Synthetic Torpor Research Articles

Synthetic torpor confers cardioprotection against myocardial ischaemia/reperfusion injury in the rat

Abstract Introduction Torpor is a hypothermic, bradycardic, hypometabolic state enabling animals, such as mice, to survive in unfavourable environmental conditions such as cold exposure or reduced food availability. It is thought to be a cardioprotective state. There is interest in developing models that mimic aspects of torpor in clinical settings such as ischaemia-reperfusion injury. The preoptic area of the hypothalamus (POA) has emerged as a key centre for triggering torpor in the mouse. Purpose Induce a synthetic torpor-like state in a species that does not naturally enter torpor (the rat) by chemoactivation of neurons in the POA that correspond to those that induce torpor in the mouse. Test the cardioprotective effects of synthetic torpor in the Langendorff ischaemia-reperfusion preparation. Methods Designer receptors exclusively activated by designer drugs (DREADDs) were used to exogenously activate excitatory neurons in the POA of the rat hypothalamus. Viral vectors (pAAV-CaMKIIa-hM3D(Gq)-mCherry) were stereotaxically injected bilaterally into the POA under ketamine and medetomidine anaesthesia, resulting in expression of DREADDs in excitatory neurons. Control animals received a vector delivering the gene for a fluorescent protein only. DREADD receptors in transfected neurons were activated by subsequent injection of Clozapine-N-Oxide (CNO, 2mg/kg IP), which results in neuronal activation and induces synthetic torpor. Heart rate and core temperature were monitored using implanted telemeters. 2 weeks after vector injection, animals were anaesthetised using 5% isoflurane and culled by cervical dislocation. Hearts were excised and ex-vivo whole heart perfusion at 37°C was performed using the Langendorff apparatus. Hearts were subjected to 30 minutes of global warm ischaemia followed by 60 minutes of reperfusion. Hearts were stained using tetrazolium triphenyl chloride, and infarct size as a proportion of the total heart area calculated. Results Chemoactivation of rat POA neurons induced synthetic torpor characterised by bradycardia (pre: 341.0 ± 17.73bpm, post: 233.4 ± 20.47bpm, paired t (4) = 5.4791, p = .0054) and hypothermia (36.2 ±0.40°C vs 31.6 ± 0.55°C (paired t (4) = 7.9023, p = .0014). CNO had no effect on heart rate or core temperature in control animals. The mean infarct size was significantly lower in synthetic torpor (21.54 ±2.84%) compared to control hearts (29.62 ±2.59%) (unpaired t (23) = 2.1054, p = .0464). Conclusions Synthetic torpor, induced centrally in a species for which torpor is not a natural behaviour, confers striking cardioprotection in an ex-vivo ischaemia-reperfusion model. This novel approach to cardioprotection against myocardial ischaemia/reperfusion injury should be further investigated as a potential therapeutic target for use in clinical practice.

Thermomechanical properties of bat and human red blood cells—Implications for hibernation

Hibernation is a widespread and highly efficient mechanism to save energy in mammals. However, one major challenge of hibernation is maintaining blood circulation at low body temperatures, which strongly depends on the viscoelastic properties of red blood cells (RBCs). Here, we examined at physiologically relevant timescales the thermomechanical properties of hundreds of thousands of individual RBCs from the hibernating common noctule bat (Nyctalus noctula), the nonhibernating Egyptian fruit bat (Rousettus aegyptiacus), and humans (Homo sapiens). We exposed RBCs to temperatures encountered during normothermia and hibernation and found a significant increase in elasticity and viscosity with decreasing temperatures. Our data demonstrate that temperature adjustment of RBCs is mainly driven by membrane properties and not the cytosol while viscous dissipation in the membrane of both bat species exceeds the one in humans by a factor of 15. Finally, our results show that RBCs from both bat species reveal a transition to a more viscous-like state when temperature decreases. This process on a minute timescale has an effect size that is comparable with fluctuations in RBC viscoelasticity over the course of the year, implying that environmental factors, such as diets, have a lower impact on the capability of RBCs to respond to different temperatures than general physical properties of the cell membrane. In summary, our findings suggest membrane viscoelasticity as a promising target for identifying mechanisms that could be manipulated to ensure blood circulation at low body temperatures in humans, which may be one first step toward safe synthetic torpor in medicine and space flight.

