Name: in vivo tracking of edema development and microvascular pathology in a model of experimental cerebral malaria using magnetic resonance imaging
Uploaded: 2017-06-08T12:30:00.000+00:00
Duration: 9 min 4 s
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

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 mosquitoes with Plasmodium berghei ANKA by feeding them for 15 min on a gametocytemic mouse. Keep the infected mosquitoes at 80% humidity and 21 &#176;C.</li>
	<li>Collect female mosquitoes from their cage 17 to 22 days after the blood meal. Place them on ice to anesthetize them.</li>
	<li>Using forceps, place three to four mosquitoes on a glass slide covered with a drop of cold RPMI medium. Place the slide under a microscope.</li>
	<li>Using forceps, carefully stretch the mosquito between the head and body. Isolate the salivary gland using a syringe and needle. Repeat this procedure with the remaining mosquitoes.</li>
	<li>Collect the salivary glands from the glass slide by sucking them up with a glass pipette and collect them in a 1.5 mL centrifuge tube. 
	NOTE: Depending on the infection rates, usually 8,000 to 15,000 infectious sporozoites can be obtained per salivary gland.</li>
	<li>For approximately 3 min, smash the isolated salivary glands within the centrifuge tube with a small, plastic stick to isolate the sporozoites from the salivary gland tissue.</li>
	<li>Centrifuge for 3 min at 1,000 x g and 4 &#176;C to purify the sporozoites from the remaining tissue.</li>
	<li>Pipette the supernatant, which contains the sporozoites (SPZ), to a new centrifuge tube and count the purified sporozoites in a Neubauer hemocytometer.</li>
	<li>Adjust the concentration of purified sporozoites to 10,000/mL by adding phosphate-buffered saline.</li>
	<li>Inject a total of 1,000 sporozoites (0.1 mL) into the tail veins of inbred C57BL/6 mice to initiate infection. To facilitate the injections, place the C57BL/6 mice in a restrainer and put the tails into warm (approximately 37 &#176;C) water to assist with the visualization of the tail veins; 
	NOTE: The injection itself is a short procedure that can be performed within a few seconds.</li>
	<li>Once daily, check blood-stage parasitemia on blood smears from day 3 onwards after the SPZ infection. 
	NOTE: Monitoring parasitemia has been previously visualized in a JoVE article by Mueller et al.8</li>
	<li>Assess the mice once daily with the Rapid Murine Coma and Behavior Scale (RMCBS) score, starting from day 5 after the sporozoite injection. 
	NOTE: A detailed description of this procedure, including a video demonstration, has been published by Caroll et al.6</li>
	<li>Assess the mice with MRI imaging according to the RMCBS score and the research question to be addressed.6</li>
</ol>
2. Magnetic Resonance Imaging Setup
<ol>
	<li>Perform MRI on a 9.4T small animal scanner using a volume resonator for radiofrequency transmission and a 4-channel-phased-array surface receiver coil. Turn on the temperature-controlled water bath to 42 &#176;C in order to maintain the body temperature of the mouse.</li>
	<li>Induce anesthesia in a chamber using 2% isoflurane and compressed air until the mouse no longer reacts to a toe pinch. Maintain the anesthesia at 1-1.5%.</li>
	<li>Place a tail vein catheter into the tail vein of the mouse. Position the mouse for 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.</li>
	<li>Connect a contrast agent injection system to the tail vein catheter. Use a custom-made injection system filled with a Gd-DTPA (0.3 mmol/kg) syringe or use PE tubing connected to a Gd-DTPA (0.3 mmol/kg) syringe.</li>
	<li>Apply dexpanthenol eye ointment to both eyes. Place the 4-channel-phased-array surface receiver head coil onto the head of the mouse. Place a breathing pad onto the back of the mouse and connect it to a respiration monitoring device.</li>
</ol>
3. Imaging Protocol
NOTE: Choose imaging sequences from the protocol listed below according to the research questions to be addressed. All listed parameters are valid for the MRI software but might need to be adjusted if other software programs are used.
<ol>
	<li>Begin by performing a localizing scan to make sure that the mouse brain is in the isocenter of the magnet.</li>
	<li>To qualitatively assess the vasogenic edema, use 3D T2-weighted imaging by selecting a multi-slice RARE sequence.
