Mild hypothermia reduces lipopolysaccharide-induced microglial activation via down-regulation of Tent5c.

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-1ß, 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.

Urine Proteomic Signatures of Mild Hypothermia Treatment in Cerebral Ischemia-Reperfusion Injury in Rats.

Mild hypothermia (MH) is an effective measure to alleviate cerebral ischemia-reperfusion (I/R) injury. However, the underlying biological mechanisms remain unclear. This study set out to investigate dynamic changes in urinary proteome due to MH in rats with cerebral I/R injury and explore the neuroprotective mechanisms of MH. A Pulsinelli's four-vessel occlusion (4-VO) rat model was used to mimic global cerebral I/R injury. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to profile the urinary proteome of rats with/without MH (32 °C) treatment after I/R injury. Representative differentially expressed proteins (DEPs) associated with MH were validated by western blotting in hippocampus. A total of 597 urinary proteins were identified, among which 119 demonstrated significant changes associated with MH. Gene Ontology (GO) annotation of the DEPs revealed that MH significantly enriched in endopeptidase activity, inflammatory response, aging, response to oxidative stress and reactive oxygen species, blood coagulation, and cell adhesion. Notably, changes in 12 DEPs were significantly reversed by MH treatment. Among them, 8 differential urinary proteins were previously reported to be closely associated with brain disease, including NP, FZD1, B2M, EPCR, ATRN, MB, CA1and VPS4A. Two representative proteins (FZD1, B2M) were further validated by western blotting in the hippocampus and the results were shown to be consistent with urinary proteomic analysis. Overall, this study strengthens the idea that urinary proteome can sensitively reflect pathophysiological changes in the brain, and appears to be the first study to explore the neuroprotective effects of MH by urinary proteomic analysis. FZD1 and B2M may be involved in the most fundamental molecular biological mechanisms of MH neuroprotection.

A Comparison of Passive Rewarming Systems Following Cold Water Immersion.

INTRODUCTION: We studied field rewarming using a typical winter sleeping bag versus two heated hypothermia wrap systems in a semi-realistic lab simulation. METHODS: 10 participants (8 M, 2 F) were cooled to 36.1°C core temperature through 10.5-11.5°C water immersion, then performed 60 min of passive rewarming in 0°C air. The rewarming methods tested were: 1) a -9°C rated mummy-style Sleeping Bag; 2) Doctor Down Rescue Wrap; and 3) Thermal Yielding Vascular Airway Capsule (TYVAC) system; the latter two methods included vapor barriers and two heating pads. Rectal and skin temperatures, along with metabolic heat production calculated via indirect calorimetry, were measured throughout rewarming. RESULTS: One male participant was removed from analysis due to lack of sufficient cooling. Rectal temperature decreased in the remaining participants by ∼1.1-1.2°C to 36.1°C during the initial immersion phase. Over the 60 min of rewarming, rectal temperature changes were Δ0.0 ± 0.6°C in a sleeping bag, Δ+0.2 ± 0.3°C in Doctor Down, and Δ+0.2 ± 0.3°C in TYVAC, with no significant differences across methods. Mean skin temperatures, metabolic heat production, and perceptual measures were also similar across methods with no method×time interactions. CONCLUSIONS: After 60 min of passive rewarming in cold conditions, all three rewarming methods were able to stall continued core cooling to levels at or slightly above post-immersion temperatures. With no differences in any physiological measures, it appears that all three rewarming methods are equally viable options for wilderness responders, and the choice should come down to environmetal conditions, availability, convenience, and ergonomics rather than rewarming efficacy.

Mild hypothermia alleviates oxygen-glucose deprivation/reperfusion-induced apoptosis by inhibiting ROS generation, improving mitochondrial dysfunction and regulating DNA damage repair pathway in PC12 cells.

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 (Δψm), 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.

Role of PERK-mediated pathway in the effect of mild hypothermia after cerebral ischaemia/reperfusion.

