Nos gustaría agradecer el apoyo de EE.UU. Institutos Nacionales de Salud subvenciones R01NS057476, P50NS010828, P01NS055104, R01EB000790, K99NS067050, R01HL081273 y R01EB007279 y la American Heart Association conceder 0855772D.

1. Amplio campo de visión de imágenes de la pO2 en la vasculatura cortical de una rata <ol><li> Las ratas (250 g -350 g) son inicialmente anestesiados con isoflurano, y la arteria femoral y la vena se cateteriza para controlar la frecuencia cardiaca, presión arterial y los gases en sangre, así como para la infusión intravenosa. La temperatura corporal se mantiene a 37 ± 0,1 ° C. Traqueotomía se realiza, y las ratas son ventilados con una mezcla de aire y oxígeno. </li><li> Las muestras de sangre para medir los gases en sangre se toman cada 30-45 minutos y la ventilación y la anestesia se ajustan para mantener los gases de la sangre dentro del rango fisiológico normal. </li><li> Una ventana cerrada del cráneo en el hueso parietal 4 mm x 4 mm de tamaño se prepara para la imagen. El hueso y la duramadre se eliminan y la ventana del cráneo está lleno de agarosa al 1,5% y se sella con un cubreobjetos microscopio. Un adicional de 1 mm 2 trepanación en el hueso frontal se utiliza para inducir la CDS por microinyección intracortical de KCl (~ 10 l, 1 M). Deben extremarse las precauciones para evitar cualquier daño de los vasos corticales. El exceso de calor, presión mecánica, o breve período de hipoxia puede comprometer la barrera sanguínea del cerebro y causar fugas de la tintura de los vasos al espacio intersticial. </li><li> Una máscara de material ópticamente opaco se coloca alrededor de las ventanas del cráneo. El propósito de la máscara es la de absorber la señal de fosforescencia que viene de la tinta que se filtró en el tejido de los bordes de la materia dura. El medio de contraste en el espacio intersticial es la fuente de la fosforescencia muy brillante que puede echar a perder la medición. El aumento de brillo de la tinta en el espacio intersticial es el resultado de la acumulación de la tinta en el medio ambiente con baja presión de oxígeno y la baja velocidad de enfriamiento. El daño de los vasos en el experimento es por lo tanto, fácilmente reconocible por la aparición de manchas brillantes fosforescencia, en cuyo caso se rechaza los datos experimentales. </li><li> Después de terminar la cirugía, el isoflurano se interrumpe y la anestesia se pasó a alfacloralosa (50 mg / kg en bolo intravenoso seguido de 40 mg / (kg h) de infusión). </li><li> Animal es trasladado en un carro con la presión arterial ventilador, y controlar la temperatura. Mientras se respira el aire de la habitación, el animal se mueve rápidamente hacia la configuración de imagen y vuelve a conectar el medidor con la mezcla adecuada de aire y oxígeno. </li><li> El marco estereotáxico que tiene el animal se encuentre en el objetivo. La ventana del cráneo se centra en el campo de visión en el objetivo y se coloca paralela al plano focal. </li><li> El láser pulsado se enciende y se puso a la energía de pulso mínimo. El haz óptico se dirige hacia medidor de energía de pulso y pulso de energía entregada a la muestra se ajusta a no más de 10 mJ / cm 2. La ISS rayo centrado en la ventana del cráneo con un ángulo de incidencia oblicua de ~ 60 grados y la ISS láser apagado. </li><li> Mediciones de gases en sangre y los ajustes de la ventilación y la anestesia se llevan a cabo hasta que todos los parámetros fisiológicos de los animales estuvieron dentro del rango fisiológico normal. </li><li> Suponiendo que el volumen de sangre es de aproximadamente el 7% del peso corporal, una cantidad adecuada de la sonda R3 Oxyphor fosforescencia se disuelve en 1 ml de solución salina para alcanzar 4 x 10 -5 M la concentración sanguínea de la sonda. La solución de la sonda se inyecta a través de la vena femoral. </li><li> Una solución 1 M de cloruro de potasio se prepara y ~ 1 ul se inyecta con una jeringa a través de la trepanación en el hueso frontal para inducir a la CDS. </li><li> El láser se enciende y la imagen se inicia inmediatamente después de la inyección de la solución de KCl. Imágenes de fosforescencia se lleva a cabo durante aproximadamente 10 min. </li><li> Otra medición de gases en sangre se realiza para confirmar que los parámetros fisiológicos de los animales se encuentran dentro del rango normal. </li><li> Vidas fosforescencia se obtienen para todos los píxeles mediante el ajuste de la decadencia exponencial con una sola conexión cuadrados no lineal con ponderación estadística en Matlab. Fosforescencia vidas se convierten en los valores de pO2 con una empírica de Stern Volmer-como la relación (vide infra). </li></ol> 2. Alta resolución de dos fotones pO2 imagen en la vasculatura cortical micro de un ratón <ol><li> Los ratones se anestesian con la arteria femoral y isofluorene se cateteriza para controlar la frecuencia cardiaca, presión arterial y los gases en sangre, así como para la administración del medio de contraste. La temperatura corporal se mantiene a 37 ± 0,1 ° C y los animales respiran de manera espontánea una mezcla de aire y oxígeno. </li><li> Una ventana craneal se prepara de acuerdo a la técnica desarrollada originalmente por David Kleinfeld y Winfried Denk. Un buen cuidado se debe tomar para evitar cualquier daño de los vasos para evitar posibles fugas del medio de contraste en el espacio intersticial. </li><li> A pocas muestras de sangre para medir los gases en sangre se toman durante un procedimiento de preparación y de ventilación y anestesia se ajustan para mantener el gas en la sangrees dentro del rango fisiológico normal. </li><li> El animal se mueve a la configuración de imágenes. Un marco estereotáxico modificado que tiene el animal se encuentre en el objetivo. La ventana del cráneo se centra en el campo de visión del objetivo Olympus 4X y se coloca paralela al plano focal. </li><li> Una imagen de la ventana del cráneo es tomada con la cámara digital a través de los oculares, y el objetivo de 4X se reemplaza con el objetivo de Olympus 20X (NA = 0,95). </li><li> Mediciones de gases en sangre y los ajustes de la ventilación y la anestesia se llevan a cabo hasta que todos los parámetros fisiológicos de los animales dentro de un rango fisiológico normal. </li><li> Suponiendo que el volumen de sangre es de aproximadamente el 7% del peso corporal, una cantidad apropiada de la fosforescencia de la sonda PTP-C343 se disuelve en 0,2 ml de solución salina para alcanzar la concentración sanguínea de aproximadamente 15 M de la sonda. La solución de la sonda se inyecta a través de la arteria femoral. </li><li> Comenzar el experimento mediante el establecimiento de los intervalos de tiempo deseado para la excitación del tinte y la recolección de emisión en cada píxel. </li><li> Adquirir un escáner encuesta que es un proceso lento de 2 dimensiones barrido de trama de la decadencia intensidad fosforescencia que muestra la estructura vascular a la profundidad actual. El objetivo se mueve perpendicular a la ventana del cráneo (eje Z) y lento exploraciones estudio de la intensidad de fosforescencia en 2D se realizan usando la potencia del láser en un mínimo de 840 nm para encontrar los microvasos en el plano de la imagen. </li><li> En cada una profundidad de imagen, basado en la exploración encuesta de fosforescencia a esa profundidad, un conjunto de puntos dentro de la vasculatura son seleccionados, y el registro de fosforescencia decae en cada punto se repite para un número predefinido de promedio. </li><li> Establecer la cantidad deseada de un promedio, intervalo de medición, y la duración de la prueba y empezar la medición. PO 2 mediciones se adquieren en los lugares seleccionados en el intervalo de medición especificado para la duración del experimento. </li><li> Durante las mediciones, el software de re-dirige el láser de excitación de las ubicaciones seleccionadas por el cambio de la posición de los espejos galvanómetro de escaneado. Control de los escáneres galvanómetro, modulador óptico electro, y el resto del equipo se realizan por software a medida en LabView. </li><li> Vidas fosforescencia se obtienen todos los puntos seleccionados mediante el ajuste de la decadencia exponencial con una sola conexión cuadrados no lineales en Matlab. Fosforescencia vidas se convierten en los valores de pO2 con una empírica de Stern Volmer-como la relación (vide infra). </li><li> Después de recoger los datos en diferentes profundidades corticales, inyectar dextrano conjugado con fluoresceína en la vasculatura para obtener imágenes de la estructura de la microvasculatura. </li><li> Obtener una pila de imágenes estructurales de los vasos mediante la realización de la imagen de dos fotones de FITC fluorescencia utilizando un canal verde en el detector de cuatro canales. </li><li> Los experimentos con animales se realizaron de acuerdo con las directrices y regulaciones establecidas por el Subcomité Hospital General de Massachusetts en Investigación en Atención Animal. </li></ol> 3. Los resultados representativos: <img src="/files/ftp_upload/1694/1694fig1.jpg" alt="Figura 1" /> Figura 1. Panel (a) en el lado izquierdo de esta figura se muestra un amplio campo de la imagen de vista de la presión de oxígeno antes de la llegada de una ola de CSD. El panel (b) en el lado derecho muestra la evolución temporal de la presión de oxígeno promedio durante la propagación de la CDS en la región de interés marcados en el panel (a). Figura 2 (la película de AVI): Esta película muestra la evolución temporal de la presión de oxígeno en la ventana del cráneo entero durante la propagación de la onda de la CDS. Barra de escala indica la presión de oxígeno en milímetros de mercurio. <img src="/files/ftp_upload/1694/1694fig3.jpg" alt="Figura 3" /> Figura 3. Proyección en 3D de la pila de vasos imagen. Los tonos de gris representan una máscara de vasos volumétricos, creado en base a la imagen estructural. Medido valores de pO 2 están codificados por color. Barra de escala es de 200 micrómetros. Reproducido con permiso de Nature Publishing Group. 7

