Abnormal permeability of inner and outer mitochondrial membranes contributes independently to mitochondrial dysfunction in the liver during acute endotoxemia

OBJECTIVE: This study was designed to determine the role played by the mitochondrial permeability transition in the pathogenesis of mitochondrial damage and dysfunction in a representative systemic organ during the acute phase of endotoxemia. DESIGN: A well-established, normotensive feline model was employed to determine whether pretreatment with cyclosporine A, a potent inhibitor of the mitochondrial permeability transition, normalizes mitochondrial ultrastructural injury and dysfunction in the liver during acute endotoxemia. SETTING: The Ohio State University Medical Center research laboratory. SUBJECTS: Random source, adult, male conditioned cats. INTERVENTIONS: Hemodynamic resuscitation and maintenance of acid-base balance and tissue oxygen availability were provided, as needed, to minimize the potentially confounding effects of tissue hypoxia and/or acidosis on the experimental results. Treatment groups received isotonic saline vehicle (control; n = 6), lipopolysaccharide (3.0 mg/kg, intravenously; n = 8), or cyclosporine A (6.0 mg/kg, intravenously; n = 6) or tacrolimus (FK506, 0.1 mg/kg, intravenously; n = 4) followed in 30 mins by lipopolysaccharide (3.0 mg/kg, intravenously). Liver samples were obtained 4 hrs posttreatment, and mitochondrial ultrastructure, function, and cytochrome c, Bax, and ceramide contents were assessed. MEASUREMENTS AND MAIN RESULTS: As expected, significant mitochondrial injury was apparent in the liver 4 hrs after lipopolysaccharide treatment, despite maintenance of regional tissue oxygen availability. Namely, mitochondria demonstrated high-amplitude swelling and exhibited altered respiratory function. Cyclosporine A pretreatment attenuated lipopolysaccharide-induced mitochondrial ultrastructural abnormalities and normalized mitochondrial respiratory control, reflecting protection against inner mitochondrial membrane damage. However, an abnormal permeability of outer mitochondrial membranes to cytochrome c was observed in all lipopolysaccharide-treated groups and was associated with increased mitochondrial concentrations of Bax and ceramide. CONCLUSIONS: These studies confirm that liver mitochondria are early targets of injury during endotoxemia and that inner and outer mitochondrial membrane damage occurs through different mechanisms. Inner mitochondrial membrane damage appears to relate to the mitochondrial permeability transition, whereas outer mitochondrial membrane damage can occur independent of the mitochondrial permeability transition. Preliminary evidence suggests that Bax may participate in lipopolysaccharide-induced outer mitochondrial membrane damage, but further investigations are needed to confirm this.     

Mitochondrial disorders in NSAIDs-induced small bowel injury

Recent studies using small bowel endoscopy revealed that non-steroidal anti-inflammatory drugs including low-dose aspirin, can often induce small bowel injury. Non-steroidal anti-inflammatory drugs-induced small bowel mucosal injury involves various factors such as enterobacteria, cytokines, and bile. Experimental studies demonstrate that both mitochondrial disorders and inhibition of cyclooxygenases are required for development of non-steroidal anti-inflammatory drugs-induced small bowel injury. Mitochondrion is an organelle playing a central role in energy production in organisms. Many non-steroidal anti-inflammatory drugs directly cause mitochondrial disorders, which are attributable to uncoupling of oxidative phosphorylation induced by opening of the mega channel called mitochondrial permeability transition pore on the mitochondrial membrane by non-steroidal anti-inflammatory drugs. Bile acids and tumor necrosis factor-α also can open the permeability transition pore. The permeability transition pore opening induces the release of cytochrome c from mitochondrial matrix into the cytosol, which triggers a cascade of events that will lead to cell death. Therefore these mitochondrial disorders may cause disturbance of the mucosal barrier function and elevation of the small bowel permeability, and play particularly important roles in early processes of non-steroidal anti-inflammatory drugs-induced small bowel injury. Although no valid means of preventing or treating non-steroidal anti-inflammatory drugs-induced small bowel injury has been established, advances in mitochondrial studies may bring about innovation in the prevention and treatment of this kind of injury.     