BACKGROUND: Hypothermia is an effective method of reducing brain injury caused by a variety of neurological insults. It is aimed to elucidate whether a change in the expression of PERK-mediated pathway proteins is an indicator of the neuroprotective effect of mild hypothermia after cerebral ischaemia/reperfusion. METHODS: One hundred and ninety-two male C57BL/6 mice were randomly divided into three groups: a sham group, a cerebral normothermic ischaemia/reperfusion (I/R) group and a cerebral hypothermic I/R group. A cerebral ischaemia model was established by ligating the bilateral common carotid artery for 15 min. Mice in the hypothermia group stayed in a cage that was set at 33°C, sprayed with a spray of 70% ethanol, and blown with two high-speed fans. The state of neurons was assessed on micropreparations stained with haematoxylin-eosin and TUNEL. The expressions of GRP78, p-perk, p-eif2α, ATF4 and CHOP were measured by western blot analysis 6, 12, 24 and 72 h after reperfusion. RESULTS: The number of surviving cells was significantly higher in the hypothermia group than in the group without hypothermia (p < .05). The GRP78 expression in the hypothermia group was statistically higher (p < .05) than in the ischaemia/reperfusion group. Optical densities of p-perk, p-eif2α and ATF4 in hippocampus CA1 neurons ischaemia were statistically significantly lower in the hypothermia group than in the ischaemia/reperfusion group (p < .05). The CHOP expression in the hypothermia group was statistically lower (p < .05) than in the ischaemia/reperfusion group. CONCLUSION: Mild hypothermia for 6 h promoted moderate neuroprotection by mediating the expression of GRP78, p-PERK, p-eIF2α, ATF4 and CHOP.

Mild Hypothermia Alleviates Complement C5a-Induced Neuronal Autophagy During Brain Ischemia-Reperfusion Injury After Cardiac Arrest.

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, S100ß, 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.

Study on the protective effect of RNA-binding motif protein 3 in mild hypothermia oxygen-glucose deprivation/reoxygenation cell model.

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.

[A neonatal intelligent regulation system based on the combination of mild hypothermia mattress and hyperbaric oxygen chamber: introduction to a patent]. / 基于亚低温床垫与高压氧舱结合的新生儿智能调控系统.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a common disease that affects brain function in neonates. At present, mild hypothermia and hyperbaric oxygen therapy are the main methods for the treatment of neonatal HIE; however, they are independent of each other and cannot be combined for synchronous treatment, without monitoring of brain function-related physiological information. In addition, parameter setting of hyperbaric oxygen chamber and mild hypothermia mattress relies on the experience of the medical practitioner, and the parameters remain unchanged throughout the medical process. This article proposes a new device for the treatment of neonatal HIE, which has the modules of hyperbaric oxygen chamber and mild hypothermic mattress, so that neonates can receive the treatment of hyperbaric oxygen chamber and/or mild hypothermic mattress based on their conditions. Meanwhile, it can realize the real-time monitoring of various physiological information, including amplitude-integrated electroencephalogram, electrocardiogram, and near-infrared spectrum, which can monitor brain function, heart rate, rhythm, myocardial blood supply, hemoglobin concentration in brain tissue, and blood oxygen saturation. In combination with an intelligent control algorithm, the device can intelligently regulate parameters according to the physiological information of neonates and give recommendations for subsequent treatment.

[Numerical Study on the Process of Human Brain Cooling Treated by Hemoperfusion Mild Hypothermia].

Mild hypothermia, as a common means of intraoperative nerve protection, has been used in clinical practice. Compared with the traditional methods such as freezing helmet and nasopharyngeal cooling, hypothermic blood perfusion is considered to be a promising treatment for mild hypothermia, but it lacks experimental and theoretical verification of its cooling effect. In this study, the commercial finite element simulation software COMSOL combined the Pennes equation with the cerebrovascular network model to construct a new simplified human brain model, which was further used to simulate the cooling process of cerebral hypothermic blood perfusion. When the hypothermic blood perfusion was 33 ℃, the human brain could enter the mild hypothermic state within 4 minutes. By comparing with helmet cooling, the feasibility and efficiency of the blood perfusion scheme were verified. By comparing with the calculation results based on Pennes equation, the rationality of the model constructed in this study were verified. This model can non-intrusively predict the changes of brain temperature during surgery, and provide a reference for the setting of treatment parameters such as blood temperature, so as to provide personalized realization of safer and more effective mild hypothermia neuro protection.

