Morphological and immunohistochemical analysis of proteins CASPASE 3 and XIAP in rats subjected to cerebral ischemia and chronic alcoholism

Abstract Purpose: To evaluate histopathological and ultrastructural changes and expression of proteins related to apoptosis CASPASE 3 and XIAP after experimental induction of temporary focal cerebral ischemia (90 minutes) due to obstruction of the middle cerebral artery in alcoholism model. Methods: Forty adult Wistar rats were used, subdivided into 5 experimental groups: control group (C); Sham group (S); Ischemic group (I); Alcoholic group (A); and Ischemic and Alcoholized group (I+A): animals submitted to the same treatment of group A and after four weeks were submitted to focal cerebral ischemia during 90 minutes, followed by reperfusion of 48 hours. Were processed for histopathological analysis and immunohistochemistry (for the protein expression of CASPASE -3 and XIAP). Results: Greater histopathological changes were observed in the animals of groups I and I+A in the three areas analyzed. The neuronal loss was higher in the medial striatum region of the animals of groups I and I + A. The protein expression of CASPASE -3 was higher than that of XIAP in the groups I and I + A for both proteins. Conclusion: The expression of XIAP was slightly higher where the histopathological changes and expression of CASPASE -3 was less evident.

Current concepts on magnetic resonance imaging (MRI) perfusion-diffusion assessment in acute ischaemic stroke: a review & an update for the clinicians.

Recently, several medical societies published joint statements about imaging recommendations for acute stroke and transient ischaemic attack patients. In following with these published guidelines, we considered it appropriate to present a brief, practical and updated review of the most relevant concepts on the MRI assessment of acute stroke. Basic principles of the clinical interpretation of diffusion, perfusion, and MRI angiography (as part of a global MRI protocol) are discussed with accompanying images for each sequence. Brief comments on incidence and differential diagnosis are also included, together with limitations of the techniques and levels of evidence. The purpose of this article is to present knowledge that can be applied in day-to-day clinical practice in specialized stroke units or emergency rooms to attend patients with acute ischaemic stroke or transient ischaemic attack according to international standards.

Atorvastatin protects GABAergic and dopaminergic neurons in the nigrostriatal system in an experimental rat model of transient focal cerebral ischemia / La atorvastatina protege las neuronas gabérgicas y dopaminérgicas del sistema nigroestriatal en un modelo experimental de isquemia cerebral focal transitoria en ratas

Introduction: Cerebral ischemia is the third leading cause of death and the primary cause of permanent disability worldwide. Atorvastatin is a promising drug with neuroprotective effects that may be useful for the treatment of stroke. However, the effects of atorvastatin on specific neuronal populations within the nigrostriatal system following cerebral ischemia are unknown. Objective: To evaluate the effects of atorvastatin on dopaminergic and GABAergic neuronal populations in exofocal brain regions in a model of transient occlusion of the middle cerebral artery. Materials and methods: Twenty-eight male eight-week-old Wistar rats were used in this study. Both sham and ischemic rats were treated with atorvastatin (10 mg/kg) or carboxymethylcellulose (placebo) by gavage at 6, 24, 48 and 72 hours post-reperfusion. We analyzed the immunoreactivity of glutamic acid decarboxylase and tyrosine hydroxylase in the globus pallidus, caudate putamen and substantia nigra. Results: We observed neurological damage and cell loss in the caudate putamen following ischemia. We also found an increase in tyrosine hydroxylase immunoreactivity in the medial globus pallidus and substantia nigra reticulata, as well as a decrease in glutamic acid decarboxylase immunoreactivity in the lateral globus pallidus in ischemic animals treated with a placebo. However, atorvastatin treatment was able to reverse these effects, significantly decreasing tyrosine hydroxylase levels in the medial globus pallidus and substantia nigra reticulata and significantly increasing glutamic acid decarboxylase levels in the lateral globus pallidus. Conclusion: Our data suggest that post-ischemia treatment with atorvastatin can have neuro-protective effects in exofocal regions far from the ischemic core by modulating the GABAergic and dopaminergic neuronal populations in the nigrostriatal system, which could be useful for preventing neurological disorders.

