Pesquisa reportada nesta publicação foi apoiada pelo Instituto Nacional de dentária e Craniofacial Research (NIDCR) e o National Cancer Institute (NCI) do institutos nacionais da saúde sob prêmio número R56 DE025098, DE027695 UG3 e CA206296 F30. O conteúdo é exclusivamente da responsabilidade dos autores e não representa necessariamente a opinião oficial do institutos nacionais da saúde. Este trabalho também foi apoiado pela NSF DMR 1206219 e a inovação IADR no prêmio de cuidado Oral (2016).
Gostaríamos de agradecer o Dr. Eri Maruyama e Andrew Hollomon por sua ajuda com a coleta de saliva. Gostaríamos de agradecer sua assistência com dissecação de glândula Pei-Lun Weng. Gostaríamos de agradecer a Matthew Ingalls por sua assistência na preparação de figura. Gostaríamos de agradecer o Dr. Elaine Smolock e Emily Wu para leitura crítica deste manuscrito.

Todos na vivo os procedimentos descritos abaixo foram aprovados pela Comissão Universidade recursos animais na Universidade de Rochester, Rochester. NY.
1. preparação
<ol><li>Usando uma balança analítica, pese 20 mg de pilocarpina. Dissolvê-lo em 2 mL de solução salina estéril em um tubo de microcentrifugadora. Nota: Porque pilocarpina é sensível à luz e perde atividade ao longo do tempo, esta solução deve ser preparada no dia da injeção e protegida da luz até administrado.</li>
	<li>Usando uma balança analítica, pesar e identificar todos os tubos de coleção e capilares de vidro. O número de tubos de coleção e capilares de vidro deve corresponder ao número de ratos.</li>
</ol>2. traqueostomia
<ol><li>Pese de camundongos C57/BL6 usando uma balança analítica.</li>
	<li>Utilizando uma seringa de 0,5 mL com agulha 29 x ½", anestesia os ratos com uma solução salina estéril intraperitonealmente injetado de 100 mg/kg quetamina e 10 mg/kg de xilazina. Prossiga para a etapa seguinte quando o mouse não responde mais aos estímulos (ou seja, ausência do reflexo de retirada de pedal), que geralmente ocorre dentro de 5 a 10 min após a injeção.</li>
	<li>Depois da preparação de lubrificante em um aplicador com ponta de algodão, delicadamente aplicam-se aos olhos e posicione o mouse em uma posição supina no palco. O animal anestesiado deve ser mantido na superfície quente. Nota: Todas as etapas são realizadas à temperatura ambiente.</li>
	<li>Proteger o nariz, os membros e cauda no palco usando fita cirúrgica.</li>
	<li>Limpar e molhar a garganta com uma compressa com álcool. Nota: Isto vai ajudar a impedir a entrada de incisões subsequentes de peles.</li>
	<li>Levantar a pele do pescoço na linha média ventral com fórceps, fazer um corte pequeno, superficial, usando tesouras de dissecação. Orientar a tesoura na abertura e abra lentamente as lâminas por via subcutânea para separar tecido aviões.</li>
	<li>Mantendo a pele levantada, cuidadosamente, corte 1 cm abaixo da boca.</li>
	<li>Faça duas incisões laterais em aspectos inferiores e superiores do primeiro corte. Usando fórceps, delicadamente afastado refletem a pele para permitir o acesso a estruturas de cabeça e pescoço.</li>
	<li>Usando o microscópio de dissecação na ampliação de X 8, Visualizar as glândulas submandibulares (mediana: Figura 1).</li>
	<li>Usando a pinça fina, levante suavemente as glândulas para expor os quatro músculos infra-hioideos (cinta) sobrejacente a traqueia. Evite rasgar ou interromper a vasculatura circundante ou ductos excretores.</li>
	<li>Se a coleta de saliva de mais de um rato, repita passos 2.1-2.10 com cada rato antes de continuar. Nota: Este procedimento pode ser realizado com segurança em até 5 ratos simultaneamente.</li>
	<li>Com tesouras pequenas de dissecação, remova a porção medial dos músculos enquanto permanecer tão próximo quanto possível na linha mediana. Só corte quanto for necessário para visualizar a traqueia e manter músculos fora do caminho durante o procedimento. Evite roubar qualquer navios nas proximidades, porque se houver depleção de volume secundário a hemorragia significativa, a pilocarpina não será eficaz na indução de secreção.</li>
