この作品は、H＆H·リー外科レジデント研究奨学プログラム（JAN）にすることにより、AO財団スタートアップ（LSM）の助成金S-12-03Mと（LSM）は国立衛生研究所の助成金R01-AI078910をサポートされていました。

<ol>
	<li>Dothager, R. S., 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=Advances+in+bioluminescence+imaging+of+live+animal+models.">Advances in bioluminescence imaging of live animal models.</a> Curr Opin Biotechnol. 20, 45-53 (2009).</li><li>Badr, C. E., Tannous, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+progress+and+applications.">Bioluminescence imaging progress and applications.</a> Trends Biotechnol. 29, 624-633 (2011).</li><li>Luker, G. D., Luker, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+current+applications+and+future+directions.">Optical imaging current applications and future directions.</a> J Nucl Med. 49, 1-4 (2008).</li><li>Ntziachristos, V., Ripoll, J., Wang, L. V., Weissleder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+and+listening+to+light+the+evolution+of+whole-body+photonic+imaging.">Looking and listening to light the evolution of whole-body photonic imaging.</a> Nat Biotechnol. 23, 313-320 (2005).</li><li>Reumann, M. K., Weiser, M. C., Mayer-Kuckuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Musculoskeletal+molecular+imaging+a+comprehensive+overview.">Musculoskeletal molecular imaging a comprehensive overview.</a> Trends Biotechnol. 28, 93-101 (2010).</li><li>Snoeks, T. J., Khmelinskii, A., Lelieveldt, B. P., Kaijzel, E. L., Lowik, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+advances+in+skeletal+imaging+applied+to+bone+metastases.">Optical advances in skeletal imaging applied to bone metastases.</a> Bone. 48, 106-114 (2011).</li><li>Sjollema, J., 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=The+potential+for+bio-optical+imaging+of+biomaterial-associated+infection+in+vivo.">The potential for bio-optical imaging of biomaterial-associated infection in vivo.</a> Biomaterials. 31, 1984-1995 (2010).</li><li>Del Pozo, J. L., Patel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+practice.+Infection+associated+with+prosthetic+joints.">Clinical practice. Infection associated with prosthetic joints.</a> N Engl J Med. 361, 787-794 (2009).</li><li>Parvizi, J., Adeli, B., Zmistowski, B., Restrepo, C., Greenwald, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+periprosthetic+joint+infection+the+current+knowledge+AAOS+exhibit+selection.">Management of periprosthetic joint infection the current knowledge AAOS exhibit selection.</a> J Bone Joint Surg Am. 94, e104 (2012).</li><li>Arciola, C. R., Campoccia, D., Speziale, P., Montanaro, L., Costerton, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm+formation+in+Staphylococcus+implant+infections.+A+review+of+molecular+mechanisms+and+implications+for+biofilm-resistant+materials.">Biofilm formation in Staphylococcus implant infections. A review of molecular mechanisms and implications for biofilm-resistant materials.</a> Biomaterials. 33, 5967-5982 (2012).</li><li>Zimmerli, W., Moser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+treatment+concepts+of+orthopaedic+biofilm+infections.">Pathogenesis and treatment concepts of orthopaedic biofilm infections.</a> FEMS Immunol Med Microbiol. 65, 158-168 (2012).</li><li>Cram, P., 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=Total+knee+arthroplasty+volume+utilization+and+outcomes+among+Medicare+beneficiaries+1991-2010.">Total knee arthroplasty volume utilization and outcomes among Medicare beneficiaries 1991-2010.</a> JAMA. 308, 1227-1236 (2012).</li><li>Wolf, B. R., Lu, X., Li, Y., Callaghan, J. J., Cram, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adverse+outcomes+in+hip+arthroplasty+long-term+trends.">Adverse outcomes in hip arthroplasty long-term trends.</a> J Bone Joint Surg Am. 94, (2012).</li><li>Bernthal, N. M., 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=A+mouse+model+of+post-arthroplasty+Staphylococcus+aureus+joint+infection+to+evaluate+in+vivo+the+efficacy+of+antimicrobial+implant+coatings.">A mouse model of post-arthroplasty Staphylococcus aureus joint infection to evaluate in vivo the efficacy of antimicrobial implant coatings.</a> PLoS ONE. 5, (2010).