Induction of brain injury and depression in rats by chronic unpredictable stress associated with the augmentation of nitrosative stress and apoptosis / Inducción de lesión cerebral y depresión en ratas por estrés crónico impredecible asociado con el aumento del estrés nitrosativo y la apoptosis

SUMMARY: Repeated stress is a risk factor for memory impairment and neurological abnormalities in both humans and animals. We sought to investigate the extent of (i) brain tissue injury; (ii) nitrosative and oxidative stress in brain tissue homogenates; (iii) apoptotic and survival biomarkers in brain tissue homogenates; and (iv) immobility and climbing abilities, induced over a period of three weeks by chronic unpredictable stress (CUS). Wistar rats were either left untreated (Control group) or exposed to a variety of unpredictable stressors daily before being sacrificed after 3 weeks (model group). Assessment of depression-like behavior was performed and animals were then culled and harvested brain tissues were stained with basic histological staining and examined under light microscopy. In addition, brain tissue homogenates were prepared and assayed for these parameters; inducible nitric oxide synthase (iNOS), malondialdehyde (MDA), superoxide dismutase (SOD), caspase-3, and B-cell lymphoma 2 (Bcl-2). Histology images showed CUS induced profound damage to the cerebral cortex as demonstrated by severe neuronal damage with shrunken cells, disrupted atrophic nuclei, perineuronal vacuolation and swollen glial cells. CUS also significantly (p<0.05) induced iNOS, MDA, and caspase-3, whereas SOD and Bcl-2 brain tissue levels were inhibited by CUS. In addition, data from the depression-like behavior, forced swimming test showed significant (p<0.05) increase in animal immobility and decrease in climbing ability in the model group of rats. Thus, here we demonstrated a reliable rat model of chronic stress-induced brain injury, which can further be used to investigate beneficial drugs or agents used for a period of three weeks to protect against CUS-induced brain damage.

RESUMEN: El estrés crónico es un factor de riesgo para el deterioro de la memoria y las anomalías neurológicas tanto en humanos como en animales. Intentamos investigar el alcance de lesión del tejido cerebral; (ii) estrés nitrosativo y oxidativo en homogeneizados de tejido cerebral; (iii) biomarcadores apoptóticos y de supervivencia en homogeneizados de tejido cerebral; y (iv) inmovilidad y habilidades de escalada, inducidas durante un período de tres semanas por estrés crónico impredecible (ECI). Se dejaron sin tratamiento (grupo control) ratas Wistar, o se expusieron a una variedad de factores estresantes impredecibles diariamente antes de ser sacrificadas después de 3 semanas (grupo modelo). Se realizó una evaluación del comportamiento similar a la depresión y luego se sacrificaron los animales y se tiñeron los tejidos cerebrales con tinción histológica básica y se examinaron con microscopía óptica. Además, se prepararon homogeneizados de tejido cerebral y se analizaron los siguientes parámetros; óxido nítrico sintasa inducible (iNOS), malondialdehído (MDA), superóxido dismutasa (SOD), caspasa- 3 y linfoma de células B 2 (Bcl-2). Las imágenes histológicas mostraron que el CUS indujo un daño profundo en la corteza cerebral como lo demuestra el daño neuronal severo con células encogidas, núcleos atróficos alterados, vacuolación perineuronal y células gliales inflamadas. ECI también indujo significativamente (p <0,05) iNOS, MDA y caspase-3, mientras que los niveles de tejido cerebral SOD y Bcl-2 fueron inhibidos por ECI. Además, los datos del comportamiento similar a la de- presión, la prueba de natación forzada mostró un aumento significativo (p <0,05) en la inmovilidad animal y una disminución en la capacidad de escalada en el grupo modelo de ratas. Por lo tanto, aquí demostramos un modelo confiable de daño cerebral crónico en rata inducido por el estrés, que se puede utilizar para investigar medicamentos o agentes beneficiosos usados durante un período de tres semanas para proteger el daño cerebral inducido por ECI.

