この作品はR01AR062207、R01AR061484、R56AI100901、K01AR053210、そして病気が（CJ劉へ）リウマチ研究財団から研究助成を狙っNIHの研究助成金によって部分的にサポートされていました。

動物に関連する手術手順のすべては、手術による痛みや不快感を極力配慮の努力で、地元の機関の動物管理倫理委員会の承認を得なければならない。 1。準備<ol><li>手術のため、約25グラムの体重で8〜12週齢のオスのC57BL / 6マウスを選択します。 </li><li>キシラジン（5 mg / kgの）およびケタミン（40 mg / kg）の両方を含むカクテルの腹腔内注射により動物に麻酔。 </li><li>カミソリで膝を剃るし、外科的に動物をドレープし、ベータダイン及びアルコール（3×）で手術部位を消毒し、軟膏でマウスの眼をカバーしています。 </li></ol> 2。外科的プロセス<ol><li>膝関節を露出させ1センチメートル縦内側パラ膝蓋骨切開する。 </li><li>膝蓋骨の横方向の脱臼と膝蓋靱帯を通してゆっくりと膝関節を開きます。 <ol><li>縦方向に膝関節包をカットステップ2.1の内側パラ膝蓋骨切開により。 </li><li>ピンセットで膝関節包を解剖。 </li><li>左手で後肢の先端部をつかみ、そしてピンセットで膝蓋骨と膝蓋靭帯の横方向の転位を行う。優しく後肢を持って、足に外傷を避けるようにしてください。関節包を切開し、より良い、より少ない力が転位を行うために必要とされる。 </li></ol></li><li>軟骨表面の乾燥を避けるために、動作中の関節軟骨の表面に滅菌生理食塩水を滴下する。 </li><li>脛骨プラトーに内側半月板を固定する内側meniscotibial靭帯を切断。内側半月の下に軟骨を痛めないようにしてください。 <ol><li>大腿骨の内側顆と脛骨の内側高原の間に位置内側半月板を特定します。 </li><li>脛骨の顆間隆起と内側半月板の側面を接続meniscotibial靭帯を識別します。 </li><li>こんにちはを開催ND手で軽くPAW、及び第10外科用メスを使用して丁寧に内側meniscotibial靭帯を切断。関節軟骨や他の靭帯にけがの原因にならないことを確認してください。多くの場合、被覆脂肪パッド側に引っ張られる必要がなく、靭帯を同定するために除去されるべきではない。 </li></ol></li><li> 6-0吸収性縫合糸で膝関節包を閉じます。 </li><li> 6-0絹縫合糸で皮膚を閉じます。 </li></ol> 3。術後ケア<ol><li>術後の痛みを最小限に抑えるために、各マウスの手術部位に0.25％ブピバカインの一滴を適用する。 </li><li>水や食料を得るために自由に操作したマウスを残す。 </li></ol> 4。外科DMMモデルの組織学的スコアリング<ol><li>示された時点で、マウスを生け贄に捧げる手術後（ 例えば 4週間、8週間、12週間。ここでは、代表として8週間の結果を示した）。 </li><li>全体膝関節は、脱灰​​し、embe固定されているパラフィンでddedしてから5ミクロン間隔で連続的に切断した。 <ol><li>第10ブレードを全体後肢を切った。 </li><li>慎重に後肢の皮膚や筋肉を解剖し、RTに3日間、4％PFAでサンプルを固定します。 </li><li> PFAを外し、水でサンプルを清掃してください。その後、2週間、4℃で、EDTAのサンプルを脱灰。 </li><li>エタノール勾配でサンプルを脱水する。具体的には、エタノールを変更するO / Nの70％エタノールの新しいセット内のサンプルを維持、1時間70％エタノール中でサンプルを保つその後、結果として80％および90％エタノールに試料を入れて、O / Nを100％エタノール、続いて、それぞれ、1時間に残し</li><li>エタノールを除去。 1時間o-キシレン中のサンプルを保持し、o-キシレンの新しいセットに変更します。このステップ3回繰り返します。 </li><li>ライカ埋め込むセンターを使用してパラフィン型の中にサンプルを埋め込む。その後、試料を回転式ミクロトーム（ライカRM225を使用して5μmに区画され、5、ドイツ）、次いでガラススライド上に集めた。 </li><li>シリアル矢状セクションは内側顆の中心に外側顆の中心から領域に及ぶ各サンプルのために切断されています。 </li></ol></li><li>サフラニンO染色は、以前に行わ25記載のようにOARSI採点システムを介して採点し、統計分析が続く。 <ol><li>まず、o-キシレン中でスライドを脱パラフィンし、蒸留水に対するエタノールの勾配でそれらを水和する。その後、スライドをワイゲルト鉄ヘマトキシリン​​溶液、0.05％ファストグリーン（FCF）溶液、1％酢酸溶液、および0.1％サフラニンO溶液を介して染色する。樹脂製のメディアを使用してスライドをマウントします。 </li><li>組織学的スコアの統計分析を行う、軟骨と軟骨構造の破壊の割合のプロテオグリカン（赤色）の損失に基づいてサフラニンO染色スライドのスコア。 </li></ol></li></ol>

