Drebrin expression patterns in patients with refractory temporal lobe epilepsy and hippocampal sclerosis.

OBJECTIVE: Drebrins are crucial for synaptic function and dendritic spine development, remodeling, and maintenance. In temporal lobe epilepsy (TLE) patients, a significant hippocampal synaptic reorganization occurs, and synaptic reorganization has been associated with hippocampal hyperexcitability. This study aimed to evaluate, in TLE patients, the hippocampal expression of drebrin using immunohistochemistry with DAS2 or M2F6 antibodies that recognize adult (drebrin A) or adult and embryonic (pan-drebrin) isoforms, respectively. METHODS: Hippocampal sections from drug-resistant TLE patients with hippocampal sclerosis (HS; TLE, n = 33), of whom 31 presented with type 1 HS and two with type 2 HS, and autopsy control cases (n = 20) were assayed by immunohistochemistry and evaluated for neuron density, and drebrin A and pan-drebrin expression. Double-labeling immunofluorescences were performed to localize drebrin A-positive spines in dendrites (MAP2), and to evaluate whether drebrin colocalizes with inhibitory (GAD65) and excitatory (VGlut1) presynaptic markers. RESULTS: Compared to controls, TLE patients had increased pan-drebrin in all hippocampal subfields and increased drebrin A-immunopositive area in all hippocampal subfields but CA1. Drebrin-positive spine density followed the same pattern as total drebrin quantification. Confocal microscopy indicated juxtaposition of drebrin-positive spines with VGlut1-positive puncta, but not with GAD65-positive puncta. Drebrin expression in the dentate gyrus of TLE cases was associated negatively with seizure frequency and positively with verbal memory. TLE patients with lower drebrin-immunopositive area in inner molecular layer (IML) than in outer molecular layer (OML) had a lower seizure frequency than those with higher or comparable drebrin-immunopositive area in IML compared with OML. SIGNIFICANCE: Our results suggest that changes in drebrin-positive spines and drebrin expression in the dentate gyrus of TLE patients are associated with lower seizure frequency, more preserved verbal memory, and a better postsurgical outcome.

Myosins and DYNLL1/LC8 in the honey bee (Apis mellifera L.) brain.

Honey bees have brain structures with specialized and developed systems of communication that account for memory, learning capacity and behavioral organization with a set of genes homologous to vertebrate genes. Many microtubule- and actin-based molecular motors are involved in axonal/dendritic transport. Myosin-Va is present in the honey bee Apis mellifera nervous system of the larvae and adult castes and subcastes. DYNLL1/LC8 and myosin-IIb, -VI and -IXb have also been detected in the adult brain. SNARE proteins, such as CaMKII, clathrin, syntaxin, SNAP25, munc18, synaptophysin and synaptotagmin, are also expressed in the honey bee brain. Honey bee myosin-Va displayed ATP-dependent solubility and was associated with DYNLL1/LC8 and SNARE proteins in the membrane vesicle-enriched fraction. Myosin-Va expression was also decreased after the intracerebral injection of melittin and NMDA. The immunolocalization of myosin-Va and -IV, DYNLL1/LC8, and synaptophysin in mushroom bodies, and optical and antennal lobes was compared with the brain morphology based on Neo-Timm histochemistry and revealed a distinct and punctate distribution. This result suggested that the pattern of localization is associated with neuron function. Therefore, our data indicated that the roles of myosins, DYNLL1/LC8, and SNARE proteins in the nervous and visual systems of honey bees should be further studied under different developmental, caste and behavioral conditions.

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: myosin-V assay.

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 ºC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.

Comparative analysis of two immunohistochemical methods for antigen retrieval in the optical lobe of the honeybee Apis mellifera: Myosin-v assay

The present study compared two heating methods currently used for antigen retrieval (AR) immunostaining: the microwave oven and the steam cooker. Myosin-V, a molecular motor involved in vesicle transport, was used as a neuronal marker in honeybee Apis mellifera brains fixed in formalin. Overall, the steam cooker showed the most satisfactory AR results. At 100 ºC, tissue morphology was maintained and revealed epitope recovery, while evaporation of the AR solution was markedly reduced; this is important for stabilizing the sodium citrate molarity of the AR buffer and reducing background effects. Standardization of heat-mediated AR of formalin-fixed and paraffin-embedded tissue sections results in more reliable immunostaining of the honeybee brain.

Assessment of vitamin A nutritional status in newborn preterm infants.

