Vitamin D3 mediates spatial memory improvement through nitric oxide mechanism in demyelinated hippocampus of rat

Abstract Studies have revealed beneficial role of vitamin D3 in neuro-cognitive function. There is also supporting evidence on the involvement of nitric oxide (NO) in the neuro-protective action. However, its over production could contribute to brain disorders. In this study, demyelination was induced by ethidium bromide (EB) injection into the right side of the hippocampus area of male rats. Vitamin D3 was administered to rats for 7 and 28 days prior to behavioral experiments using Morris water maze (MWM). Travelled distance, time spent to reach the platform, and time spent in target zone, were considered for learning and spatial memory evaluation. Nitrite oxide (NO2-) concentration was measured as an indicator for nitric oxide production. The time spent to reach the platform and the travelled distance were decreased significantly by 28 days of vitamin D3 administration (compared to 7 days experiment). Time spent in target quadrant was significantly lowered by administered vitamin on day 28. Therefore, considering a number of studies that have shown the effect of vitamin D3 on cognition, these findings could support their potential effect. Besides, nitric oxide concentration significantly differed in 28 days of vitamin D3 treated group compared with the groups treated with EB or 7 days of vitamin D3.

The effect of intermittent fasting diet on the hippocampus of adult male mouse after inducing demyelination by ethidium bromide injection / Efecto de la dieta de ayuno intermitente en el hipocampo de ratón macho adulto después de inducir la desmielinización por inyección de bromuro de etidio

Intermittent fasting diet (IF) as a restrictive regimen prevents neural degeneration and stimulates overexpression of various neurotropic factors in the hippocampus of animal models. This study evaluates the potential effect of the IF in the prevention of learning and memory dysfunction and improving the alterations in the number and volume of neurons in an ethidium bromide (EB) induced mouse model of demyelination.Mice were randomly assigned into N group (Normal Diet and normal saline injection), F group (IF and normal saline injection), EBN group (Normal Diet and EB injection), EBF group (IF and EB injection). The hidden platform test was carried out based on path length, escape latency and swim speeds of mice. Stereological studies were determined by the Cavalieri and the Optical Dissector technique. Maintenance of mice on the IF results in significantly decreased the body weight and biochemical parameters, increased total number of neurons and volume of the hippocampus, and improved learning and memory parameters of adult male mice. However, IF in EBF group did not show as excellently as F group. The EBF group displayed significantly spatial memory improvement than that in EBN group. There were no statistically significant differences between EBF and EBN groups in stereological and learning parameters, though the EBF group displayed faster escape latencies, and swam faster and shorter path lengths than the EBN group in these parameters. Therefore as a conclusion, The IF fairly improved some adverse effects of EB in experimental demyelination models.

La dieta de ayuno intermitente (AI) como régimen restrictivo, previene la degeneración neural y la estimación de la presencia de diversos factores neurotrópicos en el hipocampo de modelos animales. Este estudio evalúa el efecto potencial de la AI en la prevención del aprendizaje y la disfunción de la memoria y mejora las alteraciones en el número y el volumen de las neuronas en un modelo de desmielinización, en ratón, inducido con bromuro de etidio (BE). Los ratones se asignaron al azar en el grupo N (dieta normal e inyección salina normal), Grupo A (AI e inyección salina normal), Grupo BEN (dieta normal e inyección BE), Grupo EBF (inyección AI y BE). La prueba de la plataforma oculta se llevó a cabo en función de la longitud del trayecto, la latencia de escape y la velocidad de nado de los ratones. Los estudios estereológicos fueron determinados por la técnica de Cavalieri y la técnica del disector óptico. En el grupo AI disminuyeron significativamente el peso corporal de los ratones, los parámetros bioquímicos, el número total de neuronas y el volumen del hipocampo, y los parámetros de aprendizaje y la memoria de los ratones machos adultos. Sin embargo, el grupo AI en BEF no se mostró tan bien como el grupo A. El grupo EBF mostró una mejora en la memoria espacial significativamente mayor que la del grupo BEN. No hubo diferencias estadísticamente significativas entre los grupos A, BE y BEN en los parámetros estereológicos y de aprendizaje, aunque el grupo EBF mostró latencias de escape más rápidas, y nado en las rutas más rápidas y más cortas que el grupo BEN en estos parámetros. Por lo tanto, como conclusión, el grupo AI mejoró bastante algunos efectos adversos de la BE en los modelos de desmielinización experimental.

