Intracellular Signaling: Mediators and Protective Responses

Preconditioning

Preconditioning of the brain and other organs is an adaptive response to a noxious but nonlethal experience that activates an intracellular reaction rendering the tissue resistant to a subsequent potentially lethal event. ^, The ability of cells to increase their tolerance to a damaging stress was first described in the heart by Murry et al. and was followed shortly thereafter by studies in the brain by Schurr et al. Since these seminal studies, research in laboratories around the world demonstrated the protective effects of preconditioning at the organismal, tissue, and cellular levels and in numerous species. Up to now, an adaptive endogenous protective response has been revealed in the retina, skeletal muscle, liver, kidney, small intestine, and pancreas. Preconditioning is also observed in nature where certain species of animals appear to exist in a continuous preconditioned state, allowing them to survive in extreme environmental conditions. For example, a few species of turtles such as the freshwater turtles of the genus Trachemys and Chrysemys and the marine loggerhead turtle Caretta carreta , as well as the freshwater fish, Carassius carassius , are known to tolerate extended periods of anoxia. In fact, tolerance to an extreme or otherwise lethal environment is found ubiquitously in nature with the first published accounts of anoxia tolerance in reptiles reported by Belkin in the 1960s. ^, Metabolic adaptation is also seen in higher euthermic animals such as the Arctic ground squirrel, Spermophilus parryii , which experiences extreme reductions in cerebral blood perfusion during months of hibernation, ^, and the reperfusion-like return of blood flow during awakening where no brain damage occurs. ^, This tolerance of the Arctic ground squirrel is not just attributed to hibernation since the Arctic ground squirrel tolerates hypoxia even when not hibernating, suggesting that resistance to ischemia/reperfusion injury in the Arctic ground squirrel could persist when the animal is active.

Preconditioning is a term applied to the noxious but not lethal event that activates the protective response. The state of enhanced resistance to subsequent lethal events is termed tolerance. The clinical utility of preconditioning is not yet apparent. In the heart, retrospective studies demonstrate that patients that experience angina prior to an acute myocardial infarction have smaller infarct size, a reduction in ventricular dysfunction and arrhythmias, and a better in-hospital outcome following thrombolytic therapy than patients without preinfarction angina. ^, Similarly, retrospective studies in patients with a history of transient ischemic attack suggests that preconditioning and tolerance occur in the human brain as these patients have a more favorable outcome with smaller infarct size, milder clinical impairment, and decreased morbidity following stroke. Despite a more favorable outcome, patients who experience transient ischemic attack have a tenfold or higher increased risk of stroke, ^, and these retrospective studies did not assess patients who did not survive their stroke. In addition, doubts have been raised about whether this type of intrinsic protection prevails with age. ^, However, these data provide a provocative possibility that the human brain and heart can be preconditioned and experience a state of tolerance. Thus, understanding this process may provide new therapies targeted toward patients at risk of stroke and myocardial infarction.

Windows of Preconditioning

Preconditioning consists of early and delayed windows of protection that are characterized not only by their temporal profiles of protection but also by the mechanisms of activation and the robustness of the neuroprotective response ( Fig. 6.1 ). The early or rapid window of protection is activated immediately following the preconditioning stimuli and lasts approximately 1 hour. This rapid onset of protection is mediated by the combination of releasable factors and metabolites, altering intercellular signaling cascades. The second window of protection is activated 24–48 hours following preconditioning and lasts for at least several days. This delayed window of protection is characterized by epigenetic reprogramming of the cell, allowing for a more robust and sustained state of tolerance when compared to the early window of protection. ^, Two new windows have been recently uncovered. A 2-week repetitive hypoxic preconditioning (RHP) protocol afforded protection against transient focal stroke, which persisted for 8 weeks. Also, a pharmacologic preconditioning paradigm using a single administration of resveratrol afforded neuroprotection against focal cerebral ischemia 2 weeks later.

The preconditioning response is initiated by various trigger signals that activate receptors and downstream effector pathways leading to both the early and delayed windows of protection. Numerous trigger mechanisms have been described and include neuroactive cytokines, glutamate, adenosine, adenosine triphosphate (ATP)-sensitive potassium (K+ATP) channels, and hypoxia to name a few. Our laboratory and others have shown that both adenosine and post-synaptic N-methyl-D-aspartate (NMDA) receptors are required for ischemia-triggered preconditioning. ^{, ,} Adenosine is the final product of ATP metabolism and is released following ischemic preconditioning (IPC) where it activates adenosine A1 receptors (A1R). Activation of both NMDA and A1 receptor pathways lead to the activation of the novel protein kinase c family member PKC epsilon (PKCε). PKCε is central to the preconditioning response and is sufficient in and of itself to induce tolerance. Numerous other signaling pathways have also been characterized during the induction of tolerance including AKT, the MAP kinase signaling cascade, extracellular signal-regulated kinase (Erk), JNK, and p38. The activation of numerous signaling cascades following preconditioning suggests that there is likely cross talk between the prosurvival kinase cascades such that tolerance may be the result of the coordination of several signaling pathways that act in concert to target many of the pathophysiologic mechanisms leading to cell damage. Numerous reviews are written that provide in-depth overviews of these signaling pathways and their effect on IPC

Induction of Preconditioning

As mentioned, preconditioning can be activated by nearly any stressful stimulus. In cell culture preconditioning can be triggered by a wide variety of stimulations including adenosine, ^, norepinephrine (noradrenaline), ^, calcium, bradykinin, ^, heat shock, ^, mitochondrial uncouplers, ^, chemical inhibition of oxidative phosphorylation, exposure to excitotoxins, ^, cytokines, ^, ceramide ^, nitric oxide (NO), ^, a variety of other inhaled gases (argon, carbon monoxide, ^, hydrogen, and hydrogen sulfide ^, ), potassium chloride, ^, low-dose NMDA, ^{, ,} hypoxia, ^, anoxia, ^, exercise, hyperthermia, ^{, ,} hypothermia, ^, ɣ-radiation, dietary changes, exposure to enriched environments, remote conditioning, lipopolysaccharide (LPS), ^, and oxygen glucose deprivation.

