in absorbance of the solutions kept at 20 ℃ were as follows: 0 mM Zn2+ -0.002, 0.1 mM Zn2+ 0.007, 0.3 mM Zn2+ -0.011, 1.0 mM Zn2+ -0.0195. The corresponding absorbance changes in the solutions at 0 ℃ for 10 days were as follows: 0 mM Zn2+ -0.009, 0.1 mM Zn2+ 0.0015, 0.3 mM Zn2+ -0.005, 1.0 mM Zn2+ -0.0105. Thus, at both temperatures, only the highest Zn2+ concentration, 1 mM, produced a detectable, albeit modest, change in absorbance at 315 nm, and that change was in the direction opposite that which would indicate pyruvate dimerization. Conclusions: Over the course of 10 days, no appreciable pyruvate dimerization could be detected by spectrophotometry. Thus, pyruvate appeared to be stable in aqueous solution, even over 10 days at 20 ℃. To confirm these results, we are currently measuring pyruvate concentrations at 0, 1, 3, 7 and 14 days by a spectrophotometric assay at 340 nm utilizing the lactate dehydrogenase reaction, in which pyruvate reduction to lactate consumes NADH. This method will enable us to verify that the apparent absence of parapyruvate is accompanied by a stable concentration of pyruvate over time. Future studies will test the impact of other divalent metals, including Mg2+, an important component of many cardioplegia formulations.
N-ACETYL CYSTEINE: A MODULATOR OF THE ARTERIOBAROREFLEX IN OBSTRUCTIVE SLEEP APNEA.
(2013-04-12) Jouett, Noah
Purpose: The CDC estimates that 18 million Americans have Obstructive Sleep Apnea (OSA). Although much is known clinically about OSA, the mechanisms which cause long term detrimental effects have not been fully elucidated. Prior research demonstrates that OSA causes a hypersympathetic state with decreased responsiveness to changes in blood pressure, i.e. decreased sensitivity of the arterial baroreflex (ABR). However, the mechanistic pathways underlying this decrease in ABR sensitivity remain to be identified. The goal of this study was to evaluate the potential role of reactive oxygen species (ROSs) in the modulation of the ABR in OSA. N-acetyl cysteine (NAC), a known free radical scavenger in the central nervous system (CNS), should diminish the depressive effects of intermittent hypoxia on ABR function. Thus, our hypothesis is that NAC will attenuate the sympathoexcitation observed after IH and, consequently, blunt the increase in HR typically observed. Methods: Positive and negative pressures were applied at the carotid sinus of 10 subjects using a malleable neck collar. Carotid baroreflex function (CBR) was assessed by comparing changes in heart rate for a given altered carotid sinus pressure. Blood pressure was non-invasively recorded using a finometer and heart rate was monitored by electrocardiography. After baseline measurements were recorded, the subjects were given a drink containing either NAC or a placebo treatment. After ingestion, the subjects were allowed to rest for 1 hour after which HR and CBR function were assessed. The subjects were then intermittent hypoxia trained (IHT) and HR and ABR function were obtained immediately, 30 min and 1 hour post IHT. Results: HR was found to increase with time under both placebo and NAC treatment conditions (two-way ANOVA, p ≥ 0.712). There were also no significant changes in CBR function as assessed by the operating or response ranges and maximum gain of the reflex. Conclusions: CBR control of HR, a measure of autonomic function, was not significantly altered in NAC treated subjects. This does not indicate an inability of NAC to modulate ABR function, but rather suggests other underlying mechanisms.
NEUROPLASTICITY IN THE NUCLEUS TRACTUS SOLITARIUS PRECEDES DEVELOPMENT OF FUNCTIONAL CHANGES IN AUTONOMIC REGULATION OF ARTERIAL PRESSURE IN OBESE ZUCKER RATS.
