Impact of the timing of metoprolol administration during STEMI on infarct size and ventricular function

Pre-reperfusion administration of intravenous (IV) metoprolol reduces infarct size in ST-segment elevation myocardial infarction (STEMI). This study sought to determine how this cardioprotective effect is influenced by the timing of metoprolol therapy having either a long or short metoprolol bolus-to-reperfusion interval. We performed a post hoc analysis of the METOCARD-CNIC (effect of METOprolol of CARDioproteCtioN during an acute myocardial InfarCtion) trial, which randomized anterior STEMI patients to IV metoprolol or control before mechanical reperfusion. Treated patients were divided into short- and long-interval groups, split by the median time from 15 mg metoprolol bolus to reperfusion. We also performed a controlled validation study in 51 pigs subjected to 45 min ischemia/reperfusion. Pigs were allocated to IV metoprolol with a long (−25 min) or short (−5 min) pre-perfusion interval, IV metoprolol post-reperfusion (+60 min), or IV vehicle. Cardiac magnetic resonance (CMR) was performed in the acute and chronic phases in both clinical and experimental settings. For 218 patients (105 receiving IV metoprolol), the median time from 15 mg metoprolol bolus to reperfusion was 53 min. Compared with patients in the short-interval group, those with longer metoprolol exposure had smaller infarcts (22.9 g vs. 28.1 g; p = 0.06) and higher left ventricular ejection fraction (LVEF) (48.3% vs. 43.9%; p = 0.019) on day 5 CMR. These differences occurred despite total ischemic time being significantly longer in the long-interval group (214 min vs. 160 min; p < 0.001). There was no between-group difference in the time from symptom onset to metoprolol bolus. In the animal study, the long-interval group (IV metoprolol 25 min before reperfusion) had the smallest infarcts (day 7 CMR) and highest long-term LVEF (day 45 CMR). In anterior STEMI patients undergoing primary angioplasty, the sooner IV metoprolol is administered in the course of infarction, the smaller the infarct and the higher the LVEF. These hypothesis-generating clinical data are supported by a dedicated experimental large animal study.

Bcl-X(L) translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

BACKGROUND: The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X(L), pro-apoptotic Bax and Bad). METHODS: Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (Western immunoblots, densitometry, immunoelectron microscopy). RESULTS: Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X(L) and Bax, but not Bcl-2 or Bad, was identified in control distal cells. Bcl-X(L) and Bax had nonsignificant increases (P> 0.05) in these cells. Bcl-2, Bax, and Bcl-X(L), but not Bad, were endogenously expressed in control proximal cells. Bcl-X(L) was significantly decreased in treated proximal cultures (P < 0.05), with Bax and Bcl-2 having nonsignificant increases (P> 0.05). Immunoelectron microscopy localization indicated that control and treated but surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X(L) from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-X(L) expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. CONCLUSION: The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X(L) in proximal cells, as well as translocation of Bcl-X(L) protein to mitochondria within the surviving distal cells.

Dynamic, invivo, real-time detection of retinal oxidative status in a model of elevated intraocular pressure using a novel, reversibly responsive, profluorescent nitroxide probe

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders including cancer, Ischemia-reperfusion (I/R) injury and neurodegeneration. In times of metabolic stress e.g. ischaemia/reperfusion, reactive oxygen species (ROS) production overwhelms the intrinsic antioxidant capacity of the cell, damaging vital cellular components. The ability to quantify ROS changes in vivo, is therefore essential to understanding their biological role. Here we evaluate the suitability of a novel reversible profluorescent probe containing a redox-sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN), as an in vivo, real-time reporter of retinal oxidative status. The reversible nature of the probe's response offers the unique advantage of being able to monitor redox changes in both oxidizing and reducing directions in real time. After intravitreal administration of the ME-TRN probe, we induced ROS production in rat retina using an established model of complete, acute retinal ischaemia followed by reperfusion. After restoration of blood flow, retinas were imaged using a Micron III rodent fundus fluorescence imaging system, to quantify the redox-response of the probe. Fluorescent intensity declined during the first 60 min of reperfusion. The ROS-induced change in probe fluorescence was ameliorated with the retinal antioxidant, lutein. Fluorescence intensity in non-Ischemia eyes did not change significantly. This new probe and imaging technology provide a reversible and real-time response to oxidative changes and may allow the in vivo testing of antioxidant therapies of potential benefit to a range of diseases linked to oxidative stress