Introducción. La isquemia cerebral es la tercera causa de muerte y la primera de discapacidad permanente en el mundo. La atorvastatina es un fármaco neuroprotector prometedor para el tratamiento de la apoplejía; sin embargo, su acción sobre las poblaciones neuronales del sistema nigroestriatal después de la isquemia aún se desconoce. Objetivo. Evaluar el efecto de la atorvastatina sobre poblaciones gabérgicas y dopaminérgicas en regiones exofocales en un modelo de oclusión transitoria de la arteria cerebral media. Materiales y métodos. Se utilizaron 28 ratas Wistar macho de ocho semanas de edad. Los ejemplares con isquemia simulada y los ejemplares sometidos a isquemia fueron tratados con atorvastatina (10 mg/kg) y carboximetilcelulosa (placebo) administrados por medio de sonda a las 6, 24, 48 y 72 horas después de la reperfusión. Se analizó la inmunorreacción de la descarboxilasa del ácido glutámico y de la tirosina hidroxilasa en el globo pálido, el putamen caudado y la sustancia negra. Resultados. Los datos confirmaron el daño neurológico y la pérdida celular en el putamen caudado. Se incrementó la inmunorreacción de la tirosina hidroxilasa en el globo pálido medial y la sustancia negra pars reticulata , disminuyendo la inmunorreacción de la descarboxilasa del ácido glutámico en el globo pálido lateral de los animales isquémicos tratados con placebo; sin embargo, el tratamiento con atorvastatina pudo revertirla, lo que logró una disminución significativa de la tirosina hidroxilasa en el globo pálido medial y la sustancia negra pars reticulata y aumentando los niveles de descarboxilasa del ácido glutámico en el globo pálido lateral. Conclusión. Nuestros datos sugieren que la atorvastatina en el tratamiento posterior a la isquemia ejerce neuroprotección en las zonas exofocales, modulando las poblaciones neuronales gabérgicas y dopaminérgicas del sistema nigroestriatal, lo que podría prevenir trastornos neurológicos.

Pre, intra and post-ischemic hypothermic neuroprotection in temporary focal cerebral ischemia in rats: morphometric analysis / Neuroproteção hipotérmica pré, intra e pós-isquêmica na isquemia cerebral focal temporária em ratos: análise morfométrica

OBJECTIVE: To evaluate the neuroprotection of mild hypothermia, applied in different moments, in temporary focal cerebral ischemia in rats. METHODS: Rats was divided into Control (C), Sham (S), Ischemic-control(IC), Pre-ischemic Hypothermia (IH1), Intra-ischemic Hypothermia (IH2), and Post-ischemic Hypothermia (IH3) groups. Morphometry was performed using the KS400 software (Carl Zeiss®) in coronal sections stained by Luxol Fast Blue. Ischemic areas and volumes were obtained. RESULTS: Statistically, blue areas showed difference for C vs. IC, IC vs. IH1 and IC vs. IH2 (p=0.0001; p=0.01; p=0.03), and no difference between C vs. S, IC vs. IH3 and IH vs. IH2 (p=0.39; p=0.85; p=0.63). Red areas showed difference between C vs. IC, IC vs. IH1 and IC vs. IH2 (p=0.0001; p=0.009; p=0.03), and no difference between C vs. S, IC vs. IH3 and IH1 vs. IH2 (p=0.48; p=0.27; p=0.68). Average ischemic areas and ischemic volumes showed difference between IC vs. IH1 and IC vs. IH2 (p=0.0001 and p=0.0011), and no difference between IC vs. IH3 and IH1 vs. IH2 (p=0.57; p=0.79). CONCLUSION: Pre-ischemic and intra-ischemic hypothermia were shown to be similarly neuroprotective, but this was not true for post-ischemic hypothermia.

