Serendipitous neurotransmitter findings in a frontline defender participating in a magnetic resonance spectroscopy study of first responders and military personnel with PTSD by David Crompton OAM in Journal of Clinical Case Reports Medical Images and Health Sciences

 Serendipitous neurotransmitter findings in a frontline defender participating in a magnetic resonance spectroscopy study of first responders and military personnel with PTSD by David Crompton OAM in Journal of Clinical Case Reports Medical Images and Health Sciences

Abstract

We report a case study of a patient with a clinical diagnosis of Post-traumatic Stress Disorder (PTSD) referred to a neurochemistry Magnetic Resonance Spectroscopy (MRS) study. The clinical evaluation prior to scanning suggested the presence of PTSD, Major Depressive Disorder (MDD) accompanied by suicidal ideation. MRS prior to and at the completion treatment and 12-weeks post treatment identified neurotransmitters consistent with a diagnosis of PTSD and MDD. The neurochemistry evaluation immediately post-treatment identified levels that compared to healthy subjects while at 12-weeks posttreatment the neurochemistry reflected a recurrence of depressive symptoms that were described by the patient. These results offer the potential for neurochemical markers to monitor the response of an individual to therapy.

Introduction 

To date evaluation of mental disorders such as Major Depressive Disorder (MDD) and post-traumatic stress disorder (PTSD) have relied on clinical assessment and questionnaires. While the use of Gold Standard measures such as the Clinician Administered PTSD Scale (CAPS-5)(1) and Structured Clinical Interview for DSM-5 (SCID)(2) have improved diagnostic reliability and validity, a degree of subjectivity remains. Diagnostic challenges are particularly marked in complex cases, even for experienced clinicians with respect to both diagnosis and response to treatment. This is exacerbated when comorbidity is present. Complicating the diagnostic challenges are the differing criteria for mental disorders as defined by the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) and International Classification of Diseases (ICD-11)(3). In contrast to the similarities DSM-5 and ICD-11 criteria for MDD(4) there are considerable differences between the two classification systems with respect to the mental health disorders that may occur following exposure to traumatic events. The ICD-11(3) suggests two outcomes: PTSD and Complex PTSD (CPTSD) with CPTSD characterised by higher levels of depression, dissociation, anxiety, suicidal ideation, self-harm behaviour and impairment. People living with CPTSD have often experienced multiple traumas and/or events that are described as threatening or prolonged. In contrast, DSM-5 provides only one category of PTSD following trauma(4-8). Whilst the symptoms of MDD, PTSD and CPTSD have been well described the neurochemical characteristics of these disorders are poorly understood. Evidence to date supports GABAergic (inhibitory function) and Glutamatergic (excitatory function) dysregulation and alteration in prefrontal binding of benzodiazepine-GABA receptors in those with PTSD(9). Clinical and pre-clinical evidence suggests these pathways play a role in the variability to the stress response to traumatic events, symptom presentations in MDD, PTSD and CPTSD, adaptability and response to interventions(10, 11). One-dimensional (1D) MR spectroscopy studies in patients with MDD have revealed decreased glutamate and glutamine levels in the dorsolateral and other parts of the prefrontal cortex and increased glutamate levels in the occipital cortex(12). Glutamine and gammaaminobutyric acid (GABA) concentrations and activity, suggest that dysfunction in excitatory and/or inhibitory neurotransmitter signalling mechanisms may play a critical role in depression(13). The molecules measured by the in vivo MR spectroscopy method are mobile on the MR timescale. Thus, they are in active pools or have a high level of molecular motion. Two-dimensional MR spectroscopy has a significant advantage over one-dimensional (1D) MR spectroscopy as it unambiguously assigns the molecules in a second magnetic frequency(14). Two-dimensional (2D) MR spectroscopy can provide an unambiguous assignment of the neurochemistry of an individual(15). Recently new assignments have been made possible using the 2D-COSY protocol. These include the assignment of seven fucose-α(1–2)-glycans, and the substrates α-L-fucose, in the human brain(16, 17). From animal studies, a growing body of literature implicates these fucose-α (1–2)-glycans in the molecular mechanisms that underlie neuronal development, learning, and memory in the brain(18-20). Moreover, from rat neuronal cultures and immunocytochemistry, the fucosylated glycans are seen at the synapse of the neurons and are implicated in neuronal signalling processes (20). A recent study, evaluated PTSD patients, from first responders and the military, using in vivo neuro 2D COSY(15). When compared with age and gender-matched healthy controls, 2D COSY spectroscopy identified statistically significant increases of 31% in the total spectral region containing both free substrate fucose and fucosylated glycans, two fucose-α(1–2)-glycans (Fuc 4 and 6) were elevated by 48%, and 41%, respectively, imidazole was increased by 12%, and lipid unsaturation was increased by 12.5%. Other statistically significant differences recorded include an increase in imidazole from either histamine, histidine or homocarnosine and an increase in the level of unsaturation in a lipid fatty acyl chain. No significant differences were recorded for NAA, glutamine, glutamate, myo-inositol, or GABA using the 2D-L-COSY method. This cohort (CAPS-5 38.4 ± 10.45) also demonstrated a correlation between the PTSD subjects with the ratio IMI-1/Cr in the PCG region of the brain and evidence of fucose-α(1–2)- glycan involvement in the PTSD association between IMI-1/ Cr and hyperarousal, reactivity symptoms and severity.

