Integrative Physiology

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    Effect of Sigma-1 Receptor Activation on Renal Injury and Hypertension in Female Mice with Lupus
    (2023) Dinh, Viet; Chaudhari, Sarika; Young-Stubbs, Cassandra M.; Shimoura, Caroline; Tucker, Selina; Essajee, Salman; Warne, Cooper; Luedtke, Robert R.; Mathis, Keisa W.
    Systemic lupus erythematosus (SLE) is a female-dominant autoimmune disease with prominent renal injury and hypertension, contributing to its morbidity and mortality. Novel therapies to reduce these detrimental outcomes could be beneficial to SLE patients. The sigma-1 receptor (S1R) is a cytoprotective ligand-regulated chaperone protein that decreases protein aggregation, cellular stress, and cell death, thus preventing tissue injury. S1R activation with pharmacological ligands enhances cytoprotection in autoimmune diseases like multiple sclerosis and Huntington’s disease; however, the efficacy of S1R agonists in SLE is unknown. We hypothesize that S1R activation via the agonist LS-1-127 will reduce renal injury and halt the progression of hypertension in SLE mice. Female SLE (NZBWF1) and control (NZW) mice were weighed and urine collected via metabolic cages weekly starting at 30 weeks of age. Albuminuria was measured via dipsticks. At 33 weeks of age, SLE and control mice were treated with LS-1-127 (10 mg/kg IP) or equal volume of vehicle (10% DMSO; IP) three times a week for two weeks. At 35 weeks, mean arterial pressure (MAP) was measured in conscious mice using indwelling carotid catheters for two consecutive days and then mice were euthanized. Wire myography was used to assess potassium chloride (KCl)-induced contraction and acetylcholine (ACh)-induced relaxation in excised aorta. Markers of renal injury – urinary neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), and creatinine – as well as plasma double-stranded (ds)DNA autoantibodies were measured by ELISA. Albuminuria was present in 44.4% (4 of 9) of SLE mice and no controls. LS-1-127 did not improve albuminuria in SLE mice (50%; 3 of 6). NGAL:creatinine ratio (ng/mg) was higher in SLE mice compared to controls (327.3 ± 119.8 vs 63.2 ± 4.3 ng/mg; n=9–12; P=0.0007). LS-1-127 did not significantly alter NGAL:creatinine ratio in SLE mice (484.3 ± 209.0; n=6) or controls (71.7 ± 5.2; n=10). KIM1:creatinine ratio (ng/mg) did not differ between groups. dsDNA autoantibodies were higher in SLE mice compared to controls (6.9e5 ± 1.1e5 vs. 1.4e5 ± 3.1e4 U/mL; n=9–10; P<0.0001). LS-1-127 did not significantly alter dsDNA autoantibodies in SLE mice (7.1e5 ± 1.2e5; n=6) or controls (1.5e5 ± 4.0e4; n=10). MAP was higher in SLE mice compared to controls (146 ± 4 vs. 123 ± 3 mmHg; n=9–10; P <0.0001). LS-1-127 did not significantly alter MAP in SLE mice (150 ± 8; n=6) or controls (124 ± 2; n=10). KCl-induced aortic contraction was similar in SLE and controls (21 ± 7 vs. 25 ± 4 mM, n=3–4). Sensitivity to KCl after LS-1-127 treatment was 11 ± 3 and 21 ± 2 mM in SLE and controls (n=2–4). ACh-induced aortic relaxation did not differ between groups. In conclusion, two weeks of S1R activation with LS-1-127 did not significantly alter markers of renal injury, autoimmunity, blood pressure, or vascular reactivity in female SLE mice with advanced disease. Further inquiry into the effect of LS-1-127 on the expression of renal proinflammatory cytokines will be conducted. S1R activation at different stages of SLE disease progression also warrants future investigation.
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    Effect of selective ganglion stimulation on hypertension in systemic lupus erythematosus
    (2023) Johnson, Keanna; Dinh, Viet; Young-Stubbs, Cassandra M.; Shimoura, Caroline; Chaudhari, Sarika; Mathis, Keisa W.
