Los autores desean reconocer la Liga Francesa contra l&#39;épilepsie (MA-L), Institut National de la Santé et de la Recherche Médicale de Grant AVENIR R12087FS (FJ), beca de la Universidad de Montpellier (FJ) y la subvención de la Federación para la Investigación sobre Le Cerveau (a NM). Reconocemos la asistencia técnica de Chrystel Lafont en el IPAM instalación de plataforma de núcleo formador de imágenes in vivo de Montpellier, en. Agradecemos también a María Vernov (Weill Cornell Medical College) para la corrección del manuscrito.

Los ratones se les permite el acceso ad libitum a la comida, el agua, y se mantuvieron en un ciclo de luz-oscuridad de 12 horas. Todos los procedimientos con animales de laboratorio se ajustaban a las leyes nacionales y europeas y fueron aprobados por el Ministerio Francés de Educación e Investigación Científica (CEEA-LR-00651-01). Un total de 6 transgénico 5xFAD y ratones de control 4 camada de tipo salvaje (WT) se utilizaron para este procedimiento. Preparación 1. Pre-operatorio <ol><li> Por vía intraperitoneal (IP) inyectar metoxi-X04 (10 mg / kg) 48 horas antes de la cirugía para etiquetar depósitos Aß 11. </li><li> Preparar fluido estéril cefalorraquídeo artificial (LCRa) (NaCl 120 mM, 26,2 mM NaHCO 3, KCl 2,5 mM, 1 mM NaH 2 PO 4, 1,3 mM MgCl 2, glucosa 10 mM; burbuja con 95% O2 / 5% de CO 2 antes la adición de 2,5 mM CaCl 2). </li><li> Esterilizar instrumentos quirúrgicos (tijeras, pinzas, cuchillas de afeitar y la broca), utilizando un autoclave o esterilizador de cuentas caliente antes asépticacirugía. </li><li> Limpiar el banco y la placa de microscopio quirúrgico con 70% de etanol y cubierta con un paño absorbente limpio. </li><li> Inyectar ketamina / xilazina (75 mg / kg y 10 mg / kg, respectivamente) y el ketoprofeno analgésico (2,5 mg / kg) IP. </li><li> Comprobar el nivel apropiado de la anestesia con la pata o la cola pellizcos. </li><li> Aplique una pomada oftálmica estéril para los ojos y afeitar el pelo (nariz para los oídos), evitando los bigotes. Una crema depilatoria se puede utilizar siempre que es seguido de un enjuague con agua cuidadosamente para evitar la irritación de la piel. Afeitarse el pelo sobrante con una cuchilla de afeitar. Esterilizar la piel con una solución antiséptica de povidona yodada. </li><li> Coloque el ratón bajo el microscopio binocular y hacer una incisión en la piel de las orejas a la nariz. Tire de la piel hacia los lados para exponer el cráneo. Incisión en el periostio y enjuagar varias veces el cráneo con ACSF estéril para detener el sangrado sea posible. </li></ol> 2. El etiquetado y la vasculatura del cráneo DiluidoPreparación de la ventana (40 min) <ol><li> Retire el ungüento oftálmico con una punta de algodón. Aplicar una gota de anestésico tópico oftálmico (tetracaína al 1%) y presionar suavemente la piel alrededor de la cuenca del ojo para lograr la protrusión. </li><li> Inyectar isotiocianato de fluoresceína (FITC) conjugado con dextrano (70 kDa) (100 mg / kg o 50 l de una solución 50 mg / ml; insulina aguja hipodérmica) en el seno retro-orbital siguiendo el procedimiento descrito por Yardeni y colegas 12. Aplique una pomada oftálmica estéril para los ojos. </li><li> Asegúrese de que la piel se retrae y el cráneo esté limpio y seco en todo el área de imagen seleccionada. Aplicar una pequeña cantidad de pegamento cianoacrılico alrededor de la ventana de la cabeza dispositivo de montaje (que consiste en hojas de afeitar apilados, véase la figura 1), y el pegamento alrededor de la región de destino aplicando una presión suave durante varios segundos 10. </li><li> Tire de los extremos de la piel hasta