Role of thrombin in coronary artery bypass grafting

Thrombin is a multifunctional protease, which has a central role in the development and progression of coronary atherosclerotic lesions and it is a possible mediator of myocardial ischemia-reperfusion injury. Its generation and procoagulant activity are greatly upregulated during cardiopulmonary bypass (CPB). On the other hand, activated protein C, a physiologic anticoagulant that is activated by thrombomodulin-bound thrombin, has been beneficial in various models of ischemia-reperfusion. Therefore, our aim in this study was to test whether thrombin generation or protein C activation during coronary artery bypass grafting (CABG) associate with postoperative myocardial damage or hemodynamic changes. To further investigate the regulation of thrombin during CABG, we tested whether preoperative thrombophilic factors associate with increased CPB-related generation of thrombin or its procoagulant activity. We also measured the anticoagulant effects of heparin during CPB with a novel coagulation test, prothrombinase-induced clotting time (PiCT), and compared the performance of this test with the present standard of laboratory-based anticoagulation monitoring. One hundred patients undergoing elective on-pump CABG were studied prospectively. A progressive increase in markers of thrombin generation (F1+2), fibrinolysis (D-dimer), and fibrin formation (soluble fibrin monomer complexes) was observed during CPB, which was further distinctly propagated by reperfusion after myocardial ischemia, and continued to peak after the neutralization of heparin with protamine. Thrombin generation during reperfusion after CABG associated with postoperative myocardial damage and increased pulmonary vascular resistance. Activated protein C levels increased only slightly during CPB before the release of the aortic clamp, but reperfusion and more significantly heparin neutralization caused a massive increase in activated protein C levels. Protein C activation was clearly delayed in relation to both thrombin generation and fibrin formation. Even though activated protein C associated dynamically with postoperative hemodynamic performance, it did not associate with postoperative myocardial damage. Preoperative thrombophilic variables did not associate with perioperative thrombin generation or its procoagulant activity. Therefore, our results do not favor routine thrombophilia screening before CABG. There was poor agreement between PiCT and other measurements of heparin effects in the setting of CPB. However, lower heparin levels during CPB associated with inferior thrombin control and high heparin levels during CPB associated with fewer perioperative transfusions of blood products. Overall, our results suggest that hypercoagulation after CABG, especially during reperfusion, might be clinically important.

Treatment of illicit opioid and γ-hydroxybutyrate overdose by Helsinki emergency medical services

