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Cytochrome P450 (CYP) 2Ds are drug metabolizing enzymes found in brain and liver which metabolize numerous centrally acting drugs. Inhibition and induction of CYP2D-mediated metabolism in rodent brain alters brain drug and metabolite concentrations and resulting drug response. In female rats, brain CYP2D metabolism varies across the estrous cycle and with exogenous estrogen, changing brain drug concentrations and response. In this study harmine-induced hypothermia was lower in humanized CYP2D6 transgenic female mice during estrus compared to diestrus. Pretreatment into the cerebral ventricles with propranolol, a selective irreversible inhibitor of human CYP2D6 in brain, increased hypothermia in estrus but not in diestrus. In vivo enzyme activity was higher in brains of transgenic mice in estrus compared to diestrus and was lower after pretreatment with inhibitor in estrus, but not in diestrus. Hepatic activity and plasma harmine concentrations were unaffected by either estrous phase or inhibition of brain CYP2D6. In wild-type female mice, harmine-induced hypothermia was unaffected by either estrous phase or inhibitor pretreatment. Male mice were used as positive controls, where pretreatment with inhibitor increased harmine-induced hypothermia in transgenic but not wild-type, mice. This study provides evidence for female hormone cycle-based regulation of drug metabolism by human CYP2D6 in brain and resulting drug response. This suggests that brain CYP2D6 metabolism may vary, for example, during the menstrual cycle, pregnancy, or menopause, or while taking oral contraceptives or hormone therapy. This variation could contribute to individual differences in response to centrally acting CYP2D6-substrate drugs by altering local brain drug and/or metabolite concentrations., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Recent studies indicate that nitrous oxide (N 2 O), a gaseous anesthetic and an NMDA (N-methyl-D-aspartate) receptor antagonist, produces rapid antidepressant effect in patients suffering from treatment-resistant depression. Our recent work implies that hypothermia and reduced energy expenditure are connected with antidepressant-induced activation of TrkB neurotrophin receptors - a key regulator of synaptic plasticity. In this study, we demonstrate that a brief exposure to N 2 O leads to a drop in body temperature following the treatment, which is linked to decreased locomotor activity; enhanced slow-wave electroencephalographic activity; reduced brain glucose utilization; and increased phosphorylation of TrkB, GSK3β (glycogen synthase kinase 3β), and p70S6K (a kinase downstream of mTor (mammalian target of rapamycin)) in the medial prefrontal cortex of adult male mice. Moreover, preventing the hypothermic response in a chronic corticosterone stress model of depression attenuated the antidepressant-like behavioral effects of N 2 O in the saccharin preference test. These findings indicate that N 2 O treatment modulates TrkB signaling and related neurotrophic signaling pathways in a temperature-dependent manner, suggesting that the phenomenon driving TrkB activation - altered thermoregulation and energy expenditure - is linked to antidepressant-like behavioral responses., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Okko Alitalo reports financial support was provided by Instrumentarium Science Foundation. Okko Alitalo reports financial support was provided by University of Helsinki Doctoral Programme in Drug Research. Samuel Kohtala reports financial support was provided by Orion Research Fundation. Samuel Kohtala reports financial support was provided by Sigrid Jusélius Foundation. Samuel Kohtala reports financial support was provided by Finnish Cultural Foundation. Tomi Rantamaki reports financial support was provided by Research Council of Finland. Tomi Rantamaki reports financial support was provided by Sigrid Jusélius Foundation. Tomi Rantamaki reports financial support was provided by Business Finland. Tomi Rantamaki reports equipment, drugs, or supplies was provided by Oy Woikoski Ab. Marko Rosenholm reports financial support was provided by Finnish Cultural Foundation. Tomi Rantamäki reports financial support was provided by the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Amphiregulin (AREG) is a growth factor linked to cardioprotection and heart pathology during myocardial stress. Our aim was to investigate cardiac AREG expression, its potential as a postmortem hypothermia marker and its possible stress hormone dependency in different types of deaths., Materials and Methods: Heart RNA was isolated from hypothermic, cardiac and non-cardiac deaths. Relative AREG mRNA levels and urine stress hormone concentrations were measured by qPCR and enzyme-linked immunosorbent assays from eight different death cause groups. Receiver operating characteristic curve was used to evaluate a cut-off point for AREG expression as a hypothermia marker. Regulatory elements were predicted by PROMO., Results: The AREG mRNA levels were significantly higher in hypothermic deaths than in most cardiac and non-cardiac deaths. AREG expression indicated hypothermic deaths with nearly 70% sensitivity and specificity. However, high expression levels were also detected in non-ischaemic deaths. The highest concentrations of adrenaline and cortisol were detected in hypothermic deaths, while the highest noradrenaline concentrations associated with atherosclerotic heart disease (AHD) deaths with acute myocardial infarction and trauma deaths. There were no significant correlations between stress hormones and AREG mRNA in hypothermic and non-cardiac deaths, whereas moderate-to-high associations were detected in cardiac deaths. Putative response elements for cortisol and catecholamines were found in AREG ., Conclusions: Severe hypothermia activates cardiac AREG expression practicable as a postmortem hypothermia marker. Cortisol and catecholamines may act as transcriptional modifiers of this gene, especially in long-term ischaemic heart disease. However, the exact role of these hormones in upregulation of AREG during hypothermia remains unclear.

Cold-inducible RNA-binding protein (CIRBP) and RNA binding motif protein 3 (RBM3) are both members of the cold shock protein family expressed in response to low-temperature induction. However, their usefulness in the diagnosis of fatal hypothermia in forensic has not been reported. In this study, we report the case of a female who died of fatal hypothermia. Through detailed case investigation, scene examination, autopsy testing and tissue observation, we ultimately determined that the woman died of hypothermia. We further examined the expression of CIRBP and RBM3 in the postmortem brain, heart, lung and kidney via immunohistochemistry. The high expression of CIRBP and RBM3 in the brain, lungs, and kidneys suggested the possibility of hypothermia. This study may provide new auxiliary diagnostic indicators for the diagnosis of fatal hypothermia in forensic medicine., Competing Interests: Declaration of competing interest No conflict of competing interest exits in the manuscript, and manuscript is approved by all authors for publication., (Copyright © 2024 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

The mild hypothermia response (MHR) maintains organismal homeostasis during cold exposure and is thought to be critical for the neuroprotection documented with therapeutic hypothermia. To date, little is known about the transcriptional regulation of the MHR. We utilize a forward CRISPR-Cas9 mutagenesis screen to identify the histone lysine methyltransferase SMYD5 as a regulator of the MHR. SMYD5 represses the key MHR gene SP1 at euthermia. This repression correlates with temperature-dependent levels of histone H3 lysine 26 trimethylation (H3K36me3) at the SP1 locus and globally, indicating that the mammalian MHR is regulated at the level of histone modifications. We have identified 37 additional SMYD5-regulated temperature-dependent genes, suggesting a broader MHR-related role for SMYD5. Our study provides an example of how histone modifications integrate environmental cues into the genetic circuitry of mammalian cells and provides insights that may yield therapeutic avenues for neuroprotection after catastrophic events., Competing Interests: Declaration of interests H.T.B. is a consultant for Mahzi Therapeutics and founder of Kaldur Therapeutics. S.R. and H.T.B. have a European patent application (23167505.9) on a therapeutic strategy to activate the MHR., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Postmortem metabolomics holds promise for identifying crucial biological markers relevant to death investigations and clinical scenarios. We aimed to assess its applicability in diagnosing hypothermia, a condition lacking definitive biomarkers. Our retrospective analysis involved 1095 postmortem femoral blood samples, including 150 hypothermia cases, 278 matched controls, and 667 randomly selected test cases, analyzed using UHPLC-QTOF mass spectrometry. The model demonstrated robustness with an R2 and Q2 value of 0.73 and 0.68, achieving 94% classification accuracy, 92% sensitivity, and 96% specificity. Discriminative metabolite patterns, including acylcarnitines, stress hormones, and NAD metabolites, along with identified pathways, suggest that metabolomics analysis can be helpful to diagnose fatal hypothermia. Exposure to cold seems to trigger a stress response in the body, increasing cortisol production to maintain core temperature, possibly explaining the observed upregulation of cortisol levels and alterations in metabolic markers related to renal function. In addition, thermogenesis seems to increase metabolism in brown adipose tissue, contributing to changes in nicotinamide metabolism and elevated levels of ketone bodies and acylcarnitines, these findings highlight the effectiveness of UHPLC-QTOF mass spectrometry, multivariate analysis, and pathway identification of postmortem samples in identifying metabolite markers with forensic and clinical significance. The discovered patterns may offer valuable clinical insights and diagnostic markers, emphasizing the broader potential of postmortem metabolomics in understanding critical states or diseases., (© 2024. The Author(s).)

