1。動物の倫理マウスで長時間温熱療法を行う前に手順が施設内動物のケアと使用委員会によって承認されていることを確認してください。このホワイトペーパーで説明とそれに付随する映像で示されているすべての手順は、免疫学の国立研究所の動物実験倫理委員会によって承認された。 2。理解ハイパーサーミア全身温熱療法（WBH）は39.5に維持インキュベーターに置かれたケージ℃に温めておいた動物を置くことによって、配信されプロトコルの標準化の間に、温度センサは、外科的にマウスに移植され、彼らの体温は、異なる実験条件で監視した。それが加熱された環境で動物を置くことによって、それらの温度は徐々に37まで増加することが観察された℃〜最大42℃まで100分（ 図1）の期間にわたって。木曜日内部体温が外部環境温度よりも高い温度で撃たとして &quot;スパイク&quot;結果の電子は興味をそそられる。 体温が40℃を超えた場合、動物はストレスや鼻の周りに濡れなど脱水症状の兆候を示すでしょう。 40を超えて体温を高める°Cは、湿潤かつ不可逆的なストレスあるいは熱応力に起因する動物の死につながる。だから温熱療法の過程で温度の急上昇を防ぐことが重要です。 高温スパイクのこの現象は、温熱療法、露光中に動物の物理的な多動性に関連付けられています。同様の温度上昇が、あまりにも他のグループによって観測された、より高い温度10を有する環境に導入される時に動物の物理的な多動と関連していた。 我々のプロトコルでは、マウスの温熱療法露光中に固定化されている。として図2に示すように、動物の固定化は、温度の急上昇を防ぎます。 我々は、このプロトコルを実証するための8-12週齢のBALB / CJマウスを使用している。プロトコルは、特定のタイプ/マウスの系統に限定されるものではない。 3。実験の準備 3.1インキュベーター彼らは優れた温度制御を持っているとして、この実験は通常のCO 2インキュベーター中で行うことができる。 CO 2ガスの供給を切断し、CO 2インキュベーターの保水トレイを取り外します。インキュベーターの一部のモデルでは、CO 2のコントローラをオフに切り替えることができます。マウスケージは便利な内側に配置することができるようにインキュベーターの棚を配置します。 39.5℃でインキュベーターの温度を設定し 3.2ケージ典型的には、温熱実験を一度に4匹のマウスで実行されます。二つのケージが必要です。上eはインキュベーターの中に置かれ、他は常温で外に配置されている。寝具のパッチが入っているケージの不均一な温度をもたらす時間でウェットになるとケージの寝具は、回避されます。外ケージは食物ペレットと水のボトルを持っている必要があります。 3.3マウスの固定化チューブ温熱マウスの最初の4サイクルの途中で穴のあいたチューブに固定化されている。これらの固定化チューブはそのすべての体の上にピアスをして50ミリリットル &quot;遠心分離機&quot;チューブから3mmの穴を作ることができ、このようなチューブの4つが必要となります。 50ミリリットル管は便利に8週間以上経過したBALB / CJマウスを固定することができます。適切なサイズのチューブは、マウスのいくつかの異常に異なる株にまたは非常に若いか、50週以上経過したマウスが必要な場合があります。 場所固定化チューブやインキュベーター内部ケージは39.5℃に維持ケージのように少なくとも2時間待つとimmobilization管はインキュベーターの温度を達成する。 4。温熱療法の最初のサイクルマウスは温熱露光中に脱水症状が発生する可能性があります。したがって、実験を開始する前に、すべてのマウスに0.5ミリリットル生理食塩水を腹腔内投与する。 1mlシリンジで26 Gの針を使用します。 迅速場所、各固定化チューブ中1動物をインキュベーターに入れ、ケージを取り出して、インキュベーターにケージ背面に配置します。時間を記録します。それはインキュベーター内の同じ場所で毎回ケージをおくことが非常に重要です。 90分後、インキュベーターからケージを取り出します。固定管から動物を解放し、食料や水に簡単にアクセスでき、常温でケージの中に置いてください。インキュベーターに戻し、空の固定化チューブを置きます。 15分後、温熱療法の第2サイクルは0を提供することによって開始されます。すべてのマウスに腹腔内に生理食塩水5ミリリットル。 彼らはその後50ミリリットルあき固定化チューブに固定し、インキュベーター内に配置されます。 5。その後の温熱サイクル最初の4サイクルでは、それはハイパーサーミア露光中に、最初の2つのサイクルのためにマウスを固定化するために不可欠であり、生理食塩水注入は温熱療法の期間を開始する前に、与えられている。 残り続いて温熱療法のこれらのサイクルが完了した後、マウスを慣らす取得し、温熱暴露中に非常に活発になることはありません。マウスが容易に休憩時間中に食べ物や水を取る場合の生理食塩水を腹腔内注射は必要ないかもしれません。 6。すべてのサイクルが完了した後、最後のステップでは、動物を調べることです温熱療法サイクル中にいずれかの死があってはいけません。すべての動物は、最初は非常に注意深く監視する必要があります数サイクル。急速に鼻周辺で湿潤を探してください。この症状は、脱水の署名である。このような症状を持つマウスはすぐに室温で0.5ミリリットル生理食塩水ipと休息を与えられるべきである。固定化されたマウスのほとんどは、任意の追加のケアを必要としませんし、正常にストレスの温熱療法のご負担になります。 必要なサイクルを完了した後、動物は弱いと脱水見えるかもしれませんが、彼らはすぐに数時間以内に回復します。 <img src="/files/ftp_upload/3801/3801fig1.jpg" alt="図1" /> 図1ケージは39.5℃のインキュベーター中に置いたマウスの深部体温の変化90分後、ケージをインキュベーターから取り出し、25℃に置か℃、赤丸は高温スパイクを示しています。 <img src="/files/ftp_upload/3801/3801fig2.jpg" alt="図2" /> 図2コアボディtemperatuのバリエーション温熱療法サイクル中に固定化されたマウスの再。動物を固定した後、高温のスパイクはケージが39.5℃に置かれたときに観察されなかった

