Oxidative Stress in Ischemia/Reperfusion Injuries following Acute Ischemic Stroke

These are all causes of the neuronal cascade of death. SOLVE THEM!  I don't care that there have been thousands of failures.

These failures of clinical trials have been known for years. WHOM  is cataloging and solving them to make the next ones better?

This is not going to be easy as Dr. Michael Tymianski, of the Toronto Western Hospital Research Institute in Canada states;  over the last half-century, there have been more than 1,000 drugs (So what are they?)aimed at preventing brain damage that have failed to work in people, even though they worked well in mice or rats.

The latest here:

Oxidative Stress in Ischemia/Reperfusion Injuries followingAcute Ischemic Stroke

Anamaria Jurcau 1,2,* and Adriana Ioana Ardelean 3,4 1 Department of Psycho-Neurosciences and Rehabilitation, Faculty of Medicine and Pharmacy, University of Oradea, 410087 Oradea, Romania 2 Department of Neurology, Clinical Municipal Hospital Oradea, Louis Pasteur Street nr 26, 410054 Oradea, Romania 3 Department of Preclinical Sciences, Faculty of Medicine and Pharmacy, University of Oradea, Universitatii Street nr 1, 410087 Oradea, Romania; adriana_toadere@yahoo.com 4 Department of Cardiology, Clinical Emergency County Hospital Oradea, Gh. Doja Street nr 65, 410169 Oradea, Romania * Correspondence: anamaria.jurcau@gmail.com; Tel.: +40-744-600-833 

Abstract: 

Recanalization therapy is increasingly used in the treatment of acute ischemic stroke. However, in about one third of these patients, recanalization is followed by ischemia/reperfusion injuries, and clinically to worsening of the neurological status. Much research has focused on unraveling the involved mechanisms in order to prevent or efficiently treat these injuries. What we know so far is that oxidative stress and mitochondrial dysfunction are significantly involved in the pathogenesis of ischemia/reperfusion injury. However, despite promising results obtained in experimental research, clinical studies trying to interfere with the oxidative pathways have mostly failed. The current article discusses the main mechanisms leading to ischemia/reperfusion injuries, such as mitochondrial dysfunction, excitotoxicity, and oxidative stress, and reviews the clinical trials with antioxidant molecules highlighting recent developments and future strategies. 

Keywords: 

