Integrative Physiology

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    Sex Differences in Responses of Nucleus Tractus Solitarii Neurons to Acute Tissue Hypoxia
    (2019-03-05) Mifflin, Steve; Paundralingga, Obed
    Obstructive sleep apnea (OSA) is a risk factor associated with cardiovascular diseases, such as hypertension. Females are protected from the hypertensive and tachycardia effects of chronic intermittent hypoxia model of OSA. The present study examined the change in nucleus tractus solitarius (nTS) intracellular calcium level response to exposure to acute hypoxia in male rats and in female rats and the role of voltage gated calcium channel (VGCC) in mediating the response. Adult rat brainstem slices (250 μm thick) containing commissural and caudal nTS were incubated for 45 min with 10 μM Fura-2AM and 30 μL of F127 at room temperature and then washed for 20 min in artificial cerebrospinal fluid (aCSF) bubbled with 95% O2/5%CO2. A single slice was transferred to the recording chamber on an upright epifluorescent microscope and superfused with normal aCSF bubbled with 20% O2/5%CO2 balanced with N2 at a rate of 2.5 ml/min. Acute hypoxia was established by exposing hindbrain slices to aCSF bubbled with 95% N2/5% CO2. In slices where the role of VGCC was being studied, the slice was preincubated with 20 µM nifedipine in aCSF before the recording and was exposed to hypoxia in the presence of the same drug during the recording period. Fluorescence of Fura-2AM was excited by epi-illumination with light filtered alternatively at 340 or 380 nm while the emitted light passed through a barrier filter (510 nm). Pairs of 340 and 380 nm images were acquired at intervals of 5 s and analyzed off-line with NIS-Elements AR 3.2 software to yield 340/380 ratio. A total of 22 sections were examined from 3 male and 3 female rats and an average of 6 cells/slice were analyzed in each section. Five minutes of hypoxic aCSF triggered a greater increase from baseline in slices from male rats compared to female rats (males: 3±0.16%, n=49 vs females: 2.2+0.19%, n=40; P2+]i as a response to acute tissue hypoxia in caudal NTS is differentially regulated between male and female rats and is also dependent, at least in part, upon Ca2+ influx through L-type voltage gated calcium channel in the males. As hypoxia-induced elevations in intracellular Ca2+ are likely to alter caudal NTS neuronal function under hypoxic conditions, its differential regulation in males and females might mediate some of the protection seen at organismal level.
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    Peak Analysis of Cerebral Blood Velocity Responses to Forced Low Frequency Oscillations during Simulated Hemorrhagic Stress in Humans
    (2019-03-05) Anderson, Garen; Rosenburg, Alexander; Park, Flora; Sprick, Justin; Rickards, Caroline; Barnes, Haley J.
    Peak Analysis of Cerebral Blood Velocity Responses to Forced Low Frequency Oscillations during Simulated Hemorrhagic Stress in Humans Haley J. Barnes, B.S., Garen K. Anderson, M.S., Alexander J. Rosenberg, Ph.D., Flora S. Park, M.S., Justin D. Sprick, Ph.D., Caroline A. Rickards, Ph.D Purpose: Tolerance to blood loss injuries (actual and simulated) varies across individuals. Higher amplitude of low frequency oscillations (10-s cycle; ~0.1 Hz) in brain blood flow and arterial pressure have been associated with higher tolerance to simulated hypovolemic episodes using lower body negative pressure (LBNP). We have previously demonstrated that forcing oscillations in cerebral blood flow and arterial pressure at 0.1 Hz and 0.05 Hz with oscillatory LBNP (OLBNP) protects cerebral oxygenation during central hypovolemia. However, there was no protection of mean cerebral blood flow (indexed via mean middle cerebral artery velocity, MCAv) with these oscillatory conditions. We hypothesize that the peak mean MCAv will be higher in the 0.05 Hz and 0.1 Hz OLBNP conditions compared to the 0 Hz condition, which may account for the protection of cerebral tissue oxygenation. Methods: Fourteen healthy human subjects (3 female/11 male) were randomly exposed to 10-min of non-oscillatory (0 Hz) and oscillatory (0.05 Hz and 0.1 Hz) LBNP conditions with an average LBNP chamber pressure of -60 mmHg. Measurements included MCAv via transcranial Doppler ultrasound, frontal lobe cerebral oxygenation (ScO2) via near infrared spectroscopy, and stroke volume and arterial pressure via finger photoplethysmography. Peak analysis was performed in 10-s and 5-s windows for the 0.05 Hz and 0.1 Hz profiles, respectively. Peak responses to the three LBNP conditions were compared using a linear mixed model for repeated measures with Tukey post hoc tests. Results: As previously reported, tolerance to the two OLBNP conditions was higher compared to the 0 Hz condition (P ≤ 0.09 for both vs. 0 Hz). In partial support of our hypothesis, when compared to the 0 Hz profile, the peak MCAv was higher with 0.05 Hz OLBNP (51.0±4.2 cm/s vs. 46.3±3.4 cm/s; P = 0.004) but not with the 0.1 Hz profile (49.0±3.9 cm/s; P = 0.11 vs. 0 Hz). Conclusions: The higher peak MCAv during the 0.05 Hz OLBNP profile may contribute to the attenuated decrease in cerebral oxygenation. These findings demonstrate the potential contribution of oscillatory peaks in cerebral blood flow to the protection of cerebral oxygenation and increased tolerance to simulated hemorrhage.
