Effects of selective inhibition of nNOS and iNOS on neuropathic pain in rats.

Various animal models have been employed to understand the pathogenic mechanism of neuropathic pain. Nitric oxide (NO) is an important molecule in nociceptive transmission and is involved in neuropathic pain. However, its mechanistic actions remain unclear. The aim of this study was to better understand the involvement of neuronal and inducible isoforms of nitric oxide synthase (nNOS and iNOS) in neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve in rats. We evaluated pain sensitivity (mechanical withdrawal thresholds using Randall and Selitto, and von Frey tests, and thermal withdrawal thresholds using Hargreaves test) prior to CCI surgery, 14 days post CCI and after intrathecal injections of selective nNOS or iNOS inhibitors. We also evaluated the distribution of NOS isozymes in the spinal cord and dorsal root ganglia (DRG) by immunohistochemistry, synthesis of iNOS and nNOS by Western blot, and NO production using fluorescent probe DAF-2 DA (DA). Our results showed higher number of nNOS and iNOS-positive neurons in the spinal cord and DRG of CCI compared to sham rats, and their reduction in CCI rats after treatment with selective inhibitors compared to non-treated groups. Western blot results also indicated reduced expression of nNOS and iNOS after treatment with selective inhibitors. Furthermore, both inhibitors reduced CCI-evoked mechanical and thermal withdrawal thresholds but only nNOS inhibitor was able to efficiently lower mechanical withdrawal thresholds using von Frey test. In addition, we observed higher NO production in the spinal cord and DRG of injured rats compared to control group. Our study innovatively shows that nNOS may strongly modulate nociceptive transmission in rats with neuropathic pain, while iNOS may partially participate in the development of nociceptive responses. Thus, drugs targeting nNOS for neuropathic pain may represent a potential therapeutic strategy.

Systemic and Gastrohepatic Mucormycosis in Dogs.

We describe two cases of mucormycosis with systemic and gastrohepatic involvement in two male poodles. Respiratory, neurological and gastrointestinal signs progressed to death within 3 and 19 days of the onset of clinical signs, respectively. In case 1, there was systemic disease affecting the lungs, heart and brain. The lesions were characterized by yellow or red, raised, irregular areas that extended into deeper tissue from the surface. In case 2, there was gastric rupture; the margins of the rupture and the gastric mucosa were covered by a thick, white, friable material. In the liver, there were multiple yellow-white cavitated nodules. Histologically, pyogranulomas occurred in the affected organs and were associated with vasculitis, thrombosis and fungal hyphae. The diagnosis of mucormycosis was based on the characteristic microscopical lesions together with the morphology and staining features of the fungus. The hyphae were strongly labelled by monoclonal antibody specific for Rhizopus arrhizus on immunohistochemistry. Underlying immunosuppression was suspected in both cases.

Aspectos clínicos e terapêuticos de infecção por Mycobacterium bovis em gato doméstico - relato de caso / Clinical and therapeutic aspects of infection by Mycobacterium bovis in domestic cat - case report

Este artigo tem como objetivo relatar um caso de infecção por Mycobacterium bovis em um gato doméstico, com seis meses de idade, enfatizando seus aspectos clínicos e terapêuticos. O felino apresentava como histórico a ingestão de leite de vaca não fervido e posteriormente desenvolveu sinais de anorexia, tosse não produtiva e linfadenomegalia. Para elucidação do diagnóstico, foram realizados exames complementares de imagem, hematológicos, citológicos e reação em cadeia da polimerase (PCR). Diante da confirmação de infecção por Mycobacterium, o tratamento foi instituído por meio da associação de três antimicrobianos (enrofloxacino 5mg/kg, claritromicina 10mg/kg e rifampicina 15mg/kg), por via oral, a cada 12 horas, aos quais o animal respondeu positivamente, apresentando significativa diminuição dos sinais clínicos; entretanto, após 60 dias de tratamento, foi a óbito devido a um atropelamento. Conclui-se que a realização do tratamento em gatos com tuberculose pode ocasionar melhora dos sinais clínicos e que, embora de ocorrência rara no Brasil, essa enfermidade deve ser incluída como diagnóstico diferencial das enfermidades infecciosas nessa espécie.(AU)