Aims: To examine the characteristics, incidence, treatment and outcome of presumed opioid, γ-hydroxybutyrate (GHB) and γ-butyrolactone (GBL) overdoses involving users of illicit drugs in Helsinki. GHB/GBL were included in this study, despite not being opioids, due to the relative ease with which they can cause potentially fatal respiratory depression. The incidence and time interval of recurrent opioid toxicity after prehospital administration of naloxone, an opioid antagonist, was studied in presumed heroin overdose patients. Naloxone has been reported to have many adverse effects and the effects of naloxone administered during an opioid overdose on the cardiovascular system and catecholamine levels in piglets were studied. Materials and methods: Patients included in these published retrospective studies were from the following time periods: Study I: 1995-2002, II: 1997-2000, III: 1995-2000, V: 2006-2007. Presumed opioid overdose patients were examined in studies I, II and III. GHB/GBL overdoses among injecting drug users was examined in study V. Recurrent opioid toxicity after prehospital naloxone administration in heroin overdose patients was examined in study III. The effects of naloxone (80 μg/kg i.v.) on the cardiovascular system and catecholamine levels administered during morphine overdose (8mg/kg i.v.) and under propofol anesthesia with spontaneous breathing were studied in eight piglets (IV). In this thesis, previously unpublished data on the incidence of opioid overdose between 2001-2007 and comparison of the characteristics of buprenorphine and heroin overdose patients encountered in 1995-2005 are also included. Results: Helsinki Emergency Medical Service (EMS) ambulances were dispatched annually to 34,153- 45,118 calls from 1995 to 2007. Of them, 7-8% were coded as intoxications or overdoses. During this time, 436 patients were treated by the EMS for presumed opioid overdose. The peak incidence of opioid overdoses was in the year 2000 (113 cases), after which they declined to 6-26 cases annually. The annual incidence of buprenorphine related overdoses increased from 4 (4% of opioid overdoses) in the year 2000 to 8 (30% of opioid overdoses) in 2007. The annual number of GHB related overdose patients treated by Helsinki EMS increased from 21 to 73 between 2004-2007. There appeared to be a peak in the incidence of both GHB/GBL and opioid related overdoses on Saturdays. Characteristics of opioid overdose patients The median age of opioid overdose patients was 28 years (22;33, 25- and 75-percentiles), and 84% were male. Buprenorphine overdose patients had more polydrug, such as alcohol and/or benzodiazepines, use in comparison with heroin overdose patients, 70% versus 33%, respectively. Severe respiratory depression was reported less often with buprenorphine overdoses compared to heroin overdoses, in 67.0% versus 85.4%, respectively. Outcome of heroin overdose patients with cardiac arrest Ninety four patients suffered cardiac arrest due to acute drug poisoning/overdose and were thus considered for resuscitation. Resuscitation was attempted in 72 cases. Cardiac arrest was caused by heroin overdose for 19 patients of which three (16%) were discharged alive. Other agents also induced cardiac arrest in 53 patients, of which six (11%) were discharged alive. The arrest was either EMS witnessed or occurring after the emergency call for all survivors of heroin induced cardiac arrest. Characteristics of GHB/GBL overdose patients The records of 100 GHB/GBL related overdose patients from 2006-2007 were retrieved. The median age of GHB/GBL overdose patients encountered on weekend nights was 24 years (22;27, 25- and 75-percentiles) and 49% were male. Polydrug use was reported in 62-80% of the cases. Thirty nine patients were encountered on Friday-Saturday or Saturday-Sunday night between 11 pm-6 am. The remaining sixty one patients were outside this time frame. There was a statistically significant difference between these two groups in history of chronic injecting drug use (33% vs. 59%, respectively, p=0.012). Recurrent heroin toxicity after prehospital naloxone administration Study III included 145 presumed heroin overdose patients. After prehospital care, 84 patients refused further care and were not transported to an Emergency Department (ED). Seventy one (85%) of them were administered naloxone by the EMS. During a 12-h follow up period, none of these patients developed severe recurrent opioid toxicity. The remaining 61 patients were transported to an ED. Prior to transportation, 52 (85%) patients were administered naloxone by the EMS. Fifteen of them were administered naloxone also in the ED and recurrent opioid toxicity was evident either on arrival at the ED or shortly thereafter. Prehospital naloxone was administered either intravenously, intramuscularly (i.m.) or subcutaneously (s.c.). There was a tendency for more frequent recurrent heroin toxicity among the patients with only intravenous administration of prehospital naloxone (13/36) compared with the patients with intramuscular or subcutaneous prehospital naloxone (2/16), p=0.106. The effects of naloxone on the cardiovascular system and catecholamine levels in piglets The administration of morphine to piglets resulted in an obvious respiratory depression, which was reversed by naloxone. Two severely hypoxemic piglets developed cardiac arrest after naloxone administration. In the other six animals, the administration of naloxone did not provoke arrhythmias, cardiac ischemia or visible evidence of pulmonary edema. There was a statistically significant (p=0.012) increase in norepinephrine levels after morphine administration and before naloxone administration: from 1.9 (1.3-2.3) ng/ml at baseline, to 31.7 (8.3-83.0) ng/ml (median, 25 and 75 percentiles parentheses) after morphine administration. After the administration of naloxone, the catecholamine levels continued to increase in only one of the animals. Conclusions: The incidence of buprenorphine related overdoses increased during the study period, but was still lower in comparison to those involving heroin. Injecting drug users have also started to use GHB/GBL. While recreational drug users use GHB/GBL during weekend nights, a GHB/GBL overdose patient encounter during weekdays has a more probable history of injecting drug use. Patients with cardiac arrest after heroin overdose have a poor prognosis. It appears to be safe to leave heroin overdose patients on scene after prehospital treatment with naloxone. Although no statistically significant difference was observed, it seems prudent to administer part of the total naloxone dose s.c. or i.m. to reduce the risk of recurrent respiratory depression. If transported to an ED, an observation period of one to two hours after the last naloxone dose seems adequate. The treating physician must be vigilant, however, due to the high prevalence of polydrug use and high morbidity after non fatal heroin overdose. Furthermore, care should be taken regarding possible chronic disorders and drug rehabilitation should be addressed. In the experimental animal study, two animals developed cardiac arrest after receiving naloxone while in hypoxemia and bradycardia. Further studies are required to assess the effect of naloxone during opioid-induced hypercapnia and hypoxemia in animals addicted to opioids.