Disruption of mitochondrial calcium homeostasis after chronic alpha-naphthylisothiocyanate administration: relevance for cholestasis.

BACKGROUND: Hepatocyte dysfunction caused by impaired mitochondrial function has been pointed out as a probable leading cause of cholestatic liver injury. The aim of this study was to evaluate liver mitochondrial bioenergetics that followed repeated in vivo administration of alpha-naphthylisothiocyanate, a known cholestatic agent. METHODS: Serum markers of liver injury and endogenous adenine nucleotides were measured in alpha-naphthylisothiocyanate-treated rats (intraperitoneally, 100 mg/Kg/wk x 6 wk). Changes in membrane potential, mitochondrial respiration, as well as alterations in mitochondrial calcium homeostasis were monitored. RESULTS: In rats injected with alpha-naphthylisothiocyanate, liver injury with cholestasis developed within 48 hours, as indicated by both serum enzyme activities and total bilirubin concentration. However, 1 week after the last injection, serum enzyme activity returned to control levels. In addition, after chronic alpha-naphthylisothiocyanate administration, no alterations in mitochondrial respiratory function and membrane potential were observed. Associated with these parameters, mitochondria from treated animals exhibited increased susceptibility to disruption of mitochondrial calcium homeostasis by calcium phosphate and by bile acids, which was probably caused by induction of permeability transition pore. CONCLUSIONS: Our data suggest that chronic cholestasis in rats leads to impaired mitochondrial function due to the disruption of mitochondrial calcium homeostasis. The initiating event is the induction of a cyclosporine A-sensitive release of calcium. This event may be an important determinant of the progression of cholestatic liver injury and associated liver cirrhosis. In addition, in the present study we observed that impairment of mitochondrial function is potentiated by chenodeoxycholate, a bile acid that is known to be toxic. Ursodeoxycholate (the beta- epimer of chenodeoxycholate) is approved for the treatment of chronic cholestatic liver disease. Interestingly, we show that the susceptibility to the cyclosporine A-sensitive release of calcium was increased by the combination of both bile acids. These results indicate that the reported improvement of biochemical parameters in cholestatic patients treated with ursodeoxycholate would not prevent the associated mitochondrial dysfunction. This may explain the progression of the histological stage and the maintenance of symptoms during cholestasis.     

Cellular mechanisms underlying acquired epilepsy: the calcium hypothesis of the induction and maintainance of epilepsy

Epilepsy is one of the most common neurological disorders. Although epilepsy can be idiopathic, it is estimated that up to 50% of all epilepsy cases are initiated by neurological insults and are called acquired epilepsy (AE). AE develops in 3 phases: (1) the injury (central nervous system [CNS] insult), (2) epileptogenesis (latency), and (3) the chronic epileptic (spontaneous recurrent seizure) phases. Status epilepticus (SE), stroke, and traumatic brain injury (TBI) are 3 major examples of common brain injuries that can lead to the development of AE. It is especially important to understand the molecular mechanisms that cause AE because it may lead to innovative strategies to prevent or cure this common condition. Recent studies have offered new insights into the cause of AE and indicate that injury-induced alterations in intracellular calcium concentration levels [Ca(2+)](i) and calcium homeostatic mechanisms play a role in the development and maintenance of AE. The injuries that cause AE are different, but they share a common molecular mechanism for producing brain damage-an increase in extracellular glutamate concentration that causes increased intracellular neuronal calcium, leading to neuronal injury and/or death. Neurons that survive the injury induced by glutamate and are exposed to increased [Ca(2+)](i) are the cellular substrates to develop epilepsy because dead cells do not seize. The neurons that survive injury sustain permanent long-term plasticity changes in [Ca(2+)](i) and calcium homeostatic mechanisms that are permanent and are a prominent feature of the epileptic phenotype. In the last several years, evidence has accumulated indicating that the prolonged alteration in neuronal calcium dynamics plays an important role in the induction and maintenance of the prolonged neuroplasticity changes underlying the epileptic phenotype. Understanding the role of calcium as a second messenger in the induction and maintenance of epilepsy may provide novel insights into therapeutic advances that will prevent and even cure AE.     