(2013-04-12) Fu, Guangchao
Purpose: In juvenile OZR (7 wks) splanchnic sympathetic nerve activity (SNA), mean arterial pressure (MAP), and arterial baroreflex-mediated control of SNA are comparable to LZR. In contrast, in adult OZR (12-14 wks), baseline SNA and MAP are elevated and baroreflexes are blunted compared to age-matched LZR. Baroreceptor afferent nerves of adult OZR respond normally to rises in MAP, but direct stimulation of the afferent fibers evokes blunted decreases in SNA and MAP, suggesting changes in the brain contribute to impaired baroreflexes. Activation of the nucleus of the solitary tract (NTS), the brain stem site that receives baroreceptor inputs, by microinjection of glutamate evokes comparable decreases in SNA and MAP in juvenile OZR and LZR but blunted responses in adult OZR. In contrast, activation of the brain stem target of the NTS, the caudal ventrolateral medulla, evokes comparable responses in adult OZR and LZR, suggesting the NTS is the major site that is impaired in adult OZR. The present study sought to determine the time course and the phenotype of NTS neurons that may be altered in OZR to contribute to impaired physiological responses. Methods: Zucker rats (5-6 wk, 7-8 wk and 11-12 wk, 6/group) were transcardially perfused with 4% paraformaldehyde. Brains were harvested and fixed for 48 hours. Then the brains were cut in the coronal plane (30 microns) using a Vibratome and stored in cryoprotectant for later histological processing. To identify neurons undergoing neuroplasticity in the intermediate NTS, we performed immunohistochemistry for ΔFosB revealed by 3, 3'-diaminobenzidine. Results: At 5 wks OZR and LZR had comparable numbers of ΔFosB+ neurons in the NTS (203±30; 195±22, P=0.85). In contrast, at 7 wks and 12 wks OZR had significantly more ΔFosB+ neurons versus age-matched LZR (7 wks: 514.5±70; 207±24.5, P <0.05). (12 wks: 177±15.38; 88±15.76, P <0.05). The ΔFosB positive neurons were not catecholaminergic (immunoreactive for tyrosine hydroxylase). Conclusions: These results suggest noncatecholaminergic NTS neurons undergo neuroplasticity in the OZR prior to the onset of hypertension or impaired baroreflexes, and these changes persist in adulthood.
OXIDATIVE STRESS NEGATIVELY INFLUENCES THE EFFECTS OF ANDROGENS ON DOPAMINE NEURONAL VIABILITY
(2013-04-12) Holmes, Shaletha S.
Purpose: Parkinson's disease (PD) is a neurodegenerative disease characterized by oxidative stress and loss of dopaminergic neurons in the nigrostriatal pathway, in which men have a higher risk than women. The mechanisms involved in this gender bias remains elusive, one possibility may be that oxidative stress converts the neuronal response to androgens, which is toxic. Specifically, we hypothesize that in an oxidative stress environment, androgens such as testosterone compromises the viability of dopamine neurons. Methods: To test our hypothesis, we exposed a dopaminergic cell line (N27 cells) to a sublethal concentration of the pro-oxidant, tert-butyl-hydrogen peroxide (H202) for 24 hours and assessed cell viability in the presence or absence of testosterone. Results: Physiologically relevant concentrations of the androgen, testosterone (0, 1, 10, 100 nM) failed to compromise cell viability in non-oxidatively stressed cells. In contrast, testosterone and testosterone conjugated to BSA (T-BSA) did promote cell death in the H202 pre-treated cells. Interestingly, androgen pre-treatment protected dopamine cells from H202-induced cell death. Supporting the role of oxidative stress as a switch in this effect, the antioxidant, N-acetyl cysteine, prevented the damage promoting effects of testosterone in H202 pretreated cells. Neither the androgen receptor nor the estrogen receptor antagonists, flutamide (10 uM) and ICI 182, 780(1 uM), respectively, altered the death promoting effect of testosterone. Conclusions: Coupled with the observation that the membrane-impermeable T-BSA mimicked the effects of testosterone, we suggest that the cell death promoting effects may be mediated by a putative membrane-associated androgen receptor. Overall, these results indicate that oxidative stress acts as a molecular switch in dopamine neurons that can reverse the neuroprotective effects of androgens to that, which is neurotoxic. Thus, the interplay between oxidative stress and androgens on dopamine neuronal viability may underlie the male gender bias found in PD.
