Ce travail a été soutenu par des subventions du National Institutes of Health, National Institute of General Medical Sciences (R01GM063075) et le Centre national de la médecine complémentaire et alternative (R01AT05076).

1. Mise en place d&#39;un modèle animal de la septicémie <ol><li> Les souris sont anesthésiées avec de la kétamine (75 mg / kg, par voie intramusculaire, im) et de xylazine (10 mg / kg, im), et placé en position couchée. </li><li> Fixer les pieds de la souris avec du ruban adhésif afin d&#39;assurer une position stable. </li><li> Nettoyez l&#39;abdomen avec 3 gommages alternance de bétadine ou autre désinfectant de la peau et de l&#39;alcool. Ensuite, faire une incision médiane de 15 mm pour exposer le caecum. </li><li> Ligaturer le caecum avec un fil de suture de soie 4-0 à environ 5,0 mm de la pointe du caecum, puis la perforation de la souche ligaturé caecale une fois avec une aiguille de calibre 22 pour permettre l&#39;extrusion de matières fécales. </li><li> Le caecum est immédiatement replacé à sa position normale la position intra-abdominale. </li><li> Fermez le site de l&#39;incision en deux couches, en commençant par la fermeture de la musculature de l&#39;abdomen avec des sutures résorbables, puis la fermeture de la peau avec des clips ou des plaies de sutures non résorbables. </li><li> Réanimer la souris avec 0,5 ml de solution saline normale et un seul fairesoi de l&#39;imipénème (0,5 mg / souris, sous-cutanée) immédiatement après l&#39;achèvement de la procédure chirurgicale. </li><li> Retour de la souris à une cage propre avec un accès libre à la nourriture et l&#39;eau. </li><li> Au moment après CLP divers points, extrait de fines herbes ou d&#39;un composant est administré par voie intrapéritonéale. </li><li> La survie des animaux est surveillée pendant plus de deux semaines. Les animaux moribonds présentant des difficultés de se tenir, la respiration agonique, une atrophie musculaire et un saignement incontrôlé devraient être euthanasiés par inhalation de dioxyde de carbone surdosage. </li><li> Il est important de noter que les taux circulants de cytokines sont des paramètres importants dans ces études. Divers analgésiques ont été démontré qu&#39;elle affecte la libération de cytokines et des activités, et ont donc été délibérément évité de soins post-opératoires. </li></ol> 2. Préparation de l&#39;extrait de fines herbes <ol><li> Extrait des herbes dans l&#39;eau chaude (85 ° C) pendant 1-4 h (1 h pour les feuilles, et 4 h pour les racines). </li><li> Centrifuger l&#39;eau-de sorteluble fraction (3300 g, 20 minutes, 4 ° C) pour éliminer les particules insolubles. </li><li> Filtrat la fraction de surnageant à travers un filtre de 0,2 um. </li><li> Le clair soluble dans l&#39;eau fraction de filtrat est ensuite fractionné par ultrafiltration en utilisant Centriprep YM-10 filtre centrifuge (Catalogue no. 4305, Millipore). </li><li> Le résultat faible (&lt;10 kDa) et élevé (&gt; 10 kDa) fractions de poids moléculaire (MWF) sont sélectionnés pour HMGB1-inhibant les activités utilisant des cultures de macrophages. </li><li> L&#39;administration intrapéritonéale de HMGB1 inhibiteurs extrait à base de plantes ou de composants à 24 heures après CLP afin d&#39;évaluer leur efficacité thérapeutique. </li></ol> 3. Isolement des macrophages péritonéale <ol><li> Bouillon au thioglycolate (4%, 2,0 ml) est administré par voie intrapéritonéale à des souris normales. </li><li> Primaires des macrophages péritonéaux sont récoltées à 3 jours, comme décrit précédemment 10. </li><li> Les macrophages sont des pré-cultivée dans du milieu DMEM (Gibco BRL, Grand Island, NY) supplémenté avec 10% de sérum de veau fœtal (FBS), 2 mmol / L-glutamine, et 1% de pénicilline. </li><li> Macrophages adhérents sont lavés en douceur avec, et cultivées dans, sans sérum OPTI-MEM I moyen de deux heures avant la stimulation par l&#39;endotoxine bactérienne (lipopolysaccharide, LPS, la bactérie E. coli 0111: B4, Sigma-Aldrich). </li><li> À 16 heures après la stimulation LPS, les niveaux de HMGB1 dans le milieu de culture sont déterminés par analyse Western blot 11. </li><li> Le intensité de la bande a été quantifiée par rapport à l&#39;aide du NIH image 1.59 du logiciel pour déterminer les niveaux de HMGB1 en référence à des courbes d&#39;étalonnage générées avec purifié HMGB1. </li></ol> 4. Les résultats représentatifs 1. CLP induit une inflammation systémique et locale. En quelques heures de la chirurgie CLP, les animaux présentent des signes cliniques de septicémie qui incluent horripilation, la léthargie, se blottir, et la diminution de l&#39;apport en nourriture et en eau. Animaux en voie de développement d&#39;une péritonite sévère avec systémique consécutivel&#39;infection normalement mourir dans les 48 à 96 h. Cependant, même âge, du sexe et de génétique de fond appariés animaux peuvent répondre à la chirurgie CLP d&#39;une manière distincte dans le cadre de la septicémie expérimentale. Par exemple, à 48 heures après CLP, tandis que certains animaux peuvent avoir approché l&#39;état moribond (tel que défini dans la figure 1), d&#39;autres peuvent rester à un état ​​non-moribonde. Des études complètes sur cytokines circulantes ont révélé des différences dramatiques dans les niveaux de cytokines plusieurs (par exemple IL-6, KC, MIP-2, et sTNFR1) entre les souris dans les Etats moribonds et non moribondes (Figure 1) 12. Notamment, ces médiateurs inflammatoires ont été classés comme des marqueurs de substitution de septicémie parce que leurs taux circulants sont des indicateurs fiables de une issue fatale dans experimental 12-14 ou septicémie clinique 15. En outre, CLP a également induit la libération locale de diverses cytokines pro-et anti-inflammatoire et des chimiokines. Par exemple, à48 h après CLP, des quantités importantes de cytokines (par exemple, IL-6) et des chimiokines (KC et MIP-2) peut encore être mesurée non seulement par voie systémique dans le sang, mais aussi localement dans le liquide de lavage péritonéal (figure 2). 