Abnormal shifts in global climate, leading to extreme weather, significantly threaten the safety of individuals involved in outdoor activities. Hypothermia-induced coma or death frequently occurs in clinical and forensic settings. Despite this, the precise mechanism of central nervous system injury due to hypothermia remains unclear, hindering the development of targeted clinical treatments and specific forensic diagnostic indicators. The GEO database was searched to identify datasets related to hypothermia. Post-bioinformatics analyses, DEGs, and ferroptosis-related DEGs (FerrDEGs) were intersected. GSEA was then conducted to elucidate the functions of the Ferr-related genes. Animal experiments conducted in this study demonstrated that hypothermia, compared to the control treatment, can induce significant alterations in iron death-related genes such as PPARG, SCD, ADIPOQ, SAT1, EGR1, and HMOX1 in cerebral cortex nerve cells. These changes lead to iron ion accumulation, lipid peroxidation, and marked expression of iron death-related proteins. The application of the iron death inhibitor Ferrostatin-1 (Fer-1) effectively modulates the expression of these genes, reduces lipid peroxidation, and improves the expression of iron death-related proteins. Severe hypothermia disrupts the metabolism of cerebral cortex nerve cells, causing significant alterations in ferroptosis-related genes. These genetic changes promote ferroptosis through multiple pathways.

Hibernation and torpor are not passive responses caused by external temperature drops and fasting but are active brain functions that lower body temperature. A population of neurons in the preoptic area was recently identified as such active torpor-regulating neurons. We hypothesized that the other hypothermia-inducing maneuvers would also activate these neurons. To test our hypothesis, we first refined the previous observations, examined the brain regions explicitly activated during the falling phase of body temperature using c-Fos expression, and confirmed the preoptic area. Next, we observed long-lasting hypothermia by reactivating torpor-tagged Gq-expressing neurons using the activity tagging and DREADD systems. Finally, we found that about 40-60% of torpor-tagged neurons were activated by succeeding isoflurane anesthesia and by icv administration of an adenosine A1 agonist. Isoflurane-induced and central adenosine-induced hypothermia is, at least in part, an active process mediated by the torpor-regulating neurons in the preoptic area., (© 2024. The Author(s).)

Abstract: There is evidence to suggest that the hypothermia observed in the most severe cases of systemic inflammation or sepsis is a regulated response with potential adaptive value, but the mechanisms involved are poorly understood. Here, we investigated the interplay between brain oxygenation (assessed by tissue P o2 ) and the development of hypothermia in unanesthetized rats challenged with a hypotension-inducing dose of bacterial LPS (1 mg/kg i.v.). At an ambient temperature of 22°C, oxygen consumption (V̇O 2 ) began to fall only a few minutes after the LPS injection, and this suppression in metabolic rate preceded the decrease in core temperature. No reduction in brain P o2 was observed prior to the development of the hypometabolic, hypothermic response, ruling out the possibility that brain hypoxia served as a trigger for hypothermia in this model. Brain P o2 was even increased. Such an improvement in brain oxygenation could reflect either an increased O 2 delivery or a decreased O 2 consumption. The former explanation seems unlikely because blood flow (cardiac output) was being progressively decreased during the recording period. On the other hand, the decrease in V̇O 2 usually preceded the rise in P o2 , and an inverse correlation between V̇O 2 and brain P o2 was consistently observed. These findings do not support the existence of a closed-loop feedback relationship between brain oxygenation and hypothermia in systemic inflammation. The data are consistent with a feedforward mechanism in which hypothermia is triggered (possibly by cryogenic inflammatory mediators) in anticipation of changes in brain oxygenation to prevent the development of tissue hypoxia., Competing Interests: The authors report no conflicts of interest., (Copyright © 2024 by the Shock Society.)

Aims: Systemic administration of ammonium chloride (NH 4 Cl), an acidifying agent used in human patients and experimental conditions, causes hypothermia in mice, however, the mechanisms of the thermoregulatory response to NH 4 Cl and whether it develops in other species remained unknown., Main Methods: We studied body temperature (T b ) changes in rats and mice induced by intraperitoneal administration of NH 4 Cl after blockade of transient receptor potential vanilloid-1 (TRPV1) or ankyrin-1 (TRPA1) channels., Key Findings: In rats, NH 4 Cl decreased T b by 0.4-0.8°C (p < 0.05). The NH 4 Cl-induced hypothermia also developed in Trpv1 knockout (Trpv1 -/- ) and wild-type (Trpv1 +/+ ) mice, however, the T b drop was exaggerated in Trpv1 -/- mice compared to Trpv1 +/+ controls with maximal decreases of 4.0 vs. 2.1°C, respectively (p < 0.05). Pharmacological blockade of TRPV1 channels with AMG 517 augmented the hypothermic response to NH 4 Cl in genetically unmodified mice and rats (p < 0.05 for both). In contrast, when NH 4 Cl was infused to mice genetically lacking the TRPA1 channel, the hypothermic response was significantly attenuated compared to wild-type controls with maximal mean T b difference of 1.0°C between the genotypes (p = 0.008). Pretreatment of rats with a TRPA1 antagonist (A967079) also attenuated the NH 4 Cl-induced T b drop with a maximal difference of 0.7°C between the pretreatment groups (p = 0.003)., Significance: TRPV1 channels limit, whereas TRPA1 channels exaggerate the development of NH 4 Cl-induced hypothermia in rats and mice, but other mechanisms are also involved. Our results warrant for regular T b control and careful consideration of NH 4 Cl treatment in patients with TRPA1 and TRPV1 channel dysfunctions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Advanced in vivo imaging techniques have facilitated the comprehensive visual exploration of animal biological processes, leading to groundbreaking discoveries such as the glymphatic system. However, current limitations of macroscopic imaging techniques impede the precise investigation of physiological parameters regulating this specialized lymphatic transport system. While NIR-II fluorescence imaging has demonstrated advantages in peripheral lymphatic imaging, there are few reports regarding its utilization in the glymphatic system. To address this, a noninvasive transcranial macroscopic NIR-II fluorescence imaging model is developed using a cyanine dye-protein coupled nanoprobe. NIR-II imaging with high temporal and spatial resolution reveals that hypothermia can increase the glymphatic influx by reducing the flow rate of cerebrospinal fluid. In addition, respiratory rate, respiratory amplitude, and heart rate all play a role in regulating the glymphatic influx. Thus, targeting the glymphatic influx may alter the trajectory of immune inflammation following brain injury, providing therapeutic prospects for treating brain injury with mild hypothermia.

Mitochondrial transfer occurs between cells, and it is important for damaged cells to receive healthy mitochondria to maintain their normal function and protect against cell death. Accumulating evidence suggests that the functional mitochondria of astrocytes are released and transferred to oxygen-glucose deprivation/reoxygenation (OGD/R)-injured neurons. Mild hypothermia (33 °C) is capable of promoting this process, which partially restores the function of damaged neurons. However, the pathways and mechanisms by which mild hypothermia facilitates mitochondrial transfer remain unclear. We are committed to studying the role of mild hypothermia in neuroprotection to provide reliable evidences and insights for the clinical application of mild hypothermia in brain protection. Tunneling nanotubes (TNTs) are considered to be one of the routes through which mitochondria are transferred between cells. In this study, an OGD/R-injured neuronal model was successfully established, and TNTs, mitochondria, neurons and astrocytes were double labeled using immunofluorescent probes. Our results showed that TNTs were present and involved in the transfer of mitochondria between cells in the mixed-culture system of neurons and astrocytes. When neurons were subjected to OGD/R exposure, TNT formation and mitochondrial transportation from astrocytes to injured neurons were facilitated. Further analysis revealed that mild hypothermia increased the quantity of astrocytic mitochondria transferred into damaged neurons through TNTs, raised the mitochondrial membrane potential (MMP), and decreased the neuronal damage and death during OGD/R. Altogether, our data indicate that TNTs play an important role in the endogenous neuroprotection of astrocytic mitochondrial transfer. Furthermore, mild hypothermia enhances astrocytic mitochondrial transfer into OGD/R-injured neurons via TNTs, thereby promoting neuroprotection and neuronal recovery., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The Ang-(1-7)/MasR axis is critically involved in treating several diseases; For example, Ang-(1-7) improves inflammatory response and neurological function after traumatic brain injury and inhibits post-inflammatory hypothermia. However, its function in traumatic brain injury (TBI) combined with seawater immersion hypothermia remains unclear. Here, we used a mice model of hypothermic TBI and a BV2 cell model of hypothermic inflammation to investigate whether the Ang-(1-7)/MasR axis is involved in ameliorating hypothermic TBI. Quantitative reverse transcription PCR, western blotting assay, and immunofluorescence assay were performed to confirm microglia polarization and cytokine regulation. Hematoxylin-eosin staining, Nissl staining, and immunohistochemical assay were conducted to assess the extent of hypothermic TBI-induced damage and the ameliorative effect of Ang-(1-7) in mice. An open field experiment and neurological function scoring with two approaches were used to assess the degree of recovery and prognosis in mice. After hypothermic TBI establishment in BV2 cells, the Ang-(1-7)/MasR axis induced phenotypic transformation of microglia from M1 to M2, inhibited IL-6 and IL-1β release, and upregulated IL-4 and IL-10 levels. After hypothermic TBI development in mice, intraperitoneally administered Ang-(1-7) attenuated histological damage and promoted neurological recovery. These findings suggest that hypothermia exacerbates TBI-induced damage and that the Ang-(1-7)/MasR axis can ameliorate hypothermic TBI and directly affect prognosis., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2024 Ye et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Microglial activation is a critical factor in the pathogenesis and progression of neuroinflammatory diseases. Mild hypothermia, known for its neuroprotective properties, has been shown to alleviate microglial activation. In this study, we explore the differentially expressed (DE) mRNAs and long non-coding RNAs (lncRNAs) in BV-2 microglial cells under different conditions: normal temperature (CN), mild hypothermia (YT), normal temperature with lipopolysaccharide (LPS), and mild hypothermia with LPS (LPS + YT). Venn analysis revealed 119 DE mRNAs that were down-regulated in the LPS + YT vs LPS comparison but up-regulated in the CN vs LPS comparison, primarily enriched in Gene Ontology terms related to immune and inflammatory responses. Furthermore, through Venn analysis of YT vs CN and LPS + YT vs LPS comparisons, we identified 178 DE mRNAs and 432 DE lncRNAs. Among these transcripts, we validated the expression of Tent5c at the protein and mRNA levels. Additionally, siRNA-knockdown of Tent5c attenuated the expression of pro-inflammatory genes (TNF-α, IL-1β, Agrn, and Fpr2), cellular morphological changes, NLRP3 and p-P65 protein levels, immunofluorescence staining of p-P65 and number of cells with ASC-speck induced by LPS. Furthermore, Tent5c overexpression further potentiated the aforementioned indicators in the context of mild hypothermia with LPS treatment. Collectively, our findings highlight the significant role of Tent5c down-regulation in mediating the anti-inflammatory effects of mild hypothermia., Competing Interests: Declaration of competing interest All the authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

