Este trabalho foi suportado por concessões do National Institutes of Health, National Institute of General Medical Science (R01GM063075) e do Centro Nacional de Medicina Complementar e Alternativa (R01AT05076).

1. Estabelecimento de Modelo Animal de Sepse <ol><li> Os ratos são anestesiados com cetamina (75 mg / kg, por via intramuscular, im) e xilazina (10 mg / kg, im), e colocado em decúbito dorsal. </li><li> Fixe os pés do mouse com fita, para garantir uma posição estável. </li><li> Limpe o abdômen com 3 esfrega alternadas de betadine ou outro desinfetante da pele e do álcool. Em seguida, fazer uma incisão na linha média 15 milímetros para expor o ceco. </li><li> Ligar o ceco com uma sutura de seda 4-0 a cerca de 5,0 milímetros a partir da ponta cecal e, em seguida punção do coto ligadura cecal uma vez com uma agulha de calibre 22 para permitir a extrusão de fezes. </li><li> O ceco é imediatamente substituído de volta para sua posição intra-abdominal normal. </li><li> Feche o local da incisão em duas camadas, por primeiro fechar a musculatura do abdómen com suturas absorvíveis, e, em seguida, fechando a pele com grampos de sutura ou suturas não absorvíveis. </li><li> Ressuscitar rato com 0,5 ml solução normal salina e fazer um únicoSE de imipenem (0,5 mg / rato, por via subcutânea) imediatamente após a conclusão do procedimento cirúrgico. </li><li> Retornar o rato de volta para uma gaiola limpa com livre acesso a comida e água. </li><li> Em vários pontos CLP pós tempo, extrato de ervas ou componente é administrado por via intraperitoneal. </li><li> Sobrevivência dos animais é monitorizado durante mais de duas semanas. Os animais moribundos exibem dificuldades em repouso, respiração, atrofia muscular agonia intensa e sangramento descontrolado devem ser sacrificados por overdose inalação de dióxido de carbono. </li><li> É importante notar que os níveis circulantes de citocinas são parâmetros importantes nestes estudos. Analgésicos Vários foram mostrados para afetar a liberação de citocinas e atividades, e, portanto, ter sido deliberadamente evitado nos cuidados pós-operatório. </li></ol> 2. Preparação do extrato de plantas medicinais <ol><li> Extrair ervas em água quente (85 ° C) por 1-4 horas (1 hora para as folhas, e 4 horas para as raízes). </li><li> Centrifugar a água, de modofracção luble (3300 g, 20 min, 4 ° C) para remover partículas insolúveis. </li><li> Filtrado a fracção sobrenadante através de um filtro de 0,2 uM. </li><li> A clara solúvel em água fracção filtrado é então fraccionado por ultrafiltração utilizando Centriprep YM-10 filtro centrífugo (catálogo n °. 4305, Millipore). </li><li> O resultado de baixo custo (menos de 10 kDa) e elevado (mais de 10 kDa) fracções de peso molecular (MWF) são rastreados para HMGB1 inibidores actividades utilizando culturas de macrófagos. </li><li> Administração intraperitoneal de HMGB1 inibidores extrato de ervas ou componentes em 24 horas pós-CLP para avaliar sua eficácia terapêutica. </li></ol> 3. Isolamento de macrófagos peritoneais <ol><li> Caldo tioglicolato (4%, 2,0 ml) é administrado por via intraperitoneal em ratos normais. </li><li> Primárias macrófagos peritoneais foram colhidos menos 3 dias tal como anteriormente descritos 10. </li><li> Os macrófagos são pré-cultivadas em meio DMEM (Gibco BRL, Grand Island, NY) suplementado com 10% de soro fetal bovino (FBS), glutamina 2 mmol / L, e 1% de penicilina. </li><li> Macrófagos aderentes são suavemente lavados com, e cultivadas em, isento de soro OPTI-MEM I de duas horas antes da estimulação com endotoxina bacteriana (lipopolissacárido, LPS, E. coli 0111: B4, Sigma-Aldrich). </li><li> Às 16 horas após a estimulação com LPS, os níveis de HMGB1 no meio de cultura são determinados por análise de Western blotting 11. </li><li> A intensidade da banda relativa foi quantificada usando a imagem NIH 1,59 software para determinar os níveis de HMGB1 com referência às curvas padrão geradas com purificado HMGB1. </li></ol> 4. Os resultados representativos 1. CLP induz inflamação sistêmica e local. Dentro de algumas horas de cirurgia CLP, os animais apresentam sinais clínicos de sepse que incluem piloereção, letargia, encolhendo-se, e diminuição na ingestão de alimentos e água. Animais desenvolvimento de uma peritonite grave com sistêmica consecutivoinfecção normalmente morrem dentro de 48-96 hr. No entanto, mesmo fundo pareados idade, sexo, e genética de animais pode responder à CLP cirurgia de uma maneira distinguíveis no decurso da sépsis experimental. Por exemplo, em 48 horas pós-CLP, enquanto alguns animais podem ter abordado o estado moribundo (conforme definido na Figura 1), outros podem permanecer em um estado não-moribundo. Inquéritos abrangentes de citocinas circulantes revelou