Integrative Physiology

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    (2021) Cushen, Spencer; Ricci, Contessa; Bradshaw, Jessica L.; Silzer, Talisa; Blessing, Alexandra M.; Sun, Jie; Scroggins, Sabrina; Santillan, Mark; Santillan, Donna; Phillips, Nicole; Goulopoulou, Styliani
    PURPOSE: Cell-free circulating mitochondrial DNA (CFCmtDNA) is a damage-associated molecular pattern (DAMP) that activates Toll-like receptor-9 (TLR-9). Previous studies suggested that CFCmtDNA may be a potential pathogenic trigger or a contributor to the maintenance of preeclampsia. The main objectives of this study were 1) to determine absolute concentrations of CFCmtDNA, in membrane-bound and -unbound states, independent of nuclear DNA (nDNA) changes, in cases with preeclampsia and healthy controls and 2) to implement a penalized regression analysis to establish the contribution of CFCmtDNA to preeclampsia diagnosis and its interaction with commonly collected patient characteristics. METHODS: Plasma CFCmtDNA (MT-ND5 gene) concentrations were quantified using an absolute quantification protocol. DNase I concentrations in maternal plasma were measured using an enzyme-linked immunosorbent assay and TLR-9 activity was monitored using SEAP reporter 293 cells expressing the human TLR-9 gene. RESULTS: Concentrations of CFCmtDNA were reduced in preeclampsia compared to healthy controls both in lysis buffer-treated samples (P=0.02) and in samples not treated with lysis buffer (P< 0.0001). Even though CFCmtDNA concentrations were reduced, plasma from women with preeclampsia induced greater TLR-9 activation than plasma from gestational age matched controls (P< 0.01). Multivariate analysis showed that high concentrations of nDNA and DNase I, a prior history of preeclampsia, and a lower concentration of CFCmtDNA are predictors of preeclampsia diagnosis. CONCLUSIONS: In conclusion, our data demonstrate an increased immunostimulatory potential of CFCmtDNA and upregulation of DNA degradation mechanisms in women with preeclampsia at the third trimester.
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    Early Life Stress in Summer Months Accelerates the Progression of Autoimmunity in Female Lupus-Prone Mice
    (2021) Hartman, Rusty; Mathis, Keisa W.
    Systemic lupus erythematosus (SLE) is an autoimmune disorder with a high prevalence of renal disease. The progression of SLE is tracked by plasma double-stranded (ds) DNA autoantibodies. We have used an established model of SLE, female NZBWF1 mice, to determine mechanisms associated with SLE-induced renal disease. Typically, mice arrive by truck from Maine at 5-6 weeks of age and are not manipulated until 30-35 weeks when renal inflammation/injury is evident. Based on anecdotal observations in different cohorts of SLE mice, we hypothesized that seasonal factors, along with the stress associated with travel in early life, accelerate disease course in aged SLE mice. We performed a retrospective analysis of 5-6 week old female NZBWF1 mice ordered between 2015-2019 and divided them into two groups based on their date of arrival: summer (April-September; n=38) and winter (October-March; n=51). Average temperature on dates of arrival was higher in summer (78.5 ± 1.8 vs. 52.5 ± 1.9 °F; p< 0.001). Plasma dsDNA autoantibodies at 34-35 weeks was higher in SLE mice that arrived in summer (6.0e5 ± 8.6e4 vs. 4.1e5 ± 5.1e4 U/mL; p=0.049) and this coincided with higher indices of renal injury in SLE mice that arrived in summer (urinary albumin: 13.3e3 ± 2.6e3 vs. 6.0e3 ± 1.5e3 ug/mL; p=0.0096). Our findings suggest early life stress compounded with seasonal factors modulate autoimmunity. Future studies will investigate biochemical processes associated with warmer temperatures and their impact on the progression of SLE-associated renal disease.
