Integrative Physiology

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    Elevated Renal Oxidative Stress and Na+ Transporters are Associated with Hypertension in Postpartum Preeclamptic Rats
    (2024-03-21) Smith, Savanna; Castillo, Angie; Jones, Kylie; Smith, Jonna; Hart, Savannah; Powell, Madison; Cunningham, Mark
    Approximately 5-10% of US pregnancies result in preeclampsia (PE). PE is characterized by new onset hypertension (HTN) during pregnancy and is usually accompanied by end-organ damage, especially in the kidneys. Postpartum (PP) women and dams that had PE have an increased risk of developing HTN and chronic kidney disease (CKD) later in life. However, mechanisms linking PE to the long-term development of HTN and CKD are unknown. One aspect that may contribute to renal injury in PP PE women and dams is oxidative stress. Elevated concentrations of oxidative stress have been shown to augment the abundance and activity of renal transporters to increase sodium (Na+) reabsorption and blood volume. These alterations in renal transporters can consequently facilitate HTN. We hypothesize that at 6 weeks PP (~3 human years), PE dams will display oxidative stress, renal Na+ transporter abundance, and elevated blood pressure (BP). Pregnant Sprague Dawley rats were assigned to two groups: normal (CON) and PE dams. On gestational day 14, the reduced uterine perfusion pressure surgery was performed to generate a model of PE. Dams gave birth naturally and weaned for 3 weeks. After 6 weeks PP, BP was measured via carotid catheterization, and kidneys were removed and sectioned. Western blots were used to quantify renal Na+ transporters: Na+ K+ 2Cl-transporter (NKCC2) in the kidney medulla (KM) and epithelial Na+ channel (ENaC) in both the kidney cortex (KC) and KM. Oxidative stress was evaluated by heat shock protein 1 (HSP-1), copper zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) via Western blots. Hydrogen peroxide (H2O2) and antioxidant capacity concentrations were assessed via colorimetric assays. PP PE dams had increased BP (126.3±6.18vs105.7±3.74 mmHg, p<0.05) at 6 weeks after birth. KC HSP- 1, H2O2, MnSOD, and antioxidant capacity were unchanged between groups. However, KC CuZnSOD protein abundance was decreased in PP PE dams (69.51±11.64vs100±5.73 IU/Protein/Control%, p<0.05). In the KM, HSP-1 abundance (113.7±3.3vs100±5.07 IU/Protein/Control%, p=0.06) and H2O2 concentrations (1.97±0.11vs1.31± 0.38 nM H2O2/mg Protein, p=0.08) were elevated in PP PE dams. MnSOD, CuZnSOD, and antioxidant capacity were unchanged between groups in the KM. No changes occurred in KC and KM ENaC protein abundance. However, NKCC2 protein abundance was elevated by ~50% in PP PE dams (151.71±22.17vs100±5.59 IU/Protein/Control%, p=0.06). In summary, BP, oxidative stress, and NKCC2 were elevated in PP PE dams at 6 weeks. The presence of oxidative stress in the KM may lead to increased NKCC2 abundance. However, more studies are warranted to make this conclusion. NKCC2 elevation may result in increased Na+ and water reabsorption, leading to an increase in BP. Future studies will assess renal oxidative stress regulation of Na+ transporters in PP PE dams and determine the timeline PP in which changes in oxidative stress, Na+ transporters, and BP occur. This study is clinically relevant, because it indicates oxidative stress and NKCC2 in the KM, separately or together, may have a formative role in the pathogenesis of HTN and CKD in PP PE women later in life.
