Browsing by Author "Mallet, Robert T."
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Item A Celebration of the Extraordinary Life of Late Professor Tatiana V. Serebrovskaya (Kyiv, Ukraine) in Advancing Hypoxia Science and Medicine(Mary Ann Liebert, Inc., 2022-08-03) Swenson, Erik R.; Mallet, Robert T.; Xi, Lei; Manukhina, Eugenia B.; Downey, Fred; Burtscher, Johannes; Ehrenreich, Hannelore; Burtscher, MartinItem Acetoacetate: A Cardioprotective Antioxidant(2002-05-01) Squires, Jeffrey E.; Mallet, Robert T.; Caffrey, James L.; Carroll, JoanSquires, Jeffrey E., Acetoacetate: A Cardioprotective Antioxidant. Master of Science, June 2002, 100 pp., 1 table, 18 illustrations, bibliography, 70 titles. The purpose of this study was to test the effectiveness of acetoacetate and β-hydroxybutyrate as myocardial protectants following peroxide injury and to determine acetoacetate’s ability to potentiate β-adrenergic responsiveness following ischemia-reperfusion injury. This study utilized antegradely perfused isolated working hearts exercised from male guinea pigs and sustained with glucose-fortified Krebs-Henseleit. Hearts were challenged by either 10 min perfusion with 100 μM H2O2 or 45 min of low flow ischemia exacerbated by ι-norepinephrine infusion. H2O2-challenged hearts were treated with 5 mM acetoacetate or β-hydroxybutyrate, whereas hearts injured by ischemia/reperfusion were treated with 5 mM acetoacetate. In the case of the ischemically injured hearts, acetoacetate treatment was combined with 2 nM isoproterenol to delineate acetoacetate’s ability to enhance β-andrenergic responsiveness to submaximal inotropic stimulation. Data were compared to non-injured time control hearts and injured untreated hearts to determine the impact of ketone body treatment. Acetoacetate increased citrate and glucose 6-phosphate content, nearly restored power, and increased the glutathione antioxidant redox potential (GSH/GSSG) by 140% in H2O2-injured myocardium. Although β-hydroxybutyrate increased citrate, an activator of NADPH-generating pathways, and glucose 6-phosphate, the substrate for the hexose monophosphate shunt to the same extent as acetoacetate, β-hydroxybutyrate raised GSH/GSSG by only 60% and did not enhance cardiac power. Therefore, acetoacetate enhances contractile function by augmenting the glutathione redox potential, and does so by additional mechanisms independent of the citrate and hexose monophosphate pathway. In hearts stunned by ischemia/reperfusion, acetoacetate and isoproterenol each increased power and glutathione redox potential three-to-fourfold, but phosphocreatine potential was 70% higher in acetoacetate hearts. Combined, acetoacetate + isoproterenol synergistically increased power and GSH/GSSG 16- and 17- fold respectively, doubled {NADPH/NADP+}, and increased cyclic AMP content 30%. These findings support the conclusion that acetoacetate enhances myocardial sensitivity to β-adrenergic stimulation possibly by enhancing GSH/GSSG.Item Adenosine Receptor Blockade Increases Lactate and Purine Release But Does Not Affect Functional Recovery in Isolated Rabbit Myocardium(1995-12-01) Wang, Sheng; Downey, H. Fred; He, Miao-Xiang; Mallet, Robert T.Wang, Sheng, Adenosine Receptor Blockade Increases Lactate and Purine Release but does not Affect Functional Recovery in Isolated Rabbit Myocardium Master of Science (Biomedical Sciences), December 1995; 67 pp; 3 tables; 8 figures; bibliography, 121 titles. This study tests the hypothesis that endogenous adenosine mediates recovery of cardiac function in ischemia/reperfused rabbit hearts. Isolated isovolumic rabbit hearts perfused at constant pressure was subjected to mild ischemia (perfusion pressure 50 cm H2) or moderate ischemia (perfusion pressure 30 cm H2O) for 90 min followed by 60 min of reperfusion. In treated hearts, infusion of 100 μM 8-p-sulfophenyl theophylline (SPT) was initiated 20 min before ischemia and maintained throughout the experiment. Adenosine receptor blockade did not affect left ventricular function assessed from pressure-heart rate product (PRP). Lactate release increased to 152 ± 24% of baseline during mild ischemia and 259 ± 26% of baseline during moderate ischemia in untreated hearts. Lactate release was markedly elevated at baseline, ischemia and reperfusion by SPT treatment (p [less than] 0.05 compared to untreated). Purine nucleoside release was 4.1 ±0.7 nmol · min-1 · g-1 in SPT treated group and 1.8 ± 0.24 nmol · min-1 · g-1 in untreated group during moderate ischemia (P [less than] 0.05). Myocardial efficiency was significantly lower in the SPT treated hearts (240 ± 11 mmHg · g=1 · μl-1 O2) compared to untreated hearts (300 ± 22 mmHg · g-1 · μl-1 O2) during reperfusion after moderate ischemia. In conclusion, adenosine receptor blockade stimulates glycolysis in normoxic and ischemic myocardium, but does not affect post-ischemic functional recovery.Item Cardiac Arrest Induces Lung Inflammation 3 days after Cardiac Arrest in Domestic Pigs(2015-03) Dietemann, Daniel; Nguyen, Anh Q.; Olivencis-Yurvati, Albert H.; Mallet, Robert T.Introduction: Cardiac arrest, a leading cause of death in the United States, is associated with detrimental damages, which includes