Decontamination of genetically modified mice strains by embryo transfer for obtaining SPF colonies in a Brazilian animal facility

The introduction of new strains of mice in specific pathogen-free (SPF) animal facilities should be performed carefully to avoid breaking sanitary barriers. To meet this need, animals should be rederived to reduce infection risk and thus avoid research interference caused by loss of animal health status and welfare. The objective of this study was to implement mice embryo transfer in the laboratory mouse facility of the Department of Immunology at the Institute of Biomedical Sciences/University of São Paulo, Brazil. Embryo transfers were performed to rederive genetically modified mouse strains with undefined sanitary status, received from different research and educational institutions. Fertilized eggs at two-cell stage were obtained by natural means and transferred into the oviducts of SPF pseudo-pregnant female mice. All surgical procedures were performed under aseptic conditions. A total of 625 embryos were transferred into therecipients. 148 pups were born, of which 140 were reared. Viruses, bacteria and intestinal protozoa were eliminated using this technique. The improvement in the microbiological status of mice allowed their expansion in our SPF facility. With these results, we can stimulate the use of embryo transfer technique between rodent facilities in Brazil and thus encourage the distribution of better models to our scientific community.A introdução de novas linhagens de camundongos em biotérios livres de patógenos específicos (SPF) deve ser realizada com critérios para evitar a quebra das barreiras sanitárias. Dessa forma, os animais devem ser rederivados para reduzir os riscos de infecção e evitar as interferências provocadas pela perda do status sanitário e do bem-estar dos animais. O objetivo deste estudo foi implementar a transferência de embriões murinos no Biotério do Departamento de Imunologia do Instituto de Ciências Biomédicas da Universidade de São Paulo, Brasil. As transferências embrionárias foram realizadas para rederivar linhagens de camundongos geneticamente modificadas com status sanitário não conhecido, recebidas de diferentes instituições de pesquisa e de ensino. Os embriões em duas células foram obtidos pelos métodos naturais e transferidos para os ovidutos de fêmeas de camundongos SPF pseudoprenhas. Todos os procedimentos cirúrgicos foramrealizados sob condições assépticas. Um total de 625 embriões foram transferidos para as receptoras. Foram obtidos 148 filhotes nascidos vivos, destes 140 foram desmamados. Por meio desta técnica, foram eliminados vírus, bactérias e protozoários intestinais. A melhora no status microbiológico dos camundongos permitiu a expansão destes em nossa colônia SPF. Com esses resultados, podemos promover o uso da técnica de transferência de embriões entre os biotérios brasileiros e assim incentivar a distribuição de modelos mais adequados para a nossa comunidade científica

Perfis hematológico e bioquímico de ratos (Rattus norvegicus) mantidos sob sistema de ventilação microambiental

Previous studies reported that rats (Rattus norvegicus) kept under microenvironmental ventilation systems (MEV) present better productive and health parameters when compared to animals kept under general diluting ventilation (GDV). The objective of the present research trial was to evaluate hematological and biochemical profiles of rats kept under MVS. In order to achieve this objective, two different trials were designed: Trail 1 (E1), in which it was evaluated the reproductive performance of males and females submitted to two different air speed limits - FV1, from 0.03 to 0.26 m / sec and FV2, from 0.27 to 0.80 m / sec. In Trial 2 (E2) it was evaluated different bed change intervals (3, 5, 7 and 9 days), for males kept under constant air speed (0.5 m / sec). Values for hemogram and biochemical patterns of these animals were compared to those of rats kept under GDV. Results show statistical differences in some of the studied parameters not only for the comparison between GVD and E1 and GVD and E2, but also between both groups submitted to MEV (E1 and E2). However, values found for all studied parameters are inside the normal range reported for this species, what indicates that MEV does not induce important changes in the physiological parameters evaluated.Em estudos anteriores, demonstrou-se que ratos mantidos em sistema de Ventilação Microambiental (VMA) apresentaram parâmetros de produtividade e padrão sanitário melhores do que aqueles mantidos em sistema de Ventilação Geral Diluidora (VGD). Outra etapa dos experimentos foi determinar os parâmetros fisiológicos destes animais. O presente estudo foi realizado para avaliar os perfis hematológico e bioquímico de ratos mantidos sob o sistema de VMA. Para tanto, foram realizados dois experimentos diferentes, com ratos mantidos em VMA, quais sejam: Experimento 1 (E1), no qual foi avaliado o desempenho reprodutivo de machos e fêmeas, sob duas faixas de velocidade de ar (FV1 - de 0,03 a 0,26 m/s, e FV2 - de 0,27 a 0,80 m/s); Experimento 2 (E2), no qual foram avaliados diferentes intervalos de troca de cama (3, 5, 7 e 9 dias), para ratos machos mantidos a uma velocidade de ar constante de 0,5 m/s. Os valores do hemograma e de parâmetros bioquímicos destes animais foram comparados com os valores encontrados em ratos mantidos sob VGD. Os resultados obtidos demonstraram diferenças estatísticas em alguns dos parâmetros observados, tanto entre os sistemas VGD e VMA, como entre os diferentes grupos de VMA. Contudo, os valores encontrados em todos os parâmetros avaliados encontram-se dentro de faixas de variação normal para a espécie estudada, como é descrito na literatura. Isto indica que o emprego do sistema de VMA não induz alterações relevantes nos parâmetros fisiológicos estudados