	<li>Refletem os músculos da cinta da traqueia.</li>
	<li>Visualize a laringe, traqueia e glândula tireoide. Certifique-se de que a traqueia é clara de sobrejacente tecido.</li>
	<li>Faça uma incisão horizontal na traqueia inferior/posterior a tireoide usando tesouras pequenas de dissecação. Certifique-se de que as vias aéreas é patente e clara de fluido.</li>
</ol>3. saliva coleção
<ol><li>Remova a fita perto palco de dissecação boca e ângulo para baixo (45°, cranialmente) para ajudar com o fluxo de saliva.</li>
	<li>Utilizando uma seringa de 0,5 mL com agulha 29 x ½", execute a injeção intraperitoneal de 10 pilocarpina de peso de corpo µ l/g para uma dose total de 100 mg/kg.</li>
	<li>Inicie um timer. Se vários mouses de dosagem, reduza o tempo de chumbo movendo-se rapidamente e por ter um assistente injetar simultaneamente os ratos restantes.</li>
	<li>Usando pinças padrão, abra a boca para acesso de tubo capilar.</li>
	<li>Depois de observa-se uma gota de saliva na boca, coloca a extremidade proximal do tubo capilar no fluido, com a extremidade distal, colocada em um tubo de coletando posicionado abaixo do palco dissecação (ambos os tubos pre-pesados). Nota: Em ratos fêmeas C57/BL6 6-10 semanas de idade, salivação bruta ocorre dentro de 2 min de injeção de pilocarpina.</li>
	<li>Se saliva está construindo na boca, mas não flui através do tubo, tente novamente o passo anterior. Certifique-se de que o tubo não é colocado diretamente contra uma superfície da mucosa, que pode obstruir a entrada.</li>
	<li>Colete a saliva para um total de 12 min após estimulação de pilocarpina. Garantir que o estoma permanece claro. Nota: Aumento do volume tidal e secreções (salivares, urinárias, fecais) são normais durante este tempo.</li>
	<li>Se os ratos têm deprimido função de saliva (ex., devido a lesão ou a intervenção), transfira o líquido restante da boca para o tubo de coleta usando uma micropipeta P200. Se a recolha de vários mouses, certifique-se de que as dicas são alteradas.</li>
	<li>Registro do peso da saliva coletada mais tubos de 12 min após a injeção de pilocarpina.</li>
</ol>4. a glândula dissecação
<ol><li>Eutanásia em ratos pela eutanásia de CO2 (conforme descrito nas orientações AVMA para eutanásia de animais: edição de 2013) seguido por toracotomia, tendo o cuidado de preservar as estruturas da cabeça e do pescoço. Por esta razão, evite deslocamento cervical como um método de eutanásia.</li>
	<li>Coloque o mouse em uma posição supina no palco. Reposicione as glândulas no site original sobre a traqueia (Figura 1).</li>
	<li>Usando um microscópio dissecação sob magnificação de X 8, Visualizar as glândulas maiores: o PG, SMG e SLG. Para distinguir as glândulas durante a dissecção, identifica que o PG é difuso, lateral para a SMG e SLG a mentir. Nota: Se a dissecar o PG, isto deve ser feito primeiro porque é o menos definidas e mais susceptível de ser rasgado.</li>
	<li>Agarrar a cauda do PG com fórceps, puxando-o longe de estruturas subjacentes e aplicar suavemente a tensão antes de cortar a cabeça do PG com tesoura de dissecação.</li>
	<li>Ruptura da cápsula que rodeia os SMGs usando fórceps. Separe delicadamente SMG a torto e a direito.</li>
	<li>Livre o dorso da SMG de tecido nonglandular aderido, usando fórceps.</li>
	<li>Remova o SMG do pescoço pelo ducto de Wharton tenso de desenho com fórceps e cortar o duto com tesoura de dissecação.</li>
	<li>Suavemente separa o SLG a SMG usando fórceps. Nota: A SIG é o aspecto súpero a SMG.</li>
	<li>Usando uma balança analítica, pesos registros de SMG.</li>
</ol>

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Os autores não têm nada para divulgar.