Cyclosporin A ameliorates mitochondrial ultrastructural injury in the ileum during acute endotoxemia

OBJECTIVE: This study was designed to determine the role, if any, of the mitochondrial permeability transition in the pathogenesis of mitochondrial injury in a representative systemic organ during the acute phase of endotoxemia. DESIGN: A well-established, normotensive feline model was employed to determine whether pretreatment with cyclosporin A, a potent inhibitor of the mitochondrial permeability transition, reduces the severity of mitochondrial injury in the ileum during acute endotoxemia. SETTING: The Ohio State University Medical Center research laboratory.SUBJECTS Adult, male conditioned cats. INTERVENTIONS: Volume resuscitation and maintenance of acid/base balance and tissue oxygen availability were provided, as needed, to minimize the potentially confounding effects of tissue hypoxia and/or acidosis on the experimental results. Following isotonic saline vehicle (control; n = 6), lipopolysaccharide (3.0 mg/kg, intravenously; n = 10), or cyclosporin A (6 mg/kg, intravenously; n = 7) followed in 60 mins by lipopolysaccharide (3.0 mg/kg, intravenously) administration, ileal samples were obtained at 4 hrs posttreatment, and mitochondrial ultrastructure was assessed. Objective comparisons of mitochondrial ultrastructural morphology were performed by using digital image analyses. MEASUREMENTS AND MAIN RESULTS: As expected, significant mitochondrial injury was apparent in the ileal tissues by 4 hrs following LPS treatment, despite maintenance of regional tissue oxygen availability. Objective evaluation of mitochondrial morphology demonstrated characteristics consistent with high-amplitude swelling. Cyclosporin A pretreatment protected against the development of these LPS-induced mitochondrial ultrastructural abnormalities, an effect not attributable to the suppression of lipopolysaccharide-induced tumor necrosis factor-alpha production. CONCLUSIONS: These investigations are the first to demonstrate a protective effect of cyclosporin A against mitochondrial injury in a representative systemic organ during acute endotoxemia. We propose that mitochondrial injury likely related to induction of the mitochondrial permeability transition may participate in the pathogenesis of systemic organ injury and organ failures during acute sepsis.     

Diglycolic acid,the toxic metabolite of diethylene glycol,chelates calcium and produces renal mitochondrial dysfunction in vitro

Context: Diethylene glycol (DEG) has caused many cases of acute kidney injury and deaths worldwide. Diglycolic acid (DGA) is the metabolite responsible for the renal toxicity, but its toxic mechanism remains unclear. Objective: To characterize the mitochondrial dysfunction produced from DGA by examining several mitochondrial processes potentially contributing to renal cell toxicity. Materials and methods: The effect of DGA on mitochondrial membrane potential was examined in normal human proximal tubule (HPT) cells. Isolated rat kidney mitochondria were used to assess the effects of DGA on mitochondrial function, including respiratory parameters (States 3 and 4), electron transport chain complex activities and calcium-induced opening of the mitochondrial permeability transition pore. DGA was compared with ethylene glycol tetraacetic acid (EGTA) to determine calcium chelating ability. DGA cytotoxicity was assessed using lactate dehydrogenase leakage from cultured proximal tubule cells. Results: DGA decreased the mitochondrial membrane potential in HPT cells. In rat kidney mitochondria, DGA decreased State 3 respiration, but did not affect State 4 respiration or the ADP/O ratio. DGA reduced glutamate/malate respiration at lower DGA concentrations (0.5?mmol/L) than succinate respiration (100?mmol/L). DGA inhibited Complex II activity without altering Complex I, III or IV activities. DGA blocked calcium-induced mitochondrial swelling, indicating inhibition of the calcium-dependent mitochondrial permeability transition. DGA and EGTA reduced the free calcium concentration in solution in an equimolar manner. DGA toxicity and mitochondrial dysfunction occurred as similar concentrations. Discussion: DGA inhibited mitochondrial respiration, but without uncoupling oxidative phosphorylation. The more potent effect of DGA on glutamate/malate respiration and the inhibition of mitochondrial swelling was likely due to its chelation of calcium. Conclusion: These results indicate that DGA produces mitochondrial dysfunction by chelating calcium to decrease the availability of substrates and of reducing equivalents to access Complex I and by inhibiting Complex II activity at higher concentrations.     

