The expert technical assistance of Miriam Reinig is gratefully acknowledged. AH received funding from a postdoctoral stipend of the Medical Faculty of the University of Heidelberg. MP is supported by a memorial stipend from the Else-Kro&#776;ner-Fresenius Foundation. AKM is a recipient of a maternity leave stipend by the DZIF Academy of the German Center for Infection Research (DZIF). JP is the recipient of a Heidelberg Research Center for Molecular Medicine (HRCMM) Career Development Fellowship. We furthermore gratefully acknowledge Julia M. Sattler and Friedrich Frischknecht for providing an exemplary movie of sporozoite movement.

All animal experiments reported in this article were conducted according to the Federation for Laboratory Animal Science Associations (FELASA) category B and the Society of Laboratory Animal Science (GV-SOLAS) standard guidelines and were approved by the local German authorities in Karlsruhe (Regierungspr&#228;sidium Karlsruhe, Germany). Please note that biosavety level 2 applies to mosquito and Plasmodium berghei ANKA sporozoite work.
1. Infection
<ol>
	<li>Infect Anopheles stephensi</e...

<ol>
	<li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World Malaria Report. , (2014).</li><li>Seydel, K. B., 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=Brain+swelling+and+death+in+children+with+cerebral+malaria.">Brain swelling and death in children with cerebral malaria.</a> N Engl J Med. 372 (12), 1126-1137 (2015).</li><li>Penet, M. F., 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+experimental+cerebral+malaria+in+vivo:+significant+role+of+ischemic+brain+edema.">Imaging experimental cerebral malaria in vivo: significant role of ischemic brain edema.</a> J Neurosci. 25 (32), 7352-7358 (2005).</li><li>de Souza, J. B., Riley, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+malaria:+the+contribution+of+studies+in+animal+models+to+our+understanding+of+immunopathogenesis.">Cerebral malaria: the contribution of studies in animal models to our understanding of immunopathogenesis.</a> Microbes Infect. 4 (3), 291-300 (2002).</li><li>Curfs, J. H., van der Meide, P. H., Billiau, A., Meuwissen, J. H., Eling, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodium+berghei:+recombinant+interferon-gamma+and+the+development+of+parasitemia+and+cerebral+lesions+in+malaria-infected+mice.">Plasmodium berghei: recombinant interferon-gamma and the development of parasitemia and cerebral lesions in malaria-infected mice.</a> Exp Parasitol. 77 (2), 212-223 (1993).</li><li>Carroll, R. W., 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=A+rapid+murine+coma+and+behavior+scale+for+quantitative+assessment+of+murine+cerebral+malaria.">A rapid murine coma and behavior scale for quantitative assessment of murine cerebral malaria.</a> PLoS One. 5 (10), (2010).</li><li>Hoffmann, 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+Spreads+along+the+Rostral+Migratory+Stream.">Experimental Cerebral Malaria Spreads along the Rostral Migratory Stream.</a> PLoS Pathog. 12 (3), e1005470 (2016).</li><li>Mueller, A. K., Behrends, J., Blank, J., Schaible, U. E., Schneider, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+to+study+tuberculosis-malaria+coinfection+upon+natural+transmission+of+Mycobacterium+tuberculosis+and+Plasmodium+berghei.">An experimental model to study tuberculosis-malaria coinfection upon natural transmission of Mycobacterium tuberculosis and Plasmodium berghei.</a> J Vis Exp. (84), e50829 (2014).</li><li>Hynynen, K., McDannold, N., Sheikov, N. A., Jolesz, F. A., Vykhodtseva, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+and+reversible+blood-brain+barrier+disruption+by+noninvasive+focused+ultrasound+at+frequencies+suitable+for+trans-skull+sonications.">Local and reversible blood-brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications.</a> Neuroimage. 24 (1), 12-20 (2005).</li><li>Nag, N., Mellott, T. J., Berger-Sweeney, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+postnatal+dietary+choline+supplementation+on+motor+regional+brain+volume+and+growth+factor+expression+in+a+mouse+model+of+Rett+syndrome.">Effects of postnatal dietary choline supplementation on motor regional brain volume and growth factor expression in a mouse model of Rett syndrome.</a> Brain Res. 1237, 101-109 (2008).</li><li>Giri, 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=T2+quantification+for+improved+detection+of+myocardial+edema.">T2 quantification for improved detection of myocardial edema.</a> J Cardiovasc Magn Reson. 11, 56 (2009).</li><li>Engwerda, C., Belnoue, E., Gruner, A. 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. 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=Perivascular+Arrest+of+CD8++T+Cells+Is+a+Signature+of+Experimental+Cerebral+Malaria.">Perivascular Arrest of CD8+ T Cells Is a Signature of Experimental Cerebral Malaria.</a> PLoS Pathog. 11 (11), e1005210 (2015).</li><li>Potchen, M. J., 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=Acute+brain+MRI+findings+in+120+Malawian+children+with+cerebral+malaria:+new+insights+into+an+ancient+disease.">Acute brain MRI findings in 120 Malawian children with cerebral malaria: new insights into an ancient disease.</a> AJNR Am J Neuroradiol. 33 (9), 1740-1746 (2012).</li></ol>

