Normothermic Conditions Research Articles

Reduced perforin release from T cells as a mechanism underlying hypothermia‐mediated neuroprotection

AbstractObjectiveThe underlying mechanisms of therapeutic hypothermia, which protects neurons after severe brain damage, are partially understood. T cells infiltrate the brain within days after cerebral ischemia and play essential roles in exacerbating the delayed phase of brain injury by producing cytotoxic factors, which were also systematically shown to be involved in brain damage. Periphery brain abnormalities are interesting, because the periphery might constitute a pathway to the central nervous system and therefore could also be a target of therapeutic hypothermia. We elucidated the effects of hypothermia and hyperthermia on peripheral T cell‐derived perforin (Pfn), and the underlying mechanism of Pfn‐mediated neurotoxicity and death of brain microvascular endothelial cells (BMVECs).MethodsWe determined the levels of Pfn produced by activated CD4+ and CD8+ T cells obtained from healthy humans under hypothermic, normothermic and hyperthermic conditions. The viability in response to Pfn treatment was assessed in neuronal Neuro‐2a and brain microvascular endothelial bEnd.3 cells.ResultsCompared with normothermia, Pfn release in both CD4+ and CD8+ T cells was reduced by hypothermia, but augmented by hyperthermia. Pfn caused the death of Neuro‐2a cells, and induced both apoptosis and necrosis in bEnd.3 cells; both effects were concentration‐dependent.ConclusionsHypothermia reduces T cell‐derived release of Pfn, which mediates neuronal cell death and apoptosis/necrosis of BMVECs, suggesting that therapeutic hypothermia elicits protection in the neurons and BMVECs in the delayed phase of brain injury by directly reducing the release of T cell‐derived Pfn. BMVECs protection can aid in reduced vascular permeability and blood–brain barrier protection.

Short-Term Fever-Range Hyperthermia Accelerates NETosis and Reduces Pro-inflammatory Cytokine Secretion by Human Neutrophils.

Fever is a hallmark of infections and inflammatory diseases, represented by an increase of 1–4°C in core body temperature. Fever-range hyperthermia (FRH) has been shown to increase neutrophil recruitment to local sites of infection. Here, we evaluated the impact of a short period (1 h) of FRH (STFRH) on pro-inflammatory and bactericidal human neutrophil functions. STFRH did not affect neutrophil spontaneous apoptosis but reverted the lipopolysaccharide (LPS)-induced anti-apoptotic effect compared with that under normothermic conditions. Furthermore, STFRH accelerated phorbol myristate acetate (PMA)-induced NETosis evaluated either by the nuclear DNA decondensation at 2 h post-stimulation or by the increase in extracellular DNA that colocalized with myeloperoxidase (MPO) at 4 h post-stimulation. Increased NETosis upon STFRH was associated with an increase in reactive oxygen species (ROS) production but not in autophagy levels. STFRH also increased NETosis in response to Pseudomonas aeruginosa challenge but moderately reduced its phagocytosis. However, these STFRH-induced effects did not influence the ability of neutrophils to kill bacteria after 4 h of co-culture. STFRH also significantly reduced neutrophil capacity to release the pro-inflammatory cytokines chemokine (C-X-C motif) ligand 8/interleukin 8 (CXCL8/IL-8) and IL-1β in response to LPS and P. aeruginosa challenge. Altogether, these results indicate that a short and mild hyperthermal period is enough to modulate neutrophil responses to bacterial encounter. They also suggest that fever spikes during bacterial infections might lead neutrophils to trigger an emergency response promoting neutrophil extracellular trap (NET) formation to ensnare bacteria in order to wall off the infection and to reduce their release of pro-inflammatory cytokines in order to limit the inflammatory response.

Whole lung lavage using a rapid infusion system to treat a patient with pulmonary alveolar proteinosis.