Scheduled hypometabolism and hypothermia at the crossroads of deep space manned mission and longevity

Spaceflight is a mysterious ageing/rejuvenation transition. It is associated with factors of evolutionary unfamiliar to the earth's inhabitants — microgravity, cosmic rays and solar wind irradiation, failure of circadian rhythms and numerous other singularities. For still unknown reasons, spaceflight provokes quick acceleration of almost all hallmarks of ageing and age-related diseases. The alterations have a transitory character and disappear in the post-landing adaptation period. The posited ageing/rejuvenation transition could be repeated over multiple flights. This should be confirmed by epigenetic or other types of ageing clocks. Hypometabolism and hypothermia are considered efficient protectors from the hazards of space missions while saving energy and food intake. The two most discussed approaches are based on the idea of dormancy (synthetic torpor) or excessive sleeping (shallow metabolic depression). We suggest another model of metabolic depression induced by a rebreathing hypoxic-hypercapnic environment (HHE), which is accompanied by ‘voluntary’ calorie restriction. Crewmembers' work schedules can be designed to allow crewmembers to maintain normoxic levels during energy-demanding activities and transition to HHE during rest periods. A computerized tracking system can harmonize daily schedules with personalized HHE. The resulting scheduled hypometabolothermia (SHMT) could optimise energy expenditure without compromising productivity and be applicable during spaceflight and upon arrival at a destination and subsequent planetary exploration. We envision SHMT as a major human lifestyle on Earth as well. Of course, only further thorough explorations will reveal all the advantages and pitfalls of HHE and SHMT on Earth and in space. ________________________________________________________________________________________Keywords: hypoxia and hypercapnia; metabolic suppression; food consumption; space mission; longevity

Sleep deprivation soon after recovery from synthetic torpor enhances tau protein dephosphorylation in the rat brain.

Neuronal Tau protein hyperphosphorylation (PPtau) is a hallmark of tauopathic neurodegeneration. However, a reversible brain PPtau occurs in mammals during either natural or "synthetic" torpor (ST), a transient deep hypothermic state that can be pharmacologically induced in rats. Since in both conditions a high sleep pressure builds up during the regaining of euthermia, the aim of this work was to assess the possible role of post-ST sleep in PPtau dephosphorylation. Male rats were studied at the hypothermic nadir of ST, and 3-6h after the recovery of euthermia, after either normal sleep (NS) or total sleep deprivation (SD). The effects of SD were studied by assessing: (i) deep brain temperature (Tb); (ii) immunofluorescent staining for AT8 (phosphorylated Tau) and Tau-1 (non-phosphorylated Tau), assessed in 19 brain structures; (iii) different phosphorylated forms of Tau and the main cellular factors involved in Tau phospho-regulation, including pro- and anti-apoptotic markers, assessed through western blot in the parietal cortex and hippocampus; (iv) systemic factors which are involved in natural torpor; (v) microglia activation state, by considering morphometric variations. Unexpectedly, the reversibility of PPtau was more efficient in SD than in NS animals, and was concomitant with a higher Tb, higher melatonin plasma levels, and a higher frequency of the microglia resting phenotype. Since the reversibility of ST-induced PPtau was previously shown to be driven by a latent physiological molecular mechanism triggered by deep hypothermia, short-term SD soon after the regaining of euthermia seems to boost the possible neuroprotective effects of this mechanism.

Transcriptome Profiling Reveals Enhanced Mitochondrial Activity as a Cold Adaptive Strategy to Hypothermia in Zebrafish Muscle.