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    Examining the Sex Effect on Oxidative Stress during Simulated Hemorrhage Induced by Lower Body Negative Pressure
    (2019-03-05) Luu, My-Loan; Kay, Victoria; Sprick, Justin; Rosenburg, Alexander; Mallet, Robert T.; Rickards, Caroline; Park, Flora
    Purpose Traumatic hemorrhage is one of the leading causes of death in both the military and civilian settings. Massive blood loss is known to elicit an increase in oxidative stress as a consequence of tissue ischemia and hypoxia. We have recently demonstrated that simulated hemorrhage via application of lower body negative pressure (LBNP) also elicits an increase in oxidative stress (indexed by circulating F2-isoprostanes). It is not clear, however, whether oxidative stress responses to stimulated hemorrhage are differentiated based on sex. The aim of this study was to assess sex differences in the oxidative stress response during simulated hemorrhage via application of pre-syncopal LBNP. Methods Fifteen healthy human subjects (11 M, 4 F) participated in a LBNP step protocol until presyncope (-15, -30, -45, -60, -70, -80, -90, -100 mmHg LBNP for 5-min each). Venous blood samples were collected at baseline and at presyncope then analyzed for F2-isoprostanes. Stroke volume and mean arterial pressure were obtained via finger photoplethysmography, while muscle oxygen saturation was measured via a near infrared spectroscopy device attached to the forearm. Time to reach presyncope was measured in seconds. Results The following results are only preliminary based on the small number of female subjects tested (N=4). There was no difference in tolerance to LBNP between males and females (Males: 1616 ± 132 s vs. Females: 1486 ± 216 s; P=0.63). F2-isoprostane concentrations were similar between the sexes at baseline (P=0.27), and there was no statistical difference in the % change in concentration with application of maximal LBNP (Males: 37.0 ± 15.4 % vs. Females: 5.0 ± 10.1; P=0.11). The decreases in stroke volume (Males: -52.4 ± 5.3 % vs. Females: -56.5 ± 5.5 %; P=0.50),mean arterial pressure (Males: -11.9 ± 3.4 % vs. Females: -14.6 ± 4.2 %; P=0.63), and muscle oxygen saturation (Males: -9.1± 1.7 % vs. Females: -9.4 ±2.3 %; P=0.91) were also similar between males and females. Conclusions These preliminary data indicate that there is no effect of sex on the oxidative stress response induced by application of simulated hemorrhage with maximal LBNP. This analysis is limited and inconclusive, however, as there were only 4 females and 11 males in this group of subjects. In our current study, we plan to recruit equal numbers of males and females to further explore whether biological sex plays a role in the oxidative stress response to blood loss injuries.
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    Caspase Lesions of PVN-Projecting MnPO Neurons Blocks the Sustained Component of CIH-Induced Hypertension in Adult Male Rats
    (2019-03-05) Wang, Lei; Little, Joel; Cunningham, J. Thomas; Marciante, Alexandria B.
