Bu çalışma Ulusal Sağlık Enstitüleri, Ulusal Genel Tıp Bilimi (R01GM063075) Enstitüsü ve Tamamlayıcı ve Alternatif Tıp Ulusal Merkezi (R01AT05076) hibe ile desteklenmiştir.

1. Sepsis Hayvan Modeli Kurulması <ol><li> Fareler ketamin (75 mg / kg, kas içi, im) ve Xylazine (10 mg / kg, im) ile anestezi uygulanmıştır, ve yatar konumda yerleştirilmiştir. </li><li> Istikrarlı bir pozisyon sağlamak için bant ile fare ayakları Fix. </li><li> Betadin veya diğer cilt dezenfektan ve alkol 3 alternatif scrubs ile karın temizleyin. Sonra, bir 15 mm ensizyon çekum maruz olun. </li><li> Dışkı ekstrüzyon sağlamak için 22-gauge iğne ile bir kez bağlanan çekal güdük çekal ucundan yaklaşık 5.0 mm az bir 4-0 ipek sütür ile çekum Ligate ve sonra ponksiyon. </li><li> Çekum hemen normal karın içi konumuna geri değiştirilir. </li><li> İlk emilebilir dikişlerle karın kas kapatılması ve ardından yara klipleri veya emilemeyen sütürler ile cilt kapatılarak, iki kat kesi kapatın. </li><li> 0.5 ml serum fizyolojik solüsyonu ile fare diriltmek ve tek yapmakhemen cerrahi prosedürün tamamlanmasından sonra imipenem se (subkutan olarak 0.5 mg / fare). </li><li> Yiyecek ve su serbest erişim ile temiz bir kafes geri dönmek fare. </li><li> Farklı zaman noktalarında yazılan CLP anda, bitki özü veya bileşen intraperitoneal edilir. </li><li> Hayvan hayatta fazla iki hafta izlenir. Durmak zorluklar, agonal solunum, ciddi kas atrofi ve kontrolsüz kanama sergileyen Moribund hayvanların aşırı dozda karbon dioksit solunum yoluyla ötenazi edilmelidir. </li><li> Bu sitokin düzeylerinin dolaşan bu çalışmalarda önemli parametreler olduğunu not etmek önemlidir. Çeşitli analjezikler sitokin salınımı ve faaliyetleri etkilediği görülmüştür ve bu nedenle kasıtlı postoperatif bakım kaçınılmıştır. </li></ol> 2. Bitkisel Extract hazırlanması <ol><li> 1-4 saat (yaprak için 1 saat, ve kökleri için 4 saat) için sıcak su (85 ° C) otlar ayıklayın. </li><li> Su böylece santrifüjeluble fraksiyon (3.300 g, 20 dak, 4 ° C) çözünmeyen parçacıkların çıkarmak için kullanılır. </li><li> Süzülen madde bir 0.2 um bir filtreden süpernatan fraksiyonu. </li><li> Berrak suda eriyen süzüntü fraksiyonu sonra Centriprep YM-10 Santrifüj Filtresi (Katalog No. 4305, Millipore) kullanarak ultrafiltrasyon tarafından parçalandı olduğunu. </li><li> (&lt;10 kDa ve yüksek (10 kDa) düşük)&gt; molekül ağırlıklı fraksiyonları (MWF) makrofaj kültürleri kullanarak HMGB1 önleyici faaliyetler için taranmaktadır sonuçlandı. </li><li> 24 saat sonrası CLP az HMGB1 önleyici bitki özü veya bileşenleri intraperitoneal bunların tedavi etkinliğini değerlendirmektir. </li></ol> 3. Periton Makrofaj İzolasyon <ol><li> Thioglycollate çorbası (4,% 2.0 ml), normal farelere intraperitonal olarak uygulanır. </li><li> İlköğretim peritoneal makrofajlar daha önce açıklandığı gibi 10 3 günde hasat edilir. </li><li> Makrofajlar 