Hypothermic Conditions Research Articles

Hypothermic Oxygenated Perfusion Versus Static Cold Storage for Expanded Criteria Donors in Liver and Kidney Transplantation: Protocol for a Single-Center Randomized Controlled Trial.

BackgroundExtended criteria donors (ECD) are widely utilized due to organ shortage, but they may increase the risk of graft dysfunction and poorer outcomes. Hypothermic oxygenated perfusion (HOPE) is a recent organ preservation strategy for marginal kidney and liver grafts, allowing a redirect from anaerobic metabolism to aerobic metabolism under hypothermic conditions and protecting grafts from oxidative species–related damage. These mechanisms may improve graft function and survival.ObjectiveWith this study, we will evaluate the benefit of end-ischemic HOPE on ECD grafts for livers and kidneys as compared to static cold storage (SCS). The aim of the study is to demonstrate the ability of HOPE to improve graft function and postoperative outcomes of ECD kidney and liver recipients.MethodsThis is an open-label, single-center randomized clinical trial with the aim of comparing HOPE with SCS in ECD kidney and liver transplantation. In the study protocol, which has been approved by the ethics committee, 220 patients (110 liver recipients and 110 kidney recipients) will be enrolled. Livers and kidneys assigned to the HOPE group undergo machine perfusion with cold Belzer solution (4-10°C) and continuous oxygenation (partial pressure of oxygen of 500-600 mm Hg). In the control group, livers and kidneys undergoing SCS are steeped in Celsior solution and stored on ice. Using the same perfusion machine for both liver and kidney grafts, organs are perfused from the start of the back-table procedure until implantation, without increasing the cold ischemia time. For each group, we will evaluate clinical outcomes, graft function tests, histologic findings, perfusate, and the number of allocated organs. Publication of the results is expected to begin in 2021.ResultsDynamic preservation methods for organs from high-risk donors should improve graft dysfunction after transplantation. To date, we have recruited 108 participants. The study is ongoing, and recruitment of participants will continue until January 2020.ConclusionsThe proposed preservation method should improve ECD graft function and consequently the postoperative patient outcomes.Trial RegistrationClinicalTrials.gov NCT03837197; https://clinicaltrials.gov/ct2/show/NCT03837197 ; Archived by WebCite® at http://www.webcitation.org/76fSutT3RInternational Registered Report Identifier (IRRID)DERR1-10.2196/13922

Optimisation of Storage and Transportation Conditions of Cultured Corneal Endothelial Cells for Cell Replacement Therapy

As the cornea is one of the most transplanted tissues in the body it has placed a burden on the provision of corneas from cadaveric donors. Corneal endothelial dysfunction is the leading indication for cornea transplant. Therefore, tissue engineering is emerging as an alternative approach to overcome the global shortage of transplant-grade corneas. The propagation and expansion of corneal endothelial cells has been widely reported. However, one obstacle to overcome is the transport and storage of corneal endothelial cells. In this study we investigated whether tissue engineered corneal endothelial cells can be preserved in hypothermic conditions. Human corneal endothelial cells (HCEnCs) were exposed to various temperatures (4 °C, 23 °C, and 37 °C) in both adherent and suspension storage models. Optimal storage media and storage duration was tested along with post-storage viability. Following storage and subsequent recovery at 37 °C, cell phenotype was assessed by immunofluorescence, gene and protein expression, and proliferative capacity analysis. Functionality was also assessed within a rabbit model of bullous keratopathy. Our data support our hypothesis that functional HCEnCs can be preserved in hypothermic conditions.

Hypothermia Effects on Liver and Kidney Oxidative Stress Parameters in an Experimental Model of Sepsis in Rats.