Intervention strategies for neonatal hypoxic-ischemic cerebral injury

BACKGROUND: Accumulating evidence points to an evolving process of brain injury after intrapartum hypoxia-ischemia that initiates in utero and extends into a recovery period. It is during this recovery period that the potential for neuroprotection exists. OBJECTIVE: This discussion briefly reviews the cellular characteristics of hypoxic-ischemic cerebral injury and the current and future therapeutic strategies aimed at ameliorating ongoing brain injury after intrapartum hypoxia-ischemia. METHODS: As part of the Newborn Drug Development Initiative, the National Institute of Child Health and Human Development and the US Food and Drug Administration cosponsored a workshop held March 29 and 30, 2004, in Baltimore, Maryland. Information for this article was gathered during that workshop. Literature searches of MEDLINE (Ovid) and EMBASE (1996-2005) were also conducted; search terms included newborn, infant, hypoxia-ischemia, hypoxic-ischemic encephalopathy, asphyxia, pathogenesis, treatment, reperfusion injury, and mechanisms, as well as numerous interventions (ie, therapeutic hypothermia, magnesium, and barbiturates). RESULTS: The acute brain injury results from the combined effects of cellular energy failure, acidosis, glutamate release, intracellular calcium accumulation, lipid peroxidation, and nitric oxide neurotoxicity that serve to disrupt essential components of the cell, resulting in death. Many factors, including the duration or severity of the insult, influence the progression of cellular injury after hypoxia-ischemia. A secondary cerebral energy failure occurs from 6 to 48 hours after the primary event and may involve mitochondrial dysfunction secondary to extended reactions from primary insults (eg, calcium influx, excitatory neurotoxicity, oxygen free radicals, or nitric oxide formation). Some evidence suggests that circulatory and endogenous inflammatory cells/mediators also contribute to ongoing brain injury. The goals of management of a newborn infant who has sustained a hypoxic-ischemic insult and is at risk for injury should include early identification of the infant at highest risk for evolving injury, supportive care to facilitate adequate perfusion and nutrients to the brain, attempts to maintain glucose homeostasis, and consideration of interventions to ameliorate the processes of ongoing brain injury. Recent evidence suggests a potential role for modest hypothermia (ie, a reduction in core body temperature to -34 degrees C) administered to high-risk term infants within 6 hours of birth. Either selective (head) or systemic (body) cooling reduces the incidence of death and/or moderate to severe disability at 18-month follow-up. Additional strategies-including the use of oxygen free radical inhibitors and scavengers, excitatory amino acid antagonists, and growth factors; prevention of nitric oxide formation; and blockage of apoptotic pathways-have been evaluated experimentally but have not been replicated in a systematic manner in the human neonate. Other avenues of potential neuroprotection that have been studied in immature animals include platelet-activating factor antagonists, adenosinergic agents, monosialoganglioside GM1, insulin-like growth factor-1, and erythropoietin. CONCLUSIONS: Much progress has been made toward understanding the mechanisms contributing to ongoing brain injury after intrapartum hypoxia-ischemia. This should facilitate more specific pharmacologic intervention strategies that might provide neuroprotection during the reperfusion phase of injury.     

