Hypothermic Conditions Research Articles

The forensic significance of core temperature in identifying primary and secondary hypothermia as a cause of death: A pilot study on Wistar rats

IntroductionHypothermia is defined as a body core temperature below 35 °C and can be caused by internal or external stress. Primary hypothermia is caused by excessive exposure to low environmental temperature without any medical conditions prior to that. Secondary hypothermia is caused by alteration in thermoregulation by disease, trauma, surgery, drugs, or infections. The aim of the research is to investigate core temperature values in rats subjected to specific water temperatures at five different time points. It focuses on distinguishing between primary and secondary hypothermia in these rats. MethodsThe total 21 Wistar rats were divided into three experimental groups as: Control group rats exposed only to hypothermic condition (n = 7); Alcohol + hypothermia (n = 7); and Benzodiazepines + hypothermia (n = 7). The temperature spots analyzed in the study were: normal core temperature, core temperature during injection of 0,3 ketamine, temperature of immersion and the temperature at the onset of hypothermia and temperature at the time of death. ResultsIn our study the comparative analysis of body temperatures at various time points following submersion in water revealed significant differences among the study groups treated with either alcohol or benzodiazepines and the control group. Notable differences were observed in baseline temperature, post-anesthesia induction temperature, and immediate post-submersion temperature. Specifically, significant differences were discovered among the alcohol and benzodiazepine groups (p < 0.001) and ranging from the alcohol and control groups (p < 0.001). The analysis of survival times following induced hypothermia revealed a statistically significant difference among the three experimental groups (p = 0.04), though subsequent post-hoc comparisons did not demonstrate significant differences in mean survival times. ConclusionThere is a difference in survival time between primary and secondary hypothermia groups, depending on consumption and intoxication with alcohol or benzodiazepines. The analysis of survival times following induced hypothermia showed a statistically significant difference among the groups.

A meta-analysis of perfusion parameters affecting weight gain in exvivo perfusion.

Ex vivo machine perfusion (EVMP) has been established to extend viability of donor organs. However, EVMP protocols are inconsistent. We hypothesize that there is a significant relationship between specific parameters during EVMP and perfusion outcomes. A meta-analysis of literature was conducted in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis (PRISMA) Statement. The search encompassed articles published before July 25, 2023. PubMed, Embase, and CENTRAL databases were screened using search terms "ex-vivo," "ex-situ," "machine," and "perfusion." Weight gain, an indicator of organ viability, was chosen to compare outcomes. Extracted variables included perfused organ, warm and cold ischemia time before perfusion, perfusion duration, perfusate flow, pressure, temperature, perfusate composition (presence of cellular or acellular oxygen carrier, colloids, and other supplements) and percent weight change. Data were analyzed using SPSS statistical software. Overall, 44 articles were included. Red blood cell-based perfusates resulted in significantly lower weight gain compared to acellular perfusates without oxygen carriers (11.3% vs. 27.0%, p < 0.001). Hemoglobin-based oxygen carriers resulted in significantly lower weight gain compared to acellular perfusates (16.5% vs. 27%, p = 0.006). Normothermic perfusion led to the least weight gain (14.6%), significantly different from hypothermic (24.3%) and subnormothermic (25.0%) conditions (p < 0.001), with no significant difference between hypothermic and subnormothermic groups (24.3% vs. 25.0%, p = 0.952). There was a positive correlation between flow rate and weight gain (ß = 13.1, R = 0.390, p < 0.001). Oxygen carriers, low flow rates, and normothermic perfusate temperature appear to improve outcomes in EVMP. These findings offer opportunities for improving organ transplantation outcomes.

Pancreas Preservation: Hypothermic Oxygenated Perfusion to Improve Graft Reperfusion.

