Hypoxic preconditioning: effect, mechanism and clinical implication (Part I)

Guo-wei LU , Guo SHAO,

1. Institute for Hypoxia Medicine, 2. Basic Medical College, Capital Medical University, Beijing 100069;

3. Biomedicine Research Center, Baotou Medical College, Baotou 014060, China

Contents:

1. Introduction

2. Conceptual development of HPC

2.1. Whole body and single organ HPC

2.2. Rapid and delayed HPC

2.3. Pharmacological and physico-chemical HPC

2.4. Remote and cross HPC

2.5. Parameter and timing of HPC

3. Biological significance of HPC

3.1. Tolerance to hypoxia/ischemia

3.2. Protection agsinst hypoxia/ischemia insult

4. Energy metabolic accommodation during HPC

4.1. Metabolic suppression

4.2. Metabolic switch

4.3. Decrease in energy supply and demand

5. Cerebral morpho-functional modification during HPC

5.1. Maintenance of brain microorganization

5.2. Suppression of cerebral electrical activity

5.3. Improvement of spatial cognitive ability

6. Neurochemical acclimatization during HPC

6.1. HIF and its target gene

6.2. Signaling molecule

6.3. Anti-apoptosis and apoptosis molecule

6.4. Antioxidant enzyme

6.5. Excitatory and inhibitory amino acid and their receptor

6.6. Others

6.7. Down-/up- regulation of detrimental/ beneficial neurochemical element

7. Underlying mechanism of HPC

7.1. Remarkable remarks on HPC mechanism

7.2. A tentative framework of mechanism for HPC

7.3. Phylogenetical consideration on HPC

8. Clinical potential application of HPC

8.1. Application of HPC in surgery

8.2. HPC: An alternative strategy for fighting against hypoxia

9. Conclusions and perspectives

Introduction

Hypoxia is a common and important problem in both clinic and extreme environment. Adaptation to hypoxia has been investigated to understand acclimatization to high altitude and the effect of chronic hypoxia. Active physiological responses of the whole organism, systems, organs, and cells to maintain homeostasis under condition of hypoxia are widely considered as adaptation to hypoxia. However,the increase of physiological responses to hypoxia at system-organ level is not always suきcient to explain the hypoxic tolerance and protection in animal and human. The increase of physiological activity is not always effective to endure hypoxia when people are under an environment of low oxygen or suffering from hypoxia-ischemia diseases. No increase in physiological activity is associated with native people who have adapted to highland. Blood pressure of adult animals drastically reduced to zero within 5 min afer a trachea occlusion though their respiratory and circulatory responses are strong; whereas the blood pressure of 3-days newborn animals droped only to 67 % of the normal value up to 17 min afer the occlusion while no apparent functional responses happened. The pregnant cats die rapidly under carbon monoxide or cyanide intoxicationwhereas their embryos are still alive when they are taken out by laparotomy. After decapitation, heart beat of adult animals quickly stopped whereas heart beat of newborns might last for 1 h [1].

These facts are difficult to be explained by traditional knowledge based on adaptive responses of respiratory and cardiovascular systems. Haldane viewed this condition "a phenomenon diきcult to be understandable by physicochemical brain" [2]. The highly unusual phenomenon was understood by us in 1963 as "a kind of acquired tolerance of tissues-cells to hypoxia developed through evolution" and termed as a tissue-cell adaptation to hypoxia [1]. Since then an animal model of repeated autohypoxia was developed and the eあect and mechanism of hypoxic preconditioning (HPC) have systematically studied on the model in terms of behavior, neurophysiology,neurochemistry, neuromorphology and molecular biology.

The brain requires continuously supply oxygen and glucose to maintain its viability and function.Mechanisms allowing the brain to survive in hypoxia are key adaptation for hypoxic tolerance. Coupled to the redistribution of blood flow, hypometabolism allows survival by sparing precious oxygen in favor of the life-supporting organs such as the brain in particular. Brain plasticity, the life long capability of the brain to modify its function and organization according to particular experience of the body, is focused during the research on HPC. Modulation and plasticity are not mutually exclusive. Modulation may play a key role in the initiation and/or maintenance of plasticity. Energy saving and brain plasticity in particular are thought to be involved in the development of hypoxic tolerance and protection of tissue-cell during HPC. The HPC is thought to be an intrinsic cytoprotection theoretically [3-5]and a novel strategy for prevention and treatment of hypoxia-ischemia [4, 6].