This article aims to report a case of Mycobacterium bovis infection in a six-month-old domestic cat, emphasizing its clinical and therapeutic aspects. The feline presented as history the intake of unboiled cow's milk and later developed signs of anorexia, non-productive cough and lymphadenomegaly. Additional imaging, hematological, cytological and polymerase chain reaction (PCR) tests were performed to elucidate the diagnosis. In the presence of Mycobacterium infection, treatment was instituted by the combination of three antimicrobials (Enrofloxacin 5mg/kg, Clarithromycin 10mg/kg, and Rifampicin 15mg/kg) orally every 12 hours, to which the animal responded positively, presenting significant decrease of the clinical signs, however after 60 days of treatment the cat died accidentally, hit by a car on the street. It is concluded that the treatment in cats affected by tuberculosis can cause improvement of clinical signs and that although rare in Brazil, this disease should be included as a differential diagnosis of infectious diseases in this species.(AU)

Social stress in adolescents induces depression and brain-region-specific modulation of the transcription factor MAX.

MAX is a conserved constitutive small phosphoprotein from a network of transcription factors that are extensively studied in tumorigenesis and whose functions affect cell proliferation, differentiation and death. Inspired by its higher expression during development and in regions involved in emotional behaviors, we hypothesized its involvement in cerebral changes caused by early-life stress. We studied the effects of repeated social stress during adolescence on behaviors and on MAX and its putative partner MYC. Thirty-day-old C57BL/6 male mice underwent brief daily social defeat stress from an adult aggressor for 21 days. Following social stress episodes and housing in social groups after each defeat, adolescent mice exhibit depressive-like, but not anxiety-like behaviors and show higher MAX nuclear immunoreactivity in hippocampal (HC) but not prefrontal cortical (PFC) neurons. Conversely, MAX immunoreactivity is lower in the striatum (ST) of defeated adolescents. The positive correlation between MAX and MYC levels in the PFC revealed disruptions in both the HC and ST. The changes in MAX protein levels are not due to differential gene expression or protein degradation in those regions, suggesting that posttranscriptional modifications occurred. These findings indicate that repeated, brief social defeat in adolescent male mice, combined with group housing, is a useful protocol to study a subtype of depression that is dissociated from generalized (non-social) anxiety. To our knowledge, this is the first report of an association between dysregulation of the MAX-MYC network in the brain and a behavior, suggesting a novel approach for exploiting the neuroplasticity associated with depression.

A survey of tick-borne pathogens in dogs and their ticks in the Pantanal biome, Brazil.

Tick and blood samples collected from domestic dogs in the Brazilian Pantanal were tested by molecular methods for the presence of tick-borne protozoa and bacteria. Among 320 sampled dogs, 3.13% were infected by Babesia vogeli (Piroplasmida: Babesiidae), 8.75% by Hepatozoon canis (Eucoccidiorida: Hepatozoidae), 7.19% by Anaplasma platys (Rickettsiales: Anaplasmataceae), and 0.94% by an unclassified Anaplasma sp. In three tick species collected from dogs, the following tick-borne agents were detected: (a) B. vogeli, An. platys and Ehrlichia canis (Rickettsiales: Anaplasmataceae), infecting Rhipicephalus sanguineus sensu lato (Ixodida: Ixodidae) ticks; (b) H. canis, an unclassified Anaplasma sp. and Rickettsia amblyommii (Rickettsiales: Rickettsiaceae), infecting Amblyomma cajennense sensu lato (Ixodida: Ixodidae) ticks, and (c) Rickettsia sp. strain Atlantic rainforest, an emerging human pathogen, infecting Amblyomma ovale ticks. Molecular analysis, based on a mitochondrial gene, revealed that the Am. cajennense s.l. ticks of the present study corresponded to Amblyomma sculptum, a member of the Am. cajennense species complex, and that Rh. sanguineus s.l. belonged to the tropical lineage. Whereas dogs are exposed to a number of tick-borne bacterial and protozoan agents in the Pantanal biome, humans are potentially exposed to infection by spotted fever group rickettsiae (e.g. R. amblyommii and Rickettsia sp. strain Atlantic rainforest) because both Am. sculptum and Am. ovale are among the most important human-biting ticks in Brazil.