the lungs. Reperfusion of ischemic tissue following cardiac arrest leads to systemic inflammation inflicted by free radical production. Pyruvate , a cellular metabolite and an antioxidant, has a demonstrated protective anti-oxidative effect on the heart through enhancement of the natural anti-oxidative glutathione system. Purpose: Using Myeloperoxidase activity as a marker for neutrophilic inflammation, we sought to quantify the extent of the inflammation in lung tissue, and to determine pyruvate’s protective effect against ischemia/reperfusion injury in the lung. Methods: Fourteen juvenile male Yorkshire swine were divided into three groups, a sham group(n=3) which underwent no cardiac arrest or CPR protocol, a CPR group(n=5) which received an intravenous infusion of sodium chloride solution, and a CPR + Pyruvate group which instead received an infusion of pyruvate solution(n=6). The CPR groups were placed in cardiac arrest via ventricular fibrillation for six minutes, followed by four minutes of cardiopulmonary compression and subsequent defibrillating counter shocks. The respective infusions were delivered at a rate of 0.1 mmol/kg/min throughout CPR and for 60 minutes following cardioversion. After three days, left lung samples were collected and assayed for protein content and myeloperoxidase activity. Results: Three days following the procedure, Myeloperoxidase (MPO) activity was elevated in animals subjected to cardiac arrest compared to sham, although the result was not statistically significant (P=0.15). Pyruvate did not dampen MPO activity in the left lung.Item Cardioprotective Intermittent Hypoxia Conditioning Induces Glyoxalase-1 in Rat Left Ventricle(2016-03-23) Scott, Gary; Williams, Arthur Jr.; Ryou, Myoung-Gwi; Mallet, Robert T.; Ramani, AzaanIntermittent, normobaric hypoxia (IH) conditioning provides significant cardioprotection, including dramatically increasing myocardial resistance to ischemia-reperfusion. The cardioprotective mechanisms of IH are unknown. Myocardial ischemia-reperfusion generates methylglyoxal, a potent and toxic glycating agent. Our previous studies1 demonstrated that reactive oxygen species (ROS) generated during IH cycles contribute to cardioprotection. We hypothesize that the generation of ROS during IH cycles induce the transcription factor Nrf2 to activate expression of genes encoding cytoprotective proteins. Here we evaluated IH induction of glyoxylase 1 (GLO-1), a major enzyme responsible for methylglyoxal detoxification. Sprague-Dawley rats were conditioned by a 20 day IH program consisting of 5-8 daily, 5-10 min cycles of hypoxia (9.5-10% inspired O2) with intervening 4 min room air exposures, previously shown to produce robust cardioprotection.1 Control rats were sham-conditioned using 21% O2. After completion of the conditioning protocol, the left ventricle was isolated and extracted for enzyme analysis. The activities of cytoprotective enzymes were analyzed by spectrophotometric assays. GLO-1 activity (U/mg protein) increased threefold in IH conditioned (1.05±0.16) vs. sham (0.35±0.11) rats. Glutathione reductase activity (U/mg protein) was unchanged between IH conditioned (0.015±0.0047) vs. sham rats (0.023±0.016). IH augmentation of the anti-glycation enzyme GLO-1 may contribute to the heart’s increased resistance to ischemic injury.Item Cardiovascular Response to Endotoxin-Mediated Sepsis: A Dose-Response Study(2022) Aguirre, David Salinas; Martinez, Richard; Warne, Cooper; Mallet, Robert T.; Dick, Gregory; Tune, Johnathan; Hodge, LisaPurpose: Our long-term goal is to advance our understanding and treatment of sepsis, a potentially life-threatening condition that occurs when the response to infection causes tissue and organ damage. Sepsis can be caused by lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria. We used a swine model of LPS-induced sepsis to study the impact of the lymphatic and immune systems on the disease progression. Our first experiments were aimed at determining the optimal dose of Escherichia coli LPS in order to study the effect of sepsis on the cardiovascular system. We hypothesized that 2-hour intravenous infusions of 1- 50 µg/kg LPS would reveal dose- and time-dependent changes in hemodynamic parameters that are consistent with sepsis. Methods: Yorkshire pigs (61 ± 4 kg, n = 4, 2 male) were sedated, intubated, and ventilated. Femoral artery and venous lines were placed to allow measurement of blood pressure, infusion of LPS, and blood gas sampling. A thoracotomy was performed in order to secure a Transonic flow probe around the left anterior descending coronary artery and to insert a sampling catheter in the anterior cardiac vein. A dose of LPS (1, 5, 25, and 50 µg/kg) was given to each pig over 2 hours. Blood samples were collected immediately before LPS infusion and for every 30 minutes during and after LPS infusion for blood gas measurements. Vital signs were recorded as the animals developed sepsis. Results: Only the pig given the lowest dose of LPS (1 µg/kg) survived the full 6 hours (mean survival time in remaining 3 pigs was 180 ± 30 min). At 150 min, a ≈55% decrease in mean arterial pressure was observed (107 ± 4 to 48 ± 13 mmHg), resulting