<ol>
	<li>Kleinfeld, D., Friedman, B., Lyden, P. D., Shih, A. Y., Chen, J., Xu, Z., Xu, X., Zhang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+occlusion+to+surface+and+deep+vessels+in+neocortex+via+linear+and+nonlinear+optical+absorption,+Animal+Models+of+Acute+Neurological+Injuries.">Targeted occlusion to surface and deep vessels in neocortex via linear and nonlinear optical absorption, Animal Models of Acute Neurological Injuries.</a> Contemporary Neuroscience Series. , (2007).</li><li>Mostany, R., Portera-Cailliau, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Craniotomy+Surgery+Procedure+for+Chronic+Brain+Imaging.">A Craniotomy Surgery Procedure for Chronic Brain Imaging.</a> J Vis Exp. , (2008).</li><li>Lebedev, A. Y., Cheprakov, A. V., Sakadzic, S., Boas, D. A., Wilson, D. F., Vinogradov, S. A., A, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+Phosphorescent+Probes+for+Oxygen+Imaging+in+Biological+Systems.">Dendritic Phosphorescent Probes for Oxygen Imaging in Biological Systems.</a> Applied Materials &#38; Interfaces. , (2009).</li><li>Finikova, O. S., Lebedev, A. Y., Aprelev, A., Troxler, T., Gao, F., Garnacho, C., Muro, S., Hochstrasser, R. M., Vinogradov, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxygen+microscopy+by+two-photon-excited+phosphorescence.">Oxygen microscopy by two-photon-excited phosphorescence.</a> Chemphyschem. 9, 1673-1679 (2008).</li><li>Sakad&#382;i&#263;, S., Yuan, S., Dilekoz, E., Ruvinskaya, S., Vinogradov, S. A., Ayata, C., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+imaging+of+cerebral+partial+pressure+of+oxygen+and+blood+flow+during+functional+activation+and+cortical+spreading+depression.">Simultaneous imaging of cerebral partial pressure of oxygen and blood flow during functional activation and cortical spreading depression.</a> Appl Opt. 48, D169-D177 (2009).</li><li>Yaseen, M. A., Srinivasan, V. J., Sakadzic, S., Wu, W., Ruvinskaya, S., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+monitoring+of+oxygen+tension+in+cortical+microvessels+with+confocal+microscopy.">Optical monitoring of oxygen tension in cortical microvessels with confocal microscopy.</a> Opt Express. 17, 22341-22350 (2009).</li><li>Sakadzic, S., Roussakis, E., Yaseen, M. A., Mandeville, E. T., Srinivasan, V. J., Arai, K., Ruvinskaya, S., Devor, A., Lo, E. H., Vinogradov, S. A., Boas, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+high-resolution+measurement+of+partial+pressure+of+oxygen+in+cerebral+vasculature+and+tissue.">Two-photon high-resolution measurement of partial pressure of oxygen in cerebral vasculature and tissue.</a> Nat Methods. 7, 755-759 (2010).</li></ol>

