What are the molecular mechanisms that make the turtle brain more resistant to hypoxia?

What are the molecular mechanisms that make the turtle brain more resistant to hypoxia?

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I know that turtle brains, particularly those of individual species that hibernate and burrow are particularly resilient to hypoxia and any tissue damage secondary to a hypoxic event.

What are the known molecular mechanisms behind this resiliency? Does it have to do with the ability of their blood to store/release oxygen over extended periods, or is it molecular factors in the neural tissue that protect it from insult?

There is a bunch of literature on the topic. A good starting point is probably a short description with lots of references in this thesis (page 8), not to talk about other articles, which pop up in google scholar: 1, 2.

The mechanisms are multifaceted and involve principally decrease in oxygen and ATP demands: reduced neuronal activity, lower density of ion channels (but hyper-polarization of the membranes) and so on. Concerning blood flow: "Brain blood flow was continued or increased, and oxygen and creatine phosphate (PCr) stores offered some immediate protection. As PCr declined, turtle brain became increasingly reliant upon anaerobic glycolysis."

Transcriptomic Responses of the Heart and Brain to Anoxia in the Western Painted Turtle

Painted turtles are the most anoxia-tolerant tetrapods known, capable of surviving without oxygen for more than four months at 3°C and 30 hours at 20°C. To investigate the transcriptomic basis of this ability, we used RNA-seq to quantify mRNA expression in the painted turtle ventricle and telencephalon after 24 hours of anoxia at 19°C. Reads were obtained from 22,174 different genes, 13,236 of which were compared statistically between treatments for each tissue. Total tissue RNA contents decreased by 16% in telencephalon and 53% in ventricle. The telencephalon and ventricle showed ≥ 2x expression (increased expression) in 19 and 23 genes, respectively, while only four genes in ventricle showed ≤ 0.5x changes (decreased expression). When treatment effects were compared between anoxic and normoxic conditions in the two tissue types, 31 genes were increased (≥ 2x change) and 2 were decreased (≤ 0.5x change). Most of the effected genes were immediate early genes and transcription factors that regulate cellular growth and development changes that would seem to promote transcriptional, translational, and metabolic arrest. No genes related to ion channels, synaptic transmission, cardiac contractility or excitation-contraction coupling changed. The generalized expression pattern in telencephalon and across tissues, but not in ventricle, correlated with the predicted metabolic cost of transcription, with the shortest genes and those with the fewest exons showing the largest increases in expression.

Citation: Keenan SW, Hill CA, Kandoth C, Buck LT, Warren DE (2015) Transcriptomic Responses of the Heart and Brain to Anoxia in the Western Painted Turtle. PLoS ONE 10(7): e0131669.

Editor: Todd Adam Castoe, The University of Texas Arlington, UNITED STATES

Received: March 11, 2015 Accepted: June 5, 2015 Published: July 6, 2015

Copyright: © 2015 Keenan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper, Supporting Information files, or available through NCBI (accession numbers SRS385157-71). Annotation files may be downloaded from:

Funding: This work was funded by National Science Foundation (IOS 1253939) to DEW, and Natural Sciences and Engineering Research Council of Canada to LTB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.


Snorted, smoked, or injected, cocaine rapidly enters the bloodstream and penetrates the brain. The drug achieves its main immediate psychological effect—the high𠅋y causing a buildup of the neurochemical dopamine.

Dopamine acts as a pacesetter for many nerve cells throughout the brain. At every moment of our lives, dopamine is responsible for keeping those cells operating at the appropriate levels of activity to accomplish our needs and aims. Whenever we need to mobilize our muscles or mind to work harder or faster, dopamine drives some of the involved brain cells to step up to the challenge.

Dopamine originates in a set of brain cells, called dopaminergic (dopamine-making) cells, that manufacture dopamine molecules and launch them into their surroundings. Some of the free-floating dopamine molecules latch onto receptor proteins on neighboring (receiving) cells. Once attached, the dopamine stimulates the receptors to alter electrical impulses in the receiving cells and thereby alter the cells’ function.

The more dopamine molecules come into contact with receptors, the more the electrical properties of the receiving cells are altered. To keep the receiving cells in each brain region functioning at appropriate intensities for current demands—neither too high nor too low—the dopaminergic cells continually increase and decrease the number of dopamine molecules they launch. They further regulate the amount of dopamine available to stimulate the receptors by pulling some previously released dopamine molecules back into themselves.

Cocaine interferes with this latter control mechanism: It ties up the dopamine transporter, a protein that the dopaminergic cells use to retrieve dopamine molecules from their surroundings. As a result, with cocaine on board, dopamine molecules that otherwise would be picked up remain in action. Dopamine builds up and overactivates the receiving cells.

Although cocaine also inhibits the transporters for other neurotransmitter chemicals (norepinephrine and serotonin), its actions on the dopamine system are generally thought to be most important. To understand the powerful nature of cocaine’s actions, it is helpful to realize that dopamine pathways in the brain are very old in evolutionary terms. Early rudiments are found in worms and flies, which take us back 2 billion years in evolution. Thus, cocaine alters a neural circuit in the brain that is of fundamental importance to survival. Such alterations affect the individual in profound ways that scientists are still trying to understand.


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The epaulette shark

Tropical hypoxia

The best-studied examples of hypoxia- and anoxia-tolerant vertebrates,notably the crucian carp, goldfish and freshwater turtles, have evolved their hypoxia tolerance in response to overwintering in freshwater at temperatures close to 0°C. At these low temperatures, the periods they can survive anoxia are counted in months. Probably as a side-effect of the capacity for anoxic overwintering, these animals are also able to tolerate anoxia for a few hours or days at higher temperatures (20–25°C). Since this is at least partly an unnatural situation, the mechanisms utilised to survive at such high temperatures may be less well coordinated. Indeed, goldfish have been found to display neuronal apoptosis after 4 h of anoxia at 22°C(Poli et al., 2003). By contrast, the epaulette shark has evolved to tolerate repeated exposure to severe hypoxia (5% of normoxia) and even anoxia at 25–30°C(Wise et al., 1998 Renshaw et al., 2002) without suffering brain damage, including delayed neuronal apoptosis(Renshaw and Dyson, 1999 Renshaw et al., 2002). Only a few other vertebrates, including the toadfish (Opsanus tau Ultsch et al., 1981), the Oscar cichlid (Astronotus occellatus Muuse et al., 1998) and the tilapia (Oreochromis niloticus Fernandez and Rantin, 1989), are known to tolerate anoxia at temperatures above 25°C.

Hypoxia tolerance has been studied in the epaulette shark inhabiting the reef platform surrounding Heron Island – a small and low coral cay situated close to the southern end of the Great Barrier Reef. At nocturnal low tides, the water on the huge (∼3×10 km) reef platform becomes cut off from the surrounding ocean, essentially forming a very large tide pool. When this happens on calm nights with little water movements, the respiration of the coral and all associated organisms can cause the water [O2]to fall below 18% of air saturation(Routley et al., 2002).

General physiological responses to hypoxia

Like the crucian carp, the hypoxic epaulette shark maintains its ability to move, at least initially, during hypoxia or anoxia. However, as pointed out below, an extended period of anoxia may drive the epaulette shark into a deeper metabolic depression where it loses much of its responsiveness to external stimuli. On the respiratory level, there is a change in the gill perfusion pattern in the epaulette shark that may serve to give improved oxygen uptake (K.-O. Stensløkken, L. Sundin, G. E. Nilsson and G. M. C. Renshaw, unpublished observations) and ventilatory frequency increases to achieve short-term tolerance to moderate hypoxia(Routley et al., 2002). Interestingly, several other basic physiological responses of the epaulette shark to hypoxia appear to be different from those of other vertebrates,including those that readily tolerate hypoxia. Thus, unlike many other animals, the epaulette shark does not increase blood glucose levels or haematocrit during acute or chronic hypoxia. Indeed, its haematocrit is quite low (10–15% Routley et al.,2002). Moreover, its cerebral blood flow is maintained rather than increased during hypoxia(Söderström et al.,1999b). In virtually all other vertebrates examined, from teleost fishes and frogs to crocodiles, turtles and mammals, brain blood flow is stimulated by hypoxia (Söderström et al., 1999a,b Söderström-Lauritzen et al.,2001). Still, there appears to be a hypoxia-induced cerebral vasodilation in the epaulette shark brain, since the shark displays a 50%decrease in systemic blood pressure (accompanied by bradycardia) during hypoxia (Söderström et al.,1999b). However, unlike most other vertebrates, adenosine does not seem to be involved in the hypoxic cerebral vasodilation(Söderström et al.,1999b).

Hypoxic-preconditioning primes metabolic and respiratory responses

Exposure to a non-lethal episode of hypoxia increases hypoxia tolerance in both tolerant (Prosser et al.,1957) and non-tolerant species(Dirnagl et al., 2003 Samoilov et al., 2003). Interestingly, the way the epaulette shark is exposed to hypoxia on its reef appears to be a natural parallel to the hypoxic pre-treatment regimen, termed hypoxic-preconditioning in biomedical science. Initially during a period of spring tides, the tides become lower and lower on subsequent nights. Consequently, the epaulette shark will experience longer and longer periods of hypoxia (Fig. 3).