IntroductionOxidative stress (OS) seems to be an important mediator of cellular injury, from which sepsis can proceed. Studies have demonstrated the protective effect of controlled hypothermia in sepsis. This study aimed to evaluate its effects on OS parameters in rat hepatic and renal tissue septic after caecal ligation and puncture (CLP).Material and MethodsThree groups were appointed (10 rats/group): C (control), SN (sepsis normothermic), and SH (sepsis hypothermic). Ten hours from CLP, the liver and kidneys were harvested and total protein concentration, superoxide dismutase (SOD), glutathione peroxidase (GPx) activities, lipid peroxidation level (malondialdehyde (MDA), carbonylated proteins (2,4-dinitrophenylhydrazine (DNPH), and fatty acid profile were analysed.ResultsSepsis significantly increased SOD and GPx activities in the liver, regardless of the temperature. In renal tissue, GPx activity increased significantly in normothermic conditions and SOD tended to decrease in hypothermic conditions. MDA and DNPH concentrations increase in both tissues after CLP. Hypothermia significantly lowered MDA in the liver but only changed it insignificantly in the kidneys. The DNPH in the liver and kidneys was significantly lower in hypothermic conditions. The unsaturated-to-saturated fatty acids ratio was significantly lower in sepsis, and the fall in temperature raised this ratio.ConclusionExperimentally induced sepsis in rats enhances OS in the liver and kidneys. The effect of hypothermia on OS indices is dependent on the type of tissue.

Can the impacts of cold-water pollution on fish be mitigated by thermal plasticity?

Increasingly, cold-water pollution (CWP) is being recognised as a significant threat to aquatic communities downstream of large, bottom-release dams. Cold water releases typically occur during summer when storage dams release unseasonably cold and anoxic hypolimnetic waters, which can decrease the temperature of downstream waters by up to 16°C. Depending on the release duration, these hypothermic conditions can persist for many months. The capacity of ectothermic species to tolerate or rapidly adjust to acute temperature changes may determine the nature and magnitude of the impact of CWP on affected species. This study assessed the impacts of an acute reduction in water temperature on the physiological function and locomotor performance of juvenile silver perch (Bidyanus bidyanus) and examined their capacity to thermally compensate for the depressive effects of low temperatures via phenotypic plasticity. Locomotor performance (Ucrit and Usprint) and energetic costs (routine and maximum metabolic rate) were measured at multiple points over a 10-week period following an abrupt 10°C drop in water temperature. We also measured the thermal sensitivity of metabolic enzymes from muscle samples taken from fish following the exposure period. Cold exposure had significant depressive effects on physiological traits, resulting in decreases in performance between 10% and 55%. Although there was partial acclimation of Ucrit (~35% increase in performance) and complete compensation of metabolic rate, this occurred late in the exposure period, meaning silver perch were unable to rapidly compensate for the depressive effects of thermal pollution. The results of this study have substantial implications for the management of cold water releases from large-scale dams and the conservation of native freshwater fish species, as this form of thermal pollution can act as a barrier to fish movement, cause reduced recruitment, ecological community shifts and disruptions to timing and success of reproduction.

Tau hyperphosphorylation induced by the anesthetic agent ketamine/xylazine involved the calmodulin-dependent protein kinase II.

Tau hyperphosphorylation is a major neuropathological hallmark of many neurodegenerative disorders such as Alzheimer's disease. Several anesthetics have been shown previously to induced marked tau hyperphosphorylation. Although the ketamine/xylazine mixture is one of the most commonly used anesthetic agents in animal research and veterinary practice, the effect of this anesthetic agent on tau phosphorylation still remains to be determined. Here, we found that ketamine-/xylazine-induced a rapid and robust hyperphosphorylation of tau in a dose-dependent manner under normothermic and hypothermic conditions in mice. When used together, ketamine and xylazine exerted a synergistic action on tau phosphorylation most strongly not only on epitopes S396 and S262, but also on other residues (T181, and S202/T205). We observed that activation of the calmodulin-dependent protein kinase II (CaMKII) is the major upstream molecular event leading to tau hyperphosphorylation following ketamine/xylazine anesthesia in mice. Moreover, we observed that intracerebroventricular injection of the selective CaMKII inhibitor KN93 attenuated tau hyperphosphorylation. Since ketamine/xylazine also had a marked impact on other key molecular signaling pathways involving the MAP/microtubule affinity-regulating kinase (MARK), extracellular signal-regulated kinase (ERK), and glycogen synthase kinase-3 (GSK3), our study calls for high caution and careful monitoring when using this anesthetic agent in laboratory animal settings across all fields of biological sciences in order to avoid artifactual results.