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私たちはここに、我々は利害関係のないことを宣言します。

これは、マウスの異なる株はC57BL6、129/SvInJ、その後FVB続く、129/SvEv株では最高レベルで、DMMの後にOAの重症度を変化させてきたようにマウスの系統は、DMMモデルの誘導のために非常に重要であることが報告されている/ N 26。 DMMに比較的影響を受けやすい我々は本研究で使用したマウス、 - 導入遺伝子の大部分は、PGRN - /などのC57BL6マウスで確立されている。導入遺伝子は、このよう?...

また、変形性関節症として知られている変形性関節症（OA）は、世界の人口の15％、米国内4600万人以上の人々に影響を与え、滑膜炎、軟骨の変性、および骨棘形成1によって特徴づけられる。それは、遺伝的な代謝、生体力学的および生化学的要因の複雑な相互作用の結果であり得る。 OAの基礎となるメカニズムは、科学界を回避し続けています。 2,3 OAの病因を模倣することができる多数の動物モデルが現在存在する。それがために、様々な遺伝子改変マウスの可用性および実験の費用対効果の両方のマウスにおいて、動物モデルを確立することが重要である。実験のOAモデルのさまざまな種類の中では、内側半月板（DMM）モデルの外科的に誘発される不安定化は、その良好な再現性やOA開発中に比較的遅く進行の広く受け入れられたOAモデルです。これらの属性の両方を持っているさまざまな治療法や導入遺伝子3-8のOA進行を評価するための鍵となって。しかしながら、外科OAモデルの一貫性は、手術中に様々な要因によって影響され、その結果、外科用マウスモデルの適用が制限される。 プログラニュリン（PGRN）は、様々な細胞で発現多機能成長因子である。なお、PGRNは、創傷治癒9、10腫瘍形成、および炎症11-15のような種々の生理学的および疾患プロセスにおいて重要な役割を果たすことが知られている。研究はまた、PGRNの不足は、ヒトおよびマウスの両方において16-18神経系の変性疾患を引き起こす可能性があることを示す。なお、PGRNは、ヒト関節軟骨において発現されることが知られており、そのレベルは有意にOA 19および慢性関節リウマチを有する患者の軟骨において上昇する。さらに、PGRNも軟骨細胞増殖20、分化および軟骨性に重要な役割を果たしている開発21,22の間の成長板の骨化。最近では、PGRNは、TNF受容体への結合を介して、TNF-α拮抗し、炎症性関節炎モデル13,14,23,24において抗炎症機能を示すことを報告した。しかしながら、OAにおけるPGRNの役割は、特にインビボで 、謎のままである。ここで、我々は、外科DMMモデルを誘導するための手順を提示し、WTおよびPGRN - / DMMでモデルを構築を通してOA開発におけるPGRNの役割を調査 - マウス。

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			<td height="21" style="height:21px;width:216px;">No. 10 Surgical blades</td>
			<td style="width:135px;">FEATHER</td>
			<td style="width:119px;">25-2976#10</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;">6-0 suture</td>
			<td>APPLIED DENTAL</td>
			<td>WG-N53133</td>
			<td></td>
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</table>

establishment of a surgically-induced model in mice to investigate the protective role of progranulin in osteoarthritis

内側半月（DMM）モデルの不安定化は、 生体内での変形性関節症（OA）の発症に非常に多くの関節炎に関連する分子の病態生理学的役割を研究するための重要なツールです。しかし、マウスでは、この複雑なモデルを確立するための詳細な、特に可視化プロトコルは、使用できません。 - / - マウスではDMMモデルを誘導するためのプロトコルを導入する例として、マウス、およびこの外科的に誘発されたモデルの確立に続いて、OAの発症を比較して、ここに、我々は野生型およびプログラ（PGRN）を利用しました。マウスで実行された操作だけmenisco脛骨靭帯を切って内側半月の不安定化を引き起こし、関節包、またはDMMの操作を、開かれた偽手術、のいずれかであった。変形性関節症の重症度は、組織学的ア ​​ッセイ（ 例えばサフラニンO染色）、OA関連遺伝子の発現、軟骨の細胞外マトリックス分子の分解、および骨棘のフォームを用いて評価したATION。マウス、およびこの外科的に誘導されたモデルでは、より深刻なのOAの表現型につながっPGNR成長因子の損失 - DMM操作が正常に野生型およびPGRN - /の両方でのOA開始および進行を誘導した。

DMMモデルが正常マウスに設立され、PGRNの欠乏は外科的に誘発されるOAの開発を誇張した。 マウス-偽とDMMの操作（ 図1）は 、WTおよびPGRN - /で行った。 8週間運転した後、マウスを屠殺し、サフラニンO染色は、組織学に基づいて、関節炎スコアの統計的分析、続いて、膝関節の切片上で実施した。 図2Aに示すように、同様のベースラ?...

