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

Bu yayında bildirilen araştırma diş Ulusal Enstitüsü ve Fasiyal araştırma (NIDCR) ve ulusal sağlık Enstitüleri Ödülü numarası R56 DE025098, UG3 DE027695 ve F30 CA206296 altında Ulusal Kanser Enstitüsü (ncı) tarafından desteklenmiştir. İçeriği yalnızca yazarlar sorumludur ve mutlaka Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmiyor. Bu eser de NSF DMR 1206219 ve IADR yenilik Oral bakım Ödülü (2016) tarafından desteklenmiştir.
Dr. Eri Maruyama ve Andrew Hollomon tükürük koleksiyonu ile yardım için teşekkür etmek istiyorum. Pei-Lun Weng bezi diseksiyon ile yaptığı yardım için teşekkür etmek istiyorum. Matthew Ingalls şekil hazırlık yaptığı yardım için teşekkür etmek istiyorum. Dr. Elaine Smolock ve Emily Wu bu el yazması eleştirel okuma için teşekkür etmek istiyorum.

Aşağıda verilen tüm vivo içinde yordamlar hayvan kaynakları, Rochester Üniversitesi, Rochester Üniversitesi Komitesi tarafından kabul edildi. NY.
1. hazırlık
<ol><li>Bir analitik denge kullanarak, 20 mg Pilokarpin tartın. 2 ml steril serum microcentrifuge tüp içinde çözülür. Not: Pilokarpin ışığa duyarlı olduğunu ve zaman içinde etkinliğini kaybeder çünkü bu çözüm enjeksiyon gününün hazırlıklı olup yönetilen kadar ışıktan korunan.</li>
	<li>Bir analitik denge kullanarak, tartmak ve tüm koleksiyon tüpler ve cam kılcal tanımlayın. Koleksiyon tüpler ve cam kılcal fareler sayısı eşleşmelidir.</li>
</ol>2. trakeostomi
<ol><li>C57/BL6 fareler bir analitik denge kullanarak tartın.</li>
	<li>0.5 mL 29G x ½" iğne ile kullanarak, fareler 100 mg/kg ketamin ve 10 mg/kg xylazine intraperitoneally enjekte bir steril serum fizyolojik solüsyonu ile anestezi. Fareyi artık genellikle 5-10 dk içinde enjeksiyon takip oluşur (Yani yokluğu pedal çekilme refleks), uyaranlara yanıt verdiğinde aşağıdaki adıma geçin.</li>
	<li>Pamuk uçlu aplikatör üzerinde yağ dağıtımı sonra yavaşça gözlerini uygulamak ve fare sırtüstü pozisyonda Sahne Alanı'nda yer. İmzalat hayvan sıcak yüzey üzerinde tutulmalıdır. Not: Oda sıcaklığında tüm adımlar gerçekleştirilir.</li>
	<li>Burun, bacaklarda ve kuyruk cerrahi bant kullanarak sahne için güvenli.</li>
	<li>Temiz ve boyun bir alkol bezle ıslak. Not: Bu kürk sonraki insizyon girmesini önlemeye yardımcı olur.</li>
	<li>Ventral orta hat boyun deri Forseps ile yükselterek, diseksiyon makası kullanarak bir küçük, yüzeysel kesim yapmak. Makas içine açılış kılavuz ve subkutan doku uçaklar ayırmak için bıçaklar yavaş açılıyor.</li>
	<li>Kaldırdı cilt tutulması, dikkatle ağız aşağıda 1 cm kesin.</li>
	<li>İki yanal insizyon ilk kesim düşük kaliteli ve üstün yönleri olun. Forseps kullanarak, yavaşça uzakta baş ve boyun yapılarına erişim sağlamak için cilt yansıtmak.</li>
	<li>8 X büyütme oranında diseksiyon mikroskop kullanarak görselleştirmek submandibular bezleri (orta hat: şekil 1).</li>
	<li>İyi forseps kullanarak, bezleri Trakea örten dört infrahyoid (Askı) kasları ortaya çıkarmak için yavaşça kaldır. Yırtılma veya çevre damarlara veya boşaltım kanallarını engellemeden kaçının.</li>
	<li>Birden fazla fare tükürük toplama, adımları 2.1-2,10 her fare ile devam etmeden önce işlemini yineleyin. Not: Bu yordam güvenli bir şekilde en fazla 5 fareler aynı anda gerçekleştirilebilir.</li>