This progress-review article is to succinctly integrate our major researches for the past decades in conjunction with its development in other laboratories in the world. そe content of the review is not to include of all that is known regarding HPC since many excellent and comprehensive reviews are available [7-19]. Authors of the present article are pleased to learn from and draw on these important articles and could not help to “present Buddha with flowers given by another” (a Chinese saying). Conceptual development and biological significance of HPC are well documented and thus briefly reviewed in the present article. A unique animal model of repetitive autohypoxia was developed in adult mice. そe eあect and mechanism of HPC have been systematically studied on the model in our laboratory since the early 1960s. The animals’ tolerance to hypoxia and protection from hypoxia injury is significantly increased. The adult mice behave like hypoxic intolerant mammalian newborns and hypoxic tolerant adult animals during their exposure to repetitive autohypoxia.The overall energy supply and demand decreased,the microorganization of the brain maintained and the spacial learnig and memory ability improved but not impaired, the detrimental neurochemicals such as free radicals down- regulated and the beneficial neurochemicals such as adenosine(ADO)and antihypoxic genes/factors (AHGs/AHFs) upregulated. A tentative framework of underlying mechanism of HPC is proposed in this progressreview article for future substantiation. In the article evolutional consideration on and clinical implication of HPC are also discussed in order to guide future investigation.

Conceptual development of HPC

Hypoxia is defined as a decrease oxygen concentration below normal in tissue or ambient environment. Ischemia is defined as a decrease in blood flow to tissue preventing adequate delivery of oxygen, glucose and other nutrients to tissuecell. Stroke or infarction is defined as the death of neuron and glial as well as vascular cellular elements in the brain. It is well known that sublethal ischemic/hypoxic exposure is able to improve the tolerance of organism, organ, tissue or cell to a subsequent lethal ischemic/hypoxic insult. そe phenomenon has called ischemic/hypoxic preconditioning(I/HPC) and well documented in the heart and brain. そe words,preconditioning (PC) and tolerance, were firstly seen in Janoff’s paper in 1964 [20]. The terms ischemia and hypoxia are ofen used interchangeably although ischemia is a lack of perfusion, characterized not only by hypoxia but also by insufficient nutrient supply. Both IPC and HPC or tissue-cell adaptation,however, might preferentially be termed as HPC since the essence of ischemia or stroke is hypoxia in fact.

Whole body and single organ HPC

HPC refers to exposure of organisms, systems,organs, tissues or cells to moderate hypoxia/ischemia that is able to result in a resistance to subsequent severe hypoxia/ischemia in tissues and cells.[1, 3, 21-27]. In general, two types of HPC, whole body HPC(WbHPC) and single organ HPC (SoHPC), can be categorized [4, 28, 29]. WbHPC can protect animals against acute lethal hypoxia and involve preservation of vital organ functions [30].

A unique animal model of repetitive autohypoxia,as a model of type WbHPC, was developed on adult mice in our laboratory in early 1960s [1, 3].Experiments were conducted on BALB/C mice. そe animal was anesthetized with pentobarbital, placed in a jar and sealed with a rubber plug. In this way,the animal inside the jar was exposed to hypoxia, a condition of autohypoxia induced by the animal's own oxygen consumption in the sealed environment.The animal was removed and switched to another similar jar as soon as gasping breath appeared. The procedure was performed once (H1) and repeated 2,3, 4, or 5 runs (H2, H3, H4 or H5). Animals exposed to hypoxia for 4 or 5 runs, group H4 and H5, were judged as hypoxic preconditioned or hypoxia tolerant/resistant animals. The animals exposed once, group H1, and the animals had no exposure to hypoxia, group H0, were acted as experimental and normal blank controls respectively[1, 3-5](Fig. 1).

Many subtypes of type SoHPC have been studied in the heart, brain, lung, liver, kidney,intestine, and other organs [31-33]. Murry et al originally tried to create a larger area of myocardial infarction by performing several brief episodes of myocardial ischemia prior to protracted ischemia,paradoxical results, however, happened [34]. The protective eあect of HPC in the heart can be assessed by limiting infarct size, reducing myocardial stunning, preventing arrhythmias, or accelerating the recovery of myocardial function after ischemia[35-40]. The protection provided by IPC against ischemia/reperfusion (I/R) injuries of the lung has demonstrated in models of guinea pig [41],canine [42, 43] and rabbit[23, 44, 45]. Blockade of the pulmonary hilar blood flow, IPC was able to attenuate the pulmonary dysfunction related to exposure to subsequent I/R. Similar eあect is achieved in the rat model [46]. IPC could reduce both liver and lung damage following liver transplantation [47,48]. IPC was also able to protect against I/R injury after renal arterial occlusion or graft preservation during transplantation [49]. PC was also found to be beneficial in delaying the development of tissue necrosis and reduction of bacterial translocation during subsequent I/R in intestine [50, 51].

It is generally recognized that the PC phenomenon was first described in the heart in 1980s [34, 52] and not until 1990 in the brain.; whole body traumatic stress producing "resistance" to subsequent trauma was described in 1943 [53] in addition to the brain.Whereas the experiments regarding trauma might involve multiple organ’s tolerance to ischemia were introduced in 1964. Pre-exposure of anoxia protected against subsequent prolonged anoxic exposure in 1964 [54]. Schurr et al demonstrated adaptation of the brain to anoxia in vitro in 1986 [55]. In canine spinal cord IPC model, with their thoracic aortas being cross-clamped for 20 min followed by formal ischemia with aorta being cross clamped for 60 min, the incidence of paraplegia due to spinal cord ischemia was found to be significantly attenuated in animals undergoing ischemic IPC in comparison with sham control group [56]. IPC with short periods of both global and focal ischemia induced tolerance in the brain [57]. To investigate the pathophysiology of perinatal asphyxial brain damage in infants,Vannucci developed a model in the postnatal day 7 rats [58].