ischemic stroke; reactive oxygen species; mitochondria; oxidative stress; antioxidants; nanoparticles; stem cells 1. Introduction Although the proper management of vascular risk factors and increasing use of prophylactic measures between the 1970s and early 2000s resulted in an annual 1–1.5% decrease in stroke incidence in high income countries [1], stroke is still the main cause of disability in adults and the second leading cause of death worldwide. Moreover, the increasing prevalence of diabetes mellitus [2] and obesity [3], together with aging of the population, will probably increase the incidence of stroke [4]. The treatment of ischemic stroke entered a new era in 1995, with the release of the results of the National Institute of Neurological Disorders and Stroke trial with recombinant tissue plasminogen activator (r-tPA) [5], which showed that reestablishing blood flow in the first 3 h after stroke onset is able to salvage much of the hypoperfused cerebral tissue and improve patient outcome. Subsequent trials refined the recanalization methods by extending the time window to 4.5 h in certain subsets of patients [6], using intra-arterial thrombolysis [7], ultrasound-enhanced thrombolysis [8], or various devices for mechanical clot extraction [9] within 24 h from stroke onset [10]. However, still only 2–20% of acute ischemic stroke patients are eligible for recanalization treatments [11]. In addition, successful recanalization rates vary around 46% for intravenous thrombolysis, 63% for intra-arterial thrombolysis, or 83% for mechanical thrombectomy, and recanalization does not always translate into efficient reperfusion of the tissue at risk, leading to neurological worsening of the patient through cerebral edema, hemorrhagic transformation, or ischemia/reperfusion injuries (I/R injuries) [12]. Oxidative stress and neuroinflammation have been shown to significantly contribute to these complications. Thus, understanding the mechanisms of I/R injuries and finding ways to prevent them would significantly improve the outcome of ischemic stroke patients [13]. In the following sections we will review the literature on the pathophysiology of these injuries, focusing on oxidative stress, sources of reactive oxygen species (ROS), and neurotoxic oxidative and neuroprotective antioxidative pathways in the central nervous system (CNS). The second part reviews the studies done so far with antioxidants in ischemic stroke and discusses promising novel antioxidant approaches. 2. Oxidative Stress in the Pathophysiology of Ischemia/Reperfusion Injuries after Acute Ischemic Stroke Oxidative stress is an imbalance between the rate of generation of ROS and the biological system’s ability to clear these highly reactive molecules [14]. The cerebral tissue is particularly sensitive to oxidative stress due to a series of features, such as [15–18]: - It has the highest metabolic activity per unit weight compared to other organs; - It has low levels of antioxidant enzymes, such as superoxide dismutase, catalase, glutathione peroxidase, heme oxygenase-1; - Upon release, neurotransmitters contribute to cellular calcium overload and, through their metabolism, generate ROS; - Brain cells have a higher membrane surface/cytoplasmic volume ratio, and the plasmalemma is rich in cholesterol, is arranged in lipid rafts, has polyunsaturated fatty acids, and is very susceptible to oxidative damage; - The brain has lower levels of cytochrome c oxidase, leading to increased superoxide generation during adenosine triphosphate (ATP) generation; - Iron, released from damaged cerebral tissue, can catalyze the generation of free radicals. Restoration of blood supply to ischemic tissue, although necessary for restoration of aerobic metabolism, will also result in ROS production, which overwhelms the ability of cerebral tissue to neutralize these ROS and leads to increased oxidative stress. Research has shown that cerebral ischemia is accompanied by increased serum concentrations of markers of oxidative stress [19–21]. The main ROS are superoxide anions, (O2 −), hydroxyl radicals (OH−), and hydrogen peroxide (H2O2) [22], stemming from the activity of mitochondria, cyclooxygenases, lipoxygenases, nitric oxide synthases (NOSs), NADPH oxidase (NOX), and xanthine oxidase [23]. Once generated, ROS interact with various biological molecules: - ROS oxidize, degrade, or cleave proteins, leading to protein aggregation, modifications in ion channel activities, and enzyme inactivation [24]. - By attacking the carbon–carbon bonds of polyunsaturated fatty acids, ROS initiate lipid peroxidation, a self-propagating chain of events leading to the generation of unstable lipid radicals which further react with oxygen to form lipid peroxyl radicals [25]. Peroxidation of membrane lipids alters the bi-layer thickness, membrane fluidity, and membrane permeability. - ROS can directly damage deoxyribonucleic acids (DNA) by causing double strand breaks, structural changes, DNA mutations, or protein-DNA cross-links [26]. - They also regulate several apoptosis and necrosis signaling cascades. ROS can activate p53, a key molecule in ROS-induced cell death [27], which, in turn, upregulates PUMA (p53 upregulated modulator of apoptosis). ROS can open the mitochondrial permeability transition pore (MPTP), leading to mitochondrial swelling and cytochrome c release, thereby initiating apoptosis [28]. The MAPK (mitogen activated protein kinase) pathway, also triggered by ROS, has 3 main members: c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK 1/2), and p38 MAPK. While ERK 1/2 has a controversial role in cell death and appears to be rather neuroprotective against ischemia/reperfusion injuries [15], JNK and p38 MAPK, activated by ROS through ASK1 (apoptosis signal-regulating kinase 1), significantly contribute to apoptosis during reperfusion after an ischemic insult [29,30]. 2.1. Mitochondria as a Source of ROS and Their Implication in Cerebral Ischemia/Reperfusion Injuries Mitochondria, the powerhouse of the cell, generate over 90% of the ATP in the brain through beta-oxidation of fatty acids, the Krebs cycle, and oxidative phosphorylation (OxPhos) [31]. They also use pyruvate from cytosolic glycolysis to reduce flavin adenine dinucleotide and nicotinamide adenine dinucleotide, which serves in energy transfer to the electron transport chain (ETC) [32]. The mitochondrial electron transport chain (ETC) consists of a series of protein complexes situated in the inner mitochondrial membrane which use the electrons removed by reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) from the Krebs cycle to pump protons from the matrix into the intermembrane space, thereby generating a potential gradient across the inner mitochondrial membrane, which will be used in the final step of OxPhos to synthesize ATP [33]. NADH binds to NADH dehydrogenase (complex I), located on the inner mitochondrial membrane, and donates two electrons which will be passed down to ubiquinone to produce ubiquinol, a process coupled with the translocation of four protons from the matrix through the inner mitochondrial membrane [34]. Complex II, or succinate dehydrogenase, also participates in the Krebs cycle and contains FAD as a prosthetic group. It oxidizes succinate to fumarate and reduces ubiquinone [35]. Ubiquinol diffuses through the inner mitochondrial membrane and donates its electrons to cytochrome c reductase (complex III), which passes these electrons onto two molecules of cytochrome c while translocating two protons from the mitochondrial matrix and depositing an additional two protons in the intermembrane space [36]. At complex IV (cytochrome c oxidase), four cytochrome c molecules donate each one electron which will serve to form two H2O molecules from one O2 molecule, a process coupled with pumping of four protons from the matrix into the intermembrane space [37]. The final step is the synthesis of ATP from ADP and phosphate, achieved by ATP synthase (complex V), which uses the energy of the proton electrochemical gradient in a complex process, the elucidation of which led Boyer and Walker to achieve the 1997 Nobel Prize in Chemistry [38]. The transfer of protons from the mitochondrial matrix to the intermembrane space by the reactions of complexes I, III, and IV establishes a negative potential difference (∆Ψm) of 150–180 mV (with respect to the cytosol) across the inner mitochondrial membrane, which, together with the pH difference, drives complex V to generate ATP and cytosolic calcium ions to accumulate via the mitochondrial calcium uniporter in the matrix [32,39], where calcium stimulates the activity of dehydrogenases in the Krebs cycle and modulates the function of complexes IV and V [40]. As such, the balance between phosphorylation and dephosphorylation of the OxPhos complexes as well as intramitochondrial calcium concentrations maintain the cellular respiration rate and the ∆Ψm [41] by interfering with their electron transfer kinetics and allosteric regulation by ATP and ADP (adenosine diphosphate) [42]. Under normal conditions, more than 90% of oxygen is reduced to water, while about 2% of electrons may leak from complexes I and III and react with oxygen, generating superoxide anions [41]. During ischemia, the intramitochondrial calcium levels increase [42], activating mitochondrial phosphatases and leading to dephosphorylation of the OxPhos complexes, especially of cytochrome c and of cytochrome c oxidase [43], and ultimately to loss of allosteric inhibition by ATP [41]. Because oxygen as the final electron acceptor is lacking, OxPhos is maximally activated in a feed-forward mechanism. Upon restoration of oxygen supply, increased OxPhos activity restores ∆Ψm within 1 min and cellular levels of ATP within 15 min [44], after which it hyperpolarizes the mitochondrial membrane potential with dramatic effects on ROS production. Research has shown that a 10 mV increase in the ∆Ψm above 140 mV leads to a 70–90% increase in the generation of ROS [45].

 

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