Biofilm exopolysaccharides alter sensory-neuron-mediated sickness during lung infection.

Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.

Hypothermic Protection in Neocortex Is Topographic and Laminar, Seizure Unmitigating, and Partially Rescues Neurons Depleted of RNA Splicing Protein Rbfox3/NeuN in Neonatal Hypoxic-Ischemic Male Piglets.

The effects of hypothermia on neonatal encephalopathy may vary topographically and cytopathologically in the neocortex with manifestations potentially influenced by seizures that alter the severity, distribution, and type of neuropathology. We developed a neonatal piglet survival model of hypoxic-ischemic (HI) encephalopathy and hypothermia (HT) with continuous electroencephalography (cEEG) for seizures. Neonatal male piglets received HI-normothermia (NT), HI-HT, sham-NT, or sham-HT treatments. Randomized unmedicated sham and HI piglets underwent cEEG during recovery. Survival was 2-7 days. Normal and pathological neurons were counted in different neocortical areas, identified by cytoarchitecture and connectomics, using hematoxylin and eosin staining and immunohistochemistry for RNA-binding FOX-1 homolog 3 (Rbfox3/NeuN). Seizure burden was determined. HI-NT piglets had a reduced normal/total neuron ratio and increased ischemic-necrotic/total neuron ratio relative to sham-NT and sham-HT piglets with differing severities in the anterior and posterior motor, somatosensory, and frontal cortices. Neocortical neuropathology was attenuated by HT. HT protection was prominent in layer III of the inferior parietal cortex. Rbfox3 immunoreactivity distinguished cortical neurons as: Rbfox3-positive/normal, Rbfox3-positive/ischemic-necrotic, and Rbfox3-depleted. HI piglets had an increased Rbfox3-depleted/total neuron ratio in layers II and III compared to sham-NT piglets. Neuronal Rbfox3 depletion was partly rescued by HT. Seizure burdens in HI-NT and HI-HT piglets were similar. We conclude that the neonatal HI piglet neocortex has: (1) suprasylvian vulnerability to HI and seizures; (2) a limited neuronal cytopathological repertoire in functionally different regions that engages protective mechanisms with HT; (3) higher seizure burden, insensitive to HT, that is correlated with more panlaminar ischemic-necrotic neurons in the somatosensory cortex; and (4) pathological RNA splicing protein nuclear depletion that is sensitive to HT. This work demonstrates that HT protection of the neocortex in neonatal HI is topographic and laminar, seizure unmitigating, and restores neuronal depletion of RNA splicing factor.

Neutrophil extracellular traps formation and clearance is enhanced in fever and attenuated in hypothermia.

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.

Administration of selective brain hypothermia using a simple cooling device in neonatal rats.

BACKGROUND: The interruption of oxygen and blood supply to the newborn brain around the time of birth is a risk factor for hypoxic-ischemic encephalopathy and may lead to infant mortality or lifelong neurological impairments. Currently, therapeutic hypothermia, the cooling of the infant's head or entire body, is the only treatment to curb the extent of brain damage. NEW METHOD: In this study, we designed a focal brain cooling device that circulates cooled water at a steady state temperature of 19 ± 1 °C through a coil of tubing fitted onto the neonatal rat's head. We tested its ability to selectively decrease brain temperature and offer neuroprotection in a neonatal rat model of hypoxic-ischemic brain injury. RESULTS: Our method cooled the brain to 30-33 °C in conscious pups, while keeping the core body temperature approximately 3.2 °C warmer. Furthermore, the application of the cooling device to the neonatal rat model demonstrated a reduction in brain volume loss compared to pups maintained at normothermia and achieved a level of brain tissue protection the same as that of whole-body cooling. COMPARISON WITH EXISTING METHODS: Prevailing methods of selective brain hypothermia are designed for adult animal models rather than for immature animals such as the rat as a conventional model of developmental brain pathology. Contrary to existing methods, our method of cooling does not require surgical manipulation or anaesthesia. CONCLUSION: Our simple, economical, and effective method of selective brain cooling is a useful tool for rodent studies in neonatal brain injury and adaptive therapeutic interventions.

Mild Hypothermia via External Cooling Improves Lung Function and Alleviates Pulmonary Inflammatory Response and Damage in Two-Hit Rabbit Model of Acute Lung Injury.