Mild hypothermia ameliorates hepatic ischemia reperfusion injury by inducing RBM3 expression.

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.

Protective Effect of Mild Hypothermia on Spinal Cord Ischemia-Induced Delayed Paralysis and Spinal Cord Injury.

To explore the mechanism regarding the regulation of spinal cord ischemia (SCI) in rats by mild hypothermia. A SCI rat model was established through aorta occlusion, and in some cases, the rats were intervened with mild hypothermia, after which motor function, microglia activation, and M1/M2 polarization in rats were measured. Also, the expression of inflammatory cytokines (IL-1ß, IL-6 and TNF-α) and neuronal apoptosis were examined. Lipopolysaccharide (LPS)-induced M1 microglia and IL-4-induced M2 microglia were intrathecally injected into rats to evaluate the effect of microglial polarization on SCI. In in vitro experiments, primary microglial cells were treated under hypothermic condition, in which M1/M2 polarization and microglia apoptosis, the levels of iNOS, CD86, CD206, Arg-1 and inflammatory cytokines were assessed. Western blot analysis detected the activation of the TLR4/NF-κB pathway to investigate the role of this pathway in M1/M2 polarization. SCI treatment impaired motor function, induced higher M1 microglia proportion, and increased the levels of pro-inflammatory cytokines in rats, and mild hypothermic treatment attenuated these trends. Moreover, injection of M1 microglia increased M1 microglia proportion and increased the levels of pro-inflammatory cytokines, while injection of M2 microglia induced the reverse results, i.e. decreased M1 microglia proportion and reduced pro-inflammatory cytokine levels. In LPS-induced microglial cells, mild hypothermia treatment increased M2 microglia proportion and decreased pro-inflammatory cytokine levels, relative to normothermia. Mild hypothermia inactivated the TLR4/NF-κB pathway in LPS-treated microglia. TLR4 overexpression reversed the function of mild hypothermia in LPS-stimulated microglia, and under normal condition, TLR4/NF-κB pathway suppressed microglial M2 polarization. Mild hypothermia inhibits TLR4/NF-κB pathway and promotes microglial M2 polarization, thus attenuating SCI-induced injury and inflammation.

Mild controlled hypothermia for necrotizing enterocolitis treatment to preterm neonates: low technology technique description and safety analysis.

We performed a quality improvement project to necrotizing enterocolitis (NEC) and published our results about the initiative in 2021. However, aspects on the safety of the cooling and how to do therapeutic hypothermia with low technology to preterm infants are not described in this previous reporter. Thus, we aim to describe the steps and management to apply hypothermia in preterm infants using low technology and present the safety aspects regarding the initiative. We performed a quality improvement project to NEC in a reference hospital for neonatology (intensive care unit). Forty-three preterm infants with NEC (modified Bell's stage II/III) were included: 19 in the control group (2015-2018) and 24 in the hypothermic group (2018-2020). The control group received standard treatments. The hypothermia group received standard treatment and underwent passive cooling (35.5 °C, used for 48 h after NEC diagnosis). We reported cooling safety to NEC, assessing hematological and gasometrical parameters, coagulation disorders, clinical instability, and neurological disorders. We described how to perform cooling to preterm infants using incubators' servo-control and the occurrence and management of dysthermia during the cooling. We turn-off the incubator and used the esophageal probe to monitor the temperature every 15 min; if the temperature dropped, the incubator was turned on with a rewarming speed of 0.5 °C/h. The participants' average weights and gestational ages were 1186 g and 32 weeks, respectively. There were no differences among hematological indices, serum parameters (sodium, potassium, creatinine, lactate, and bicarbonate), pH, pCO2, and pO2/FiO2 between the groups during treatment and after rewarming. We did not observe dysthermia, bradycardia, hemodynamic instability, apnea, seizure, bleeding, peri-intraventricular hemorrhage, or any alterations in ventilatory parameters due to the cooling technique in preterm babies. This simple technique was performed without intercurrences through a rigorous team evaluation, with a target cooling speed of 0.5 °C/h. The target temperature was successfully reached between the second and third hours of life with the incubator control in 21 children; ice bags were used in only three cases. The temperature was maintained at the expected level during the programmed cooling period. CONCLUSION: Mild controlled hypothermia for preterm infants with NEC is safe. The cooling of preterm infants could be performed through passive methods, using the servo-control of the incubators for temperature management. WHAT IS KNOWN: • Mild controlled hypothermia to NEC treatment is feasible and associated with a decrease in NEC surgery, short bowel, and death. • Mild controlled hypothermia to preterm is feasible and can be performed through low technology and passive cooling. WHAT IS NEW: • Mild controlled hypothermia to preterm is safe and does not associate with safety adverse effects during and after the cooling. • Preterm infants can be cooled through passive methods by just using the servo control of the incubator, presenting acceptable temperature variance, without dysthermia, achieving and remaining at the target temperature with a proper cooling speed. Mild controlled temperature for preterm infants does not need an additional cooling device.