Hypoxic-preconditioning on a coral reef platform like that of Heron Island.(A) At very low tides, the water on the platform gets cut off from the surrounding ocean, essentially forming a very large tide pool. If this happens at night, the respiration of the reef organisms will make the water hypoxic,particularly on calm nights with little wave action. (B) The tide chart shows a period where the tides become lower and lower over a few days. As a result,the time that the water on the reef platform is disconnected from the ocean will increase in length for each subsequent night, causing the nocturnal hypoxic episodes to become longer and longer and therefore increasingly severe. Such `natural preconditioning periods' occur once or twice per month.

Hypoxic-preconditioning on a coral reef platform like that of Heron Island.(A) At very low tides, the water on the platform gets cut off from the surrounding ocean, essentially forming a very large tide pool. If this happens at night, the respiration of the reef organisms will make the water hypoxic,particularly on calm nights with little wave action. (B) The tide chart shows a period where the tides become lower and lower over a few days. As a result,the time that the water on the reef platform is disconnected from the ocean will increase in length for each subsequent night, causing the nocturnal hypoxic episodes to become longer and longer and therefore increasingly severe. Such `natural preconditioning periods' occur once or twice per month.

An experimental regimen of hypoxic-preconditioning prior to respirometry shows that metabolic characteristics of the epaulette shark are significantly altered. The rate of normoxic oxygen consumption is lowered by ∼30% and there is a significant ∼20% drop in the shark's critical [O2],bringing it close to the critical [O2] of the crucian carp and goldfish (Routley et al.,2002). (The critical [O2] is the lowest oxygen concentration where the routine rate of oxygen consumption can be maintained.)Another study has shown that the deeper neural depression that the shark will finally enter during anoxia (see below) is reached sooner if the shark has been pre-exposed to anoxia (Renshaw et al., 2002). However, sharks retain the ability to enter metabolic and ventilatory depression in response to anoxia even when they have been away from the preconditioning effects of their natural environment for more than 6 months (G. M. C. Renshaw, unpublished).

Adenosine and metabolic depression in the epaulette shark

An elevated adenosine level acts as a trigger to disengage energy-expensive cellular processes (Newby,1984), regulate glycolytic rate, stimulate cerebral blood flow and initiate metabolic depression in hypoxia- and anoxia-tolerant species(Nilsson, 1991 Nilsson and Lutz, 1992 Perez-Pinzon et al., 1993 Boutilier, 2001 Lutz et al., 2003). Adenosine's net effect slows energy use while increasing anaerobic ATP production to extend survival time.

While cerebral blood flow is not stimulated by adenosine during anoxia in the epaulette shark (see above), it seems to play a role in the metabolic depression of anoxic epaulette sharks. Exposing the epaulette shark to anoxia resulted in a 3.5-fold increase in brain adenosine levels when compared with normoxic controls (Renshaw et al.,2002). Moreover, after ∼40 min in anoxia, the epaulette sharks became unresponsive and lost their righting reflex while they still successfully defended their brain ATP levels. Thus, at this stage they appear to enter into a deeper phase of metabolic depression. Adenosine may be particularly important for entering this second stage since sharks treated with aminophylline, an adenosine receptor blocker, lost their righting reflex much later, at a point when brain ATP levels had started to fall(Renshaw et al., 2002). Interestingly, this first anoxic episode appeared to prime the sharks neural depression, since a second anoxic episode 24 h later led to unresponsiveness(with maintained brain [ATP]) within 20 min rather than 40 min(Renshaw et al., 2002).

Glutamate and GABA in the epaulette shark brain

The ability to maintain brain glutamate homeostasis in response to low oxygen levels distinguishes hypoxia- and anoxia-tolerant vertebrates from intolerant species, which respond with a surge in extracellular glutamate levels that ultimately culminates in neuronal death (see Lutz et al., 2003 for a review). In addition, hypoxia-tolerant species, as mentioned, show a neuroprotective increase in GABA levels(Nilsson, 1990 Nilsson et al., 1990, 1991 Nilsson and Lutz, 1993). Histological staining for glutamate in epaulette shark brain(Fig. 4A) indicates that glutamate homeostasis is either maintained or significantly lowered in descending axon tracts such as the median longitudinal fasciculus and the fasciculus of Steida in the brainstem after exposure to hypoxia (5% of air saturation G. Wise and G. M. C. Renshaw, unpublished observations). In the median longitudinal fasciculus, this was concomitant with a significant increase in GABA, localised to small GABAergic neurons (J. M. Mulvey and G. M. C. Renshaw, unpublished observations Fig. 4B,C). These observations suggest that the epaulette shark may utilise a changed balance between the GABA and glutamate transmitter systems to induce metabolic depression in selected brain areas. Where glutamate levels are maintained, these may be needed to re-establish neuronal activity once oxygen is restored (Milton et al.,2002).

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Brain Mitochondria and HIF-1 Transcriptional Activity in Hypoxia

According to current concepts, the leading role in development of adaptation to hypoxia belongs to hypoxia-inducible factor 1 (HIF-1), a specific protein factor induced by hypoxia. This factor discovered in the early 1990s (Wang and Semenza, 1993 Semenza, 2002, 2007, 2009) functions as the major regulator of oxygen homeostasis. HIF-1 is a mechanism that the body uses to respond to hypoxia by controlling expression of proteins responsible for oxygen delivery to cells, i.e., HIF-1 mediates cell adaptive responses to changes in tissue oxygenation.

HIF-1 is a heterodimeric redox-sensitive protein consisting of two subunits, the cytoplasmic inducible, oxygen-sensitive α subunit (Semenza, 2002, 2007, 2009), which is expressed in practically all mammalian cells, and the constitutive α subunit. The HIF-1 activity depends primarily on the HIF-1α subunit whose synthesis is controlled by the MAPK and P13K signaling systems activated by the tyrosine kinase receptor. The receptor agonists include tyrosine hydroxylase, cytokines, growth factors (such as insulin-like factor), and succinate. Normally, the intracellular level of HIF-1α subunit is low because this subunit undergoes proteasomic degradation in oxygen-dependent reactions of prolyl hydroxylation and ubiquitination. Hypoxia creates prerequisites for inactivation of prolyl hydroxylase reactions and thereby provides HIF-1α stabilization and accumulation, induction of HIF-1α transcription and translocation to the nucleus, HIF-1α heterodimerization with the HIF1 β/ARNT subunit, formation of the active transcription complex HRE, expression of HIF-1 dependent target genes, and synthesis of protective, adaptive proteins (Semenza, 2002, 2007, 2009 Kim et al., 2006).

Our investigations have shown that under hypoxic preconditioning, neither free-radical processes nor cytokines and NO perform the function of signaling mechanisms for immediate adaptation responsible for the accumulation of HIF-1α in the early posthypoxic period, and they are likely to be only secondary messengers playing an important role in the formation of delayed adaptation (Kirova et al., 2013, 2014 Lukyanova, 2014).

At the same it is known that oxygen-dependent process of HIF-1α prolyl-hydroxylation and proteasomic degradation occurring in the cytosol of normoxic cells is coupled with utilization of the NAD-dependent substrate of TAC cycle, α-ketoglutarate, while another TAC cycle substrate, succinate, is an allosteric inhibitor of this process (Semenza, 2002, 2007, 2009 Hewitson et al., 2007). Hypoxia inhibits the malate-aspartate bypass, which provides α-ketoglutarate to the cytosol, whereas succinate synthesis is intensified. This creates prerequisites (along with O2 and Fe 2+ shortage) for inactivation of prolyl hydroxylase reactions and HIF-1α stabilization, accumulation and potentiation of HIF-1α transcriptional activity.

Now it is proved that functioning of the mitochondrial respiratory chain is coupled with the hypoxia-induced transcriptional expression of HIF-1α. It was shown that even a partial (20%) suppression of C-II activity almost completely inhibited the hypoxic induction of HIF-1α. However it recovered in the presence of succinate (Vaux et al., 2001 Paddenberg et al., 2003 Napolitano et al., 2004 Selak et al., 2005 Hewitson et al., 2007 Koivunen et al., 2007).

We have also shown, that induction of HIF-1α requires a low C-1 activity and a high C-II activity, i.e., potentiation of succinate oxidation (Lukyanova et al., 2008b, 2009b, 2011 Kirova et al., 2013, 2014 Lukyanova, 2014). If that is the case, a relationship should exist between activation of the succinate oxidase oxidation pathway and HIF-1α formation in hypoxia (Figure 2).

Figure 2. Interaction of succinate oxidase-mediated oxidation (C-II) and HIF-1α transcriptional activity in hypoxia. Activation of C-II contributes to inhibition of prolyl hydroxylase-mediated reactions (PHD) HIF-1α accumulation and translocation to the nucleus, and expression of HIF-1α-dependent adaptation genes. C-II, C-III, C-IV - mitochondrial enzyme complexes TAC, tricarbonic acid cycle PHD, prolyl hydroxylase-mediated reaction.