Salicylic Acid Reduces OmpF Expression, Rendering Salmonella enterica Serovar Typhimurium More Resistant to Cephalosporin Antibiotics / 전남의대학술지

Salmonella enterica serovar Typhimurium is one of the most important bacterial pathogens causing diarrhea. The resistance of S. typhimurium to antimicrobial agents, which has recently been isolated from patients, is causing serious problems. We investigated the effects of salicylic acid (Sal) and acetyl salicylate (AcSal) on the susceptibility of S. typhimurium to cephalosporin antibiotics, which are known to increase resistance to cephalosporin and quinolone antibiotics. The MIC of cephalosporin antibiotics was higher than that of the media without Sal. The rate of accumulation of ethidium bromide (EtBr) in the bacteria by the outer membrane protein (Omp) was not different from that of the bacteria cultured in the medium containing Sal. However, Carbonyl cyanide-m-chlorophenylhydrazone (CCCP), an inhibitor of bacterial efflux pumps, significantly reduced the rate of accumulation of EtBr in bacteria cultured on Sal containing medium. In the medium containing CCCP, the MIC of the antimicrobial agent tended to decrease as compared with the control. In addition, the MIC of the bacteria treated with CCCP and Sal was higher than that of the antimicrobial agent against the CCCP treated experimental bacteria. These results suggest that Sal decreases the expression of OmpF in the Omp of S. typhimurium and reduces the permeability of cephalosporin antibiotics to bacteria, which may induce tolerance to cephalosporin antibiotics.

Curcumin decreases astrocytic reaction after gliotoxic injury in the rat brainstem / Curcumina reduz a reação astrocitária após injúria gliotóxica no tronco encefálico de ratos

ABSTRACT Recent studies have demonstrated that curcumin (Cur) has antioxidant, anti-inflammatory and anti-fibrotic effects. Ethidium bromide (EB) injections into the central nervous system (CNS) are known to induce local oligodendroglial and astrocytic loss, resulting in primary demyelination and neuroinflammation. Peripheral astrogliosis is seen around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). This investigation aimed to evaluate the effect of Cur administration on astrocytic response following gliotoxic injury. Wistar rats were injected with EB into the cisterna pontis and treated, or not, with Cur (100 mg/kg/day, intraperitoneal route) during the experimental period. Brainstem sections were collected at 15, 21 and 31 days after EB injection and processed for GFAP immunohistochemical staining. Astrocytic reactivity was measured in a computerized system for image analysis. In Cur-treated rats, the GFAP-stained area around the lesion was significantly smaller in all periods after EB injection compared to untreated animals, showing that Cur reduces glial scar development following injury.

RESUMO Estudos recentes têm demonstrado que a curcumina (Cur) possui efeitos antioxidantes, anti-inflamatórios e antifibróticos. Sabe-se que a injeção de brometo de etídio (EB) no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização primária e neuroinflamação. Astrogliose periférica é observada ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). A presente investigação objetivou avaliar o efeito da Cur sobre a resposta astrocitária após injúria gliotóxica. Ratos Wistar foram injetados com EB na cisterna basal e tratados ou não com Cur (100 mg/kg/dia, via intraperitoneal) durante o período experimental. Amostras do tronco encefálico foram coletadas aos 15, 21 e 31 dias pós-injeção de EB e processadas para estudo imuno-histoquímico para a GFAP. A reatividade astrocitária foi medida em um sistema computadorizado para análise de imagem. Nos ratos tratados com Cur, a área marcada para GFAP foi significantemente menor em todos os períodos pós-injeção de EB, indicando que a Cur reduz o desenvolvimento da cicatriz glial após injúria.

Aerobic Swim Training Restores Aortic Endothelial Function by Decreasing Superoxide Levels in Spontaneously Hypertensive Rats

OBJECTIVE: We aimed to determine whether aerobic training decreases superoxide levels, increases nitric oxide levels, and improves endothelium-dependent vasodilation in the aortas of spontaneously hypertensive rats. METHODS: Spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) were distributed into 2 groups: sedentary (SHRsd and WKYsd, n=10 each) and swimming-trained (SHRtr, n=10 and WKYtr, n=10, respectively). The trained group participated in training sessions 5 days/week for 1 h/day with an additional work load of 4% of the animal’s body weight. After a 10-week sedentary or aerobic training period, the rats were euthanized. The thoracic aortas were removed to evaluate the vasodilator response to acetylcholine (10-10 to 10-4 M) with or without preincubation with L-NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; 10-4 M) in vitro. The aortic tissue was also used to assess the levels of the endothelial nitric oxide synthase and nicotinamide adenine dinucleotide oxidase subunit isoforms 1 and 4 proteins, as well as the superoxide and nitrite contents. Blood pressure was measured using a computerized tail-cuff system. RESULTS: Aerobic training significantly increased the acetylcholine-induced maximum vasodilation observed in the SHRtr group compared with the SHRsd group (85.9±4.3 vs. 71.6±5.2%). Additionally, in the SHRtr group, superoxide levels were significantly decreased, nitric oxide bioavailability was improved, and the levels of the nicotinamide adenine dinucleotide oxidase subunit isoform 4 protein were decreased compared to the SHRsd group. Moreover, after training, the blood pressure of the SHRtr group decreased compared to the SHRsd group. Exercise training had no effect on the blood pressure of the WKYtr group. CONCLUSIONS: In SHR, aerobic swim training decreased vascular superoxide generation by nicotinamide adenine dinucleotide oxidase subunit isoform 4 and increased nitric oxide bioavailability, thereby improving endothelial function.

