Hiroyuki Uchino

Amelioration by cyclosporin A of brain damage in transient forebrain ischemia in the rat

The immunosuppressant drug cyclosporin A (CsA) is considered to be inherently protective in conditions of ischemia, e.g. in hepatic and cardiac tissue. However, investigations of effects of CsA on neuronal tissue have been contradictory, probably because the blood-brain barrier (BBB) is virtually impermeable to CsA. In the present study, we exploited the finding that the insertion of a syringe needle into brain parenchyma obviously disrupts the BBB and allows influx of CsA, and explored whether CsA, given as intraperitoneal injections daily for 1 week before and 1 week after forebrain ischemia of 7 or 10 min duration, ameliorates the damage incurred to the hippocampal CA 1 sector. In other experiments, the needle insertion and the first i.p. injection of CsA were made 30 min after the start of recirculation, with continued daily administration of CsA during the postinsult week. In animals which were injected with CsA in daily doses of 10 mg kg-1, but in which no needle was inserted, the drug failed to ameliorate CA1 damage, whether the ischemia had a duration of 7 or 10 min. Likewise, needle insertion had no effect on CA1 damage if CsA was not administered. In contrast, when CsA was given to animals with a needle insertion, CA1 damage was dramatically ameliorated, whether treatment was initiated 1 week before ischemia, or 30 min after the start of recirculation. The effect of CsA seemed larger than that of any other drug proposed to have an anti-ischemic effect in forebrain/global ischemia. Injection of tritiated CsA in one animal with BBB disruption lead to detectable radioactivity throughout the ventricular system, suggesting a generalised increase of the entry of CsA across the BBB. The results demonstrate that immunosuppressants of the type represented by CsA markedly ameliorate delayed neuronal damage after transient forebrain ischemia, provided that they can pass the BBB. It is discussed whether the effect of the drug is one involving calcineurin, a protein phosphatase, or if CsA counteracts a permeability transition of the inner mitochondrial membrane, assumed to occur in response to adverse conditions, e.g. gradual accumulation of Ca2+ in the mitochondria in the postischemic period.

Neuroprotective properties of propofol and midazolam, but not pentobarbital, on neuronal damage induced by forebrain ischemia, based on the GABAA receptors

Background: The mechanism of the neuroprotective effects of propofol was compared to two other types of intravenous (i.v.) anesthetics (i.e., benzodiazepine; midazolam and barbiturate; pentobarbital) using Mongolian gerbils focusing on GABA receptor subtypes.

Amelioration by cyclosporin A of brain damage following 5 or 10 min of ischemia in rats subjected to preischemic hyperglycemia

It has recently been shown that the immunosuppressant cyclosporin A (CsA) dramatically ameliorates the selective neuronal necrosis which results from 10 min of forebrain ischemia in rats. Since CsA is a virtually specific blocker of the mitochondrial permeability transition (MPT) pore which is assembled under adverse conditions, such as mitochondrial calcium accumulation and oxidative stress, the results suggest that the delayed neuronal death is due to an MPT. In the present study we explored whether CsA can also ameliorate the aggravated brain damage which is observed in hyperglycemic subjects, and which encompasses rapidly evolving neuronal lesions, edema, and postischemic seizures. Anaesthetised rats with a plasma glucose concentration of approximately 13 mM were subjected to 10 min of forebrain ischemia, and allowed a recovery period of 7 days. In these animals, CsA prevented seizure from occurring and virtually eliminated neuronal necrosis. In order to allow even higher plasma glucose values (approximately 20 mM) to be studied, with long-term recovery, the duration of ischemia had to be reduced to 5 min. Again, CsA suppressed seizure activity and reduced neuronal damage. However, the effects were not as marked or consistent as in the 10 min group, suggesting that excessive tissue acidosis recruits mechanisms of damage which are not sensitive to CsA.

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.

Changes in the bioenergetic state of rat hippocampus during 2.5 min of ischemia, and prevention of cell damage by cyclosporin A in hyperglycemic subjects.

Abstract A recent study from this laboratory has shown that brief transient ischemia (2 min 30 s) in normo- and hyperglycemic rats leads to moderate neuronal necrosis in CA1 cells of the hippocampus, of equal density in the two groups. However, hyperglycemic animals failed to depolarize during the ischemia, nor did they show a decrease in extracellular calcium concentration. The present study was undertaken to study the metabolic correlates to these unexpected findings. Normoglycemic (plasma glucose ∼6 mM) and hyperglycemic (∼20 mM) rats were subjected to ischemic periods of 1 min and 2 min 15 s (2 min 30 s with freezing delay considered), and their brains were frozen in situ. Samples of dorsal hippocampus were dissected at –22°C and extracted for the measurement of phosphocreatine (PCr), creatine, ATP, ADP, AMP, glucose, glycogen, and lactate. Normoglycemic animals showed rapid depletion of PCr, ATP, glucose, and glycogen, and a rise in lactate content to 10–12 mM·kg–1 during the ischemia. Hyperglycemic animals displayed a more moderate rate of fall of PCr and ATP, with ATP values exceeding 50% of control after 2 min 30 s. Glycogen stores were largely maintained, but degradation of glucose somewhat enhanced the lactic acidosis. The results demonstrate that hyperglycemic rats maintained ATP at levels sufficient to prevent cell depolarization and calcium influx during the ischemic period. However, the metabolic perturbation observed must have been responsible for the delayed neuronal damage. We speculate that lowered ATP, increased inorganic P, and oxidative stress triggered a delayed mitochondrial permeability transition (MPT), which led to delayed neuronal necrosis. This assumption was supported by a second series of experiments in which CA1 damage in hyperglycemic rats was prevented by cyclosporin A, a virtually specific inhibitor of the MPT.