Improved tolerance of Escherichia coli to oxidative stress by expressing putative response regulator homologs from Antarctic bacteria.

Response regulator (RR) is known a protein that mediates cell's response to environmental changes. The effect of RR from extremophiles was still under investigation. In this study, response regulator homologs were mined from NGS data of Antarctic bacteria and overexpressed in Escherichia coli. Sixteen amino acid sequences were annotated corresponding to response regulators related to the two-component regulatory systems; of these, 3 amino acid sequences (DRH632, DRH1601 and DRH577) with high homology were selected. These genes were cloned in pRadGro and expressed in E. coli. The transformant strains were subjected to various abiotic stresses including oxidative, osmotic, thermal stress, and acidic stress. There was found that the robustness of E. coli to abiotic stress was increased in the presence of these response regulator homologs. Especially, recombinant E. coli overexpressing drh632 had the highest survival rate in oxidative, hypothermic, osmotic, and acidic conditions. Recombinant E. coli overexpressing drh1601 showed the highest tolerance level to osmotic stress. These results will be applicable for development of recombinant strains with high tolerance to abiotic stress.

Cardiac myocyte functional and biochemical changes after hypothermic preservation in vitro. Protective effects of storage solutions.

In this study, we evaluated cardiac myocyte viability and function under hypothermic conditions with four types of storage solutions, saline solution, Euro-Collins solution, University of Wisconsin solution, and MCDB 107 medium. Cardiac myocytes were isolated from neonatal rat ventricles by collagenase dispersion and cultured for 4 days with MCDB 107 medium. A total of 12.5 x 10(5) myocytes per culture dish was used and the myocytes were incubated at 4 degrees C for 6, 12, 18, and 24 hours in the various storage solutions. After each incubation time, creatine kinase and lactate dehydrogenase were measured in the storage solutions. The myocytes were then incubated for 24 hours at 37 degrees C to evaluate the recovery of the myocyte beating rate. In the MCDB 107 group (n = 7), the recovery ratio of myocyte beating rate was complete by 12 hours, then decreased to 44.8% of control (beating rate before hypothermic incubation) at 24 hours. The saline, Euro-Collins, and University of Wisconsin groups (n = 7 each) had significantly lower recovery ratios than the MCDB 107 group (at 12 hours: 61.0%, 32.2%, and 48.9%; at 18 hours: 0.0%, 5.5%, and 15.1% of control, respectively). Release of creatine kinase and lactate dehydrogenase in the MCDB 107 group gradually increased and at 24 hours was 143.2 mIU/flask and 486.2 mIU/flask, respectively. However, the saline and University of Wisconsin groups had significantly increased creatine kinase and lactate dehydrogenase values at 24 hours (creatine kinase: 334.6 and 319.6 mIU/flask; lactate dehydrogenase: 821.6 and 654.4 mIU/flask, respectively). The Euro-Collins group showed the greatest increase in both markers (creatine kinase: 1587.5, lactate dehydrogenase: 2106.9 mIU/flask). In summary, saline and University of Wisconsin solutions showed a beneficial effect on recovery of myocyte viability at 12 hours compared with Euro-Collins solution, however MCDB 107 medium had the best overall protective effect on cultured myocytes. Accordingly, alternate hypothermic storage solutions, such as cell-culture medium, may have protective characteristics that are suitable for cardiac preservation.

