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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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 (Regierungspräsidium Karlsruhe, Germany). Please note that biosavety level 2 applies to mosquito and Plasmodium berghei ANKA sporozoite work.

1. Infection

1. 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 °C.
2. Collect female mosquitoes from their cage 17 to 22 days after the blood meal. Place them on ice to anesthetize them.
3. 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.
4. 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.
5. 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.
6. 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.
7. Centrifuge for 3 min at 1,000 x g and 4 °C to purify the sporozoites from the remaining tissue.
8. Pipette the supernatant, which contains the sporozoites (SPZ), to a new centrifuge tube and count the purified sporozoites in a Neubauer hemocytometer.
9. Adjust the concentration of purified sporozoites to 10,000/mL by adding phosphate-buffered saline.
10. 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 °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.
11. 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
12. 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
13. Assess the mice with MRI imaging according to the RMCBS score and the research question to be addressed.⁶

2. Magnetic Resonance Imaging Setup

1. 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 °C in order to maintain the body temperature of the mouse.
2. 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%.
3. 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.
4. 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.
5. 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.

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.

1. Begin by performing a localizing scan to make sure that the mouse brain is in the isocenter of the magnet.
2. To qualitatively assess the vasogenic edema, use 3D T2-weighted imaging by selecting a multi-slice RARE sequence.
   1. 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°; (spin-echo), and rare factor = 8. Start the sequence and wait 10 min 48 s to acquire the raw images.
3. To quantitatively assess vasogenic edema perform T2 relaxometry by selecting a multi-slice, multiple spin echo sequence.
   1. 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.
4. 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.
   1. 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, δ = 3 ms, Δ = 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.
5. To assess microhemorrhages, use 3D T2*-weighted imaging. Select a flow-compensated FLASH sequence.
   1. 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.
6. To assess arterial patency, use time of flight angiography by selecting a 3D FLASH sequence.
   1. 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.
7. 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.
   1. 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°;. Start the sequence and wait 1 min 14s until the images are acquired.

4. Image Processing and Analysis

1. 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
2. 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
3. Vasogenic edema
   1. 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).
   2. 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.
4. To analyze microhemorrhage volume, delineate microhemorrhages, which appear as ovoid, dark foci on T2*-weighted datasets, using the segmentation editor.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
Isoflurane	Baxter	1001747	for anesthesia
Dotarem	Guebert	1086923	Gd-DTPA contrast agent; 0.5 mmol/mL
Amira (Image Processing Program)	FEI Group		Version Amira 5.3.2
MATLAB	The MathWorks, Inc.,		Release 2012b
FDT toolbox	FMRIB's Software Library	http://www.fmrib.ox.ac.uk/fsl/fdt/index.html