The clinical standard for pancreas preservation for transplantation is static cold storage (SCS). Oxygenation during preservation has been shown to be advantageous in clinical studies. This study evaluates the efficiency of different oxygenation modalities during hypothermic pancreas preservation. Thirty-two porcine pancreases were procured in a controlled donation after circulatory death model and were divided to be preserved in 8 groups: (1) SCS, (2) hypothermic machine perfusion (HMP), (3) hypothermic oxygenated machine perfusion (HOPE) with 21% oxygen, (4) HOPE and 100%, (5) SCS and oxygen carrier, M101, (6) HMP and M101, (7) HOPE 21% and M101, and (8) HOPE 100% and M101. All the groups underwent 24 h of hypothermic preservation, followed by 2 h of normothermic reperfusion. Oxygen partial pressures were assessed using parenchymal probes. Perfusion parameters, perfusate samples, and tissue biopsies were analyzed. This study showed that HMP was linked to higher tissue oxygen partial pressures, lower succinate levels, and better reperfusion parameters. Furthermore, the addition of M101 to either SCS or HMP was associated with lower succinate and creatinine phosphokinase accumulation, suggesting a protective effect against ischemia. Our research has demonstrated the efficacy of machine perfusion in hypothermic conditions in providing oxygen to the pancreas during preservation and conditioning the pancreatic microvasculature for reperfusion during transplantation. Furthermore, the addition of M101 suggests a protective effect on the graft from ischemia.

Comparative Analysis of Endothelial Cell Density and Central Thickness of Corneas Used for Endothelial Keratoplasty Procedures Using Various Storage Solutions.

The goal of this study was to evaluate the effect of various Food and Drug Administration-approved storage solutions on endothelial cell density (ECD) and central corneal thickness (CCT). We analyzed 6220 tissues used for endothelial keratoplasty procedures between January 2022 and June 2023 that were stored in either Life4°C, Optisol-GS, Kerasave, or Eusol-C under hypothermic conditions. We analyzed preprocessing CCT, success rate of meeting surgeon's Descemet stripping automated endothelial keratoplasty (DSAEK) thickness preferences, and preprocessing and postprocessing ECD. Results were analyzed using one-way analysis of variance, followed by multiple pairwise comparisons using the Tukey test. Mean preprocessing CCT was significantly lower in the Life4°C group (532 μm) than in Optisol-GS (549 μm), Kerasave (582 μm), and Eusol-C (589 μm) groups (all P < 0.0001). Preprocessing CCT in the Optisol-GS group was significantly lower than in Kerasave and Eusol-C groups (F (3, 3273) = 153.1, all P < 0.0001). Success rate of meeting surgeon DSAEK preferences was statistically similar among all 4 groups. Preprocessing ECD of the Kerasave group (2821 cells/mm2) was numerically higher than of the Eusol-C (2791 cells/mm2), Life4°C (2759 cells/mm2), and Optisol-GS (2768 cells/mm2) groups (P = 0.3232, 0.0004, and 0.0015, respectively). Corneal tissues stored in Kerasave and Eusol-C are significantly thicker than those in Life4°C and Optisol-GS. However, the success rate of meeting surgeon DSAEK preferences is similar among all 4 storage solutions.

Computational insights into survival durations and prehospital interventions in accidental cold-water immersion: A comprehensive analysis of fresh and saltwater temperatures

This study examines the complex relationship between scenarios of cold-water immersion, survival durations, and prehospital interventions. It utilizes computational modeling methods to shed light on how different water temperatures affect individuals facing accidental cold-water immersion incidents. The analysis reveals significant variations in survival times based on water temperature. For example, subjects immersed in water at temperatures of 5 °C, 2 °C, and 0 °C had average survival times of 136, 113, and 100 min, respectively, under stable conditions. In flowing water at the same temperatures, survival times decreased to 119, 92, and 81 min, indicating the impact of water movement on cooling rates and survival durations. Likewise, individuals immersed in saltwater at temperatures of 5 °C, 2 °C, 0 °C, and −2 °C showed average survival times of 111, 88, 80, and 66 min, respectively, in static conditions. In flowing saltwater at the same temperatures, survival times decreased to 98, 74, 68, and 57 min, highlighting the influence of water flow on cooling rates and survival durations. A comparison between immersion in pure water and saltwater at 2 °C revealed survival times of 113 and 88 min under stable conditions and 92 and 74 min under dynamic conditions, emphasizing the role of water composition in survival outcomes. The study also challenges the notion that the demise of the Titanic's passengers and crew resulted from hypothermia, asserting instead that severe thermal shock was the primary cause. These numerical findings underscore the importance of considering water temperature, flow dynamics, and prompt medical responses in cold-water emergencies to enhance survival prospects. The study identifies water within the range of 41–43 °C as the most effective active external rewarming fluid for critical hypothermal conditions. By quantifying the impact of these variables on survival times, the study provides data-driven recommendations to improve emergency protocols and outcomes for individuals facing cold-water immersion incidents.

