Application Of Mild Hypothermia Research Articles

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.

The CAG promoter maintains high-level transgene expression in HEK293 cells.

The vast majority of therapeutic recombinant proteins are produced in mammalian cell lines. However, proteins generated in nonhuman cell lines, such as Chinese hamster ovary (CHO) cells, are decorated with human-like glycan structures that differ from those of human cells, and these may induce immunogenic responses in human cells. Human embryonic kidney cells (HEK293F) are also extensively used as hosts for the expression of recombinant therapeutic proteins, but their utility is limited by the low expression of transgenes in these cells. Here, we investigated recombinant protein expression from eight frequently used promoters in transfected HEK293F cells. The expression levels and stability of the transgenes were evaluated by flow cytometry and qRT-PCR. The most efficient expression (in terms of both mRNA and protein yields) was achieved using a cytomegalovirus (CMV) major immediate-early enhancer combined with the chicken beta-actin promoter (CAG) promoter, as compared to all other tested promoters under both transient and stable transfection conditions. In addition, application of mild hypothermia (i.e., 33°C) after transfection improved the positive effect of the CMV enhancer fused to the chicken beta-actin promoter (CAG promoter) on enhanced green fluorescent protein (eGFP) expression. Although the temperature sensitivity of the CMV promoter is greater than that of CAG promoter, recombinant protein levels were still highest when expression was driven by the CAG promoter. When eGFP was replaced with hepatitis B surface antigen, the CAG promoter still showed the highest transgene expression. In conclusion, our data show that the CAG promoter is a strong promoter for recombinant protein expression in HEK293F cells.

Protective effect of mild hypothermia at different starting times on organ function in patient with exertional heat stroke

To investigate the protective effect of mild hypothermia at different starting times on the physiological functions of the viscera of exertional heat stroke (EHS). A prospective randomized controlled trial was conducted. EHS patients admitted to intensive care unit of the 159th Hospital of People's Liberation Army and the First Affiliated Hospital of Zhengzhou University from June 2015 to June 2017 were enrolled. The patients were divided into 2, 4, 6 hours start hypothermia treatment groups according to the random number table method, the mild hypothermia was initiated at 2, 4 and 6 hours after the disease onset respectively, and the methods were the same in each group. After treatment of 2, 12, 24 hours, the venous blood in the three groups was collected to detect serum cardiac troponin I (cTnI) with chemiluminescence method, MB isoenzyme of creatine kinase (CK-MB) with immunosuppressive method, creatinine (Cr) with creatine oxidase method, β2-microglobulin (β2-MG) with turbidimetry, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) with enzyme method. Multiple organ dysfunction syndrome (MODS) within 24 hours after treatment was recorded. Linear regression analysis of the correlation between mild hypothermia start-up time and MODS was done. Ninety-three cases of EHS were included, with 32, 31 and 30 patients in 2, 4, 6 hours start treatment groups respectively. There were no significant differences in gender, age, core temperature, onset time to admission, Glasgow coma scale (GCS), acute physiology and chronic health evaluation system II (APACHE II) score at admission among the three groups. There were no significant differences in the levels of serum cTnI, CK-MB, Cr, β2-MG, ALT and AST at 2 hours after treatment. But with the prolongation of the treatment time, all indicators gradually increased. And the earlier start of the mild hypothermia, the less significant of the above indexes. All indexes in 2 hours start treatment group were significantly lower than those of 2 hours and 6 hours start treatment groups at 24 hours after treatment [cTnI (ng/L): 49.53±9.25 vs. 56.52±10.05, 64.57±11.21; CK-MB (U/L): 51.47±11.83 vs. 57.87±7.43, 64.40±7.93; Cr (μmol/L): 140.97±11.33 vs. 148.16±10.39,155.57±8.65; β2-MG (mg/L): 10.28±1.46 vs. 11.58±2.13, 12.93±1.98; ALT (U/L): 248.53±75.47 vs. 341.42±129.58, 425.77±101.23; AST (U/L): 197.25±42.59 vs. 292.81±58.49, 351.20±60.41, all P < 0.05]. There was significant difference in the incidence of MODS in 2, 4, 6 hours start treatment groups [43.75% (14/32), 64.52% (20/31), 80.08% (24/30), χ2 = 8.761, P = 0.013]. Linear regression analysis showed that the earlier onset time of mild hypothermia, the lower incidence of MODS (R2 = 0.915, P = 0.013). The application of mild hypothermia in 2 hours can effectively protect the physiological function of EHS organs and reduce the incidence of MODS.

