Caitlin Fowler1, Dan Madularu2, Masoumeh Dehghani2, Gabriel Devenyi2, and Jamie Near3
1Biological and Biomedical Engineering, McGill University - Douglas Hospital Research Centre, Verdun, QC, Canada, 2Douglas Hospital Research Centre, Verdun, QC, Canada, 3Psychiatry, McGill University - Douglas Hospital Research Centre, Montréal, QC, Canada
Synopsis
To better understand pathological
aging, we must first have a thorough understanding of changes that occur in the
brain during healthy aging. This project employs Magnetic Resonance
Spectroscopy to characterize longitudinal changes in the neurochemical profile
of healthy Fischer 344 rats. In addition to the commonly reported metabolites,
nine macromolecular resonances were included in the basis set and quantified,
based on parameterization of a group-averaged metabolite-nulled spectrum. For
the first time, longitudinal age-related changes in metabolites and individual macromolecules
were simultaneously characterized in the Fischer rat. Sex-specific differences
were also identified in metabolites and macromolecules.
Introduction
To better understand pathological aging, we must first thoroughly
characterize the neurological changes that occur in healthy aging. Magnetic
resonance spectroscopy (MRS) allows longitudinal detection of neurochemical
changes related to specific cellular mechanisms1–3, and can be used for translational
research in both animal models and human subjects.3,4 The present study reports normal
age-related changes in both metabolite and macromolecule (MM) levels in the hippocampus of the Fischer
344 rat – an inbred strain commonly used in aging research5 – measured by short echo-time
(TE) proton MRS (1H-MRS) at 7T. To quantify MM, individual MM basis
spectra were derived from in vivo metabolite-nulled spectra and included
in quantification basis sets. Accounting for the MM contribution in this way
not only improves metabolite quantification but may also provide valuable
information regarding chemical changes occurring in the aging brain.6 Finally, we also examined
sex-specific differences in metabolite and MM concentrations, as previous human
and animal studies have indicated such differences in the progression
of neurochemical changes with both age and disease presentation.1,7,8Methods
Proton MRS
acquisition: All 1H-MRS data were acquired on a 7 Tesla Bruker
Biospec 70/30 scanner. Automated localized shimming was performed in a region
of interest (ROI) in the dorsal hippocampus (31mL) using the FASTMAP
method10 (water linewidth 9.9±1.0
Hz). Water suppressed (256 averages) and unsuppressed scans (8 averages) from the
ROI were acquired using PRESS (TR/TE = 3000/11ms). Macromolecule parameterization:
metabolite-nulled inversion recovery (IR, TI = 800ms) scans were obtained in 8 10
month old rats from the same hippocampal ROI as above. A
group-averaged MM spectrum was generated using
FID-A11 and exported into JMRUI for parameterization using AMARES.12 Specifically, the chemical shift, linewidth and relative
amplitudes of nine prominent macromolecule resonances were estimated. Spectral
quantification: The frequency and linewidth for each MM resonance
was used to simulate MM basis functions in FID-A; the MM amplitudes relative to
MM1 (0.9ppm) were used as soft constraints within LCModel analysis. The complete LCModel13 basis set generated in FID-A comprised 20 metabolites and
9 macromolecules. Data pre-processing
was also performed in FID-A. Absolute Concentration Calculations:
Water T1 and T2 were estimated in a subset of 10 month old rats (n=7) using
measured TE-series and TI-series, respectively. Metabolite T1 and T2 relaxation
constants at 7T were estimated based on
previously published data at 4T and 9.4 T14. Absolute concentrations, in mmol/L, were estimated
by referencing to water and adjusting for T1 and T2 relaxation (Figure 5). Statistical
analysis: A linear mixed-effects models with fixed effects for age, sex, and
water linewidth, and a random effect of subject was used to fit the data in
RStudio (RStudio Team). Weighting based on the LCModel absolute standard
deviation of each measurement was introduced to account for differences in
fitting reliability. Any metabolite for which 50% of observations had very low
reliability (i.e. CRLB > 100%) was excluded entirely (Scyllo and GPC).
Scaling on all continuous variables was performed to make beta values
immediately interpretable. FDR correction (0.05) for multiple comparisons was
applied, with p-values and q-values summarized in Figure 4. Results and Discussion
Our approach to parameterize and quantify each
of the individual MM resonances allowed improved quantification of metabolite
signals, while simultaneously providing valuable information on MM changes with
aging.6,15,16 Nine MM resonances were quantified in this study
(MM1, 0.9 ppm; MM2, 1.20 ppm; MM3, 1.39 ppm; MM4, 1.66 ppm; MM5, 2.02 ppm; MM6,
2.26 ppm; MM7, 2.97 ppm; MM8, 3.18 ppm; MM9, 3.84 ppm) in addition to an array
of 18 standard metabolites. Linear modelling of neurochemical concentrations
revealed effects of age and/or sex in key metabolites associated with the aging
process such as increased myo-inositol, and decreased glutathione and glutamate
(Figures 2 and 4). No change was seen in other commonly reported metabolites
such as N-acetylaspartate (NAA) and total choline (tCho) (Figure 4); reports
have been inconsistent as to whether NAA decreases with age and tCho increases
with age, or whether they remain stable1,2,7. It should be noted that the final timepoint of our
study was at 20-months of age, at which point rats are reaching
late-middle-age; it is possible that we might have seen more significant
neurochemical changes had we extended this study. Finally, changes due to sex
and/or age were seen in 6 of the 9 macromolecules, with the most notable being
a significant increase with age in MM4 and MM9, and an increase with age
combined with an increase in males relative to females in MM8 (Figure 3). Aside
from the knowledge that the MM resonances can be assigned to cytosolic proteins
and that regional differences in MM concentrations have been reported2,6,7, the physiological meaning behind changes in
individual MMs remains to be investigated. Conclusion
The present study reports, for the first time, longitudinal
modifications in metabolite and macromolecule levels in the hippocampus of the
aging Fischer rat, measured at 7T. These results present the basal
neurochemical profile of the hippocampus in aging Fischer rats, and therefore confirm
the value of MRS measurements as non-invasive biomarkers for detection and
monitoring of neurologic disorders and/or treatment efficacy. Acknowledgements
No acknowledgement found.References
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