LITHUANIAN UNIVERSITY OF HEALTH SCIENCES MEDICAL ACADEMY
Olga Suchadolskienė
A NEW SWINE MODEL OF GLOBAL
INCOMPLETE CEREBRAL ISCHEMIA FOR
NEUROPROTECTANTS RESEARCH
Doctoral Dissertation, Biomedical Sciences,
Medicine (06B)
The dissertation was prepared at the Lithuanian University of Health Scien-ces during the period of 2009–2014.
Scientific Supervisor:
Prof. Dr. Dinas Vaitkaitis (Lithuanian University of Health Sciences, Medical Academy, Biomedical Sciences, Medicine – 06B)
TABLE OF CONTENT
ABREVIATIONS ... 5
1. INTRODUCTION ... 6
2. AIM AND TASKS OF THE STUDY ... 10
2.1. The aim of the study ... 10
2.2. Tasks of the study ... 10
3. NOVELTY OF THE RESEARCH ... 11
4. REVIEW OF LITERATURE ... 12
4.1. Stroke ... 12
4.2. Postcardiac arrest brain injury ... 13
4.3. Pathological mechanisms associated with global cerebral ischemia ... 14
4.4. The role mitochondria in cerebral ischemia ... 18
4.5. Why targeting mitochondria? ... 21
4.6. Animal models of cerebral ischemia ... 22
4.7. Neuroprotection research ... 27
4.8. Cyclosporine A as a neuroprotective agent ... 29
4.9. Methylene blue (MB) as neuroprotective agent ... 31
5. METHODS ... 33
5.1. Study design... 33
5.2. Modeling of global cerebral ischaemia in pigs ... 34
5.2.1. Anesthesia and surgical preparation ... 34
5.2.2. Experimental groups ... 36
5.2.3. Hemodynamic and blood gas measurements ... 36
5.2.4. Microcirculatory evaluation ... 37
5.2.5. Assessment of mitochondrial function ... 38
5.2.6. Histological analysis ... 38
5.2.7. TUNEL assay ... 39
5.3. Evaluation of neuroprotectants ... 40
5.3.1. Anesthesia and surgical preparation ... 41
5.3.2. Experimental groups ... 41
5.3.3. Hemodynamic and blood gas measurements ... 41
5.3.4. Assessment of mitochondrial function ... 41
5.3.5. Histological analysis ... 42
5.3.6. TUNEL assay ... 42
5.4. Statistical analysis ... 42
6. RESULTS ... 43
6.1. Modeling of global cerebral ischemia ... 43
6.1.1. Assessment of mitochondrial function ... 43
6.1.2. Histological analysis and TUNEL assay ... 45
6.1.3. Microcirculatory evaluation ... 46
6.1.4. Hemodynamic and blood gas measurements. ... 48
6.2. Detection of the primary target of cerebral ischemia ... 50
6.3. Evaluation of neuroprotectants ... 51
6.3.1. Assessment of mitochondrial function ... 51
6.3.2. Histological analysis and TUNEL assay ... 53
6.2.3. Hemodynamic and blood gas measurements ... 56
7. DISCUSSION ... 58
8. LIMITATIONS OF THE STUDY ... 71
9. CONCLUSIONS ... 72 10. RECOMMENDATIONS ... 74 11. REFERENCES ... 75 12. LIST OF PUBLICATIONS ... 91 13. ACKNOWLEGMENTS ... 93 4
ABREVIATIONS
MB – Methylene blueCsA – Cyclosporin A CA – cardiac arrest
OMM – outer mitochondrial membrane IMM – inner mitochondrial membrane IMC – intermembrane space
ETC – electron transport chain OXPHOS – oxidative phosphorylation RCI – respiratory control index ADP – adenosine diphosphate ATP – adenosine triphosphate 4VO – four-vessel occlusion 2VO – two-vessel occlusion CCA – common carotid arterial CBF – cerebral blood flow
MCAO – middle cerebral artery occlusion CCAO – common carotid artery occlusion
MERCI – mechanical embolus removal in cerebral ischemia FDA – Federal Drug Agency
rt-PA – recombinant tissue Plasminogen Activator ROSC – return of spontaneous circulation
ROS – reactive oxygen species COX-2 – cyclooxygenase-2 SDF – side dark field ECG – electrocardiogram TIA – transient ischemic attack AHA – American Heart Association CPR – cardiopulmonary resuscitation BBB – blood-brain barrier
1. INTRODUCTION
Cerebral ischemia is one of the foremost causes of high mortality and morbidity for both developed and developing countries. Cerebral ischemia occurs commonly in patients who have a variety of clinical conditions including cardiac arrest (CA), shock, and asphyxia and in patients undergoing complex cardiac surgery [1-3]. Stroke is an important cause of morbidity and mortality in industrialized countries and few therapies exist thus far. Despite recent advances in out-of-hospital cardiac arrest resusci-tation, hypoxic-ischemic brain damage still causes considerable mortality and morbidity. Although initial return of spontaneous circulation (ROSC) from cardiac arrest is achieved in about 30 to 40% of cases, only 10 to 30% of the patients admitted to the hospital will be discharged with good outcome [4]. Of the patients who survive to discharge, only 20% or fewer will have good neurologic function at the end of 1 year [5]. Stroke is the third leading cause of death in the United States with approximately 5.8 million current cases and approximately 780,000 new cases every year [6] and 1/6 of all humans beings will suffer at least one stroke in their lives [7]. Stroke is one of the leading cause of morbidity, mortality and disability in Lithuania [8, 9], especially in older population[10]. Even if patients survive the acute episode of cerebral ischemia, approximately 15–30% of these patients remain disabled at 3 months after onset of stroke, and approxima-tely 20% require permanent medical care in nursing and supportive care institutions [11–17]. These major health issues require urgent development of novel and more effective therapies. To this day, treatment modalities for stroke are limited and mainly targeted at preventing complications and reduction of risk factors. Effective stroke therapies require recanalization of occluded cerebral blood vessels. Apart from the use of fibrinolytic agents (recombinant tissue Plasminogen Activator or rt-PA), there is no medical therapy that restores neural function and protects from ischemic damage. There is still extremely limited pharmacologic treatment for ischemic stro-ke. Currently there is thromobolytic treatment and mechanical thrombecto-my. The recently FDA-approved the MERCI devices for intra-arterial clot removal in ischemic stroke can be used at 8 hours after stroke, especially for patients ineligible for intravenous rt-PA [18, 19]. Recanalization rate using MERCI retriever can reach 48% [20]. Use of these treatments is also limited by the narrow therapeutic window associated with stroke, during which the-re is hope of salvaging neurological integrity [15]. However, the-reperfusion can cause neurovascular injury, leading to cerebral edema, brain hemor-rhage, and neuronal death by apoptosis/necrosis [21]. These complications,
which result from excess production of reactive oxygen species in mito-chondria, significantly limit the benefits of stroke therapies. It is indeed widely accepted that not all brain cells die immediately after the insult. Surrounding a core of severe and rapid tissue injury, brain cell death spreads more slowly in a heterogeneous region called the penumbra that could still be salvaged [22]. Mitochondria are centrally involved in the development of this tissue injury due to modifications of their major role in supplying ATP and to changes in their properties that can contribute to the development of apoptotic and necrotic cell death. In animal models of stroke, the limited availability of glucose and oxygen directly impairs oxidative metabolism in severely ischemic regions of the affected tissue and leads to rapid changes in ATP and other energy-related metabolites. In the less-severely ischemic “penumbral” tissue, more moderate alterations develop in these metabolites, associated with near normal glucose use but impaired oxidative metabolism. This tissue remains potentially salvageable for at least the first few hours following stroke onset. Early restoration of blood flow can result in substantial recovery of energy-related metabolites throughout the affected tissue. However, glucose oxidation is markedly decreased due both to lower energy requirements in the post-ischemic tissue and limitations on the mitochondrial oxidation of pyruvate. A secondary deterioration of mito-chondrial function subsequently develops that may contribute to progression to cell loss. Mitochondrial release of multiple apoptogenic proteins has been identified in ischemic and post-ischemic brain, mostly in neurons.