    Effect of selective ganglion stimulation on hypertension in systemic lupus erythematosus Keanna K. Johnson1, Viet Dinh2, Cassandra M. Young-Stubbs2, Caroline G. Shimoura2, Sarika Chaudhari2, Keisa W. Mathis2 1School of Public Health,2Department of Physiology and Anatomy, University of North Texas Health Science Center, Fort Worth, Texas Purpose: Systemic lupus erythematous (SLE) is a female-dominant autoimmune disease that causes widespread inflammation in various organs. Inflammation precedes the prevalent hypertension in the disease. The cholinergic anti-inflammatory pathway (CAP) is an endogenous neuroimmune pathway that reduces inflammation upon stimulation. We hypothesize that stimulation of the CAP by selective activation of the superior cervical ganglion will halt disease progression and hypertension in SLE. Methods: Female SLE (NZBWF1) and control (NZW) mice received unilateral microinjections of pAAV-hSyn-hM3D(Gq)-mCherry, a designer receptor exclusively activated by designer drug (DREADD), or pAAV-hSyn-mCherry (vehicle) at the superior cervical ganglion (SCG) at 32 weeks of age. SCG DREADD injections generate muscarinic receptors on SCG neurons that are activated by the designer drug, clozapine N-oxide (CNO), ultimately leading to neuronal stimulation and potentially activation of the CAP. At 33 weeks of age, mice with SCG DREADD received a daily s.c. injection of CNO (3mg/kg) for two weeks. At 35 weeks, mice received a catheter implant in the carotid artery to measure mean arterial pressure (MAP) for two consecutive days followed by euthanasia and tissue collection. Plasma was collected biweekly via retro-orbital bleeding. Plasma samples were used to quantify double-stranded (ds) DNA autoantibodies. Results: dsDNA autoantibodies were higher in SLE than control mice (8.6e5 ± 1.8e5 vs. 5.1e4 ± 1.1e4 U/mL; p=0.0002; n=13). SCG DREADD did not change dsDNA autoantibody levels in SLE mice (1.1e6 ± 2.6e5 U/mL; p=0.1410; n=6) or control mice (5.4e4 ± 1.5e4 U/mL; p=0.6549; n=9). MAP was significantly higher in SLE mice compared to control mice (150 ± 9 vs. 122 ± 3 mmHg; p=0.0009; n=5–9). SCG DREADD did not change MAP in SLE mice (137 ± 2 mmHg; p=0.2572; n=6) or controls (127 ± 3 mmHg; p=0.7678; n=9). Conclusion: These data suggest that selective activation of the CAP at the level of the SCG using DREADD did not significantly alter disease severity or blood pressure in female SLE mice with advanced disease. Future studies will determine the effect of selective ganglion stimulation on inflammatory outcomes. Funding: Research reported in this abstract was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number R25HL125447. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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    A Modified Kidney Perfusion System for Improved Data Acquisition and Assessment of Renal Function and Metabolism
    (2023) Wade, Michael; Farmer, George; Yurvati, Albert; Mallet, Robert T.
    PURPOSE In the United States, there are over 88,000 candidates on the waitlist for kidney donation as of December 2022 with less than 42,0000 transplant procedures being recorded in the same year. Coinciding with this continued disparity has been a growing interest in the technology to preserve and improve the function of kidneys for transplantation, most notably machine perfusion (MP). Studies have shown that MP is superior to static cold storage, the most common method of kidney preservation used globally, which has allowed transplant programs to expand the current donor pool by use of marginal donor kidneys. Despite these advancements, machine perfusion technology used clinically is limited in its use for obtaining critical information regarding the real-time assessment of renal function, fluid dynamics, and metabolism. Here we discuss the development of a modified kidney perfusion system that allows for enhanced data acquisition of perfusion parameters, renal arterial and venous sampling, and organ monitoring within a controlled environment. METHODS The modified kidney perfusion system was assembled to house and monitor a singular organ. The organ is placed in a chamber that allows for maximal cooling to 2˚C - 6˚C or temperature regulation up to room temperature. Renal artery and vein cannulation allow for accurate perfusate sampling in different parts of the system without disruption of organ perfusion. Continuous monitoring of pressure, flow, and resistance is performed using pressure and flow transducers in line with the polyethylene tubing. In line with the system includes an air trap to prevent gas emboli, modifiable filter, and screen for large particulate matter. A specialized rotor allows for pulsatile perfusion into the kidney while a servomotor is used to set a desired pressure or flow into the system. Values for flow, pressure, resistance are recorded in real time using Spike2 software and the corresponding waveforms displayed in the program. RESULTS & CONCLUSION The limitations of machine perfusion technology currently used clinically hinder the ability to obtain data for interpretation of pre-transplant renal function. This modified kidney perfusion system improves upon many of these limitations and provides entirely new methods to gain insight on kidney function. Assessment of renal metabolite production and injury markers can be obtained from multiple sites throughout the perfusion circuit and instantaneous recording of both pressure and flow waveforms throughout the duration of machine perfusion.