2. Le dépistage de HMGB1 inhibiteurs à base de plantes ou de composants extrait. En utilisant des cultures de macrophages, nous étions en mesure d&#39;évaluer les capacités des extrait de fines herbes diverses composantes ou dans l&#39;inhibition induite par l&#39;endotoxine communiqué HMGB1 (Figure 3). À la lumière de leur capacité dans le communiqué de HMGB1 inhiber, nous avons encore exploré leur efficacité dans des modèles animaux septicémie. Compte tenu de la cinétique de la fin et prolongée de l&#39;accumulation de la protéine HMGB1 dans le sepsis expérimental 8, la première dose de HMGB1 inhibiteurs a été donnée 24 heures après le début de la septicémie - un point de temps lorsque les souris ont développé des signes clairs de septicémie y compris la léthargie, la diarrhée, et l&#39;horripilation. Comme le montre la figure 4, retardée et l&#39;administration répétée d&#39;une grande c thé vertomponent, l&#39;épigallocatéchine-3-gallate (EGCG), à partir de 24 h après l&#39;apparition de la septicémie, de manière significative sauvé les souris d&#39;une septicémie mortelle 12. Même lorsqu&#39;il est administré par voie orale, l&#39;EGCG souris encore sauvés de septicémie mortelle, en augmentant sensiblement le taux de survie des animaux de 16% à 44% 16. Il serait important de déterminer si les thérapies combinatoires avec plusieurs composants à base de plantes pourrait atteindre une protection significative de mieux dans des modèles animaux d&#39;infection. Pris ensemble, ces données expérimentales validé notre approche à l&#39;écran de nouveaux agents thérapeutiques en utilisant un modèle animal de CLP-induite septicémie. 5. Les résultats représentatifs <img alt="Figure 1" src="/files/ftp_upload/3926/3926fig1.jpg" /> Figure 1. Les taux circulants de marqueurs de substitution sont considérablement plus élevés chez les animaux septiques approchant état ​​moribond. Souris Balb / C ont été soumis à une septicémie par le CLP et surveillés pour l&#39;apparition de signes de maladie. Lors d&#39;unestade tardif de l&#39;infection (52 heures après CLP), le sang a été recueilli à partir de 3 souris normales (CLP-), 3 souris septiques qui approchent de l&#39;état moribond, et 3 souris septiques à l&#39;état non-moribonde. Le sérum a été mis en commun de chaque groupe, et analysés pour le profil des cytokines par les puces à anticorps. Notez la différence dramatique dans les niveaux relatifs de plusieurs marqueurs de substitution entre les différents groupes. Adapté de doi: 10.1371/journal.pone.0001153.g006 avec la permission accordée par l&#39;éditeur. <img alt="Figure 2" src="/files/ftp_upload/3926/3926fig2.jpg" /> Figure 2. Détection des cytokines locales et systémiques et des chimiokines à 48 heures après CLP. Souris Balb / C ont été soumis à une septicémie par le CLP, et sang ou de liquide péritonéal ont été récoltés à 48 h après CLP. Les niveaux relatifs de plusieurs cytokines et des chimiokines dans le liquide de lavage en commun le sérum ou le péritoine ont été mesurés par des réseaux d&#39;anticorps cytokine, et exprimées en unités arbitraires (UA). Comme fluide contrôles, de sang et sa péritonéalemples ont été obtenus à partir d&#39;animaux sains normaux (CLP-) sans laparotomie. <img alt="Figure 3" src="/files/ftp_upload/3926/3926fig3.jpg" /> Figure 3. Composante à base de plantes dose-dépendante induite par l&#39;endotoxine inhibée HMGB1 communiqué dans les cultures primaires de macrophages. Primaires macrophages péritonéaux murins ont été stimulées par le LPS, en l&#39;absence ou la présence de la composante à base de plantes (par exemple, l&#39;EGCG, 15 uM). À 16 heures après stimulation par le LPS, les niveaux de HMGB1 dans le milieu de culture ont été déterminés par analyse Western blot. Adapté de doi: 10.1371/journal.pone.0001153.g006 avec la permission accordée par l&#39;éditeur. <img alt="Figure 4" src="/files/ftp_upload/3926/3926fig4.jpg" /> Figure 4. Composante à base de plantes a sauvé les souris contre une septicémie mortelle. Souris Balb / C ont été soumis à une septicémie mortelle par le CLP, et le composant à base de plantes (EGCG) était administré par voie intraperitoneale à +24, +48, +72 heures poster le début de la septicémie. Unla survie iMAL a été suivie pendant jusqu&#39;à deux semaines. La méthode de Kaplan-Meier a été utilisée pour comparer les différences dans les taux de mortalité entre les groupes. *, P &lt;0,05 par rapport à une solution saline. Adapté de doi: 10.1371/journal.pone.0001153.g006 avec la permission accordée par l&#39;éditeur.

