Special thanks to the Gidday lab for their work in developing the RHP protocol, as well as the Neuro-Models Facility (UTSW) for their assistance in the tMCAo surgeries. This work was supported by grants from the American Heart Association (AMS), The Haggerty Center for Brain Injury and Repair (UTSW; AMS), and The Spastic Paralysis Research Foundation of the Illinois-Eastern Iowa District of Kiwanis International (JMG).

The authors have nothing to disclose.

Una sola exposición a la hipoxia sistémica (es decir, 2 hr de 11% O 2) en ratones protege &quot;transitoriamente&quot; el cerebro de tMCAo, 29 es decir, la respuesta epigenética al desafío de preacondicionamiento hipóxica es de corta duración, y el fenotipo de la línea de base se restablece dentro de día. Presentaciones repetitivo del estímulo hipóxico preacondicionamiento se extienden dramáticamente la duración del fenotipo neuroprotector. 6 Muchos estudios han demo...

Como la principal causa de discapacidad en los adultos y la cuarta causa principal de muerte, accidente cerebrovascular es uno de los estados de enfermedad más debilitantes que enfrenta la población adulta de los Estados Unidos. 1 Los modelos animales de accidente cerebrovascular permiten para la investigación experimental de nuevos métodos de reducción de la lesión isquémica y mejorar la recuperación después de un accidente cerebrovascular. Una vía novedosa para este tipo de investigación traslacional se preacondicionamiento. Preacondicionamiento es el uso intencional de un estímulo no perjudicial para reducir el daño de una posterior, y más grave, lesiones. 2 hipóxico preacondicionamiento se ha demostrado que producen cambios pleiotrópicos en el cerebro que proporcionan protección contra el accidente cerebrovascular en tanto in vivo e in vitro . 3 Sin embargo, una sola exposición a la hipoxia sólo ofrece neuroprotección corto plazo, lo que induce a menos de 72 horas de la tolerancia frente a la isquemia en ratones adultos. 4 incluso después de cuatro semanas de exposiciones diarias 14 hr a la hipoxia hipobárica, Lin et al. found neuroprotección que solamente se mantuvo durante una semana. 5 precondicionamiento hipóxico repetitiva (RHP) se caracteriza por las variaciones estocásticas en la frecuencia, duración e intensidad de las exposiciones hipóxicas. En contraste con un único reto preacondicionamiento, RHP induce un fenotipo cerebroprotectora que dura hasta ocho semanas en ratones. 6 RHP redujo los volúmenes de infarto, barrera hematoencefálica (BBB) ​​perturbación, inflamación vascular y diapedesis de leucocitos durante semanas después de la exposición hipóxica definitiva . RHP reduce específicamente la inflamación en el cerebro isquémico mediante la reducción de las células T, monocitos, macrófagos y las poblaciones, mientras que el mantenimiento de las poblaciones de células B en el hemisferio isquémico. 7 De hecho, RHP induce un fenotipo inmunosupresor en ratones antes de cualquier lesión del SNC, incluido el accidente cerebrovascular. Células B tratados con RHP aisladas de ratones sanos tratados-RHP exhibieron un fenotipo antiinflamatorio único, con una regulación a la baja tanto de la presentación de antígenos y la producción de anticuerpos. Losreducción general en mecanismos inmunes adaptativos pro-inflamatorias hace RHP una excelente metodología para inducir inmunosupresión endógena no sólo para enfermedades inflamatorias del SNC-específicos, sino también modelos de lesión o enfermedad sistémica que incluyen una patología pro-inflamatorio. RHP reduce tanto el volumen del infarto y la disrupción de la BHE tras una oclusión transitoria de la arteria cerebral media (tMCAo). Los modelos animales de accidente cerebrovascular, tales como el comúnmente usado tMCAo, mejorar dramáticamente la comprensión de la fisiopatología del accidente cerebrovascular, así