	<ol>
		<li>Enter the following parameters into the MRI software: repetition time = 2.000 ms, echo time = 22 ms, isotropic resolution = 0.1mm, field of view = 20 x 10 x 12 mm3; matrix = 200 x 100 x 120, flip angle = 90-180&#176;; (spin-echo), and rare factor = 8. Start the sequence and wait 10 min 48 s to acquire the raw images.</li>
	</ol>
	</li>
	<li>To quantitatively assess vasogenic edema perform T2 relaxometry by selecting a multi-slice, multiple spin echo sequence.
	<ol>
		<li>Use the following parameters: repetition time = 3.100 ms, echo time = 8-136 ms in increments of 8 ms, number of slices = 17, slice thickness = 0.7 mm, in plane resolution = 0.116 mm x 0.116 mm, field of view 20 x 20 mm2, matrix = 172 x 172, flip angle = 90-180 degrees; (spin-echo). Start the sequence and wait 8 min 53 s to acquire the raw images.</li>
	</ol>
	</li>
	<li>To quantitatively assess both vasogenic edema and cytotoxic edema, carry out diffusion-weighted imaging/apparent diffusion coefficient (ADC) mapping by selecting a spin-echo EPI diffusion sequence.
	<ol>
		<li>Use the following parameters: repetition time = 3.400 ms, echo time = 20 ms, slice thickness = 0.7 mm, number of slices = 17, number of diffusion sensitized directions = 30, b value = 1.500 s/mm2, &#948; = 3 ms, &#916; = 9 ms, partial Fourier encoding acceleration factor = 1.51, field of view = 12 x 15 mm2, matrix = 96 x 128, in-plane resolution = 0.125 mm x 0.117 mm, flip angle = 90-180 degrees, and number of saturation slices (sagittal) = 1. Start the sequence and wait 7 min 56 s until the raw images are acquired.</li>
	</ol>
	</li>
	<li>To assess microhemorrhages, use 3D T2*-weighted imaging. Select a flow-compensated FLASH sequence.
	<ol>
		<li>Enter the following parameters into the MRI software: repetition time = 2.000 ms, echo time = 22 ms, isotropic resolution 0.08 mm, field of view = 32 x 15 x 8 mm3, matrix size = 400 x 188 x 100, and flip angle = 12 degrees. Start the sequence and wait 15 min 40 s to acquire the raw images.</li>
	</ol>
	</li>
	<li>To assess arterial patency, use time of flight angiography by selecting a 3D FLASH sequence.
	<ol>
		<li>Use the following parameters in the MRI software: repetition time = 16 ms, echo time = 3.5 ms, slice thickness = 0.07 mm, in-plane resolution = 0.104 x 0.104 mm, field of view = 20 x 20 x 10 mm3, matrix = 192 x 192 x 142, and flip angle = 15 degrees. Start the sequence and wait 7 min 16 s until the images are acquired.</li>
	</ol>
	</li>
	<li>To assess blood-brain barrier disruption, use 3D T1-weighted imaging before and after a contrast agent injection of 0.3 mmol/kg. Select a radio frequency-spoiled FLASH sequence with a global radio frequency excitation.
	<ol>
		<li>Use the following sequence parameters in the MRI software: repetition time = 5 ms, echo time = 1.9 ms, isotropic resolution = 0.156 mm, field of view 20 x 18.7 x 18.7 mm3, matrix 128 x 120 x 120 and flip angle = 8.5&#176;;. Start the sequence and wait 1 min 14s until the images are acquired.</li>
	</ol>
	</li>
</ol>
4. Image Processing and Analysis
<ol>
	<li>To analyze blood-brain barrier disruption, subtract 3D non-enhanced T1-weighted images from enhanced T1-weighted images with the arithmetic tool or image calculator tool in ImageJ. Evaluate the subtraction images for an increase of signal, which corresponds to blood-brain barrier disruption.9</li>
	<li>To analyze brain volume, use native 3D T1- or 3D T2-weighted datasets. Delineate the brain from the olfactory bulb to the cerebellum using the segmentation editor.10</li>
	<li>Vasogenic edema
	<ol>
		<li>Process the T2 relaxometry data with MRI software or use a nonlinear least-squares fit procedure.11 Process diffusion-weighted data to obtain ADC maps using MRI software or FDT toolbox (see Materials Table).</li>
		<li>Place regions of interest manually into the different anatomical regions. 
		NOTE: Automatic placement of regions of interest may register incorrectly due to significant brain swelling. The T2 times and ADC values of the chosen anatomical regions are obtained in this fashion.</li>
	</ol>
	</li>
	<li>To analyze microhemorrhage volume, delineate microhemorrhages, which appear as ovoid, dark foci on T2*-weighted datasets, using the segmentation editor.</li>
</ol>