<ol>
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我々は、開示することは何もない。

<html>にまでさかのぼる5000 BCとして、エジプトの医師は熱で腫瘍を治療した。ギリシア人はいくつかの医学的治療における熱の価値を認識した。モーセの律法の中で最も古いテキストは、治療的に体温を上昇させる温泉（創世記36:24）を言及する。 以前の技術は温熱療法1の誘導のための発熱や温水浴の誘導のための発熱性微生物抽出物の使用のいずれかを採用し?...

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protocol for long duration whole body hyperthermia in mice

温熱療法は、通常、上記の中核体温の上昇を定義するために使用される一般的な用語です。それは頻繁に発熱時に観察される増加中核体温を記述するために使用されます。アジュバントとしての温熱療法の使用は癌生物学の分野での腫瘍退縮のための有望な手法として浮上している。この目的のために、最も重要な要件は、信頼性が高く、均一な加熱·プロトコルを持つことです。我々は、マウスの温熱療法（全身）のためのプロトコルを開発してきました。このプロトコルでは、動物は、15分の休止期間が続く90分間の温熱療法のサイクルにさらさ​​れている。この期間中にマウスが食料や水への容易なアクセスを持っています。最初のいくつかの温熱曝露サイクル中にマウスで、高体温の上昇が、動物を固定化することによって防止される。また、生理食塩水は、脱水の影響を最小限に抑えるため、最初の数サイクルで投与される。このプロトコルは、最大12〜24時間にマウスでの条件のような発熱をシミュレートすることができます。我々は持っているプロトコルを実証するために8〜12週齢のBALB / CJ雌マウスを使用していました。

Watch this Scientific Journal Video about Protocol for Long Duration Whole Body Hyperthermia in Mice at JoVE.com

Protocol for Long Duration Whole Body Hyperthermia in Mice

本稿では、最大12〜24時間の条件のように熱を刺激することができ、マウスにおける全身温熱療法のためのプロトコルについて説明します。

マウスの長い持続時間の全身温熱療法のためのプロトコル

Hyperthermia is a general term used to define the increase in core body temperature above normal. It is often used to describe the increased core body ...