Fig. 1 A: Hypoxia preconditioning mouse model. B: Tolerance time in the different exposure runs. *P＜0.05, **P＜0.01 vs the preceding runs.

Rapid and delayed HPC

PC triggers a fundamentally different adaptive response, and in mammals, this response is characterized by at least two distinct periods of induced tolerance relative to the preconditioning stimulus and the subsequent ischemia. A shortlasting protective phenotype, rapid PC, is induced in an early phase from several minutes to several hours after sublethal ischemia / hypoxia loading[4, 55, 59, 60], as a result of changes in ion channel permeabilities, protein phosphorylation and other post-translational modifications.

The other, delayed HPC, is induced in a delayed phase after several days and best appreciated as 'classical preconditioning' that requires gene activation and de novo protein synthesis; this requires many hours or even days to become fully manifest.Diverse families of pro-survival genes are activated and, in turn, encode proteins that serve to enhance the brain's resistance to ischaemia. Protection is achieved by the attenuation of broad categories of injury-inducing mechanisms and engaging innate survival mechanisms and enhancing endogenous repair processes [7].

The two subtypes of HPC differ in the induction mechanism and duration [61, 62]. The HPC phenomenon in the early phase is transient and may reflect changes in cellular metabolism and the one in the delayed phase is sustained and may reflect changes in gene expression. Despite of the mechanism of two kinds of HPC is diあerent, they are eliminated by protein or RNA synthesis inhibition[63] indicating that new synthesis of protein or RNA involved in neuroprotection. The new synthesis of protein or RNA may regulate other gene expression and play roles in protection directly. The detailed mechanisms of HPC vary in diあerent animal species or organ systems, although in general these could be classified as the immediate acquisition which is related to post-translational modification, or delayed induction related to new protein synthesis[27].

Murry et al [34] first described IPC in which sublethal repeated ischemia protects the heart against subsequent sustained ischemia and reperfusion injury. Later reports have described "late preconditioning," in which repeated brief episodes of ischemia exert cardioprotective effects not only immediately after, but also 24 h after, induction of sublethal ischemia in a biphasic manner [35, 64,65]. The “biphasic” phenomena of HPC have been observed in brain, heart, and liver tissues in human or other animals [21, 25, 48, 66-70]. A biphasic phenomenon in myocardial HPC was revealed with an early phase of protection that develops within minutes from the initial assault and lasts 2 to 3 h, and a delayed phase that becomes apparent 12 to 24 h later and lasts 3 to 4 d [64, 65]. Unlike the early HPC of myocardium, which can protect against infarction only, the delayed HPC can also protect against myocardial stunning [66, 71]. そe cascade of delayed HPC in the myocardium can be subdivided into the following three major components: (1) Triggers: the molecules that are generated during the first ischemic challenge and responsible for the initial adaptation.(2) Mediators: the molecules that are expressed in the 24 to 72 h later and responsible for conferring protection during the index ischemic challenge,and (3) そe intercellular signaling pathways that are activated by the triggers [66]. Late preconditioning can also be induced by treatment with heat stress [72,73] and cytokines[74, 75] and has been shown to be related to the expression of stress proteins such as HSPs [65, 73] and antioxidant enzymes [76-78].

Pharmacological and physico-chemical HPC

Traditional model of hyperthermic PC in rat is 41°C for 15 min [70, 79], which would result in more cellular damage. A less severe thermal PC, using water bath to elevate core temperature by 1°C for 15 min for 5 consecutive days, can also achieve protective effects of PC [50]. Hyperthermal PC can attenuate pulmonary dysfunction afer various animal models of injuries mimicking clinical conditions,such as organ transplantation, pulmonary air emboli,hemorrhagic shock, or abdominal surgery.