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	<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. 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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. 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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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The authors declare that they have no conflicts of interest.

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. Anesthesia should also be kept to a minimum. If mice with mild disease are imaged, the best time point for MRI is 12-24 h prior to the expected onset of the first clinical symptoms. Mild vasogenic edema in the olfactory bulb is visible in mice that do not exhibit any clinical signs of ECM (RMCBS score of 20).
The length of the MRI protocol itself can be adapted according to the specific research question. The minimal protocol should include a T2-weighted sequence or T2 relaxometry and a T2*-weighted sequence in order to judge the presence of vasogenic edema and microhemorrhage load/microvascular impairment. To get good-quality images, motion artefacts should be kept to a minimum. Motion will be reduced through the proper positioning of the mouse and by keeping the breathing rate at about 60-80 breaths per min.
Other in vivo imaging methods, such as intravital microscopy, show promise in visualizing pathological alterations in ECM on a cellular level.14,15,16,17 Compared to MRI, intravital microscopy can offer high spatial resolution at the cost of only covering a limited cortical area. In contrast, MRI is able to cover the whole brain at a resolution of up to 80 &#181;m. MRI can therefore guide further cellular investigations by identifying the predilection sites-the beginning of disease-as well as the stage of the disease, which can then be assessed using further methodologies. MRI findings in ECM can be compared to pathological alterations detected with MRI in human cerebral malaria. Both human and experimental cerebral malaria show vasogenic edema in areas of neurogenesis, as well as microvascular pathology, such as secondary cerebral infarcts in watershed areas.7,18 White and grey matter are affected in a similar fashion, and brain swelling in both human and experimental cerebral malaria involves the brainstem in severe disease, explaining the comatose state.2,7
We hope that in vivo imaging will help unravel the underlying pathological mechanism of cerebral malaria, which currently remains elusive. As edema and brain swelling are barely visible ex vivo, methods to visualize this pathology in vivo are key and will help to improve the evaluation of novel therapies and vaccinations.

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.

<table><tbody><tr><td>Isoflurane</td><td>Baxter</td><td>1001747</td><td>for anesthesia</td></tr><tr><td>Dotarem</td><td>Guebert</td><td>1086923</td><td>Gd-DTPA contrast agent; 0.5 mmol/mL</td></tr><tr><td>Amira (Image Processing Program)</td><td>FEI Group</td><td></td><td>Version Amira 5.3.2</td></tr><tr><td>MATLAB&amp;nbsp;</td><td>The MathWorks, Inc.,</td><td></td><td>Release 2012b</td></tr><tr><td>FDT toolbox&amp;nbsp;</td><td>FMRIB&#039;s Software Library</td><td>&lt;a href=&quot;http://www.fmrib.ox.ac.uk/fsl/fdt/index.html&quot;&gt;http://www.fmrib.ox.ac.uk/fsl/fdt/index.html&lt;/a&gt;</td><td></td></tr></tbody></table>