Whole lung lavage (WLL) is a therapeutic procedure to remove accumulated material by infusing and draining the lungs with lavage fluid. This procedure has been regarded as the current standard of care to treat pulmonary alveolar proteinosis. However, the WLL protocol has not yet been standardized and the technique has been refined and modified a number of times. A rapid infusion system is a device used to infuse blood or other fluids at precise rates and normothermic conditions. This device is not typically used in WLL, which relies on the passive infusion of fluids using the gravitational force. However, in this study we performed WLL using a rapid infusion system, since we aimed to take advantage of its shorter operation time and greater degree of control over fluid volume and temperature. The patient’s symptoms improved without the occurrence of any complications.

Hepatic Metabolic Derangements Triggered by Hyperthermia: An In Vitro Metabolomic Study.

Background and aims: Liver toxicity is a well-documented and potentially fatal adverse complication of hyperthermia. However, the impact of hyperthermia on the hepatic metabolome has hitherto not been investigated. Methods: In this study, gas chromatography-mass spectrometry (GC-MS)-based metabolomics was applied to assess the in vitro metabolic response of primary mouse hepatocytes (PMH, n = 10) to a heat stress stimulus, i.e., after 24 h exposure to 40.5 °C. Metabolomic profiling of both intracellular metabolites and volatile metabolites in the extracellular medium of PMH was performed. Results: Multivariate analysis showed alterations in levels of 22 intra- and 59 extracellular metabolites, unveiling the capability of the metabolic pattern to discriminate cells exposed to heat stress from cells incubated at normothermic conditions (37 °C). Hyperthermia caused a considerable loss of cell viability that was accompanied by significant alterations in the tricarboxylic acid cycle, amino acids metabolism, urea cycle, glutamate metabolism, pentose phosphate pathway, and in the volatile signature associated with the lipid peroxidation process. Conclusion: These results provide novel insights into the mechanisms underlying hyperthermia-induced hepatocellular damage.

Serum, erythrocyte and urinary concentrations of iron, copper, selenium and zinc do not change during an incremental test to exhaustion in either normothermic or hyperthermic conditions.

The aim of this study was to evaluate the effect of the performance of an incremental exercise test until exhaustion in normothermic and hyperthermic conditions on serum, erythrocyte and urine concentrations of Iron (Fe), Copper (Cu), Selenium (Se) and Zinc (Zn). Nineteen adult males (age: 22.58 ± 1.06 years) performed two maximum incremental exercise tests on a cycloergometer in normothermia (22 ± 2 °C) and hyperthermia (42±2 °C) separated by 48 h. Urine, serum and erythrocyte samples were collected before and after each test. Serum Se (p < 0.01) and Cu (p < 0.05) levels were altered after each test, but the significance disappeared with the correction for haematocrit. The rest of the values did not undergo alterations in either condition. It seems that a higher stimulus is necessary to obtain changes in these minerals. The study reveals the need to correct serum concentrations concerning possible changes in these volumes after an acute effort.

Risperidone Treatment after Transient Ischemia Induces Hypothermia and Provides Neuroprotection in the Gerbil Hippocampus by Decreasing Oxidative Stress.

Compelling evidence from preclinical and clinical studies has shown that mild hypothermia is neuroprotective against ischemic stroke. We investigated the neuroprotective effect of post-risperidone (RIS) treatment against transient ischemic injury and its mechanisms in the gerbil brain. Transient ischemia (TI) was induced in the telencephalon by bilateral common carotid artery occlusion (BCCAO) for 5 min under normothermic condition (37 ± 0.2 °C). Treatment of RIS induced hypothermia until 12 h after TI in the TI-induced animals under uncontrolled body temperature (UBT) compared to that under controlled body temperature (CBT) (about 37 °C). Neuroprotective effect was statistically significant when we used 5 and 10 mg/kg doses (p < 0.05, respectively). In the RIS-treated TI group, many CA1 pyramidal neurons of the hippocampus survived under UBT compared to those under CBT. In this group under UBT, post-treatment with RIS to TI-induced animals markedly attenuated the activation of glial cells, an increase of oxidative stress markers [dihydroethidium, 8-hydroxy-2′ -deoxyguanosine (8-OHdG), and 4-Hydroxynonenal (4-HNE)], and a decrease of superoxide dismutase 2 (SOD2) in their CA1 pyramidal neurons. Furthermore, RIS-induced hypothermia was significantly interrupted by NBOH-2C-CN hydrochloride (a selective 5-HT2A receptor agonist), but not bromocriptine mesylate (a D2 receptor agonist). Our findings indicate that RIS-induced hypothermia can effectively protect neuronal cell death from TI injury through attenuation of glial activation and maintenance of antioxidants, showing that 5-HT2A receptor is involved in RIS-induced hypothermia. Therefore, RIS could be introduced to reduce body temperature rapidly and might be applied to patients for hypothermic therapy following ischemic stroke.