Biological Efficacy of Ionizing Radiation Sources on 3D Organotypic Tissue Slices Assessed by Fluorescence Microscopy.

Traditional cell-based radiobiological methods are inadequate for assessing the toxicity of ionizing radiation exposure in relation to the microstructure of the extracellular matrix. Organotypic tissue slices preserve the spatial organization observed in vivo, making the tissue easily accessible for visualization and staining. This study aims to explore the use of fluorescence microscopy of physiologically compatible 3D tissue cultures to assess the effects of ionizing radiation. Organotypic tissue slices were obtained by vibratome, and their mechanical properties were studied. Slices were exposed by two ionizing radiation sources; electron beams (80 Gy and 4 Gy), and soft gamma irradiation (80 Gy and 4 Gy). Two tissue culture protocols were used: the standard (37°C), and hypothermic (30°C) conditions. A qualitative analysis of cell viability in organotypic tissue slices was performed using fluorescent dyes and standard laser confocal microscopy. Biological dosimetry is represented by differentially stained 200-µm thick organotypic tissue sections related to living and dead cells and cell metabolic activity. Our results underscore the ability of fluorescence laser scanning confocal microscopy to rapidly assess the radiobiological effects of ionizing radiation in vitro on 3D organotypic tissue slices.

Impaired autophagy following ex vivo cooling of simulated hypothermic temperatures in peripheral blood mononuclear cells from young and older adults

Hypothermia is a critical consequence of extreme cold exposure that increases the risk of cold-related injury and death in humans. While the initiation of cytoprotective mechanisms including the process of autophagy and the heat shock response (HSR) is crucial to cellular survival during periods of stress, age-related decrements in these systems may underlie cold-induced cellular vulnerability in older adults. Moreover, whether potential sex-related differences in autophagic regulation influence the human cold stress response remain unknown. We evaluated the effect of age and sex on mechanisms of cytoprotection (autophagy and the HSR) and cellular stress (apoptotic signaling and the acute inflammatory response) during ex vivo hypothermic cooling. Venous blood samples from 20 healthy young (10 females; mean [SD]: 22 [2] years) and 20 healthy older (10 females; 66 [5] years) adults were either isolated immediately (baseline) for peripheral blood mononuclear cells (PBMCs) or exposed to water bath temperatures maintained at 37, 35, 33, 31, or 4 °C for 90 min before PBMC isolation. Proteins associated with autophagy, apoptosis, the HSR, and inflammation were analyzed via Western blotting. Indicators of autophagic initiation and signaling (LC3, ULK1, and beclin-2) and the HSR (HSP90 and HSP70) increased when exposed to hypothermic temperatures in young and older adults (all p ≤ 0.007). Sex-related differences were only observed with autophagic initiation (ULK1; p = 0.015). However, despite increases in autophagic initiators ULK1 and beclin-2 (all p < 0.001), this was paralleled by autophagic dysfunction (increased p62) in all groups (all p < 0.001). Further, apoptotic (cleaved-caspase-3) and inflammatory (IL-6 and TNF-α) signaling increased in all groups (all p < 0.001). We demonstrated that exposure to hypothermic conditions is associated with autophagic dysfunction, irrespective of age or sex, although there may exist innate sex-related differences in cytoprotection in response to cold exposure as evidenced through altered autophagic initiation.

The neurophysiological effect of mild hypothermia in gyrencephalic brains submitted to ischemic stroke and spreading depolarizations.