Comparative study on the efficacy of non-steroidal, steroid and non-use of anti-inflammatory in the treatment of acute epidemic conjunctivitis

Abstract Purpose To evaluate the effects of prednisolone against sodium diclofenac both with ciprofloxacin compared to artificial tears on the symptoms and signs of acute viral conjunctivitis. Methods Study included 37 patients diagnosed with acute conjunctivitis and distributed by three groups: A (1% prednisolone acetate + ciprofloxacin (0.3%); B (Sodium diclofenac (0.1%) + ciprofloxacin (0.3%) and C (artificial tears + ciprofloxacin (0.3%). Patients received medication 6/6 hours daily. Signs and symptoms (e.g. lacrimation, burning, photophobia, etc.) were scored at baseline and on the first, third, fifth and seventh days and in the end of treatment using a standardized questionnaire and slit lamp anterior segment examination. Results All three groups demonstrated an improvement in the signs and symptoms of conjunctivitis in their follow-up visits. There was no significant difference in symptom and sign scores between Group A and B and B and C in the study visits ( p >0.05). However, the comparison between groups A and C showed a clinical trend (p=0.05) on third evaluation suggesting better clinical action using the corticosteroids. Conclusion The prednisolone acetate was not superior to the use of sodium diclofenac or artificial tears in relieving the signs and symptoms of viral conjunctivitis.

Safety and efficacy of low intensity shockwave (LISW) treatment in patients with erectile dysfunction

ABSTRACT The primary goal in the management strategy of a patient with ED would be to determine its etiology and cure it when possible, and not just to treat the symptoms alone. One of the new therapeutic strategies is the use of low intensity extracorporeal shockwave (LISW) therapy. The mechanism of shockwave therapy is not completely clear. It is suggested that LISW induces neovascularization and improvement of cavernosal arterial flow which can lead to an improvement of erectile function by releasing NO, VEGF and PCNA. Materials and Methods: 31 patients between February and June 2013 with mild to severe ED and non-Phosphodiesterase 5 inhibitors responders were enrolled. Patients underwent four weekly treatment sessions. During each session 3600 shocks at 0.09mJ/ mm2 were given, 900 shocks at each anatomical area (right and left corpus cavernosum, right and left crus). Improvement of the erectile function was evaluated using the International Index of Erectile Function (IIEF-EF), the Sexual Encounter Profile (SEP) diaries (SEP-Questions 2 and 3) and Global Assessment Questions (GAQ-Q1 and GAQ-Q2). Results: At 3-month follow-up IIEF-EF scores improved from 16.54±6.35 at baseline to 21.03±6.38. Patients answering ‘yes’ to the SEP-Q2 elevated from 61% to 89% and from 32% to 62% in the SEP-Q3. A statistically significant improvement was reported to the Global Assessment Questions (GAQ-Q1 and GAQ-Q2). Conclusion: In conclusion, we can affirm that LISW is a confirmed therapeutic approach to erectile dysfunction that definitely needs more long-term trials to be clarified and further verified.

Mesenteric lymph reperfusion exacerbates spleen injury caused by superior mesenteric artery occlusion shock

The intestinal lymph pathway plays an important role in the pathogenesis of organ injury following superior mesenteric artery occlusion (SMAO) shock. We hypothesized that mesenteric lymph reperfusion (MLR) is a major cause of spleen injury after SMAO shock. To test this hypothesis, SMAO shock was induced in Wistar rats by clamping the superior mesenteric artery (SMA) for 1 h, followed by reperfusion for 2 h. Similarly, MLR was performed by clamping the mesenteric lymph duct (MLD) for 1 h, followed by reperfusion for 2 h. In the MLR+SMAO group rats, both the SMA and MLD were clamped and then released for reperfusion for 2 h. SMAO shock alone elicited: 1) splenic structure injury, 2) increased levels of malondialdehyde, nitric oxide (NO), intercellular adhesion molecule-1, endotoxin, lipopolysaccharide receptor (CD14), lipopolysaccharide-binding protein, and tumor necrosis factor-α, 3) enhanced activities of NO synthase and myeloperoxidase, and 4) decreased activities of superoxide dismutase and ATPase. MLR following SMAO shock further aggravated these deleterious effects. We conclude that MLR exacerbates spleen injury caused by SMAO shock, which itself is associated with oxidative stress, excessive release of NO, recruitment of polymorphonuclear neutrophils, endotoxin translocation, and enhanced inflammatory responses.

