この出版物で報告された研究は、国立歯科研究所と顔面研究 (NIDCR) ・賞数 R56 DE025098、UG3 DE027695、F30 CA206296 の下で健康の国民の協会の国立癌研究所 (NCI) によって支えられました。内容は著者の責任と国立衛生研究所の公式見解を必ずしも表さない。この作品も、NSF DMR 1206219 と口腔ケア賞 (2016), 技術革新によって支えられました。
IVIS 実験を行うことで彼女の援助のジェーン ・ Gavrity に感謝したいと思います。我々 は彼女の入力と EM を実行する支援のためカレン ベントレーを感謝したいです。IHC と彼の援助の Pei Lun 翁に感謝したいと思います。我々 は図の準備の彼の援助のマシュー ・ インガルスを感謝したいです。この原稿の批判的読解の博士 Elaine Smolock とエミリー呉に感謝したいと思います。

すべては、生体内での下記手続は、ロチェスターの大学、ロチェスター、ニューヨーク動物資源の大学委員会によって承認されました。
1. 準備
<ol><li>ワイヤー インセット 32 G 頭蓋内カテーテル チューブを使用して、斜めの端、長い軸に約 45 ° を形成するチューブの 3 cm をカットします。ワイヤーは少なくとも 1 cm チューブよりも長いことを確認します。</li>
	<li>ハミルトンの注射器に 50 μ L PSMA ナノ粒子溶液 (図 1) のまたは他の注入材料を読み込みます。Barotrauma の確率を減らすため射出時に、カテーテル チューブを付削除、注射器にスタイレットとデッド ボリュームを追放します。</li>
	<li>ナノ粒子が完全に投与閉塞を防ぐために溶媒和ように注射液を検査します。</li>
	<li>アトロピン 0.1 mg/mL のソリューションを準備します。 注: アトロピン光に敏感、時間の経過とともに低下するため、このソリューション必要があります予防接種、させられる投与までの光から保護します。</li>
</ol>2. アクセスして管のエントリ ポイントを可視化
<ol><li>分析用天秤を用いた C57/BL6 マウスの重量を量る。</li>
	<li>29 x ½"針で 0.5 mL シリンジを使用して、100 mg/kg 10 mg/kg とケタミン ・ キシラジンの腹腔内注入滅菌生理食塩液をマウスを麻酔します。マウスはもはや刺激に反応、一般的に、投与 5 〜 10 分以内に発生するときは、次の手順に進みます。 注: この手順はイソフルランの下で行なうことも可能ですが、口腔内へのアクセスを許可するカスタム ノーズコーンが必要になります。</li>
	<li>プロシージャの間に乾燥を防ぐため、目に潤滑剤を適用し、カスタム ステージ上腹臥位でマウスを配置します。 注: 口腔内のプロシージャの適切な条件を維持するためにツールする必要がありますされる消毒または滅菌を使う前に。</li>
	<li>金属線に上顎前歯を保護することによって口腔内を開き、弾性バンドを使用して (図 2 a) の下顎前歯部の後ろ下方の緊張を適用します。</li>
	<li>顎の基部が視覚化されるように解剖顕微鏡の下にマウスを合わせます。</li>
	<li>口を広げるには、両側頬粘膜に張力を適用するのにカスタム、湾曲した鋼リトラクターを使用します。</li>
	<li>顎下腺の乳頭を視覚化するには、把握し、鈍い鉗子を使用して口の床から舌をそっと持ち上げます。 注: 乳頭舌 (図 2 b) の下にある 2 つの薄い突起として表示されます。</li>
	<li>口腔内で可視化しをさらに操作を容易にするためには、舌と頬の粘膜の間の綿を配置します。</li>
</ol>3. 管挿管および線の配置
<ol><li>カーブタイプ鉗子、罰金を使用して、ワイヤーの挿入カテーテル チューブを把握します。穿刺時に最適な手動制御、鉗子 (図 3 a) の曲率を持つチューブを合わせます。</li>