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    Induced Blood Flow Oscillations at 0.1 Hz Protects Oxygenation of Severely Ischemic Tissue
    (2023) Davis, K. Austin; Bhuiyan, Nasrul; McIntyre, Benjamin; Rickards, Caroline
    Purpose: Early interventions that improve vital organ perfusion will reduce the number of lives lost from blood loss injuries. We have shown that generating 10 second (~0.1 Hz) fluctuations or "oscillations” in arterial pressure and blood flow during simulated hemorrhage protects cerebral tissue oxygenation. Lower body negative pressure (LBNP) was used to both simulate hemorrhage, and induce the hemodynamic oscillations in these previous studies. However, the magnitude of cerebral tissue ischemia is limited to 20-30% with LBNP due to the onset of pre-syncopal symptoms. To examine the effect of 0.1 Hz hemodynamic oscillations on blood flow delivery and tissue oxygenation of severely ischemic tissues, we developed a limb ischemia model. Hypothesis: Oscillatory arterial pressure and blood flow will attenuate reductions in brachial artery blood flow and forearm tissue oxygenation in a severely ischemic limb. Methods: Nine healthy human subjects (5M, 4F; 27.2 ± 4.1 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 (BA) blood velocity by ~70-80% from baseline. In the oscillation condition (OSC), 0.1 Hz oscillations in mean arterial pressure (MAP) and BA blood flow were then induced by inflating and deflating bilateral thigh cuffs every 10 seconds (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. BA blood flow was measured via duplex ultrasound, forearm muscle tissue oxygenation (SmO2) was measured via near infrared spectroscopy, and arterial pressure was measured via finger photoplethysmography. Results: The magnitude of forearm ischemia, indexed by the reduction in BA blood velocity, was matched between protocols (CON: -75.2 ± 8.4 % vs. OSC: -78.3 ± 7.8 %, p=0.20). Power spectral density of 0.1 Hz oscillations in MAP (CON: 19.4 ± 22.8 mmHg2 vs. OSC: 716.8 ± 514.6 mmHg2; p<0.001) and BA blood velocity (CON: 0.7 ± 1.0 cm/s2 vs. OSC: 10.6 ± 7.1 cm/s2, p=0.02) were greater with oscillatory thigh cuff compression compared with the control condition. While oscillatory thigh cuff compression during forearm ischemia had no effect on absolute MAP (CON: 94.3 ± 6.6 mmHg vs. OSC: 94.4 ± 10.8 mmHg, p=0.99), BA blood flow (CON: 9.7 ± 5.8 ml/min vs. OSC: 9.5 ± 7.3 ml/min, p=0.82), or BA conductance (CON: 0.10 ± 0.06 ml/min/mmHg vs. OSC: 0.09 ± 0.06 ml/min/mmHg, p=0.39), the reduction in SmO2 was attenuated (CON: -38.7 ± 8.3 % vs. OSC: -28.4 ± 9.7 %; p=0.04). These data provide further evidence for the use of 0.1 Hz hemodynamic oscillations as a therapeutic intervention for conditions associated with severe vital organ ischemia such as hemorrhage, stroke, myocardial infarction, and sepsis.