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    Immunological alterations in murine systemic lupus erythematosus following α7-nicotinic acetylcholine receptor antagonism
    (2021) Kulp, Dennis; Mathis, Keisa W.; Stubbs, Cassandra
    Systemic lupus erythematosus (SLE) is an autoimmune disease associated with pathology of multiple organs and the morbidity and mortality that ensues can be reduced with anti-inflammatory strategies. The cholinergic anti-inflammatory pathway, is an endogenous neuroimmune reflex that inhibits pro-inflammatory cytokine release through stimulation of α7 nicotinic acetylcholine receptors (α7nAChR) on splenic immune cells. Our published studies demonstrate anti-inflammatory capability of α7nAChR activation via nicotine, but loss-of-function studies are needed to further delineate the role of this receptor in SLE. Thus, we hypothesized that α7-nicotinic receptor antagonism with methyllycaconitine would exacerbate SLE's inflammatory cascade. Female SLE (NZBWF1) mice were administered methyllylcaconitine (MLA; 10 mg/kg/day IP) or saline for 14 consecutive days starting at 33 weeks of age and were then euthanized with their spleen and bone marrow processed for flow cytometry (n = 4/group). The percentage of splenic CD3+CD8+ T cells was higher in SLE mice treated with MLA (27.9±4.8 vs. 20.4±3.5%; P=NS), but CD3+CD4+ T cells (66.6±4.3 vs. 73.8±3.2%; P=NS) and CD11c dendritic cells (1.9±4.8 vs. 9.2±5.0%; P=NS) were lower in MLA-treated compared to saline-treated SLE mice. CD3+CD8+ T cells were also higher in the bone barrow of MLA-treated SLE (21.3±0.3% vs. 13.9±3.9 %; P=NS). Although not significant, these results suggest blockade of α7nAChRs potentiates the cytotoxic inflammatory profile in SLE mice. Future work will confirm the role of the α7nAChR in potentiating inflammation and end organ damage in SLE.
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    Acute Intermittent Optogenetic Stimulation of Median Preoptic Nucleus (MnPO) Induces Sympathetic Long-term Facilitation (sLTF)
    (2021) Paundralingga, Obed; Gusson Shimoura Almeida Lima, Caroline; Cunningham, Joseph
    Acute intermittent hypoxia can produce a ramp increase in sympathetic nerve activity (SNA). The induction of this increase, termed sympathetic long-term facilitation (sLTF), involves the paraventricular nucleus (PVN). The median preoptic nucleus (MnPO) projects to the PVN and might mediate the induction of sLTF. This study used an intersectional viral approach to test the ability of acute intermittent optogenetic (AIO) stimulation of PVN-projecting MnPO neurons to induce sLTF. Male Sprague-Dawley rats were microinjected with CRE-containing retrograde-AAV in PVN and with either Channelrhodopsin 2-containing AAV or control virus in MnPO. This resulted in CRE-dependent expression of either ChR2 or mCherry in MnPO-PVN neurons. Following a 4-wk recovery, rats underwent AIO experiments where SNA, blood pressure (BP), and heart rate (HR) were recorded under anesthesia. MnPO terminals in the PVN were stimulated with a train of 5 Hz stimuli. A single optogenetic duty cycle did not alter splanchnic SNA, renal SNA, blood pressure, nor heart rate. AIO significantly increased splanchnic SNA (P=0.0163) in rats with ChR2 as compared to rats injected with the control vector 1h post-AIO. There were no differences in renal SNA, BP, and between rats injected with ChR2 as compared to those injected with the control vector 1h post-AIO. These results suggest that intermittent activation of PVN-projecting MnPO neurons may induce sLTF in the splanchnic SNA but not in renal SNA. Further study is needed to clarify the mechanisms responsible for the differential effects of MnPO-PVN AIO.
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    Store-operated calcium entry mediated high glucose-induced podocyte injury and mitochondrial impairment
    (2021) Tao, Yu; Chaudhari, Sarika; Yazdizadeh Shotorbani, Parisa; Ma, Rong
    Purpose: Diabetic Nephropathy (DN) is a major complication of diabetes mellitus, a metabolic disease initiated by high glucose (HG). Podocyte injury is associated with progression of DN. However, the mechanisms underlying podocyte injury induced by HG is poorly understood. Store-operated calcium entry (SOCE) is a multifunctional signaling pathway in many cell types. However, its role in podocyte injury in DN is not known. The aim of the present study was to determine if SOCE mediated HG-induced podocyte injury by impairing mitochondria. Methods: Immortalized human podocytes were used for all experiments. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and nephrin (a podocyte specific protein). Calcium imaging was used to analyze SOCE. TMRE fluorescence was used to probe the mitochondria membrane potential (MMP).Results: HG (25mM) treatment for time periods ranging from 2 to 12 hours significantly increased Orai1 protein abundance. This HG effect was dose dependent. Consistently, Ca2+ imaging showed that HG (25 mM for 12 hours) significantly enhanced SOCE. Furthermore, the protein abundance of nephrin decreased in podocytes exposure to HG (25 mM), indicating podocyte injury by ambient HG. BTP2 (4 µM), a selective SOCE inhibitor blunted the HG-induced nephrin reduction. Moreover, HG (25 mM for 24 hours) decreased MMP, indicating mitochondria damage by HG. The MMP decrease was prevented by BTP2, suggesting the contribution of SOCE to the detrimental effect of HG.Conclusion: An upregulated SOCE contributes to HG-induced podocyte injury, possibly by impairing mitochondrial function.