Pharmacological agents, following the discovery of related molecular mechanisms of HPC, gradually replace ischemia or hyperthermia, as the specific“targeted” pretreatment methods to protect organs from various types of injuries. PC with diethylmaleate, an intracellular pro-oxidant agent,can protect against I/R-induced lung injury in rat model [80]. In rat model stress PC with geldanamycin could restore a normal function of the alveolar epithelium in the early phase following hemorrhagic shock by attenuating nitric oxide (NO) mediated oxidative stress to the lung epithelium [81]. IPC with intravenous administration of 3-nitropropionate, an inhibitor of the mitochondrial complex II, in isolated lung perfusion of rat mode[82], or N-acetyl-L-cysteine, an oxidant scavenger promoting glutathione(GSH) [83], is able to prevent lung from cold or warm I/R injury. Lung reperfusion injury from ischemia of remote organ, such as intestine, can also be ameliorated by PC with pharmacological agents,such as doxorubicin, an inducer of heme oxygenase-1(HO-1, a stress protein) [84]. そese protective eあects could be abolished by the administration of HO-1 inhibitor such as tin protoporphyrin [85]. NO, a potent vasodilator, could also be administered as a method of pulmonary PC. In porcine lung in situ normothermic I/R model, PC with NO inhalation 10～15 min prior to ischemia was found to be able to protect against pulmonary hypertension, impaired gas exchange, and the inflammatory response of pulmonary I/R injury [86]. This NO PC maintains endothelial integrity in a subsequent I/R largely responsible to the nonvasodilatory and non-cGMP-related mechanisms [87]. Several other types of PC have been described in vivo and in vitro including hyperthermia, hypothermia [61, 88-91], chemical PC by blocking the Krebs cycle or respiratory chain[92] , glutamate and seizures [93, 94], linoleic acid[94], erythropoietin [95], tumor necrosis factor(TNF) [96], ceramide [96, 97], desferrioxamine and cobalt [98, 99], isoflurane[100], and thrombin[101,102]. Jiang et al showed that electro-acupuncture preconditioning abrogates the elevation of c-Fos and c-Jun expression in neonatal hypoxic-ischemic rat brains induced by glibenclamide, an ATP-sensitive potassium channel blocker [103].

Remote and cross HPC

In the past, attention has mainly paid to HPC-induced tolerance to hypoxia - ischemia on local in situ organ - tissue exposed directly to hypoxia ( local in situ HPC, li HPC ). The phenomena of HPC are now extended to remote organs-tissues other than local- in situ organ itself and stresses or pathological processes other than hypoxia-ischemia. Interest in studying the remote-ectopic HPC (ReHPC) and the cross-pluripotential HPC (CpHPC) is increasing (Fig.2)[5].

Remote ectopic HPC

IPC, in its original conception, describes intramyocardial protection which could be relayed from the myocardium served by one coronary artery to another. It soon became apparent that myocardial infarct size could be dramatically reduced by applying brief ischemia and reperfusion to an organ or tissue remote from the heart before the onset of myocardial infarction. そe concept of remote organ protection has now been extended beyond that of solely protecting the heart to providing a general form of inter-organ protection against ischemiareperfusion injury[104].

The concept of remote preconditioning was first described by Przyklenk et al [105]. Transient ischemia of the left circumflex territory was shown to reduce the effects of subsequent potentially lethal ischemia in the left anterior descending artery territory in dogs. Further studies in rodent models demonstrated that ischemia of the kidney and intestine may induce myocardial protection[106, 107]. Now a concept of remote ectopic preconditioning (ReHPC) can be raised(Fig. 2A)[5]. It describes a intriguing phenomenon: transient non-lethal ischemia and reperfusion of one organ or tissue induce resistance to a subsequent episode of lethal ischemia reperfusion injury in remote organs or tissues. ReHPC provids a strategy for harnessing the whole body's endogenous protective capabilities against the injury incurred by ischemic preconditioning stressor.

Fig. 2 A: Limb transiently ischemia can induce ischemic tolerance in a distant organ; B: The effects of hypoxic preconditioning (HPC).

Skeletal muscle ischemia has been shown to be a potent remote preconditioning stimulus in humans and larger animals [108]. Four 5-min episodes of limb ischemia induced by inflation of a blood-pressure cuff prevented ischemic endothelial dysfunction in the forearm in normal volunteers and reduced infarct size in a porcine model of myocardial infarction. The same stimulus, when applied to the recipient, protects the donor heart against IR injury in a cardiac transplant model [109] and has been shown to modify expression of proinflammatory genes in circulating human neutrophils [110].In a porcine model of cardiopulmonary bypass(CPB), remote IPC afforded myocardial and pulmonary protection [111]. This was evidenced by lower levels of troponin I and shorter duration of lactic acidosis, in addition to lower pulmonary vascular resistance, and significantly less change in pulmonary vascular resistance and lower peak inspiratory pressure after CPB in comparison with control patients. Open-heart surgery in children results in a predictable IR injury with a welldocumented systemic inflammatory reaction. One of the obvious advantages of the technique of remote preconditioning is its non-invasive nature and ease of application. Furthermore, in contrast to local ischemic preconditioning, the effects of transient skeletal muscle ischemia are relatively benign, there being no myocardial dysfunction, risk of arrhythmia,low cardiac output, or secondary organ injury. The"non-local" effect of remote IPC may afford more widespread protection against IR injury and the CPB-induced systemic inflammatory response[8].Ischemia-reperfusion injury and CPB during cardiac surgery are associated with a predictable systemic inflammatory response, myocardial dysfunction,and pulmonary endothelial dysfunction. Human neutrophils following recipient IPC, induced by four 5-min cycles of upper-limb ischemia, demonstrated modulation of genes coding for key proteins involved in cytokine synthesis, leukocyte chemotaxis,adhesion and migration, exocytosis, innate immunity-signaling pathways, and apoptosis [110].Although local ischemic preconditioning, induced by short-lived non-fatal ischemia in the target tissue, has been shown to be of benefit in patients undergoing coronary angioplasty and surgical revascularization in some studies[112, 113], other studies [114, 115]have been less conclusive. Pulmonary dysfunction after various types of reperfusion injuries, such as cardiopulmonary bypass (CPB), off-pump coronary arterial bypass (OPCAB) surgery, liver transplantation, hemorrhagic shock are attenuated when IPC is made in remote heart, liver, or limb[81]. IPC or pharmacological PC was also proved to protect the limbs as well as the remote heart and lung organs against injuries from I/R occurred in various clinical diseases or situations [68, 116, 117].