Apresentamos um método várias etapas para avaliar a função de glândula salivar, que pode ser aplicada para estudar terapêutica e lesão da glândula. Nosso procedimento envolve a traqueostomia, coleta de saliva e dissecação de glândula, cada qual com aplicações experimentais que podem suportar um estudo integrado da biologia de glândula salivar. Por exemplo, traqueostomia murino tem sido usada para gestão geral das vias respiratórias durante os procedimentos de obstruir a cavidade oral.
<p class="jove_c...

Distúrbios da glândula salivar incluem síndromes de desregulação ou secreção prejudicada, levando a subprodução (xerostomia e hipossalivação) de saliva1ou superprodução (sialorrhea). Em ambos os casos, há interesse em melhorar a nossa compreensão da biologia da glândula salivar com o objectivo final de desenvolvimento terapêutico2.
As glândulas salivares são órgãos altamente radiosensitive e muitas vezes são danificadas durante a radioterapia de câncer de cabeça e pescoço, levando a permanente boca seca (xerostomia)3,4. Ao contrário de outros tecidos radiosensitive, no entanto, a taxa de rotatividade das glândulas salivares é relativamente baixa, e o mecanismo de perda secretora é mal compreendido5,6. Neste cenário de lesão única, estratégias de regeneração e protecção contra as radiações de tecido requerem avaliação funcional salivar. Experimentalmente, coleta de saliva murino é uma ferramenta particularmente valiosa em avaliar a resposta de glândula a radiação e agentes terapêuticos.
Aqui, apresentamos um método para executar e quantificar a secreção de saliva estimulada usando pilocarpine, um potente agonista muscarínico7. Pilocarpina estimula o sistema nervoso autônomo para induzir a secreção de glândula8,9. Para completar este teste apropriadamente, uma traqueostomia é necessária para garantir que o rato mantém uma via aérea patente durante todo o procedimento e para reduzir os riscos de asfixia e aspiração de secreções em pool na cavidade oral10.
Este é um procedimento terminal, culminando na remoção de uma das três principais glândulas salivares: as parótidas (PG), a submandibular (SMG) e a sublingual (SLG). Para estudos funcionais, glândula pesos são registados e são frequentemente usados para normalizar a saliva medição11,12,13. Este dados são particularmente importantes em estudos de radiação, em que a atrofia da glândula é um resultado esperado14,15
Há variabilidade na literatura em relação à secreção de saliva como estimulada é executada e relatou16. Por exemplo, doses de pilocarpina dentro da literatura extensão pelo menos três ordens de magnitude17,18,19,20,21,22,23. Aqui, apresentamos um protocolo de pilocarpina otimizada de alta dose com a intenção de melhor reprodutibilidade na execução do método, bem como fornecendo uma plataforma modular de técnicas (traqueostomia, coleta de saliva e dissecação de glândula) que pode ser adaptado como precisava.
Além de demonstração de protocolo, incluímos dados funcionais representativos do fluxo de saliva em 2 semanas seguintes à radiação fracionada (4 doses de Gy 6.85) para a região SMG.

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			<td>Capillary tubes</td>
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			<td>Refresh Lacri-Lube</td>
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			<td height="42" style="height:42px;width:219px;">Goat polyclonal anti-Nkcc1</td>
			<td style="width:128px;">Santa Cruz Biotech</td>
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			<td>Nkcc1 Antibody</td>
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			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>DAPI</td>
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			<td height="21" style="height:21px;width:219px;">GraphPad Prism</td>
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			<td style="width:119px;">ver6.0</td>
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			<td>Applicator for eye ointment</td>
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			<td style="width:128px;">BD</td>
			<td style="width:119px;">7629</td>
			<td>Syringe for intraperitoneal injection</td>
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murine salivary functional assessment via pilocarpine stimulation following fractionated radiation

Hipossalivação é comumente observada na reação auto-imune da síndrome de Sjögren ou seguir lesão de radiação para as glândulas salivares maiores. Nestes casos, perguntas permanecem sobre a patogênese da doença e de intervenções eficazes. A técnica otimizada que permite a avaliação funcional das glândulas salivares é inestimável para investigação terapêutica, disfunção e biologia de glândula exócrina. Aqui, apresentamos uma abordagem passo a passo para executar pilocarpina estimulou a secreção de saliva, incluindo a traqueostomia e a dissecação das três principais murino glândulas salivares. Também detalhamos a anatomia cabeça e pescoço murino apropriada acessada durante estas técnicas. Esta abordagem é escalável, permitindo vários mouses para serem processados simultaneamente, melhorando assim a eficiência do fluxo de trabalho. Nosso objetivo é melhorar a reprodutibilidade destes métodos, cada qual tem mais aplicativos dentro do campo. Além da recolha de saliva, discutimos métricas para quantificar e normalizando a capacidade funcional desses tecidos. Dados representativos são incluídos a partir de glândulas submandibulares com função deprimida glândula salivar 2 semanas seguintes à radiação fracionada (4 doses de 6.85 Gy).