como el diseño de Neurotherapeutics más eficaces. Primero desarrollado por Koizumi et al., En 1986, el procedimiento tMCAo 8 es un método ampliamente utilizado de inducir accidente cerebrovascular en roedores y uno de los métodos preferidos para la investigación de la inflamación después de la reperfusión. Como los métodos para tMCAo evolucionan, el uso más reciente de filamentos recubiertos de silicona reducir aún más el riesgo de hemorragia subaracnoidea en comparación con otros modelos de 9,10 &lt;/ Sup&gt; y mejorar la fiabilidad, aunque por desgracia tMCAo menudo produce una amplia variación en los volúmenes de infarto. 11-13 La mayor parte de estos estudios delinear regiones de infarto en el cerebro secciones coronales mediante tinción con cloruro de 2,3,5- trifeniltetrazolio (TTC), considerado un estándar de oro para la cuantificación del infarto debido a que es una manera simple y barata de producir vivos, resultados replicables. TTC sirve como un sustrato de deshidrogenasas presentes en las mitocondrias. Cuando rodajas de cerebro están expuestos a la solución de TTC, TTC se toma selectivamente en células vivas donde su producto de reducción no soluble, formazán, precipita a un color rojo intenso en la mitocondria viables. Debido a la disfunción mitocondrial en el tejido isquémico, este tejido permanece blanco, lo que permite la diferenciación del tejido dañado y sano. 14 RHP también reduce la acreditación interrupción en el hemisferio isquémico. 6 Por lo tanto, la doble cuantificación de acreditación integridad dentro de la misma blluvias como el volumen del infarto basado en TTC Determinaciones 15 proporcionaría información útil sobre la eficacia plena de la protección endógena, y posibles relaciones causales entre la disrupción de la BHE y el infarto en los animales no tratados y tratados. La afluencia de sangre periférica a través de una acreditación interrumpido, secundaria a un accidente cerebrovascular, aumenta poblaciones de leucocitos, citoquinas pro-inflamatorias, estrés oxidativo, edema vasogénico y transformación hemorrágica en el hemisferio isquémico, en última instancia, el aumento de las tasas de infección y mortalidad en pacientes con ictus isquémico 16,17. Un método común de medir la disrupción de la BHE en modelos animales es a través de la cuantificación de azul de Evans (EB) de fugas de tinte en el cerebro. 15,18-21 EB se une selectivamente a la albúmina sérica, una proteína globular (MW = 65 kDa) que no atraviesa la BBB en animales no lesionados. 22 Tras el accidente cerebrovascular isquémico, EB se infiltra en el cerebro, y emite fluorescencia a 620 nm, lo que permite la medición de la densidad óptica within el parénquima lesionado perfundido. 22 La densidad óptica es directamente proporcional a la permeabilidad de la BBB cuando EB ha sido lavada fuera de la vasculatura cortical post-mortem por perfusión transcardiaca. Con el procesamiento inmediato de los cerebros teñidas con TTC en animales con administración EB, tanto el volumen del infarto y la disrupción de la BHE se pueden cuantificar de manera efectiva. Cabe señalar, sin embargo, que el daño neuronal y la disrupción de la BHE no son procesos concomitantes en el cerebro post-accidente cerebrovascular, 23,24 lo que la selección de tiempo de sacrificio es una consideración importante. El protocolo que sigue detalla el método RHP, el método tMCAo para inducir una oclusión arterial temporal que los modelos de las oclusiones de la arteria cerebral media en pacientes humanos, y los métodos histológicos duales para determinar neurales y vasculares puntos finales de lesiones accidente cerebrovascular. TTC mide la muerte celular y el daño tisular acumulativa, lo que permite la cuantificación de un infarto vol generalume, mientras que EB ofrece para la cuantificación hemisférica de acreditación daños.