diferenças dramáticas em vários níveis de citocinas (por exemplo, IL-6, KC, MIP-2, e sTNFR1) entre ratinhos nos estados moribundos e não-moribundos (Figura 1) 12. Notadamente, esses mediadores inflamatórios têm sido classificados como marcadores de sepse porque seus níveis circulantes são indicadores confiáveis ​​de desfecho letal em 12-14 experimental ou sepse clínica 15. Além disso, CLP também induziu a libertação local de várias citocinas pró e anti-inflamatória e quimiocinas. Por exemplo, no48 horas após a CLP, quantidades significativas de citocinas (por exemplo, IL-6) e quimiocinas (KC e MIP-2) ainda pode ser medida, não só sistemicamente no sangue, mas também localmente no fluido de lavagem peritonial (Figura 2). 2. Triagem para HMGB1 inibidores extrato de ervas ou componentes. Usando culturas de macrófagos, fomos capazes de avaliar as capacidades de extracto de plantas medicinais ou vários componentes em inibir a liberação HMGB1 induzida pela endotoxina (Figura 3). À luz da sua capacidade em inibir a liberação de HMGB1, nós ainda explorada a sua eficácia na sepse modelos animal. Dada a cinética tardias e prolongada de HMGB1 acumulação na sépsis experimental 8, a primeira dose de HMGB1 inibidores foi determinado 24 horas após o início da sépsis - um ponto de tempo quando os ratinhos desenvolveram sinais claros de sépsis, incluindo letargia, piloerecção, diarreia e. Como mostrado na Figura 4, atrasado e administração repetida de uma grande chá verde component, epigalocatequina-3-galato (EGCG), com início às 24 h após o início da sepse, significativamente resgatado camundongos com sepse letal 12. Mesmo quando administrado por via oral, EGCG ratos ainda resgatados de sepsis letal, aumentando significativamente as taxas de sobrevivência de animais de 16% para 44% 16. Seria importante para determinar se as terapias combinações com vários componentes à base de plantas poderia alcançar uma protecção significativa melhor em modelos animais de sépsis. Em conjunto, estes dados experimentais validado nossa abordagem à tela novos agentes terapêuticos, utilizando um modelo animal de CLP-induzida a sepse. 5. Os resultados representativos <img alt="A Figura 1" src="/files/ftp_upload/3926/3926fig1.jpg" /> Figura 1. Os níveis circulantes de marcadores são significativamente maior em animais sépticos aproximam estado moribundo. Balb / C camundongos foram submetidos a sepse por CLP e monitorados para o desenvolvimento de sinais de doença. Numafase tardia da sepse (52 horas pós-CLP), foi coletado sangue de ratos normais (3-CLP), 3 ratos sépticos que se aproximam do estado moribundo e 3 ratos sépticos em estado não-moribundo. O soro foi reunidas a partir de cada grupo, e ensaiado para o perfil de citocinas por matrizes de anticorpos. Observe a diferença dramática nos níveis relativos de vários marcadores entre diferentes grupos. Adaptado de doi: 10.1371/journal.pone.0001153.g006 com a permissão concedida a partir do editor. <img alt="A Figura 2" src="/files/ftp_upload/3926/3926fig2.jpg" /> Figura 2. Detecção de citocinas locais e sistémicos e quimiocinas às 48 horas após a CLP. Balb / C ratinhos foram submetidos a sépsis por CLP, e sangue ou líquido peritoneal foram colhidas às 48 h pós CLP. Níveis relativos de várias citocinas e quimiocinas em reunidas soro ou fluido peritoneal lavagem foram medidos por matrizes de anticorpos de citocina, e expressos como unidades arbitrárias (UA). Como controlos sangue, fluido peritoneal e samples foram obtidos de animais saudáveis ​​normais (-CLP), sem laparotomia. <img alt="A Figura 3" src="/files/ftp_upload/3926/3926fig3.jpg" /> Figura 3. Componente à base de plantas dose-dependente inibida induzida pela endotoxina HMGB1 libertação em culturas de macrófagos primários. Primários macrófagos peritoneais murinos foram estimulados com LPS na ausência ou presença de componente à base de plantas (por exemplo, EGCG, 15 uM). Às 16 horas após a estimulação com LPS, os níveis de HMGB1 no meio de cultura foram determinados por análise de Western blotting. Adaptado de doi: 10.1371/journal.pone.0001153.g006 com a permissão concedida a partir do editor. <img alt="A Figura 4" src="/files/ftp_upload/3926/3926fig4.jpg" /> Figura 4. Componente à base de plantas resgatado a partir de ratinhos sépsis letal. Balb / C ratinhos foram submetidos a sépsis letal por CLP, e componente à base de plantas (EGCG) foi administrado intraperitonealmente em 24, +48, +72 horas após o início da sépsis. Umsobrevivência máxima foi monitorado por até duas semanas. O método de Kaplan-Meier foi utilizado para comparar as diferenças nas taxas de mortalidade entre os grupos. *, P &lt;0,05 versus salina. Adaptado de doi: 10.1371/journal.pone.0001153.g006 com a permissão concedida a partir do editor.