Commentary on selected aspects of cardioprotection

Three aspects of cardioprotection are discussed in this article. The first is myocyte death as a function of the duration and severity of ischemia in experimental acute myocardial infarction in the dog heart. The short period of time during which reperfusion with arterial blood will salvage myocytes is demonstrated along with data showing that this period diminishes significantly if collateral flow is very low or absent. The second topic is a discussion of potential mechanisms underlying postconditioning. It begins with a review of the changes that lead to irreversible injury during acute ischemia in the dog heart along with a discussion of the genesis of contraction band necrosis and no reflow when myocardium is salvaged by unrestricted reperfusion with arterial blood in order to provide a basis to discuss the potential mechanisms underlying postconditioning, a situation in which reflow is intermittent and restricted. Postconditioning is reported to achieve greater myocyte salvage than unrestricted reflow. Potential explanations for this beneficial effect include: first, sufficient sarcolemmal repair occurring during the intermittent reflow (reoxygenation) to prevent cell death by explosive cell swelling, and second, prevention of the opening of the mitochondrial permeability transition pore, thereby preventing mitochondrial failure and cell death in the reperfused tissue. Since there is no way available to identify and specifically study the myocytes that would have died if not protected by postconditioning, direct demonstration of mechanisms is difficult or impossible. Finally, the third topic in this commentary is an analysis of the obstacles faced by investigators using small rodent hearts to establish cardioprotective mechanisms. Such studies provide valid data but the relationship of the changes and the proposed mechanisms underlying these changes are not necessarily directly transferable to ischemic large animal hearts including the heart of man.     

Non-neurologic organ dysfunction in acute brain injury

Patients with acute brain injury are a distinct group within the ICU who may develop non-neurologic organ dysfunction in the absence of systemic injury or infection. This dysfunction may arise directly as a result of the brain injury or indirectly with complications of brain-specific therapies. This article reviews the current literature with respect to the incidence of organ dysfunction or failure and its association with outcome in patients with acute brain injury. Organ system-specific etiologic considerations and management are discussed.     

Clinically approved heterocyclics act on a mitochondrial target and reduce stroke-induced pathology

Substantial evidence indicates that mitochondria are a major checkpoint in several pathways leading to neuronal cell death, but discerning critical propagation stages from downstream consequences has been difficult. The mitochondrial permeability transition (mPT) may be critical in stroke-related injury. To address this hypothesis, identify potential therapeutics, and screen for new uses for established drugs with known toxicity, 1,040 FDA-approved drugs and other bioactive compounds were tested as potential mPT inhibitors. We report the identification of 28 structurally related drugs, including tricyclic antidepressants and antipsychotics, capable of delaying the mPT. Clinically achievable doses of one drug in this general structural class that inhibits mPT, promethazine, were protective in both in vitro and mouse models of stroke. Specifically, promethazine protected primary neuronal cultures subjected to oxygen-glucose deprivation and reduced infarct size and neurological impairment in mice subjected to middle cerebral artery occlusion/reperfusion. These results, in conjunction with new insights provided to older studies, (a) suggest a class of safe, tolerable drugs for stroke and neurodegeneration; (b) provide new tools for understanding mitochondrial roles in neuronal cell death; (c) demonstrate the clinical/experimental value of screening collections of bioactive compounds enriched in clinically available agents; and (d) provide discovery-based evidence that mPT is an essential, causative event in stroke-related injury.     

Hyperoxia: good or bad for the injured brain?

PURPOSE OF REVIEW: For decades it was assumed that cerebral ischemia was a major cause of secondary brain injury in traumatic brain injury, and management focused on improving cerebral perfusion and blood flow. Following the observation of mitochondrial dysfunction in traumatic brain injury and the widespread use of brain tissue oxygen tension (P(br)O(2) monitoring, however, recent work has focused on the use of hyperoxia to reduce the impact of traumatic brain injury. RECENT FINDINGS: Previous work on normobaric hyperoxia utilized very indirect measures of cerebral oxygen metabolism (intracranial pressure, brain oxygen tension and microdialysis) as outcome variables. Interpretation of these measures is controversial, making it difficult to determine the impact of hyperoxia. A recent study, however, utilized positron emission tomography to study the impact of hyperoxia on patients with acute severe traumatic brain injury and found no improvement on cerebral metabolic rate for oxygen with this intervention. SUMMARY: Despite suggestive data from microdialysis studies, direct measurement of the ability of the brain to utilize oxygen indicates that hyperoxia does not increase oxygen utilization. This, combined with the real risk of oxygen toxicity, suggests that routine clinical use is not appropriate at this time and should await appropriate prospective outcome studies.     