However, it should be kept in mind that excessive tissue accumulation of succinate in pathological conditions related with impairment of the SDH oxidative function or deficiency of this enzyme may result in excessively high tissue content of HIF-1α and, eventually, uncontrolled potentiation of proliferation, encephalomyopathy, and tumors (Chávez et al., 2000). Thus, succinate dehydrogenase mutations were shown to induce renal, gastric, and thyroid carcinoma, and degeneration of striatal spiny neurons (Huntington's disease) (Baysal, 2003 Selak et al., 2005).

Molecular Mechanisms of the Redox Regulation of the Na,K-ATPase

This review considers the molecular mechanisms involved in the redox regulation of the Na,K-ATPase. The enzyme creates a transmembrane gradient of sodium and potassium ions, which is necessary for the vital activity of all animal cells, and acts as a receptor of cardiotonic steroids (CTSs), which regulate cell proliferation and apoptosis. The function of the Na,K-ATPase depends on the cell’s redox status. Although oxidative stress was initially found to inhibit the enzyme, it is clear now that the redox regulation of the Na,K-ATPase activity is a complex process that cannot be explained only by oxidative damage to the protein. Na,K-ATPase activity is maximal at physiological oxygen concentrations and decreases by both hypoxia and hyperoxia, as well as due to decrease or increase of intracellular glutathione concentrations. Thus, a specific range of redox conditions provides maximal activity of the Na,K-ATPase. Now it is obvious that a disturbance of the Na,K-ATPase activity in a number of pathologies such as hypoxia, ischemia, diabetes, Alzheimer’s disease is associated with a change in redox status in the cells. The receptor function of the Na,K-ATPase also depends on the cell redox status and it isshould be taken into account when studying the effects of cardiotonic steroids on cells and tissues. The very special point of this review is the redox modifications of thiol groups in Na,K-ATPase subunits and the regulatory processes in which they are involved in normal and pathological conditions. Insight into the molecular mechanisms of redox regulation provides a better understanding of what is necessary for preventing Na,K-ATPase dysfunction in pathological conditions and thus reducing cell damage.

Prenatal Hypoxia in Rats

In our studies for more than two decades we have developed and intensively utilized a model of normobaric hypoxia using laboratory Wistar rats at various days of pregnancy which is described in detail in our early work (Zhuravin, 2002 Lavreneva et al., 2003). For this we use a 100 L chamber supplied with gas analysis equipment, thermoregulation, and facility for removal of excess CO2. The hypoxic conditions are achieved by replacing oxygen with nitrogen down to 7% O2 concentration (or other desired level) during 10 min and then remaining at this level for 3 h. This paradigm provides a reliable and reproducible setting for maintaining hypoxic conditions and obtaining the material for further experiments either from the fetuses or rat pups during different stages of their postnatal development. The detailed analysis of the data obtained in these studies has recently been reviewed in Zhuravin et al. (2018). Below we shall discuss the main effects of prenatal hypoxia on rat brain anatomical, biochemical and functional properties (Figure 2) comparing the results of our studies with the data of other research groups employing different hypoxia paradigms.

Figure 2. Postnatal deficits observed in the rat model of prenatal hypoxia.

Structural Changes in Rat Brain After Prenatal Hypoxia

There is a significant amount of data demonstrating that prenatal hypoxia results in a set of physiological changes in rat embryos leading to functional and behavioral changes in the postnatal period including reduced body weight of newborn pups (Gross et al., 1981 Olivier et al., 2005 Dubrovskaya and Zhuravin, 2010). Although some authors have not observed significant changes in brain weight of rat pups after prenatal hypoxia (Gross et al., 1981 Liu Z. H. et al., 2011) they have reported changes in the brain to body weight ratio (Liu Z. H. et al., 2011) as well as DNA/protein ratio (Gross et al., 1981). In the experiments with chronic prenatal hypoxia (10.5% O2, E4-E21) the decreased brain weight has been reported both in the fetuses and 6-week old offspring (Wei et al., 2016). However, other authors reported an increased brain weight in male offspring of Sprague-Dawley rats, subjected to maternal hypoxia (10.5% oxygen) on gestational day 21 (Zhang et al., 2016).

Obstructive sleep apnoea during pregnancy, and especially in late gestation, is a rather common complication in women. Intermittent hypoxia, to which the fetus is subjected during apnoea episodes, induces metabolic dysfunction which can be detected as increased body weight and higher adiposity index in adult male offspring. This suggests differential sex-dependent effects of the condition on expression of fetal genes (Khalyfa et al., 2017).

The major anatomical and structural alterations in rat brain after prenatal hypoxia are manifested at the level of the cellular composition of various brain structures (the cortex, hippocampus, striatum, cerebellum, etc.), including degeneration of neuronal cells, gliosis and apoptosis (Rees and Inder, 2005 Golan and Huleihel, 2006 Zhuravin et al., 2006 Liu Z. H. et al., 2011 Wang et al., 2017). Increased levels of apoptosis in rat brain after hypoxia correlated with upregulation of caspases, in particular of active caspase-3, which contributed to alteration in neuronal composition of different cortical layers (Vasilev et al., 2016a). Activation of apoptotic events caused by perinatal hypoxia modeling birth asphyxia was also shown in the cortex and CA1 area of the hippocampus in rat pups during the first 2 weeks after the insult resulting in reduced cell density and the accumulation of cells with nuclear fragmentation specific for apoptosis (Daval and Vert, 2004).

Importantly, it was also shown that prenatal hypoxia affects the cells in brain neurogenic zones and, in particular, the levels of expression of the protein paired box 6 (Pax6) which plays an important role in neurogenesis, cell proliferation, differentiation and survival during the development of the central nervous system (Simpson and Price, 2002). Although in the fetuses subjected to prenatal hypoxia the levels of Pax6 were increased in the subventricular zone and subgranular zone of the hippocampal dentate gyrus, they were significantly decreased in the cerebral cortex (So et al., 2017). This finding correlates with the reduced number of neuronal cells in rat cortex during the first month of postnatal life observed in our studies (Vasiliev et al., 2008).

Using light and electron microscopic techniques it was demonstrated that prenatal hypoxia caused a delay in differentiation of neurones and formation of synaptic contacts in rat neuropil as well as affecting myelination of nerve fibers at the ultra-structural levels both in the neocortex and basal ganglia (Zhuravin et al., 2006 Vasiliev et al., 2008 Vasilɾv et al., 2010). In particular, on postnatal days P10-30 in the brain cortex there was a significant decrease in the total number of pyramidal neurones (Vasiliev et al., 2008). However, this decrease was observed only during the first month of rat postnatal development and only in the group subjected to prenatal hypoxia on E14, but not on E18 (Dubrovskaya and Zhuravin, 2010 Vasilev et al., 2016b). Changes in cell composition have also been observed in the dorsal hippocampus of hypoxic rats, especially in the CA1 with increased number of neurones possessing retracted apical dendrites (Zhuravin et al., 2009a).

The effects of prenatal hypoxia were more profound when it was applied at mid rather than late gestation and became less apparent with development of rats in the postnatal period (Nyakas et al., 1996 Dubrovskaya and Zhuravin, 2010). Because formation of main brain anatomical architecture starts on embryonic day 12 (E12) and the precursors of cortical and striatal neurons actively proliferate on E14 and of the hippocampal neurons on E15 (Rice and Barone, 2000), the timing of hypoxia or other insults determines their impact on cellular composition and structure of specific brain regions and therefore affects formation of the physiological functions related to them. For example, prenatal hypoxia on E14 and E18 resulted in different outcomes of neuronal migration into the cortical layers of rat cortex and performance of behavioral tasks in postnatal life (Vasilev et al., 2016b).

Changes in Development of Brain Functions

There are many tests available to assess the development of brain integrity in rats at very early stages after birth including habituation, exploratory behavior, reactivity and motor coordination. The most commonly employed tests include the body-righting reflex, negative geotaxis, placing, homing, head elevation, ascending on a wire mesh, which are extensively reviewed in Rice and Barone (2000). In our model of prenatal hypoxia in rats we also observed a delay in pup maturation (reduced body weight during the first month of life, delayed eye opening time and the onset of separation of the external ear from the skin of the head) and development of various sensory-motor reactions including the body-righting reflex, negative geotaxis, forelimb placing reaction, maintenance of balance on a rotating grid etc (Dubrovskaya and Zhuravin, 2010). Some developmental features such as later separation of the external ear from the scull, the forepaw-placing reaction and the whisker placing reaction were found to be delayed only after prenatal hypoxia on E14 and not on E18 (Dubrovskaya and Zhuravin, 2010 Vasilev et al., 2016b).

Although the deficit in innate motor reactions of new-born rats after prenatal hypoxia, observed in our studies, becomes less pronounced with pup development during the first month of postnatal life, execution of more skilful movements, e.g., reaching and pushing, and learning new motor reflexes were still compromised in adulthood (Zhuravin et al., 2002). This correlates with the observations that motor and coordination abilities remained partially impaired in the old rats subjected to prenatal hypoxia, especially under high oxygen demand (Jänicke and Coper, 1994). Some authors link the cause of motor deficit observed after prenatal hypoxia with a failure in the migration and maturation of oligodendroglial progenitor cells causing delay of myelination in the cerebellum (Barradas et al., 2016).

