DOI: 10.5061/dryad.79cnp5hwp
title: Data from: Early detection of cerebrovascular pathology and protective antiviral immunity by MRI

description: Central nervous system (CNS) infections are a major cause of human
 morbidity and mortality worldwide. Even patients that survive CNS
 infections can have lasting neurological dysfunction resulting from immune
 and pathogen induced pathology. Developing approaches to noninvasively
 track pathology and immunity in the infected CNS is crucial for patient
 management and development of new therapeutics. Here, we develop novel
 MRI-based approaches to monitor virus-specific CD8+ T cells and their
 relationship to cerebrovascular pathology in the living brain. We studied
 a relevant murine model in which a neurotropic virus (vesicular stomatitis
 virus) was introduced intranasally and then entered the brain via
 olfactory sensory neurons – a route exploited by many pathogens in humans.
 Using T2*-weighted high-resolution MRI, we identified small cerebral
 microbleeds as the earliest form of pathology associated with viral entry
 into the brain. Mechanistically, these microbleeds occurred in the absence
 of peripheral immune cells and were associated with infection of vascular
 endothelial cells. We monitored the adaptive response to this infection by
 developing methods to iron label and track individual virus specific CD8+
 T cells by MRI. Transferred antiviral T cells were detected in the brain
 within a day of infection and were able to reduce cerebral microbleeds.
 These data demonstrate the utility of MRI in detecting the earliest
 pathological events in the virally infected CNS as well as the therapeutic
 potential of antiviral T cells in mitigating this pathology.
description: <strong>MRI study and
 quantification of bleeding in the nasal turbinates and
 brain.</strong> MRI experiments were performed at 11.7 T on a Bruker
 Biospec MRI system (Bruker BioSpin). T2<sup>*</sup>-weighted
 3D gradient-recalled echo (GRE) sequences were used for acquisitions. For
 <em>in-vivo</em> imaging, the following parameters were used:
 isotropic resolution = 75 mm, TE/TR = 10/30 ms, FA = 10°, NA = 3. After
 <em>in-vivo</em> acquisitions, mice were perfused
 transcardially with 5% formalin. The heads were post-fixed with 10%
 formalin. 24-hr before <em>ex-vivo</em> MRI imaging
 acquisition, heads were transferred to PBS buffer. For
 <em>ex-vivo</em> MRI: isotropic resolution = 50 mm, TE/TR =
 20/40 ms, FA = 15°, NA = 12. <em>Ex-vivo</em>
 high-resolution MRI was used to quantify nasal and brain bleeding in VSV
 infected mice. Because bleeding in the turbinates was so extensive,
 volumes were measured. Hypointensity volumes were obtained by manually
 drawn serial voxels of interest (VOI) using the Medical Image Processing,
 Analysis, and Visualization (MIPAV) program (http://mipav.cit.nih.gov)
 (1). Hypointensities in the brain were more focal so the number of spots
 was counted manually. The volume of hypointensities and number of
 hypointensity spots from normal healthy mice were used as background and
 subtracted from the measurements for each infected mouse. N = 5-7 mice per
 group. <strong>Quantitative real-time
 PCR (RT-qPCR) for VSV titers.</strong> Total RNA was isolated from
 OB using miRNeasy Mini Kit (Qiagen). Subsequently, 1 μg of RNA was treated
 with amplification grade DNAse I (Life Technologies) to remove any
 contaminating genomic DNA. We used 250 ng of total RNA for cDNA synthesis
 using iScript cDNA Synthesis Kit (Bio-Rad). All Q-PCRs were performed in
 20 μL volumes using SYBR Green PCR master mix (Applied Biosystems) in a
 96-well optic tray using a CFX96 Real-Time PCR machine (Bio-Rad). The
 reactions were conducted in duplicate, and samples without reverse
 transcriptase were used as non-template controls. Forward primer (specific
 for the VSV genes): TGA TGA TGC ATG ATC CAG CTC T; reverse primer: ACA CAC
 CTC CAA TGG AAG GGT. Q-PCR reactions were run with an initial denaturation
 temperature of 95 °C for 10 min, followed by 40 cycles of three-step
 amplification (denaturation at 95 °C for 10 s, 60 °C annealing for 15s and
 extension at 72 °C for 20 s). Actin RNA was used as internal standard. N =
 4-6 mice per group. <strong>Quantification of CD8
 T cells and microbleeds.</strong> Quantifications were carried out
 under 40x view (0.2 mm x 0.2 mm). N = 4-6 mice per group. 6 views were
 counted manually per mouse. <strong>Quantification of
 vessel VCAM-1 and ICAM-1 coverage and intensity of CD68
 signals.</strong> Images obtained under 20x view (0.4 mm x 0.4 mm)
 were used for quantification. ImageJ was used for quantification. During
 quantifications of vessel coverage, the CD31 area was taken as 100% and
 the VCAM-1 or ICAM-1 positive area was expressed as a percentage
 normalized to CD31 area. N = 3-4 mice per group. 2-4 views were counted
 manually per mouse. <strong>Statistical
 analysis.</strong> Statistical analysis was carried out with the
 Student's t-test. A p &lt; 0.05 was considered statistically
 significant. <strong>References</strong> 1.         Saar G, Cheng N, Belluscio L, Koretsky AP. Laminar specific detection of APP induced neurodegeneration and recovery using MEMRI in an olfactory based Alzheimer's disease mouse model. Neuroimage. 2015;118:183-92. doi: 10.1016/j.neuroimage.2015.05.045. PubMed PMID: WOS:000360630200018.