Name: establishment of deep hypothermic circulatory arrest in rats
Uploaded: 2022-12-16T12:30:00
Description: Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Os autores agradecem a Liang Zhang por ajudar a coletar os dados de vídeo durante o experimento. Este estudo foi apoiado pela Fundação Nacional de Ciências Naturais da China (número de subvenção: 82070479) e os Fundos de Pesquisa Fundamental para as Universidades Centrais (número de subvenção: 3332022128).

Os protocolos passaram por uma revisão institucional e receberam aprovação do Comitê Institucional de Cuidados e Uso de Animais, Hospital Fuwai, Academia Chinesa de Ciências Médicas (FW-2021-0005). Todos os procedimentos experimentais foram realizados de acordo com o Guia para o Cuidado e Uso de Animais de Laboratório publicado pelos Institutos Nacionais de Saúde.
NOTA: Ratos Sprague-Dawley machos (peso: 500-600 g, idade: 12-14 semanas) foram mantidos em condições laboratoriais padr?...

<ol>
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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+profiles+of+circular+RNAs+in+the+rat+hippocampus+after+deep+hypothermic+circulatory+arrest.">Differential expression profiles of circular RNAs in the rat hippocampus after deep hypothermic circulatory arrest.</a> Artificial Organs. 45 (8), 866-880 (2021).</li><li>Linardi, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+versus+fast+rewarming+after+hypothermic+circulatory+arrest:+effects+on+neuroinflammation+and+cerebral+oedema.">Slow versus fast rewarming after hypothermic circulatory arrest: effects on neuroinflammation and cerebral oedema.</a> European Journal of Cardiothoracic Surgery. 58 (4), 792-780 (2020).</li><li>Engelman, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Society+of+Thoracic+Surgeons,+The+Society+of+Cardiovascular+Anesthesiologists,+and+The+American+Society+of+ExtraCorporeal+Technology:+Clinical+practice+guidelines+for+cardiopulmonary+bypass--Temperature+management+during+cardiopulmonary+bypass.">The Society of Thoracic Surgeons, The Society of Cardiovascular Anesthesiologists, and The American Society of ExtraCorporeal Technology: Clinical practice guidelines for cardiopulmonary bypass--Temperature management during cardiopulmonary bypass.</a> Annals of Thoracic Surgery. 100 (2), 748-757 (2015).</li><li>Jenke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AdipoRon+attenuates+inflammation+and+impairment+of+cardiac+function+associated+with+cardiopulmonary+bypass-induced+systemic+inflammatory+response+syndrome.">AdipoRon attenuates inflammation and impairment of cardiac function associated with cardiopulmonary bypass-induced systemic inflammatory response syndrome.</a> Journal of the American Heart Association. 10 (6), 018097 (2021).</li></ol>

A canulação é o procedimento mais fundamental para o estabelecimento de DHCA em ratos. Antes da canulação, a imersão da artéria com 0,5 mL de lidocaína a 2% facilitará a canulação. Após a canulação, a heparinização com 500 UI/kg de heparina via veia jugular externa é necessária para evitar a formação de microtrombos17. Descobrimos repetidamente que esta dose de heparina pode atingir o objetivo de um tempo de coagulação ativado (TCA) >480 s. O período de reaqueciment...