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    Role of A1/A2 Neurons in the Dysregulation of Vasopressin Release and Dilutional Hyponatremia in Liver Disease
    (2021) Aikins, Ato; Little, Joel; Cunningham, Joseph
    Abstract Purpose: Inappropriate release of arginine vasopressin (AVP) has been linked to dilutional hyponatremia in patients with cirrhosis. Elevated Plasma AVP causes water retention, hypoosmolality, ascites formation, and a perceived decrease in plasma volume. The perceived decrease in plasma volume is sensed by the A1/A2 norepinephrine neurons in the caudal ventrolateral medulla (CVLM) and the nucleus tractus solitarius (NTS) respectively. We propose that these neurons provide the initial stimuli that activates AVP-secreting neurons in the supraoptic nucleus (SON) leading to inappropriate AVP release and dilutional hyponatremia. Method: Adult male rats were bile duct ligated (BDL) to model cirrhosis. Selective lesioning of the SON-projecting A1/A2 norepinephrine neurons was achieved using anti-DβH-Saporin [IT-03] (Advanced Targeting Systems). Plasma copeptin concentration was measured as a surrogate marker for AVP using ELISA. Plasma osmolality and hematocrit measurements were also taken. Immunohistochemistry for delta FosB and dopamine beta-hydroxylase (DβH) was performed on brain slices. Results: Lesions of the A1/A2 neurons projecting to SON (Saporin/BDL n=9) was associated with decreased copeptin as compared to BDL controls (Vehicle/BDL, n=6, p< 0.05). However, the number of delta FosB immunoreactive A1/A2 cells was not significantly different. While A1/A2 lesions seemed to normalize osmolality and hematocrit in the BDL rats, the trends were not statistically significant. Conclusion: The result suggests that A1/A2 neurons could contribute to increased plasma AVP seen in male BDL rats, but there could be other contributing factors preventing a recovery of plasma osmolality.
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    Sympathetic Response to Resistance Breathing During Simulated Hemorrhage in Humans
    (2021) Davis, Kenneth; Sprick, Justin; Kay, Victoria; Rickards, Caroline
    Background: Resistance breathing increases venous return and stroke volume by amplifying the respiratory pump during inspiration. Breathing with resistance devices augments reflex compensatory mechanisms to improve tolerance to hypovolemic stress, such as hemorrhage. We tested the hypothesis that protection of arterial pressure with resistance breathing during simulated hemorrhage is due to the mechanical effect of the respiratory pump, and not due to increases in sympathetic nervous system activity. Methods: Lower body negative pressure (LBNP) was used to simulate hemorrhage in eight human subjects (4M, 3F; 27.5±1.6 y). Two experiments were conducted (randomized order): 1) a control condition in which ramp LBNP was applied at 3 mmHg/min until the onset of presyncope, and 2) a resistance breathing condition in which the same LBNP protocol was used, but subjects breathed through a resistance device during the final stages of the protocol. Mean arterial pressure and stroke volume were monitored continuously, and blood samples were collected every 5-min for measurement of plasma norepinephrine and epinephrine. Results: There were no differences in the reduction in stroke volume (control, -36.9±13.8% vs. resistance breathing, -33.5±17.1%, P=0.31) or mean arterial pressure (control, -5.5±4.2% vs. resistance breathing, -6.3±7.4%, P=0.63) between conditions. LBNP induced increases in both norepinephrine (P=0.03) and epinephrine (P=0.003), but there was no main effect of resistance breathing on these responses (norepinephrine, P=0.8; epinephrine, P=0.48). Conclusion: These data support the hypothesis that, based on circulating catecholamine responses, resistance breathing has no direct effect on sympathetic drive to compensate for central hypovolemia.