el borde de la ventana de la cabeza dispositivo de montaje, asegurándose de que la zona estáseco. Asegure la piel y el borde de la ventana con una pequeña cantidad de pegamento veterinaria cianoacrılico grado y esperar hasta que se seque (Figura 1). </li><li> Asegure el animal en la etapa de utilizar el dispositivo de montaje en la cabeza. Enjuague con ACSF estéril y asegurar que el pozo entre la piel y el dispositivo de la cabeza de montaje está perfectamente sellados. Envolver el ratón en una manta de supervivencia para mantener la normotermia. </li><li> Realizar adelgazamiento del cráneo en solución LCRa como se describió anteriormente 10. Tenga en cuenta que el cuero cabelludo no se elimina completamente. Cambiar regularmente las ACSF para limpiar restos de hueso y tejido para evitar el sobrecalentamiento. Cualquier restos de hueso restante se puede quitar con soplos de aire breves. La solución LCRa también atenúa las vibraciones creadas por la perforación. NOTA: El espesor del cráneo del ratón varía entre 80 a 150 micras dependiendo de la edad del animal y la región de interés. </li><li> Usando un taladro dental (a velocidad media y 0,7 mm rebabas) inicia el adelgazamiento de la superficie del cráneo en una zona 1.0 mm de diámetro utilizando un movimiento regular vertical paralelo al cráneo y eliminar la mayoría del hueso esponjoso (primero 40 - 70 micras). recuerde que debe sustituir a ACSF cada 20-30 segundos y limitar la perforación ininterrumpido a 3-4 seg. Tenga en cuenta que el hueso cerca de suturas es altamente vascularizado. Aplicar LCRa remojado previamente espuma de gel para detener un eventual sangrado. </li><li> Utilice una hoja de microcirugía oftálmica desechable afilado para continuar con el adelgazamiento del cráneo, teniendo cuidado de no aplicar demasiada presión. La región final es de alrededor de 0,5 mm de diámetro y con un espesor de 20 - 35 micras. NOTA: Debido a la regeneración del hueso y la cicatrización, es necesario más delgada la ventana en cada sesión de imágenes. </li></ol> 3. microscopía de dos fotones (45 min) <ol><li> Si es necesario, inyectar con 19 mg de ketamina / kg para mantener la anestesia adecuada. </li><li> Transferir el ratón (fijo a la etapa headmount) bajo el microscopio de láser de dos fotones. El uso de un obj inmersión 20X aguareflexivo con una apertura numérica de 1,0, ubicar la ventana craneal adelgazada en el centro del campo óptico usando la lámpara de epi-fluorescencia. Asegúrese de que el objetivo siempre está inmerso en ACSF. </li><li> Iniciar el escaneado láser con un láser pulsado modo bloqueado (Ti-zafiro 680-1040nm). Ajuste de longitud de onda de excitación a 750 nm para detectar la fluorescencia emitida de la metoxi-xO4 y FITC-dextrano en los canales azul y verde, respectivamente. NOTA: El microscopio tiene dos divisores de haz en 506 nm y 555 nm. La luz azul es capturado por un detector de NDD equipado con un filtro de 470 ± 12 nm. La luz verde es capturado por un detector de alta sensibilidad GaAsP equipado con un filtro de 525 ± 25 nm. La potencia del láser máxima emitida por el objetivo no debe ser superior a 10 mW para evitar el daño tisular inducido por láser. </li><li> Adquirir una pila bajo aumento (500 m x 500 m, 512 x 512 píxeles; 2 micras paso) a la de zoom numérico 0.7X para crear un mapa en 3D para la relocalización precisa del retorno de la inversiónen puntos de tiempo posteriores. </li><li> Adquirir un mosaico de 4 imágenes de alta amplificación digital con un zoom de 2X numérica (200 m x 200 m, 512 x 512 píxeles; 1 micra paso). Usando el software de imágenes mover la ventana en 200 micras pasos para