in a ≈60% increase in heart rate (91 ± 9 to 146 ± 14 beats/min). Coronary blood flow and myocardial oxygen consumption decreased ≈28% (0.53 ± 0.06 to 0.38 ± 0.02 ml/min/g) and ≈33% (60 ± 6 to 44 ± 2 µl/min/g), respectively. Ventricular fibrillation was the cause of death in the 3 non-surviving pigs. Conclusion: A dose of 1 µg/kg appears to be an optimal dose for future studies, as this dose was survivable in the desired time frame, while causing hypotension and tachycardia. In future studies, this model will allow us to study the effect of novel therapeutics during acute sepsis.Item Characterization of the Role of PKN in TGF-Beta 1-Mediated Differentiation of Vascular Smooth Muscle Cells(2004-05-01) Deaton, Rebecca Ann; Dillon, Glenn; Shepard, Allan; Mallet, Robert T.Rebecca Ann Deaton, Characterization of the role of PKN in TGF-beta 1-mediated differentiation of vascular smooth muscle cells. Doctor of Philosophy (Biomedical Sciences), May 2004, 178 pp, 5 tables, 34 illustrations, references, 197 titles. Differentiated vascular smooth cells (SMCs) exhibit a work phenotype characterized by expression of several well-documented contractile apparatus-associated proteins. However, when exposed to mitogens such as serum or growth factors. SMCs retain the ability to de-differentiate into an “immature” proliferative phenotype, in which they lack contractile myofilaments. Proliferation of SMCs is involved in the formation of atherosclerotic plaques as well as arterial restenosis following balloon angioplasty. Thus, understanding the mechanism involved in maintain SMC differentiation process is critical to the development of therapies and treatments for the abnormal growth seen in these disease states. In this study, the molecular mechanisms through which transforming growth factor-beta 1 (TGF-B1) induces differentiation of SMCs were examined. The data presented demonstrate that TGF-B1 stimulates actin re-organization, up-regulation of SM-specific marker gene expression and inhibition of cell proliferation of PAC-1 SMCs. These characteristics are indicative of the differentiated phenotype. The effects of TGF-B1 can be blocked by pretreatment of the cells with either HA1077 or Y-27632, which inhibit the functions of the kinases downstream of RhoA. Moreover, TGF-B1 induced differentiation is correlated with an increase in the activity of RhoA and its downstream target, PKN. Over-expression of active PKN alone is sufficient to increase the transcriptional activity of the SM a-actin, SM-MHC and SM22 promoters in PAC-1 cells. In addition, the activity of SRF-GATA and MEF2, three transcription factors that are known to regulate expression of SM-specific marker genes, are also increased by PKN. Finally, examination of MAPK signaling cascades demonstrates that TGF-B1 increases the activity of MKK3/6 and p38 MAPK and decreases the activity of ERK1/2 and JNK ½. Co-expression of dominant negative p38 MAPK is sufficient to abolish PNK-mediated activation of SRF, GATA and MEF2 as well as PKN-mediated activation of SMC marker gene promoters. Taken together, these results identify components of an important intracellular signaling pathway through which TGF-B1 activates RhoA and PKN to promote differentiation of SMCs.Item 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.Item Cytokine Response in an Endotoxin-Mediated Sepsis Model(2022) Martinez, Richard; Aguirre, David Salinas; Warne, Cooper; Dick, Gregory; Mallet, Robert T.; Tune, Johnathan; Hodge, LisaPurpose: Sepsis is a life-threatening condition that develops secondary to infection and can manifest acute organ dysfunction due to the body's overactive systemic response. Sepsis affects approximately 1.7 million US adults and claims 270,000 lives as a result. The long-term goal of our project is to gain a better understanding of the roles of the lymphatic and immune systems in the progression of sepsis. The purpose of this study is to collect pilot data using a translational swine model of endotoxin-mediated sepsis. We chose a swine model because it closely mimics how sepsis progresses in humans. Sepsis was induced by infusion of lipopolysaccharide (LPS) from Escherichia coli. LPS was chosen because it is a key mediator in the activation of the immune system and the development of sepsis. We hypothesized that the administration of LPS would increase the concertation of the proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-?) in a dose-dependent manner over time. Methods: Yorkshire pigs (61 � 4 kg, n = 4, 2 male) were sedated, intubated, and ventilated. Thoracotomy was performed under anesthesia to record flow data and sample cardiac blood for use in another study. Femoral artery and venous lines were placed to allow measurement of blood pressure and infusion of LPS. Specifically, LPS was prepared at 1, 5, 25, or 50 ?g/kg (pig body weight) in sterile saline. LPS was infused into anesthetized pigs over a 2-hour period. Blood samples were collected immediately prior to LPS administration and at 30 min intervals during 2 hours of LPS infusion up to 4 hours following LPS infusion. The plasma was analyzed via enzyme-linked immunosorbent immunoassay (ELISA) for the concentrations of IL-6 and TNF-? using