Hypoxia-ischemia (HI) insults can have profound effects on the immature brain, impairing development and leaving survivors with lifelong physical and cognitive deficits. Improvements in neonatal care have resulted in more newborns surviving HI, but effective treatments for the long-term consequences of this disorder have yet to be established. Using the Rice-Vannucci model of hypoxia-ischemia at postnatal day (PND) 7, we investigated the effects of metformin and enriched rehabilitation on short and long-term motor and cognitive outcome in both male and female Sprague-Dawley rats. A battery of behavioural tests was used to assess early development and motor function from PND 8-21, while long-term motor and cognitive function was assessed from PND 49 onwards. Metformin, administered from PND 8-49, improved several aspects of early development that were compromised following HI (weight gain, neurological reflexes). However, it worsened motor impairments in the adhesive strip removal task and Montoya staircase. Enriched rehabilitation, beginning at PND 21, improved motor function in the adhesive strip removal task, open field and Montoya staircase. Additionally, it enhanced cognition in the Barnes maze and Morris water maze. Our results indicated that, despite early beneficial effects on development, metformin was not effective at improving long-term outcome. Enriched rehabilitation led to significant improvements in several aspects of motor and cognitive function, even when administered 2 weeks post-injury. This data suggests that enriched rehabilitation, but not metformin, may be a valuable intervention for treating behavioural impairments resulting from episodes of perinatal hypoxia-ischemia.

Made available in DSpace on 2014-10-09T12:33:46Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:04:36Z (GMT). No. of bitstreams: 0 / As células necessitam do contato com outras células e com a matriz extracelular, para a formação de tecidos. As junções gap são estreitos canais que conectam o citoplasma de células adjacentes, promovendo a passagem de íons orgânicos, aminoácidos, nucleotídeos e outros metabólitos. Estas junções são compostas por dois conexons ou hemicanais, que atravessam a membrana plasmática da célula a que pertencem, e são compostos por seis proteínas integrais de membrana denominadas conexinas (Cxs). A Cx43 é a mais expressa, e é fosforilada ao longo do ciclo de vida, sofrendo mudanças conformacionais, resultando em diferentes isoformas (P0, P1 e P2), apresentando propriedades distintas. A Cx43 apresenta-se distribuída em todo o rim adulto. A injúria renal aguda (IRA) é uma síndrome metabólica em que ocorre redução aguda da função renal e rápida diminuição da taxa de filtração glomerular, sendo hipóxia decorrente da isquemia sua causa principal. A restrição de oxigênio e nutrientes, e o acúmulo de metabólitos, resultam na injúria das células epiteliais tubulares. A depleção dos níveis de ATP, aumento nos níveis de cálcio intracelular, alterações na membrana e deformações no citoesqueleto caracterizam esta injúria. A reoxigenação tecidual atua como agressão adicional devido à liberação de radicais livres. Estudos sugerem que a ativação de hemicanais de Cx43, resultante da desfosforilação da proteína, durante depleção de ATP, esteja envolvida na IRA. Este trabalho verificou o envolvimento da Cx43 em modelo murino desta injúria, ocasionada por isquemia/reperfusão. Foram utilizados camundongos machos da linhagem C57BL/6J. A isquemia foi induzida por clampeamento das artérias renais por 45 minutos. A reperfusão ocorreu durante 24 horas após cirurgia. Foram utilizados 6 animais por grupo (isquêmicos, reperfundidos e controle). Após sacrifício, fragmentos dos rins foram submetidos a ensaios de western blot, PCR em tempo real, imuno-histoquímica e imunofluorescência. O modelo experimental foi validado através da dosagem de uréia e creatinina plasmática. As análises estatísticas foram realizadas pela análise de variância (ANOVA), seguido do teste de Bonferroni. Observou-se aumento significativo dos níveis de uréia e creatinina nos animais isquêmicos e reperfundidos, em relação ao controle. A expressão gênica apresentou aumento significativo apenas nos rins de camundongos reperfundidos (1,9 vezes; P<0,01 vs controle). No western blot verificou-se aumento na quantidade da isoforma hiperfosforilada da Cx43 (P2) em rins isquêmicos (2,73 vezes; P<0,05 vs controle), com diminuição significativa nos reperfundidos (2,37 vezes; P<0,05 vs isquêmico). Nas isoformas menos fosforiladas (P1/P0), observou-se aumento nos rins isquêmicos (2,33 vezes; P<0,05 vs controle), com diminuição nos reperfundidos (10 vezes; P<0,01 vs isquêmico). Nos ensaios imuno-histológicos verificou-se diferentes localizações da Cx43 nas células epiteliais de túbulos corticais nos grupos comparados. Nos controles verificou-se distribuição difusa, e nos isquêmicos observou-se intensa marcação em superfície celular apical. Nos rins reperfundidos, a distribuição da Cx43 foi basolateral. As alterações observadas na expressão gênica, fosforilação protéica e distribuição da Cx43 nos rins foram semelhantes às mudanças observadas na isquemia cardíaca. Este estudo mostrou pela primeira vez a regulação da Cx43 em níveis transcricionais e pós-traducionais, e sua localização celular na IRA ocasionada por isquemia/reperfusão, indicando sua participação neste processo. / Dissertação (Mestrado) / IPEN/D / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Background: Cerebral ischemia is a leading cause of death and disability with limited treatment options. Although inflammatory and immune responses participate in ischemic brain injury, the molecular regulators of neuroinflammation after ischemia remain to be defined. Translocator protein 18 kDa (TSPO) mainly localized to the mitochondrial outer membrane is predominantly expressed in glia within the central nervous system during inflammatory conditions. This study investigated the effect of a TSPO agonist, etifoxine, on neuroinflammation and brain injury after ischemia/reperfusion. Methods: We used a mouse model of middle cerebral artery occlusion (MCAO) to examine the therapeutic potential and mechanisms of neuroprotection by etifoxine. Results: TSPO was upregulated in Iba1(+) or CD11b(+) CD45(int) cells from mice subjected to MCAO and reperfusion. Etifoxine significantly attenuated neurodeficits and infarct volume after MCAO and reperfusion. The attenuation was pronounced in mice subjected to 30, 60, or 90 min MCAO. Etifoxine reduced production of pro-inflammatory factors in the ischemic brain. In addition, etifoxine treatment led to decreased expression of interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and inducible nitric oxide synthase by microglia. Notably, the benefit of etifoxine against brain infarction was ablated in mice depleted of microglia using a colony-stimulating factor 1 receptor inhibitor. Conclusions: These findings indicate that the TSPO agonist, etifoxine, reduces neuroinflammation and brain injury after ischemia/reperfusion. The therapeutic potential of targeting TSPO requires further investigations in ischemic stroke.