	<li>解剖顕微鏡を使用して、ビューのフィールドに鉗子とワイヤーを移動します。 注: ワイヤーはチューブから突出する必要があります。</li>
	<li>小さく、表面、粘膜パンク (0.076 mm 径) カテーテル チューブ (直径 0.25 mm) の後のエントリを容易にするを生成する嵌め込む式ワイヤーを使用して 1 つの顎下腺乳頭のベースに軽く押さえます。抵抗が発生した場合は、鋭い解剖はさみでチューブとワイヤ埋め込みの両方で新鮮な斜めのヒントをカットします。</li>
	<li>次のエントリ、スタイレットを撤回し、解剖顕微鏡を使用して、穿刺部位にある唾液の存在を確認します。スタイレットの出血を引き起こす可能性がありますまたは管の整合性が損なわれることの説得力がある、または突然の動き (撤退または挿入) を避けてください。</li>
	<li>チューブ (図 3 b) にスタイレットを撤回します。</li>
	<li>チュービングは、ウォートンのダクトの開口部に適合するには、以前作った穿刺 (図 2 C) に剛体ガイドとして、スタイレットを含むチューブを挿入します。 注: すぐに実行されない場合、ローカルの腫れが再挿入をできない場合があります。</li>
	<li>長期にわたる閉塞からの背圧を防ぐためには、チューブを撤回します。顎下腺の乳頭の開口部、顕微鏡では、表示を見ることができることを確認するを確認します。目に見える出血が発生した場合、スタイレットを削除し、ステップ 3.2 反対の顎下腺乳頭からやり直します。</li>
	<li>マウスを動かさずに 1 mg/kg のアトロピン ソリューション、プロシージャの間に唾液の分泌を減らすための腹腔内注入を管理します。5-10 分を待ちます。</li>
	<li>注射器のチューブの端をつかみ、オリフィスの内径 (図 3) を使用して挿入します。抵抗が発生した場合は、チューブと再試行に新鮮な角度付き端部をカットします。</li>
	<li>管は顎下腺乳頭内の箇所で、一度ゆっくりとダクトに 3-5 mm を進めます。鉗子からチューブを離します。</li>
	<li>チューブと顎下腺の乳頭間のシールを高めるためには、軽く 1 分のガーゼでしみが付くことによってインターフェイスを乾燥します。</li>
	<li>チューブの位置が乾燥中にシフトしているいないことを確認するを確認します。</li>
</ol>4. 注入
<ol><li>10 μ L/分マウス鎮静のまま苦痛 (図 2 D) の兆候が表示されないことを確認する検査の速度で材料を注入します。 注: 15 50 μ L の注射が忍容性良好。大量の注入は、barotrauma で起因できます。</li>
	<li>次の注射、ウォートンのダクトと SMG (図 4) 内にある材料の保持を改善するために 5 分のための注射器圧力を維持します。その管が管のオリフィスを終了しないように定期的に顎下腺の乳頭を検査します。</li>
	<li>微細鉗子を使用して、つかみ、軽く顎下腺の乳頭からチューブを撤回します。 メモ: 乳頭からいくつかの流体の出口を観察するは正常です。</li>
	<li>リトラクターと綿を口腔内からステージからマウスを移動する前に削除します。 注: 動物残さないように無人までそれは胸骨の横臥を維持するために十分な意識を取り戻しました。さらに、完全に回復するまで他のマウスとマウスが収納されていませんことを確認します。</li>
</ol>5. 検証・解析
注: (として示されている 1 h、図 5に注射後 24 h) 注入蛍光ナノ粒子の保持を評価するため生体内イメージング システム (IVIS) を使用できます。
<ol><li>皮膚を通して SMG 内蛍光信号を見やすく、剃毛か化学脱毛を使用して SMGs を覆う腹の毛皮を削除します。 注: 次の安楽死、SMG 組織も収穫することができます、修正 (4% パラホルムアルデヒド) で一晩、および注入 (図 6) 1 日の蛍光に分類された NP の永続性を確認するため免疫組織化学を用いたステンド グラスします。</li>
</ol>