Early predictors of brain injury in patients with acute carbon monoxide poisoning and the neuroprotection of mild hypothermia.

INTRODUCTION: Carbon monoxide (CO) poisoning can cause serious neurological sequelae. However, there is neither effective treatment strategy nor reliable indicators to determine the prognosis of patients with CO poisoning. The present study aimed to observe the changes of neurological function score, disease severity score, cerebral oxygen utilization (O2UCc), bispectral (BIS) index and neuron-specific enolase (NSE) concentration, and to elucidate the clinical significance of these potential indicators and the neuroprotective effect of mild hypothermia on brain injury in patients with severe acute CO poisoning. MATERIALS AND METHODS: A total of 277 patients with acute severe CO poisoning from 2013 to 2018 were enrolled in our hospital. Patients were divided into three groups according to their body temperature on the day of admission and their willingness to treat: a fever group (n = 78), a normal temperature group (NT group, n = 113), and a mild hypothermia group (MH group, n = 86). All patients were given hyperbaric oxygen therapy, while those in the MH group received additional mild hypothermia treatment. The severity of the disease, the neurobehavioral status, the incidence of delayed encephalopathy after acute carbon monoxide poisoning (DEACMP), and other indicators including BIS, O2UCc, NSE were further evaluated in all patients at given time-points. RESULTS: Mild hypothermia therapy improved the prognosis of patients with CO poisoning, significantly decreased the value of O2UCc and NSE, and up-regulated BIS. The incidence of DEACMP at 6 months was 27% in the fever group, 23% in the NT group, and 8% in the MH group. The values of Glasgow-Pittsburgh coma scale (G-P score), BIS index and NSE were closely related to the occurrence of DEACMP, the cutoff values were 12.41, 52.17 and 35.20 ng/mL, and the sensitivity and specificity were 79.3%, 77.6%, 79.3% and 67.6%, 89.5%, 88.6% in the receiver operating characteristic curve (ROC), respectively. CONCLUSIONS: Early mild hypothermia treatment could significantly reduce the severity of brain injury after CO poisoning, and might be further popularized in clinic. G-P scores, NSE and BIS index can be regarded as the prediction indicators in the occurrence and development of DEACMP. CLINICAL TRIAL REGISTRATION: The study protocol was granted from Qingdao University Research Ethics Committee (Clinical trial registry and ethical approval number: QD81571283).

The Role of Sphingomyelin Metabolism in the Protection of Rat Brain Microvascular Endothelial Cells by Mild Hypothermia.