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AES e HW são co-inventores de pedidos de patentes relacionadas com HMGB1 inibidores (tanshinones) como potenciais agentes terapêuticos para sepse.

No laboratório, vários modelos animais de sépsis têm sido empregues para compreender a patogénese da sépsis, a fim de desenvolver novas terapias potenciais. Sua relevância clínica continua a ser um objecto de debate antes da tradução bem sucedida de estudos em animais para aplicações clínicas de sepse. Embora os anticorpos neutralizantes contra o citocinas precoces (por exemplo, o TNF) foram protector em modelos animais de endotoxemia / bacteremia 17,18, eles piorar a sobrevivência em modelo ani...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> Betadine </td><td> Purdue Products LP </td><td> 25655-41-8 </td></tr><tr><td> imipenem </td><td> Merck &amp; Co., Inc. </td><td> 9882821 </td></tr><tr><td> A ketamina HCl </td><td> Hospira Inc. </td><td> RL-0065 </td></tr><tr><td> Xilazina </td><td> Lloyd Laboratories </td><td> 4821 </td></tr><tr><td> Autoclip </td><td> Becton Dickinson </td><td> 427631 </td></tr><tr><td> Sutura de seda 4-0 </td><td> Roboz </td><td> SUT-15-2 </td></tr><tr><td> Surflo Cateter IV </td><td> Terumo </td><td> SR * OX2419CA </td></tr><tr><td> Rato RayBio matriz anticorpo citocina </td><td> RayBiotech, Inc. </td><td> AAM-CYT-3 </td></tr><tr><td> Tioglicolato </td><td> Becton Dickinson </td><td> 211716 </td></tr></tbody></table>

