This work was supported by NIH HL-10342

注：すべての動物のプロトコルはジョンズ·ホプキンス大学動物実験委員会によって承認された。 1.動物の準備<ol><li>以下のステップ2.3に概説されているようケタミンとキシラジンでそれらを麻酔により、DFCO測定に6 C57BL / 6対照マウスを準備します。 </li><li>対照と同様の手順を使用して、表1に示す異なる肺病変を有する他のすべてのマウスを準備する。これらのモデルのそれぞれを確立するために必要な具体的な詳細は、関連文献に見出される。対照マウスと他の病理学的コホートのものは、年齢のすべての6-12週間です。 </li></ol>一酸化炭素に対する拡散係数の2.測定（DFCO） <ol><li>窒素、酸素、ネオン、及び一酸化炭素のピークを測定する機械で供給されたガスクロマトグラフモジュールを設定する。このアプリケーションでは唯一のネオンとCOデータを使用する。 注：この装置は、分子のSを使用しています12.00ミクロンフィルム、320.00ミクロンID及び長さ10mで、キャリアガスとしてヘリウムを用いてカラムをIEVE。クロマトグラフカラムは、0.8ミリリットルの容量を持っていますが、我々は、サンプルとの接続配管の適切なクリアを確実にするために2ミリリットルを使用していました。 </li><li>各実験日の開始時に、マウスからのサンプルの測定を行う前に、約0.5％のNeと、0.5％のCOを含むガス混合物バッグから直接の2mlサンプルを取り、空気バランスをとる、および較正するために、このサンプルを使用ガスクロマトグラフ。 </li><li>ケタミン（90ミリグラム/ kg）およびキシラジン（15ミリグラム/ kg）でマウスを麻酔し、反射運動の欠如によって麻酔を確認する。乾燥を防ぐために、目に獣医軟膏を適用します。 （非常に若いマウスでは成人で18 Gまたは20 G）スタブ針カニューレをマウスにTracheostomize。 注：DFCOは麻酔後に10分未満で完了し、すべての機械的な換気または他の手順の前にされている。 </li><li> 6週齢よりも大きいマウスでは3 mlシリンジtを使うO混合ガスバッグからのガス0.8mlの撤回。気管カニューレにシリンジを接続し、すぐに肺を膨らませる。メトロノームを使用して、9秒をカウントしてから、すぐに0.8ミリリットル（呼気）を撤回。 </li><li>この撤回0.8ミリリットルが室内空気との2ミリリットルに空気を吐き出さ希釈し、それは少なくとも15秒間休むことができます。その後、分析のためにガスクロマトグラフに全サンプルを注入する。 </li><li>この第DFCO試料を分析しながら、ガス混合バッグから第0.8ミリリットルでマウス肺を膨張し、最初のサンプルと同じでこのサンプルを処理する。 2 DFCO測定を平均化する。 注：0.8 mlの体積が非常に若いマウスの肺で測定を行うには大きすぎるので、2週齢の幼いマウスにおいて測定のために、0.4 mlの容量を使用する。これは、6週間以上経過したマウスの0.8容を使用することをお勧めし、0.4mlの容量がいくつかのマウスのために必要とされる場合、それは研究されてコホート内のすべてのマウスについて一貫して使用されるべきである。 </li><li> DFCOを計算（9 CO / CO c）は/（Neが9 / Neとc）は、cおよび9添字注入校正ガスの濃度は、それぞれ、9秒の息止め時間後に除去したガスを参照する- 1である。 </li><li>分析し、一方向ANOVAとの差を比較し、すべてのコホートマウスにおける多重比較のためのテューキーの補正との有意水準を評価する。 P &lt;0.05として重要な価値を考えてみましょう。 注：ここで使用したマウスのすべてがここで報告されていない肺換気、力学、肺洗浄液、または組織学のいくつかのその後の測定を伴う実験研究の一部であった。この方法は、対照マウスに上記行ったように、すべての実験モデルにおいて同様であるのでさらに、種々の病理学的モデルからの結果のみを提示する。これらのモデルの詳細については、補足表に示されている。 </li><li>深いanestheにより動物を安楽死させるチック過剰摂取は、頸椎脱臼または断頭が続く。必要な場合には、さらに生物学的または組織学的処理および分析のために死亡したマウスからの肺細胞および/または組織を取り除く。 </li></ol>