Reconstruction of magnetic source images using the Wiener filter and a multichannel magnetic imaging system.

A system for imaging magnetic surfaces using a magnetoresistive sensor array is developed. The experimental setup is composed of a linear array of 12 sensors uniformly spaced, with sensitivity of 150 pT*Hz(-1/2) at 1 Hz, and it is able to scan an area of (16 × 18) cm(2) from a separation of 0.8 cm of the sources with a resolution of 0.3 cm. Moreover, the point spread function of the multi-sensor system is also studied, in order to characterize its transference function and to improve the quality in the restoration of images. Furthermore, the images are generated by mapping the response of the sensors due to the presence of phantoms constructed of iron oxide, which are magnetized by a pulse of 80 mT. The magnetized phantoms are linearly scanned through the sensor array and the remanent magnetic field is acquired and displayed in gray levels using a PC. The images of the magnetic sources are reconstructed using two-dimensional generalized parametric Wiener filtering. Our results exhibit a very good capability to determine the spatial distribution of magnetic field sources, which produce magnetic fields of low intensity.

Evaluation of serological cross-reactivity between canine visceral leishmaniasis and natural infection by Trypanosoma caninum.

In order to evaluate if the presence of Trypanosoma caninum can lead to a confuse diagnosis of canine visceral leishmaniasis (CVL), we investigated the serological status of dogs infected by T. caninum and assessed the serological cross-reactivity with CVL. A set of 117 serum samples from dogs infected by T. caninum, Leishmania chagasi and not infected dogs (n=39 in each group) was tested using commercial kits--indirect immunofluorescence (IFI-LVC), ELISA (EIE-LVC) and immunochromatographic test (DPP)--and in house tests with T. caninum (IIF-Tc and ELISA-Tc) and L. chagasi antigens (IIF-Lc and ELISA-Lc). IIF-Tc and ELISA-Tc presented sensitivity of 64.1% and 94.9% and specificity of 23.1% and 35.9%, respectively. The sensitivity of the IFI-LVC, EIE-LVC and DPP tests was 100% and the specificity was 70.5%, 68% and 97.5% respectively. The concordance between the tests was considered as satisfactory. The specificities of IFI-LVC, EIE-LVC and DPP were higher when the group Tc was excluded, with significant values for IFI-LVC (χ2=4.36, P-value=0.036), thus suggesting that the infection by T. caninum can confuse the diagnosis of CVL.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection.

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 microg in 50 microL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed's lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48 percent by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469 percent). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20 percent), reaching the greatest increase (60 percent) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells.

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+]i) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of Galphaq/11-coupled M1, M3 and M5 receptors and intracellular calcium stores, whereas Galphai/o-protein coupled M2 receptor activity mediated neuronal differentiation.

Hernioplastia diafragmática em cão com pericárdio bovino conservado em solução supersaturada de açúcar / Diaphragmatic hernioplasty in dogs with bovine pericardium preserved in supersaturated sugar solution

Foram utilizados sete cães adultos, três machos e quatro fêmeas, sem raça definida, com pesos entre 10 e 22kg, para avaliação do processo cicatricial do músculo diafragma, na presença do implante de pericárdio bovino conservado em solução supersaturada de açúcar a 300 por cento. Foi criado um defeito na porção muscular do hemidiafragma esquerdo de dimensões 8,0 x 5,0cm. Após a toracotomia no oitavo espaço intercostal esquerdo, o implante heterógeno foi fixado com fio poliamida nº 3-0 por meio de sutura simples contínua. Decorrido o período pré-estabelecido de pós-operatório, os animais foram submetidos a exames radiográficos simples e contrastado e a estudos macroscópico e histológico. Na avaliação radiográfica, foi verificada presença das silhuetas diafragmática e cardíaca, sem evidências de vísceras abdominais no interior do tórax. Macroscopicamente, notou-se a formação de tecido conjuntivo fibroso semi-transparente que ocluia o defeito diafragmático. O segmento de pericárdio bovino conservado em solução supersaturada de açúcar a 300 por cento, em temperatura ambiente, é substituído por uma fina camada de tecido conjuntivo fibroso e promove a restauração do defeito no músculo diafragma de cão