commercially available kits. Results: As hypothesized, the infusion of LPS increased the concentration of the inflammatory mediators IL-6 and TNF-? over time compared to pre-LPS infusion. Specifically, the greatest increase in IL-6 was seen at 180 minutes in both the 50 and 25 ?g/kg LPS infused pigs. TNF-? concentration peaked between 30 to 90 minutes during LPS infusion in both the 50 and 25 ?g/kg LPS infused pigs. The lower doses of 1 and 5 ?g/kg LPS produced little to no IL-6 or TNF-?. Furthermore, we discovered that the pigs who received 50 or 25 ?g/kg of LPS died from septic shock within 180 minutes of LPS infusion, whereas the pigs that received 1or 5 ?g/kg of LPS survived longer. Conclusion: In this study, we identified the impact of increasing the doses of LPS on the production of IL-6 and TNF-? in swine. Our preliminary results suggest a dose range of 10-20 ?g/kg of LPS may be ideal to study the inflammatory response in this model. The acquisition of these data are essential to pursue our long-term research objective, which is to identify the role of the lymphatic and immune systems during sepsis.Item Cytoprotective and Anti-Glycation Defenses in Porcine Brain after Cardiac Arrest and Cardiocerebral Resuscitation(2016-03-23) Ryou, Myoung-Gwi; Hollrah, Roger; Williams, Jr., Arthur; Scott, Gary; Olivencia-Yurvati, Albert; Mallet, Robert T.; Nguyen, Anh; Nguyen, AnhCardiac arrest (CA) is often lethal, and survivors often face sequelae that greatly impact quality of life due to brain injury inflicted by ischemia-reperfusion. Effective cardiocerebral resuscitation (CCR) is essential for survival and recovery from CA. The complexity of the injury cascades ignited by CA and the presence of the blood-brain barrier challenge the development of pharmacological interventions to protect the brain. Our goal is to identify a blood-brain barrier-permeable intervention that mitigates CA-induced brain damage and, thus, fosters neurological recovery. Pyruvate, a cellular metabolite, energy substrate and antioxidant, has been found to be neuroprotective in a rat stroke model via induction of the cytoprotective cytokine erythropoietin (EPO). Hypothesis: Pyruvate treatment during CCR and post resuscitation decreases brain injury by upregulating cellular defense mechanisms including hypoxia inducible factor-1α (HIF-1α), EPO, the product of HIF-1 α’s gene program, and glyoxalase-1, the principal component of the brain’s defenses against the powerful glycating agent and glycolytic byproduct, methylglyoxal. Methods: Yorkshire swine (25-35 kg, both genders) were subjected to sham protocol (n = 6) or pacing-induced CA (n = 12). CCR was administered by precordial chest compressions (100/min) at 10-14 min CA, and transthoracic countershocks were applied to restore sinus rhythm. NaCl (n = 6) or Na-pyruvate (n = 6) was infused iv (0.1 mmol/kg/min) during CCR and the first 60 min recovery. At 4 h recovery, brain was cross-perfused with 0.9% NaCl, and then brain biopsies were freeze-clamped for biochemical analysis or fixed in 10% formalin for immunohistochemistry. Results: In hippocampus, activity of the oxyradical-sensitive TCA cycle enzyme aconitase fell by 50% (PItem DELAYED NEURONAL DEATH IN SWINE FOLLOWING CARDIAC ARREST AND RESUSCITATION(2014-03) Nguyen, Anh Q.; Cherry, Brandon H.; Myoung-Gwi, Ryou; Williams, Arthur G.; Hollrah, Roger; Baker, Charla L.; Choudhury, Gourav; Olivencia-Yurvati, Albert H.; Mallet, Robert T.Purpose (a): Cardiac arrest, a leading cause of death in the U.S., kills >90% of its victims, and survivors often are disabled by permanent brain injury inflicted by ischemia-reperfusion. Purkinje cells of the cerebellum and CA1 neurons of the hippocampus are especially vulnerable to post-ischemic neuronal death. We tested the hypothesis that cardiac arrest in a swine model caused delayed neuronal death. Methods (b): Yorkshire swine (25-35 kg) were subjected to cardiac arrest-resuscitation (n = 9) or non-arrest sham (n = 5) protocols. Ventricular fibrillation was induced by electrical pacing. Precordial compressions (100/min) were given at 6-10 min arrest, and then sinus rhythm was restored with transthoracic countershocks. NaCl was infused iv at 0.1 mmol/kg/min during CPR and the first 60 min after return of spontaneous circulation (ROSC). At 7 d ROSC, brain regions were fixed in 4% paraformaldehyde and H&E stained. Results (c): More than 70% of the Purkinje cells were shrunken, lacked dendrites and displayed condensed cytoplasm at 7 d ROSC; in contrast, in shams the majority of Purkinje cells retained the characteristic thick dendrites and well-defined nuclei. Conclusions (d): Thus, cardiac arrest-resuscitation produced marked changes in cerebellar neurons evident 7d after acute insult.Item Does Intermittent Hypoxia Training Augment Antioxidant and Anti-Glycation Enzymes in Rat Brain?