Mechanisms Of Ischemic Cell Death Research Articles

Photothrombotic Stroke as a Model of Ischemic Stroke.

The search of effective anti-stroke neuroprotectors requires various stroke models adequate for different aspects of the ischemic processes. The photothrombotic stroke model is particularly suitable for the study of cellular and molecular mechanisms underlying neurodegeneration, neuroprotection, and neuroregeneration. It is a model of occlusion of small cerebral vessels, which provides detailed study of molecular mechanisms of ischemic cell death and useful for search of potential anti-stroke agents. Its advantages include well-defined location and size of ischemic lesion that are determined by the aiming of the laser beam at the predetermined brain region; easy impact dosing by changing light intensity and duration; low invasiveness and minimal surgical intervention without craniotomy and mechanical manipulations with blood vessel, which carry the risk of brain trauma; low animal mortality and prolonged sensorimotor impairment that provide long-term study of stroke consequences including behavior impairment and recovery; independence on genetic variations of blood pressure and vascular architecture; and high reproducibility. This review describes the current application of the photothrombotic stroke model for the study of cellular and molecular mechanisms of stroke development and ischemic penumbra formation, as well as for the search of anti-stroke drugs.

In Memoriam: Bo K Siesjö, 1930–2013

Bo K Siesjo was an outstanding brain researcher and will be remembered as such. He was born in Sweden on 8 January 1930 and died in his home country on the 27 June 2013. He graduated from medical school at the Lund's University in 1958 and defended his PhD thesis at the same university in 1962. His postgraduate training took place in Sweden and Cambridge, and in the late 1980s he was a visiting scientist in Nice. After his graduation from medical school, he had appointments at the Lund's University and University Hospital until his retirement in 1995. In Lund he created the Laboratory for Experimental Brain Research, and the Swedish Research Council established a special professorship for him in 1974. He, subsequently, became the chief of the research department at Lund's University Hospital. After his retirement he went to Hawaii to build up a neuroscience research department at Queens Medical Center in Honolulu. Siesjo's research focus was on cerebral metabolism in the normal and injured brain, and an early important topic of his interest was the regulation of pH in the extra- and intracellular fluids in the brain. He published a textbook on the brain's energy metabolism and authored or co-authored more than 500 publications listed on PubMed. In 1970s his research focused on the ischemic metabolic cascade, the mechanisms of ischemic cell death, and the energy reserves in the brain, work that bore rich fruit. Two classic papers from this period stand out for their influence on the field, ie, his 1981 paper with Astrup and Symon on the concept of the ischemic penumbra,1 and his synthesis paper on the mechanisms of ischemic cell death.2 In his later years of research he also investigated the pathophysiology of chronic brain diseases, such as dementia of the Alzheimer's type. In his research carrier, Siesjo became one of the most highly cited scientists in his field. Bo Siesjo was the main catalyst for the founding in 1981 of the Journal of Cerebral Blood Flow and Metabolism and the organization of its parent professional society, the International Society of Cerebral Blood Flow and Metabolism. He was also the recipient of a number of awards that include the Mihara Award in 1989, the J Allan Taylor International Prize in Medicine in 1992, the KJ Zulch Prize in 1992, and the Lifetime Achievement Award by the International Society for Cerebral Blood Flow and Metabolism in 2003. Siesjo's research in Lund was renowned worldwide, and many young scientists from all over the world came to work with him. Many of them, who got their scientific start in his laboratory, later became well-established researchers in their home countries; more than 20 of them achieved full professorships in institutions worldwide. In addition to his scientific interests, Bo Siesjo had an intense interest in art that led him to establish an art collection concentrated mainly on modern national art. Bo was married with Bodil, and they had three children, Peter, Viveka, and Bjorn. All honor to his name.

