Imaging Reactive Oxygen Species (ROS) using CEST MRI
Rong-Wen Tain1,2, Alessandro Scotti2,3, Weiguo Li4,5, Xiaohong Joe Zhou1,2,3,6, and Kejia Cai1,2,3

1Radiology, College of Medicine, University of Illinois, Chicago, IL, United States, 23T Research Program, Center for MR Research, College of Medicine, University of Illinois, Chicago, IL, United States, 3Bioengineering, College of Engineering, University of Illinois, Chicago, IL, United States, 4Research Resource Center, University of Illinois, Chicago, IL, United States, 5Radiology, Northwestern University, Chicago, IL, United States, 6Neurosurgery, College of Medicine, University of Illinois, Chicago, IL, United States

Synopsis

It is extremely challenging to non-invasively measure tissue ROS due to its low concentration and short lifetime. This study aims to demonstrate a fully non-invasive CEST MRI method to measure ROS concentration. CEST Z-spectra were acquired from egg white samples with and without hydrogen peroxide treatment. In addition, proton exchange rate, T1, and T2 relaxation time maps were acquired for further clarification on CEST contrast origin. We have demonstrated that ROS is paramagnetic and can greatly enhance proton exchange rate leading to reduced CEST contrast.

Target audience

Scientists or clinicians interested in oxidative stress, reactive oxygen species, and CEST MRI.

Purpose

Oxidative stress due to elevated reactive oxygen species (ROS) is a critical contributor in various processes including normal aging, Alzheimer's disease, stroke, cardiac arrest, cancer, and diabetes (1-3). Direct and non-invasive approaches for quantifying tissue ROS will provide useful information in early diagnosis, disease stratification, and treatment response. Given that ROS has an extremely short lifetime (~10-9 s for hydroxyl radicals) (4), and its in-vivo concentration is in µM level(5), it has been difficult to perform direct measurement. Currently there is no direct and non-invasive method to assess ROS production (6-9). In this study, we aim at developing a fully non-invasive method to measure tissue ROS concentration based on endogenous MRI contrasts including the chemical exchange saturation transfer (CEST) contrast that origins from exchangeable protons associated with specific metabolites.

Methods

We investigated CEST MRI properties of ROS produced by adding hydrogen peroxide (H2O2) into ex-vivo tissues. Fresh egg white tissues (n=5) treated with various concentrations of H2O2 (0.25, 0.1, 0.05, and 0.025 v/v%) were scanned at different time points(0.5, 1, and 3 hours) at room temperature. Fluorescence studies using the hydroxyphenyl fluorescein (HPF) dye were performed to measure ROS production from the samples treated within 1 hour. CEST Z-spectra were collected on a horizontal 9.4-T MRI scanner using a customized sequence with a frequency-selective rectangle saturation pulse (B1=50 Hz, 3 s) followed by a Fast Low-Angle Shot (FLASH) readout. The entire Z-spectra contained 49 saturation offsets ranging from -5 to 5 ppm with steps of 0.25 ppm, and at ±10, ±20, ±50, and ±100 ppm. CEST asymmetry effect was computed at 3.5 ppm. In a separate study, the proton exchange rates of untreated and treated (0.1 v/v% H2O2) egg white samples (n=4) were assessed based on the method previously described (10). The steady-state CEST images were acquired with a 4 s long saturation pulse at various saturation amplitudes B1 from 50 up to 450 Hz. Data were fitted with a polynomial function to determine the amplitude leading to the maximal steady-state CEST contrast. In addition, T1, T2, and pH were measured.

Results and Discussion

Compared to untreated egg white samples, H2O2 treatment modulated the entire CEST Z-spectra (Figure 1A), resulting in reductions in the CEST contrast. The CEST contrast reduction was proportional to H2O2 concentration (Figure 1B). Fluorescence studies showed that ROS production was also proportional to H2O2 treatment concentrations (Figure 1C). Interestingly, the required saturation amplitude for the maximum steady-state CEST contrast was much higher for the treated samples than the untreated, indicating that ROS greatly enhances proton exchange rate (Figure 2). Since CEST contrast relies on slow-to-intermediate proton exchange, we believe that ROS reduces CEST contrast by substantially raising the proton exchange rate. In addition, pH measurements showed only minor changes between baseline and at 1 hour post-treatment with 0.25 v/v% of H2O2 (pH = 9.00 ± 0.01 vs. 8.92 ± 0.01). These suggest that ROS likely serves as an independent exchange modulator from pH. We also found that T1 relaxation time in egg white reduced due to H2O2 treatments, while the reduction in T2 relaxation time was negligible (Figure 3). This indicates that ROS has a strong paramagnetic effect that can be detected by T1-weighted MRI. In contrast, intact H2O2 (in PBS) does not affect T1 significantly. At 3 hours post treatment, CEST contrast recovered almost back to the pre-treatment level (Figure 4), indicating all the H2O2 was converted to ROS within this time. We modeled the dynamics of CEST contrast as an exponential decay function and estimated that the ROS concentration (with 0.025 v/v% H2O2 at 1 hour post-treatment) was about 50 µM, which is within the in-vivo pathological or physiological range (5, 11, 12). We are currently working toward the in-vivo applications of this technique.

