Diese Arbeit wurde durch Mittel der National Institutes of Health, National Institute of General Medical Science (R01GM063075) und das Nationale Zentrum für Komplementär-und Alternativmedizin (R01AT05076) unterstützt.

1. Gründung Tiermodell der Sepsis <ol><li> Mäuse sind mit Ketamin (75 mg / kg, intramuskulär, im) und Xylazin (10 mg / kg, im), und in Rückenlage. </li><li> Befestigen der Füße der Maus mit Band, um eine stabile Position zu sichern. </li><li> Reinigen Sie den Bauch mit 3 abwechselnd von Scrubs Betadin oder andere Haut Desinfektionsmittel und Alkohol. Dann, mit 15 mm Mittellinienschnitt bis zum Zökum aussetzen. </li><li> Ligieren des Blinddarms mit einem 4-0 Seidenfaden bei ca. 5,0 mm von der Blinddarm-Spitze, und dann Punktion der ligierten cecal Stumpf einmal mit einer 22-Gauge-Nadel zur Extrusion von Stuhl zu ermöglichen. </li><li> Der Blinddarm wird sofort wieder in seine normale Position intraabdominalen ersetzt. </li><li> Schließen Sie die Inzisionsstelle in zwei Schichten, indem Sie zuerst Schließen der Muskulatur des Bauches mit resorbierbaren Fäden und dann Schließen der Haut mit Wundklammern oder nicht resorbierbaren Fäden. </li><li> Resuscitate Maus mit 0,5 ml physiologischer Kochsalzlösung und eine einzelne tunsich von Imipenem (0,5 mg / Maus, subkutan) unmittelbar nach Beendigung des chirurgischen Eingriffs. </li><li> Kehren Sie die Maustaste wieder auf einem sauberen Käfig mit freiem Zugang zu Nahrung und Wasser. </li><li> Zu verschiedenen Zeitpunkten nach der CLP wird pflanzliche Extrakt oder eine Komponente intraperitoneal verabreicht. </li><li> Das Überleben der Tiere wird für mehr als zwei Wochen überwacht. Sterbende Tiere ausstellen Schwierigkeiten zu stehen, agonale Atmung, schwere Muskelatrophie und unkontrollierte Blutungen sollten durch Überdosierung Kohlendioxidinhalation eingeschläfert werden. </li><li> Es ist wichtig zu beachten, dass zirkulierende Cytokin Ebenen sind in diesen Studien. Verschiedene Analgetika hat sich gezeigt, Zytokinfreisetzung und Aktivitäten beeinflussen, und deshalb wurden absichtlich in der postoperativen Betreuung vermieden werden. </li></ol> 2. Vorbereitung der Kräuter-Extrakt <ol><li> Auszug Kräuter in heißem Wasser (85 ° C) für 1-4 Std. (1 Std. für Blätter und Wurzeln für 4 Std.). </li><li> Zentrifugieren Sie die Wasser-soUnlöslich Fraktion (3300 g, 20 min, 4 ° C) zur Entfernung unlöslicher Teilchen. </li><li> Filtrat der Überstand Fraktion durch eine 0,2 um-Filter. </li><li> Die klare wasserlösliche Filtratfraktion wird dann fraktioniert durch Ultrafiltration unter Verwendung von Centriprep YM-10 Filterzentrifuge (Katalog-Nr. 4305, Millipore). </li><li> Das führte zu niedrig (&lt;10 kDa) und hoch (&gt; 10 kDa) Molekulargewichtsfraktionen (MWF) für HMGB1-hemmenden Aktivitäten mit Makrophagen-Kulturen gescreent werden. </li><li> Intraperitoneale Gabe von HMGB1-hemmenden Kräuterextrakt oder Komponenten bei 24 Stunden nach CLP zu evaluieren ihre therapeutische Wirksamkeit. </li></ol> 3. Peritonealen Makrophagen Isolierung <ol><li> Thioglycolat-Brühe (4%, 2,0 ml) intraperitoneal in normale Mäuse verabreicht. </li><li> Primäre Peritonealmakrophagen werden zu 3 Tage früher als 10 beschrieben geerntet. </li><li> Makrophagen sind vorkultiviert in DMEM-Medium (Gibco BRL, Grand Island, NY), ergänzt mit 10% fötalem Rinderserum (FBS), 2 mmol / l Glutamin und 1% Penicillin. </li><li> Adhärente Makrophagen vorsichtig mit gewaschen und kultiviert, serumfreien OPTI-MEM I Medium zwei Stunden vor der Stimulation mit bakteriellem Endotoxin (Lipopolysaccharid, LPS, E. coli 0111: B4, Sigma-Aldrich). </li><li> 16 Stunden nach der LPS-Stimulation, die Spiegel des HMGB1 in dem Kulturmedium durch Western-Blot-Analyse 11 bestimmt. </li><li> Die relative Bandenintensität wurde unter Verwendung des NIH-Image-Software 1,59 HMGB1 Ebenen mit Bezug auf Standardkurven mit gereinigtem HMGB1 erzeugt bestimmen quantifiziert. </li></ol> 4. Repräsentative Ergebnisse 1. CLP induziert systemische und lokale Entzündung. Innerhalb von wenigen Stunden von CLP Chirurgie, zeigen Tiere klinische Symptome einer Sepsis, die Piloerektion, Lethargie, kauern, und die Abnahme der Futter-und Wasseraufnahme sind. Tiere entwickeln eine schwere Bauchfellentzündung mit konsekutiver systemischer96 Stunden - Infektion in der Regel innerhalb von 48 sterben. Allerdings kann auch das Alter, das Geschlecht und genetischen Hintergrund abgestimmt Tieren reagieren Operation in einer unterscheidbaren Weise im Verlauf der experimentellen Sepsis CLP. Zum Beispiel bei 48 Stunden nach der CLP, während einige Tiere dem schlechten Zustand (wie in 1 definiert sind) Ansätze möglich sind, können andere in einem Nicht-moribunde Zustand bleiben. Umfassende Erhebungen über zirkulierende Zytokine zeigten dramatische Unterschiede bei den verschiedenen Zytokinen (zB IL-6, KC, MIP-2, und sTNFR1) zwischen Mäusen in den maroden und nicht-sterbenden Staaten (Abbildung 1) 12. Bemerkenswerterweise haben diese Entzündungsmediatoren als Surrogat-Marker der Sepsis klassifiziert worden, weil ihre Blutspiegel zuverlässige Prädiktoren für tödlichem Ausgang sind in der experimentellen oder klinischen Sepsis 12-14 15. Darüber hinaus CLP auch lokale Freisetzung von verschiedenen pro-und anti-inflammatorische Zytokine und Chemokine induziert. Zum Beispiel bei der48 h nach der CLP, signifikante Mengen von Cytokinen (zB IL-6) und Chemokine (KC und MIP-2) noch nicht nur systemisch im Blut gemessen werden, sondern auch lokal im Peritoneallavage Fluid (2). 