</li><li>Bernthal, N. M., 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=Protective+role+of+IL-1beta+against+post-arthroplasty+Staphylococcus+aureus+infection.">Protective role of IL-1beta against post-arthroplasty Staphylococcus aureus infection.</a> J Orthop Res. 29, 1621-1626 (2011).</li><li>Niska, J. A., 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=Monitoring+bacterial+burden,+inflammation+and+bone+damage+longitudinally+using+optical+and+&#181;CT+imaging+in+an+orthopaedic+implant+infection+in+mice.">Monitoring bacterial burden, inflammation and bone damage longitudinally using optical and &#181;CT imaging in an orthopaedic implant infection in mice.</a> PLoS ONE. 7, e47397 (2012).</li><li>Niska, J. A., 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=Daptomycin+and+tigecycline+have+broader+effective+dose+ranges+than+vancomycin+as+prophylaxis+against+a+Staphylococcus+aureus+surgical+implant+infection+in+mice.">Daptomycin and tigecycline have broader effective dose ranges than vancomycin as prophylaxis against a Staphylococcus aureus surgical implant infection in mice.</a> Antimicrob Agents Chemother. 56, 2590-2597 (2012).</li><li>Niska, J. A., 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=Vancomycin-Rifampin+Combination+Therapy+has+Enhanced+Efficacy+Against+an+Experimental+Staphylococcus+aureus+Prosthetic+Joint+Infection.">Vancomycin-Rifampin Combination Therapy has Enhanced Efficacy Against an Experimental Staphylococcus aureus Prosthetic Joint Infection.</a> Antimicrob Agents Chemother. 57, 5080-5086 (2013).</li><li>Pribaz, J. R., 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=Mouse+model+of+chronic+post-arthroplasty+infection+noninvasive+in+vivo+bioluminescence+imaging+to+monitor+bacterial+burden+for+long-term+study.">Mouse model of chronic post-arthroplasty infection noninvasive in vivo bioluminescence imaging to monitor bacterial burden for long-term study.</a> J Orthop Res. 30, 335-340 (2012).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+green+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages.</a> Blood. 96, 719-726 (2000).</li><li>Kadurugamuwa, J. L., 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=Direct+continuous+method+for+monitoring+biofilm+infection+in+a+mouse+model.">Direct continuous method for monitoring biofilm infection in a mouse model.</a> Infect Immun. 71, 882-890 (2003).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J Biomed Opt. 12, 024007 (2007).</li><li>Kim, M. H., 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=Neutrophil+survival+and+c-kit+-progenitor+proliferation+in+Staphylococcus+aureus-infected+skin+wounds+promote+resolution.">Neutrophil survival and c-kit+-progenitor proliferation in Staphylococcus aureus-infected skin wounds promote resolution.</a> Blood. 117, 3343-3352 (2011).</li><li>Deirmengian, C. A., Lonner, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What's+new+in+adult+reconstructive+knee+surgery.">What's new in adult reconstructive knee surgery.</a> J Bone Joint Surg Am. 94, 182-188 (2012).</li><li>Ning, X., 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=Maltodextrin-based+imaging+probes+detect+bacteria+in+vivo+with+high+sensitivity+and+specificity.">Maltodextrin-based imaging probes detect bacteria in vivo with high sensitivity and specificity.</a> Nat Mater. 10, 602-607 (2011).</li><li>Panizzi, P., 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=In+vivo+detection+of+Staphylococcus+aureus+endocarditis+by+targeting+pathogen-specific+prothrombin+activation.">In vivo detection of Staphylococcus aureus endocarditis by targeting pathogen-specific prothrombin activation.</a> Nat Med. 17, 1142-1146 (2011).</li><li>van Oosten, M., 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=Realtime+in+vivo+imaging+of+invasive+and+biomaterial+associated+bacterial+infections+using+fluorescently+labelled+vancomycin.">Realtime in vivo imaging of invasive and biomaterial associated bacterial infections using fluorescently labelled vancomycin.</a> Nat Commun. 4, 2584 (2013).</li><li>Kong, Y., 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=Imaging+tuberculosis+with+endogenous+beta+lactamase+reporter+enzyme+fluorescence+in+live+mice.">Imaging tuberculosis with endogenous beta lactamase reporter enzyme fluorescence in live mice.</a> Proc Natl Acad Sci USA. 107, 12239-12244 (2010).</li></ol>