Conclusion

In summary, we have observed that ROS is paramagnetic. ROS also greatly enhances proton exchange, independent to pH, leading to reduced CEST contrast in general. We have showed that CEST combined with T1-weighted contrast has the potential to improve the specificity for imaging ROS or oxidative stress in tissue. This novel MRI technique may serve as a longitudinal imaging tool for biochemical analysis of ROS production in ex-vivo tissue or other experimental set-ups. It may also allow for non-invasively quantifying and mapping ROS production in vivo with high temporal and spatial resolutions.

Acknowledgements

No acknowledgement found.

References

1. Cook JA, Gius D, Wink DA, Krishna MC, Russo A, Mitchell JB. Oxidative stress, redox, and the tumor microenvironment. Seminars in Radiation Oncology.14(3):259-66. doi: 10.1016/j.semradonc.2004.04.001. 2. Barnham KJ, Masters CL, Bush AI. Neurodegenerative diseases and oxidative stress. Nat Rev Drug Discov. 2004;3(3):205-14. 3. Allen RG, Tresini M. Oxidative stress and gene regulation. Free Radical Biology and Medicine. 2000;28(3):463-99. doi: http://dx.doi.org/10.1016/S0891-5849(99)00242-7. 4. Pryor WA. Oxy-radicals and related species: their formation, lifetimes, and reactions. Annu Rev Physiol. 1986;48:657-67. doi: 10.1146/annurev.ph.48.030186.003301. PubMed PMID: 3010829. 5. Nagababu E, Rifkind JM. Reaction of Hydrogen Peroxide with Ferrylhemoglobin:  Superoxide Production and Heme Degradation. Biochemistry. 2000;39(40):12503-11. doi: 10.1021/bi992170y. 6. Li LZ, Zhou R, Xu HN, Moon L, Zhong T, Kim EJ, Qiao H, Reddy R, Leeper D, Chance B, Glickson JD. Quantitative magnetic resonance and optical imaging biomarkers of melanoma metastatic potential. Proceedings of the National Academy of Sciences. 2009;106(16):6608-13. doi: 10.1073/pnas.0901807106. 7. Tsitovich PB, Burns PJ, McKay AM, Morrow JR. Redox-activated MRI contrast agents based on lanthanide and transition metal ions. Journal of Inorganic Biochemistry. 2014;133(0):143-54. doi: http://dx.doi.org/10.1016/j.jinorgbio.2014.01.016. 8. Ratnakar SJ, Viswanathan S, Kovacs Z, Jindal AK, Green KN, Sherry AD. Europium(III) DOTA-tetraamide Complexes as Redox-Active MRI Sensors. Journal of the American Chemical Society. 2012;134(13):5798-800. doi: 10.1021/ja211601k. 9. Ratnakar SJ, Soesbe TC, Lumata LL, Do QN, Viswanathan S, Lin C-Y, Sherry AD, Kovacs Z. Modulation of CEST Images in Vivo by T1 Relaxation: A New Approach in the Design of Responsive PARACEST Agents. Journal of the American Chemical Society. 2013;135(40):14904-7. doi: 10.1021/ja406738y. 10. Woessner DE, Zhang S, Merritt ME, Sherry AD. Numerical solution of the Bloch equations provides insights into the optimum design of PARACEST agents for MRI. Magnetic Resonance in Medicine. 2005;53(4):790-9. doi: 10.1002/mrm.20408. 11. Gunther MR, Sampath V, Caughey WS. Potential roles of myoglobin autoxidation in myocardial ischemia-reperfusion injury. Free Radical Biology and Medicine. 1999;26(11–12):1388-95. doi: http://dx.doi.org/10.1016/S0891-5849(98)00338-4. 12. Svistunenko DA, Patel RP, Voloshchenko SV, Wilson MT. The Globin-based Free Radical of Ferryl Hemoglobin Is Detected in Normal Human Blood. Journal of Biological Chemistry. 1997;272(11):7114-21. doi: 10.1074/jbc.272.11.7114.

Figures

Z-spectra (A), CEST contrasts (B), and fluorescent signal (C) from egg white samples treated with various concentrations of H2O2 for 1 hour.

(A) CEST contrast from un- and treated (0.1 v/v% for 1 hour) egg white samples obtained with various B1. (B) Representative exchange rate maps from un- and treated samples.

ROS produced by H2O2 treatment greatly reduces T1 relaxation time in egg white, while H2O2 itself does not affect T1 significantly.

The reduction of CEST contrasts over time due to treatments with various H2O2 concentrations. After 3 hours, CEST contrasts recovered almost back to the pre-treatment levels.



Proc. Intl. Soc. Mag. Reson. Med. 24 (2016)
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