	<li>Küçük parçalara makasla kayış kasları medial kısmı kalırken orta hat gibi mümkün olduğunca yakın kaldırın. Sadece nefes borusu görselleştirmek ve kayış kasları yoldan işlem sırasında tutmak gerektiği kadar kesti. Önemli kanama için ikincil birim tükenmesi ise Pilokarpin salgı inducing etkili olmayacağı için herhangi bir yakın gemi nicking kaçının.</li>
	<li>Trakea uzak kayış kasları yansıtır.</li>
	<li>Larinks, trakea ve tiroid bezi görselleştirin. Trakea doku örten açık olduğundan emin olun.</li>
	<li>Yatay bir kesi borusunda inferior/arka küçük diseksiyon makası kullanarak tiroid olun. Hava yolu patent ve sıvı açık olduğundan emin olun.</li>
</ol>3. tükürük toplama
<ol><li>Ağız ve açı diseksiyon sahne aşağı yakınındaki bandı çıkarın (45°, cranially) tükürük akışı ile yardımcı olmak için.</li>
	<li>0.5 mL 29G x ½" iğne ile kullanarak, 10 µL/g vücut ağırlığı Pilokarpin mayi enjeksiyon toplam doz 100 mg/kg için gerçekleştirin.</li>
	<li>Bir sayacı başlatmak. Birden çok fareler dozaj hızlı hareket ve aynı anda kalan fareler enjekte bir asistan sahip sağlama süresini azaltın.</li>
	<li>Standart forseps kullanarak, kılcal tüp erişim için ağzını aç.</li>
	<li>Tükürük bir boncuk ağızda görülmektedir kez proksimal sonuna kılcal tüp distal ucu diseksiyon sahne (önceden tartılır her iki tüpler) altında konumlandırılmış bir toplama tüp içine yerleştirilmiş olan sıvı yerleştirin. Not: C57/BL6 dişi farelerde 6-10 hafta-in yaş, brüt salya Pilokarpin enjeksiyon 2 dk içinde oluşur.</li>
	<li>Tükürük ağız bina ancak tüpten akan değil, önceki adımı deneyin. Tüp giriş engel doğrudan bir Mukozal yüzey karşı yerleştirilmez emin olun.</li>
	<li>Tükürük toplam 12 dk Pilokarpin stimülasyon takip toplamak. Ağzının açık kalmasını sağlamak. Not: Bu dönemde artan gelgit hacmi ve salgıları (tükürük, idrar, dışkı) normaldir.</li>
	<li>Fareler tükürük işlevi depresif Eğer (örn., yaralanma veya müdahale nedeniyle), kalan sıvı P200 micropipette kullanarak toplama tüp ağzından aktarmak. Birden çok fareler toplama ipuçları değiştirilir olun.</li>
	<li>Kayıt toplanan tükürük artı borular Pilokarpin enjeksiyon sonra 12 dk ağırlığı.</li>
</ol>4. bezi diseksiyon
<ol><li>Fareler CO2 ötenazi tarafından ötenazi (ötenazi hayvanlar için travma kurallarda açıklandığı gibi: 2013 Edition) baş ve boyun yapıları korumak için dikkat çekici Torakotomi tarafından takip. Bu nedenle, ötenazi servikal çıkık bir yöntem olarak kaçının.</li>
	<li>Fare, Sahne Alanı'nda bir sırtüstü pozisyonda yerleştirin. Bezleri özgün sitedeki Trakea (şekil 1) yeniden konumlandırın.</li>
	<li>Diseksiyon mikroskop altında 8 X büyütme, kullanarak görselleştirmek büyük bezleri: PG, SMG ve SLG. Bezlerinin diseksiyon sırasında ayırt etmek için SMG ve SLG yanal yalan PG diffüz, tanımlayın. Not: az tanımlanmış ve parçalanmış olması büyük olasılıkla çünkü PG anatomi, önce yapılması gerekir.</li>
	<li>Forseps, temel yapılarını uzak çekerek ile PG kuyruğunun kavramak ve yavaşça gerginlik makas diseksiyon ile PG kafa kesme önce uygulayın.</li>
	<li>İhlali forseps kullanarak SMGs çevreleyen kapsül. Yavaşça sol ve sağ SMG ayırın.</li>
	<li>Forseps kullanarak yapıştırılan nonglandular dokusundan SMG dorsal yönünü ücretsiz.</li>
	<li>SMG boynundan Wharton'ın kanal gergin Forseps ile çizerek ve makas diseksiyon ile kanal kesme kaldırın.</li>
	<li>Yavaşça SLG forseps kullanarak SMG ayırın. Not: SMG superolateral yönü SLG var.</li>
	<li>Analitik bir denge, kayıt SMG ağırlıkları kullanarak.</li>
</ol>