capturar todas las regiones. Profundidad de pilas es típicamente 250 m, a partir de la superficie pial en cada imagen del mosaico. </li><li> Antes de retirar el ratón del microscopio, tomar una foto o hacer un mapa 2D dibujado a mano de la vasculatura pial para la futura relocalización del campo de la imagen. </li></ol> 4. Recuperación y reconstrucción de imagen <ol><li> Retire el dispositivo de la cabeza de montaje aplicando tracción mientras sostiene el cráneo debajo. Si el pegamento permanece unida al cráneo o de la piel, raspar cuidadosamente con unas pinzas finas. Suturar el cuero cabelludo y colocar el ratón en una jaula calienta a seguimiento de la recuperación de la anestesia antes de devolverlo a la jaula de alojamiento. Aplicar crema antibiótica tópica a la sutura e inyectar ketoprofeno (/ kg 2,5 mg) IP los siguientes 2 días. </li><li> Repita los pasos 1,1 a 2,5 para las sesiones de formación de imágenes repetidas. Si es necesario, afeitar el hueso con una cuchilla microquirúrgica para asegurar una calidad óptima de la imagen. NOTA: adelgazamiento adicional con el taladro dental puede ser necesaria cuando el intervalo entre las sesiones de formación de imágenes es más de un mes. </li><li> Coloque el ratón debajo del microscopio de epifluorescencia usando luz de acuerdo con el mapa en 2D de la vasculatura pial tomada de la sesión anterior (paso 3.6). </li><li> Iniciar el escaneo láser de dos fotones y ajustar platina del microscopio para lograr la alineación en la escala del micrómetro usando el mapa 3D obtenida en el paso 3.4. Proceder a la imagen detallada como se describe en el paso 3.5. </li></ol> 5. posteriores a la adquisición de tres dimensiones-Reconstrucción y Análisis de Imágenes <ol><li> Obtener reconstrucciones tridimensionales de la vasculatura y las deposiciones de amiloide utilizando el software de análisis de imágenes 3D como Imaris (versión 8.0 y utiliza 7.7.2). </li&gt; <li> Utilizar el plugin de capa Normalizar en el menú de procesamiento de imágenes y el submenú cambiar el contraste para obtener el contraste ideal de los dos canales en toda la profundidad de la pila de imágenes. </li><li> Abrir las imágenes de la misma región en todos los puntos de forma simultánea y siempre les procesar en paralelo con el fin de comparar los diferentes puntos de tiempo. </li><li> vasos traza utilizando el módulo de trazador de filamento. <ol><li> Haga clic en el icono de filamentos para crear un nuevo filamento, omita el trazador automático y elegir el método de auto-ruta. </li><li> Asegúrese de que el canal seleccionado se corresponde con la imagen vascular y el uso de la función de selección del puntero, haga clic en una bifurcación buque que se conserva a través de las sesiones de formación de imágenes, mientras que la celebración de las teclas shift + control para determinar un punto de partida. Utilice la función de selección del puntero y haga clic mientras mantiene pulsada la tecla de mayúsculas para seleccionar los puntos extremos de los filamentos. NOTA: La comparación de la obteesqueletos nidas mostrarán los cambios estructurales de la vasculatura. </li></ol></li><li> Realizar plano por análisis de avión para realizar un seguimiento de nuevas placas. Aplicar módulo de superficie para el canal azul para el seguimiento del crecimiento de las placas de Aß. Haga clic en el icono de la superficie para crear una nueva superficie y proceder a la creación de superficies automatizado. Utilice el método de umbral para establecer y restar la señal de fondo. El menú de estadísticas proporciona datos respecto a la superficie de los depósitos de amiloide individuales. </li></ol>