Propentofylline reduces glial scar development following gliotoxic damage in the rat brainstem / Propentofilina reduz o desenvolvimento da cicatriz glial após dano gliotóxico no tronco encefálico de ratos

ABSTRACT Propentofylline is a xanthine derivative that depresses activation of glial cells, whose responses contribute to neural tissue damage during inflammation. Ethidium bromide injection into the central nervous system induces local oligodendroglial and astrocytic loss, resulting in primary demyelination, neuroinflammation and blood-brain barrier disruption. Surviving astrocytes present a vigorous reaction around the injury site with increased immunoreactivity to glial fibrillary acidic protein (GFAP). Objective This study aimed to evaluate the effect of propentofylline administration on astrocytic response following gliotoxic injury. Method Wistar rats were injected with ethidium bromide into the cisterna pontis and treated or not with propentofylline (12.5mg/kg/day, intraperitoneal) during the experimental period. Brainstem sections were collected from 15 to 31 days after gliotoxic injection and processed for GFAP immunohistochemistry. Results and Conclusion Results demonstrate that propentofylline decreased astrocytic activation until the 21st day, suggesting that this drug may have a role in reducing glial scar development following injury.

RESUMO A propentofilina é uma xantina que deprime a ativação das células gliais, cujas respostas contribuem para o dano neural durante inflamação. A injeção de brometo de etídio no sistema nervoso central induz a perda oligodendroglial e astrocitária, resultando em desmielinização, neuroinflamação e ruptura da barreira hematoencefálica. Os astrócitos sobreviventes apresentam vigorosa reação ao redor da lesão com aumento da imunorreatividade à proteína glial fibrilar ácida (GFAP). Objetivo Este estudo objetivou avaliar o efeito da propentofilina sobre a resposta astrocitária após injúria gliotóxica. Método Ratos Wistar foram injetados com brometo de etídio na cisterna basal e tratados ou não com propentofilina (12.5mg/kg/dia, intraperitoneal). Amostras do tronco encefálico foram coletadas dos 15 aos 31 dias pós-injeção do gliotóxico e processadas para estudo ultraestrutural e imuno-histoquímico para GFAP. Resultados e Conclusão Os resultados demonstram que a propentofilina reduziu a ativação astrocitária até o 21o dia, sugerindo que essa droga pode atuar na redução da cicatriz glial após injúria.

Hormonal therapy with estradiol and drospirenone improves endothelium-dependent vasodilation in the coronary bed of ovariectomized spontaneously hypertensive rats

Drospirenone (DRSP) is a progestin with anti-aldosterone properties and it reduces blood pressure in hypertensive women. However, the effects of DRSP on endothelium-dependent coronary vasodilation have not been evaluated. This study investigated the effects of combined therapy with estrogen (E2) and DRSP on endothelium-dependent vasodilation of the coronary bed of ovariectomized (OVX) spontaneously hypertensive rats. Female spontaneously hypertensive rats (n=87) at 12 weeks of age were randomly divided into sham operated (Sham), OVX, OVX treated with E2 (E2), and OVX treated with E2 and DRSP (E2+DRSP) groups. Hemodynamic parameters were directly evaluated by catheter insertion into the femoral artery. Endothelium-dependent vasodilation in response to bradykinin in the coronary arterial bed was assessed using isolated hearts according to a modified Langendorff method. Coronary protein expression of endothelial nitric oxide synthase and estrogen receptor alpha (ER-α) was assessed by Western blotting. Histological slices of coronary arteries were stained with hematoxylin and eosin, and morphometric parameters were analyzed. Oxidative stress was assessed in situ by dihydroethidium fluorescence. Ovariectomy increased systolic blood pressure, which was only prevented by E2+DRSP treatment. Estrogen deficiency caused endothelial dysfunction, which was prevented by both treatments. However, the vasodilator response in the E2+DRSP group was significantly higher at the three highest concentrations compared with the OVX group. Reduced ER-α expression in OVX rats was restored by both treatments. Morphometric parameters and oxidative stress were augmented by OVX and reduced by E2 and E2+DRSP treatments. Hormonal therapy with E2 and DRSP may be an important therapeutic option in the prevention of coronary heart disease in hypertensive post-menopausal women.