Unlike motor functions which are practically compensated and restored during the first month of pups' development, the cognitive deficits caused by prenatal hypoxia on E14 or E18 remain detectable at all tested stages of postnatal life. For example, various types of rat memory (working, short and long-term memory) assessed by 8-arm maze and novel object recognition were compromised and correlated with the structural changes observed in the hippocampus (Zhuravin et al., 2011 Nalivaeva et al., 2012 Cunha-Rodrigues et al., 2018). Some studies link working memory impairment observed both in juvenile and adult rats subjected to prenatal hypobaric hypoxia with increased levels of phosphatidylinositol 4,5-diphosphates and phosphatidylinositol 4-phosphates in the hippocampus and upregulated expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) (Tyul'kova et al., 2015). Maternal hypoxia on days 15� was shown to result in increased activity of metalloproteinases and significant cell death in the hippocampus of rat pups on days 0𠄷 after birth, which correlated with worsened development of their neurobehavioral functions (Tong et al., 2010). On the other hand learning deficits in adult rats subjected to prenatal hypoxia was shown to correlate with a significant reduction in the number of neurones positive to the polysialylated markers in the dentate granular zone of the hippocampus (Foley et al., 2005).

Prenatal transient systemic hypoxia-ischaemia created in Sprague-Dawley rats by occlusion of uterine arteries for 60 min on E18 has recently been reported to cause a sustained motor deficit and poor social interaction in young adult rats, which were accompanied by impaired white matter microstructure and diffusion abnormalities in the hippocampus, striatum and thalamus (Robinson et al., 2018). In a similar model of prenatal hypoxia on E18 adult rat offspring also demonstrated increased anxiety behavior and reduced spatial exploration and deficit in habituation memory (Sab et al., 2013). Prenatal ischaemia induced by unilateral ligation of the uterine artery on E17 was also shown to induce motor hyperactivity and deficits in information encoding, and short- and long-term memory in adult offspring (P40 to P80) although no impairments in spatial learning or working memory were observed when animals were tested in the Morris water maze (Delcour et al., 2012).

According to our data, rats subjected to prenatal hypoxia demonstrate reduced ability to learn new instrumental reflexes. Thus, on postnatal days 20-30 the number of rats in the experimental groups capable of learning to push a piston inside a narrow tube was 30% lower compared to the control group and at the age of 3 months, the number of hypoxic rats capable of learning this reflex for a certain duration was 40% lower than in the control group (Zhuravin et al., 2002). Analysing the ability of rats to remember the learnt task after a 5 week interval it was found that control rats were able to remember the learnt duration of the reinforced movements while hypoxic rats returned to the level before training, which implies a significant memory deficit caused by prenatal hypoxia.

Impaired learning abilities of rats were also reported in other paradigms of prenatal hypoxia. Thus, a 30-min hypoxic insult by complete clamping of the uterine vasculature on E17 was found to impair spatial memory in the Morris water maze and caused learning deficits in the passive avoidance test during the first month of development (Cai et al., 1999). These abnormalities the authors linked to the reduction in NOS expression and activity in the affected brain areas. On the contrary, gestational intermittent hypoxia induced by computer-controlled exposure of pregnant Sprague-Dawley rats either to room air or to 10% O2 alternately every 90 seconds starting on E5 until delivery did not result in any changes in acquisition and retention of a spatial memory both at 1 and 4 months of age (Gozal et al., 2003). This outcome, however, might be attributed to the development of tolerance to hypoxia in the fetal brain caused by repeated short episodes of maternal hypoxia.

Synaptic Plasticity

Existing literature suggests that impaired brain functions caused by prenatal hypoxia are related to impaired neurotransmitter circuits and synaptic plasticity (Herlenius and Lagercrantz, 2004 Barradas et al., 2016 McClendon et al., 2017). In rats submitted to prenatal hypoxia on E14 we have also observed a significant reduction in the number of synaptopodin-positive dendritic spines (Zhuravin et al., 2011 Vasilev et al., 2016b) which are fundamental for the formation of synaptic contacts and memory (Martin et al., 2000 Zito et al., 2009 Segal, 2010). The decrease in the number of synaptopodin-positive dendritic spines was particularly evident in the molecular layer of the neocortex and in the CA1 area of the hippocampus which correlated with impaired working memory (Zhuravin et al., 2009b). This decrease in the number of labile dendritic spines in the CA1 area of the hippocampus might be related to the changes in the entorhinal cortex which, in humans, is considered to be the earliest event in the development of Alzheimer's disease (Killiany et al., 2002). Damage in the medial and lateral entorhinal cortices correlating with impaired memory have indeed been reported in adult rats subjected to prenatal hypoxia on E17 (Delcour et al., 2012). The reduction of the number of synaptopodin-positive spines along with decreased ability for learning is also observed in normally aging rats, which could be one of the reasons for cognitive decline related to advanced age, and in the sporadic form of Alzheimer's disease (Zhuravin et al., 2011 Arnold et al., 2013).

The mechanisms of impairment of neuronal interactions caused by prenatal hypoxia in rat brain are more complex and do not involve only the changes in the number of dendritic spines and neuronal contacts but also result in disruption in the development of various mediator systems in the postnatal period (Nyakas et al., 1994 Gerstein et al., 2005 Tyulkova et al., 2011). As we have also observed, prenatal hypoxia on E14 resulted in a decrease in the number of VAChT-positive cholinergic terminals which form synapses on the bodies of the pyramidal neurones in layers V-VI of the parietal cortex. On the other hand, the EAAT levels were found to be much higher in hypoxic animals resulting in spontaneous epileptogenic activity and increased kindling in response to pharmacological agents and other external stimuli (Zhuravin et al., 2018) and even a weak electric shock could induced seizure episodes in 1.5 years old rats subjected to prenatal hypoxia on E14 with more pronounced average duration than in control animals (Kalinina et al., 2015).

Changes at the Molecular Level

Structural and functional changes in rat brain after prenatal hypoxia are underlined by significant alterations in its biochemical characteristics including various classes of molecules (nucleic acids, proteins and lipids) and metabolic pathways (Gross et al., 1981 White and Lawson, 1997 Peyronnet et al., 2000 Beltran-Parrazal et al., 2010 Camm et al., 2011). For example, acute prenatal hypoxia on E14 affects activities of the different forms (cytosolic, membrane-bound, and soluble) of acetyl- and butyryl-cholinesterases (AChE and BChE) in the sensorimotor cortex detected at various stages of postnatal ontogenesis (Lavreneva et al., 2003 Kochkina et al., 2015). The increase in brain BChE activity might have a compensatory effect on the stress response of the brain due to the enzyme's ability for hydrolysing various toxic agents (for review see Lockridge, 2015). However, with aging it can lead to neurodegeneration and is considered as an indicator of Alzheimer's disease in humans (Greig et al., 2002). Changes in AChE and BChE activities after prenatal hypoxia are also observed in blood plasma of rats at various stages of postnatal development, which might affect their immune and stress responses (Kozlova et al., 2018).

Prenatal hypoxia on E14 also affected the levels of brain expression and activity of such peptidases as neprilysin and endothelin-converting enzymes (Nalivaeva et al., 2004, 2012) and altered the adenylate cyclase system (Zhuravin et al., 2002). In particular, the enzyme activity of adenylate cyclase in the striatum, which reversely correlates with the ability of rats to learn instrumental reflexes, was much higher in rats subjected to prenatal hypoxia and correlated with their learning deficits.

Hypoxia, and prenatal hypoxia in particular, are known to regulate expression of APP whose gene has a hypoxia-responsive element (Lahiri et al., 2003). This protein plays an important role in development of the nervous system (Young-Pearse et al., 2007) and the Aβ peptide produced from its precursor has a causative role in development of Alzheimer's disease (Hardy and Selkoe, 2002). Analysis of the content of APP in rats subjected to prenatal hypoxia also revealed significant changes in the levels of this protein in the sensorimotor cortex (Nalivaeva et al., 2003). Prenatal hypoxia not only led to an increase in the content of the membrane bound form of APP at different postnatal stages of rat development but also reduced production of its soluble forms (sAPP) which have protective neuritogenic properties (for review see Chasseigneaux and Allinquant, 2012). Moreover, the most significant changes after prenatal hypoxia on E14 were observed on P10-P30 when formation of rat brain neuronal networks is the most active and any deficit of neuritogenic factors might underlie cognitive dysfunctions. These data also indirectly testify that prenatal hypoxia might modify the activity of α-secretase enzymes, which are important for releasing sAPPα and hence preventing formation of Aβ. The deficit of α-secretase after prenatal hypoxia might also explain the decreased production of soluble AChE since this activity can also be involved in AChE secretion (Nalivaeva and Turner, 1999). Moreover, maternal hypoxia in rats was shown to result in an increase in the activity of matrix metallopeptidases (MMPs) and decreases in the expression of tissue inhibitor of metalloproteinases (TIMPs) in the brain of neonatal rats, which can also underlie remodeling of neuronal circuits during brain development (Tong et al., 2010).