Papel dos receptores β-adrenérgicos no reparo cutâneo de lesões crônicas em camundongos: modelo não invasivo de lesão por isquemia e reperfusão

Os receptores β1- e β2-adrenérgicos estão presentes em inúmeras células que participam do processo de reparo como fibroblastos, queratinócitos, células inflamatórias e células endoteliais. Diversos trabalhos demonstram que estes receptores modulam o processo de reparo tecidual. Entretanto, nenhum trabalho demonstrou se o bloqueio destes receptores compromete o reparo de úlceras de pressão. O objetivo deste estudo foi avaliar o efeito do bloqueio dos receptores β1- e β2-adrenérgicos no reparo de úlceras de pressão em camundongos, para isto utilizamos um modelo não invasivo de lesão por isquemia e reperfusão. No presente estudo, utilizamos animais que foram tratados por gavagem com propranolol (um antagonista não seletivo dos receptores β1- e β2-adrenérgicos). A administração do antagonista teve início um dia antes do início dos ciclos de isquemia e reperfusão e se manteve diariamente até a eutanásia. Para desenvolver a úlcera de pressão, um par de magnetos foi aplicado no dorso dos animais previamente depilado. O período de permanência do magneto é caracterizado como período de isquemia, enquanto sua retirada é caracterizada como período de reperfusão. Os ciclos de isquemia e reperfusão foram repetidos duas vezes, e ao final do último ciclo, duas úlceras circulares foram criadas no dorso dos animais. Os animais foram mortos 3, 7, 14 e 21 dias após a lesão. Após o último ciclo de isquemia, o fluxo sanguíneo da área comprimida foi nulo, 7 horas após a compressão o fluxo sanguíneo estava elevado, com níveis superiores ao da pele normal. Após 24 e 48 horas, o fluxo sanguíneo estava reduzido e abaixo dos níveis da pele normal. O bloqueio dos receptores β1- e β2-adrenérgicos aumentou os níveis de peróxidos lipídicos 3 dias após a lesão, comprometeu a migração dos queratinócitos, levando a um aumento da proliferação epitelial, resultando em uma re-epitelização atrasada. O retardo na formação da neo-epiderme induzido pelo bloqueio destes receptores prejudicou a remoção do tecido necrótico. O bloqueio dos receptores β1- e β2-adrenérgicos aumentou o número de células inflamatórias (neutrófilos e macrófagos), os níveis proteicos de elastase neutrofílica 3 dias após a lesão e reduziu os níveis proteicos de MCP-1 3 dias após a lesão e os níveis proteicos de MMP-12 7 dias após a lesão. O bloqueio dos receptores β1- e β2-adrenérgicos aumentou a proliferação celular e apoptose no tecido conjuntivo 7 dias após a lesão e aumentou a densidade de vasos sanguíneos 14 e 21 dias após a lesão. O bloqueio dos receptores β1- e β2-adrenérgicos retardou a diferenciação miofibroblástica e reduziu os níveis proteicos de TGF-β 1/2/3 3 dias após a lesão e a contração da lesão. Vinte e um dias após a lesão, o bloqueio dos receptores β1- e β2-adrenérgicos aumentou a espessura da neo-epiderme e a expressão de tenascina-C em fibroblastos e reduziu a deposição de colágeno. Em conclusão, o bloqueio dos receptores β1- e β2-adrenérgicos atrasa o reparo tecidual em úlceras de pressão.

Efeito do pré-condicionamento isquêmico remoto em modelo de lesão hepática por isquemia-reperfusão em ratos