(2017-03-14) Cherry, Brandon; Williams, Arthur Jr.; Marianna, Jung; Myoung-Gwi, Ryou; Mallet, Robert T.; Nguyen, JulianHypothesis: Intermittent hypoxia training (IHT) has been found to minimize damage in the brain of rats subjected to ischemic stroke and alcohol intoxication-withdrawal, but the neuroprotective mechanisms are unclear. The finding that antioxidant treatments during IHT blunt the protection identifies a pivotal role of reactive oxygen species (ROS). Because ROS activate expression of antioxidant and anti-glycation enzymes, this study addressed the hypothesis that IHT augments these enzymes in rat brain. Specifically, the cerebral activities of anti-glycation (glyoxalase-1, i.e. GLO1), anti-oxidant (glucose 6-phosphate dehydrogenase, i.e. G6PDH) and hypoxia-inert (lactate dehydrogenase, i.e. LDH) enzymes were analyzed in IHT and non-hypoxic rats. Material and Methods: Ten rats (5 males) completed a 20 day IHT program (5-8 daily cycles of 5-10 min exposures to 9.5-10% O2 followed by 4 min room air exposures), and another 10 rats (5 males) were sham-conditioned by cyclic exposures to 21% O2. One day after completing the IHT or sham programs, the rats were isoflurane-anesthetized and decapitated. The cerebra were harvested, flash-frozen in liquid N2, pulverized in a mortar under liquid N2, homogenized in phosphate buffer, and centrifuged. Enzyme activities in supernatants were analyzed by spectrophotometry, and total protein content by colorimetric Bradford assay. Results: Activities of GLO1 were 78 ± 8 and 62 ± 7 mU/mg protein in the IHT and sham groups, respectively (P = 0.23). G6PDH activities were 21 ± 2 and 24 ± 2 mU/mg protein in the IHT and sham groups (P = 0.32). As expected, LDH activities were similar in the two groups: 899 ± 49 mU/mg protein in the IHT rats, and 940 ± 58 mU/mg protein in the sham rats (P = 0.60). Thus, IHT did not produce a statistically significant treatment effect on these enzymes. Conclusions: The 20 day IHT program, which exerts robust cerebroprotection against ischemia-reperfusion and ethanol intoxication-withdrawal, did not augment activities of selective antioxidant and anti-glycation enzymes. The impact of IHT on other antioxidant (e.g. glutathione peroxidase, superoxide dismutase, catalase) and anti-glycation (e.g. glyoxalase-2) enzymes remains to be evaluated. It also is possible that IHT activates other cytoprotective mechanisms, including signaling cascades that ameliorate mitochondrial permeability transition, oxidative stress and other mechanisms of neural injury. Such alternative mechanisms merit investigation.Item Effects of pyruvate fortified cardiopulmonary resuscitation on myocardial injury after cardiac arrest(2015-03) Ray, George; Batliwala, Shehzad; Nguyen, Anh; Estrada, Juan; Olivencia-Yurvati, Albert H.; Mallet, Robert T.Effects of pyruvate fortified cardiopulmonary resuscitation on myocardial injury after cardiac arrest BACKGROUND: Cardiac arrest kills 400,000 Americans annually. Over 90% of victims outside the hospital and 76% of those inside the hospital succumb to the destructive effects of cardiac arrest on the vital organs. To save the victims, forceful precordial chest compressions and trans-thoracic countershocks are applied, but the use of metabolic substrates as adjuvant treatments for cardiac arrest has not been tested clinically. Pyruvate, a natural intermediary metabolite, energy substrate and antioxidant, has been found to be cardioprotective during ischemia. Pyruvate accomplishes this cardioprotection through several mechanisms including enhancement of myocardial energy reserves and antioxidant defenses, and reduction of reactive oxygen species. Pyruvate-fortified cardioplegic solution administered during cardiopulmonary bypass hastened recovery of cardiac function and shortened hospitalization in patients undergoing coronary revascularization. Despite these discoveries, pyruvate’s effects on myocardial structural damage and inflammation after cardiopulmonary resuscitation and defibrillatory countershocks treated cardiac arrest have not been reported. This study tested the hypothesis that intravenous pyruvate administration minimizes left ventricular structural damage and inflammation that result from myocardial ischemia-reperfusion, precordial chest compressions, and trans-thoracic countershocks. METHODS: Isoflurane-anesthetized Yorkshire swine, 25-40 kg of both genders were studied. The heart was arrested by rapid pacing. After 6 minutes of arrest, precordial compressions were applied at a rate of 100/min for 4 minutes while sodium pyruvate or NaCl control was infused iv to a concentration of 4 mM. Transthoracic countershocks (200-300 J) were administered until spontaneous cardiac rhythm was restored, and infusions maintained until 60 min recovery. The pigs were recovered for 72 hours, and then transmural biopsies of left ventricular free wall were collected, formalin-fixed and paraffin-embedded. Non-arrested sham experiments also were performed and compared with pyruvate- and NaCl-treated cardiac arrest-resuscitation. Sections were cut, stained with hematoxylin and eosin, and 20 random high power (100x) fields were examined and scored by an investigator blinded to the protocol. Structural endpoints included extracellular expansion, neutrophil invasion and hypercontracted tissue. RESULTS: Sections of left ventricular myocardium are currently being analyzed from the 6 sham, 6 cardiac arrest, and 6 pyruvate treated cardiac arrest experiments for neutrophil infiltration, edema, hypercontraction and other