Although not studied in the models of prenatal hypoxia there is evidence that hypoxic conditions can alter expression of the γ-secretase complex (Liu et al., 2016) which not only regulates animal development via Notch signaling but also is a major enzyme involved in production of Aβ and Alzheimer's disease pathogenesis (Hartmann et al., 2001). Studies in transgenic mice modeling Alzheimer's disease have confirmed that prenatal hypoxia accelerates development of the pathology (Zhang et al., 2013).

One of the important factors which predisposes to formation of the sporadic form of Alzheimer's disease is the deficit of amyloid clearance (for review see Baranello et al., 2015). Our and other studies have shown that prenatal hypoxia leads to a significant deficit of the major amyloid-degrading enzyme neprilysin in rat brain at various stages of postnatal development (Nalivaeva et al., 2004, 2012 Wang et al., 2014). Together with the deficits of other amyloid-degrading enzymes e.g., endothelin-converting enzyme, angiotensin-converting enzyme and insulin-degrading enzyme, which are also affected by prenatal hypoxia or ischaemia (Nalivaeva et al., 2004), reduced NEP activity can lead to permanent insufficiency of amyloid clearance over the years and hence predispose to development of Alzheimer's disease pathology in later life (Nalivaeva et al., 2008 Wang et al., 2010).

On the other hand we observed an increased level of TTR expression in the choroid plexus of rat pups subjected to prenatal hypoxia (Vasilev et al., 2018). TTR is suggested to play a contributory role in regulation of the levels of brain Aβ (Li and Buxbaum, 2011 Du et al., 2012). Since APP expression in rat brain is also increased after prenatal hypoxia (Nalivaeva et al., 2004) it is possible that TTR increase might also function as a measure to protect the brain from potential accumulation of neurotoxic levels of Aβ and partially compensate for any reduction in NEP activity. However, TTR itself might undergo protein misfolding and aggregation leading to TTR amyloidosis (Coles and Young, 2012).

The naked mole-rat as an animal model in biomedical research: current perspectives

Abstract: The naked mole-rat (NMR) is a subterranean rodent that has gained significant attention from the biomedical research community in recent years as molecular mechanisms underlying its unusual biology start to be unraveled. With very low external mortality, NMRs have an unusually long lifespan while showing no signs of aging, such as neurodegeneration or cancer. Furthermore, living underground in large colonies (100 to 300 animals), results in comparatively high carbon dioxide and low oxygen levels, from which NMRs have evolved extreme resistance to both hypoxia and hypercapnia. In this paper we have summarized the latest developments in NMR research and its impact on biomedical research, with the aim of providing a sound background that will inform and inspire further investigations.

Keywords: naked mole-rat, longevity, cancer, hypoxia, nociception, pain

The naked mole-rat (NMR) (Heterocephalus glaber) is a subterranean mammal, which has recently gained interest from scientists across a variety of research fields. Unlike the majority of mammals, NMRs are poikilothermic and eusocial, ie, are cold-blooded and have a single breeding female within a colony. 1 In addition to these features, which have limited biomedical translatability, NMRs have also evolved several physiological adaptations to habituate to their extreme environmental conditions, which have led researchers to study this mammal with the hypothesis that by understanding the extreme biology of NMRs, more will be understood about normal mammalian physiology. Although studied for several decades, it has been recent advances in sequencing technology that have helped fuel the current surge in research dedicated to understanding more about the molecular mechanisms underlying NMR biology, and thus making it a major model in biomedical research.

In this paper, we will firstly describe the unusual physiological features of NMRs compared to other mammals, before focusing on recent findings that demonstrate the significant impact that this relatively new model species is having on biomedical research in areas such as longevity, cancer, hypoxic brain injury, and nociception. Finally, we will discuss the limitations of NMRs as a laboratory model in biomedical studies, as well as highlighting some future perspectives for further understanding the extraordinary biology of NMRs.

NMR eusociality and poikilothermy

NMRs belong to the Bathyergidae African mole-rat family and are found in arid regions of East Africa, mainly in Somalia, Kenya, and Ethiopia. The bathyergids are endemic to Sub-Saharan Africa and live in a variety of habitats that differ with regards to their relative humidity, soil types, and vegetation diversity. 2 According to recent molecular-based phylogenetic analysis, the bathyergids can be divided into six main genera: Bathyergus, Georychus, Heliophobius, Fukomys, Cryptomys and Heterocephalus. 3 Species among these genera can be split into either solitary species (eg, the Cape mole-rat, Georychus capensis and the silvery mole-rat, Heliophobius argenteocinereus), social species (eg, the Natal mole-rat, Cryptomys hottentotus natalensis, and the Mahali mole-rat, Cryptomys hottentotus mahali), and eusocial species: (eg, the NMR, H. glaber, and the Damaraland mole-rat [DMR], Fukomys damarensis). 2,4

Like social insects, such as bees or termites, NMRs are eusocial and live in colonies of up to 300 animals. 1,4,5 Strict eusociality, defined by overlapping generations within a colony, cooperative offspring care, and division of labor between reproductive and non-reproductive groups of animals, is extremely rare among mammals and only NMRs and DMRs fulfill all of these characteristics. 1 Within each colony, a single breeding female gives birth to pups and remains reproductively active into old age. 6,7 Morphologically, the breeding female is usually larger than the other mole-rats of the colony, 8 and actively suppresses the reproductive ability of both male and females colony members, 9 except for one to three breeding males. Importantly, although maintained in a prepubescent stage while subjected to reproduction suppression by the breeding female, 10 non-breeder individuals are able to effectively mate and reproduce when the opportunity arises, eg, after the death of the breeding female, or when split off as a breeding pair in captivity. 7

As will be discussed in the following sections, this highly social lifestyle has an important impact on the biology of NMRs, as eusociality is thought to favor the occurrence of longevity, but can also worsen hypoxic/hypercapnic conditions encountered by NMRs within the burrows.

The underground network of chambers and tunnels where NMRs live can extend for up to three kilometers, depending on the food availability and the number of individuals composing the colony. 11 Like many subterranean mammals, NMRs chiefly feed on roots, tubers, and corms of various geophyte plant species. 11 This subterranean habitat, in addition to offering good protection against predation, also provides stable climatic conditions, allowing for an ambient temperature and humidity levels to be maintained in the burrows temperature varies from 28°C to 32°C depending upon burrow depth with little seasonal change, while humidity is uniformly high (up to 90%). 11,12 This high constant ambient temperature is a critical factor for NMRs, as they have a poor thermoregulatory capacity. NMRs exhibit a surprisingly low body temperature (Tb) (approximately 32°C), 12 even in comparison to other fossorial mammals (eg, the lesser blind mole-rat, Spalax Leucodon: 37°C the silvery mole-rat, H. argentiocinereus: 35°C and the southeastern pocket gopher, Geomys pinetis: 36.1°C), 12 which also exhibit lower Tb than other mammals, due to higher rates of heat loss and increased risks of overheating and water loss during digging activities in the relatively higher temperatures of burrows than aboveground. 12,13 When exposed to temperatures outside their thermoneutral zone (31°C to 34°C), NMRs do not maintain their Tb, and thus Tb follows the ambient temperature 12,14 (a fact that anyone receiving a shipment of NMRs can attest to – a box of barely moving animals is transformed after 30 minutes in a 32°C incubator, into a box of highly energetic animals). NMRs are thus defined as poikilotherms, 14 and indeed one might predict this phenotype when considering some of their morphologic characteristics such as thick, hairless skin, and little subcutaneous fat. 15 However, they possess brown adipose tissue which is a similar property of homoeothermic species, 16 and use their social environment to regulate their Tb, notably through huddling in groups. 17

To our knowledge, NMRs are the only poikilothermic mammal. This poor thermoregulatory capacity, possibly due to their low thyroid hormone levels, 18 may have significant implications for other aspects of NMR physiology, such as longevity or hypoxia tolerance.

The physiological adaptations to their extreme living conditions, as well as their highly unusual mammalian traits, such as eusociality and poikilothermy, establish NMRs as a unique animal model for biomedical studies.

