Questo lavoro è stato supportato da sovvenzioni da parte del National Institutes of Health, National Institute of General Medical Science (R01GM063075) e il Centro Nazionale di Medicina Complementare e Alternativa (R01AT05076).

1. Istituzione di un modello animale di sepsi <ol><li> I topi sono anestetizzati con ketamina (75 mg / kg, intramuscolare, IM) e xilazina (10 mg / kg, im), e posto in posizione supina. </li><li> Fissare i piedini del mouse con il nastro per garantire una posizione stabile. </li><li> Pulire l&#39;addome con 3 scrub alternati di betadine o altro disinfettante cutaneo e alcool. Quindi, una incisione mediana 15 mm per esporre il cieco. </li><li> Legare il cieco con una sutura di seta 4-0 a circa 5,0 millimetri dalla punta cecale, e poi perforare il moncone ligato cecale una volta con un 22-gauge per consentire estrusione di feci. </li><li> Il cieco viene immediatamente sostituito alla sua normale posizione intra-addominale. </li><li> Chiudere il sito di incisione in due strati, chiudendo prima la muscolatura dell&#39;addome con suture assorbibili, e poi chiudendo la pelle con clips ferite o suture assorbibili. </li><li> Resuscitare il mouse con 0,5 ml di soluzione salina normale e una singola farese di imipenem (0,5 mg / topo, sottocutanea) immediatamente dopo il completamento della procedura chirurgica. </li><li> Riportare il mouse avanti ad una gabbia pulita con libero accesso a cibo e acqua. </li><li> Al momento dopo CLP vari punti, estratto di erbe o componente viene somministrato per via intraperitoneale. </li><li> Sopravvivenza degli animali è monitorato per più di due settimane. Gli animali moribondi che presentano difficoltà di stare in piedi, respiro agonico, grave atrofia muscolare e sanguinamento incontrollato dovrebbero essere l&#39;eutanasia per inalazione di anidride carbonica overdose. </li><li> È importante notare che i livelli circolanti di citochine sono parametri importanti in questi studi. Diversi analgesici hanno dimostrato di influire rilascio di citochine e le attività, e quindi sono stati volutamente evitati in cura post-operatoria. </li></ol> 2. Preparazione di Estratto di erbe <ol><li> Estrarre le erbe in acqua calda (85 ° C) per 1-4 ore (1 ora per le foglie, e 4 ore per le radici). </li><li> Centrifugare d&#39;acqua in modofrazione luble (3300 g, 20 min, 4 ° C) per rimuovere le particelle insolubili. </li><li> Filtrare il surnatante attraverso un filtro da 0,2 um. </li><li> Il chiaro frazione solubile in acqua filtrato viene poi frazionata mediante ultrafiltrazione usando Centriprep YM-10 filtro centrifugo (Catalog no. 4305, Millipore). </li><li> Il risultato basso (&lt;10 kDa) e alta (&gt; 10 kDa), frazioni di peso molecolare (MWF) sono sottoposti a screening per HMGB1 che inibiscono le attività che utilizzano le culture macrofagi. </li><li> La somministrazione intraperitoneale di estratti di erbe HMGB1-inibitori o dei componenti a 24 ore dopo CLP per valutare la loro efficacia terapeutica. </li></ol> 3. Macrofagi isolamento peritoneale <ol><li> Thioglycollate brodo (4%, 2.0 ml) viene somministrato per via intraperitoneale in topi normali. </li><li> Macrofagi peritoneali primari vengono raccolte