<ol>
	<li>Miranda-Rius, J., Brunet-Llobet, L., Lahor-Soler, E., Farre, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salivary+Secretory+Disorders,+Inducing+Drugs,+and+Clinical+Management.">Salivary Secretory Disorders, Inducing Drugs, and Clinical Management.</a> International Journal Of Medical Sciences. 12 (10), 811-824 (2015).</li><li>Acauan, M. D., Figueiredo, M. A. Z., Cherubini, K., Gomes, A. P. N., Salum, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy-induced+salivary+dysfunction:+Structural+changes,+pathogenetic+mechanisms+and+therapies.">Radiotherapy-induced salivary dysfunction: Structural changes, pathogenetic mechanisms and therapies.</a> Archives of Oral Biology. 60 (12), 1802-1810 (2015).</li><li>Dirix, P., Nuyts, S., Vander Poorten, V., Delaere, P., Van den Bogaert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+xerostomia+after+radiotherapy+on+quality+of+life.">The influence of xerostomia after radiotherapy on quality of life.</a> Supportive Care in Cancer. 16 (2), 171-179 (2008).</li><li>Vissink, 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=Clinical+management+of+salivary+gland+hypofunction+and+xerostomia+in+head-and-neck+cancer+patients:+successes+and+barriers.">Clinical management of salivary gland hypofunction and xerostomia in head-and-neck cancer patients: successes and barriers.</a> International Journal of Radiation Oncology Biology Physics. 78 (4), 983-991 (2010).</li><li>Delporte, C., 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=Increased+fluid+secretion+after+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+to+irradiated+rat+salivary+glands.">Increased fluid secretion after adenoviral-mediated transfer of the aquaporin-1 cDNA to irradiated rat salivary glands.</a> Proceedings of the National Academy of Sciences. 94 (7), 3268-3273 (1997).</li><li>Samuni, Y., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+in+salivary+glands:+from+the+bench+to+the+clinic.">Gene delivery in salivary glands: from the bench to the clinic.</a> Biochimica et Biophysica Acta. 1812 (11), 1515-1521 (2011).</li><li>Beahm, D. D., 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=Surgical+approaches+to+the+submandibular+gland:+A+review+of+literature.">Surgical approaches to the submandibular gland: A review of literature.</a> International Journal of Surgery. 7 (6), 503-509 (2009).</li><li>Zheng, C., Shinomiya, T., Goldsmith, C. M., Di Pasquale, G., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convenient+and+reproducible+in+vivo+gene+transfer+to+mouse+parotid+glands.">Convenient and reproducible in vivo gene transfer to mouse parotid glands.</a> Oral diseases. 17 (1), 77-82 (2011).</li><li>Zheng, C., 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=Prevention+of+Radiation-Induced+Salivary+Hypofunction+Following+hKGF+Gene+Delivery+to+Murine+Submandibular+Glands.">Prevention of Radiation-Induced Salivary Hypofunction Following hKGF Gene Delivery to Murine Submandibular Glands.</a> Clinical Cancer Research. 17 (9), 2842-2851 (2011).</li><li>Okazaki, 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=Acceleration+of+rat+salivary+gland+tissue+repair+by+basic+fibroblast+growth+factor.">Acceleration of rat salivary gland tissue repair by basic fibroblast growth factor.</a> Archives of Oral Biology. 45 (10), 911-919 (2000).