Propentofylline reverses delayed remyelination in streptozotocin-induced diabetic rats

Objective The diabetic state induced by streptozotocin injection is known to impair oligodendroglial remyelination in the rat brainstem following intracisternal injection with the gliotoxic agent ethidium bromide (EB). In such experimental model, propentofylline (PPF) recently showed to improve myelin repair, probably due to its neuroprotective, antiinflammatory and antioxidant effects. The aim of this study was to evaluate the effect of PPF administration in diabetic rats submitted to the EB-demyelinating model. Materials and methods Adult male rats, diabetic or not, received a single injection of 10 microlitres of 0.1% EB solution into the cisterna pontis. For induction of diabetes mellitus the streptozotocin-diabetogenic model was used (50 mg/kg, intraperitoneal route – IP). Some diabetic rats were treated with PPF (12.5 mg/kg/day, IP route) during the experimental period. The animals were anesthetized and perfused from 7 to 31 days after EB injection and brainstem sections were collected for analysis of the lesions by light and transmission electron microscopy. Results Diabetic rats injected with EB showed larger amounts of myelin-derived membranes in the central areas of the lesions and considerable delay in the remyelinating process played by surviving oligodendrocytes and invading Schwann cells after the 15th day. On the other hand, diabetic rats that received PPF presented lesions similar to those of non-diabetic animals, with rapid remyelination at the edges of the lesion site and fast clearance of myelin debris from the central area. Conclusion The administration of PPF apparently reversed the impairment in remyelination induced by the diabetic state. Arch Endocrinol Metab. 2015;59(1):47-53 .

Observation of radiobiological characteristics in a HepG2 cell line with mitochondrial DNA deletion / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion.</p><p><b>METHODS</b>HepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay.</p><p><b>RESULTS</b>HepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/β value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness.</p><p><b>CONCLUSION</b>HepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.</p>

Genotypic and phenotypic detection of efflux pump in Rhodococcus equi

The req_39680 gene, associated to a putative efflux system, was detected in 60% (54/90) of R. equi isolates by PCR. The phenotypic expression of efflux mechanism was verified in 20% of the isolates using ethidium bromide. For the first time, the expression of efflux mechanism was demonstrated in R. equi.

Excretion product of shigella dysenteriae (sdyep) induced cell death in early larval stage of zebrafish (danio rerio): acridine orange and ethidium bromide (AO/EB) in vivo staining / Producto de excreción de shigella dysenteriae (pesdy) induce muerte celular en larvas de pez cebra (danio rerio): marcaje in vivo con naranja de acridina y bromuro de etidio (NA/BE)

In the field of studies of acute toxicity induced by bacterial agents, Shiga toxins have been relevant due to the severity of the extra-intestinal diseases they cause. Numerous studies have shown that Shiga toxin induced apoptosis in different cell types; however, this important process has been little studied in vivo experimental models. In this study, the effects of excretion products of Shigella dysenteriae, in which Shiga toxin is present, were investigated on early larval stages of Zebrafish, an animal model with many advantages over other in vivo experimental models traditionally used. Both the collection of eggs and larvae of Zebrafish, and the product from excretion from Shigella dysenteriae (SdyEP) were performed according to laboratory standards. Also, toxicity bioassay, larvae treatment with pure and diluted solution, 10-1, 10-2, 10-3 , 10-4 and 10-5, v/v SdyEP and cell death in vivo using Acridine Orange (AO) and Ethidium Bromide (EB) were applied. The excretion product of Shigella dysenteriae (SdyEP) effect was expressed in terms of larval mortality and dependent dilution rather than incubation time. The larval population surviving treatment with Shigella excretion product presents severe morphological effects. The larval population generally presents notable severe morphological damage, the necrosis state is represented by the opacity of the larvae after being treated for 24 h (b) compared to control. Other changes associated with larval anatomy were also observed; particularly the caudal end curvature was significant into 10%. The use of AO/EB revealed a distribution pattern from fluorescence into green and orange in surviving larvae SdyEP poisoning, there was a large population of dead cells around the anal and caudal region as evidenced by the presence of orange nuclei in greater numbers as controls in the larvae. The results support the application of coloring AO/EB in Zebrafish experimental models for the evaluation of the toxic action of new molecules and new products with therapeutic potential.