BACKGROUND: Sphingomyelin, composed of ceramide (CER), sphingosine (Sph), and sphingosine-1-phosphate (S1P), is an essential structural component of cellular membranes and plays an important role in the signal transduction regulating cell proliferation, differentiation, and apoptosis. CER is mainly metabolized to Sph, and under the action of sphingosine kinases (SphKs), Sph produces S1P, which can be converted back to Sph by S1P phosphatase. It is suggested that the fate of cells is controlled partly by the interconversion of CER and intracellular S1P. SphK2 is considered the main kinase of S1P synthesis in the central nervous system. The objective of this study was to explore the hypothesis that SphK2 and sphingomyelin metabolism participated in the process of cell apoptosis and the protection of mild hypothermia. METHODS: Rat brain microvascular endothelial cells were divided into groups for intervention of SphK2 inhibitor, SphK2 small interfering RNA (SiRNA) transfection, ischemia-reperfusion, and mild hypothermia. After interventions, cell apoptosis was detected by 4,6-diamino-2-phenyl indole (DAPI) and flow cytometry, the expression of apoptosis-related protein was detected by Western Blot, and SphK2 enzyme activity and the content of sphingomyelin were determined. RESULTS: ABC294640 and transfection of SphK2 SiRNA could increase apoptosis, accompanied by the increase of the expression of proapoptotic genes Caspase3 and Bax and the decrease of the expression of BCL-2. This effect could be partially reversed with mild hypothermia. Ischemia-reperfusion injury, transfection of SphK2 SiRNA, and the addition of ABC294640 could significantly inhibit the activity of SphK2, accompanied by the increase of CERs and the decrease of S1P. Mild hypothermia could reverse the changes of sphingolipids but have no significant effect on the activity of sphk2. CONCLUSIONS: Mild hypothermia can inhibit the occurrence of apoptosis and reverse the changes of apoptosis-related genes and sphingomyelin content induced by ischemia-reperfusion injury, but the effect on sphk2 enzyme activity was not significant.

Mild hypothermia in rat with acute myocardial ischaemia-reperfusion injury complicating severe sepsis.

Myocardial ischemia-reperfusion injury (MIRI) with concurrent severe sepsis has led to substantial mortality. Mild hypothermia (MHT) has been proved to have a therapeutic effect in either MIRI or severe sepsis, which suggests it might be beneficial for MIRI complicating severe sepsis. In this study, Sprague-Dawley rats with MIRI complicating severe sepsis were allotted in either MHT (33 ± 0.5°C) group or normothermia (NT, 37 ± 0.5°C) group; as control, rats receiving sham surgery and normal saline were kept at NT. After 2h of temperature maintenance, blood and heart tissue were acquired for detections. Lactate dehydrogenase (LDH) and MB isoenzyme of creatine kinase (CK-MB) in blood, triphenyl tetrazolium chloride and Evans blue staining, hematoxylin and eosin staining for myocardium were employed to detect myocardial damage. Tumor necrosis factor (TNF)-α and caspase-3 was performed by immunohistochemistry to exam myocardial inflammation and apoptosis. Detection of NADPH oxidase (NOX) 2 was for myocardial oxidative stress. In MHT group, systolic blood pressure was improved significantly compared with NT group. Myocardial infarct size, morphological change, LDH and CK-MB levels were attenuated compared to NT group. Moreover, less expressions of TNF-α, caspase-3 and NOX2 in MHT group were presented compared with NT group. MHT showed cardioprotection by improving cardiac dysfunction, reducing myocardial infarct size and attenuating myocardial injury, inflammation, apoptosis and oxidative stress.

Mild hypothermia pretreatment extenuates liver ischemia-reperfusion injury through Rab7-mediated autophagosomes-lysosomes fusion.

Liver ischemia-reperfusion (IR) injury is an unavoidable pathological process in transplantation, closely related to poor prognosis. To date, there has been no clear therapeutic measure. We previously reported that mild hypothermia (MH), a widely used therapy, can exert significant protective effect against liver IR injury. Among the multiple mechanisms underlying the therapeutic effect of MH, autophagy flux drew our special attention. In this study, we evaluated the role of autophagy flux in IR injury and thereby explored the relationship between MH and autophagy flux in IR injury. We developed in vivo and in vitro models for hepatic IR injury. By autophagy flux assay with Western blotting and immunofluorescence, we found that MH restricts heavy accumulation of autophagosomes (APs) during IR injury. Activation and blocking of the autophagy flux unraveled that accumulation of APs further aggravated IR injury. Further, MH reduces APs accumulation to restore autophagy flux by regulating the fusion of APs and lysosomes. Besides, MH upregulated the level of Rab7 protein expression that was seriously impaired during IR injury. Inhibition of Rab7 expression increased apoptosis of liver cells and reduced the degree of overlap between APs and lysosomes. The results were reversed upon activation of Rab7. In conclusion, MH can alleviate liver IR injury by regulating the Rab7-mediated APs-lysosomes fusion that reduces APs accumulation. This can provide a theoretical basis for the further application of MH in related clinical diseases.

