Investigación en esta publicación fue apoyada por el Instituto Nacional de odontología y el Instituto Nacional de cáncer (NCI) de los institutos nacionales de salud Premio número R56 DE025098, DE027695 UG3 y F30 CA206296 Craniofacial Research (NIDCR). El contenido es responsabilidad exclusiva de los autores y no representan necesariamente las opiniones oficiales de los institutos nacionales de salud. Este trabajo también fue apoyado por la NSF DMR 1206219 y la innovación de la IADR en Premio de Cuidado Oral (2016).
Nos gustaría agradecer al Dr. Eri Maruyama y Andrew Hollomon su asistencia con la colección de saliva. Nos gustaría agradecer su ayuda con la disección de la glándula Weng Pei-Lun. Nos gustaría agradecer a Matthew Ingalls por su ayuda en la preparación de la figura. Nos gustaría agradecer al Dr. Elaine Smolock y Emily Wu lectura crítica de este manuscrito.

Todo en vivo los procedimientos descritos a continuación fueron aprobados por el Comité de la Universidad sobre los recursos de la Universidad de Rochester, Rochester. NUEVA YORK.
1. preparación
<ol><li>Utilizando una balanza analítica, pesan 20 mg de pilocarpina. Disolver en 2 mL de solución salina estéril en un tubo de microcentrífuga. Nota: Porque pilocarpina es sensible a la luz y pierde actividad con el tiempo, esta solución debe ser preparada el día de la inyección y protegida de la luz hasta que administra.</li>
	<li>Utilizando una balanza analítica, pesar e identificar todos los tubos y capilares de vidrio. El número de tubos y capilares de vidrio debe coincidir con el número de ratones.</li>
</ol>2. traqueostomía
<ol><li>Pesar ratones C57/BL6 utilizando una balanza analítica.</li>
	<li>Utilizando una jeringa de 0.5 mL con aguja 29 x ½", anestesiar ratones con una solución salina estéril inyectada por vía intraperitoneal de 100 mg/kg ketamina y 10 mg/kg xilacina. Proceder al paso siguiente cuando el ratón deja de responder a los estímulos (es decir, ausencia del reflejo pedal retirada), que ocurre generalmente dentro de 5 a 10 minutos después de la inyección.</li>
	<li>Después de dispensar lubricante en un aplicador con punta de algodón, suavemente aplique a los ojos y coloque el ratón en una posición supina en el escenario. El animal anestesiado debe mantenerse en la superficie caliente. Nota: Todas las medidas se realizan a temperatura ambiente.</li>
	<li>Asegure la nariz, extremidades y cola a la etapa con cinta quirúrgica.</li>
	<li>Limpiar y mojar el cuello con un algodón empapado en alcohol. Nota: Esto ayudará a prevenir la piel entren en incisiones posteriores.</li>
	<li>Levantando la piel del cuello del midline ventral con pinzas, hacer un corte pequeño y superficial con tijeras de disección. Guía de las tijeras en la abertura y abra lentamente las hojas por vía subcutánea para separar tejidos planos.</li>
	<li>Mantener la piel levantada, cuidadosamente corte a 1 cm por debajo de la boca.</li>
	<li>Hacer dos incisiones laterales en los aspectos inferior y superiores del primer corte. Suavemente con unas pinzas, reflejan a la piel para permitir el acceso a las estructuras de cabeza y cuello.</li>
	<li>Usando el microscopio de disección con 8 aumentos, visualizar las glándulas submaxilar (media: figura 1).</li>
	<li>Con unas pinzas finas, levante suavemente las glándulas para exponer los cuatro músculos del infrahyoid (correa) que cubre la tráquea. Evitar desgarros o interrumpir la vasculatura circundante o conductos excretores.</li>
	<li>Si recoger saliva de más de un ratón, repita los pasos 2.1-2.10 con cada ratón antes de continuar. Nota: Este procedimiento puede realizar con seguridad en hasta 5 ratones al mismo tiempo.</li>
	<li>Con pequeñas tijeras de disección, quitar la porción medial de los músculos de la correa durante su estancia lo más cerca posible de la línea media. Corte solamente tanto como sea necesario visualizar la tráquea y mantener los músculos de la correa del camino durante el procedimiento. Evitar mellar cualquier buques cercanos porque si hay depleción de volumen secundaria a hemorragia significativa, pilocarpina no será eficaz en la inducción de secreción.</li>
