We thank Dr. John Chang (Radiation Oncology, LSU Health Science Center) for assistance with radiation dose measurements. The study was supported by the American Cancer Society (Grant number: 116945-RSG-09-038-01-CCE), National Institute of Health (Grant number: R21CA173162) and the Feist-Weiller Cancer Center.

모든 절차는 LSU 건강, 슈 리브 포트, 동물 관리 및 사용위원회의 승인 관리 및 실험 동물의 사용에 대한 NIH의 지침에 따라이었다 하였다. 쥐 턱밑 침샘의 1 캐 뉼러 <ol><li> 주사기 - 튜브 어셈블리의 제조 <ol><li> 메스로 PE10 폴리에틸렌 튜브의 10cm 길이를 잘라. 집게 손가락과 엄지 손가락 사이의 관의 양쪽 끝을 잡고. 부드러운 불꽃 상기 튜브의 중간 부분을 가열, 부드럽게 양쪽을 잡아 당겨 길이를 두 배로 연화 튜브를 스트레칭. </li><li> 테이퍼 끝 각 - 2 캐 뉼러를 얻기 위해 45 오의 각도로 메스와 중간에 튜브를 잘라. 0.5 CC의 인슐린 주사기의 29 G 바늘 이상을 맞춤으로써 캐 뉼러의 비 테이퍼 끝을 확장합니다. </li><li> 정맥을 제거하고 주사기로 생리 식염수 220 μl를 그립니다. 공기 방울을 제거하기 위해 누릅니다. cannul를 장착바늘을 통해, 공기를 배출하고 정맥을 통해 솔루션의 자유로운 흐름을 보장하기 위해 주사기 플런저를 밀어 넣습니다. 200 μL에 볼륨을 조정합니다. </li></ol></li><li> 턱밑 글 랜드 덕트의 내부 구강 캐 뉼러 참고 : 오토 클레이브 악기 절차 이전에, 뜨거운 구슬 살균기 절차 사이에 소독. <ol><li> 스프 라그 돌리 쥐의 무게를 측정하고, 피하 주사기에서 계산 된 케타민의 볼륨 (42 ㎎ / ㎏) / 자일 라진 (8 ㎎ / ㎏) / 아세 프로 마진 (1.4 ㎎ / ㎏) 혼합 분배. </li><li> 검지 한 손의 엄지 손가락 사이의 꼬리의 기부를 파악하고 파악 몸체 위에 다른 손으로 밀어 동물을 억제. 엄지 손가락과 나머지 손가락으로 몸통을 누르고있는 동안 머리의 측면을 따라 색인과 중간 손가락을 휴식. </li><li> 뒷다리 근육에 마취제를 주입한다. 발가락 핀치와 눈꺼풀 반사에 의한 마취의 깊이를 확인합니다. 건조의 동안을 방지하기 위해 눈 윤활제를 적용동물 마취입니다. </li><li> 특수하게 설계된 플랫폼 (그림 1)에 동물을 배치하고 가로 막대의 위쪽 앞니의 치아를 참여. 아래 앞니 주위에 고무 밴드를 반복하고 플랫폼에 고정하여 아래 턱을 아래로 당깁니다. </li><li> 혀를 통해 멸균 봉합을 전달하고 구강의 바닥 인상을 올립니다. 