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de células, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excreción de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recolección de los huevos y larvas de pez cebra, así como la obtención del producto de la excreción, se realizaron de acuerdo a los estándares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 101, 10-2, 10-3, 10-4 y 10-5, v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del SdyEP, se expresó como dependiente de la concentracion y del tiempo de exposición. La población de larvas sobrevivientes, presentaron curvatura troncal en un 10%, en relación a los controles. La necrosis se puso en evidencia a través de la opacidad de las larvas después de 24 h. El uso de NA/BE reveló un patrón de distribución de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una granpoblación de células muertas, alrededor de la región anal y caudal, se ponen en evidencia por la presencia de núcleos de naranja en mayor número que en los controles. Los resultados apoyan la aplicación de la coloración NA/BE en modelo experimental de pez cebra, para la evaluación de la acción tóxica de nuevas moléculas y nuevos productos de excreción bacterial con potencial terapéutico.

Efflux-mediated resistance identified among norfloxacin resistant clinical strains of group B Streptococcus from South Korea / 한국역학회지

OBJECTIVES: Group B Streptococcus (GBS), a common bowel commensal, is a major cause of neonatal sepsis and an emerging cause of infection in immune-compromised adult populations. Fluoroquinolones are used to treat GBS infections in those allergic to beta-lactams, but GBS are increasingly resistant to fluoroquinolones. Fluoroquinolone resistance has been previously attributed to quinolone resistance determining regions (QRDRs) mutations. We demonstrate that some of fluoroquinolone resistance is due to efflux-mediated resistance. METHODS: We tested 20 GBS strains resistant only to norfloxacin with no mutations in the QRDRs, for the efflux phenotype using norfloxacin and ethidium bromide as substrates in the presence of the efflux inhibitor reserpine. Also tested were 68 GBS strains resistant only to norfloxacin not screened for QRDRs, and 58 GBS strains resistant to ciprofloxacin, levofloxacin or moxifloxacin. Isolates were randomly selected from 221 pregnant women (35-37 weeks of gestation) asymptomatically carrying GBS, and 838 patients with GBS infection identified in South Korea between 2006 and 2008. The VITEK II automatic system (Biomerieux, Durham, NC, USA) was used to determine fluoroquinolone resistance. RESULTS: The reserpine associated efflux phenotype was found in more than half of GBS strains resistant only to norfloxacin with no QRDR mutations, and half where QRDR mutations were unknown. No evidence of the efflux phenotype was detected in GBS strains that were resistant to moxifloxacin or levofloxacin or both. The reserpine sensitive efflux phenotype resulted in moderate increases in norfloxacin minimum inhibitory concentration (average=3.6 fold, range=>1-16 fold). CONCLUSIONS: A substantial portion of GBS strains resistant to norfloxacin have an efflux phenotype.

Uso alternativo del colorante Gelreden la tinción de ácidos nucleicos / Alternative use of gelred dye in the staining nucleic acids

Objetivo: Determinar la reproducibilidad en la tinción del ADN en geles de agarosa mediante el colorante GelRedTM con el fin de reemplazar el uso del bromuro de etidio en la tinción de ácidos nucleicos. Materiales y Métodos: Se realizó extracción de ADN total de sangre periférica a nueve muestras de pacientes con cáncer colorrectal,posteriormente se amplificó un fragmento de ADN de 494 pb asociado al exón 15 delgen APC y de seis fragmentos entre 150 pb y 350 pb asociados a los genes hMLH-1 y MSH-2; ambas preparaciones, ADN total y ADN amplificado fueron visualizadas mediante el uso del colorante GelRed y Bromuro de Etidio. Resultados: El uso del colorante GelRed para la tinción de ácidos nucleicos (ADN) permitió visualizar los de forma eficiente. Conclusión: El reactivo GelRed es altamente reproducible y se recomienda usarlo incluido en las muestras de ADN, para disminuir la contaminaciónen los laboratorios de biología molecular.

Excretion product of shigella dysenteriae (sdyep) induced cell death in early larval stage of zebrafish (danio rerio): acridine orange and ethidium bromide (ao/eb) in vivo staining / El producto de excreción de la shigella dysenteriae (pesdy) Induce muerte celular en larvas de pez cebra (danio rerio): marcaje in vivo con naranja de acridina y bromuro de etidio (na/be)

