INTRODUCTION

Chemical Exchange Saturation Transfer (CEST) imaging provides data in the form of Z-spectrum, which in vivo contains the contributions from CEST and rNOE (relayed Nuclear Overhouser Effect) effects of labile proteins and metabolites, as well as from magnetization transfer (MT) from semi-solid macromolecules and direct water saturation effect (DE)¹. In this study we aimed to assess the differences in Z-spectrum in rat brain hippocampus and cortex before and after post-mortem CLARITY lipids removal procedure (Clear, Lipid-exchanged, Acrylamide-hybridized Rigid, Imaging/immunostaining compatible, Tissue hydrogel)². As the rNOE signal is known to originating from lipids³ and manifesting in Z-spectrum as broad peak upfield the water resonance, we hypothesized that CLARITY procedure would influence mostly this effect.

METHODS

CLARITY procedure: Five mm slice of rat brain (Fig. 1 A) was fixed with a hydrogel solution containing acrylamide, paraformaldehyde, bis-acrylamide and thermal initiator VA-044, as described in². For clearing procedure the sodium dodecyl sulphate and boric acid clearing solution was used (Fig. 1 B).
Imaging: MRI (7T PharmaScan 70/16 US scanner, Bruker, Germany) was performed twice: in vivo and post-mortem after CLARITY procedure. Five Z-spectra were acquired from single-slice axial (covering hippocampus and cortex) MT-prepared EPI sequence (TE 37ms, TR 5s, NA 3, FOV 30×30mm², slice thickness 1mm), with block RF saturation pulses of 4,900 ms duration, and five different B₁ peak amplitudes: 0.5 and 0.75 µT in a frequency offsets range of -6 to +6 ppm (139 offsets for in vivo scans, and 424 offsets every 10 Hz post-mortem) for CEST imaging, 3.0 and 5.0 µT (at 32 offsets between +300 to -300 ppm) for MT-sensitive effects, and 0.1 µT at 24 offsets between +0.5 and -0.5 ppm for DE measurement. High flip angle 3D FLASH scans and inversion recovery RARE scans were performed for B₁ scale correction and R₁ mapping, respectively.
Data analysis: The Z-spectra were derived from segmented hippocampal and cortical area (Fig. 1 C), B₀‑corrected and averaged in ROI. Z-spectra with saturations B₁s of 3 and 5 µT, and the average R₁ were fitted to a two-pool MT model⁴, giving four quantitative MT parameters: the transverse relaxation times of the liquid (T_2,A) and macromolecular (T_2,B) pools, the initial magnetization of the macromolecular pool (M_0,B) and the exchange rate between liquid and macromolecular pools (R). The estimated model parameters were used for simulating MT semisolid macromolecular pool contributions to the CEST Z-spectra acquired with B₁s of 0.5 and 0.75 µT.

RESULTS

The lipids removal almost nulled-out MT macromolecular-originating signal measured with B₁s of 3 and 5 µT as compared to in vivo data (Fig. 2). Fitting two-pool model to these data revealed prolonged T₂ times from both liquid (T_2,A) and macromolecular (T_2,B) pools after CLARITY, and reduced both R₁ and R exchange rate (Table 1).
The CLARITY procedure also resulted in substantially decreased CEST signal within the whole range of spectrum received with both saturation B₁ powers of 0.5 and 0.75 μT (Fig. 3). After CLARITY the z-spectra were flattened as compared to in vivo, with remaining distinct peaks at +3.5, +2.8, +2 and -3.5 ppm. For the saturation impulse power 0.5 μT, an average signal decrease was 12 ± 3% and for B₁ of 0.75 μT an average signal decrease was 21 ± 4%. The simulated for B₁s of 0.5 and 0.75 μT MT signal contribution to the Z-spectra was distinctly substantial in the in vivo data, as opposite to post-CLARITY measurements.

DISCUSSION & CONCLUSIONS

The study showed that the macromolecular MT contribution into in vivo z-spectra originates mostly from lipids, since the CLARITY technique removed the MT component from the spectrum. The additionally observed drop-down of the CEST effect was associated mostly with increased water content in comparison to molecules, as the CLARITY procedure resulted in swelling of the tissue sample (Fig. 1 B). Our results suggest that lipid-originating signal manifests not only in the rNOE part of the Z-spectrum, but has also significant contribution to the resonances downfield the water. The above conclusions are in agreement with biological data, from which lipids are known as the biggest group of macromolecules (around 11%) of the brain tissue⁵.

Acknowledgements

This work was supported by the National Science Centre, Poland (2015/17/B/NZ4/02986) and Canadian Institute for Health Research, Canada (PJT148660).