1 ile desteklenmiş DMEM ortamı (Gibco BRL, Grand Island, NY) içinde ön-kültür analizi% 0 fetal bovin serumu (FBS), 2 mmol / L glutamin, ve% 1 penisilin. </li><li> Yapışık makrofajlar hafifçe ile yıkanmış, ve kültür edilir, serumsuz iki saat bakteriyel endotoksin ile uyarılması daha önce OPTI-MEM I ortamı (lipopolisakkarid, LPS, E. coli 0111: B4, Sigma-Aldrich). </li><li> LPS uyarıldıktan sonra 16 saat sonra, kültür ortamı içinde HMGB1 seviyeleri Western blot analizi 11 tarafından belirlenir. </li><li> Göreceli bant yoğunluğu HMGB1 saflaştırılmış ile oluşturulan standart eğrileri referans HMGB1 düzeylerini belirlemek için NIH Image 1.59 yazılımı kullanılarak ölçüldü. </li></ol> 4. Temsilcisi Sonuçlar 1. CLP sistemik ve lokal inflamasyon neden olur. CLP cerrahi bir kaç saat içinde, hayvanlar piloerection, letarji, huddling, gıda ve su alımında azalma, sepsis klinik belirtiler gösterirler. Ardışık sistemik ağır bir peritonit gelişen Hayvanlar96 saat - Enfeksiyon normal olarak 48 saat içinde ölür. Ancak, hatta yaş, cinsiyet ve genetik arka plan olarak eşleştirilmiş hayvanların deneysel sepsis sırasında ayırt edici bir şekilde ameliyat CLP yanıt verebilir. Bazı hayvanların can çekişen devlet (Şekil 1&#39;de tanımlanan) yaklaştı olabilecekken Örneğin, 48 saat sonrası CLP de, diğerleri olmayan bir can çekişen devlet olarak kalabilir. Dolaşımdaki sitokinlerin kapsamlı araştırmalar can çekişmekte olan ve olmayan can çekişmekte olan devletler (Şekil 1) 12 fareler arasında birçok sitokin düzeylerinde dramatik farklılıklar (örn., IL-6, KC, MIP-2 ve sTNFR1) saptandı. Bunların dolaşımdaki seviyeleri 12-14 deneysel veya klinik sepsis 15 ölümcül sonucu güvenilir yordayıcıları çünkü Özellikle, bu inflamatuar mediatörlerin sepsis taşıyıcı belirteç olarak sınıflandırılmıştır. Ayrıca, CLP ayrıca çeşitli pro-ve anti-inflamatuar sitokinler ve kemokinlerin yerel yayın bağlı. At, örneğin48 saat sonrası CLP, önemli bir sitokin miktarları (örn., IL-6) ve kemokin (KC ve MIP-2) hala ayrıca lokal olarak periton lavaj (Şekil 2), kan içinde sadece sistemik olarak ölçülen, fakat edilebilir. 2. HMGB1 önleyici bitki özü veya bileşenler için tarama. Makrofaj kültürleri kullanarak, inhibe endotoksin bağımlı HMGB1 sürümde çeşitli bitki özü veya bileşenlerinin kapasiteleri (Şekil 3) değerlendirmeyi başardık. Inhibe HMGB1 sürümde kapasiteleri ışığında, başka hayvan modellerinde sepsis etkinliğini araştırdı. Bir zaman noktasında farelerde letarji, ishal ve piloerection dahil olmak üzere sepsis net işaretler geliştirdi - Deneysel sepsis 8 HMGB1 birikimi geç ve uzun süreli kinetiği göz önüne alındığında, HMGB1 inhibitörlerinin ilk doz sepsisin başlangıcından sonra 24 saat verildi. Gibi, Şekil 4&#39;te gösterildiği geciktirilen ve büyük bir yeşil çay c tekrarlanan idaresiomponent, epigallocatechin-3-gallate (EGCG), sepsis başlamasından 