지혈과 봉합을 고정하고 가로 가로대 위를 전달합니다. </li><li> 사용자 정의 내장 뺨 스프레더 (그림 1)과 함께 뺨을 확장하고 해부 현미경 입의 바닥에 설하 용의자를 찾습니다. </li><li> 섬세한 집게를 사용하여 예비 성형 PE10 튜브의 테이퍼 끝을 잡고. 조심스럽게 설하 유두의 유관 구멍에 정맥의 끝을 조작 할 수 있습니다. 이 3 스레드에 의해 정맥의 위치를​​ 확인 - 덕트에 5mm를; 이 장애물없이 통과 있는지 확인합니다. </li></ol></li><li> 턱밑 글 랜드 Instillation <ol><li> 목의 목덜미에 피하 아트로핀 (0.5 ㎎ / ㎏)을 주입하고, 타액 분비의 감소를 위해 10 분을 기다립니다. </li><li> 시아 노 아크릴 레이트 (접착제)의 드롭 덕트 구멍을 캐 뉼러를 고정하고, 건조 할 수 있습니다. 천천히 50 μL / 분 ~의 속도로 시린지 플런저를 가압하여 마개 (200 μL / 샘)의 용액을 주입. </li><li> 지혈제와 튜브를 분쇄, 조심스럽게 주사기를 제거합니다. 동물이 의식을 회복 할 때까지 60 분 - 30 덕트에 튜브를 유지. 혀를 보유하고 봉합사를 제거합니다. </li><li> 별도의 케이지에 동물을 전송하고 복구하는 동안 따뜻한 유지하기 위해 가열 램프를 사용합니다. 이 흉골 드러 누움을 유지하기 위해 의식을 회복 할 때까지 무인 동물을 두지 마십시오. </li><li> 동물이 완전히 외래되면, 음식과 물에 대한 무제한 접근 할 수있는 동식물 사육장에서 그것을 집. </li></ol></li></ol> 2. 현지화 된 분획 조사턱밑 글 랜드의 <ol><li> 전술 한 바와 같이 동물을 억제하고, 뒷다리의 근육 내 케타민의 투여 (33 ㎎ / ㎏) / 자일 라진 (6 ㎎ / ㎏) / 아세 프로 마진 (1 ㎎ / ㎏)으로 마취 믹스. 마취의 깊이를 확인하고 눈에 윤활제를 적용합니다. </li><li> 선형 가속기 탁상에 동물 부정사를 놓고 머리를 기울여 목을 확장합니다. 흉골의 상단 하악골의 하부 경계에서의 영역을 포함하는 방사선 필드 (3cm 슬릿 폭)을 시준. </li><li> 이 지역에 걸쳐 1cm 조직 - 상응하는 덩어리를 놓고 방사선 소스와 100cm에 덩어리의 상단 사이의 거리를 조정합니다. </li><li> 선형 가속기의 6 MV 광자 빔을 사용하여 동물 (2.5 Gy의)을 조사한다. 노출 시간을 지시한다 표면 루스하도록 방사선 소스로부터 레이트, 필드 크기 및 거리 복용량. 설정된 전류, 동물 ~ 1 Gy의 / 분의 투여 량 비율을 조사 하였다. </li><li> 노출을 반복; 8 개의 2.5 Gy의 / 일일; 사일 / 사이에 2 일 간격으로 주. 복구하는 동안 동물을 따뜻하게 유지. 완전히 외래 후 사육장으로 동물을 이동. </li><li> 선 기능 (14)을 측정하는 방사선 후 자극 턱밑 글 랜드 타액 8 주 수집합니다. 차가운 4 % 파라 포름 알데히드 / 인산염 완충 식염수 산도 7.2의 심장 재관류에 의해 마취하에 동물을 안락사. 조직 학적 및 면역 조직 화학 (14)를 분석하기위한 턱밑 샘을 근절. </li></ol>