in 3 giorni come descritto in precedenza 10. </li><li> I macrofagi sono pre-coltivate in mezzo DMEM (Gibco BRL, Grand Island, NY) supplementato con 10% di siero fetale bovino (FBS), 2 mmol / L glutammina e 1% di penicillina. </li><li> Macrofagi aderenti sono delicatamente lavate con, e coltivate in, privo di siero OPTI-MEM I due ore prima della stimolazione con endotossina batterica (lipopolisaccaride, LPS, E. coli 0111: B4, Sigma-Aldrich). </li><li> A 16 ore dopo la stimolazione con LPS, livelli di HMGB1 nel terreno di coltura sono determinati mediante Western blotting 11. </li><li> L&#39;intensità relativa banda è stata quantificata utilizzando l&#39;immagine NIH 1,59 software per determinare i livelli HMGB1 con riferimento alle curve standard generata con purificato HMGB1. </li></ol> 4. Risultati rappresentativi 1. CLP induce infiammazione sistemica e locale. Entro poche ore di intervento chirurgico CLP, gli animali mostrano segni clinici di sepsi che comprendono piloerezione, letargia, stringendosi, e la diminuzione dell&#39;assunzione di cibo e acqua. Animali in via di sviluppo uno peritonite grave con sistemica consecutivol&#39;infezione di solito muoiono entro 48-96 ore. Tuttavia, anche età, sesso, e sfondo genetico-matched animali possono rispondere a CLP chirurgia in modo distinguibile nel corso di sepsi sperimentale. Per esempio, a 48 ore dopo CLP, mentre alcuni animali possono aver avvicinato lo stato moribondo (come definito nella Figura 1), altri possono rimanere in uno stato di non-moribonda. Indagini complete di citochine circolanti rivelato differenze drammatiche nei livelli di diverse citochine (per esempio, IL-6, KC, MIP-2, e sTNFR1) tra topi negli stati moribonde e non moribondo (Figura 1) 12. In particolare, questi mediatori infiammatori sono stati classificati come marcatori surrogati di sepsi perché i loro livelli circolanti sono indicatori attendibili di esito letale nel 12-14 sperimentale o clinico sulla sepsi 15. Inoltre, CLP anche indotto rilascio locale di varie citochine pro-e anti-infiammatorie e chemochine. Ad esempio, a48 ore dopo CLP, notevoli quantità di citochine (ad esempio, IL-6) e chemochine (KC e MIP-2) possono ancora essere misurato non solo sistemico nel sangue, ma anche localmente nel liquido di lavaggio peritoneale (Figura 2). 2. Screening per l&#39;estratto di erbe HMGB1-inibitori o componenti. Utilizzando colture di macrofagi, siamo stati in grado di valutare le capacità dei vari estratto di erbe o di componenti di inibire endotossina-indotto rilascio HMGB1 (Figura 3). Alla luce della loro capacità di inibire la liberazione HMGB1, abbiamo ulteriormente esplorato la loro efficacia in modelli animali sepsi. Date le cinetiche di ritardo e prolungato accumulo di HMGB1 nella sepsi sperimentale 8, la prima dose di HMGB1 inibitori è stata data 24 ore dopo l&#39;insorgenza di sepsi - un punto di tempo in cui topi hanno sviluppato chiari segni di sepsi compresi letargia, diarrea e piloerezione. Come mostrato in figura 4, ritardata e somministrazione ripetuta di un importante tè verde component, epigallocatechina-3-gallato (EGCG), con inizio alle 24 ore dopo l&#39;insorgenza di sepsi, salvato in modo significativo