Nagao Ishii

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition.

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.

Spinal cord mitochondria display lower calcium retention capacity compared with brain mitochondria without inherent differences in sensitivity to cyclophilin D inhibition

The mitochondrial permeability transition (mPT) is a potential pathogenic mechanism in neurodegeneration. Varying sensitivity to calcium‐induced mPT has been demonstrated for regions within the CNS possibly correlating with vulnerability following insults. The spinal cord is selectively vulnerable in e.g. amyotrophic lateral sclerosis and increased mPT sensitivity of mitochondria derived from the spinal cord has previously been demonstrated. In this study, we introduce whole‐body hypothermia prior to removal of CNS tissue to minimize the effects of differential tissue extraction prior to isolation of spinal cord and cortical brain mitochondria. Spinal cord mitochondria were able to retain considerably less calcium when administered as continuous infusion, which was not related to a general increased sensitivity of the mPT to calcium, its desensitization to calcium by the cyclophilin D inhibitor cyclosporin‐A, or to differences in respiratory parameters. Spinal cord mitochondria maintained a higher concentration of extramitochondrial calcium during infusion than brain mitochondria possibly related to an increased set‐point concentration for calcium uptake. A hampered transport and retention capacity of calcium may translate into an increased susceptibility of the spinal cord to neurodegenerative processes involving calcium‐mediated damage.

Probing the molecular mechanisms of neuronal degeneration: importance of mitochondrial dysfunction and calcineurin activation

Cerebral injury is a critical aspect of the management of patients in intensive care. Pathological conditions induced by cerebral ischemia, hypoxia, head trauma, and seizure activity can result in marked residual impairment of cerebral function. We have investigated the potential mechanisms leading to neuronal cell death in pathological conditions, with the aim of discovering therapeutic targets and methods to minimize neuronal damage resulting from insults directed at the central nervous system (CNS). Over the years, deeper understanding of the mechanisms of neuronal cell death has indeed evolved, enabling clinical critical care management to salvage neurons that are at the brink of degeneration and to support recovery of brain function. However, no substantial breakthrough has been achieved in the quest to develop effective pharmacological neuroprotective therapy directed at tissues of the CNS. The current situation is unacceptable, and preservation of function and protection of the brain from terminal impairment will be a vital medical issue in the twenty-first century. To achieve this goal, it is critical to clarify the key mechanisms leading to neuronal cell death. Here, we discuss the importance of the calcineurin/immunophilin signal transduction pathway and mitochondrial involvement in the detrimental chain of events leading to neuronal degeneration.

Calcineurin and cyclophilin D are differential targets of neuroprotection by immunosuppressants CsA and FK506 in ischemic brain damage

The search for an effective treatment for global ischemia following cardiac arrest has proved to be very difficult. However, studies by Uchino et al. show that the immunosuppressant cyclosporin A (CsA), when administered in such a way that the drug can bypass the blood brain barrier (BBB), dramatically reduces ischemic damage in rat forebrain preparations. An alternative immunosuppressant, FK506, is apparently less efficacious. Both CsA and FK506 are specific inhibitors of immunophilins, (CsA inhibits cyclophilins, FK506 inhibits FKBPs), and of calcineurin, a type 2B Ser/Thr phosphatase that is abundant in the central nervous system. The superiority of CsA may be partly attributable to its selective amelioration of mitochondrial damage, as assayed in vivo and in vitro. Our results suggest that pathways involving calcineurin and cyclophilins, particularly mitochondrial cyclophilin D, play pivotal roles in the development of ischemic brain damage. The present findings may inform the search for new drugs in the treatment of global ischemic damage to the brain, and in other organs.

A novel neuroprotective compound FR901459 with dual inhibition of calcineurin and cyclophilins

Brain ischemia leads to severe damage in the form of delayed neuronal cell death. In our study, we show that the marked neuroprotection of the new immunosuppressant FR901495 in forebrain ischemia is due not only to inhibition of calcineurin, but also to protection against mitochondrial damage caused by mitochondrial permeability transition pore formation through cyclophilin D, one of the prolyl cis/trans isomerase family members. These findings shed light on the clinical application and development of new drugs for the treatment of ischemic damage in the brain as well as in the heart and liver.