	<li>Reflejo de los músculos de la correa de la tráquea.</li>
	<li>Visualizar la laringe, la tráquea y la glándula tiroides. Asegúrese de que la tráquea esté libre de tejido sobrepuesto.</li>
	<li>Hacer una incisión horizontal en la tráquea inferior posterior de la tiroides usando pequeñas tijeras de disección. Asegurar la vía aérea patente y claro del líquido.</li>
</ol>3. saliva colección
<ol><li>Retire la cinta junto a la boca y el ángulo de fase de disección hacia abajo (45°, cranially) para ayudar a flujo de la saliva.</li>
	<li>Utilizando una jeringa de 0.5 mL con aguja 29 x ½", realizar la inyección intraperitoneal de 10 μl/g cuerpo peso la pilocarpina para una dosis total de 100 mg/kg.</li>
	<li>Iniciar un temporizador. Si la dosis múltiples ratones, reducir tiempo moviendo rápidamente y por tener un ayudante inyecta simultáneamente los ratones restantes.</li>
	<li>Con unas pinzas estándar, abrir la boca para el acceso del tubo capilar.</li>
	<li>Una vez que se observa una gota de saliva en la boca, coloque el extremo proximal del tubo capilar en el líquido, con el extremo distal se coloca en un tubo colector colocado debajo de la etapa de disección (ambos tubos previamente pesados). Nota: En los ratones hembra C57/BL6 6-10 semanas de edad, bruta salivación ocurre dentro de 2 minutos de la inyección de pilocarpina.</li>
	<li>Si saliva en la boca del edificio, pero no fluye a través del tubo, reintentar el paso anterior. Asegúrese de que el tubo no esté directamente contra una superficie mucosa, que puede obstruir la entrada.</li>
	<li>Recoger saliva para un total de 12 minutos después del estímulo la pilocarpina. Asegúrese de que el estoma permanece claro. Nota: Aumento del volumen tidal y secreciones (salivales, urinarias, fecales) son normales durante este tiempo.</li>
	<li>Si los ratones han presionado función de saliva (ej., debido a lesión o intervención), transferir el líquido restante de la boca en el tubo de la colección mediante una micropipeta de P200. Si recogida de varios ratones, asegúrese de que los consejos han cambiado.</li>
	<li>Registrar el peso de saliva recogida más tubos 12 min después de la inyección de pilocarpina.</li>
</ol>4. glándula disección
<ol><li>Eutanasia a ratones por CO2 eutanasia (como se describe en las pautas de AVMA de eutanasia de los animales: edición 2013) seguido de toracotomía, teniendo cuidado de preservar las estructuras de la cabeza y el cuello. Por esta razón, evitar la dislocación cervical como método de eutanasia.</li>
	<li>Coloque el ratón en una posición supina en el escenario. Vuelva a colocar las glándulas en el sitio original sobre la tráquea (figura 1).</li>
	<li>Utilizando un microscopio de disección bajo 8 aumentos, visualizar las glándulas principales: el PG, SMG y SLG. Para distinguir las glándulas durante la disección, identificar que el PG es difusa, lateral a la SMG y SLG. Nota: Si el PG de disección, esto debe hacerse en primer lugar porque es el menos definido y más probable ser rasgado.</li>
	<li>Agarre la cola de la PG con pinzas, tirando lejos de las estructuras subyacentes y suavemente aplique tensión antes de cortar la cabeza de la PG con tijeras de disección.</li>
	<li>Incumplimiento la cápsula que rodea el metralletas con unas pinzas. Separe suavemente el SMG de izquierda y derecho.</li>
	<li>Libre de la cara dorsal de la SMG del tejido nonglandular adherido con unas pinzas.</li>
	<li>Retire el SMG del cuello tenso del conducto de Wharton con pinzas y corte el tubo con tijeras de disección.</li>
	<li>Separe suavemente el SLG de SMG con unas pinzas. Nota: La SLG es en el aspecto superolateral del SMG.</li>
	<li>Con una balanza analítica, registre pesos SMG.</li>
</ol>

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Los autores no tienen nada que revelar.