Case Study

 We report a case study of a member of the Australian Defence Force who was referred to the study with a clinical diagnosis of PTSD (DSM-5). Ethics approval for the study was provided and the patient provided informed consent. The patient reported a 51-month history of PTSD symptoms. Psychological assessment using the SCID-5 and CAPS-5 prior to the initial scan supported a diagnosis of PTSD and comorbid MDD with chronic suicidality/self-harm behaviour. The index trauma was a traumatic deployment to an overseas war zone. The CAPS-5 score based on fulfilling criteria B to E and a total severity score of 45 met the criteria for PTSD(1, 21). The patient’s psychological history revealed a history of childhood sexual abuse. The clinical assessment was consistent with an ICD-11 diagnosis of CPTSD with comorbid MDD. The patient was scanned before the commencement of treatment, at the completion of 20 sessions of repetitive Transcranial Magnetic Stimulation (rTMS) and concurrent Eye Movement Desensitisation Reprocessing (EMD-R therapy) (day 14) and 12 weeks after the second scan. EMD-R is an evidence based treatment for PTSD(22). rTMS is a non-invasive stimulation of the region of the brain associated with the development of depression involved in mood control. Studies have demonstrated rTMS efficacy in the treatment of depression (23, 24). The patient an oral self-report description of current symptoms prior to each scan. The patient, prior to treatment, was psychologically unwell and reported a recent episode of self-harm behaviour and described ongoing suicidal ideations. During the initial scanning session, the patient exhibited a low affect, minimal eye contact and reported feeling exhausted. The radiographer was concerned for the patient’s psychological well-being and sought clinical input prior to the scan. On the day of the second scan (day 14), the patient reported feeling happy and enthusiastic, better able to engage with people and feeling the “the best I have in years”. Imaging staff also noted the marked difference in the patient’s affect at the time of the second scan. At the 12 weeks follow up scan the patient reported still feeling “ok” but was starting to feel down again. The patient, when comparing pre and post treatment states, described a feeling of “slipping back to about halfway in-between”. The clinical record indicated the patient was still able to carry out normal duties. An assessment by the Clinical Psychologist also noted a CAPS-5 score of 34.