<table border="0" cellpadding="0" cellspacing="0" width="830">
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			<td height="42" style="height:42px;width:249px;">Material/ Equipment</td>
			<td style="width:171px;">Company</td>
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			<td style="width:283px;">Comments/Description</td>
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		<tr height="220">
			<td height="220" style="height:220px;width:249px;">Flowmeters, regulators</td>
			<td style="width:171px;">VetEquip, Inc</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;">Four flowmeters are attached to 6.0 mm flexible PVC tubing which connects to the inlet port on each induction chamber with a plastic female connector. These flowmeters are bolted to a 6.5&#34; x 1&#34; x 1&#34; metal bar. This metal bar is bolted to a MI-246-P pressure gauge with a DISS outlet. This pressure gauge and flowmeter equipment can be attached to each new gas cylinder with a wrench.</td>
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		<tr height="26">
			<td height="26" style="height:26px;width:249px;">21% O2 tank</td>
			<td style="width:171px;">AirGas</td>
			<td style="width:127px;">OX USP200</td>
			<td style="width:283px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:249px;">11% O2 tank</td>
			<td style="width:171px;">AirGas</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;"></td>
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		<tr height="26">
			<td height="26" style="height:26px;width:249px;">8% O2 tank&#160;</td>
			<td style="width:171px;">AirGas</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">15L induction chambers</td>
			<td style="width:171px;">VetEquip</td>
			<td style="width:127px;">941454</td>
			<td style="width:283px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Moor Laber Dopper Flow&#160;</td>
			<td style="width:171px;">Moor Instruments&#160;</td>
			<td style="width:127px;">moorVMS-LDF1-HP</td>
			<td style="width:283px;">0.8mm diameter probe&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">High Intensity Illuminator&#160;</td>
			<td style="width:171px;">Nikon</td>
			<td style="width:127px;">NI-150</td>
			<td style="width:283px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Zoom Stereo Microscope&#160;</td>
			<td style="width:171px;">NIkon</td>
			<td style="width:127px;">SMZ800</td>
			<td style="width:283px;">Other surgical microscopes may be used.&#160;</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:249px;">Kent Scientific Right Temperature CODA</td>
			<td style="width:171px;">Kent Scientific Corporation</td>
			<td style="width:127px;">Discontinued</td>
			<td style="width:283px;">Recommended replacement is PhysioSuite with RightTemp Temperature Monitoring and Homeothermic Control (Kent Scientific, #PS-RT).</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:249px;">Hovabator Incubator</td>
			<td style="width:171px;">Stromberg&#39;s</td>
			<td style="width:127px;">2362-E</td>
			<td style="width:283px;">Our model is the 2362N. 2362E is a later model and includes an electronic thermostat.&#160;</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:249px;">V010 Anesthesia system&#160;</td>
			<td style="width:171px;">VetEquip</td>
			<td style="width:127px;">901807</td>
			<td style="width:283px;">Includes: ten foot high-pressure oxygen hose, frame, flowmeter, oxygen flush assembly, vaporizer, breathing circuit, chamber, nosecones, waste gas evacuation tubing and two VapoGuard filters</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">250 mL isoflurane&#160;</td>
			<td style="width:171px;">Butler Schein</td>
			<td style="width:127px;">NDC-11695</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">D-6 Vet Trim Animal Cordless Trimmer&#160;</td>
			<td style="width:171px;">Andis&#160;</td>
			<td style="width:127px;">#23905</td>
			<td style="width:283px;">Replacement blades are available from Andis (#23995)</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Betadine&#160;</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">19-898-867&#160;</td>
			<td style="width:283px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Q-tips</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
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			<td height="20" style="height:20px;width:249px;">Gauze Pads</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">67622</td>
			<td style="width:283px;"></td>
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			<td height="20" style="height:20px;width:249px;">Surgical drape</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">GM300&#160;</td>
			<td style="width:283px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Silk Sutures&#160;</td>
			<td style="width:171px;">Look/Div Surgical Specialties</td>
			<td style="width:127px;">SP115</td>
			<td style="width:283px;"></td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Nylon Sutures</td>
			<td style="width:171px;">Look/Div Surgical Specialties</td>
			<td style="width:127px;">SP185</td>
			<td style="width:283px;"></td>
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			<td height="20" style="height:20px;width:249px;">Durmont #5 forceps (2)&#160;</td>
			<td style="width:171px;">Fine Science Tools&#160;</td>
			<td style="width:127px;">11251-35</td>
			<td style="width:283px;">Angled 45&#176;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Surgical Scissors</td>
			<td style="width:171px;">Fine Science Tools&#160;</td>
			<td style="width:127px;">14028-10</td>
			<td style="width:283px;"></td>
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			<td height="40" style="height:40px;width:249px;">3mm Vannas</td>
