Name: in vivo tracking of edema development and microvascular pathology in a model of experimental cerebral malaria using magnetic resonance imaging
Uploaded: 2017-06-08T12:30:00
Description: Watch this Scientific Journal Video about In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging at JoVE.com

Se agradece la asistencia técnica de expertos de Miriam Reinig. AH recibió fondos de una beca postdoctoral de la Facultad de Medicina de la Universidad de Heidelberg. MP es apoyado por un estipendio conmemorativo de la Fundación Else-Kröner-Fresenius. AKM es un beneficiario de una licencia de maternidad estipendio por la Academia DZIF del Centro Alemán de Investigación de Infecciones (DZIF). JP es el ganador de una Beca de Desarrollo Profesional de la Medicina Molecular (HRCMM) de Heidelberg. Además, agradecemos a Julia M. Sattler y Friedrich Frischknecht por brindar una película ejemplar de movimiento de esporozoitos.

Todos los experimentos con animales descritos en este artículo se llevaron a cabo de acuerdo con las directrices estándar de la Federación de Asociaciones de Científicos de Laboratorio (FELASA) y de la Sociedad de Laboratorios de Zoología (GV-SOLAS) y fueron aprobados por las autoridades alemanas locales en Karlsruhe (Regierungspräsidium Karlsruhe , Alemania). Tenga en cuenta que el nivel 2 de biosavianza se aplica al trabajo con esporozoítos de mosquitos y Plasmodium berghei ANKA. 1. I...

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En este artículo, describimos un protocolo de resonancia magnética cerebral completo para delinear los cambios en la malaria cerebral experimental. Creemos que la RM ha sido subutilizada en la investigación de la malaria hasta la fecha y esperamos que nuestros protocolos ayuden a otros investigadores. Nos gustaría describir algunos puntos adicionales que pueden ser útiles. Si los ratones gravemente enfermos son imágenes, el posicionamiento es crucial. Debido al aumento de la presión i...

La malaria es un importante problema de salud mundial. 1 La malaria severa se caracteriza en parte por la afectación cerebral y es a menudo un factor pronóstico deficiente. La afectación cerebral es frecuente en niños menores de cinco años en áreas de alta transmisión de la malaria y representa la principal causa de muerte relacionada con el paludismo en ese grupo de edad. 1 Aunque el tratamiento agresivo puede salvar vidas, la detección de la malaria cerebral, especialmente en las primeras etapas, puede ser difícil. Los procesos patológicos implicados en la malaria cerebral incluyen la interrupción microvascular y el edema cerebral, que puede conducir a la hinchazón cerebral severa. En este artículo, presentamos un protocolo de resonancia magnética (MRI) que permite el cerebro entero de imágenes in vivo de la malaria experimental cerebral (ECM). Métodos de imágenes de alta resolución de cerebro entero han sido ampliamente subutilizados en esta enfermedad, a pesar de que se sabe poco sobre cómo ECM inicia en el centroSistema nervioso o qué mecanismos específicos conducen a la enfermedad. La RMN in vivo , que abarca todo el cerebro, representa una importante herramienta de investigación para obtener una mejor comprensión de la patología de la ECM. La RM es capaz de evaluar la inflamación cerebral cerebral global, que recientemente se ha reconocido que es un importante predictor de la muerte no sólo en ECM, sino también en la malaria cerebral humana. La inflamación cerebral grave ocurre en la enfermedad mortal y representa una de varias características patológicas entre los modelos ECM y la enfermedad humana, una enfermedad que se caracteriza por alteraciones tanto inflamatorias como microvasculares. 4 La ECM puede inducirse en ratones CBA o C57BL mediante la infección con el ANKA letal de Plasmodium berghei. 5 El inicio de la MEC ocurre típicamente entre los días 6 y 10 después de la infección y resulta en ajuste, ataxia, dificultad respiratoria y coma, que conducen al rapMuerte. 4 La Escala Rápida de Coma y Comportamiento Murino (RMCBS) es una puntuación útil para evaluar los síntomas clínicos de ECM. Consta de 10 parámetros, cada uno puntuado de 0 a 2, con una puntuación máxima posible de 20. 6 Recientemente, hemos mostrado un buen acuerdo entre la gravedad de las puntuaciones RMCBS en ratones ECM y los cambios patológicos demostrados por la RM. En este protocolo, describimos la inducción de ECM en ratones y la resonancia magnética in vivo de ratones con ECM.

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			<td>Isoflurane</td>
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			<td colspan="2">FMRIB&#39;s Software Library</td>
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in vivo tracking of edema development and microvascular pathology in a model of experimental cerebral malaria using magnetic resonance imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

En los ratones C57BL / 6, los primeros síntomas clínicos de ECM pueden observarse entre los días 6 y 10 después de la infección con esporozoitos de P. berghei ANKA . ECM se desarrolla en 60-80% de los ratones infectados y rápidamente progresa al coma y la muerte en 24 a 48 h. En contraste, los ratones que no desarrollan ECM mueren después de la segunda semana después de la infección de la anemia grave debido a la hiperparasitemia. 12 <p class...