OBJETIVO: Avaliar a neuroproteção da hipotermia leve, aplicada em diferentes momentos, durante isquemia cerebral focal temporária em ratos. MÉTODOS: Ratos foram divididos em grupos: Controle (C), Sham (S), Controle-isquêmico (IC), Hipotermia Pré-isquêmica (IH1), Hipotermia Intra-isquêmica (IH2) e Hipotermia Pós-isquêmica (IH3). A morfometria foi realizada em secções coronais coradas por Luxol Fast Blue através do programa KS400 (Carl Zeiss®). Foram calculados áreas e volumes isquêmicos. RESULTADOS: Estatisticamente, áreas azuis demonstraram diferença entre os grupos C vs. IC, IC vs. IH1 e IC vs. IH2 (p=0,0001; p=0,01; p=0,03), e nenhuma diferença entre C vs. S, IC vs. IH3 e IH vs. IH2 (p=0,39; p=0,85; p=0,63). Áreas vermelhas demonstraram diferença entre C vs. IC, IC vs. IH1 e IC vs. IH2 (p=0,0001; p=0,009; p=0,03), e nenhuma diferença entre C vs. S, IC vs. IH3 e IH1 vs. IH2 (p=0,48; p=0,27; p=0,68). Áreas isquêmicas médias e volumes isquêmicos demonstraram diferença entre os grupos IC vs. IH1 e IC vs. IH2 (p=0,0001 and p=0,0011), e nenhuma diferença entre IC vs. IH3 and IH1 vs. IH2 (p=0,57; p=0,79). CONCLUSÃO: Hipotermias pré-isquêmica e intra-isquêmica demonstraram neuroproteção em grau semelhante, o que não ocorreu com hipotermia pós-isquêmica.

Transient Ischemic Attack and Stroke Can Be Differentiated by Analyzing the Diffusion Tensor Imaging

OBJECTIVE: We wanted to differentiate between transient ischemic attack (TIA) and minor stroke using fractional anisotropy and three-dimensional (3D) fiber tractography. MATERIALS AND METHODS: The clinical data, conventional magnetic resonance imaging (MRI), diffusion weighted imaging (DWI) and diffusion tensor imaging (DTI) were obtained for 45 TIA patients and 33 minor stroke patients. The fractional anisotrophy ratio (rFA) between the lesion and the mirrored corresponding contralateral normal tissue was calculated and analyzed. The spatial relationship between the lesion and the corticospinal tract (CST) was analyzed and the lesion sizes in the minor stroke patients and TIA patients were compared. RESULTS: Twenty-two of the 45 TIA patients (49%) revealed focal abnormalities following DWI. The rFA was significantly lower (p < 0.05) in the stroke patients (0.71 +/- 0.29) compared to that of the TIA patients (1.05 +/- 0.37). The CST was involved in almost all stroke lesions, but it was not involved in 68% of the TIA lesions. The TIA patients had significantly lower CST injury scores (3.25 +/- 1.75) than did the stroke patients (8.80 +/- 2.39) (p = 0.004). CONCLUSION: Our data indicate that TIA and minor stroke can be identified by analyzing the rFA and the degree of CST involvement, and this may also allow more accurate prediction of a patient's long-term recovery or disability.

Iron mediates endothelial cell damage and blood-brain barrier opening in the hippocampus after transient forebrain ischemia in rats

Blood cells are transported into the brain and are thought to participate in neurodegenerative processes following hypoxic ischemic injury. We examined the possibility that transient forebrain ischemia (TFI) causes the blood-brain barrier (BBB) to become permeable to blood cells, possibly via dysfunction and degeneration of endothelial cells in rats. Extravasation of Evans blue and immunoglobulin G (IgG) was observed in the hippocampal CA1-2 areas within 8 h after TFI, and peaked at 48 h. This extravasation was accompanied by loss of tight junction proteins, occludin, and zonula occludens-1, and degeneration of endothelial cells in the CA1-2 areas. Iron overload and mitochondrial free radical production were evident in the microvessel endothelium of the hippocampus before endothelial cell damage occurred. Administration of deferoxamine (DFO), an iron chelator, or Neu2000, an antioxidant, blocked free radical production and endothelial cell degeneration. Our findings suggest that iron overload and iron-mediated free radical production cause loss of tight junction proteins and degeneration of endothelial cells, opening of the BBB after TFI.