In the field of studies of acute toxicity induced by bacterial agents, Shiga toxins have been relevant due to the severity of the extra-intestinal diseases they cause. Numerous studies have shown that Shiga toxin induced apoptosis in different cell types; however, this important process has been little studied in vivo experimental models. In this study, the effects of excretion products of Shigella dysenteriae, in which Shiga toxin is present, we investigated on early larval stages of Zebrafish, an animal model with many advantages over other in vivo experimental models traditionally used. Both the collection of eggs and larvae of Zebrafish, and the product from excretion from Shigella dysenteriae (SdyEP) were performed according to laboratory standards. Also, toxicity bioassay, larvae treatment with pure and diluted solution, 10-1, 10-2, 10-3 , 10-4 and 10-5 v/v SdyEP and cell death in vivo using Acridine Orange (AO) and Ethidium Bromide (EB) were applied. The excretion product of Shigella dysenteriae (SdyEP) effect was expressed in terms of larval mortality and dependent dilution rather than incubation time. The larval population surviving treatment with Shigella excretion product presents severe morphological effects. The larval population generally presents notable severe morphological damage, the necrosis state is represented by the opacity of the larvae after being treated for 24 h (b) compared to control. Other changes associated with larval anatomy were also observed; particularly the caudal end curvature was significant into 10%. The use of AO/EB revealed a distribution pattern from fluorescence into green and orange in surviving larvae SdyEP poisoning, there was a large population of dead cells around the anal and caudal region as evidenced by the presence of orange nuclei in greater numbers as controls in the larvae. The results support the application of coloring AO/EB in Zebrafish experimental...

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de células, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excreción de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recolección de los huevos y larvas de pez cebra, así como la obtención del producto de la excreción, se realizaron de acuerdo a los estándares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 10-1, 10-2, 10-3 , 10-4 and 10-5 v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del PESdy, se expresó como dependiente de la concentración y del tiempo de exposición. La población de larvas sobrevivientes, presentaron curvatura troncal en un 10 por ciento, en relación a los controles. La necrosis se puso en evidencia a través de la opacidad de las larvas después de 24 h. El uso de NA/BE reveló un patrón de distribución de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una gran población de células muertas, alrededor de la región anal y caudal, se ponen en evidencia por la presencia de núcleos de naranja en mayor número que en los controles. Los resultados apoyan la aplicación de la coloración NA/BE en modelo experimental...

Decreased astrocytic GFAP expression in streptozotocin-induced diabetes after gliotoxic lesion in the rat brainstem / Expressão astrocitária diminuída de GFAP no diabetes induzido por estreptozotocina após lesão gliotóxica no tronco encefálico de ratos

OBJECTIVE: The aim of this study was to evaluate the effect of diabetic hyperglycemia on astrocyte function, estimated by means of glial fibrillary acidic protein - GFAP - immunohistochemical expression. MATERIALS AND METHODS: Adult male rats received a single intravenous injection of streptozotocin (50 mg/kg) and were submitted 10 days later to a single injection of 10 microlitres 0.1% EB solution or 0.9% saline solution into the cisterna pontis. Ten microliters of 0.1% EB or 0.9% saline solution were also injected in non-diabetic rats. Animals were anesthetized and perfused through the heart 15 and 31 days after EB or saline injection, and brainstem sections were collected for ultrastructural analysis and GFAP immunohistochemical staining. RESULTS: The GFAP brown-stained areas were evaluated by colorimetry using a computerized image analysis system and the results have shown that diabetes hindered the increase of GFAP astrocyte expression in the EB-injected group compared to non-diabetic animals. However, diabetes did not affect GFAP response in the saline-injected group or in control animals. CONCLUSION: Streptozotocin-induced diabetic condition reduced astrocytic GFAP expression following gliotoxic injury.

OBJETIVO: O objetivo deste estudo foi avaliar o efeito da hiperglicemia na função astrocitária, estimada pela expressão imuno-histoquímica da proteína glial fibrilar ácida - GFAP. MATERIAIS E MÉTODOS: Ratos machos adultos receberam uma injeção intravenosa única de estreptozotocina (50 mg/kg) e foram submetidos, 10 dias após, à injeção de 10 microlitros de solução de BE 0,1% ou de salina 0,9% na cisterna pontina. Dez microlitros de BE 0,1% ou salina 0,9% foram também injetados em ratos não diabéticos. Os animais foram anestesiados e perfundidos por via intracardíaca aos 15 e 31 dias pós-injeção de BE ou salina, e amostras de tronco encefálico foram coletadas para estudo ultraestrutural e análise imuno-histoquímica para a GFAP. RESULTADOS: Utilizando um sistema computadorizado de análise de imagens, os resultados das áreas coradas em marrom pela GFAP, medidas por colorimetria, mostram que o diabetes reduziu o aumento de expressão dessa proteína no grupo injetado com BE em comparação aos animais não diabéticos, mas não alterou a resposta no grupo injetado com salina ou nos controles diabéticos. CONCLUSÃO: O estado diabético induzido pela estreptozotocina reduziu a expressão astrocitária de GFAP após dano gliotóxico.