A parada circulatória hipotérmica profunda (DHCA) tem sido utilizada em cirurgia cardíaca desde 19531. A DHCA envolve a redução da temperatura central do paciente para níveis profundamente hipotérmicos (15-22 °C) antes de interromper globalmente o fluxo sanguíneo para o corpo2. A parada circulatória pode fornecer um campo operacional relativamente sem sangue. A hipotermia profunda diminui o metabolismo, principalmente no cérebro e no miocárdio, que é um método eficaz de proteção contra isquemia3. O DHCA é comumente aplicado durante cirurgias para cardiopatia congênita complexa, doença do arco aórtico e até mesmo tumores renais ou adrenais com trombo de veia cava 4,5. Portanto, o estabelecimento de modelos animais DHCA fornece uma referência importante para o refinamento do procedimento e a prevenção de complicações em ambientes clínicos.
Embora modelos possam ser estabelecidos com caninos6, coelhos7 e outros animais, é preferível o uso de ratos devido à sua operacionalidade e baixo custo. O modelo DHCA em ratos foi descrito pela primeira vez em 2006 por Jungwirth et al.8. Verificou-se que a duração da parada circulatória teve impacto nos desfechos neurológicos. Desde então, os modelos de ratos DHCA têm sido investigados amplamente. Esclareceu-se que o DHCA poderia provocar a síndrome da resposta inflamatória sistêmica (SRIS)9. Em estudos subsequentes, farmacologistas descobriram que a neuroinflamação relacionada ao DHCA induzida pela SIRS poderia ser atenuada pelo resveratrol10 e triptolide11. Nossa equipe também descobriu que a neuroinflamação relacionada ao DHCA poderia ser atenuada inibindo a proteína de ligação ao RNA induzível a frio12. No sistema cardiovascular, a superóxido dismutase tem efeito cardioprotetor nas lesões de isquemia/reperfusão (I/R) durante a DHCA13. Esses resultados expandiram a compreensão dos processos fisiopatológicos relacionados ao DHCA e ofereceram novas direções para melhorar os resultados do DHCA. No entanto, os resultados em relação à endotoxemia, estresse oxidativo e autofagia após DHCA são inconclusivos. A DHCA utiliza a mesma tecnologia operacional da circulação extracorpórea (CEC)14, mas sua estratégia de manejo é diferente, e as etapas para gerar a DHCA diferem entre as várias equipes 8,9,10,11. O presente estudo tem como objetivo fornecer um método para estabelecer o procedimento DHCA em ratos.

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establishment of deep hypothermic circulatory arrest in rats

A parada circulatória hipotérmica profunda (DHCA) é rotineiramente aplicada durante cirurgias para cardiopatia congênita complexa e doença do arco aórtico. O presente estudo tem como objetivo fornecer um método para o estabelecimento de DHCA em ratos. Para avaliar o impacto do processo DHCA sobre os sinais vitais, foi utilizado como controle um modelo de revascularização miopulmonar (CEC) de temperatura normal sem parada circulatória. Como esperado, o DHCA levou a uma diminuição significativa da temperatura corporal e da pressão arterial média. A gasometria indicou que o DHCA aumentou os níveis de ácido láctico, mas não influenciou o pH sanguíneo e as concentrações de hemoglobina, hematócrito, Na+, Cl−, K+ e glicose. Além disso, em comparação com os ratos CEC de temperatura normal, os resultados da microscopia eletrônica de transmissão mostraram um leve aumento nos autofagossomos hipocampais nos ratos DHCA.

Como grupo controle, os ratos CEC de temperatura normal (NtCPB) sem parada circulatória apresentaram pressão arterial (PAM) média estável e temperatura corporal durante todo o procedimento, enquanto a PAM dos ratos DHCA diminuiu durante a parada cardíaca (p < 0,01, Figura 3A). A temperatura dos ratos DHCA caiu rapidamente durante a fase de resfriamento e se recuperou gradualmente durante a fase de reaquecimento. Ao desmamar os ratos dos circuitos DHCA, a temperatura dos ratos D...

Watch this Scientific Journal Video about Establishment of Deep Hypothermic Circulatory Arrest in Rats at JoVE.com

Establishment of Deep Hypothermic Circulatory Arrest in Rats

Este protocolo apresenta o estabelecimento de parada circulatória hipotérmica profunda em ratos, que pode ser aplicada para investigar a síndrome da resposta inflamatória sistêmica, lesão de isquemia/reperfusão, estresse oxidativo, neuroinflamação, etc.

Estabelecimento de Parada Circulatória Hipotérmica Profunda em Ratos

Deep hypothermic circulatory arrest (DHCA) is routinely applied during surgeries for complex congenital heart disease and aortic arch disease. The present ...