Evaluation of the encephalitis etiopathogenesis caused by equine herpesvirus type 1 using a mouse model of neuroinfection

O herpesvirus equino tipo 1 (EHV-1) é um importante patógeno que causa doença respiratória, abortamento e desordens neurológicas em equinos. O presente estudo foi realizado visando estabelecer um modelo murino de infecção pelo EHV-1 para investigar a resposta do hospedeiro frente à infecção viral e as alterações neurológicas causadas por esse agente. Camundongos das linhagens BALB/c, BALB/c nude, C3H/HeJ, C57BL/6, C57BL/6 CD4-/- e C57BL/6 CD8-/- foram inoculados por via intranasal com as estirpes brasileiras A4/72, A9/92 e A3/97 do EHV-1. Neste estudo, associou-se a histopatologia, a imunoistoquímica e o método de transcrição reversa seguida pela PCR quantitativa em tempo real para investigar a relação entre a infecção pelo vírus com o desenvolvimento de lesões e a resposta de citocinas pró-inflamatórias no SNC de camundongos das diferentes linhagens. As estirpes brasileiras A4/72 e A9/92 do EHV-1 causaram infecção aguda e letal nas diferentes linhagens de camundongos isogênicos. Os sinais clínicos e neurológicos, tais como perda de peso, pelos arrepiados, postura arqueada, apatia, descarga nasal e ocular, dispnéia, desidratação e sialorréia apareceram entre o 2º e 3º dpi. Essas manifestações foram acompanhadas pelo aumento da sensibilidade a estímulos externos, convulsões, recumbência e morte. O vírus foi consistentemente isolado do SNC, pulmões, fígado, baço e timo de todos os camundongos com sinais neurológicos. As alterações histopatológicas consistiram de leptomeningite, hemorragia focal, ventriculite, degeneração e necrose neuronal, neuronofagia, inflamação não supurativa, gliose multifocal e infiltração perivascular de células polimorfonucleares e mononucleares. A análise imunoistoquímica demonstrou que as estirpes A4/72 e A9/92 do EHV-1 replicaram-se nos neurônicos do bulbo olfatório, cortex cerebral e no hipocampo. Ao contrário, os camundongos inoculados com a estirpe A3/97 do EHV-1 não apresentaram perda de peso ou quaisquer sinais clínicos ou neurológicos; entretanto, o vírus foi isolado dos pulmões no 3º dpi. As estirpes A4/72 e A9/92 do EHV-1 apresentaram tropismo pelo tecido nervoso com capacidade de neuroinvasão e neurovirulência. A estirpe A3/97 do EHV-1 não foi neurovirulenta, apesar de ter sido reisolada do SNC de camundongos BALB/c nude infectados. Detectou-se aumento da expressão de mRNA para TNF-α, IL-6 e CCL2 no SNC dos camundongos infectados pelo EHV-1 com 2 e 3 dpi; entretanto, não houve expressão de mRNA para IFN-γ. Os camundongos com o fundo genético C57BL/6, que apresentam predominantemente resposta do tipo Th1, mostraram níveis mais altos de expressão de mRNA para TNF-α, IL-6 e CCL2, quando comparados com os BALB/c. A gravidade dos sinais observados em camundongos infectados pode ser correlacionada com o pico destas citocinas pró-inflamatórias (TNF-α e IL-6) e da quimiocina CCL2, que são produzidas logo após a infecção viral por células residentes da glia e/ou infiltrativas no SNC. Esses achados indicam que as diferentes linhagens de camundongos isogênicos são susceptíveis a infecção por estirpes neuropatogênicas do EHV-1 e poderiam servir como modelo para o estudo da patogênese e dos mecanismos que contribuem no desenvolvimento da mieloencefalopatia herpética equina.Equid herpesvirus type 1 (EHV-1) is a major pathogen which causes respiratory disease, abortions and neurological disorders in horses. The present study was carried out to establish a murine model of EHV-1 infection and investigate host response against the virus and neurological disorders caused by this pathogen. BALB/c, BALB/c nude, C3H/HeJ, C57BL/6, C57BL/6 CD4-/- and C57BL/6 CD8-/- mice were intranasally inoculated with EHV-1 A4/72, A9/92 and A3/97 Brazilian strains. In this study, we combined histopathology, immunohistochemistry, and a quantitative real-time RT-PCR method to investigate the relationship between virus infection and the development of lesions and cytokine responses in the CNS of different strains of mice. Intranasal inoculation of EHV-1 A4/72 and A9/92 induced acute and lethal meningoencephalitis in mice. Clinical and neurological signs appeared between the 2nd and 3rd dpi and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was consistently recovered from the CNS and visceral organs of all mice with neurological symptoms. Histopathological changes consisted of leptomeningitis, focal hemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multi-focal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, cortical regions and hippocampus. In contrast, mice inoculated with the EHV-1 strain A3/97 showed neither weight loss nor apparent clinical or neurological signs of the disease; however, the virus was recovered from their lungs at 3 dpi. While EHV-1 strains A4/72 and A9/92 exhibited a high degree of tropism for the CNS with robust neuroinvasiveness and neurovirulence, the EHV-1 strain A3/97 was not neurovirulent despite being detected in the CNS of infected BALB/c nude mice. Increased mRNA levels of TNF-α, IL-6 and CCL2 were detected in the nervous tissue of EHV-1 infected mice at 2 and 3 dpi; however, IFN-γ mRNA was not consistently expressed. Mice with the background C57BL/6, which exhibit predominantly Th1-type responses, showed the highest levels of TNF-α, IL-6 and CCL-2 mRNA in the CNS, when compared to BALB/c mice. The severity of signs observed in infected mice could be correlated with the peak of these proinflammatory cytokines (TNF-α and IL-6) and the chemokine CCL2, which are produced early after viral infection by both cells infiltrating into the CNS from the periphery and/or glial resident cells. These findings indicate that several inbred mouse strains are susceptible to neuopathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to equine herpes myeloencephalopathy in horses

