著者は、セムズ - マーフィークリニック、メンフィス、テネシー州だけでなく、メンフィス、テネシー州のテネシー大学健康科学センターの脳神経外科部門でのMRI施設のスタッフに感謝したいと思います。

1。ゲルファントムの作製と色素<ol><li>脱イオン水1000ml中の0.1％アガロース粉末2gを溶解させて、0.2％アガロースゲルを調製する。適切な混合を確実にするために約1分間溶液を撹拌し;とすぐにインターバルの間、攪拌、9分間または透明になるまで3分間隔でソリューションを電子レンジ</li><li>アガロースゲルは液体であるが、5センチメートル×5センチ×5cmの容器にソリューションを注ぐ。水を追加し、アガロースゲルを冷却し、沈降させるために、容器の上部にあるスペースを確保してください。 </li><li>アガロースゲルが固化したら（約1〜2時間）、ゲルと冷蔵の上に水を1cmの追加。それは、混合の24〜48時間以内にゲルを使用するのが最適ですが、10を冷蔵週まで保存することができます。 </li><li> 0.017パーセントブロモフェノールブルー色素（BPB）の50ミリリットル、およびガドテリドール放射性造影剤の2 Mmからなる60ミリリットル注射器で放射性造影剤を準備します。 <ol><li> 50メートルにBPB色素の8.5 mgの組み合わせLは0.017パーセントBPBソリューションを作成するために脱イオン水。 </li><li> 2mMのガドテリドールソリューションを作成するために50ミリリットル0.017パーセントBPB溶液に株式の0.2ミリリットルの0.5Mガドテリドールを追加します。 </li></ol></li></ol>注入システムの2。準備<ol><li>シリンジポンプ注入システム（好ましい方法）：シリンジポンプ調製のために、輸液ラインのデッドボリュームを低減すること、圧力センサを介して注射器に直接注入カテーテルを取り付ける。シリンジポンプのパージ機能は、10μL/分の速度で、カテーテルのプライミング体積よりも大きなボーラスを用いて空気のラインをクリアするために使用されてもよい。 </li><li>チューブポンプ注入システム（代替法）：注入ポンプに放射性造影剤を含む注射器を接続します。 IVのモニターに接続トランスデューサとポンプ出口に圧力センサーを取り付けます。圧力センサの開口端部に16gの注入カテーテルを取り付けます。注：16のG注入カテーテルの先端が内径を持っている0.2ミリメートルと0.35ミリメートルの外径測定器。先端は、溶融シリカで形成され、先​​端の長さは3mmである。約0.75ミリメートルに増加し、15ミリメートル継続、カテーテルはその後、1.6ミリメートルまたは16 Gにテーパー形でステップアップ</li><li>任意の気泡を除去するために16.667μL/分で約15分間システムをパージすることによって注入の準備をします。マシンは、高いライン圧に注入を中止するように、16.667μL/分の流量を超えないようにしてください。輸液ポンプ、輸液ポンプの「ボーラス」機能を使用することにより、空気のパージラインを出るラインに注入カテーテルを取り付ける以下。 </li><li>ゲルファントムコンテナ（5センチメートル×5センチ×5cm）に及びMRIにおける場所に注入カテーテルマウント取り付け、軌道フレーム。 </li></ol> 3。CEDジェル注入およびMRスキャン<ol><li>注入を開始する前に、IVモニターによって記録された圧力値（mmHgのを）ゼロ。 </li><li>アガロースゲルWに注入カテーテルを挿入しますi番目の輸液ポンプは、このケース1.667μL/分で、可能な限り低い流速で実行されている。 </li><li> 表1に示すパラメータを用いて、MRスキャンを開始し、1.667μL/分の速度で注入し続ける。注入された全量を60μl（約38分）に達するまで一定の速度でゲルを注入する。 </li><li> 3分50秒間隔で連続的にゲルをスキャンします。圧力値ごとに60秒を記録します。注入されたボリュームが60μLに達すると、注入ポンプをオフにする。完全なMRスキャンは、圧力測定値を記録し続けている。 </li></ol> 4。磁気データ解析<ol><li> MR画像を解析するために、ROIセグメンテーション機能を備えた適切なDICOMビューアを使用しています。 </li><li> 図1に示すように、カテーテルの断面積によってマークされ、各スキャンで正しいフレームを選択します。 </li><li>ツール、いずれも含まれていないゲルの最大部分を選択 - 「四角形のROI」を使用した注入部位の一部分。ソフトウェアが出力されます標準偏差の平均画素密度。平均から3標準偏差に対応する値を見つけます。この値は、コントラストは99.7％の信頼度で存在するときに判定するための閾値として使用される。 </li><li> 「ROI - サークル」を使用してツールを、十分な大きさの円で注入部位を囲み、この一意の名前を付けます。 </li><li>円を選択し、使用して - &quot;現在の値よりも大きい場合：「ツール、ステップ4.3で見つかった入力された閾値に「ROIに設定した画素値を「これだけの行ボックスとチェックマーク。そして、「この新しい値に「箱、大きな値（25000）を入力します。以前に定義されたしきい値に包含される範囲を選択するために画素密度をリセットします。 </li><li> &quot; - 成長領域（2D/3Dセグメンテーション）、ROI」ツール、初期半径パラメータ= 2、ブラシの投資収益率と2D産地、信頼アルゴリズムを選択次に、使用して。 O総面積を計算するためのソフトウェアのための注入部位の内側をクリックこの領域F。 </li><li>球状注入雲を仮定すると、以下の式を介した地域からの拡散の体積を計算する：V =4/3π（√（面積/π））3 </li></ol>