2. Screening für HMGB1-hemmenden Kräuterextrakt oder Komponenten. Mit Makrophagen-Kulturen, konnten wir die Kapazitäten der verschiedenen Kräuterextrakt oder Komponenten bei der Hemmung der Endotoxin-induzierten Freisetzung HMGB1 (Abbildung 3) zu bewerten. Angesichts ihrer Kapazität bei der Hemmung der Freisetzung HMGB1 wir weiter erforscht ihre Wirksamkeit in Tiermodellen Sepsis. Einem Zeitpunkt, wenn Mäuse deutliche Anzeichen einer Sepsis entwickelt einschließlich Lethargie, Durchfall und Piloerektion - Angesichts der späten und verlängerte Kinetik der Akkumulation HMGB1 in der experimentellen Sepsis 8, wurde die erste Dosis von HMGB1-Hemmern 24 Stunden nach dem Auftreten der Sepsis gegeben. Wie in 4 gezeigt ist, verzögert und wiederholte Verabreichung einer großen grünen Tee cls Bestandteil, Epigallocatechin-3-Gallat (EGCG), beginnend bei 24 h nach Beginn einer Sepsis, deutlich gerettet Mäuse vor tödlichen Sepsis 12. Selbst wenn es oral verabreicht, EGCG noch zu retten Mäuse vor tödlichen Sepsis, die deutliche Steigerung Tier-Überlebensraten von 16% auf 44% 16. Es wäre wichtig, um festzustellen, ob kombinatorische Therapien mit verschiedenen pflanzlichen Komponenten konnte eine signifikante besseren Schutz im Tiermodell der Sepsis zu erzielen. Zusammengenommen sind diese experimentellen Daten unserer Herangehensweise an Bildschirm neuartigen therapeutischen Wirkstoffen mit einem Tiermodell der CLP-induzierten Sepsis validiert. 5. Repräsentative Ergebnisse <img alt="1" src="/files/ftp_upload/3926/3926fig1.jpg" /> Abbildung 1. Zirkulierenden Spiegel Surrogatmarker sind dramatisch höher in septischen Tieren nähern moribunden Zustand. Balb / C-Mäuse wurden zu einer Sepsis durch CLP unterworfen und überwacht werden für die Entwicklung von Anzeichen einer Krankheit. An einspäten Stadium der Sepsis (52 Stunden nach der CLP) wurde Blut aus 3 normalen Mäusen (-CLP), 3 septischen Tieren nähert dem schlechten Zustand, und 3 septischen Tiere in der nicht-moribunde Zustand gesammelt. Das Serum wurde aus jeder Gruppe vereinigt, und im Hinblick auf die Zytokin-Profils von Antikörper-Arrays. Notieren Sie sich den dramatischen Unterschied in der relativen Pegel von mehreren Surrogat-Marker zwischen verschiedenen Gruppen. Übernommen aus doi: 10.1371/journal.pone.0001153.g006 mit Erlaubnis des Herausgebers. <img alt="2" src="/files/ftp_upload/3926/3926fig2.jpg" /> Abbildung 2. Nachweis von lokalen und systemischen Cytokine und Chemokine bei 48 Stunden nach CLP. Balb / C-Mäuse wurden von Sepsis durch CLP unterzogen, und Blut oder Peritonealflüssigkeit wurden nach 48 h nach CLP geerntet. Relative Ebenen mehrerer Zytokine und Chemokine im gepoolten Serum oder peritoneale Lavage wurden von Zytokin-Antikörper-Arrays gemessen und ausgedrückt in willkürlichen Einheiten (AU). Als Kontrollen, Blut-und Peritonealflüssigkeit SAmples wurden aus normalen gesunden Tieren (-CLP) ohne Laparotomie erhalten. <img alt="Abbildung 3" src="/files/ftp_upload/3926/3926fig3.jpg" /> Abbildung 3. Herbal Komponente dosisabhängig gehemmt Endotoxin-induzierten Freisetzung HMGB1 in primären Makrophagen-Kulturen. Primären murinen peritonealen Makrophagen mit LPS in Abwesenheit oder Anwesenheit von Kräuter-Komponente (zB EGCG, 15 pM) stimuliert wurden. 16 Stunden nach der LPS-Stimulation wurden Ebenen von HMGB1 in dem Kulturmedium durch Western-Blot-Analyse bestimmt. Übernommen aus doi: 10.1371/journal.pone.0001153.g006 mit Erlaubnis des Herausgebers. <img alt="Abbildung 4" src="/files/ftp_upload/3926/3926fig4.jpg" /> Abbildung 4. Herbal Komponente gerettet Mäuse aus tödliche Sepsis. Balb / C-Mäuse wurden zu tödlichen Sepsis durch CLP unterzogen und Kräuter-Komponente (EGCG) wurde intraperitoneal bei +24 verabreicht wird, posten 48, 72 Stunden das Auftreten von Sepsis. Einimal Überleben wurde für bis zu zwei Wochen beobachtet. Die Kaplan-Meier-Methode wurde verwendet, um die Unterschiede in der Sterblichkeit zwischen den Gruppen zu vergleichen. *, P &lt;0,05 im Vergleich zu Kochsalzlösung. Übernommen aus doi: 10.1371/journal.pone.0001153.g006 mit Erlaubnis des Herausgebers.