    Purpose: Obstructive Sleep Apnea (OSA) is characterized by cessations in respiration that leads to development of hypertension and persists into the waking period even during normal respiratory patterns. Previous studies show that experimental models of chronic intermittent hypoxia (CIH) produces sustained hypertension similar to that with OSA. It has been proposed that peripheral and CNS renin-angiotensin systems contribute to hypertension associated with CIH. Our working hypothesis is that increased circulating angiotensin II feeds into the forebrain, increasing excitatory signaling through the hypothalamus and hindbrain, creating a vicious cycle. The median preoptic nucleus (MnPO) is an integrative forebrain region that contributes to blood pressure regulation. The MnPO has projections to the paraventricular nucleus (PVN) of the hypothalamus, which contains pre-autonomic centers that project to regions in the hindbrain and regulate sympathetic outflow. We hypothesize that lesioning pathway specific projections from the MnPO to the PVN could attenuate CIH hypertension. Methods: Adult male Sprague-Dawley rats (250-300g) were anesthetized with isoflurane and stereotaxically injected bilaterally in the PVN with a retrograde AAV containing Cre (AAV9.CMV.HI.eGFP-Cre.WPRE.SV40) and with the caspase-3 (AAV5-flex-taCasp3-TEVp) or control virus (AAV5-hSyn-DIO-mCherry) in the MnPO. After 1-week recovery, rats were instrumented with aortic radio telemetry and allowed an additional week recovery. Rats were then moved to new homecages and underwent baseline recording before undergoing our 7-day CIH protocol. Results: The control group exposed to CIH developed chronic hypertension, however, caspase lesions blunted the sustained hypertension. Brain tissue processed for FosB immunohistochemistry showed decreased expression with caspase-induced inhibition in the MnPO and downstream autonomic regulating nuclei. CIH significantly increased plasma advanced oxidative protein products (AOPP) levels in controls. This increase in AOPP levels was blocked in caspase-lesioned rats comparable to normoxic control concentrations. In situ hybridization experiments indicate a reduction in angiotensin type 1a receptors (AT1aR) expression in the caspase-lesioned group exposed to CIH compared to CIH controls. Conclusion: The results indicate that MnPO neurons that project to the PVN play a significant role in blood pressure regulation and in the development of persistent CIH hypertension.
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    Acute Leg Heating Protects Against Vascular Ischemia-Reperfusion Injury in Humans
    (2019-03-05) Romero, Steven; Engelland, Rachel
    Purpose: Reperfusion that follows a period of ischemia paradoxically reduces vasodilator function in humans, a phenomenon referred to as vascular ischemia-reperfusion (I/R) injury. Acute whole-body hot water immersion protects against vascular I/R injury in young healthy humans. However, the effect of acute leg heating on I/R injury is unclear. Therefore, the purpose of this study was to test the hypothesis that acute lower leg heating prevents the attenuation in macro- and microvascular dilator function following I/R injury in young healthy humans. Methods: Three healthy male subjects (age 25 ± 4 years; height 177 ± 10 cm; weight 86 ± 16 kg; mean ± SD) immersed their lower legs into a circulated water bath for 60 min under two thermal conditions: 1) thermoneutral immersion (~33 °C); 2) hot water immersion (~42 °C). The order of thermal conditions was randomized and counterbalanced. Macrovascular (brachial artery flow-mediated dilation) and microvascular (reactive hyperemia area under the curve) dilator function were assessed using Doppler ultrasound at three time points: 1) pre-immersion; 2) 60 min post-immersion; 3) post-I/R. Vascular I/R injury was induced by occluding brachial artery blood flow for 20 min, followed by 20 min of reperfusion. Results: Compared with pre-immersion (6.1 ± 0.6%), macrovascular dilator function was decreased 60 min following thermoneutral immersion (4.3 ± 0.6%; P P P = 0.4) and following I/R injury (5.3 ± 0.6%; P = 0.4 vs. pre-immersion). Microvascular dilator function was decreased 60 min following thermoneutral immersion (pre-immersion 3.3 ± 0.1 mL mmHg-1 vs. 60 min post-immersion 2.5 ± 0.3 mL mmHg-1; P -1; P = 0.1 vs. pre-immersion). However, microvascular dilator function did not differ from pre-immersion (3.1 ± 0.5 mL mmHg-1) to 60 min post hot water immersion (3.0 ± 0.3 mL mmHg-1; P = 0.7), but still tended to decrease following I/R injury (2.5 ± 0.3 mL mmHg-1; P = 0.1). Conclusions: Taken together, acute leg heating appears to prevent the decrease in macrovascular dilator function that occurs following I/R injury in young healthy humans. Further evidence is needed to determine if acute leg heating equally protects the microvasculature following I/R injury.