Characterize the neurophysiological effects of mild hypothermia on stroke and spreading depolarizations (SDs) in gyrencephalic brains. Left middle cerebral arteries (MCAs) of six hypothermic and six normothermic pigs were permanently occluded (MCAo). Hypothermia began 1 h after MCAo and continued throughout the experiment. ECoG signals from both frontoparietal cortices were recorded. Five-minute ECoG epochs were collected 5 min before, at 5 min, 4, 8, 12, and 16 h after MCAo, and before, during, and after SDs. Power spectra were decomposed into fast (alpha, beta, and gamma) and slow (delta and theta) frequency bands. In the vascular insulted hemisphere under normothermia, electrodes near the ischemic core exhibited power decay across all frequency bands at 5 min and the 4th hour after MCAo. The same pattern was registered in the two furthest electrodes at the 12th and 16th hour. When mild hypothermia was applied in the vascular insulted hemispheres, the power decay was generalized and seen even in electrodes with uncompromised blood flow. During SD analysis, hypothermia maintained increased delta and beta power during the three phases of SDs in the furthest electrode from the ischemic core, followed by the second furthest and third electrode in the beta band during preSD and postSD segments. However, in hypothermic conditions, the third electrode showed lower delta, theta, and alpha power. Mild hypothermia attenuates all frequency bands in the vascularly compromised hemisphere, irrespective of the cortical location. During SD formation, it preserves power spectra more significantly in electrodes further from the ischemic core.

Comparing Hypothermic and Thermal Neutral Conditions to Induce Metabolic Suppression.

Suppressing metabolism in astronauts could decrease CO2 production. It is unknown whether active cooling is required to suppress metabolism in sedated patients. We hypothesized that hypothermia would have an additive effect with dexmedetomidine on suppressing metabolism. This is a randomized crossover trial of healthy subjects receiving sedation with dexmedetomidine and exposure to a cold (20°C) or thermal neutral (31°C) environment for 3 hours. We measured heart rate, blood pressure, core temperature, resting oxygen consumption (VO2), resting carbon dioxide production (VCO2), and resting energy expenditure (REE) at baseline and each hour of exposure to either environment. We also evaluated components of the Defense Automated Neurobehavioral Assessment (DANA) Brief to evaluate the effect of metabolic suppression on cognition. Six subjects completed the study. Heart rate and core temperature were lower during the cold (56 bpm) condition than the thermal neutral condition (67 bpm). VO2, VCO2, and REE decreased between baseline and the 3-hour measurement in the cold condition (Δ = 0.9 mL/min, 56.94 mL/min, 487.9 Kcal/D, respectively). DANA simple response time increased between baseline and start of recovery in both conditions (20°C 136.9 cognitive efficiency [CE] and 31°C 87.83 CE). DANA procedural reaction time increased between baseline and start of recovery in the cold condition (220.6 CE) but not in the thermal neutral condition. DANA Go/No-Go time increased between baseline and start of recovery in both conditions (20°C 222.1 CE and 31°C 122.3 CE). Sedation and cold environments are required for metabolic suppression. Subjects experienced decrements in cognitive performance in both conditions. A significant recovery period may be required after metabolic suppression before completing mission critical tasks.

Abstract TP288: Selective Brain Hypothermia Induced by Thermoelectric Cooling

Background: Stroke or Traumatic Brain Injury (TBI) are leading causes of disability and death in the United States. Hypothermia is a known neuronal protectant. Current hypothermia interventions include intravascular, intranasal, and surface brain cooling technologies but these interventions have not been well utilized clinically. Cooling the brain a few degrees below normothermia (T&lt;36°C) has been shown to yield significant neuroprotective effects. Noninvasive surface cooling methods are most desirable given their less patient complications, provided the desired therapeutic hypothermia effect (T 33-36°C) can be maintained. Method: These investigators propose a novel noninvasive solid state technology that employs addressable Peltier thermoelectric cooling elements to generate hypothermia. ThermoPelt’s closed-looped microcontroller design provides the ability to address individual Peltier elements to selectively reduce the temperature of localized regions of the brain, thus allowing precision therapeutic hypothermic conditions to be realized. Results: Assessment of ThermoPelt performance was accomplished with use of virtual prototype (engineering mathematical model representations of the brain) and early, in vitro experimental physical prototype testing. Preliminary results obtained from virtual and physical prototype tests verify ThermoPelt feasibility to achieve hypothermic conditions. Conclusion: In this new method of generating hypothermia, the preliminary findings demonstrate that our noninvasive ThermoPelt technology is capable of reducing brain temperature at controlled rates to selectively target specific head and neck regions.