EXAMINING PYRUVATE S ANTI-INFLAMMATORY ACTIONS IN BRAIN FOLLOWING CARDIAC ARREST
(2013-04-12) Nguyen, Anh
Purpose: Approximately 0.5 million Americans suffer cardiac arrest each year. Cardiac arrest is usually lethal, and survivors often face severe neurological disability due to irreversible brain injury inflicted by global ischemia/reperfusion. Brain ischemia and reperfusion activates matrix metalloproteinases (MMPs) that disrupt the blood-brain barrier (BBB), enabling inflammatory cells to invade the brain parenchyma. We have shown that pyruvate, a metabolic intermediate and antioxidant, prevents death of hippocampal and cerebellar neurons and preserves neurobehavioral function in dogs after cardiac arrest. However, pyruvate's neuroprotective mechanism is elusive. We are testing the hypothesis that pyruvate preserves BBB integrity following cardiac arrest by suppressing MMPs and evoking expression of protective proteins hypoxia-inducible factor (HIF)-1ɑ and heat shock protein (Hsp)-70. Methods: Yorkshire swine (30-40 kg) were subjected to cardiac arrest-resuscitation or non-arrest sham protocols. Ventricular fibrillation was induced by a train of electric impulses transmitted to the right ventricle via a pacing wire. Precordial compressions were given at 6-10 min arrest, and then sinus rhythm was restored with defibrillatory transthoracic countershocks. NaCl or Na-pyruvate was infused iv at the rate of 0.1 mmol/kg/min during chest compressions and the first 60 min post-defibrillation. Cerebral cortex was snap-frozen in liquid nitrogen for immunoblot or fixed in 4% paraformaldehyde for immunohistochemistry (IHC) at 4 h after defibrillation. HIF-1ɑ and Hsp-70 contents were assessed by immunoblots and IHC. Four groups were studied: cardiac arrest + NaCl (n = 9) or Na-pyruvate (n = 7), or sham + NaCl (n = 5) or Na-pyruvate (n = 5). Results: Although immunoblots did not reveal appreciable differences in temporal cortex HIF-1ɑ content among the groups, IHC revealed increased HIF-1ɑ content in pyruvate-treated compared to NaCl-treated sham and cardiac arrest groups. Hsp-70 content also was similar among the groups, suggesting that this cytoprotective protein was not augmented within 3 h after 60 min pyruvate treatment. Conclusions: Current studies are examining neurobehavioral function and brain proteins over 7 days post-arrest. MMP activities in brain are being measured by gel zymography. Future experiments will determine plasma concentrations of neurofilament light chain, which, when elevated up to 7 days post-cardiac arrest, is associated with poor neurological outcomes.
PEDIATRICIAN CARDIOVASCULAR SCREENING RESEARCH
(2013-04-12) Mou, Margaret
Purpose: The objective of this project is to understand general pediatricians' screening and treatment processes in children with a variety of cardiovascular disease risk factors. We believe that less than 50% of general pediatricians follow current national guidelines for screening cholesterol in children ages 9-11. Methods: Pediatricians in the Cook Children's network were given a questionnaire via email from the Medical Staff, where the data was collected electronically. Results: 1. 29% of respondents gave cholesterol screenings 100% of the time in 9-11 year old children in routine check-ups 2. 21% of respondents gave cholesterol screenings 0% of the time in 9-11 year old children in routine check-ups 3. More respondents were likely to have a child's cholesterol screened if they had other conditions, such as Diabetes Mellitus, are obese, and have a family history of cardiovascular disease Conclusions: The sample size of this project was too small to make conclusive results, but it gives an idea of where general pediatricians come from and the mentality behind universal screening for cardiovascular health in children ages 9-11 years old.
IMPROVED DRAINAGE DEVICE FOR CARDIOTHORACIC SURGERY
(2013-04-12) Cherry, Brandon
Purpose: The objectives of this preclinical study are (1) to compare the effectiveness of a novel, Y-shaped, split chest tube vs. conventional, separate tubes to drain pleural and mediastinal effusions following midsternal thoracotomy and thoracic closure; (2) to evaluate the impact of fluid viscosity on the respective abilities of these two tubing configurations to drain the thorax. Methods: A Y-shaped chest tube was developed that allowed for a single incision site while still accommodating separate intrathoracic drainage sites. The split chest drain vs. conventional dual chest drains were compared based on the rate and efficiency of fluid drainage. After sternotomy, pericardiotomy, and left pleurotomy, three domestic pigs (50-60 kg) were fitted sequentially with the Y-shaped tubing and then the separate chest drains to test both configurations in the same animal. The intrathoracic openings of both configurations were positioned in the mediastinum and in the costodiaphragmatic recess, where post-thoracotomy effusions usually accumulate. One liter of 0.58 M sucrose solution, with a viscosity 1.8 times that of pure water and approximating that of plasma, was infused into the thorax and drainage suction was applied at -20 cm H2O for 30 min. Drained volume (mean ± SD) was measured at 1, 5, 10, 20, and 30 minutes to evaluate the efficiency of fluid drainage. Results: Within one minute, the split drain evacuated more fluid (743 ± 556 ml) than the separate drains (630 ± 135 ml). By 30 minutes, the split drain had removed more fluid (1123 ± 71 ml) and left a lower residual volume (22 ± 19 ml) than the separate drains (1120 ± 27 and 43 ± 38 ml, respectively). Results of the current study are being compared with our previous study of low-viscosity D5W in which the Y-shaped tube drained 442 ± 251 ml in the first minute and 1056 ± 60 ml after 30 minute, with a residual volume of 19 ± 24 ml, versus respective values of 194 ± 163, 886 ± 280 and 208 ± 213 ml with separate drains. Conclusions: Initial results of this ongoing study suggest the split chest tube drained the thoracic cavity more effectively than the conventional separate chest tubes, and that the split tube afforded more efficient drainage than the separate tubes when draining fluids of two different viscosities. This new drainage device could potentially reduce postoperative complications of tamponade and residual pleural effusions.