The neuroprotective impact of the leak potassium channel TASK1 on stroke development in mice

Oxygen depletion (O 2) and a decrease in pH are initial pathophysiological events in stroke development, but secondary mechanisms of ischemic cell death are incompletely understood. By patch-clamp recordings of brain slice preparations we show that TASK1 and TASK3 channels are inhibited by pH-reduction (42 ± 2%) and O 2 deprivation (36 ± 5%) leading to membrane depolarization, increased input resistance and a switch in action potential generation under ischemic conditions. In vivo TASK blockade by anandamide significantly increased infarct volumes at 24 h in mice undergoing 30 min of transient middle cerebral artery occlusion (tMCAO). Moreover, blockade of TASK channels accelerated stroke development. Supporting these findings TASK1 −/− mice developed significantly larger infarct volumes after tMCAO accompanied by worse outcome in functional neurological tests compared to wild type mice. In conclusion, our data provide evidence for an important role of functional TASK channels in limiting tissue damage during cerebral ischemia.

Aging Blunts Ischemic-Preconditioning-Induced Neuroprotection Following Transient Global Ischemia in Rats

The present study examines the hypothesis that aging defined by the 50% survival age compromises neuroprotection afforded by ischemic preconditioning (IPC). Sixty-four male F344 rats aged 4- and 24-months, respectively, were subjected to IPC, (3-min ischemia) or sham-surgery followed by 10-min (full) ischemia or sham-surgery 2 days later. There were 4 groups at each age: sham-surgery-sham-surgery (SS), preconditioning-sham-surgery (PS), preconditioning-ischemia (PI) and sham-surgery-ischemia (SI) groups. Assessments of histology and immunoreactivities of N-methyl-D-aspartic acid receptor 1 (NMDAr1) and caspase-3 active peptide (C3AP) in the hippocampal CA1 region were performed 8 days after full ischemia. The CA1 "living cell ratio" was greater in the aged SI group than in the young SI group (32+/-6% vs. 17+/-5%, p<0.05), whereas the degree of protection against full ischemia afforded by IPC was reduced in the aged compared with the young (53+/-17% vs. 241+/-25%, P<0.0001). The basal level of NMDAr1 immunofluorescence was significantly higher in young animals, while the numbers of C3AP-positive cells were greater in all three aged ischemic groups as compared to respective young groups (p<0.01, p=0.055 and p<0.05). A fourth method of assessing cell damage using Fluoro Jade C labeled degenerating neurons that were also intensively eosinophilic. Counts of Fluoro Jade C-positive cells were higher in the young SI group than in the aged SI group (P<0.05), suggesting that mechanisms of ischemic cell death may change with aging. In conclusion, aging alters mechanisms of ischemic cell death in CA1 neurons and ischemic tolerance mechanisms are blunted by aging.

Neuroprotection against ischemic/hypoxic brain damage: blockers of ionotropic glutamate receptor and voltage sensitive calcium channels.

The growing number of cellular and molecular pathways believed to be involved in mechanisms of ischemic cell death in the brain has spurred a similar growth in the number of potential neuroprotective modalities, the majority of which are pharmacological in nature. Preventing or minimizing the first few steps in the cascade of events leading to ischemic cell death would have a more profound effect on the postischemic outcome than intervention at later steps in that cascade. This logic is, of course, at the heart of the urgency in providing the stroke or cardiac arrest patient with the earliest possible neuroprotective treatment. For the purpose of assessing potential neuroprotective modalities, the use of a well-established cerebral hypoxic/ischemic model system is a prerequisite. In our studies, we have used two major approaches, in vitro and in vivo. We evaluated both agonists and antagonists of ionotropic glutamate receptor channels (IGRC) and their effects in exacerbating and attenuating, respectively, the posthypoxic/ischemic outcome. Other drugs were tested for their ability to block the L-type voltage-sensitive calcium channels (VSCC), which are responsible for calcium influx and overload upon hypoxia/ischemia. These two membrane protein entities, the IGRC and the VSCC, are believed to be involved in the early stages of the cellular cascade that leads to the demise of neurons posthypoxia/ischemia. Some of the drugs were also tested for possible interaction with each other searching for possible synergy. These and other published studies in the field are reviewed here.

Proceedings of the Princeton Conference on Cerebrovascular Disease. April 2-4, 2004, Baltimore, Maryland, USA.