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 signs of ECM are visible in the olfactory bulb, which belongs to the olfactory system of the mouse and is located in the rostral part of the brain.7,13 An increase in blood-brain barrier disruption (seen on contrast-enhanced T1-weighted images) and a fluid increase (increase of T2 signal on T2-weighted images, T2 relaxometry, and diffusion-weighted imaging) indicate early vasogenic edema in the olfactory bulb that is already present in mild disease (RMCBS score 20-16) and starts to spread as the severity of the disease increases. Relative T1 signal images help to detect subtle blood-brain barrier disruption (Figure 1). Blood-brain barrier disruption and vasogenic edema progress in a specific fashion. These pathological changes spread along the rostral migratory stream, a migrating route for neuroblasts, towards the brainstem, which is reached in very severe disease (RMCBS sore 9-0). On T2 maps, the severity of vasogenic edema can be quantified by drawing regions of interest into specific anatomical regions, as demonstrated in Figure 2. For each region, a T2 relaxation time that increases with increasing edema can be obtained. Along with vasogenic edema and blood-brain barrier disruption, brain swelling starts as the ECM severity increases. Brain volume indicative of edema starts to significantly increase in moderately sick mice as compared to baseline and further increases in severely sick mice (Figure 3).
Microvascular pathology, as evidenced by microhemorrhages and an increase of vessel susceptibility contrast (corresponding to slow flow), occurs after the first signs of blood-brain barrier disruption and vasogenic edema and can be visualized with T2*-weighted imaging. Microhemorrhages predominantly occur in the olfactory bulb (Figure 4). The number of microhemorrhages increases with progressive disease. With progressive disease microhemorrhages are also evident in cortex, basal ganglia, cerebellum, white matter and brainstem.
Decreased arterial patency is seen by time-of-flight angiography as a sign of macrovascular impairment in parallel to microvascular changes. Cerebral infarction is not a key finding in ECM. In very severe disease, however, focal areas of ADC decrease can be identified, indicating cerebral infarctions.
<img alt="Figure 1" src="/files/ftp_upload/55334/55334fig1.jpg" /> 
Figure 1: Blood-brain Barrier Disruption. (A) A representative coronal 3D T1-weighted image. An increase in T1 signal is visible in the central region of the olfactory bulb. (B) On the corresponding T1 relative signal image (T1 pre-contrast image subtracted from the T1 post-contrast image), contrast agent extravasation is easier to delineate. Contrast agent extravasation is visible in the frontal cortical regions (white arrows). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55334/55334fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55334/55334fig2.jpg" /> 
Figure 2: T2 Relaxometry. Sample T2 maps at different heights from one severely sick mouse with an RMCBS score of 5 (A) and from the baseline scan (B) are displayed in color scale, in grey scale, and in grey scale with ROIs as colored overlays. Dark blue = RMS, green = cortex, turquoise = external capsule, yellow = basal ganglia, light blue = DMS, orange = thalamus, and red = brainstem <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55334/55334fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55334/55334fig3.jpg" /> 
Figure 3: Brain Volume Measurements. Brain volume significantly increases in moderately sick mice (RMCBS 15-10). Representative values of 10 mice are shown. Data is presented as the mean standard deviation. A paired Student&#39;s t-test was used to analyze the differences in brain swelling between groups. In moderately/severely sick mice, the brain volume was significantly increased compared to baseline (p = 0.021/0.001). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55334/55334fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55334/55334fig4.jpg" /> 
Figure 4: Microhemorrhages. Representative T2*-weighted images of a mouse before the onset of blood-stage infection (A) and on day 8 after infection with an RMCBS score of 14 (B and C) are shown. In the first image, no microhemorrhges are evident (A), while in the second and third images, several microhemorrhages are visible, with a predominance in the olfactory bulb (B: plane coronal image; C: 3D projection with segmented microhemorrhages).