INTERMITTENT HYPOXIA RELATED GENERATION OF REACTIVE OXYGEN SPECIES AND BAROREFLEX REGULATION OF BLOOD PRESSURE
(2013-04-12) Lane, Aphton
Purpose: The intermittent hypoxia related generation of reactive oxygen species (ROS) associated with obstructive sleep apnea (OSA) increases sympathetic activity. However, the specific role of ROS in establishing the hypertension associated with chronic OSA has not been identified. We hypothesize that the intermittent hypoxia (IH) induced production of ROS will reset the operating point of the Carotid Baroreflex to a higher mean arterial pressure (MAP). Methods: Human subjects (n = 11, 5 female) underwent neck pressure/suction (NP/NS) to assess carotid baroreflex (CBR) function before and after intermittent hypoxia training with or without the antioxidant, N-acetyl cysteine (NAC). During NP/NS, mean arterial pressure was recorded non-invasively using a finometer. Following baseline measurements for hemodynamic variables and CBR function, subjects were asked to ingest a drink containing either NAC or a placebo. One hour after ingestion, hemodynamic variables and CBR function were again measured, following which the subjects were intermittent hypoxia trained (IHT). Immediately after IHT, hemodynamic and CBR function measurements were repeated three times 30 minutes apart. A two-way analysis of variance (ANOVA) was used to analyze differences between treatment groups across time. Results: There were no changes in CBR function with NAC versus placebo across time (p=0.791). In addition, the assessment of the operating point of the MAP with NAC versus placebo across time was not different (p=0.62). Conclusions: These data indicate that the known scavenging effect of ROS on central Nitric Oxide enabling a greater central sympathetic outflow was counteracted by an unidentified local vasodilation resulting in an unchanged MAP. The lack of increase in resting MAP following IHT is in agreement with previous studies that report no change in MAP after IHT.
MEDIAN PREOPTIC NUCLEUS NEURONS ACTIVATED FOLLOWING CHRONIC INTERMITTENT HYPOXIA PROJECT TO THE PARAVENTRICULAR NUCLEUS
(2013-04-12) Faulk, Katelynn
Purpose: Chronic Intermittent Hypoxia (CIH) is a model for the arterial hypoxemia seen in sleep apnea and is associated with hypertension and increased sympathetic tone. In rats, the MnPO demonstrates increased FosB expression following CIH. A major projection from the MnPO is to the paraventricular nucleus (PVN). This projection may serve as one possible pathway for increased sympathetic tone and sustained hypertension in CIH. The purpose of this study is to further characterize MnPO neurons transcriptionally activated by CIH and determine if they project to the PVN. Methods: Isoflurane anesthetized adult male rats were microinjected with 200nl of a retrograde tracer, flourogold, unilaterally into PVN. Rats were then exposed to CIH for 7 days through 3 minute periods of hypoxia (10% oxygen) and 3 minute periods of normoxia (21% oxygen) for 8 hours per day (0800-1600 h). Controls were not exposed to to room air. Forebrains were fixed through paraformaldehyde perfusion and later processed for immunohistochemistry. Antibodies used were goat polyclonal FosB (1:2000). The sections were imaged using an Olympus DSU confocal microscope. Results: In the MnPO, CIH exposure increased FosB positive cells (Control 7.75± l.l; CIH 25.7±4.4; P<0.05) and colocalization of FosB with flourogold positive cells (Control 0.67±0.4; CIH 7.25±1.2; P<0.05). Conclusions: These results show that MnPO neurons transcriptionally activated by CIH project to the PVN. P01 HL88052
INTRAVENOUS PYRUVATE FOR CARDIAC ARREST DOES NOT CAUSE PERSISTENT HYPERNATREMIA
(2013-04-12) Cherry, Brandon
Purpose: Intravenous infusion of sodium pyruvate can protect internal organs from ischemia-reperfusion imposed by cardiac arrest and resuscitation, but may produce hypernatremia due to the sodium load. The purpose of this study was to examine the effects of Na-pyruvate infusion on plasma pyruvate, bicarbonate and sodium concentrations in order to test the hypothesis that pyruvate infusion does not cause persistent hypernatremia. Methods: Swine were subjected to 6 min cardiac arrest, 4 min closed-chest CPR, defibrillation and 4 h recovery. Na-pyruvate (n=7) or NaCl control (n=9) were infused iv (0.1 mmol/kg/min) during CPR and the first 60 min recovery. Results: Pyruvate infusion produced a sustained increase in plasma bicarbonate concentration (44.2 ± 1.2 mM in pyruvate-treated vs. 27.5 ± 2.6 mM in NaCl-treated group at 3 hours post-treatment; P<0.05), which may potentially offset post-arrest acidemia. Although pyruvate and NaCl infusions similarly increased plasma sodium concentrations (146 ± 2 mM and 148 ± l mM, respectively), the hypernatremia resolved to pre-arrest concentrations by 3 h post-pyruvate (140 ± 1 mM), but persisted 3 h post-NaCl (147 ± 2 mM; P<0.05). Conclusions: This study confirms the hypothesis that pyruvate administration after cardiac arrest did not cause persistent hypernatremia.