Toll-like receptors (TLRs) play a critical role in the induction of innate immune responses which have been implicated in neuronal death induced by global cerebral ischemia/reperfusion (GCI/R). The present study investigated the role and mechanisms-of-action of TLR4 signaling in ischemia-induced hippocampal neuronal death. Neuronal damage, activation of the TLR4 signaling pathway, expression of pro-inflammatory cytokines and activation of the PI3K/Akt signaling pathway in the hippocampal formation (HF) were assessed in wild type (WT) mice and TLR4 knockout (TLR4-/-) mice after GCI/R. GCI/R increased expression of TLR4 protein in the hippocampal formation (HF) and other brain structures in WT mice. Phosphorylation of the inhibitor of kappa B (p-Ik{cyrillic}B) as well as activation of nuclear factor kappa B (NFk{cyrillic}B) increased in the HF of WT mice. In contrast, there were lower levels of p-Ik{cyrillic}B and NFk{cyrillic}B binding activity in TLR4-/- mice subjected to GCI/R. Pro-inflammatory cytokine expression was also decreased, while phosphorylation of Akt and GSK3β were increased in the HF of TLR4-/- mice after GCI/R. These changes correlated with decreased neuronal death/apoptosis in TLR4-/- mice following GCI/R. These data suggest that activation of TLR4 signaling contributes to ischemia-induced hippocampal neuronal death. In addition, these data suggest that modulation of TLR4 signaling may attenuate ischemic injury in hippocampal neurons.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21415号 / 医博第4405号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 坂井 義治, 教授 川口 義弥 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Background - The role of protein phosphatases (PPs) during ischemic preconditioning in the rabbit heart was examined. Methods and Results - Fostriecin, a potent inhibitor of PP2A, was administered to isolated rabbit hearts starting either 15 minutes before or 10 minutes after the onset of a 30-minute period of regional ischemia and continuing until the onset of reperfusion. After 2 hours of reperfusion, infarct size was measured with triphenyltetrazolium chloride. In a second study with isolated rabbit cardiomyocytes, the effect of fostriecin pretreatment was assessed by measuring changes in cell osmotic fragility during simulated ischemia. PP1 and PP2A activities of isolated control and ischemically preconditioned cells were also measured. In a third series of experiments, left ventricular biopsies of isolated rabbit hearts were obtained before and at selected times during 60 minutes of global ischemia, and the tissue was assayed for PP1 and PP2A activities. In isolated hearts pretreated with fostriecin, only 8% of the ischemic zone infarcted, significantly less than that in untreated control hearts (33%; P<0.001) but comparable to that in ischemically preconditioned hearts (9%; P<0.001 versus control). Significant protection was also observed in the hearts treated only after the onset of ischemia (18% infarction; P<0.05 versus control). In isolated myocytes, fostriecin also provided protection comparable to that produced by metabolic preconditioning. Preconditioning had no apparent effect on the activity of either PP1 or PP2A in isolated ventricular myocytes or ventricular tissue obtained from heart biopsies. Conclusions - Fostriecin, a potent inhibitor of PP2A, can protect the rabbit heart from infarction even when administered after the onset of ischemia. But inhibition of either PP1 or PP2A does not appear to be the mechanism of protection from ischemic preconditioning.