GFRAL-expressing neurons actuate aversion and nausea, are targets for obesity treatment, and may mediate metformin effects by long-term GDF15-GFRAL agonism. Whether GFRAL + neurons acutely regulate glucose and energy homeostasis is, however, underexplored. Here, we report that cell-specific activation of GFRAL + neurons using a variety of techniques causes a torpor-like state, including hypothermia, the release of stress hormones, a shift from glucose to lipid oxidation, and impaired insulin sensitivity, glucose tolerance, and skeletal muscle glucose uptake but augmented glucose uptake in visceral fat. Metabolomic analysis of blood and transcriptomics of muscle and fat indicate alterations in ketogenesis, insulin signaling, adipose tissue differentiation and mitogenesis, and energy fluxes. Our findings indicate that acute GFRAL + neuron activation induces endocrine and gluco- and thermoregulatory responses associated with nausea and torpor. While chronic activation of GFRAL signaling promotes weight loss in obesity, these results show that acute activation of GFRAL + neurons causes hypothermia and hyperglycemia., Competing Interests: Declaration of interests F.B. is a co-founder and shareholder of Roxbiosens, Inc., and Implexion Pharma AB, receives research funding from Biogaia AB, and is a member of the scientific advisory board of Bactolife A/S., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Integration of a point mutation to correct or edit a gene requires the repair of the CRISPR-Cas9-induced double-strand break by homology-directed repair (HDR). This repair pathway is more active in late S and G2 phases of the cell cycle, whereas the competing pathway of nonhomologous end-joining (NHEJ) operates throughout the cell cycle. Accordingly, modulation of the cell cycle by chemical perturbation or simply by the timing of gene editing to shift the editing toward the S/G2 phase has been shown to increase HDR rates. Using a traffic light reporter in mouse embryonic stem cells and a fluorescence conversion reporter in human-induced pluripotent stem cells, we confirm that a transient cold shock leads to an increase in the rate of HDR, with a corresponding decrease in the rate of NHEJ repair. We then investigated whether a similar cold shock could lead to an increase in the rate of HDR in the mouse embryo. By analyzing the efficiency of gene editing using single nucleotide polymorphism changes and loxP insertion at three different genetic loci, we found that a transient reduction in temperature after zygote electroporation of CRISPR-Cas9 ribonucleoprotein with a single-stranded oligodeoxynucleotide repair template did indeed increase knockin efficiency, without affecting embryonic development. The efficiency of gene editing with and without the cold shock was first assessed by genotyping blastocysts. As a proof of concept, we then confirmed that the modified embryo culture conditions were compatible with live births by targeting the coat color gene tyrosinase and observing the repair of the albino mutation. Taken together, our data suggest that a transient cold shock could offer a simple and robust way to improve knockin outcomes in both stem cells and zygotes.

Primary cilia act as cellular sensors for multiple extracellular stimuli and regulate many intracellular signaling pathways in response. Here we investigate whether the cold-shock proteins (CSPs), CIRP and RBM3, are present in the primary cilia and the physiological consequences of such a relationship. R28, an immortalized retinal precursor cell line, was stained with antibodies against CIRP, RBM3, and ciliary markers. Both CSPs were found in intimate contact with the basal body of the cilium during all stages of the cell cycle, including migrating with the centrosome during mitosis. In addition, the morphological and physiological manifestations of exposing the cells to hypothermia and shear stress were investigated. Exposure to moderately cold (32°C) temperatures, the hypothermia mimetic small molecule zr17-2, or to shear stress resulted in a significant reduction in the number and length of primary cilia. In addition, shear stress induced expression of CIRP and RBM3 in a complex pattern depending on the specific protein, flow intensity, and type of flow (laminar versus oscillatory). Flow-mediated CSP overexpression was detected by qRT-PCR and confirmed by Western blot, at least for CIRP. Furthermore, analysis of public RNA Seq databases on flow experiments confirmed an increase of CIRP and RBM3 expression following exposure to shear stress in renal cell lines. In conclusion, we found that CSPs are integral components of the centrosome and that they participate in cold and shear stress sensing., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Dexmedetomidine is widely used to induce sedation in the perioperative period. This study examined the effect of hypothermia (33 and 25 °C) on dexmedetomidine-induced contraction in an endothelium-intact aorta with or without the nitric oxide synthase inhibitor N W -nitro-L-arginine methyl ester (L-NAME). In addition, the effect of hypothermia on the contraction induced by dexmedetomidine in an endothelium-denuded aorta with or without a calcium-free Krebs solution was examined. The effects of hypothermia on the protein kinase C (PKC), myosin light chain (MLC 20 ) phosphorylation, and Rho-kinase membrane translocation induced by dexmedetomidine were examined. Hypothermia inhibited dexmedetomidine-induced contraction in the endothelium-intact aorta with L-NAME or endothelium-denuded aorta. Hypothermia had almost no effect on the dexmedetomidine-induced contraction in the endothelium-denuded aorta with the calcium-free Krebs solution; however, the subsequent contraction induced by the addition of calcium was inhibited by hypothermia. Conversely, the transition from profound hypothermia back to normothermia reversed the hypothermia-induced inhibition of subsequent calcium-induced contractions. Hypothermia inhibited any contraction induced by KCl, PDBu, and NaF, as well as PKC and MLC 20 phosphorylation and Rho-kinase membrane translocation induced by dexmedetomidine. These results suggest that hypothermia inhibits dexmedetomidine-induced contraction, which is mediated mainly by the impediment of calcium influx and partially by the attenuation of pathways involving PKC and Rho-kinase activation.

Therapeutic hypothermia (TH) mitigates damage in ischemic stroke models. However, safer and easier TH methods (e.g., pharmacological) are needed to circumvent physical cooling complications. This study evaluated systemic and pharmacologically induced TH using the adenosine A 1 receptor agonist, N 6 -cyclohexyladenosine (CHA), with control groups in male Sprague-Dawley rats. CHA was administered intraperitoneally 10 minutes following a 2-hour intraluminal middle cerebral artery occlusion. We used a 1.5 mg/kg induction dose, followed by three 1.0 mg/kg doses every 6 hours for a total of 4 doses, causing 20-24 hours of hypothermia. Animals assigned to physical hypothermia and CHA-hypothermia had similar induction rates and nadir temperatures, but forced cooling lasted ∼6 hours longer compared with CHA-treated animals. The divergence is likely attributable to individual differences in CHA metabolism, which led to varied durations at nadir, whereas physical hypothermia was better regulated. Physical hypothermia significantly reduced infarction (primary endpoint) on day 7 (mean reduction of 36.8 mm 3 or 39% reduction; p  = 0.021 vs. normothermic animals; Cohen's d  = 0.75), whereas CHA-induced hypothermia did not ( p  = 0.33). Similarly, physical cooling improved neurological function (physical hypothermia median = 0, physical normothermia median = 2; p  = 0.008) and CHA-induced cooling did not ( p  > 0.99). Our findings demonstrate that forced cooling was neuroprotective compared with controls, but prolonged CHA-induced cooling was not neuroprotective.

