Introduction

Ebola virus disease (EVD) is a severe and often lethal disease caused by Ebola virus (EBOV) ¹. Despite reports of headaches, cognitive impairment, hypoactivity or hyperactivity in EVD patients ², there is little in vivo neuroimaging evidence to support central nervous system (CNS) involvement. Characterization of CNS pathophysiology in the setting of EBOV infection is necessary to better understand the long term sequelae of the infection in survivors. Using quantitative T1 and R2* relaxometry techniques, we evaluated brain changes in a small-particle aerosol nonhuman primate model of EVD. Quantitative Imaging findings were then correlated with plasma, brain tissue, and cerebral spinal fluid (CSF) viral load.

Methods

Magnetic resonance (MR) imaging was performed on an Achieva 3.0T clinical MR scanner (Philips Healthcare, Cleveland, OH, USA) equipped with an 8-channel pediatric neuro-spine coil. A total of seven rhesus macaques (6.93±3.78 yr; two males, five females) were studied. All animals were exposed via small-particle aerosol to an EBOV Makona variant (particle size range 0.5–3.0 µm, averaged inhaled dose 1,150 plaque-forming units). Subjects were intubated, immobilized using isoflurane, and positioned supine on the scanner bed. For quantitative assessment of the acute viral effect on MR signal changes, MR images were obtained prior to and 8-9 days following exposure. Plasma, brain tissue, and CSF viral loads were measured using real-time reverse transcription polymerase chain reaction (RT-qPCR).
Image Acquisition
T1-weighted (T1w) anatomical whole-brain images were acquired using a magnetization‑prepared rapid gradient echo (MP-RAGE) sequence (Repetition time (TR) = 9.8 ms, echo time (TE) = 4.7 ms, Inversion time (TI) = 1,100 ms, flip angle (FA) = 8°, resolution = isotropic 0.5 mm, 2 average, field-of-view (FOV) = 96 mm x 96 mm x 68 mm, and acquisition time = 4.5 min). For T1 measurement, a fast field echo (FFE) sequence with a dual-flip-angle method ³ was performed (TR/TE = 27 ms/3.4 ms, FA 1/FA 2 = 5°/27°, resolution = isotropic 0.5 mm, 1 average, 136 slices, acquisition time = 12 min). For R2* measurement, T1w FFE sequence was performed (TR/TE = 27 ms/34 ms, FA = 5°, resolution = isotropic 0.5 mm, 1 average, and acquisition time = 6 min), and principles of echo shifting with R2* (PRES R2*) in combination with T1 imaging data (FA=5°) was used to create R2* maps.
Image Processing and Statistical Analysis
For the quantitative analysis, all MR images were processed using an in-house built pipeline, comprising non-linear image registration to an INIA19 template, skull stripping, Gaussian smoothing (1.5 mm full width at half maximum (FWHM)), and voxel-based statistical analysis within a general linear model. A paired t-test was applied for group differences between pre-and post-EBOV exposure and the false discovery rate was applied for correcting multiple comparisons. A statistical significance was considered at p < 0.05. Finally, Pearson correlation (r) was used to examine the relationship between quantitative MR results and viral RNA load.

Results

Comparison of the averaged pre-exposure and post-exposure T1 maps (n = 7) (Fig. 1) showed increasing T1 values in multiple brain regions, including the cerebellum, occipital lobe, and frontal cortex, which was confirmed by voxel-based whole-brain analysis (p < 0.005, Fig. 2). The averaged R2* maps (n = 7) showed increased values post-exposure compared to pre-exposure (Fig. 3). Voxel-based R2* analysis displayed a widespread increase in R2* values (Fig. 4), including gray matter, white matter, and ventricles. Viral RNA was detected in brain tissue samples, CSF, and plasma (Fig 5). R2* changes overlapping the central venous system were positively correlated with the viral load in CSF (r = 0.79, p = 0.018, Fig. 5).

Discussion and Conclusion

We demonstrated acute alterations of quantitative T1 and R2* values in rhesus macaque brain 8–9 days after small-particle aerosol exposure to EBOV. Several pathological EVD features may cause a change in brain tissue characteristics, contributing to T1 and R2* changes. Pathological features contributing to the T1 changes may include endothelial injury and edema induced by EBOV infection ⁴. The significant R2* elevation after infection may reflect the altered microenvironment in the brain tissue, such as accumulation of iron-containing although it’s more likely those changes are due to venous congestion (data not shown here). In line with the increased viral load in brain tissue samples, CSF, and plasma, a significant correlation between the central venous R2* changes and the CSF viral load reinforces in vivo imaging evidence of CSF involvement proportional to disease severity. Taken together, these results present evidence of CNS involvement in EBOV infection and provide new insights into the pathogenesis of aerosol-induced EBOV disease.

Acknowledgements

The authors thank David Thomasson and Margaret Lentz and the Integrated Research Facility at Fort Detrick (IRF-Frederick) team for their support. With the U.S. National Institute of Allergy and Infectious Diseases (NIAID), this work was supported in part through the prime contract of Laulima Government Solutions, LLC, under contract (HHSN272201800013C), Tunnell Government Services, a subcontractor of Laulima Government Solutions, LLC under contract (HHSN272201800013C), and Battelle Memorial Institute’s former prime contract under contract (HHSN272200700016I). This work was further supported in part by federal funds from the National Cancer Institute and National Institutes of Health (NIH) under contract (75N91019D00024, Task Order no. 75N91019F00130). The content of this publication does not necessarily reflect the views or policies of the U.S. Department of Health and Human Services or of the institutions and companies affiliated with the authors, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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