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    Sex differences in the activation of central autonomic control regions and neuroinflammation in chronic intermittent hypoxia
    (2023) Appiah, Cephas; Little, Joel; Mabry, Steve; Cunningham, Rebecca L.; Cunningham, J. Thomas
    Purpose: Obstructive sleep apnea (OSA) is an independent risk factor for hypertension. Chronic interment hypoxia (CIH), which models episodic hypoxemia of OSA, produces daytime hypertension, oxidative stress, and activation of central autonomic regions that regulate mean arterial pressure (MAP) in male Sprague Dawley (SD) rats. Unlike gonadally intact females, gonadectomized females and males develop CIH hypertension. Lesioning of median preoptic nucleus (MnPO) in males prevents CIH hypertension. We hypothesize that the sex difference observed in CIH hypertension is due sex differences in neuroinflammation and activation of central autonomic regions that support MAP in CIH. Methods: Gonadally intact adult male and female SD rats (250-300g) were continuously exposed to normoxia (CON) or CIH (10% O2 every 3mins cycling 21% O2 every 3mins, 8h/day) for 7 days. Radiotelemetry transmitters were implanted in rats to record MAP and heart rate (HR). After one week of baseline recording, the rats were exposed to either continuous normoxia or CIH and were euthanized (inactin 100 mg/kg ip) on the 8th day for immunohistochemistry and blood analysis. All forebrain sections were stained for FosB/ΔFosB and either neuronal nitric oxide synthase (NOS1) or IBA1 to identify active microglia. Results: CIH males exhibited significantly increased hematocrit indicating erythropoiesis compared to control males (CON 42.1% ± 0.6, n=10; CIH 44.6% ± 0.7, n = 10. P = 0.0173). CIH males exhibited an increase in the average number of FosB positive neurons (CON male 20 ± 2 cells/section, CIH male 35 ± 3; CON female 11 ± 1, CIH female 12 ± 2,) and colocalization of FosB and NOS1 (CON male 10 ± 1 cells/section, CIH male 18 ± 4; CON female 5 ± 1, CIH female 6 ± 1) in the MnPO. CIH females showed a trend for an increase in the average number of IBA1 immunoreactive microglial cells in MnPO (CON 187 ± 16, n = 2; CIH 218 ± 18, n = 4). Conclusion: CIH is associated with increased FosB staining in the MnPO of male rats as opposed to female rats which is consistent with our working hypothesis. In addition, FosB positive MnPO neurons also contained NOS1. In female rats, CIH is associated with a trend for an increase in the numbers of IBA1 positive microglia, indicating increased neuroinflammation in females that is independent of hypertension. CIH was associated with increased FosB staining in NOS1 positive MnPO neurons suggesting that they may be contributing to the sustained hypertension reported in male rats. The research is funded by NIH grant RO1 HL155977.
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    Colloid-Enhanced Flush Limits Initial Edema but Exacerbates Subsequent Edema During Hypothermic Machine Perfusion of Porcine Kidneys
    (2023) Tamayo, Jesus; Mohammad, Moath; Wade, Michael; Tran, Amanda; O'Hara, Collin; Yurvati, Albert; Mallet, Robert T.
    Purpose: The epidemic of end-stage renal disease (ESRD) has steadily increased demand for transplantable kidneys, and the widening disparity between organ supply and demand is a major public health concern. Hypothermic machine perfusion (HMP) is widely used to preserve deceased donor kidneys for transplantation. Kidneys are harvested and flushed with crystalloid solution before HMP. This study tested the hypothesis that flushing kidneys with solution containing a colloid, hydroxyethyl starch (HES), minimizes edema and improves organ perfusion during subsequent HMP. Methods: Kidneys harvested from anesthetized Yorkshire swine were flushed for 15 min with ice-cold Ringer’s solution ± 50 g/l HES, and then either biopsied or installed in a LifePort organ preservation system for 72 h hypothermic (2-4°C) machine perfusion (HMP) before biopsy. ATP contents in renal cortical biopsies were analyzed by UV-Vis spectrophotometry. Results: Kidneys gained 49 ± 5% of initial mass during flush with control solution, but only another 3% to 52 ± 5% of initial mass over 72 h HMP. Kidneys flushed with HES-enriched solution gained only 18 ± 3% of initial mass (P<0.001 vs control) during flush, but over 72 h HMP gained another 72% to 90 ± 7% above initial mass (P<0.001 vs. control). Tissue water contents paralleled the respective weight gains (Figure). The HES-flushed kidneys experienced steeper declines in perfusion during HMP than the controls. Cortical ATP content (mmol/g dry mass) fell over 72 h HMP from 2.36 ± 1.40 to 0.44 ± 0.44 (mean ± SD) in control kidneys, and from 1.36 ± 1.31 to 0.38 ± 0.16 in HES-flushed kidneys (both groups: P<0.05, pre- vs. post-HMP). Conclusion: Flushing kidneys with HES-enriched solution minimized edema before HMP, but exacerbated edema during subsequent machine perfusion, failed to preserve ATP, and was associated with a steeper decline in organ perfusion during HMP. The mechanisms responsible for edema exacerbation by HES-enhanced flush are under investigation.