Presentamos un método de varios pasos para evaluar la función de la glándula salival, que puede ser aplicada para el estudio de lesiones de la glándula y terapéutica. Nuestro procedimiento consiste en traqueostomía, la recolección de saliva y disección de la glándula, que tiene aplicaciones experimentales que soporten un estudio integrado de la biología de la glándula salival. Por ejemplo, traqueotomía murino se ha utilizado para la gestión general de la vía aérea durante procedimientos de obstrucción de ...

Trastornos de la glándula salival incluyen síndromes de altera o deteriora la secreción hacia la superproducción (sialorrea) o insuficiente (xerostomía e hiposalivación) de saliva1. En ambos casos, hay un interés por mejorar nuestra comprensión de la biología de la glándulas salivales hacia la meta final del desarrollo terapéutico2.
Las glándulas salivales son órganos altamente radiosensibles y a menudo se dañan durante la radioterapia de cáncer cabeza y cuello, hacia la permanente boca seca (xerostomía)3,4. Sin embargo, a diferencia de otros tejidos radiosensibles, índice de rotación de la glándula salival es relativamente baja, y el mecanismo de la pérdida de secreción es mal entendido5,6. En este escenario de lesiones únicas, estrategias de regeneración y protección radiológica de tejido requieren evaluación funcional salival. Experimentalmente, colección de saliva murino es una herramienta especialmente valiosa en la evaluación de la respuesta de la glándula a la radiación y los agentes terapéuticos.
Aquí, presentamos un método para realizar y cuantificar la secreción de saliva estimulada con pilocarpina, un potente agonista muscarínico7. La pilocarpina estimula el sistema nervioso autónomo para inducir la secreción de la glándula8,9. Para completar correctamente esta prueba, una traqueotomía se requiere para asegurarse de que el ratón mantenga una vía aérea patente durante todo el procedimiento y para reducir los riesgos de asfixia y aspiración de secreciones agrupadas en la cavidad oral10.
Este es un procedimiento terminal, culminando en la eliminación de las tres glándulas salivales mayores: parótida (PG), el submaxilar (SMG) y sublingual (SLG). Para los estudios funcionales, glándula pesos se registran y se utilizan a menudo para normalizar saliva medición11,12,13. Esta información es particularmente importante en estudios de radiación, en la que la atrofia de la glándula es un resultado esperado14,15
Hay variabilidad en la literatura con respecto a la secreción de saliva estimulada cómo se realiza y divulga16. Por ejemplo, dosis de pilocarpina en la literatura palmo por lo menos tres órdenes de magnitud17,18,19,20,21,22,23. Aquí, presentamos un protocolo de pilocarpina optimizado de dosis alta con la intención de mejora reproducibilidad en ejecución método, así como proporcionar una plataforma modular de técnicas (traqueostomía, la recolección de saliva y disección de la glándula) que pueden ser adaptados como necesario.
Además de la demostración del Protocolo, incluimos datos funcionales representativos de flujo de la saliva a las 2 semanas después de la radiación fraccionada (4 dosis de 6.85 Gy) a la región SMG.

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			<td height="21" style="height:21px;width:219px;">Pilocarpine hydrochloride</td>
			<td style="width:128px;">Sigma Aldrich</td>
			<td style="width:119px;">P6503</td>
			<td style="width:225px;">Pilocarpine</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Student Vannas Spring Scissors</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">91500-9</td>
			<td>Spring Scissors for Tracheostomy</td>
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			<td height="21" style="height:21px;width:219px;">Sterile Saline Solution</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">RDI30296H</td>
			<td>Saline</td>
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			<td height="19" style="height:19px;width:219px;">Dumont #7 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11274-20</td>
			<td>Curved Forceps</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:219px;">Dumont #5 Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">11251-10</td>
			<td>Straight Forceps</td>
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			<td height="17" style="height:17px;width:219px;">Standard Pattern Forceps</td>
			<td style="width:128px;">Fine Science Tools</td>
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			<td>Blunt Forceps</td>
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			<td height="21" style="height:21px;width:219px;">Fine Scissors- Tungsten Carbide</td>
			<td style="width:128px;">Fine Science Tools</td>
			<td style="width:119px;">14568-09</td>
			<td>Dissection Scissors</td>
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			<td height="42" style="height:42px;width:219px;">Microhematocrit Heparinized Capillary Tubes</td>
			<td style="width:128px;">Fisher Scientific</td>
			<td style="width:119px;">22362566</td>
			<td>Capillary tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">Lubricant Eye Ointment</td>
			<td style="width:128px;">Refresh</td>
			<td style="width:119px;">N/A</td>
			<td>Refresh Lacri-Lube</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">Goat polyclonal anti-Nkcc1</td>
			<td style="width:128px;">Santa Cruz Biotech</td>
			<td style="width:119px;">SC-21545</td>
			<td>Nkcc1 Antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:128px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td>DAPI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:219px;">GraphPad Prism</td>
			<td style="width:128px;">GraphPad</td>
			<td style="width:119px;">ver6.0</td>
			<td>Statistical Software</td>
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			<td height="21" style="height:21px;width:219px;">Cotton tipped applicator</td>
			<td style="width:128px;">Medline</td>
			<td style="width:119px;">MDS202000</td>
			<td>Applicator for eye ointment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:219px;">0.5cc Insulin Syringe, 29G x 1/2&#34;</td>
			<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