Exploring and exploiting unique properties of the hippocampal dentate gyrus for post-stroke therapy: astrocytes link ischemic resistance with neurogenic potential.

Acute cerebral ischemia can occur secondary to embolism, cardiac arrest, hemorrhage, traumatic brain injury, edema, vascular compression, or any physiologic condition resulting in low cardiac output state. Survivors of cerebral ischemic events frequently suffer from profound disability, accounting for > $70 billion in 2010 for treatment of embolic stroke in the US alone. More strikingly, the cost to treat stroke in the US is anticipated to soar to > $180 billion by 2030, according to estimates from the American Heart Association (Ovbiagele et al., 2013). Despite hundreds of promising pre-clinical studies in animal models, effective clinical treatments for the most common forms of cerebral ischemia remain limited: standard treatment for embolic stroke is restricted to thrombolytic therapy or clot retrieval during a narrow therapeutic window, while post-resuscitation treatment following cardiac arrest remains the (controversial) application of mild hypothermia. The massive failure to translate experimental findings to the development of any successful clinical intervention suggests that redirecting investigational resources to developing new therapies to improve long-term recovery in survivors of stroke may be a logical and necessary next step.

Reduction of autophagy: Another potential mechanism of cardioprotective effect of mild hypothermia?

We read with great interest the recent article “Hypothermia may attenuate ischemia/reperfusion-induced cardiomyocyte death by reducing autophagy” by Cheng et al. [1] in the International Journal of Cardiology. The authors demonstrated that application of mild hypothermia could reduce the cell autophagy in H9c2 cardiomyocytes subjected to ischemia/reperfusion (I/R). To the best of our knowledge, autophagy, as one of the forms of programmed cell death, was firstly reported by the authors to be a potential mechanism for the cardioprotection provided by mild hypothermia.

Fundamental research progress of mild hypothermia in cerebral protection

Through the years, the clinical application of mild hypothermia has been carried out worldwide and is built from the exploration and cognition of neuroprotection mechanisms by hypothermia. However, within the last decade, extensive and fundamental researches in this area have been conducted. In addition to aspects of the previous findings, scholars have discovered several new contents and uncertain results. This article reviews and summarizes this decade’s progression of mild hypothermia in lowering the cerebral oxygen metabolism, protecting the blood–brain-barrier, regulating the inflammatory response, regulating the excessive release of neurotransmitters, inhibiting calcium overload, and reducing neuronal apoptosis. In many aspects, particularly in regulating inflammatory reverse reaction, various results have been reported and therefore guide scholars to conduct more detailed analysis and investigation in order to discover the inherent theories surrounding the effect of mild hypothermia, and for better clinical services.

Mild hypothermia during global cardiac ischemia opens a window of opportunity to develop heart donation after cardiac death

Although heart donation after cardiac death (DCD) could greatly improve graft availability, concerns regarding warm ischemic damage typically preclude transplantation. Improving tolerance to warm ischemia may thus open a window of opportunity for DCD hearts. We investigated the hypothesis that, compared with normothermia, mild hypothermia (32°C) initiated after ischemic onset improves cardiac functional recovery upon reperfusion. Isolated, working hearts from adult, male Wistar rats underwent global, no-flow ischemia, and reperfusion (n=28). After ischemic onset, temperature was maintained at either 37°C for 20 or 30min or reduced to 32°C for 40, 50, or 60min. Recovery was measured after 60-min reperfusion. Following normothermic ischemia, recovery of rate-pressure product (RPP; per cent of preischemic value) was almost complete after 20-min ischemia (97±9%), whereas no recovery was detectable after 30-min ischemia. After mildly hypothermic ischemia (32°C), RPP also recovered well after 40min (86±4%). Markers of metabolism and necrosis were similar in 37°C/20min and 32°C/40min groups. Simple reduction in cardiac temperature by a few degrees after the onset of global ischemia dramatically prolongs the interval during which the heart remains resistant to functional deterioration. Preservation of hemodynamic function is associated with improved metabolic recovery and reduced necrosis. The application of mild hypothermia may be a simple first step towards development of clinical protocols for DCD heart recovery.