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    Postpartum preeclamptic rats have hypertension and elevated cerebral oxidative stress
    (2023) Smith, Savanna; Smith, Jonna; Jones, Kylie; Castillo, Angie; McCafferty, Adair; Wiemann, Natalia; Owen, Malissa; Srivastava, Prakriti; Cunningham, Mark
    Background: Postpartum (PP) preeclamptic (PE) women have an increased risk for developing hypertension (HTN), cerebrovascular diseases, and chronic kidney diseases later in life. The timing and mechanisms that contribute to a rise in blood pressure (BP), cerebrovascular and kidney dysfunction in PP PE women is unknown and the focus of this study. Previous studies in our lab (PMID: 34727994) indicate PP PE rats at 10 weeks have HTN and decreased antioxidant capacity (AC). Our current study examines BP and oxidative stress (OS) in PP PE rats at an earlier time point, 6 weeks (PP6). Understanding changes in cerebral and renal OS may reveal the pathophysiology of HTN, cerebrovascular, and renal disease development in PP PE women. We hypothesize that BP, renal, and cerebral OS will increase in PP6 PE rats. Methods: Pregnant Sprague Dawley rats were divided into 2 groups: control (CON) normal pregnant rats, and PE rats, derived from the surgically induced placental ischemic (reduced uterine perfusion pressure) model of PE. All rats gave birth and weaned for 3 weeks. At PP6, BP was measured via carotid catheterization. Brain and kidney tissues were collected to measure OS (HSP-1, Cu/ZnSOD, and MnSOD proteins and AC) through colorimetric assays and western blots. Results: PP6 PE vs CON rats, BP was elevated (128±6 vs 106±4mmHg, p<0.05) and AC was decreased in systemic circulation (28.5±5.1 vs 36.9±4.5mM Trolox/mg protein, ns). In the brain, both HSP-1 and Cu/ZnSOD were unchanged between PP6 PE and CON rats, while the levels of MnSOD (88.9±2.0 vs 100±2.5 IU/protein/CON %, p<0.05) and AC were decreased (619.1±179.2 vs 850.2±50.3 mM Trolox/mg protein, ns) in PP6 PE vs CON rats. In the kidney, HSP-1 decreased (88.7±3.0 vs 100±4.0 IU/protein/CON %, ns) in PP6 PE vs CON, while Cu/ZnSOD levels remained unchanged. However, kidney MnSOD levels significantly increased (124.3±8.0 vs 100±2.7 IU/protein/CON %, p<0.05) alongside an increase in AC (791.0±165.4 vs 587.0±64.5mM Trolox/mg protein, ns) in PP6 PE vs CON rats. Conclusion: PP6 PE rats have HTN and increased cerebral OS. Despite changes in the brain, kidneys appear to be protected from OS due to a decrease in a reactive OS protein (HSP-1) and increases in antioxidant capacity and protein (MnSOD). Future studies will determine the relationship between brain OS, HTN, and cerebral damage/dysfunction to PP PE rats. Furthermore, future studies will be designed to elucidate the protective mechanisms of the kidney in PP6 PE rats. Findings of this study are clinically relevant and could be used to improve the maternal health of women after PE pregnancies. In addition, therapy designed to target organ specific OS may be helpful in preventing HTN, cerebrovascular, and chronic kidney diseases later in life for women who have experienced PE.