The utilisation of synthetic torpor for interplanetary travel once seemed farfetched. However, mounting evidence points to torpor-induced protective benefits from the main hazards of space travel, namely, exposure to radiation and microgravity. To determine the radio-protective effects of an induced torpor-like state we exploited the ectothermic nature of the Danio rerio (zebrafish) in reducing their body temperatures to replicate the hypothermic states seen during natural torpor. We also administered melatonin as a sedative to reduce physical activity. Zebrafish were then exposed to low-dose radiation (0.3 Gy) to simulate radiation exposure on long-term space missions. Transcriptomic analysis found that radiation exposure led to an upregulation of inflammatory and immune signatures and a differentiation and regeneration phenotype driven by STAT3 and MYOD1 transcription factors. In addition, DNA repair processes were downregulated in the muscle two days' post-irradiation. The effects of hypothermia led to an increase in mitochondrial translation including genes involved in oxidative phosphorylation and a downregulation of extracellular matrix and developmental genes. Upon radiation exposure, increases in endoplasmic reticulum stress genes were observed in a torpor+radiation group with downregulation of immune-related and ECM genes. Exposing hypothermic zebrafish to radiation also resulted in a downregulation of ECM and developmental genes however, immune/inflammatory related pathways were downregulated in contrast to that observed in the radiation only group. A cross-species comparison was performed with the muscle of hibernating Ursus arctos horribilis (brown bear) to define shared mechanisms of cold tolerance. Shared responses show an upregulation of protein translation and metabolism of amino acids, as well as a hypoxia response with the shared downregulation of glycolysis, ECM, and developmental genes.

Synthetic torpor triggers a regulated mechanism in the rat brain, favoring the reversibility of Tau protein hyperphosphorylation.

Introduction: Hyperphosphorylated Tau protein (PPTau) is the hallmark of tauopathic neurodegeneration. During "synthetic torpor" (ST), a transient hypothermic state which can be induced in rats by the local pharmacological inhibition of the Raphe Pallidus, a reversible brain Tau hyperphosphorylation occurs. The aim of the present study was to elucidate the - as yet unknown - molecular mechanisms underlying this process, at both a cellular and systemic level. Methods: Different phosphorylated forms of Tau and the main cellular factors involved in Tau phospho-regulation were assessed by western blot in the parietal cortex and hippocampus of rats induced in ST, at either the hypothermic nadir or after the recovery of euthermia. Pro- and anti-apoptotic markers, as well as different systemic factors which are involved in natural torpor, were also assessed. Finally, the degree of microglia activation was determined through morphometry. Results: Overall, the results show that ST triggers a regulated biochemical process which can dam PPTau formation and favor its reversibility starting, unexpectedly for a non-hibernator, from the hypothermic nadir. In particular, at the nadir, the glycogen synthase kinase-β was largely inhibited in both regions, the melatonin plasma levels were significantly increased and the antiapoptotic factor Akt was significantly activated in the hippocampus early after, while a transient neuroinflammation was observed during the recovery period. Discussion: Together, the present data suggest that ST can trigger a previously undescribed latent and regulated physiological process, that is able to cope with brain PPTau formation.

Investigating the effects of chronic low-dose radiation exposure in the liver of a hypothermic zebrafish model

Mankind’s quest for a manned mission to Mars is placing increased emphasis on the development of innovative radio-protective countermeasures for long-term space travel. Hibernation confers radio-protective effects in hibernating animals, and this has led to the investigation of synthetic torpor to mitigate the deleterious effects of chronic low-dose-rate radiation exposure. Here we describe an induced torpor model we developed using the zebrafish. We explored the effects of radiation exposure on this model with a focus on the liver. Transcriptomic and behavioural analyses were performed. Radiation exposure resulted in transcriptomic perturbations in lipid metabolism and absorption, wound healing, immune response, and fibrogenic pathways. Induced torpor reduced metabolism and increased pro-survival, anti-apoptotic, and DNA repair pathways. Coupled with radiation exposure, induced torpor led to a stress response but also revealed maintenance of DNA repair mechanisms, pro-survival and anti-apoptotic signals. To further characterise our model of induced torpor, the zebrafish model was compared with hepatic transcriptomic data from hibernating grizzly bears (Ursus arctos horribilis) and active controls revealing conserved responses in gene expression associated with anti-apoptotic processes, DNA damage repair, cell survival, proliferation, and antioxidant response. Similarly, the radiation group was compared with space-flown mice revealing shared changes in lipid metabolism.

