Synopsis

Keywords: Parkinson's Disease, CEST & MT

Reveal alterations in molecules and CSF-tissue water exchange could enable interactive interventions in PD. Here we applied CEST and MISL to study the changes in a PD model at 3T. We observed significant changes (P<0.05) at 2.6ppm on day 28 and at -3.5ppm on day 7, which could indicate a substantial decrease of dopamine and lipid-related pathology at the substantia nigra, respectively. Along the nigrostriatal pathway, we observed a gradual MISL drop at the ventricles, which was significantly lower at day 28 (P<0.05) and might imply the CSF-tissue-related pathologies. Our findings could serve as potential biomarkers for early PD detection.

INTRODUCTION

Parkinson's disease (PD) is an age-related neurodegenerative disease, with key pathologies including the loss of dopaminergic neurons at the substantia nigra pars compacta (1,2) and the deposition of unwanted Lewy bodies in the brain (3). Although levodopa has been well-established for alleviating PD symptoms at the clinical level (3,4), no single treatment approach can cure or reverse the disease status. One of the key hurdles comes from the ambiguity of PD pathology at the molecular level.
With the advances in PD-related research, different animal models have been developed to study the etiologies and reveal subtle molecular changes (5). Recent studies have shown that aquaporin 4 (AQP4) may play a role in PD (6,7). As such, we proposed to study the molecular changes of an MPTP-induced PD mouse model with Chemical Exchange Saturation Transfer (CEST) MRI. CEST is an MRI technique that can image molecular changes up to the millimolar range (8). The recently developed Magnetization transfer Indirect Spin Labeling (MISL) protocol can quantify cerebrospinal fluid (CSF) water exchange with the surrounding macromolecules in the brain and AQP4-related diseases (9). Since the MPTP model is well established for preclinical PD research (1,5,10,11), we will use this model to analyze the underlying molecular changes. We anticipate this can provide valuable information for early diagnosis and treatment planning.

METHODS

A set of dopamine phantoms was prepared to study the CEST properties. In brief, 1 mg/mL of dopamine hydrochloride (Sigma) was dissolved in PBS with different pH.
For in vivo study, 3 C57BL/6 male mice (8-10 weeks) received 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, purity ≥ 98%, Sigma) via intraperitoneal injection (18 mg/kg per dose, diluted in saline) four times in 24 hours with an interval of at least 2 hours (1,5,10,11). The control group received the same volume of saline for the same period.
The phantom and the mice were scanned by a horizontal bore 3T Bruker BioSpec animal scanner (Bruker, Germany) with a 40-mm volume transceiver coil. The CEST sequence was referenced to our previously reported protocol (12). In addition, a MISL sequence was acquired referencing to the protocol from Li et al. (9). In brief, TR=5000ms, TE=250ms, B₁=2.0μT, and t_sat=3s. We used -10ppm as the labeled MT signal and 300ppm as the control image. The MISL images were calculated by subtracting the labeled MT signal from the control, followed by normalizing the control image. The MRI readout and other parameters were identical to the in vivo CEST sequence. The scan time of the MISL acquisition was about 4 min. The PD group was longitudinally imaged before, 7 days, and 28 days after injection. For the control mice, the scan was only performed on day 7.
One-way ANOVA analyzed longitudinal comparisons, and the comparisons with control were analyzed by Student’s t-test, all by GraphPad Prism 8.0.1.

RESULTS & DISCUSSION

The exchangeable protons of dopamine generated CEST contrast at 1.2ppm and 2.6ppm (Figure 1A and B). The peaks were sensitive to acidic pH (Figure 1C). We further studied the B₁ dependence of the two peaks (Figure 1D-F). Since 2.6ppm is further away from water, we used it to monitor the dopamine change in vivo.
Our in vivo study analyzed the longitudinal CEST change at the substantia nigra with three different offsets, i.e. 2.6ppm, 3.5ppm for amide protons, and -3.5ppm for aliphatic protons (Figure 2). As expected, the signal at 2.6ppm decreased by 18.4% on day 28 (Figure 2B, P<0.05). Signals at 3.5ppm (Figure 2C) and -3.5ppm (Figure 2D) changed over time, yet the changes were insignificant. We compared the PD mice with the control on day 7 since a stabilized loss of dopaminergic neurons was reported at this timepoint (10). Interestingly, a significant signal increase, around 12.9%, was observed at -3.5ppm (Figure 2G, P<0.05).
We further studied the CSF-tissue water exchange along the nigrostriatal pathway. Two consecutive slices anterior to the substantia nigra, covering the third and lateral ventricles, were imaged by MISL (Figure 3). Figure 3B showed that the MISL signal gradually decreased after MPTP intoxication (-13.7% on day 7) and reached a minimum on day 28 (-21.1%, P<0.05). We did not observe a significant difference in the MISL on day 7 (Figure 3C). The slice 3mm anterior to the substantia nigra also showed a decrease in MISL signal longitudinally (Figure 3E), and day 7 showed a 10.6% reduction compare to the control (Figure 3F, -10.6%). Representative MISL maps from the 1.5mm anterior slice were shown in Figure 3G.
Although we observed some trends in regional CEST and MISL signals, conclusions could not be drawn due to limited number of animals. A large cohort of animal study together with validation by histology is underway to further study the changes in these imaging parameters and the underlying neuropathology.
To summarize, this study demonstrated the detectability of dopamine at 1.2ppm and 2.6ppm. CEST detects changes at 2.6ppm, 3.5ppm, and -3.5ppm in the PD model's both regionally and longitudinally. A significant decrease at 2.6ppm was observed on day 28 after disease induction (P<0.05). Moreover, MISL indicated a significant decrease in MISL on day 28 (P<0.05), which could indicate CSF-tissue-related pathologies. Our findings could provide valuable information for early PD detection.

Acknowledgements

Authors would like to acknowledge the funding supports from Research Grants Council (11102218, PDFS2122-1S01, 11200422, RFS2223-1S02, C1134-20G); City University of Hong Kong (7005433, 7005626, 9239070, 9609307, 9610560); National Natural Science Foundation China (81871409); Tung Biomedical Sciences Centre; Hong Kong Centre for Cerebro-cardiovascular Health Engineering.