Quando realizar pilocarpina dose alta estimulada coleta de saliva, é importante manter as vias aéreas para evitar aspiração ou asfixia de secreções na cavidade oral. Um esquema da traqueostomia é fornecido (Figura 1). Após incisão traqueal, o estoma deve ficar claro de tecidos e fluidos.
Para aprimorar a ação capilar durante a coleta de saliva, ratos devem ser posicionados de cabeça para...

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

Murino salivar avaliação funcional através de pilocarpina estimulação seguinte fracionada de radiação

Apresentamos uma abordagem detalhada para realizar coleta de saliva, incluindo murino traqueostomia e o isolamento dos três glândulas salivares maiores.

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

Research

JoVE Journal

Medicine

11.0K visualizações.  University of Rochester. Apresentamos uma abordagem detalhada para realizar coleta de saliva, incluindo murino traqueostomia e o isolamento dos três glândulas salivares maiores.

Vídeo: 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.

Canulação da glândula submandibular do rato através do duto salivar de Wharton

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

Medicina

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.

Um modelo murino de treinamento muscular por eletroestimulação neuromuscular

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.

Infarto do Miocárdio e Avaliação de Resultados Funcional em porcos

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

Controlling Parkinson's Disease With Adaptive Deep Brain Stimulation

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time according to fluctuating disease and medication state. In the present realization of adaptive DBS we record and stimulate from the DBS electrodes implanted in the subthalamic nucleus of patients with Parkinson&#39;s disease in the early post-operative period. Local field potentials are analogue filtered between 3 and 47 Hz before being passed to a data acquisition unit where they are digitally filtered again around the patient specific beta peak, rectified and smoothed to give an online reading of the beta amplitude. A threshold for beta amplitude is set heuristically, which, if crossed, passes a trigger signal to the stimulator. The stimulator then ramps up stimulation to a pre-determined clinically effective voltage over 250 msec and continues to stimulate until the beta amplitude again falls down below threshold. Stimulation continues in this manner with brief episodes of ramped DBS during periods of heightened beta power.
Clinical efficacy is assessed after a minimum period of stabilization (5 min) through the unblinded and blinded video assessment of motor function using a selection of scores from the Unified Parkinson&#39;s Rating Scale (UPDRS). Recent work has demonstrated a reduction in power consumption with aDBS as well as an improvement in clinical scores compared to conventional DBS. Chronic aDBS could now be trialed in Parkinsonism.

Adaptive deep brain stimulation (aDBS) is effective for Parkinson&#8217;s disease, improving symptoms and reducing power consumption compared to conventional deep brain stimulation (cDBS). In aDBS we track a local field potential biomarker (beta oscillatory amplitude) in real time and use this to control the timing of stimulation.

Controlar a doença de Parkinson com Adaptive Estimulação Cerebral Profunda

Adaptive deep brain stimulation (aDBS) has the potential to improve the treatment of Parkinson&#39;s disease by optimizing stimulation in real time ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Difusa espectroscopia óptica para a avaliação quantitativa da Aguda de Radiação Ionizante toxicidade cutânea induzida pelo uso de um modelo de rato

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

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.

Retroductal submandibular Glândula instilação e localizada fracionado irradiação em um modelo de rato de salivar Hipofunção

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

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Avaliação eletrofisiológica de murino átrios com mapeamento óptico de alta resolução

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Development and Functional Characterization of Murine Tolerogenic Dendritic Cells

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating na&#239;ve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.

Here, we present a protocol to develop and characterize tolerogenic dendritic cells (TolDCs) and evaluate their immunotherapeutic utility.

Desenvolvimento e caracterização funcional de células dendríticas Tolerogenic murino

The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state ...

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.

Tratamento de radiação de culturas organotypic de glândulas salivares submandibulares e parótidas modelos chave in vivo características

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