It was found that organs- tissues other than the brain such as the liver are also protected from hypoxic injury by repetitive exposure to hypoxia,suggesting the effect of HPC is not limited to the exposed organ - tissue itself [118]. The tolerance to hypoxia-ischemia is also significantly increased in remote organs-tissues more than the increase in local in situ organ-tissue itself[68, 108, 110, 118-125]. For example, the tolerance of other remote and ectopic organs-tissues is enhanced by repetitive occlusion of coronary, carotid common and thoracic arteries in addition to tolerance increase in heart,brain, and spinal cord, respectively. As shown in Fig.2 A, repetitive ischemia of a limb in one side can result in increased tolerance to ischemia not only in theipsilateral and contralateral limb, but also in brain, heart, lung, kidney, intestine and other organstissues.

Cross pluripotential HPC

More interesting is the phenomenon of cross pluripotential HPC (CpHPC), by which a primary exposure to a stressful stimulus results in an adaptive response whereby the cell, tissue, organ or organism is resistant to a subsequent stress that is different from the initial stress ( i.e., exposure to heat stress leading to resistance to oxidant stress) (Fig. 2B)[5]. Cross-tolerance, CpHPC, or the ability of one stressor to transiently increase tolerance to a second heterologous stressor, is thought to involve the induction of heat shock proteins. The heat shock response is one of the more commonly described examples of stress adaptation and is characterized by the rapid expression of a unique group of proteins,heat shock proteins, suggesting the possible relevance of this response in nature.

The protection against stresses or pathological processes other than ischemia- hypoxia is proposed to be markedly enhanced by liHPC of an organtissue. Since hypoxia is associated with almost all pathological processes induced by variety of stressors, a cross and pluripotential tolerance of HPC is thus induced in other stresses or pathological processes (Fig.2B) [5, 96, 110, 119, 121, 123, 124]. For example, inflammatory genes of white blood cells are significantly inhibited by repetitive limb ischemia in human[110] as shown above. Pain threshold of skin of a limb could be significantly increased with the increased tolerance to ischemia when blood supply of the limb is repeatedly occluded. Intriguingly,a significant anticancer ability by blood serum of hypoxic preconditioned mice was also recently found in our laboratory(unpublished data).

Parameter and timing of HPC

The specific stimulus paradigm is critical in many models of HPC. Its duration, intensity, pattern, and history of stimulation are important. The duration and intensity of stimulation often determine the duration and direction of the HPC. Differences in the parameter of HPC, such as intensity, duration,and/or frequency of a particular stress stimulus determine whether that stimulus is too weak to elicit any response, of sufficient magnitude to serve as a preconditioning trigger, or too robust and therefore harmful[126]. Molecules known to cause ischemic brain injury — such as glutamate, reactive oxygen species, inflammatory cytokines and caspases —might, at the lower levels achieved in response to a particular preconditioning stimulus, trigger adaptive rather than deleterious responses in resident brain cells[127-137]. There was consensus on the timings of IPC satrting usually immediately before the index I/R. However, the duration or number of cycles of IPC varied. In rabbit in vivo lung ischemia model,lung ischemia was performed by blocking the hilum of the left lung for 10 min and then release for 15 min [44]. In similar rabbit model, 15 min of IPC is better than 5 min. Repeated cycles appear slightly better than single episode of HPC [23] and repeated hypoxic exposure even significantly better than single exposure(Fig.1B) [3]. In canine model, the duration of IPC was 10 min [42], however, another study showed that in rabbit model IPC 5 min was better than 10 min for the protection of subsequent I/R[43]. IPC, in which the pulmonary hilum is usually clamped for 10 min with subsequent 10 min of reperfusion, has also been applied in clinical conditions, such as people undergoing major lung resection[138, 139] or isolated lung perfusion with chemotherapeutic agents for unresectable cancer or metastatic sarcoma [140]. It has been documented in various preparations including the brain, in vitro brain slice, and cultured neurons[27, 55, 59, 89, 126,141-144]. Majority of studies have been focused on the delayed IPC/HPC neuroprotection, several studies performed on the whole brain, spinal cord,and hippocampal slice[55, 145-150] showing a rapid IPC/HPC-induced protection, in which the protection develops within 60 minutes after the preconditioning. Ma et al found that delayed HPC protects cultured cortical neurons from subsequent insults [151]. Several signal pathways have been suggested for delayed IPC/HPC protection[142, 152].