OBJECTIVE: This study assessed the vitamin A nutritional status of preterm infants determined by the vitamin A relative dose-response test (RDR) compared with serum levels of vitamin A, retinol-binding protein (RBP), transthyretin (TTR), and retinol relations with carrier proteins. METHODS: Serum levels of retinol, RBP, and TTR and retinol/RBP, retinol/TTR, and RBP/TTR molar ratios were determined in 120 infants at 7 d and in 92 at 28 d. For the determination of the performance of the tests, the RDR was considered the reference method. The sensitivity and specificity for all possible cutoff values were determined by constructing receiver operator characteristic curves. The areas under the curves were used to estimate the overall accuracy of the tests. The best cutoff values to be used for the calculation of sensitivity and specificity were determined with 95% confidence intervals. RESULTS: RDR indicated vitamin A deficiency in 60% of the infants at 7 d and in 51.1% at 28 d. In the receiver operator characteristic curves, the best area under the curve was 0.710 obtained for serum retinol at 28 d of postnatal age and considered moderately accurate. The least inadequate cutoff level was set at 25 mg/dL, but no value was considered adequate due to low sensitivity and/or low specificity. CONCLUSION: Compared with RDR, the determination of serum levels of retinol, RBP, and TTR and their molar ratios are not adequate to assess nutritional vitamin A status in preterm infants.

Histological composition and progression of carotid plaque.

BACKGROUND: To analyse histological composition and progression of carotid plaque. METHODS: Thirty-one patients (22 males, mean age 68.03 +/- 7.3 years) admitted for carotid endarterectomy for extracranial high-grade internal carotid artery stenosis (>or= 70% luminal narrowing) were enrolled. The patients were divided into 2 groups according to symptomatology (group I, 17 symptomatic patients; and group II, 14 asymptomatic patients). A histological analysis and inflammatory cell quantification of each excised carotid plaque was made. Nine carotid arteries were removed from human cadavers that were not preselected for carotid artery disease. These specimens were used as a control tissue without any macroscopic signs of atherosclerotic plaques. RESULTS: Fifty eight percent of all carotid plaques were classified as complex plaque with possible surface defect, hemorrhage or thrombus. The inflammatory cells concentration did not differ between the two groups. All specimens from human cadavers were classified as preatheroma with extracellular lipid pools. CONCLUSION: Asymptomatic and symptomatic patients could have the same histological components on their carotid plaques. Fibrotic and calcific plaques could become vulnerable as complex plaques with surface defect, hemorrhage and thrombus could remain silent. Asymptomatic carotid stenosis should be followed close with no invasive diagnostic methods and clinical evaluation.

Análise videodensitométrica da placa carotídea vulnerável: correlação histopatológica / Videodensitometric analysis of vulnerable carotid plaque: correlation with histological examination

Objetivos: Estabelecer a correlação entre a avaliação quantitativa, através da caracterização tecidual videodensitométrica, de imagens ultra-sonográficas bidimensionais digitalizadas e a análise histopatológica de placas carotídeas vulneráveis removidas cirurgicamente da artéria carótida, com estenose acima de 70 por cento, foram submetidos à caracterização tecidual ultra-sônica da placa antes do procedimento cirúrgico. Um software de computador (CaPAS - Carotid Plaque Analysis Software) foi especificamente desenvolvido para a análise videodensitométrica das imagens bidimensionais digitalizadas das placas carotídeas através de índice estatísticos de primeira (NMC - nível médio de cinza e DP - desvio padrão) e segunda (Entopia, Energia e Homogeneidade) ordens. Os pacientes foram divididos em 3 grupos de acordo com a sintomatologia: GRUPO I(n igual 6), pacientes com sintomas até 3 meses antecedendo o procedimento cirúrgico, GRUPO II(n igual 9), pacientes com sintomas acima de 3 meses antecedendo o procedimento cirúrgico e GRUPO III(n igual 10), pacientes assintomáticos. Os espécimes cirúrgicos foram analisados histopatologicamente para determinar as percentagens de gordura/necrose, fibrose e cálcio. Resultados: O NMC (primeira ordem) e os parâmetros de segunda ordem (entropia, energia e homogeneidade) diferenciaram os grupos clínicos, mas não se correlacionaram com os achados histopatológicos, mostrando apenas um tendência no NMC (primeira ordem) em refletir tais achados. Conclusão: A análise videodensitométrica de imagens bidimensionais digitalizadas pode ser usada para identificar placas vulneráveis e potencialmente instáveis, porém não se correlaciona com a proporção de componentes estruturais básicos avaliados histologicamente.