Development of a Rat Liver Machine Perfusion System for Normothermic and Subnormothermic Conditions

Ex vivo liver machine perfusion (MP) is a promising alternative for preservation of liver grafts from extended criteria donors. Small animal models can be used to evaluate different perfusion conditions. We here describe the development of a miniaturized ex vivo MP system for rat liver grafts, evaluating cell-free and erythrocyte-based perfusion solutions, subnormothermic and normothermic temperatures, and dialysis. A perfusion chamber was designed after a suitable liver position was identified. Normothermic ex vivo liver perfusion (NEVLP) required supplementation of erythrocytes to reduce cell damage. Perfusion with erythrocytes led to rising potassium levels after 12 h (NEVLP, 16.2 mM, interquartile range [IQR]: 5.7 and subnormothermic ex vivo liver perfusion [SNEVLP], 12.8 mM, IQR: 3.5), which were normalized by dialysis using a laboratory dialysis membrane (NEVLP, 6.2 mM, IQR: 0.5 and SNEVLP, 5.3 mM, IQR: 0.1; p = 0.004). Livers treated with NEVLP conditions showed higher bile production (18.52 mg/h/g, IQR: 8.2) compared to livers perfused under SNEVLP conditions (0.4 mg/h/g, IQR: 1.2, p = 0.01). Reducing the perfusion volume from 100 to 50 mL allowed for higher erythrocyte concentrations, leading to significantly lower transaminases (15.75 U/L/mL, IQR: 2.29 vs. 5.97 U/L/mL, IQR: 18.07, p = 0.002). In conclusion, a well-designed perfusion system, appropriate oxygen carriers, dialysis, and miniaturization of the perfusion volume are critical features for successful miniaturized ex vivo liver MP. Impact Statement Ex vivo liver machine perfusion (MP) is an emerging preservation alternative to static cold storage. Even though clinical studies have shown benefits for extended criteria donor grafts, standardized systems for perfusion of rat liver grafts are not available, which are inevitable for large-scaled studies on liver reconditioning by ex vivo MP. We here comprehensively describe the development of an ex vivo rat liver perfusion system that can be used as modular setting in various approaches of liver MP. We describe pitfalls and techniques that might be of interest when establishing such perfusion systems for basic and translational research.

Implementation of an experimental isolated lung perfusion model on surgically resected human lobes

Isolated lung perfusion (ILP) is an ideal model to study treatment effects on a variety of pathologies. As published research mostly relies on rejected donor lungs or animal organs, this study investigates the use of surgically resected human lobes as an alternative and novel model for personalized experimental research. Ten surgically resected lobes were perfused in acellular and normothermic condition. The indication for surgery was lung cancer. Perfusion and ventilation were adapted to the size of the lobes and both functional and metabolic parameters were assessed during ILP. Patients (age 67.5 y (59–81)|♀n = 3|♂n = 7) underwent anatomic pulmonary lobectomy. Ischemic time between arterial ligation and ILP was 226 minutes (161–525). Median duration of ILP was 135 (87–366) minutes. Gas exchange and mechanical respiratory parameters remained steady during ILP (pulmonary venous pO2 196(151–219) mmHg | peak AWP: 14.5(11–22) cmH2O). Metabolism stayed constant during ILP (Glucose consumption: 1.86 mg/min/LTLC (95%CI: −2.09 to −1.63) | lactate production: 0.005 mmol/min/ LTLC (95%CI: 0.004 to 0.007)). ILP of surgically resected human lobes is a feasible and promising method. By maintaining a near physiological setting, this model may pave the way for future experimental lung research including cancer research, transplantation, physiology, pharmacology and mechanical ventilation.