OBJECTIVES: Targeted temperature management (TTM) with therapeutic hypothermia (TH) has an organ-protective effect by mainly reducing inflammatory response. Here, our objective was to determine, for the first time, whether mild TH with external cooling, a simple and inexpensive method, could be safe or even beneficial in two-hit rabbit model of acute lung injury/acute respiratory distress syndrome (ALI/ARDS). METHODS: Twenty-two New Zealand rabbits (6-month-old) were randomly divided into healthy control (HC) with conventional ventilation, but without injury, model group (ALI), and hypothermia group with external cooling (ALI-HT). After induction of ALI/ARDS through mild lung-lavages followed by non-protective ventilation, mild hypothermia was started in ALI-HT group (body temperature of 33-34 °C). All rabbits were conventionally ventilated for an additional 6-h by recording respiratory parameters. Finally, lung histopathology and inflammatory response were evaluated. RESULTS: Hypothermia was associated with higher oxygen saturation, resulting in partial improvement in the P/F ratio (PaO2/FiO2), oxygenation index, mean airway pressure, and PaCO2, but did not affect lactate levels. The ALI-HT group had lower histopathological injury scores (hyperemia, edema, emphysema, atelectasis, and PMN infiltration). Further, tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and -8 levels in lung tissue and serum samples markedly reduced due to hypothermia. CONCLUSION: Mild TH with external cooling reduced lung inflammation and damage, whereas it resulted in partial improvement in gas exchanges. Our findings highlight that body temperature control may be a potentially supportive therapeutic option for regulating cytokine production and respiratory parameters in ALI/ARDS.

Coronavirus Disease 2019 Temperature Trajectories Correlate With Hyperinflammatory and Hypercoagulable Subphenotypes.

OBJECTIVES: Body temperature trajectories of infected patients are associated with specific immune profiles and survival. We determined the association between temperature trajectories and distinct manifestations of coronavirus disease 2019. DESIGN: Retrospective observational study. SETTING: Four hospitals within an academic healthcare system from March 2020 to February 2021. PATIENTS: All adult patients hospitalized with coronavirus disease 2019. INTERVENTIONS: Using a validated group-based trajectory model, we classified patients into four previously defined temperature trajectory subphenotypes using oral temperature measurements from the first 72 hours of hospitalization. Clinical characteristics, biomarkers, and outcomes were compared between subphenotypes. MEASUREMENTS AND MAIN RESULTS: The 5,903 hospitalized coronavirus disease 2019 patients were classified into four subphenotypes: hyperthermic slow resolvers (n = 1,452, 25%), hyperthermic fast resolvers (1,469, 25%), normothermics (2,126, 36%), and hypothermics (856, 15%). Hypothermics had abnormal coagulation markers, with the highest d-dimer and fibrin monomers (p < 0.001) and the highest prevalence of cerebrovascular accidents (10%, p = 0.001). The prevalence of venous thromboembolism was significantly different between subphenotypes (p = 0.005), with the highest rate in hypothermics (8.5%) and lowest in hyperthermic slow resolvers (5.1%). Hyperthermic slow resolvers had abnormal inflammatory markers, with the highest C-reactive protein, ferritin, and interleukin-6 (p < 0.001). Hyperthermic slow resolvers had increased odds of mechanical ventilation, vasopressors, and 30-day inpatient mortality (odds ratio, 1.58; 95% CI, 1.13-2.19) compared with hyperthermic fast resolvers. Over the course of the pandemic, we observed a drastic decrease in the prevalence of hyperthermic slow resolvers, from representing 53% of admissions in March 2020 to less than 15% by 2021. We found that dexamethasone use was associated with significant reduction in probability of hyperthermic slow resolvers membership (27% reduction; 95% CI, 23-31%; p < 0.001). CONCLUSIONS: Hypothermics had abnormal coagulation markers, suggesting a hypercoagulable subphenotype. Hyperthermic slow resolvers had elevated inflammatory markers and the highest odds of mortality, suggesting a hyperinflammatory subphenotype. Future work should investigate whether temperature subphenotypes benefit from targeted antithrombotic and anti-inflammatory strategies.

Hypothermia is not therapeutic in a neonatal piglet model of inflammation-sensitized hypoxia-ischemia.