Hiposalivación se observa comúnmente en la reacción autoinmune síndrome de Sjögren o siguiente lesión por radiación a las glándulas salivales mayores. En estos casos, quedan preguntas con respecto a la patogénesis de la enfermedad y las intervenciones eficaces. Una técnica optimizada que permite la evaluación funcional de las glándulas salivales es muy valiosa para la investigación terapéutica, disfunción y biología de la glándula exocrine. Aquí, presentamos un enfoque paso a paso para realizar la pilocarpina estimula la secreción de saliva, incluyendo la disección de las glándulas salivales murinas principales tres y traqueostomía. También detallamos la anatomía cabeza y cuello murina apropiada accedida durante estas técnicas. Este enfoque es escalable, permitiendo múltiples ratones ser procesados simultáneamente, mejorando así la eficiencia del flujo de trabajo. Nuestro objetivo es mejorar la reproducibilidad de estos métodos, cada uno de ellos tiene más aplicaciones en el campo. Además de la colección de saliva, discutimos métricas para cuantificar y normalización de la capacidad funcional de estos tejidos. Datos representativos se incluyen de las glándulas submaxilares con función deprimida de la glándula salival 2 semanas después de la radiación fraccionada (4 dosis de 6.85 Gy).

Cuando realizar pilocarpina dosis altas estimula la colección de saliva, es importante mantener la vía aérea para prevenir la aspiración o asfixia de las secreciones en la cavidad bucal. Un esquema de la traqueotomía se proporciona (figura 1). Tras la incisión traqueal, el estoma debe permanecer claro de tejido y fluidos.
Para mejorar la acción capilar durante la recogida de saliva, ratones d...

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

Evaluación funcional salival murino mediante pilocarpina estímulo siguiente fraccionado radiación

Presentamos un enfoque detallado para realizar la colección de saliva, incluyendo traqueotomía murino y el aislamiento de tres glándulas salivales mayores.

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 vistas.  University of Rochester. Presentamos un enfoque detallado para realizar la colección de saliva, incluyendo traqueotomía murino y el aislamiento de tres glándulas salivales mayores.

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.

Canulación de la glándula salival submaxilar del ratón a través del conducto 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 ...

Investigación

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.

Un modelo murino de entrenamiento de los músculos mediante la estimulación eléctrica 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.

Recopilación de la saliva y la medición de cortisol salival y alfa-amilasa en Frail comunitarios residentes adultos mayores a través de Cuidadores de Familia

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 de miocardio y la evaluación de resultados funcionales en los cerdos

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.

El control de la enfermedad de Parkinson con Adaptive estimulación 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.

Espectroscopia óptica difusa para la evaluación cuantitativa de la radiación ionizante aguda La toxicidad cutánea inducida utilizando un modelo de ratón

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 Glándula instilación y con localización fraccionado irradiación en un modelo de rata de hipofunción salival

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.

Evaluación electrofisiológica de Atria murino con mapeo óptico alta resolución

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.

Desarrollo y caracterización funcional de células dendríticas tolerogénicas 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.

Tratamiento de radiación de cultivos organotípico de glándulas salivales submandibulares y parótidas modelos clave en 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 ...