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Yazarlar ifşa gerek yok.

Biz bezi yaralanma ve tedavi uygulanan Tükürük bezi işlevi değerlendirmek için multistep bir yöntem mevcut. Bizim yordamı trakeostomi, tükürük toplama ve bezi diseksiyon, her biri bir tümleşik çalışma Tükürük bezi Biyoloji destekleyen deneysel uygulamaları sahip içerir. Örneğin, fare trakeostomi ağız boşluğu tıkayan işlemler sırasında genel hava yolu yönetimi için kullanılmıştır.
Uygun diseksiyon ve trakeal kesi Pilokarpin için gerekli teşvik tükürük s...

Tükürük bezi bozuklukları sendromlar dysregulated veya Engelli salgı aşırı üretimi (sialorrhea) veya tükürük1eksik (xerostomia ve hyposalivation) içerir. Her iki durumda da, terapötik geliştirme2sonu hedefi doğru Tükürük bezi biyoloji anlayışımız iyileştirilmesi bir ilgi vardır.
Tükrük bezleri son derece radiosensitive organları ve genellikle kalıcı ağız kuruluğu (xerostomia)3,4' e lider baş ve boyun kanser radyoterapi sırasında hasar. Radiosensitive diğer dokular, aksine Ancak, Tükürük bezi Ciro oranı nispeten düşüktür ve salgı kaybı kötü anlaşılan5,6mekanizmadır. Bu benzersiz yaralanma ayarında doku rejenerasyonu ve radioprotection stratejileri tükürük işlevsel değerlendirme gerektirir. Deneysel, fare tükürük toplama radyasyon ve terapötik ajanlar bezi cevaben değerlendirilmesinde özellikle değerli bir araçtır.
Burada, performans ve uyarılmış tükürük salgısı Pilokarpin, güçlü muscarinic agonist7kullanarak miktarının bir yöntem mevcut. Pilokarpin bezi salgısının8,9ikna etmek için otonom sinir sistemini uyarır. Bu test uygun şekilde tamamlamak için bir trakeostomi fareyi bir patent hava yolu yordamı boyunca tutar emin olmak için ve boğulma ve aspirasyon biriken salgıları ağız boşluğu10üzerinden riskleri azaltmak için gereklidir.
Bu üç büyük tükürük bezleri kaldırılmasına sonuçlanan bir terminal, bir uygulamadır: parotid (PG), çene (SMG) ve hapı (SLG). Fonksiyonel çalışmalar, bezi ağırlık kaydedilir ve tükürük ölçüm11,12,13normalleştirmek için sıklıkla kullanılır. Bu veriler, neyin bezi Atrofi beklenen sonucu14,15 yaşında radyasyon çalışmalarda, özellikle önemlidir
Edebiyat nasıl uyarılmış tükürük salgısı açısından değişkenlik gerçekleştirilir ve16bildirdi. Örneğin, edebiyat yayılma alanı en az üç büyüklük17,18,19,20,21,22,23içinde doz Pilokarpin. Burada, bir en iyi duruma getirilmiş yüksek doz Pilokarpin protokolü ile yöntem yürütme hem teknikleri olarak adapte edilebilir (trakeostomi, tükürük toplama ve bezi diseksiyon) modüler bir platform sağlayan geliştirilmiş tekrarlanabilirlik amacıyla mevcut ihtiyacım vardı.
İletişim kuralı gösteri ek olarak 2 hafta fraksiyonlara radyasyon (4 doz 6.85 Gy) SMG bölgesine takip tükürük akışı temsilcisi fonksiyonel verileri içerir.