BACKGROUND: Perinatal inflammation combined with hypoxia-ischemia (HI) exacerbates injury in the developing brain. Therapeutic hypothermia (HT) is standard care for neonatal encephalopathy; however, its benefit in inflammation-sensitized HI (IS-HI) is unknown. METHODS: Twelve newborn piglets received a 2 µg/kg bolus and 1 µg/kg/h infusion over 52 h of Escherichia coli lipopolysaccharide (LPS). HI was induced 4 h after LPS bolus. After HI, piglets were randomized to HT (33.5 °C 1-25 h after HI, n = 6) or normothermia (NT, n = 6). Amplitude-integrated electroencephalogram (aEEG) was recorded and magnetic resonance spectroscopy (MRS) was acquired at 24 and 48 h. At 48 h, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive brain cell death, microglial activation/proliferation, astrogliosis, and cleaved caspase-3 (CC3) were quantified. Hematology and plasma cytokines were serially measured. RESULTS: Two HT piglets died. aEEG recovery, thalamic and white matter MRS lactate/N-acetylaspartate, and TUNEL-positive cell death were similar between groups. HT increased microglial activation in the caudate, but had no other effect on glial activation/proliferation. HT reduced CC3 overall. HT suppressed platelet count and attenuated leukocytosis. Cytokine profile was unchanged by HT. CONCLUSIONS: We did not observe protection with HT in this piglet IS-HI model based on aEEG, MRS, and immunohistochemistry. Immunosuppressive effects of HT and countering neuroinflammation by LPS may contribute to the observed lack of HT efficacy. Other immunomodulatory strategies may be more effective in IS-HI. IMPACT: Acute infection/inflammation is known to exacerbate perinatal brain injury and can worsen the outcomes in neonatal encephalopathy. Therapeutic HT is the current standard of care for all infants with NE, but the benefit in infants with coinfection/inflammation is unknown. In a piglet model of inflammation (LPS)-sensitized HI, we observed no evidence of neuroprotection with cooling for 24 h, based on our primary outcome measures: aEEG, MRS Lac/NAA, and histological brain cell death. Additional neuroprotective agents, with beneficial immunomodulatory effects, require exploration in IS-HI models.

The impact of therapeutic hypothermia on peripheral blood cell in newborns with hypoxic ischemic encephalopathy

Abstract The effect of hypothermia treatment on white blood cell (WBC), neutrophil-to-lymphocyte ratio (NLR), lymphocyte-to-monocyte ratio (LMR) and platelet-to-lymphocyte ratio (PLR) values as an indicator of inflammation was evaluated in newborns with hypoxic ischemic encephalopathy (HIE). The study was performed that the before-therapeutic hypothermia (TH) and after-TH WBC, lymphocytes, neutrophils, monocytes and NLR, LMR and PLR values of the complete blood cell count were retrospectively evaluated. The results of the patient group were compared with the results of healthy newborns. A total of 78 patients who underwent TH were evaluated in our study. Mean values before and after TH were NLR3.8/2.7, LMR 5.6/8.6, and PLR 60.3/67.1 respectively. A statistical significance was present for NLR values before and after TH in those with seizure in our study (4.15±2.95/3.01±2.54) but no statistical significance was found for LMR or PLR. In neonates with HIE, effect of TH on complete blood cell count and inflammatory mechanisms (mediated neutrophil and lymphocyte) may be minimal.

Death following extreme temperature exposure: Histological, biochemical and immunohistochemical markers.

INTRODUCTION: Defining extreme temperatures as the cause of death remains challenging. It is mostly based on circumstantial, macroscopic and microscopic features. METHODS: We retrospectively compared groups of cases of fatal hypothermia, fatal hyperthermia and non-extreme temperature-related deaths. We analysed specific histological findings, focusing on samples from the liver, pancreas and kidney. RESULTS: Between 1 January 2013 and 31 December 2016, 15 autopsies were performed for deaths related to extreme temperatures. They included 11 cases of fatal hypothermia (group A), four cases of fatal hyperthermia (group B) and eight controls (group C). Perinuclear hepatocyte vacuolisation was observed in seven cases of hypothermia, one case of hyperthermia and four controls. Pancreatic cytoarchitecture was well preserved in two cases of hypothermia, one case of hyperthermia and two controls. No particular microscopic feature was found in pancreatic samples. Renal epithelial tubular cell vacuolisation was observed in seven cases of hypothermia and one case of hyperthermia, while it was absent in all controls. Chromogranin A (CgA) was markedly positive in the pancreatic tissue of five cases of fatal hypothermia and one control, and mildly positive in one case of fatal hyperthermia. No significant p-values were observed for any comparisons (p > 0.05), except when hypothermia cases group were compared to the control group for the Armanni-Ebstein phenomenon test (p = 0.0078). CONCLUSIONS: Although our study did not find a specific microscopic marker, hepatocyte vacuolisation, the Armanni-Ebstein phenomenon and pancreatic CgA positivity, taken together, may be useful tools to confirm hypo- and hyperthermia-related deaths, in addition to circumstantial and macroscopic findings.