Reduçäo fechada e fixaçäo esquelética externa tipo I para tratamento de fraturas de tibiotarso em pombos domésticos (Columba livia) / Closed reduction and type-I external skeletal fixation for treatment of tibiotarsus fractures in domestic pigeons (Columba livia)

Estudaram-se a reduçäo fechada e a fixaçäo esquelética externa tipo I para o tratamento de fraturas de tibiotarso em 10 pombos domésticos (Columba livia) adultos. As aves foram anestesiadas com a associaçäo de xilazina e cetamina e em seguida foi realizada fratura do tibiotarso direito por pressäo digital sobre a diáfise do membro. Quatro pinos de Kirschner, dois proximais e dois distais à linha da fratura, foram inseridos percutaneamente através de ambas as corticais ósseas e, após reduçäo fechada da fratura, conectados externamente por uma barra de acrílico autopolimerizável na face lateral do membro. Em três aves foi observado radiograficamente desvio ósseo angular, porém, a funçäo do membro näo foi afetada. O tempo médio e o desvio-padräo para a cicatrizaçäo óssea foram 23,4± 3,0 dias. Os resultados demonstram que a reduçäo fechada e a aplicaçäo de fixador esquelético externo tipo I é um método efetivo para o tratamento de fraturas de tibiotarso em pombos domésticos (Columba livia)

Hepatitis B vaccine in infants: a randomized controlled trial comparing gluteal versus anterolateral thigh muscle administration.

A significantly diminished antibody response to hepatitis B vaccine has been demonstrated in adults when the buttock is used as the injection site. However, in Brazil, the buttock continues to be recommended as site of injection for intramuscular administration of vaccines in infants. In this age group, there are no controlled studies evaluating the immunogenicity of the hepatitis B vaccine when administered at this site. In the present study, 258 infants were randomized to receive the hepatitis B vaccine either in the buttock (n = 123) or in the anterolateral thigh muscle (n = 135). The immunization schedule consisted of three doses of hepatitis B vaccine (Engerix Bregister mark or target, 10 microg) at 2, 4 and 9 months of age. There were no significant differences in the proportion of seroconversion (99.3% x 99.2%), or in the geometric mean titer of ELISA anti-HBs (1,862.1 x 1,229.0 mIU/mL) between the two groups. This study demonstrates that a satisfactory serological response can be obtained when the hepatitis B vaccine is administered intramuscularly into the buttock.

Differential localization of acetylcholinesterase in relation to pre- and postsynaptic nicotinic receptors in the chick brain.

Immunofluorescence and confocal microscopy were combined to study the distribution of acetylcholinesterase in relation to the localization of the beta2 subunit of the nicotinic acetylcholine receptors in the chick brain. In several areas where the beta2 subunit is recognizably part of presynaptic receptors, the localization of acetylcholinesterase appeared not to overlap the localization of beta2. On the other hand, acetylcholinesterase and the beta2 subunit exhibited a strictly matching localization in areas where postsynaptic nicotinic receptors are known to be present. These data may represent a morphological substrate for possible differential actions of acetylcholinesterase at presynaptic and postsynaptic nicotinic sites.

Elimination of biliary stones through the urinary tract: a complication of the laparoscopic cholecystectomy.

The introduction and popularization of laparoscopic cholecystectomy has been accompanied with a considerable increase in perforation of gallbladder during this procedure (10% - 32%), with the occurrence of intraperitoneal bile spillage and the consequent increase in the incidence of lost gallstones (0.2% - 20%). Recently the complications associated with these stones have been documented in the literature. We report a rare complication occurring in an 81-year-old woman who underwent laparoscopic cholecystectomy and developed cutaneous fistula to the umbilicus and elimination of biliary stones through the urinary tract. During the cholecystectomy, the gall bladder was perforated, and bile and gallstones were spilled into the peritoneal cavity. Two months after the initial procedure there was exteriorization of fistula through the umbilicus, with intermittent elimination of biliary stones. After eleven months, acute urinary retention occurred due to biliary stones in the bladder, which were removed by cystoscopy. We conclude that efforts should be concentrated on avoiding the spillage of stones during the surgery, and that no rules exist for indicating a laparotomy simply to retrieve these lost gallstones.