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    The Effect of 0.1 Hz Blood Flow Oscillations on Microvascular Blood Flow Responses Following Severe Ischemia
    (2024-03-21) Davis, K. Austin; Bhuiyan, Nasrul; McIntyre, Benjamin; Rickards, Caroline
    Background: We have shown that inducing 10 second (0.1 Hz) oscillations in arterial pressure and blood flow protects against reductions in tissue oxygenation during ischemia, independent of changes in macrovascular blood flow. However, it is unknown whether 0.1 Hz hemodynamic oscillations impacts microvascular function and vasodilatory capacity following severe ischemia. To examine this question, we assessed the reactive hyperemic response following a prolonged peripheral limb ischemia protocol with and without induced 0.1 Hz hemodynamic oscillations. Hypothesis: 0.1 Hz oscillations in blood pressure and blood flow will increase microvascular blood flow, assessed via reactive hyperemia following a 10-min period of ischemia. Methods: Thirteen healthy human participants (6M, 7F; 27.3 ± 4.2 y) completed two experimental protocols separated by ≥48 h. In both conditions, ischemia of the forearm was induced with a pneumatic cuff on the upper arm to decrease brachial artery blood velocity by ~70-80% from baseline. In the oscillation condition (OSC), 0.1 Hz oscillations in mean arterial pressure (MAP) and brachial artery blood flow were induced by inflating and deflating bilateral thigh cuffs every 5-s (10-s cycles; 0.1 Hz) throughout the forearm ischemia period. In the control condition (CON), the thigh cuffs were in place, but were inactive throughout the forearm ischemia period. Beat to beat arterial pressure was measured via finger photo plethysmography, and brachial artery diameter and blood velocity were measured via duplex Doppler ultrasound during baseline, ischemia, and the reperfusion period. The maximum mean brachial artery blood velocity, and 3-min area under the curve (AUC) of mean brachial artery blood velocity were used to determine the reactive hyperemia response. Results: The magnitude of forearm ischemia, indexed by the reduction in brachial artery conductance, was matched between conditions (CON: -74.8 ± 10.4% vs. OSC: -75.6 ± 6.7%, p=0.39). Reactive hyperemia was not different between conditions as indexed by maximum mean brachial artery blood velocity (CON: 36.4 ± 12.4 cm/s vs. OSC: 39.3 ± 11.2 cm/s, p=0.53) or 3-min brachial artery blood velocity AUC (CON: 1495 ± 744 (cm/s)2 vs. OSC: 1596 ± 804 (cm/s)2, p=0.74). Conclusion: Inducing 0.1 Hz hemodynamic oscillations during severe ischemia does not affect microvascular function, indexed by reactive hyperemia following release of the ischemic stimulus. A more direct measure of microvascular blood flow is needed to examine whether 0.1 Hz hemodynamic oscillations improves microvascular perfusion during ischemia.
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    Renal Oxidative Stress May Explain Sex Differences in Blood Pressure in Adult Offspring Exposed to an Impoverished Environment
    (2024-03-21) Castillo, Angie; Smith, Savanna; Smith, Jonna; Jones, Kylie; Powell, Madison
    Nearly 40 million people experience poverty in the U.S. Poverty is linked to adverse childhood experiences (ACEs) which affects ~64% of adults in the U.S. Previous studies indicate that those who experience ACEs are at a higher risk of developing hypertension (HTN) and cardiovascular diseases (CVDs) later in life, with a greater severity and earlier onset in males. The mechanisms behind the ACEs-attributed development of sex differences in HTN later in life is unknown. One plausible mechanism for this sex difference is increased oxidative stress. One rodent model to mimic poverty as an ACE is the limited bed and nesting (LBN) model. This model simulates an impoverished and low resource environment, as observed in poverty, in which the nesting material during weaning is reduced. We hypothesize that male offspring exposed to LBN will have elevated blood pressure and oxidative stress, while females will have no change in blood pressure and reduced oxidative stress. Pregnant Sprague Dawley rats gave birth naturally and weaned their offspring for 3 weeks. During the weaning period, on Days 2-9, the dams and their respective pups were divided into 2 groups: LBN and control (CON). After LBN treatment, all rats received normal bedding. After weaning, offspring were divided by sex and experimental status: LBN male (n=5), LBN female (n=5), CON male (n=6), and CON female (n=6). At 16-17 weeks, mean arterial pressure (MAP) was measured via carotid