Quality of Adherence to the ARRIVE Guidelines in the Material and Methods Section in Studies Where Swine Were Used as Surgical Biomodels: A Systematic Review (2013–2018)

Over the last two decades, pigs have become animal biomodels widely used for the investigation and practice of surgical techniques because of their great physiological and anatomical similarities to humans. Even though many of these studies must be carried out later in humans, the description of basic information is limited, making exact repetitions of the reported experimental methods impossible. In this review, 108 studies from 2013 to 2018 were considered to determine the quality of adherence to the ARRIVE guidelines in the reports of the methodologies. The majority of the studies lacked the details recommended in the ARRIVE guidelines regarding data directly related to the welfare of animals undergoing surgery and those about anesthetic protocols and analgesics. Information related to sample size calculation and housing and husbandry conditions was also very limited. We believe that the ARRIVE guidelines are an excellent tool for good-quality reporting. We encourage scientists to consistently use them as a tool to improve the quality of their scientific reports and, consequently, ensure animal welfare

Effect of the analgesics dipyrone, tramadol, and meloxicam on the behavior of laboratory rats

Awareness about the ethical use of laboratory animals has increased over recent decades alongside the development of strict guidelines and legislation concerning the welfare of animals in scientific research. Although minimization of animal suffering during experimentation is of paramount importance, it is sometimes neglected because of the presumed interference of analgesics on behavioral patterns. For this reason, comprehensive research with multiple pharmacological agents is vital if analgesics are to be included in experimental procedures. In this context, we aimed to evaluate the effects of the analgesics dipyrone (DIP), tramadol (TRA), meloxicam (MEL), and combinations thereof on the behavioral parameters of naïve laboratory rats. Forty-eight SPF male Wistar-Han rats were divided randomly into 6 equal groups (n = 8 per group) and treated by intraperitoneal injection with DI (178 mg/kg), TRA (17.8 mg/kg), MEL (1.5 mg/kg), DIP+TRA (178 + 17.8 mg/kg), MEL+TRA (1.5 + 17.8 mg/kg) or saline (SAL; control). The behavior of the treated animals was assessed 30 minutes after injection of analgesic by sequential submission to Open Field, Elevated Plus Maze, and Grooming Transfer tests. Treatment with DIP+TRA provoked the most significant alterations in rat behavior by reducing locomotion, rearing, and grooming. Although treatment with DI alone diminished exploratory behavior, the effect on grooming was not remarkable, thereby suggesting that DI has a suppressive motor effect, possibly caused by its action on the endocannabinoid system, which is potentiated by TRA. In the Open Field tests, the grooming behavior of all TRA-treated animals was reduced while treatment with MEL increased locomotion. Given that analgesia is an ethical duty, the experimental design should always consider the behavioral effects of the drugs themselves.Universidad de Costa Rica/[723-B9-197]/UCR/Costa RicaUniversidad de Costa Rica/[837-B7-603]/UCR/Costa RicaUniversidad de Costa Rica/[837-B8-123]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Laboratorio de Ensayos Biológicos (LEBI)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Neurociencias (CIN)UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Cirugía y Cáncer (CICICA)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