<ol>
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著者は、彼らが競合する経済的利益を持っていないことを宣言します。

<html>注入の成功を確保するための重要なステップは次のとおり、空気の注入ラインをパージするアガロースゲルを混合し、MRデータを解析し、小さな内側カテーテル直径を使用して、使用して逆流を最小限にするためにカテーテル設計段とが感じる圧力を最小化する薬剤が注入されてその中にゲルまたは組織。先に述べたように、注入の成功への主な不利益は、輸液ラインの空気である。正?...

対流増加送達（CED）は、悪性脳腫瘍、てんかん、代謝障害（例えば、パーキンソン病など）、神経変性疾患1、脳卒中、外傷2を含む神経疾患の広域スペクトルに対する治療選択肢として提案されている。 CEDは、薬物または他の注入剤の配布のための正圧バルクフローを採​​用しています。 CEDは、臨床的に関連するボリュームが3で、ローからハイまで、高分子量化合物の、安全で信頼性が高く、均質な配信を提供します。脳組織への伝統的な薬物送達が厳しく、血液脳関門4によって制限される。脳実質に進入する血液脳関門ブロックが極性および高分子量分子、脳内の毛細血管を構成する内皮細胞間の密着結合によって形成されている。 CEDを介して直接的実質内脳注入は、以前の治療用薬剤送達モダリティの限界を克服することができるおよび血液脳関門を通過しないであろう治療剤の使用が可能になり、したがって生存可能な治療の選択肢5として以前は利用できていた。 米国国立衛生研究所（NIH）の研究者らは、拡散のみによる6-8よりも高い治療薬物濃度を達成する手段として、1990年代初頭に、CEDを説明した。 CEDの第一の方法は、脳内に一つまたは複数のカテーテルを移植カテーテルに注入ポンプを接続し、標的領域に直接治療剤をポンプ関与する。増加した分配画分と比較的安定濃度は、注入ポンプにより正圧が組織を拡張9および薬物の浸透を可能にさせるように起こることが報告されている。 CEDのための基本的な技術は、主に、最初に説明したのと同じままです。カテーテルの設計10、注入技術の進歩<suのp&gt; 11、脳シフト12,13を補正する複数の同一線上の注入14を最適化し、注入液の損失15を監視するライン圧モニタリング2、およびリアルタイムMRIモニタリングは、治療10の安全性および有効性を増加している。付加的な重要性は、流量を含むカテーテルのデザインおよび輸液戦略に配置されている。成功したCEDは、限られたカテーテル還流し、組織損傷に、カテーテルの設計および注入速度と相関している。細径カテーテル先端16で脳カテーテル界面に沿って逆流だけでなく、制限の被害を最小限に抑えるため、低注入速度を備えたカテーテルを使用すること。また、輸液逆流または異常な配達17の補正を可能にしながらMRイメージングは、視覚的な注入カテーテルの配置のための正しい位置を確認し、このような薬物送達を提供しています。 MR画像は、分布の容積を近似し、追跡するためにも使用することができる（Vdを）注入された薬物の。 Vdを18分割するための閾値として、周囲の非注入されたゲルからの平均を上回る標準偏差の3倍を超えるMRイメージング信号強度値を用いて算出される。それは脳内に分布した薬物の量を表すため、Vdは、CEDのための有用な測定である。 （Viに）注入容積と共に、比率が注入された薬物によって覆わ体積を定量する（Vdを/ Viに）生成することができる。 Vdは、ゲルカテーテルの相互作用、多孔質弾性特性、および注入クラウド形態10：アガロースゲルファントムのようなCEDを理解する上で重要なヒトの脳のいくつかの重要な機械的特性を模倣する。 0.2％アガロースゲルの混合物は、CEDにゲル膨張に起因する局所細孔画分における生体内変化を模倣することが示されている。人間の脳に似気孔分率は、同様の相互作用とVdの19の正確な測定を促進する。のさらに、同様の濃度例えば、0.6％、0.8％などgaroseゲルは、脳20と同様に、注入圧力のプロファイルを示している。また、半透明のアガロースゲルはカテーテル留置および注入還流のリアルタイム可視化の利点を提供する。アガロースゲルファントムは製造が比較的安価である。アガロースゲルファントムのコストは、神経学的な手術を通して将来広範な訓練の鍵となることがあります。ため、これらのプロパティに、アガロースゲルは、脳組織を使用することなく、人間の脳注入の重要な属性の多くを複製、有用な代理を提供する。 画像誘導CEDがアガロースゲルモデルに、前述したように、試験、研究、訓練のためのin vitro法で有益を提供しています。この記事の目的は、アガロースゲルファントムを再作成する方法を説明するために、適切なCEDのテストと分析のプロトコルの概要を説明し、神経疾患の治療のためのCED注入の間に直面する一般的なエラーに対処することである。

