In this study, a novel head-restraining system was successfully implemented for the first time to obtain metabolic information using MRS in an awake rodent model for the first time. Using this preparation, it is possible to obtain localized 1H MR spectra in several brain regions of interest (anterior cingulate cortex, somatosensory cortex, hippocampus and thalamus) without the need for any post-processing scheme to correct for possible motion during data acquisition. In all four regions, most brain metabolites or the combinations were reliably detected (CRLB < 18 %), except for some metabolites in the thalamus. Because of the use of a surface coil, the rostral regions of the anterior cingulate cortex, somatosensory cortex, and hippocampus, exhibited high signal-to-noise ratio (~22) and the spectra obtained were highly reliable (CRLB < 18%). The overall signal to noise of the spectra for thalamus was lower (~13) as this region was at the periphery of the reception field of the four-element surface coil. In this region, metabolites with low concentration provided less reliable spectra (GABA, Gln, and Tau; CRLB = 21–29 %; ).
The concentrations of the metabolites quantified in this study are within the range specified in the rat brain (see Table 3 in 43). It is important to note however that the GABA concentration may be overestimated in this study because there can be a significant overlap with the macromolecules in the GABA region of the spectrum. In addition, the Tau concentration may be underestimated because of its long spin-lattice relaxation time and short TR (44
However, the concentrations of metabolites appear to be different from those reported in Xin et al
. and Rao et al
). In our study, MRS experiments were performed in 14-week old female rats to assess metabolic profiles in the somatosensory cortex, anterior cingulate cortex, hippocampus and the thalamus. In Xin et al
) the animal gender was different (adult male rats) and although the regions of interest studied included the somatosensory cortex and hippocampus, the voxel sizes used were significantly different [somatosensory cortex: 6 × 1.5 × 2 mm3
vs. 1.8 × 4.5 × 4.0 mm3
in this study; hippocampus: 3 × 2 × 2 mm3
vs. 2.5 × 4.0 × 3.0 mm3
). In Rao et al
) the age and gender of the animals (8-week old, male) were also different. The methodological differences between the current study and the studies referenced above may explain the differences in metabolites’ concentrations. It is well documented that metabolic profiles depend on the age and gender of the animals as well as the region studied (46
A major concern when performing experiments in restrained animals is the stress induced (39
) and its effect on MRS results. To minimize this concern, we adopted similar methods to those described in King et al.
) and acclimated and handled the animals from a young age before performing the experiments. We also assessed changes in mechanical withdrawal thresholds over time and monitored breathing during the MRS experiments. The acclimation and training procedure proved effective in reducing animal stress as evident from the lack of change in mechanical withdrawal thresholds and the stable and normal breathing rate. Future experiments should test if awake MRS experiments can be performed using shorter training paradigms as those described in Becerra et al
). In addition, the experiments should assess the state of the animal and investigate whether stress was induced or not. Such experiments will benefit from quantifying serum cortisone levels during the imaging procedures, and monitoring the heart rate (23
MRS acquisitions require that the main magnetic field, B0
, remain very stable during data acquisition. The movement of the animal during the data acquisition can significantly change the value of the local B0
over time. B0
fluctuations can broaden the line width of the observed metabolites in the MR spectrum thereby losing fidelity in resolving two closely resonating spins. In a 1
H MRS study on awake non-human primates at 7 T (33
), line-width changed from 15.6 Hz at the beginning of the study to 19.5 Hz for the averaged spectrum. These changes in the spectral line width were due to major body movement of the awake animal during data acquisition. However, respiration and small body movement did not show a significant spatial B0
dependence and shim effect. To correct for movement, the authors applied a scheme which rejected periods of major body motion and corrected frequency/phase of each of the single acquisition of the spectra before averaging (33
). Using this correction scheme the line width of the spectrum was reduced to 15.8 Hz. In our experimental design a motion correction scheme was not performed. The shimming procedure routinely resulted in line widths of 11–15 Hz of the single 1
H metabolite resonance (0.037–0.050 ppm) and a good separation of peaks that resonate closely was possible (e.g; glutamate (2.35 ppm) and glutamine (2.45 ppm). Furthermore, repeated acquisition of the spectra over two different days were highly reproducible (CV <11%) suggesting that preparing the animal in this manner leads to negligible motion and provides a stable environment for obtaining quality MRS. However, the quality of the raw spectra acquired can be further improved by performing additional data preprocessing steps as those described in Pfeuffer et al
). Correcting for animal movement and frequency/phase of single acquisitions will reduce the minimum detectable differences in metabolite concentrations and increase reproducibility and therefore improve the quality of the acquired spectra.
Rodents are used extensively in neuroscience research to study disease processes that involve cognition and emotion such as pain, addiction, depression and psychiatric disorders. These conditions cannot be studied reliably in the presence of agents that may alter consciousness, cognition, and neuronal transmission. Similarly, MRS has proven very useful in studying the pathophysiologic mechanisms of a large range of neurological and psychiatric disorders (2
) and many of these studies are not possible if the animals are not restrained to prevent movement. An awake rodent MRS preparation as demonstrated here allows one to study these conditions and correlate changes in brain metabolic profiles during resting and functional states with changes in neuronal physiology, chemistry, and behavior without the confounds of anesthetic and paralytic agents.
We conclude that high-resolution and high-quality 1H MRS can be obtained from unanesthetized rat brains at 7 T. With proper training and restraint apparatus, several proton metabolites can be reliably measured even without post processing correction schemes. The current development offers a novel approach to study major brain metabolites in awake rodents. This method circumvents the effects of anesthesia, and allows for longitudinal experiments for prolonged periods of time to study progression of disease especially in the field of chronic pain research. It will also open the door for translational research that bridges the gap between animal and human studies.