<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

Hyposalivation otoimmün reaksiyon Sjoegren Sendromu veya aşağıdaki radyasyon hasarı büyük tükürük bezleri için yaygın olarak görülmektedir. Bu gibi durumlarda, hastalığın patogenezinde ve etkili müdahaleler ile ilgili sorular kalır. Ekzokrin bezi biyoloji, disfonksiyon ve tedavi soruşturma için çok değerli tükürük bezlerinin işlevsel değerlendirme sağlar en iyi duruma getirilmiş bir tekniktir. Burada, Pilokarpin gerçekleştirmek için adım adım bir yaklaşım mevcut uyarılmış tükürük salgısı, trakeostomi ve üç büyük fare tükürük bezlerinin diseksiyon dahil olmak üzere. Biz de bu teknikler sırasında erişilen uygun fare baş ve boyun anatomisine ayrıntı. Ölçeklenebilir, böylece iş akışı verimliliği artırmak aynı anda, işlenmek üzere birden çok fareler için izin veren bir yaklaşımdır. Bu yöntemlerin her biri daha fazla alan içinde uygulamaları olan tekrarlanabilirlik geliştirmek hedefliyoruz. Tükürük toplama ek olarak, biz miktarının ve bu dokuların fonksiyonel kapasite normalleştirme için ölçümler tartışıyorlar. Temsilcisi veri 2 hafta fraksiyonlara radyasyon (4 doz 6.85 Gy) takip depresif Tükürük bezi fonksiyonu ile çene bezlerinden dahil edilir.

Yüksek doz Pilokarpin gerçekleştirme tükürük toplama uyardığında, aspirasyon önlemek için hava yolu korumak önemli veya oral kavite içinde salgıları den boğulma. Trakeostomi şematik (şekil 1) sağlanır. Trakeal kesi, stoma doku ve sıvı açık kalmalıdır.
Kılcal eylem tükürük toplama sırasında geliştirmek için fare başlarını aşağıya doğru 45 ° açıyla yerleşti...

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

Fare tükürük işlevsel değerlendirme Pilokarpin stimülasyon aşağıdaki yolu ile radyasyon şeker

Biz tükürük toplama, fare trakeostomi ve üç büyük tükürük bezleri yalıtımı da dahil olmak üzere performans detaylı bir yaklaşım sunuyoruz.

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 Görüntüleme.  University of Rochester. Biz tükürük toplama, fare trakeostomi ve üç büyük tükürük bezleri yalıtımı da dahil olmak üzere performans detaylı bir yaklaşım sunuyoruz.

Video: 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.

Wharton Kanalı üzerinden Fare Submandibular Tükürük Bezi kanülasyonu

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

Tıp

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.

Nöromüsküler Elektriksel Stimülasyon tarafından Kas Eğitimi A Mürin Modeli

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.

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.

Domuzlar miyokard enfarktüsü ve Fonksiyonel Sonuç Değerlendirmesi

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.

Fare Tükürük Bezi Kök Hücre İzolasyonu

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 ...

Biyoloji

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.

Fare Submandibular bezi Retroductal nanopartikül enjeksiyon

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

Biyomühendislik

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.

Tükürük hipofonksiyonunun bir Sıçan Modelinde Retroductal Submandibuler Bezi damlatma ve yerelleştirilmiş fraksiyonlara Işınlama

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.

Tükürük bezi işlevi farelerde ölçülmesi için bir yöntem

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

İmmünoloji ve Enfeksiyon

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.

Submandibular ve parotis tükürük bezlerinden Organotipik kültürlerin radyasyon tedavisi anahtar In vivo özellikleri

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

Fare Submandibular Bez Rejenerasyonunun Gerçek Zamanlı Canlı Görüntülenmesi

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.

Yazar Spotlight: Yaralanma Sonrası Tükürük Bezi Rejenerasyonunda Makrofaj-Epitel Hücre Etkileşimlerinin İncelenmesi 

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.

Tükürük bezindeki hücre-hücre etkileşimlerinin ex vivo canlı hücre görüntüleme ile sorgulanması

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