use of animal model of sepsis to evaluate novel herbal therapies

Sépsis refere-se a uma síndrome de resposta inflamatória sistémica resultante de uma infecção microbiana. Tem sido rotineiramente simulado em animais por várias técnicas, incluindo a infusão de toxina bacteriana exógena (endotoxemia) ou bactérias (bacteremia), bem como a perfuração cirúrgica do ceco por ligação e perfuração cecal (CLP) 1-3. CLP permite que as bactérias derramamento e contaminação fecal da cavidade peritoneal, imitando a doença humana clínico de apendicite perfurada ou diverticulite. A gravidade da sépsis, tal como reflectido pelas taxas de mortalidade eventuais, podem ser controlados cirurgicamente variando o tamanho da agulha usada para punção cecal 2. Em animais, a CLP induz hemodinâmica, similar bifásico respostas cardiovasculares, metabólicas e imunológicas, como observado durante a evolução clínica da sepse humana 3. Assim, o modelo CLP é considerado como um dos modelos mais clinicamente relevantes para sépsis experimental 1-3. &lt;/ P&gt; Vários modelos animais têm sido utilizados para elucidar os mecanismos subjacentes à intrincados patogénese da sépsis experimental. A consequência letal de sépsis é atribuível parcialmente a uma acumulação excessiva de citocinas iniciais (tais como TNF, IL-1 eo IFN-γ) 4-6 e mediadores pró-inflamatórios tardia (por exemplo, HMGB1) 7. Comparado com os primeiros citocinas pró-inflamatórias, de ação tardia mediadores têm uma ampla janela terapêutica para aplicações clínicas. Por exemplo, a administração retardada de HMGB1 anticorpos neutralizadores começando 24 horas após CLP, ainda resgatado a partir de ratinhos letalidade 8,9, estabelecendo HMGB1 como um mediador tardia da sépsis letal. A descoberta de HMGB1 como um mediador de ação tardia deu início a um novo campo de investigação para o desenvolvimento de terapias que utilizam sepse medicina tradicional chinesa. Neste artigo, descrevemos um procedimento de CLP-induzida a sepse, e seu uso na seleção de ervas medicinais para a HMGB1 de metas terapêuticas.

Watch this Scientific Journal Video about Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies at JoVE.com

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepse se refere a uma síndrome de resposta inflamatória sistêmica resultante de uma infecção microbiana, e pode ser simulado por uma técnica cirúrgica chamada ligadura e perfuração cecal (CLP). Aqui nós descrevemos um método para usar CLP-induzida modelo animal para a tela ervas medicinais para agentes terapêuticos.

Uso de Modelo Animal de Sepse para avaliar novas terapias à base de plantas

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

use-of-animal-model-of-sepsis-to-evaluate-novel-herbal-therapies

Research

JoVE Journal

Medicine

19.3K visualizações.  North Shore – LIJ Health System. Sepse se refere a uma síndrome de resposta inflamatória sistêmica resultante de uma infecção microbiana, e pode ser simulado por uma técnica cirúrgica chamada ligadura e perfuração cecal (CLP). Aqui nós descrevemos um método para usar CLP-induzida modelo animal para a tela ervas medicinais para agentes terapêuticos.

Vídeo: Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

The CYP2D6 Animal Model: How to Induce Autoimmune Hepatitis in Mice

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease 3-5. However, in various models the induction of disease was rather complex and often hepatitis was only transient3-5. Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger6. Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-27,8. Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field9 for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFN&gamma;-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Infection of mice with an Adenovirus expressing the major human autoantigen cytochrome P450 2D6 (hCYP2D6) recognized by sera of patients suffering from type 2 autoimmune hepatitis results in a persistent form of autoimmune-mediated liver disease characterized by extensive hepatitis, fibrosis and generation of a CYP2D6-specific immune response.

