NaSA-CEST: MR monitoring of brain inflammation using an aspirin metabolite as contrast agent
Xiaolei Song1, Yanrong Chen1,2, Chenwang Jin1,3, Chengyan Chu1, Irina Shats1, Yuguo Li1, Yue Yuan1, Xiaowei He2, Piotr Walzcak1, and Jeff W.M Bulte1

1Johns Hopkins Univeristy, Baltimore, MD, United States, 2Northwest University, Xi'an, Shaanxi, China, 3Xi'an Jiaotong University, Xi'an, Shaanxi, China


Sodium salicylate (NaSA), a nonsteroidal anti-inflammatory drug and the main metabolite of aspirin, can accumulate specifically in inflamed tissue. We investigated the use of NaSA-enhanced CEST MRI for in vivo mapping of brain inflammation, induced by intrastriatal injection of lipopolysaccharide (LPS). Inflamed mice exhibited an increase of ~8% NaSA-CEST signal on the AUC10-30min images, with signals for the ipsilateral striatum significantly higher than those for the mirrored contralateral regions. While for the non-inflamed sham mice, which received saline instead of LPS, no such NaSA-CEST signal could be observed. Our NaSA-CEST approach could possibly lead to the development of inflammation-specific theranostics.


Sodium salicylate (NaSA), a nonsteroidal anti-inflammatory drug and the main metabolite of aspirin, can accumulate specifically in inflamed tissue1, where it can exert analgesic, antipyretic, and anti-inflammatory effects. We previously showed that NaSA can accumulate ex vivo in lipopolysaccharide (LPS)-treated mouse organotypic brain slice cultures2, and that NaSA can be detected with Chemical Exchange Saturation Transfer (CEST) MRI in vivo at millimolar concentrations3. In this study, we investigated the use of NaSA-enhanced CEST MRI for in vivo mapping of brain inflammation.

Materials and Methods

Focal neuroinflammation was induced by a unilateral intrastriatal injection of 2 ul of 5 mg/ml lipopolysaccharide (LPS) in C57/Bl6 mice (8-10 weeks, n=8)4. For another group of mice called sham mice, saline was injected instead of LPS using the same surgical procedure. 24 hours later, mice underwent MR scans on a 11.7 T horizontal scanner, with tail vein injection of 100 ul NaSA as CEST MRI contrast agent (100 mg/Kg body weight). A single-slice RARE sequence was used to acquire pre- and post-NaSA injection images, with a saturation frequency offset at 9.3 ppm (peak frequency for NaSA). Dynamic Salicylate Enhancement (DSE) at time-point t was quantified as DSE = [S(+9.3ppm, t)-S(+9.3ppm, 0)]/S0. DSE images from 10 min to 30 min post-injection of NaSA were pooled to form an area-under-the curve (AUC) image, representing the NaSA-CEST contrast4. At the end of the imaging experiment, Gd-enhanced T1-w images were collected to verify the blood-brain-barrier status. Post-mortem immunohistology was performed using H&E staining, microglia (anti-IBA1) staining, and anti-COX1 staining. To validate the accumulation of NaSA in inflamed brain, ex vivo 11.7 T CEST MRI and LC/MS-MS were performed on brain tissue, excised 30 min post-injection of NaSA. For LC/MS-MS, the two hemispheres were separated, and NaSA was quantified as previously described2. All image analysis was performed using MATLAB, and statistical analysis was performed using Prism and a non-parametric Wilcoxon signed rank test.


Inflamed mice exhibited an increase of ~8% NaSA-CEST signal on the AUC10-30min images. The enhanced region was more diffuse than the T2w or Gd-enhanced T1w lesions (Fig. 1A). Regions of interest (ROIs) were plotted on the enhanced cortex and striatum in the ipsilateral hemisphere as well as their contralateral counterparts. The DSE signals for all the four ROIs showed a similar trend, increasing at ~10–15 min post-injection of NaSA and a gradual decay after 40 min (Fig. 1B). The DSE images at different time points are shown in Fig. 1C. For all mice induced with LPS, the DSE signal at 30 min was significantly higher in ROI 1 compared to ROI 2 (Fig. 1D), but not between ROI 3 and ROI 4. In non-inflamed sham mice (Fig.2), which received saline instead of LPS, no NaSA-CEST signal could be observed following NaSA injection, and lesions were seen on T2w- and Gd-enhanced images except for the needle track. Inflammation was validated by immunohistochemical staining, showing the presence of (Iba1+) activated microglia (Fig.3A) and COX1 enzyme, which is one of the main molecules involved in the NaSA pathway. COX1 co-localized with the nuclei of many cell types including microglia, infiltrating lymphocytes, and neurons. LC-MS/MS measurements also indicated that the inflamed mouse brains contained more NaSA than non-inflamed sham mice (Fig.3B) Ex vivo CEST Z-spectra further validated that the region near LPS-injection site displays a peak at the NaSA saturation frequency offset (~9.3 ppm) while the lower cortex region did not (Fig. 3C). Similar to the in vivo DSE signal intensity at 30 min, the ex vivo NaSA-CEST images showed a ~4% enhanced region using the area quantification due to lack of pre-image. We also collected DSE images on the inflamed mice upon i.v. injection of saline, it does not show a signal trend like in Fig.1B. (not shown)