Animal models represent important tools for investigating the patho-genesis of human disease and developing appropriate treatment strategies. From the late 1970s, animal models of cerebral ischemia were developed with the aim of identifying mechanisms that cause tissue damage and to provide the basis for the development, at a preclinical level, of new thera-pies for cerebral ischemia. Several recent animal models have been designed specifically to address specific risk factors, to determine neural repair pro-cesses, to test new neuroprotective and recanalizing strategies. Most fre-quently for cerebral ischemia development are used rats, mouses and other small animals. Nevertherless, it is very important to perform preclinical stu-dies of potencial neuroprotectors in large animal with gyrencephalic brain.
Neuroprotection is a therapeutic approach that aims to prevent or atte-nuate neuronal degeneration and loss of function in neurological diseases. Unfortunately, despite promising results from preclinical studies, outcomes of clinical neuroprotection trials have been repeatedly disappointing [23, 24]. Neuroprotective strategies, including but not limited to free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades [25]. Assessment of the
efficacy of neuroprotective compounds against ischemia-reperfusion injury is an important in vivo experimental strategy relevant to ischemic stroke. There are a number of experimental models of global cerebral ischemia used for preclinical studies of pharmacological interventions. Experimental evidence supports the concept that establishing reperfusion alone is not enough to cease ischemic injury and each step of the ischemic cascade may be a genuine target for therapeutic intervention. A large number of potentially neuroprotective agents directed at different harmful mechanisms in the ischemic cascade have been investigated in experimental animal stroke studies. But majority of the substances which were found to be neuroprotective in animals have failed in clinical trials. Potential reasons of this translation failure from laboratory to the clinic have been extensively discussed elsewhere [26–32]. Pharmacological agents used in these studies include estrogens [33, 34], antiepileptic drugs [35], COX-2 inhibitors [36], free radical scavengers [37] and tissue plasminogen activator [38]. Except for the use of mild hypothermia after cardiac arrest, currently recommended therapy in the 2005 and 2010 guidelines of the European Resuscitation Council [39, 40], clinical neuroprotection practice rests solely on extrapo-lation from animal experimental work or weak clinical studies [41]. For protection of the brain and other organs, hypothermia is a helpful therapeu-tic approach in patients who remain comatose (usually defined as a lack of meaningful response to verbal commands) after ROSC [40].
Multiple clinical trials for the treatment of acute ischemic stroke were conducted in last years, but all were unsuccessful. Can animal models truly replicate clinical stroke? The answer is complex, and it should be recog-nized that, in the end, these are only models of stroke, not stroke itself, which is a multifactorial human disease [32, 42–44]. That is very huge challenge- to create useful model of cerebral ischemia for neuroprotective compounds research. STAIR [45] recommends use pigs for neuroprotection research in preclinical studies. It is very important to understand processes in ischemia-injured cells and to find first target of injury. The additional treatment with neuroprotective agents theoretically could extend the time window and limit the tissue damage following ischemia. The cascade of injury by ischemic stroke is very complex and multifaceted and therefore, different neuroprotective agents have been tested to block the progression of injury signals at different steps. But unfortunately up to now, no clinical study could demonstrate the efficacy of neuroprotective drugs [46].
The growing interest in mitochondrial targeting is revealing new avenues to identify both novel therapeutics as well as potential sources of toxicity for existing medicines. Increasing evidence has indicated that mito-chondrial dysfunction is a common pathological mechanism that underlines
many neuropathological conditions. Mitochondria are key organelles that perform essential cellular functions and play pivotal roles in cell death and survival signaling. Mitochondrial dysfunction is one of the major events responsible for activation of neuronal cell death pathways during cerebral ischemia [47]. Methylene Blue and Cyclosporin A has been shown to pro-tect neurons in various diseases. Mitochondrial respiration represent an attractive target for drugs to treat metabolic, degenerative and hyperproli-ferative cerebral diseases. Targeting mitochondria with organelle-specific agents or drugs has proven to be an effective therapeutic strategy. More specifically, controling the cellular ROS balance via selective delivery of an antioxidant “payload” into mitochondria is an elegant emerging therapeutic concept. Several reviews of neuroprotection in both ischemic and hemor-rhagic stroke have already been published in the last few years [48, 49]. Reasons for the unsuccessful translation of neuroprotective therapies from animal to human are probably multiple [49]. This has led the Stroke Aca-demic Industry Roundtable (STAIR) to make recommendations to improve the quality of preclinical studies of purported acute stroke therapies [50, 51]. One aspect concerns the preclinical stage of the drug development where insufficient dose-response or time-window studies, inappropriate drug deli-very protocol, or brain penetration issues are often encountered. In vivo analysis of the mechanism targeted by the drug is also among the aspects that should be improved.
A better understanding of clinical and cell physiological alteration is crucial to develop therapeutic strategies. A new model of global cerebral ischemia using pig could help to understand development of cerebral ischemia, found the first ischemical target in brain cell and evaluate effect of neuroprotectants. Stroke and cerebral ischemia continues to kill millions people each year, and the development of safe and effective treatment is a major challenge to experimental and clinical neuroscience.
2. AIM AND TASKS OF THE STUDY
2.1. The aim of the studyTo create a new experimental swine model of global incomplete cerebral ischemia and to check neuroprotective effect of Methylene blue and Cyclosporine A in created model.
2.2. Tasks of the study
1. To evaluate mitochondrial respiration, histological and microcir-culatory changes in experimental models of global cerebral ischemia.
2. To evaluate mechanisms and identify the primary target of ischemia in brain cell in experimental models.
3. To evaluate neuroprotective effect of Methylene blue and Cyclo-sporin A in created swine model of global cerebral ischemia.
3. NOVELTY OF THE RESEARCH
This multidiscipline cerebral ischemia research incorporates neurology, biochemistry, histology, videomicroscopy analysis. Simultaneous use of histological, mitochondrial respiration, and microcirculatory evaluation techniques allows detailed study of the mechanisms involved in the deve-lopment of cerebral ischemia and create useful experimental model of global incomplete cerebral ischemia. In this study, we performed detailed and complex investigation of changes in mitochondrial respiration, micro-circulation and histology during global cerebral ischemia in the pig. A new swine model of global incomplete cerebral ischemia allowed to find the primary target of ischemia injury in neurons and check neuroprotective effect of methylene blue and cyclosporine A.
The Stroke Academic Industry Roundtable (STAIR) recommends to use large animals for high quality preclinical studies [50, 51]. In this study was used pigs- animal with gyrencephalic brains and performed detailed evaluation of global incomplete cerebral ischemia.
For the first time in a single study it was performed detailed investigation of global cerebral ischemia, created new swine model of global incomplete ischemia and evaluated neuroprotective effects of Methylene blue and Cyclosporine A in swine model.
4. REVIEW OF LITERATURE
4.1. StrokeStroke is the 3rd most common cause of death after coronary heart disease and cancer in developed countries and the most frequent cause of long-term disability in the adult population worldwide [15]. Stroke is an increasingly prevalent clinical condition, especially with a gradually aging population. Nearly 800,000 strokes occur in the United States each year, ranking it as the fourth leading cause of death behind heart disease, cancer and chronic lower respiratory disease [52, 53]. A stroke is clinically defined as the sudden loss of oxygen to brain tissue in a localized area due to inadequate blood flow. The American Heart Association (AHA) classifies stroke into two categories: ischemic (clots), hemorrhagic (bleed). Ischemic strokes are the most common type of stroke and accounting for approxi-mately 85–87% of all strokes while non-traumatic hemorrhage account for up to 13–15%. Although ischemic stroke is the third most common cause of death in the United States and Europe, the only currently approved medical treatment is the administration of intravenous recombinant tissue plasmi-nogen activator within 4.5 hours of stroke onset (according to the European Stroke Organisation guidelines), aimed at restoring cerebral blood flow [11, 54].