Biological significance of HPC

Tolerance to hypoxia/ischemia

As shown above, the first landmark paper on cardiac preconditioning in dogs by brief coronary ischaemia was published in 1986 [34]. The ability of brief hyperthermia to protect against subsequent focal stroke was documented shortly thereafter[153].Rather, it was the dramatic finding that the delayed neuronal death of gerbil hippocampal CA1 pyramidal cells after global ischemia could be completely prevented if carotid blood flow was briefly interrupted 2 days earlier [154]. そe finding launched the field of ischemic tolerance research in the brain.A number of robust and reproducible experimental models of cerebral ischemic tolerance are widely recognized[27, 88, 126, 141, 155]. Although rodent and neuronal cell culture models serve as the foundation for the field so far, emerging evidence indicates that ischemic tolerance is an evolutionarily conserved form of cerebral plasticity that occurs in invertebrates and vertebrates, including humans.

HPC with protective roles in many organ systems against various types of injuries has been well documented. With our WbHPC model, the appearance of gasping was judged as the limit of animal's tolerance to hypoxia in each run. The tolerance was significantly increased run by run.Tolerance time in run 2, 3, 4, and 5 was close to 2, 4,6, and 8 times of the first run(Fig.1B)[3]. Under the condition of hypobaric chamber with PO2at 2.7 kPa and lethal dose cyanide administration, the survival time of preconditioned animals was respectively 10 and 4 times than those of normal controls. When animals were randomly paired based on their body weight and sex, normal animals survived only for 1.7 min on average inside the chamber, whereas the preconditioned ones kept alive for 146 min on average, 86 times the survival time as long as their normal partners. Afer decapitation, residual activity of the isolated medulla oblongata and the spinal cord of hypoxia tolerant mice lasted 124 and 66 sec on average, respectively, 5 and 3 times that in control mice with no exposure to hypoxia [3, 4].

Protection against hypoxia/ischemia insult

そe remarkable eあect of I/HPC on neuroprotection under ischemic/hypoxic condition has been widely and clearly demonstrated [144, 156-159]. Tolerance has been hypothesized to have a neuroprotective role in cerebral ischemia [160]. The first in vivo study of cerebral preconditioning documented an acute increase in the capacity of the rat brain for anaerobic glycolysis after brief anoxia, which increased the survival time of the animal following a subsequent exposure to prolonged anoxia [54]. そe ability of brief hyperthermia to protect against subsequent focal stroke was documented shortly thereafer[153]. Now,a number of robust and reproducible experimental models of cerebral ischemic tolerance are recognized[27, 88, 126, 141, 155].

In our model, the survival time (24.3±6.2 min)of normal mice injected with brain homogenate supernatant taken from hypoxic preconditioned mice in vivo inside the hypobaric chamber was about double that of saline control group (13.2±4.8 min) and the group H1 (11.6±3.5 min). Methyl thiazolyl tetrazolium (MTT) assay showed that PC12 cells co-cultured with brain extract taken from preconditioned animals were significantly more alive than those co-cultured with that taken from control animals. The leakage of lactic dehydrogenase(LDH)in cortical synaptosomes co-cultured with brain extract taken from preconditioned animals was progressively decreased, indicating the synaptosomes are well protected from hypoxic injury by the extract.そese in vivo and in vitro protective actions exerted by preconditioned animals' brain extract suggest that something of great importance happened to neurochemicals in the brain of preconditioned animals[3, 4].

Energy metabolic accommodation during HPC

Metabolic suppression

During hypoxia, the drop in metabolic rate, a well known phenomenon in invertebrates and lower vertebrates, is also a frequent occurrence in mammals, both newborns and adults, and seems to be largely related to the control of thermoregulation[161]. Many animals respond to hypoxia by reducing body temperature and metabolic rate[162]. Since the 1950s, this was known to occur in a broad range of small mammals [163] and in neonates of larger mammals, including humans [164].This hypoxia-induced drop in body temperature and metabolic depression serves a protective role by reducing oxygen demand, eliminating costly thermogenesis, improving blood oxygen aきnity, and reducing the costs of ventilation[165-167]. Numerous studies support the conclusion that hypoxia resets the hypothalamic thermoregulatory set point to a lower level[168, 169], suggesting that it is a regulated process.