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AES et HW sont co-inventeurs de demandes de brevet liées à HMGB1 inhibiteurs tanshinones) comme agents thérapeutiques potentiels pour le sepsis.

Dans le laboratoire, plusieurs modèles animaux d&#39;infection ont été employées pour comprendre la pathogenèse de l&#39;infection afin de développer de nouvelles thérapies potentielles. Leur pertinence clinique reste un sujet de débat avant l&#39;application réussie des études sur les animaux dans des applications cliniques pour le sepsis. Bien que des anticorps neutralisants contre des cytokines précoce (par exemple, le TNF) jouaient un rôle protecteur dans des modèles animaux de bactériémie / endotoxé...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Betadine </td><td> Produits Purdue LP </td><td> 25655-41-8 </td></tr><tr><td> imipénème </td><td> Merck &amp; Co., Inc </td><td> 9882821 </td></tr><tr><td> La kétamine HCl </td><td> Hospira Inc </td><td> RL-0065 </td></tr><tr><td> La xylazine </td><td> Laboratoires Lloyd </td><td> 4821 </td></tr><tr><td> Autoclip </td><td> Becton Dickinson </td><td> 427631 </td></tr><tr><td> Suture en soie 4-0 </td><td> Roboz </td><td> SUT-15-2 </td></tr><tr><td> Surflo IV cathéter </td><td> Terumo </td><td> SR * OX2419CA </td></tr><tr><td> La souris RayBio cytokine réseau d&#39;anticorps </td><td> RayBiotech, Inc </td><td> AAM-CYT-3 </td></tr><tr><td> Thioglycollate </td><td> Becton Dickinson </td><td> 211716 </td></tr></tbody></table>