The Princeton Conference has a one-half century history as a unique, highly focused forum for the presentation and discussion of current scientific information and future directions in stroke research. The 24th Princeton Conference on Cerebrovascular Disease was held in Baltimore, Maryland, April 2 to 4, 2004, at the Baltimore Marriott Waterfront Hotel. This conference focused on the current status and future directions of stroke pathophysiology, diagnosis and treatment, with emphasis on cellular and molecular mechanisms of ischemic cell death and cell repair, and clinical aspects of imaging, risk factors, and therapeutic strategies in stroke. There were 10 major areas of presentation and discussion. The meeting began with a discussion of vascular dementia and included discussion of amyloid and epidemiology of Alzheimer disease and vascular dementias. The next discussion concerned multimodal imaging and its future in stroke. The role of sex steroids in stroke was the next area of discussion. Estrogen and progesterone were discussed as potential neuroprotection agents, and their differences as neuroprotectants in animal models was discussed in light of the recent major negative clinical trials. The ischemic penumbra, what it is, how to image it and its molecular identification …

Preface [Hot Topic: Molecular and Cellular Mechanisms of Ischemic Cell Death in the Brain (Executive Editor: Rita Sattler)

Preface [Hot Topic: Molecular and Cellular Mechanisms of Ischemic Cell Death in the Brain (Executive Editor: Rita Sattler)

Preface [Hot Topic: Molecular and Cellular Mechanisms of Ischemic Cell Death in the Brain (Executive Editor: Rita Sattler)

Preface [Hot Topic: Molecular and Cellular Mechanisms of Ischemic Cell Death in the Brain (Executive Editor: Rita Sattler)

The Ischemic Penumbra: Identification, Evolution and Treatment Concepts

The concept of the ischemic penumbra is an important one for both basic investigators of cerebral ischemia and for clinicians who treat stroke patients. The ischemic penumbra has been defined in a variety of ways, but the most clinically relevant definition is that portion of the ischemic territory that is still potentially salvageable, if an appropriate treatment is given. Currently, three main challenges persist for those interested in the ischemic penumbra concept: how can this ischemic region be most accurately identified in stroke patients, what mechanisms of ischemic cell death are most important for progression from penumbra towards irreversible injury and what therapeutic modalities are most likely to impede the development of infarction? Much important information regarding each of these topics has become available recently and will be the focus of this paper.

Clinical Relevance of Ischemic Preconditioning

The mechanisms of ischemic cell death and reperfusion injury in the myocardium and the ways to limit these have been under extensive research for decades. The discovery of the phenomenon of ischemic preconditioning, i.e. endogenous protection against ischemia-reperfusion injury obtained by one or more brief preceding episodes of ischemia, really boosted this research 15 years ago. Even though extensive research in experimental animals has provided data on the cellular mechanisms of ischemic preconditioning, such as adenosine receptor activation, opening of mitochondrial adenosine triphosphate (ATP)-sensitive potassium channels and production of endogenous protective stress proteins, direct clinical applications are still missing. The purpose of this study is to summarize the latest progress in solving the cellular and molecular mechanisms of the phenomenon, as well as the evidence for the existence of this phenomenon in humans and its clinical relevance.

Morphologic criteria and detection of apoptosis.