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: This study evaluated the difference in brain metabolite profiles between normothermia and hypothermia reaching 25°C in humans in vivo., Methods: Thirteen patients who underwent thoracic aorta surgery under moderate hypothermia were prospectively enrolled. Plasma samples were collected simultaneously from the arteries and veins to estimate metabolite uptake or release. Targeted metabolomics based on liquid chromatographic mass spectrometry and direct flow injection were performed, and changes in the profiles of respective metabolites from normothermia to hypothermia were compared. The ratios of metabolite concentrations in venous blood samples to those in arterial blood samples (V/A ratios) were calculated, and log 2 transformation of the ratios [log 2 (V/A)] was performed for comparison between the temperature groups., Results: Targeted metabolomics were performed for 140 metabolites, including 20 amino acids, 13 biogenic amines, 10 acylcarnitines, 82 glycerophospholipids, 14 sphingomyelins, and 1 hexose. Of the 140 metabolites analyzed, 137 metabolites were released from the brain in normothermia, and the release of 132 of these 137 metabolites was decreased in hypothermia. Two metabolites (dopamine and hexose) showed constant release from the brain in hypothermia, and 3 metabolites (2 glycophospholipids and 1 sphingomyelin) showed conversion from release to uptake in hypothermia. Glutamic acid demonstrated a distinct brain metabolism in that it was taken up by the brain in normothermia, and the uptake was increased in hypothermia., Conclusion: Targeted metabolomics demonstrated various degrees of changes in the release of metabolites by the hypothermic brain. The release of most metabolites was decreased in hypothermia, whereas glutamic acid showed a distinct brain metabolism., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2024 The Korean Academy of Medical Sciences.)

Global cerebral ischemia (GCI) caused by clinical conditions such as cardiac arrest leads to delayed neuronal death in the hippocampus, resulting in physical and mental disability. However, the mechanism of delayed neuronal death following GCI remains unclear. To elucidate the mechanism, we performed a metabolome analysis using a mouse model in which hypothermia (HT) during GCI, which was induced by the transient occlusion of the bilateral common carotid arteries, markedly suppressed the development of delayed neuronal death in the hippocampus after reperfusion. Fifteen metabolites whose levels were significantly changed by GCI and 12 metabolites whose levels were significantly changed by HT were identified. Furthermore, the metabolites common for both changes were narrowed down to two, adenosine monophosphate (AMP) and xanthosine monophosphate (XMP). The levels of both AMP and XMP were found to be decreased by GCI, but increased by HT, thereby preventing their decrease. In contrast, the levels of adenosine, inosine, hypoxanthine, xanthine, and guanosine, the downstream metabolites of AMP and XMP, were increased by GCI, but were not affected by HT. Our results may provide a clue to understanding the mechanism by which HT during GCI suppresses the development of delayed neuronal death in the hippocampus., (© 2024. The Author(s).)

Cold stress prompts an increased prevalence of cardiovascular morbidity yet the underneath machinery remains unclear. Oxidative stress and autophagy appear to contribute to cold stress-induced cardiac anomalies. Our present study evaluated the effect of heavy metal antioxidant metallothionein on cold stress (4 °C)-induced in cardiac remodeling and contractile anomalies and cell signaling involved including regulation of autophagy and mitophagy. Cold stress (3 weeks) prompted interstitial fibrosis, mitochondrial damage (mitochondrial membrane potential and TEM ultrastructure), oxidative stress (glutathione, reactive oxygen species and superoxide), lipid peroxidation, protein injury, elevated left ventricular (LV) end systolic and diastolic diameters, decreased fractional shortening, ejection fraction, Langendorff heart function, cardiomyocyte shortening, maximal velocities of shortening/relengthening, and electrically stimulated intracellular Ca 2+ rise along with elongated relaxation duration and intracellular Ca 2+ clearance, the responses of which were overtly attenuated or mitigated by metallothionein. Levels of apoptosis, cell death (Bax and loss of Bcl2, IL-18), and autophagy (LC3BII-to-LC3BI ratio, Atg7 and Beclin-1) were overtly upregulated with comparable p62 under cold stress. Cold stress also evoked elevated mitophagy (decreased TOM20, increased Parkin and FUNDC1 with unaltered BNIP3). Cold stress overtly dampened phosphorylation of autophagy/mitophagy inhibitory molecules Akt and mTOR, stimulated and suppressed phosphorylation of ULK1 and eNOS, respectively, in the absence of altered pan protein levels. Cold stress-evoked responses in cell death, autophagy, mitophagy and their regulatory domains were overtly attenuated or ablated by metallothionein. Suppression of autophagy and mitophagy with 3-methyladenine, bafilomycin A1, cyclosporine A, and liensinine rescued hypothermia-instigated cardiomyocyte LC3B puncta formation and mechanical anomalies. Our findings support a protective nature for metallothionein in deep hypothermia-evoked cardiac abnormalities associated with regulation of autophagy and mitophagy., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Post-mortem diagnosis of fatal hypothermia (FHT) is challenging in forensic practice because traditional morphological and biochemical methods lack specificity. Recent studies have reported that brown adipose tissue (BAT) is activated during cold-induced non-shivering thermogenesis in mammals, but BAT has not been used to diagnose FHT. The aim of this study was to identify novel biomarkers in BAT for FHT based on morphological changes and differential protein expression. Two FHT animal models were created by exposing mice to 4 or -20 °C at 50% humidity. Morphologically, the unilocular lipid droplet content was significantly increased in BAT of FHT model mice compared with that of control mice. Proteomics analysis revealed a total of 283 and 266 differentially expressed proteins (DEPs) between the 4 or -20 °C FHT subgroups and control group, respectively. In addition, 140 proteins were shared between the FHT subgroups. GO and KEGG analyses revealed that the shared DEPs were mainly enriched in pathways associated with metabolism, oxidative phosphorylation, and thermogenesis. Further screening (|log 2 FC| > 1.6, q-value (FDR) < 0.05) identified GMFB, KDM1A, DDX6, RAB1B, SHMT-1, CLPTM1, and LMF1 as candidate biomarkers of FHT. Subsequent validation experiments were performed in FHT model mice using classic immunohistochemistry and western blotting. RAB1B and GMFB expression was further verified in BAT specimens from human cases of FHT. The results demonstrate that BAT can be used as a target organ for FHT diagnosis employing RAB1B and GMFB as biological markers, thus providing a new strategy for the post-mortem diagnosis of FHT in forensic practice., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Brachial plexus avulsion (BPA) is a combined injury involving the central and peripheral nervous systems. Patients with BPA often experience severe neuropathic pain (NP) in the affected limb. NP is insensitive to the existing treatments, which makes it a challenge to researchers and clinicians. Accumulated evidence shows that a BPA-induced pain state is often accompanied by sympathetic nervous dysfunction, which suggests that the excitation state of the sympathetic nervous system is correlated with the existence of NP. However, the mechanism of how somatosensory neural crosstalk with the sympathetic nerve at the peripheral level remains unclear. In this study, through using a novel BPA C7 root avulsion mouse model, we found that the expression of BDNF and its receptor TrκB in the DRGs of the BPA mice increased, and the markers of sympathetic nervous system activity including α1 and α2 adrenergic receptors (α1-AR and α2-AR) also increased after BPA. The phenomenon of superexcitation of the sympathetic nervous system, including hypothermia and edema of the affected extremity, was also observed in BPA mice by using CatWalk gait analysis, an infrared thermometer, and an edema evaluation. Genetic knockdown of BDNF in DRGs not only reversed the mechanical allodynia but also alleviated the hypothermia and edema of the affected extremity in BPA mice. Further, intraperitoneal injection of adrenergic receptor inhibitors decreased neuronal excitability in patch clamp recording and reversed the mechanical allodynia of BPA mice. In another branch experiment, we also found the elevated expression of BDNF, TrκB, TH, α1-AR, and α2-AR in DRG tissues from BPA patients compared with normal human DRGs through western blot and immunohistochemistry. Our results revealed that peripheral BDNF is a key molecule in the regulation of somatosensory-sympathetic coupling in BPA-induced NP. This study also opens a novel analgesic target (BDNF) in the treatment of this pain with fewer complications, which has great potential for clinical transformation., (© 2023. Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences.)

Cold stimulation dynamically remodels mitochondria in brown adipose tissue (BAT) to facilitate non-shivering thermogenesis in mammals, but what regulates mitochondrial plasticity is poorly understood. Comparing mitochondrial proteomes in response to cold revealed FAM210A as a cold-inducible mitochondrial inner membrane protein. An adipocyte-specific constitutive knockout of Fam210a (Fam210a AKO ) disrupts mitochondrial cristae structure and diminishes the thermogenic activity of BAT, rendering the Fam210a AKO mice vulnerable to lethal hypothermia under acute cold exposure. Induced knockout of Fam210a in adult adipocytes (Fam210a iAKO ) does not affect steady-state mitochondrial structure under thermoneutrality, but impairs cold-induced mitochondrial remodeling, leading to progressive loss of cristae and reduction of mitochondrial density. Proteomics reveals an association between FAM210A and OPA1, whose cleavage governs cristae dynamics and mitochondrial remodeling. Mechanistically, FAM210A interacts with mitochondrial protease YME1L and modulates its activity toward OMA1 and OPA1 cleavage. These data establish FAM210A as a key regulator of mitochondrial cristae remodeling in BAT and shed light on the mechanism underlying mitochondrial plasticity in response to cold., (© 2023. Springer Nature Limited.)