Hemos demostrado dos aplicaciones de la medición de pO2 en la microvasculatura cortical basada en el oxígeno-dependiente de extinción de la fosforescencia. Aunque el primer método basado en imágenes CCD ofrece amplio campo de vista del control pO2, la medición de la presión parcial de oxígeno en la microvasculatura cortical sobre la base de dos fotones microscopía capilar ofrece una resolución de imagen y permite en profundidad. Ambos métodos proporcionan alta velocidad y las mediciones de relación señal-ru...

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<td>Glycopyrrolate</td>
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<td>Alpha Chloralose</td>
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<td>Fluorescein isothio-cyanate&#x2013;dextran</td>
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cerebral blood oxygenation measurement based on oxygen-dependent quenching of phosphorescence

Seguimiento de las características espacio-temporales de la sangre cerebral y la oxigenación tisular es crucial para una mejor comprensión de la relación neuro-metabólico-vasculares. Desarrollo de nuevas modalidades de medición pO2 con un control simultáneo de pO2 en grandes campos de visión con mayor resolución espacial y / o temporal que permitirá una mayor penetración en el funcionamiento del cerebro normal y también tendrá un impacto significativo en el diagnóstico y el tratamiento de las enfermedades neurovasculares como accidente cerebrovascular, la enfermedad de Alzheimer, y lesiones en la cabeza. Modalidades de imágenes ópticas han demostrado un gran potencial para proporcionar alta resolución espacio-temporal y de imagen cuantitativa de pO2 basado en la absorción de la hemoglobina en el rango visible e infrarrojo cercano del espectro óptico. Sin embargo, la medición multiespectral de la oxigenación sanguínea cerebral se basa en la migración de fotones a través del tejido cerebral de alta dispersión. Estimación de parámetros y modelado de tejidos ópticos, que pueden sufrir cambios dinámicos durante el experimento, que normalmente se requiere una estimación precisa de la oxigenación de la sangre. Por otro lado, la estimación de la presión parcial de oxígeno (pO2) basada en el oxígeno-dependiente de extinción de la fosforescencia no debe verse afectado significativamente por los cambios en los parámetros ópticos de los tejidos y proporciona una medida absoluta de pO2. Sistemas experimentales que utilizan tintes sensibles al oxígeno se ha demostrado en estudios in vivo del tejido perfundido, así como para monitorear el contenido de oxígeno en cultivos de tejidos, demostrando que la fosforescencia de enfriamiento es una tecnología potente capaz de obtener imágenes precisas de oxígeno en el rango fisiológico pO2. Aquí se demuestra con dos modalidades de imágenes diferentes de cómo realizar la medición de la pO2 en la vasculatura cortical sobre la base de imágenes de toda la vida fosforescencia. En la primera demostración se presenta amplio campo de visión de imágenes de la pO2 en la superficie cortical de una rata. Esta modalidad de imagen tiene la configuración experimental relativamente simple basado en una cámara CCD y un láser pulsado verde. Un ejemplo de control de la propagación de la depresión cortical sobre la base de toda la vida fosforescencia de tinte R3 Oxyphor fue presentado. En segunda manifestación se presenta una alta resolución de dos fotones pO2 imagen en la vasculatura cortical micro de un ratón. El dispositivo experimental incluye una hecha a la medida de 2 fotones con microscopio láser de femtosegundo, modulador electro-óptico, y el fotón-contando tubo foto multiplicador. Se presenta un ejemplo de imagen de la heterogeneidad pO2 en la microvasculatura corticales incluyendo capilares, utilizando un nuevo punto a punto-C343 colorante con una mejora de dos fotones sección transversal de excitación. Haga clic aquí para ver el artículo relacionado <a href="http://www.jove.com/Details.php?ID=1731">Síntesis y caracterización de nanosondas fosforescentes de imágenes de oxígeno en los sistemas biológicos.</a>