<ol>
	<li>Harb, R., Whiteus, C., Freitas, C., Grutzendler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+cerebral+microvascular+plasticity+from+birth+to+death.">In vivo imaging of cerebral microvascular plasticity from birth to death.</a> J Cereb Blood Flow Metab. 33 (1), 146-156 (2013).</li><li>Whiteus, C., Freitas, C., Grutzendler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbed+neural+activity+disrupts+cerebral+angiogenesis+during+a+postnatal+critical+period.">Perturbed neural activity disrupts cerebral angiogenesis during a postnatal critical period.</a> Nature. 505 (7483), 407-411 (2014).</li><li>Masamoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+sprouting,+extension,+and+creation+of+new+capillary+connections+with+adaptation+of+the+neighboring+astrocytes+in+adult+mouse+cortex+under+chronic+hypoxia.">Microvascular sprouting, extension, and creation of new capillary connections with adaptation of the neighboring astrocytes in adult mouse cortex under chronic hypoxia.</a> J Cereb Blood Flow Metab. 34 (2), 325-331 (2014).</li><li>Marchi, N., Lerner-Natoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrovascular+remodeling+and+epilepsy.">Cerebrovascular remodeling and epilepsy.</a> Neuroscientist. 19 (3), 304-312 (2013).</li><li>Giannoni, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebrovascular+pathology+during+the+progression+of+experimental+Alzheimer's+disease.">Cerebrovascular pathology during the progression of experimental Alzheimer's disease.</a> Neurobiol Dis. 88, 107-117 (2016).</li><li>Kimbrough, I. F., Robel, S., Roberson, E. D., Sontheimer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+amyloidosis+impairs+the+gliovascular+unit+in+a+mouse+model+of+Alzheimer's+disease.">Vascular amyloidosis impairs the gliovascular unit in a mouse model of Alzheimer's disease.</a> Brain. 138 (Pt 12), 3716-3733 (2015).</li><li>Herzig, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abeta+is+targeted+to+the+vasculature+in+a+mouse+model+of+hereditary+cerebral+hemorrhage+with+amyloidosis.">Abeta is targeted to the vasculature in a mouse model of hereditary cerebral hemorrhage with amyloidosis.</a> Nat Neurosci. 7 (9), 954-960 (2004).</li><li>Oakley, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraneuronal+beta-amyloid+aggregates,+neurodegeneration,+and+neuron+loss+in+transgenic+mice+with+five+familial+Alzheimer's+disease+mutations:+potential+factors+in+amyloid+plaque+formation.">Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation.</a> J Neurosci. 26 (40), 10129-10140 (2006).</li><li>Liston, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+glucocorticoid+oscillations+promote+learning-dependent+synapse+formation+and+maintenance.">Circadian glucocorticoid oscillations promote learning-dependent synapse formation and maintenance.</a> Nat Neurosci. 16 (6), 698-705 (2013).</li><li>Yang, G., Pan, F., Parkhurst, C. N., Grutzendler, J., Gan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinned-skull+cranial+window+technique+for+long-term+imaging+of+the+cortex+in+live+mice.">Thinned-skull cranial window technique for long-term imaging of the cortex in live mice.</a> Nat Protoc. 5 (2), 201-208 (2010).</li><li>Klunk, W. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Abeta+plaques+in+living+transgenic+mice+with+multiphoton+microscopy+and+methoxy-X04,+a+systemically+administered+Congo+red+derivative.">Imaging Abeta plaques in living transgenic mice with multiphoton microscopy and methoxy-X04, a systemically administered Congo red derivative.</a> J Neuropathol Exp Neurol. 61 (9), 797-805 (2002).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retro-orbital+injections+in+mice.">Retro-orbital injections in mice.</a> Lab Anim (NY). 40 (5), 155-160 (2011).</li><li>Cao, V. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+two-photon+imaging+of+experience-dependent+molecular+changes+in+cortical+neurons.">In vivo two-photon imaging of experience-dependent molecular changes in cortical neurons.</a> J Vis Exp. (71), (2013).</li><li>Holtmaat, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+term,+high-resolution+imaging+in+the+mouse+neocortex+through+a+chronic+cranial+window.">Long term, high-resolution imaging in the mouse neocortex through a chronic cranial window.</a> Nat Protoc. 4 (8), 1128-1144 (2009).</li><li>Heppner, F. L., Ransohoff, R. M., Becher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+attack:+the+role+of+inflammation+in+Alzheimer+disease.">Immune attack: the role of inflammation in Alzheimer disease.</a> Nat Rev Neurosci. 16 (6), 358-372 (2015).</li><li>Marker, D. F., Tremblay, M. E., Lu, S. M., Majewska, A. K., Gelbard, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thin-skull+window+technique+for+chronic+two-photon+in+vivo+imaging+of+murine+microglia+in+models+of+neuroinflammation.">A thin-skull window technique for chronic two-photon in vivo imaging of murine microglia in models of neuroinflammation.</a> J Vis Exp. (43), (2010).</li><li>Joseph-Mathurin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+immunization+worsens+iron+deposits+in+the+choroid+plexus+and+cerebral+microbleeds.">Amyloid beta immunization worsens iron deposits in the choroid plexus and cerebral microbleeds.</a> Neurobiol Aging. 34 (11), 2613-2622 (2013).</li><li>Sadowski, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+prion+amyloid+deposits+in+vivo.">Targeting prion amyloid deposits in vivo.</a> J Neuropathol Exp Neurol. 63 (7), 775-784 (2004).</li></ol>