catheterization and the kidneys, brain, heart, and plasma were collected to measure antioxidant capacity (AC) via colorimetric biochemical assays. LBN males had a significant increase (18 mmHg) in MAP compared to CON males (128.17±3.93 vs 110.72± 3.93 mmHg, P>0.001), while female LBN and CON rodents displayed no differences. In males, there were no changes in antioxidant capacity in the brain, heart, and plasma. However, there was a significant 2-fold decrease in renal antioxidant capacity (233±14.0 vs 442±12.3 mM Trolox/mg protein, p>0.0001). In females, there were no changes in the kidneys, heart, and brain antioxidant capacity. Conversely, LBN females showed a trending increase in plasma antioxidant capacity compared to CON (3.33±0.16 vs 2.53±0.35 mM Trolox/mg protein, p=0.053). Males exposed to an impoverished environment during weaning have elevated blood pressure, while females do not. This difference in blood pressure may be explained by decreases in renal AC in males only. On the other hand, females may be protected from elevated blood pressure because they experience a slight increase in systemic AC. Future studies will examine the role of antioxidants in blood pressure regulation in the kidneys. This is clinically relevant because ACEs affect a large percentage of the American population with ~17% of adults experiencing 4 or more ACEs, with minorities at a greater risk. Understanding the mechanisms on how ACEs contribute to HTN may alleviate some of the racial and ethnic disparities for people with HTN and CVDs. Perhaps, organ and sex-specific antioxidant therapies may prevent or reduce the development of HTN in adults that were exposed to ACEs, like poverty during childhood.
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    Pulsatile Perfusion Therapy at 0.1 Hz Improves Survival Following Severe Hemorrhage in Rats
    (2024-03-21) Dinh, Viet; Farmer, George; Rickards, Caroline
    PURPOSE: Oscillations in arterial pressure and blood flow at 0.1 Hz are associated with protection of tissue oxygenation during conditions of reduced tissue perfusion. Pulsatile Perfusion Therapy (PPT) is a method we have developed to induce these oscillations, and has been associated with increased tolerance to simulated hemorrhage via lower body negative pressure in humans. However, it is unknown how effective this therapy would be in an actual hemorrhage model. The aim for this study was to test the efficacy of PPT in a rat model of severe hemorrhage. We hypothesized that PPT at 0.1 Hz would protect arterial pressure and cerebral blood flow following severe hemorrhage in rats, subsequently improving survival. METHODS: Eleven adult Sprague-Dawley rats (six female, five male) were anesthetized via isoflurane, then underwent bilateral carotid artery catheterization for assessment of arterial pressure and carotid artery blood flow, while heart rate was assessed via lead II ECG. Following a 15-min baseline period, all animals were hemorrhaged to 50% of their estimated blood volume over 30-min. Rats were then randomly assigned to the PPT group (3 female, 3 male) or the control group (CON; 3 female, 2 male). PPT was administered via inflatable cuffs attached to both hind limbs, oscillating between 0 mmHg and 250 mmHg every 5-s (10-s cycles or 0.1 Hz) for 30-min immediately following hemorrhage. For the CON group, the leg cuffs were also attached but were not inflated for this 30-min period. All animals were then monitored for an additional 150-min recovery period post-hemorrhage, or until death – defined as the absence of ventricular function on the ECG. Survival time and peak mean arterial pressure (MAP), carotid blood flow, and heart rate were assessed to determine the effectiveness of PPT in protecting hemodynamic responses following hemorrhage. RESULTS: PPT increased survival time (P = 0.02), with 3 of 6 (50%) rats in the PPT group and 0 of 5 (0%) rats in the CON group surviving the entire 180-min recovery period following hemorrhage. During recovery, PPT protected MAP (PPT: −46.5 ± 12.9% vs. CON: −72.6 ± 19.5% from baseline; P = 0.07) and carotid blood flow (PPT: −61.5 ± 17.7% vs. CON: −83.7 ± 6.7% from baseline; P = 0.04), but did not affect heart rate (PPT: 0.11 ± 6.58% vs. CON: −18.70 ± 29.34% from baseline, P = 0.20), although responses were highly variable between subjects. CONCLUSIONS: PPT protected arterial pressure and carotid blood flow in rats following severe hemorrhage, subsequently improving survival. These data add further evidence for the use of 0.1 Hz hemodynamic oscillations as a therapeutic intervention for the treatment of hemorrhage.