i topi da sepsi letale 12. Anche quando somministrato per via orale, EGCG topi ancora salvati dalla sepsi letale, aumentando in modo significativo i tassi di sopravvivenza degli animali dal 16% al 44% 16. Sarebbe importante per determinare se le terapie combinatorie con diversi ingredienti erboristici potrebbe ottenere una significativa protezione migliore in modelli animali di sepsi. Presi insieme, questi dati sperimentali convalidato il nostro approccio alla schermata nuovi agenti terapeutici utilizzando un modello animale di CLP-indotta da sepsi. 5. Risultati rappresentativi <img alt="Figura 1" src="/files/ftp_upload/3926/3926fig1.jpg" /> Figura 1. I livelli circolanti di marcatori surrogati sono drammaticamente più elevato negli animali settici che si avvicinano stato moribondo. Balb / C topi sono stati sottoposti a sepsi da CLP e monitorati per lo sviluppo di segni di malattia. Afase avanzata di sepsi (52 hr dopo CLP), il sangue è stato raccolto da 3-topi normali (CLP), 3 topi settici che si avvicinano allo stato moribondo, e 3 topi settiche nel settore non-statale moribonda. Siero è stato riunito da ciascun gruppo, e analizzati per il profilo di citochine da parte array di anticorpi. Si noti la differenza drammatica i relativi livelli di alcuni marcatori surrogati tra i diversi gruppi. Adattato da doi: 10.1371/journal.pone.0001153.g006 con il permesso rilasciato dal publisher. <img alt="Figura 2" src="/files/ftp_upload/3926/3926fig2.jpg" /> Figura 2. Rilevamento di citochine locali e sistemiche e chemochine a 48 ore post-CLP. Balb / C topi sono stati sottoposti a sepsi da CLP, e sangue o liquido peritoneale sono state raccolte a 48 ore dopo CLP. Relativi livelli di diverse citochine e chemochine in pool di siero o nel liquido di lavaggio peritoneale sono stati misurati mediante array di anticorpi citochine, ed espresso in unità arbitrarie (UA). Come fluido sa controlli, sangue e peritonealemples sono stati ottenuti da animali sani (normali-CLP) senza laparotomia. <img alt="Figura 3" src="/files/ftp_upload/3926/3926fig3.jpg" /> Figura 3. Componente di Herbal dose-dipendente inibito endotossina-indotto HMGB1 rilascio in colture primarie di macrofagi. Primarie macrofagi peritoneali murini sono state stimolate con LPS in assenza, o presenza di componenti a base di erbe (per esempio, EGCG, 15 pm). A 16 ore dopo la stimolazione con LPS, livelli di HMGB1 nel terreno di coltura sono stati determinati mediante Western blotting. Adattato da doi: 10.1371/journal.pone.0001153.g006 con il permesso rilasciato dal publisher. <img alt="Figura 4" src="/files/ftp_upload/3926/3926fig4.jpg" /> Figura 4. Componente di Herbal salvato i topi da sepsi letale. Balb / C topi sono stati sottoposti a sepsi letale da CLP, e la componente a base di erbe (EGCG) è stato somministrato per via intraperitoneale a +24, +48, +72 ore dopo l&#39;insorgenza di sepsi. Unsopravvivenza IMAL è stata monitorata per un massimo di due settimane. Il metodo di Kaplan-Meier è stato utilizzato per confrontare le differenze nei tassi di mortalità tra i gruppi. *, P &lt;0,05 rispetto salina. Adattato da doi: 10.1371/journal.pone.0001153.g006 con il permesso rilasciato dal publisher.