<table border="0" cellpadding="0" cellspacing="0" width="590">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Prohance</td>
			<td style="width:71px;">Bracco</td>
			<td style="width:119px;"></td>
			<td style="width:195px;">Gadoteridol radio contrast media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Bromophenol Blue Dye</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-0404</td>
			<td>Dye for infusate visualization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Agarose Gel Powder</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;">161-3101EDU</td>
			<td>Agarose powder for creating gels</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Veris MR Vital Signs Monitor</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pressure monitor</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">16 Gauge SmartFlow Catheter</td>
			<td style="width:71px;">SurgiVision</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Medrad Continuum MR Infusion System</td>
			<td style="width:71px;">Medrad</td>
			<td style="width:119px;"></td>
			<td>MR safe infusion pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">SMART Frame MRI Guided Trajectory Frame</td>
			<td style="width:71px;">ClearPoint</td>
			<td style="width:119px;"></td>
			<td>Infusion catheter frame</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:205px;">Osirix Imaging Software and DICOM Viewer</td>
			<td style="width:71px;">Osirix Imaging Software</td>
			<td style="width:119px;"></td>
			<td>OsiriX 32-bit DICOM Viewer</td>
		</tr>
	</tbody>
</table>

image-guided convection-enhanced delivery into agarose gel models of the brain

対流増加送達（CED）は、神経疾患の広い範囲の治療選択肢として提案されている。 NeuroinfusionカテーテルCEDは、従来の薬物送達方法より頭蓋内のターゲットに治療薬のより大量に配信するために正圧バルク流れを可能にします。 CED（RCED）をガイド付きリアルタイムMRIの臨床的有用​​性を正確に、ターゲット療法を監視し、合併症を識別するための能力にある。トレーニング、RCEDは効率的であり、合併症を最小限に抑えることができる。脳のアガロースゲルモデルはCED試験、研究、訓練のためのアクセス可能なツールを提供しています。また、注入の視覚的なフィードバックを提供しながら、シミュレートされた脳RCEDモック手術の練習が可能になります。注入の分析は、ヒト脳組織と比較して、被訓練者は、モデルの類似性を検証することを可能に分配画分（Vdを/ Viの）の計算を可能にする。この記事では、我々のアガロースゲル脳ファントムを説明し、重要な私を概説神経疾患の治療のためにCED注入中直面する共通の落とし穴に対処しながら、CED注入および分析プロトコルの間trics。

CED注入を解釈し、分析することは、流通画分とした注入液の還流のようないくつかの重要な要因を含んでいる。分配割合の計算は、VDの計算に大きく依存している。そのため、MR画像の正確な解釈は非常に重要です。我々は、上記のように、確実にこれらの測定値を再生するための半自動化方法を提案する。これらのメソッドは、客観的に注入剤雲とおおよそ半径の断面積を決定する。変数?...