A lesão por isquemia-reperfusão (I/R) é o mecanismo fisiopatológico central no desenvolvimento da insuficiência hepática pós-operatória. Diversas estratégias para minimizar suas consequências estão sendo desenvolvidas, mas ainda sem resultados satisfatórios. Recentemente o pré-condicionamento isquêmico remoto (PCIR), método em que ciclos breves de I/R aplicados em um órgão ou membro é capaz de atenuar os resultados da I/R em um órgão distante, vem sendo utilizado, em modelos experimentais, com resultados promissores. No entanto seu mecanismo de ação ainda não foi esclarecido. Um dos mecanismos propostos é a modulação na expressão das citocinas sintetizadas durante a resposta inflamatória que acompanha o processo de I/R. Foram utilizados 36 ratos (Rattus norvegicus), machos, com peso entre 250 e 280 g, divididos em três grupos: Grupo Sham, cirurgia simulada; Grupo IR, isquemia de 70% do fígado por 45 minutos e reperfusão; e Grupo PCIR, pré-condicionamento isquêmico remoto do fígado através de seis ciclos de isquemia-reperfusão da pata do animal, com quatro minutos de isquemia e quatro minutos de reperfusão em cada ciclo, seguido de isquemia hepática semelhante ao do Grupo IR. Terminado os procedimentos cirúrgicos, metade dos animais foi morta decorridos 60 minutos de reperfusão, e a outra metade após 180 minutos. Foi coletado tecido hepático do lobo submetido à isquemia, para estudo histopatológico, utilizando o índice de injúria hepática modificado; e sangue, para dosagem plasmática de TNF-α, IL-6, IL-10 e ALT. A análise histopatológica mostrou que a necrose celular foi significativamente reduzida no Grupo PCIR quando comparado com Grupo IR (p <0,0001). As transaminases mostraram o mesmo padrão com redução significativa dos seus valores no Grupo PCIR quando comparados com o Grupo I-R (p <0,0001). A dosagem das interleucinas mostrou redução significativa na expressão da IL-6 no Grupo PCIR quando comparado com o Grupo IR (p<0,001). Houve aumento da expressão de IL-10 nos grupo PCIR, porém não atingiu significância estatística. Não foi identificada diferença na dosagem de TNF-α nos grupos estudados. O PCI-R foi eficaz na redução na necrose celular resultante da lesão por I-R nos grupos estudados. A redução na síntese de IL-6 segue o padrão observado em outros estudos.

Efeitos de modificação dietética associada ou não a uma rotina de exercícios aeróbicos na microcirculação em modelo animal de obesidade exógena

A obesidade atinge proporções epidêmicas em países industrializados e está relacionada a uma série de doenças metabólicas e circulatórias. Nesse contexto, a atividade física, tratamento não farmacológico da obesidade, acessível a diversas populações e está relacionada com a redução do risco cardiovasvascular mesmo. O objetivo deste trabalho foi avaliar, após mudança ou não da dieta, associação ou não a um programa de treinamento aeróbico (PTA) durante 8 semanas, a possível reversibilidade dos danos causados por uma dieta hiperlipídica por 12 semanas. Para tal, 120 hamsters machos da espécie Mesocricetus auratus, com massa corporal de 60 g, foram distribuídos em quatro grupos, cada um subdividido em três subgrupos, com dez animais para diferentes análises. Os grupos Obeso Controle (OBC) e Obeso Exercitado (OBEX) receberam a ração hiperlipídica por 20 semanas, com adição do PTA ao grupo OBEX nas últimas 8 semanas. Os Obeso Ração Padrão (OBRP) e Obeso Ração Padrão/Exercício (OBRP/EX) tiveram a ração modificada para comercial padrão e adição do PTA ao grupo OBRP/EX após as 12 semanas iniciais. Para as análises microcirculatórias, a bolsa da bochecha foi usada para determinação do número máximo de extravasamentos induzidos por 30 min de isquemia seguida de reperfusão e da reatividade microvascular após a aplicação tópica de acetilcolina e nitroprussiato de sódio. No sangue coletado foi avaliado o perfil lipídico, glicemias quinzenais e leptina. As expressões de eNOS e iNOS foram determinadas na aorta por imunoblotting e a composição corporal avaliada nos tecidos adiposos visceral, urogenital e retroperitoneal, retirados no dia do experimento. Os resultados foram analisados com os métodos o teste estatístico de análise de variância (One Way ANOVA - Teste de Kruskal-Wallis), seguido pelo pós-teste de Dunn. Resultados mostram que a modificação dietética, associada ou não ao PTA, reduziu significativamente a massa corporal (p<0,0001), comprimento naso-anal (p=0,0011) e tecido adiposo (visceral [p<0,0001], urogenital [p=0.0004] e retroperitoneal [p= 0,0083]). Nas análises sanguíneas não foram encontradas diferenças com relação ao perfil lipídico e glicemia, já na leptina houve uma redução significativa (p=0,0039). A análise da reatividade microvascular mostrou melhora significativa na vasodilatação endotélio-dependente nos grupos submetidos à modificação dietética associada ou não ao PTA. Nas medidas de permeabilidade a macromoléculas houve redução significativa no número de extravasamentos nos grupos submetidos à modificação dietética associada ou não ao PTA, (5 min [p= 0,0207] e 10 min [p= 0,0057]). Houve um aumento na expressão de eNOS nos grupos submetidos à modificação dietética associada ou não ao PTA, em comparação ao grupo OBC (p=0,0352). Os resultados mostraram que a modificação dietética, associada ao protocolo de treinamento aeróbico melhora a vasodilatação endotélio-dependente, aumenta a expressão da óxido nítrico sintase endotelial e reduz o número de extravasamentos induzidos por isquemia e reperfusão, mesmo sem melhoras nos marcadores bioquímicos tradicionais como glicemia e perfil lipídico.