O modelo animal CYP2D6: Como induzir hepatite auto-imune em ratos

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology1,2. In the past many attempts have been made to ...

Medicina

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

Um tumor modelo de estresse cirúrgico para explorar os mecanismos de pós-operatório Imunossupressão e Avaliar Novel perioperatórios Imunoterapias Rato

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold standard treatment for the high-grade non-muscle invasive bladder cancer (NMIBC). However, since the treatment-related side effects are relevant, newer biological response modifiers with a better benefit/side effects ratio are needed.
The tumour microenvironment can influence both tumour development and therapy efficacy. In order to obtain a good model, it is desirable to implant tumour cells in the organ from which the cancer originates.
In this protocol, we describe a method for establishing a tumour in the bladder cavity of female mice and subsequent delivery of therapeutic agents; the latter are exemplified by our use of Helicobacter pylori neutrophil activating protein (HP-NAP). A preliminary chemical burn of the mucosa, followed by the injection of mouse urothelial carcinoma cell line MB49 via urethral catheterization, enables the cells to attach to the bladder mucosa. After a period, required to allow an initial proliferation of the cells, mice are treated with HP-NAP, administrated again via catheterization. The anti-tumour activity of HP-NAP is evaluated comparing the tumour volume, the extent of necrosis and the degree of vascularization between vehicle- and HP-NAP-treated animals.

Evaluation of the Efficacy of the H. pylori Protein HP-NAP as a Therapeutic Tool for Treatment of Bladder Cancer in an Orthotopic Murine Model

Here the method to establish a syngeneic mouse model of orthotopic bladder tumour to evaluate the anti-tumour efficacy of the bacterial protein HP-NAP is described.

Avaliação da Eficácia da<em&gt; H. pylori</em&gt; Protein HP-NAP como uma ferramenta terapêutica para o tratamento de cancro da bexiga em um Modelo murino Orthotopic

Bladder cancer is one of the most common malignancies of the urogenital tract. Intravesical injection of Bacillus Calmette-Gu&#233;rin (BCG) is the gold ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

Bioluminescência imagem de um modelo animal para imunocompetentes Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, normothermic ex vivo liver perfusion (NEVLP) has been introduced as a method to evaluate and modify organ function. NEVLP has many advantages in comparison to hypothermic and subnormothermic perfusion including reduced preservation injury, restoration of normal organ function under physiologic conditions, assessment of organ performance, and as a platform for organ repair, remodeling, and modification. Both murine and porcine NEVLP models have been described. We demonstrate a rat model of NEVLP and use this model to show one of its important applications &#8212; the use of a therapeutic molecule added to liver perfusate. Catalase is an endogenous reactive oxygen species (ROS) scavenger and has been demonstrated to decrease ischemia-reperfusion in the eye, brain, and lung. Pegylation has been shown to target catalase to the endothelium. Here, we added pegylated-catalase (PEG-CAT) to the base perfusate and demonstrated its ability to mitigate liver preservation injury. An advantage of our rodent NEVLP model is that it is inexpensive in comparison to larger animal models. A limitation of this study is that it does not currently include post-perfusion liver transplantation. Therefore, prediction of the function of the organ post-transplantation cannot be made with certainty. However, the rat liver transplant model is well established and certainly could be used in conjunction with this model. In conclusion, we have demonstrated an inexpensive, simple, easily replicable NEVLP model using rats. Applications of this model can include testing novel perfusates and perfusate additives, testing software designed for organ evaluation, and experiments designed to repair organs.

A Small Animal Model of Ex Vivo Normothermic Liver Perfusion

There is a significant liver donor shortage, and criteria for liver donors have been expanded. Normothermic ex vivo liver perfusion (NEVLP) has been developed to evaluate and modify organ function. This study demonstrates a rat model of NEVLP and tests the ability of pegylated-catalase, to mitigate liver preservation injury.

