Severe hypothermia and hibernation are two extreme metabolic conditions where, despite the presence of much reduced metabolic rates, it is possible to recover without irreversible cerebral lesion.
The possibility of inducing hypometabolic states following acute cerebral ischemia by lowering body temperature as well as oxygen consumption may help increase the window of therapeutic opportunity of thrombolysis, thus contributing to making it accessible to an increased number of patients with ischemic stroke. 
INTRODUCTION

Current treatment of acute ischemic cerebrovascular accidents (CVA) includes arterial reperfusion using rt-PA (Recombinant tissue-plasminogen activator) and treatment at CVA Units. However, numerous neuroprotective substances have presented advantages during pre-clinical studies, both in terms of final volume and in functional result. Unfortunately, to date none has demonstrated unequivocal clinical benefit. The reasons advanced for the difficulties of extrapolation to human beings include the need for excessively early administration of drugs, which presents difficulties within a clinical context, the disparity of measurement instruments, including functional scales [1], or the differences in the acquisition of imaging techniques during the acute stage, such as the definition of the penumbra[2,3].

There is evidence that, if untreated, the penumbra area bordering the ischemic lesion will soon become part of the central infarct zone, increasing the volume of the initial lesion, compromising functional prognosis in the short and medium terms.

One of the factors associated with functional improvement is early intervention, in order to prevent irreversible brain cell ischemia; early intervention has a higher impact in terms of functional results than interventions performed at later stages. This is one of the reasons behind the recommendation to treat CVA as an emergency situation. Another reason is the short therapeutic window mediating between the start of symptoms and the possibility of reperfusion. Measures enabling the widening of that window allow an increasing number of patients to benefit from brain reperfusion techniques. Procedures aiming to slow down or interrupt the metabolic processes involved in the so-called “ischemic cascade” have the potential of extending the time available to perform the reperfusion.

HYPOTHERMIA
Inducing a drop in body temperature may reduce deterioration following acute ischemic lesion, if induced early after the start of ischemia symptoms and if carried out for the right period of time.

The use of moderate hypothermia in the context of the acute stage of CVA is currently at Phase II of clinical trials.
In animal models of acute ischemia, induced hypothermia has presented benefit, both in terms of reducing the infarct and in functional results.

There are two levels of reducing body temperature that have therapeutic interest for translational studies: mild hypothermia (33ºC – 35ºC) and moderate hypothermia (30ºC – 33ºC). Mild hypothermia is safer and technically easier to perform[4]. In humans, it is possible to reach the desired temperature without resorting to sedation or mechanical ventilation, which potentially allows for extended clinical use. Nonetheless, the neuroprotective effect does not seem to be the same with regard to different temperatures, during the hypothermia and the gap between the start of ischemia symptoms and hypothermia induction; it was noted that maximum efficacy occurs when cooling is moderate (=31ºC) and over a prolonged period of time (12h a 48h) [5].

However, it recently has been confirmed that with even with small reductions of basal body temperature of around 2ºC or less, there is a neuroprotective effect with histological and behavioural evidence[6].

The mechanisms which make hypothermia neuroprotective are not entirely clear yet. With regard to mild hypothermia, neuroprotection was exclusively associated with the induced metabolic changes (the drop in temperature reduces metabolic needs). However, protective biological processes, such as detoxification and repair, would also be inhibited, which makes the final balance difficult to establish. Currently, there seems to be evidence that the pathophysiology of hypothermia also includes changes in the cerebral blood flow, in cell membrane stability, energetic metabolism, calcium metabolism and intracellular signalling, in excitotoxicity mechanisms, in acid-base balance, in the formation of free radicals, and in apoptosis mechanisms[7].

Accordingly, this makes hypothermia a protector of tissues, due to the fact it boosts resistance to deleterious effects (including interfering with glucose metabolism, diverging it from the glycolysis pathway to the pentose phosphate pathway, which is potentially a neuroprotective pathway, and reducing the levels of lactate tissue and intracellular acidosis, freeing excitatory amino acids, the formation of free radicals, the mitochondrial release of cytochrome C and kinase C, the activation of microglia, the inflammation and behaviour of the hematoencephalic barrier, reducing its deterioration and rupture) and inducing the inhibition of at least one cell death pathway, covering several routes of lesion mechanisms and contributing to the preservation of both cell function and structure, thus improving the final tissue result [8].