Synthetic torpor protects rats from exposure to accelerated heavy ions

Hibernation or torpor is considered a possible tool to protect astronauts from the deleterious effects of space radiation that contains high-energy heavy ions. We induced synthetic torpor in rats by injecting adenosine 5′-monophosphate monohydrate (5′-AMP) i.p. and maintaining in low ambient temperature room (+ 16 °C) for 6 h immediately after total body irradiation (TBI) with accelerated carbon ions (C-ions). The 5′-AMP treatment in combination with low ambient temperature reduced skin temperature and increased survival following 8 Gy C-ion irradiation compared to saline-injected animals. Analysis of the histology of the brain, liver and lungs showed that 5′-AMP treatment following 2 Gy TBI reduced activated microglia, Iba1 positive cells in the brain, apoptotic cells in the liver, and damage to the lungs, suggesting that synthetic torpor spares tissues from energetic ion radiation. The application of 5′-AMP in combination with either hypoxia or low temperature environment for six hours following irradiation of rat retinal pigment epithelial cells delays DNA repair and suppresses the radiation-induced mitotic catastrophe compared to control cells. We conclude that synthetic torpor protects animals from cosmic ray-simulated radiation and the mechanism involves both hypothermia and hypoxia.

Effects of sleep deprivation in the process of Tau protein dephosphorylation following synthetic torpor in the rat

Effects of sleep deprivation in the process of Tau protein dephosphorylation following synthetic torpor in the rat

Melatonin mediates the reversibility of brain hyperphosphorylated tau protein induced by synthetic torpor in rats.

The hyperphosphorylation of tau protein (PPtau) in the brain is the main pathophysiological marker of tauopathies. Recently was found that when induced by a "synthetic torpor" (ST)1 condition (induced on rats), PPtau accumulations is reversible, as observed in hibernators2 . Thus, ST uncover a latent physiological mechanism able to cope with PPtau and not specifically evolved with hibernation. Aim of the present work was to describe it. We induced ST as already reported2 on 12 Sprague-Dawley rats. Hippocampal and plasma samples were collected at the following experimental conditions: nadir of hypothermia (N); early recovery (ER), as soon as animals reached normothermia following N; 6h following ER (R6). Control (C) animals were also included. Levels of AT8 (p[S020/T205]-tau), p[S9]-GSK3β (inhibited form of the main kinase targeting tau) and plasma melatonin were determined. To better understand in vivo experimental results, we performed in silico simulations of melatonin-tubulin interactions3 . Figure 1 shows, at N, a huge amount of AT8 and high levels of p[S9]-GSK3β and melatonin in respect to C. All factors returned to normal at R6. These paradoxical results (i.e. the coexistence of high levels of PPtau and p[S9]-GSK3β) could be interpreted considering the destabilization of microtubules (MTs) induced by hypothermia as the main trigger of the whole process, then eliciting a neuroprotective physiological response mediated by melatonin, also interacting with MTs. To sustain this hypothesis, we also provide computational analysis of the microtubule stability as a function of temperature and other factors, such as melatonin binding. The molecular docking simulation shows that melatonin did not bind to 1sa0 structure, but it binds to one site of 1jff structure on the α-tubulin monomer (Figure 2). This is further elucidated using a molecular fingerprint representation (Figure 3), showing the binding site of melatonin with respect to those well-known binding locations. Our results could pave the way for an effective new strategy to contrast tauopathies, with next-step studies aimed to pharmacologically interacting with this process at physiological temperature.