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    Visium Spatial Transcriptomics Reveal Sex Differences in Supraoptic Nucleus Gene Expression of Adult Rats Related to Cell Signaling and Ribosomal Pathways
    (2023) Nguyen, Dianna; Phillips, Nicole; Cunningham, J. Thomas
    Purpose: There are many well-known sexually dimorphic regions of the hypothalamus; however, sex differences in gene expression in the supraoptic nucleus (SON), a region crucial in the regulation of body fluid homeostasis, has been relatively unexplored. Our previous spatial transcriptomics study revealed gene cluster analysis successfully differentiated myelinated fiber tracts from nuclei and identified several distinct neuronal populations in the coronal brain sections from both male and female rats. Our current study aims to interrogate the sex differences in SON gene expression using two unique methods of differential gene expression (DGE), gene ontology, and pathway analyses. The first DGE approach used Loupe Browser, an application developed by 10x Genomics specifically for their transcriptomics workflow, while the second approach used DESeq2, a more traditional DGE analysis method. Methods: Gonadally-intact adult male (n=4) and female (n=4) Sprague-Dawley rats were anesthetized with isoflurane (2-3% in 95% O2) and their brains were removed and flash frozen. Each brain was sectioned at 10μm thickness and sections (~4x4mm) containing the SON and other brain structures were mounted in capture areas on Visium slides containing probes that bind mRNA. All sections underwent the following workflow: 1) sample staining and imaging, 2) cDNA library preparation, 3) sequencing, and 4) analysis/data visualization. Data were analyzed using 10x Genomics’ Loupe Browser application and other bioinformatic tools. Results: Using Loupe Browser, DGE analysis of the SON identified 116 genes (e.g., Avp and Oxt) common to both sexes, 31 genes unique to the males, and 73 genes unique to the females. DGE analysis using DESeq2 revealed 183 significant differentially expressed genes between the two groups. Gene Ontology (GO) Enrichment and pathway analyses using significant genes identified via Loupe Browser revealed GO terms and pathways related to: 1) neurohypophyseal hormone activity, regulation of peptide hormone secretion, and regulation of ion transport for the significant genes common to both males and females, 2) Gi signaling/G-protein mediated events for the significant genes unique to males, and 3) potassium ion transport/voltage gated potassium channels for the significant genes unique to females, as some examples. In contrast, GO/pathway analyses using significant genes identified via DESeq2 comparing female vs. male groups revealed GO terms/pathways related to ribosomal structure/function. Conclusions: The two DGE analysis approaches elucidated different aspects of sex differences in SON gene expression. Loupe Browser-based analysis identified genes related more to cell signaling pathways, while DESeq2 identified genes associated with ribosomal structure/function. Future spatial transcriptomic studies will investigate changes in SON gene expression that contribute to sex differences in cellular mechanisms involved in body fluid homeostasis and possibly pathophysiology.
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    Pyruvate-enriched solution limits lactate and creatine accumulation in hypothermic machine-perfused kidney
    (2023) Mohammad, Moath; Tamayo, Jesus; Wade, Michael; Tran, Amanda; O'Hara, Collin; O-Yurvati, Albert; Mallet, Robert T.