Hypothermia Attenuates Neuronal Damage via Inhibition of Microglial Activation, Including Suppression of Microglial Cytokine Production and Phagocytosis.

Although therapeutic hypothermia (TH) provides neuroprotection, the cellular mechanism underlying the neuroprotective effect of TH has not yet been fully elucidated. In the present study, we investigated the effect of TH on microglial activation to determine whether hypothermia attenuates neuronal damage via microglial activation. After lipopolysaccharide (LPS) stimulation, BV-2 microglia cells were cultured under normothermic (37 °C) or hypothermic (33.5 °C) conditions. Under hypothermic conditions, expression of pro-inflammatory cytokines and inducible nitric oxide synthase (iNOS) was suppressed. In addition, phagocytosis of latex beads was significantly suppressed in BV-2 cells under hypothermic conditions. Moreover, nuclear factor-κB signaling was inhibited under hypothermic conditions. Finally, neuronal damage was attenuated following LPS stimulation in neurons co-cultured with BV-2 cells under hypothermic conditions. In conclusion, hypothermia attenuates neuronal damage via inhibition of microglial activation, including microglial iNOS and pro-inflammatory cytokine expression and phagocytic activity. Investigating the mechanism of microglial activation regulation under hypothermic conditions could contribute to the development of novel neuroprotective therapies.

Acacetin suppresses the electrocardiographic and arrhythmic manifestations of the J wave syndromes.

BACKGROUND: J wave syndromes (JWS), including Brugada (BrS) and early repolarization syndromes (ERS), are associated with increased risk for life-threatening ventricular arrhythmias. Pharmacologic approaches to therapy are currently very limited. Here, we evaluate the effects of the natural flavone acacetin. METHODS: The effects of acacetin on action potential (AP) morphology and transient outward current (Ito) were first studied in isolated canine RV epicardial myocytes using whole-cell patch clamp techniques. Acacetin's effects on transmembrane APs, unipolar electrograms and transmural ECGs were then studied in isolated coronary-perfused canine RV and LV wedge preparations as well as in whole-heart, Langendorff-perfused preparations from which we recorded a 12 lead ECG and unipolar electrograms. Using floating glass microelectrodes we also recorded transmembrane APs from the RVOT of the whole-heart model. The Ito agonist NS5806, sodium channel blocker ajmaline, calcium channel blocker verapamil or hypothermia (32°C) were used to pharmacologically mimic the genetic defects and conditions associated with JWS, thus eliciting prominent J waves and provoking VT/VF. RESULTS: Acacetin (5-10 µM) reduced Ito density, AP notch and J wave area and totally suppressed the electrocardiographic and arrhythmic manifestation of both BrS and ERS, regardless of the experimental model used. In wedge and whole-heart models of JWS, increasing Ito with NS5806, decreasing INa or ICa (with ajmaline or verapamil) or hypothermia all resulted in accentuation of epicardial AP notch and ECG J waves, resulting in characteristic BrS and ERS phenotypes. Phase 2-reentrant extrasystoles originating from the RVOT triggered VT/VF. The J waves in leads V1 and V2 were never associated with a delay of RVOT activation and always coincided with the appearance of the AP notch recorded from RVOT epicardium. All repolarization defects giving rise to VT/VF in the BrS and ERS models were reversed by acacetin, resulting in total suppression of VT/VF. CONCLUSIONS: We present experimental models of BrS and ERS capable of recapitulating all of the ECG and arrhythmic manifestations of the JWS. Our findings provide definitive support for the repolarization but not the depolarization hypothesis proposed to underlie BrS and point to acacetin as a promising new pharmacologic treatment for JWS.