EFFECTS OF INSPIRATORY RESISTANCE ON CEREBRAL BLOOD VELOCITY DURING ORTHOSTASIS WITH DEHYDRATION
(2013-04-12) Moralez, Gilbert
Purpose: Inspiratory resistance increases low frequency (LF) middle cerebral artery blood velocity (MCAv) oscillations and delays symptoms of pre-syncope during lower body negative pressure, despite reductions in absolute MCAv. We tested the hypothesis that during central hypovolemia induced by combined dehydration and orthostasis (via head-up tilt, HUT), inspiratory resistance would increase MCAv oscillations and attenuate pre-syncopal symptoms. Methods: 10 subjects were tilted to 70° for 10-min under euhydration (EUH) and dehydration (DEH; 40 mg of furosemide/fluid restriction) conditions, with and without inspiratory resistance (Impedance Threshold Device, ITD) in a randomized, cross-over design. Results: Arterial pressure (AP) and MCAv were measured continuously. DEH decreased blood (6.5±0.9%) and plasma (11.2±1.4%) volume (both P<0.01), but did not impact AP or MCAv (P≥0.25) at rest. HUT decreased AP and MCAv under both EUH and DEH (P≤0.016) with no difference between hydration conditions (P=0.498). ITD breathing did not attenuate the fall in AP or MCAv under either hydration condition (P≥0.123), and subjective pre-syncopal symptoms were similar across all conditions (P=0.78). Conclusions: ITD breathing increased MCAv LF oscillations under both hydration conditions during HUT (P≤0.05). While ITD breathing increased MCAv LF oscillations during DEH and HUT, it did not effect MCAv or the reporting of pre-syncopal symptoms.
ROLE OF CATECHOLAMINERGIC A2 NEURONS OF NUCLEUS OF THE SOLITARY TRACT (NTS) IN CARDIOVASCULAR AND RESPIRATORY ADAPTATIONS TO CHRONIC INTERMITTENT HYPOXIA (CIH) IN RATS
(2013-04-12) Bathina, Chandra Sekhar
Purpose: To understand the effect of CIH on the mRNA expression levels of AT1a, AT1b and excitatory amino acid (EAAs) receptor subunits in the A2 neurons of NTS and to assess the effect of tyrosine hydroxylase (TH) knockdown in A2 neurons on the cardiovascular responses to CIH Methods: Adult male Sprague-Dawley rats (250-350g) were injected with adeno-associated viruses, either (AAV-GFP-TH) to label the A2 neurons or (AAV-TH-shRNA) to knockdown TH in A2 neurons, into NTS. The control rats for (AAV-TH-shRNA) were injected with virus having scrambled RNA (AAV-sc). All the (AAV-TH-shRNA) and (AAV-sc) injected rats were implanted with radiotelemetry transmitters, 7 days prior to receiving virus injections, to monitor the changes in mean arterial pressure (MAP), heart rate (HR) and respiratory frequency (RF). 14 days after virus injections 7 of AAV-GFP-TH injected rats, all AAV-TH-shRNA and AAV-sc injected rats were exposed to 7 days of CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). 7 of AAV-GFP-TH injected rats were used as controls and were exposed to room air. Laser capture microdissection (LCM) was performed to capture 7-10 A2 neurons for RNA extraction. Gene expression for different genes were assessed by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and compared between the control and CIH rats using 2-ΔΔct method. Hindbrains collected from AAV-TH-shRNA and AAV-sc injected rats were utilized for immunohistochemistry and western blot analysis Results: qRT-PCR study showed that CIH decreased AT1a (p=0.002; control - 1.08 ± 0.13, n=7; CIH - 0.48 ± 0.07, n= 6) and AMPA receptor subunit GluR2 (p=0.03; control - 1.11 ± 0.24, n=7; CIH- 0.52 ± 0.12, n= 6) and increase transcription factor FosB (p=0.03; control - 1.14 ± 0.25, n=7; CIH- 1.97 ± 0.25, n= 5) mRNA expression levels in the A2 neurons. CIH increased MAP and HR during the day in both the scRNA (n= 14, p<.001 MAP; p<.001HR) and shRNA (n=13, p<.001 MAP; p<.001 HR) groups. During the night, MAP and HR remained elevated in the scrambled rats (p<0.001 MAP; p<0.001 HR) but not in the shRNA group which can be attributed to the significant reduction in TH-immunoreactivity in NTS (shRNA: 28±lcells/section; scRNA: 35±lcells/section) Conclusions: These results suggest that A2 neurons play a role in the cardiovascular responses to CIH and following CIH, NTS neurons may become more calcium permeable, as GluR2 is found to resist calcium permeability
COMPREHENSIVE MODEL OF AUTONOMIC CONTROL OF HEART RATE DURING EXERCISE
(2013-04-12) White, Daniel