use of animal model of sepsis to evaluate novel herbal therapies

Septicémie se réfère à un syndrome de réponse inflammatoire systémique résultant d&#39;une infection microbienne. Il a été régulièrement simulée chez les animaux par des techniques plusieurs, notamment de perfusion de toxine bactérienne exogène (endotoxémie) ou bactéries (bactériémie), ainsi que la perforation chirurgicale du caecum par ligation du caecum et la perforation (CLP) 1-3. CLP permet déversement des bactéries et la contamination fécale de la cavité péritonéale, imitant la maladie clinique chez l&#39;homme de l&#39;appendicite perforée ou diverticulite. La gravité de la septicémie, comme en témoignent les taux de mortalité éventuels, peut être contrôlée chirurgicalement en faisant varier la taille de l&#39;aiguille utilisée pour la ponction du caecum 2. Chez les animaux, CLP induit similaire, hémodynamique biphasique réponses cardiovasculaires, métaboliques et immunologiques comme observé au cours de l&#39;évolution clinique de l&#39;infection humaine 3. Ainsi, le modèle CLP est considéré comme l&#39;un des modèles les plus pertinents sur le plan clinique pour le sepsis expérimental 1-3. &lt;/ P&gt; Divers modèles animaux ont été utilisés pour élucider les mécanismes sous-jacents complexes la pathogenèse de la septicémie expérimentale. La conséquence mortelle de l&#39;infection est imputable en partie à une accumulation excessive de cytokines début (comme le TNF, l&#39;IL-1 et l&#39;IFN-γ) 4-6 et des médiateurs pro-inflammatoires (par exemple la fin, HMGB1) 7. Par rapport à début cytokines pro-inflammatoires, la fin d&#39;action des médiateurs ont une plus large fenêtre thérapeutique pour les applications cliniques. Par exemple, l&#39;administration retardée de HMGB1-anticorps neutralisants commençant 24 heures après CLP, toujours sauvé les souris de létalité 8,9, établissant HMGB1 en tant que médiateur de la septicémie mortelle fin. La découverte de HMGB1 en tant que médiateur à action retard a lancé un nouveau champ d&#39;investigation pour le développement de thérapies utilisant septicémie médecine traditionnelle chinoise à base de plantes. Dans cet article, nous décrivons une procédure de CLP-induite septicémie, et son utilisation dans le dépistage médecine par les plantes pour HMGB1-ciblage des thérapies.

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

Sepsis se réfère à un syndrome de réponse inflammatoire systémique résultant d&#39;une infection microbienne, et peut être simulé par une technique chirurgicale appelée ligature du caecum et à la perforation (CLP). Nous décrivons ici une méthode pour utiliser un modèle animal CLP-induite pour cribler des herbes médicinales pour les agents thérapeutiques.

L&#39;utilisation de modèle animal de sepsis pour évaluer de nouvelles thérapies à base de plantes

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 vues.  North Shore – LIJ Health System. Sepsis se réfère à un syndrome de réponse inflammatoire systémique résultant d'une infection microbienne, et peut être simulé par une technique chirurgicale appelée ligature du caecum et à la perforation (CLP). Nous décrivons ici une méthode pour utiliser un modèle animal CLP-induite pour cribler des herbes médicinales pour les agents thérapeutiques.

Vidéo: 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.

Le modèle du CYP2D6 animale: Comment provoquer une hépatite auto-immune chez la souris

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 ...

Recherche

Médecine

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.

Une tumeur modèle murin de stress chirurgical à explorer les mécanismes de l&#39;immunosuppression postopératoire et évaluer de nouvelles immunothérapies périopératoires

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.

Évaluation de l&#39;efficacité de la<em&gt; H. pylori</em&gt; Protéines HP-NAP comme outil thérapeutique pour le traitement du cancer de la vessie dans un modèle murin orthotopique

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.

Bioluminescence imagerie d&#39;un modèle animal pour immunocompétentes glioblastome

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.

Un petit modèle Animal de Perfusion hépatique normothermique 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.

Mesure de la contractilité isométrique de l’artère mésentérique de souris à l’aide de fil myographie

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.

Nouvelles méthodes pour l’Administration intranasale sous anesthésie par Inhalation pour évaluer des nez-de-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 ...

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.

Évaluation fonctionnelle du cœur du donneur pendant la perfusion ex situ : aperçu des boucles pression-volume et de l’échocardiographie de surface

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.

Ligature partielle du nerf sciatique : un modèle murin de douleur neuropathique chronique pour étudier l’effet antinociceptif de nouvelles thérapies

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

Mongolian Gerbils as an Animal Model of Wound Healing

Corneal wound healing studies have been conducted for a long time and have helped to reduce suffering and develop treatments that contribute to improving patients' eye health. Historically, corneal healing has been studied in rodents such as mice and rats, but these models might not completely mimic human disorders. However, information on other rodents such as Mongolian gerbils (Meriones unguiculatus) is scant in corneal research.
Here, we describe a technique to develop a novel animal model for studying corneal healing after photorefractive keratectomy. Due to the limited literature available on the cornea of M. unguiculatus, we also describe a histological analysis of the normal cornea. These research techniques can also be employed in the study of eye diseases because of the similarity between the corneas of Mongolian gerbils and humans in terms of genetics, anatomy, and physiology.

This article describes a new animal model developed to study the anatomy and histology of the cornea and its healing processes. This new animal model uses the Mongolian gerbil, which has a cornea with many similarities to the human cornea.

Les gerbilles de Mongolie comme modèle animal de cicatrisation des plaies

Corneal wound healing studies have been conducted for a long time and have helped to reduce suffering and develop treatments that contribute to improving ...