</li><li>Arany, S., Benoit, D. S., Dewhurst, S., Ovitt, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-mediated+gene+silencing+confers+radioprotection+to+salivary+glands+in+vivo.">Nanoparticle-mediated gene silencing confers radioprotection to salivary glands in vivo.</a> Molecular Therapy. 21 (6), 1182-1194 (2013).</li><li>Cotrim, A. P., Sowers, A., Mitchell, J. B., Baum, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+irradiation-induced+salivary+hypofunction+by+microvessel+protection+in+mouse+salivary+glands.">Prevention of irradiation-induced salivary hypofunction by microvessel protection in mouse salivary glands.</a> Molecular Therapy. 15 (12), 2101-2106 (2007).</li><li>Redman, R. S., Ball, W. D., Mezey, E., Key, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispersed+donor+salivary+gland+cells+are+widely+distributed+in+the+recipient+gland+when+infused+up+the+ductal+tree.">Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree.</a> Biotechnic &amp; Histochemistry. 84 (6), 253-260 (2009).</li><li>Grundmann, O., Fillinger, J. L., Victory, K. R., Burd, R., Limesand, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+radiation+therapy-induced+salivary+gland+dysfunction+in+mice+by+post+therapy+IGF-1+administration.">Restoration of radiation therapy-induced salivary gland dysfunction in mice by post therapy IGF-1 administration.</a> BMC Cancer. 10, 417-417 (2010).</li><li>Limesand, K. 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=Insulin-Like+Growth+Factor-1+Preserves+Salivary+Gland+Function+After+Fractionated+Radiation.">Insulin-Like Growth Factor-1 Preserves Salivary Gland Function After Fractionated Radiation.</a> International Journal of Radiation Oncology Biology Physics. 78 (2), 579-586 (2010).</li><li>Marmary, 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=Radiation-induced+loss+of+salivary+gland+function+is+driven+by+cellular+senescence+and+prevented+by+IL-6+modulation.">Radiation-induced loss of salivary gland function is driven by cellular senescence and prevented by IL-6 modulation.</a> Cancer Research. , (2016).</li><li>Baum, B. 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=Early+responses+to+adenoviral-mediated+transfer+of+the+aquaporin-1+cDNA+for+radiation-induced+salivary+hypofunction.">Early responses to adenoviral-mediated transfer of the aquaporin-1 cDNA for radiation-induced salivary hypofunction.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (47), 19403-19407 (2012).</li><li>Arany, 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=Pro-apoptotic+gene+knockdown+mediated+by+nanocomplexed+siRNA+reduces+radiation+damage+in+primary+salivary+gland+cultures.">Pro-apoptotic gene knockdown mediated by nanocomplexed siRNA reduces radiation damage in primary salivary gland cultures.</a> Journal of Cellular Biochemistry. 113 (6), 1955-1965 (2012).</li><li>Benoit, D. S. W., Henry, S. M., Shubin, A. D., Hoffman, A. S., Stayton, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-responsive+polymeric+siRNA+carriers+sensitize+multidrug+resistant+ovarian+cancer+cells+to+doxorubicin+via+knockdown+of+polo-like+kinase+1.">pH-responsive polymeric siRNA carriers sensitize multidrug resistant ovarian cancer cells to doxorubicin via knockdown of polo-like kinase 1.</a> Molecular pharmaceutics. 7 (2), 442-455 (2010).