evidence of structural damage. CONCLUSIONS: Evidence of decreased neutrophil count, extracellular volume and cardiomyocyte hypercontraction in pyruvate-treated vs. control myocardium 72 h after cardiac arrest will be taken as evidence supporting the hypothesis. Such outcomes would argue for the acute use of pyruvate based interventions during initial treatment to promote post-cardiac arrest myocardial structural integrity.Item 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, FloraPurpose 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.Item Hydroxyethyl Starch-Enhanced Flush Initially Minimizes but Ultimately Exacerbates Edema of Machine Perfused Porcine Kidneys(2022) Wade, Michael; Ramos, Katherine; Reyad, Ashraf; Williams, Arthur; Yurvati, Albert; Mallet, Robert T.; Horani, CaseyHydroxyethyl Starch-Enhanced Flush Initially Minimizes but Ultimately Exacerbates Edema of Machine Perfused Porcine Kidneys. Michael Wade, Katherine Ramos, Casey Horani, Ashraf Reyad, Arthur G. Williams Jr., Albert H. O-Yurvati, Robert T. Mallet. PURPOSE: Improved preservation of explanted kidneys is essential to narrow the supply vs. demand disparity for transplantable human kidneys. Edematous expansion of the explanted organ may make explanted kidneys unsuitable for transplant. This study evaluated the impact of initial flushing of porcine kidneys with a solution containing hydroxyethyl starch (HES), an osmolyte commonly used in preservation solutions for kidney transplantation, on edema during subsequent hypothermic machine perfusion METHODS: This study utilized kidneys from Yorkshire swine due to the anatomical and functional similarities of porcine and human kidneys. Left kidneys were harvested from isoflurane-anesthetized pigs via laparotomy. The renal artery was cannulated and the organ was flushed for 10 min with 400 mL of control Ringer's solution (group A) or with Ringer's solution containing 50 g/L hydroxyethyl starch (group B). Kidneys then underwent hypothermic machine perfusion (2-4°C) for 21-72 hr in a LifePort organ preservation system, with flow rates and resistance recorded throughout perfusion. The kidneys were weighed before and after flush of the organ, weighed again after machine perfusion, and then biopsied for histological analysis of renal cortex and medulla. RESULTS: Group A kidney mass increased by 49 ± 13% (mean ± standard deviation) during initial flush (n =7) and by 52 ± 14% after flush and 72 h machine perfusion (n=8); thus, 95% of the organ expansion occurred during the initial flush. In contrast, Group B kidney mass increased by 19 ± 9% during initial organ flush (n=6), a 60% reduction vs. Group A (P < 0.001), but total weight gain was 83 ± 21% after machine perfusion (n=3). Machine perfusion of 2 of the 3 Group B kidneys failed at 21 hr and 23 hr perfusion, and only 1 was perfused for the entire 72 hours. In both groups, histology revealed preserved tubular and glomerular architecture, but appreciable cellular edema following machine perfusion. Accumulation of debris in the tubular lumina and vacuolization of tubular epithelial cells was evident in Group B, but not Group A. CONCLUSION: In explanted kidneys flushed with colloid-free Ringer's and machine-perfused for 72 h, almost the entire increase in mass occurred during the initial organ flush. As hypothesized, the addition of HES colloid sharply lowered the initial organ expansion, but unexpectedly exacerbated organ expansion during subsequent hypothermic machine perfusion, such that the HES-flushed kidneys gained more mass than the controls flushed without HES. Thus, the potential benefit of including HES in the initial flush solution was lost during machine perfusion. Arguably, the excessive expansion may have contributed to the impaired machine perfusion of these kidneys. Animal Use Protocol: IACUC-2020-0011Item Iatrogenic Ureter Injury and Repair: Comparing Suture Versus Suture and Glue in Ureter End-to-End Anastomosis(2023) Iloani, Nwamaka Amy; Wade, Michael; Joseph, Matthew; Martinez, Maria; Ntekim, Nedeke; Mallet, Robert T.; Yurvati, AlbertFunding: Dallas Southwest Osteopathic Physicians The ureter is a delicate structure due to its size and anatomical location. It is found deep within the retroperitoneal space and the lower third of the ureter lies adjacent to numerous pelvic structures such as the uterine artery, cervix, vagina, colon, and iliac vessels, and due to its proximity, the ureter is subject to unintentional accidental injury during diagnostic or medical procedures, termed iatrogenic. This paper and associated aim to explore the odds of iatrogenic ureteral injuries (IUI) when comparing different surgical methods, discuss the best preventative options and review the current repair measures, as well as any associated complications. The aim of this study focused on ureter transection that require surgical repair using a ureterouretal anastomosis, a procedure that ligates the free ends of the transected ureter together. Ureters were obtained from anesthetized Yorkshire pigs via standard laparotomy [protocol: IACUC-20-0011]. Ureters were transected laterally midway along the length of the harvested ureter. In Group A ureters, 2 standard stay sutures were placed 180˚ degrees opposite one another to anchor the ureter and ligated using a simple continuous running suture technique. In Group B, ureters were anchored in a similar fashion with stay sutures and ligated using glue to perform the anastomosis. 