			<td style="width:171px;">Kent Scientific Corporation</td>
			<td style="width:127px;">INS600177</td>
			<td style="width:283px;">Straight blade</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Hartman Hemostats&#160;</td>
			<td style="width:171px;">Fine Scientific Tools</td>
			<td style="width:127px;">13002-10</td>
			<td style="width:283px;"></td>
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		<tr height="60">
			<td height="60" style="height:60px;width:249px;">Occluding filaments</td>
			<td style="width:171px;">Washington University</td>
			<td style="width:127px;">Specialty order</td>
			<td style="width:283px;">Filaments are silicone coated at Washington Univeristy and provided to UTSW facilities for a fee.&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Evans Blue</td>
			<td style="width:171px;">Sigma Aldritch</td>
			<td style="width:127px;">E2129-10G</td>
			<td style="width:283px;"></td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Filter Paper&#160;</td>
			<td style="width:171px;">Sigma Aldritch</td>
			<td style="width:127px;">WHA1001150</td>
			<td style="width:283px;">150 mm, circles, Grade 1&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Weigh Boats</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">02-202-101</td>
			<td style="width:283px;">2.5&#34; diameter</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:249px;">0.9% Sodium Chloride Injection USP&#160;</td>
			<td style="width:171px;">Baxter Pharmaceutics&#160;</td>
			<td style="width:127px;">2B1321</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">0.3cc insulin syringe with 29 g needle</td>
			<td style="width:171px;">Becton Dickinson Labware</td>
			<td style="width:127px;">309301</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Flat bottom restrainer&#160;</td>
			<td style="width:171px;">Braintree Scientific&#160;</td>
			<td style="width:127px;">FB&#160;M</td>
			<td style="width:283px;">2.0&#34; diameter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">TTC</td>
			<td style="width:171px;">Sigma</td>
			<td style="width:127px;">T8877</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">10X PBS, pH 7.4</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">BP399-20</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:249px;">Water Bath</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;">Scintillation tubes with TTC may be manually held under running warm water as an alternative to the water bath.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Styrofoam board</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Large Syringe Kit</td>
			<td style="width:171px;">PumpSystems Inc</td>
			<td style="width:127px;">P-SYRKIT-LG</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Perfusion Pump</td>
			<td style="width:171px;">PumpSystems Inc</td>
			<td style="width:127px;">NE-300&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">60 cc syringe</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">NC9203256</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">27g winged infusion set</td>
			<td style="width:171px;">Kawasumi Laboratories, Inc</td>
			<td style="width:127px;">D3K1-25G 1</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">20 ml scintillation vial</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">50-367-126</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Stainless steel spatula</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">14-373-25A</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Alto acrylic 1.0 mm mouse brain, coronal</td>
			<td style="width:171px;">CellPoint Scientific&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">0.21 mm stainless steel blades, 25 pk</td>
			<td style="width:171px;">CellPoint Scientific&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;">Reusable cryostat blades are an inexpensive alternative.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">4% paraformaldehyde</td>
			<td style="width:171px;">Santa Cruz Biotechnology&#160;</td>
			<td style="width:127px;">SC-281692</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Superfrost microscope slides&#160;</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">12-550-15</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">HP Scanjet G4050</td>
			<td style="width:171px;">Multiple sellers&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;">Other commercial scanners are suitable for this step in the protocol.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">ImageJ&#160;</td>
			<td style="width:171px;">National Institute of Health</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Analytical Balance</td>
			<td style="width:171px;">Mettler Toledo&#160;</td>
			<td style="width:127px;">XSE 205U</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Precision Compact Oven&#160;&#160;</td>
			<td style="width:171px;">Thermo Scientific&#160;</td>
			<td style="width:127px;">PR305225M</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">1.7 mL microcentrifuge tubes (Eppendorfs)</td>
			<td style="width:171px;">Denville Scientific&#160;</td>
			<td style="width:127px;">C2170</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">Formamide</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">BP228-100</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:249px;">96-well plates</td>
			<td style="width:171px;">Fisher Scientific</td>
			<td style="width:127px;">07-200-9</td>
			<td style="width:283px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:249px;">Epoch Microplate Spectrophotometer&#160;</td>
			<td style="width:171px;">BioTek&#160;</td>
			<td style="width:127px;">Catalog number not available&#160;</td>
			<td style="width:283px;"></td>
		</tr>
	</tbody>
</table>