Ischemic stroke results from the blockade of an artery to the brain from in situ thrombosis or an embolus from another artery or the heart. Ischemic stroke results in macrophage infiltration, blood brain barrier breakdown and cellular dysregulation and infarction, followed by formation of edema [55-58]. Hemorrhagic stroke occurs due to a weakened blood vessel rupturing due to aneurysm or arteriovenous malformations, which results in bleeding into the surrounding brain tissue and subsequent tissue compression. TIA, as the name suggests, caused by transient blockage usually less than 24 h (typically <5 min) of a vessel, and is often considered a warning stroke (by the AHA). TIAs are typically not associated with damage due to the short duration of the blockage. Blood flow deficit inhibits the delivery of oxygen, glucose and other nutrients from the blood, resulting in an expanding infarct core with a time-sensitive, salvageable periinfarct penumbra that is the primary target for treatment strategies [59–61]. Penumbral tissue is potentially salvageable because this region still exhibits partial blood flow, oxygenation and metabolic activity [62]. Cellular integrity and function are still preserved to varying degrees within this area [63]. Due to the brain’s limited capacity to store glucose and the limited ability to utilize anaerobic
metabolism, rapid neurodegeneration occurs. The capacity of damage to the brain is determined by a number of factors including the location and dura-tion of blockage, state of the circulatory system, collateral flow within the affected region, and the presence of other disease states [64–66]. A primary goal of developing therapeutic interventions in acute ischemic stroke is to preserve the ischemic, but still viable, cerebral tissue. The other potential approach to acute stroke treatment is to try to interfere the ischemic cascade by targeting various components of the cascade that are deemed to be of importance. This latter approach is called the neuroprotection strategy [67].
4.2. Postcardiac arrest brain injury
Post cardiac arrest brain injury is a common cause of morbidity and mortality worldwide [68]. Every year approximately 330 000 individuals in the United States [6, 69, 70] and 700 000 in Europe [71] suffer an episode of sudden cardiac arrest outside the hospital. Efforts to reestablish life are formidably challenging, requiring not only that cardiac activity be reestab-lished but that injury to vital organs be prevented, minimized, or reversed. Current resuscitation methods yield an average survival rate to hospital discharge with intact neurological function that approaches only 5% [41]. Efficient Emergency Medical Services systems can initially reestablish cardiac activity in approximately 30% of victims [72–74] with over 30% dying before hospital admission [75]. Of those admitted to a hospital, nearly 75% die before hospital discharge suffering variable degrees of myocardial dysfunction, neurological dysfunction, systemic inflammation, intercurrent illnesses, or a combination thereof [75-77]. Thus, initial reestablishment of cardiac activity using current resuscitation techniques does not ensure ultimate survival. In the course of cardiac arrest, global cerebral blood flow is severely impaired with the consequent risk of ischemic damage of brain cells, which magnitude seems to be associated with the cumulative time staying in cardiac arrest. Thus, most deaths (60%) during the post-resuscita-tion period have been attributed to extensive brain injury and neuronal damage that develops as a consequence of alteration of cell processes triggered by cerebral ischemia and reperfusion, during and after cardiac arrest. In addition, it is known that transient interruption or reduction of blood flow in the whole brain, are main causes of permanent brain damage and functional disruptions in human beings, and near around a half of surviving patients show permanent impairment of cognitive functions, such as learning and memory, attention, and executive functioning, and only a small proportion (less than 10%) of those survivors are able to reassume
their former usual life styles [78, 79]. Novel resuscitation approaches are needed to increase the rate of initial resuscitation and subsequent survival with intact organ function. After successful CPR and restoration of spontaneous circulation (ROSC) neuronal death initiated by ischemia during CA is increased also during reperfusion leading to secondary postischemic-anoxic encephalopathy [80], part of the so-called postresuscitation synd-rome [81, 82]. Cerebral recovery is dependent on duration of arrest and cardiopulmonary resuscitation (CPR), and numerous factors related to basic, advanced, and prolonged life support [83, 84].
Although resuscitation requires reperfusion of ischemic tissue with oxygenated blood to restore aerobic metabolism and organ function, reper-fusion concomitantly activates multiple pathogenic mechanisms, collecti-vely known as “reperfusion injury” [85]. At the center of reperfusion injury are mitochondria, playing a critical role as effectors and targets of injury.
4.3. Pathological mechanisms associated with global cerebral ischemia
Cerebral ischemia may be defined as a condition of reduction in blood supply to the brain, which leads to decreased availability of glucose and oxygen to brain cells. This can be due to a lack of blood flow caused by stroke or a cardiac arrest. Glucose and oxygen depletion after ischemia cau-se disruption of cell ion homeostasis, leading to increacau-sed intracellular cal-cium and neuronal cell death [86]. Interruption of blood flow and hence, of glucose and oxygen supply to the brain, results in an immediate severe energy failure in terms of ATP depletion that leads to alterations of the cell membrane ionic gradients and a severe breakdown in cellular homeostasis. Several mechanisms of neuronal damage are triggered and evolve both in cascade and as parallel pathways [79, 87] (Fig. 1). In particular, a massive accumulation of intracellular calcium and sodium occurs because of failure of their energy-dependent efflux processes, and anoxic depolarization. This further leads to accumulation of lactate and hydrogen ions, and as a consequence, to decreased pH.
Fig. 1. Potencial mechanisms of injury from ischemia [100].
The pathophysiology of stroke is complex and involves numerous processes including energy failure, loss of ion homeostasis, overload of intracellular calcium ion (Ca2+) concentrations, excitotoxicity, activation of free radicals, release of cytokines, disruption to the blood-brain barrier (BBB), activation of glial cells and inflammation; these series of cascading events are well interrelated and coordinated, and they ultimately lead to necrosis and apoptosis (Fig. 1). Global cerebral ischemia is generally cha-racterized by the sudden loss of blood circulation in the brain, resulting in a corresponding loss of neurologic function, complications, and death.
Glucose taken up from the blood is metabolized through two major pathways: glycolysis and oxidative phosphorylation. Glycolysis, which occurs in cytosol, is the metabolism of glucose to pyruvate and lactate, and has a low-energy yield. In contrast, oxidative phosphorylation is the major pathway of ATP synthesis. It is driven by energy derived from electron transport in the mitochondria and is responsible for ~92% of total ATP production. In an attempt to repair cell damage, numerous energy-consum-ptive pathways are activated, which may further contribute to cell death. The resultant disruption to delivery of glucose and oxygen leads to a greatly reduced ATP generation. The mechanisms involve a complex series of
pathophysiological events that are dependent on the severity, duration, and location of the ischemia within the brain. A simple overview of these pathophysiological mechanisms is that energy failure results in neuron depolarization, which causes activation of glutamate receptors, which in turn alters ionic gradients of Na+, Ca++, Cl–, and K+. As glutamate increases in the extracellular space, peri-infarct depolarization occurs. Then, as water shifts occur, cells swell with resulting cerebral edema. The result of increasing intracellular Ca++ is an upregulation of a variety of enzyme systems such as lipases, proteases, and endonucleases. As a result, free O2 radicals are generated via a variety of biochemical pathways, and apoptotic cell death occurs. Free radicals also induce formation of a variety of inflammatory mediators such as platelet and endothelium selectins, a variety of molecules, platelet activating factor, tumor necrosis factor α, and an assortment of interleukins. Ionic gradients across the plasma membrane quickly dissipate resulting in marked losses of intracellular potassium and large shifts of calcium into cells [88–90]. Excessive intracellular calcium activate abnormal cell processes promoting functional derangements of mitochondria and an increased production of free radicals, exceeding the neuronal antioxidant reserves, and imposing risks to the structural and functional integrity of neuronal cells. The brain is highly susceptible to oxidative damage as a consequence of its high lipid and metal content, as well as other biochemical characteristics [91, 92]. Reperfusion and reoxygenation of the ischemic tissue, which must be reestablished within minutes in an effort to prevent severe neurological damage and favor survival of individuals, also may provide chemical substrates for further increasing cellular alterations, neuronal death and neurological deficits [91].