Estabelecimento de parada circulatória hipotérmica profunda em ratos. Parada circulatória hipotérmica profunda, o DHCA é rotineiramente aplicado durante cirurgias de cardiopatia congênita complexa e doença do arco aórtico. O presente estudo tem como objetivo fornecer um método para o estabelecimento de DHCA em ratos.Os protocolos receberam uma revisão institucional e obtiveram aprovação do Comitê Institucional de Cuidados e Uso de Animais, Hospital Fuwai, Academia Chinesa de Ciências Médicas. Trabalhos preparatórios. Certifique-se de que os circuitos de CEC contenham um reservatório modificado a partir de transformadores conta-gotas, bomba de rolos, trocador de calor, oxigenador de membrana, tubo de conexão e um tanque de água conectado ao circuito e faça 12 mililitros de hidroxiamido com um mililitro de heparina sódica e um mililitro de solução salina preparar o circuito com solução de escorva de 40 mililitros com a bomba de rolos girando suavemente.Dois, anestesia e canulação. Conecte o tubo com o ventilador e os parâmetros laterais. Coloque um cobertor de aquecimento elétrico e o direito e encare o direito com fita adesiva.Aplique pomada oftálmica nos olhos para evitar a secura. Raspe o cabelo na região inguinal direita, região cervical direita e a cauda com o barbeador. Em seguida, desinfete a pele com aldeído e álcool.Corte a pele e a região inguinal esquerda e disseque o músculo e o tecido osso mole para expor a veia e artéria femoral esquerda. Separe a artéria com cuidado. Canulado cateter intravenoso calibre 22 na artéria femoral esquerda.Artéria ligada e cateter com seda. Use solução salina contendo heparina para lavar a cânula para evitar a coagulação. Conecte o cateter com o sensor de pressão para monitorar a pressão arterial.Corte a pele da cauda e, em seguida, use um bisturi para cortar a amostra superficial da artéria da cauda para expor a artéria da cauda que está no meio do campo cirúrgico. Anular a artéria caudal com o cateter intravenoso calibre 22. Artéria e cateter ligados.Use solução salina contendo heparina para lavar o cateter para evitar a coagulação. Corte a pele da jugular direita da veia e, em seguida, separe o músculo e o tecido para expor a veia. Insira um cateter intravenoso multiidade de calibre 16 na jugular externa direita da veia e coloque-o na veia cava inferior direita ou no átrio direito com cuidado.Administrar heparina sódica onde a veia externa direita cobre cada região canulada com bolas úmidas para evitar a contaminação. Três, conecte o circuito com o cateter na artéria da cauda e mantenha a bomba baixa taxa de um a dois mililitros por minuto. Em seguida, conecte o reservatório com o cateter na veia jugular externa direita.Certifique-se de que há sempre um nível sanguíneo de cerca de um centímetro no reservatório. Ligue o tanque de água e defina a temperatura da água em 37 centígrados primeiro. Depois que a pressão arterial estiver estável, aumente suavemente a bomba de fluxo até 80 a 100 mililitros por minuto.Quatro, arrefecimento. Defina a temperatura ambiente para cerca de 20 centígrados. Coloque cubos de gelo nestas luvas descartáveis e, em seguida, coloque-os na cabeça e nos lados direitos.A temperatura do tanque foi sugerida em tempo real de acordo com a temperatura retal do arroz. Alterar os parâmetros relevantes do ventilador adequadamente de acordo com os resultados da análise de gases no sangue. Cinco, hipotermia profunda parada circulatória.Em seguida, a temperatura retal cai para 15 a 20 centígrados. Troque o bloco de gelo e descarte as luvas para garantir a manutenção da hipotermia profunda durante a parada circulatória. Pare a bomba de roletes.Mantenha o reservatório em contato com o ambiente e drene o sangue lentamente da veia jugular externa para o reservatório. Preste atenção à reforma da pressão arterial. Depois que não houver pressão arterial e batimentos cardíacos, pare a drenagem e mantenha o reservatório fechado.Desligue o ventilador. Seis, aquecimento e reperfusão. Retire todas as luvas descartáveis e aumente a temperatura ambiente para 25 centígrados.Restaure a memória de oxigênio interventilação, mantendo o corte do tubo de drenagem venosa. Ligue a bomba de roletes para se certificar de que o sangue no reservatório está lentamente de volta ao corpo direito. Ligue o ventilador depois que o nível sanguíneo no reservatório permanecer um centímetro.Usando o tubo de drenagem e drene o sangue do átrio direito para o reservatório lentamente. Ligue a lâmpada de aquecimento, a almofada de aquecimento e o tanque de água. Defina a temperatura do tanque de água para 25 centígrados.Em seguida, ajuste-o para a temperatura esquerda oportunamente. Remova a lâmpada de aquecimento depois que a temperatura retal retornar a 34 centígrados. Sete, desmame da CEC.Reduza lenta e gradualmente o rolo da taxa de fluxo da bomba e ajuste a velocidade do gênero da veia na taxa de fluxo reduzida para um mililitro por minuto. Mantenha o reservatório em contato com o meio ambiente. Infundir o sangue restante em um circuito com uma taxa de fluxo de um mililitro por minuto.Pare a oxigenação da memória e a bomba de roletes. Eutanasiado após um período de ventilação mecânica e anestesia profunda. Resultados representativos.Como o grupo controle, a temperatura normal CEC, NtCPB, rato. Sem a parada circulatória houve média estável da PAM arterial arterial e da temperatura corporal durante todo o procedimento. Enquanto a PAM de ratos DHCA diminuiu durante a parada cardíaca.A temperatura dos ratos DHCA caiu rapidamente durante a fase de resfriamento e se recuperou gradualmente durante a fase giratória. O desmame do circuito DHCA a temperatura dos ratos DHCA voltou ao normal. Os dados acima indicaram que nosso procedimento de DHCA imitou as mudanças reais de temperatura e PAM dos pacientes nas clínicas.O efeito do processador e aumento de DHCA foi investigado pela análise de gases sanguíneos. Após o contato do sangue total com a solução primária, a concentração de hemoglobina foi superior a seis gramas por decilitor em ambos os grupos. O desmame da superfície do DHCA aumentou para nove gramas por decilitter por causa da reinfusão de sangue remanescente no circuito de CEC à direita.O hematócrito, HCT, apresentou tendência semelhante à HB. Após a reperfusão de DHCA, o ácido lactato aumentou rapidamente, o que foi mais pronunciado no grupo DHCA. O pH diminuiu após o procedimento de DHCA, que provavelmente foi o resultado do acúmulo de ácido lactato. Durante todo o experimento, a concentração de sódio, cloreto, íons potássio e glicose não apresentou diferenças significativas em cada 10 pontos.Este resultado sugere que o DHCA apenas induziu o aumento para ácido lactato, mas não influenciou o pH sanguíneo e a concentração de hemoglobina, hematócrito, íons sódio, íons cloreto, íons potássio e glicose. Para avaliar o impacto do DHCA na autofagia neuronal, utilizamos o eletromicroscópio de transmissão e surpreendentemente encontramos e aumentamos o número de autofagossomos no hipocampo de ratos DHCA. Os dados acima indicaram que nosso modelo DHCA imitou as mudanças reais de temperatura e PAM dos pacientes nas clínicas.Este modelo pode ser aplicado para investigar a síndrome da resposta inflamatória sistêmica, neuroinflamação, etc.