Los autores no tienen intereses financieros en competencia.

La técnica y el cráneo abierto para microscopía in vivo de dos fotones ofrece la ventaja de las sesiones de formación de imágenes ilimitadas de grandes campos de imagen 13,14. Sin embargo, esta técnica también produce inflamación en la región de interés 14, a menudo incompatibles o impactar neuro-vascular 15 salidas lectura. Por el contrario, la técnica de cráneo transcraneal adelgazada no resulta en neuro-inflamación, lo que permite de imagen fiable de las estructu...

La vasculatura del cerebro es una estructura multicelular, que está anatómicamente y funcionalmente acoplado a las neuronas. Una remodelación dinámica de los vasos se produce en todo el desarrollo del cerebro y durante la progresión de patologías del sistema nervioso central (CNS) 1,2. Es ampliamente aceptado que el daño cerebrovascular es una característica de varias enfermedades del sistema nervioso central, incluyendo la epilepsia, la enfermedad de Alzheimer (EA), lesión cerebral traumática y la encefalitis de 3,4. Por lo tanto, el seguimiento de los cambios cerebrovasculares en vivo se vuelve significativo cuando se modela enfermedades del SNC, desde el inicio y en fases crónicas. Como modificaciones cerebrovasculares ocurren a menudo en forma concomitante con el daño neuronal o plasticidad, la imagen de la neuro-vascular representa un punto de partida clave para descifrar SNC fisiopatología de la enfermedad. Este protocolo describe un procedimiento basado en dos fotones longitudinal para realizar un seguimiento de la remodelación de la vasculatura cerebral en un modelo de ratón deAD, una patología progresiva caracterizada por defectos en los vasos cerebrovasculares calibre grandes y pequeñas, debido a la deposición de placas amiloidogénica 5-7. Este procedimiento permite la visualización de los depósitos de amiloide y el seguimiento de su posición y el crecimiento con respecto a la remodelación neurovascular durante todo el curso de la enfermedad. Colorantes fluorescentes vitales se inyectan antes de cada sesión de formación de imágenes para la visualización de las placas de amiloide y vasculatura cerebral en ratones transgénicos AD 8. Sesiones de formación de imágenes repetidas de un retorno de la inversión a través de una ventana cráneo transcraneal adelgazada es no invasivo y el método de elección para evaluar la remodelación neurovascular en el cerebro de ratón de estar 2,5,9,10. En el procedimiento siguiente se describe el protocolo quirúrgico, adquisición y procesamiento de imágenes. La progresión temprana de la angiopatía amiloide cerebral (CAA) en su mayoría en general leptomeníngeo y arteriolas penetrantes se caracteriza.