Because of contributions to perfusion from adjacent vessels, a lesser ischemia develops in tissue surrounding the core. This “penumbral” or “perifocal” tissue typically exhibits reductions to approximately 20 to 40% of normal flow [93–95]. These are interrelated and coordinated events, which can lead to ischemic necrosis, which occurs in the severely affected ischemic-core regions. Within a few minutes of a cerebral ischemia, the core of brain tissue exposed to the most dramatic blood flow reduction, is mortally injured, and subsequently undergoes necrotic cell death. This necrotic core is surrounded by a zone of less severely affected tissue which is rendered functionally silent by reduced blood flow but remains meta-bolically active [96]. Necrosis is morphologically characterized by initial cellular and organelle swelling, subsequent disruption of nuclear, organelle, and plasma membranes, disintegration of nuclear structure and cytoplasmic organelles with extrusion of cell contents into the extracellular space [96]. The region bordering the infarct zone, known as the ischemic penumbra,
comprises as much as half of the total lesion volume during the initial stages of ischemia, and represents the region in which there is opportunity for salvage via post-stroke therapy [97]. Less severe ischemia, as occurs in the penumbra region of a focal ischemic infarct, evolves more slowly, and depends on the activation of specific genes and may ultimately result in apoptosis [87, 98]. Recent research has revealed that many neurons in the ischemic penumbra, or periinfarct zone, may undergo apoptosis only after several hours or days, and thus they are potentially recoverable for some time after the onset of stroke.
It has been known that mechanisms of cellular damage, repair and plasticity may be the same, in general, both if reduction of blood flow to the brain tissue results from occlusion of one of the main cerebral arteries as would occur in focal ischemia, and if it is the result of reduction of blood flow to the whole brain as it would occur after a cardiorrespiratory arrest.
Unless rapidly reversed, the occlusion of a major artery usually pro-duces tissue infarction, in which affected parts of the brain exhibit a non-selective loss of all cells including neurons, astrocytes, oligodendrocytes, microglia and endothelial cells. The size and location of these infarcts are important determinants of the long-term functional deficits resulting from ischemic stroke. During stroke, the diminished supply of oxygen and glucose to the brain leads to reduced cellular metabolism and depletion of energy stores. Neuronal injury may also result in necrotic and apoptotic cell death. Combined with tissue damage due to mechanisms such as those mentioned above, cell death by either necrosis or apoptosis may be initiated. In contrast to necrosis (cell death by acute injury), apoptosis is a well-regulated physiological process. In the context of intervention, apoptosis is preferable to necrosis because it can be blocked by various treatments, allowing damaged tissue to be rescued. Cells within the core infarct ty-pically die by necrosis, whereas those in the penumbra die by apoptosis. The primary factor in determining which mechanism of cell death occurs is the level of ATP within the cell [48]. ATP is required for the process of apop-tosis, and cells with insucient ATP stores will die by necrosis instead. Apoptosis can occur by several pathways. The mitochondrial pathway can proceed through either caspase dependent or caspase independent mecha-nisms. Alternatively, apoptosis may be induced by the death receptor path-way. In the caspase dependent pathway of mitochondrial apoptosis, release of cytochrome C from mitochondria results in activation of caspase 3, which initiates a caspase cascade leading to the degradation of cellular components and cell death. Caspase 3 activity is commonly used as an indicator of apop-tosis. Cells undergoing apoptosis are characterized by cytoplasmic shrin-kage, nuclear condensation, and formation of membrane-bound vesicles.
Key elements of the apoptotic pathway include changes in mitochondrial respiration [47, 99]. Mitochondrial dysfunction triggers the cell death sig-naling cascade and results in organ failure and disease. An excellent review of ischemia injury cascades is provided by Dirnagl et al. in 1999 [98].
It is critical to comprehend these complicated pathophysiological casca-des of molecular and cellular events resulting from ischemia completely because one of the goals of utilizing animal models of cerebral ischemia is to dissect apart these various mechanisms of injury to arrive at a potential target site for treatment of ischemic injury (i.e., neuroprotection).
4.4. The role of mitochondria in cerebral ischemia
Mitochondria have the primary function of providing cellular chemical energy in the form of ATP by oxidative phosphorylation via the electron transport chain, and as such they have been termed the cells “powerhouse”.
Fig. 2. Transmission electron microscope image of a thin section cut
through an area of mammalian lung tissue. The high magnification image shows a mitochondria (http://remf.dartmouth.edu/imagesindex.html).
The mitochondrion is a discreet organelle present in most eukaryotic cells (Fig. 2). Mitochondrial dysfunction triggers the cell death signaling cascade and results in organ failure and disease Mitochondria contain two major membranes (Fig. 3). Its unusual structure is comprised of four distinct
compartments that carry out specialized functions: the outer mitochondrial membrane (OMM), the intermembrane space (IMS), the inner rial membrane (IMM), and the mitochondrial matrix. The outer mitochond-rial membrane fully surrounds the inner membrane, with a small inter-membrane space in between. The outer inter-membrane has many protein-based pores that are big enough to allow the passage of ions and molecules as large as a small protein. In contrast, the inner membrane has much more restricted permeability, much like the plasma membrane of a cell. The inner membrane is also loaded with proteins involved in electron transport and ATP synthesis. The IMM is highly folded into cristae, which house the protein complexes of the electron transport chain (ETC) and ATPase, controlling the fundamental rates of cellular metabolism. This membrane surrounds the mitochondrial matrix, where the citric acid cycle produces the electrons that travel from one protein complex to the next in the inner membrane.
Fig. 3. Structure of mitochondrion (from www.cronodon.com).
This essential role of the mitochondrion is responsible for its reference as the “power plant of the cell”. However, the function of mitochondria is not limited to supplying cellular energy [99]. Adenosine triphosphate (ATP) production through the oxidative phosphorylation (OXPHOS) process requires a continuous flow of electrons. As such, mitochondria are the major source of reactive oxygen species (ROS, i.e. superoxide and H2O2), generated as byproducts of the ETC [101, 102]. At the end of this electron transport chain, the final electron acceptor is oxygen, and this ultimately forms water
(H2O). At the same time, the electron transport chain produces ATP. This is why the the process is called oxidative phosphorylation.) During electron transport, the participating protein complexes push protons from the matrix out to the intermembrane space. This creates a concentration gradient of protons that another protein complex, called ATP synthase, uses to power synthesis of the energy carrier molecule ATP. Mitochondria are essential for cellular bioenergetics by way of energy production in the form of ATP through the process of oxidative phosphorylation. This crucial task is executed by five multi-protein complexes of which mitochondrial NADH: ubiquinone oxidoreductase or complex I is the largest and most complicated one. Together with these unique properties, mitochondria hold a central position in cellular bioenergetics. The most important mitochondrial energy-yielding reaction is performed by the oxidative phosphorylation system (OXPHOS). This system can be resolved into five large multi-subunit complexes (CI-CV) and is embedded in the inner mitochondrial membrane. Thirteen of the about 80 essential OXPHOS subunits are encoded by maternally inherited mitochondrial DNA and the remainder by nuclear DNA. Electron transfers from OXPHOS substrates to molecular oxygen results in the translocation of protons across the inner mitochondrial membrane at CI, CIII and CIV, which creates a substantial electrochemical gradient. This gradient is utilized for ATP synthesis, ion translocation and protein import.