Insights into longevity and healthy aging

Across species, maximum lifespan (MLSP) is positively correlated with body mass, such that as body mass increases, extrinsic mortality (eg, predation) decreases, and species can invest more in maintaining fecundity and long-term survival. 19 The NMR seems to be one of the few exceptions to this rule: similar in mass to the laboratory mouse, C57/BL6, Mus musculus (adult NMR: 35󈞷 g, and adult C57/BL6 mouse: 25󈞏 g), NMRs have an MSLP of 32 years compared to 4 years for mice. 20 It has been suggested that the fossorial lifestyle of NMRs, and other long-lived rodents, is a contributor to the extended lifespan, since it also strongly decreases extrinsic mortality. A recent analysis of age and body mass data of vertebrates, taking into account habitat and ecology, showed a clear correlation between fossorial, or volant lifestyles, and longer life. 19 However, it has been argued that a more important driver of longevity is eusociality. Using data that included loosely eusocial species (such as wolf, jackal, and coyote), as well as NMR and DMR, it was concluded that while body mass accounts for about 30% of the variation in MLSP, sociality affected 3.3%, while habitat only explained 0.01% of the effect. 21

In addition to long life, NMRs also fulfill all criteria associated with negligible senescence: the queen reproduces life-long with fully maintained fecundity, and there is no age-related change in physiological functions or gradual change in mortality rate, as is usually observed in most other species which exhibit a gradual, life-long deterioration. 22 NMRs also show no age-related changes in basal metabolic rate, body fat, bone mineral density, and only very small changes in cardiovascular and gastrointestinal function. 23,24 NMRs do however, show a fast decrease in functions when they reach MLSP and age very rapidly just before death. 22,25

Investigations into the molecular adaptions and metabolic changes of NMRs have unearthed some clues as to why this small rodent lives a long and healthy life. Contradictory to the notion that oxidative damage is detrimental to health, NMRs exhibit elevated levels of DNA oxidative damage from a young age, but appear able to deal with this damage more efficiently than other organisms. 25 It has been suggested that the NMR genome has a low background mutation rate and low nucleotide diversity, which would point to more efficient DNA damage control, but it is unclear if this observation is not just a consequence of the small gene pool and extreme inbreeding, since it can also be observed in the eusocial DMR. 26,27 Interestingly, in DMRs, it has been observed that oxidative damage is lower in longer-lived breeding females than in other colony members, 28 which suggests that breeding status is likely of importance. Indeed, evidence indicates that the breeding female in DMR colonies live for longer than non-breeding females, 29 and it would be interesting to determine if similar is true for NMRs.

Compared to mice, NMR proteins have higher levels of cysteine residues, which have been suggested to act as a buffer of oxidative damage. While aging organisms accumulate proteins that exhibit both irreversibly oxidized cysteine and polyubiquitination, 30 NMRs show no age-related changes in their overall low levels of either, which indicates that their proteins are kept in a healthy state throughout their life. 25 Also, the NMR genome shows an expansion of heat shock protein HSP70 and HSP90 protein families, which could play a role in preventing protein misfolding. 31 Interestingly, protein unfolding in response to treatment with urea was much more pronounced in mouse than in NMR, and whereas the amount of unfolded protein increased with age in mice, it did not in NMRs. 25 Furthermore, RING domain ubiquitin ligases have been shown to be enriched for pseudogenes in the NMR genome, suggesting that there are lower levels of ubiquitination. 32 NMR hepatic cells also have a higher cytosolic proteasome activity than mice, which suggests enhanced protein degradation and turnover. 33,34 In addition, NMR hepatic cells show increased autophagy, which have previously been associated with longer lifespans in birds, and might contribute to a healthier cell metabolism. 35 A contributing factor to healthy protein metabolism might be the observed change in ribosome processing, where NMR 28S rRNA is cleaved into two molecules. 36 Further to this break, NMR cells have higher translation fidelity, which is consistent with the earlier observation that there is less accumulation of ubiquitinated proteins or age-related aggregates of misfolded protein. 36 It has been speculated that this change in 28S rRNA processing was present in the common ancestor of the hystricognath clade (to which Bathyergidae belong), since it is also present in the South American tuco tuco (Ctenomys brasiliensis), and Degu (Octodon degus), but not in the South American guinea pig or African DMR, 27 suggesting that it was present before a geographical dispersion occurred even though it has been lost by species on both continents.

NMRs show no signs of neurodegeneration, but they express higher levels of amyloid-β and a heavily phosphorylated version of tau protein, both of which are associated with Alzheimer’s disease, when compared with a transgenic mouse model of Alzheimer’s disease. 37,38 However, no aggregations of these proteins or plaque formation has been observed, and it was suggested that high amyloid-β and phosphorylated tau might function as neural regulators in an environment of high oxidative stress. 38 The maintenance of neuronal integrity has also been attributed to neuregulin 1 and its receptor ErbB4, which are both elevated in long-lived rodents, NMRs exhibiting the highest observed levels. 39 Neuregulin 1 has also been indicated as cardio-protective, 40 and it should be noted that NMRs exhibit no significant cardiovascular aging. 24 Another transcription factor implicated in cardiovascular health and longevity is nuclear factor erythroid 2-related factor (NRF2). 41,42 NRF2 regulates the transcription of antioxidants, chaperones, and other cytoprotective molecules. 43 Two regulators of NRF2, Keap1 and βTrCP, which target NRF2 for degradation, 44,45 are negatively correlated with MLSP and the high levels of NRF2 activity in underground species may have resulted from convergent evolution. 24,46

Mouse models of longevity are associated with caloric restriction and lowered insulin-like growth factor signaling. 47,48 These animals are usually smaller, which suggests that within a species, smaller body mass is correlated to a longer lifespan. 49 Taking into account insulin-like growth factor 1 receptor (IGF1R) levels in tissues of 16 rodent species with different body sizes and MLSP, it was shown that IGF1R levels in the brain, but not in other tissues, are strongly negatively correlated with MLSP. 50 Hystricognaths, including guinea pigs, crested porcupines, DMR, and NMR have evolved an alternative glucose metabolism that does not rely on insulin and insulin receptor, but instead makes use of IGF2R and its binding protein, which closely resembles a mode of glucose handling usually observed in the fetus, 51 which is in agreement with the observation of lowered IGF1R signaling in calorie restricted rodent models. 47

Molecular mechanisms of cancer resistance

According to Cancer Research UK, 52 there were over 330,000 new cases of cancer in 2011 and over 160,000 cancer-related deaths in 2012 in the UK (Ӭ.5% and Ӭ.2% of the UK population, respectively). These statistics indicate the importance of increasing our understanding of cancer pathogenesis in order to identify novel therapeutic targets. The NMR, alongside other cancer-resistant species, offers a unique insight into cancer pathogenesis, as a cancer resistant species, as it presumably has a plethora of different mechanisms that biomedical research might be able to leverage for the treatment of cancer in patients.

NMRs, and other long-lived rodents such as DMRs, and the blind mole-rat (BMR) (Spalax galili), appear not to develop cancer throughout their long lives, and furthermore, cancer cannot be artificially induced. 53󈞣 Moreover, upon oncogene transformation with SV40 large T antigen (LT) and Ras G12V, which commonly leads to tumor formation in mice and rat cells, NMR cells undergo crisis when transplanted into immunodeficient mice (crisis is a terminal state resulting in necrosis due to DNA damage and chromosome dysfunction). 56,57 It has thus been proposed that crisis might act as a tumor suppressor mechanism in NMR cells. 57

One common mechanism of cancer prevention in long-lived organisms, such as humans, is replicative senescence, the suppression of telomerase activity in somatic cells during adult life. 58 In replicative cells, telomerase prevents the shortening of the chromosomes’ telomere regions, and in organisms with replicative senescence this activity is restricted to adult stem cells. However, in rodents, telomerase activity seems to coevolve with body mass rather than with lifespan, and long-lived small rodents such as NMRs and the Eastern grey squirrel, Sciurus carolinensis, retain telomerase activity in somatic cells, suggesting that other anti-cancer mechanisms must exist. 59 It has been observed that in most long-lived rodents (eg, NMR, muskrat, and Eastern grey squirrel), fibroblast proliferation rate in vitro negatively correlates with longevity, and fibroblasts from these animals exhibit slow proliferation rates, compared to short-lived small rodents such as mice. 60,61