Avaliação dos testículos de ratos submetidos à torção testicular antes, durante e após a puberdade, e o efeito do tratamento com L-arginina

Torção testicular (TT) é uma síndrome urológica comumente encontrada em recém nascidos, crianças e adolescentes. Neste trabalho foi estudada as lesões morfológicas e a função reprodutiva em ratos adultos que sofreram TT, em diferentes idades de maturidade sexual e o efeito protetor da L-arginina, contra os danos causados pela isquemia/reperfusão na torção testicular. Dezoito ratos pré-púberes (4 semanas de vida) dezessete púberes (6 semanas de vida) e dezessete adultos (9 semanas de vida) foram submetidos à TT. Sob anestesia o testículo direito foi rotacionado em 720 e fixado, sendo então destorcido após 4 horas. Vinte e quatro ratos (ARG4, n=8, ARG6, n=8 e ARG9 n=8) foram submetidos ao tratamento com 650mg/kg de L-arginina, por via oral, durante 7 dias. Outros trinta ratos de mesma idade sofreram cirurgia simulada (SH4, n=10, SH6, n=10 e SH9, n=10). Com 12 semanas de idade, foram submetidos ao acasalamento controlado com 3 fêmeas e ao vigésimo dia de gestação o número de fetos, corpos lúteos, absorções e implatações foram contados. Na 14 semana, os ratos foram mortos e os espermatozóides coletados da cauda dos epidídimos, foi anotado o peso corporal, o peso e volume testicular. O soro foi usado para dosagem de testosterona. Foram avaliadas a concentração, motilidade e a viabilidade espermática. Os testículos coletados foram fixados em Bouin, pós-fixados em formalina e processados em parafina. Utilizando o programa de imagem Image J, lâminas coradas com HE, mensuramos a altura do epitélio, densidade volumétrica e diâmetro do túbulo seminífero. Para avaliar a integridade do epitélio seminífero foi utilizado a frequência dos estágios do ciclo do epitélio e o escorre de Johnsen. Também foi avaliado a proliferação do compartimento tubular e intertubular, através da imunomarcação com PCNA. Os dados foram tabulados e as médias dos grupos comparadas pelo teste de ANOVA com pós-teste de Bonferroni ou teste de Kruskal-Wallis com pós teste de Dunns (programa Graphpad Prism, com p < 0,05). Os resultados revelaram grandes danos produzidos pela injuria testicular, no testículo ipsilateral, com diminuição da capacidade reprodutiva, da concentração, viabilidade e mobilidade, do peso e volume testicular. Animais TT9 não apresentaram espermatozoides nas amostras coletas. Houve diminuição do diâmetro dos túbulos seminíferos e da altura do epitélio, aumento do compartimento intertubular, com ênfase dos vasos sanguíneos, aumento da proliferação celular estromal e diminuição da proliferação epitelial. Houve diminuição da concentração sérica de testosterona no grupo TT4, quando comparado como grupo TT9. Em geral, os animais submetidos à torção na fase adulta foram os mais acometidos. Não foram encontradas alterações dignas de nota, nos testículo contralaterais. Animais tratados com L-arginina obtiveram melhora dos índices reprodutivos, com aumento da potência em todas as idades. Houve aumento da concentração espermática no testículo contralateral de ARG4 e ARG6, mostrando que a L-arginina atuou como antioxidante. Não houve proteção para as lesões causadas pela torção na maioria dos grupos, mas os animais tratados ARG9 apresentaram concentração espermática mensuravel, quando comparados aos ratos TT9, que tinham azospermia.