Watch this Scientific Journal Video about Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis at JoVE.com

Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

我々は、マウスにおける内側半月（DMM）モデル、変形性関節症（OA）研究のための効果的なツールの不安定化のためのプロトコルを記述します。加えて、我々はプログラがOAの病因において保護的な役割を果たしていることを示す、このモデルを用いてprogranulincan誇張のOAの発症および進行の欠乏を示した。

変形性関節症におけるプログラの保護的役割を調査するためにマウスの外科的誘発モデルの確立

Destabilization of medial meniscus (DMM) model is an important tool for studying the pathophysiological roles of numerous arthritis associated molecules ...

establishment-surgically-induced-model-mice-to-investigate-protective

Research

JoVE Journal

Bioengineering

27.3K ビュー.  NYU Hospital for Joint Diseases. 我々は、マウスにおける内側半月（DMM）モデル、変形性関節症（OA）研究のための効果的なツールの不安定化のためのプロトコルを記述します。加えて、我々はプログラがOAの病因において保護的な役割を果たしていることを示す、このモデルを用いてprogranulincan誇張のOAの発症および進行の欠乏を示した。 

ビデオ: Establishment of a Surgically-induced Model in Mice to Investigate the Protective Role of Progranulin in Osteoarthritis

Multiparametric Optical Mapping of the Langendorff-perfused Rabbit Heart

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not have been accomplished by other approaches1. With the use of the calcium- and voltage-sensitive dyes, optical mapping allows measurement of transmembrane action potentials and calcium transients with high spatial resolution without the physical contact with the tissue. This makes measurements of the cardiac electrical activity possible under many conditions where the use of electrodes is inconvenient or impossible1. For example, optical recordings provide accurate morphological changes of membrane potential during and immediately after stimulation and defibrillation, while conventional electrode techniques suffer from stimulus-induced artifacts during and after stimuli due to electrode polarization1. 
The Langendorff-perfused rabbit heart is one of the most studied models of human heart physiology and pathophysiology. Many types of arrhythmias observed clinically could be recapitulated in the rabbit heart model. It was shown that wave patterns in the rabbit heart during ventricular arrhythmias, determined by effective size of the heart and the wavelength of reentry, are very similar to that in the human heart2. It was also shown that critical aspects of excitation-contraction (EC) coupling in rabbit myocardium, such as the relative contribution of sarcoplasmic reticulum (SR), is very similar to human EC coupling3. Here we present the basic procedures of optical mapping experiments in Langendorff-perfused rabbit hearts, including the Langendorff perfusion system setup, the optical mapping systems setup, the isolation and cannulation of the heart, perfusion and dye-staining of the heart, excitation-contraction uncoupling, and collection of optical signals. These methods could be also applied to the heart from species other than rabbit with adjustments to flow rates, optics, solutions, etc.
Two optical mapping systems are described. The panoramic mapping system is used to map the entire epicardium of the rabbit heart4-7. This system provides a global view of the evolution of reentrant circuits during arrhythmogenesis and defibrillation, and has been used to study the mechanisms of arrhythmias and antiarrhythmia therapy8,9. The dual mapping system is used to map the action potential (AP) and calcium transient (CaT) simultaneously from the same field of view10-13. This approach has enhanced our understanding of the important role of calcium in the electrical alternans and the induction of arrhythmia14-16.

This article describes the basic procedures for conducting optical mapping experiments in the Langendorff-perfused rabbit heart using the panoramic imaging system, and the dual (voltage and calcium) imaging modality.

ランゲンドルフ灌流ウサギの心臓のMultiparametric光学マッピング

Optical imaging and fluorescent probes have significantly advanced research methodology in the field of cardiac electrophysiology in ways that could not ...

