MRI demonstrates glutamine antagonist-mediated reversal of cerebral malaria pathology in mice

Abstract

The deadliest complication of Plasmodium falciparum infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood-brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.

Keywords: MRI; Plasmodium falciparum; cerebral malaria; experimental cerebral malaria; glutamine antagonist.

Fig. 1.

T₂-weighted coronal images of a mouse brain at multiple planes. (A) Diagram showing the planes of imaging in a mouse brain, starting with the olfactory bulbs and continuing through the cerebellum. (B) Representative scans of one uninfected mouse and one PbA-infected mouse on day 6 p.i. The infected animal shows high signal intensity in the white matter of the olfactory bulbs, along the corpus callosum (mainly inferior aspect) and the external capsules (white arrows) consistent with edematous changes.

Fig. 2.

Quantitative analysis of cross-sectional T₂ relaxometry and ADC values in different regions of the brain. (A) Cross-sectional T₂ values for different regions of the brain in uninfected mice (n = 4), and mice day 5 p.i. (n = 5, average clinical score = 3) and day 6 p.i. (n = 4, average clinical score = 4). The olfactory bulbs show a significant increase in T₂ values between uninfected and day 6 p.i. animals (Dunn’s post hoc, P = 0.025) while the striatum shows a significant increase between day 5 and day 6 p.i. animals (Dunn’s post hoc, P = 0.012). Kruskal–Wallis analysis: olfactory bulbs, P = 0.0068; cortex, P > 0.1; corpus callosum, P > 0.1; and striatum, P = 0.0022. (B) Cross-sectional ADC values for different regions of the brain in uninfected mice (n = 4) and infected mice day 5 p.i. (n = 4, average clinical score = 3) and day 6 p.i. (n = 4, average clinical score = 2). The olfactory bulbs show a significant increase in ADC values between uninfected mice and infected mice day 6 p.i. (Dunn’s post hoc, P = 0.043). Kruskal–Wallis analysis: olfactory bulbs, P = 0.022; cortex, P > 0.1; corpus callosum, P > 0.1; and striatum, P > 0.1. Error bars represent mean ± SD. Dunn’s post hoc analysis: *P < 0.05.

Fig. 3.

Pre- and postcontrast coronal T₁ images at different planes in the mouse brain. Representative cross-sectional pre- and postcontrast T₁-weighted images in (A) an uninfected and (B) an infected mouse. Postcontrast images in the infected animal show increased signal intensity (solid white arrows) in the olfactory bulbs, underneath the corpus callosum, and external capsules. Physiologic contrast leakage into the ventricular system is seen in the uninfected animal due to prolonged imaging time. Ventricular contrast leakage in the infected animal is more marked, suggesting blood–CSF barrier disruption (arrowheads). Multiple microhemorrhagic changes are seen in the infected animal (hollow white arrows).

Fig. 4.

Quantification of cross-sectional T₁ signal enhancement (between pre- and postcontrast images) in different regions of the brain. Cross-sectional T₁ values shown are for uninfected mice (n = 4), day 5 p.i. mice (n = 5, average clinical score = 3); post initial treatment, day 6 p.i. mice (n = 8, average clinical score = 4); and post second treatment day 7 p.i. mice (n = 6, average clinical score = 2). Treated day 6 p.i. animals were scanned immediately following initial treatment on the morning of day 6 p.i., while treated day 7 p.i. animals were imaged 12+ h after receiving second treatment. Kruskal–Wallis analysis: olfactory bulbs, P = 0.0012; cortex, P = 0.0012; corpus callosum, P = 0.0031; and striatum, P = 0.0008. Error bars represent mean ± SD. Dunn’s post hoc analysis: *P < 0.05, **P < 0.005.

Fig. 5.

Longitudinal, coronal, T₂-weighted images of JHU-083–treated animals. (A) Coronal T₂-weighted images in a representative animal that survived long-term imaged after treatment at 6:00 AM on day 6 p.i. with JHU-083. Abnormal high signal intensity foci in the OBs appear on day 6 p.i. (solid white arrows) and then resolve by day 12 p.i. (B) Quantification of T₂ values and (C) ADC values in the OBs of infected animals that responded to treatment (n = 4, black symbols) and two that progressed rapidly and did not survive despite treatment (n = 2, red circles) are shown. T₂: paired t test comparison between day 5 and 8 p.i., P = 0.043; and ADC: paired t test comparison between day 5 and 8 p.i., P > 0.05. Dashed lines denote mean ± SD for T₂ and ADC values in uninfected animals (T₂, 39.1 ± 2.2 and ADC 0.64 ± 0.05). (D) Corresponding clinical score data vs. day postinfection for the imaged animals.

Fig. 6.

Longitudinal, coronal postcontrast T₁-weighted images of JHU-083–treated animals. (A) Postcontrast T₁-weighted images showing reversal of abnormal enhancement (solid white arrows) in the OBs, CC, and external capsules in a representative treated animal scanned on days 6, 8, and 14 p.i. (B) Quantitative decrease in percent signal enhancement for infected animals measured on day 6 and 8 p.i. (n = 7). Paired t test in the olfactory bulbs, P < 0.0001; cortex, P = 0.0038; corpus callosum, P = 0.0010; and striatum, P = 0.0017. Dashed lines denote mean ± SD for uninfected animals in a given region (olfactory bulbs 3.5 ± 2.3; cortex 2.5 ± 2.3; corpus callosum 4.0 ± 2.1; and striatum 0.75 ± 1.5). (C) Corresponding clinical scoring vs. day postinfection for the same seven animals. All animals were treated at 6:00 AM on day 6 p.i.

Fig. 7.

Immunofluorescent staining of sagittal sections of mice infected with PbA (untreated and treated) and control uninfected mice. (A) A representative full sagittal section of the stained mouse brain. (B) Magnified images obtained in the olfactory bulbs (red square in A) of an infected untreated mouse that was euthanized 6 d p.i. (Left column), a treated mouse euthanized 8 day p.i. (Middle column), and an uninfected mouse (Right column). Positive staining for fibrinogen (bright green), red blood cells (TER119, red), endothelial cells (CD31, yellow), and GFP-labeled parasites (light gray) show evidence of extravascular protein and blood extravasation in both infected mice compared with the control animal. Regions of protein accumulation colocalize with extravasated, parasite-infected RBCs (33.3% magnification). (Scale bar: 100 µm.)