Genetic influences on outcome following acute neurological insults

PURPOSE OF REVIEW: To examine the evidence for a genetic influence on clinical outcome after a variety of acute neurologic events. RECENT FINDINGS: Clinical outcome after brain injury is variable and cannot easily be predicted. It has been proposed that genetic polymorphisms may have an important role in determining outcome from a number of conditions, including acute neurologic events. Apolipoprotein E, an important mediator of cholesterol and lipid transport in the brain, is coded by a polymorphic gene (APOE). The APOE epsilon4 allele has been associated with unfavorable outcome after traumatic brain injury (TBI), hemorrhagic stroke and subarachnoid hemorrhage (SAH). Genes involved in other pathophysiological processes, such as cytokine genes in neuroinflammation, are now being implicated. For example interleukin-6 (IL-6) promoter polymorphisms are a risk factor for poor outcome after ischemic stroke, and may have an effect after traumatic brain injury. The emerging importance of a number of other gene polymorphisms is outlined in the review. SUMMARY: There is evidence demonstrating the epsilon4 allele of APOE predisposes to poor outcome after TBI, hemorrhagic stroke and SAH, but not ischemic stroke. The reason for this difference is unclear but it suggests there may be differences in the key mechanisms underlying the response to different types of insult. The role of other gene polymorphisms is being increasingly explored but there is still a need for larger prospective studies looking at larger panels of gene polymorphisms.     

Antioxidant gene therapy against neuronal cell death

Oxidative stress is a common hallmark of neuronal cell death associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, as well as brain stroke/ischemia and traumatic brain injury. Increased accumulation of reactive species of both oxygen (ROS) and nitrogen (RNS) has been implicated in mitochondrial dysfunction, energy impairment, alterations in metal homeostasis and accumulation of aggregated proteins observed in neurodegenerative disorders, which lead to the activation/modulation of cell death mechanisms that include apoptotic, necrotic and autophagic pathways. Thus, the design of novel antioxidant strategies to selectively target oxidative stress and redox imbalance might represent important therapeutic approaches against neurological disorders. This work reviews the evidence demonstrating the ability of genetically encoded antioxidant systems to selectively counteract neuronal cell loss in neurodegenerative diseases and ischemic brain damage. Because gene therapy approaches to treat inherited and acquired disorders offer many unique advantages over conventional therapeutic approaches, we discussed basic research/clinical evidence and the potential of virus-mediated gene delivery techniques for antioxidant gene therapy.     

Erratum to "Cellular mechanisms underlying acquired epilepsy: the calcium hypothesis of the induction and maintenance of epilepsy." [Pharmacol. Ther. 105(3) (2005) 229-266

Epilepsy is one of the most common neurological disorders. Although epilepsy can be idiopathic, it is estimated that up to 50% of all epilepsy cases are initiated by neurological insults and are called acquired epilepsy (AE). AE develops in 3 phases: (1) the injury [central nervous system (CNS) insult]. (2) epileptogenesis (latency), and (3) the chronic epileptic (spontaneous recurrent seizure) phases. Status epilepticus (SE), stroke, and traumatic brain injury (TBI) are 3 major examples of common brain injuries that can lead to the development of AE. It is especially important to understand the molecular mechanisms that cause AE because it may lead to innovative strategies to prevent or cure this common condition. Recent studies have offered new insights into the cause of AE and indicate that injury-induced alterations in intracellular calcium concentration levels ([Ca(2+)](i)) and calcium homeostatic mechanisms play a role in the development and maintenance of AE. The injuries that cause AE are different, but the share a common molecular mechanism for producing brain damage--an increase in extracellular glutamate and are exposed to increased [Ca(2+)](i) are the cellular substrates to develop epilepsy because dead cells do not seize. The neurons that survive injury sustain permanent long-term plasticity changes in [Ca(2+)](i) and calcium homeostatic mechanisms that are permanent and are a prominent feature of the epileptic phenotype. In the last several years, evidence has accumulated indicating that the prolonged alteration in neuronal calcium dynamics plays an important role in the induction and maintenance of the prolonged neuroplasticity changes underlying the epileptic phenotype. Understanding the role of calcium as a second messenger in the induction and maintenance of epilepsy may provide novel insights into therapeutic advances that will prevent and even cure AE.     