Mathematical Modelling of Acetaminophen Release in HPC/PAAm Hydrogel: Synthesis and Application

Hydrogels are commonly used as Drug Delivery Systems (DDS) as patches due to its ability to store drug molecules within their structures. The release can be activated under certain stimuli, such as temperature and pH. In this paper, the mathematical modelling of acetaminophen release in hydroxypropyl cellulose with polyacrylamide (HPC/PAAm) is reported. The HPC/PAAm gel was synthesized in proportions of 25/75 wt% and was characterized by FTIR, DSC, optical microscopy, SEM, and TGA, with and without acetaminophen. The release tests were performed for hypothermic, normal, and febrile human body conditions, at 35, 37, and 39°C, respectively, on two release media: water and phosphate buffer solution. In order to describe the release of acetaminophen in HPC/PAAm gel, a genetic programming algorithm was used to accomplish Multigene Symbolic Regression (MSR). Characterization results showed that the drug was crystallized on the surface of the HPC/PAAm gel. Release test results showed that several simultaneous processes occurred in the acetaminophen diffusion phenomenon. A unique mathematical model was obtained by MSR. This model was able to describe the release of acetaminophen in HPC/PAAm gel with high values of R2 and adjusted R2 and to simulate the drug release at times beyond the end of the experiment. High values of R2 and low values of Coefficient of Variation (CV), Root-Mean-Square Error (RMSE), and Mean Absolute Error (MAE) were obtained from the comparison between the simulated and the experimental data. This allows to conclude that the mathematical model is reliable to represent and simulate the acetaminophen release in HPC/PAAm gel at 35, 37, and 39°C.

Factors limiting the duration of artificially induced torpor in mice

The possibility of artificial induction of a torpid state in animals that do not naturally do so, as well as in humans, offers a great potential in biomedicine and in human spaceflight. However, the mechanisms of action that provide a coordinated and concomitant downregulation with a safe recovery from this state are poorly understood. In our previous study, we demonstrated that the metabolic rate of mice can be reduced by nearly 94% and can remain stable under hypothermic conditions for a prolonged period of up to 11 h. The present study was carried out in order to test the limitations and identify potential factors that can enable the safe and reversible arousal of non-hibernating mice from deep artificially-induced torpor to an active state. Results demonstrate that the energy budget may be a limiting factor for the prolongation and safe recovery from the hypometabolic state. While the continuation of torpor may be possible for additional hours, we found that a reduction of 40% or more in the plasma glucose level increases the risk of heart fibrillation, which results in death during arousal. Therefore, the plasma glucose level could be a component of the criteria indicating the reversibility of torpor.Another important factor complementing the energetic necessity that may limit the duration of torpor in mice is a gradual reduction in body mass during torpor. Under the conditions of our experiment, body mass declines by nearly 15% after 16 h from the initiation of torpor and may continue to decline if the mice are allowed to remain in torpor longer. Extrapolation of this data suggests that there may be a critical mass relating to animal mortality and thus limiting the duration of torpor. Control and maintenance of the body mass and glucose level in a torpid animal may extend the longevity of torpor and mitigate the risk of cardiac failure during rewarming to the metabolically active state. The cardiac complications that occur during arousal from torpor in many cases could be mitigated and even avoided by applying appropriate temperature-arising kinetics and providing a sufficient dynamic range to maintain cardiac output.