Fever and hypothermia represent two opposite strategies for fighting systemic inflammation. Fever results in immune activation; hypothermia is associated with energy conservation. Systemic Inflammatory Response Syndrome (SIRS) remains a significant cause of mortality worldwide. SIRS can lead to a broad spectrum of clinical symptoms but importantly, patients can develop fever or hypothermia. During infection, polymorphonuclear cells (PMNs) such as neutrophils prevent pathogen dissemination through the formation of neutrophil extracellular traps (NETs) that ensnare and kill bacteria. However, when dysregulated, NETs also promote host tissue damage. Herein, we tested the hypothesis that temperature modulates NETs homeostasis in response to infection and inflammation. NETs formation was studied in response to infectious ( Escherichia coli , Staphylococcus aureus ) and sterile (mitochondria) agents. When compared to body temperature (37°C), NETs formation increased at 40°C; interestingly, the response was stunted at 35°C and 42°C. While CD16+ CD49d+ PMNs represent a small proportion of the neutrophil population, they formed ~45-85% of NETs irrespective of temperature. Temperature increased formyl peptide receptor 1 (FPR1) expression to a differential extent in CD16+ CD49d- vs . CD49d+ PMNSs, suggesting further complexity to neutrophil function in hypo/hyperthermic conditions. The capacity of NETs to induce Toll-like receptor 9 (TLR9)-mediated NF-κB activation was found to be temperature independent. Interestingly, NET degradation was enhanced at higher temperatures, which corresponded with greater plasma DNase activity in response to temperature increase. Collectively, our observations indicate that NETs formation and clearance are enhanced at 40°C whilst temperatures of 35°C and 42°C attenuate this response. Targeting PMN-driven immunity may represent new venues for intervention in pathological inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Janko, Bečka, Kmeťová, Hudecová, Konečná, Celec, Bajaj-Elliott and Pastorek.)

Mild hypothermia is proven neuroprotective in clinical practice. While hypothermia leads to the decrease of global protein synthesis rate, it upregulates a small subset of protein including RNA-binding motif protein 3 (RBM3). In this study, we treated mouse neuroblastoma cells (N2a) with mild hypothermia before oxygen-glucose deprivation/reoxygenation (OGD/R) and discovered the decrease of apoptosis rate, down-regulation of apoptosis-associated protein and enhancement of cell viability. Overexpression of RBM3 via plasmid exerted similar effect while silencing RBM3 by siRNAs partially reversed the protective effect exerted by mild hypothermia pretreatment. The protein level of Reticulon 3(RTN3), a downstream gene of RBM3, also increased after mild hypothermia pretreatment. Silencing RTN3 weakened the protective effect of mild hypothermia pretreatment or RBM3 overexpression. Also, the protein level of autophagy gene LC3B increased after OGD/R or RBM3 overexpression while silencing RTN3 decreased this trend. Furthermore, immunofluorescence observed enhanced fluorescence signal of LC3B and RTN3 as well as a large number of overlaps after RBM3 overexpressing. In conclusion, RBM3 plays a cellular protective role by regulating apoptosis and viability via its downstream gene RTN3 in the hypothermia OGD/R cell model and autophagy may participate in it., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Periventricular leukomalacia (PVL), characterized by distinctive form of white matter injury, often arises after neonatal cardiac surgery. Proven therapies for PVL are absent. In this study, we designed to quest therapeutic effects of delayed mild hypothermia on PVL and its mechanism in a neonatal rat brain slice model. With the increase of delayed mild hypothermia-treating time, the reduced expression of myelin basic protein and loss of preoligodendrocytes were significantly attenuated after oxygen-glucose deprivation. In addition, the proportion of ionized calcium binding adapter molecule 1 (Iba-1)-positive cells and the expression of Iba-1 were apparently reduced with the increased duration of mild hypothermia treatment. Furthermore, the levels of tumor necrosis factor alpha and interleukin-6 reduced after the mild hypothermia treatment relative to the control. Inhibition of microglial activation with prolonged mild hypothermia may be a potential strategy for white matter protection during cardiopulmonary bypass and hypothermic circulatory arrest.

Divers are at enhanced risk of hypothermia, due to the independent action of the inspired inert gases on thermoregulation. Thus, narcosis induced by acute (≤2 h) exposure to either hyperbaric nitrogen or normobaric nitrous oxide (N 2 O) impairs shivering thermogenesis and accelerates body core cooling. Animal-based studies, however, have indicated that repeated and sustained N 2 O administration may prevent N 2 O-evoked hypometabolism. We, therefore, examined the effects of prolonged intermittent exposure to 30% N 2 O on human thermoeffector plasticity in response to moderate cold. Fourteen men participated in two ∼12-h sessions, during which they performed sequentially three 120-min cold-water immersions (CWIs) in 20°C water, separated by 120-min rewarming. During CWIs, subjects were breathing either normal air or a normoxic gas mixture containing 30% N 2 O. Rectal and skin temperatures, metabolic heat production (via indirect calorimetry), finger and forearm cutaneous vascular conductance (CVC; laser-Doppler fluxmetry/mean arterial pressure), and thermal sensation and comfort were monitored. N 2 O aggravated the drop in rectal temperature ( P = 0.01), especially during the first (by ∼0.3°C) and third (by ∼0.4°C) CWIs. N 2 O invariably blunted the cold-induced elevation of metabolic heat production by ∼22%-25% ( P < 0.001). During the initial ∼30 min of the first and second CWIs, N 2 O attenuated the cold-induced drop in finger ( P ≤ 0.001), but not in forearm CVC. N 2 O alleviated the sensation of coldness and thermal discomfort throughout ( P < 0.001). Thus, the present results demonstrate that, regardless of the cumulative duration of gas exposure, a subanesthetic dose of N 2 O depresses human thermoregulatory functions and precipitates the development of hypothermia. NEW & NOTEWORTHY Human thermoeffector plasticity was evaluated in response to prolonged iterative exposure to 30% N 2 O and moderate cold stress. Regardless of the duration of gas exposure, N 2 O-induced narcosis impaired in a persistent manner shivering thermogenesis and thermoperception.

Thermal homeostasis is vital for mammals and is controlled by brain neurocircuits. Yet, the neural pathways responsible for cold defense regulation are still unclear. Here, we found that a pathway from the lateral parabrachial nucleus (LPB) to the dorsomedial hypothalamus (DMH), which runs parallel to the canonical LPB to preoptic area (POA) pathway, is also crucial for cold defense. Together, these pathways make an equivalent and cumulative contribution, forming a parallel circuit. Specifically, activation of the LPB → DMH pathway induced strong cold-defense responses, including increases in thermogenesis of brown adipose tissue (BAT), muscle shivering, heart rate, and locomotion. Further, we identified somatostatin neurons in the LPB that target DMH to promote BAT thermogenesis. Therefore, we reveal a parallel circuit governing cold defense in mice, which enables resilience to hypothermia and provides a scalable and robust network in heat production, reshaping our understanding of neural circuit regulation of homeostatic behaviors., (© 2023. Springer Nature Limited.)

After restoration of spontaneous circulation (ROSC) following cardiac arrest, complements can be activated and excessive autophagy can contribute to the brain ischemia-reperfusion (I/R) injury. Mild hypothermia (HT) protects against brain I/R injury after ROSC, but the mechanisms have not been fully elucidated. Here, we found that HT significantly inhibited the increases in serum NSE, S100β, and C5a, as well as neurologic deficit scores, TUNEL-positive cells, and autophagic vacuoles in the pig brain cortex after ROSC. The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen-glucose deprivation/reoxygenation. HT could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Our findings demonstrated that C5a could bind to C5aR1 to induce neuronal autophagy during the brain I/R injury, which was associated with the inhibited PI3K/Akt/mTOR pathway. HT could inhibit C5a-induced neuronal autophagy by regulating the C5a-C5aR1 interaction and the PI3K/Akt/mTOR pathway, which might be one of the neuroprotective mechanisms underlying I/R injury. The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen-glucose deprivation/reoxygenation. Mild hypothermia (HT) could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Proposed mechanism by which HT protects against brain I/R injury by repressing C5a-C5aR1-induced excessive autophagy. Complement activation in response to brain I/R injury generates C5a that can interact with C5aR1 to inactivate mTOR, probably through the PI3K-AKT pathway, which can finally lead to autophagy activation. The excessively activated autophagy ultimately contributes to cell apoptosis and brain injury. HT may alleviate complement activation and then reduce C5a-induced autophagy to protect against brain I/R injury. HT, mild hypothermia; I/R, ischemia reperfusion., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Increased gut permeability is implicated in the initiation and extent of the cytokine inflammatory response associated with exertional heat stroke (EHS). The primary objective of this study was to determine if a five amino acid oral rehydration solution (5AAS), specifically designed for the protection of the gastrointestinal lining, would prolong time to EHS, maintain gut function and dampen the systemic inflammatory response (SIR) measured during EHS recovery. Male C57/BL6J mice instrumented with radiotelemetry were gavaged with 150 μL of 5AAS or H 2 O, and ≈12 h later were either exposed to an EHS protocol where mice exercised in a 37.5°C environmental chamber to a self-limiting maximum core temperature (Tc,max) or performed the exercise control (EXC) protocol (25°C). 5AAS pretreatment attenuated hypothermia depth and length (p < 0.005), which are indicators of EHS severity during recovery, without any effect on physical performance or thermoregulatory responses in the heat as determined by percent body weight lost (≈9%), max speed (≈6 m/min), distance (≈700 m), time to Tc,max (≈160 min), thermal area (≈550°C∙min), and Tc,max (42.2°C). EHS groups treated with 5AAS showed a significant decrease in gut transepithelial conductance, decreased paracellular permeability, increased villus height, increased electrolyte absorption and changes in tight junction protein expression pattern suggestive of improved barrier integrity (p < 0.05). No differences were witnessed between EHS groups in acute phase response markers of liver, circulating SIR markers, or indicators of organ damage during recovery. These results suggest that a 5AAS improves Tc regulation during EHS recovery through maintaining mucosal function and integrity., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