<table border="0" cellpadding="0" cellspacing="0" width="627">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">methoxy-X04</td>
			<td style="width:125px;">tocris</td>
			<td align="right" style="width:119px;">4920</td>
			<td style="width:167px;">use 10 mg/Kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-Dextran 70Kda</td>
			<td style="width:125px;">sigma</td>
			<td align="right" style="width:119px;">46945</td>
			<td>use 100 mg/Kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gelfoam/Bloxang</td>
			<td style="width:125px;">Bausch and Lomb</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">micorsurgical blade</td>
			<td style="width:125px;">surgistar</td>
			<td align="right" style="width:119px;">6900</td>
			<td>must be sharp and not dented</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">povidone-iodine</td>
			<td style="width:125px;">betadine</td>
			<td style="width:119px;"></td>
			<td>antisceptic solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">binocular stereomicroscope</td>
			<td style="width:125px;">olympus</td>
			<td style="width:119px;">SX10</td>
			<td>optimal image contrast is crucial for this procedure</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-photon microscope</td>
			<td style="width:125px;">zeiss</td>
			<td style="width:119px;"></td>
			<td>Zeiss LSM 710mp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fine scissors-toughcut</td>
			<td style="width:125px;">Fine science tools</td>
			<td>14058-09</td>
			<td>this scissors are optimized for cutting skin and soft tissue</td>
		</tr>
	</tbody>
</table>

longitudinal in vivo imaging of the cerebrovasculature: relevance to cns diseases

Remodelación de la vasculatura del cerebro es un rasgo común de las patologías cerebrales. In vivo técnicas de imagen son fundamentales para detectar la plasticidad cerebrovascular o daños que se produzcan las horas extraordinarias y en relación con la actividad neuronal o el flujo sanguíneo. In vivo microscopía de dos fotones permite el estudio de la plasticidad estructural y funcional de las grandes unidades celulares en el cerebro vivo. En particular, la preparación de ventana cráneo adelgazada permite la visualización de regiones corticales de interés (ROI) sin inducir inflamación significativa en el cerebro. sesiones de formación de imágenes repetitivas de ROI cortical son factibles, proporcionando la caracterización de características distintivas de la enfermedad con el tiempo durante la progresión de numerosas enfermedades del SNC. Esta técnica acceder a las estructuras pial dentro de 250 micras del cerebro se basa en la detección de sondas fluorescentes codificadas por marcadores celulares genéticos y / o colorantes vitales. Este último (por ejemplo, dextranos fluorescentes) se utilizan para mapear la Luminal compartimiento de las estructuras cerebrovasculares. Germane con el protocolo descrito en el presente documento es el uso de un marcador in vivo de depósitos de amiloide, metoxi-O4, para evaluar la enfermedad de Alzheimer progresión (AD). También se describe el tratamiento de la imagen posterior a la adquisición utilizado para realizar un seguimiento de los cambios vasculares y los depósitos amiloides. Aunque se centra actualmente en un modelo de AD, el protocolo descrito es relevante para otros trastornos del sistema nervioso central donde se producen cambios patológicos cerebrovasculares.

Este protocolo describe un método para la visualización de la vasculatura cerebral y los depósitos de amiloide horas extras. Se inyectaron tintes fluorescentes para etiquetar las deposiciones de amiloide (metoxi-xO4) 11 y para llenar el lumen cerebrovasculares (FITC-dextrano) 1. módulos de software de análisis de imágenes 3D se utilizan para crear imágenes en 3D de un campo constante de vista capturado en los puntos de tiempo consecutivos. Imágenes represent...