Purpose: Exercise Physiology textbooks illustrate the autonomic control of heart rate (HR) at the beginning of dynamic exercise as an immediate and selective withdrawal of parasympathetic (PS) control of the Sino-Atrial node resulting in a rise in HR up to 100 beats per min. The appearance of norepinephrine in the blood after 100 bpm was interpreted as the time at which the sympathetic nervous system was activated. In the past ten years animal investigations indicate that increases in sympathetic outflow are involved in the increase in HR at exercise onset. Therefore, we hypothesized that re-analysis of previous investigations utilizing pharmacologic blockade of the autonomic nervous system and our carotid baroreflex modeling techniques of humans during dynamic exercise would identify the role of sympathetic activity in exercise. Methods: Comprehensive analysis of HR data presented in 8 "autonomic neural control of heart rate" research articles and book chapters in both humans and animals coupled with our own neck pressure/neck suction published data was used to generate a novel model of the autonomic control of HR during exercise. Results: Throughout the progression of exercise (HR = 100, 117, 150), neck pressure stimuli induce reflexive increases in HR (+6, 3, 1), and pharmacological blockade with glycopyrrolate induces increases in HR (+24, 21, 12). Comprehensive regression modeling of the increases in heart rate during exercise identified that sympathetic activity establishes the exercise intensity related HR and it is the PS control of the heart that expresses the baroreflex response. Conclusions: These data provide compelling evidence that cardiac sympathetic and PS activities were continuously active throughout exercise. Furthermore, there was no evidence of an abrupt shift from PS withdrawal to sympathetic activation in autonomic control of HR during exercise but rather a steady rise in HR due to increasing cardiac sympathetic tone. Contrary to there being a PS withdrawal at the onset of exercise previous work identify that during exercise onset increases in PS tone dampen the sharp rise in HR in response to the immediate increase in cardiac sympathetic activity.
MINERALOCORTICOID RECEPTOR IN NTS STIMULATES SALT INTAKE DURING 4TH VENTRICULAR INFUSIONS OF ALDOSTERONE
(2013-04-12) Koneru, Bhuvaneswari
Purpose: The purpose of this study was to determine if neurons within the NTS that possess the mineralocorticoid receptor (MR) play a role in aldosterone stimulation of salt intake. Methods: Adult WKY rats received microinjections of Adeno associated virus (AAV) with either short hairpin RNA for the MR (shRNA; n=9) or a scrambled RNA (scRNA; n=8) into the NTS. The vector used to knock-down the MR is AAV1/2-U6-Rat.Nr3c3/MR.shRNAterminator-CAG-EGFP-WPRE-BGH-polyA. Immediately after the viral construct injections, aldosterone-filled osmotic mini-pumps were implanted subcutaneously and connected to tubing within the 4th ventricle to infuse aldosterone (dissolved in aCSF) at a rate of 20ng/h. Prior to and after surgeries, rats had ad libitum access to food and two graduated drinking bottles filled with distilled water and 0.3M NaCl respectively. Salt intake was expressed as 100 X the ratio of 0.3M NaCl intake to total fluid intake (salt plus water). To verify that the shRNA reduced MR levels in the NTS, immunohistochemistry was performed for the MR and HSD2. Cells demonstrating immunoreactivity were counted in NTS sections from each rat and expressed as an average number per section. Results: Prior to surgeries, basal salt intake was 4.7% ± 2.9% in the scRNA group and 3.91% ± 2.6% in the shRNA group. 24 days post-surgery salt intake was 20.6% ± 2.9% in the scRNA group and 4.3% ± 2.8% in the shRNA group. Post-mortem immunohistochemistry revealed a significant reduction in the number of NTS neurons exhibiting immunoreactivity for the MR (scRNA 33 ± 2 cells/section; shRNA 23 ± 1 cells/section; p=.008) with no significant change in the number of HSD2 immunoreactive neurons. Conclusions: These results indicate 4th ventricular infusions of aldosterone stimulate salt intake and that at least part of the stimulation of salt intake during 4th ventricular infusions of aldosterone is mediated by hindbrain NTS neurons that possess the MR.