Involvement of the brain-derived neurotrophic factor/TrkB pathway in neuroprotecive effect of cyclosporin A in forebrain ischemia

Recent studies have shown that cyclosporin A, a specific antagonist of calcineurin, a phosphatase, ameliorates neuronal cell death in the CA1 sector of the hippocampus after forebrain ischemia in animal models. The mechanism of this neuroprotective effect, however, has not yet been established. Brain-derived neurotrophic factor (BDNF), a member of the neurotrophins, is one of the potent survival and developmental factors whose expression is regulated by cyclic AMP-response element-binding protein (CREB). Activation of CREB is dependent on its phosphorylation at Ser(133), and calcineurin has been reported to dephosphorylate CREB via protein phosphatase 1. Based on these observations, we attempted to investigate how cyclosporin A treatment would affect the changes of phosphorylated CREB (pCREB), BDNF and its receptor tyrosine kinase B (TrkB) after forebrain ischemia in rats. Phosphorylation of CREB was kept augmented throughout the time course examined in cyclosporin A-treated animals, while it ceased without cyclosporin A. Reverse transcription-polymerase chain reaction revealed prolonged maintenance of BDNF mRNA expression in the CA1 sector of cyclosporin A-treated animals. The protein expression of BDNF and TrkB appeared to be up-regulated in cyclosporin A-treated animals, whereas it was transiently up-regulated but decreased to the marginal level of expression without cyclosporin A.From these results we suggest that cyclosporin A induces pCREB by an inhibition of calcineurin, resulting in the induction of BDNF. The mechanisms by which cyclosporin A protects the CA1 region from neuronal cell death in forebrain ischemia may involve the interaction of pCREB, BDNF and TrkB.

Mitochondrial respiration in human viable platelets-Methodology and influence of gender, age and storage.

Studying whole cell preparations with intact mitochondria and respiratory complexes has a clear benefit compared to isolated or disrupted mitochondria due to the dynamic interplay between mitochondria and other cellular compartments. Platelet mitochondria have a potential to serve as a source of human viable mitochondria when studying mitochondrial physiology and pathogenic mechanisms, as well as for the diagnostics of mitochondrial diseases. The objective of the present study was to perform a detailed evaluation of platelet mitochondrial respiration using high-resolution respirometry. Further, we aimed to explore the limits of sample size and the impact of storage as well as to establish a wide range of reference data from different pediatric and adult cohorts. Our results indicate that platelet mitochondria are well suited for ex-vivo analysis with the need for minute sample amounts and excellent reproducibility and stability.

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.

Cyclophilin D-Sensitive Mitochondrial Permeability Transition in Adult Human Brain and Liver Mitochondria

The mitochondrial permeability transition (mPT) is considered to be a major cause of cell death under a variety of pathophysiological conditions of the central nervous system (CNS) and other organs. Pharmacological inhibition or genetic knockout of the matrix protein cyclophilin D (CypD) prevents mPT and cell degeneration in several models of brain injury. If these findings in animal models are translatable to human disease, pharmacological inhibition of mPT offers a promising therapeutic target. The objective of this study was to validate the presence of a CypD-sensitive mPT in adult human brain and liver mitochondria. In order to perform functional characterization of human mitochondria, fresh tissue samples were obtained during hemorrhage or tumor surgery and mitochondria were rapidly isolated. Mitochondrial calcium retention capacity, a quantitative assay for mPT, was significantly increased by the CypD inhibitor cyclosporin A in both human brain and liver mitochondria, whereas thiol-reactive compounds and oxidants sensitized mitochondria to calcium-induced mPT. Brain mitochondria underwent swelling upon calcium overload, which was reversible upon calcium removal. To further explore mPT of human mitochondria, liver mitochondria were demonstrated to exhibit several classical features of the mPT phenomenon, such as calcium-induced loss of membrane potential and respiratory coupling, as well as release of the pro-apoptotic protein cytochrome c. We concluded that adult viable human brain and liver mitochondria possess an active CypD-sensitive mPT. Our findings support the rationale of CypD and mPT inhibition as pharmacological targets in acute and chronic neurodegeneration.

Increased potassium conductance of brain mitochondria induces resistance to permeability transition by enhancing matrix volume.

Modulation of K+ conductance of the inner mitochondrial membrane has been proposed to mediate preconditioning in ischemia-reperfusion injury. The mechanism is not entirely understood, but it has been linked to a decreased activation of mitochondrial permeability transition (mPT). In the present study K+ channel activity was mimicked by picomolar concentrations of valinomycin. Isolated brain mitochondria were exposed to continuous infusions of calcium. Monitoring of extramitochondrial Ca2+ and mitochondrial respiration provided a quantitative assay for mPT sensitivity by determining calcium retention capacity (CRC). Valinomycin and cyclophilin D inhibition separately and additively increased CRC. Comparable degrees of respiratory uncoupling induced by increased K+ or H+ conductance had opposite effects on mPT sensitivity. Protonophores dose-dependently decreased CRC, demonstrating that so-called mild uncoupling was not beneficial per se. The putative mitoKATP channel opener diazoxide did not mimic the effect of valinomycin. An alkaline matrix pH was required for mitochondria to retain calcium, but increased K+ conductance did not result in augmented ΔpH. The beneficial effect of valinomycin on CRC was not mediated by H2O2-induced protein kinase Cϵ activation. Rather, increased K+ conductance reduced H2O2 generation during calcium infusion. Lowering the osmolarity of the buffer induced an increase in mitochondrial volume and improved CRC similar to valinomycin without inducing uncoupling or otherwise affecting respiration. We propose that increased potassium conductance in brain mitochondria may cause a direct physiological effect on matrix volume inducing resistance to pathological calcium challenges.