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    The Effect of Sex on GABAA Receptor Activation in Vasopressin Neurons from the Supraoptic Nucleus
    (2021) Brock, Courtney; Bachelor, Martha E.; Little, Joel; Farmer, George; Balapattabi, Kirthikaa; Cunningham, Joseph
    Purpose: Arginine Vasopressin (AVP) is important in maintaining proper fluid balance and plasma osmolality. Disruption in its regulation occurs in patients with chronic heart failure (CHF) and liver failure, which leads to poorer patient outcomes. AVP neurons from the supraoptic nucleus (SON) receive input from GABA, yet it is unknown what effects GABAA receptor activation has on these neurons under pathophysiological conditions or whether the effects are sex-specific. What is known is that under pathophysiological conditions, AVP neurons are unaffected by negative feedback which leads to excessive AVP release. Understanding the role of the GABAA receptor in these conditions is important. Here, we investigate whether activation of the GABAA receptor leads to sex dependent effects. Methods: Adult, intact, Sprague Dawley rats were anesthetized and bilaterally injected with the AAV2-0VP1-ClophensorN virus directly into the SON. After a two-week recovery, the animals were sacrificed and the brains were rapidly removed. Cells from the SON were dissociated and incubated for two hours. After incubation, recordings were taken using ratiometric live cell imaging. Selected neurons were sequentially excited at 445nm and 556nm and then emission data was collected between 500-550nm and 580-653nm respectively. After 40 cycles of 3-second recordings, muscimol (100nM), a GABAA receptor agonist was transiently applied to the cells. Results: In both males and females, application of muscimol resulted in chloride influx, which implies neuronal inhibition. Conclusion: Under normal physiological conditions, GABAA receptor activation does not show sex specific effects in neurons from the SON.
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    Impact of Sleep Quality on Cardiovascular Responses to Simulated Hemorrhage in Humans
    (2021) Hua, Vincent; Barnes, Haley J.; Rosenberg, Alexander; Anderson, Garen K.; Luu, My-Loan; Rickards, Caroline
    Poor sleep quality may limit cardiovascular responsiveness to physiological stress. We hypothesized that subjects with poor sleep quality would be less tolerant to simulated hemorrhage, which would be associated with lower arterial pressure and cerebral blood flow, and higher heart rates compared to subjects with good sleep quality. Hemorrhage was simulated in 20 human subjects with lower body negative pressure (LBNP). Sleep quality was classified as POOR in 5 subjects (Global Pittsburgh Sleep Quality Index (PSQI) score ≥5), and GOOD in 15 subjects (Global PSQI score < 5). Markers of cardiovascular function were measured continuously throughout the LBNP protocol. Sleep quality had no effect on LBNP tolerance (POOR: 1453±223 s vs. GOOD: 1535±88 s; P=0.34), and there were no differences in the magnitude of central hypovolemia at presyncope (%Δ stroke volume, POOR: -53±8 % vs. GOOD: -49±4 %; P=0.32). However, there were differences in the magnitude of hypotension (%Δ mean arterial pressure, POOR: -18±3 % vs. GOOD: -22±2 %; P=0.08), cerebral blood flow reduction (%Δ MCAv, POOR: -19±6 % vs. GOOD: -28±2 %; P=0.03), and reflex tachycardia (% Δ heart rate, POOR: 103±30 % vs. GOOD: 72±9 %; P=0.09). There was a moderate association between sleep quality and the magnitude of MCAv reduction at presyncope (r=0.53; P=0.02). Sleep quality did not affect tolerance to simulated hemorrhage in healthy human subjects. While there were differences in hemodynamic responses, this may be related to premature termination of the protocol due to early onset of subjective presyncopal symptoms.
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    Peaks and Valleys - Oscillatory cerebral blood flow at high altitude
    (2021) Anderson, Garen K.; Rosenberg, Alexander; Barnes, Haley J.; Bird, Jordan D.; Pentz, Brandon; Byman, Britta; Jendzjowsky, Nicholas; Wilson, Richard; Day, Trevor; Rickards, Caroline
    An oscillatory pattern in cerebral blood flow (at ~0.1 Hz) protects cerebral tissue oxygen saturation (ScO2) under conditions of cerebral hypoperfusion. In this study, we hypothesized that inducing oscillations in cerebral blood flow at 0.1 Hz would protect cerebral blood flow and ScO2 during exposure to combined simulated hemorrhage and sustained hypobaric hypoxia. Eight healthy human subjects (4 M:4 F, 30.1 ± 7.6 y) participated in two lower body negative pressure (LBNP) experiments (simulating hemorrhage) at high altitude (3800 m): 1) 0 Hz control condition (CTRL) and 2) 0.1 Hz oscillatory LBNP (OLBNP) condition. Measurements included internal carotid artery (ICA) blood flow via duplex Doppler ultrasound, middle cerebral artery velocity (MCAv) via transcranial Doppler ultrasound, and ScO2 via near-infrared spectroscopy. Mean MCAv waveforms were fast Fourier transformed to verify oscillations were generated at ~0.1 Hz. Low frequency power (0.07-0.15 Hz) in mean MCAv increased during OLBNP vs. CTRL (P = 0.05). OLBNP did not protect ICA flow (OLBNP: -32.5±12.2 Δ%; CTRL: -20.2±24.3 Δ%; P = 0.18) or mean MCAv (OLBNP: -26.5±13.8 Δ%; CTRL: -17.7±15.7 Δ%; P = 0.58), but ScO2 was protected (OLBNP: -0.89±0.61 Δ%; CTRL: -3.99±2.2 Δ%; P = 0.007). These results support our hypothesis that inducing oscillatory blood flow leads to protection of cerebral tissue oxygenation, despite no differences in ICA blood flow or mean MCAv. Overall, these data suggest that therapies using oscillatory perfusion may help preserve cerebral tissue oxygen saturation under conditions of reduced oxygen delivery.