A CASE FOR A STANDARDIZED METHOD FOR THE NORMALIZATION OF MUSCLE SYMPATHETIC NERVE ACTIVITY AMPLITUDE
(2013-04-12) White, Daniel
Purpose: MSNA has no agreed upon standard for analysis. For example, mean burst strength (MBS), total activity (TA) and total MSNA (TMSNA) rely on amplitude measurements assigned arbitrary units which are highly dependent on the method of normalization. We investigated the hypothesis that computer algorithms used for automatic normalization of MSNA amplitudes differ from those obtained with manual normalization leading to erroneous interpretations. Methods: MSNA from healthy human subjects (n=8, 2 female) was obtained by microneurography of the peroneal nerve while resting quietly in a 30* semi-recumbent position. Data was analyzed by 3 different normalization methods (Min-Max Auto (MM), Mean Auto (MA), and Rectified Manual (RM)). MSNA is quantified as burst frequency (BF), burst incidence (BI), MBS, TA, and TMSNA. Results: There were no differences in BF or BI between the three analysis methods. However, significant differences were detected between MM and RM in MBS, TA and TMSNA (p=0.002, p=0.009, and p=0.004). Rankings according to activity were not different between MM and RM but were different in MA compared to either MM or RM. Conclusions: There is an overwhelming need to standardize the analysis of MSNA. The current study points out that the variables BF and BI can be compared across studies. However, our data identify that the RM normalization is the only method to quantify MBS, TA and TMSNA for cross-study comparisons.
THE ROLE OF SLEEP APNEA INDUCED REACTIVE OXYGEN SPECIES IN SYMPATHOEXCITATION
(2013-04-12) McKay, Pilar
Purpose: The obstructive sleep apnea (OSA) patient experiences multiple hypopneas and apneas, or intermittent hypoxia (IH) throughout the night. In healthy subjects, one 20s to 30s bout of IH has been reported to increase muscle sympathetic nervous activity (MSNA) for periods of 3hrs. Results from animal and human investigations indicate that apnea induced elevations in reactive oxygen species (ROS) are associated with increases in MSNA. Increases in sympathetic activity result in increases in the operating point of the arterial baroreflex's control of blood pressure. We hypothesized that the IH induced increase in ROS and sympathoexcitation would attenuate arterial baroreflex (ABR) control of stroke volume (SV). Methods: Healthy human subjects (n = 11, 5 female) underwent neck pressure/suction (NP/NS) to assess carotid baroreflex (CBR) function before and after intermittent hypoxia training with or without the antioxidant, N-acetyl cysteine (NAC). During NP/NS, mean arterial pressure and stroke volume were recorded non-invasively using a finometer. Following baseline measurements for hemodynamic variables and CBR function, subjects were asked to ingest a drink containing either NAC or a placebo. One hour after ingestion, hemodynamic variables and ABR function were measured, following which the subjects were intermittently hypoxia trained (IHT) using 20s of intermittently breathing nitrogen and breath holding resulting in IH. After that, measurements of SV were recorded during NP/NS (immediately 30 minutes and 1 hour post IHT). A two way repeated measures analysis of variance (ANOVA) was used to analyze differences between treatment groups across time. Results: Repeated measures (ANOVA) comparing stroke volume between treatment groups identified that there were no differences in SV over time (p = 0.332). There was no difference in CBR function (p = 0.891) between placebo and NAC conditions. Conclusions: These data suggest that there is no significant influence of IHT or ROS on the CBR control of stroke volume.
THE ROLE OF DIFFERENTIAL OXYGEN DISTRIBUTION BETWEEN THE BRAIN AND PERIPHERAL TISSUES ON TOLERANCE TO INDUCED CENTRAL HYPOVOLEMIA
(2013-04-12) Kay, Victoria
Purpose: Maintenance of cerebral perfusion and oxygenation is essential for survival from hemorrhage. As individuals vary in tolerance to reduced blood volume (e.g., central hypovolemia), we hypothesized that subjects with high tolerance (HT) will maintain cerebral oxygenation compared to low tolerant (LT) subjects, which will be associated with greater peripheral vasoconstriction. Methods: 13 human subjects were instrumented for cerebral (ScO2) and muscle (SmO2) oxygenation (near-infrared spectroscopy, NIRS), then completed a step-wise presyncopal-limited lower body negative pressure (LBNP) protocol. Total hemoglobin (HbT) was calculated as an index of local peripheral resistance (inverse relationship) at the muscle. Results: Progressive decreases in ScO2 were observed in LT subjects (n=5) from -45 mmHg LBNP (P≤0.01 compared to baseline), while Sc02 did not change throughout LBNP for the HT subjects (n=8; P≥0.44). Sm02 decreased at -30 and -60 mmHg LBNP (P≤0.01) in the HT group with concomitant increases in resistance (decreases in HbT; P≤0.03). In contrast, both Sm02 (P=0.01) and HbT (P=0.06) only decreased at -60 mmHg LBNP in the LT group. Conclusions: In support of our hypothesis, increased tolerance to progressive central hypovolemia was associated with maintained cerebral oxygenation, due, in part, to earlier onset of peripheral vasoconstriction.