500 mL saline was then flushed through the ureter to assess the structural integrity of the ureterouretal anastomosis. Group A ureters showed less stricture formation and fluid leakage at the site of anastomoses, indicating that the standard suture technique used in ureter repair is a safe and reasonable gold standard. In contrast, Group B ureters showed stricture formation and some fluid leakage at suture/glue line, indicating the method of ureter anastomoses was inferior. When IV saline was pushed in Group B ureters, the fluid often exited the ureter at both the suture site and the end portion of the ureter, more so than the Group A ureters. Future study needs to be conducted in suture/glue method as it is promising. However, it is not at the level where it can replace current ureter repair end to end anastomoses gold standard. *These authors are solely listed in alphabetical order Research reported in this publication 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.Item Impact of Age on Cognitive Testing Practice Effects and Cardiorespiratory Responses(Sage Publications, 2024-02-27) Reddy, Priyanka M.; Abdali, Kulsum; Ross, Sarah E.; Davis, Sandra; Mallet, Robert T.; Shi, XiangrongObjective: This study tested the hypothesis that healthy aging attenuates cognitive practice effects and, consequently, limits the familiarity-associated reductions in heart rate (HR) and breathing frequency (BF) responses during retesting. Methods: Twenty-one cognitively normal older and younger adults (65 +/- 2 vs. 26 +/- 1 years old) participated in the study. Mini-Mental State Examination (MMSE), Digit-Span-Test (DST), Trail Making Test (TMT-B), and California Verbal Learning Test (CVLT-II) were administered twice at 3-week intervals, while HR and BF were monitored by electrocardiography and plethysmography, respectively. Results: Cognitive performances were not affected by the age factor, and the retest factor only affected CVLT-II. HR and BF increased only in the younger adults (p < .01) during cognitive tests; retesting attenuated these responses (retest factor p < .01). Long-delay free-recall in CVLT-II was unchanged in cognitively normal older versus younger adults. Healthy aging did not diminish short-term memory assessed by DST and CVLT-II short-delay or long-delay free-recalls. Conclusions: Only CVLT-II, but not MMSE, DST or TMT-B, demonstrated cognitive retesting practice effects in the younger and older adults. Cognitive testing at 3-week intervals in cognitively normal older and younger subjects revealed divergent cardiorespiratory responses to MMSE, DST, and TMT-B cognitive testing, particularly HR, which increased only in younger adults and to a lesser extent during retesting despite the absence of practice effects.Item Inline flow sensor for ventriculoperitoneal shunts: Experimental evaluation in swine(2018-03-14) Qin, Chuchu; Williams, Arthur Jr.; Dasgupta, Pernandu; Mallet, Robert T.; Yurvati, Albert; Eskildsen, DaneBackground: Hydrocephalus is a potentially life-threatening disorder in which cerebrospinal fluid (CSF) fails to circulate properly, causing a dangerous buildup of pressure in the cerebral ventricles and the surrounding brain tissue. It is seen most often in infants with congenital abnormalities of the CSF tract, and in patients with traumatic brain injury. The only treatment in most cases is the placement of a tubing to drain the excess CSF from the brain to the abdomen. These shunts are prone to blockage that requires invasive replacement surgery, so a reliable flow sensor is needed to detect shunt failure at its early stages. Currently available flow sensors often fail to detect blockage. Objective: The purpose of this project is to evaluate an advanced in-line electronic flow sensor capable of monitoring CSF flow over time for use in the treatment of hydrocephalus. This project evaluated the performance of this sensor in domestic swine. Methods/Materials: Ventriculo-peritoneal shunts were installed in the third cerebral ventricle of juvenile Yorkshire pigs, and routed to the peritoneal space in the abdomen. The flow sensor was positioned halfway between the cephalic and peritoneal ends of the shunt. Data were acquired on a laptop computer. Shunt flows were obtained at 30 s intervals. The sensor alternately heated the shunt fluid for 5 s and then monitored temperature decline, the rate of which was proportional to flow, for 25 s. The fluid was diverted into pre-weighed vials for 1- or 5-min to determine flow gravimetrically. At regular intervals, 5-20 ml boluses of artificial CSF were injected into the third ventricle. Flows reported by the sensor were compared to concomitant gravimetric flows by linear regression. Results: Over 4300 sensor measurements of flow were obtained in 6 experiments. The flow sensor reliably reports shunt flows up to 35 ml/min, the highest rate produced by 20 ml CSF injections. Four experiments showed strong linear correlations (r2 ³ 