Fig. 4. The electrochemical proton gradient and ATP synthase
(from www.nature.com)
At the inner mitochondrial membrane, a high energy electron is passed along an electron transport chain (Fig. 4.). The energy released pumps hyd-rogen out of the matrix space. The gradient created by this drives hydhyd-rogen
back through the membrane, through ATP synthase. As this happens, the enzymatic activity of ATP synthase synthesize ATP from ADP.
Mitochondria are key regulators of death in most cells in species ranging from C elegans to mammals [103]. Mitochondria have been impli-cated as central players in the development of ischemic cell death both through impairment of their normal role in generating much of the ATP for neural cell function and as key mediators in cell death pathways [47, 104]. It is very important to know about the current understanding of the mito-chondrial responses to cerebral ischemia and the contributions of these organelles to tissue damage. Additional aspects of this topic and further discussion of some of the earlier studies can be found in reviews [105-109]. They play a major role in necrosis, apoptosis and autophagic cell death [110, 111] and several recent reviews provide detailed information on the general mechanisms involved [112]. Furthermore, mitochondria are impli-cated in most degenerative diseases in the CNS [113]. In the immature brain, NMDA/AMPA receptor activation, increased intracellular Ca2+ and accumulation of reactive oxygen species exert stress on mitochondria after cerebral ischemia. Indeed, blockade of NMDA receptors improves mito-chondrial respiration and reduces injury [114–116]. These upstream events combine with increased pro- vs. anti-apoptotic B cell lymphoma 2 (Bcl-2) family protein balance [117, 118] and inflammatory activation of death receptors [119], to induce mitochondrial permeabilization. This, leads to release of cytochrome c and apoptosis-inducing factor resulting in activation of caspase-[120] and non-caspase-dependent [121] pathways, further impai-red respiratory and Ca2+ regulatory capacity and ultimately to cell death. For gaining comprehensive understandings on the role of mitochondria in the immature brain injury, it is essential to create a useful model of cerebral ischemia. A better understanding of clinical and cell physiological altera-tionsis crucial to develop therapeutic strategies.
4.5. Why targeting mitochondria?
Despite tremendous progress in preclinical studies, none of the treat-ment options has proven efficacious in clinical studies. Some of the mole-cular events that can be targeted by neuroprotectants include: glutamate release, glutamate receptor activation, excitotoxicity, Ca2+ influx into cells, mitochondrial dysfunction, activation of many intracellular enzymes, free radical production, nitric oxide production, inflammation, necrosis and apoptosis. Apoptosis or programmed cell death, a process associated with genomic fragmentation, is characterized by cell shrinkage, chromatin
gation, and preservation of cell membrane integrity and mitochondria wi-thout inflammation and injury to surrounding tissue [98].
Increasing evidence has indicated that mitochondrial dysfunction is a common pathological mechanism that underlines many neuropathological conditions. The oxidative damage is one of the major causes of nervous cells death in ischemia/hypoxia/brain reoxygenation [87, 122] and metabo-lic characteristics of nervous tissue make it inherently more vulnerable to the damaging effects of ROS. ROS reflect the level of cellular oxidative stress, causing severe damage to macromolecules when overproduced. Con-sequently, according to the Harman’s oxidative stress theory, they have been linked to aging, age-related pathologies, and death [123]. However, when produced in a controlled amount, ROS may also play important signaling roles in various redox-dependent processes, including apoptosis [124, 125], cell proliferation [126] and hypoxia [127]. Furthermore, mito-chondria are active players in cellular calcium homeostasis [128]. Mito-chondrial Ca2+ accumulation regulates functions as diverse as aerobic metabolism and induction of cell death [129]. Mutations in mitochondrial DNA (mtDNA) are responsible for many mitochondrial metabolic disor-ders, and are thought to contribute to aging by promoting apoptosis [130, 131]. Thus, because of their pivotal role in controling cell life and death [132-134], mitochondria represent an attractive target for mitochondrial gene therapy [135] as well as drugs treating either degenerative or hyperpro-liferative diseases.
There is growing evidence that special to mitochondria targeted drugs can have neuroprotective effects in some patients with neurological injury. The growing interest in mitochondrial targeting is revealing new avenues to identify novel therapeutics.
4.6. Animal models of cerebral ischemia
There are a number of experimental models of global cerebral ischemia used for preclinical studies of pharmacological interventions, including two or four vessel occlusion or cardiac arrest induction. Animal models of global cerebral ischemia allow studying, at different levels of biological organization of the central nervous system, the development and temporal course of those processes that may result in irreversible ischemic neuronal damage, as well as in the subsequent cell repair and plasticity underlying either permanent cerebral functional impairment or recovery as a result of intrinsic brain mechanisms or neuroprotective procedures. Thus, animal-related factors (species, strain, age, sex, co-morbidities), animal-
related factors (choice of ischemic model, anesthetic procedures, duration of ischemia, reperfusion, survival, possibility of monitoring of physiological parameters), selective vulnerability of specific neuron types in several brain structures, outcome assessment (histopathological, biochemical, functional, parameters of brain injury in specific cerebral structures), short- or long-term experimental design, pharmacological characteristics of the presump-tive neuroprotecpresump-tive agent itself, timing and dose-response of neuropro-tective drug administration with reference to starting and ending of the ischemic episode, may account for the relevance of results from these investigations. The goal of cerebral ischemia (focal and global) models is to reduce oxygen and glucose supply to brain tissue. This process produces brain injury via a variety of cellular and molecular mechanisms that impair the energetics required to maintain ionic gradients. Thus, biochemical, electrophysiological, histological, and behavioral parameters of ischemic brain damage have been included in experimental designs to evaluate the efficacy and safety of pharmacological and non pharmacological neuro-protective procedures against brain injury resulting from the significant reduction of blood supply to the whole brain, in several animal models of global cerebral ischemia [23].
Models of global cerebral ischemia have been performed in both large (monkeys, sheep, dogs, pigs, cats, rabbits) and small animals (gerbils, rats, mice). Among these, both advantages and disadvantages can be recognized according to several practical aspects: main objectives of the model; moni-toring procedures to be used; nature, number and timing of simultaneous parameters to be recorded in order to evaluate the ischemic brain injury and recovery; degree of similarity of structural and functional characteristics of brains of experimental animals to those of the human brain; and updated ethical outlines for the use of experimental animals in research protocols.
Clinical variability of stroke, mainly in terms of causes, duration, locali-zation, and severity of ischemia and coexisting systemic diseases, raises are need for very large patient group sizes in clinical research to avoid confoun-ding effects of the diversity. The pig is often the primary biomedical model for a number of diseases, for surgical research and for organ transplantation owing to the similarity in size, anatomy and physiology between pigs and humans [136].