The effects of nickel chloride on papillary muscle contractility under normothermic and hypothermic conditions: Comparison of active and hibernating ground squirrels (Urocitellus undulatus) with Wistar rats.

Extracellular Ca2+ plays a pivotal role in the regulation of cardiac contractility under normal and extreme conditions. Here, by using nickel chloride (NiCl2), a non-specific blocker of extracellular Ca2+ influx, we studied the input of extracellular Ca2+ on the regulation of papillary muscle (PM) contractility under normal and hypothermic conditions in ground squirrels (GS), and rats. By measuring isometric force of contraction, we studied how NiCl2 affects force-frequency relationship and the rest effect in PM of these species at 30 °C and 10 °C. We found that at 30 °C 1.5 mM NiCl2 significantly reduced force of contraction across entire frequency range in active GS and rats, whereas in hibernating GS force of contraction was reduced at low and high frequency range. Additionally, NiCl2 evoked spontaneous contractility in rats but not GS PM. The rest effect was significantly reduced by NiCl2 for active GS and rats but not hibernating GS. At 10 °C, NiCl2 fully reduced contractility in active GS and, to a lesser extent, in rats, whereas in hibernating GS it was significant only at 0.3 Hz. The rest effect was significantly reduced by NiCl2 in both active and hibernating GS, whereas it was unmasked in rats that had high contractility under hypothermic conditions in control. Our results show a significant contribution of extracellular Ca2+ to myocardial contractility in GS not only in active but also in hibernating states, especially under hypothermic conditions, whereas limitation of extracellular Ca2+ influx in rats under hypothermia can play protective role for myocardial contractility.

Clinical outcome of organ‐cultured preloaded DMEK compared to standard DMEK ‐ A single center study

The use of ready‐to‐use grafts from specialized eye banks might provide a number of benefits, including graft quality control, assured availability at a certain operation time, a decreased likelihood of case cancellation, and a shorter and less sophisticated DMEK surgery, with a resulting lower risk of graft damage. However, it is critical to thoroughly establish the clinical safety of employing these preloaded tissues. Especially since most of the studies were prepared and stored under hypothermic conditions. There are only a few studies on preprepared tissues in organ culture, which are partly controversial.In this prospective study we included patients who received DMEK surgery at the Knappschaft Eye Clinic Sulzbach between 05/2021–12/2022. Patients received either a preloaded DMEK (pDMEK), prepared 5 days before surgery in the eye bank, or a conventional, directly before surgery, surgeon‐prepared DMEK (sDMEK).The preliminary data shows that preloaded DMEK is a safe procedure, but there is a trend towards more frequent rebubbling in the pDMEK group and a statistically non‐significant lower postoperative endothelial cell count compared to the sDMEK group. However, visual acuity and corneal thickness reduction were similar in both groups.

CRISPR Deletion of miR-27 Impacts Recombinant Protein Production in CHO Cells.

MicroRNAs represent an interesting group of regulatory molecules with the unique ability of a single miRNA able to regulate the expression of potentially hundreds of target genes. In that regard, their utility has been demonstrated as a strategy to improve the cellular phenotypes important in the biomanufacturing of recombinant proteins. Common approaches to stably deplete miRNAs are the use of sponge decoy transcripts or shRNA inhibitors, both of which require the introduction and expression of extra genetic material in the cell. As an alternative, we implemented the CRISPR/Cas9 system in our laboratory to generate CHO cells which lack the expression of a specific miRNA for the purpose of functional studies. To implement the system, miR-27a/b was chosen as it has been shown to be upregulated during hypothermic conditions and therefore may be involved in influencing CHO cell growth and recombinant protein productivity. In this chapter, we present a protocol for targeting miRNAs in CHO cells using CRISPR/Cas9 and the analysis of the resulting phenotype, using miR-27 as an example. We show that it is possible to target miRNAs in CHO cells and achieved ≥80% targeting efficiency. Indel analysis and TOPO-TA cloning combined with Sanger sequencing showed a range of different indels. Furthermore, it was possible to identify clones with no detectable expression of mature miR-27b. Depletion of miR-27b led to improved viability in late stages of batch and fed-batch cultures, making it a potentially interesting target to improve bioprocess performance of CHO cells.