Watch this Scientific Journal Video about Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases at JoVE.com

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Este manuscrito describe un procedimiento para realizar un seguimiento de la remodelación de la vasculatura cerebral durante la acumulación de placa amiloide in vivo usando longitudinal microscopía de dos fotones. Una preparación-cráneo adelgazada permite la visualización de los tintes fluorescentes para evaluar la progresión del daño cerebrovascular en un modelo de ratón de la enfermedad de Alzheimer.

Longitudinal<em&gt; En Vivo</em&gt; Obtención de imágenes de la vasculatura cerebral: Importancia de las enfermedades del sistema nervioso central

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

longitudinal-vivo-imaging-cerebrovasculature-relevance-to-cns

Research

JoVE Journal

Medicine

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

7.0K vistas.  Institute of Functional Genomics.  Este manuscrito describe un procedimiento para realizar un seguimiento de la remodelación de la vasculatura cerebral durante la acumulación de placa amiloide in vivo usando longitudinal microscopía de dos fotones. Una preparación-cráneo adelgazada permite la visualización de los tintes fluorescentes para evaluar la progresión del daño cerebrovascular en un modelo de ratón de la enfermedad de Alzheimer. 

Video: Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

La medición y tratamiento de la represión en la ambliopía

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Investigación

Medicina

The Use of Pharmacological-challenge fMRI in Pre-clinical Research: Application to the 5-HT System

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel underlying neurobiological mechanisms behind drug action and neurotransmitter-related disorders, such as depression and ADHD. Like most of the imaging methods (PET, SPECT, CT) it represents a progress in the investigation of brain disorders and the related function of neurotransmitter pathways in a non-invasive way with respect of the overall neuronal connectivity. Moreover it also provides the ideal tool for translation to clinical investigations. MRI, while still behind in molecular imaging strategies compared to PET and SPECT, has the great advantage to have a high spatial resolution and no need for the injection of a contrast-agent or radio-labeled molecules, thereby avoiding the repetitive exposure to ionizing radiations. Functional MRI (fMRI) is extensively used in research and clinical setting, where it is generally combined with a psycho-motor task. phMRI is an adaptation of fMRI enabling the investigation of a specific neurotransmitter system, such as serotonin (5-HT), under physiological or pathological conditions following activation via administration of a specific challenging drug.
The aim of the method described here is to assess brain 5-HT function in free-breathing animals. By challenging the 5-HT system while simultaneously acquiring functional MR images over time, the response of the brain to this challenge can be visualized. Several studies in animals have already demonstrated that drug-induced increases in extracellular levels of e.g. 5-HT (releasing agents, selective re-uptake blockers, etc) evoke region-specific changes in blood oxygenation level dependent (BOLD) MRI signals (signal due to a change of the oxygenated/deoxygenated hemoglobin levels occurring during brain activation through an increase of the blood supply to supply the oxygen and glucose to the demanding neurons) providing an index of neurotransmitter function. It has also been shown that these effects can be reversed by treatments that decrease 5-HT availability16,13,18,7. In adult rats, BOLD signal changes following acute SSRI administration have been described in several 5-HT related brain regions, i.e. cortical areas, hippocampus, hypothalamus and thalamus9,16,15. Stimulation of the 5-HT system and its response to this challenge can be thus used as a measure of its function in both animals and humans2,11.

The goal of this technique is to assess serotonin (5-HT) neurotransmitter function in the live and free-breathing animal with pharmacological magnetic resonance imaging (phMRI) and an intravenous challenge with a selective serotonin reuptake inhibitor (SSRI), fluoxetine.