The brain ischemia/reperfusion (I/R) injury has a great impact on human life and property safety. As far as we know, mild hypothermia (MH) is an effective measure to reduce neuronal injury after I/R. However, the precise mechanism is not extremely clear. The purpose of this study was to investigate whether mild therapeutic hypothermia can play a protective role in nerve cells dealing with brain I/R injury and explore its specific mechanism in vitro. A flow cytometer, cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release assay were performed to detect apoptotic rate of cells, cell viability and cytotoxicity, respectively, reactive oxygen species (ROS) assay kit, JC-1 fluorescent methods, immunofluorescence and western blot were used to explore ROS, mitochondrial transmembrane potential ( Δ ψm), mitochondrial permeability transition pore (MPTP) and protein expression, respectively. The result indicated that the cell activity was decreased, while the cytotoxicity and apoptosis rate were increased after treating with oxygen-glucose deprivation/reperfusion (OGD/R) in PC12 cells. However, MH could antagonize this phenomenon. Interestingly, treating with OGD/R increased the release of ROS and the transfer of Cytochrome C (Cyt-C) from mitochondria to cytoplasm. In addition, it up-regulated the expression of γH2AX, Bax and Clv-caspase3, down-regulated the expression of PCNA, Rad51 and Bcl-2, and inhibited the function of mitochondria in PC12 cells. Excitingly, the opposite trend was observed after MH treatment. Therefore, our results suggest that MH protects PC12 cells against OGD/R-induced injury with the mechanism of inhibiting cell apoptosis by reducing ROS production, improving mitochondrial function, reducing DNA damage, and enhancing DNA repair., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mild hypothermia has been proven to inhibit microglia activation after TBI. Exosomal microRNA derived from microglia played a critical role in promoting neurite outgrowth and synapse recovery. Here, we aimed to investigate the role of microRNAs in microglial exosomes after hypothermia treatment on neuronal regeneration after TBI. For in vitro study, stretch-injured neurons were co-cultured with microglial exosomes. For in vivo study, C57BL/6 mice were under controlled cortical impact and injected with microglial exosomes. The results showed that MG-LPS-EXO HT increased the number of dendrite branches and total length of dendrites both in vitro and in vivo, elevated the expression levels of PSD-95 and GluR1 in stretch-injured neurons, and increased spine density in the pericontusion region. Moreover, MG-LPS-EXO HT improved motor function and motor coordination. A high-throughput sequencing showed that miR-20b-5p was upregulated in MG-LPS-EXO HT . Elevating miR-20b-5p promoted neurite outgrowth and synapse recovery of injured neurons both in vitro and in vivo. Following mechanistic study demonstrated that miR-20b-5p might promote neurite outgrowth and synapse recovery by directly targeting PTEN and activating PI3K-AKT pathway. In conclusion, mild hypothermia could modify the microRNA prolife of exosomes derived from LPS activated BV2 cells. Furthermore, high level of microglial exosomal miR-20b-5p induced by mild hypothermia could transfer into injured neurons and promote neurite outgrowth and synapse recovery after TBI via activating the PI3K-AKT pathway by suppressing PTEN expression., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Inc.)

Brain injury remains a major problem in patients suffering cardiac arrest (CA). Disruption of the blood-brain barrier (BBB) is an important factor leading to brain injury. Therapeutic hypothermia is widely accepted to limit neurological impairment. However, the efficacy is incomplete. Hydrogen sulfide (H 2 S), a signaling gas molecule, has protective effects after cerebral ischemia reperfusion injury. This study showed that combination of hypothermia and H 2 S after resuscitation was more beneficial for attenuated BBB disruption and brain edema than that of hypothermia or H 2 S treatment alone. CA was induced by ventricular fibrillation for 4 min. Hypothermia was performed by applying alcohol and ice bags to the body surface under anesthesia. We used sodium hydrosulphide (NaHS) as the H 2 S donor. We found that global brain ischemia induced by CA and cardiopulmonary resuscitation (CPR) resulted in brain edema and BBB disruption; Hypothermia or H 2 S treatment diminished brain edema, decreased the permeability and preserved the structure of BBB during the early period of CA and resuscitation, and more importantly, improved the neurologic function, increased the 7-day survival rate after resuscitation; the combination of hypothermia and H 2 S treatment was more beneficial than that of hypothermia or H 2 S treatment alone. The beneficial effects were associated with the inhibition of matrix metalloproteinase-9 expression, attenuated the degradation of the tight junction protein occludin, and subsequently protected the structure of BBB. These findings suggest that combined use of therapeutic hypothermia and hydrogen sulfide treatment during resuscitation of CA patients could be a potential strategy to improve clinical outcomes and survival rate., (© 2022. The Author(s).)

Background: Most platelets are present in peripheral blood, but some are stored in the spleen. Because the tissue environments of peripheral blood vessels and the spleen are quite distinct, the properties of platelets present in each may also differ. However, no studies have addressed this difference. We previously reported that hypothermia activates splenic platelets, but not peripheral blood platelets, whose biological significance remains unknown. In this study, we focused on platelet-derived microvesicles (PDMVs) and analyzed their biological significance connected to intrasplenic platelet activation during hypothermia., Methods: C57Bl/6 mice were placed in an environment of -20 °C, and their rectal temperature was decreased to 15 °C to model hypothermia. Platelets and skeletal muscle tissue were collected and analyzed for their interactions., Results: Transcriptomic changes between splenic and peripheral platelets were greater in hypothermic mice than in normal mice. Electron microscopy and real-time RT-PCR analysis revealed that platelets activated in the spleen by hypothermia internalized transcripts, encoding tissue repairing proteins, into PDMVs and released them into the plasma. Plasma microvesicles from hypothermic mice promoted wound healing in the mouse myoblast cell line C2C12. Skeletal muscles in hypothermic mice were damaged but recovered within 24 h after rewarming. However, splenectomy delayed recovery from skeletal muscle injury after the mice were rewarmed., Conclusions: These results indicate that PDMVs released from activated platelets in the spleen play an important role in the repair of skeletal muscle damaged by hypothermia., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Cardiac arrest (CA) and return of spontaneous circulation (ROSC), a global ischemia and reperfusion event, lead to neuronal damage and/or death in the spinal cord as well as the brain. Hypothermic therapy is reported to protect neurons from damage and improve hindlimb paralysis after resuscitation in a rat model of CA induced by asphyxia. In this study, we investigated roles of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) in the lumbar spinal cord protected by therapeutic hypothermia in a rat model of asphyxial CA. Male Sprague-Dawley rats were subjected to seven minutes of asphyxial CA (induced by injection of 2 mg/kg vecuronium bromide) and hypothermia (four hours of cooling, 33 ± 0.5 °C). Survival rate, hindlimb motor function, histopathology, western blotting, and immunohistochemistry were examined at 12, 24, and 48 h after CA/ROSC. The rats of the CA/ROSC and hypothermia-treated groups had an increased survival rate and showed an attenuated hindlimb paralysis and a mild damage/death of motor neurons located in the anterior horn of the lumbar spinal cord compared with those of the CA/ROSC and normothermia-treated groups. In the CA/ROSC and hypothermia-treated groups, expressions of cytoplasmic and nuclear Nrf2 and HO-1 were significantly higher in the anterior horn compared with those of the CA/ROSC and normothermia-treated groups, showing that cytoplasmic and nuclear Nrf2 was expressed in both motor neurons and astrocytes. Moreover, in the CA/ROSC and hypothermia-treated group, interleukin-1β (IL-1β, a pro-inflammatory cytokine) expressed in the motor neurons was significantly reduced, and astrocyte damage was apparently attenuated compared with those found in the CA/ROSC and normothermia group. Taken together, our results indicate that hypothermic therapy after CA/ROSC attenuates CA-induced hindlimb paralysis by protecting motor neurons in the lumbar spinal cord via activating the Nrf2/HO-1 signaling pathway and attenuating pro-inflammation and astrocyte damage (reactive astrogliosis).