The key to NMR cancer resistance might be a phenomenon termed early contact inhibition (ECI), that involves the retinoblastoma (Rb) and p53 pathways and p16 (Figure 1). 54 The Rb pathway contains regulators of cell cycle control, specifically the G1 progression, and p53 is involved in apoptosis and cell cycle arrest mutations in both oncogenes can be found in a majority of cancers. 62 Mammalian cell lines grown in culture stop proliferating when they form a monolayer through a mechanism called contact inhibition, that uses the cyclin-dependent kinase inhibitor p27 to arrest the cell cycle in the G1 phase. 63 NMR fibroblasts stop proliferation at a cell density that is three times lower than that of mouse fibroblasts, as soon as cell-cell contacts are established. 54 This ECI is mediated by cell cycle arrest through the Rb and p16 pathway, 64 and an apoptotic response using the p53 pathway. 65 Inhibition of both of these factors using LT abolishes ECI in NMR cells, but when ECI is lost, NMR cells rely on the upregulation of p27 for contact inhibition if either one of the factors is blocked, cells start growing more densely, but ultimately undergo apoptosis. 54 Assuming that ECI is an important mechanism for cancer resistance in NMRs, then determining how it is triggered could be an important step forward in identifying a potential mechanism for inhibition tumor growth in humans. It has recently been suggested that ECI is initiated by hyaluronic acid (HA), an unbranched disaccharide glucuronic acid/N-acetylglucosamine polymer of the extracellular matrix. 66 Isolated NMR fibroblasts produce a high molecular mass HA (HMM-HA) (NMR: 6 to 12 MDa, compared to mouse HA: 0.5 to 3 MDa), and an enzyme responsible for HA synthesis, HA synthase HAS2, is overexpressed in adult NMR skin fibroblasts compared to its levels in mouse and human fibroblasts. 67 Furthermore, NMR HAS2 has two amino acid changes that might account for its high activity and the enzyme that breaks HA down, hyaluronidase, displays lower activity. 67 It has been suggested that HMM-HA acts via its receptor, CD44, to activate p16 elevated expression of which coincides with ECI. Interestingly, the genes encoding CD44 and HMMR (another HA receptor), show signs of positive selection in NMRs, 68 and NMR cells treated with CD44 blockers, or grown with hyaluronidase, show an absence of ECI and reduced levels of p16, while human embryonic kidney (HEK) cells overexpressing NMR HAS2 started to secrete HMM-HA. 67 However, it has not been tested whether these HEK cells have elevated levels of p16 or show any ECI properties. The BMR has also been shown to secrete HMM-HA, though its cells do not exhibit ECI, 69 and it is not known if they have elevated levels of p16 or p27. DMRs share one of two amino acid changes found in NMR HAS2, and also secrete HMM-HA, but whether they show ECI remains to be investigated. 27 It has been suggested that HAS2 is undergoing purifying selection in other mammals, but whether or not this can be linked to cancer resistance remains to be investigated. 70 BMRs do however, have a mutation in the DNA-binding domain of the p53 gene, which leads to decreased p53 activity and the downstream pro-apoptotic pathway, 71 and which might contribute to the observed necrotic cell death. NMRs do not show changes in p53 expression, they do however show positive selection in the locus of p53 and additional proline motives in a proline-rich domain, which they share with humans and which might be a stabilizing mechanism co-evolved with an extended lifespan and enhanced DNA damage response. 68 More recently, it has been found that NMRs express an additional product from the inhibitors of cyclin dependent kinase 4 (INK4) locus (Figure 1). 72 In addition to p15, p16, and alternate reading frame, alternative splicing of the p15 exon 1, and p16 exons 2 and 3 creates pALT (named for alternative splicing), which is present in NMR cells and tissues, but is not found in mouse or human cells. Interestingly, its expression is strongly induced during ECI and stressors, such as ultra-violet radiation, and the INK4a/b locus was strongly upregulated by cell crowding and the build-up of HMM-HA, 72 suggesting that it plays a role in the cellular defense mechanism of NMRs. When expressed in human cells, pALT achieved a stronger induction of cell-cycle than p15 or p16 alone. 72 See Figure 1 for a summary of proposed cancer resistance mechanisms in NMR.

Figure 1 Early contact inhibition (ECI) in the NMR.
Notes: ( A ) Transcription of the NMR INKa/b locus gives rise to a novel splice variant, pALT, consisting of the first exon of p15 and the second and third exon of p16. ( B ) With increasing cell density, levels of pALT and p16 expression rise, while HMM-HA is secreted from cells, together these events participate in ECI. In other organisms, contact inhibition is likely signaled through a rise in p27 expression and occurs at a higher cell density. Adapted from Seluanov A, Hine C, Azpurua J, et al. Hypersensitivity to contact inhibition provides a clue to cancer resistance of naked mole-rat. Proc Natl Acad Sci U S A. 2009106(46):19352� 54 and Tian X, Azpurua J, Ke Z, et al. INK4 locus of the tumor-resistant rodent, the naked mole rat, expresses a functional p15/p16 hybrid isoform. Proc Natl Acad Sci U S A. 2015112(4):1053�. 72
Abbreviation: NMR, naked mole-rat.

Hypoxia tolerance as an adaptation to a subterranean habitat

Hypoxia, a low level of oxygen, is involved in numerous pathological conditions, including cerebral ischemia (eg, stroke), heart defects, cancer, and neurodegenerative disorders, such as Alzheimer’s disease. 73󈞷 One feature of ischemia is that it produces cell death because cells primarily use oxygen during aerobic metabolism to produce adenosine tri-phosphate (ATP), and thus oxygen deprivation starves cells of the ability to produce sufficient ATP. The central nervous system is particularly sensitive to hypoxic insults due to its high energetic requirements compared to its low energy reserves. Most of the ATP used by neurons is dedicated to maintenance of ion gradients and membrane potentials during synaptic transmission. 76,77 Rapid reduction of cellular ATP levels during hypoxic/ischemic insults results in disruption of ion and neurotransmitter homeostasis, 78,79 increased intracellular calcium levels, 80 and leads to irreversible neuronal damage and cellular death. 81 Moreover, accumulation of toxic byproducts created during anaerobic metabolism, such as lactic acid and protons, induce tissue acidosis which can worsen neurotoxicity through activation of acid-sensing ion channels (ASICs). 82

Using hypoxia-sensitive animal models, such as laboratory mice and rats, has advanced our understanding of some of the mechanisms mediating hypoxia-induced neuronal damage and thus, has identified certain molecular therapeutic targets. However, it is equally sensible to study how hypoxia-tolerant organisms cope with low levels of oxygen, as these organisms have evolved successful physiological, cellular, and/or molecular strategies to survive hypoxic insults, and it is possible that mechanistic understanding of these strategies could identify new targets for the prevention, and/or treatment of hypoxia-related injury.

Numerous vertebrates experience periods of hypoxia as part of their normal activity, 83,84 such as diving and hibernating mammals (eg, deep-diving hooded seals, Cystophora cristata, and arctic ground squirrels, Spermophilus parryii), reptiles (eg, the Western painted turtle, Chrysemys picta), fishes (eg, the crucian carp, Carassius carassius), and amphibians (eg, the common frog, Rana temporaria). Additionally, subterranean species have also received attention for their extreme resistance to hypoxia, 84󈟂 including NMRs, which likely encounter oxygen levels as low as 6%, 12,87 due to a combination of their subterranean, poorly ventilated habitat, and large colony size.

The ability to deal with sustained low levels of oxygen is aided by NMR hemoglobin, which has higher oxygen affinity than mice, 88 thus securing oxygen delivery under low oxygen conditions. Moreover, NMR hypoxia-tolerance can also be explained by its surprisingly low basal metabolic rate (between 0.27 to 1 mL O2/g/h within its thermoneutral range), 12,14,23,85,89 compared to the mouse (ɭ.2 mL O2/g/h), 90,91 enabled by its low Tb. During 3% hypoxia, NMR metabolic rate is even further reduced. 85 Metabolic suppression is a common physiological adaptation of hypoxia-tolerant species, 92 in order to reduce ATP consumption, so that the supply matches demand when pools of available ATP are reduced during hypoxic periods. 93 Moreover, existence of close interactions between Tb and hypoxia have been known about for several years. 94 For example, hypothermia is thought to be neuroprotective in hypoxic insults, notably by reducing brain metabolic rates, 95 although clinical benefits of therapeutic hypothermia are still debated. 96 Thus, the peculiar poikilothermic thermoregulation of NMRs establishes them as a unique mammalian model to study metabolic suppression in hypoxia, 97 and the role of thermoregulation in development of hypoxia-mediated pathological conditions.

In addition to physiological adaptations at the organism level, NMRs have developed hypoxia-tolerance at a neuronal level. In the hippocampus of hypoxia-sensitive mice, oxygen depletion induced a rapid decline in synaptic transmission while anoxia lead to neuronal death within ten minutes. 98 By contrast, hippocampal slices from NMRs maintained synaptic activity during hypoxia and, more drastically, within the first 30 to 40 minutes of anoxia. 98 Moreover, a significant number of NMR slices (approximately 75%), recovered functional synaptic activity upon returning to normoxia, whereas no recovery was observed in mouse slices. 98 Additionally, less cell death occurs in organotypic hippocampal slices from NMRs compared to slices from rats after oxygen-glucose deprivation. 99 These findings strongly support the fact that NMR neurons are able to face severe hypoxic insults with attenuated neurotoxicity compared to the mouse. In fact, NMR hippocampal slices perfused with hypoxic solutions accumulated less calcium than mouse slices, 100 suggesting an adaptive mechanism to decrease intracellular calcium signaling and avoid the resulting neurotoxicity.

Modulation of neuronal NMDA receptors (NMDARs), is a major player of excitotoxicity, in order to reduce calcium-mediated neurotoxicity, which is common in hypoxia-tolerant animals. 101� In hypoxia-tolerant neonatal mice, differential expression of the GluN2 NMDAR subunits is associated with hypoxia sensitivity. 106 Expression of the GluN2D subunit is transient during development and is thought to confer hypoxia-tolerance to neonates as it shortens the opening time of the channels, and decreases neuronal calcium entry. 106 By contrast, NMRs maintain high levels of the GluN2D subunit during adulthood, implying that they retain the ability to reduce hypoxia-mediated calcium accumulation throughout their life. 107 Similar up-regulation of GluN2D subunits is also found in the BMR, 103 suggesting evolutionary convergence of these hypoxia-tolerance molecular mechanisms. Thus, NMRs are one of the few mammals known to modulate their glutamatergic activity to successfully deal with hypoxic challenges, 84,103 although the molecular mechanisms of such regulation is not fully understood. Further investigations using hypoxia-tolerant NMRs will improve our knowledge of molecular mechanisms reducing glutamate-mediated and calcium-mediated neurotoxicity in hypoxic insults, and may open novel avenues for therapeutic strategies.