JAM、BNT、EL、NZ、KPFはXen29生物発光S.を提供し 、イメージング機器を製造してパーキンエルマーの従業員が支払われる黄色ブドウ球菌株は、このビデオ、記事の出版費用を支払った。残りの著者は、開示することは何もない。

そのようなμCTイメージングと組み合わせてインビボ光学イメージングに利用するイメージング技術としてマルチモダリティイメージングは、解剖学的文脈1-4 3Dビジュアライゼーション、定量および生物学的プロセスの長手方向の監視を可能にする新たな技術的アプローチを提供します。本研究におけるプロトコルは、非侵襲的に長さ方向にわたって骨に細菌負荷、好中球性炎症および解剖学的変化を監視するために、マウスにおける整形外科補綴インプラント感染モデルにμCTイメージングと組み合わせることができるかのin vivo生物発光と蛍光イメージングの詳細な情報を提供する時間。まとめると、光学的および構造的イメージングを組み合わせることによって得られた情報は、特に筋骨格系に影響を与える生物学的プロセスおよび病理学的状態を研究するために適してもよい主要な技術的進歩を表す。 One関心指摘されるべきであるる知見は、私たちがEGFP-好中球の蛍光シグナルは、14〜21日までにバックグラウンドレベルまで減少し、生物発光細菌の存在にもかかわらず、実験の期間中、バックグラウンドレベルのままであったことを観察することである。私たちは、非照射マウス19における好中球の信号の類似の動態を観察し、X線照射の影響を受けた好中球の生存可能性は低い。私たちの以前の研究ではSのモデルを含む黄色ブドウ球菌に感染した傷、好中球の浸潤は循環から強固な好中球動員、感染部位、彼らはローカルで好中球23を成熟を ​​生じる膿瘍にKIT +前駆細胞のホーミングで長期の好中球生存のフォーメーションを採用。これは、同様の処理が整形外科用インプラントSに好中球浸潤に寄与した可能性がある黄色ブドウ球菌感染モデル。それは不明であるが、好中球の信号は、整形外科において減少した理由aedic感染モデルは、この感染症は慢性感染への急性から進行し、これが今後の調査の対象であり、免疫応答は経時的に変化しているかもしれません。 注目すべきである整形外科人工関節感染およびin vivoでのマルチモダリティイメージングのこのマウスモデルでは制限があります。まず、このマウスモデルは、ヒト24に整形外科手術で使用される実際の手順および材料の過度の単純化である。それにもかかわらず、このモデルは、慢性感染を再現し、人間の整形外科用インプラント感染8,9に見られる骨および関節組織の炎症が続くん。また、μCT画像を得るために、X線照射の比較的低用量は、感染の経過の間、動物の健康への悪影響を最小限にするために使用された。 μCTイメージングに使用することができる骨の良好な分解能、X線放射のより高用量のために安楽死にnimals。しかしながら、これは、非侵襲的かつ縦方向に実験の期間にわたって骨の変化を監視する能力を排除する。 結論として、解剖学的μCTイメージングを用いたインビボ動物全体光学イメージングの組み合わせを含むマルチモダリティイメージングは、感染症や炎症反応についてのより総合的な情報が可能になった。また、これらの技術は、骨および関節組織における感染および炎症の影響の評価を可能にした。今後の課題は、私たちが14〜18を調査し始めているように抗菌療法、免疫応答、疾患の病因と骨中の反応性の変化の有効性を評価するためにマルチモダリティイメージングを利用することができます。また、マルチモダリティイメージングは​​、以前に動物モデルにおいて大腿感染症、心内膜炎、肺感染し、記載のように感染の存在を診断するためのプローブおよびトレーサーを評価することができイオンや生体材料の感染25-28。最後に、マルチモダリティイメージングの使用は、感染症を超えて拡大し、そのような骨格癌、転移性疾患、骨折や関節炎5-7のような筋骨格系に影響を与える他の条件を調査するために整形外科、リウマチ学および腫瘍学などの学問分野、全体で使用することができる。