Adaptive laboratory evolution of stable insect cell lines for improved HIV-Gag VLPs production

Adaptive laboratory evolution (ALE) has been extensively used to modulate the phenotype of industrial model organisms (e.g. Escherichia. coli and Saccharomyces cerevisae) towards a specific trait. Nevertheless, its application to animal cells, and in particular to insect cell lines, has been very limited.In this study, we describe employing an ALE method to improve the production of HIV-Gag virus-like particles (VLPs) in stable Sf-9 and High Five cell lines. Serial batch transfer was used for evolution experiments. During the ALE process, cells were cultured under controlled hypothermic conditions (22 °C instead of standard 27 °C) for a prolonged period of time (over 3 months), which allowed the selection of a population of cells with improved phenotype. Adapted cells expressed up to 26-fold (Sf-9 cells) and 10-fold (High Five cells) more Gag-VLPs than non-adapted cells cultured at standard conditions. The production of HIV Gag-VLPs in adapted, stable insect Sf-9 cell lines was successfully demonstrated at bioreactor scale. The Gag-VLPs produced at 22 °C and 27 °C were comparable, both in size and morphology, thus confirming the null impact of adaptation process and hypothermic culture conditions on VLP’s quality.This work demonstrates the suitability of ALE as a powerful method for improving yields in stable insect cell lines producing VLPs.

Reduced perforin release from T cells as a mechanism underlying hypothermia‐mediated neuroprotection

AbstractObjectiveThe underlying mechanisms of therapeutic hypothermia, which protects neurons after severe brain damage, are partially understood. T cells infiltrate the brain within days after cerebral ischemia and play essential roles in exacerbating the delayed phase of brain injury by producing cytotoxic factors, which were also systematically shown to be involved in brain damage. Periphery brain abnormalities are interesting, because the periphery might constitute a pathway to the central nervous system and therefore could also be a target of therapeutic hypothermia. We elucidated the effects of hypothermia and hyperthermia on peripheral T cell‐derived perforin (Pfn), and the underlying mechanism of Pfn‐mediated neurotoxicity and death of brain microvascular endothelial cells (BMVECs).MethodsWe determined the levels of Pfn produced by activated CD4+ and CD8+ T cells obtained from healthy humans under hypothermic, normothermic and hyperthermic conditions. The viability in response to Pfn treatment was assessed in neuronal Neuro‐2a and brain microvascular endothelial bEnd.3 cells.ResultsCompared with normothermia, Pfn release in both CD4+ and CD8+ T cells was reduced by hypothermia, but augmented by hyperthermia. Pfn caused the death of Neuro‐2a cells, and induced both apoptosis and necrosis in bEnd.3 cells; both effects were concentration‐dependent.ConclusionsHypothermia reduces T cell‐derived release of Pfn, which mediates neuronal cell death and apoptosis/necrosis of BMVECs, suggesting that therapeutic hypothermia elicits protection in the neurons and BMVECs in the delayed phase of brain injury by directly reducing the release of T cell‐derived Pfn. BMVECs protection can aid in reduced vascular permeability and blood–brain barrier protection.

Target temperature in post-cardiac-arrest complex intensive care: arguments for mild therapeutic hypothermia

Long term survival of successfully resuscitated patients is primarily determined by their post-cardiac arrest neurological function. If the patient undergoes a long-term resuscitation or remains comatose as part of the post-cardiac arrest syndrome (PCAS), organ-specific intensive care is urged to aim hemodynamic stabilisation, normalisation of organ perfusion and prevention of injuries at cellular level. One of the basic measures of PCAS intensive care is to prevent hypoxic brain injury by mild therapeutic hypothermia (THT). The physiological changes of the human body at hypothermic conditions require high level monitoring and specially focused intensive care limiting its implementation. The multicentric, controlled, randomized targeted temperature management (TTM) trial published in 2013 compared the TTM against the THT in the treatment of PCAS patients. The equal outcome of the 2 methods has partly changed the practice of the intensivists in the treatment of such patients. This manuscript gives the pros and cons for each therapeutic method in post-resuscitation therapy. Nevertheless, the author shows the possible implementations and the DRG (diagnosis-related group) reimbursement of the method in Hungary. Orv Hetil. 2019; 160(46): 1840-1844.

Manipulation of the dually thermoresponsive behavior of peptide-based vesicles through modification of collagen-like peptide domains.