Ultrastructural study of neuronal death in rat hippocampus after transient and permanent focal cerebral ischemia

Morphological changes of CA1 neurons in rat hippocampus after transient and permanent focal cerebral ischemia were studied to clarify the nature of postischemic cell death in the subfield. Male adult rats were divided into 3 groups: Control [Shamoperated], transient ischemic group [30 minutes of MCAO followed by 48 hours of reperfusion], and permanent ischemic group [48 hours of MCAO]. After the mentioned times, deep anesthesia was induced in the rats and their brains were removed and processed for transmission electron microscopy [TEM] and evaluation. Electron-microscopic examination on day 2 showed key morphological signs of apoptosis in the permanent ischemic group, while morphological signs of necrosis were observed in the transient ischemic group. These results suggest necrosis [as dominant mechanism of neuronal death after transient ischemia] and apoptosis [after permanent ischemia] to be involved in neuronal death

Trombolisis intravenosa con rtPA en el Stroke Isquémico Agudo. Eficacia y seguridad del programa instalado en el Hospital Universitario de la Fundación Favaloro / Intravenous thrombolysis with recombinant tissue plasminogen activator in the acute ischemic stroke

The study of the National Institute of Neurological Disorders and Stroke (NINDS) proved that the use of the recombinant tissue plasminogen activator (rtPA) within the first 3 hours since the beginning of the symptomatology of the acute ischemis stroke (AIS) is as well safe as effective...These preliminary data reported in our study show that a strict protocol of thrombolysis IV with rtPA in AIS is feasible to be carried on with good results in a high complexity Center.

Progesterone: Protective effects on the cat hippocampal neuronal damage due to acute global cerebral ischemia

Background. Barbiturates, benzodiazepines, and synthetic steroids having anesthetic properties, by enchacing the inhibitory GABAergic neurotransmission to the neruronal circuits of cerebral structures vulnerable to ischemia, reduce the damage induced by this condition. Some endogenous steroids resulting from progesterone (P4) biotransformation in the brain exert GABAaergic effects, thus inhibiting neuronal excitability. Hence, P4 administration both before and after an experimentally induced ischemic episode may prevent or decrease the ischemic cerebral damage. Methods. Ovariectomized adult cats were treated sc with either P4 (10 mg/kg/day) or corn oil during 7 days before and 7 days after being subjected to a period of acute global cerebral ischemia by 15 min of cardiorespiratory arrest followed by 4 min of reanimation. After 14 days of survival, animals were sacrificed and the brains perfused in situ and formalin-fixed for histological examination. Results. Acute global cerebral ischemia resulted in a severe loss of neurons (54-85 percent), mainly in CA1 and CA2 subfields of oil-treated cats. Progesterone significantly reduced the neuronal loss in those areas (21-49 percent). Conclusions. Overall results suggest that progesterone exerts protective effects against the neuronal cerebral damage induced by acute global cerebral ischemia

Os radicais livres derivados do oxigênio na isquemia cerebral transitória: estudo experimental / Free oxigen-derived radicals in transient ischemia: experimental study