Initial perfusate purification during subnormothermic machine perfusion for porcine liver donated after cardiac death.

Improvement of machine perfusion (MP) technologies is required to enhance organ quality for donor after cardiac death (DCD) grafts. Installing a dialyzer or a filter into the perfusion circuit to maintain the perfusate condition has some advantages. However, the consequences of purification perfusate during subnormothermic machine perfusion (SNMP) remain unexplained. In this study, the effects of initial purification perfusate with simple method of replacing the first 0.5-L perfusate during SNMP were investigated to consider installation effect of the filter or the dialyzer. Porcine liver grafts, which have 60-min warm ischemia time, were procured to imitate the DCD graft condition. Purified SNMP (PSNMP) results were compared with simple cold storage and conventional SNMP. In PSNMP, initial perfusate of 0.5L was removed to substitute for purification. After preservation process, the preserved grafts were reperfused with diluted autologous blood for 2h under normothermic machine perfusion condition to evaluate the liver function using an isolated reperfusion model. The vascular pressures, enzyme release rates and the metabolic indexes during reperfusion were analyzed. The pressures in the hepatic artery after reperfusion 60min were significantly lower in PSNMP group compared with cold storage (CS) and SNMP groups. In addition, lactate dehydrogenase and alkaline phosphatase were significantly lower after PSNMP than after the CS or SNMP. Also, the metabolic indexes of hyaluronic acid and lactate were significantly decreased by purifying the perfusate in MP preservation than in CS or SNMP. The effectiveness of initial purification perfusate during SNMP was investigated.

Stratifying comatose postanoxic patients for somatosensory evoked potentials using routine EEG

IntroductionMultimodal neurological prognostication is recommended for comatose patients after cardiac arrest. The absence of cortical N20-potentials in a somatosensory evoked potential (SSEP) examination reliably predicts poor outcome, but presence of N20-potentials have limited prognostic value. A benign routine electroencephalogram (EEG) may identify patients with a favourable prognosis who are likely to have present N20-potentials. ObjectiveTo investigate whether a routine EEG can identify patients where SSEP is unnecessary to perform. MethodsIn a multi-centre trial, comatose patients after cardiac arrest were randomised to a controlled temperature of 33 °C or 36 °C. A routine EEG was protocolised and SSEP performed at the clinicians’ discretion, both during normothermic conditions. EEGs were categorised into benign, malignant or highly malignant based on standardised terminology. A benign EEG was defined as a continuous normal-voltage background without abundant discharges. The N20-potentials were reported as absent (bilaterally) or present (bilaterally or unilaterally). ResultsBoth EEG and SSEP were performed in 161 patients. EEG was performed before SSEP in 60%. A benign EEG was seen in 29 patients and 100% (CI 88–100%) had present N20-potentials. For the 69 patients with a malignant EEG and the 63 patients with a highly malignant EEG, 67% (CI 55–77%) and 44% (CI 33–57%) had present N20-potentials, respectively. ConclusionsAll patients with a benign EEG had present N20-potentials, suggesting that SSEP may be omitted in these patients to save resources. SSEP is useful in patients with a malignant or highly malignant EEG since these patterns are associated with both present and absent N20-potentials.

A 2.5 min cold water immersion improves prolonged intermittent sprint performance