Regional ischemia after head injury

PURPOSE OF REVIEW: To examine the evidence of regional cerebral ischemia after traumatic brain injury. RECENT FINDINGS: This review describes the mechanisms responsible for secondary brain injury and the similarities between traumatic and ischemic neuronal cell death. Cerebral ischemia is defined, and the difficulties of quantifying the burden of cerebral ischemia in the context of clinical head injury are presented. Recent clinical data obtained from monitoring brain tissue oxygenation, tissue metabolites using microdialysis, and cerebral blood flow, blood volume, oxygen metabolism, and oxygen extraction fraction using oxygen-15 positron emission tomography are discussed. These data highlight that significant episodes of regional ischemia occur within the acute phase after injury and are associated with poor outcome. Although various monitoring tools are capable of detecting significant episodes of regional ischemia, each of the currently available techniques is limited in its clinical application. SUMMARY: There is increasing evidence to suggest that a small but significant volume of brain tissue is at risk of ischemic injury after trauma. Future studies should examine the pathophysiology underlying such ischemia and how monitoring techniques can be used to direct appropriate therapy and influence outcome.     

Apoptosis Induced by Microbubble-Assisted Acoustic Cavitation in K562 Cells: The Predominant Role of the Cyclosporin A-Dependent Mitochondrial Permeability Transition Pore

Acoustic cavitation of microbubbles has been described as inducing tumor cell apoptosis that is partly associated with mitochondrial dysfunction; however, the exact mechanisms have not been fully characterized. Here, low-intensity pulsed ultrasound (1 MHz, 0.3-MPa peak negative pressure, 10% duty cycle and 1-kHz pulse repetition frequency) was applied to K562 chronic myelogenous leukemia cells for 1 min with 10% (v/v) SonoVue microbubbles. After ultrasound exposure, the apoptotic index was determined by flow cytometry with annexin V–fluorescein isothiocyanate/propidium iodide. In addition, mitochondrial membrane potential (ΔΨ_m) was determined with the JC-1 assay. Translocation of apoptosis-associated protein cytochrome c was evaluated by Western blotting. We found that microbubble-assisted acoustic cavitation can increase the cellular apoptotic index, mitochondrial depolarization and cytochrome c release in K562 cells, compared with ultrasound treatment alone. Furthermore, mitochondrial dysfunction and apoptosis were significantly inhibited by cyclosporin A, a classic inhibitor of the mitochondrial permeability transition pore; however, the inhibitor of Bax protein, Bax-inhibiting peptide, could not suppress these effects. Our results suggest that mitochondrial permeability transition pore opening is involved in mitochondrial dysfunction after exposure to microbubble-assisted acoustic cavitation. Moreover, the release of cytochrome c from the mitochondria is dependent on cyclosporin A–sensitive mitochondrial permeability transition pore opening, but not formation of the Bax-voltage dependent anion channel complex or Bax oligomeric pores. These data provide more insight into the mechanisms underlying mitochondrial dysfunction induced by acoustic cavitation and can be used as a basis for therapy.     

The endothelium in acute lung injury/acute respiratory distress syndrome

PURPOSE OF REVIEW: Since pulmonary edema from increased endothelial permeability is the hallmark of acute lung injury, a frequently encountered entity in critical care medicine, the study of endothelial responses in this setting is crucial to the development of effective endothelial-targeted treatments. RECENT FINDINGS: From the enormous amount of research in the field of endothelial pathophysiology, we have focused on work delineating endothelial alterations elicited by noxious stimuli implicated in acute lung injury. The bulk of the material covered deals with molecular and cellular aspects of the pathogenesis, reflecting current trends in the published literature. We initially discuss pathways of endothelial dysfunction in acute lung injury and then cover the mechanisms of endothelial protection. Several experimental treatments in animal models are presented, which aid in the understanding of the disease pathogenesis and provide evidence for potentially useful therapies. SUMMARY: Mechanistic studies have delivered several interventions, which are effective in preventing and treating experimental acute lung injury and have thus provided objectives for translational studies. Some of these modalities may evolve into clinically useful tools in the treatment of this devastating illness.     