Imunoexpressão hepática de marcadores inflamatórios e de resistência insulínica na esteato-hepatite não alcóolica / Immunohistochemistry pattern of hepatic inflammatory and insulin resistance markers in experimental model of nonalcoholic steatohepatitis

Introduction: The pathophysiology of nonalcoholic steatohepatitis (NAS) includes, basically, insulin resistance, inflammation and oxidative stress. Thus, a study of immunostaining for liver insulin, adiponectin, tumor necrosis factor alpha (TNF-α), and inducible nitric oxide synthase (iNOS) receptors was conducted. Objective: To expand the knowledge about the pathophysiological and molecular mechanisms underlying the experimental model of steatohepatitis in rats fed a high-fat diet. Method: Twenty Wistar rats were divided into two groups: G1 (control, fed a standard diet), and G2 (fed a high-fat diet containing 58% of energy derived from fat, 18% from protein and 24% from carbohydrate). After eight weeks the animals were sacrificed. Blood glucose, insulin, total cholesterol, high-density lipoprotein (HDL), the very low-density lipoproteins (VLDL), triglycerides, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) were determined. The liver tissue was submitted to histopathological analysis, using a NAS score. In immunohistochemistry, we studied the expression of the insulin receptor, adiponectin, TNF-α and iNOS by tissue microarray method. Results and conclusion: There was marked cytoplasmic immunostaining for TNF-α and iNOS mediators in the group on a fat diet. Regarding insulin and adiponectin molecular markers, a reduction of cytoplasmic immunoreactivity of these antigens was observed in the group on a fat diet, reflecting, respectively, the state of hepatocellular inflammation (steatohepatitis) and insulin resistance in this experimental model of fat liver disease...

Introdução: Os mecanismos fisiopatológicos da esteato-hepatite não alcoólica incluem basicamente resistência insulínica, processo inflamatório e estresse oxidativo. Desta forma, um estudo sobre o padrão de imunoexpressão hepática para receptores de insulina, adiponectina, fator de necrose tumoral alfa (TNF-α) e sintase indutível do óxido nítrico (iNOS) foi conduzido. Objetivo: Ampliar os conhecimentos sobre os mecanismos moleculares subjacentes, em modelo experimental de esteato-hepatite. Método: Vinte ratos Wistar com dois meses de idade, pesando de 250 a 300 mg foram subdivididos em dois grupos: G1 (controle normal, submetido à dieta padrão) e G2 (grupo-controle, submetido à dieta hiperlipídica contendo 58% de energia derivada de gorduras, 18% de proteínas e 24% de carboidratos). Após oito semanas, os animais foram sacrificados; o sangue, submetido à análise bioquímica; e o fígado, removido e fixado em formalina tamponada e emblocado em parafina para estudo histopatológico. Para estudo imuno-histoquímico, foi utilizada a técnica de microarranjo de tecido. As lâminas obtidas foram submetidas à incubação com os anticorpos contra adiponectina, receptor de insulina, TNF-α e iNOS. Resultados e conclusão: Observou-se marcada imunoexpressão citoplasmática para os mediadores TNF-α e iNOS no grupo submetido à dieta hiperlipídica. No que diz respeito aos marcadores moleculares insulina e adiponectina, observou-se uma redução da imunoexpressão citoplasmática desses anticorpos no grupo submetido à dieta hiperlipídica, traduzindo, respectivamente, o estado de inflamação hepatocelular (esteato-hepatite) e resistência insulínica, desenvolvidos nesse modelo experimental de doença hepática gordurosa...

Nitric oxide levels in cochlear fluids of guinea pig

PURPOSE: To describe a technique to obtain guinea pigs cochlear fluids and measure nitric oxide (NO) concentration. METHODS: Six guinea pigs were used and sacrificed. The cochlear fluids collected for measurement of NO, performed by chemiluminescence (NOA 280). RESULTS: Through the chemiluminescence was possible to analyze the concentration of NO in cochlear fluids obtained. Average levels of nitric oxide from guinea pigs was 12.55 µM. CONCLUSION: It is possible to obtain nitric oxide cochlear fluids, with this technique and nitric oxide concentration measure by chemiluminescence, a quantitative and more precise method.