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    Interactions Between Carotid Arterial Stiffness, Amplitude of Cerebral Blood Flow Oscillations, and Cerebral Tissue Oxygenation During Simulated Hemorrhage in Humans
    (2024-03-21) Hudson, Lindsey; Davis, Austin; Anderson, Garen; Rosenberg, Alexander; McKeefer, Haley; Bird, Jordan; Pentz, Brandon; Byman, Britta; Jendzjowsky, Nicholas; Wilson, Richard; Day, Trevor; Rickards, Caroline
    Introduction: Inducing 0.1 Hz (10-s cycle) oscillations in cerebral blood flow attenuates the reduction in cerebral tissue oxygenation during simulated hemorrhage in humans. It is unknown, however, how stiffness of the cerebral feed arteries influences the magnitude of cerebral blood flow oscillations, and/or the protection of cerebral tissue oxygenation. When 0.1 Hz oscillations are induced during simulated hemorrhage, we hypothesize that: 1) arterial stiffness of the internal carotid artery (ICA) will increase from rest; 2) the amplitude of 0.1 Hz oscillations in cerebral blood flow will be higher in individuals with stiffer arteries, and; 3) the reduction in cerebral tissue oxygenation will be smaller with higher amplitude of cerebral blood flow oscillations. Methods: 8 healthy human participants (age: 30.1±7.6 y) underwent a 10-min hypovolemic oscillatory lower body negative pressure (OLBNP) protocol, where chamber pressure oscillated every 5-s between -30 mmHg and -90 mmHg (i.e., 0.1 Hz). ICA beta stiffness index was calculated from measurements of ICA diameter (via ultrasound imaging), and arterial pressure (via finger photoplethysmography). Middle cerebral artery velocity (MCAv) was measured using transcranial doppler ultrasound, and cerebral tissue oxygenation (ScO2) was measured with near infrared spectroscopy. Fast Fourier transformation was used to quantify oscillations in mean MCAv at ~0.1 Hz. Results: While Mean MCAv 0.1 Hz oscillations increased from baseline to OLBNP (N=8, 34.0±33.9 (cm/s)2vs. 104.7±58.1(cm/s)2, p=0.01), ICA beta stiffness did not increase (N=5, 6.1±0.7 au vs. 8.2±2.7 au, p=0.21). There was no relationship between baseline ICA beta stiffness and the percent change in mean MCAv 0.1 Hz oscillations (N=5; r=0.44, p=0.46). ScO2 decreased from baseline to OLBNP (N=8, 66.5±2.9 % vs. 64.8±2.9%,p=0.03), but there was also no relationship between the percent change in mean MCAv 0.1 Hz oscillations and the decrease in ScO2(r=0.28, p=0.50). Conclusions: Based on these data, 0.1 Hz OLBNP does not affect ICA stiffness, and there is no relationship between ICA stiffness, amplitude of induced 0.1 Hz cerebral blood flow oscillations, and the reduction in cerebral tissue oxygenation during simulated hemorrhage. However, as this analysis was performed retrospectively, and arterial stiffness was not initially an outcome measure, there was limited data available for analysis. This limitation will be addressed in a project currently in progress in our laboratory.
