Purpose

The chronic forced swimming stress (CFSS) depression-like animals have been used to investigate the pathophysiology of depression focusing on the monoamine system. An in vivo proton magnetic resonance spectroscopy (¹H MRS) has been used to investigate metabolic alterations, other than the monoamine systems, including glutamate (Glu), choline-compounds, and myo-inositol (mIns), in patients with major depressive disorder (MDD). However, recent in vivo ¹H MRS studies were conducted at clinical field strengths which had limitations in the quantification of metabolites with low concentrations, and/or spectral overlaps and strong J-coupling. High-field (7T) and relatively short-TE (16.3 ms) in vivo ¹H MRS can improve the value of in vivo ¹H MRS in investigating MDD. Thus, the goal of this study was to investigate the CFSS-induced effects in the prefrontal cortex (PFC) of depression-like animals using high-field and short-TE in vivo ¹H MRS.

Materials and Methods

Ten male Wistar rats underwent MRI and ¹H MRS (pre-CFSS). Figure 1 shows experimental protocols including the MRI/MRS, forced swim test (FST), and CFSS. For the FST, the rats were placed in water tank (5 min; 30 °C). The rats underwent the CFSS to simulate depressive conditions (15 min; 25 °C). After the last CFSS (post-CFSS), MRI/MRS and FST were performed. MRI/MRS were acquired using a Bruker PharmaScan 7T MRI system (Bruker BioSpin) with a 72-mm birdcage volume coil and a surface coil. T2-weighted images (T2WIs) were acquired in rat brain (rapid-acquisition-with-relaxation-enhancement (RARE); TR/TE, 4000/11 ms; RARE factor, 8; field of view, 30 × 30 mm²; matrix size, 256 × 256; slice thicknesses, 1.5/1 mm (axial/coronal)). In vivo ¹H MRS was acquired in the voxel (1.5 × 5 × 3 mm³) containing the PFC using point-resolved spectroscopy (TR/TE: 5000/16.3 ms, average: 256, spectral bandwidth: 5000 Hz). Water signals were suppressed by variable pulse power and optimized relaxation delays. Unsuppressed water signals were obtained in the same condition (average: 8). The FID signals were processed using linear combination of model spectra (LCModel, version 6.3) software to estimate the concentration of the following 19 metabolites: alanine, aspartate, creatine (Cr), phosphocreatine (PCr), γ-aminobutyric acid, glucose, Gln (glutamine), Glu, glycerophosphocholine (GPC), phosphocholine (PCh), glutathione, mIns, lactate, N-acetyl aspartate (NAA), N-acetyl-aspartyl-glutamate, scyllo-inositol, total creatine (tCr; Cr + PCr), total choline (tCho; PCh + GPC) and taurine, and scaled by the factor of receiver gain (Gainunsuppressed/Gainsuppressed). In order to assess the spectral overlap between Glu and Gln, a geometrical overlap ratio was computed from the intersection area of the LCModel fitted spectra of Glu and Gln as a percentage of the union area of Glu and Gln ((Glu ∩ Gln)/(Glu ∪ Gln) × 100%). The range of 2.25–2.55 ppm was selected for assessment, to include the main resonance for quantification of Glu (2.33–2.35 ppm) and Gln (2.43–2.45 ppm). Spectral overlaps between Glu and Gln were assessed using numerical simulation and in vitro measurements parametrically matched with in vivo experiments. Reliability of the quantification was assessed by Cramér-Rao lower bound (CRLB) values. The differences in metabolite levels and swimming behaviors were statistically analyzed using a paired sample t-test.

Results

CFSS significantly changed swimming behaviors of the rats in FST. Figure 2 illustrates representative (a-b) axial/coronal T2WI of the rat brain with (c) the MRS voxel. Representative in vivo ¹H MRS are shown in Figure 3(a): the raw data (blue), Glu (black), Gln (red), and residues (top). Figure 3(b-c) represents the results of simulation and in vitro measurements. Although the Glu and Gln spectra obtained with in vivo ¹H MRS had slightly lower signal-to-noise ratios (SNRs) and larger line broadening, the spectral shapes showed good agreements. The levels of mIns, tCho, NAA, mIns/tCr, and tCho/tCr were significantly increased after the CFSS. Table 1 lists the geometrical overlap ratios, SNRs, and CRLB values.

Discussion and Conclusion

In this study high-field and short-TE in vivo ¹H MRS were used to assess CFSS-induced metabolic effects in the PFC in depression-like rats. With spectral simulations and in vitro experiments, we confirmed that in vivo ¹H MRS could reliably assess Glu metabolism. Although significant alterations in Glu and Gln levels were not observed, increased prefrontal mIns, tCho, and NAA levels were partially consistent with the results found in patients with MDD, which suggested that the CFSS-induced behavioral despair and metabolic alterations were similar to those found in human patients with depressive disorders. We expect our findings to contribute to the investigation of the alterations in metabolic systems in MDD using in vivo ¹H MRS, in addition to the monoaminergic system, and provide alternative insights into the pathophysiology and treatment strategies for patients with MDD.

Acknowledgements

This study was supported by grants (2012-007883) from the Mid-career Researcher Program though the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP) of Korea.