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AES und HW sind Miterfinder der Patentanmeldungen im Zusammenhang mit HMGB1-Inhibitoren (tanshinones) als potenzielle Therapeutika für die Sepsis.

Im Labor wurden verschiedene Tiermodelle der Sepsis eingesetzt worden, um die Pathogenese der Sepsis zu verstehen, um mögliche neue Therapien zu entwickeln. Deren klinische Relevanz bleibt ein Thema der Debatte vor der gelungene Übersetzung von Tierversuchen in klinische Anwendungen für die Sepsis. Obwohl neutralisierende Antikörper gegen den frühen Zytokinen (zB TNF) waren Schutzschicht in Tiermodellen von Bakteriämie / Endotoxämie 17,18 sie tatsächlich verschlechtern Überleben im Tiermodell der Sep...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </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> Ketamin HCl </td><td> Hospira Inc. </td><td> RL-0065 </td></tr><tr><td> Xylazin </td><td> Lloyd Laboratories </td><td> 4821 </td></tr><tr><td> Autoclip </td><td> Becton Dickinson </td><td> 427631 </td></tr><tr><td> 4-0 Seidenfaden </td><td> Roboz </td><td> SUT-15-2 </td></tr><tr><td> Surflo IV-Katheter </td><td> Terumo </td><td> SR * OX2419CA </td></tr><tr><td> RayBio Maus Zytokin-Antikörper-Array </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