Mild hypothermia on reperfusion after warm ischemia improves guinea pig isolated heart function

Application of mild hypothermia during the initial critical period of reperfusion after cardiac ischemia may slow mitochondrial and cell metabolism sufficiently to attenuate cardiac damage and better preserve mechanical function. To test this we isolated guinea pig hearts for perfusion at constant pressure with Krebs‐Ringer's solution at pH 7.4. Left ventricular pressure (LVP), coronary flow (CF), and O2 consumption were measured and continuous NADH/FAD and mitochondrial (m) [Ca2+] were assessed in the LV wall by fluorescence spectrophotometry in the absence or presence of indo 1 + MnCl2 to quench cell Ca2+. Hearts were subjected to 30 min ischemia at 37ºC, and then reperfused at either 32ºC or 37ºC for 60 min followed by additional 60 min reperfusion at 37ºC. Values up to reperfusion were similar in both groups. Developed LVP and m[Ca2+] were both lower at 32ºC vs. 37ºC. At the end of reperfusion the 32ºC group showed improved developed LVP (62 % of baseline) when compared to the non‐treatment group (50 %). There were no noticeable improvements in CF, NADH, FAD, and mCa2+ loading during later perfusion. Lower contractility and m[Ca2+] by hypothermia during the critical early reperfusion period may protect the heart during later warm reperfusion.

Effect of therapeutic mild hypothermia initiated by cold fluids after cardiac arrest on haemostasis as measured by thrombelastography

Application of mild hypothermia (32–33°C) has been shown to improve neurological outcome in patients with cardiac arrest, and is therefore a class IIa recommendation in the treatment of those patients. However, hypothermia affects haemostasis, and even mild hypothermia has been found to be associated with bleeding and increased transfusion requirements in patients undergoing surgery [1]. On the other hand, crystalloid hemodilution has been shown to enhance the coagulation onset [2]. Currently, it is unknown in which way the induction of mild therapeutic hypothermia by a bolus infusion of cold crystalloids affects the coagulation system of patients with cardiac arrest.

Anti‐inflammatory effects of sevoflurane and mild hypothermia in endotoxemic rats

Volatile anesthetics and hypothermia attenuate the inflammatory response. We aimed to compare the anti-inflammatory effects of sevoflurane and mild hypothermia during experimental endotoxemia in the rat. Anesthetized, ventilated Sprague-Dawley (SD) rats were randomly treated as follows (n = 6 per group): lipopolysaccharide (LPS) only, animals received LPS [LPS 5 mg/kg, intravenously (i.v.)] with no further treatment. In the LPS-hypothermia group, rats were cooled down to a temperature of 33 degrees C 15 min after LPS-injection (LPS 5 mg/kg i.v.). In animals of the LPS-sevoflurane group, sevoflurane inhalation (1 MAC) was initiated 15 min after induction of endotoxemia. The LPS-sevoflurane-hypothermia group received combined sevoflurane and hypothermia 15 min after induction of endotoxemia. A Sham group served as control without endotoxemia or treatment. After 4 h of endotoxemia, plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-10 were measured. Alveolar macrophages (AM) were ex vivo cultured for nitrite assay. Inhalation of sevoflurane significantly attenuated plasma levels of TNF-alpha (-60%, P < 0.05) and IL-1beta (-68%, P < 0.05) as compared with the LPS-only group. Hypothermia and its combination with sevoflurane significantly reduced TNF-alpha levels (-46% and -58%, each P < 0.05), but not IL-1beta. Application of mild hypothermia and also its combination with sevoflurane resulted in a significant increase in plasma IL-10 as compared with endotoxemic controls. Nitrite release from AM was found to be significantly suppressed by sevoflurane (-83%), hypothermia (-73%) and by the combination of both (-67%) (P < 0.05, each). Our data suggest that sevoflurane and mild hypothermia attenuate the inflammatory response during endotoxemia in vivo thus contributing to their beneficial role in clinical organ protection.