Experimental global cerebral ischemia models have been developed to mimic human stroke or postcardiac arrest cerebral ischemia and serve as an indispensable tool in the cerebral ischemia research field. In a cerebral ischemia model, variables may take under strict control and researchers may address specific questions about either pathologic events occurring after cerebral ischemia and how to develop novel neuroprotective therapies. The
number and diversity of experimental global cerebral ischemia models have increased over the recent decades and animal studies have been provided most of our knowledge on pathophysiological mechanism involved in glo-bal cerebral ischemia. Cardiac arrest (induced by injection of KCl, electric shock, thoracic compression, asphyxia, and mechanical obstruction of the ascending aorta) followed by cardiopulmonary resuscitation (by artificial ventilation, closed chest massage and electrical defibrillation), both in large experimental animals (formerly a common model, but nowadays rarely used) and also in rodents, has been a technique to produce global cerebral ischemia in an attempt to closely resemble the clinical situation of cardiac arrest, including complete ischemia and reperfusion in renal, splachnic and other peripheral organs. This technique seemed to be an excellent model of global cerebral ischemia, but it is expensive when large experimental ani-mals are used, and intensive care (cardiopulmonary support under uncon-sciousness, control of blood pressure, pH, body fluids, and temperature) must be provided to the animals, especially during the first 24–48 h after the cardiac arrest. Complete acute global cerebral ischemia during cardiac arrest (8–20 min) and a variable period of incomplete cerebral ischemia during reperfusion, even after a successful cardiopulmonary resuscitation, as well as damage in those brain structures most vulnerable to ischemia, can be expected from this model [137, 138]. In particular, models of global cereb-ral ischemia in mice are currently of interest because of the availability of transgenic and knock-out strains for identification of cellular pathways of ischemic damage, and for neuroprotection studies.
Today reliable animal models for global cerebral ischemia are available in a variety of species including primates, pigs, sheep, dogs, cats, mongolian gerbils, rabbits, rats and mice. The pig has been used as an important large animal model for human disease for decades. The animal has a long lifespan of 10–15 years, so disease progression is more similar to that seen in humans. Furthermore, as already discussed, the pig shares anatomical and physiological characteristics with humans that make it a unique and viable model for biomedical research [139]. Because of the similarity in body mass of pigs to humans, the pig has become a model of choice for tissue enginee-ring and imaging studies[140]. Their large size also makes them ideal models for study in such medical fields as surgery, imaging, chemotherapy and radiation, which cannot be accurately tested in small animal models. Their cardiovascular anatomy and physiology, in combination with the pig’s response to atherogenic diets, have made them a universally standard model for the study of atherosclerosis, myocardial infarction and general cardio-vascular studies. Their gastrointestinal anatomy has some significant diffe-rences from that of humans; however, the physiology of their digestive
processes has made them a valuable model for digestive diseases. The urinary system of swine is similar to humans in many ways, especially in the anatomy and function of the kidneys [141]. Even STAIRS [45, 51] recommend in preclinical studies use large animal such pig.
Fig. 5. Pathophysiological mechanisms involved
in ischemia within the brain.
Global ischemia occurs when cerebral blood flow (CBF) is reduced throughout most or all of the brain, whereas focal ischemia is represented by a reduction in blood flow to a very distinct, specific brain region. With complete ischemia, global blood flow has ceased completely; whereas with incomplete ischemia, global blood flow is severely reduced but the amount of flow is insufficient to maintain cerebral metabolism and function. In focal cerebral ischemia, there may be absolutely no blood flow in the very central core of the ischemia, but usually there is some flow that reaches the area via collateral circulation. Thus, there is usually a gradient of blood flow from the inner core reaching out to the limits of the ischemic area.
Cerebral ischemia experimental models are characterized as global and focal ischemia (Fig. 5). Global ischemia occurs when cerebral blood flow (CBF) is reduced throughout most or all of the brain, whereas focal ische-mia is represented by a reduction in blood flow to a very distinct, specific brain region. Focal ischemia is characterized by a reduction of cerebral blood flow in a distinct region of the brain, whereas in global ischemia the reduction of blood flow affects the entire brain or forebrain [100, 142]. In focal cerebral ischemia, there may be absolutely no blood flow in the very central core of the ischemia, but usually there is some flow that reaches the area via collateral circulation. Thus, there is usually a gradient of blood flow from the inner core reaching out to the limits of the ischemic area. With complete ischemia, global blood flow has ceased completely; whereas with incomplete ischemia, global blood flow is severely reduced but the amount of flow is insufficient to maintain cerebral metabolism and function. Global cerebral ischemia, characterized by the critical reduction of cerebral blood flow in the whole brain, induces selectively neuronal injury in the CA1 region of the hippocampus as long as the duration of ischemia is limited. Upon extension of ischemia, other brain areas get involved, and at a critical
duration a so-called no-reflow phenomenon takes place in which the restoration of blood flow is unsuccessful [143–145].
Global ischemia can be induced by means of different approaches. The so called ‘four vessel occlusion method’ (4VO) consists of a reversible CCA occlusion, which, combined with permanent interruption of the vertebral arteries via electro cauterization, results in bilateral forebrain and brainstem ischemia with a highly predictable brain damage [146]. The four-vessel occlusion (4-VO) and the two-four-vessel occlusion with hypotension (2-VO) models in rats became, nowadays, the most widely used animal models that simulate the reduction of blood flow, as it would occur by effect of cardiac arrest, on the forebrain. The 4-VO model [146] provides a method of reversible forebrain ischemia in awake, freely moving rats (but also in anesthetized rats). In a first step of the model procedures, vertebral arteries are permanently occluded and 24 or 48 hours later, the ischemia is produced through transient (10–20 min) occlusion of the common carotid arteries under light inhaled anesthesia so that the ischemic episode occurs while the animal is unanesthetized. In this way, a reduction in cerebral blood flow to less than 5% of control values, which is followed by hyperemia during 5 to 15 min after reperfusion, and subsequent hypoperfusion lasting for 24 hr result in main ischemic neuronal damage in hippocampus, neocortex and striatum, along hours to days after ischemia, its magnitude relating to the duration of the ischemia. The effects of this insult are, however, quite variable between rat strains, as well as between those individuals surviving (survival rate, 50−75%) after having fulfilled the criteria required to be included in the experimental groups.
As alternative to the 4VO method, global ischemia can also be induced by the occlusion of the two common carotid arteries, i.e., by two-vessel occlusion (2VO) together with induction of hypotension for a limited time period. In this forebrain ischemia model, selective injury in the CA1 of the hippocampus, the caudate putamen and neocortex is observed [147]. Mouse models of global cerebral ischemia have been developed through bilateral common carotid occlusion and controlled pulmonary ventilation [100]. It is known that animal models of global cerebral ischemia require adequate control of certain variables, such as careful control of animal’s temperature and blood glucose concentration, in order to achieve consistent pathophysio-logical effects and brain injury [87]. Hyperthermia and hyperglycemia increase brain injury, while hypothermia results in neuroprotection by itself.
Experimental global cerebral ischemia models have been developed to mimic human stroke or postcardiac arrest cerebral ischemia and serve as an indispensable tool in the cerebral ischemia research field. Nevertherless, it is very important to create a useful model of cerebral ischemia for injury
mechanisms and neuroprotective compounds investigation. Before moving to clinical trials, drugs should be reevaluated based on animal studies.
4.7. Neuroprotection research
It is generally acknowledged that many agents, proven neuroprotective in experimental models, fail in clinical practice, possibly because they cannot respond to the complex multifaceted nature of the ischemic cascade after stroke [23, 48, 148]. Despite progress in preclinical studies, none of the treatment options has proven efficacious in clinical studies. Some of the molecular events that can be targeted by neuroprotectants include: glutamate release, glutamate receptor activation, excitotoxicity, Ca2+ influx into cells, mitochondrial dysfunction, activation of many intracellular enzymes, free radical production, nitric oxide production, inflammation, necrosis and apoptosis.