    With a 5% annual increase in incidence, end-stage renal disease (ESRD) is a mounting epidemic. Kidney transplantation is the only definitive treatment for ESRD, but the growing demand for transplantable kidneys greatly exceeds the supply, thus improved methods of organ preservation are urgently needed. Recently, renewed interest in hypothermic machine perfusion (HMP) has prompted refinements of perfusion solutions to improve graft performance and transplant outcomes. In previous studies, solutions containing the intermediary metabolite pyruvate prevented ATP depletion, detoxified reactive oxygen species, suppressed inflammation and optimized protection of ischemic myocardium and brain. This study aimed to examine the hypothesis that pyruvate-enriched preservation fluids provide robust preservation of histological structure and energy metabolism in porcine kidneys during 72 hours HMP. The study used 7 Yorkshire swine kidneys, which were harvested and immediately flushed with ice-cold Ringer's solution and then perfused with either control or 20 mM pyruvate-enriched Ringer's solution for 72 hours in a LifePort organ preservation system. Values for renal artery flow and resistance were recorded over time. After 72 hours HMP, the renal cortex was biopsied and analyzed for metabolite content via spectrophotometry, while the cortex and medulla were biopsied for histological evaluation. The results showed that pyruvate-enriched preservation fluids lowered creatine content by 89% (P < 0.05), and surprisingly lowered lactate content while increasing glucose-6-phosphate content, a source for maintaining antioxidant reducing power. Flow and resistance were comparable between the two groups, and histological analysis revealed interstitial and intracellular edema, varying stages of acute tubular necrosis and variable loss of overall glomerular architecture in both groups. In conclusion, pyruvate-enriched preservation fluids stabilized the cellular energy state, supplied glucose-6-phosphate for sustaining antioxidants, and shunted metabolites from the glycolytic pathway leading to lower lactate accumulation in the renal cortex. Further research is warranted to understand pyruvate's impact on oxidative stress and inflammation during HMP.
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    Hemodynamic Responses to Oscillatory Thigh Cuff Inflations
    (2023) McIntyre, Benjamin; Davis, K. Austin; Bhuiyan, Nasrul; Rickards, Caroline
    Background. In the clinical setting, individuals have varying tolerance to hypovolemia induced by blood loss. Experimental generation of 0.1 Hz oscillations (~10-s cycle) in arterial pressure and cerebral blood flow via oscillatory lower body negative pressure (OLBNP) increases tolerance to this simulated hemorrhage, and protects cerebral tissue oxygenation. However, use of OLBNP as a method of inducing hemodynamic oscillations in the clinical setting is limited as: 1) it is a large and cumbersome technique, and; 2) it induces central hypovolemia, which would only worsen the magnitude of hemorrhage. In this study we evaluated a more clinically applicable method of inducing 0.1 Hz oscillations in arterial pressure and cerebral blood flow, using intermittent inflation of bilateral thigh cuffs. We hypothesized that the amplitude of arterial pressure and cerebral blood flow oscillations at 0.1 Hz would increase in response to repeated thigh cuff inflations at 0.1 Hz when compared with a baseline control condition. Methods. Ten healthy human subjects were tested (6 male, 4 female; 26.8 ± 4.1 y). Middle cerebral artery velocity (MCAv) was measured via transcranial doppler ultrasound, arterial pressure was measured via finger photoplethysmography, and end tidal CO2 (etCO2) was measured via capnography. Following a 10-min baseline period, intermittent thigh cuff inflations at 0.1 Hz and 230 mmHg (5-s inflation, 5-s deflation) were performed for 10-min ("oscillations”). 0.1 Hz oscillatory amplitude of mean arterial pressure and mean MCAv were quantified using Fast Fourier transformation during the last 5-min of baseline and the oscillatory period, and compared via two-tailed paired t-tests. Results. The amplitude of 0.1 Hz oscillations increased during the oscillatory period vs. baseline for mean arterial pressure (baseline: 1.7 ± 1.0 mmHg2 vs. oscillations: 9.0 ± 6.2 mmHg2; P = 0.004) and mean MCAv (baseline: 1.1 ± 0.6 (cm/s)2 vs. oscillations: 3.4 ± 3.1 (cm/s)2; P = 0.04). Absolute mean arterial pressure was similar between baseline and the oscillatory period (baseline: 97.2 ± 8.1 mmHg vs. oscillations: 99.1 ± 15.0 mmHg; P = 0.54), but absolute mean MCAv was lower during the oscillatory period (baseline: 61.7 ± 14.6 cm/s vs. oscillations: 53.2 ± 13.1 cm/s; P = 0.02). This reduction in mean MCAv was most likely due to hypocapnia (indexed by etCO2) induced by pacing the breathing of all subjects at ≥10 breaths/min (baseline: 33.2 ± 4.8 mmHg vs. oscillations 27.2 ± 4.5 mmHg; P = 0.005). Conclusions. Intermittent thigh cuff inflations at 0.1 Hz induced 0.1 Hz oscillations in both arterial pressure and cerebral blood flow when compared to baseline. These findings indicate that intermittent thigh cuff inflations could be developed as a method to induce pulsatile perfusion as a potential new therapy for individuals experiencing major blood loss.