Prodigiosin found in Serratia marcescens y2 initiates phototoxicity in the cytomembrane

Background: Light can be absorbed by bacterial pigment and affects its growth. Prodigiosin is a red pigment found in various bacterial species. The purpose of this study was to investigate the impacts of light on prodigiosin production, biomass formation, and membrane integrity of Serratia marcescens y2. Results: S. marcescens y2 grew better and produced more intracellular prodigiosin in darkness than in illumination. The pigment leakage ratio from cells was detected more in light than in darkness conditions. Ethidium bromide uptake assay could visually prove the prodigiosin-related loss of membrane integrity under illumination. A higher concentration of malondialdehyde (MDA) was detected in light-treated culture than in darkness. Tests of different light treatments (red, yellow, blue and green) showed that the maximum extracellular pigment and the minimum biomass formation and intracellular pigment were obtained in green light. Conclusions: Prodigiosin could absorb light, and then initiate phototoxicity damage of the cytomembrane.

Blood-brain barrier breakdown and repair following gliotoxic drug injection in the brainstem of streptozotocin-diabetic rats / Ruptura e reparo da barreira hematoencefálica pós-injeção de droga gliotóxica no tronco encefálico ratos diabéticos

Ethidium bromide (EB) causes local astrocytic disappearance, with glia limitans disruption and blood-brain barrier (BBB) breakdown. The aim of this study was to evaluate the BBB integrity after the injection of 0.1 percent EB or 0.9 percent saline solution into the cisterna pontis of Wistar rats submitted or not to the streptozotocin diabetogenic model. Brainstem sections were collected from 24 hours to 31 days post-injection for ultrastructural analysis and glial fibrillary acidic protein immunohistochemical staining. Some animals received colloidal carbon ink by intravenous route at the same periods. In rats injected with EB, results revealed astrocyte disappearance and leakage of carbon particles beginning at 48 hours and persisting for 7 days in non-diabetic rats and for 15 days in the diabetic ones, although, in both groups, several areas remained devoid of astrocytic processes up to 31 days. In rats injected with saline, there was no sign of astrocytic loss or carbon particles leakage.

O brometo de etídio (BE) determina o desaparecimento local de astrócitos, com ruptura da glia limitans e dano na barreira hematoencefálica (BHE). Este estudo visou avaliar a integridade da BHE após injeção de solução de BE a 0,1 por cento ou de salina a 0,9 por cento na cisterna pontis de ratos Wistar submetidos ou não ao modelo diabetogênico da estreptozotocina. Fragmentos do tronco encefálico foram coletados das 24 horas aos 31 dias pós-injeção para estudo ultraestrutural e marcação imuno-histoquímica para proteína glial fibrilar ácida. Alguns animais receberam carvão coloidal por via intravenosa nos mesmos períodos. Nos grupos injetados com BE, os resultados mostraram desaparecimento astrocitário e extravasamento de partículas de carvão nas lesões a partir das 48 horas, persistindo por até sete dias nos animais não diabéticos e 15 dias nos diabéticos, embora, em ambos os grupos, diversas áreas permanecessem destituídas de astrócitos até 31 dias após. Nos ratos injetados com salina, diabéticos ou não, não houve sinal de perda astrocitária nem de extravasamento vascular de carvão.

Antioxidant activities of licorice-derived prenylflavonoids

Glycyrrhiza uralensis (or licorice) is a widely used Oriental herbal medicine from which the phenylflavonoids dehydroglyasperin C (DGC), dehydroglyasperin D (DGD), and isoangustone A (IsoA) are derived. The purpose of the present study was to evaluate the antioxidant properties of DGC, DGD, and IsoA. The three compounds showed strong ferric reducing activities and effectively scavenged DPPH, ABTS+, and singlet oxygen radicals. Among the three compounds tested, DGC showed the highest free radical scavenging capacity in human hepatoma HepG2 cells as assessed by oxidant-sensitive fluorescent dyes dichlorofluorescein diacetate and dihydroethidium bromide. In addition, all three compounds effectively suppressed lipid peroxidation in rat tissues as well as H2O2-induced ROS production in hepatoma cells. This study demonstrates that among the three phenylflavonoids isolated from licorice, DGC possesses the most potent antioxidant activity, suggesting it has protective effects against chronic diseases caused by reactive oxygen species as well as potential as an antioxidant food additive.

Evaluation of DNA damage in oral precancerous and squamous cell carcinoma patients by single cell gel electrophoresis.