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The authors have nothing to disclose.

침샘은 종종 머리 목 암, 목 노드의 선택 과목 절제, 또는 지역 혈액 종양에 대한 방사선 치료를받은 환자에서 조직 복구의 임계 값을 초과 방사선 량을받을 수 있습니다. 그랜드 유체 분비 선포 세포는 말기 분화되어 있지만, 이들은 역설적 방사선에 민감하다. 분비 기능은 만성 낮은 타액 출력에 복사하고, 돌이킬 수없는 선 피해 결과의 첫 주 이내에 삭제합니다. 가난한 선 기능과 계속된다 구강...

건강한 조직의 담보 파괴는 암 치료의 해로운 부작용의 다수를 차지한다. 방사선 필드 거짓말 주요 침샘의 일부 또는 전부가 불가피하게 파괴된다. 따라서, 골수 이식 전에 경부암, 자궁 경관 임파종 또는 전신 조사를위한 방사선 치료를받은 대부분의 환자들은 방사선, 침샘의 기능 저하 2-6의 일반적인 영구 부작용을 겪는다. 침샘의 유체 생산 선방 세포는 방사선에 심하게 민감하다. 침샘의 손상은 타액의 흐름과 같은 타액 기능 저하라고하는 조건의 급격한 감소하는 발생합니다. 타액의 흐름에서 만성 감소는 저작, 연하, 음성, 맛,하지만 강렬한 통증, 점막 눈물, 연하 곤란, 기회 감염의 병적 인 후유증으로 키 구강 활동을 손상 및 충치는을 악화환자의 건강과 기능 2,3. 방사선 관련 타액선 세포 손실 비가역 때문에, 구강 건조증의 더 교정 치료는 없다. 인공 타액 대체 및 prosecretory 약물로 증상을 assuaging에 초점을 맞추고 현재 치료는 장기적인 구호 6 비효율적이다. 개선 된 방사 전달 기술은 증상의 중증도를 감소 도움이되지만, 정상 조직에 독성 및 부작용은 암 치료에 -6,7- 제한 요소 남아있다. 방사선 - 관련 합병증을 예방하는 선제 대책 따라서, 표준이되고있다. DNA 수리 자유 라디칼 산소 종, 위탁 셀 다시 채우기를 청소할 또는 향상 라디오 - 보호제는 타액 기능 저하 8-11을 피하기 위해 탐구되고있다. 외분비 침샘의 분비는 주요 배설 덕트를 통해 입으로 배출. 일의 내 경구 삽관조영제의 주입을위한 전자 배설 덕트는 외래 환자 절차로 정기적으로 수행됩니다. 유사한 방법을 이용 침샘 직접 국소 치료 (12)를 대상으로 할 수있다. 이 외에 전신 부작용의 위험을 감소에서, retroductal 선 점안 혜택을 추가했다. 도관 트리 주위 타액선 세포의 단층 배열은 모든 타액 상피 세포 표적화 수 있고, 장벽 그랜드 행위 섬유상 캡슐 불필요한 치료 확산을 줄이기 위해. 본질적으로, 침샘은 방사선에 의한 타액의 기능 저하 등의 글 랜드 고통의 표적 치료를위한 최적 적합하다. 주 동안 - (2.5 Gy를 / 분수 / 일, 주 5 일 1.8) 암 치료에 대한 기존의 방사선은 작은 용량으로 제공됩니다. 따라서, 실험 모델의 장기화 조사 방식에 대한 효능을 보여줍니다 라디오 보호 치료는 큰 임상 베어링을 보유하고 있습니다. COMPRO분획 후 방사능 mised 타액 함수 작은 동물에 기록하지만, 방사선 소스, 도즈 비율 및 사용되는 프로토콜은 다양한 9,10,13이다되었다. 이 보고서에 retroductal 배달 및 환자 관련 방사선 소스 및 용량 부분을 사용하여 쥐 턱밑 샘의 지역화 된 방사선하는 방법을 설정합니다.

<table><tbody><tr><td>Intramedic Polyethylene tubing (PE10)</td><td>Becton Dickson</td><td>427401</td><td></td></tr><tr><td>1/2 cc Insulin Syringe U-100</td><td>Becton Dickson</td><td>309306</td><td></td></tr><tr><td>Artificial Tears</td><td>Miller Vet Supply&amp;nbsp;</td><td>5098-9840-64</td><td></td></tr><tr><td>Hot Bead Sterilizer</td><td>Fine Science Tools</td><td>18000-45</td><td></td></tr><tr><td>Perma-Hand silk suture</td><td>Ethicon</td><td>K833H</td><td></td></tr><tr><td>Graefe forcep</td><td>Fine Science Tools</td><td>11051-10</td><td></td></tr><tr><td>Olympus SZX16 Stereo Microscope</td><td>Hunt Optics and Imaging</td><td></td><td></td></tr><tr><td>6 MV Linear Accelerator</td><td>Elekta</td><td></td><td></td></tr><tr><td>Bolus - Skinless&amp;nbsp;</td><td>Civco</td><td>MTCB410</td><td></td></tr><tr><td>Heat Lamp</td><td>Braintree Scientific</td><td>HL-1&amp;nbsp;110V</td><td></td></tr></tbody></table>

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.

최소한의 침습적 sialography 기술을 적응, 주요 침샘의 지역 치료는 가능하다. 쥐 턱밑 침샘에서 Retroductal 정부는 와튼의 덕트의 내부 경구 삽관 (그림 2)에 의해 시도되었다. 입의 바닥에있는 하위 언어 용의자에 열려 와튼의 타액 덕트,하지만 구멍은 쉽게 볼 수 없습니다. 캐 뉼러의 삽입은, 그러므로, 부드러운 프로빙에 의해 수행되었다. 캐 뉼러를 배치하는 동...

Watch this Scientific Journal Video about Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction at JoVE.com

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

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.

타액 기능 저하의 쥐 모델에서 Retroductal 턱밑 글 랜드 점적 및 현지화 된 분획 조사

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

retroductal-submandibular-gland-instillation-localized-fractionated

Research

JoVE Journal

Medicine

11.3K 조회수.  Louisiana State University Health Sciences Center. 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.