Avaliação estereológica glomerular em rins de ratos após isquemia-reperfusão por clampeamento arteriovenoso e arterial

A técnica de isquemia-reperfusão tem sido utilizada em cirurgias conservadoras do rim como a nefrectomia parcial e em transplantes renais. Para se realizar a isquemia pode-se fazer o bloqueio do fluxo sanguíneo da artéria renal ou o bloqueio simultâneo da artéria e da veia renal. O evento isquêmico acarreta em dano celular ao rim principalmente pelo estresse oxidativo local e a liberação de radicais livres assim como o aumento da resposta inflamatória. Diversos autores verificaram lesão renal após a isquemia-reperfusão, porém, apenas testes funcionais foram realizados até o momento. Os autores que tentaram avaliar a lesão morfológica do rim apenas fizeram a quantificação de escores subjetivos. O nosso objetivo é avaliar por quantificação estereológica o dano causado pela isquemia-reperfusão comparando o clampeamento somente arterial com o clampeamento arteriovenoso. Utilizamos 24 ratos wistar, machos, de quatro meses de idade. Os animais foram divididos em três grupos: o grupo Sham (n=8), o grupo de clampeamento somente da artéria renal (n=8) e o grupo de clampeamento simultâneo da artéria e da veia renal (n=8). Os animais foram submetidos a laparotomia mediana. Os animais do grupo Sham permaneceram os 60 minutos anestesiados mas sem obstrução do fluxo sanguíneo de seus vasos renais. Os animais do grupo de clampeamento arterial foram submetidos à clampeamento de sua arterial renal esquerda por 60 minutos e os animais do grupo de clampeamento arterial e venoso tiveram seus vasos renais esquerdos clampeados simultaneamente e em bloco pelo mesmo tempo. Após os 60 minutos os animais foram suturados e mantidos por 30 dias em caixas próprias sendo mortos por sobredose anestésica após decorrido esse tempo. Os rins foram coletados e mantidos em solução de formalina tamponada e posteriormente processados para análise histológica e estereológica. Foram analisados a densidade volumétrica (Vv) dos glomérulos, o número de glomérulos/mm3(Nv) e o volume glomerular médio (VGM). A Vv e Nv se encontrou reduzida nos rins esquerdos submetidos à isquemia mas foi somente significativa nos animais do grupo de clampeamento arterial e venoso. Mesmo usando o rato como modelo animal experimental, a partir de nossos resultados recomendamos o uso do clampeamento somente arterial nos casos em que mínina lesão ao rim é imperiosa.

Effects of aniracetam on extracellular levels of transmitter amino acids in the hippocampus of the conscious gerbils: an intracranial microdialysis study

The effects of aniracetam on extracellular amino acid levels in the hippocampus of conscious gerbils, with or without transient cerebral ischemia/reperfusion, were measured by microdialysis and reverse phase-high performance liquid chromatography. Increased extracellular levels of aspartate and glutamate that were observed in the hippocampus of conscious gerbils during transient global forebrain ischemia were reversed by aniracetam. In contrast, the level of extracellular gamma-aminobutyric acid was increased, while taurine was maintained at a higher level than other amino acids by administration of aniracetam (100 mg/kg, p.o.) 60 min before ischemia. Further, in contrast to ischemic animals, administration of aniracetam (100 mg/kg, p.o.) enhanced the release of glutamate and aspartate in the normal gerbil hippocampus. The results suggest that these effects might be due to a partial calcium agonist activity of aniracetam, and that the effects of aniracetam on amino acid levels might be a mechanism of protection against delayed neuronal death in the ischemic hippocampus, thereby improving memory dysfunction induced by ischemia/reperfusion. (C) 2003 Elsevier Science Ireland Ltd. All rights reserved.

C-Kit(+) Cells Isolated from Developing Kidneys Are a Novel Population of Stem Cells with Regenerative Potential

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit(+) cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit(+) cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex vivo expanded c-kit(+) cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together, these findings document a novel neonatal rat kidney c-kit(+) stem cell population that can be isolated, expanded, cloned, differentiated, and used for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. STEM Cells 2013;31:1644-1656

Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential.