Um pequeno modelo Animal de perfusão de fígado normotérmica Ex Vivo

There is a significant shortage of liver allografts available for transplantation, and in response the donor criteria have been expanded. As a result, ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Medição de contratilidade isométrica da artéria mesentérica Mouse usando fio miografia

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Novel Methods for Intranasal Administration Under Inhalation Anesthesia to Evaluate Nose-to-Brain Drug Delivery

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain barrier. However, there have been few reports regarding not only the quantitative analysis but also optimal administration conditions and dosing regimens for investigations of nose-to-brain delivery. The limited progress in research on nose-to-brain pathway mechanisms using rodents represents a significant impediment in terms of designing nose-to-brain delivery systems for candidate drugs.
To gain some headway in this regard, we developed and evaluated two novel methods of stable intranasal administration under inhalation anesthesia for experimental animals. We also describe a method for the evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radio-labeled [14C]-inulin (molecular weight: 5,000) as a model substrate of water-soluble macromolecules.
Initially, we developed a pipette-based intranasal administration protocol using temporarily openable masks, which enabled us to perform reliable administration to animals under stable anesthesia. Using this system, [14C]-inulin could be delivered to the brain with little experimental error.
We subsequently developed an intranasal administration protocol entailing reverse cannulation from the airway side through the esophagus, which was developed to minimize the effects of mucociliary clearance (MC). This technique led to significantly higher levels of [14C]-inulin, which was quantitatively detected in the olfactory bulb, cerebrum, and medulla oblongata, than the pipette method. This appears to be because retention of the drug solution in the nasal cavity was substantially increased by active administration using a syringe pump in a direction opposite to the MC into the nasal cavity.
In conclusion, the two methods of intranasal administration developed in this study can be expected to be extremely useful techniques for evaluating pharmacokinetics in rodents. The reverse cannulation method, in particular, could be useful for evaluating the full potential of nose-to-brain delivery of drug candidates.

Here, we describe two novel methods of stable intranasal administration under inhalation anesthesia with minimal physical stress for experimental animals. We also describe a method for quantitative evaluation of drug distribution levels in the brain via the nose-to-brain pathway using radiolabeled [14C]-inulin as a model substrate of water-soluble macromolecules.

Novos métodos para a Administração Intranasal sob anestesia por inalação para avaliar a entrega da droga de nariz-de-Brain

Intranasal administration has been reported to be a potential pathway for nose-to-brain delivery of therapeutic agents that circumvents the blood-brain ...

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing (Neo)adjuvant Therapies

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant or neo-adjuvant therapies. This occurs when surgical margins are insufficiently tumor-free, resulting in residual cancer cells. From a biological and immunological perspective, surgery is not a null event; the wound healing environment is known to induce both pro- and anti-tumorigenic pathways. As a consequence, preclinical models for drug development aimed at preventing local relapse should incorporate surgical resection when testing new (neo)adjuvant therapies, to model the clinical settings in patients treated with surgery.
Here, we describe a mouse model of incomplete surgical resection of WEHI 164 soft tissue sarcoma that allows testing of (neo)adjuvant therapies in the setting of a wound healing response. In this model, 50% or 75% of the tumor is removed, leaving behind some cancer tissue in situ to model gross residual disease after surgery in the clinical setting. This model allows testing therapies in the context of surgery while also considering the wound healing response, which may affect the efficacy of (neo)adjuvant treatments. The incomplete surgical resection results in reproducible regrowth of the tumor in all mice in the absence of adjuvant therapy. Adjuvant treatment with checkpoint blockade results in reduced tumor regrowth. This model is thus appropriate for testing therapies in the context of debulking surgery and its associated wound healing response and can be extended to other types of solid cancer.

A Mouse Model of Incompletely Resected Soft Tissue Sarcoma for Testing Neoadjuvant Therapies

In this protocol, we describe a mouse model of incomplete surgical resection of soft tissue sarcoma for testing (neo)adjuvant therapies.