Contemporary biomedical engineering perspective on volitional evolution for human radiotolerance enhancement beyond low-earth orbit.

A primary objective of the National Aeronautics and Space Administration (NASA) is expansion of humankind’s presence outside low-Earth orbit, culminating in permanent interplanetary travel and habitation. Having no inherent means of physiological detection or protection against ionizing radiation, humans incur capricious risk when journeying beyond low-Earth orbit for long periods. NASA has made large investments to analyze pathologies from space radiation exposure, emphasizing the importance of characterizing radiation’s physiological effects. Because natural evolution would require many generations to confer resistance against space radiation, immediately pragmatic approaches should be considered. Volitional evolution, defined as humans steering their own heredity, may inevitably retrofit the genome to mitigate resultant pathologies from space radiation exposure. Recently, uniquely radioprotective genes have been identified, conferring local or systemic radiotolerance when overexpressed in vitro and in vivo. Aiding in this process, the CRISPR/Cas9 technique is an inexpensive and reproducible instrument capable of making limited additions and deletions to the genome. Although cohorts can be identified and engineered to protect against radiation, alternative and supplemental strategies should be seriously considered. Advanced propulsion and mild synthetic torpor are perhaps the most likely to be integrated. Interfacing artificial intelligence with genetic engineering using predefined boundary conditions may enable the computational modeling of otherwise overly complex biological networks. The ethical context and boundaries of introducing genetically pioneered humans are considered.

Reversible Tau Phosphorylation Induced by Synthetic Torpor in the Spinal Cord of the Rat.

Tau is a key protein in neurons, where it affects the dynamics of the microtubule system. The hyperphosphorylation of Tau (PP-Tau) commonly leads to the formation of neurofibrillary tangles, as it occurs in tauopathies, a group of neurodegenerative diseases, including Alzheimer's. Hypothermia-related accumulation of PP-Tau has been described in hibernators and during synthetic torpor (ST), a torpor-like condition that has been induced in rats, a non-hibernating species. Remarkably, in ST PP-Tau is reversible and Tau de-phosphorylates within a few hours following the torpor bout, apparently not evolving into pathology. These observations have been limited to the brain, but in animal models of tauopathies, PP-Tau accumulation also appears to occur in the spinal cord (SpCo). The aim of the present work was to assess whether ST leads to PP-Tau accumulation in the SpCo and whether this process is reversible. Immunofluorescence (IF) for AT8 (to assess PP-Tau) and Tau-1 (non-phosphorylated Tau) was carried out on SpCo coronal sections. AT8-IF was clearly expressed in the dorsal horns (DH) during ST, while in the ventral horns (VH) no staining was observed. The AT8-IF completely disappeared after 6 h from the return to euthermia. Tau-1-IF disappeared in both DH and VH during ST, returning to normal levels during recovery. To shed light on the cellular process underlying the PP-Tau pattern observed, the inhibited form of the glycogen-synthase kinase 3β (the main kinase acting on Tau) was assessed using IF: VH (i.e., in motor neurons) were highly stained mainly during ST, while in DH there was no staining. Since tauopathies are also related to neuroinflammation, microglia activation was also assessed through morphometric analyses, but no ST-induced microglia activation was found in the SpCo. Taken together, the present results show that, in the DH of SpCo, ST induces a reversible accumulation of PP-Tau. Since during ST there is no motor activity, the lack of AT8-IF in VH may result from an activity-related process at a cellular level. Thus, ST demonstrates a newly-described physiological mechanism that is able to resolve the accumulation of PP-Tau and apparently avoid the neurodegenerative outcome.