Watch this Scientific Journal Video about Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence at JoVE.com

Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Se presenta un procedimiento experimental para la medición de la presión parcial de oxígeno (pO2) en la vasculatura cerebral basada en el oxígeno-dependiente de extinción de la fosforescencia. Preparación de animales y los procedimientos de imagen se describe, tanto para el campo visual amplio CCD basado en imágenes de la pO2 de las ratas y 2-fotones de imágenes basado en la excitación de la pO2 de ratones.

Medición de la oxigenación sanguínea cerebral basada en el oxígeno-dependiente de enfriamiento de fosforescencia

Monitoring of the spatiotemporal characteristics of cerebral blood and tissue oxygenation is crucial for better understanding of the 
...

cerebral-blood-oxygenation-measurement-based-on-oxygen-dependent

Research

JoVE Journal

Neuroscience

14.6K vistas.  Massachusetts General Hospital and Harvard Medical School. Se presenta un procedimiento experimental para la medición de la presión parcial de oxígeno (pO2) en la vasculatura cerebral basada en el oxígeno-dependiente de extinción de la fosforescencia. Preparación de animales y los procedimientos de imagen se describe, tanto para el campo visual amplio CCD basado en imágenes de la pO2 de las ratas y 2-fotones de imágenes basado en la excitación de la pO2 de ratones.

Video: Cerebral Blood Oxygenation Measurement Based on Oxygen-dependent Quenching of Phosphorescence

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body by mass, it utilizes approximately one quarter of the glucose used by the body at rest1. The function of non rapid eye movement sleep (NREMS), the largest portion of sleep by time, is uncertain. However, one salient feature of NREMS is a significant reduction in the rate of cerebral glucose utilization relative to wakefulness2-4. This and other findings have led to the widely held belief that sleep serves a function related to cerebral metabolism. Yet, the mechanisms underlying the reduction in cerebral glucose metabolism during NREMS remain to be elucidated.
	One phenomenon associated with NREMS that might impact cerebral metabolic rate is the occurrence of slow waves, oscillations at frequencies less than 4 Hz, in the electroencephalogram5,6. These slow waves detected at the level of the skull or cerebral cortical surface reflect the oscillations of underlying neurons between a depolarized/up state and a hyperpolarized/down state7. During the down state, cells do not undergo action potentials for intervals of up to several hundred milliseconds. Restoration of ionic concentration gradients subsequent to action potentials represents a significant metabolic load on the cell8; absence of action potentials during down states associated with NREMS may contribute to reduced metabolism relative to wake.
	Two technical challenges had to be addressed in order for this hypothetical relationship to be tested. First, it was necessary to measure cerebral glycolytic metabolism with a temporal resolution reflective of the dynamics of the cerebral EEG (that is, over seconds rather than minutes). To do so, we measured the concentration of lactate, the product of aerobic glycolysis, and therefore a readout of the rate of glucose metabolism in the brains of mice. Lactate was measured using a lactate oxidase based real time sensor embedded in the frontal cortex. The sensing mechanism consists of a platinum-iridium electrode surrounded by a layer of lactate oxidase molecules. Metabolism of lactate by lactate oxidase produces hydrogen peroxide, which produces a current in the platinum-iridium electrode. So a ramping up of cerebral glycolysis provides an increase in the concentration of substrate for lactate oxidase, which then is reflected in increased current at the sensing electrode. It was additionally necessary to measure these variables while manipulating the excitability of the cerebral cortex, in order to isolate this variable from other facets of NREMS.
	We devised an experimental system for simultaneous measurement of neuronal activity via the elecetroencephalogram, measurement of glycolytic flux via a lactate biosensor, and manipulation of cerebral cortical neuronal activity via optogenetic activation of pyramidal neurons. We have utilized this system to document the relationship between sleep-related electroencephalographic waveforms and the moment-to-moment dynamics of lactate concentration in the cerebral cortex. The protocol may be useful for any individual interested in studying, in freely behaving rodents, the relationship between neuronal activity measured at the electroencephalographic level and cellular energetics within the brain.