ObjectivesWe investigated if cold water immersion (CWI) affects exercise performance during a prolonged intermittent sprint test (IST), designed to mimic activity patterns of team-sports. DesignRandomized-crossover design. MethodsTen male team-sport players completed 3 IST protocols (two 40-min “halves” of repeated 2-min blocks consisting of a 8-s “all-out” sprint, 100-s active recovery and 12-s rest) on a cycle ergometer at normothermic conditions. Each “half” was separated by a 15 min recovery period of either: (i) passive rest, (ii) 5-min CWI at 8 °C (CWI-5) or (iii) 2.5-min CWI at 8 °C (CWI-2.5), in a random counterbalanced order. ResultsPhysical performance, core temperature (Tcore) and heart rate were not different among conditions in the first half. In the passive rest trial, total work (TW) and peak power (PP) were lower during the second half (TW: 5.04 ± 1.11 kJ; PP: 929 ± 286 W) than the first half (TW: 5.66 ± 1.02 kJ; PP: 1009 ± 266 W); while TW and PP were not different between halves following CWI-5 (first half, TW: 5.34 ± 1.02 kJ, PP: 1016 ± 283 W; second half, TW: 5.19 ± 1.38 kJ; PP: 996 ± 318 W) and CWI-2.5 (first half, TW: 5.47 ± 1.19 kJ, PP: 966 ± 261 W; second half, TW: 5.25 ± 1.17 kJ; PP: 952 ± 231 W). Tcore was lower until the 20th minute of the second half after CWI-5 and CWI-2.5 compared with passive rest. ConclusionsA post-exercise 2.5–5-min CWI attenuates the reductions in prolonged sprint performance that occur in the second half of team sports, due, at least partly, to reductions in core temperature and associated increase in heat storage.

Effect of heat exposure and physical exercise until exhaustion in normothermic and hyperthermic conditions on serum, sweat and urinary concentrations of magnesium and phosphorus.

The aim of this survey was to ascertain the difference in the levels of Magnesium (Mg) and Phosphorus (P) after an exercise test in normothermia and hyperthermia before and after heat acclimation in comparison to their respective pre-test values. Twenty-nine male university students were divided into an Experimental Group (EG) (n = 15) and a Control Group (CG) (n = 14). All of them voluntarily participated in this investigation. Both groups performed an incremental test until exhaustion on a cycloergometer in normothermia (22 °C) and hyperthermia (42 °C). EG underwent 9 sessions of heat acclimation (100 °C) in a sauna (Harvia C105S Logix Combi Control; 3-15 W; Finland). Once the experimental period was completed, all initial measurements were carried out again under identical conditions. Urine and blood samples were obtained before and after each trial. Sweat samples were collected at the end of every test performed in hyperthermia. The samples were frozen at -80 °C until further analysis by ICP-MS. Lower seric Mg levels were observed in both groups at the end of pre-acclimation tests. After acclimation, only EG experimented a decrease of Mg in serum after testing (p < .01). The urinary excretion was unaffected in the pre-acclimated period, but EG experimented an increase in Mg after trials in the post-acclimation evaluation (p < .01). Mg sweat loss decreased significantly after heat acclimation (p < .05). P did not undergo changes, except in its urinary excretion, which was elevated after the normothermia trial in the post-acclimation period (p < .05). It seems that exercise in hyperthermia altered Mg status but not P homeostasis. Additionally, heat acclimation reduces Mg losses in sweat while increasing its loss in urine. Thus, Mg supplementation should be considered in unacclimated and acclimated subjects if physical exercise is going to be performed in hyperthermic conditions.

Rewarming Injury after Cold Preservation

Organ dysfunction pertinent to tissue injury related to ischemic ex vivo preservation during transport from donor to recipient still represents a pivotal impediment in transplantation medicine. Cold storage under anoxic conditions minimizes metabolic activity, but eventually cannot prevent energetic depletion and impairment of cellular signal homeostasis. Reoxygenation of anoxically injured tissue may trigger additional damage to the graft, e.g., by abundant production of oxygen free radicals upon abrupt reactivation of a not yet equilibrated cellular metabolism. Paradoxically, this process is driven by the sudden restoration of normothermic conditions upon reperfusion and substantially less pronounced during re-oxygenation in the cold. The massive energy demand associated with normothermia is not met by the cellular systems that still suffer from hypothermic torpor and dys-equilibrated metabolites and eventually leads to mitochondrial damage, induction of apoptosis and inflammatory responses. This rewarming injury is partly alleviated by preceding supply of oxygen already in the cold but more effectively counteracted by an ensuing controlled and slow oxygenated warming up of the organ prior to implantation. A gentle restitution of metabolic turnover rates in line with the resumption of enzyme kinetics and molecular homeostasis improves post transplantation graft function and survival.

The development of an extended normothermic ex vivo reperfusion model of the sheep uterus to evaluate organ quality after cold ischemia in relation to uterus transplantation.