Hypothermia Advocates Functional Mitochondria and Alleviates Oxidative Stress to Combat Acetaminophen-Induced Hepatotoxicity.

For years, moderate hypothermia (32 °C) has been proposed as an unorthodox therapy for liver injuries, with proven hepatoprotective potential. Yet, limited mechanistic understanding has largely denied its acceptance over conventional pharmaceuticals for hepatoprotection. Today, facing a high prevalence of acetaminophen-induced liver injury (AILI) which accounts for the highest incidence of acute liver failure, hypothermia was evaluated as a potential therapy to combat AILI. For which, transforming growth factor-α transgenic mouse hepatocytes (TAMH) were subjected to concomitant 5 mM acetaminophen toxicity and moderate hypothermic conditioning for 24 h. Thereafter, its impact on mitophagy, mitochondrial biogenesis, glutathione homeostasis and c-Jun N-terminal kinase (JNK) signaling pathways were investigated. In the presence of AILI, hypothermia displayed simultaneous mitophagy and mitochondrial biogenesis to conserve functional mitochondria. Furthermore, antioxidant response was apparent with higher glutathione recycling and repressed JNK activation. These effects were, however, unremarkable with hypothermia alone without liver injury. This may suggest an adaptive response of hypothermia only to the injured sites, rendering it favorable as a potential targeted therapy. In fact, its cytoprotective effects were displayed in other DILI of similar pathology as acetaminophen i.e., valproate- and diclofenac-induced liver injury and this further corroborates the mechanistic findings of hypothermic actions on AILI.

Lethal hypothermia due to impalement.

Impalement injuries in the region of large blood vessels can lead to extensive and even lethal blood loss. However, they can also lead to forced positions from which the affected persons cannot free themselves. This 85-year-old woman was found dead in a prone position in her garden. A metal bar had penetrated deeply into the front of her right thigh, while the other end of the bar was stuck firmly in the soil. The metal bar had merely displaced the woman's muscles and the larger blood vessels without causing major blood loss. There were typical findings of lethal hypothermia, including Wischnewski spots of the gastric mucosa and frost erythema on both knees and the left lower leg. The fall onto the metal bar caused an impalement injury leading to a forced position from which the woman could not free herself where she finally succumbed to lethal hypothermia.

Respiratory Characteristics of Rat Liver Mitochondria Depend on the Duration of Moderate Hypothermia.

The development of pathological or compensatory-and-adaptive reactions in homoeothermic animals during various periods of hypothermia can be caused by shifts in the respiratory functions of the mitochondria. Short-term hypothermia promoted an increase in the rates of the glutamate- and succinate-dependent respiration of mitochondria. Phosphorylation rate increased as well, while oxidative phosphorylation coefficient (P/O), respiratory control, and 2,4-DNP sensitivity depended. Changes in respiratory characteristics in the dynamics of prolonged hypothermia depends on the type of substrate. Prolongation of hypothermia to 1 h was associated with further intensification of succinate-dependent respiration, decrease in P/O and respiratory control, while prolongation of hypothermia to 3 h led to their normalization. The majority of respiratory characteristics of glutamate-dependent respiration did not change under these conditions and their levels were the same as during short-term hypothermia.

Low myocardial transcript variant alt-a of cyclin dependent kinase inhibitor p21 expression differentiates hypothermia from cardiac/respiratory causes of death.

Gene expressions in the myocardium have been shown to vary between different causes of death, which can be utilized in the recognition of varied processes. Our previous work with a limited number of cases showed a high messenger ribonucleic acid expression of the transcript variant alt-a of cyclin dependent kinase inhibitor p21 (p21 alt-a) in chronic cardiac ischemia deaths and a low expression in hypothermia deaths and acute myocardial ischemia deaths. In present work, p21 alt-a expression in the myocardium of human cadavers was calculated using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference gene. In this collection of 143 samples, the p21 alt-a expression was significantly lower in hypothermia than in chronic cardiac ischemic heart disease with (P < .001) or without (P < .001) acute myocardial infarction and in other cardiac and respiratory disease deaths (P < .000). Chronic ischemic heart disease in hypothermia cases did not increase the expression. The p21 alt-a expression did not correlate with postmortem interval, quality of RNA or with the age of the deceased. The p21 alt-a referenced to GAPDH expression in cadaver myocardium has apparent potential as a marker distinguishing between hypothermia and cardiac/respiratory diseases as causes of death.