protocol-for-long-duration-whole-body-hyperthermia-in-mice

Research

JoVE Journal

Medicine

11.5K ビュー.  National Institute of Immunology. 本稿では、最大12〜24時間の条件のように熱を刺激することができ、マウスにおける全身温熱療法のためのプロトコルについて説明します。

ビデオ: Protocol for Long Duration Whole Body Hyperthermia in Mice

Protocols for Assessing Radiofrequency Interactions with Gold Nanoparticles and Biological Systems for Non-invasive Hyperthermia Cancer Therapy

Cancer therapies which are less toxic and invasive than their existing counterparts are highly desirable. The use of RF electric-fields that penetrate deep into the body, causing minimal toxicity, are currently being studied as a viable means of non-invasive cancer therapy. It is envisioned that the interactions of RF energy with internalized nanoparticles (NPs) can liberate heat which can then cause overheating (hyperthermia) of the cell, ultimately ending in cell necrosis.
In the case of non-biological systems, we present detailed protocols relating to quantifying the heat liberated by highly-concentrated NP colloids. For biological systems, in the case of in vitro experiments, we describe the techniques and conditions which must be adhered to in order to effectively expose cancer cells to RF energy without bulk media heating artifacts significantly obscuring the data. Finally, we give a detailed methodology for in vivo mouse models with ectopic hepatic cancer tumors.

We describe the protocols used to investigate the interactions of 13.56 MHz radiofrequency (RF) electric-fields with gold nanoparticle colloids in both non-biological and biological systems (in vitro/vivo). These interactions are being investigated for applications in cancer therapy.

非侵襲ハイパーサーミアがん治療のための金ナノ粒子と生体系とラジオ波の相互作用を評価するためのプロトコル

Cancer therapies  which are less toxic and invasive than their existing counterparts are highly  desirable. The use of RF electric-fields that penetrate ...

医学

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

マウスの並体結合：詳細なプロトコル

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Carotid Artery Infusions for Pharmacokinetic and Pharmacodynamic Analysis of Taxanes in Mice

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study model. As the use of mouse models are often a vital step in preclinical drug discovery and drug development1-8, it is necessary to design a system to introduce drugs into mice in a uniform, reproducible manner. Ideally, the system should permit the collection of blood samples at regular intervals over a set time course. The ability to measure drug concentrations by mass-spectrometry, has allowed investigators to follow the changes in plasma drug levels over time in individual mice1, 9, 10. In this study, paclitaxel was introduced into transgenic mice as a continuous arterial infusion over three hours, while blood samples were simultaneously taken by retro-orbital bleeds at set time points. Carotid artery infusions are a potential alternative to jugular vein infusions, when factors such as mammary tumors or other obstructions make jugular infusions impractical. Using this technique, paclitaxel concentrations in plasma and tissue achieved similar levels as compared to jugular infusion. In this tutorial, we will demonstrate how to successfully catheterize the carotid artery by preparing an optimized catheter for the individual mouse model, then show how to insert and secure the catheter into the mouse carotid artery, thread the end of the catheter out through the back of the mouse&#8217;s neck, and hook the mouse to a pump to deliver a controlled rate of drug influx. Multiple low volume retro-orbital bleeds allow for analysis of plasma drug concentrations over time.

This method was developed with the goal of delivering a steady drug solution via the carotid artery, to assess the pharmacokinetics of novel drugs in mouse models.

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

When proposing the use of a drug, drug combination, or drug delivery into a novel system, one must assess the pharmacokinetics of the drug in the study ...

