Name: murine salivary functional assessment via pilocarpine stimulation following fractionated radiation
Uploaded: 2018-05-04T14:30:00.000+00:00
Duration: 9 min 10 s
Description: Watch this Scientific Journal Video about Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation at JoVE.com

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。
唾コレクション援助の博士えり丸山とアンドリュー ホローモンに感謝したいと思います。腺解剖して彼の支援の 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、ロチェスター動物資源の大学委員会によって承認されました。NY。
1. 準備
<ol><li>分析用天秤を使用すると、ピロカルピンの 20 mg の重量を量る。微量遠心チューブの滅菌生理食塩水 2 mL に溶かします。 注: ピロカルピンが光に敏感で、時間をかけて活動を失うのでこのソリューション必要があります準備注入の日と投与までの光から保護されました。</li>
	<li>分析用天秤を使用すると、重量を量るし、すべてのコレクション チューブ、ガラス管を識別します。コレクション チューブ、ガラス管の数には、マウスの数を一致する必要があります。</li>
</ol>2. 気管切開
<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。マウスが応答する刺激 (すなわちペダル逃避反射の不在) に一般的に投与 5 〜 10 分以内に発生するときは、次の手順に進みます。</li>
	<li>綿の先端アプリケータに潤滑剤を塗布後軽く目に適用され、仰臥位でステージ上にマウスを置きます。麻酔下の動物は、暖かい表面に保たれなければなりません。 注: すべての手順は、室温で実行されます。</li>
	<li>鼻、手足、サージカル テープを使用して、ステージに尾を保護します。</li>
	<li>きれいにしアルコール ワイプで首を濡れています。 注: これは入力後切開から毛皮を防ぐため役立ちます。</li>
	<li>鉗子で腹側正中線の首の皮膚を引き上げ、解剖はさみを使用して小さい、表面的なカットをします。開口部にはさみをご案内し、ゆっくりと皮下組織面を分離するブレードを開きます。</li>
	<li>口の下に 1 cm 上げた肌に保つ、慎重にカットします。</li>
	<li>最初のカットの劣ると優れた側面を 2 つの横切開を確認します。鉗子を使用すると、頭と首の構造へのアクセスを有効にする皮膚反映まで優しく。</li>
	<li>顎下腺 8 倍の倍率で解剖顕微鏡を用いた可視化 (正中線:図 1)。</li>
	<li>微細鉗子を使用すると、気管を覆う 4 つの舌骨下 (ストラップ) 筋肉を公開する腺をそっと持ち上げます。涙や周囲の血管や導管を中断することを避けるため。</li>
	<li>1 つ以上のマウスから唾液を収集する続行する前に各マウスを使って手順 2.1 2.10 を繰り返します。 メモ: この手順が安全で実行できます 5 マウスまで同時に。</li>
	<li>小さな解剖はさみでできるだけ正中線に近い滞在中ストラップの筋肉の内側の部分を削除します。気管を視覚化し、プロシージャの間に方法のストラップの筋肉を保つために必要な量だけカットのみ。ピロカルピンが分泌を誘導する効果的ではない重要な出血にセカンダリ ボリュームの枯渇がある場合ために任意の近くの血管を傷つけないでください。</li>
	<li>気管からストラップの筋肉を反映してください。</li>
	<li>喉頭・気管・甲状腺を視覚化します。気管がない組織を覆うことを確認します。</li>
	<li>小さな解剖はさみを使って甲状腺に劣る/後部気管内横切開を加えます。気道が特許と流体の明確なことを確認します。</li>
</ol>3. 唾液コレクション
<ol><li>口と角解離ステージ下方近くテープを削除 (45 ° 頭側) 唾液の流れを支援します。</li>
	<li>29 x ½"針で 0.5 mL シリンジを使用して、100 mg/kg の総線量の 10 μ L/g 本体重量ピロカルピンの腹腔内投与を実行します。</li>
	<li>タイマーを起動します。複数のマウスを投与している場合、は、迅速に移動することによって、アシスタント残りのマウスを同時に注入することでリードタイムを短縮します。</li>
	<li>通常鉗子を使用すると、キャピラリー チューブのアクセスのための口を開きます。</li>
	<li>口の中で唾液のビードを観察すると、遠位端で、流体にキャピラリー チューブの近位端の場所が (両方のチューブが事前の重量を量った) 解離性の段階の下に収集の管に配置。 注: C57/BL6 雌ネズミの 6-10 週齢総唾液分泌に発生しますピロカルピン注射の 2 分以内。</li>
	<li>唾液は、口の中でビル管を通って流れていない場合、前の手順を再試行します。入口を妨げることができる粘膜表面に対して直接チューブが配置されていないことを確認します。</li>
	<li>ピロカルピン刺激 12 分の合計のため唾液を収集します。ストーマは明確に残ることを確認します。 注: この時間の間に、通常一回換気量の増加や分泌物 (唾液、尿、便) が。</li>
	<li>マウスは唾液の機能に踏み込んだ場合 (e.g。、ケガや介入のため)、P200 ピペットを使用してコレクションの管口から残りの流体に転送。複数のマウスから収集する場合は、ヒントを変更することを確認します。</li>
	<li>ピロカルピン注射後採取唾液プラス チューブ 12 分の重量を記録します。</li>
</ol>4. 腺解剖
<ol><li>CO2安楽死によってマウスを安楽死させる (動物の安楽死の AVMA のガイドラインに従って: 2013 年版) 開胸、頭と首の構造を維持するために世話が続きます。このため、安楽死の方法として頚部転位を避けます。</li>
	<li>仰臥位でステージ上にマウスを置きます。気管 (図 1) を元のサイトで腺の位置を変更します。</li>
	<li>8 倍の倍率で解剖顕微鏡を用いた視覚化大腺: PG、SMG、SLG。解剖中腺を区別するために、PG がびまん性、SMG と SLG に横で横になっていることを特定します。 注: PG を解剖する場合これされるべき最初だから少なくとも定義され引き裂かれるらしい。</li>
	<li>基になる構造から引き離し、鉗子で PG の尾をつかみ、優しく解剖はさみで PG の頭を切断する前に緊張を適用します。</li>
	<li>鉗子を用いた SMGs を覆う被膜を破る。左と右の SMG を優しきます。</li>
	<li>無料の鉗子を用いた付着 nonglandular 組織から SMG の背側面。</li>
	<li>鉗子でピンと張ったウォートンのダクトを描画し、解剖はさみとダクトを切断、首から SMG を削除します。</li>
	<li>SMG は鉗子を使用してから、SLG を優しきます。 注: SLG は、SMG の上外側面では。</li>
	<li>分析のバランスを使用して、SMG の重みを記録します。</li>
</ol>