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    Brain mitochondrial dysfunction in postpartum preeclamptic rodents
    (2023) Jones, Kylie; Smith, Jonna; Smith, Savanna; Castillo, Angie; McCafferty, Adair; Wiemann, Natalia; Owen, Malissa; Srivastava, Prakriti; Cunningham, Mark
    Purpose: Pre-eclampsia (PE), new-onset hypertension during pregnancy, impacts 3-8% of all births in the USA yearly and causes significant neurological damage to the mother during and after pregnancy. Studies show postpartum PE women to have increased risks of hypertension (HTN) and cerebrovascular dysfunction (CVD). Although the cause of HTN and cerebral damage is unknown, mitochondrial dysfunction (mtDys) may play a role. MtDys includes reduced mitochondria-specific antioxidants, raised mitochondrial reactive-oxygen species, changes in mitochondrial fission and fusion proteins, and reduced efficiency of the electron transport chain (ETC). Previous studies in our lab indicate associations between cardiac mtDys and the reduced uterine perfusion pressure (RUPP) rat model of PE with HTN at 10 weeks postpartum (PMID: 34727994). However, cerebral mtDys has not been examined in RUPP rats postpartum. This study aims to examine cerebral mitochondrial functional proteins in hypertensive RUPP postpartum rats at six weeks. We hypothesize that RUPP postpartum rats will have lower amounts of cerebral mitochondrial functional proteins compared to control (CON) postpartum rats. Methods: We divided pregnant Sprague Dawley rats into two groups: CON normal pregnant (NP, n = 4) and RUPP (n = 4). Then, the RUPP surgery was performed on gestational day 14. Pregnant rats gave birth naturally and weaned for three weeks. Six weeks after giving birth, rats were euthanized for brain collections to measure functional proteins via Western Blot analysis, including ETC complexes (Complexes I-V), fusion proteins (OPA-1 and MFN-2), fission protein (DRP-1), and mitochondria-specific antioxidant (MnSOD). Results: In the brain, RUPP postpartum rats have significantly reduced Complex I proteins compared to NP postpartum rats (91 ± 2.27 vs. 100 ± 2.45 IU/protein/CON %, p < 0.05) with slight decreases in Complexes II (93 ± 4.14 % vs. 100 ± 7.57 IU/protein/CON %, ns), III (91 ± 3.18 vs. 100 ± 6.11 IU/protein/CON %, ns), IV (86 ± 11.25 vs. 100 ± 7.95 IU/protein/CON %, ns), and V (92 ± 3.99 vs. 100 ± 6.33 IU/protein/CON %, ns). RUPP and NP postpartum rats have no significant differences in fusion proteins OPA-1 (102 ± 2.56 vs. 100 ± 2.02 IU/protein/CON %, ns) and MFN-2 (106 ± 18.25 vs. 100 ± 14.35 IU/protein/CON %, ns). Fission protein DRP-1 has an increase in RUPP postpartum rats compared to NP postpartum rats (111 ± 6.92 vs. 100 ± 4.55 IU/protein/CON %, ns). RUPP postpartum rats have significantly decreased MnSOD in comparison to NP postpartum rats (89 ± 2.00 vs. 100 ± 2.45 IU/protein/CON %, p < 0.05). Conclusion: RUPP postpartum rats have cerebral mtDys indicated by decreased ETC complexes, especially Complex I. RUPP postpartum rat brains have reduced MnSOD, which suggests elevated mitochondrial oxidative stress. Furthermore, raised mitochondrial fission in the brain supports the presence of mitochondrial damage and mtDys. Future studies will examine the role of cerebral mtDys in causing HTN and CVD in RUPP postpartum rats. This study is clinically relevant because our findings provide a possible mechanism for the pathophysiology of CVD in postpartum PE women and novel targets for cerebral mitochondrial therapy.