この作品は、コーク南診療所ビクトリア大学病院乳ファンド、アイルランド癌協会（CRI07TAN）とコークがん研究センターで賄われていた。安定的にルシフェラーゼを発現するルイス肺癌細胞株は、セルジェネシス社、サンフランシスコ、アメリカ、ヨースカリン博士から親切な贈り物だった。

<html>はじめ肝臓は多くの場合、転移性疾患の最初のサイトであり、先進的な疾患を有する患者のように多くの40 30％程度の広がりの唯一のサイトかもしれません。しかし、転移性肝腫瘍を治療するためのオプションは、切除するのに適した少数の患者にはいくつかあり、化学療法への唯一の23％が主要なレスポンダー。この分野の臨床研究は、分離された肝疾患を研究するために適切なマウスモデルの不在によって制限されています。我々は確実に均一な、びまん性肝転移を確立するために使用することができる分割脾臓のアプローチを説明します。脾臓のこの半分の切除は、脾腫瘍の増殖の影響を回避し、脾臓の残りの半分は、脾臓の免疫保護特性を維持するためにそのままのポータル循環に戻されます。我々が説明するモデルは、実行するために非常に簡単ですし、単一の切開を通じて迅速に行うことができます。腫瘍細胞株の生物発光特性は、動物の犠牲なしに、治療に対する反応の正確な定量が可能になります。 プロトコル I.細胞の調製<ol><li>ルイス肺癌細胞を37℃、10％ウシ胎児血清を添加したダルベッコ改変必須培地° C、5％CO 2を成長させ、トリプシン処理するときにサブコンフルエントによって収穫されています。 </li><li>細胞は無血清培地で調製したNucleocounter（ケモmetec、デンマーク）と200μlの1 × 10 6細胞の単一の細胞懸濁液を用いてカウントした。 </li></ol> II。マウスの準備注：すべての手順は、コーク大学の倫理委員会によって承認された。私たちのラボでは、マウスを麻酔注射麻酔薬を使用して、別の方法として、同じ効果を達成するために吸入麻酔薬を使用することができます。我々が使用するマウス系統は、ハーランラボラトリーズ（オックスフォード、英国）から得られた無毛のMF1 NU / NU株である。 16〜22グラムと年齢の6-8週の重量を量るメスMF1 nu / nuマウスは、この実験に含まれていました。このようなC57およびBALB / cのような他の株は、適当な腫瘍細胞株で使用できます。 <ol><li>麻酔は1.5 mgのケタミンと300 UGのキシラジンの100ulの腹腔内注射によって送達された。 </li><li>麻酔の深さはつま先のピンチを利用して評価されます。つま先のピンチでは撤退の反射はないはずです。 </li><li>麻酔マウスが正しく配置し、加熱パッドを介してテープで固定されています。 </li><li>腹部は10％betadine液で整形処理されています。 </li><li>腹部と手術部位を無菌的に覆われている。 </li></ol> III。腹部切開<ol><li>小さな肋骨下の切開は、皮膚で作られています。 </li><li>切開は2〜3 cmに拡張されます。 </li><li>腹壁の筋肉組織が分けられ、腹腔が入力されました。 </li></ol> IV。曝露と脾臓の課<ol><li>脾臓を動員し、優しくプレカット滅菌ガーゼで体外される。 </li><li>二つの4から0 vicryl合字は、脾臓の半ばに体の周りに関連付けられています。 </li><li>脾臓は、2つの半脾臓を作成する2つの合字の間に分かれています。 </li></ol> V.ヘミ-脾臓への細胞の注入<ol><li> 27 Gは、1つの半球脾臓に細胞を100μLの量を注入するために使用されます。 </li><li>腫瘍細胞は注入された細胞の大部分が肝臓に到達するのに十分な時間を提供する10分のために肝臓への排出を許可されます。 </li></ol> VI。ヘミ - 脾臓、腹部閉鎖およびリカバリの切除<ol><li>注入された半脾臓を摘出されています。 </li><li>他の半脾臓は腹腔内に返されます。 </li><li>筋層は、単純なランニングステッチを使用して4から0までのPDS縫合糸で閉じられます。 </li><li>腹部の皮膚が中断ステッチを使用して4から0 Proleneで閉じられます。 </li><li>流体のボーラスが投与され、手術後の鎮痛、Carpofen（5 mg / kg）を24時間の手術後のためにIP注射によって投与される。 </li><li>マウスは、麻酔から回復するために許可されています</li></ol> VII。 IVIS全身生物発光イメージング<ol><li>マウスは注射麻酔薬を使用して麻酔です。 </li><li>ルシフェリン基質は、10分イメージを作成する前に腹腔内注射で与えられる。 </li><li>マウスがステージ上に配置され、IVIS全身イメージングシステムを用いて3分間撮像する</li><li>肝臓からの発光は、関心のツールの領域を使用して測定することができます。 </li></ol> VIII。マウスの肝臓の暴露<ol><li>マウスは注射麻酔薬を使用して麻酔です。 </li><li>屋上腹部切開は、 生体内のびまん性肝転移の可視化が可能になります。 </li><li>マウスを頚椎脱臼により安楽死され、肝臓は、さらなる分析のために摘出されることがあります。 </li></ol>_upload/1977/1977fig1.jpg&quot;altが=&quot;図1&quot;/&gt; 図1：新鮮なマウス未処理および腫瘍浸潤肝臓を摘出。 <img src="/files/ftp_upload/1977/1977fig2.jpg" alt="図2" /> 図2。IVIS全身イメージングによって証明されるように肝腫瘍の成長の速度論。