2D L-COSY Acquisition

 and Analysis All scans were performed on a 60 cm bore 3T PRISMA scanner (Siemens, Erlangen, Germany, software version VE11C) with a 64-channel head and neck coil (Siemens, Erlangen) at the Princess Alexandra Hospital (QLD, Australia). Accurate MRS voxel placement and the 2D L-COSY spectrum acquisition was undertaken using techniques described by Tosh et al. (2019). Scans were performed at the same time of the day to avoid diurnal variation effects. All raw data were pre-processed as reported previously(17). Each prominent diagonal and cross peak was selected and integrated to determine the peak chemical shift, intensity, and volume. These values were internally referenced using the total creatine methyl diagonal peak at 3.02 ppm. The 2D COSY spectra (0-5ppm), recorded prior to treatment and following treatment are shown in Figure 1. The expanded region of the 2D COSY (F2: 4.1-4.5ppm, F1:1.1-1.6ppm) where the fucose-α(1–2)-glycans and substrate α-L-fucose resonate are shown in Figure 2. The aromatic region of the 2D COSY spectra (5 – 9ppm ppm) are shown in Figure 3. The diagonal and crosspeak values that were measured are summarised in Table 1 where the average value for the healthy cohort is compared with the patients’ data at each timepoint. Prior to treatment the concentration of some neurochemicals associated with brain activity were severely depleted compared to the healthy subjects (Table 1). The crosspeak volume differences (Table 2) were calculated as a percentage difference from the healthy cohort. Many of the neurochemicals recorded for this patient were reduced compared to the control cohort. The exception is the lipid C=C, at 5.32ppm, which increased by 46%. Notably GABA was reduced by 43% and Glutamatergic dysfunction was a 12% decrease in the composite glutamine/glutamate crosspeak, and the biochemically associated aspartate level was reduced by 10% compared to the control cohort. Glutathione was normal at 4% less than the healthy cohort. The total fucose region was reduced by 16% prior to treatment compared to the control cohort. The fucoseα(1–2)-glycan, denoted “Fuc 2”, (F:2 4.28, F1:1.14ppm) crosspeak, not usually recorded in the healthy brain, Figure 1: In Vivo L-COSY of the human brain (Post Cingulate Gyrus) acquired at 3T (Prisma) using a 64-channel head and neck coil; voxel size 30x30x30 mm3, increment size 0.8ms, increments 96, 8 averages per increment, TR 1.5 sec, total experimental time 19.12 min, acquired vector: 1024 points, acquisition time: 512 ms, spectral width in F2: 2000 Hz, spectral with in F1: 1250 Hz. A. Patient pre therapy B. Patient immediately post therapy. C. Patient 12 weeks after therapy These can be compared with a typical healthy control in Mountford et al(16) Abbreviations: N-acetyl aspartate (NAA), choline (Cho); creatine (Cr); glutamate and glutamine together (Glx); aspartate (Asp); myo-inositol (m-Ino); histidine (His); lactate (Lac); γ-aminobutyric acid (GABA); macro molecule (MM); glutathione (GSH); threonine (Thr); phosphoryl ethanolamine (PE). The region highlighted by the green box, contains the fucosylated glycans, and is expanded in Figure 2.Figure 2: Expanded region of the L-COSY spectrum in Figure 1 (F2: 4.1-4.5ppm, F1:1.1-1.6ppm), with the assignments of the fucoseα(1–2)-glycans, Fuc 1 to 7 denoted(16, 17). A. Patient pre therapy B. Patient immediately post therapy. C. Patient 12 weeks after therapy.was visible in the 2D COSY spectrum pre-treatment. The fucose-α (1–2)-glycan, denoted “Fuc 5” (F2: 4.40, F1: 1.36) was reduced by 53% and the fucose-α (1–2)-glycan, denoted “Fuc 7” (F2:4.29, F1:1.36) by 7%. Both α-L-fucose 1 and 2 substrates were reduced by 26% and 21% respectively at pre-treatment compared to the healthy control cohort suggesting that the fucose-α(1–2)-glycans could not repopulate at the normal rate during the illness. The neurochemistry at the time of the second scan differed markedly from the pre-treatment scan. GABA levels had increased by 24%. Lipid unsaturation as measured by the C=C at 5.3ppm increased by a further 27%. The glutathione Figure 3: Expanded region of the L-COSY spectrum (F2: 4.5- 9.0ppm, F1: 4.5-9.0ppm), with the assignment of phenylalanine (Phe) at 7.23ppm and the lipid C=C at 5.23ppmm marked. A. Patient pre-therapy B. Patient immediately post-therapy C. Patient 12 weeks after therapy level was at 7% less than normal. Phenylalanine, which was within the normal range before treatment, decreased by a factor of 25%. Glutamine and glutamate decreased by a further 15% and aspartate by a further 37%. The fucoseα(1–2)-glycan ’Fuc 2” (F:2 4.28, F1:1.14ppm) was no longer detectable. The substrates α-L-fucose were recorded for the first time, in this patient, and the fucose-α(1–2)-glycan “Fuc 5” was starting to repopulate (Tables 1 &2). The fucoseα(1–2)-glycans response over the period of treatment is shown in Figures 4. At the 12-week follow up scan when the patient reported negative changes in her mental state there were Table 1: Significant neurochemicals measured using In Vivo 2D L-COSY of a patient pre-therapy, post-therapy and 12 week follow up. Patient peak volumes are referenced to creatine (F2, F1-3.02, 3.02) and are compared to average healthy control peak volumes (N=39).again noticeable shifts in her neurochemistry with the phenylalanine level increased by 15%, the GABA level started to return towards normal levels and Glutamine and glutamate were recorded as moving towards pre-treatment levels. The most notable difference was a reduction in glutathione to 51% less than normal. The substrates α-Lfucose had stabilised but remained much lower than those reported in healthy subjects and the glycans were still repopulating and fucose-α(1–2)-glycan “Fuc 2” was also starting to appear again.