Local lymphocytes and nitric oxide synthase in the uterine cervical stroma of patients with grade III cervical intraepithelial neoplasia

OBJECTIVES: Precancerous and cancerous cells can trigger an immune response that may limit tumor development and can be used as a prognostic marker. The aims of the present study were to quantify the presence of B and T lymphocytes, macrophages and cells expressing inducible nitric oxide synthase (iNOS) in the cervical stroma of women with grade III cervical intraepithelial neoplasia (CIN III) or in the intratumoral and peritumoral tissue of women with stage I invasive carcinoma. METHODS: Cervical tissue specimens were obtained from 60 women (20 each from control tissues, CIN III and invasive carcinomas). The average ages in the control, CIN III and invasive groups were 43.9 (± 4.3), 35.5 (± 9.5), and 50 (± 11.2) years, respectively. The specimens were immunohistochemically labeled with antibodies to identify T lymphocytes (CD3), cytotoxic lymphocytes (CD8), B lymphocytes (CD20), macrophages (CD68) and iNOS. We evaluated the markers in the stroma above the squamocolumnar junction (control), at the intraepithelial lesion (CIN cases), and in the nfiltrating tumor. Two independent observers performed the immunohistochemical analysis. RESULTS: T lymphocytes, B lymphocytes, macrophages and iNOS were present more frequently (P<0.05) in the stroma of peritumoral invasive tumors compared to the controls and intratumoral invasive cancer samples. CD3+ and CD20+ lymphocytes were present more frequently in CIN III patients compared to samples from patients with intratumoral invasive cancer (P<0.05). CONCLUSION: High numbers of T and B lymphocytes, macrophages and iNOS-expressing cells in the peritumoral stroma of the invasive tumors were observed. Cell migration appeared to be proportional to the progression of the lesion.

Ongoing angiogenesis in blood vessels of the abdominal aortic aneurysm

Pathogenesis of the abdominal aortic aneurysm has been attributed to neovascularization of the aortic wall. However, it is not clear whether angiogenesis persists in the aneurysm. In sections of aneurysms, we determined the immunohistochemical distributions of the alpha v beta 3 integrin, tenascin and endothelial nitric oxide synthase (eNOS), which are markers respectively, of angiogenesis, matrix remodeling and vasoregulatory function. In addition, we used reverse transcription followed by in situ PCR, to determine the distribution of alpha v mRNA. All aneurysm specimens exhibited extensive increases of wall vascularization as compared with the control aortic wall, and showed the presence of perivascular inflammatory exudates containing macrophages and lymphocytes. The neovascularization consisted of thick-walled vessels in the media and adventitia, and capillaries in the subintima. The majority of vessels stained positively for the alpha v beta 3 antigen and eNOS. Tenascin was deposited as bands that circumscribed thick-walled vessels. The distribution of av mRNA was extensive and was positive even in those vessels that failed to stain for the alpha v beta 3 protein. No staining was evident in control aortas for the alpha v beta 3 antigen, tenascin or alpha v mRNA. The upregulation of av mRNA and the alpha v beta 3 integrin in blood vessels surrounded by a matrix expressing tenascin, indicates that angiogenesis is an ongoing process in the mature aortic aneurysm.

Sensory Neuronal Change after Intravesical Electrical Stimulation in Spinailized Rat