<ol>
	<li>Kasuya, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+subcutaneous+spleen+reservoir+for+multiple+portal+venous+injections+to+treat+liver+malignancies.">Mouse models of subcutaneous spleen reservoir for multiple portal venous injections to treat liver malignancies.</a> Cancer Res. 65, 3823-3827 (2005).</li><li>Liu, Q. Z., Tuo, C. W., Wang, B., Wu, B. Q., Zhang, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+metastasis+models+of+human+colorectal+carcinoma+established+in+nude+mice+by+orthotopic+transplantation+and+their+biologic+characteristic.">Liver metastasis models of human colorectal carcinoma established in nude mice by orthotopic transplantation and their biologic characteristic.</a> World J Gastroenterol. 4, 409-411 (1998).</li><li>Li, Z. Y., Ni, S., Yang, X., Kiviat, N., Lieber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograft+models+for+liver+metastasis:+Relationship+between+tumor+morphology+and+adenovirus+vector+transduction.">Xenograft models for liver metastasis: Relationship between tumor morphology and adenovirus vector transduction.</a> Mol Ther. 9, 650-657 (2004).</li><li>Cashman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+gene+therapy+as+a+neoadjuvant+to+surgical+excision+to+control+metastatic+cancers.">Immune gene therapy as a neoadjuvant to surgical excision to control metastatic cancers.</a> Cancer Lett. , (2008).</li></ol>

利害の衝突は宣言されません。

肝転移の研究のためのin vivoモデル信頼性の発展は新たな治療戦略の出現により、重要な増加になってきています。新たな戦略の正確な研究のために、動物モデルでは、実行することは比較的容易である転移のかなりの数を形成して、転移カスケードのすべてのステップを含んでおり、転移性細胞の限られた数の検出及び計数を可能にするレポーターシステムを持つ必要があります。前...

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<td>DMEM</td>
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<td>D6429</td>
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<td>D8537</td>
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<td>Carprofen</td>
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murine bioluminescent hepatic tumour model

このビデオはその後、生物発光イメージングによって解析することができるマウスモデルにおける肝転移の成立を説明します。腫瘍細胞は、脾臓の各半分の茎をそのまま血管を残して、二つに脾臓及び分割の動員を経由して、ローカライズされた肝腫瘍を誘発する肝臓に特異的に投与されています。構成的にホタルルシフェラーゼ遺伝子（luc1）を発現ルイス肺癌細胞は、その後10分後に切除されているものヘミ脾臓に接種されています。他の半脾臓はそのままにして腹部に返されます。肝臓の腫瘍の成長は、IVIS全身イメージングシステムを用いて生物発光イメージングによって監視することができます。 IVISを用いて腫瘍の成長の定量的なイメージングが可能な腫瘍細胞の正確な定量を提供します。薬物治療による腫瘍細胞死と壊死を​​早期に生物発光信号の減少によって示されます。このマウスモデルは、転移性腫瘍細胞の生存と成長のメカニズムを調査することができますし、肝転移の治療薬の評価に使用することができます。

Watch this Scientific Journal Video about Murine Bioluminescent Hepatic Tumour Model at JoVE.com

Murine Bioluminescent Hepatic Tumour Model

この記事では、マウスで同所生物発光性肝腫瘍の誘発、およびライブ全身の発光イメージングを使用して肝臓に限局する腫瘍の成長のその後の分析のための手順を説明します。

マウス生物発光肝腫瘤モデル

This video describes the establishment of liver metastases in a mouse model that can be subsequently analysed by bioluminescent imaging. Tumour cells are ...

murine-bioluminescent-hepatic-tumour-model

Research

JoVE Journal

Medicine

17.1K ビュー.  University College Cork. この記事では、マウスで同所生物発光性肝腫瘍の誘発、およびライブ全身の発光イメージングを使用して肝臓に限局する腫瘍の成長のその後の分析のための手順を説明します。