Sepsis bezieht sich auf eine Systemic Inflammatory Response Syndrome, die aus einer mikrobiellen Infektion. Es wurde routinemäßig simulierten bei Tieren durch mehrere Techniken, einschließlich der Infusion von exogenem bakterielles Toxin (Endotoxämie) oder Bakterien (Bakteriämie), sowie chirurgische Perforation des Blinddarms von Blinddarm Ligation und Punktion (CLP) 1-3. CLP ermöglicht es Bakterien Verschütten und fäkalen Verunreinigung der Bauchhöhle, Nachahmung der menschlichen klinischen Erkrankung der perforierten Appendizitis oder Divertikulitis. Die Schwere der Sepsis, wie durch die eventuelle Sterblichkeit reflektiert, können chirurgisch durch Variieren der Größe der Nadel zum Blinddarm Punktion 2 verwendet gesteuert werden. Bei Tieren induziert CLP ähnlich, biphasische hämodynamischen kardiovaskulären, metabolischen und immunologischen Reaktionen, wie bei den klinischen Verlauf der menschlichen Sepsis 3 beobachtet. So wird die CLP-Modell als eines der klinisch relevanten experimentellen Modellen für Sepsis 3.1 berücksichtigt. &lt;/ P&gt; Verschiedene Tiermodelle wurden verwendet, um die komplizierten Mechanismen der Pathogenese der experimentellen Sepsis aufzuklären. Die tödliche Folge von Sepsis basiert teils auf einer übermäßigen Ansammlung von frühen Zytokine (wie TNF, IL-1 und IFN-γ) 4-6 und späten proinflammatorischen Mediatoren (zB HMGB1) 7. Im Vergleich mit früher proinflammatorische Zytokine, haben Ende wirkenden Mediatoren eine größere therapeutische Fenster für klinische Anwendungen. So verzögerte Gabe von HMGB1-neutralisierende Antikörper beginnend 24 Stunden nach CLP, noch zu retten Mäuse aus Letalität 8,9, Gründung HMGB1 als späte Vermittler von tödlichen Sepsis. Die Entdeckung von HMGB1 als spät wirkender Mediator hat ein neues Feld der Forschung für die Entwicklung einer Sepsis Therapien mit traditionellen chinesischen Kräutermedizin eingeleitet. In diesem Beitrag beschreiben wir ein Verfahren der CLP-induzierten Sepsis, und seine Verwendung in Screening Kräutermedizin für HMGB1-Targeting-Therapien.

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 bezieht sich auf eine systemische inflammatorische Reaktion, die sich aus einer mikrobiellen Infektion, und simuliert werden können durch eine Blinddarm-OP-Technik bezeichnet Ligatur und Punktion (CLP). Hier beschreiben wir eine Methode zur CLP-induzierte Tiermodell zu verwenden, um Heilkräuter für therapeutische Wirkstoffe zu screenen.

Die Verwendung Tiermodell für Sepsis zu neuartigen Therapien zu beurteilen Herbal

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 Aufrufe.  North Shore – LIJ Health System. Sepsis bezieht sich auf eine systemische inflammatorische Reaktion, die sich aus einer mikrobiellen Infektion, und simuliert werden können durch eine Blinddarm-OP-Technik bezeichnet Ligatur und Punktion (CLP). Hier beschreiben wir eine Methode zur CLP-induzierte Tiermodell zu verwenden, um Heilkräuter für therapeutische Wirkstoffe zu screenen.

Serie: 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.

Die CYP2D6-Tiermodell: Wie Autoimmune Hepatitis in Mäusen zu induzieren

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 ...

Forschung

Medizin

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.

Ein Maus-Tumormodell der operativen Belastung zu Entdecken Sie die Mechanismen der postoperative Immunsuppression und Bewerten Novel Perioperative Immuntherapien

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.

Bewertung der Wirksamkeit der<em&gt; H. pylori</em&gt; Protein HP-NAP als therapeutisches Werkzeug zur Behandlung von Blasenkrebs in einem orthotopen Mausmodell

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.

Biolumineszenz-Bildgebung von einem immunkompetenten Tiermodell für 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.

Ein kleines Tier Modell der Ex Vivo Normothermic Leber Perfusion

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.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht 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.

Neuartige Methoden für intranasale Verabreichung unter Inhalation Anästhesie auszuwertende Nase zu Gehirn 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.

Funktionsbeurteilung des Spenderherzens während der Ex-situ-Perfusion : Erkenntnisse aus Druck-Volumen-Schleifen und Oberflächenechokardiographie

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.

Partielle Ischiasnervligatur: Ein Mausmodell für chronische neuropathische Schmerzen zur Untersuchung der antinozizeptiven Wirkung neuartiger Therapien

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.

Mongolische Rennmäuse als Tiermodell der Wundheilung

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