Long-term Blood Pressure Measurement in Freely Moving Mice Using Telemetry

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. Indeed, research focusing on the discovery of new potential therapeutic targets using genetically altered mice requires a reliable, long-term assessment of the systemic arterial pressure variation. Currently, the gold standard for obtaining long-term measurements of blood pressure in ambulatory mice uses implantable radio-transmitters, which require&#160;artery cannulation. This technique eliminates the need for tethering, restraining, or anesthetizing the animals which introduce stress and artifacts during data sampling. However, arterial blood pressure monitoring in mice via catheterization can be rather challenging due to the small size of the arteries. Here we present a step-by-step guide to illustrate the crucial key passages for a successful subcutaneous implantation of radio-transmitters and carotid artery cannulation in mice. We also include examples of long-term blood pressure activity taken from freely moving mice after a period of post-surgery recovery. Following this procedure will allow reliable direct blood pressure recordings from multiple animals simultaneously.

The goal of this protocol is to assess systemic blood pressure in conscious freely moving mice using implantable radio-telemetry devices.

テレメトリを使用して自由に動くマウスにおける長期血圧測定

During the development of new vasoactive agents, arterial blood pressure monitoring is crucial for evaluating the efficacy of the new proposed drugs. ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

身体組成と高脂肪給電マウスにおける代謝ケージ分析

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Protocol to Create Chronic Wounds in Diabetic Mice

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ~6.5M people and cost ~$40B/year in the US alone. Although a significant effort has been invested in understanding how chronic wounds develop in humans, fundamental questions remain unanswered. Recently, we developed a novel mouse model for diabetic chronic wounds that have many characteristics of human chronic wounds. Using db/db-/- mice, we can generate chronic wounds by inducing high levels of oxidative stress (OS) in the wound tissue immediately after wounding, using a one-time treatment with inhibitors specific to the antioxidant enzymes catalase and glutathione peroxidase. These wounds have high levels of OS, develop biofilm naturally, become fully chronic within 20 days after treatment and can remain open more for more than 60 days. This novel model has many features of diabetic chronic wounds in humans and therefore can contribute significantly to advancing fundamental understanding of how wounds become chronic. This is a major breakthrough because chronic wounds in humans cause significant pain and distress to patients and result in amputation if unresolved. Moreover, these wounds are very expensive and time-consuming to treat, and lead to significant loss of personal income to patients. Advancements in this field of study through the use of our chronic wound model can significantly improve health care for millions who suffer under this debilitating condition. In this protocol, we describe in great detail the procedure to cause acute wounds to become chronic, which has not been done before.

Chronic wounds are developed from acute wounds on a diabetic mouse model by inducing high levels of oxidative stress after a full-thickness cutaneous wound. The wound is treated with inhibitors for catalase and glutathione peroxidase, resulting in impaired healing and biofilm development by bacteria present in the skin microbiome.

糖尿病マウスの慢性創傷を作成するためのプロトコル

Chronic wounds develop as a result of defective regulation in one or more complex cellular and molecular processes involved in proper healing. They impact ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

ハンドヘルド金属探知器検診小児における金属異物摂取

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to expend energy has made it a potential target for novel therapies to ameliorate obesity and associated metabolic disorders in humans. Though this tissue has been well studied in small animals, BAT&#39;s thermogenic capacity in humans remains largely unknown due to the difficulties of measuring its volume, activity, and distribution. Identifying and quantifying active human BAT is commonly performed using 18F-Fluorodeoxyglucose (18F-FDG) positron emission tomography and computed tomography (PET/CT) scans following cold-exposure or pharmacological activation. Here we describe a detailed image-analysis approach to quantify total-body human BAT from 18F-FDG PET/CT scans using an open-source software. We demonstrate the drawing of user-specified regions of interest to identify metabolically active adipose tissue while avoiding common non-BAT tissues, to measure BAT volume and activity, and to further characterize its anatomical distribution. Although this rigorous approach is time-consuming, we believe it will ultimately provide a foundation to develop future automated BAT quantification algorithms.

Whole Body and Regional Quantification of Active Human Brown Adipose Tissue Using 18F-FDG PET/CT

Using free, open-source software, we have developed an analytical approach to quantify total and regional brown adipose tissue (BAT) volume and metabolic activity of BAT using 18F-FDG PET/CT.

人間の活動の地域の定量化と全身褐色脂肪組織18F-FDG PET/CT を使用

In endothermic animals, brown adipose tissue (BAT) is activated to produce heat for defending body temperature in response to cold. BAT&#39;s ability to ...