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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palifermin+induces+alveolar+maintenance+programs+in+emphysematous+mice.">Palifermin induces alveolar maintenance programs in emphysematous mice.</a> Am J Respir Crit Care Med. 181 (7), 705-717 (2010).</li><li>Collins, S. L., Chan-Li, Y., Hallowell, R. W., Powell, J. D., Horton, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pulmonary+vaccination+as+a+novel+treatment+for+lung+fibrosis.">Pulmonary vaccination as a novel treatment for lung fibrosis.</a> PLoS One. 7 (2), e31299 (2012).</li><li>Alessio, F. 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利害の対立はなく、開示することは何もない。

本研究では、マウス肺の能力を交換するガスを定量化するための新しいメトリックを定義した。このメトリックは、拡散能、すなわち、肺の主要な機能を測定する一般的な臨床測定、ガスを交換する能力に類似している。拡散容量は、簡単かつ迅速に、マウスおよびヒトの両方で行うことができる唯一の​​肺の機能的測定である。マウスにおける肺疾患の検出のために、主要な目的は、対?...

マウスは、現在の肺疾患の種々のモデル化に使用される主要な動物である。そのような病状をunderlyメカニズムを研究するために、表現型の方法は、それを病理学的変化を定量化することが必要とされている。換気力学が測定されている多くのマウス試験がありますが、これらの測定値は一般的に、通常、ヒトで行われ、肺機能の標準的評価とは無関係である。マウスおよびヒト対象において同等の測定を実行する能力は、ヒト疾患のマウスモデルの結果の翻訳を容易にすることができるので、これは残念なことである。 ヒト被験体における最も一般的かつ容易に行われる測定の一つは、一酸化炭素（DLCO）-1,2-ための拡散能であるが、この測定は、まれにしかマウスモデルで行われていない。手順は、しばしば扱いにくいまたはreqができるので、それは3-7報告されているこれらの研究では、部分的には、何のフォローアップ研究が行われていない複雑な装置uire。別のアプローチは、意識のあるマウスにおけるCOの拡散を測定することができるという利点を有する定常状態のシステムにおいて、CO再呼吸法を使用することである。しかし、この方法は非常に煩雑であり、結果は、マウスの換気のレベル並びにO 2およびCO 2濃度8.9で変化することができる。これらの困難は、そのいくつかの利点にもかかわらず、マウスでは肺の病理を検出する能力を拡散する日常的な使用を妨げているように見える。 マウスにおいて、拡散能力の測定に伴う問題を回避するために、マウスでそれを測定するための簡単な手段の詳細については、最近10に報告されている。手続きはすぐにガスを触発全体に等しい体積をサンプリングすることによって汚染されていない肺胞ガスをサンプリングする困難な問題を排除します。非常に再現性のある測定では、この手順の結果は、パのホストに敏感である一酸化炭素（DFCO）用の拡散係数と呼ばれる肺の表現型のthologic変化。 （CO 9 / CO c）に /（Neが9 / Neとc）は、cおよび9添字注入校正ガスおよび9秒の息止め時間後に除去したガスの濃度を表す、 - DFCOこうして1のように計算されるそれぞれ。 DFCO 1はCOのない取り込みを反映していない完全なすべてのCOの摂取、及び0を反映して、0と1の間で変化する無次元の変数である。 この発表では、この拡散能測定を行う方法を示し、そしてどのようにそれが気腫、線維症、急性肺損傷、およびウイルスおよび真菌感染を含むほぼすべての既存のマウス肺疾患モデルの変更を文書化するために使用することができる。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Gas Chromatograph</td>
			<td>Inficon</td>
			<td>Micro GC Model 3000A</td>
			<td>Agilent makes a comparable model</td>
		</tr>
		<tr>
			<td>18 g Luer stub needle</td>
			<td>Becton Dickenson</td>
			<td></td>
			<td>Several other possible vendors</td>
		</tr>
		<tr>
			<td>3 mL plastic syringe</td>
			<td>Becton Dickenson</td>
			<td></td>
			<td>Several other possible vendors</td>
		</tr>
		<tr>
			<td>Polypropylene gas sample bags</td>
			<td>SKC</td>
			<td>1 or 2 liter capacity works well</td>
			<td>Other gas tight bags will work well</td>
		</tr>
		<tr>
			<td>Gas tank, 0.3% Ne,0.3% CO, balance air; (size ME)</td>
			<td>Airgas, Inc</td>
			<td>Z04 NI785ME3012</td>
			<td>This is the standard mixture used for DLCO in humans</td>
		</tr>
		<tr>
			<td>25 TCID50/mouse of influenza virus A/PR8 diluted in phosphate buffered saline.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Porcine pancreatic elastase</td>
			<td>Elastin Products, Owensville, MO</td>
			<td></td>
			<td>5.4 U</td>
		</tr>
		<tr>
			<td>Bleomycin</td>
			<td>APP Pharmaceuticals, Schaumburg, IL</td>
			<td></td>
			<td>0.25 U</td>
		</tr>
		<tr>
			<td>Escherichia coli LPS8</td>
			<td>Sigma L2880</td>
			<td></td>
			<td>3 &#956;g/g body weight; O55:B5</td>
		</tr>
		<tr>
			<td>Aspergillus fumigatus (isolate Af293) conidia were collected from mature colonies grown on potato dextrose agar.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