El uso de la fMRI farmacológico-reto en la investigación preclínica: la aplicación al sistema de 5-HT

Pharmacological MRI (phMRI) is a new and promising method to study the effects of substances on brain function that can ultimately be used to unravel ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Administración intranasal de la terapéutica del SNC para Ratones Awake

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are intensively and increasingly recruited in septic joints from the early phases of infection and the infiltration is supposed to regress once efficient removal of the pathogens is obtained. The ability to identify in vivo macrophage activity in an infected joint can therefore provide two main applications: early detection of acute synovitis and monitoring of therapy.
In vivo noninvasive detection of macrophages can be performed with magnetic resonance imaging using iron nanoparticles such as ultrasmall superparamagnetic iron oxide (USPIO). After intravascular or intraarticular administration, USPIO are specifically phagocytized by activated macrophages, and, due to their magnetic properties, induce signal changes in tissues presenting macrophage infiltration. A quantitative evaluation of the infiltrate is feasible, as the area with signal loss (number of dark pixels) observed on gradient echo MR images after particles injection is correlated with the amount of iron within the tissue and therefore reflects the number of USPIO-loaded cells.
We present here a protocol to perform macrophage imaging using USPIO-enhanced MR imaging in an animal model of septic arthritis, allowing an initial and longitudinal in vivo noninvasive evaluation of macrophages infiltration and an assessment of therapy action.

In vivo Macrophage Imaging Using MR Targeted Contrast Agent for Longitudinal Evaluation of Septic Arthritis

We demonstrate how to perform macrophage MR imaging using ultrasmall superparamagnetic contrast agent (USPIO) in septic arthritis, allowing an initial and longitudinal in vivo non-invasive evaluation of macrophages infiltration and an assessment of therapy efficacy.

En vivo los macrófagos de imágenes utilizando MR Agente de contraste dirigida para la Evaluación Longitudinal de Artritis séptica

Macrophages are key-cells in the initiation, the development and the regulation of the inflammatory response to bacterial infection. Macrophages are ...

Diffusion Tensor Magnetic Resonance Imaging in the Analysis of Neurodegenerative Diseases

Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls. 	
DTI data analysis is performed in a variate fashion, i.e. voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies. The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e. differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders. Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels.	
In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis.

Diffusion tensor imaging (DTI) basically serves as an MRI-based tool to identify in vivo the microstructure of the brain and pathological processes due to neurological disorders within the cerebral white matter. DTI-based analyses allow for application to brain diseases both at the group level and in single subject data.

Tensor de difusión por resonancia magnética en el análisis de las enfermedades neurodegenerativas

Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Trasplante en la cámara anterior del ojo para Longitudinal, no invasiva<em&gt; En vivo</em&gt; Imagen con una sola célula de resolución en tiempo real

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo MRI de 19F para el seguimiento de la célula

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Trauma cerebral aguda en ratones seguido de Longitudinal dos fotones de imágenes

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

En vivo de imágenes y Seguimiento de tecnecio-99m Etiquetada células madre mesenquimales de médula ósea en Equinos tendinopatía

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to image cellular events in an intact animal. Additionally, the ability to induce physiological disease states such as stroke in vivo increases its utility. The technique described herein allows for physiological assessment of cellular responses within the CNS following a stroke and can be adapted for other pathological conditions being studied. The technique presented uses laser excitation of the photosensitive dye Rose Bengal in vivo to induce a focal ischemic event in a single blood vessel. 
The video protocol demonstrates the preparation of a thin-skulled cranial window over the somatosensory cortex in a mouse for the induction of a Rose Bengal photothrombotic event keeping injury to the underlying dura matter and brain at a minimum. Surgical preparation is initially performed under a dissecting microscope with a custom-made surgical/imaging platform, which is then transferred to a confocal microscope equipped with an inverted objective adaptor. Representative images acquired utilizing this protocol are presented as well as time-lapse sequences of stroke induction. This technique is powerful in that the same area can be imaged repeatedly on subsequent days facilitating longitudinal in vivo studies of pathological processes following stroke.

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Here, we describe a semi-invasive optical microscopy approach for the induction of a Rose Bengal photothrombotic clot in the somatosensory cortex of a mouse in vivo. The technical aspects of the imaging procedure are described from induction of a photothrombotic event to application and data collection.

Rosa de Bengala Photothrombosis por imagen óptica confocal<em&gt; En Vivo</em&gt;: Un modelo de solo recipiente Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...