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腺傷害や治療研究に適用可能な唾液腺機能を評価するためにマルチ ステップ法を提案します。私たちの手順には、気管、唾液のコレクション、および唾液腺の生物学の統合研究をサポートすることができます実験アプリケーションを持つ腺解剖が含まれます。たとえば、マウスの気管は、口腔内を妨害する手順の中で一般的な気道管理のため使用されています。
ピロカルピンの適切な郭清と気管切開が必要で 100 mg/kg の唾液の分泌を刺激します。または、削減ピロカルピン投与気管切開の必要性を避けることができるし、唾液分泌16の縦断的評価のため。
唾液の分泌を記録に加えこのプロトコルは、追加の分析のため唾液を収集するために正確で一貫した手段を提供します。このような研究には、レオロジー、プロテオミクス、酵素活性、唾浸透圧25,26,27、およびバイオ マーカー探索28,29が含まれます。
最後に、標準郭清法大唾液腺唾液コレクションを超えてユーティリティがあります。腺の発達と傷害への応答を理解することに関心があります。効果的な調査の主要な唾液腺の一貫性のある、きれいな解剖を行うことが重要です。きれいにし、効率的な腺分離に由来する、機械的および酵素によって解離や大唾液腺30,31, からの一次電池文化の重要な組織学的および免疫組織化学準備。また、腺組織切片における分泌タンパク質の染色しているとき、は、考慮し、これらの製品の可能性が枯渇のため制御が重要です刺激唾液採取します。
けがや介入の調査で唾液腺機能の定量化は重要な実験手法です。腺組織は有益でありの重要な観測を引き出す可能性がありますまだ結論が明確な機能データでサポートされる最高。