生物工学

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

タンパク質、細胞閉じ込めのための二次元加工基板を作成する

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

のための周期的圧力のバイオリアクターの設計生体外で</em大動脈心臓弁の>研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

パラメータ化拡張期充満フォーマリズムを経由Transmitral流れの運動学的モデルベースの​​解析によるグローバル拡張期機能の定量化

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

3Dコラーゲンマトリックス中の走化性を調査するために、平面グラデーション拡散システム

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

アン<em&gt;インビトロ</emマウス椎間板の&gt;器官培養モデル

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Creating a Structurally Realistic Finite Element Geometric Model of a Cardiomyocyte to Study the Role of Cellular Architecture in Cardiomyocyte Systems Biology

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal microscopy, the scientific community has unprecedented access to large datasets at sub-micrometer resolution that characterize the architectural remodeling that accompanies changes in cardiomyocyte function in health and disease. However, these datasets have been under-utilized for&#160;investigating the role of cellular architecture remodeling in cardiomyocyte function. The purpose of this protocol is to outline how to create an accurate finite element model of a cardiomyocyte using high resolution electron microscopy and confocal microscopy images. A detailed and accurate model of cellular architecture has significant potential to provide new insights into cardiomyocyte biology, more than experiments alone can garner. The power of this method lies in its ability to computationally fuse information from two disparate imaging modalities of cardiomyocyte ultrastructure to develop one unified and detailed model of the cardiomyocyte. This protocol outlines steps to integrate electron tomography and confocal microscopy images of adult male Wistar (name for a specific breed of albino rat) rat cardiomyocytes to develop a half-sarcomere finite element model of the cardiomyocyte. The procedure generates a 3D finite element model that contains an accurate, high-resolution depiction (on the order of ~35 nm) of the distribution of mitochondria, myofibrils and ryanodine receptor clusters that release the necessary calcium for cardiomyocyte contraction from the sarcoplasmic reticular network (SR) into the myofibril and cytosolic compartment. The model generated here as an illustration does not incorporate details of the transverse-tubule architecture or the sarcoplasmic reticular network and is therefore a minimal model of the cardiomyocyte. Nevertheless, the model can already be applied in simulation-based investigations into the role of cell structure in calcium signaling and mitochondrial bioenergetics, which is illustrated and discussed using two case studies that are presented following the detailed protocol.

This protocol outlines a novel method to create a spatially detailed finite element model of the intracellular architecture of cardiomyocytes from electron microscopy and confocal microscopy images. The power of this spatially detailed model is demonstrated using case studies in calcium signaling and bioenergetics.

心筋細胞のシステム生物学における細胞構造の役割を研究する心筋細胞の構造的に現実的な有限要素の幾何学的なモデルを作成します。

With the advent of three-dimensional (3D) imaging technologies such as electron tomography, serial-block-face scanning electron microscopy and confocal ...

Calorespirometry: A Powerful, Noninvasive Approach to Investigate Cellular Energy Metabolism

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic respiration. However, the extent to which both pathways contribute to the landscape of cellular energy production is consistently overlooked. Transformed cells cultured in saturating levels of glucose often show a decreased dependency on oxidative phosphorylation for ATP production, which is compensated by an increase in substrate-level phosphorylation. This shift in metabolic poise allows cells to proliferate despite the presence of mitochondrial toxins. In neglecting the altered metabolic poise of transformed cells, results from a pharmaceutical screening may be misinterpreted since the potentially mitotoxic effects may not be detected using model cell lines cultured in the presence of high glucose concentrations. This protocol describes the pairing of two powerful techniques, respirometry and calorimetry, which allows for the quantitative and noninvasive assessment of both aerobic and anaerobic contributions to cellular ATP production. Both aerobic and anaerobic respirations generate heat, which can be monitored via calorimetry. Meanwhile, measuring the rate of oxygen consumption can assess the extent of aerobic respiration. When both heat dissipation and oxygen consumption are measured simultaneously, the calorespirometric ratio can be determined. The experimentally obtained value can then be compared to the theoretical oxycaloric equivalent and the extent of the anaerobic respiration can be judged. Thus, calorespirometry provides a unique method to analyze a wide range of biological questions, including drug development, microbial growth, and fundamental bioenergetics under both normoxic and hypoxic conditions.

This protocol describes calorespirometry, the direct and simultaneous measurement of both heat dissipation and respiration, which provides a noninvasive approach to assess energy metabolism. This technique is used to assess the contribution of both aerobic and anaerobic pathways to energy utilization by monitoring the total cellular energy flow.

Calorespirometry: 細胞のエネルギー代謝を調査する強力な非侵襲的アプローチ

Many cell lines used in basic biological and biomedical research maintain energy homeostasis through a combination of both aerobic and anaerobic ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

オレイン酸のトリペプチド安定ナノエマルション

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

マトリックス細胞代謝クロストークを研究するためのヒト3D細胞外マトリックス-脂肪細胞培養モデル

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...