24 saat sonra başlayan, önemli ölçüde ölümcül sepsis 12 fareleri kurtardı. Ağızdan verilen dahi, önemli ölçüde% 16 44% 16 hayvan sağkalım oranları artan ölümcül sepsis hala kurtarıldı farelerin EGCG. Birkaç bitkisel bileşenler ile kombinezonsal tedaviler sepsis hayvan modellerinde önemli bir iyi bir koruma elde edebiliriz olmadığını belirlemek için önemli olurdu. Birlikte ele alındığında, bu deneysel veriler CLP-indüklenen sepsis bir hayvan modeli kullanarak ekran terapötik ajanlar bizim yaklaşım geçerli. 5.. Temsilcisi Sonuçlar <img alt="Şekil 1" src="/files/ftp_upload/3926/3926fig1.jpg" /> Şekil 1.. Göstergelerinin kandaki düzeylerinin can çekişen devlet yaklaşırken septik hayvanlarda önemli ölçüde yüksektir. Balb / C fareler CLP sepsis tabi ve hastalık belirtileri gelişimi açısından takip edildi. Bir andasepsis (52 saat sonrası CLP) ve geç evre, kan 3 normal farelerin (-CLP), can çekişen devlet yaklaşan 3 septik fareler ve olmayan can çekişen devlet 3 septik fareler toplanmıştır. Serum her gruptan toplanmış, ve antikor dizileri tarafından sitokin profil için tahlil edilmiştir. Farklı gruplar arasında çeşitli göstergelerinin göreli düzeylerinde dramatik farka dikkat edin. Yayıncının izni ile 10.1371/journal.pone.0001153.g006: DOI uyarlanmıştır. <img alt="Şekil 2" src="/files/ftp_upload/3926/3926fig2.jpg" /> Şekil 2. 48 saat sonrası CLP. Balb / C fareler yerel ve sistemik sitokin ve kemokinlerin Algılama CLP sepsis tabi tutuldu ve kan veya periton sıvısı 48 saat sonrası CLP hasat edildi. Çeşitli sitokinler ve toplanan serum veya peritoneal lavaj sıvısında kemokinlerin Bağıl sitokin düzeylerinin antikor dizileri ile ölçülen, ve keyfi birim (AU) olarak ifade edildi. Kontrolleri, kan ve periton sıvısı gibi samples laparotomisiz normal sağlıklı hayvanlar (-CLP) elde edilmiştir. <img alt="Şekil 3" src="/files/ftp_upload/3926/3926fig3.jpg" /> Şekil 3. Bitkisel bileşeni doza bağlı olarak primer kültürler olarak makrofaj endotoksin bağımlı HMGB1 yayın inhibe etti. Primer mürin periton makrofajlar yokluğunda LPS veya bitkisel bileşen mevcudiyetinde (örneğin, EGCG, 15 uM) ile stimüle edildi. LPS uyarıldıktan sonra 16 saat sonra, kültür ortamı içinde HMGB1 seviyeleri Western blot analizi ile tayin edildi. Yayıncının izni ile 10.1371/journal.pone.0001153.g006: DOI uyarlanmıştır. <img alt="Şekil 4" src="/files/ftp_upload/3926/3926fig4.jpg" /> Şekil 4. Bitkisel bileşeni Balb / C fareler CLP ölümcül sepsis tabi tutuldu. Ölümcül sepsis fareler kurtarıldı ve bitkisel bileşen (EGCG) +24 az intraperitoneal oldu, +48, 72 saat sepsis başlamadan gönderin. Birolmuş bölgeler hayatta iki haftaya kadar açısından gözlenmiştir. Kaplan-Meier yöntemi gruplar arasında ölüm oranları farklılıkları karşılaştırmak için kullanıldı. * P &lt;0.05 karşı salin. Yayıncının izni ile 10.1371/journal.pone.0001153.g006: DOI uyarlanmıştır.