phenotyping mouse pulmonary function in vivo with the lung diffusing capacity

マウスは、現在の肺疾患の種々のモデル化に使用される主要な動物である。このような病状の根底にあるメカニズムを研究するために、表現型の方法は、病理学的変化を定量化することが必要とされている。さらに、マウスモデルへの並進の関連性を提供するために、そのような測定を容易にヒトおよびマウスの両方で行うことができるテストであるべきである。残念ながら、現在の文献に肺機能のいくつかの表現型の測定は、ヒトへの直接のアプリケーションがあります。唯一の例外は、日常的にヒトにおいて行われた測定である一酸化炭素拡散能である。今回の報告では、迅速かつ簡単にマウスではこの拡散能を測定するための手段を説明する。手順は、1分ガス分析時間が続く、麻酔したマウスにおけるトレーサガスと短い肺膨張を伴う。私たちは、肺気腫、線維症、急性肺損傷、およびインフルエンザを含むいくつかの肺の病変を検出するためのこの方法の能力をテストし、真菌の肺感染症、ならびに若い仔における肺成熟をモニタする。結果は、すべての重要な肺病変の減少、ならびに肺成熟と拡散容量の増加を示す。肺拡散能のこの測定は、このように既存の病的な肺モデルのほとんどと表現型の構造変化を検出する能力を有する広い用途を有する肺機能試験を提供する。

図1は、アスペルギルス属およびインフルエンザ感染症の両方に有意な減少、ならびに線維症の有意な減少、気腫、急性があったグループA、B、C、D、Eにおける成体マウスからDFCO測定値を示しており、F.肺損傷モデル。 図2に、2-6週間のマウスの年齢などの経時DFCOでグループG発達的変化を示している。この時間経過にわたって肺の発達と、わずかではあるが有意な増?...

Watch this Scientific Journal Video about Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity at JoVE.com

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

表現型マウス肺機能<em&gt;インビボ</em&gt;肺拡散能力を持つ

The mouse is now the primary animal used to model a variety of lung diseases. To study the mechanisms that underlie such pathologies, phenotypic methods ...

phenotyping-mouse-pulmonary-function-vivo-with-lung-diffusing

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JoVE Journal

Biology

Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

10.5K ビュー.  Johns Hopkins University Bloomberg School of Public Health. We describe a means to quickly and simply measure the lung diffusing capacity in mice and show that it is sufficiently sensitive to phenotype changes in multiple common lung pathologies. This metric thus brings direct translational relevance to the mouse models, since diffusing capacity is also easily measured in humans.

ビデオ: Phenotyping Mouse Pulmonary Function In Vivo with the Lung Diffusing Capacity

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

生体イメージングおよび卵巣癌の同所性マウスモデルにおける治療的処置の

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

生物学

Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0, the characteristic impedance ZC, and the index of wave reflection RW - provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.

The following protocol outlines the process of isolating, ventilating and instrumenting mouse lungs to measure steady or pulsatile pulmonary vascular pressure-flow relationships in order to quantify the effects of blood flow, airflow, airway changes and vascular changes on right ventricular afterload.

絶縁型、ベンチレーテッド、および計装のマウス肺の特性評価は、拍動流を灌流

The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

人工多能性幹細胞に由来するマウスの作製

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

大型単層小胞とBcl-2ファミリーの機能を調べる

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

エア·液界面での人間の鼻上皮細胞の培養

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

無傷骨格筋線維内のカルシウムスパークスの評価

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum &#8220;cast&#8221; intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies.

Basic techniques and refinements of freeze-fracture processing of biological specimens and nanomaterials for examination by transmission electron microscopy are described. This technique is a preferred method for revealing ultrastructural features and specializations of biological membranes and for obtaining ultrastructural level dimensional and spatial data in materials sciences and nanotechnology products.

生物学電子顕微鏡でのフリーズフラクチャー/フリーズエッチングの基本技術要素

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated ...

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

ハイスループットマイクロプレート呼吸測定のためのマウスの骨格筋の最小量からミトコンドリアの単離

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

C-FOSタンパク質免疫組織学的検出：特定の生理反応に関与する中央経路のマーカーとして有用ツール<em&gt;インビボ</em&gt;と<em&gt; ex vivoで</em

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

表現型マウス肺機能

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