Our NaSA-CEST MRI approach for mapping local inflammation uses a non-metallic, organic metabolite of an over-the-counter available anti-inflammatory agent (aspirin). These initial results, if further validated, could possibly lead to the development of inflammation-specific theranostics, where NaSA and similar compounds may diagnose and suppress inflammation simultaneously.


NIH R21EB020905


1. Brune K. Biodistribution of salicylates: a clue to the understanding of some effects and side effects. Agents and actions Supplements 1977;2:163-177.

2. Schaffhauser M. MA, Marx J, Bulte J, McMahon M, Fatemi A, Song X. Assessment of salicylate accumulation in LPS-treated organotypic mouse brain slices using CEST-MRI” , New York City. 2016; New York City. p 2493111.

3. Song X, Walczak P, He X, Yang X, Pearl M, Bulte JW, Pomper MG, McMahon MT, Janowski M. Salicylic acid analogues as chemical exchange saturation transfer MRI contrast agents for the assessment of brain perfusion territory and blood-brain barrier opening after intra-arterial infusion. J Cereb Blood Flow Metab 2016;36(7):1186-1194.

4. Xu X, Chan KW, Knutsson L, Artemov D, Xu J, Liu G, Kato Y, Lal B, Laterra J, McMahon MT, van Zijl PC. Dynamic glucose enhanced (DGE) MRI for combined imaging of blood-brain barrier break down and increased blood volume in brain cancer. Magn Reson Med 2015;74(6):1556-1563.


Fig.1 In vivo NaSA-CEST MRI 24 hours after intrastriatal-injection of LPS. (A) From left to right: illustration of experimental procedure, T2w- and Gd-enhanced images showing hyper-intense areas around the needle track (arrow), Gd-enhanced image indicating the opened BBB area; the NaSA-CEST image using AUC10-30min depicting a more diffusive enhanced area mostly in the right hemisphere ; (B) DSE ROIs, as indicated in A; (C) DSE images at several time points post NaSA injection. (D) The averaged DSE signals at 30 min show significant difference for ipsilateral striatum and contralateral striatum,. (n = 8, * p < 0.01, error bar: standard error).

Fig.2 In vivo NaSA-CEST for (Sham) mice after 24 hours of intrastriatal-injection of saline. (A) From Left to Right, illustration of experimental procedure, T2w image, Gd-enhanced image showing slight only enhancement near the injection site; NaSA-CEST image suggests no obvious enhancement except for the vertical region; (B) DSE signals for four ROIs, as in Fig.1A; (C) DSE images at several time points post NaSA injection. (D) The averaged DSE signals at 30 min are ~ 0% and did not show statistical difference between ROIs of the ipsilateral side and the contral lateral side. (n = 6, error bar: standard error)

Fig.3 Validation of NaSA accumulation through immunohistochemical stainings (A), LC/MS-MS (B) and ex vivo CEST MRI (C). (A) A fluorescence image at region near LPS-injection site, depicting cell nucleus (blue, DAPI), the activated microglia using Iba1 (green) and COX1 enzyme (red). (B) the LC-MS/MS measurement indicates the inflamed mouse brains contain more NaSA than those normal mice, but no significant difference between the two hemisphere properly due to the small sample size. (C) ex vivo NaSA-CEST with mouse brain harvested at 30 min post-injection. Z-spectrum clearly displays a peak at 9.3 ppm in the enhanced region of NaSA-CEST image.

Proc. Intl. Soc. Mag. Reson. Med. 26 (2018)