Phosphorylated Tau protein in the myenteric plexus of the ileum and colon of normothermic rats and during synthetic torpor

Tau protein is of primary importance for neuronal homeostasis and when hyperphosphorylated (PP-Tau), it tends to aggregate in neurofibrillary tangles, as is the case with tauopathies, a class of neurodegenerative disorders. Reversible PP-Tau accumulation occurs in the brain of hibernating rodents and it was recently observed in rats (a non-hibernator) during synthetic torpor (ST), a pharmacological-induced torpor-like condition. To date, the expression of PP-Tau in the rat enteric nervous system (ENS) is still unknown. The present study immunohistochemically investigates the PP-Tau expression in the myenteric plexus of the ileum and colon of normothermic rats (CTRL) and during ST, focusing on the two major subclasses of enteric neurons, i.e., cholinergic and nitrergic.Results showed that both groups of rats expressed PP-Tau, with a significantly increased percentage of PP-Tau immunoreactive (IR) neurons in ST vs. CTRL. In all rats, the majority of PP-Tau-IR neurons were cholinergic. In ST rats, the percentage of PP-Tau-IR neurons expressing a nitrergic phenotype increased, although with no significant differences between groups. In addition, the ileum of ST rats showed a significant decrease in the percentage of nitrergic neurons. In conclusion, our findings suggest an adaptive response of ENS to very low core body temperatures, with changes involving PP-tau expression in enteric neurons, especially the ileal nitrergic subpopulation. In addition, the high presence of PP-Tau in cholinergic neurons, specifically, is very interesting and deserves further investigation. Altogether, these data strengthen the hypothesis of a common cellular mechanism triggered by ST, natural hibernation and tauopathies occurring in ENS neurons.

Hibernation as a Tool for Radiation Protection in Space Exploration.

With new and advanced technology, human exploration has reached outside of the Earth’s boundaries. There are plans for reaching Mars and the satellites of Jupiter and Saturn, and even to build a permanent base on the Moon. However, human beings have evolved on Earth with levels of gravity and radiation that are very different from those that we have to face in space. These issues seem to pose a significant limitation on exploration. Although there are plausible solutions for problems related to the lack of gravity, it is still unclear how to address the radiation problem. Several solutions have been proposed, such as passive or active shielding or the use of specific drugs that could reduce the effects of radiation. Recently, a method that reproduces a mechanism similar to hibernation or torpor, known as synthetic torpor, has started to become possible. Several studies show that hibernators are resistant to acute high-dose-rate radiation exposure. However, the underlying mechanism of how this occurs remains unclear, and further investigation is needed. Whether synthetic hibernation will also protect from the deleterious effects of chronic low-dose-rate radiation exposure is currently unknown. Hibernators can modulate their neuronal firing, adjust their cardiovascular function, regulate their body temperature, preserve their muscles during prolonged inactivity, regulate their immune system, and most importantly, increase their radioresistance during the inactive period. According to recent studies, synthetic hibernation, just like natural hibernation, could mitigate radiation-induced toxicity. In this review, we see what artificial hibernation is and how it could help the next generation of astronauts in future interplanetary missions.

Human torpor: translating insights from nature into manned deep space expedition.

During a long-duration manned spaceflight mission, such as flying to Mars and beyond, all crew members will spend a long period in an independent spacecraft with closed-loop bioregenerative life-support systems. Saving resources and reducing medical risks, particularly in mental heath, are key technology gaps hampering human expedition into deep space. In the 1960s, several scientists proposed that an induced state of suppressed metabolism in humans, which mimics 'hibernation', could be an ideal solution to cope with many issues during spaceflight. In recent years, with the introduction of specific methods, it is becoming more feasible to induce an artificial hibernation-like state (synthetic torpor) in non-hibernating species. Natural torpor is a fascinating, yet enigmatic, physiological process in which metabolic rate (MR), body core temperature (Tb ) and behavioural activity are reduced to save energy during harsh seasonal conditions. It employs a complex central neural network to orchestrate a homeostatic state of hypometabolism, hypothermia and hypoactivity in response to environmental challenges. The anatomical and functional connections within the central nervous system (CNS) lie at the heart of controlling synthetic torpor. Although progress has been made, the precise mechanisms underlying the active regulation of the torpor-arousal transition, and their profound influence on neural function and behaviour, which are critical concerns for safe and reversible human torpor, remain poorly understood. In this review, we place particular emphasis on elaborating the central nervous mechanism orchestrating the torpor-arousal transition in both non-flying hibernating mammals and non-hibernating species, and aim to provide translational insights into long-duration manned spaceflight. In addition, identifying difficulties and challenges ahead will underscore important concerns in engineering synthetic torpor in humans. We believe that synthetic torpor may not be the only option for manned long-duration spaceflight, but it is the most achievable solution in the foreseeable future. Translating the available knowledge from natural torpor research will not only benefit manned spaceflight, but also many clinical settings attempting to manipulate energy metabolism and neurobehavioural functions.