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 ...

in-vivo-tracking-edema-development-microvascular-pathology-model

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JoVE Journal

Medicine

6.4K Views.  Heidelberg University Hospital. 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. 

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

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neuroscience

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Immunology and Infection

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Continuous advancements in noninvasive imaging modalities such as magnetic resonance imaging (MRI) have greatly improved our ability to study physiological or pathological processes in living organisms. MRI is also proving to be a valuable tool for capturing transplanted cells in vivo. Initial cell labeling strategies for MRI made use of contrast agents that influence the MR relaxation times (T1, T2, T2*) and lead to an enhancement (T1) or depletion (T2*) of signal where labeled cells are present. T2* enhancement agents such as ultrasmall iron oxide agents (USPIO) have been employed to study cell migration and some have also been approved by the FDA for clinical application. A drawback of T2* agents is the difficulty to distinguish the signal extinction created by the labeled cells from other artifacts such as blood clots, micro bleeds or air bubbles. In this article, we describe an emerging technique for tracking cells in vivo that is based on labeling the cells with fluorine (19F)-rich particles. These particles are prepared by emulsifying perfluorocarbon (PFC) compounds and then used to label cells, which subsequently can be imaged by 19F MRI. Important advantages of PFCs for cell tracking in vivo include (i) the absence of carbon-bound 19F in vivo, which then yields background-free images and complete cell selectivityand(ii) the possibility to quantify the cell signal by 19F MR spectroscopy.

Monitoring Dendritic Cell Migration using 19F / 1H Magnetic Resonance Imaging

Tracking of cells using MRI has gained remarkable attention in the past years. This protocol describes the labeling of dendritic cells with fluorine (19F)-rich particles, the in vivo application of these cells, and monitoring the extent of their migration to the draining lymph node with 19F/1H MRI and 19F MRS.

Continuous  advancements in noninvasive imaging modalities such as magnetic resonance  imaging (MRI) have greatly improved our ability to study ...

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are particularly devastating, difficult to treat, and confer a poor prognosis. While the precise incidence of brain metastases in the United States remains hard to estimate, it is likely to increase as extracranial therapies continue to become more efficacious in treating cancer. Thus, it is necessary to identify and develop novel therapeutic approaches to treat metastasis at this site. To this end, intracranial injection of cancer cells has become a well-established method in which to model brain metastasis. Previously, the inability to directly measure tumor growth has been a technical hindrance to this model; however, increasing availability and quality of small animal imaging modalities, such as magnetic resonance imaging (MRI), are vastly improving the ability to monitor tumor growth over time and infer changes within the brain during the experimental period. Herein, intracranial injection of murine mammary tumor cells into immunocompetent mice followed by MRI is demonstrated. The presented injection approach utilizes isoflurane anesthesia and a stereotactic setup with a digitally controlled, automated drill and needle injection to enhance precision, and reduce technical error. MRI is measured over time using a 9.4 Tesla instrument in The Ohio State University James Comprehensive Cancer Center Small Animal Imaging Shared Resource. Tumor volume measurements are demonstrated at each time point through use of ImageJ. Overall, this intracranial injection approach allows for precise injection, day-to-day monitoring, and accurate tumor volume measurements, which combined greatly enhance the utility of this model system to test novel hypotheses on the drivers of brain metastases.

Intracranial brain metastasis modeling is complicated by an inability to monitor tumor size and response to treatment with precise and timely methods. The presented methodology couples intracranial tumor injection with magnetic resonance imaging analysis, which when combined, cultivates precise and consistent injections, enhanced animal monitoring, and accurate tumor volume measurements.

Metastatic spread of cancer is an unfortunate consequence of disease progression, aggressive cancer subtypes, and/or late diagnosis. Brain metastases are ...