At the cellular level, adaptation to hypoxia is brought about on one hand by increasing the efficiency of energy-producing pathways, mainly through increased anaerobic glycolysis activity,and on the other hand by decreasing energyconsuming processes[170]. Ion-motive ATPases and protein synthesis are the dominant energyconsuming process of cells at standard metabolic rate, making up to more than 90% of the ATP consumption in rat skeletal muscle and as much as 66% in rat thymocytes[171]. Reallocation of cellular energy between essential and nonessential ATP demand processes as ATP supply becomes limiting is thus the key for cells to survive decreased oxygen levels. Studies on hepatocytes have shown that protein synthesis is largely inhibited in response to hypoxia[172]. In fact, the ATP consuming processes are arranged in a hierarchy, with protein synthesis and RNA/DNA synthesis being the first to be inhibited as energy becomes limiting and with Na+/K+pumping and Ca2+cycling taking the greatest priority.This phenomenon, known as oxygen conformance,involves precise regulatory mechanisms notably at the level of translation initiation [173].

Differential sensitivity to hypoxia-induced cell death in diあerent cell types seems at least in part to be due to the extent of their electric activity, ie, to the relative importance of the ATPase ionic pumps versus other ATP-consuming processes. This ion pumping represents as much as 80% of ATP consumption in neurons while only 20% in skeletal muscle cells.In case of severe oxygen limitation, most excitable cells cannot continue to meet the energy demands of ion transporting systems, leading to cell death[174]. On the other hand, the metabolic suppression response is particularly well characterized in intact heart, where decreases in myocardial oxygen delivery result in decreased contractile activity and oxygen consumption in a phenomenon called "hibernating myocardium. " [175]. Active changes also accompany hibernation: metabolism and blood flow are lowered to levels less than 10% of baseline[176], body temperature and white cell counts are reduced, and stress kinases and heat shock proteins are activated.Unique lipid and protein sequestration patterns have been noted in neuronal endoplasmic reticulum[177]. The mechanisms by which hibernating animals handle the rapid increase in cerebral blood flow and severe oxidative stress during arousal are poorly understood, but it is possible that they could be applied in a translational way to meet the similar challenges faced by the human brain during postischemic reperfusion.

Metabolic switch

Several responses are developed by cells and tissues facing with a hypoxic challenge: (1) increase in ventilation and cardiac output, (2) a switch from aerobic to anaerobic metabolism, (3) promotion of improved vascularization, and (4) enhancement of the oxygen carrying capacity of the blood. Most of these processes take place very early at the onset of hypoxia and occur through the activation of already present proteins; however, in the longer term, all of them are also mediated by up-regulation of genes encoding key actors of these responses. Such genes,for example, are: (1) tyrosine hydroxylase, which is involved in dopamine synthesis in carotid body type I cells; (2) the glycolytic enzymes phosphoglycerate kinase 1, pyruvate kinase, phosphofructokinase,aldolase A, glyceraldehyde 3-phosphate dehydrogenase, enolase 1, and glucose transporters Glut-1 and Glut-4; (3) VEGF, platelet-derived growth factor (PDGF) to induce angiogenesis, and inducible NO synthase that increases vasodilation;and (4) erythropoietin and transferrin receptors that favor erythrocyte production. These transcriptional responses are mediated in large part by the action of hypoxia-inducible factor-1 (HIF-1)[178].

In addition to the energy-balanced metabolic suppression, cells turn to glycolysis to meet their energetic demands in hypoxia. The switch between the two pathways of ATP regeneration from the oxygen-dependent mitochondrial respiration to the oxygen-independent glycolysis was first noted by Pasteur in the late 19th century, hence its name"Pasteur effect". Although glycolysis is less efficient than oxidative phosphorylation in the generation of ATP, in the presence of suきcient glucose, glycolysis can sustain ATP production due to increases in the activity of the glycolytic enzymes. そis is manifested within minutes in the allosteric regulation of phosphofructokinase activity and chronically, in the HIF-1-dependent overexpression of many glycolytic enzymes. Phosphofructokinase is considered to be the major regulator controlling carbon flux through glycolysis. It is allosterically activated by ADP and AMP and inhibited by ATP, hence setting the glycolytic rate according to the energy demand.However, the most potent allosteric activator is fructose-2,6-biphosphate [179].

Other metabolic enzymes are also regulated by AMP, ADP, and ATP. Atkinson proposed that most of the branch points between anabolism and catabolism would be controlled by these nucleotides in the 1960s[180]. This was termed the "adenylate control". In 1987, Hardie’s laboratory [181] identified AMPK as the primary sensor of cellular energy charge, and most of the eあects of imbalance between ATP and AMP. AMPK is a heterotrimeric protein comprising α , ß, and γ subunits. [182].

The activated kinase switches on catabolic pathways that generate ATP and switches off anabolic pathways that consume ATP [183, 184]. It is achieved both acutely via direct phosphorylation and chronically via effects of gene expression. The phosphorylation of PFK-2 is one such example.AMPK activation has also been reported to stimulate translocation of the glucose transporter Glut-4 to plasma membrane and consequent glucose uptake. In the longer term, it increases the expression of Glut-4, hexokinase, and mitochondrial enzymes involved in the tricarboxylic acid cycle and respiratory chain. On the other hand, AMPK directly inhibits fatty acid, triglyceride, and sterol synthesis and the expression of enzymes of fatty acid synthesis and gluconeogenesis [182].