</li><li>Malcolm, D. W., Varghese, J. J., Sorrells, J. E., Ovitt, C. E., Benoit, D. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Effects+of+Biological+Fluids+on+Colloidal+Stability+and+siRNA+Delivery+of+a+pH-Responsive+Micellar+Nanoparticle+Delivery+System.">The Effects of Biological Fluids on Colloidal Stability and siRNA Delivery of a pH-Responsive Micellar Nanoparticle Delivery System.</a> ACS Nano. , (2017).</li><li>Baranello, M. P., Bauer, L., Benoit, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(styrene-alt-maleic+anhydride)-based+diblock+copolymer+micelles+exhibit+versatile+hydrophobic+drug+loading,+drug-dependent+release,+and+internalization+by+multidrug+resistant+ovarian+cancer+cells.">Poly(styrene-alt-maleic anhydride)-based diblock copolymer micelles exhibit versatile hydrophobic drug loading, drug-dependent release, and internalization by multidrug resistant ovarian cancer cells.</a> Biomacromolecules. 15 (7), 2629-2641 (2014).</li><li>Wang, 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=Fracture-Targeted+Delivery+of+&#946;-Catenin+Agonists+via+Peptide-Functionalized+Nanoparticles+Augments+Fracture+Healing.">Fracture-Targeted Delivery of &#946;-Catenin Agonists via Peptide-Functionalized Nanoparticles Augments Fracture Healing.</a> ACS Nano. 11 (9), 9445-9458 (2017).</li><li>Baranello, M. P., Bauer, L., Jordan, C. T., Benoit, D. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micelle+Delivery+of+Parthenolide+to+Acute+Myeloid+Leukemia+Cells.">Micelle Delivery of Parthenolide to Acute Myeloid Leukemia Cells.</a> Cellular and Molecular Bioengineering. 8 (3), 455-470 (2015).</li><li>Kuriki, 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=Cannulation+of+the+Mouse+Submandibular+Salivary+Gland+via+the+Wharton's+Duct.">Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct.</a> Journal of Visualized Experiments. (51), e3074 (2011).</li><li>Nair, R. P., Zheng, C., Sunavala-Dossabhoy, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroductal+Submandibular+Gland+Instillation+and+Localized+Fractionated+Irradiation+in+a+Rat+Model+of+Salivary+Hypofunction.">Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction.</a> Journal of Visualized Experiments. (110), (2016).</li><li>Wang, Y., Malcolm, D. W., Benoit, D. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+and+sustained+delivery+of+siRNA/NPs+from+hydrogels+expedites+bone+fracture+healing.">Controlled and sustained delivery of siRNA/NPs from hydrogels expedites bone fracture healing.</a> Biomaterials. 139 (Supplement C), 127-138 (2017).</li><li>Hoffman, M. D., Van Hove, A. H., Benoit, D. S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradable+hydrogels+for+spatiotemporal+control+of+mesenchymal+stem+cells+localized+at+decellularized+bone+allografts.">Degradable hydrogels for spatiotemporal control of mesenchymal stem cells localized at decellularized bone allografts.</a> Acta Biomaterialia. 10 (8), 3431-3441 (2014).</li><li>Nguyen, C. Q., Yin, H., Lee, B. H., Chiorini, J. A., Peck, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL17:+potential+therapeutic+target+in+Sjogren's+syndrome+using+adenovirus-mediated+gene+transfer.">IL17: potential therapeutic target in Sjogren's syndrome using adenovirus-mediated gene transfer.</a> Laboratory Investigation. 91 (1), 54-62 (2011).</li></ol>