Context: Single cell gel electrophoresis (SCGE) or "comet assay" is a rapid and very sensitive fluorescent microscopic method for detecting various forms of DNA damage at individual cell level. Aims: The aim of the present study was to detect the extent of DNA damage in oral cancer, oral submucous fibrosis (OSMF) and leukoplakia in comparison to normal individual. Settings and Design: A total of 44 consecutive patients with oral cancer (n=26), leukoplakia (n=12) and OSMF (n=6) and 10 healthy normal volunteers with normal oral epithelia (controls) were recruited from Dr. R. Ahmed Dental College and Hospital and were assessed for the extent of DNA damage using SCGE following clinical diagnosis. Materials and Methods: Peripheral blood was collected by venepuncture and comet assay was performed using SCGE. Mean tail length was compared between diagnostic groups and between different oral habit groups using t-tests and analysis of variance (ANOVA). Pearson's product moment correlation was used to examine the linear association between the extent of DNA damage and oral habit pack-years. Scheffe's pair-wise test was employed to adjust for multiple comparisons. Results: None of the controls were associated with any oral habits. Mean (±SD) tail lengths (in mm) for cancer (24.95 ± 5.09) and leukoplakia (12.96 ± 2.68) were significantly greater than in controls (8.54 ± 2.55, P<0.05). After adjustment, well-, moderately, and poorly differentiated carcinomas had significantly greater tail length than controls. Whereas the extent of DNA damage in cancer cases was significantly greater in leukoplakia than in compared to OSMF (11.03 ± 5.92), the DNA damage in latter was not different from controls. DNA damage for people with any oral habit (19.78 ± 7.77) was significantly greater than those with no habits (8.54 ± 2.55; P<0.0001). Conclusions: DNA damage measured by SCGE is greater in leukoplakia and squamous cell carcinoma, but not in OSMF. Deleterious oral habits are also associated with greater DNA damage.

Semi-quantitative analysis of the effects of cyclosporine on remyelination following gliotoxic injection in the brainstem / Análise semiquantitativa dos efeitos da ciclosporina na remielinização após injeção gliotóxica no tronco encefálico

The use of cyclosporine (CsA) has shown to induce an increase in the density of oligodendrocytes near remyelinating areas following the injection of ethidium bromide (EB), a demyelinating agent, in the rat brainstem. This study was designed in order to evaluate if CsA has the capacity of increasing remyelination. In this context, a comparison between the final balance of myelin repair in CsA treated and non-treated rats was assessed using a semi-quantitative method developed for documenting the extent and nature of remyelination in gliotoxic lesions. Wistar rats were submitted to intracisternal injection of 10 microliters of 0.1 percent EB. Some were treated during 31 days with CsA (group III - 10 mg/kg/day by 7 days and, thereafter, 3 times a week, with a minimal interval of 48 hours) by intraperitonial route. Others were not treated with CsA (group I). A control group was planned receiving into the cisterna pontis 10 microliters of 0.9 percent saline solution and following after that the same CsA administration protocol (group II). Results clearly demonstrate that in vivo administration of CsA after EB-demyelinating lesions stimulated oligodendrocyte remyelination (mean remyelination scores of 3.72±0.25 for oligodendrocytes and 1.04±0.39 for Schwann cells) compared to non-treated animals (3.13±0.71 and 1.31±0.62, respectively), although the mechanisms by which this positive CsA effect occurs are unclear.

O uso de ciclosporina (CsA) mostrou induzir um aumento na densidade de oligodendrócitos próximos a áreas de remielinização após injeção de brometo de etídio (EB), um agente desmielinizante, no tronco encefálico de ratos. Este estudo foi desenvolvido a fim de avaliar se a CsA possui a capacidade de acelerar a remielinização. Neste contexto, foi feita uma comparação entre o balanço final de reparo mielínico em ratos tratados ou não com CsA usando-se um método semiquantitativo desenvolvido para documentação da extensão e natureza da remielinização em lesões gliotóxicas. Ratos Wistar foram submetidos à injeção intracisternal de EB a 0,1 por cento. Alguns foram tratados durante 31 dias com CsA (grupo III - 10 mg/kg/dia por 7 dias e, após, 3 vezes por semana, com um intervalo mínimo de 48 horas entre as aplicações) por via intraperitoneal. Outros não foram tratados com CsA (grupo I). Um grupo controle foi desenvolvido recebendo, na cisterna pontina, 10 microlitros de solução salina e seguindo após o mesmo protocolo de administração de CsA (grupo II). Os resultados mostram claramente que a administração in vivo de CsA após lesões desmielinizantes induzidas pelo EB estimulou a remielinização por oligodendrócitos (escores médios de remielinização de 3,72±0,25 para oligodendrócitos e 1,04±0,39 para células de Schwann) em comparação aos animais não-tratados (3,13±0,71 e 1,31±0,62, respectivamente), embora os mecanismos pelos quais este efeito positivo da CsA ocorre sejam desconhecidos.