Gene expression in the liver of the rat induced in synthetic torpor

Torpor/hibernation is a survival strategy used by several mammals in periods of unfavorable environmental conditions, such as food shortage and low ambient temperature. It is characterized by an active reduction in metabolic rate followed by a decrease in body temperature. The decrease of metabolism and therefore in oxygen demand could be beneficial in many clinical conditions, such as, but not only, transplant and stroke. Recently, rats (non‐hibernating mammals) were successfully induced in a torpor‐mimicking state called synthetic torpor (Cerri, Annu Rev Physiol., 2017) by central inhibition of Raphe Pallidus (RPa), a key brainstem area involved in thermoregulation (Cerri et al., J Neurosci., 2013). Even though this model is very promising, it is still not known how this condition of induced etherotermy acts on a cellular level on a non‐hibernating mammal.Eight male Sprague‐Dawley rats, adapted to a standard Light‐Dark cycle of 12h‐12h (Light: 07 AM–07 PM), were used. Under general anesthesia (Diazepam, 5mg/kg, i.m., Ketamine, 100 mg/kg, i.p.), animals were implanted with a hypothalamic thermistor and a microcannula within the RPa. After recovery from surgery, starting at 7 AM, rats were divided in two groups: i) animals induced in a state of synthetic torpor by the repeated microinjections of the GABA‐A agonist muscimol (1mM, 120nl), within the RPa for 9 hours (Synthetic torpor, n=4); ii) homeothermic animals, injected with artificial cerebrospinal fluid (aCSF) within the RPa (Control, n =4), for 9 hours. At 4 PM, rats were euthanized and the liver was dissected and processed for gene expression analysis by RNA‐Seq.Analysis of the Differential Expression (DE) shows that in synthetic torpor a total of 1107 mRNA sequences were significantly up‐regulated, among which genes involved in the metabolic switch to beta‐oxidation of free fatty acids (fatty acid binding proteins (FABP), Peroxisome proliferator‐activated receptor (PPAR) family, Pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), Carnitine Palmitoyltransferase 1α (CPT1α)) and 1337 significantly down‐regulated.In a preliminary analysis, we report that several upregulated genes in synthetic torpor are known to be upregulated in hibernation as well. These preliminary findings suggest that the specific adaptation observed in natural torpor may be replicated also in non hibernators. Significantly upregulated mRNA expression of genes involved in fatty acid metabolism, in rats induced in synthetic torpor (Synthetic torpor, n=4) vs normothermic ones (Control, n=4) expressed in values of log2FC (FC=fold change). On the right side, their corresponding known function, and a list of hibernating species where a significant upregulation of the genes has been observed in literature. mRNA log2FC Function Upregulated in Fatty‐acid binding proteins (FABP) Facilitation of β‐oxidation in mitochondria Myotis lucifugus (Eddy& Storey, 2004) Ictidomys tridecemlineatus (Epperson et al., 2010) FABP5 1.05 FABP9 1.65 FABP12 1.42 Peroxisome proliferator‐activated receptor (PPAR) family Safe deposition and consumption of fatty acid, avoiding the associated inflammatory risks (Nunn et al. 2007). Linked to the integration of nutritional information and circadian rhythms (Kohsaka & Bass, 2007; Green et al. 2008). Spermophilus tridecemlineatus (Eddy et al., 2004) Myotis lucifugus (Eddy& Storey, 2004) PPARGC1a 3.40 PPARGC1b 1.80 Pyruvate dehydrogenase lipoamide kinase isozyme 4 Shift away from the oxidation of carbohydrates and toward the combustion of stored fatty acids as the primary source of energy during torpor (Buck et al., 2002) Ictidomys Tridecemlineatus (Buck et al., 2002) PDK4 4.03 Carnitine Palmitoyltransferase 1α Fatty acid catabolism Urocitellus parryii (Williams et al., 2011) CPT1α 1.08