Uterus transplantation has recently proved that infertility in women with uterine factor infertility can be cured. It is still an experimental procedure with numerous critical details remaining to be established, including tolerance to warm and cold ischemic insults. In preparation for human uterus transplantation trials, most teams use the sheep as a model system for research and team training, since the vasculature and the uterus is of similar size as in the human. We, therefore, aimed to develop an ex vivo sheep uterus reperfusion platform that mimics the reperfusion situation so that initial assessments and comparisons can be performed without the need for costly and labor-intensive in vivo transplantation experiments. Isolated sheep uteri were perfused with the preservation solution IGL-1 and were then exposed to cold ischemia for either 4(n=6) or 48hours (n=7). Uteri were then reperfused for 48hours under normothermic conditions with an oxygenated recirculating perfusate containing growth factors and synthetic oxygen carriers. Histological and biochemical analysis of the perfusate was conducted to assess reperfusion injury. Quantification of cell density indicated no significant edema in the myometrium or in the endometrium of uteri exposed to 4hours cold ischemia and then a normothermic ex vivo reperfusion for 48hours. Only the outer serosa layer and the inner columnar luminal epithelial cells were affected by the reperfusion. However, a much faster and severe reperfusion damage of all uterine layers were evident during the reperfusion experiment following 48hours of cold ischemia. This was indicated by major accumulation of extracellular fluid, presence of apoptotic-labeled glandular epithelial layer and vascular endothelium. A significant accumulation of lactate was measured in the perfusate with a subsequent decrease in pH. We developed a novel ex vivo sheep uterus model for prolonged perfusion. This model proved to be able to distinguish reperfusion injury-related differences associated to organ preservation. The experimental setup is a platform that can be used to conduct further studies on uterine ischemia- and reperfusion injury that may lead to improved human uterus transplantation protocols.

Neural activity and network during cognitive tasks in heat‐stressed individuals

Reductions in cerebral blood flow accompanying heat stress‐induced hypocapnia via hyperventilation are likely to decrease the cognitive function, but the mechanism remains unknown. Using electroencephalographic event‐related potentials (EEG‐ERP) and functional magnetic resonance imaging (fMRI), we evaluated temporal (EEG) and spatial (fMRI) characteristics during cognitive tasks in heat‐stressed humans. Healthy young volunteers performed a visual cognitive task (Go/No‐go task) before (i.e., normothermia) and during heat stress on separate days. The external canal temperature during heat stress was increased by ~ 1.1°C from the normothermic condition. Decreased cerebral blood flow during heat stress was confirmed by reductions in middle cerebral artery blood velocity (MCAV) and internal carotid blood flow in the EEG‐ERP trial. The amplitudes of the Go‐P300 (i.e., execution) and No‐go‐P300 (i.e., inhibition) components were decreased during heat stress. In the fMRI trial, we observed broadly activated brain regions, including the dorsolateral and ventrolateral prefrontal cortices, and motor‐related areas such as the supplementary motor area and premotor area during heat stress rather than during normothermia. These results suggest that heat stress increases the load of neural activity while performing cognitive tasks, relative to normothermia, while heat stress reduced cerebral blood flow and neural activity.Support or Funding InformationJSPS KAKENHI 18H03166This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

High intensity interval exercise reduces tolerance to a simulated hemorrhagic challenge relative to steady state exercise in heat stressed individuals