The clinical benefits of intravesical electrical stimulation (IVES) in patients with increased residual urine or reduced bladder capacity have been reported. However, studies on the underlying mechanism of IVES has been limited to the A delta afferent and parasympathetic neurons. This study investigated the changes in the calcitonin gene-related peptide (CGRP), substance P (SP), and nitric oxide synthase (NOS) expression in the thoracolumbar and lumbosacral dorsal root ganglia (DRG) of spinalized rats to determine the effect of IVES on the C fiber afferent nerve. Forty Sprague-Dawley rats were divided into normal controls (n=10); IVES treated normal rats (n=10), spinalized rats (n=10), and IVES treated spinalized rats (n=10). IVES was performed for 2 weeks (5 days a week). IVES was started 3 weeks after spinalization in the spinalized animals. All animals had the DRG removed at the thoracolumbar (T13-L2) and lumbosacral (L5-S1) level. Changes in the CGRP, SP and n-NOS levels at the DRG were measured by western-blot analysis. The relative density of the CGRP and SP following spinalization was significantly higher compared to the controls in both the T13-L2 and L5-S1 DRG. However, IVES in the spinalized rat significantly decreased the relative density of the CGRP and SP compared to the rats with spinalization alone. A significant increase in the relative density of n-NOS was detected in the L5-S1 DRG following spinalization. However, the density of n-NOS was significantly lower after IVES in both the T13-L2 and L5-S1 DRGs. In conclusion, IVES significantly reduced the CGRP, SP and n-NOS levels in the DRG of spinalized rats. CGRP, SP and n-NOS are the main factors that contribute to the hyperexcitability of the micturition reflex after spinal cord injury. These results suggest that the bladder C fiber afferent is also involved in modulating the micturition reflex by IVES.

Immunohistochemical study of constitutive neuronal and inducible nitric oxide synthase in the central nervous system of goat with natural listeriosis

The expression of both constitutive and inducible forms of nitric oxide synthase (NOS) was investigated by immunohistochemical staining of formalin-fixed paraffin-embedded sections in normal and Listeria monocytogenes-infected brains of goats. In normal control goats, a small number of neurons showed immunoreactivity of both iNOS and nNOS, and the number of iNOS-positive neurons was higher than the number of nNOS-positive neurons. In natural listeriosis, listeria antigens were easily immunostained in the inflammatory cells of microabscesses. In this lesion, the immunoreactivity of iNOS in neurons was more intense than the control, but nNOS was not. In microabscesses, nNOS was weakly visualized in macrophages and neutrophils, while iNOS was expressed in macrophages, but not in neutrophils. These findings suggest that normal caprine brain cells, including neurons, constitutively express iNOS and nNOS, and the expressions of these molecules is increased in Listeria monocytogenes infections. Furthermore, inflammatory cells, including macrophages, expressing both nNOS and iNOS may play important roles in the pathogenesis of bacterial meningoencephalitis in goat.

Análise das alteraçöes do óxido nítrico em pacientes com megacólon chagásico / Analysis of the alterations of nitric oxide in the patient with megacolon in Chaga's disease

A fisiopatologia da Doença de Chagas ainda näo está completamente esclarecida. O óxido nítrico (NO) tem sido citado como neurotransmissor responsável pelo relaxamento do esfíncter interno do ânus no indivíduo normal. Neuronal nicotinamida adenina dinucleotídeo fosfato (NADPH) diaforase pode ser usado como marcador neuronal do NO. Objetivos: Examinar as alteraçöes nos neurônios produtores de NO do cólon de pacientes submetidos à ressecçäo por megacólon avançado e comparar com o intestino delgado dos mesmos pacientes e com controles. Métodos: Espécimes obtidos da ressecçäo do reto e biópsias extramucosas do intestino delgado de 11 pacientes chagásicos foram comparadas a 10 pacientes controles com câncer de cólon. Os tecidos foram fixados em soluçäo de Zamboni e submetidos à histoquímica para neurônios contendo NADPH diaforase. A reatividade foi avaliada utilizando-se uma escala de 0 a 4 nas diversas camadas da parede intestinal: musculatura lisa longitudinal (ML), plexo mioentérico (PM), musculatura lisa circular (MC), plexo submucoso (PSM), e mucosa (M). Resultados: Os casos controles mostraram os plexos mioentérico e submucoso bem corados e uma rica rede de terminaçöes nervosas nas camadas musculares. Os espécimes provenientes de doentes chagásicos revelaram uma diminuiçäo da reatividade e da coloraçäo em todas as camadas do intestino. De maneira geral, houve uma diminuiçäo estatisticamente significante nos neurônios contendo NADPH diaforase. O intestino delgado clinicamente näo envolvido também demonstrou diminuiçäo da reatividade, porém em menor grau. Conclusöes: A atividade da NADPH diaforase está diminuída em pacientes com megacólon avançado, especialmente no plexo mioentérico e na camada muscular lisa; 2. Houve também uma diminuiçäo da atividade neuronal do NO no jejuno clinicamente näo envolvido pela doença, mas em menor grau