Watch this Scientific Journal Video about Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain at JoVE.com

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

対流増加送達（CED）は、神経疾患の広い範囲の治療選択肢として提案されている。 CEDの適用のためのヘルスケアの専門家を調製するために、アクセス可能なトレーニングモデルが必要とされている。私たちは、試験、研究、訓練のための人間の脳のようなモデルとして、アガロースゲルの使用を記載している。

脳のアガロースゲルモデルに画像誘導対流増加送達

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

image-guided-convection-enhanced-delivery-into-agarose-gel-models

Research

JoVE Journal

Medicine

11.3K ビュー.  University of Tennessee Health Science Center. 対流増加送達（CED）は、神経疾患の広い範囲の治療選択肢として提案されている。 CEDの適用のためのヘルスケアの専門家を調製するために、アクセス可能なトレーニングモデルが必要とされている。私たちは、試験、研究、訓練のための人間の脳のようなモデルとして、アガロースゲルの使用を記載している。 

ビデオ: Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

MRI and PET in Mouse Models of Myocardial Infarction

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ideal model for testing novel therapeutic strategies.
In vivo magnetic resonance imaging (MRI) gives excellent views of the heart noninvasively with clear anatomical detail, which can be used for accurate functional assessment. Contrast agents can provide basic measures of tissue viability but these are nonspecific. Positron emission tomography (PET) is a complementary technique that is highly specific for molecular imaging, but lacks the anatomical detail of MRI. Used together, these techniques offer a sensitive, specific and quantitative tool for the assessment of the heart in disease and recovery following treatment.
In this paper we explain how these methods are carried out in mouse models of acute myocardial infarction. The procedures described here were designed for the assessment of putative protective drug treatments. We used MRI to measure systolic function and infarct size with late gadolinium enhancement, and PET with fluorodeoxyglucose (FDG) to assess metabolic function in the infarcted region. The paper focuses on practical aspects such as slice planning, accurate gating, drug delivery, segmentation of images, and multimodal coregistration. The methods presented here achieve good repeatability and accuracy maintaining a high throughput.

We describe how to perform MRI and PET imaging of the mouse heart. The protocol is tailored to assess treatment efficacy in models of myocardial infarction and heart failure.

Myocardial infarction is one of the leading causes of death in the Western world. The similarity of the mouse heart to the human heart has made it an ...

医学

DNBS/TNBS Colitis Models: Providing Insights Into Inflammatory Bowel Disease and Effects of Dietary Fat

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently host immune responses to microbial and environmental agents. Dinitrobenzene sulfonic acid (DNBS)-induced colitis allows one to study the pathogenesis of IBD associated environmental triggers such as stress and diet, the effects of potential therapies, and the mechanisms underlying intestinal inflammation and mucosal injury. In this paper, we investigated the effects of dietary n-3 and n-6 fatty acids on the colonic mucosal inflammatory response to DNBS-induced colitis in rats. All rats were fed identical diets with the exception of different types of fatty acids [safflower oil (SO), canola oil (CO), or fish oil (FO)] for three&#160;weeks prior to exposure to intrarectal DNBS. Control rats given intrarectal ethanol continued gaining weight over the 5 day study, whereas, DNBS-treated rats fed lipid diets all lost weight with FO and CO fed rats demonstrating significant weight loss by 48&#160;hr and rats fed SO by 72&#160;hr. Weight gain resumed after 72&#160;hr post DNBS, and by 5 days post DNBS, the FO group had a higher body weight than SO or CO groups. Colonic sections collected 5&#160;days post DNBS-treatment showed focal ulceration, crypt destruction, goblet cell depletion, and mucosal infiltration of both acute and chronic inflammatory cells that differed in severity among diet groups. The SO fed group showed the most severe damage followed by the CO, and FO fed groups that showed the mildest degree of tissue injury. Similarly, colonic myeloperoxidase (MPO) activity, a marker of neutrophil activity was significantly higher in SO followed by CO fed rats, with FO fed rats having significantly lower MPO activity. These results demonstrate the use of DNBS-induced colitis, as outlined in this protocol, to determine the impact of diet in the pathogenesis of IBD.

DNBS/TNBS induced colitis offers an alternative and inexpensive method to study the pathobiology of inflammatory bowel disease. The protocol discussed in this paper outlines the successful application of DNBS to induce colitis in mice and rats and allows one to thoroughly study host-mediated intestinal responses.