唾液腺の障害には、調節不全の過剰産生 (sialorrhea) や唾1の生産不足 (口腔乾燥症と唾液) の分泌障害症候群が含まれます。どちらの場合も、治療開発2の最終目標に向けて唾液腺の生物学の私達の理解の向上に関心があります。
唾液腺は放射線感受性非常器官、頭頸部がんの放射線治療、永久的なドライマウス (口腔乾燥症)3,4につながる間しばしば破損しています。他の放射線感受性の組織とは異なりただし、唾液腺の離職率は比較的低いと分泌の損失のメカニズムはかり5,6。このユニークなけが設定で組織再生と放射線防護戦略唾液の機能評価が必要です。実験的マウス唾液コレクションは腺に対する放射線と治療薬の評価に特に貴重なツールです。
実行して、ピロカルピン、強力なムスカリン作動薬7を使用して刺激唾液分泌の定量化の方法を紹介します。ピロカルピンは、腺分泌8,9を誘導するために自律神経を刺激します。このテストを適切に完了するには、気管切開は、マウスがプロシージャ中気道を維持するために口腔内10でプールされた分泌物から誤嚥・窒息のリスクを低減する必要があります。
これは、3 つの大唾液腺の除去で最高潮に達する、ターミナル手順: (PG) 耳下腺、顎下腺 (SMG)、舌下腺 (SLG)。機能性研究腺重量は記録され、唾液測定11,12,13を正規化する頻繁に使用されます。このデータは、前記腺萎縮が予想した結果14,15放射線研究で特に重要です。
どのように刺激唾液の分泌に関して文献の変動が実行され、16の報告があります。たとえば、ピロカルピン投与量文学スパン少なくとも 3 桁17,18,19,20,21,22,23内。今回、最適化された高用量ピロカルピン プロトコルとして合わせることができる技術 (気管、唾液のコレクション、および腺郭清) のモジュール式プラットフォームを提供することと同様に、メソッドの実行、再現性向上の目的で必要があります。
プロトコル デモに加えて、我々 は SMG 領域に分別された放射線 (6.85 Gy の 4 用量) に続く 2 週間で唾液の流れの代表的な機能のデータを含めます。

<table><tbody><tr><td>Pilocarpine hydrochloride</td><td>Sigma Aldrich</td><td>P6503</td><td>Pilocarpine</td></tr><tr><td>Student Vannas Spring Scissors</td><td>Fine Science Tools</td><td>91500-9</td><td>Spring Scissors for Tracheostomy</td></tr><tr><td>Sterile Saline Solution</td><td>Medline</td><td>RDI30296H</td><td>Saline</td></tr><tr><td>Dumont #7 Forceps</td><td>Fine Science Tools</td><td>11274-20</td><td>Curved Forceps</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11251-10</td><td>Straight Forceps</td></tr><tr><td>Standard Pattern Forceps</td><td>Fine Science Tools</td><td>11000-12</td><td>Blunt Forceps</td></tr><tr><td>Fine Scissors- Tungsten Carbide</td><td>Fine Science Tools</td><td>14568-09</td><td>Dissection Scissors</td></tr><tr><td>Microhematocrit Heparinized Capillary Tubes</td><td>Fisher Scientific</td><td>22362566</td><td>Capillary tubes</td></tr><tr><td>Lubricant Eye Ointment</td><td>Refresh</td><td>N/A</td><td>Refresh Lacri-Lube</td></tr><tr><td>Goat polyclonal anti-Nkcc1</td><td>Santa Cruz Biotech</td><td>SC-21545</td><td>Nkcc1 Antibody</td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td>DAPI</td></tr><tr><td>GraphPad Prism</td><td>GraphPad</td><td>ver6.0</td><td>Statistical Software</td></tr><tr><td>Cotton tipped applicator</td><td>Medline</td><td>MDS202000</td><td>Applicator for eye ointment</td></tr><tr><td>0.5cc Insulin Syringe, 29G x 1/2&quot;</td><td>BD</td><td>7629</td><td>Syringe for intraperitoneal injection</td></tr></tbody></table>

murine salivary functional assessment via pilocarpine stimulation following fractionated radiation

唾液は、シェーグレン症候群や大唾液腺に次の放射線障害の自己免疫の反作用でよく観察されます。これらの場合、病気の発症と効果的介入に関する質問のまま。唾液腺の機能評価ができる最適化されたテクニックは外分泌腺の生物学、機能不全、および治療の調査のため貴重です。ピロカルピンを実行するステップ バイ ステップ アプローチを提案するここでは、気管切開と 3 つの主要なマウス唾液腺の解剖など、唾液の分泌を刺激します。我々 はまた、これらのテクニックの中にアクセス適切なマウス頭頸部解剖の詳細します。このアプローチは、スケーラブルな仕事の流れの効率を上げる複数のマウスを同時に処理を可能にします。それぞれはさらにフィールド内でアプリケーションがこれらのメソッドの再現性の向上を目指しています。唾コレクションに加えて指標を定量化し、これらの組織の機能の正常化をについて説明します。代表的なデータは分別された放射線 (6.85 Gy の 4 用量) に続く 2 週間落ち込んで唾液腺機能と顎下腺から含まれます。