Nociceptors are sensory neurons that can be activated by noxious stimuli, commonly perceived as pain, such as heat, cold, mechanical force, or chemicals. 108� The ability of an organism to detect noxious stimuli is critical to its survival, a point validated by the commonality of nociceptors to organisms within the animal kingdom, 109,111� but chronic pain, that often serves no survival benefit, is widespread with 19% of the adult human population expected to experience chronic pain at some point in their life, and the majority of these patients describe their pain medication as inadequate. 115 Recent studies using the NMR as a novel model in nociception research, have helped to identify some of the molecular mechanisms that drive pain, which thus reinforces the validity in using this species in research with a biomedical focus, as will now be discussed.

In mammals, most nociceptors are unmyelinated C-fibers, but there are also thinly myelinated Aδ-fibers. 109,110,116 C-fibers can be broadly categorized into non-peptidergic and peptidergic populations, the latter of which express the neurotransmitters substance P (SP), and calcitonin gene-related peptide. 117 NMRs are peculiar in that their cutaneous nerves display a relative paucity of C-fibers, 118 but more oddly still, cutaneous NMR C-fibers seem to completely lack SP and calcitonin gene-related peptide. 119 Investigation of nocifensive behaviors has found that NMRs respond with avoidance behaviors to noxious thermal and mechanical stimuli similar to mice. 120,121 NMRs develop mechanical, but not thermal hyperalgesia upon injection of complete Freund’s adjuvant, and do not display nerve growth factor-induced thermal hyperalgesia. 121 NMRs are also behaviorally insensitive to capsaicin, ammonia fumes, and histamine-induced itch, all of which are known to activate peptidergic C-fibers in other mammals. 122,123 Interestingly, isolated dorsal root ganglion (DRG) neurons respond to both capsaicin and histamine, suggesting that the lack of behavior is not down to insensitivity of sensory neurons, and capsaicin activates sensory neurons in the in vitro skin nerve preparation. 121,123 However, intrathecal infusion of SP, prior to capsaicin or histamine administration, rescues nocifensive and scratching behaviors, respectively. 121,124 These results suggest that SP neurokinin-1 receptors are present and functional in NMR spinal cord circuitry and, as in mice, neurokinin-1 receptors are expressed in the superficial dorsal horn of the NMR spinal cord (Figure 2). 121

Figure 2 Pain circuitry in the NMR.
Notes: ( A ) Mouse and NMR have capsaicin-sensitive and capsaicin-insensitive C-fibers. Subcutaneous injection of capsaicin, the substance that makes chili papers taste hot, into the skin of the paw induces pain behaviors (licking) in mice (top panel), but not NMR (middle panel) however, intrathecal administration of SP prior to capsaicin injection rescues behavioral capsaicin sensitivity and induces pain (bottom panel). ( B ) Domain IV of the NMR NaV1.7 contains an amino acid motif that confers acid-insensitivity (EKE), a motif that is shared by other fossorial animals and those that live in high numbers in caves, but lacking in other species. Adapted from Park TJ, Lu Y, Jüttner R, et al. Selective inflammatory pain insensitivity in the African naked mole-rat (Heterocephalus glaber). PLoS Biol. 20086(1):e13. 121 Smith ES, Omerbašić D, Lechner SG, Anirudhan G, Lapatsina L, Lewin GR. The molecular basis of acid insensitivity in the African naked mole-rat. Science. 2011334(6062):1557�. 126 Fang X, Seim I, Huang Z, et al. Adaptations to a subterranean environment and longevity revealed by the analysis of mole rat genomes. Cell Rep. 20148(5):1354�. 27
Abbreviations: DMR, Damaraland mole-rat NaV, voltage-gated sodium NMR, naked mole-rat SP, substance P.

NMRs are also insensitive to acid-induced pain and acidic fumes. 121,125 Using the in vitro skin-nerve preparation, acid failed to excite NMR sensory neurons, 121 thus suggesting that the lack of nocifensive response to acid is that NMR neurons are insensitive to acid. Further investigation found that isolated NMR DRG neurons do respond to acid and that NMR proton sensors, such as ASIC1a, ASIC1b and transient receptor potential vanilloid 1 have similar proton sensitivity to mouse homologs. 126 How then do NMR neurons not fire action potentials in response to acid? We identified that voltage-gated sodium (NaV) currents were more strongly inhibited by acid in NMR DRG neurons than mouse DRG neurons. Subsequent cloning and functional analysis demonstrated that NMR NaV1.7 has an amino acid variation resulting in more negatively charged amino acids that render the channel more sensitive to proton block: 126 acid acts like an anesthetic, rather than an activator, of NMR sensory neurons. Interestingly, the acid-resistance NaV1.7 motif is shared between the NMR and other underground or cave-dwelling species, 126,127 suggesting that this is an adaption to an environment that is high in carbon dioxide (Figure 2). In the blood, carbon dioxide dissociates into bicarbonate and protons, and an excess of carbon dioxide can lead to acidification of tissues, 128 resulting, besides from other effects on metabolism, in acid pain.

NMRs also exhibit extreme changes in the opioid system compared to other mammals. In rats, opioids such as morphine or codeine induce a strong antinociceptive response, reducing for example response latency during a hotplate test. 129 In NMRs, opioids lead to hyperactive and aggressive behavior, such as animals killing each other. 120 Further investigations revealed that both mu and delta opioid agonists cause naloxone reversible (an opioid antagonist) hyperalgesia in NMRs in the hot plate test, while only a kappa opioid specific agonist caused analgesia, 130 suggesting that some aspects of nociception are signaled by a non-opioid nociceptive system. In the formalin test however, morphine and synthetic agonists to mu, delta and kappa opioid receptors were antinociceptive, 131,132 indicating that for some aspects of peripheral nociception, opioid systems play a role in the NMR.

Future perspectives and limitations

NMRs constitute an incredible resource for biomedical research with potential health care translatability due to what these mammals have evolved in order to cope with their extreme habitat (eg, hypoxia/hypercapnia), and by showing numerous adaptations to biological stressors (eg, longevity and cancer resistance). Although some of these phenotypes are shared with other vertebrate species (eg, tolerance to hypoxia/hypercapnia in hibernating mammals and others mole-rat species), NMRs possess a combination of characteristics, which designate them as a singular animal model (Figure 3). However, it should be noted that the majority of studies in current literature have compared NMRs with the mouse or rat, and that there is a relative lack of knowledge about species that, from a phylogenetic point of view, are more closely related to the NMRs, eg, the dassie rat (Petromus typicus), and the cane rat (Thyronomys), which, along with bathyergids, belong to the infraorder Phiomorpha. Further cross species comparisons will enable a better understanding of just how unique NMRs are, and will thus assist in establishing the molecular mechanisms that underlie NMR physiology.

Figure 3 Physiological adaptations of the NMR.
Notes: Schematic summary of the physiological adaptations of NMRs. Solid lines between boxes represent known interactions between physiological traits, dashed lines indicate putative links. Biomedical fields or diseases to which each physiological adaptation can be related are in red. Photo courtesy of author LN Schuhmacher.
Abbreviation: NMR, naked mole-rat.

Importantly, the physiological adaptations studied in NMR with respect to one particular biomedical field, often overlap with secondary research areas. For example, hypoxia-mediated pathways are involved in tumorigenesis, 133 and hypoxic insults worsen neurodegenerative diseases 134 thus it could be profitable to explore these aspects in NMRs, which in the wild, live in a chronically hypoxic environment without developing these pathologies. In addition, NMR cellular tolerance to high oxidative stress, primarily examined with respect to their extreme longevity, 25,135 may be involved in other aspects of its physiology, as reactive oxygen species, notably generated during hypoxia, act on molecular pathways involved in cancer, inflammation and neurodegenerative diseases. 136,137 Some of the most striking interactions between NMR adaptations are summarized in Figure 3.

Nevertheless, using NMRs as a standard laboratory animal model has some experimental limitations. Firstly, like commonly used laboratory mice and rats, NMRs are rodents and therefore one can argue about the relative gap separating these species from humans, in order to develop appropriate translational therapeutic strategies. More specifically, breeding NMRs in the laboratory is more uncertain and time consuming than for mice/rat colonies, notably due to the fact that there is only one breeding female per colony and the gestational period is far longer than mice. 7,138 Thus, development of transgenic NMRs would be hard to achieve in the near future, but with rapid advances in genome editing technology, such as CrispR, and the ever-increasing simplicity and decreasing costs of making transgenic mice, the potential for generating knock-in mice that express genes of interest from NMRs is a definite possibility this line of investigation is aided by the NMR genome having been sequenced. Lastly, there is also the logistical element to consider: NMRs are poikilothermic, eusocial mammals, which means that to house this species a research facility must have sufficient space to accommodate a large caging system (interconnected by tunnels), that does not fit in standard cage racking systems it is also necessary to be able to control both temperature and humidity within this space.

In summary, we believe that studying NMRs and their extreme biology will further our understanding of normal biology, which in turn will aid our investigation of mechanisms underlying various biological diseases which are having an increasing impact on modern society, such as neurodegenerative diseases, aging, cancer, and stroke.

The authors report no conflicts of interest in this work.

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Watch the video: Oxygen Deficiency In Body - Hypoxia Signs And Symptoms (May 2022).


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