Materials that respond to temporally defined exogenous cues continue to be an active pursuit of research toward on‐demand nanoparticle drug delivery applications, and using one or more exogenous temperature stimuli could significantly expand the application of nanoparticle‐based drug delivery formulations under both hyperthermal and hypothermal conditions. Previously we have reported the development of a biocompatible and thermoresponsive elastin‐b‐collagen‐like polypeptide (ELP‐CLP) conjugate that is capable of self‐assembling into vesicles and encapsulating small molecule therapeutics that can be delivered at different rates via a single temperature stimulus. Herein we report the evaluation of multiple ELP‐CLP conjugates, demonstrating that the inverse transition temperature (T t) of the ELP‐CLPs can be manipulated by modifying the melting temperature (T m) of the CLP domain, and that the overall hydrophilicity of the ELP‐CLP conjugate also may alter the T t. Based on these design parameters, we demonstrate that the ELP‐CLP sequence (VPGFG)6‐(GPO)7GG can self‐assemble into stable vesicles at 25°C and dissociate at elevated temperatures by means of the unfolding of the CLP domain above its T m. We also demonstrate here for the first time the ability of this ELP‐CLP vesicle to dissociate via a hypothermic temperature stimulus by means of exploiting the inverse transition temperature (T t) phenomena found in ELPs. The development of design rules for manipulating the thermal properties of these bioconjugates will enable future modifications to either the ELP or CLP sequences to more finely tune the transitions of the conjugates for specific biomedical applications.

The Effect of Hypothermia on Some Structural and Functional Characteristics of Lactate Dehydrogenase of the Rat Brain

Abstract—We studied the activity and some physicochemical parameters of LDH in the rat brain under normal and hypothermic conditions. It was found that during hypothermia the activity of LDH in the rat brain increases, while this increase is less pronounced in an enzyme preparation purified from ballast proteins. A study of LDH self-fluorescence showed a decrease in the intensity of total fluorescence of LDH, in which the main contribution is made by tryptophan residues. Analysis of the second derivatives of the fluorescence spectra showed that hypothermia affects tryptophanyls, which are located on the periphery of the LDH molecule. Study of the binding kinetics of the fluorescent probe ANS with LDH showed the presence of at least two types of binding sites of the probe, which differ in polarity. Hypothermia leads to a decrease in the fluorescence intensity of the ANS, a decrease in the number of binding sites, an increase in the probe dissociation constants, and a shift in the inflection position on the graph of the temperature dependence of the ANS fluorescence to higher temperatures. The study of the content of sulfhydryl and carbonyl groups in the LDH molecule suggests that one of the causes of the changes in the activity and spectral characteristics of LDH in the brain of hypothermic rats is the modification of the enzyme by reactive oxygen species.

Multivariate analysis of metabolic parameters and optimization of antibody production using high cell density hybridoma in hollow fiber bioreactors.

The relationships of manipulation of culture temperature and medium circulation rate on the metabolic parameters were regressed by multiple linear regression analysis in hollow fiber bioreactors (HFB). The high circulation rate could significantly enhance the oxygen consumption of the hybridoma cells and the medium's oxidation-reduction potential. A mildly hypothermic condition of 36°C and a circulation rate of 182.5mL/min could support the hybridoma had the maximal antibody titer of 60.75μg/mL for 20days. When the ammonium ion was 65ppm or lactate close to 2.6g/L, the medium was replaced to maintain the stable and healthy cells at the high cell concentration of 3.33 × 108/mL for continuous antibody production. Two serum-free media could be successfully applied to this perfusion system and maintain hybridoma growth and antibody production. The single-use HFBs could provide the advantages including high cell density, low shear stress, and continuous antibody production.