No / Objectives Ischaemia modified albumin (IMA) has been used as a marker of myocardial ischaemia but little is known about its production during ischaemia of other tissues. The clinical models of patients with intermittent claudication and major arterial surgery were used to investigate IMA production from ischaemic skeletal muscle. Materials and methods IMA was measured pre-operatively, at end ischaemia, and 5min, 4, 24, 48, 72 and 144h post-surgery in patients undergoing (a) revascularisation for intermittent claudication (IC, n=15), (b) abdominal aortic aneurysm repair (AAA, n=12) and controls (n=16). Results The median pre-operative IMA concentration in IC patients was significantly higher than the AAA group (88.3 versus 83.5U/ml, p=0.036) and controls (88.3 versus 80.3U/ml, p=0.031). IMA concentrations increased significantly during arterial clamping in both IC and AAA groups (88.3 versus 120.0U/ml, p=0.001; 83.5 versus 118.8U/ml, p=0.002, respectively) consistent with increased skeletal muscle ischaemia. In contrast, there was only a mild perioperative increase in the controls (80.3 versus 91.6U/ml, p=0.012). Conclusions Patients with intermittent claudication have significantly elevated IMA and skeletal muscle ischaemia during arterial surgery results in significantly increased circulating IMA. When IMA is used to detect myocardial ischaemia, ischaemic skeletal muscle must be excluded.

Published Article / Magnesium is the second most abundant intracellular cation with many control and regulatory functions. It regulates energy production and utilization and modulates activity of membrane ionic channels. Magnesium has direct control effects on cardiac myocyte ion channels making it useful in certain arrhythmias. Calcium is responsible for pacemaker excitation and for excitation-contraction coupling in myocytes but increased intracellular calcium produces early and late afterdepolarisations initiating arrhythmias. Magnesium regulates calcium channel activity preventing raised intracellular levels. Potassium channel activity is enhanced by magnesium hyperpolarizing the cell reducing arrhythmia generation. Magnesium is effective against long QT Torsade de Pointes. In rapid atrial fibrillation magnesium produces rate control slowing AV nodal conduction. Magnesium prevents digitalis toxicity due to associated hypomagnesemia.

Persufflation (PSF; gaseous oxygen perfusion) is an organ preservation technique with a potential for use in donor heart preservation. Improved heart preservation with PSF may improve outcomes by maintaining cardiac tissue quality in the setting of longer cold ischemia times and possibly increasing the number of donor hearts available for allotransplant. Published data suggest that PSF is able to extend the cold storage times for porcine hearts up to 14 hours without compromising viability and function, and has been shown to resuscitate porcine hearts following donation after cardiac death. This review summarizes key published work on heart PSF, including prospective implications and future directions for PSF in heart transplantation. We emphasize the potential impact of extending preservation times and expanding donor selection criteria in heart allotransplant. Additionally, the key issues that need to be addressed before PSF were to become a widely utilized preservation strategy prior to clinical heart transplantation are summarized and discussed.

published_or_final_version / abstract / Physiology / Master / Master of Philosophy