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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</ol>

著者が明らかに何もありません。

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?...

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?...

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 de Cartagena UDEC

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

Quantifying Cognitive Decrements Caused by Cranial Radiotherapy

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For many patients afflicted with CNS and non-CNS malignancies, radiotherapy and chemotherapy offer the best options for disease control. These treatments however come at a cost, and nearly all cancer survivors (~11 million in the US alone as of 2006) incur some risk for developing cognitive dysfunction, with the most severe cases found in patients subjected to cranial radiotherapy (~200,000/yr) for the control of primary and metastatic brain tumors1. Particularly problematic are pediatric cases, whose long-term survival plagued with marked cognitive decrements results in significant socioeconomic burdens2. To date, there are still no satisfactory solutions to this significant clinical problem.
We have addressed this serious health concern using transplanted stem cells to combat radiation-induced cognitive decline in athymic rats subjected to cranial irradiation3. Details of the stereotaxic irradiation and the in vitro culturing and transplantation of human neural stem cells (hNSCs) can be found in our companion paper (Acharya et al., JoVE reference). Following irradiation and transplantation surgery, rats are then assessed for changes in cognition, grafted cell survival and expression of differentiation-specific markers 1 and 4-months after irradiation. To critically evaluate the success or failure of any potential intervention designed to ameliorate radiation-induced cognitive sequelae, a rigorous series of quantitative cognitive tasks must be performed. To accomplish this, we subject our animals to a suite of cognitive testing paradigms including novel place recognition, water maze, elevated plus maze and fear conditioning, in order to quantify hippocampal and non-hippocampal learning and memory. We have demonstrated the utility of these tests for quantifying specific types of cognitive decrements in irradiated animals, and used them to show that animals engrafted with hNSCs exhibit significant improvements in cognitive function3.
The cognitive benefits derived from engrafted human stem cells suggest that similar strategies may one day provide much needed clinical recourse to cancer survivors suffering from impaired cognition. Accordingly, we have provided written and visual documentation of the critical steps used in our cognitive testing paradigms to facilitate the translation of our promising results into the clinic.

Cognitive impairment resulting from the radiotherapeutic management of brain tumors represents a clinically intractable condition that adversely impacts quality of life. The capability to critically evaluate potential interventions for ameliorating radiation-induced cognitive decrements ultimately depends on the capability to undertake rigorous quantitative assessments of cognition.

頭蓋放射線療法によって引き起こされた定量認知デクリメント

With the exception of survival, cognitive impairment stemming from the clinical management of cancer is a major factor dictating therapeutic outcome. For ...

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

焦点放射線阻害と大人の視床下部ニューロン新生の機能的な尋問

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

神経科学

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

ピロカルピン誘導てんかんマウスにおける記憶機能の評価

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

行動学

Adapting Human Videofluoroscopic Swallow Study Methods to Detect and Characterize Dysphagia in Murine Disease Models

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research. Successful outcomes are dependent upon three critical components: test chambers that permit self-feeding while standing unrestrained in a confined space, recipes that mask the aversive taste/odor of commercially-available oral contrast agents, and a step-by-step test protocol that permits quantification of swallow physiology. Elimination of one or more of these components will have a detrimental impact on the study results. Moreover, the energy level capability of the fluoroscopy system will determine which swallow parameters can be investigated. Most research centers have high energy fluoroscopes designed for use with people and larger animals, which results in exceptionally poor image quality when testing mice and other small rodents. Despite this limitation, we have identified seven VFSS parameters that are consistently quantifiable in mice when using a high energy fluoroscope in combination with the new murine VFSS protocol. We recently obtained a low energy fluoroscopy system with exceptionally high imaging resolution and magnification capabilities that was designed for use with mice and other small rodents. Preliminary work using this new system, in combination with the new murine VFSS protocol, has identified 13 swallow parameters that are consistently quantifiable in mice, which is nearly double the number obtained using conventional (i.e., high energy) fluoroscopes. Identification of additional swallow parameters is expected as we optimize the capabilities of this new system. Results thus far demonstrate the utility of using a low energy fluoroscopy system to detect and quantify subtle changes in swallow physiology that may otherwise be overlooked when using high energy fluoroscopes to investigate murine disease models.

This study successfully adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating translational dysphagia research.

マウス疾患モデルで嚥下障害を検出し、特徴づけるために、人間のVideofluoroscopicツバメ調査方法を適応させる

This study adapted human videofluoroscopic swallowing study (VFSS) methods for use with murine disease models for the purpose of facilitating ...

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 ...