A 37-year-old woman with a six-year history of chronic TMJ was recruited for this study and examined by a clinician specializing in TMJ pain. The participant's data was statistically compared to n = 14 healthy controls (100% F, mean 35.71 +- S.D. 6.58). The patient had experienced a level of pain that made it difficult to concentrate and had a history of successful outcomes from treatment with Botulinum Toxin A, with a cessation of pain within 24 h.
The healthy controls were recruited from a number of sources including social media and local advertising. Participants were included if they were aged between 18 and 65 years. They were assessed by the Structured Clinical Interview for DSM V (SCID) 28 and included if they had had no current DSM-V Axis I disorder and no history of an anxiety or mood disorder, current or past history of neurological disease; major head injury; currently or possibly pregnant or had any contraindications to MRI scanning.
Healthy controls were screened by a Clinical Psychologist using a combination of clinical interview and psychometric tests. These tests included a screen to assess symptoms of post-traumatic stress disorder (post-traumatic stress disorder checklist), mood disorders, anxiety disorders (depression, anxiety and stress scale), alcohol-related disorders (alcohol use disorders identification test) and history of head injury (Ohio State University TBI checklist). Following the psychological assessment, neuroimaging was conducted.
The TMJ participant was imaged prior to Botulinum Toxin A treatment and again 6 days after treatment. All scans were performed on a 3T Prisma scanner (Siemens, Erlangen, Germany, software version VD13D and VE11C) with a 64-channel head and neck coil (Siemens, Erlangen) at the Princess Alexandra Hospital (QLD, Australia).
Magnetic resonance imaging including T1 and T2 weighted images were obtained for diagnostic reporting and anatomical localization. A 3D T1-weighted magnetization-prepared rapid gradient-echo (MPRAGE) was acquired (TR/TE/TI = 2,530/3.5/1,100 ms, flip angle = 7 , field of view = 256 x 256 mm, IPAT = 3, acquisition time 4:28 min), for accurate MRS voxel placement.
On T1 and T2 weighted imaging an 11 cm sebaceous cyst on the left side of the skull vault was identified. This was considered an incidental finding that was present on T1 weighted imaging at each time point.
L-COSY data were acquired from a 3 x 3 x 3 cm3 voxel positioned in the posterior cingulate gyrus (PCG), Figure 1. In this article, as in previous work, data is collected from the PCG because it can be routinely shimmed to achieve sub-15 Hz linewidths from a large voxel. L-COSY was acquired with the following parameters: RF carrier frequency at 2.0 ppm; TR 1,500 ms; TE 30 ms; water suppression using WET; 96 t1 increments; with 8 averages per increment, acquired vector size 1,024 points; acquisition time 512 ms; spectral width in F2 2,000 Hz and spectral width in F1 1,250 Hz (0.8 ms increment size). Time of acquisition was 19 min, 12 s. Localized shimming was undertaken by adjustment of zero- and first-order shim gradients using the automatic B0 field mapping technique supplied by the vendor (Siemens AG). Each voxel was manually shimmed to ensure that FWHM was not >15 Hz as per the system reported results. All participant data obtained was acceptable for analysis.
Data was processed and analyzed using MATLAB (R2017b, The MathWorks, Inc., Natick, Massachusetts, USA), and FelixNMR (2007, San Diego, USA), a 2D NMR processing software. The post-processing parameters used in Felix were: F2domain (skewed sine-squared window, zero-filling to 2,048 points), F1 domain (sine-squared window, linear prediction to 96 points, zero-filling to 512 points) (Lin et al.,). In Felix, each prominent diagonal and cross peak was selected and integrated to determine the peak chemical shift, intensity, and volume. These values were internally normalized using the total creatine methyl diagonal peak at 3.02 ppm. Peak and cross-peak assignments were manually adjusted, to ensure the region of integration was centered on the peak, then exported for further analysis. Each spectrum was referenced to the creatine cross peak (3.02, 3.02 ppm) and specifying a constant number of contour levels (set to 28), as well as a constant level multiplier, set to 1.05 (Lin et al.,).
The average chemical shift, in ppm, of each cross-peak position from the 14 healthy volunteers was then calculated and compared to the patient before and after treatment. The z-score was calculated on the healthy cohort distribution (mean and standard deviation) to see how much the pre- and post-deviated from the healthy cohort. We took the peaks in the pre- and post-treatment with z-score of >1.959, which is the equivalent of a p-value of <5%.
The patient was evaluated by a pain physician specializing in temporomandibular disorders (TMD), and recorded a pain score of 8/10 at the time of the first scan. The treatment for this subject with severe myofascial pain of the temporomandibular region used botulinum toxin injections, administered at another site 2 days after the first scan. There are extensive studies describing the efficacy and safety of botulinum toxin for TMD (Mor et al.,; Munoz Lora et al.,). The procedure followed a standard clinical protocol of 10 botulinum units (BU) per injection site. Botulinum was administered to three sites spread 1 cm apart in the masseter and temporalis muscles on each side. The subject reported significant pain relief from the treatment.
The patient's TMJ pain resolved within 24 h of treatment and the pain score was 0/10. The patient was re-scanned 6 days later when the pain score remained at 0/10.