Apoptosis is an organized, energy dependent process, which leads to cell death. Its definition is based on distinct morphological features [10] and demonstration of internucleosomal DNA degradation [27], executed by selectively activated DNAses [4, 22]. The morphologic hallmarks of apoptosis include chromatic margination, nuclear condensation and fragmentation, and condensation of the cell with preservation of organelles. The process is followed by fragmentation of the cell into membrane-bound apoptotic bodies, which undergo phagocytosis by nearby cells without associated inflammation [10, 11]. Apoptosis characteristically occurs in insolated single cells. The duration of apoptosis is estimated to be from 12 to 24 hours, but in cell culture visible morphologic changes are accomplished in less than two hours [10, 16]. Non-apoptotic cell death, a prototype of which is cell death due to ischemia (oncosis), is characterized by depletion of intracellular ATP stores, swelling of the cell with disruption of organelles and rupture of the plasma membrane [15]. Groups of necrotic cells and inflammation are found in tissues [10, 15]. The significance of apoptosis has mostly been studied using the TUNEL assay that detects DNA strand breaks in tissue sections and allows quantification of apoptotic cells by light microscopy [6]. Common experience seems to be that the TUNEL assay is prone to false positive or negative findings. This has been explained by the dependence of the staining kinetics on the reagent concentration [17], fixation of the tissue [2] and the extent of proteolysis [17]. Active RNA synthesis [12] and DNA damage in necrotic cells [17, 19] may cause non-specific staining. To obtain reliable and reproducible results, TUNEL assay should be carefully standardized by using tissue sections treated with DNAse (positive control of apoptosis). Quantification of apoptosis should include enough microscopic fields and identification of the cell type undergoing apoptosis. The specificity of the results can be substantiated by combining other methods with TUNEL, such as assessment of the pattern of DNA fragmentation or evaluation of the morphological features. Even though there is high variation in the results obtained in consecutive studies under the same circumstances, increasing evidence shows that TUNEL-positive cardiomyocytes and internucleosomal DNA fragmentation are associated with various cardiac diseases, including acute myocardial infarction and heart failure [reviewed in 5, 9]. Some morphological features of apoptosis have been observed in TUNEL-positive cardiomyocytes using light microscopy (Figure 1) or confocal microscopy [20]. Electron microscopic evidence of apoptosis has been found in the degenerating conduction system [7], in experimental heart failure [23], and in human hibernating myocardium [3]. In acutely ischemic myocardium the interpretation of the findings remains controversial, since only non-apoptotic cell morphology has been found in electron microscopy [8, 19]. One explanation might be abortion of the apoptotic program due to the lack of ATP before the morphologic features are fully evident [14]. Another explanation is the possibility that non-apoptotic cell death and apoptosis share common mechanisms in the early phases of the processes [14, 19]. The exact mechanisms of ischemic cell death remain to be clarified and the classification between apoptosis and non-apoptosis cell death to be specified. Recently, caspase activation has emerged as the central molecular event leading to apoptosis, preceding DNA degradation and the development of apoptotic morphology [22, 25]. New methods have been developed to demonstrate caspase activation [1, 13]. Inhibition of caspase may be an efficient way to prevent apoptotic cardiomyocyte death as well as to define and specifically probe the significance of apoptotic cell death in cardiac diseases.

Glutamate-mediated selective vulnerability to ischemia is present in organotypic cultures of hippocampus

Ischemic damage to the brain, whether induced experimentally or observed clinically, often produces a pattern of delayed selective cell death in subfield CA1 of hippocampus which has been associated with significant neurologic deficits. The present study demonstrates that this selective vulnerability of CA1 neurons to ischemia, with relative preservation of their neighbors, is expressed in organotypic tissue culture and is prevented by the N- methyl- d-aspartate (NMDA) receptor blocker, MK-801. These data provide conclusive evidence that this selective cells death does not have a vascular etiology but is mediated by factors intrinsic to the hippocampal neurons and/or local circuitry. This model system provides an opportunity both to examine mechanisms of ischemic cell death in an avascular environment and to study methods of prevention in the absence of systemic variables.

Quantifying cell death in the myocardium: Myosin specific antibody in the evaluation of membrane defects

Methods for evaluating cardiac myocyte necrosis utilizing antibodies specific for the heavy or light chains of cardiac myosin are reviewed. Cell death, associated with sarcolemmal disruption, results in the leakage of myosin light chains from the cytoplasm as well as the accessibility of myosin heavy chains to exogenous specific antibodies. Measurement of plasma light chain concentration has been useful in the diagnosis of myocardial infarction, though more recently, patients with congestive cardiomyopathy associated with an inflammatory infiltrate have been identified by an elevated plasma light chain concentration. The binding of myosin heavy chains to necrotic myocytes has been useful in the study of mechanisms of ischemic cell death in cell culture, in the diagnosis and quantification of myocardial infarction, both experimentally and clinically, and more recently in the study of experimental myocarditis and cardiac transplantation. It is hoped that these methods may evolve as useful clinical tools in the identification of those cardiomyopathy patients whose course is characterized by rapid myocyte loss.