Expression regulation of cold-inducible protein RBM3 by FAK/Src signaling for neuroprotection against rotenone under mild hypothermia.

Mild hypothermia is a well-established technique for alleviating neurological injuries in clinical surgery. RNA-binding protein motif 3 (RBM3) has been identified as a crucial factor in mediating hypothermic neuroprotection, providing its induction as a promising strategy for mimicking therapeutic hypothermia. However, little is known about molecular control of RBM3 and signaling pathways affected by hypothermia. In the present study, human SH-SY5Y neuroblastoma cells were used as a neural cell model. Screening of signaling pathways showed that cold exposure led to inactivation of ERK and AMPK pathways, and activation of FAK and PLCγ pathways, with activities of p38, JNK and AKT pathways moderately changed. Next, various small molecule inhibitors specific to these signaling pathways were applied. Interestingly, only FAK-specific inhibitor exhibited a significant inhibitory effect on hypothermia-induced RBM3 gene transcription and protein expression. Likewise, FAK silencing using siRNA technique significantly abrogated the induction of RBM3 by hypothermia. Moreover, FAK inhibition accounted for an inactivation of Src, a known kinase downstream of FAK. Next, either the silencing of Src by siRNA or its inactivation by a chemical inhibitor, strongly blocked the induction of RBM3 by cooling. Notably, in HEK293 and PC12 cells, FAK/Src activation was also shown to be indispensable for hypothermia-stimulated RBM3 expression. Lastly, the CCK8 and Western blot assays showed that both FAK/Src inacitivation and their knockdown substantially abrogate the neuroprotective effects of mild hypothermia against rotenone in SH-SY5Y cells. These data suggest that FAK/Src signaling axis regulates the transcription of Rbm3 gene and mediates neuroprotective effects of mild hypothermia.

Icariin enhance mild hypothermia-induced neuroprotection via inhibiting the activation of NF-κB in experimental ischemic stroke.

Therapeutic hypothermia (TH) is a promising neuroprotective agent for treating stroke. However, its clinical application was limited by the impractical duration. Icariin (ICA) were reported to have therapeutic effect on cerebral ischemia. In this research, our aim was to investigate whether the combination of TH and ICA had better neuroprotective effects on ischemic stroke. An ischemia-reperfusion rat model was established and treated with mild hypothermia, ICA or JSH-23 (inhibitor of NF-κB). Thermistor probe, 2'3'5'-triphenyl tetrazolium chloride (TTC), 5/12-score system, and ELISA were used to detect temperature (rectum, cortex, striatum), infarct volume, neurological deficit, and cerebral cell death of these rats. The expressions of tumor necrosis factor (TNF)-α, Interleukin- 6 (IL-6), nuclear factor-kappa B (NF-κB), nuclear factor erythroid2-related factor (Nrf2), peroxisome proliferator activated receptor gamma (PPARα), PPARγ, Janus kinase 2 (JAK2), p-JAK2, signal transducers and activators of transduction-3 (STAT3), and p-STAT3 were detected by Western blot or q-PCR. Mild hypothermia, ICA, and JSH-23 reduced the cerebral infarct volume, neurological deficit, cerebral cell death of rats, downregulated the expressions of TNF-α, IL-6, C-Caspase 3 and Bax, and the activation of PPARs/Nrf2/NF-κB and JAK2/STAT3 pathways, but elevated the expression of Bcl-2. ICA promoted the effect of mild hypothermia on infarct volume, neurological deficit, and cerebral cell death. Moreover, ICA also enhanced the regulatory effect of mild hypothermia on apoptosis/inflammation factors expressions and activation of PPARs/Nrf2/NF-κB and JAK2/STAT3 pathways. ICA could promote mild hypothermia-induced neuroprotection by inhibiting the activation of NF-κB through the PPARs/Nrf2/NF-κB and JAK2/STAT3/NF-κB pathways in experimental stroke.