著者が明らかに何もありません。

Retroductal 注入は、唾液腺にローカライズされた薬物送達のため重要です。このテクニックには、シェーグレン症候群と・ リフの9,10,28を含む条件のための治療薬のスクリーニングでアプリケーションがあります。Retroductal 注入を介して SMG に直接薬剤投与は、免疫活性化11を含むオフターゲット効果を減ら...

唾液腺機能不全、シェーグレン症候群、機能的な分泌組織と照射誘起唾液その他)、頭頸部癌放射線治療1の一般的な sequella の自己免疫を介した損失を含む多くの病因。いずれかの条件のための唾液の機能の損失口腔と全身感染、虫歯、消化、嚥下機能障害、音声障害、大鬱病1,2,3個人の素因となります。その結果、生活の質が大幅低下、介入治療4ではなく、症状の緩和に限定とします。生体内での新規治療法を調査、生物活性化合物の唾液腺に直接管理に興味のです。
Retroductal 注入は、唾液腺に直接生理活性化合物を提供し、病気、傷害、または正常組織の恒常性の下での有効性をテストする貴重な方法です。3 つの大唾液腺は耳下腺 (PG)、顎下腺 (SMG)、舌下腺 (SLG) 導管を通じて口腔内には、空のすべて。痙攣の解剖学 SMG 舌5の下に口の階にウォートンのダクトのカニュレーションを介して直接アクセスを許可します。次の穿刺、溶媒和の薬は、SMG に直接管理できます。次の retroductal 配信外腺の拡散は周囲の構造6の材料の交換を調整する周囲組織のカプセルによって制限されます。SMG とそのダクト同様に、人間の構造は、SMG 手術と sialoendoscopy7の中に日常的にアクセスされます。人間、マウス、PG は頬粘膜8Stensen のダクトを介してアクセス可能な同様に。
・ リフのマウスモデルで SMG retroductal 注入は、成長因子、一次電池、アデノ、サイトカイン、および損傷への細胞応答を調整すると、結果を減らす抗酸化化合物を含む治療を提供に使用されています組織損傷5,9,10、11,12,13,14,15,16。Retroductal 注射の最も注目すべき臨床成功は直接水チャネル (アクアポリン 1 の発現するアデノウイルスベクターの管理AQP1) 患者の頭頸部の癌のための17の放射線に続きます。
以前は、我々 を開発し、・ リフ11,18,19,20から唾液腺の機能を保護するために retroductally を注入した高分子ナノ粒子 siRNA システムの有効性を示します。私たちの過去の作品の拡張として、示す retroductal SMG 注射、蛍光ナノ粒子を用いた (NP) の読み込みと難溶性の提供以外のことができる当社のプロトコル薬21,22,23。
ポリ (スチレン alt マレイン酸 anhydride)-b-poly(styrene) (PSMA) 可逆加算鎖断片化 (いかだ) 重合、21を前述のようから成るジブロック共重合体から NP を合成しました。溶媒交換を通じてこれらのポリマー自発的に自己組み立てる疎水性インテリアと親水性外装21NP ミセル構造に。NPs は、動物を犠牲にせず腺に NP 配信の検証を許可するテキサス赤蛍光体が付いています。動物の画像をライブ、1 h で次の注入 1 日 SMG 免疫組織化学を示します。
これが更新され、再現可能なカニュレーション プロトコル必要があります retroductal 注入を達成するために他人を有効にします。この洗練されたテクニックが重要な生体内での研究、治療開発24,25になることを見込んでいます。

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			<td>Syringe for intraperitoneal injection</td>
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retroductal nanoparticle injection to the murine submandibular gland

唾液腺治療の 2 つの共通の目標は、予防と次のいずれかの組織の機能不全の治療自己免疫または放射線障害。ローカル唾液腺に生物活性化合物を提供すること、によって、全身投与と組織濃度を安全に実現できます。さらに、標的組織を材料の外腺に蓄積することによる効果は大幅に削減することができます。この点で、retroductal 注入、唾液腺の生物学・病態生理学を調査するため広く使用されている方法です。成長因子、一次電池、アデノ ウイルスのベクトル、小分子薬の Retroductal 管理は、傷害の設定で腺機能をサポートする示されています。私たちは以前照射腺の機能を維持するために注入した retroductally ナノ粒子 siRNA 戦略の有効性を示しています。ウォートンのダクトをマウスの顎下腺にナノ材料を管理するための非常に効果的かつ再現性のある方法の詳細ここでは、(図 1)。口腔内にアクセスしてプロシージャ全体で品質チェックとしてより詳細な観察と cannulate のウォートンのダクトに必要な手順の概要を説明します。

Retroductal 注射は、マウス SMG (図 1) に、NPs を管理に使用できます。ここでは、50 μ g PSMA NPs テキサス赤蛍光ラベルの付いたお届け。
マウスの適切な配置により、安易なアクセスなど (図 2 aB) 口の中の床の可視化。顎下腺の乳頭は舌の下に 2 つの肉質の突起として識?...