Hypothermia Attenuates Neurotoxic Microglial Activation via TRPV4.

Therapeutic hypothermia (TH) provides neuroprotection. However, the cellular mechanisms underlying the neuroprotective effects of TH are not fully elucidated. Regulation of microglial activation has the potential to treat a variety of nervous system diseases. Transient receptor potential vanilloid 4 (TRPV4), a nonselective cation channel, is activated by temperature stimulus at 27-35°C. Although it is speculated that TRPV4 is associated with the neuroprotective mechanisms of TH, the role of TRPV4 in the neuroprotective effects of TH is not well understood. In the present study, we investigated whether hypothermia attenuates microglial activation via TRPV4 channels. Cultured microglia were incubated under normothermic (37°C) or hypothermic (33.5°C) conditions following lipopolysaccharide (LPS) stimulation. Hypothermic conditions suppressed the expression of pro-inflammatory cytokines, inducible nitric oxide synthase, and the number of phagocytic microglia. AMP-activated protein kinase (AMPK)-NF-κB signaling was inhibited under hypothermic conditions. Furthermore, hypothermia reduced neuronal damage induced by LPS-treated microglial cells. Treatment with TRPV4 antagonist in normothermic culture replicated the suppressive effects of hypothermia on microglial activation and microglia-induced neuronal damage. In contrast, treatment with a TRPV4 agonist in hypothermic culture reversed the suppressive effect of hypothermia. These findings suggest that TH suppresses microglial activation and microglia-induced neuronal damage via the TRPV4-AMPK-NF-κB pathway. Although more validation is needed to consider differences according to age, sex, and specific central nervous system regions, our findings may offer a novel therapeutic approach to complement TH.

Enhancing productivity of Chinese hamster ovary (CHO) cells: synergistic strategies combining low-temperature culture and mTORC1 signaling engineering

Introduction: The growing demand for recombinant proteins in medicine has prompted biopharmaceutical companies to seek ways to maximize the manufacturing process. Despite its known negative impact on cell growth, temperature shift (TS) has emerged as a cost-effective strategy to enhance protein quantity and quality in Chinese Hamster Ovary cells (CHO). As cells adapt their growth and protein synthesis rate to the environment through influencing mTOR complex 1 (mTORC1), here we evaluated the potential of mTORC1 signaling engineering to improve the production of granulocyte-macrophage colony-stimulating factor (GM-CSF) protein in stable CHO cells at low temperature.Methods: First, the expression of genes that negatively control mTORC1 functions in response to environmental fluctuations, including TSC1, AMPK, MAPKAPK5, and MARK4 genes, was assessed via real-time qPCR in CHO-K1 after a temperature shift from 37°C to 30°C. Then, plasmids harboring the shRNAs targeting these genes were constructed into the PB513B-1 plasmid with expression driven by either the constitutive CMV promoter or the cold-inducible HSP90 promoter. Finally, the impact of transient gene downregulation was evaluated on GM-CSF and mTOR proteins productivity in GM-CSF-producing CHO-K1 cells using ELISA and Western-blot assays, respectively. The growth rate of the transfected cells at the two temperatures was evaluated using flow cytometry.Results: Hypothermic conditions promote the upregulation of mTORC1 inhibitor genes, especially TSC1 and MAPKAPK5, while downregulating S6K, a key effector of the mTORC1 signaling pathway, in CHO-K1 cells. Transcription and protein levels of mTOR increased upon transfection, “pB513-b CMV-P/4shRNAs/GFP” plasmid, “pB513-bHSP90-P/4sh-RNAs/GFP” and pB513B-1 plasmid as mock group in GM-CSF-producing CHO-K1 cells (approximately 60%), along with a high transcript level of S6K. Cell growth-related characteristics were improved, albeit with distinct effects at different temperatures. Notably, these changes were more efficient at 30°C when utilizing the HSP90 promoter, resulting in a three-fold increase in GM-CSF production after 3 days.Conclusion: This study highlights the importance of temperature regulation and mTORC1 modulation in CHO cellular processes, particularly in recombinant protein production. Understanding these mechanisms paves the way for developing innovative strategies to enhance cell growth, protein synthesis, and overall bioprocess performance, particularly in manufacturing human therapeutic proteins.