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AES HW e sono co-inventori di domande di brevetto relative a HMGB1 inibitori tanshinones) come potenziali agenti terapeutici per la sepsi.

In laboratorio, modelli animali di sepsi sono stati impiegati per comprendere la patogenesi di sepsi, al fine di sviluppare nuove terapie potenziali. La loro rilevanza clinica rimane un argomento di dibattito prima della traduzione con successo studi su animali in applicazioni cliniche per la sepsi. Sebbene anticorpi neutralizzanti contro citochine precoci (ad esempio, TNF) sono stati protettivo in modelli animali di batteriemia / endotossemia 17,18, in realtà peggiorano sopravvivenza nel modello animale di ...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </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> 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> Seta filo di sutura 4-0 </td><td> Roboz </td><td> SUT-15-2 </td></tr><tr><td> Surflo IV Catetere </td><td> Terumo </td><td> SR * OX2419CA </td></tr><tr><td> RayBio del mouse citochine serie di anticorpi </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

Sepsi si riferisce ad una sindrome da risposta infiammatoria sistemica risultante da una infezione microbica. È stato simulato routine negli animali con varie tecniche, tra cui l&#39;infusione di esogeno tossina batterica (endotossemia) o batteri (batteriemia), nonché la perforazione chirurgica del cieco mediante ligazione cecale e puntura (CLP) 1-3. CLP permette fuoriuscite batteri e contaminazione fecale della cavità peritoneale, imitando la malattia clinica nell&#39;uomo di appendicite perforata o diverticolite. La gravità di sepsi, come risulta dalle eventuali tasso di mortalità, può essere controllata chirurgicamente variando la dimensione dell&#39;ago utilizzato per la puntura cecale 2. Negli animali, CLP induce simile, emodinamica bifasica risposte cardiovascolari, metaboliche, immunologiche e di come è stato osservato durante il decorso clinico di sepsi umana 3. Pertanto, il modello CLP viene considerato come uno dei modelli più clinicamente rilevanti per sepsi sperimentale 1-3. &lt;/ P&gt; Vari modelli animali sono stati utilizzati per illustrare i meccanismi complessi alla base della patogenesi di sepsi sperimentale. La conseguenza letale di sepsi è attribuibile in parte ad un accumulo eccessivo di citochine precoci (ad esempio TNF, IL-1 e IFN-γ) 4-6 e mediatori proinfiammatori ritardo (ad esempio, HMGB1) 7. Rispetto ai primi citochine proinfiammatorie, tarda ad azione mediatori hanno una finestra terapeutica più ampia per le applicazioni cliniche. Per esempio, la somministrazione ritardata di HMGB1 Anticorpi neutralizzanti che iniziano 24 ore dopo la CLP, ancora salvato i topi di letalità 8,9, stabilendo HMGB1 come mediatore di sepsi tardiva letale. La scoperta di HMGB1 come un tardo-acting mediatore ha avviato un nuovo campo di indagine per lo sviluppo di terapie che utilizzano sepsi Medicina Tradizionale Cinese di erbe. In questo articolo descriviamo una procedura di CLP-indotta la sepsi, e il suo utilizzo nello screening di medicina di erbe per HMGB1-targeting terapie.

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

Sepsi si riferisce ad una sindrome da risposta infiammatoria sistemica risultante da una infezione microbica, e può essere simulato da una tecnica chirurgica denominata legatura cecale e puntura (CLP). Qui si descrive un metodo per utilizzare CLP-indotta modello animale per lo screening erbe medicinali per gli agenti terapeutici.

Uso di un modello animale di sepsi per valutare nuove terapie a base di erbe

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 Views.  North Shore – LIJ Health System. Sepsi si riferisce ad una sindrome da risposta infiammatoria sistemica risultante da una infezione microbica, e può essere simulato da una tecnica chirurgica denominata legatura cecale e puntura (CLP). Qui si descrive un metodo per utilizzare CLP-indotta modello animale per lo screening erbe medicinali per gli agenti terapeutici.

Video: 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.

Il CYP2D6 modello animale: come indurre epatite autoimmune in topi

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 ...

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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.

Un tumore modello murino di stress chirurgico per esplorare i meccanismi di immunosoppressione post-operatorio e valutare Novel perioperatoria Immunoterapie

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.

Valutazione dell&#39;efficacia della<em&gt; H. pylori</em&gt; Proteine ​​HP-NAP come strumento terapeutico per il trattamento del cancro della vescica in un ortotopico Modello murino

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.

Bioluminescenza Imaging di un Modello immunocompetenti Animal 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.

Un piccolo modello animale di aspersione epatica normotermica 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.

Misurazione di contrattilità isometrica dell'arteria mesenterica Mouse con filo Myography

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.

Metodi innovativi per la somministrazione intranasale in anestesia per inalazione per valutare il naso--cervello 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.

Valutazione funzionale del cuore del donatore durante la perfusione ex situ : approfondimenti dai loop pressione-volume e dall'ecocardiografia di superficie

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.

Legatura parziale del nervo sciatico: un modello murino di dolore neuropatico cronico per studiare l'effetto antinocicettivo di nuove terapie

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.

Gerbilli mongoli come modello animale di guarigione delle ferite

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