In this article, we describe a whole-brain MRI protocol to delineate changes in experimental cerebral malaria. We believe that MRI has been under-utilized in malaria research to date and hope that our protocols will aid other investigators. We would like to describe some additional points that may be helpful.
If severely sick mice are imaged, positioning is crucial. Due to increased intracranial pressure, mice are susceptible to death, and thus, the cervical spine should not be stretched. Anes...

Malaria is a significant global health problem.1 Severe malaria is characterized in part by cerebral involvement and is often a poor prognostic factor. Cerebral involvement is common in children under the age of five in areas of high malaria transmission and represents the major cause of malaria related death in that age group.1 While aggressive treatment can be life-saving, the detection of cerebral malaria, especially in early stages, can be difficult. The pathological processes involved in cerebral malaria include microvascular disruption and cerebral edema, which can lead to severe brain swelling. In this article, we present a magnetic resonance imaging (MRI) protocol that allows whole-brain in vivo imaging of experimental cerebral malaria (ECM). Whole-brain high-resolution imaging methods have been widely underutilized in this disease, even though little is known about how ECM initiates in the central nervous system or what specific mechanisms lead to the disease. In vivo MRI, covering the whole brain, represents an important research tool to gain a better understanding of ECM pathology. MRI is able to assess global cerebral brain swelling, which has recently been recognized to be an important predictor of death not only in ECM, but also in human cerebral malaria.2,3 Severe brain swelling occurs in fatal disease and represents one of several pathological features between the ECM models and human disease, a disease that is characterized by both inflammatory and microvascular alterations.4
ECM can be induced in CBA or C57BL mice through infection with lethal Plasmodium berghei ANKA.5 The onset of ECM typically occurs between days 6 and 10 post-infection and results in fitting, ataxia, respiratory distress, and coma, which lead to rapid death.4 The Rapid Murine Coma and Behavior Scale (RMCBS) is a helpful score to evaluate clinical symptoms of ECM. It consists of 10 parameters, each scored from 0 to 2, with a maximum possible score of 20.6 Recently, we showed good agreement between the severity of the RMCBS scores in ECM mice and pathological changes demonstrated by MRI.7 In this protocol, we describe ECM induction in mice and in vivo magnetic resonance imaging of mice with ECM.

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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.

In C57BL/6 mice, the first clinical symptoms of ECM can be observed between days 6 and 10 after infection with P. berghei ANKA sporozoites. ECM develops in 60 - 80% of infected mice and rapidly progresses to coma and death within 24 to 48 h. In contrast, mice that do not develop ECM die after the second week post-infection from severe anemia due to hyperparasitemia.12
In MRI imaging, the earliest...

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.

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 ...

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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6.4K visualizações.  Heidelberg University Hospital. 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 se...