Um modelo de mouse de sarcoma de tecido mole incompletamente ressectado para testar (Neo)terapias adjuvantes

Surgery is often the first treatment for many solid tumors. However, local relapses frequently occur following primary tumor resection, despite adjuvant ...

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the allocation of a growing number of donor hearts with extended criteria. These marginal grafts are associated with a high risk of primary graft failure and may benefit from ex situ perfusion before transplant. This technology allows for extended organ preservation using warm oxygenated blood perfusion with continuous metabolic monitoring. The only NESP device currently available for clinical practice perfuses the organ in an unloaded non-working state, which does not allow for functional assessment of the beating heart. We therefore developed an original platform of NESP in working mode conditions with adjustment of left ventricular preload and afterload. This protocol was applied in porcine hearts. Ex situ functional assessment of the heart was achieved with intracardiac conductance catheterization and surface echocardiography. Along with a description of the experimental protocol, we herein report the main results, as well as pearls and pitfalls associated with the acquisition of pressure-volume loops and myocardial power during NESP. Correlations between hemodynamic findings and ultrasound variables are of major interest, especially for further rehabilitation of donor hearts before transplantation. This protocol aims to improve the assessment of donor hearts to both increase the donor pool and reduce the incidence of primary graft failure.

Functional Assessment of the Donor Heart During Ex Situ Perfusion: Insights from Pressure-Volume Loops and Surface Echocardiography

A reliable noninvasive approach for functional assessment of the donor heart during normothermic ex situ heart perfusion (NESP) is lacking. We herein describe a protocol for ex situ assessment of myocardial performance using the epicardial echocardiography and conductance catheter method.

Avaliação funcional do coração do doador durante a perfusão ex situ : insights de alças de pressão-volume e ecocardiografia de superfície

Heart transplantation remains the gold standard treatment for advanced heart failure. However, the current critical organ shortage has resulted in the ...

Partial Sciatic Nerve Ligation: A Mouse Model of Chronic Neuropathic Pain to Study the Antinociceptive Effect of Novel Therapies

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. Chronic neuropathic pain results from lesions or diseases in the somatosensory system. To investigate potential therapies with reduced side effects, animal pain models are the gold standard in preclinical studies. Therefore, well-characterized and well-described models are crucial for the development and validation of innovative therapies. 
Partial ligation of the sciatic nerve (pSNL) is a procedure that induces chronic neuropathic pain in mice, characterized by mechanical and thermal hypersensitivity, ongoing pain, and changes in limb temperature, making this model a great fit to study neuropathic pain preclinically. pSNL is an advantageous model to study neuropathic pain as it reproduces many symptoms observed in humans with neuropathic pain. Furthermore, the surgical procedure is relatively fast and straightforward to perform. Unilateral pSNL of one limb allows for comparison between the ipsilateral and contralateral paws, as well as evaluation of central sensitization. 
To induce chronic neuropathic hypersensitivity, a 9-0 non-absorbable nylon thread is used to ligate the dorsal third of the sciatic nerve. This article describes the surgical procedure and characterizes the development of chronic neuropathic pain through multiple commonly used behavioral tests. As a plethora of innovative therapies are now being investigated to treat chronic pain, this article provides crucial concepts for standardization and an accurate description of surgeries required to induce neuropathic pain.

Partial sciatic nerve ligation induces long-lasting chronic neuropathic pain, characterized by exaggerated responses to thermal and mechanical stimuli. This mouse model of neuropathic pain is commonly used to study innovative therapies for pain management. This article describes in detail the surgical procedure to improve standardization and reproducibility.

Ligadura parcial do nervo ciático: um modelo murino de dor neuropática crônica para estudar o efeito antinociceptivo de novas terapias

Management of chronic pain remains challenging to this day, and current treatments are associated with adverse effects, including tolerance and addiction. ...