Watch this Scientific Journal Video about Retroductal Nanoparticle Injection to the Murine Submandibular Gland at JoVE.com

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

顎下腺への薬剤の局所投与は、唾液腺の生物学を理解に、新規治療法の開発に興味のです。配信精度と実験の再現性を向上させるため、更新と詳細な retroductal 注入プロトコルを提案します。ここに提示されたアプリケーションは、高分子ナノ粒子の配信です。

マウス顎下腺への Retroductal ナノ粒子注入

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

retroductal-nanoparticle-injection-to-the-murine-submandibular-gland

Research

JoVE Journal

Bioengineering

9.9K ビュー.  University of Rochester. 顎下腺への薬剤の局所投与は、唾液腺の生物学を理解に、新規治療法の開発に興味のです。配信精度と実験の再現性を向上させるため、更新と詳細な retroductal 注入プロトコルを提案します。ここに提示されたアプリケーションは、高分子ナノ粒子の配信です。

ビデオ: Retroductal Nanoparticle Injection to the Murine Submandibular Gland

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 ...

生物工学

Synthesis, Assembly, and Characterization of Monolayer Protected Gold Nanoparticle Films for Protein Monolayer Electrochemistry

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently incorporated into film assemblies that serve as adsorption platforms for protein monolayer electrochemistry (PME). PME is utilized as the model system for studying electrochemical properties of redox proteins by confining them to an adsorption platform at a modified electrode, which also serves as a redox partner for electron transfer (ET) reactions. Studies have shown that gold nanoparticle film assemblies of this nature provide for a more homogeneous protein adsorption environment and promote ET without distance dependence compared to the more traditional systems modified with alkanethiol self-assembled monolayers (SAM).1-3 In this paper, MPCs functionalized with hexanethiolate ligands are synthesized using a modified Brust reaction4 and characterized with ultraviolet visible (UV-Vis) spectroscopy, transmission electron microscopy (TEM), and proton (1H) nuclear magnetic resonance (NMR). MPC films are assembled on SAM modified gold electrode interfaces by using a "dip cycle" method of alternating MPC layers and dithiol linking molecules. Film growth at gold electrode is tracked electrochemically by measuring changes to the double layer charging current of the system. Analogous films assembled on silane modified glass slides allow for optical monitoring of film growth and cross-sectional TEM analysis provides an estimated film thickness. During film assembly, manipulation of the MPC ligand protection as well as the interparticle linkage mechanism allow for networked films, that are readily adaptable, to interface with redox protein having different adsorption mechanism. For example, Pseudomonas aeruginosa azurin (AZ) can be adsorbed hydrophobically to dithiol-linked films of hexanethiolate MPCs and cytochrome c (cyt c) can be immobilized electrostatically at a carboxylic acid modified MPC interfacial layer. In this report, we focus on the film protocol for the AZ system exclusively. Investigations involving the adsorption of proteins on MPC modified synthetic platforms could further the understanding of interactions between biomolecules and man-made materials, and consequently aid the development of biosensor schemes, ET modeling systems, and synthetic biocompatible materials.5-8

Alkanethiolate stabilized gold colloids known as monolayer protected clusters (MPCs) are synthesized, characterized, and assembled into thin films as an adsorption interface for protein monolayer electrochemistry of simple redox protein like Pseudomonas aeruginosa azurin (AZ) and cytochrome c (cyt c).

合成、組立、および単分子膜のキャラクタリゼーションは、タンパク質単分子層の電気化学のための金ナノ粒子膜を保護

Colloidal gold nanoparticles protected with alkanethiolate ligands called monolayer protected gold clusters (MPCs) are synthesized and subsequently ...

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

マウスモデルにおける腸のティッシュエンジニアリング

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

We describe a 3D culture model of the human breast epithelium that is suitable to study hormone action.