Search for novel gene markers of traumatic brain injury by time differential microarray analysis

Neuronal and glial cell death caused by axonal injury sometimes contributes to whole brain pathology after traumatic brain injury (TBI). We show that neuroprotection by 2 types of immunosuppressants, cyclosporin A (CsA) and tacrolimus (FK506), in a cryogenic brain injury model results from inhibition of calcineurin and protection from mitochondrial damage caused by formation of a mitochondrial permeability transition pore induced by cyclophilin D (CyPD), one of the prolyl cis/trans isomerase family members. We evaluated why CsA is neuroprotective by microarray analysis of gene expression in the cryogenic brain injury rat model. Analyses of expression patterns demonstrated that expression of over 14,000 genes changed between the groups with and without CsA treatment, and about 350 genes among them were extracted showing a significant difference. We learned that the differential expression of several gene targets showed specific patterns in a time-dependent manner. These results may help elucidate the mechanisms of neuronal cell death after TBI and the neuroprotective effects of CsA after TBI.

Effects of temperature gradient reduction in three different carbon dioxide absorbents.

Background and objective Temperature gradients in CO2 absorbents may locally increase the water content by condensation. We hypothesized that temperature gradient reduction (TGR) would prevent increased water content, thus preserving the reactivity of the CO2 absorbent and thereby increasing its time to exhaustion (longevity). The purpose of this study was to compare the effects of TGR on the longevity of CO2 absorbent with three different types of CO2 absorbents. Methods We constructed a novel TGR canister. Experiments were conducted using three different types of CO2 absorbents: Drägersorb 800 Plus (D800), Drägersorb Free and Amsorb Plus. One kilogram of fresh CO2 absorbent of each type was placed into two types of canister: the conventional control canister (n = 6) and the TGR canister (n = 6). Results In the case of Drägersorb Free, the TGR canister most effectively and specifically prevented local increase in water content of the CO2 absorbent and markedly increased the longevity (30% increase) compared with the control canister. In the case of Amsorb Plus, the TGR canister also prevented local excessive water content, but the increase in longevity was smaller (17% increase). In the case of D800, the TGR canister markedly increased the longevity (27% increase), but its prevention of local excessive water content was smaller. Conclusions TGR is a useful method to prevent local increase in water content and improve the longevity of CO2 absorbent. The effectiveness of TGR on longevity and water content changes varied in the different types of CO2 absorbent.

Usefulness of galvanic skin reflex monitor in CT-guided thoracic sympathetic blockade for palmar hyperhidrosis

Computed tomography (CT)-guided thoracic sympathetic blockade with ethanol was performed while monitoring sympathetic nerve activity, with an alternating current (AC) galvanic skin reflex (GSR) monitor, in a patient with palmar hyperhidrosis in whom endoscopic thoracic sympathectomy was impossible because of pleural adhesion. Sweating was suppressed after the thoracic sympathetic blockade, and the monitor showed a significant increase in skin resistance. The effect of sympathetic blockade could be evaluated directly and in real time using a GSR monitor.

Mitochondrial permeability transition: Evaluation of putative “pore blockers”

Neuroprotective effects of ethyl pyruvate (EP), a stable derivative of pyruvate, on energy metabolism of rat brain exposed to ischemia were investigated by 31P-nuclear magnetic resonance (31P-NMR) spectroscopy.Brain slices (400 m) were incubated in standard artificial cerebrospinal fluid (ACSF) with 2 mM EP (EP+) or ACSF only (EP-) at 25 ◦C. Brain slices were exposed to ischemia by halting the perfusion for 1 h. Levels of high-energy phosphates, phosphocreatine (PCr) and -ATP, relative to pre-ischemic levels were measured by 31P-NMR.Recovery of PCr after reperfusion was significantly greater in EP+ than in EP−. When brain slices were pretreated with 100 M fluorocitrate, an astrocytic poison, such difference in recovery of PCr was not observed. These results indicated that neuroprotective effects of EP depend on the presence of astrocytes. EP might be neuroprotective via functions of astrocytes as a neuro-protectant from oxidative stress and/or a provider of lactate by glycolysis.

Changes in hemodynamics and cytokines in spetic shock patients treated by direct hemoperfusion with polymyxin B immobilized fiber (PMX-20R)

ConclusionHemoperfusion with PMX-20R may be a useful therapeutic measure in patients with septic shock. This treatment should be considered for use in patients with systemic inflammatory response syndrome (SIRS).