Except for the use of mild hypothermia after ventricular fibrillation car-diac arrest, currently recommended therapy in the 2005 and 2010 guidelines of the European Resuscitation Council [39, 40], clinical neuroprotection practice rests solely on extrapolation from animal experimental work or weak clinical studies [41]. After evaluation of its effectiveness as a neuro-protective strategy in animal models of global cerebral ischemia, hypo-thermia has been tested in clinical trials in patients having suffered cardiac arrest, the most frequent cause of global cerebral ischemia in human beings. It seems that new and better strategies to translate preclinical data suppor-ting the potential clinical usefulness of neuroprotective drugs to clinical trials, must be developed. In spite of advances in our understanding of the pathophysiology of cerebral ischemia, therapeutic options for acute ische-mic stroke remain very limited. Only one drug is approved for clinical use in the thrombolytic treatment of acute ischemic stroke in the USA, and that is intravenous administration of recombinant tissue plasminogen activator (rt-PA). When delivered within 3–4,5 h after symptom onset, rt-PA reduces neurological deficits and improves functional outcome in stroke patients. However, this improvement in recovery is achieved at the expense of an increased incidence of symptomatic intracranial hemorrhage, which occurs in approximately 6% of patients. Furthermore, the large majority of patients with acute ischemic stroke is not able to reach a hospital within 3–4,5 h after stroke onset, and most of them therefore do not receive rt-PA treat-ment. Consequently, successful treatment of acute ischemic stroke remains one of the major challenges in clinical practice. Translation of knowledge about neuroprotection obtained from models in experimental animals, to
clinical practice has not been successful. This situation has been also obser-ved in the case of focal cerebral ischemia, leading to consensus meetings [50, 51]attempting to establish the better conditions for preclinical studies of neuroprotection as to give reliable results to be applied in clinical condi-tions. If opinion of these consensuses may be recognized as applicable to preclinical studies of global cerebral ischemia, it is apparent that some fac-tors must be taken in account for designing and carrying of the respective experimental protocols. Thus, studies in animal models of global cerebral ischemia should give information on effective neuroprotective doses in the case of drugs being tested; hence, dose-response relationships should be investigated.
Increasing evidence has indicated that mitochondrial dysfunction is a common pathological mechanism that underlines many neuropathological conditions.
The role of ROS in the pathogenesis of cerebral ischemia reperfusion injury is well known. Reperfusion produces a burst in ROS formation after cerebral ischemia and has been known as one of the major mechanisms by which reperfusion worsens ischemic damage [149]. Antioxidant has been viewed as one of the most promising neuroprotective strategies for the treatment of ischemic stroke. However, the failure of the SAINT II trial has raised concerns regarding the traditional free radical trapping strategy [150]. Instead of neutralizing the free radical, the alternative electron transfer strategy blocked the overproduction of ROS generated by the inhibition of ETC complex I and III.
While the acute treatment of stroke today is highly standardized and secondary prevention is effective, an efficient protection of the cells at risk in the penumbra is lacking. Several studies have reported that inhibiting inflammatory processes such as production of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) reduces delayed increases in stroke volume in animal models. Thus, inflammatory signaling cascades are a target of translational research on stroke [151–153]. Many pro-inflamma-tion enzymes such as iNOS, COXs and xanthine oxidase participate in oxi-dative injury during cerebral ischemia [154]. Neuroprotection targeted in ischemia injured cell could help “penumbral” tissue survive.
The window of therapeutic opportunity in animal models is not neces-sarily predictive of the time window in humans, but the determination of relative windows is useful. In animal models, the time of the stroke or ischemic onset is known precisely, as is the administration of drug at precise times, whereas in humans this is less often the case. There are a number of important issues that remain unresolved regarding the translation of experi-mental developments to the clinical setting. Novel interventions will be
required to overcome hurdles associated with bench-to-bedside translation [155]. While stroke investigators have achieved progress in attempting to improve recanalization for clot-occluded intracranial arteries, the search for an effective neuroprotectant for acute stroke treatment has not been succes-sful. In an extensive review of 1026 experimental treatments that have been tested, neuroprotective efficacy was superior to control conditions in 62% of the preclinical models of focal ischemia, in 70% of preclinical models of global ischemia, and in 74% of culture models. Of these experimental treat-ments, 114 have been tested in human with little to no success [23]. No neuroprotectants have been approved for clinical use in stroke.
In our study we have chosen to evaluate the effects of two drugs (methylene blue and cyclosporine A) known to have a positive effect on the complex I of the respiratory chain in mitochondria. It was hypothesized that this treatment immediately before the induction of the brain ischemia would have a pronounced neuroprotective effect (mitochondrial targeted) in the initial phase of ischemia before the occurrence of noticeable structural damage occurs in brain tissue, similar as in case of ischemia and reperfusion during the cardiac arrest or in penumbra during the ischemic stroke in the clinical setting. Thus potentially such a treatment could decrease the extent of damage to the brain tissue and/or extend the safe therapeutical window for reperfusion therapy.
4.8. Cyclosporine A as a neuroprotective agent
Cyclosporine A (CsA), which has been used as an immunosuppressive agent in transplant medicine long time, has been proposed to block the MPTP and protect the rat brain from stroke [156, 157]. CsA has been used extensively in the solid organ and bone marrow transplant populations for > 20 years due to its other mechanism of action. The immunosuppressive action is based on its high affinity for binding to cyclophilin-A. It inhibits T-cell lymphocyte function by binding cyclophilin A, which functions to alter the formation of specific cytosolic proteins. This complex inhibits calci-neurin, a cytosolic phosphatase. Inhibition of calcineurin results in the prompt abolition of cytokine expression (such as IL-2, IFN-1 and TNF-1) that is responsible for the activation and proliferation of T-lymphocytes [158]. Interestingly, the inhibition of calcineurin may also play a role in the neuroprotective activity of CsA. Cyclosporine A has been shown in vivo to reduce swelling of isolated brain mitochondria [159]. Furthermore, experi-mental studies show that cyclosporine A inhibits opening of the MPTP by binding to cyclophilin-D [157]. This raises the possibility that maintaining
cell integrity by cyclosporine may have certain beneficial effects in neuro-protection. In animal models of traumatic brain injury, cyclosporine treat-ment has been shown to reduce axonal injury [160] and attenuate lipid pero-xidation [161]. The cardioprotection of cyclosporine was further supported by experiments in wild-type mice given cyclosporine on reperfusion and subsequent myocardial infarct size reduction compared with cyclophilin-D-deficient mice [162].The cardioprotective benefits of cyclosporine in humans have been demonstrated in adult patients who underwent percuta-neous coronary intervention for ST-elevation myocardial infarction [163]. Cyclophilin-D, a key component of the mitochondrial permeability transi-tion pore [157, 164, 165], is thus believed to be the molecular target of cyclosporine in the amelioration of postischemic reperfusion injury. In reperfusion injury, the formation of mitochondrial permeability transition pore is believed to be a key mechanism in which cyclosporine reduced injury in isolated rat hearts [164-166]. According to multiple studies, CypD-deficient mice displayed a reduction in brain infarct size after acute middle cerebral artery occlusion and reperfusion [167]. Chronic administration of cyclosporine A (for 3 days) before the ischemic insult demonstrated neuro-protection in the CA1 region at 7 days of reperfusion in a rat model of global cerebral ischemia [168].
In the past 10 years, this agent has shown neuroprotective effects in animal models of traumatic brain injury [161, 169] and a good safety profile of CsA infusion when given at the chosen dose of 5 mg/kg, infused over 24 h, during the early phase after severe head injury in humans, with the aim of neuroprotection [170]. Postresuscitation administration of cyclosporine causes bell-shaped preservation of cardiac function with an effective dose of 10 mg/kg in newborn piglets after asphyxia–reoxygenation without worse-ning renal injury [171].
Emerging evidence suggests CsA might be a promising agent in trea-ting stroke and further investigation in the clinical settrea-ting should be perfor-med [156]. Cyclosporin A (CsA) has proved to be safe and effective for use in transplantation. The potential clinical applications of CsA in stroke are enormous, not only it maybe useful in patients that are not candidates for tPA, but its neuroprotective mechanism may also be complementary to tPA treated stroke patients as well. However, the possible adverse effects of CsA should be recognized as well, and the eventual clinical role in treating stroke will be based on the risk and benefit ratio.