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    Spatial Transcriptomics of Supraoptic Nucleus and Paraventricular Nucleus of the Hypothalamus in the Adult Rat
    (2021) Nguyen, Dianna; Hales, Nicole; Cunningham, Joseph
    Purpose: Magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus release oxytocin (OXY) and arginine vasopressin (AVP) into the peripheral circulation, playing a key role in the regulation of body fluid homeostasis. Transcriptomics of MNCs have been studied; however, the data lack spatial context important for better understanding physiological functions of genes. Our study leverages spatially-resolved transcriptomics to address this limitation. Methods: 10x Genomics' Visium Spatial Gene Expression was used to obtain spatially-resolved gene expression data for SON and PVN of an adult male Sprague-Dawley rat. Briefly, the workflow includes: 1) sample preparation (section, mount, stain, and image tissue), 2) cDNA library preparation, 3) sequencing, and 4) analysis/data visualization. Results: Gene cluster analysis successfully differentiated myelinated fiber tracts from nuclei and identified several distinct neuronal populations. Clusters overlaying fiber tracts contain glia-specific markers (e.g., Mbp and Gfap), and those overlaying SON and PVN regions contain neuron-specific markers (e.g., Syn1 and Nrgn). SON clustered with magnocellular PVN, while parvocellular PVN was a different cluster. Differential expression analysis of these two clusters revealed relevant genes for the distinct regions and neuronal subpopulations in the top 20 globally distinguishing genes (e.g., Avp and Oxt for SON and magnocellular PVN cluster and Trh for parvocellular PVN cluster). Conclusions: This data allows us to obtain whole transcriptomic data without sacrificing morphological context. Future spatial transcriptomic studies will investigate transcriptomics of MNCs related to sex differences and disease models that can potentially inform pathophysiology.
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    Optimization of machine perfusion to preserve structure and function of porcine kidney
    (2021) Wade, Michael; Mallet, Robert T.; Yurvati, Albert; Williams, Arthur; Hare, Richard; Rayad, Ashraf; Chaudhari, Sarika
    Purpose: Improved preservation of explanted kidneys is essential to narrow the supply vs. demand disparity for transplantable human kidneys. This study evaluated whether the use of buffer containing mannitol, a non-toxic osmolyte that minimizes cellular edema, improves kidney preservation during prolonged hypothermic perfusion. Methods: Left kidneys were harvested from anesthetized Yorkshire pigs via laparotomy, followed by cold (2-4°C) machine perfusion for 72 h. Flow rate and resistance were recorded throughout perfusion to assess renal vascular function, then biopsy for histological analysis of hematoxylin and eosin-stained renal cortex and medulla. Results: Kidneys with comparable initial flows and resistances received standard (A) or mannitol-enriched (B) perfusion buffer. Flows (ml/min)/resistances (mm Hg/ml/min) were 65/0.40 (A) vs. 65/0.38 (B) at 5 h, 27/1.03 (A) vs. 54/0.46 (B) at 24 h, and 13/1.99 (A) vs. 49/0.50 (B) at 48 h perfusion. The mass of Kidney A increased by 47%, and that of kidney B by 39%, over 72 h. Histology revealed improved preservation of tubular and glomerular architecture in B vs. A. Conclusion: The addition of mannitol afforded appreciable improvement in renal vascular function during machine perfusion and reduced tubular necrosis when compared to mannitol-free perfusion. Preservation of renal structure using mannitol-enhanced perfusion buffer was superior to the current standard of cold machine perfusion buffer for preserving human kidneys for transplant. By reducing cellular damage and maintaining renal perfusion, the optimization of cold machine perfusion with mannitol holds the potential to increase the availability of kidneys suitable for transplantation.