Recombinant cellular model system for human muscle-type nicotinic acetylcholine receptor α12β1δε

The human muscle-type nicotinic acetylcholine receptor α12β1δε (nAChR) is a complex transmembrane receptor needed for drug screening for disorders like congenital myasthenic syndromes and multiple pterygium syndrome. Until today, most models are still using the nAChR from Torpedo californica electric ray. A simple reproducible cellular system expressing functional human muscle-type nAChR is still missing. This study addressed this issue and further tested the hypothesis that different chaperones, both biological and chemical, and posttranslational modification supporting substances as well as hypothermic incubation are able to increase the nAChR yield. Therefore, Gibson cloning was used to generate transfer plasmids carrying the sequence of nAChR or chosen biological chaperones to support the nAChR folding in the cellular host. Viral transduction was used for stable integration of these transgenes in Chinese hamster ovary cells (CHO). Proteins were detected with Western blot, in-cell and on-cell Western, and the function of the receptor with voltage clamp analysis. We show that the internalization of nAChR into plasma membranes was sufficient for detection and function. Additional transgenic overexpression of biological chaperones did result in a reduced nAChR expression. Chemical chaperones, posttranslational modification supporting substances, and hypothermic conditions are well-suited supporting applications to increase the protein levels of different subunits. This study presents a stable and functional cell line that expresses human muscle-type nAChR and yields can be further increased using the chemical chaperone nicotine without affecting cell viability. The simplified access to this model system should enable numerous applications beyond drug development.Graphical abstractGraphical abstract created with http://biorender.com

The inversion of the inotropic effect of isoproterenol in the rat myocardium during deep hypothermia

The present study examined the effects of the в-adrenergic receptor agonist, isoproterenol, on contractile activity of right ventricle papillary muscles in the rat heart at near-physiological temperature (30°C) and under conditions of deep hypothermia (10°C). Isoproterenol exerts a pronounced positive inotropic effect at 30°C. After agonist addition, the force of contraction increases from 1.2 ± 0.1 mN in control to 2.4 ± 0.4 mN that leads to a reliable acceleration of time parameters of contraction. So, time-to-maximum contraction decreased from 101 ± 6 ms to 85 ± 4 ms; time-to-50% relaxation declined from 55 ± 3 ms to 36 ± 1 ms. Under hypothermic conditions, isoproterenol produced a powerful negative inotropic effect, reducing the force of contraction from 2.2 ± 0.4 mN to 1.2 ± 0.4 mN. Similarly as at 30°C, there was a tendency for increase in contraction speed, so time-to-maximum contraction decreased from 717 ± 52 ms to 624 ± 50 ms, and time-to-50% relaxation was shortened from 667 ± 86 ms to 450 ± 40 ms. Thus, under conditions of deep hypothermia at 10°C, the isoproterenol-induced inotropy changes from positive to negative, while negative lusitropic effect remains clear.

A novel method to extend viability and functionality of living heart slices.