Cancer Research

In vivo 19F MRI for Cell Tracking

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

In vivo 19F MRI for Cell Tracking

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

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

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events of myocardial injuries, various mouse models of myocarditis have been widely used. This study involved experimental autoimmune myocarditis (EAM) induced with cardiac myosin heavy chain (Myhc)-&#945; 334-352 in A/J mice; the affected animals develop lymphocytic myocarditis but with no apparent clinical signs. In this model, the utility of magnetic resonance microscopy (MRM) as a non-invasive modality to determine the cardiac structural and functional changes in animals immunized with Myhc-&#945; 334-352 is shown. EAM and healthy mice were imaged using a 9.4 T (400 MHz) 89 mm vertical core bore scanner equipped with a 4 cm millipede radio-frequency imaging probe and 100 G/cm triple axis gradients. Cardiac images were acquired from anesthetized animals using a gradient-echo-based cine pulse sequence, and the animals were monitored by respiration and pulse oximetry. The analysis revealed an increase in the thickness of the ventricular wall in EAM mice, with a corresponding decrease in the interior diameter of ventricles, when compared with healthy mice. The data suggest that morphological and functional changes in the inflamed hearts can be non-invasively monitored by MRM in live animals. In conclusion, MRM offers an advantage of assessing the progression and regression of myocardial injuries in diseases caused by infectious agents, as well as response to therapies.

This study demonstrates the successful establishment of magnetic resonance microscopy imaging as a non-invasive tool to assess the cardiac abnormalities in mice affected with autoimmune myocarditis. The data indicate that the technique can be used to monitor the disease-progression in live animals.

Myocarditis is an inflammation of the myocardium, but only ~10% of those affected show clinical manifestations of the disease. To study the immune events ...

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes it is challenging to study them on clinical MRI scans. It was recently demonstrated that cortical microinfarcts can be depicted with MRI scanners using high magnetic field strengths (7T). Based on this experience, a proportion of these lesions is also visible on lower resolution 3T MRI. These findings were corroborated with ex vivo imaging of post-mortem human brain tissue, accompanied by histopathological verification of possible cortical microinfarcts.
Here an ex vivo imaging protocol is presented, for the purpose of validating MR observed cerebral microvascular pathology with histological evaluation. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on both in vivo 7T and 3T MR images. These guidelines provide researchers with a tool to rate cortical microinfarcts on in vivo images of larger patient samples, to further unravel their clinical relevance in cognitive decline and dementia, and establish these lesions as a novel biomarker of cerebral small vessel disease.

A high resolution ex vivo 7T MR imaging protocol is presented, to perform MR-guided histopathological validation of microvascular pathology in post-mortem human brain tissue. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on in vivo 7T as well as 3T MR images.

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes ...

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

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

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

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

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

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Generation of a Mouse Model of Experimental Cerebral Malaria Using Host Mosquito-Derived Sporozoites

Source: Hoffmann, A. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55334">In Vivo&#160;Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging.</a>&#160;J. Vis. Exp.&#160; (2017)
This video demonstrates the generation of an experimental mouse model of cerebral malaria. Sporozoites isolated from Plasmodium berghei-infected female mosquitoes are injected into a restrained mouse&#39;s tail veins, leading to blood-brain barrier disruption and brain inflammation, causing cerebral malaria. This infected mouse model offers a valuable tool to study in vivo microvascular pathology associated with cerebral malaria.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Summary

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Chapters in this video

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

Monitoring Dendritic Cell Migration using ¹⁹F / ¹H Magnetic Resonance Imaging

Modeling Brain Metastases Through Intracranial Injection and Magnetic Resonance Imaging

In vivo ¹⁹F MRI for Cell Tracking

Noninvasive Assessment of Cardiac Abnormalities in Experimental Autoimmune Myocarditis by Magnetic Resonance Microscopy Imaging in the Mouse

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images

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

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Generation of a Mouse Model of Experimental Cerebral Malaria Using Host Mosquito-Derived Sporozoites