A procedure is described for manipulating the activity of cerebral cortical pyramidal neurons optogenetically while the electroencephalogram, electromyogram, and cerebral lactate concentration are monitored. Experimental recordings are performed on cable-tethered mice while they undergo spontaneous sleep/wake cycles. Optogenetic equipment is assembled in our laboratory; recording equipment is commercially available.

Electroencefalografía simultánea, en tiempo real Medición de la concentración de lactato y Manipulación optogenético de la actividad neuronal en la corteza cerebral de roedores

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Investigación

Neurociencias

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dos registros electrofisiológicos de sinápticamente respuestas evocadas neuronal y astroglial en agudas rodajas de hipocampo

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Un modelo de rata de hipoperfusión leve intrauterina con microcoil de transcatheter estenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Aislamiento de los capilares cerebrales del cerebro humano fresco tejido

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Quantitative Autoradiographic Method for Determination of Regional Rates of Cerebral Protein Synthesis In Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it is thought that dysregulation of protein synthesis in the nervous system may be a core phenotype in some developmental disorders. Accurate measurement of rates of cerebral protein synthesis in animal models is important for understanding these disorders. The method that we have developed was designed to be applied to the study of awake, behaving animals. It is a quantitative autoradiographic method, so it can yield rates in all regions of the brain simultaneously. The method is based on the use of a tracer amino acid, L-[1-14C]-leucine, and a kinetic model of the behavior of L-leucine in the brain. We chose L-[1-14C]-leucine as the tracer because it does not lead to extraneous labeled metabolic products. It is either incorporated into protein or rapidly metabolized to yield 14CO2 which is diluted in a large pool of unlabeled CO2 in the brain. The method and the model also allow for the contribution of unlabeled leucine derived from tissue proteolysis to the tissue precursor pool for protein synthesis. The method has the spatial resolution to determine protein synthesis rates in cell and neuropil layers, as well as hypothalamic and cranial nerve nuclei. To obtain reliable and reproducible quantitative data, it is important to adhere to procedural details. Here we present the detailed procedures of the quantitative autoradiographic L-[1-14C]-leucine method for the determination of regional rates of protein synthesis in vivo.

Protein synthesis is&#160;a critical biological process for cells. In brain, it is required for adaptive changes. Measurement of rates of protein synthesis in the intact brain requires careful methodological considerations. Here we present the L-[1-14C]-leucine quantitative autoradiographic method for determination of regional rates of cerebral protein synthesis in vivo.

Método autoradiográfico cuantitativo para la determinación de las tasas regionales de síntesis de proteínas cerebrales in Vivo

Protein synthesis is required for development and maintenance of neuronal function and is involved in adaptive changes in the nervous system. Moreover, it ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Efectos de Neurotrauma inducido ráfaga en roedor a presión media de las arterias cerebrales

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

Cheek Injection Model for Simultaneous Measurement of Pain and Itch-related Behaviors

