Introduction

Experimental autoimmune encephalomyelitis (EAE) is a common mouse model, resembling the clinical manifestations of multiple sclerosis and aiding in the assessment of neuroinflammation [1]. During active neuroinflammation, inflammatory immune cells in the brain, astrocytes and microglia, are significantly activated, generating a neurotoxic microenvironment partially attributed to the release of reactive oxygen species [2,3]. We hypothesize that extracellular ROS released by reactive microglia and astrocytes during neuroinflammation will mediate the oxidation of Fe-PyC3A from the low-relaxivity Fe2+ state to the high-relaxivity Fe3+ state [4], enhancing MR signal. Here, we imaged a mouse model of EAE-induced neuroinflammation using the oxidatively activated ROS-sensing MRI contrast agent, Fe-PyC3A, for non-invasive detection of astrocytes and microglia changes.

Methods

A total of 12 C57BL/6 male mice (10-11 weeks old). EAE is induced in C57BL/6 mice by immunization with an emulsion of MOG35-55 in complete Freund’s adjuvant, followed by administration of pertussis toxin in PBS, first on the day of immunization and again on the following day (N = 8). Mice were monitored daily, and clinical disease severity was measured using the standard EAE grading scale. Control mice received i.p. injection of saline (3 mg/kg) (N = 4). On day 20 post-immunization, animals were anesthetized with isoflurane (1.5%) and imaged with a 4.7 Tesla Bruker MRI scanner equipped with a custom-built volume coil. Prior to i.v. injection of 0.3 mmol/kg Fe-PyC3A or 0.1 mmol/kg Gd-DOTA, the blood-brain barrier was transiently disrupted via i.v. injection of 25% wt/wt mannitol in the control mice. T1-weighted gradient echo images were acquired dynamically before and up to 20 minutes post-injection of Fe-PyC3A or Gd-DOTA. We selected hippocampus as regions of interest (ROIs) and analyzed the (post-pre) injection percentage change in signal intensity (SI%) from these ROIs and (post-pre) injection change in brain ROIs vs. muscle contrast to noise ratio (ΔCNR) from 2 min after Fe-PyC3A or Gd-DOTA injection. Mice were euthanized 1h post-injection, and brain tissue was harvested for immunohistochemical evaluation of microglial activation (Iba1) and astrocytes activation (glial fibrillary acidic protein (GFAP)). The proportional area of Iba-1 and GFAP expression (area%) in the hippocampus was measured in ImageJ.

Results

On day 20 after the mice were immunized, their EAE clinical score was 7 or worse (Figure 1A). Iba-1 staining demonstrates significantly greater microglial activation in EAE mice vs. controls (P = 0.027) in the hippocampus, while GFAP immunostaining shows greater astrocyte activation (P = 0.004) (Figure 1B-C). T1w images recorded prior to and after Gd-DOTA or Fe-PyC3A injection into control and EAE mice demonstrate differential hippocampal signal enhancement in the EAE mice as shown in Figure 1D. We found that significantly higher SI% in the hippocampus of EAE mice than in the controls (P=0.13) at 2 min post-Fe-PyC3A with no significant difference in the SI% between the two groups (P<0.0001) using Gd-DOTA as a non-oxidatively activated negative control probe (Figure 1E). In the EAE mice, the ΔCNR in the hippocampus induced by Fe-PyC3A was significantly higher than that using Gd-DOTA (P<0.0001). The proportional area of Iba-1 and GFAP expression significantly and positively correlated with SI% (r = 0.851, P < 0.001 and r = 0.926, P < 0.0001; respectively), and ΔCNR (r = 0.738, P = 0.006, and r = 0.828, P < 0.001; respectively), for all mice imaged with Fe-PyC3A. In contrast, no significant correlations between SI% or ΔCNR and Iba-1 or GFAP positive area were observed in the hippocampus and cortex for mice imaged with Gd-DOTA.

Conclusion

Molecular MRI using Fe-PyC3A provided robust and selective contrast enhancement in the hippocampus of EAE mice as compared to controls, potentially serving as a noninvasive tool to quantify neuroinflammation in neurological disorders. 

Image/Figure:

Click to view full size

Image/Figure Caption:

Figure 1 A) Averaged clinical EAE score. Mice were assessed daily using standard EAE scoring for 20 days post-immunization. B) Representative Iba-1 and GFAP immunohistochemistry images for control and EAE mice taken at 20×. C) Proportional area of Iba-1 and GFAP expression (microglia and astrocytes area %) in the hippocampus. D) T₁-weighted images acquired before and after injection of 0.1 mmol/kg Gd-DOTA or 0.3 mmol/kg Fe-PyC3A in control and EAE groups (C57BL/6 mice). E) Comparison of SI% and ΔCNR of the hippocampus after mice injected with Fe-PyC3A or Gd-DOTA.

Author