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.

MRI study and quantification of bleeding in the nasal turbinates and brain. MRI experiments were performed at 11.7 T on a Bruker Biospec MRI system (Bruker BioSpin). T2^*-weighted 3D gradient-recalled echo (GRE) sequences were used for acquisitions. For in-vivo imaging, the following parameters were used: isotropic resolution = 75 mm, TE/TR = 10/30 ms, FA = 10°, NA = 3. After in-vivo acquisitions, mice were perfused transcardially with 5% formalin. The heads were post-fixed with 10% formalin. 24-hr before ex-vivo MRI imaging acquisition, heads were transferred to PBS buffer. For ex-vivo MRI: isotropic resolution = 50 mm, TE/TR = 20/40 ms, FA = 15°, NA = 12.

Ex-vivo 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.

Quantitative real-time PCR (RT-qPCR) for VSV titers. 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.

Quantification of CD8 T cells and microbleeds. 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.

Quantification of vessel VCAM-1 and ICAM-1 coverage and intensity of CD68 signals. 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.

Statistical analysis. Statistical analysis was carried out with the Student's t-test. A p < 0.05 was considered statistically significant.

References

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.