Several intracellular signaling pathways mediate preconditioning. The molecular events that mediate tolerance are an active area of investigation. In vivo, preconditioning in the brain can also be experimentally induced by a variety of stimuli. Both global ischemia (occlusion of both common carotid arteries) and focal ischemia (occlusion of the middle cerebral artery) for short durations can activate tolerance within 24 hours. ^, Placing animals in a chamber and exposing them to hypoxia for 1–6 hours induces tolerance to either transient or permanent focal ischemia 1–3 days following the hypoxic incident. A small dose of LPS injected into the peritoneal cavity is sufficient to induce tolerance within 2–3 days following injection that is sustained for approximately 7 days. ^, Inhibition of oxidative phosphorylation by the irreversible inhibitor of succinate dehydrogenase, 3-nitropropionic acid, activates preconditioning in gerbils and rats that develops over 1–4 days, providing protection against transient focal ischemia. Exposure to cold or heat can trigger tolerance in experimental animals. Hypothermic (25–32°C) ^, or hyperthermic temperatures (42–43°C), ^, induce tolerance to focal ischemia 24 hours later. However, this preconditioning appears to have a shorter window of opportunity than other stressors in which tolerance is sustained from 24 to 72 hours. Temperature stress provides protection between 18 and 24 hours, but this protection resolves by 48 hours. Cortical spreading depression of slowly propagating waves of depolarization across the cortex can be triggered experimentally by the application of potassium chloride on the surface of the dura mater or the cortex. Cortical spreading depression induces a prolonged phase of ischemic tolerance that lasts 1–7 days, ^{, ,} providing protection against transient and permanent focal ischemia. Surprisingly, inhalational anesthetics can chemically precondition the brain in experimental animal models. Isoflurane, sevoflurane, and halothane when given to animals provide protection against permanent or focal ischemia. In the case of isoflurane, the protective effects are immediate and last at least 24 hours. This suggests that isoflurane can activate both acute and chronic preconditioning. These observations introduce a significant confound in exploring preconditioning in animal models since all experimental stroke studies are conducted under anesthesia. Since preconditioning can be activated by a diverse set of stressors, the molecular mechanisms initiating and sustaining the protective response are active areas of investigation. It would be simple to understand if preconditioning positively altered cerebral blood flow. However, measurements of cerebral blood flow showed that tolerance was not accompanied by an improvement of regional tissue perfusion during or after the ischemia that induced tolerance. ^, Thus, ischemic tolerance is likely a result of changes in the neurons, glia, and blood vessels of the brain at the cellular level in response to stress.

Cross-Tolerance

Numerous studies indicate that preconditioning can be activated by a wide range of insults, chemical, pharmacologic, or physical. Virtually any stimulus that can alter brain function appears to have the capacity to increase brain resistance to future injurious events. Furthermore, one stressor that activates preconditioning can induce tolerance toward a different injurious stressor such as LPS inducing tolerance against ischemia. This phenomenon is termed cross-tolerance. The degree of efficacy in cross-tolerance may be somewhat diminished in comparison to IPC inducing ischemic tolerance. Also, the window for the development of tolerance may be altered. For example, in many models, it takes between 2 and 3 days for maximal tolerance to be realized following injection of LPS rather than 1 day for ischemic tolerance. Since tolerance is observed across organ systems and tolerance can be induced by a wide variety of stressors, it is logical but perhaps simplistic to presume that these stress signals would converge onto a final common pathway to promote cellular survival. While very attractive, this hypothesis does not appear to be valid. A number of genetic analyses of tissue following exposure to different preconditioning stressors show that different gene sets are differentially expressed depending on the nature of the preconditioning stimulus. ^, Studies of individual proteins and their involvement in preconditioning and tolerance support this observation as well. ^{, ,} Conceptually, potential mechanisms could include both enhanced cellular defense functions and increased cellular surveillance improving cell maintenance through the stress response. Evidence for both pathways exists. Preconditioning can arise either by post-translational modification of proteins or by expression of new proteins. These newly expressed proteins and enhanced signaling cascades can either strengthen survival mechanisms or may inhibit cell death signaling. Activation of the cell stress response and synthesis of stress proteins increases the capacity for general cell maintenance allowing for proper cell function. The best-known stress response proteins are protein chaperones that unfold damaged or misfolded proteins to facilitate the disposal of these unneeded proteins by the cell. It is not yet known if these two strategies work in concert or independently. However, it is curious that in many experimental model systems knockdown or knockout of a single preconditioning molecule is sufficient to block the development of preconditioning, and the expression of a single molecule is often sufficient to provide protection. These observations would suggest some sort of a network response to provide protection, even if there is no final common pathway.