A Whole Body Dosimetry Protocol for Peptide-Receptor Radionuclide Therapy (PRRT): 2D Planar Image and Hybrid 2D+3D SPECT/CT Image Methods

Peptide-receptor-radionuclide-therapy (PPRT) is a targeted therapy that combines a short-range energy radionuclide with a substrate with high specificity for cancer cell receptors. After injection, the radiotracer is distributed throughout the entire body, with a higher uptake in tissues where targeted receptors are overexpressed. The use of beta/gamma radionuclide emitters enables therapy imaging (beta-emission) and post-therapy imaging (gamma-emission) to be performed at the same time. Post-treatment sequential images permit absorbed dose calculation based on local uptake and wash-in/wash-out kinetics. We implemented a hybrid method that combines information derived from both 2D and 3D images. Serial whole-body images and blood samples are acquired to estimate the absorbed dose to different organs at risk and to lesions disseminated throughout the body. A single 3D-SPECT/CT image, limited to the abdominal region, overcomes projection overlap on planar images of different structures such as the intestines and kidneys. The hybrid 2D+3D-SPECT/CT method combines the effective half-life information derived from 2D planar images with the local uptake distribution derived from 3D images. We implemented this methodology to estimate the absorbed dose for patients undergoing PRRT with 177Lu-PSMA-617. The methodology could, however, be implemented with other beta-gamma radiotracers. To date, 10 patients have been enrolled into the dosimetry study with 177Lu-PSMA-617 combined with drug protectors for kidneys and salivary glands (mannitol and glutamate tablets, respectively). The median ratio between kidney uptake at 24 h evaluated on planar images and 3D-SPECT/CT is 0.45 (range:0.32-1.23). The comparison between hybrid and full 3D approach has been tested on one patient, resulting in a 1.6% underestimation with respect to full 3D (2D: 0.829 mGy/MBq, hybrid: 0.315 mGy/MBq, 3D: 0.320 mGy/MBq). Treatment safety has been confirmed, with a mean absorbed dose of 0.73 mGy/MBq (range:0.26-1.07) for kidneys, 0.56 mGy/MBq (0.33-2.63) for the parotid glands and 0.63 mGy/MBq (0.23-1.20) for submandibular glands, values in accordance with previously published data.

A Whole Body Dosimetry Protocol for Peptide-Receptor Radionuclide Therapy PRRT: 2D Planar Image and Hybrid 2D+3D SPECT/CT Image Methods

This method estimates the absorbed dose of different structures for peptide-receptor-radionuclide-therapy (PRRT) with the possibility of avoiding organ overlap on 2D-projections. Serial whole-body planar images permit estimation of mean absorbed doses along the whole body, while the hybrid approach, combining planar images and 3D-SPECT/CT image, overcomes the limitations of structure overlapping.

ペプチド受容体放射性核種療法のための全身線種測定プロトコル(PRRT):2D平面画像とハイブリッド2D+3D SPECT/CT画像法

Peptide-receptor-radionuclide-therapy (PPRT) is a targeted therapy that combines a short-range energy radionuclide with a substrate with high specificity ...

Assessing Whole-Body Lipid-Handling Capacity in Mice

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a class of many different molecules with many pathways involved in their synthesis and metabolism. A starting point for evaluating lipid hemostasis for nutrition and obesity research is needed. This paper describes three easy and accessible methods that require little expertise or practice to master, and that can be adapted by most labs to screen for lipid-metabolism abnormalities in mice. These methods are (1) measuring several fasting serum lipid molecules using commercial kits (2) assaying for dietary lipid-handling capability through an oral intralipid tolerance test, and (3) evaluating the response to a pharmaceutical compound, CL 316,243, in mice. Together, these methods will provide a high-level overview of lipid handling capability in mice.

This paper provides three easy and accessible assays for assessing lipid metabolism in mice.

マウスにおける全身脂質処理能力の評価

Assessing lipid metabolism is a cornerstone of evaluating metabolic function, and it is considered essential for in vivo metabolism studies. Lipids are a ...