0.90) between gravimetric and sensor flows. The slope of the linear regression between the two flows was 1.05 ± 0.14 in the 6 experiments, indicating that the sensor accurately reported flows of up to 35 ml/min. Conclusions: The results of this experiment indicate that the flow sensor can report accurately ventriculo-peritoneal shunt flows over a wide range in a large animal model. Studies are planned to evaluate performance of chronically implanted shunts in ambulatory pigs.Item INTERMITTENT HYPOXIA CONDITIONING REDUCES INOS AND ENOS EXPRESSION IN RAT MYOCARDIUM: A MECHANISM FOR PROTECTION FROM ISCHEMIA AND REPERFUSION INJURY(2014-03) Downey, H. Fred; Manukhina, Eugenia B.; Goryacheva, Anna V.; Belkina, Ludmila M.; Terekhina, Olga L.; Mallet, Robert T.During a heart attack, the heart produces excessive amounts of harmful chemicals. This study showed that prior exposure of rats to short cycles of a low oxygen atmosphere reduced production of these chemicals and lessened the damage to muscle and blood vessels of the heart. This procedure may benefit humans at risk of heart attack. Purpose (a): Recently we demonstrated that intermittent, normobaric hypoxia conditioning (IHC) prevented injuries of myocardium and coronary blood vessels induced by myocardial ischemia and reperfusion (IR). This cardio- and vasoprotection of was associated with alleviation of nitric oxide overproduction. The aim of this study was to identify specific NO synthases responsible for the IR-induced NO overproduction and to determine the effects of IHC on these NO synthases. Methods (b): This research was approved by the Animal Care and Use Committee of the Institute of General Pathology and Pathology. IHC of rats was performed in a normobaric chamber (5-8 cycles/d for 20 d, FIO2 9.5 - 10% for 5 - 10 min/cycle, with intervening 4-min normoxia). IR was produced by ligation of the left anterior descending coronary artery for 30 min followed by 60-min reperfusion. The protein nitration marker, nitrotyrosine (3-NT) and neuronal (nNOS), inducible (iNOS), and endothelial (eNOS) nitric oxide synthases were measured by immunoblot. Results (c): IR induced appreciable 3-NT accumulation in the left ventricular free wall, increasing the 3-NT content by 42% (p<0.01), but not in septum. In IHC rats, 3-NT after IR was similar to that of control rats without IR. IHC decreased iNOS by 71% (p<0.05) and eNOS by 41% (p<0.05) in the left ventricular myocardium; the myocardial content of nNOS remained unchanged. Conclusions (d): IHC prevents IR-induced NO overproduction in myocardium by restricting myocardial expression of iNOS and eNOS.Item Intermittent Hypoxia Training to Foster Brain Recovery after Ischemic Stroke in rats(2018-05) Ruelas, Steven S.; Mallet, Robert T.; Jung, Marianna E.; Schreihofer, Ann M.; Das, Hriday K.Purpose: Ischemic stroke is the leading cause of disability and #5 cause of death in the US. Annually, nearly 800,000 Americans suffer an ischemic stroke, and 130,000 die. The only FDA approved treatment for stroke is recombinant tissue plasminogen activator, but this thrombolytic agent neither protects the affected tissue, nor mitigates the motor or cognitive impairments resulting from stroke. Intermittent hypoxia training (IHT) has been shown to increase cerebral blood flow, reduce oxidative stress, mobilize cerebroprotective signaling cascades and minimize behavioral deficits in a rat model of Alzheimer's Disease. Moreover, a 20 d IHT program attenuated behavioral deficits and protected neurons in ethanol-withdrawn (EW) rats, even when EW began 35 d after IHT. Therefore, we hypothesize that IHT, initiated in rats after stroke, preserves motor and cognitive function, relative to non-IHT rats. Methods: Ischemic stroke will be produced in rats by 90 min occlusion and abrupt reperfusion of the middle cerebral artery (MCA). Motor function and coordination will be evaluated by the rotarod test before and at 1 week intervals after MCA occlusion (MCAO). Rats must balance on a rotating cylinder that accelerates at a constant speed. High fall latency represents intact motor function. The Morris Water Maze (MWM) assesses spatial learning and memory. Rats are placed in an open, circular pool and must find a sunken platform within 90 s. 24 h after stroke, rats undergoing IHT will breathe moderately hypoxic gas (10% O2) for 5-8 cycles, each lasting 5-10 min, with intervening 4 min room air breathing, for 20 consecutive days. These rats will be compared to an MCAO group continuously exposed to 21% O2. At 21 d post-stroke, the brain will be harvested for analyses of infarct and neuroprotective proteins. Results: In pre-stroke testing, the time taken to solve the MWM fell progressively over 10 days, indicating spatial learning and memory, and fall latency on the rotarod lengthened over 5 days, reflecting improved coordination and possibly a training effect. These studies have established the pre-stroke baselines for assessment of IHT's impact on post-stroke recovery. Conclusions: We expect that IHT given after stroke will minimize motor and cognitive impairment by activating neuroprotective signaling cascades culminating in expression of anti-oxidant and anti-inflammatory proteins.