AT1A RECEPTOR KNOCKDOWN IN THE MEDIAN PREOPTIC NUCLEUS ATTENUATES THE SUSTAINED COMPONENT OF HYPERTENSION RESULTING FROM CHRONIC INTERMITTENT HYPOXIA
(2013-04-12) Shell, Brent
Purpose: Obstructive sleep apnea produces hypertension and increases sympathetic nerve activity during the hypoxic sleeping events as well as during the waking hours. This study examined a possible receptor contribution to the sustained component of hypertension. Methods: To model the hypoxemia experienced during sleep apnea, rodents are exposed to chronic intermittent hypoxia (CIH). The median preoptic nucleus (MnPO) is a forebrain region that contributes to this sustained increase in mean arterial pressure (MAP) from CIH. This region integrates information from forebrain circumventricular organs and projects to the paraventricular nucleus to influence sympathetic nerve activity. Our lab has previously shown that in response to CIH there is increased ΔFosB expression in the MnPO and that after a 7 day hypoxia protocol MnPO AT1a receptor mRNA is increased 7 fold whereas AT1b shows no change. In this study, we tested the hypothesis that MnPO AT1a receptors contribute to CIH hypertension using adult male Sprague Dawley rats injected with a neuron specific adenoviral vectors with either shRNA to knock down expression of AT1a receptors in the MnPO or a scrambled RNA sequence. Results: All rats showed an increase in MAP during the hypoxic light period, but rats receiving the AT1a knock down did not exhibit the sustained increase in MAP during the normoxic dark phase (P<.05). Conclusions: This data indicates that the AT1a receptors in the MnPO is necessary for the sustained component of hypertension resulting from CIH.
METABOLIC DYSFUNCTION IN CHRONIC INTERMITTENT HYPOXIA- ROLE OF HYPOTHALAMIC PEPTIDES
(2013-04-12) Nedungadi, T.Prashant
Purpose: Obstructive sleep apnea (OSA) is associated with an increase in cardiovascular risk factors that includes metabolic syndrome including hypertension. Our model of Chronic Intermittent Hypoxia (CIH) mimics the hypoxemia and the persistently elevated arterial pressure seen in OSA patients. Here we examine, if metabolic dysregulation associated with CIH is initiated through neuropeptides in central metabolic sites, arcuate nucleus (ARH) and paraventricular nucleus (PVN) of the hypothalamus. Methods: Body weight, daily food intake were measured. Biochemical assays were used for measuring hormonal levels. Metabolic studies to identify glucose tolerance, insulin tolerance and pre-disposal to diabetes were performed. Laser Capture Microdissection followed by real time RT-PCR were carried out for measurement of neuropeptide and receptor mRNA levels in the ARH and PVN along with immunohistochemical studies. Results: Body weight change was higher in the controls (CON) compared to CIH (CON: 25 ± 2.5; CIH: 16 ± 2.7). Food intake was significantly lower for CIH rats compared to controls (p < 0.03) Corticosterone (CORT) was elevated 6 fold during CIH. CIH caused reduction in fasting blood glucose levels, and improved glucose tolerance. Laser capture microdissection of ARH and PVN followed by RT-PCR showed significant increase in ΔFosB mRNA in both PVN and ARH (PVN CON: 1± 0.1 vs PVN CIH: 1.96 ± 0.14; ARH CON: 1 ± 0.18 vs ARH CIH: 2.5 ± 0.5). Neuropeptide Y mRNA levels was significantly decreased after CIH in ARH. However leptin levels were significantly higher in the PVN of CIH rats. Preliminary immunohistochemical observations suggest elevated FosB in both these regions. Conclusions: This is the first study to examine if metabolic dysfunction associated with CIH, characteristic feature of sleep apnea, is initiated through key metabolic sites in the brain. Metabolic dysregulation associated with CIH might be initiated centrally through peptides in the ARH-PVN hypothalamic circuitry.

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