quantification of neurovascular protection following repetitive hypoxic preconditioning and transient middle cerebral artery occlusion in mice

Modelos animales experimentales de ictus son herramientas muy valiosas para la comprensión de la patología de carrera y el desarrollo de estrategias de tratamiento más eficaces. Un protocolo de 2 semanas para precondicionamiento hipóxico repetitivo (RHP) induce una protección a largo plazo contra las lesiones del sistema nervioso central (SNC) en un modelo de ratón de accidente cerebrovascular isquémico focal. RHP consta de 9 exposiciones estocásticos a la hipoxia que varían tanto en duración (2 o 4 hr) y la intensidad (8% y 11% O 2). RHP reduce los volúmenes de infarto, barrera hematoencefálica (BBB) ​​perturbación, y la respuesta inflamatoria posterior al accidente cerebrovascular durante semanas después de la última exposición a la hipoxia, lo que sugiere una inducción a largo plazo de un fenotipo SNC-protectora endógena. La metodología para la cuantificación de doble el volumen del infarto y disrupción de la BHE es eficaz en la evaluación de la protección neurovascular en ratones con RHP u otros neuroprotectores putativos. Adultos ratones macho Swiss Webster fueron acondicionados previamente por RHP o exposiciones de duración equivalente al 21% de O <sub&gt; 2 (es decir, aire ambiente). Una oclusión media transitoria 60 min arteria cerebral (tMCAo) fue inducida 2 semanas después de la última exposición hipóxica. Tanto la oclusión y reperfusión fueron confirmados por transcraneal flujometría Doppler láser. Veintidós horas después de la reperfusión, azul de Evans (EB) se administró por vía intravenosa a través de una inyección en la vena de la cola. 2 horas más tarde, los animales fueron sacrificados por sobredosis y secciones cerebrales isoflurano se tiñeron con cloruro de 2,3,5- trifeniltetrazolio (TTC). Los volúmenes de infarto fueron luego cuantificados. A continuación, EB se extrajo a partir del tejido durante 48 hr para determinar disrupción de la BHE después de tMCAo. En resumen, RHP es un protocolo simple que puede ser replicado, con un costo mínimo, para inducir protección neurovascular endógeno a largo plazo de una lesión derrame cerebral en ratones, con el potencial de la traducción de otros estados de enfermedad pro-inflamatorias CNS-basados ​​y sistémicos.

