Introduction

Neurological conditions are a growing global concern and evidence-based treatments for these represent a priority area to strengthen the healthcare system. Central to evidence-based treatments are advanced neurophysiological techniques. These are used to examine neurological conditions including neurodegenerative (e.g., Alzheimer's) and psychiatric conditions (e.g., schizophrenia), and clinical pain (e.g., Fibromyalgia). Measurement of neurochemicals via proton-magnetic resonance spectroscopy (1H-MRS) provides a non-invasive, unique opportunity to examine the brain's biochemical environment. Emerging from animal and human studies there is evidence to suggest a link between glutamate and pain1–4. The cingulate cortex, consisting of the anterior, medial, and posterior divisions, is a part of the limbic system that, is consistently activated during pain, but the functional specificity of cingulate divisions has not been considered in human pain experiments 5. The distinct cingulate subdivisions can be delineated based on neurochemistry to gain insight into biochemical processes in health and disease. We have performed a metabolic map of the cingulate cortex using MRS localized with sLASER at 3T and examined the relationships to measures of pain sensitivity.

Methods

Participants
Twenty-two healthy participants were recruited for this experiment (9M/13F, mean age= 26.1 SD= 4.8 range=19-35). Participants were asked to come to the laboratory in a fasting state. The timing of the scan was kept consistent (11:30 am-12:30 pm) across participants and women were tested approximately during the follicular phase of their cycle Figure 1.
Pain Measurements
Quantitative sensory testing involved collecting participants' pain thresholds (method of limits, 32°C, ramp rate 1 °C/s, 30s ISI). Four trials were performed, with the final threshold determined as an average of the final three6. Pain sensitivity was measured with laser evoked potentials (LEPs), the participants were then fitted with an EEG cap and LEPs were performed (total 80 stimuli); the participants rated each stimulus using the 0-10 numeric pain rating scale. Participants were then given a muffin and escorted to the MRI center Figure 1.
MR Procedures
3T data were collected on a 3T Philips Ingenia Elition X, with a 32 channel SENSE head coil, and the sequences included:1) MPRAGE 3D T1 (TE/TR/TI=4.3/9.3/950ms, shot interval=2400ms, +0.8mm³ isotropic resolution, FOV (ap/rl/fh) =256/256/180mm³, scan time=5:49).2) 1H-MRS (sLASER, TE/TR=32/5000ms, NSA=64, voxel size=30/25/15mm3 =11.2mL, automated 2nd order shimming, 32-step phase cycle with water suppression using the frequency selective Excitation option. Four voxels were prescribed along the AP direction in the cingulate cortex (Figure 1), and the order of MRS acquisition in the voxels was randomized for each participant.
Data Analysis
Figure 2 shows the MRS analysis steps which included, pre-processing and absolute quantification performed using in-house MATLAB scripts. Spectral fitting was performed with LCModel version 6.37. A liner-mixed model and pairwise comparisons for each location were performed in R Studio (V1.1.442- lme4 Bates Maechler & Bolker, 2012). P-values were adjusted for comparing four estimates.

Results

One participant’s data had to be rejected, and for three other participants, we excluded metabolites from one location, due to data quality. The data quality form included participants can be seen in Figure 3 and the SNR and LW were similar across locations. The missing values can be seen in white in Figure 4. No sex differences were seen. Significant differences in metabolite concentrations were seen for glutamate (ACC - MCC β=0.97±0.28, p=0.005; ACC –dMCC β=1.34±0.27, p= <.0001; ACC-PCC β= 1.17±0.27, p=0.0004), NAA (ACC - MCC β=0.69±0.19, p=0.003; ACC–dMCC β=0.521±0.18, p=0.03), creatine (ACC-MCC β=0.67±0.23, p=0.02; ACC-dMCC β=0.74±0.22, p=0.01; ACC – PCC β=0.82±0.23, p=0.004) and myo-inositol (ACC - MCC β=0.67±0.21, p=0.01; ACC – dMCC β=1.02±0.20, p=<.0001; ACC – PCC β=0.97±0.20, p=0.0001). While choline remained constant across locations (Figure 4). A Pearson correlation revealed a significant relationship between glutamate levels and pain ratings in the ACC and a trend for pain thresholds and glutamate in the dMCC (Figure 5). The coefficient of variation of the neurochemicals per location can be seen in Figure 5.

Discussion & Conclusion

The relationship between glutamate concentrations and pain outcomes in the cingulate subdivisions reveal intriguing results fitting into an existing framework set out by Rohini Kuner’s group whereby a pathway from the MCC to the posterior insula has been demonstrated to gate adaptive sensory responses without influencing pain affect5. Whereas, the ACC has been recognized for its relationship with cognitive appraisal or pain and fear. These results suggest there may be functional specificity of MRS-visible neurochemicals in the subdivisions of the cingulate cortex. As MRS moves towards becoming a standardized clinical technique, understanding the spatial distributions and variability across regions and metabolites will provide an important basis for the understanding of the healthy human brain’s biochemical environment. These findings will be used as a basis for a planned clinical pain (i.e., Fibromyalgia) cohort where the same methodology will be applied. Practically, these results can inform other studies in decision-making regarding voxel placement depending on the respective research question.

References

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