ビデオ: Murine Bioluminescent Hepatic Tumour Model

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

ヒト乳腺線維芽細胞からの癌関連線維芽細胞の実験的生成（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

医学

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

生体デュアル基質生物発光イメージングにおける

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The cell line was originally induced by intracranial injection of 3-methyl-cholantrene into a C57BL/6 syngeneic mouse strain 1; therefore, immunologically competent C57BL/6 mice can be used. While we use GL261, the following protocol can be used for the implantation and monitoring of any intracranial mouse tumor model. GL261 cells were engineered to stably express firefly luciferase (GL261-luc). We also created the brighter GL261-luc2 cell line by stable transfection of the luc2 gene expressed from the CMV promoter. C57BL/6-cBrd/cBrd/Cr mice (albino variant of C57BL/6) from the National Cancer Institute, Frederick, MD were used to eliminate the light attenuation caused by black skin and fur. With the use of albino C57BL/6 mice; in vivo imaging using the IVIS Spectrum in vivo imaging system is possible from the day of implantation (Caliper Life Sciences, Hopkinton, MA). The GL261-luc and GL261-luc2 cell lines showed the same in vivo behavior as the parental GL261 cells. Some of the shared histological features present in human GBMs and this mouse model include: tumor necrosis, pseudopalisades, neovascularization, invasion, hypercellularity, and inflammation 1.
Prior to implantation animals were anesthetized by an intraperitoneal injection of ketamine (50 mg/kg), xylazine (5 mg/kg) and buprenorphine (0.05 mg/kg), placed in a stereotactic apparatus and an incision was made with a scalpel over the cranial midline. A burrhole was made 0.1mm posterior to the bregma and 2.3mm to the right of the midline. A needle was inserted to a depth of 3mm and withdrawn 0.4mm to a depth of 2.6mm. Two &mu;l of GL261-luc or GL261-luc2 cells (107 cells/ml) were infused over the course of 3 minutes. The burrhole was closed with bonewax and the incision was sutured.
Following stereotactic implantation the bioluminescent cells are detectable from the day of implantation and the tumor can be analyzed using the 3D image reconstruction feature of the IVIS Spectrum instrument. Animals receive a subcutaneous injection of 150&mu;g luciferin /kg body weight 20 min prior to imaging. Tumor burden is quantified using mean tumor bioluminescence over time. Tumor-bearing mice were observed daily to assess morbidity and were euthanized when one or more of the following symptoms are present: lethargy, failure to ambulate, hunched posture, failure to groom, anorexia resulting in &gt;10% loss of weight. Tumors were evident in all of the animals on necropsy.

Intracranial Implantation with Subsequent 3D In Vivo Bioluminescent Imaging of Murine Gliomas

Intracranial implantation of GL261 cells into C57BL/6 mice produces malignant gliomas that recapitulate many of the hallmarks of human glioblastoma multiforme. We used GL261 cells stably expressing luciferase to allow us to use in vivo imaging to follow tumor progression. The surgery and 3D in vivo imaging are demonstrated.

その後の3Dと頭蓋内注入インビボでの</emマウス神経膠腫の>生物発光イメージング

The mouse glioma 261 (GL261) is recognized as an in vivo model system that recapitulates many of the features of human glioblastoma multiforme (GBM). The ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

定位頭蓋内注入と<emインビボ&gt;</em多形性膠芽腫のマウスモデル系における腫瘍異種移植片の&gt;生物発光イメージング

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

創傷治癒のマウスモデル

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

膵臓癌の進行の生物発光同所性モデル

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

くも膜下出血のマウスモデル

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

肝転移の前臨床マウスモデル

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ischemia/reperfusion (I/R) injury with myriad negative consequences. Potential I/R injury in marginal organs destined for liver transplantation contributes to the current donor shortage secondary to a decreased organ utilization rate. A significant need exists to explore hepatic I/R injury in order to mediate its impact on graft function in transplantation. Rat liver hilar clamp models are used to investigate the impact of different molecules on hepatic I/R injury. Depending on the model, these molecules have been delivered using inhalation, epidural infusion, intraperitoneal injection, intravenous administration or injection into the peripheral superior mesenteric vein. A rat liver hilar clamp model has been developed for use in studying the impact of pharmacologic molecules in ameliorating I/R injury. The described model for rat liver hilar clamp includes direct cannulation of the portal supply to the ischemic hepatic segment via a side branch of the portal vein, allowing for direct segmental hepatic delivery. Our approach is to induce ischemia in the left lateral and median lobes for 60 min, during which time the substance under study is infused. In this case, pegylated-superoxide dismutase (PEG-SOD), a free radical scavenger, is infused directly into the ischemic segment. This series of experiments demonstrates that infusion of PEG-SOD is protective against hepatic I/R injury. Advantages of this approach include direct injection of the molecule into the ischemic segment with consequent decrease in volume of distribution and reduction in systemic side effects.

A unique rat liver hilar clamp model was developed for studying the impact of pharmacologic molecules in ameliorating ischemia-reperfusion injury. This model includes direct cannulation of the portal supply to the ischemic liver segment via a branch of the portal vein, allowing for direct hepatic delivery.

ラット肝臓肝門部クランプモデルを用いたダイレクトセグメント別肝臓内送達方法

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

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.

ワイヤー図法を使用してマウスの腸間膜動脈の等尺性収縮力測定

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