光学的および解剖学的情報の組み合わせを伴うマルチモダリティ前臨床イメージング技術は、3D 1-4生物学的現象の可視化および監視を可能にします。 μCTイメージングは骨の解剖学的構造の絶妙な可視化を可能にするので、光学イメージングとの組み合わせてμCTイメージングを使用すると、骨の生物学5-7を伴うプロセスを調査するために特に有用かもしれないユニークな組み合わせを表しています。例は、整形外科手術手順8,9次の悲惨な合併症を表す整形外科用インプラント感染を研究するためにこれらの技術を使用することであろう。細菌バイオフィルムは細菌10,11へのアクセスからの感染をブロック抗生物質を検知することから免疫細胞を妨げる物理的障壁として機能することにより、細菌の生存を促進する移植された異物に形成する。関節組織（敗血症性関節炎）、ANの慢性持続感染d個の骨（骨髄炎）は、人工関節の緩み、最終的故障8,9につながる骨吸収を誘導する。この得られた人工関節周囲の骨溶解が増加罹患率と死亡率の12,13に関連付けられています。 私たちの以前の研究では、in vivoでの生物発光と蛍光イメージングは、マウス14〜19における整形外科人工関節感染モデルにおけるX線およびマイクロコンピュータ断層撮影（μCT）と一緒に使用した。このモデルは、インプラントの切断端が14-19マウスの大腿骨から膝関節に伸長するように（Kワイヤ）チタンキルシュナー·ワイヤを配置関係する。手術部位が14-19を閉じる前に、黄色ブドウ球菌 （生物発光株Xen29またはXen36）の接種は、その後。膝関節におけるインプラントの表面上にピペットで移したインビボ光学イメージングは、生物発光シグナルを検出および定量化するために使用されたNUに対応した感染した関節と骨組織14〜19中の細菌のmber。また、蛍光好中球20を有しLysEGFPマウスのin vivo蛍光イメージング、Kワイヤイ ​​ンプラント14,19を含む感染した膝関節に移住した好中球の数を定量した。最後に、高分解能X線イメージングとμCT画像を含む解剖学的撮像モダリティは、私たちが任意に2,6術後週16の間、典型的には終わるだろう慢性感染症の全期間にわたって影響を受けた骨のそれぞれの2次元および3次元解剖学的画像化を可能にし、18。このモデルを使用して、局所的および全身的抗菌療法、防御免疫応答および骨における病理学的解剖学的変化の有効性は、14〜18を評価することができる。本稿では、この整形外科人工関節感染モデルにおける光とμCTイメージングモダリティのための詳細なプロトコルはrepresentatiとして提供された骨の解剖学的文脈で生物学的プロセスを研究するためのシステムをVEの。これらは、μCT画像取得および分析した3D光学画像の同時登録を、マウスにおける整形外科人工関節感染をモデル化するための外科的処置を含む2Dおよび3D インビボ光学イメージング手順（細菌性生物発光シグナルおよび蛍光好中球の信号を検出するため） μCT画像。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Xen36 bioluminescent Staphylococcus aureus strain</td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Bioluminescent Staphylococcus aureus strain derived from ATCC 49525 (Wright), a clinical isolate from a bacteremia patient</td>
		</tr>
		<tr>
			<td>Tryptic soy broth</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>211825</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto Soy Agar</td>
			<td>BD Biosciences, Franklin Lakes, NJ</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>LysEGFP knockin mouse strain</td>
			<td></td>
			<td></td>
			<td>Not commercially available. This strain contains a knockin of enhanced green fluorescence protein (EGFP) into the lysozyme M gene</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Purdue Products, Stamford, CT</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kirschner-wire (titanium, 0.8 mm diameter)</td>
			<td>Synthes, West Chester, PA</td>
			<td>492.08</td>
			<td></td>
		</tr>
		<tr>
			<td>Wire Cutter - Duracut T.C.</td>
			<td>H&#38;H Company, Ontario, Canada</td>
			<td>83-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Baxter, Deerfield, IL</td>
			<td>118718</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl 5-0 sutures (P-3 Reverse cutting)</td>
			<td>Ethicon, Summerville, NJ. Purchased through VWR International.</td>
			<td>95056-936</td>
			<td></td>
		</tr>
		<tr>
			<td>Sustained-release Buprenorphine (5 ml - 1 mg/ml)</td>
			<td>Zoopharm, Windsor, CO</td>
			<td></td>
			<td>analgesic</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/IVISSPE?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS Spectrum Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>optical in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Quantum%20FX%20%20microCT?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Quantum FX in vivo &#956;CT system</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>&#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Category/ID/Living%20Image%20Software?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">IVIS SpectrumCT Imaging System</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>combined optical and &#956;CT in vivo imaging system</td>
		</tr>
		<tr>
			<td><a href="http://www.perkinelmer.com/Catalog/Product/ID/119240?utm_source=JoVE&amp;utm_medium=display&amp;utm_content=LST-IDT2014-GLO-Q414-JoVE&amp;utm_campaign=LST-IDT2014-GLO">Living Image Software</a></td>
			<td>PerkinElmer, Hopkinton, MA</td>
			<td></td>
			<td>Image analysis software for in vivo optical imaging</td>
		</tr>
	</tbody>
</table>

combined in vivo optical and &#181;ct imaging to monitor infection, inflammation, and bone anatomy in an orthopaedic implant infection in mice