Decompressive craniectomy and mild hypothermia reduces infarction size and counterregulates Bax and Bcl-2 expression after permanent focal ischemia in rats

Both mild hypothermia (MH) and decompressive craniectomy (CE) have been shown to have neuroprotective effects in brain ischemia. We investigated a possible effect of MH and a combination of CE and MH (CE + MH) on the changes of infarction size, DNA fragmentation, and immunoreactivities for Bcl-2 and Bax after 24 h of permanent middle cerebral artery occlusion (MCAO) in rats. For the estimation of ischemic brain injury, we calculated the infarct size of the MCA region at 24 h after the MCAO. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick labeling (TUNEL) staining was performed for the detection of DNA fragmentation. Immunoreactivities for Bcl-2 and Bax were stained. Infarction size after permanent MCAO was significantly reduced by CE+MH treatment (P < 0.01). Infarction size did not change significantly by application of MH alone (P > 0.05). TUNEL staining was remarkably reduced both in MH-treated animals and in CE + MH-treated animals. Immunoreactivity for Bcl-2 was greatly induced both in MH-treated animals and in CE + MH-treated animals. Induction of immunoreactivity for Bcl-2 was obviously inhibited both in MH-treated animals and in CE + MH-treated animals. It suggests that temporary MH delays infarct evolution and ameliorates neuron apoptosis but does not significantly reduce definite infarction size. CE + MH not only ameliorates neuron apoptosis but also remarkably reduces infarction size.

Mild hypothermia reduces expression of intercellular adhesion molecule‐1 (ICAM‐1) and the accumulation of neutrophils after acid‐induced lung injury in the rat

The pathophysiology of the acute phase of acid-induced lung injury (AILI) has been elucidated. However, once acute respiratory distress syndrome (ARDS) develops, the mortality rate remains high and there is, as yet, no effective therapy. There are reports that application of mild hypothermia is an effective treatment for ARDS. In this study, we hypothesize that mild hypothermia inhibits activation of neutrophils and expression of intercellular adhesion molecule-1 (ICAM-1) in an injured lung. We studied the effects of mild hypothermia on the expression of ICAM-1 and the accumulation of neutrophils after AILI in the rat. Male Sprague-Dawley rats were randomly allocated to one of the four groups: control normothermic group, induced mild hypothermia group, acid-instilled normothermic group, and acid-instilled group with mild hypothermia. At 6 h after instillation of acid, lungs were removed to measure neutrophil activity and to detect the expression of ICAM-1 in each group. Oxygenation in acid-instilled rats was significantly impaired as compared to that in non-instilled groups, but induction of mild hypothermia gradually improved oxygenation. Expression of ICAM-1 was enhanced in the acid-instilled normothermic group. By contrast, no overexpression of ICAM-1 and its mRNA was detected in the acid-instilled hypothermic group. In addition, accumulation of neutrophils was markedly inhibited after exposure to mild hypothermia irrespective of the instillation of acid. Our data suggest mild hypothermia can inhibit the adhesion, activation, and accumulation of neutrophils during the acute phase of AILI in the rat and may have the potential to reduce ongoing inflammation of ALI or ARDS.

Differential temperature sensitivity of ischemia-induced glutamate release and eicosanoid production in rats

The effect of mild and moderate hypothermia on ischemia-induced glutamate release and eicosanoid production was evaluated in WKY rats subjected to incomplete forebrain ischemia. Under isoflurane anesthesia, microdialysis probes were inserted into the hippocampus and caudate nucleus. In four groups of rats, the intraischemic temperature was maintained at either 38°C (normothermia), 36°C, 34°C (mild hypothermia) and 30°C (moderate hypothermia). In these groups, normothermia was restored immediately upon reperfusion. In two additional groups, both intra- and post-ischemic temperatures were maintained at either 34°C or 30°C. The levels of glutamate were measured in the dialysate collected during ischemia and the levels of TxB 2, 6-keto-PGF 1α and PGF 2α were measured in dialysate collected prior to and after ischemia. As expected, hypothermia reduced ischemia-induced glutamate release in both structures. However, the application of mild hypothermia did not attenuate post-ischemic levels of all eicosanoids measured. Moderate hyothermia (30°C) attenuated the post-ischemic increase in the levels of PGF 2α. The data suggest that the processes that lead to eicosanoid formation are less sensitive to temperature reduction than those that lead to glutamate release.