Shallow metabolic depression and human spaceflight: a feasible first step.

Synthetic torpor is an induced state of deep metabolic depression (MD) in an organism that does not naturally employ regulated and reversible MD. If applied to spaceflight crewmembers, this metabolic state may theoretically mitigate numerous biological and logistical challenges of human spaceflight. These benefits have been the focus of numerous recent articles where, invariably, they are discussed in the context of hypothetical deep MD states in which the metabolism of crewmembers is profoundly depressed relative to basal rates. However, inducing these deep MD states in humans, particularly humans aboard spacecraft, is currently impossible. Here, we discuss shallow MD as a feasible first step toward synthetic torpor during spaceflight and summarize perspectives following a recent NASA-hosted workshop. We discuss methods to safely induce shallow MD (e.g., sleep and slow wave enhancement via acoustic and photoperiod stimulation; moderate sedation via dexmedetomidine), which we define as an ~20% depression of metabolic rate relative to basal levels. We also discuss different modes of shallow MD application (e.g., habitual versus targeted, whereby shallow MD is induced routinely throughout a mission or only under certain circumstances, respectively) and different spaceflight scenarios that would benefit from its use. Finally, we propose a multistep development plan toward the application of synthetic torpor to human spaceflight, highlighting shallow MD's role. As space agencies develop missions to send humans further into space than ever before, shallow MD has the potential to confer health benefits for crewmembers, reduce demands on spacecraft capacities, and serve as a testbed for deeper MD technologies.

On the immunological limitations of hibernation and synthetic torpor as a supporting technique for astronauts’ radioprotection in deep space missions

On the immunological limitations of hibernation and synthetic torpor as a supporting technique for astronauts’ radioprotection in deep space missions

Autonomic effects induced by pharmacological activation and inhibition of Raphe Pallidus neurons in anaesthetized adult pigs.

The Raphe Pallidus (RPa) is a region of the brainstem that was shown to modulate the sympathetic outflow to many tissues and organs involved in thermoregulation and energy expenditure. In rodents, the pharmacological activation of RPa neurons was shown to increase the activity of the brown adipose tissue, heart rate, and expired CO2 , whereas their inhibition was shown to induce cutaneous vasodilation and a state of hypothermia that, when prolonged, leads to a state resembling torpor referred to as synthetic torpor. If translatable to humans, this synthetic torpor-inducing procedure would be advantageous in many clinical settings. A first step to explore such translatability, has been to verify whether the neurons within the RPa play the same role described for rodents in a larger mammal such as the pig. In the present study, we show that the physiological responses inducible by the pharmacological stimulation of RPa neurons are very similar to those observed in rodents. Injection of the GABAA agonist GABAzine in the RPa induced an increase in heart rate (from 99 to 174bpm), systolic (from 87 to 170mmHg) and diastolic (from 51 to 98mmHg) arterial pressure, and end-tidal CO2 (from 49 to 62 mmHg). All these changes were reversed by the injection in the same area of the GABAA agonist muscimol. These results support the possibility for RPa neurons to be a key target in the research for a safe and effective procedure for the induction of synthetic torpor in humans.

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.