Individuals who are exposed to an increased risk of experiencing a hemorrhagic insult, such as soldiers and firefighters, are often required to complete intermittent high intensity exercise in the presence of environmental heat stress. In normothermic conditions, increases in heart rate and reductions in vascular resistance can be greater following high intensity interval exercise relative to continuous steady state exercise. These hemodynamic alterations following high intensity interval exercise may reduce the capacity to withstand a hemorrhagic insult. We investigated whether high intensity interval exercise reduces tolerance to a simulated hemorrhagic challenge (lower body negative pressure; LBNP) relative to steady state exercise in heat stressed individuals. Eight healthy participants (Age: 27 ± 5 years; Ht: 179 ± 9 cm; Wt: 78.9 ± 18.7 kg) completed two trials (Steady state and Interval). Participants performed cycling exercise either by alternating between 10 and 88% (Interval) or continuously at 38% (Steady state) of the predetermined maximal aerobic power output whilst wearing a warm water perfused suit until core temperatures increased by 1.40 ± 0.21°C. Participants then underwent progressive LBNP (−20mmHg, −30mmHg, etc.) to pre syncope. LBNP tolerance was quantified as cumulative stress index (CSI; mmHg*min). Following exercise and prior to LBNP, mean skin temperatures were similarly elevated from baseline in both trials (from: 32.35 ± 0.42 to 37.95 ± 0.59°C, P &lt; 0.05). Mean arterial pressure was not different between trials prior to LBNP (Interval: 79 ± 6 vs. Steady state: 77 ± 7 mmHg; P = 0.57) and were similarly reduced in both trials at pre syncope (to 63 ± 6 and 62 ± 6 mmHg respectively, both P &lt; 0.05). CSI was lower in the interval trial relative to the steady state trial (280 ± 204 vs. 518 ± 285 mmHg*min, respectively; P = 0.024). In heat stressed individuals, tolerance to a simulated hemorrhagic challenge was reduced following high intensity interval exercise relative to steady state exercise. These findings have implications for individuals who exercise at intermittent high intensities in hot environments and are at increased risk of experiencing a hemorrhagic injury (e.g. soldiers, firefighters and miners).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Restoration of brain circulation and cellular functions hours post-mortem.

The brains of humans and other mammals are highly vulnerable to interruptions in blood flow and decreases in oxygen levels. Here we describe the restoration and maintenance of microcirculation and molecular and cellular functions of the intact pig brain under ex vivo normothermic conditions up to four hours post-mortem. We have developed an extracorporeal pulsatile-perfusion system and a haemoglobin-based, acellular, non-coagulative, echogenic, and cytoprotective perfusate that promotes recovery from anoxia, reduces reperfusion injury, prevents oedema, and metabolically supports the energy requirements of the brain. With this system, we observed preservation of cytoarchitecture; attenuation of cell death; and restoration of vascular dilatory and glial inflammatory responses, spontaneous synaptic activity, and active cerebral metabolism in the absence of global electrocorticographic activity. These findings demonstrate that under appropriate conditions the isolated, intact large mammalian brain possesses an underappreciated capacity for restoration of microcirculation and molecular and cellular activity after a prolonged post-mortem interval.

Biochemical detection of fatal hypothermia and hyperthermia in affected rat hypothalamus tissues by Fourier transform infrared spectroscopy.

It is difficult to determinate the cause of death from exposure to fatal hypothermia and hyperthermia in forensic casework. Here, we present a state-of-the-art study that employs Fourier-transform infrared (FTIR) spectroscopy to investigate the hypothalamus tissues of fatal hypothermic, fatal hyperthermic and normothermic rats to determine forensically significant biomarkers related to fatal hypothermia and hyperthermia. Our results revealed that the spectral variations in the lipid, protein, carbohydrate and nucleic acid components are highly different for hypothalamuses after exposure to fatal hypothermic, fatal hyperthermic and normothermic conditions. In comparison with the normothermia group, the fatal hypothermia and hyperthermia groups contained higher total lipid amounts but were lower in unsaturated lipids. Additionally, their cell membranes were found to have less motional freedom. Among these three groups, the fatal hyperthermia group contained the lowest total proteins and carbohydrates and the highest aggregated and dysfunctional proteins, while the fatal hypothermia group contained the highest level of nucleic acids. In conclusion, this study demonstrates that FTIR spectroscopy has the potential to become a reliable method for the biochemical characterization of fatal hypothermia and hyperthermia hypothalamus tissues, and this could be used as a postmortem diagnostic feature in fatal hypothermia and hyperthermia deaths.

