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

L&#39;intervento tecnico esperto di Miriam Reinig è riconosciuto con gratitudine. AH ha ricevuto finanziamenti da uno stipendio postdoctorale della Facoltà di Medicina dell&#39;Università di Heidelberg. Il deputato è sostenuto da uno stipendio commemorativo della Fondazione Else-Kröner-Fresenius. AKM è un destinatario di uno stipendio per la maternità dall&#39;Accademia DZIF del Centro tedesco per la ricerca sulle infezioni (DZIF). JP è il destinatario di un Centro di Ricerca Heidelberg per la Medicina Molecolare (HRCMM) Borsa di Sviluppo per la Carriera. Inoltre riconosciamo con gratitudine Julia M. Sattler e Friedrich Frischknecht per aver fornito un film esemplare di movimento sporozoite.

Tutti gli esperimenti sugli animali riportati in questo articolo sono stati condotti secondo le linee guida della Federazione per le Associazioni di Scienza degli Animali del Laboratorio (FELASA) e le norme standard della Società di Scienze del Laboratorio (GV-SOLAS) e sono state approvate dalle autorità locali tedesche a Karlsruhe (Regierungspräsidium Karlsruhe , Germania). Si prega di notare che il livello 2 di biosavidità si applica alla zanzara e al lavoro di sporozoite Plasmodium berghei ANKA. <p class="jov...

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C., Renia, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+cerebral+malaria.">Experimental models of cerebral malaria.</a> Curr Top Microbiol Immunol. 297, 103-143 (2005).</li><li>Zhao, 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=Olfactory+plays+a+key+role+in+spatiotemporal+pathogenesis+of+cerebral+malaria.">Olfactory plays a key role in spatiotemporal pathogenesis of cerebral malaria.</a> Cell Host Microbe. 15 (5), 551-563 (2014).</li><li>Nacer, 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=Experimental+cerebral+malaria+pathogenesis--hemodynamics+at+the+blood+brain+barrier.">Experimental cerebral malaria pathogenesis--hemodynamics at the blood brain barrier.</a> PLoS Pathog. 10 (12), e1004528 (2014).</li><li>Nacer, 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=Neuroimmunological+blood+brain+barrier+opening+in+experimental+cerebral+malaria.">Neuroimmunological blood brain barrier opening in experimental cerebral malaria.</a> PLoS Pathog. 8 (10), e1002982 (2012).</li><li>Pai, S., 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=Real-time+imaging+reveals+the+dynamics+of+leukocyte+behaviour+during+experimental+cerebral+malaria+pathogenesis.">Real-time imaging reveals the dynamics of leukocyte behaviour during experimental cerebral malaria pathogenesis.</a> PLoS Pathog. 10 (7), e1004236 (2014).</li><li>Shaw, T. 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In questo articolo, descriviamo un protocollo MRI intero a cervello per delineare i cambiamenti nella malaria cerebrale sperimentale. Riteniamo che la risonanza magnetica sia stata poco utilizzata nella ricerca sulla malaria e speriamo che i nostri protocolli aiuteranno altri investigatori. Vorremmo descrivere alcuni punti aggiuntivi che possono essere utili. Se i topi gravemente malati vengono imaged, il posizionamento è cruciale. A causa di una maggiore pressione intracranica, i topi sono...

La malaria è un grave problema di salute globale. 1 La malaria grave è caratterizzata in parte dal coinvolgimento cerebrale e spesso è un fattore prognostico scarso. Il coinvolgimento cerebrale è comune nei bambini al di sotto dei cinque anni in aree di trasmissione di malaria elevata e rappresenta la causa principale della morte correlata alla malaria in quel gruppo di età. 1 Mentre il trattamento aggressivo può essere salvato, il rilevamento della malaria cerebrale, soprattutto nelle fasi iniziali, può essere difficile. I processi patologici coinvolti nella malaria cerebrale includono la disfunzione microvascolare e l&#39;edema cerebrale, che possono portare a rigonfiamenti del cervello. In questo articolo, presentiamo un protocollo di risonanza magnetica (MRI) che consente l&#39;imaging in vivo di tutta la cervello della malaria cerebrale sperimentale (ECM). I metodi di imaging ad alta risoluzione ad intero cervello sono stati ampiamente inutilizzati in questa malattia, anche se poco conosciuto su come l&#39;ECM inizia nel centroIl sistema nervoso o quali meccanismi specifici conducono alla malattia. La risonanza magnetica in vivo , che copre l&#39;intero cervello, rappresenta un importante strumento di ricerca per ottenere una migliore comprensione della patologia ECM. La MRI è in grado di valutare il gonfiore del cervello cerebrale globale, che è stato recentemente riconosciuto come un importante predittore della morte non solo in ECM, ma anche nella malaria cerebrale umana. 2 , 3 Il gonfiore cerebrale si manifesta in malattie fatali e rappresenta una delle caratteristiche patologiche tra i modelli ECM e la malattia umana, una malattia caratterizzata da alterazioni infiammatorie e microvascolari. 4 ECM può essere indotto nei topi CBA o C57BL attraverso l&#39;infezione con letale Plasmodium berghei ANKA. 5 L&#39;insorgenza di ECM si verifica tipicamente tra i giorni 6 e 10 dopo l&#39;infezione e produce risultati di attaccamento, atassia, distress respiratoria e coma, che portano a rapId di morte. 4 La Coma di Rapid Murine e la Scala del Comportamento (RMCBS) è un utile punteggio per valutare i sintomi clinici di ECM. È composto da 10 parametri, ognuno segnato da 0 a 2, con un punteggio massimo di 20 possibile. 6 Recentemente, abbiamo mostrato un buon accordo tra la gravità dei punteggi RMCBS nei topi ECM e le modificazioni patologiche dimostrate da MRI. 7 In questo protocollo, descriviamo l&#39;induzione di ECM nei topi e la rappresentazione in vivo di risonanza magnetica dei topi con ECM.

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			<td><a href="http://www.fmrib.ox.ac.uk/fsl/fdt/index.html">http://www.fmrib.ox.ac.uk/fsl/fdt/index.html</a></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.

Nei topi C57BL / 6 i primi sintomi clinici di ECM possono essere osservati nei giorni 6 e 10 dopo l&#39;infezione con sporozoites P. berghei ANKA . ECM si sviluppa nel 60-80% dei topi infetti e progredisce rapidamente al coma e alla morte entro 24 - 48 h. Al contrario, i topi che non sviluppano ECM muoiono dopo la seconda settimana dopo l&#39;infezione da anemia grave a causa di iperparastasiemia. 12 <p class="jove_content" fo:keep-together.within-pag...

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.

Tracciamento in vivo di sviluppo dell&#39;edema e patologia microvascolare in un modello di malaria cerebrale sperimentale mediante l&#39;utilizzo di immagini a risonanza magnetica

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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