4.9. Methylene blue (MB) as neuroprotective agent
Methylene blue (MB), originally discovered as a synthetic dye, easily crosses the blood-brain barrier and accumulates in nervous tissue after systemic administration[172]. Safety/FDA approved methylene Blue (MB), a cationic thiazine dye with a low toxicity profile at low doses, is efficiently trapped in the brain and its concentration is over 10 times higher in the brain than in the circulation one hour after systemic administration, indicating a rapid and extensive accumulation in the nervous system. These properties of MB have attracted many researchers to this drug, revealing MB’s potential to treat methemoglobinemia, cyanide poisoning [173], Alzheimer Disease [174], and psychosis [175]. MB, the first lead chemical structure of pheno-thiazine and other derivatives, is commonly used in diagnostic procedures and as a treatment for methemoglobinemia, malaria, and carbon monoxide and cyanide poisoning [176, 177]. Methylene blue has been experimentally proven neuroprotective in a porcine model of global ischemia−reperfusion in experimental cardiac arrest. MB counteracts the damaging effect of rotenone, an inhibitor of mitochondrial electron transfer complex I on retinal neurons [178]. MB has been used as a neuroprotective agent in drug induced encephalopathy, dementia and manic-depressive psychosis [179– 181]. Furthermore, MB exhibits promising cardio- and neuroprotective properties in experimental cardiac arrest [182, 183] and is effective in both attenuating ischemia−reperfusion (I/R) syndrome [184] and increasing short-term survival after resuscitation from cardiac arrest [185]. However, no comprehensive analyses have been conducted at mitochondrial respira-tion level in gyrencephalic brains. MB not only has great affinity for ner-vous tissue, it has also been recognized as one of the most potent chain-breaking antioxidants [186]. Recent evidence supports that MB effectively improves memory in healthy animals and humans. These enhancing effects have been shown in a variety of experimental learning and memory paradigms ranging from habituation to spatial memory. In addition, MB has been used in the therapy of mental disorders and cardiac arrest-associated brain damage [180, 183, 185, 187–189]. The data suggest that the mecha-nism for these effects is based on MB’s redox cycling properties and its effects on the energy metabolism machinery in mitochondria. This is all in addition to its well-established role as an FDA-grandfathered antidote for the treatment of methemoglobinemia, a condition characterized by elevated blood levels of an oxidized form of hemoglobin with decreased ability to release oxygen to tissues. In experimental cardiac arrest, MB has been proven to be neuroprotective and cardioprotective. The cardioprotective effect of MB has been attributed to its blocking effect on NOS and
lylcyclase [182, 191]. It seemed to stabilize the systemic circulation, increa-se cerebral cortical blood flow, decreaincrea-se lipid peroxidation and inflam-mation and cause less anoxic tissue injury in the brain and the heart [182].
Currently effects of two drugs (methylene blue and cyclosporine A) are known to have a positive effect on the complex I of the respiratory chain in mitochondria. It was hypothesized that this treatment immediately before the induction of the brain ischemia would have a pronounced neuroprotec-tive effect (ischemia targeted) in the initial phase of ischemia before the occurrence of noticeable structural damage occurs in brain tissue, similar as in case of ischemia and reperfusion during the cardiac arrest or in penumbra during the ischemic stroke in the clinical setting. Thus potentially such a treatment could decrease the extent of damage to the brain tissue and/or extend the safe therapeutical window for reperfusion therapy.
5. METHODS
To define the most appropriate experimental model of global cerebral ischemia, to identify the primary target of ischemia in ischemia-injured brain cell and to evaluate neuroprotection, Lithuanian White female pigs were used for the experiment. Animals were treated following guidelines for the care and use of experimental animals of Lithuanian university of health sciences in accordance to applicable laws. The study protocol was approved by the Lithuanian Animal Ethics Committee (SFVS Permission number 0204/0238).
5.1. Study design
Study was performed in 2 stages: 1 – modeling of cerebral ischemia and detection of the primary target of ischemia (Fig. 6); 2 – evaluation of neuroprotectants (Fig. 7).
To create new swine model of global incomplete cerebral ischemia 17 experiments were performed in the first stage of our study. The global incomplete cerebral ischemia was induced by unilateral carotid occlusion in 6 pigs (UCO group), by bilateral carotid occlusion – in 3 experimental animals (BCO group) and by bilateral carotid occlusion with hypotension - in 4 animals (BCOH group). Four animals were used for control measu-rements, when cerebral ischema was not induced. Complex investigation of cerebral ischemia induced injury was performed: we evaluated changes in mitochondrial respiration, possible microcirculation and histological alte-rations. After the first stage one model with the most notable but reversible alteration in ischemia injured brain was selected for the next stage – evaluations of neuroprotectants and all models were compared to identify the primary target of ischemia in brain cell.
Fig. 6. 1 stage of study design
To evaluate effects of methylene blue and cyclosporine A in the second stage of our study 15 experiments were performed (Fig. 7). We used our developed swine model of global incomplete cerebral ischemia for this purpose. Methylene blue was evaluated in 4 experiments, cyclosporine A – in 4 experiments, for control measurements were performed 3 experiments with ischemia and 4 experiments without ischemia. Neuroprotectant (me-thylene blue or cyclosporine A) was injected before ischemia induced. Our aim was to hit the first target of ischemia injury in cell and establish how neuroprotectants act ischemia mechanisms. It was hypothesized that this treatment immediately before the induction of the brain ischemia would have a pronounced neuroprotective effect (ischemia targeted) in the initial phase of ischemia before the occurrence of noticeable structural damage occurs in brain tissue, similar as in case of ischemia and reperfusion during the cardiac arrest or in penumbra during the ischemic stroke in the clinical setting.
Fig. 7. 2 stage of study design
5.2. Modelling of global cerebral ischemia in pigs 5.2.1. Anesthesia and surgical preparation
Seventeen Lithuanian White female pigs were used for this part of the study. Animals were fasted for 12 h before experiment, with free access to water. Anesthesia was initiated by intramuscular injection of ketamine (20 mg/kg), xylasine (2 mg/kg) and atropine (0,01 mg/kg), completed by ear vein injection of sodium thiopental (6 mg/kg). After endotracheal intuba-tion, pigs were ventilated using a volume-controlled mode (Drager, Lubeck, Germany) under the following conditions: fraction of inspired oxygen (FiO2) of 0.21 at 14–16 breaths/min and tidal volume of 10 mL/kg to maintain normocapnia.
Anesthesia was maintained by continuous infusion of sodium thiopental (5 mg/kg/h) and fentanil (0.01 mg/kg/h). Paralysis was achieved with
venous pipecuronium bromide boluses as required. Ringer’s solution (10 ml/kg/h) was administered continuously. A standard lead II electrocar-diogram (ECG) was used to monitor cardiac rhythm. To ensure an appro-priate depth of anesthesia, we monitored indirect measurements such as tail-clamping, monitoring of the corneal reflex, and lacrimation, as well as changes in hemodynamics and heart rate.
A saline-filled central venous catheter (7-French) was inserted in the right or left femoral vein for drug administration. Core body temperature was monitored continuously via the esophageal temperature probe and kept >38.0°C using warmed solutions and heating mattresses. An arterial line was placed into the left or right femoral artery to measure invasive arterial blood pressure and to obtain blood gases.
Depending on the group, the neck area was surgically opened to expose the internal carotid arteries bilaterally or unilaterally, and after placing a monofilament nylon hook around one or both arteries, the wound was closed (Fig. 8). A standard craniotomy was performed in the temporopa-rietal region, avoiding injury to the medial venous sinus, to perform direct SDF imaging and to obtain tissue samples for assessment of mitochondrial function, histology, and apoptosis. A thorough hemostasis was achieved prior to the microcirculation measurements and tissue harvesting using mo-nopolar coagulation and bone wax.
Fig. 8. Blood vessels occlusion during ischemia.