Este estudio incluyó a 25 ratones macho Swiss Webster que eran las 10 semanas de edad en el inicio de la asignación al azar en RHP (n = 10) o 21% de O 2 (n = 15) grupos. Dos semanas después de la exposición final RHP, se realizaron procedimientos quirúrgicos, con grupos cegados y contrapeso entre los días. Siguiendo tMCAo, 1 ratón murió durante la recuperación postoperatoria y 1 ratón fue excluido del estudio, ya que no cumplía con el criterio CBF reperfusión. Ambos ratones fueron excluidos del gr...

Watch this Scientific Journal Video about Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice at JoVE.com

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Cuantificación de Protección neurovascular Siguiendo repetitivo hipóxico Precondicionamiento y transitoria cerebral media oclusión de la arteria en ratones

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

quantification-neurovascular-protection-following-repetitive-hypoxic

Research

JoVE Journal

Medicine

13.6K vistas.  University of Texas Southwestern Medical Center. This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Video: Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modelado de accidente cerebrovascular en ratones - Oclusión arteria cerebral media con el modelo de filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Investigación

Medicina

Mouse Model of Middle Cerebral Artery Occlusion

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the brain (ischemic stroke) 2. Since most ischemic strokes (~80%) occur in the territory of middle cerebral artery (MCA) 3, many animal stroke models that have been developed have focused on this artery. The intraluminal monofilament model of middle cerebral artery occlusion (MCAO) involves the insertion of a surgical filament into the external carotid artery and threading it forward into the internal carotid artery (ICA) until the tip occludes the origin of the MCA, resulting in a cessation of blood flow and subsequent brain infarction in the MCA territory 4. The technique can be used to model permanent or transient occlusion 5. If the suture is removed after a certain interval (30 min, 1 h, or 2 h), reperfusion is achieved (transient MCAO); if the filament is left in place (24 h) the procedure is suitable as a model of permanent MCAO. This technique does not require craniectomy, a neurosurgical procedure to remove a portion of skull, which may affect intracranial pressure and temperature 6. It has become the most frequently used method to mimic permanent and transient focal cerebral ischemia in rats and mice 7,8. To evaluate the extent of cerebral infarction, we stain brain slices with 2,3,5-triphenyltetrazolium chloride (TTC) to identify ischemic brain tissue 9. In this video, we demonstrate the MCAO method and the determination of infarct size by TTC staining.

We demonstrate in the video a method for producing a middle cerebral artery occlusion in adult mice using an intraluminal monofilament. We also show how to evaluate the extent of cerebral infarction by 2,3,5-triphenyltetrazolium chloride (TTC) staining.

Modelo de ratón de la oclusión arteria cerebral media

Stroke is the most common fatal neurological disease in the United States 1. The majority of strokes (88%) result from blockage of blood vessels in the ...

Intraluminal Middle Cerebral Artery Occlusion (MCAO) Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United States in 20091,2. The intraluminal middle cerebral artery occlusion (MCAO) model was developed by Koizumi4 in 1986 to simulate this 
impactful human pathology in the rat. A modification of the MCAO method was later presented by Longa3. Both techniques have been widely used to identify 
molecular mechanisms of brain injury resulting from ischemic stroke and potential therapeutic modalities5. This relatively noninvasive method in rats has been 
extended to use in mice to take advantage of transgenic and knockout strains6,7. To model focal cerebral ischemia, an intraluminal suture is advanced via 
the internal carotid artery to occlude the base of the MCA. Retracting the suture after a specified period of time mimics spontaneous reperfusion, but the 
suture can also be permanently retained. This video will be demonstrating the two major approaches for performing intraluminal MCAO procedure in mice in a 
stepwise fashion, as well as providing insights for potential drawbacks and pitfalls. The ischemic brain tissue will subsequently be stained by 
2,3,5-triphenyltetrazolium chloride (TTC) to evaluate the extent of cerebral infarction8.