Watch this Scientific Journal Video about In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging at JoVE.com

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Seguimiento In Vivo del Desarrollo del Edema y Patología Microvascular en un Modelo de Malaria Cerebral Experimental usando Imágenes de Resonancia Magnética

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

The overall goal of this method is to monitor pathological changes of experimental cerebral malaria in vivo. This method can help to answer key questions in the pathogenesis of experimental cerebral malaria as it allows us to visualize in vivo how pathology of the whole brain evolves in space and over time. The technique thereby allows a detailed assessment of brain pathology, and can be used to assess the efficacy of novel treatment strategies in cerebral malaria.Performing the MRI setup will be Manuel Fischer, our lab manager. Begin by collecting female mosquitoes from their cage, 17 to 22 days after the blood meal. Use forceps to place three to four mosquitoes on a glass slide, combined with a drop of cold RPMI medium.Then, place the slide under a microscope. Carefully stretch the mosquito between the head and body with forceps, and use a syringe and needle to isolate the salivary gland. Next, collect the salivary glands from the glass slide by sucking them up with a glass pipette, and transferring them into 1.5 milliliter centrifuge tube.Then, use a small plastic stick to smash the isolated salivary glands within the centrifuge tube for three minutes in order to isolate the sporozoites from the salivary gland tissue. Centrifuge for three minutes at 1, 000 times g and four degrees Celsius to purify the sporozoites from the remaining tissue. Pipette the supernatant, which contains the sporozoites, to a new centrifuge tube, and count the purified sporozoites in a Neubauer hemocytometer.Sporozoites show a typical counterclockwise movement. Next, adjust the concentration of the purified sporozoites to 10, 000 per milliliter by adding phosphate-buffered saline. Finally, place a C57BL/6 mouse in a restrainer, and put its tail into warm water to better visualize the tail vein.Then, inject a total of 1, 000 sporozoites, or 0.1 milliliters, into the tail vein to initiate infection. Begin by bringing the mouse to a 9.4T small animal Magnetic Resonance Imaging, or MRI scanner, that uses a volume resonator for radiofrequency transmission. Then, apply dexpanthenol eye ointment to both eyes.Turn on the temperature-controlled water bath to 42 degrees Celsius in order to maintain the body temperature of the mouse. Then, place a tail vein catheter into the mouse's tail vein. Next, position the mouse for the MRI by placing it prone and with a crunched back on an animal bed equipped with a headlock and tooth bar to minimize head motion.Take care not to straighten the cervical spine of the mouse. Connect a contrast agent injection system filled with 0.3 millimole per kilogram Gd-DTPA to the tail vein catheter. Next, place the 4-Channel Phased Array Surface Receiver head coil onto the head of the mouse.Finally, place a breathing pad onto the back of the mouse and check the respiration on the monitoring device. Turn on the positional laser by pressing F2 on the control panel, and move the mouse until the horizontal positional light is in the middle of the smaller part of the coil, which covers the head of the mouse. Turn off the positional laser by pressing F2 again, and then move the mouse into the final position by pressing F3 on the control panel.Begin by performing a localizing scan to make sure that the mouse brain is in the isocenter of the magnet. Qualitatively assess vasogenic edema by using 3D T2-weighted imaging. Select a multi-slice RARE sequence, and adjust the slice position, making sure that the whole brain, from the nose to the cerebellum is covered.After adjusting the saturation slices, start the sequence, and wait 10 minutes and 48 seconds to acquire the images. Next, perform T2 relaxometry to quantitatively assess vasogenic edema by selecting multi-slice, multiple-spin echo sequence. Adjust the slice position, and start the acquisition of the sequence, which takes eight minutes and 53 seconds.To quantitatively assess both vasogenic edema and cytotoxic edema, carry out diffusion-weighted imaging by selecting a spin-echo EPI diffusion sequence. Adjust the slice position and saturation slice. Then start the sequence and wait seven minutes and 56 seconds until the raw images are acquired.Following that, assess microhemorrhages, using 3D T2 star-weighted imaging. Select a flow-compensated FLASH sequence, adjust the slice position, and start the sequence, which runs for 15 minutes and 40 seconds. Afterwards, assess arterial patency using time of flight angiography by selecting a 3D FLASH sequence.Adjust the slice position, start the sequence, and then wait seven minutes and 16 seconds until the images are acquired. Finally, assess the blood-brain barrier disruption. Select a 3D T1-weighted imaging with a FLASH sequence, adjust the slice position, and start the sequence.Wait one minute and 14 seconds until images without contrast are acquired. Then, inject contrast of 0.3 millimole per kilogram and repeat the measurement. An increase in blood-brain barrier disruption and a fluid increase indicate early vasogenic edema in the olfactory bulb that is already present in mild disease, and starts to spread towards the brain stem as the severity of the disease increases.On T2 maps, the severity of vasogenic edema can be quantified by drawing regions of interest into specific anatomical regions. For each region, a T2 relaxation time that increases with increasing edema can be obtained. Furthermore, brain volume indicative of edema starts to significantly increase in moderately sick mice as compared to baseline, and further increases in severely sick mice.Lastly, microvascular pathology, as evidenced by microhemorrhages and an increase of vessel susceptibility contrast, occurs after the first signs of blood-brain barrier disruption in vasogenic edema. These microhemmorhages predominantly occur in the olfactory bulb. If the whole MRI protocol is obtained, all images including positioning can be obtained within one hour, if it is performed properly.The minimal protocol should include a T2 weighted sequence or T2 relaxometry, and a T2 star-weighted sequence in order to judge the presence of vasogenic edema, microhemmorhage load, and microvascular impairment. After watching this video, you should have a good understanding how whole-brain MRI visualizes pathological changes in experimental cerebral malaria, including such changes as brain swelling, which is difficult to detect ex vivo.

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Medicine

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

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