The use of spreading depression waves for acute and long-term monitoring of the penumbra zone of focal ischemic damage in rats.

Slow potential recording was used for long-term monitoring of the penumbra zone surrounding an ischemic region produced by middle cerebral artery (MCA) occlusion in adult hooded rats (n = 32). Four capillary electrodes (El-E4) were chronically implanted at 2-mm intervals from AP -3, L 2 (El) to AP 0, L 5 (E4). Spontaneous or evoked slow potential waves of spreading depression (SD) were recorded during and 4 h after a 1-h MCA occlusion and at 2- to 3-day intervals afterward for 3 weeks. Duration of the initial focal ischemic depolarization was maximal at E4 and decreased with distance from the focus. SD waves in the penumbra zone were high at El and E2, low and prolonged at E3, and almost absent at E4. Amplitude of elicited SD waves was further reduced 3 days later and slowly increased in the following week. Cortical areas displaying marked reduction of SD waves in the first days after MCA occlusion either remained low or showed substantial (60%) recovery, the probability of which decreased with the duration of the initial focal ischemic depolarization and increased with the distance from the focus. It is concluded that the outcome of ischemia monitored by long-term SD recovery in the perifocal region can be partly predicted from the acute signs of MCA occlusion.

Cardioprotective effects of 70-kDa heat shock protein in transgenic mice.

Heat shock proteins are proposed to limit injury resulting from diverse environmental stresses, but direct metabolic evidence for such a cytoprotective function in vertebrates has been largely limited to studies of cultured cells. We generated lines of transgenic mice to express human 70-kDa heat shock protein constitutively in the myocardium. Hearts isolated from these animals demonstrated enhanced recovery of high energy phosphate stores and correction of metabolic acidosis following brief periods of global ischemia sufficient to induce sustained abnormalities of these variables in hearts from nontransgenic littermates. These data demonstrate a direct cardioprotective effect of 70-kDa heat shock protein to enhance postischemic recovery of the intact heart.

Discovery of adrenomedullin in rat ischemic cortex and evidence for its role in exacerbating focal brain ischemic damage.

Focal brain ischemia is the most common event leading to stroke in humans. To understand the molecular mechanisms associated with brain ischemia, we applied the technique of mRNA differential display and isolated a gene that encodes a recently discovered peptide, adrenomedullin (AM), which is a member of the calcitonin gene-related peptide (CGRP) family. Using the rat focal stroke model of middle cerebral artery occlusion (MCAO), we determined that AM mRNA expression was significantly increased in the ischemic cortex up to 17.4-fold at 3 h post-MCAO (P < 0.05) and 21.7-fold at 6 h post-MCAO (P < 0.05) and remained elevated for up to 15 days (9.6-fold increase; P < 0.05). Immunohistochemical studies localized AM to ischemic neuronal processes, and radioligand (125I-labeled CGRP) displacement revealed high-affinity (IC50 = 80.3 nmol) binding of AM to CGRP receptors in brain cortex. The cerebrovascular function of AM was studied using synthetic AM microinjected onto rat pial vessels using a cranial window or applied to canine basilar arteries in vitro. AM, applied abluminally, produced dose-dependent relaxation of preconstricted pial vessels (P < 0.05). Intracerebroventricular (but not systemic) AM administration at a high dose (8 nmol), prior to and after MCAO, increased the degree of focal ischemic injury (P < 0.05). The ischemia-induced expression of both AM mRNA and peptide in ischemic cortical neurons, the demonstration of the direct vasodilating effects of the peptide on cerebral vessels, and the ability of AM to exacerbate ischemic brain damage suggests that AM plays a significant role in focal ischemic brain injury.

Susceptibility of cloned K+ channels to reactive oxygen species.

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.

Blocking effects of polyunsaturated fatty acids on Na+ channels of neonatal rat ventricular myocytes.

Recent evidence indicates that polyunsaturated long-chain fatty acids (PUFAs) prevent lethal ischemia-induced cardiac arrhythmias in animals and probably in humans. To increase understanding of the mechanism(s) of this phenomenon, the effects of PUFAs on Na+ currents were assessed by the whole-cell patch-clamp technique in cultured neonatal rat ventricular myocytes. Extracellular application of the free 5,8,11,14,17-eicosapentaenoic acid (EPA) produced a concentration-dependent suppression of ventricular, voltage-activated Na+ currents (INa). After cardiac myocytes were treated with 5 or 10 microM EPA, the peak INa (elicited by a single-step voltage change with pulses from -80 to -30 mV) was decreased by 51% +/- 8% (P < 0.01; n = 10) and 64% +/- 5% (P < 0.001; n = 21), respectively, within 2 min. Likewise, the same concentrations of 4,7,10,16,19-docosahexaenoic acid produced the same inhibition of INa. By contrast, 5 and 10 microM arachidonic acid (AA) caused less inhibition of INa, but both n - 6 and n - 3 PUFAs inhibited INa significantly. A monounsaturated fatty acid and a saturated fatty acid did not. After washing out EPA, INa returned to the control level. Raising the concentration of EPA to 40 microM completely blocked INa. The IC50 of EPA was 4.8 microM. The inhibition of this Na+ channel was found to be dose and time, but not use dependent. Also, the EPA-induced inhibition of INa was voltage dependent, since 10 microM EPA produced 83% +/- 7% and 29% +/- 5% inhibition of INa elicited by pulses from -80 to -30 mV and from -150 to -30 mV, respectively, in single-step voltage changes. A concentration of 10 microM EPA shifted the steady-state inactivation curve of INa by -19 +/- 3 mV (n = 7; P < 0.01). These effects of PUFAs on INa may be important for their antiarrhythmic effect in vivo.

Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.

The majority of severe visual loss in the United States results from complications associated with retinal neovascularization in patients with ischemic ocular diseases such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. Intraocular expression of the angiogenic protein vascular endothelial growth factor (VEGF) is closely correlated with neovascularization in these human disorders and with ischemia-induced retinal neovascularization in mice. In this study, we evaluated whether in vivo inhibition of VEGF action could suppress retinal neovascularization in a murine model of ischemic retinopathy. VEGF-neutralizing chimeric proteins were constructed by joining the extracellular domain of either human (Flt) or mouse (Flk) high-affinity VEGF receptors with IgG. Control chimeric proteins that did not bind VEGF were also used. VEGF-receptor chimeric proteins eliminated in vitro retinal endothelial cell growth stimulation by either VEGF (P < 0.006) or hypoxic conditioned medium (P < 0.005) without affecting growth under nonstimulated conditions. Control proteins had no effect. To assess in vivo response, animals with bilateral retinal ischemia received intravitreal injections of VEGF antagonist in one eye and control protein in the contralateral eye. Retinal neovascularization was quantitated histologically by a masked protocol. Retinal neovascularization in the eye injected with human Flt or murine Flk chimeric protein was reduced in 100% (25/25; P < 0.0001) and 95% (21/22; P < 0.0001) 0.0001) of animals, respectively, compared to the control treated eye. This response was evident after only a single intravitreal injection and was dose dependent with suppression of neovascularization noted after total delivery of 200 ng of protein (P < 0.002). Reduction of histologically evident neovascular nuclei per 6-microns section averaged 47% +/- 4% (P < 0.001) and 37% +/- 2% (P < 0.001) for Flt and Flk chimeric proteins with maximal inhibitory effects of 77% and 66%, respectively. No retinal toxicity was observed by light microscopy. These data demonstrate VEGF's causal role in retinal angiogenesis and prove the potential of VEGF inhibition as a specific therapy for ischemic retinal disease.

Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures.

N-Methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity may depend, in part, on the generation of nitric oxide (NO.) and superoxide anion (O2.-), which react to form peroxynitrite (OONO-). This form of neurotoxicity is thought to contribute to a final common pathway of injury in a wide variety of acute and chronic neurologic disorders, including focal ischemia, trauma, epilepsy, Huntington disease, Alzheimer disease, amyotrophic lateral scelerosis, AIDS dementia, and other neurodegenerative diseases. Here, we report that exposure of cortical neurons to relatively short durations or low concentrations of NMDA, S-nitrosocysteine, or 3-morpholinosydnonimine, which generate low levels of peroxynitrite, induces a delayed form of neurotoxicity predominated by apoptotic features. Pretreatment with superoxide dismutase and catalase to scavenge O2.- partially prevents the apoptotic process triggered by S-nitrosocysteine or 3-morpholinosydnonimine. In contrast, intense exposure to high concentrations of NMDA or peroxynitrite induces necrotic cell damage characterized by acute swelling and lysis, which cannot be ameliorated by superoxide dismutase and catalase. Thus, depending on the intensity of the initial insult, NMDA or nitric oxide/superoxide can result in either apoptotic or necrotic neuronal cell damage.

Platelet-derived growth factor BB induces functional vascular anastomoses in vivo.