Intraluminal Middle Cerebral Artery Occlusion MCAO Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Mice

The intraluminal middle cerebral artery occlusion (MCAO) model is the most frequent used model among experimental ischemic stroke models. Here we will demonstrate the entire model in detail with the guide of Laser Doppler flowmetry, and its representative results.

Intraluminal la arteria cerebral media de oclusión (MCAO) Modelo para el ictus isquémico con Orientación flujometría láser Doppler en ratones

Stroke is the third leading cause of death and the leading cause of disability in the world, with an estimated cost of near $70 billion 
in the United ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

La endotelina-1 inducida por la arteria cerebral media oclusión Modelo para el ictus isquémico con Orientación flujometría láser Doppler en la rata

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Utilizando una ventana craneal para visualizar la arteria cerebral media Durante la endotelina-1 inducida por oclusión de la arteria cerebral media

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

Sistema optimizado para la monitorización de la perfusión cerebral en el modelo de rata de Carrera intraluminal oclusión arteria cerebral media

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Carrera Modelado en ratones: Coagulación permanente de la arteria cerebral media Distal

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Arteria embólico cerebral media oclusión (MCAO) para el ictus isquémico con homólogas coágulos de sangre en las ratas

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Surgical Approach for Middle Cerebral Artery Occlusion and Reperfusion Induced Stroke in Mice

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ischemic in origin and occur in the territory of the middle cerebral artery (MCA). Currently the only Food and Drug Administration (FDA) approved drug for the treatment of this devastating disease is tissue plasminogen activator (tPA). However, tPA has a small therapeutic window for administration (3 - 6 hr), and is only effective in 4% of the patients who actually receive it. Current research focuses on understanding the pathophysiology of stroke in order to find potential therapeutic targets. Thus, reliable models are crucial, and the MCA occlusion (MCAo) model (also termed the intraluminal filament or suture model) is deemed to be the most clinically relevant surgical model of ischemic stroke, and is fairly non-invasive and easily reproducible. Typically the MCAo model is used with rodents, especially with mice due to all the genetic variations available for this species. Here we describe (and present in the video) how to successfully perform the MCAo model (with reperfusion) in mice to generate reliable and reproducible data.

In order to understand the pathophysiology of stroke, it is important to use reliable models. This paper will describe one of the most frequently used stroke models in mice, termed the middle cerebral artery occlusion (MCAo) model (also termed the intraluminal filament or suture model) with reperfusion.

Técnica quirúrgica para la oclusión de la arteria cerebral media y la reperfusión del movimiento inducidos en ratones

Stroke is a leading cause of death worldwide and continues to be one of the major causes of long-term adult disabilities. About 87% of strokes are ...

Transient Middle Cerebral Artery Occlusion Model of Neonatal Stroke in P10 Rats

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature and mature brain. Stroke occurs commonly in the perinatal period in humans, and transient ischemia-reperfusion is the most common form of stroke in neonates. The reperfusion phase is a critical component of injury progression, which occurs over a period of days to weeks, and of the endogenous response to injury. This postnatal day 10 (p10) rat model of transient middle cerebral artery occlusion (tMCAO) creates a unilateral, non-hemorrhagic focal ischemia-reperfusion injury that can be utilized to study the mechanisms of focal injury and repair in the full-term-equivalent brain. The injury pattern that is produced by tMCAO is consistent and highly reproducible and can be confirmed with MRI or histological analyses. The severity of injury can be manipulated through changes in occlusion time and other methods that will be discussed.

Neonatal stroke is a significant cause of early brain injury requiring a translational model with consistent focal injury patterns and high reproducibility in order to enable study. This study describes the detailed surgical procedure for creating a non-hemorrhagic, unilateral focal ischemia-reperfusion injury in full-term-equivalent rodents.

Transitoria de la arteria cerebral media oclusión Modelo de accidente cerebrovascular neonatal en ratas P10

A number of animal models have been used to study hypoxic-ischemic injury, traumatic injury, global hypoxia, or permanent ischemia in both the immature ...