Myocardial hypothermia increases autophagic flux, mitochondrial mass and myocardial function after ischemia-reperfusion injury

Animal studies have demonstrated beneficial effects of therapeutic hypothermia on myocardial function, yet exact mechanisms remain unclear. Impaired autophagy leads to heart failure and mitophagy is important for mitigating ischemia/reperfusion injury. This study aims to investigate whether the beneficial effects of therapeutic hypothermia are due to preserved autophagy and mitophagy. Under general anesthesia, the left anterior descending coronary artery of 19 female farm pigs was occluded for 90 minutes with consecutive reperfusion. 30 minutes after reperfusion, we performed pericardial irrigation with warm or cold saline for 60 minutes. Myocardial tissue analysis was performed one and four weeks after infarction. Therapeutic hypothermia induced a significant increase in autophagic flux, mitophagy, mitochondrial mass and function in the myocardium after infarction. Cell stress, apoptosis, inflammation as well as fibrosis were reduced, with significant preservation of systolic and diastolic function four weeks post infarction. We found similar biochemical changes in human samples undergoing open chest surgery under hypothermic conditions when compared to the warm. These results suggest that autophagic flux and mitophagy are important mechanisms implicated in cardiomyocyte recovery after myocardial infarction under hypothermic conditions. New therapeutic strategies targeting these pathways directly could lead to improvements in prevention of heart failure.

Effectiveness of Antiplatelet Drugs Under Therapeutic Hypothermia: A Comprehensive Review.

Out-of-hospital cardiac arrest is among the most frequent causes of death worldwide. Immediate intervention, as well as dual antiplatelet therapy consisting of acetylsalicylic acid and a P2Y12 inhibitor is often recommended. In line with the growing number of reports on cardiac arrest treatment, therapeutic hypothermia (TH) has been proposed for unconscious patients to improve neurological outcomes. Nevertheless, studies report controversial and often discrepant results on the effect of hypothermia on blood coagulability and platelet reactivity. In this review, we summarize the knowledge on platelet function under diverse hypothermic conditions. Additionally, we review the current literature on the effect of systemic hypothermia on pharmacokinetic and pharmacodynamic properties of antiplatelet agents. It has been shown that TH can alter the effectiveness of antiplatelet agents, including P2Y12 inhibitors, through multiple mechanisms, hence, special attention should be paid while implementing antiplatelet therapy in patients under TH conditions.

Phosphorylation and Dephosphorylation of Tau Protein During Synthetic Torpor.

Tau protein is of primary importance for many physiological processes in neurons, where it affects the dynamics of the microtubule system. When hyperphosphorylated (PP-Tau), Tau monomers detach from microtubules and tend to aggregate firstly in oligomers, and then in neurofibrillary tangles, as it occurs in a group of neurodegenerative disorders named thauopathies. A hypothermia-related accumulation of PP-Tau, which is quickly reversed after the return to normothermia, has been shown to occur in the brain of hibernators during torpor. Since, recently, in our lab, a hypothermic torpor-like condition (synthetic torpor, ST) was pharmacologically induced in the rat, a non-hibernator, the aim of the present work was to assess whether ST can lead to a reversible PP-Tau accumulation in the rat brain. PP-Tau was immunohistochemically assessed by staining for AT8 (phosphorylated Tau) and Tau-1 (non-phosphorylated Tau) in 19 brain structures, which were chosen mostly due to their involvement in the regulation of autonomic and cognitive functions in relation to behavioral states. During ST, AT8 staining was strongly expressed throughout the brain, while Tau-1 staining was reduced compared to control conditions. During the following recovery period, AT8 staining progressively reduced close to zero after 6 h from ST. However, Tau-1 staining remained low even after 38 h from ST. Thus, overall, these results show that ST induced an accumulation of PP-Tau that was, apparently, only partially reversed to normal during the recovery period. While the accumulation of PP-Tau may only depend on the physicochemical characteristics of the enzymes regulating Tau phosphorylation, the reverse process of dephosphorylation should be actively regulated, also in non-hibernators. In conclusion, in this work a reversible and widespread PP-Tau accumulation has been induced through a procedure that leads a non-hibernator to a degree of reversible hypothermia, which is comparable to that observed in hibernators. Therefore, the physiological mechanism involved in this process can sustain an adaptive neuronal response to extreme conditions, which may however lead to neurodegeneration when particular intensities and durations are exceeded.