Neovascularization that generates collateral blood flow can limit the extent of tissue damage after acute ischemia caused by occlusion of the primary blood supply. The neovascular response stimulated by the BB homodimeric form of recombinant platelet-derived growth factor (PDGF-BB) was evaluated for its capacity to protect tissue from necrosis in a rat skin flap model of acutely induced ischemia. Complete survival of the tissue ensued, when the original nutritive blood supply was occluded, as early as 5 days after local PDGF-BB application, and the presence of a patent vasculature was evident compared to control flaps. To further evaluate the vascular regenerative response, PDGF-BB was injected into the muscle/connective tissue bed between the separated ends of a divided femoral artery in rats. A patent new vessel that functionally reconnected the ends of the divided artery within the original 3- to 4-mm gap was regenerated 3 weeks later in all PDGF-BB-treated limbs. In contrast, none of the paired control limbs, which received vehicle with an inactive variant of PDGF-BB, had vessel regrowth (P < 0.001). The absence of a sustained inflammatory response and granulation tissue suggests locally delivered PDGF-BB may directly stimulate the angiogenic phenotype in endothelial cells. These findings indicate that PDGF-BB can generate functional new blood vessels and nonsurgically anastomose severed vessels in vivo. This study supports the possibility of a therapeutic modality for the salvage of ischemic tissue through exogenous cytokine-induced vascular reconnection.

Essential role of adenosine, adenosine A1 receptors, and ATP-sensitive K+ channels in cerebral ischemic preconditioning.

Preconditioning with sublethal ischemia protects against neuronal damage after subsequent lethal ischemic insults in hippocampal neurons. A pharmacological approach using agonists and antagonists at the adenosine A1 receptor as well as openers and blockers of ATP-sensitive K+ channels has been combined with an analysis of neuronal death and gene expression of subunits of glutamate and gamma-aminobutyric acid receptors, HSP70, c-fos, c-jun, and growth factors. It indicates that the mechanism of ischemic tolerance involves a cascade of events including liberation of adenosine, stimulation of adenosine A1 receptors, and, via these receptors, opening of sulfonylurea-sensitive ATP-sensitive K+ channels.

Neurotoxicity of advanced glycation endproducts during focal stroke and neuroprotective effects of aminoguanidine.

Cerebral infarction (stroke) is a potentially disastrous complication of diabetes mellitus, principally because the extent of cortical loss is greater in diabetic patients than in nondiabetic patients. The etiology of this enhanced neurotoxicity is poorly understood. We hypothesized that advanced glycation endproducts (AGEs), which have previously been implicated in the development of other diabetic complications, might contribute to neurotoxicity and brain damage during ischemic stroke. Using a rat model of focal cerebral ischemia, we show that systemically administered AGE-modified bovine serum albumin (AGE-BSA) significantly increased cerebral infarct size. The neurotoxic effects of AGE-BSA administration were dose- and time-related and associated with a paradoxical increase in cerebral blood flow. Aminoguanidine, an inhibitor of AGE cross-linking, attenuated infarct volume in AGE-treated animals. We conclude that AGEs may contribute to the increased severity of stroke associated with diabetes and other conditions characterized by AGE accumulation.

Comparação entre a técnica femoral com dispositivo de hemostasia e a técnica radial em pacientes submetidos à estratégia invasiva precoce

A via de acesso arterial é um importante sítio de complicações após a realização de procedimentos coronários invasivos. Dentre as estratégias para a redução de complicações vasculares, encontra-se estabelecida a eficácia da técnica radial. Os dispositivos de oclusão vascular propiciam maior conforto ao paciente, reduzindo o tempo de hemostasia e repouso no leito. Entretanto, a inconsistência de dados comprovando sua segurança limita sua adoção rotineira como estratégia para redução de complicações vasculares, requerendo evidências de estudos randomizados com metodologia adequada. O objetivo deste estudo foi comparar a incidência de complicações no sítio de punção arterial entre a técnica radial e a técnica femoral com utilização de Angio-Seal em pacientes com síndrome coronariana aguda sem supradesnível do segmento ST submetidos à estratégia invasiva precoce. Trata-se de um ensaio clínico unicêntrico, de não inferioridade, no qual duzentos e quarenta pacientes foram randomizados para a técnica radial ou técnica femoral com utilização de Angio-Seal. O objetivo primário foi a ocorrência de complicações no sítio de punção arterial até 30 dias após o procedimento, incluindo sangramento grave, hematoma >= 5 cm, hematoma retroperitoneal, síndrome compartimental, pseudoaneurisma, fístula arteriovenosa, infecção, isquemia de membro, oclusão arterial, lesão de nervo adjacente ou necessidade de reparo vascular cirúrgico. Em relação às características demográficas e clínicas, houve diferença apenas quanto ao gênero, com presença maior de pacientes do sexo feminino no grupo radial (33,3% versus 20,0%, p=0,020). Não se observaram diferenças entre os grupos quanto ao diagnóstico de admissão, alterações isquêmicas presentes no eletrocardiograma, elevação de marcadores de necrose miocárdica ou escores de risco, bem como quanto à farmacoterapia antitrombótica adjunta e características da intervenção coronária percutânea. A hemostasia foi obtida na totalidade dos procedimentos do grupo radial com a utilização da pulseira compressora seletiva TR Band e em 95% dos procedimentos realizados pela técnica femoral com o Angio-Seal (p=0,029). Exceto pela maior incidência de oclusão arterial no grupo radial comparado ao femoral, não houve diferenças entre os demais desfechos analisados. Segundo o teste de não inferioridade para complicações na via de acesso arterial aos 30 dias, verificou-se que a utilização do Angio-Seal não produziu resultados inferiores ao acesso radial, considerando-se a margem de 15% (12,5% versus 13,3%, diferença -0,83%, IC 95% -9,31 - 7,65, p para não inferioridade <0,001). Os resultados principais deste estudo demonstram que, em uma população de pacientes com diagnóstico de síndrome coronariana aguda sem supradesnível do segmento ST, submetida à estratificação de risco invasiva, a utilização do dispositivo de oclusão vascular Angio-Seal confere ao procedimento efetivado pelo acesso femoral inferioridade na incidência de complicações no sítio de punção arterial aos 30 dias quando comparado ao acesso radial.