Organ ischemia or hypoperfusion causes ATP exhaustion in mitochondria, with subsequent change in cell membrane permeability, intracellular osmolality, cytoskeletal and mitochondrial damage,or even cell apoptosis or necrosis. Organ reperfusion causes further worsening of injury, which is related to the interaction between neutrophil and dysfunctional endothelial cells. Subsequent changes, such as the organ free radical production, activation or the coagulation system, or further inflammatory cells adhesion and other types of tissue injuries that occur in the reperfusion stage [185-189]. HPC can reduce the ischemia-reperfusion injury in solid organs such as the heart, liver, kidney and bones.

Decrease in energy supply and demand

Reducing demand through depression of metabolic rate is the only viable long-term strategy for a vertebrate to survive without oxygen[190, 191]. Some special hypoxia tolerant animals such as Crucian cap and freshwater turtles have superior capacity to suppress their metabolic levels[9]. Newborn animals are more eあective in down-regulating their metabolic levels than their adults[10, 12]. Metabolic depression can be well accomplished by rapidly decreasing their body temperature, heart rate, respiration rate and movements in hypoxia tolerant animals and newborn animals[12]. Metabolic suppression can also be realized by inhibition of some cellular activity such as protein synthesis and ion channels in these animals[13, 192, 193] . One of the most effective ways to make the adult hypoxia-intolerant animals tolerant to hypoxia is increasing their capacity to suppress metabolism.

Accompanying the increased hypoxic tolerance,the mice greatly down-regulates their metabolic level, which is manifested as gradually but drastically reduction in heart and respiration rates, oxygen consumption and carbon dioxide production in the animal model of WbHPC [194]. そe animal's energy supply was reduced progressively. そe rate of oxygen consumption and carbon dioxide production was exponentially declined as the exposure went on.そe dynamic PO2change in the jar showed that the reduction in PO2was gradually slowed down with the increasing number of hypoxic exposures. そe PO2in H1 was rapidly decreased from 21% to around 6.4%in about 17 min, a tolerant limit level for the mouse in H1. However, the time prolonged to about 42 min in H2 before the PO2decreased to the tolerant limit level. The time for the reduction of the PO2to the tolerant limit level was further extended to 106 min in H3, and 160 min in H4 and H5, respectively. そe body temperature of the preconditioned animals was also exponentially decreased. It was 35°C on average before the first explosure and decreased to 20°C close to room temperature immediately following the conclusion of the fifh run[3].

The level of two major forms of glucose transporters, Glut-1 and Glut-3, was down regulated in the hippocampus in group H5, showing the metabolic level is lowered in the brain of hypoxic preconditioned animals[195]. The activity of succinate dehydrogenase(SDH) and LDH was respectively decreased and increased in the hippocampus of preconditioned animals, leading to a transformation to anaerobic glycolysis from aerobic oxidation of glucose. No apparent diあerence was shown in the activity of glucose-6-phosphatase and ATPase, indicating no apparent changes happen to the activity of glycogenolysis and neuronal membrane pumps can thus be maintained under hypoenergy provided mainly by glycolysis. After injection of iodoacetic acid, the animal's survival time was significantly prolonged in comparison with malonic acid and cyanide acid injected animals,indicating the efficacy of energy via pathway of glycolysis is higher than pathways of both Krebs cycle and respiratory chain in the preconditioned animals[3].

During the repetitive exposure of the model WbHPC, the animal's energy demand gradually decreased. Spontaneous movement became less and less. During the first run, the animals' respiration quickened gradually, cyanosis gradually increased and finally spasm-like activity and gasping breath appeared. Similar behavior was evident during the second run. However, starting with the third run, the animals remained quiet most of the time and their respiration became slow, deep but regular in pattern.Cyanosis became more apparent than before and the eyeballs showed a black-violet color. Starting with the run 3, the animals kept quiet most of the time till the end of fifh run. そe animals' respiration and heart rate were also progressively decreased as the exposure repeated. Similar changes also happened to electrical activity in the cerebral cortex, spinal cord and hippocampus [194].

そe rhythm of ECG was kept regular all the time from run 1 through 5. そe average initial heart rate at beginning at each run progressively decreased from 744 beat/min in run 1 to 180 beat/min in run 5,and the ending one at the first gasping was reduced from 469 beat/min in run 1 to 157 beat/min in run 5, which was one fifth of the initial rate in run 1.The same pattern was also shown in the rhythm of respiration. The average initial respiration rate also decreased progressively from 315 breath/min in run 1 to 96 breath/min, which was also one fifth of the initial rate in run 1[194]. そese physiological changes indicate that the increase in tolerance of animals to hypoxia seems to be negatively correlated to the decrease in energy metabolism, reduction in energy demand and supply. All these changes are quite similar to those occurred in the hypoxia-tolerant animals and hypoxia-intolerant newborn mammals.

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