Oxidative stress has become widely viewed as an underlying condition in a number of diseases, such as ischemia-reperfusion disorders, central nervous system disorders, cardiovascular conditions, cancer, and diabetes. Thus, natural and synthetic antioxidants have been actively sought. Superoxide dismutase is a first line of defense against oxidative stress under physiological and pathological conditions. Therefore, the development of therapeutics aimed at mimicking superoxide dismutase was a natural maneuver. Metalloporphyrins, as well as Mn cyclic polyamines, Mn salen derivatives and nitroxides were all originally developed as SOD mimics. The same thermodynamic and electrostatic properties that make them potent SOD mimics may allow them to reduce other reactive species such as peroxynitrite, peroxynitrite-derived CO(3)(*-), peroxyl radical, and less efficiently H(2)O(2). By doing so SOD mimics can decrease both primary and secondary oxidative events, the latter arising from the inhibition of cellular transcriptional activity. To better judge the therapeutic potential and the advantage of one over the other type of compound, comparative studies of different classes of drugs in the same cellular and/or animal models are needed. We here provide a comprehensive overview of the chemical properties and some in vivo effects observed with various classes of compounds with a special emphasis on porphyrin-based compounds.

Smartphone ECG for evaluation of STEMI: Results of the ST LEUIS Pilot Study

© 2015 Elsevier Inc. All rights reserved.Background 12-lead ECG is a critical component of initial evaluation of cardiac ischemia, but has traditionally been limited to large, dedicated equipment in medical care environments. Smartphones provide a potential alternative platform for the extension of ECG to new care settings and to improve timeliness of care. Objective To gain experience with smartphone electrocardiography prior to designing a larger multicenter study evaluating standard 12-lead ECG compared to smartphone ECG. Methods 6 patients for whom the hospital STEMI protocol was activated were evaluated with traditional 12-lead ECG followed immediately by a smartphone ECG using right (VnR) and left (VnL) limb leads for precordial grounding. The AliveCor™ Heart Monitor was utilized for this study. All tracings were taken prior to catheterization or immediately after revascularization while still in the catheterization laboratory. Results The smartphone ECG had excellent correlation with the gold standard 12-lead ECG in all patients. Four out of six tracings were judged to meet STEMI criteria on both modalities as determined by three experienced cardiologists, and in the remaining two, consensus indicated a non-STEMI ECG diagnosis. No significant difference was noted between VnR and VnL. Conclusions Smartphone based electrocardiography is a promising, developing technology intended to increase availability and speed of electrocardiographic evaluation. This study confirmed the potential of a smartphone ECG for evaluation of acute ischemia and the feasibility of studying this technology further to define the diagnostic accuracy, limitations and appropriate use of this new technology.

CARBON MONOXIDE AND PREGNANCY: A SEARCH FOR A POSSIBLE THERAPEUTIC IN THE TREATMENT OF PRE-ECLAMPSIA

Pre-eclampsia (PE) is a pregnancy disorder that affects roughly 5-7% of all pregnancies and is a leading cause of both maternal and fetal/neonatal morbidity and mortality. With no present cure for the disease, researchers are interested in the lower incidence of PE observed among the cigarette smoking pregnant population. However, women who use smokeless tobacco do not experience the same decreased incidence of PE, leading to hypothesis of protection against PE from the largest combustible product of cigarette smoke, carbon monoxide (CO). Studies evaluated levels of CO in PE women and found that they were statistically lower than those of healthy pregnancy. Researchers have found CO to possess many cytoprotective and regulatory properties and specifically within the placenta, it has been found to increase perfusion pressure, decrease oxidative stress, decreases ischemia/reperfusion induced apoptosis and maintain endothelial functioning. The idea for use of CO as a possible therapeutic for PE has thus become a real possibility. This study determined CO levels in pregnant women ± smoking as well as in PE women±smoking, as to discover a possible therapeutic range for future treatments. The best correlated automated CO measurement device with blood CO levels was determined, for use in future clinical studies. This thesis also sought a possible CO delivery concentration, in order to achieve the CO levels observed in the human correlation study. A threshold level of maternal CO exposure in a murine animal model was found, for which fetal and maternal negative toxicities were not observed. The results of this thesis lend a few more pieces to the complicated puzzle involving CO and PE and offer another step toward the possibility of a therapeutic treatment/prevention using this gaseous molecule.