Molecular mechanisms of Wischnewski spot development on gastric mucosa in fatal hypothermia: an experimental study in rats.

Numerous dark-brown-coloured small spots called "Wischnewski spots" are often observed in the gastric mucosa in the patients dying of hypothermia, but the molecular mechanisms through which they develop remain unclear. We hypothesised that hypothermia may activate the secretion of gastric acid and pepsin, leading to the development of the spots. To investigate this, we performed experiments using organotypic rat gastric tissue slices cultured at 37 °C (control) or 32 °C (cold). Cold loading for 6 h lowered the extracellular pH in the culture medium. The mRNA expression of gastrin, which regulates gastric acid secretion, increased after cold loading for 3 h. Cold loading increased the expression of gastric H+,K+-ATPase pump protein in the apical canalicular membrane and resulted in dynamic morphological changes in parietal cells. Cold loading resulted in an increased abundance of pepsin C protein and an elevated mRNA expression of its precursor progastricsin. Collectively, our findings clarified that cold stress induces acidification by activating gastric H+,K+-ATPase pumps and promoting pepsin C release through inducing progastricsin expression on the gastric mucosa, leading to tiny haemorrhages or erosions of the gastric mucosa that manifest as Wischnewski spots in fatal hypothermia.

Preconditioning hippocampal slices with hypothermia promotes rapid tolerance to hypoxic depolarization and swelling: Mediation by erythropoietin.

We suggested previously that hippocampal slices were protected from hypoxic depolarization and swelling by preincubating them at room temperature (Kreisman et al., 2000). We postulated that hypothermic preconditioning induced tolerance in our slices, which protected against hypoxic depolarization and swelling. Control hippocampal slices were incubated at 34-35 °C for two hours and the response to 10 min of severe hypoxia was compared to slices which were preconditioned for two hours at room temperature (22-23 °C) prior to warming to 34-35 °C. Recordings of the extracellular DC potential provided an index of tissue depolarization and changes in tissue light transmittance provided an index of swelling. Hypothermic preconditioning significantly reduced hypoxia-induced swelling, particularly in CA3 and the dentate inner blade. Since erythropoietin (EPO) had been shown to mediate hypoxic preconditioning, we tested whether EPO also mediated hypothermic preconditioning in our slices. Recombinant rat EPO (1-10 micromolar) mitigated hypoxia-induced swelling and depolarization in dentate inner blade of unconditioned slices in a dose-dependent manner. We also blocked the protective effects of hypothermic preconditioning on hypoxic depolarization and swelling in the inner blade of the dentate gyrus by administering soluble EPO receptor in the bath and treating slices with wortmannin to block phosphorylation of PI3 kinase, a critical step in the activation of the downstream neuroprotectant, Akt. These results suggest that EPO mediates tolerance to hypoxic depolarization and swelling induced by hypothermic preconditioning. They also emphasize that various preincubation protocols used in experiments with hippocampal slices may differentially affect basal electrophysiological and metabolic properties of those slices.

Expression of Apoptosis Regulator Proteins Bcl-2 and Bad in Rat Ovarian Follicular Apparatus during Recovery after Extreme Hypothermia.

The expression of molecular and cellular regulators of apoptosis (proapoptotic protein Bad and antiapoptotic protein Bcl-2) was measured in the follicular apparatus of rat ovaries during the recovery period (days 7 and 14) after hyperthermia (up to rectal temperature 43.5°C). The Bcl-2/Bad index was calculated. The expression of Bcl-2 in the follicular apparatus of rat ovaries increased on day 7 after the exposure. The Bcl-2/Bad index also increased, which suggests that the development of apoptosis by the mitochondrial pathway in follicles was limited at this term after hyperthermia. On day 14 after hyperthermia, the area of immunohistochemical staining for the antiapoptotic protein Bcl-2 significantly decreased in cells of the ovarian follicular epithelium, but the expression of the proapoptotic protein Bad significantly increased; these changes led to a decrease in Bcl-2/Bad index, which attested to weakening of the antiapoptotic defense and activation of oocyte apoptosis by the mitochondrial pathway.

Management of accidental hypothermia: an established extracorporeal membrane oxygenation centre experience.