Este estudo experimental tem por objetivo pesquisar as açoes lesivas dos radicais livres derivados do oxigênio, liberados no encéfalo, na presença de isquemia cerebral transitória e completa, verificando os efeitos de inativadores destes radicais, através da reduçao do corante óptico Nitro Blue Tetrazolium (NBT). Utilizou-se 32 coelhos subdivididos em quatro grupos, compostos cada um de oito animais. O grupo I (controle) nao foi submetido à isquemia cerebral, mantendo-se os níveis pressóricos em níveis normais. Os coelhos dos grupos II, III e IV foram submetidos à isquemia cerebral transitória, através de hipotensao controlada, durante 60 minutos, seguidos de 30 minutos de reperfusao. Nos grupos III e IV utilizou-se, antes da fase de reperfusao, respectivamente, N-acetilcisteína e Deferoxamina. No grupo II a cor azul do corante revelou intensa liberaçao de radicais livres derivados do oxigênio e os exames histológicos em microscopia óptica e eletrônica evidenciaram lesao tecidual. Enfatiza-se a importância do ferro como catalizador das reaçoes que geram radiais livres inorgânicos e das reaçoes capazes de gerar radicais livres orgânicos a partir de produtos do metabolismo do ácido araquidônico. Finalmente, comenta-se o efeito dos bloqueadores dos radicais livres derivados do oxigênio, que exerceram açao protetora sobre o tecido cerebral nos grupos III e IV, evitando o aparecimento de lesoes histopatológicas. Tais resultados sugerem fortemente a importância do uso profilático de inativadores dos radicais livres, na isquemia cerebral transitória.

Efeito da gangliectomia cervical superior bilateral e de um inibidor do enzima conversor de angiotensina (captopril) no vasoespasmo precoce pós hemorragia meníngea / Effect of superior cervical gangliectomy and of the angiotensin converting enzyme (captopril) in the development of cerebral vasospasm after meningeal haemorrhage

O vasoespasmo cerebral é um importante fator na evoluçäo clínica dos pacientes com hemorragia meníngea pós-sangramento aneuristmático, devio à alta morbidade que apresenta. O tratamento, a prevençäo e mesmo os mecanismos do vasoespasmo näo estäo ainda bem determinados. É conhecida a açäo vasoconstritora do sistema simpático sobre o tono muscular lisodas artérias cerebrais e também da açäo da angiotensina II sobre este sistema, bem como a possível sensibilidade da vasculatura cerebral à açäo relaxadora da bradicinina. No presente trabalho foi investigado o efeito da simpatectomia e de um inibidor do enzima conversor da angiotensina, isolada e conjuntamente sobre o vasoespasmo experimental. Quarenta e oito coelhos submetidos à anestesia inalatória, em respiraçäo espontânea, receberam injeçäo intracisternal de sangue autólogo. Por meio de angiografia carotídea, documentou-se e foi medido posteriormente o calibre da artéria basilar, antes da hemorragia meníngea e dez e trinta minutos após a mesma. Imediatamente após a hemorragia os animais, por sorteio, foram incluídos em um dos seguintes grupo: grupo 1 - animais submetidos à gangliectomia cervical superior bilateral e tratados com o inibidor do enzima conversor da angiotensina, captopril; grupo 2 - submetido apenas à gangliectomia; grupo 3 - somente tratado com captopril I.A.; grupo 4 - sem tratamento nenhum; e finalmente um grupo de seis animais que näo foi submetido à hemorragia meníngea e a nenhum tratamento, grupo 5. A medida do diâmetro da artéria basilar, no material documentado, demonstrou que os coelhos submetidos à gangliectomia e tratados com captopril e os coelhos apenas tratados com captopril näo desenvolveram vasoespasmo, enquanto que os animais submetidos apenas à gangliectomia se comportaram como os do grupo-controle näo tratado, apresentando vasoespasmo significativo na angiografia executada dez minutos após a hemorragia meníngea. A partir destes resultados conclui-se que o captopril intraluminal impede o vasoespasmo precoce decorrente da hemorragia meníngea e que a secçäo bilateral dos gânglios simpáticos cervicais superiores näo exerce nenhum efeito imediato sobre o tono vascular cerebral, associado ou näo ao uso de captopril. Sugere, outrossim, que o enzima conversor de angiotensina, através da angiotensina II ou pela degradaçäo da bradicinina, participe do mecanismo do vasoespasmo precoce e que sua inibiçäo possa ser um recurso na prevençäo do mesmo.