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    Assessment of Neuroinflammation in Cognitive and Motor Brain Regions in Female Rats Exposed to Chronic Intermittent Hypoxia
    (2024-03-21) Appiah, Cephas; Little, Joel; Kunwar, Kishor; Cunningham, Tom
    Sleep apnea increases the risk of neurodegenerative disorders in postmenopausal women. In this study, we tested whether chronic intermittent hypoxia (CIH), a preclinical model of sleep apnea-associated cyclical hypoxia, can be used to identify early changes in the brain that might contribute to impair neurological function in intact (INT) and ovariectomized (OVX) female rats. To test this hypothesis, we conducted immunohistochemistry studies using markers for glial activation and neuroinflammation. We hypothesize that CIH will increase glial activation in ovariectomized (OVX) relative to intact (INT) female rats. Adult female Sprague Dawley INT (n=4) or OVX (n=4) rats that were part of a larger study and underwent 7 days of CIH (10% O2 and 21% O2 cycle, every 6 mins, 8h/day during the light phase) or continuous normoxia (CON) were euthanized on day 8, and their brains were collected. Brains were processed for microglia (IBA1) and astrocytes (GFAP) activation markers in (CA1) hippocampus, medial prefrontal cortex (mPFC), and caudate putamen (CP) striatum. Confocal images from each region are being used to optimize a fractal analysis protocol to test for changes in glial morphology. Raw images will be linearly processed in Huygens Essentials software to limit noise and optimize quality for further analysis in ImageJ. Skeletal and fractal analyses will be performed on randomly selected cells in the photomicrographs to determine cell ramification (branching, junctions, endpoint voxels, branch lengths) and complexity (fractal dimension, cell span ratio, density) to complement cell counts of immunohistochemical markers of activation. Our preliminary analysis has allowed us to determine the appropriate parameters needed for image capture and subsequent analysis. We have observed some possible qualitative changes indicative of increased activation in the CA1, CP, and mPFC regions following CIH in OVX rats relative to intact rats. The results of this study aim to contribute to our understanding of the potential impact of CIH in female rats of differing ovarian function, providing insights into sleep apnea-associated CNS dysfunction in women.
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    Evaluating the Role of Arterial Stiffness on Amplitude of Cerebral Blood Flow Oscillations
    (2024-03-21) Lal, Kevin; Davis, Austin; Anderson, Garen; Bhuiyan, Nasrul; Rickards, Caroline
    Background: Changing the pattern of cerebral blood flow by forcing oscillations in arterial pressure and blood flow at 0.1 Hz (10-second cycle) can limit reductions in cerebral tissue oxygenation during a condition of reduced cerebral perfusion. This method of inducing 0.1 Hz hemodynamic oscillations is called Pulsatile Perfusion Therapy (PPT). Sympathetic activation can increase the amplitude of 0.1 Hz hemodynamic oscillations, and acutely increase arterial stiffness. The impact of increasing carotid arterial stiffness on the magnitude of 0.1 Hz cerebral blood flow oscillations has not been examined. We hypothesize that the with application of 0.1 Hz PPT during a condition of cerebral hypoperfusion, 1) the subsequent increase in sympathetic activity will acutely increase carotid arterial stiffness, and; 2) greater carotid artery stiffness will result in a higher amplitude of oscillations in cerebral blood flow. Methods: 10 healthy participants (8 males, 2 females) were exposed to 10-min of oscillatory lower body negative pressure (OLBNP) at 0.1 Hz, which induced both a state of cerebral hypoperfusion, and 0.1 Hz hemodynamic oscillations. Middle cerebral artery velocity (MCAv), internal carotid artery (ICA) diameter, and beat-to-beat arterial pressure were measured. ICA stiffness was determined using the beta-stiffness index, incorporating ICA diameter and arterial pressure measurements. The amplitude of 0.1 Hz MCAv oscillations was assessed via fast Fourier transformation. Results: While OLBNP increased MCAv 0.1 Hz oscillations (36.1 ± 24.2 cm/s2 vs. 812.4 ± 668.0 cm/s2; P=0.01), ICA beta stiffness was not different between the baseline and OLBNP conditions (12.3 ± 4.9 au vs. 13.2 ± 5.7 au; P=0.56). There was no relationship between ICA stiffness and the amplitude of MCAv oscillations during OLBNP (r=0.17, P=0.68). Conclusions: Contrary to our hypothesis, ICA stiffness did not increase during 0.1 Hz OLBNP, and there was no correlation between ICA stiffness and the magnitude of MCAv oscillations induced at 0.1 Hz. These data suggest that ICA stiffness may not determine the magnitude of induced oscillations in cerebral blood flow. Future studies will examine these effects in older adults to determine the potential beneficial application of PPT for the treatment of low cerebral perfusion conditions (e.g., Alzheimer’s disease, stroke).