マルチモダリティイメージングは​​、両方の前臨床および臨床研究で使用される一般的な技術的アプローチとして浮上している。 インビボ光学およびμCTイメージングにおいて組み合わせる高度な技術は、解剖学的文脈における生命現象の可視化を可能にする。これらの画像診断法は、骨に影響を与える条件を研究するのに特に有用である可能性がある。具体的には、整形外科用インプラントの感染症は、臨床整形外科手術における重要な問題である。これらの感染症は、細菌のバイオフィルムは、外国外科的に移植材料に形成するので、持続的な炎症、骨髄炎、最終的には、インプラントの緩みや故障をもたらすインプラントを、周囲の骨の最終的な骨溶解を引き起こす、治療が困難である。ここで、感染した整形外科用補綴インプラントのマウスモデルは、このような方法で、大腿骨の髄内管内にキルシュナー·ワイヤ用インプラントの外科的配置を関連するその使用したインプラントeの端膝関節にXtendedでは。このモデルでは、LysEGFPマウス、EGFP蛍光好中球を有するマウス株は、自然光を発する生物発光の黄色ブドウ球菌株と組み合わせて使用した。細菌は、前に手術部位を閉鎖したマウスの膝関節に接種した。 インビボ生物発光及び蛍光撮像は、それぞれ、細菌負荷及び好中球の炎症性応答を定量した。生物発光および蛍光の光信号の3D位置は解剖学的μCT画像と共に同時登録することができるように、また、μCTイメージングは​​同じマウスで行った。経時的な骨の変化を定量化するために、遠位大腿骨の外側の骨体積は、半自動化された輪郭ベースのセグメンテーションプロセスを使用して、特定の時点で測定した。 μCTイメージングを用いたインビボ生物発光/蛍光イメージングの組み合わせをf特に有用である、一緒になってまたは感染、炎症反応と時間をかけて骨に解剖学的変化を非侵襲的に監視する。

生体内生物発光および蛍光イメージングでは、 本研究では、プロトコルは関節内に大腿骨に髄内管から延びチタンKワイヤイ ​​ンプラントの外科的配置を伴うマウス14-19整形外科人工関節感染症のこの以前に発表されたモデルで、に記載されているスペース14-19。S.黄色ブドウ球菌生物発光株Xen29（2μlのPBS中の1×10 3 CFU）を前に、手術部位16を閉じることに膝関節内のエンドチタンインプラントの上に直接ピペッティングした。視覚化し、麻酔しLysEGFPマウスにおいて非侵襲的に細菌負荷及び好中球流入を定量化するために、in vivoでの動物全体の光学イメージングは、画像を順次実施したIVISスペクトラム光学動物全体インを使用して浸潤好中球からの細菌から生物発光信号やEGFP蛍光シグナル生体内3術後日数（ すなわち、2日目、14および28）上の画像システム。 Xen29感染マウスの生物発光シグナルは、実験（ 図1A、C）16の間、偽感染マウスのバックグラウンドシグナルを超えたまま。 私たちの以前の研究は、生体内生物発光信号が密接インプラント17,18ジョイント/骨組織と接着性から分離されたex vivoでのCFUの数を近似していることを実証した。また、EGFP蛍光シグナルは、初期の時点で偽感染マウスよりも高かったが、感染の経過（ 図2B、C）16の間にバックグラウンドレベルに近づいた。 3DμCT画像とin vivoでの光信号の同時登録私は、手術後の膝関節の解剖学的コンテキストにおいて光信号（ すなわち、細菌の生物発光およびEGFP蛍光信号）を可視化するnは、3D、IVISスペクトルイメージングシステムを用いて生成された光学像は、同時登録された量子FXμCTイメージングシステムを用いて生成されたμCT画像であった。マウス撮像チャンバは、マウスが正確に同じ向きであったことを確実にするためにいずれかのマシンに挿入することができるため、この共登録は達成することができる。画像取得は、動物の身体的再配置を必要とせずに1機器に両方のモダリティを統合IVISスペクトラム-CT のin vivoイメージングシステムを用いて行われ、この正確さを確認するために、結果を比較した。 3DでμCT画像上に光学データをマップするために、拡散光トモグラフィ再構成アルゴリズム16を利用した 。結果として得られる3D再構成は（ ムービー1）が示されている。 また、μCTイメージングは​​中に発生した骨の質や寸法の必然的な変化の可視化および定量化を可能にした感染（ 図2）16。既報の通り、遠位大腿骨の外側の骨量は、実質的に時間をかけた（ 図2A）16 を増加させた。これらの変化を定量化するために、3Dボリュメトリック画像解析は、大腿骨のボニー面の先端25％で行った経時骨量の変化は、初期の骨量に対して正規化した。外側の骨量は、実質的に偽感染マウス（ 図2B）16と比較して、感染したマウスでは増加した。遠位大腿骨外側骨量の増加は、μCTイメージングと組織学的解析16を用いて観察した関節組織および骨の感染によって引き起こされる骨の損傷、と思われた。 <img alt="図1" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig1highres.jpg" width="500" /> 生体内で図1の2D </strオング&gt; 生物発光と蛍光シグナル。S. 黄色ブドウ Xen29または細菌なし（非感染）は、Kワイヤの配置およびLysEGFPマウスをIVISスペクトルイメージングシステム16を使用して画像化した後膝関節に接種した。（A）平均インビボでの生物発光シグナルを全フラックス（光子/秒）によって測定される±SEM。（B）の平均in vivoでの EGFP蛍光シグナルを全放射効率（光子/秒）によって測定されるように黒白写真の上に重ねて、生体内生物発光と蛍光シグナルにおける SEM（C）代表±/（μW/ cm 2）をマウスのイメージ。 インビボでの生物発光イメージングに用いて細菌負荷の検出限界は1×10 2〜1×10 3 CFUの間である。 * P &lt;0.05、†のP &lt;0.01、‡P &lt;偽感染マウス対0.001 Xen29感染マウス（スチューデントのt検定[両側]）。どうぞこれはXen29を使用して生成し、IVISルミナXRイメージングシステム16で画像化以前に公表されたデータが含まれている代表的な図であることに注意。 <a href="https://www.jove.com/files/ftp_upload/51612/51612fig1highres.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" fo:content-width="5in" src="/files/ftp_upload/51612/51612fig2highres.jpg" width="500" /> 図2 3DμCTイメージング。 黄色ブドウ球菌 Xen29または細菌なし（非感染）Kワイヤ配置とマウスを量子FX のin vivoμCTシステムを使用して画像化した後に膝関節に接種した。Xen29の（A）代表3DμCTレンダリング感染したマウス（上のパネル）および偽感染マウス（下のパネル）。初期TIに正規化され、外側の骨体積変化（大腿骨の遠位25％）の（B）の割合私のポイント（平均±SEM）。 * P &lt;0.05、†のP &lt;0.01、‡P &lt;偽感染マウス対0.001 Xen29感染マウス（スチューデントのt検定[両側]）。これは生物発光菌株Sを使用して生成され、以前に公開されたデータが含まれる代表的な図であることに注意してください量子FX のin vivoμCTイメージングシステム16で画像化球菌 Xen29とは<a href="https://www.jove.com/files/ftp_upload/51612/51612fig2highres.jpg" target="_blank">、この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <a href="https://www.jove.com/files/ftp_upload/51612/Movie_1.mpg" target="_blank">ムービー1</a> 。μCT画像と組み合わせたXen29の生物発光信号およびEGFP-好中球の蛍光シグナルの代表的な3D解剖共同登録。画像は、縦軸に回転させる。