Protection against cardiac ischemia-reperfusion injury by hypothermia and by inhibition of succinate accumulation and oxidation is additive

Hypothermia induced at the onset of ischemia is a potent experimental cardioprotective strategy for myocardial infarction. The aim of our study was to determine whether the beneficial effects of hypothermia may be due to decreasing mitochondria-mediated mechanisms of damage that contribute to the pathophysiology of ischemia/reperfusion injury. New Zealand male rabbits were submitted to 30 min of myocardial ischemia with hypothermia (32 °C) induced by total liquid ventilation (TLV). Hypothermia was applied during ischemia alone (TLV group), during ischemia and reperfusion (TLV-IR group) and normothermia (Control group). In all the cases, ischemia was performed by surgical ligation of the left anterior descending coronary artery and was followed by 3 h of reperfusion before assessment of infarct size. In a parallel study, male C57BL6/J mice underwent 30 min myocardial ischemia followed by reperfusion under either normothermia (37 °C) or conventionally induced hypothermia (32 °C). In both the models, the levels of the citric acid cycle intermediate succinate, mitochondrial complex I activity were assessed at various times. The benefit of hypothermia during ischemia on infarct size was compared to inhibition of succinate accumulation and oxidation by the complex II inhibitor malonate, applied as the pro-drug dimethyl malonate under either normothermic or hypothermic conditions. Hypothermia during ischemia was cardioprotective, even when followed by normothermic reperfusion. Hypothermia during ischemia only, or during both, ischemia and reperfusion, significantly reduced infarct size (2.8 ± 0.6%, 24.2 ± 3.0% and 49.6 ± 2.6% of the area at risk, for TLV-IR, TLV and Control groups, respectively). The significant reduction of infarct size by hypothermia was neither associated with a decrease in ischemic myocardial succinate accumulation, nor with a change in its rate of oxidation at reperfusion. Similarly, dimethyl malonate infusion and hypothermia during ischemia additively reduced infarct size (4.8 ± 2.2% of risk zone) as compared to either strategy alone. Hypothermic cardioprotection is neither dependent on the inhibition of succinate accumulation during ischemia, nor of its rapid oxidation at reperfusion. The additive effect of hypothermia and dimethyl malonate on infarct size shows that they are protective by distinct mechanisms and also suggests that combining these different therapeutic approaches could further protect against ischemia/reperfusion injury during acute myocardial infarction.

Identification of whole blood mRNA and microRNA biomarkers of tissue damage and immune function resulting from amphetamine exposure or heat stroke in adult male rats.

This work extends the understanding of how toxic exposures to amphetamine (AMPH) adversely affect the immune system and lead to tissue damage. Importantly, it determines which effects of AMPH are and are not due to pronounced hyperthermia. Whole blood messenger RNA (mRNA) and whole blood and serum microRNA (miRNA) transcripts were identified in adult male Sprague-Dawley rats after exposure to toxic AMPH under normothermic conditions, AMPH when it produces pronounced hyperthermia, or environmentally-induced hyperthermia (EIH). mRNA transcripts with large increases in fold-change in treated relative to control rats and very low expression in the control group were a rich source of organ-specific transcripts in blood. When severe hyperthermia was produced by either EIH or AMPH, significant increases in circulating organ-specific transcripts for liver (Alb, Fbg, F2), pancreas (Spink1), bronchi/lungs (F3, Cyp4b1), bone marrow (Np4, RatNP-3b), and kidney (Cesl1, Slc22a8) were observed. Liver damage was suggested also by increased miR-122 levels in the serum. Increases in muscle/heart-enriched transcripts were produced by AMPH even in the absence of hyperthermia. Expression increases in immune-related transcripts, particularly Cd14 and Vcan, indicate that AMPH can activate the innate immune system in the absence of hyperthermia. Most transcripts specific for T-cells decreased 50–70% after AMPH exposure or EIH, with the noted exception of Ccr5 and Chst12. This is probably due to T-cells leaving the circulation and down-regulation of these genes. Transcript changes specific for B-cells or B-lymphoblasts in the AMPH and EIH groups ranged widely from decreasing ≈ 40% (Cd19, Cd180) to increasing 30 to 100% (Tk1, Ahsa1) to increasing ≥500% (Stip1, Ackr3). The marked increases in Ccr2, Ccr5, Pld1, and Ackr3 produced by either AMPH or EIH observed in vivo provide further insight into the initial immune system alterations that result from methamphetamine and AMPH abuse and could modify risk for HIV and other viral infections.