Clinical observation of mild hypothermia combined with intravenous thrombolysis in treating patients with acute cerebral infarction.

OBJECTIVES: To investigate the clinical effect of mild hypothermia combined with intravenous thrombolysis in the treatment of acute cerebral infarction. METHODS: Eighty-eight patients with acute cerebral infarction in Binzhou People's Hospital between May 2018 and August 2019 were randomly selected and divided into a control group and an observation group according to the random number table method, with 44 patients in each group. The control group was given intravenous thrombolysis; the observation group was treated with mild hypothermia (30-35°C) in addition to intravenous thrombolytic thrombolysis. The clinical efficacy, incidence of complications, oxidative stress indexes, inflammatory factor level, neurological function, and mental state of the two groups before and after treatment were compared. RESULTS: The clinical efficacy of the observation group was significantly better than that of the control group, and the difference was statistically significant (P<0.05). There was no significant difference in the levels of oxidative stress indexes and inflammatory factors between the two groups before treatment (P<0.05). After treatment, the levels of oxidative stress indexes and inflammatory factors of the two groups significantly improved, and the improvement of the observation group was better than that of the control group; the differences were statistically significant (P<0.05). There was no significant difference in the neurological function and mental state between the two groups before treatment (P<0.05). After treatment, the neurological function and mental state of the two groups significantly improved, and the improvement of the observation group was better than that of the control group; the differences were statistically significant (P<0.05). There was no significant difference in the incidence of complications and mortality between the two groups (P>0.05). CONCLUSION: Thrombolytic therapy combined with mild hypothermia has a good efficacy in the treatment of acute cerebral infarction. The therapy can improve the neurological function of patients with acute cerebral infarction by significantly improving the oxidative stress index and relieving the inflammatory reaction. Its efficacy is better than single thrombolytic therapy.

Transcriptome analysis of mild hypothermia protection against radiation-induced rat lung injury based on RNA-seq. / 基于RNA-SeqæŠ€æœ¯çš„äºšä½Žæ¸©é˜²æŠ¤å¤§é¼ æ”¾å°„æ€§è‚ºæŸä¼¤çš„è½¬å½•ç»„å­¦åˆ†æž.

OBJECTIVES: To analyze the differentially expressed genes (DEGs) with radiation-induced rat lung injury, and to reveal the protective mechanism for mild hypothermia in the radiation-induced lung injury in rats at the transcriptome level. METHODS: A total of 10 male SD rats aged 6-8 weeks were randomly divided into 2 groups to establish a rat model of radiation-induced lung injury, and one group was treated with mild hypothermia. RNA was extracted from left lung tissue of each group, and sequenced by BGISEQ-500 platform. Significance analysis of DEGs was carried out by edgeR software. Gene ontology (GO) function enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were used to analyze the gene function. Then 5 key DEGs were verified by real-time reverse transcription PCR (real-time RT-PCR). RESULTS: There were 2 790 DEGs (false discovery rate<0.001, |log2(fold change)|>1) in the mild hypothermia group compared with the model group, in which 2 257 genes were up-regulated and 533 genes were down-regulated. When real-time RT-PCR was used to validate the 5 key genes, the result was consistent with the RNA-seq. GO functional enrichment analysis showed that these DEGs were related to cell binding, metabolic process and cell membrane structure, etc. KEGG pathway enrichment analysis showed that these genes were involved in important biological pathways such as cell adhesion molecules, mammalian target of rapamycin, tight junction, and NF-κB. CONCLUSIONS: The DEGs and pathways related to mild hypothermia protection against radiation-induced lung injury in rats are obtained, which provides an experimental basis for the protection of mild hypothermia against radiation-induced lung injury.