Watch this Scientific Journal Video about Combined In vivo Optical and ÂµCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice at JoVE.com

Combined In vivo Optical and  &amp;#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

黄色ブドウ球菌の生物発光操作された株を用いて、整形外科用インプラント感染のマウスモデルにおいて、光学およびμCTイメージングを組み合わせて、非侵襲的にかつ長手方向における細菌感染の動態、ならびに対応する炎症反応および解剖学的変化をモニターする能力を提供骨。

合わせた<em&gt;インビボ</em&gt;光とμCTイメージングマウスにおける整形外科インプラント感染で感染症、炎症、および骨解剖学を監視する

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

combined-vivo-optical-ct-imaging-to-monitor-infection-inflammation

Research

JoVE Journal

Medicine

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

16.9K ビュー.  David Geffen School of Medicine at University of California, Los Angeles (UCLA).  黄色ブドウ球菌の生物発光操作された株を用いて、整形外科用インプラント感染のマウスモデルにおいて、光学およびμCTイメージングを組み合わせて、非侵襲的にかつ長手方向における細菌感染の動態、ならびに対応する炎症反応および解剖学的変化をモニターする能力を提供骨。 

ビデオ: Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

口腔扁平上皮癌の同所性マウスモデルにおける腫瘍細胞の浸潤の多光子イメージング

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

医学

Bronchoalveolar Lavage (BAL) for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove nonadherent cells and lung lining fluid from the mucosal surface. In research environments, BAL allows sampling of innate (lung macrophage), cellular (B- and T- cells), and humoral (immunoglobulin) responses within the lung.
BAL is internationally accepted for research purposes and since 1999 the technique has been performed in &#62; 1,000 subjects in the UK and Malawi by our group.
Our technique uses gentle hand-held suction of instilled fluid; this is designed to maximize BAL volume returned and apply minimum shear force on ciliated epithelia in order to preserve the structure and function of cells within the BAL fluid and to preserve viability to facilitate the growth of cells in ex vivo culture. The research technique therefore uses a larger volume instillate (typically in the order of 200 ml) and employs manual suction to reduce cell damage.
Patients are given local anesthetic, offered conscious sedation (midazolam), and tolerate the procedure well with minimal side effects. Verbal and written subject information improves tolerance and written informed consent is mandatory. Safety of the subject is paramount. Subjects are carefully selected using clear inclusion and exclusion criteria.
This protocol includes a description of the potential risks, and the steps taken to mitigate them, a list of contraindications, pre- and post-procedure checks, as well as precise bronchoscopy and laboratory techniques.