唾コレクションに刺激高用量ピロカルピンを実行するときは、誤嚥を防ぐために気道を維持するために重要なまたは口腔内の分泌物から窒息です。切開の概略図 (図 1) を提供しています。気管切開後ストーマは組織や体液の明確なままする必要があります。
唾コレクション中に毛管作用を向上させるため、下方 45 ° の角度で自分の頭を持つマウスを配置必要があります。これらの手順を実行できます > 1 マウス、5 マウスを一度に (図 2) を超えることは推奨はしないが。コレクションが完了すると、チューブが蒸発損失を避けるために迅速に検討するをお勧めします。
次の唾コレクション、マウス、安楽死させた。頭と首の構造を損傷する可能性があります、頚部転位を実行できませんする必要があります。マウスは解離性のステージに戻り、最初に配置として、気管上腺の位置を変更する必要があります。腺解剖 (図 3) はパプア ニューギニア、その位置とびまん性の一貫性のために始まるべきであります。SMG と SLG のしっかりした、簡単に削除することができます。
次のマウスの smg 6.85 Gy 放射線の 4 分画、唾大幅縮小します 2 週間 (図 4)。両方合計唾液分泌、分泌腺の湿重量に正規化をプロットしました。いずれかのメトリックを使用して、次の照射242 週間で期待どおりに、唾液の機能容量は削減されます。
腺組織は、固定との切片の組織学的汚れや免疫組織化学的にすることができます。ヘマトキシリンとエオシン染色 (H & E) に示しますような SMG アーキテクチャ ピロカルピン刺激 (図 5) の有無の両方を総します。Nkcc1 の汚損、膜塩化ナトリウム カリウム輸送体は、ピロカルピン刺激 (図 6) に関係なく同じような細胞の形態を示しています。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57522/57522fig1.jpg"> 図 1.気管スケマティック。(A)頸部を開いたら、主要な構造を可視化します。(B)気管切開を完了するには、持ち上げてが血液の供給、神経支配、導管など近隣の構造を停止させることがなく SMGs を取り外さないで優しく。気管切開を作る前に気管軟骨を公開するストラップの筋肉をカットします。場所と全体の唾コレクション処理中に公開されている気管の SMG を残します。<a href="/files/ftp_upload/57522/57522fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57522/57522fig2.jpg"> 図 2.唾コレクション。ピロカルピン刺激マウス下の角度し、郭清のステージの下に配置されている事前重量を量られたコレクションの管に毛細管を通じて唾液を収集します。<a href="/files/ftp_upload/57522/57522fig2large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57522/57522fig3.jpg"> 図 3.主要な唾液腺解剖します。マウスに安楽死されている位置を変更、別の左と右の SMG。慎重に nonglandular 組織や血管を周囲から各腺を区切ります。PG は、拡散反射光、水平構造です。SMG、SLG の参加している鉗子できれいに分離することができます。<a href="/files/ftp_upload/57522/57522fig3large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57522/57522fig4.jpg"> 図 4.代表的な分泌データ。Smg、分別された放射 (6.85 Gy の 4 用量) を次の 2 週間ピロカルピン刺激唾液の分泌を測定しました。唾液の機能が報告される総唾液量 (A) と合計唾液量 (B) に正規化された SMG 湿重量を合計します。予想通り、そこは SMG の機能が大幅に低下 (± SD を意味 * *p < 2 尾対になっていない t 検定を用いて 0.01)。<a href="/files/ftp_upload/57522/57522fig4large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57522/57522fig5.jpg"> 図 5.ヘマトキシリンとエオシン SMG 染色します。SMG 組織制御、未処理マウス SMG (左) と比較してピロカルピン刺激 (100 mg/kg、右) 後のマウスから収穫されました。10 X で総腺構造を示しています H & E 染色 (スケール バー: 200 μ m) (A、B) と 40 X (スケール バー: 50 μ m) (C, D)。<a href="/files/ftp_upload/57522/57522fig5large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57522/57522fig6.jpg"> 図 6.Nkcc1 SMG 免疫。SMG 組織制御、未処理マウス SMG (左) と比較してピロカルピン刺激 (100 mg/kg、右) 後のマウスから収穫されました。Nkcc1 は、膜タンパク質、細胞形態を評価するために使用できます。DAPI は核の対比染色として使用されます (拡張バー: 75 μ m)。<a href="/files/ftp_upload/57522/57522fig6large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

Watch this Scientific Journal Video about Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation at JoVE.com

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

ピロカルピン刺激次を介してマウス唾液機能的アセスメント分別放射線

マウスの気管切開と 3 つの大唾液腺の分離を含む唾液のコレクションを実行する詳細なアプローチを提案します。

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

murine-salivary-functional-assessment-via-pilocarpine-stimulation

Universidad San Sebastian

Research

JoVE Journal

Medicine

11.1K ビュー.  University of Rochester. マウスの気管切開と 3 つの大唾液腺の分離を含む唾液のコレクションを実行する詳細なアプローチを提案します。