Equid herpesvirus type-1 exhibits neurotropism and neurovirulence in a mouse model

Intranasal inoculation of equid herpesvirus type-1 (EHV-1) Brazilian strains A4/72 and A9/92 induced an acute and lethal infection in four different inbred mouse strains. Clinical and neurological signs appeared between the 2nd and 3rd day post inoculation (dpi) and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was recovered consistently from the brain and viscera of all mice with neurological signs. Histopathological changes consisted of leptomeningitis, focal haemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multifocal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, the cortex and the hippocampus. In contrast, mice inoculated with the EHV-1 Brazilian strain A3/97 showed neither weight loss nor apparent clinical or neurological signs; however, the virus was recovered consistently from their lungs at 3 dpi. These three EHV-1 strains showed distinct degrees of virulence and tissue tropism in mice. EHV-1 strains A4/72 and A9/92 exhibited a high degree of central nervous system tropism with neuroinvasion and neurovirulence. EHV-1 strain A3/97 was not neurovirulent despite being detected in the brains of infected BALB/c nude mice. These findings indicate that several inbred mouse strains are susceptible to neuropathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to EHV-induced myeloencephalopathy in horses. (C) 2011 Elsevier Ltd. All rights reserved.Sao Paulo Research Foundation (FAPESP) [2009/51886-3, 2007/58861-0]Sao Paulo Research Foundation (FAPESP)National Council for Scientific and Technological Development (CNPq)National Council for Scientific and Technological Development (CNPq) [473735/2008-3

Hematological and biochemical profiles of rats (Rattus norvegicus) kept under microenvironmental ventilation system

Em estudos anteriores, demonstrou-se que ratos mantidos em sistema de Ventilação Microambiental (VMA) apresentaram parâmetros de produtividade e padrão sanitário melhores do que aqueles mantidos em sistema de Ventilação Geral Diluidora (VGD). Outra etapa dos experimentos foi determinar os parâmetros fisiológicos destes animais. O presente estudo foi realizado para avaliar os perfis hematológico e bioquímico de ratos mantidos sob o sistema de VMA. Para tanto, foram realizados dois experimentos diferentes, com ratos mantidos em VMA, quais sejam: Experimento 1 (E1), no qual foi avaliado o desempenho reprodutivo de machos e fêmeas, sob duas faixas de velocidade de ar (FV1 - de 0,03 a 0,26 m/s, e FV2 - de 0,27 a 0,80 m/s); Experimento 2 (E2), no qual foram avaliados diferentes intervalos de troca de cama (3, 5, 7 e 9 dias), para ratos machos mantidos a uma velocidade de ar constante de 0,5 m/s. Os valores do hemograma e de parâmetros bioquímicos destes animais foram comparados com os valores encontrados em ratos mantidos sob VGD. Os resultados obtidos demonstraram diferenças estatísticas em alguns dos parâmetros observados, tanto entre os sistemas VGD e VMA, como entre os diferentes grupos de VMA. Contudo, os valores encontrados em todos os parâmetros avaliados encontram-se dentro de faixas de variação normal para a espécie estudada, como é descrito na literatura. Isto indica que o emprego do sistema de VMA não induz alterações relevantes nos parâmetros fisiológicos estudados

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)