Bronchoalveolar Lavage BAL for Research; Obtaining Adequate Sample Yield

We describe a research technique for fiberoptic bronchoscopy and bronchoalveolar lavage using low pressure suction. The technique is used to harvest immune cells from the lung bronchoalveolar surfaces. Local anesthetic and mild conscious sedation (midazolam) is used. Subjects tolerate the procedure well and experience minimal side effects.

研究のための気管支肺胞洗浄（BAL）、適切なサンプル利回りの取得

We describe a research technique for fiberoptic bronchoscopy with bronchoalveolar lavage (BAL) using manual hand held suction in order to remove ...

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

実験的な人間の肺炎球菌キャリッジ

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

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

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

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

マウスにおけるタキサンの薬物動態学的および薬力学的分析のための頸動脈煎茶

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

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

磁気共鳴映像法を用いた実験的大脳マラリアモデルにおける浮腫発生と微小血管病変のin vivo追跡

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection may result in sustained inflammation, osteomyelitis, and eventual bone non-union. Establishment of a feasible, reproducible animal model is important to bone infection research and antibiotic treatment. As an in vivo model, the rabbit model is widely used in bone infection research. However, previous studies on rabbit bone infection models showed that the infection status was inconsistent, as the amount of bacteria was variable. This study presents an improved surgical method for inducing bone infection on a rabbit, by blocking the bacteria in the bone marrow. Then, multi-level evaluations can be carried out to verify the modelling method.
In general, debriding necrotic tissue and implantation of vancomycin-loaded calcium sulphate (VCS) are predominant in antibiotic treatment. Although calcium sulphate in VCS benefits osteocyte crawling and new bone growth, massive bone defects occur after debriding. Autogenous bone (AB) is an appealing strategy to overcome bone defects for the treatment of massive bone defects after debriding necrotic bone.
In this study, we used the tail bone as an autogenous bone implanted in the bone defect. Bone repair was measured using micro-computed-tomography (micro-CT) and histological analysis after animal sacrifice. As a result, in the VCS group, bone non-union was consistently obtained. In contrast, the bone defect areas in the VCS-AB group were decreased significantly. The present modeling method described a reproducible, feasible, stable method to prepare a bone infection model. The VCS-AB treatment resulted in lower bone non-union rates after antibiotic treatment. The improved bone infection model and the combination treatment of VCS and autogenous bone could be helpful in studying the underlying mechanisms in bone infection and bone regeneration pertinent to traumatology orthopedic applications.

This study presents an improved rabbit model infected with Staphylococcus aureus by blocking the same amount of bacteria in bone marrow. Vancomycin loaded calcium sulphate and autogenous bone are used for antibiotic and bone repair treatment. The protocol could be helpful for studying bone infection and regeneration.

カルシウム硫酸塩および改良されたウサギ骨感染症モデルにおける自家骨にバンコマイシンによる治療が読み込まれます

Bone infection results from bacterial invasion, which is extremely difficult to treat in clinical, orthopedic, and traumatic surgery. The bone infection ...

Rapid In Vivo Assessment of Adjuvant's Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. Therefore, adjuvants with both humoral and cellular immuno-stimulatory capabilities that are able to promote cytotoxic T lymphocytes (CTL) responses are urgently needed. Thus, faithful monitoring of adjuvant candidates that induce cross-priming and subsequently enhance CTL generation represents a crucial step in vaccine development. In here we present an application for a method that uses SIINFEKL-specific (OT-I) T cells to monitor the cross-presentation of the model antigen ovalbumin (OVA) in vivo in the presence of different adjuvant candidates. This method represents a rapid test to select adjuvants with the best cross-priming capabilities. The proliferation of CD8+ T cells is the most valuable indication of cross-priming and it is also regarded as a correlate of adjuvant-induced cross-presentation. This feature can be evaluated in different immune organs like lymph nodes and spleen. The extent of the CTL generation can also be monitored, thereby giving insights on the nature of a local (draining lymph node mainly) or a systemic response (distant lymph nodes and/or spleen). This technique further allows multiple modifications for testing drugs that can inhibit specific cross-presentation pathways and also offers the possibility to be used in different strains of conventional and genetically modified mice. In summary, the application that we present here will be useful for vaccine laboratories in industry or academia that develop or modify chemical adjuvants for vaccine research and development.