establishment-of-deep-hypothermic-circulatory-arrest-in-rats

Research

JoVE Journal

Immunology and Infection

Orthotopic Hind-Limb Transplantation in Rats

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel therapeutic entity in reconstructive surgery. However, long term high-dose multi-drug immunosuppressive therapy is required to ensure graft survival, bearing the risk of serious side effects which halters broader application. Further progression in this field may depend on better understanding of basic immunology and ischemia reperfusion injury in composite tissue grafts.
To date, orthotopic hind limb transplantation in rats has been the preferred rodent model for reconstructive transplantation (RT), however, it is an extremely demanding procedure that requires extraordinary microsurgical skills for reattachment of vasculature, bones, muscles and nerves.
We have introduced the vascular cuff anastomosis technique to this model, providing a rapid and reliable approach to rat hind limb transplantation. This technique simplifies and shortens the surgical procedure and enables surgeons with basic microsurgical experience to successfully perform the operation with high survival and low complication rates. The technique seems to be well suited for immunological as well as ischemia reperfusion injury (IRI) studies.

Here we describe the orthotopic rat hind-limb transplantation procedure, which seems to be the gold standard in vivo model for composite tissue allotransplantation research.

Transplante Limb-Hind ortotópico em ratos

Composite tissue allotransplantation (CTA) now represents a valid therapeutic option after the loss of a hand, forearm or digits and has become a novel ...

Imunologia e Infecção

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

Diagnóstico de Ecto e Endoparasitas em ratos de laboratório e Ratos

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid cell lines (LCL) in vitro. Since LCL are latently infected with EBV, they provide a model system to investigate EBV latency and virus-driven B cell proliferation and tumorigenesis1. LCL have been used to present antigens in a variety of immunologic assays2, 3. In addition, LCL can be used to generate human monoclonal antibodies4, 5 and provide a potentially unlimited source when access to primary biologic materials is limited6, 7.
A variety of methods have been described to generate LCL. Earlier methods have included the use of mitogens such as phytohemagglutinin, lipopolysaccharide8, and pokeweed mitogen9 to increase the efficiency of EBV-mediated immortalization. More recently, others have used immunosuppressive agents such as cyclosporin A to inhibit T cell-mediated killing of infected B cells7, 10-12.
The considerable length of time from EBV infection to establishment of cell lines drives the requirement for quicker and more reliable methods for EBV-driven B cell growth transformation. Using a combination of high titer EBV and an immunosuppressive agent, we are able to consistently infect, transform, and generate LCL from B cells in peripheral blood. This method uses a small amount of peripheral blood mononuclear cells that are infected in vitroclusters of cells can be demonstrated. The presence of CD23 with EBV in the presence of FK506, a T cell immunosuppressant. Traditionally, outgrowth of proliferating B cells is monitored by visualization of microscopic clusters of cells about a week after infection with EBV. Clumps of LCL can be seen by the naked eye after several weeks. We describe an assay to determine early if EBV-mediated growth transformation is successful even before microscopic clusters of cells can be demonstrated. The presence of CD23hiCD58+ cells observed as early as three days post-infection indicates a successful outcome.