Oxygen Consumption by Warm Ischemia-Injured Porcine Kidneys in Hypothermic Static and Machine Preservation

Static cold storage (SCS) and hypothermic machine perfusion (HMP) are currently standard methods for renal grafts clinical preservation. Both methods are predominantly implemented without the active delivery of oxygen, even for donation after circulatory death-like kidneys. However, even under severe hypothermia (4°C-6°C), kidneys can consume oxygen and produce ATP. What is not established, though, is to what extent and how SCS and HMP compare in terms of oxygen. Using a porcine preclinical model of renal warm ischemia (WI) to compare SCS and HMP methods, we continuously monitored and quantified oxygen level and consumption along preservation; we also determined prepreservation and postpreservation cortical ATP level; values were given as median and [min; max] range. One-hour WI reduced ATP by ∼90% (from 3.3 [1.7; 4.5] mmol/L tissue in Controls). Oxygen consumption (QO2, μmol/min per 100 g) was determined from initial solution PO2 decrease (SCS and HMP) and from arterio-venous difference (HMP). In SCS and HMP, PO2 decreased rapidly (t1/2 ∼1 h) from atmospheric levels to 52.9 [38.0; 65.9] and 8.2 [3.0, 16.0] mmHg, respectively. In HMP, QO2 was 2.7 [0.4; 3.9] versus 0.5 [0.0; 1.3] in SCS (P < 0.05); postpreservation ATP amounted to 5.8 [3.2; 6.5] in HMP versus 0.1 [0.0; 0.2] in SCS. Despite hypothermic conditions in SCS or HMP, donation after circulatory death-like renal grafts require oxygen. Increased oxygen consumption, restored ATP level, and improved histological profile in HMP might explain the established HMP superiority over SCS. These results establish a rational basis for the use of oxygen in hypothermic preservation. Optimal levels required for preservation and graft-type variants remain to be determined.

Standardized Measurement of Capillary Refill Time using Novel Technology

Introduction:In a patient going into shock, blood is redistributed from the periphery to the central circulation, making an assessment of skin perfusion useful in a prehospital setting. Capillary refill time (CRT) is the time required for a pressure blanched skin site to reperfuse. Currently, CRT is tested by manually applying pressure for 5s to the skin and observing the time before reperfusion. Guidelines state that CRT should be 2-3s in a healthy patient. Shortcomings in this procedure include lack of standardization of pressure, subjective assessment of the time for reperfusion, and not accounting for the patient’s skin temperature.Aim:To develop a standardized objective procedure for testing CRT in the prehospital setting.Method:The study protocol was approved by the Ethics Committee at Linköping University (M200-07, 2015-99-31). An electro-pneumatic device exerting constant force (9N) over 5s was developed. CRT was measured using the Tissue Viability Imager (Wheelsbridge AB, Sweden) which relies on polarization spectroscopy. To simulate hypothermic conditions, healthy volunteers were subjected to low ambient temperature (8°C). Blood loss was simulated using a custom-built lower body negative pressure (LBNP) chamber. In both scenarios, the CRT test was carried out on three test sites (finger pulp, forehead, and sternum).Results:CRT on the finger pulp and sternum was shown to be increased following the hypothermic conditions, but not on the forehead. Skin temperature on the three sites followed the same pattern, with the forehead being virtually unchanged. Tests performed during LBNP revealed an apparent effect on CRT following the simulated blood loss, with prolonged CRT for all sites tested.Discussion:A successful methodology for objective assessment of CRT was developed, which was validated on healthy volunteers following hypothermia or simulated blood loss. Ongoing work will investigate a combination of hypothermia and blood loss to more accurately simulate the prehospital setting.