NITRIC OXIDE, CARBON MONOXIDE AND HYDROGEN SULPHIDE 
Nitric Oxide (NO) and Carbon Monoxide (CO) are signalling gaseous molecules of the central nervous and cardiovascular system, which have denoted cytoprotection benefits in a contest of brain and heart ischemia [9]. Recently, especial attention has been paid to the potential biological significance of hydrogen sulphide (H2S)[10], first known for its environmental toxic properties [11]. These 3 labile biological mediators (NO, CO e H2S) are able to cross cell membranes without resorting to specific transporters.

H2S is synthesized in mammals in several tissues through two metabolic pathways of the L-Cysteine that depend on pyridoxal-5′-phosphate enzymes: cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CSE). L-Cysteine can originate from food sources or be synthesized from L-Methionine through the transsulfuration pathway, Homocysteine acting as the intermediary in the process.

The biological effects of exposure to H2S seem to depend, in particular, on its capacity to inhibit mitochondrial oxidative phosphorylation and hypothermia induction.

Nonetheless, they are controversial and depend, among other factors, on the dosage and duration of the exposure. In micro molecular concentrations, hydrogen sulphide appears to have a neural protection role in oxidative stress[12], with modulation of the intracellular caspase and kinase pathways, influencing the activity of the N-metil-d-aspartate (NMDA) receptors in physiological concentrations and modifying the induction of long term potentiation (LTP) [13]. It also induces an upregulation of cytoprotective and anti-inflammatory genes, including Heme oxygenase-1 (HO1), which allows the production of CO, which is cytoprotective and anti-inflammatory.

It is present in the central nervous system in relatively high levels of concentration (50 to 160 microM), by comparison with other organs, which suggest it has a localized physiological function[14].

OXIDATIVE PHOSPHORYLATION INHIBITORS AND HYBERNATION
During states of dormancy such as hibernation, mammals are able to survive with metabolism rates as low as 2% of their usual needs, without suffering irreversible brain lesions. During these periods, animals need to carry out adaptive transformations at brain level, which include adapting to deleterious conditions for species that do not normally hibernate.

These adaptations include not only mechanisms of metabolic suppression, but also increasing oxidative defences, in addition to lowering body temperature and immune functions[15]. Knowledge of the regulation and of the reversible induction of extreme hypometabolic conditions mechanisms can contribute to a better understanding of some aspects of ischemic lesions and reperfusion.

Curiously, it was exactly thanks to H2S that it was possible, in 2005, to induce a condition similar to hibernation in mammals that normally do not hibernate. This condition was called “suspended animation-like state”[16]. Using rats that had inhaled H2S, a significant drop in metabolic rates (which were measured through O2 consumption and CO2 elimination) and in body temperature was shown. After 6 hours of exposure to this H2S enriched atmosphere, it was stopped, and the rats returned spontaneously to their normal metabolic rates and body temperature, with no changes in their health condition in the short or medium terms; accordingly, the potential role of H2S, as a neuroprotective agent and hypometabolic inductor was advanced, probably for having a tamponading effect in oxygen consumption, changing the intracellular redox environment, preventing imbalance between the sources of energy and the search for energy[17]. The proposed mechanism, thanks to which H2S may regulated the consumption of cell O2, appears to be centred on inhibiting cytochrome C oxidase, which, at the level of the brain, reduces oxygen consumption and inhibits the recapture of the L-glutamate excitatory neurotransmitter. The cytoprotective role of hypothermia is known in the context of acute experimental ischemia. Its potential benefit has been demonstrated in animal models of acute brain ischemia, not just in terms of the size of the legion, but also at functional level. The mechanism probably involves the reduction of the deterioration following acute ischemic lesion.

Nevertheless, the controversy about the cytoprotective role of H2S still persists, with some studies with rodents showing neurotoxic effects. These discrepancies in the action of H2S have been observed within the same specie [18], between distinct species (small mouse and piglets)[19] and between distinct organs (brain ischemia and cardiac ischemia[20] ). The explanation for these results may include the fact that effect may depend on dosage (as indicated by the myocardial ischemia[21]), as well as on difference in exposure times with regard to larger mammals for purposes of manifestation of hypometabolic effects.

Severe hypothermia and hibernation are two extreme metabolic conditions where, despite the presence of much reduced metabolic rates, it is possible to recover without irreversible cerebral lesion.

The possibility of inducing hypometabolic states following acute cerebral ischemia by lowering body temperature as well as oxygen consumption, may help increase the window of therapeutic opportunity of thrombolysis, thus helping to make it accessible to an increased number of patients with ischemic CVA.

Isabel Henriques, Maria Gutiérrez, Exuperio Diez-Tejedor
Instituto de Investigation Hospital Universitário de la Paz, Madrid, Faculdade de Medicina da Universidade de Lisboa (Faculty of Medicine of the University Lisbon)
ilh.igc@gmail.com
_______________________

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