Background: Inflammation, the physiological response to infection and injury, is coordinated by the immune and nervous systems. Interleukin-1β (IL-1β) and other cytokines produced during inflammatory responses activate sensory neurons (nociceptors) to mediate the onset of pain, sickness behavior, and metabolic responses. Although nociceptors expressing Transient Receptor Potential Ankyrin-1 (TRPA1) can initiate inflammation, comparatively little is known about the role of TRPA1 nociceptors in the physiological responses to specific cytokines., Methods: To monitor body temperature in conscious and unrestrained mice, telemetry probes were implanted into peritoneal cavity of mice. Using transgenic and tissue specific knockouts and chemogenetic techniques, we recorded temperature responses to the potent pro-inflammatory cytokine IL-1β. Using calcium imaging, whole cell patch clamping and whole nerve recordings, we investigated the role of TRPA1 during IL-1β-mediated neuronal activation. Mouse models of acute endotoxemia and sepsis were used to elucidate how specific activation, with optogenetics and chemogenetics, or ablation of TRPA1 neurons can affect the outcomes of inflammatory insults. All statistical tests were performed with GraphPad Prism 9 software and for all analyses, P ≤ 0.05 was considered statistically significant., Results: Here, we describe a previously unrecognized mechanism by which IL-1β activates afferent vagus nerve fibers to trigger hypothermia, a response which is abolished by selective silencing of neuronal TRPA1. Afferent vagus nerve TRPA1 signaling also inhibits endotoxin-stimulated cytokine storm and significantly reduces the lethality of bacterial sepsis., Conclusion: Thus, IL-1β activates TRPA1 vagus nerve signaling in the afferent arm of a reflex anti-inflammatory response which inhibits cytokine release, induces hypothermia, and reduces the mortality of infection. This discovery establishes that TRPA1, an ion channel known previously as a pro-inflammatory detector of cold, pain, itch, and a wide variety of noxious molecules, also plays a specific anti-inflammatory role via activating reflex anti-inflammatory activity., (© 2022. The Author(s).)

Encouraging advances in both regenerative medicine and tissue engineering with stem cells require a short-term preservation protocol to provide enough time for quality control or the transportation of cell products from manufacturing facilities to clinical destinations. The hypothermic preservation of stem cells under refrigerated conditions (2-8 °C) in their specific culture medium provides an alternative and low-cost method for cryopreservation or commercial preservation fluid for short-term storage. However, most stem cells are vulnerable to hypothermia, which might result in cell damage from the cooling process and the lack of extracellular matrix (ECM). Herein, we report a peptide scaffold cell-culture-medium additive for mimicking in vivo ECM to enhance the storage efficiency of mesenchymal stem cells (MSCs) under hypothermic preservation. Peptide scaffolds exhibit protective effects against hypothermic injury by maintaining the viability, proliferation, migration, and differentiation capabilities of cells. The mechanistic study showed that the peptide scaffold was conducive to maintain mitochondrial function by retaining mitochondrial respiration, mitochondrial membrane potential (ΔΨm), and mass to alleviate intracellular and mitochondrial reactive oxygen species (ROS) production. Moreover, the peptide scaffold also prolonged the survival and retained the multipotency of hematopoietic stem and progenitor cells (HSPCs) under hypothermic conditions. In conclusion, these results demonstrate a feasible and convenient preservation system for stem cells that has the potential to promote the clinical application of hematopoietic stem cell therapy.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue 1,2 , but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3β) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3β is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER-LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3β have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3β is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3β associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3β-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Liver ischemia reperfusion injury (IRI) is a serious complication of certain liver surgeries, and it is difficult to prevent. As a potential drug-free treatment, mild hypothermia has been shown to promote positive outcomes in patients with IRI. However, the protective mechanism remains unclear. We established in vivo and in vitro models of hepatic ischemia reperfusion (IR) and mild hypothermia pretreatment. Hepatocytes were transfected with RNA-binding motif protein 3 (RBM3) overexpression plasmids, and IR was performed. Cell, culture medium, blood and tissue samples were collected to assess hepatic injury, oxidative stress, apoptosis and changes in RBM3 expression in the liver. Upregulation of RBM3 expression by mild hypothermia reduced the aminotransferase release, liver tissue injury and mitochondrial injury induced by liver IR. Hepatic IR-induced p38 and c-Jun N-terminal kinase (JNK) signaling pathway activation, oxidative stress injury and apoptosis could be greatly reversed by mild hypothermia. Overexpression of RBM3 mimicked the hepatoprotective effect of mild hypothermia. Mild hypothermia protects the liver from ischemia reperfusion-induced p38 and JNK signaling pathway activation, oxidative stress injury and apoptosis through the upregulation of RBM3 expression., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Adipose tissue is the largest endocrine organ. When activated by cancer cells, adipocytes secrete adipocytokines and release fatty acids, which are then transferred to cancer cells and used for structural and biochemical support. How this metabolic symbiosis between cancer cells and adipocytes affects skeletal muscle and thermogenesis during cancer cachexia is unknown. Cancer cachexia is a multiorgan syndrome and how the communication between tissues is established has yet to be determined. We investigated adipose tissue secretory factors and explored their role in crosstalk of adipocytes, muscle, and tumor during pancreatic cancer cachexia., Methods: We used a pancreatic cancer cachexia mouse model generated by syngenic implantation of pancreatic ductal adenocarcinoma (PDAC) cells (KPC) intraperitoneally into C57BL/6 mice and Lcn2-knockout mice. For in vitro studies, adipocytes (3T3-L1 and primary adipocytes), cachectic cancer cells (Panc0203), non-cachectic cancer cells (Du145 cells), and skeletal muscle cells (C2C12 myoblasts) were used., Results: To identify molecules involved in the crosstalk of adipose tissue with muscle and tumors, we treated 3T3-L1 adipocytes with conditioned medium (CM) from cancer cells. Upon screening the secretomes from PDAC-induced adipocytes, several adipocytokines were identified, including lipocalin 2 (Lcn2). We investigated Lcn2 as a potential mediator of cachexia induced by adipocytes in response to PDAC. During tumor progression, mice exhibited a decline in body weight gain, which was accompanied by loss of adipose and muscle tissues. Tumor-harboring mice developed drastic hypothermia because of a dramatic loss of fat in brown adipose tissue (BAT) and suppression of the thermogenesis pathway. We inhibited Lcn2 with an anti-Lcn2 antibody neutralization or genomic ablation in mice. Lcn2 deficiency significantly improved body temperature in tumor-bearing mice, which was supported by the increased expression of Ucp1 and β3-adrenergic receptor in BAT. In addition, Lcn2 inhibition abrogated the loss of fat and muscle in tumor-bearing mice. In contrast to tumor-bearing WT mice, the corresponding Lcn2-knockout mice showed reduced ATGL expression in iWAT and decreased the expression of muscle atrophy molecular markers MuRF-1 and Fbx32., Conclusions: This study showed that Lcn2 is causally involved in the dysregulation of adipose tissue-muscle-tumor crosstalk during pancreatic cancer cachexia. Therapeutic targets that suppress Lcn2 may minimize the progression of cachexia., (Published by Elsevier GmbH.)

Forensic diagnosis of fatal hypothermia is considered difficult because no specific findings, such as molecular markers, have been identified. Therefore, determining the molecular mechanism in hypothermia and identifying novel molecular markers to assist in diagnosing fatal hypothermia are important. This study aimed to investigate microRNA (miRNA) and mRNA expression in iliopsoas muscle, which plays a role in homeostasis in mammals, to resolve the molecular mechanism in hypothermia. We generated rat models of mild, moderate, and severe hypothermia, then performed body temperature-dependent miRNA and mRNA expression analysis of the iliopsoas muscle using microarray and next-generation sequencing. Analysis showed that rno-miR-203a-3p expression was lower with decreasing body temperature, while Socs3 expression was significantly increased only by severe hypothermia. Luciferase reporter assays suggested that Socs3 expression is regulated by rno-miR-203a-3p. Socs3 and Mex3B small interfering RNA-mediated knockdown showed that suppressing Mex3B could induce the activation of Socs3, followed by a change in caspase 3/7 activity and adenosine triphosphate levels in iliopsoas muscle cells. These findings indicate that rno-miR-203a-3p and Mex3B are deactivated by a decrease in body temperature, whereby it contributes to suppressing apoptosis by accelerating Socs3. Accordingly, the rno-miR-203a-3p-Socs3-Casp3 or Mex3B-Socs3-Casp3 axis may be the part of the biological defense response to maintain homeostasis under extreme hypothermia., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

The blood-brain barrier (BBB) presents a major obstacle in developing specific diagnostic imaging agents for many neurological disorders. In this study we aimed to generate single domain anti-mouse transferrin receptor antibodies (anti-mTfR VHHs) to mediate BBB transcytosis as components of novel MRI molecular contrast imaging agents. Anti-mTfR VHHs were produced by immunizing a llama with mTfR, generation of a VHH phage display library, immunopanning, and in vitro characterization of candidates. Site directed mutagenesis was used to generate additional variants. VHH fusions with neurotensin (NT) allowed rapid, hypothermia-based screening for VHH-mediated BBB transcytosis in wild-type mice. One anti-mTfR VHH variant was fused with an anti-amyloid-beta (Aβ) VHH dimer and labeled with fluorescent dye for direct assessment of in vivo target engagement in a mouse model of AD-related Aβ plaque pathology. An anti-mTfR VHH called M1 and variants had binding affinities to mTfR of <1nM to 1.52nM. The affinity of the VHH binding to mTfR correlated with the efficiency of the VHH-NT induced hypothermia effects after intravenous injection of 600 nmol/kg body weight, ranging from undetectable for nonbinding mutants to -6°C for the best mutants. The anti-mTfR VHH variant M1P96H with the strongest hypothermia effect was fused to the anti-Aβ VHH dimer and labeled with Alexa647; the dye-labeled VHH fusion construct still bound both mTfR and Aβ plaques at concentrations as low as 0.22 nM. However, after intravenous injection at 600 nmol/kg body weight into APP/PS1 transgenic mice, there was no detectible labeling of plaques above control levels. Thus, NT-induced hypothermia did not correlate with direct target engagement in cortex, likely because the concentration required for NT-induced hypothermia was lower than the concentration required to produce in situ labeling. These findings reveal an important dissociation between NT-induced hypothermia, presumably mediated by hypothalamus, and direct engagement with Aβ-plaques in cortex. Additional methods to assess anti-mTfR VHH BBB transcytosis will need to be developed for anti-mTfR VHH screening and the development of novel MRI molecular contrast agents., Competing Interests: The authors have declared that no competing interests exist.