Non-neurological organ dysfunction in neurocritical care: impact on outcome and etiological considerations

PURPOSE OF REVIEW: Organ dysfunction is an important determinant of outcome in critical care medicine. Patients with life threatening neurologic injury represent a distinct subset of critically ill patients in whom non-neurologic organ dysfunction may develop. In this paper the incidence and impact of non-neurologic organ dysfunction in patients with major neurologic injury will be reviewed. Further, potential etiological considerations will be addressed and management strategies discussed. RECENT FINDINGS: Non-neurologic organ dysfunction is extremely common in patients with brain injury occurring in 80-90% of patients admitted to intensive-care units. Several studies have now identified this dysfunction as an independent predictor of poor outcome in neurocritical care. This dysfunction may arise as a result of the neurologic injury or secondary to treatment. Massive catecholamine release continues to be the primary etiological theory of non-neurologic organ dysfunction due to brain injury. Currently employed therapies directed at intracranial hypertension such as maintenance of cerebral perfusion pressure and the use of hypothermia or barbiturates predispose non-neurologic organ dysfunction. SUMMARY: Non-neurologic organ dysfunction is common. This dysfunction independently predicts poor outcome following brain injury and represents a potentially modifiable risk factor. Further study is required to develop optimal management strategies.     

Hyperoxia in the intensive care unit: why more is not always better

PURPOSE OF REVIEW: Hyperoxic inspired gas is essential for patients with hypoxic respiratory failure; it is also suspected, however, as a contributor to the pathogenesis of acute lung injury. Several recent studies in humans, animals, and cell culture have identified mechanisms by which hyperoxia may exert deleterious effects on critically ill patients. This review identifies relevant new findings regarding hyperoxic lung injury in the context of providing guidance for future clinical studies. RECENT FINDINGS: Recent studies have clarified the roles of both receptor-mediated and mitochondrial cell death pathways in experimental hyperoxic lung injury. Studies in animals demonstrate that hyperoxia interacts with mechanical stretch to augment ventilator-induced lung injury. Finally, studies in humans implicate hyperoxia in impairment of host defense responses to infections. SUMMARY: Although hyperoxia has not been conclusively identified as a clinically important cause of lung injury in humans, animal data strongly implicate it. Reports of interaction effects between hyperoxia and both mechanical ventilation and host defense suggest that clinical studies of hyperoxia must take these variables into account. Accumulating data about how hyperoxia initiates cell death provide guidance for development of both biomarkers to identify hyperoxia-induced injury and pharmacological interventions to limit hyperoxia's adverse effects.     

Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies

Mitochondrial dysfunction is a major mechanism whereby drugs can induce liver injury and other serious side effects such as lactic acidosis and rhabdomyolysis in some patients. By severely altering mitochondrial function in the liver, drugs can induce microvesicular steatosis, a potentially severe lesion that can be associated with profound hypoglycaemia and encephalopathy. They can also trigger hepatic necrosis and/or apoptosis, causing cytolytic hepatitis, which can evolve into liver failure. Milder mitochondrial dysfunction, sometimes combined with an inhibition of triglyceride egress from the liver, can induce macrovacuolar steatosis, a benign lesion in the short term. However, in the long term this lesion can evolve in some individuals towards steatohepatitis, which itself can progress to extensive fibrosis and cirrhosis. As liver injury caused by mitochondrial dysfunction can induce the premature end of clinical trials, or drug withdrawal after marketing, it should be detected during the preclinical safety studies. Several in vitro and in vivo investigations can be performed to determine if newly developed drugs disturb mitochondrial fatty acid oxidation (FAO) and the oxidative phosphorylation (OXPHOS) process, deplete hepatic mitochondrial DNA (mtDNA), or trigger the opening of the mitochondrial permeability transition (MPT) pore. As drugs can be deleterious for hepatic mitochondria in some individuals but not in others, it may also be important to use novel animal models with underlying mitochondrial and/or metabolic abnormalities. This could help us to better predict idiosyncratic liver injury caused by drug-induced mitochondrial dysfunction.