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts are typically counted to evaluate itch induced by pruritogens. However, previous reports have shown that algesic substances also induce scratching behaviors in a mouse neck injection model, which is the most common test used for scratching behaviors. This finding makes it difficult to study itch in mice. &#160;In contrast, capsaicin, a common algogen, reduced scratching behaviors in some neck injection experiments. Therefore, the effect of pain on scratching behaviors remains unclear. It is thus necessary to develop a method to concurrently investigate itch and pain sensation using behavioral tests. Here, a cheek injection model is introduced which can be used to simultaneously measure pain- and itch-related behaviors. In this model, pruritogens induce scratching behaviors while algesic substances induce wiping behaviors. Using this model, lysophosphatidic acid (LPA), an itch mediator found in cholestatic patients with itch, is shown to exclusively induce itch but not pain. However, in mouse models, LPA has been reported to be both a pruritogen and an algogen. Investigation into the effects of LPA in a mouse cheek injection model showed that LPA only induced scratching, but not wiping behaviors. This indicates that LPA acts as a pruritogen similarly in mice and humans, and demonstrates the utility of a cheek injection model for itch research.

Typically, the mouse neck injection model is used for evaluate pruritogen-induced scratch behaviors. However, the model provides information only on itch, not pain. Here, a cheek injection model is introduced in mice which can be used to simultaneously measure pain and itch-related behaviors.

Modelo de inyección de mejillas para la medición simultánea del dolor y los comportamientos relacionados con la picazón

Itch was defined as &#34;an unpleasant cutaneous sensation that provokes a desire to scratch&#34;&#160;by Rothman in 1941. In mouse models, scratch bouts ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

Generación de hidrogeles a base de colágeno 3D para analizar el crecimiento y el comportamiento axonal durante el desarrollo del sistema nervioso

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Mapeo del flujo sanguíneo cerebral y los campos eléctricos: colocación de electrodos en ratones

Placement of Extracranial Stimulating Electrodes and Measurement of Cerebral Blood Flow and Intracranial Electrical Fields in Anesthetized Mice

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and variations in the substrate supply to the brain. This paper describes a protocol for measuring CBF responses to transcranial alternating current stimulation (tACS). Dose-response curves are estimated both from the CBF change occurring with tACS (mA) and from the intracranial electric field (mV/mm). We estimate the intracranial electrical field based on the different amplitudes measured by glass microelectrodes within each side of the brain. In this paper, we describe the experimental setup, which involves using either bilateral laser Doppler (LD) probes or laser speckle imaging (LSI) to measure the CBF; as a result, this setup requires anesthesia for the electrode placement and stability. We present a correlation between the CBF response and the current as a function of age, showing a significantly larger response at higher currents (1.5 mA and 2.0 mA) in young control animals (12-14 weeks) compared to older animals (28-32 weeks) (p &lt; 0.005 difference). We also demonstrate a significant CBF response at electrical field strengths &lt;5 mV/mm, which is an important consideration for eventual human studies. These CBF responses are also strongly influenced by the use of anesthesia compared to awake animals, the respiration control (i.e., intubated vs. spontaneous breathing), systemic factors (i.e., CO2), and local conduction within the blood vessels, which is mediated by pericytes and endothelial cells. Likewise, more detailed imaging/recording techniques may limit the field size from the entire brain to only a small region. We describe the use of extracranial electrodes for applying tACS stimulation, including both homemade and commercial electrode designs for rodents, the concurrent measurement of the CBF and intracranial electrical field using bilateral glass DC recording electrodes, and the imaging approaches. We are currently applying these techniques to implement a closed-loop format for augmenting the CBF in animal models of Alzheimer's disease and stroke.

Autor destacado: Enfoque basado en la estimulación para mejorar el flujo sanguíneo cerebral en el modelo de Alzheimer 

We describe a protocol for assessing dose-response curves for extracranial stimulation in terms of brain electrical field measurements and a relevant biomarker-cerebral blood flow. Since this protocol involves invasive electrode placement into the brain, general anesthesia is needed, with spontaneous breathing preferred rather than controlled respirations.

Colocación de electrodos estimulantes extracraneales y medición del flujo sanguíneo cerebral y campos eléctricos intracraneales en ratones anestesiados

The detection of cerebral blood flow (CBF) responses to various forms of neuronal activation is critical for understanding dynamic brain function and ...