We describe a method for generating transformed B cell lines using Epstein-Barr virus. We also illustrate a novel assay that can identify B cells destined to undergo transformation as early as three days after infection.

Estabelecimento de Epstein-Barr Virus Crescimento transformada linhagens de células linfoblastóides

Infection of B cells with Epstein-Barr virus (EBV) leads to proliferation and subsequent immortalization, resulting in establishment of lymphoblastoid ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Complete Thymectomy in Adult Rats with Non-invasive Endotracheal Intubation

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and pneumothorax from pleural disruption can result in a significant mortality rate. This protocol is a simple method of rat thymectomy that utilizes a mini-sternotomy and endotracheal intubation. Intubation is accomplished with a non-invasive and easily reproducible method and allows for positive pressure ventilation to prevent pneumothorax and a controlled airway that allows sufficient time for careful thymus dissection to minimize pleural disruption. A 1.5 cm sternal incision decreases contact with mediastinal vessels and pleura, while still providing full visualization of the thymus. Following exposure of the mediastinum, the thymus is removed by blunt dissection under magnification. The pleural space is then sealed by suture closure of the pre-tracheal muscles followed by the application of surgical glue. The thorax is then closed by suture closure of the sternum, followed by suture closure of the skin. All thymectomies were complete as evidenced by immunohistochemical (IHC) staining of mediastinal tissue, and absence of na&#239;ve T-cells by flow cytometry, and the procedure had a 96% survival rate. This method is suitable when complete thymectomy with minimal complications is desired for further immunological studies in athymic adult rats.

Rodent thymectomy is a valuable technique in immunological research. Here, a protocol for complete thymectomy in adult rats using a mini-sternotomy along with non-invasive intubation and positive pressure ventilation to minimize perioperative morbidity and mortality is described.

Timectomia completo em ratos adultos com não-invasiva entubação endotraqueal

Thymectomy in neonatal rodents is an established and reliable procedure for immunological studies. However, in adult rats, complications of hemorrhage and ...

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two Sprague-Dawley rats were used in this study. Animals were orally treated twice a day for two days with B. subtilis BSB3 strain or PBS. The next day after the last treatment, each group was divided and two experimental groups (one treated with PBS and one treated with B. subtilis) were placed at 45 oC for 25 min. Two control groups stayed for 25 min at room temperature. All rats were euthanized and different parameters were analyzed in all groups. Adverse effects of heat stress are registered by the decrease of villi height and total mucosal thickness in the intestinal epithelium; translocation of bacteria from the lumen; increased vesiculation of erythrocytes and elevation of the lipopolysaccharides (LPS) level in the blood. The protective efficacy of treatment is evaluated by prevention of these side effects. The protocol was set up for the oral treatment of rats with bacteria for prevention of heat stress complications, but this protocol can be modified and used for other routes of administration and for analysis of different compounds.

Prevention of Heat Stress Adverse Effects in Rats by Bacillus subtilis Strain

We described a protocol for prevention of heat stress effects in rats by oral pre-treatment with beneficial bacteria. This protocol can be modified and used for various routes of administration and for analysis of different compounds.

Prevenção de estresse térmico efeitos adversos em ratos pela<em&gt; Bacillus subtilis</em&gt; Strain

This study was designed to evaluate the protective effect of the Bacillus subtilis strain against complications related to heat stress. Thirty-two ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Estenose da veia Cava Inferior: um modelo murino de trombose venosa profunda

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

Trombose venosa profunda induzida por estase em camundongos monitorado por ultra-sonografia de alta frequência

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Profunda dérmica injeção como um modelo de Candida albicans de infecção da pele para análise histológica

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Estabelecimento de infecção Viral e análise da interação vírus-hospedeiro em Drosophila Melanogaster.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...