ヒト乳腺上皮のホルモン応答性の3D培養モデル

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

干渉のないマイクロ/ナノ粒子の細胞工学のためのマイクロ流体緩衝液交換

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Determination of Zeta Potential via Nanoparticle Translocation Velocities through a Tunable Nanopore: Using DNA-modified Particles as an Example

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and nanoparticles. Tunable resistive pulse sensing (TRPS) is a relatively recent adaptation to RPS that incorporates a tunable pore that can be altered in real time. Here, we use TRPS to monitor the translocation times of DNA-modified nanoparticles as they traverse the tunable pore membrane as a function of DNA concentration and structure (i.e., single-stranded to double-stranded DNA).
TRPS is based on two Ag/AgCl electrodes, separated by an elastomeric pore membrane that establishes a stable ionic current upon an applied electric field. Unlike various optical-based particle characterization technologies, TRPS can characterize individual particles amongst a sample population, allowing for multimodal samples to be analyzed with ease. Here, we demonstrate zeta potential measurements via particle translocation velocities of known standards and apply these to sample analyte translocation times, thus resulting in measuring the zeta potential of those analytes.
As well as acquiring mean zeta potential values, the samples are all measured using a particle-by-particle perspective exhibiting more information on a given sample through sample population distributions, for example. Of such, this method demonstrates potential within sensing applications for both medical and environmental fields.

Here we use a polyurethane tunable nanopore integrated into a resistive pulse sensing technique to characterize nanoparticles surface chemistry via the measurement of particle translocation velocities, which can be used to determine the zeta potential of individual nanoparticles.

調整可能なナノ細孔を介してナノ粒子の転ベロシティを経由してゼータ電位の決意：例として、DNA修飾された粒子を用いました

Nanopore technologies, known collectively as Resistive Pulse Sensors (RPS), are being used to detect, quantify and characterize proteins, molecules and ...

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

ウサギの乳腺への管内配達

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

キナーゼのアップコン バージョンのナノ粒子を介した光活性化によって細胞内シグナル伝達をリモートでトリガーする統合システム

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

Light-sheet Fluorescence Microscopy for the Study of the Murine Heart

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. Traditional light-sheet techniques involve the use of a single illumination beam directed orthogonally at sample tissue. Images of large samples that are produced using a single illumination beam contain stripes or artifacts and suffer from a reduced resolution due to the scattering and absorption of light by the tissue. This study uses a dual-sided illumination beam and a simplified CLARITY optical clearing technique for the murine heart. These techniques allow for deeper imaging by removing lipids from the heart and produce a large field of imaging, greater than 10 x 10 x 10 mm3. As a result, this strategy enables us to quantify the ventricular dimensions, track the cardiac lineage, and localize the spatial distribution of cardiac-specific proteins and ion-channels from the post-natal to adult mouse hearts with sufficient contrast and resolution.

This study uses a dual-sided illumination light-sheet fluorescence microscopy (LSFM) technique combined with optical clearing to study the murine heart.

マウス心臓の研究のための光シート蛍光顕微鏡

Light-sheet fluorescence microscopy has been widely used for rapid image acquisition with a high axial resolution from micrometer to millimeter scale. ...

Optical Imaging of Isolated Murine Ventricular Myocytes

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances in calcium sensitive dyes have permitted the robust optical recording of single cell calcium dynamics, recording of robust transmembrane optical voltage signals has remained difficult. Arguably, this is because of the low single to noise ratio, phototoxicity, and photobleaching of traditional potentiometric dyes. Therefore, single cell voltage measurements have long been confined to the patch clamp technique which while the gold standard, is technically demanding and low throughput. However, with the development of novel potentiometric dyes, large, fast optical responses to changes in voltage can be obtained with little to no phototoxicity and photobleaching. This protocol describes in detail how to isolate adult murine myocytes which can be used for cellular shortening, calcium, and optical voltage measurements. Specifically, the protocol describes how to use a ratiometric calcium dye, a single-excitation calcium dye, and a single excitation voltage dye. This approach can be used to assess the cardiotoxicity and arrhythmogenicity of various chemical agents. While phototoxicity is still an issue at the single cell level, methodology is discussed on how to reduce it.

We present the methodology for the isolation of murine myocytes and how to obtain voltage or calcium traces simultaneously with sarcomere shortening traces using fluorescence photometry with simultaneous digital cell geometry measurements.

単離マウス心室筋細胞の光学イメージング

The ability to isolate adult cardiac myocytes has permitted researchers to study a variety of cardiac pathologies at the single cell level. While advances ...