Caracterização tecidual ultra-sônica da placa carotídea vulnerável pela análise videodensitométrica / Ultrasonic tissue characterization of vulnerable carotid plaque by videodensitometric technique

A ruptura de placas carotideas e embolização de material atero-trombótico é o mecanismo patogênico mais comum causador de isquemia cerebral devido à estenose de artéria carótida. Vários trabalhos multicêntricos e randomizados demostraram o benefício da cirúrgia de endarterectomia da artéria carótida (CEAC) em prevenir acidente vascular cerebral(AVC) tanto em pacientes sintomáticos quanto assintomáticos. Entretanto, estes mesmos trabalhos também notaram que a maioria dos pacientes portadores de estenoses significativas das artérias carótidas permaneceram livres de eventos neurológicos apenas com a terapia medicamentosa e que um grande número e pacientes pode ser operado desnecessariamente. Assim, é necessário identificar pacientes com alto risco, em que a CEAC deve ser considerada, e pacientes com baixo risco os quais devem ser poupados deste procedimento frequentementee custoso e não isento de riscos. Estudos histológicos têm demonstrado que um núcleo lipídico grande, localizado próximo ao lúmen, com ruptura da capa fibrosa, hemorragia intraplaca e ulceração da superfície ocorrem mais frequemtemente em placas sintomáticas. A caracterização quantitativa desses achados histológicos através de métodos não invasivos permitirá que a CEAC seja realizada em pacientes portadores de placas de alto risco. O método videodensitométrico é uma técnica promissora que pode identificar placas carotídeas e potencialmente instáveis.

Modulation of eosinophil migration from bone marrow to lungs of allergic rats by nitric oxide.

Chronic blockade of nitric oxide (NO) synthesis attenuates the eosinophil infiltration into airways of allergic rats. This study was designed to investigate whether the inhibition of eosinophil influx to the lung of allergic rats reflects modifications in the pattern of cell mobilization from the bone marrow to peripheral blood and/or to lung. Male Wistar rats were treated with N(omega)-nitro-l-arginine methyl ester (l-NAME; 20mg/rat per day) for 4 weeks and sensitized with ovalbumin (OVA). In control rats, the pulmonary OVA-challenge promoted an early (24h) increase in the bone marrow eosinophil population that normalized at 48 h after OVA-challenge, at which time the eosinophils disappeared from the blood and reached the lungs in mass. In l-NAME-treated rats, an accumulation of eosinophils in bone marrow was observed at 24 and 48 h post-OVA-challenge. No variation in this cell type number was observed in peripheral blood and bronchoalveolar lavage throughout the time-course studied. In control rats, the adhesion of bone marrow eosinophils to fibronectin-covered wells was significantly increased at 24h after OVA-challenge, whereas in l-NAME-treated rats the increased adhesion was detected at 48 h. A 32% decrease in the expression of inducible nitric oxide synthase (iNOS) (but not endothelial nitric oxide synthase; eNOS) in eosinophils from l-NAME-treated rats was observed. The levels of IgE, IgG(1) and IgG(2a) were not affected by the l-NAME treatment. Our findings suggest that inhibition of NO synthesis upregulates the binding of eosinophils to extracellular matrix proteins such as fibronectin, producing a delayed efflux of eosinophils from bone marrow to peripheral blood and lungs.

Modulation of eosinophil migration from bone marrow to lungs of allergic rats by nitric oxide

Chronic blockade of nitric oxide (NO) synthesis attenuates the eosinophil infiltration into airways of allergic rats. This study was designed to investigate whether the inhibition of eosinophil influx to the lung of allergic rats reflects modifications in the pattern of cell mobilization from the bone marrow to peripheral blood and/or to lung. Male Wistar rats were treated with Nù-nitro-L-arginine methyl ester (L-NAME; 20 mg/rat per day) for 4 weeks and sensitized with ovalbumin (OVA). In control rats, the pulmonary OVA-challenge promoted an early (24 h) increase in the bone marrow eosinophil population that normalized at 48 h after OVA-challenge, at which time the eosinophils disappeared from the blood and reached the lungs in mass. In L-NAME-treated rats, an accumulation of eosinophils in bone marrow was observed at 24 and 48 h post-OVA-challenge. No variation in this cell type number was observed in peripheral blood and bronchoalveolar lavage throughout the time-course studied. In control rats, the adhesion of bone marrow eosinophils to fibronectin-covered wells was significantly increased at 24 h after OVA-challenge, whereas in L-NAME-treated rats the increased adhesion was detected at 48 h. A 32% decrease in the expression of inducible nitric oxide synthase (iNOS) (but not endothelial nitric oxide synthase; eNOS) in eosinophils from L-NAME-treated rats was observed. The levels of IgE, IgG1 and IgG2a were not affected by the L-NAME treatment. Our findings suggest that inhibition of NO synthesis upregulates the binding of eosinophils to extracellular matrix proteins such as fibronectin, producing a delayed efflux of eosinophils from bone marrow to peripheral blood and lungs.