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AES ve HW sepsis için potansiyel bir terapötik ajan olarak HMGB1 inhibitörleri (tanshinones) ile ilgili patent başvuruları arasında co-mucitleri.

Laboratuvarda, sepsis çeşitli hayvan modellerinde olası yeni tedaviler geliştirmek için sepsisinde anlamak için istihdam edilmiştir. Onların klinik sepsis klinik uygulamalar içine hayvan çalışmaları başarılı bir çeviri daha önce tartışma konusu olmaya devam etmektedir. Erken sitokinlerin karşı nötralize edici antikorlar (örneğin, TNF) bakteriyemi / endotoksemi 17,18 hayvan modellerinde koruyucu olmasına rağmen, aslında sepsis 19 hayvan modelinde hayatta kötüleştirebil...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Betadine </td><td> Purdue Ürünleri 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 A.Ş. </td><td> RL-0065 </td></tr><tr><td> Ksilazin </td><td> Lloyd Laboratuvarları </td><td> 4821 </td></tr><tr><td> AutoCLIP </td><td> Becton Dickinson </td><td> 427631 </td></tr><tr><td> 4-0 ipek sütür </td><td> Roboz </td><td> SUT-15-2 </td></tr><tr><td> Surflo IV Kateter </td><td> Terumo </td><td> SR * OX2419CA </td></tr><tr><td> RayBio fare sitokin antikor dizi </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 bir mikrobik enfeksiyon sonucu ortaya çıkan bir sistemik inflamatuvar yanıt sendromu ifade eder. Bu eksojen bakteri toksini (endotoksemi) veya bakteriler (bakteriyemi) infüzyonu yanı sıra çekal ligasyon ve delme (CLP) 1-3 ile çekum cerrahi perforasyonu da dahil olmak üzere çeşitli teknikler, hayvanların rutin olarak simüle olmuştur. CLP perfore apandisit veya divertikülit insan klinik hastalığı taklit eden, bakteri dökülme ve periton boşluğu fekal kontaminasyon sağlar. Olarak nihai ölüm oranı tarafından yansıtılan sepsis şiddeti, çekal ponksiyonu 2 için kullanılan iğne boyutu değiştirilerek cerrahi olarak kontrol edilebilir. Insan sepsis 3 klinik seyri sırasında gözlemlediği gibi hayvanlar, CLP, kardiyovasküler metabolik ve immünolojik cevapta benzeri, bifazik hemodinamik indükler. Böylece, CLP modeli 1-3 Deneysel sepsis için klinik olarak en uygun modellerden biri olarak kabul edilir. &lt;/ P&gt; Çeşitli hayvan modellerinde deneysel sepsis patogenezine yatan karmaşık mekanizmaları aydınlatmak için kullanılmıştır. Sepsis öldürücü sonucu erken sitokinlerin aşırı birikimi 4-6 ve geç proinflammatuar mediatörleri (örn., HMGB1) 7 (örneğin, TNF, IL-1 ve IFN-γ gibi), kısmen ilişkindir. Erken proinflamatuar sitokinler ile karşılaştırıldığında, geç etkili mediatörler klinik uygulamalar için daha geniş bir terapötik pencereye sahiptir. Örneğin, CLP 24 saat sonra başlayan HMGB1-nötralize edici antikorlar gecikmeli yönetim, hala ölümcül sepsis geç bir arabulucu olarak HMGB1 kuran, öldürücülüğü 8,9 farelerin kurtardı. Geç etkili arabulucu olarak HMGB1 keşfi Geleneksel Çin Bitkisel Tıp kullanarak sepsis tedavilerin geliştirilmesi için soruşturma yeni bir alan başlattı. Bu yazıda, HM için bitkisel ilaç taranmasında CLP-indüklenen sepsis bir prosedür tarif ve kullanımTedaviler GB1 hedefleme.

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 bir mikrobik enfeksiyon sonucu ortaya çıkan bir sistemik inflamatuvar yanıt sendromu ifade eder ve çekal ligasyon ve delme (CLP) diye adlandırdıkları bir cerrahi teknik ile simüle edilebilir. Burada tedavi ajanları için şifalı otlar taranması amacıyla CLP bağlı hayvan modelinde kullanılacak bir yöntem açıklanmaktadır.

Roman Bitkisel Tedaviler Değerlendirmek için Sepsis Hayvan Modeli kullanımı

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 Görüntüleme.  North Shore – LIJ Health System. Sepsis bir mikrobik enfeksiyon sonucu ortaya çıkan bir sistemik inflamatuvar yanıt sendromu ifade eder ve çekal ligasyon ve delme (CLP) diye adlandırdıkları bir cerrahi teknik ile simüle edilebilir. Burada tedavi ajanları için şifalı otlar taranması amacıyla CLP bağlı hayvan modelinde kullanılacak bir yöntem açıklanmaktadır.

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.

CYP2D6 Hayvan Modeli:, Fare Otoimmün Hepatit indükleyin nasıl

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 ...

Tıp

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.

Ameliyat sonrası Immunosupressjon&#39;un ve Mekanizmaları keşfedin ve Roman Perioperatif İmmünoterapiler Değerlendirmesinde Cerrahi Stres Fare Tümör Modeli

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.

Etkinliğinin Değerlendirilmesi<em&gt; H. pylori</emOrtotopik Fare Modelinde Mesane Kanseri Tedavisinde Terapötik Aracı Olarak&gt; Protein HP-NAP

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.

Glioblastoma&#39;nın için bir Immünkompetan Hayvan Modeli Biyoparlaklık Görüntüleme

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.

Ex Vivo Normothermic karaciğer perfüzyon küçük bir hayvan Model

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.

İzometrik Contractility ölçüm tel Myography kullanarak fare mezenterik arter

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.

Burun beyin ilaç dağıtım değerlendirmek için inhalasyon anestezi altında burunici yönetim için yeni Yöntemler

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

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

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

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

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

Test (Neo) adjuvan Tedaviler için İntam Rezeke Yumuşak Doku Sarkomu Bir Fare Modeli

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

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

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

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

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

Ex Situ Perfüzyon Sırasında Donör Kalbin Fonksiyonel Değerlendirmesi : Basınç-Hacim Döngüleri ve Yüzey Ekokardiyografisinden Elde Edilen Bilgiler

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.

Kısmi Siyatik Sinir Ligasyonu: Yeni Tedavilerin Antinosiseptif Etkisini İncelemek için Kronik Nöropatik Ağrının Bir Fare Modeli

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