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

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.

와튼의 덕트를 통해 마우스 턱밑 침샘의 Cannulation

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

연구

의학

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.

마우스 침샘에 줄기 세포의 분리

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 ...

생물학

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.

Retroductal 나노 Murine Submandibular 동맥에 주입

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

생체공학

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.

쥐에 있는 침 샘 기능의 측정 하는 방법

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

면역학 및 감염병학

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

똑바로 Intravital 현미경 Murine Submandibular 침 샘의 준비

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Ultrasound-guided Botulinum Toxin-A Injections: A Method of Treating Sialorrhea

Neurological diseases can be complicated by sialorrhea, an excessive flow of saliva. Patients suffering from moderate to severe sialorrhea have an impaired quality of life, often worsened by correlated complications such as aspiration pneumonia, oral infections, dental caries, and maceration of the skin. Diverse therapeutic approaches have been proposed for the treatment of sialorrhea, including surgery and the use of anticholinergic agents, with limited results and the possible occurrence of serious adverse events. Recently, botulinum toxin (BoNT) injection within the major salivary glands has been proposed in patients refractory to anticholinergic therapy, with the aim of inhibiting local acetylcholine release and gland activity. 
In order to obtain a better outcome in terms of reduction of saliva production, efficacy, duration, and avoidance of major adverse events, we developed an ultrasound-guided BoNT-type A injection technique accurately described in the text. Here we present a method of treating sialorrhea with bilateral parotid and submandibular gland BoNT-type A injections under ultrasound guidance. Four quadrants of the parotid gland and two quadrants of the submandibular gland are visualized and injected using two accesses and one access, respectively. 
The ultrasound-guided procedure provides a simple, non-invasive, real-time visualization of the muscular and glandular tissues and their surrounding structures, optimizing treatment efficacy and safety.

Botulinum toxin is injected into the salivary glands for the treatment of sialorrhea. The ultrasound-guided botulinum toxin injection in both the parotid and submandibular glands demonstrates a sustained efficacy and the absence of serious side effects.

보툴리눔 독소-A 주사를 초음파 유도 : Sialorrhea을 치료하는 방법

Neurological diseases can be complicated by sialorrhea, an excessive flow of saliva. Patients suffering from moderate to severe sialorrhea have an ...

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Pilocarpine 자극 다음 통해 murine 침 기능 평가 분류 한 방사선

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

Retrograde Parotid Gland Infusion through Stensen's Duct in a Non-Human Primate for Vectored Gene Delivery

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of secreting proteins into the bloodstream and are easily accessible, making them potentially superior tissue sites for replacement hormone production or vaccination by gene transfer. Suggested methods for gene delivery include transcutaneous injection and retrograde infusion through salivary ducts. We demonstrate how to perform Retrograde Salivary Gland Infusion (RSGI) in non-human primates. We describe the important anatomic landmarks including identification of the parotid papilla, an atraumatic method of cannulating and sealing Stensen&#39;s Duct utilizing basic dental tools, polyethylene tubing, and cyanoacrylate, and the appropriate rate of infusion. While this is the least traumatic method of delivery, the method is still limited by the volume able to be delivered (&#60;0.5 mL) and the potential for trauma to the duct and gland. We demonstrate using fluoroscopy that an infusate can be fully delivered into the gland, and further demonstrate by immunohistochemistry the transduction of a typical vector and expression of the delivered gene.

Salivary glands have been proposed as a tissue target site for gene therapy, especially in the area of vaccination by gene transfer. We demonstrate gene delivery in a non-human primate model utilizing retrograde parotid infusion.

벡터 유전자 전달을 위한 비인간 영장류에서 스텐센의 덕트를 통한 역행 파로티드 글랜드 주입

Salivary glands are an attractive tissue target for gene therapy with promising results already leading to human trials. They are inherently capable of ...

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.

하 악 및 파 타 액 땀 샘에서 유기 유형 배양의 방사선 치료는 생체 내 특성의 핵심

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

마우스 턱밑샘 재생의 실시간 실시간 이미징

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.

저자 스포트라이트: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury(부상 후 타액선 재생에서 대식세포-상피세포 상호작용 연구) 

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.

Ex vivo Live Cell Imaging을 통한 타액선의 세포 간 상호 작용 조사

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