INTRODUCTION: Data on management of severe accidental hypothermia published from an established high-volume extracorporeal membrane oxygenation centre are scarce. METHODS: A total of 28 patients with intravesical temperature lower than 28°C on admission were either treated with veno-arterial extracorporeal membrane oxygenation or rewarmed conservatively. RESULTS: A total of 10 patients rewarmed on veno-arterial extracorporeal membrane oxygenation (age: 37 ± 12.6 years) and 18 conservatively (age: 55.2 ± 11.2 years) were collected over a course of 5 years. The dominant cause was alcohol intoxication with exposure to cold (39%), 12 patients were resuscitated prior to admission. The admission temperature in the extracorporeal membrane oxygenation group (23.8 ± 2.6°C) was lower than in the non-extracorporeal membrane oxygenation group (26.0 ± 1.5°C, p = 0.01). The peripheral percutaneous veno-arterial extracorporeal membrane oxygenation was always cannulated in malignant arrhythmias causing refractory cardiac arrest. The typical extracorporeal membrane oxygenation blood flow was 3-4 L/minute and sweep gas flow 2 L/minute, the median extracorporeal membrane oxygenation duration was 48.3 (28.1-86.7) hours. The median rates of rewarming did not differ (0.41 (0.35-0.7)°C/hour in extracorporeal membrane oxygenation and 0.77 (0.54-0.98)°C/hour in non-extracorporeal membrane oxygenation, p = 0.46) as well as the admission arterial lactate, pH and potassium. Their development was not different between the groups except for higher pH between the third and ninth hour of rewarming in the extracorporeal membrane oxygenation group. The hospital mortality was 10% in the extracorporeal membrane oxygenation group and 11.1% in the non-extracorporeal membrane oxygenation group with the median last Glasgow Coma Scale 15 and Cerebral Performance Score 1. CONCLUSION: Veno-arterial extracorporeal membrane oxygenation for severe hypothermia shows promising outcome data collected in an extracorporeal membrane oxygenation/extracorporeal cardiopulmonary resuscitation centre located in a European urban area. Except for presence of refractory cardiac arrest, the established hypothermia-related prognostic indicators did not differ between patients in need for extracorporeal membrane oxygenation and those rewarmed without extracorporeal membrane oxygenation.

Effect of mild hypothermia on cerebral microcirculation in a murine cardiopulmonary resuscitation model.

BACKGROUND: We hypothesized that mild hypothermia may improve brain microcirculation by reducing cerebral microvascular endothelial cells apoptosis, and this effect may be maximized by moving up the initiation of mild hypothermia from after return of spontaneous circulation (ROSC) to the start of cardiopulmonary resuscitation (CPR). METHODS: A total of 35 rats were randomized into the intra-arrest hypothermia group (IAH), post-resuscitation hypothermia group (PRH), normothermia group (NT), or the sham control group. A craniotomy exposed the parietal cortex for visualization of microcirculation. Ventricular fibrillation was electrically induced and untreated for 8 minutes, followed by 8 minutes of precordial compression and mechanical ventilation. Hypothermia (33 ± 0.5°C) in the IAH and PRH group was induced and maintained for 6 hours at the beginning of CPR or after ROSC, respectively. At baseline, 1, 3, and 6 hours, hemodynamic parameters were measured and the pial microcirculations were visualized with a sidestream dark field imaging video microscope. Microvascular flow index and perfused microvessel density (PMD) were calculated. Rats were euthanized, and brain tissues were removed at 3 and 6 hours separately. Expression of Bax, Bcl-2, and Caspase 3 in brain microvascular endothelial cells was examined by Western blot. RESULTS: Microvascular flow index and PMD were significantly reduced after cardiac arrest and resuscitation (all P < 0.05), and the former was largely preserved by hypothermia regardless when the hypothermia treatment was induced (P < 0.05). Bax and Caspase 3 increased and Bcl-2 decreased significantly after resuscitation, and hypothermia treatment reversed the trend partly (all P < 0.05). A moderate correlation was observed between MFI and those proteins (Bcl-2/BAX: 3 hours: r = 0.730, P = 0.002; 6 hours: r = 0.743, P = 0.002). CONCLUSION: Mild hypothermia improves cerebral microcirculatory blood supply, partly by inhibiting endothelial cell apoptosis. Mild hypothermia induced simultaneously with CPR has shown no additional benefit in microcirculation or endothelial cell apoptosis.