Living heart slices have recently emerged as a powerful experimental model for fundamental cardiac research. By retaining the structure and function of the native myocardium while maintaining the simplicity of cell culture models, heart slices can be easily employed in electrophysiological, pharmacological, biochemical, and structural investigations. One single heart yields many slices (>20 slices for rodents, >100 slices for porcine or human hearts), however due to the low throughput of most assays and rapid slice degeneration within 24 h of preparation, many slices remain unused and are discarded at the end of the preparation day. Here we present a novel method to extend viability and functionality of living heart slices, enabling their use in experiments over several consecutive days following preparation. By combining hypothermic conditions with inhibition of myosin II ATPase using 2,3-butanedione monoxime (BDM), slices prepared from the left ventricle of porcine hearts remain viable and exhibit preserved contractile function and morphology for up to 6 days. Electrophysiological function was also confirmed over the 6 days by extracellular field potentials recordings. This simple method not only maximizes the use of slices prepared from one single heart, thus reducing the number of animals required, but also increases data reproducibility by allowing multiple electrophysiological, pharmacological, biochemical, and structural studies to be performed from the same heart.

GCN2 is required to maintain core body temperature in mice during acute cold.

Nonshivering thermogenesis in rodents requires macronutrients to fuel the generation of heat during hypothermic conditions. In this study, we examined the role of the nutrient sensing kinase, general control nonderepressible 2 (GCN2) in directing adaptive thermogenesis during acute cold exposure in mice. We hypothesized that GCN2 is required for adaptation to acute cold stress via activation of the integrated stress response (ISR) resulting in liver production of FGF21 and increased amino acid transport to support nonshivering thermogenesis. In alignment with our hypothesis, female and male mice lacking GCN2 failed to adequately increase energy expenditure and veered into torpor. Mice administered a small molecule inhibitor of GCN2 were also profoundly intolerant to acute cold stress. Gcn2 deletion also impeded liver-derived FGF21 but in males only. Within the brown adipose tissue (BAT), acute cold exposure increased ISR activation and its transcriptional execution in males and females. RNA sequencing in BAT identified transcripts that encode actomyosin mechanics and transmembrane transport as requiring GCN2 during cold exposure. These transcripts included class II myosin heavy chain and amino acid transporters, critical for maximal thermogenesis during cold stress. Importantly, Gcn2 deletion corresponded with higher circulating amino acids and lower intracellular amino acids in the BAT during cold stress. In conclusion, we identify a sex-independent role for GCN2 activation to support adaptive thermogenesis via uptake of amino acids into brown adipose.NEW & NOTEWORTHY This paper details the discovery that GCN2 activation is required in both male and female mice to maintain core body temperature during acute cold exposure. The results point to a novel role for GCN2 in supporting adaptive thermogenesis via amino acid transport and actomyosin mechanics in brown adipose tissue.

Endothelial damage inhibitor preserves the integrity of venous endothelial cells from patients undergoing coronary bypass surgery.

Despite the success of coronary artery bypass graft (CABG) surgery using autologous saphenous vein grafts (SVGs), nearly 50% of patients experience vein graft disease within 10 years of surgery. One contributing factor to early vein graft disease is endothelial damage during short-term storage of SVGs in inappropriate solutions. Our aim was to evaluate the effects of a novel endothelial damage inhibitor (EDI) on SVGs from patients undergoing elective CABG surgery and on venous endothelial cells (VECs) derived from these SVGs. SVGs from 11 patients participating in an ongoing clinical registry (NCT02922088) were included in this study, and incubated with both full electrolyte solution (FES) or EDI for 1 h and then examined histologically. In 8 of 11 patients, VECs were isolated from untreated grafts, incubated with both FES and EDI for 2 h under hypothermic stress conditions and then analysed for activation of an inflammatory phenotype, cell damage and cytotoxicity, as well as endothelial integrity and barrier function. The EDI was superior to FES in protecting the endothelium in SVGs (74 ± 8% versus 56 ± 8%, P < 0.001). Besides confirming that the EDI prevents apoptosis in SVG-derived VECs, we also showed that the EDI temporarily reduces adherens junctions in VECs while protecting focal adhesions compared to FES. The EDI protects the connectivity and function of the SVG endothelium. Our data suggest that the EDI can preserve focal adhesions in VECs during short-term storage after graft harvesting. This might explain the superiority of the EDI in maintaining most of the endothelium in venous CABG surgery conduits.