Rapid In Vivo Assessment of Adjuvant&#039;s Cytotoxic T Lymphocytes Generation Capabilities for Vaccine Development

We present here an application for a standard immunological technique (CFSE stained OT-I proliferation) intended to rapidly monitor adjuvant-mediated cytotoxic T lymphocyte (CTL) generation in vivo. This fast estimation of CTL capacities is useful for the development of prophylactic vaccines against intracellular pathogens as well as therapeutic cancer vaccines.

急速なIn Vivo評価補助の細胞傷害性 T リンパ球生成機能のワクチン開発のため

The assessment of modern sub-unit vaccines reveals that the generation of neutralizing antibodies is important but not sufficient for adjuvant selection. ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

マウスで閉じた大腿骨骨折の生成: 骨の治癒を研究するためのモデル

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

High-Throughput Analysis of Optical Mapping Data Using ElectroMap

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we present, in a step-by-step guide, the use of ElectroMap for analysis, quantification, and mapping of high-resolution voltage and calcium datasets acquired by optical mapping. ElectroMap analysis options cover a wide variety of key electrophysiological parameters, and the graphical user interface allows straightforward modification of pre-processing and parameter definitions, making ElectroMap applicable to a wide range of experimental models. We show how built-in pacing frequency detection and signal segmentation allows high-throughput analysis of entire experimental recordings, acute responses, and single beat-to-beat variability. Additionally, ElectroMap incorporates automated multi-beat averaging to improve signal quality of noisy datasets, and here we demonstrate how this feature can help elucidate electrophysiological changes that might otherwise go undetected when using single beat analysis. Custom modules are included within the software for detailed investigation of conduction, single file analysis, and alternans, as demonstrated here. This software platform can be used to enable and accelerate the processing, analysis, and mapping of complex cardiac electrophysiology.

This protocol describes the setup and use of ElectroMap, a MATLAB-based open-source software platform for analysis of cardiac optical mapping data. ElectroMap provides a versatile high-throughput tool for analysis of optical mapping voltage and calcium datasets across a wide range of cardiac experimental models.

エレクトロマップを用いた光マッピングデータのハイスループット解析

Optical mapping is an established technique for high spatio-temporal resolution study of cardiac electrophysiology in multi-cellular preparations. Here we ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They mature in the bone marrow and are released into the peripheral blood, where they circulate for about 6&#8722;8 h; however, in tissue, they can survive for days. By diapedesis through the endothelium, they leave the blood stream, enter tissues, and migrate towards the site of an infection following chemotactic gradients.&#160;Neutrophils can combat invading microorganisms by phagocytosis,&#160;degranulation, and generation of neutrophil extracellular traps (NETs). This protocol will help to detect NETs in paraffin-embedded tissue. NETs are the result of a process called NETosis, which leads to the release of nuclear, granular, and cytoplasmic components either from living (vital NETosis) or dying (suicidal NETosis) neutrophils. In vitro, NETs form cloud-like structures, which occupy a space several times larger than that of the cells from which they descended. The backbone of NETs is chromatin, to which a selection of proteins and peptides originating from granules and cytoplasm are bound. Thereby, a high local concentration of toxic compounds is maintained so that NETs can capture and inactivate a variety of pathogens including bacteria, fungi, viruses, and parasites, while diffusion of the highly active NET components leading to damage in neighboring tissue is limited. Nevertheless, in recent years it has become apparent that NETs, if generated in abundance or cleared insufficiently, do have pathological potential ranging from autoimmune diseases to cancer. Thus, detection of NETs in tissue samples may have diagnostic significance, and the detection of NETs in diseased tissue can influence the treatment of patients. Since paraffin-embedded tissue samples are the standard specimen used for pathological analysis, it was sought to establish a protocol for fluorescent staining of NET components in paraffin-embedded tissue using commercially available antibodies.

Neutrophil extracellular traps (NETs) are three-dimensional structures generated by stimulated neutrophil granulocytes. It has become clear in recent years that NETs are involved in a wide variety of diseases. Detection of NETs in tissue may have diagnostic relevance, so standardized protocols for labelling NET components are required.

パラフィン埋め込み組織におけるヒトおよびマウス好中球外細胞トラップの免疫蛍光標識

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...