RNA-binding motif protein 3 (RBM3), an outstanding cold shock protein, is rapidly upregulated to ensure homeostasis and survival in a cold environment, which is an important physiological mechanism in response to cold stress. Meanwhile, RBM3 has multiple physiological functions and participates in the regulation of various cellular physiological processes, such as antiapoptosis, circadian rhythm, cell cycle, reproduction, and tumogenesis. The structure, conservation, and tissue distribution of RBM3 in human are demonstrated in this review. Herein, the multiple physiological functions of RBM3 were summarized based on recent research advances. Meanwhile, the cytoprotective mechanism of RBM3 during stress under various adverse conditions and its regulation of transcription were discussed. In addition, the neuroprotection of RBM3 and its oncogenic role and controversy in various cancers were investigated in our review., (© 2022 Wiley Periodicals LLC.)

Aim: Cardiac surgery requiring cardiopulmonary bypass (CPB) can result in renal and cerebral injury. Intraoperative tissue hypoxia could contribute to such organ injury. Hypothermia, however, may alleviate organ hypoxia. Therefore, we tested whether moderate hypothermia (30°C) improves cerebral and renal tissue perfusion and oxygenation during ovine CPB., Methods: Ten sheep were studied while conscious, under stable anesthesia, and during 3 h of CPB. In a randomized within-animal cross-over design, five sheep commenced CPB at a target body temperature of 30°C (moderate hypothermia). After 90 min, the body temperature was increased to 36°C (standard procedure). The remaining five sheep were randomized to the opposite order of target body temperature., Results: Compared with the standard procedure, moderately hypothermic CPB reduced renal oxygen delivery (-34.8% ± 19.6%, P = 0.003) and renal oxygen consumption (-42.7% ± 35.2%, P = 0.04). Nevertheless, moderately hypothermic CPB did not significantly alter either renal cortical or medullary tissue PO 2 . Moderately hypothermic CPB also did not significantly alter cerebral perfusion, cerebral tissue PO 2 , or cerebral oxygen saturation compared with the standard procedure. Compared with the anesthetized state, the standard procedure reduced renal medullary PO 2 (-21.0 ± 13.8 mmHg, P = 0.014) and cerebral oxygen saturation (65.0% ± 7.0% to 55.4% ± 9.6%, P = 0.022) but did not significantly alter either renal cortical or cerebral PO 2 ., Conclusion: Ovine experimental CPB leads to renal medullary tissue hypoxia. Moderately hypothermic CPB did not improve cerebral or renal tissue oxygenation. In the kidney, this is probably because renal tissue oxygen consumption is matched by reduced renal oxygen delivery., (© 2022 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Background: Hypothermia (H), cardioplegia (CP), and both combined (HCP) are known to be protective against myocardial ischemia reperfusion (IR) injury. Mitochondria have molecular signaling mechanisms that are associated with both cell survival and cell death. In this study, we investigated the dynamic changes in proapoptotic and prosurvival signaling pathways mediating H, CP, or HCP-induced protection of mitochondrial function after acute myocardial IR injury., Methods: Rats were divided into five groups. Each group consists of 3 subgroups based on a specific reperfusion time (5, 20, or 60 min) after a 25-min global ischemia. The time control (TC) groups were not subjected to IR but were perfused with 37 °C Krebs-Ringer's (KR) buffer, containing 4.5 mM K + , in a specific perfusion protocol that corresponded with the duration of each IR protocol. The IR group (control) was perfused for 20 min with KR, followed by 25-min global ischemia, and then KR reperfusion for 5, 20, or 60 min. The treatment groups were exposed to 17 °C H, 37 °C CP (16 mM K + ), or HCP (17 °C + CP) for 5 min before ischemia and for 2 min on reperfusion before switching to 37 °C KR perfusion for the remainder of each of the reperfusion times. Cardiac function and mitochondrial redox state (NADH/FAD) were monitored online in the ex vivo hearts before, during, and after ischemia. Mitochondria were isolated at the end of each specified reperfusion time, and changes in O 2 consumption, membrane potential ( ΔΨ m ), and Ca 2+ retention capacity (CRC) were assessed using complex I and complex II substrates. In another set of hearts, mitochondrial and cytosolic fractions were isolated after a specified reperfusion time to conduct western blot assays to determine hexokinase II (HKII) and Bax binding/translocation to mitochondria, cytosolic pAkt levels, and cytochrome c (Cyto- c ) release into the cytosol., Results: H and HCP were more protective of mitochondrial integrity and, concomitantly, cardiac function than CP alone; H and HCP improved post-ischemic cardiac function by (1) maintaining mitochondrial bioenergetics, (2) maintaining HKII binding to mitochondria with an increase in pAkt levels, (3) increasing CRC, and (4) decreasing Cyto- c release during reperfusion. Bax translocation/binding to mitochondria was unaffected by any treatment, regardless of cardiac functional recovery., Conclusions: Hypothermia preserved mitochondrial function and cardiac function, in part, by maintaining mitochondrial bioenergetics, by retaining HKII binding to mitochondria via upstream pAkt, and by reducing Cyto- c release independently of Bax binding to mitochondria., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Jie Sun et al.)

Despite the fact that heart transplantation (HTx) is a relatively mature procedure, heart ischemic and reperfusion (I/R) injury during HTx remains a challenge. Even after a successful operation, the heart will be at risk of primary graft failure and mortality during the first year. In this study, temperature-sensitive polymer poly( N - n -propylacrylamide- co - N - tert -butyl acrylamide) (PNNTBA) was coated on diallyl trisulfide (DATS)-loaded mesoporous silica nanoparticles (DATS-MSN) to synthesize hypothermia-triggered hydrogen sulfide (H 2 S) releasing particles (HT-MSN). Because the PNNTBA shell dissolves in phosphate-buffered saline at 4 °C, the loaded DATS could continuously release H 2 S within 6 h when activated by glutathione (GSH). Furthermore, after co-culturing biocompatible HT-MSN with cardiomyocytes, H 2 S released from HT-MSN at 4 °C was found to protect cardiomyocytes from ischemic and reperfusion (I/R) injury. In detail, the rate of cell apoptosis and lactate dehydrogenase activity was decreased, as manifested by increased BCL-2 expression and decreased BAX expression. More importantly, in an isolated heart preservation experiment, HT-MSN demonstrated potent protection against cardiac I/R injury and reduced expression of inflammatory factors TNF-α and IL-1β. This study provided a new method for the controlled release of H 2 S by the donor and myocardial protection from I/R injury.

The cold-inducible RNA-binding protein (CIRBP) assists cells in adapting to new environmental conditions stabilizing specific mRNAs and promoting their translation. CIRBP participates in anti-apoptotic and anti-senescence processes, and its expression is induced by mild hypothermia, which may be advantageous to oocytes during vitrification. Several newly discovered small molecules, like zr17-2, mimic the effects of cold temperatures by increasing the expression of CIRBP at normothermia. This study aimed to evaluate the mRNA changes of a group of cold-inducible protein-encoding and apoptotic genes in response to exogenous supplementation of zr17-2 (experiment 1) or CIRBP (experiment 2) in vitro matured bovine oocytes and their cumulus cells. In experiment 1, cumulus-oocyte complexes (COCs) were randomly exposed to three concentrations of zr17-2 (1, 10, and 100 μM) during 24 h of in vitro maturation (IVM) under normothermia (38.5 °C) or mild hypothermia (34 °C) culture conditions. In experiment 2, COCs were randomly exposed to three concentrations of CIRBP (2, 4, and 6 μg/mL) or subjected to mild hypothermia (34 °C), followed by oocyte vitrification/warming after 20 h of IVM. The quantification of the selected gene transcript expression was performed separately in oocytes and cumulus cells by quantitative real-time PCR. We show that oocytes and cumulus cells exhibited similar mRNA expression responses to mild hypothermia and vitrification. However, minor differences were observed when COCs were exposed to exogenous supplementation with zr17-2 and CIRBP. In conclusion, the alterations observed in the mRNA expression in these experimental conditions may impact the quality of the cumulus-oocyte complexes in terms of vitrification and sublethal hypothermia challenges. In this sense, our results should complement other oocyte quality assessments for its application in future assisted reproductive techniques in the bovine species, including improving oocyte cryotolerance to vitrification., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)