Pré-condicionamento isquêmico remoto em portadores de claudicação intermitente de membros inferiores

INTRODUÇÃO: Pré-condicionamento isquêmico remoto (PCIR) é o fenômeno pelo qual curtos períodos de isquemia sub-letal sobre um órgão ou tecido, intercalados com reperfusão do mesmo, conferem a outros órgãos ou tecidos distantes deste, um aumento na capacidade da resistir a episódios subsequentes de isquemia, a qual os mesmos possam ser expostos. Com base nesse fato, testamos a hipótese de que o pré-condicionamento isquêmico remoto em pacientes portadores de claudicação intermitente de membros inferiores poderia aumentar a capacidade de deambulação desses pacientes, extrapolando o conceito do PCIR de aumento da capacidade de preservação da integridade celular frente à isquemia, para a manutenção da função celular, tornando a célula mais apta ao trabalho em situações de privação de oxigênio, geradas pela restrição do fluxo sanguíneo, como ocorre nos pacientes com claudicação intermitente de membros inferiores, durante o exercício. OBJETIVOS: Avaliar se o PCIR aumenta a distância de início de claudicação e/ou a distância total de claudicação em pacientes com doença arterial obstrutiva periférica. MÉTODOS: Foram estudados 52 pacientes ambulatoriais que apresentavam queixa de claudicação intermitente dos membros inferiores, associada a um pulso arterial ausente ou reduzido no membro sintomático e/ou um índice tornozelo-braço <0,90. Estes pacientes foram randomizados em três grupos (A, B e C). Todos os pacientes foram submetidos a dois testes de caminhada em esteira de acordo com o protocolo de Gardner. O grupo A fez o primeiro teste de esteira sem o pré-condicionamento isquêmico remoto e, após 7 dias, foi submetido a um novo teste de esteira, agora precedido pelo pré-condicionamento isquêmico remoto. O grupo B foi submetido ao pré-condicionamento isquêmico remoto antes do primeiro teste de esteira e, após 7 dias, realizou novo teste de esteira, agora sem o pré-condicionamento isquêmico remoto. Já no Grupo C (grupo controle), ambos os testes de esteira foram realizados sem pré-condicionamento isquêmico remoto, também com 7 dias de intervalo. RESULTADOS: Os grupos A e C mostraram um aumento na distância de início de claudicação, no segundo teste, em comparação com o primeiro teste. O grupo A teve um aumento estatisticamente significativo, em relação ao grupo C (grupo controle). Com relação à distância total de claudicação, todos os grupos (A, B e C), mostraram um aumento estatisticamente significativo a favor do segundo teste, porém não foi observada diferença entre os grupos (A, B e C). CONCLUSÕES: O pré-condicionamento isquêmico remoto aumentou a distância inicial de claudicação em pacientes com claudicação intermitente, no entanto, ele não afetou a distância total de claudicação dos pacientes portadores de claudicação intermitente de membros inferiores

Trends in Socioeconomic Inequalities in Ischemic Heart Disease Mortality in Small Areas of Nine Spanish Cities from 1996 to 2007 Using Smoothed ANOVA

The aim of this study was to analyze the evolution of socioeconomic inequalities in mortality due to ischemic heart diseases (IHD) in the census tracts of nine Spanish cities between the periods 1996–2001 and 2002–2007. Among women, there are socioeconomic inequalities in IHD mortality in the first period which tended to remain stable or even increase in the second period in most of the cities. Among men, in general, no socioeconomic inequalities have been detected for this cause in either of the periods. These results highlight the importance of intra-urban inequalities in mortality due to IHD and their evolution over time.

Valor da enolase neuroespecífica (NSE) como marcador prognóstico neurológico, em doentes pediátricos, no contexto de hipoxia-isquémia cerebral

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Portal cavernoma cholangiopathy : case report and systematic review

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014