DNBS / TNBS大腸炎モデル：食事脂肪の炎症性腸疾患への洞察とエフェクトを提供

Inflammatory Bowel Diseases (IBD), including Crohn&#39;s Disease and Ulcerative Colitis, have long been associated with a genetic basis, and more recently ...

Heterotopic Mucosal Engrafting Procedure for Direct Drug Delivery to the Brain in Mice

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high molecular weight compounds from the bloodstream and into brain tissue. Some direct delivery success in humans has been achieved via implantation of transcranial catheters; however this method is highly invasive and associated with numerous complications. A less invasive alternative would be to dose the brain through a surgically implanted, semipermeable membrane such as the nasal mucosa that is used to repair skull base defects following endoscopic transnasal tumor removal surgery in humans. Drug transfer though this membrane would effectively bypass the BBB and diffuse directly into the brain and cerebrospinal fluid. Inspired by this approach, a surgical approach in mice was developed that uses a donor septal mucosal membrane engrafted over an extracranial surgical BBB defect. This model has been shown to effectively allow the passage of high molecular weight compounds into the brain. Since numerous drug candidates are incapable of crossing the BBB, this model is valuable for performing preclinical testing of novel therapies for neurological and psychiatric diseases.

A mouse model of human endoscopic skull base reconstruction has been developed that creates a semipermeable interface between the brain and nose using nasal mucosal grafts. This method allows researchers to study delivery to the central nervous system of high molecular weight therapeutics which are otherwise excluded by the blood-brain barrier when administered systemically.

マウスの脳に直接薬剤送達のための異粘膜移植手順

Delivery of therapeutics into the brain is impeded by the presence of the blood-brain barrier (BBB) which restricts the passage of polar and high ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

マウス腎臓移植：同種移植片拒絶のモデル

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

新生児マウスの脳室下帯へのプラスミドDNAのトランスポゾン媒介統合は、神経膠芽腫の新規モデルを生成する

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

マウスを用いた胃食道逆流の外科モデル

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Ivacaftor、主要な代謝産物および嚢胞性線維症患者の体液中の Lumacaftor の臨床サンプルの高スループット分析のための最適化された LC-MS/MS 法

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

体外血液脳関門ブタ脳血管内皮細胞に基づくモデルの確立のための改良法

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Image-Guided Resection of Glioblastoma and Intracranial Implantation of Therapeutic Stem Cell-seeded Scaffolds

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in part to the inability of the current treatment, consisting of surgical resection followed by radiation and chemotherapy, to eliminate invasive tumor foci. Treatment of these foci may be improved with tumoricidal human mesenchymal stem cells (MSCs). MSCs exhibit potent tumor tropism and can be engineered to express therapeutic proteins that kill tumor cells. Advancements in preclinical models indicate that surgical resection induces premature MSC loss and reduces therapeutic efficacy. Efficacy of MSC treatment can be improved by seeding MSCs on a biodegradable poly(lactic acid) (PLA) scaffold. MSC delivery into the surgical resection cavity on a PLA scaffold restores cell retention, persistence, and tumor killing. To study the effects of MSC-seeded PLA implantation on GBM, an accurate preclinical model is needed. Here we provide a preclinical surgical protocol for image-guided tumor resection of GBM in immune-deficient mice followed by MSC-seeded scaffold implantation. MSCs are engineered with lentiviral constructs to constitutively express and secrete therapeutic TNF&#945;-related apoptosis-inducing ligand (TRAIL) as well as green fluorescent protein (GFP) to allow fluorescent tracking. Similarly, the U87 tumor cells are engineered to express mCherry and firefly luciferase, providing dual fluorescent/luminescent tracking. While currently used for investigating stem cell mediated delivery of therapeutics, this protocol could be modified to investigate the impact of surgical resection on other GBM interventions.

Tumor-seeking therapeutic mesenchymal stem cells (MSCs) show promise as a treatment for invasive glioblastoma. Optimal transplantation involves delivery of MSCs into the tumor resection cavity on scaffolds. Here, preclinical techniques to study MSC treatment of glioblastoma are provided including: image-guided tumor resection; implantation of MSC-seeded scaffolds; and postoperative therapy tracking.

膠芽腫の治療の幹細胞シード足場の頭蓋内注入画像誘導切除

Glioblastoma (GBM), the most common and aggressive primary brain cancer, carries a life expectancy of 12-15 months. The short life expectancy is due in ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

機械学習を用いた画像誘導治療治療に対する反応を予測: 肝細胞癌における経動脈的治療の例

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...