ビデオ: Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

ワルトン管を経由してマウス顎下唾液腺のカニュレーション

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

医学

A Murine Model of Muscle Training by Neuromuscular Electrical Stimulation

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional capacity. Transcutaneous surface stimulation of skeletal muscle involves a current flow between a cathode and an anode, thereby inducing excitement of the motor unit and the surrounding muscle fibers. 
NMES is an attractive modality to evaluate skeletal muscle adaptive responses for several reasons. First, it provides a reproducible experimental model in which physiological adaptations, such as myofiber hypertophy and muscle strengthening6, angiogenesis7-9, growth factor secretion9-11, and muscle precursor cell activation12 are well documented. Such physiological responses may be carefully titrated using different parameters of stimulation (for Cochrane review, see 13). In addition, NMES recruits motor units non-selectively, and in a spatially fixed and temporally synchronous manner14, offering the advantage of exerting a treatment effect on all fibers, regardless of fiber type. Although there are specified contraindications to NMES in clinical populations, including peripheral venous disorders or malignancy, for example, NMES is safe and feasible, even for those who are ill and/or bedridden and for populations in which rigorous exercise may be challenging. 
Here, we demonstrate the protocol for adapting commercially available electrodes and performing a NMES protocol using a murine model. This animal model has the advantage of utilizing a clinically available device and providing instant feedback regarding positioning of the electrode to elicit the desired muscle contractile effect. For the purpose of this manuscript, we will describe the protocol for muscle stimulation of the anterior compartment muscles of a mouse hindlimb.

A murine model of neuromuscular electrical stimulation (NMES), a safe and inexpensive clinical modality, to the anterior compartment muscles is described. This model has the advantage of modifying a readily available clinical device for the purpose of eliciting targeted and specific muscle contractions in mice.

神経筋電気刺激による筋力トレーニングのマウスモデル

Neuromuscular electrical stimulation (NMES) is a common clinical modality that is widely used to restore1, maintain2 or enhance3-5 muscle functional ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

唾液を収集し、家族介護者を経由して高齢者を居住する虚弱コミュニティ唾液コルチゾールおよびα-アミラーゼの測定

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Myocardial Infarction and Functional Outcome Assessment in Pigs

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One important prerequisite is a good understanding of all safety and efficacy aspects obtained in a large animal model that validly reflect the human scenario of myocardial infarction (MI). Pigs are widely used in this regard since their cardiac size, hemodynamics, and coronary anatomy are close to that of humans. Here, we present an effective protocol for using the porcine MI model using a closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. This approach is based on 90 min of myocardial ischemia, inducing large left ventricle infarction of the anterior, septal and inferoseptal walls. Furthermore, we present protocols for various measures of outcome that provide a wide range of information on the heart, such as cardiac systolic and diastolic function, hemodynamics, coronary flow velocity, microvascular resistance, and infarct size. This protocol can be easily tailored to meet study specific requirements for the validation of novel cardioregenerative biologics at different stages (i.e. directly after the acute ischemic insult, in the subacute setting or even in the chronic MI once scar formation has been completed). This model therefore provides a useful translational tool to study MI, subsequent adverse remodeling, and the potential of novel cardioregenerative agents.

This protocol describes the porcine myocardial infarction (MI) model using a 90 min closed-chest coronary balloon occlusion of the left anterior descending artery (LAD), followed by reperfusion. Furthermore, the protocol for several outcome parameters, such as cardiac function, hemodynamics, microvascular resistance, and infarct size, are also presented.

豚における心筋梗塞および機能的結果の評価

Introduction of newly discovered cardiovascular therapeutics into first-in-man trials depends on a strictly regulated ethical and legal roadmap. One ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

マウス唾液腺幹細胞の単離

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

生物学

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

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 ...

生物工学

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

唾液機能低下のラットモデルにおけるRetroductal顎下腺点滴とローカライズされた分割照射

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

マウスの唾液腺機能の測定法

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

免疫・感染学

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

顎下および耳下腺唾液腺からの Organotypic 培養の放射線治療は生体内特性をモデル化する

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...

マウス顎下腺再生のリアルタイムライブイメージング

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

著者スポットライト:損傷後の唾液腺再生におけるマクロファージ-上皮細胞相互作用の研究 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

唾液腺における細胞間相互作用の Ex Vivo Live Cell Imaging(英語)

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...