 Discussion 

This patient, referred to the program with a diagnosis of PTSD. Based on her clinical symptoms and assessments using the CAPS-5 and SCID-5 fulfilled the ICD-11 criteria for CPTSD and comorbid MDD. Compared to a study of a healthy population she recorded only 80% of the MR visible neurochemical levels(15). The level of glutamatergic activity was 12% below normal levels and GABA 43% below normal when the patient reported suicidal ideations. This is consistent with the report by Lener(13) of glutamate and GABA pathophysiology in MDD. Following treatment, the GABA levels continued to rise over the 12-week period. The glutamine levels continued to decrease following treatment but started to rise again when the patient at 12 weeks reported herself as “slipping back“. Pre-treatment phenylalanine level was the same as that reported in a group of healthy subjects but was reduced by 25% at the second scan when the patient reported feeling well. The phenylalanine level rose again at the 12 week follow up scan when she was reporting increased emotional distress. A change in phenylalanine levels indicate that the tyrosine kinase pathway is affected. The change in phenylalanine levels is linked with alteration in serotonin and dopamine neurotransmitter levels. The tyrosine kinase pathway and serotonin and dopamine neurotransmitters are important for mood, learning, memory, and motivation(25). Glutathione (GSH) is one of the most abundant thiol antioxidants in cells. Many chronic and age-related diseases are associated with a decline in cellular GSH(26). The level of Glutathione prior to treatment was only 4% below the levels reported in a study of healthy subjects and after treatment decreased to 51% below normal. It is thought that these changes may be a protective mechanism related to regeneration of activity at the neuronal synapse. The level of the spectral region encompassing the α-Lfucose substrates and the fucose-α(1–2)-glycans was 20- 25% less than the healthy control cohort. The fucosylated glycans and substrates α L-fucose responded immediately post therapy as can be seen in Figure 2. The substrates α-L-fucose increased following treatment but dropped at 12 weeks. The levels remain considerably lower than in the control cohort. The fucose-α(1–2)-glycan “Fuc 2”, which has been associated with pain (27) was increased in concentration. Fucose-α(1–2)-glycan “Fuc 2”, in this patient was reduced post treatment and increased again at 12 weeks when she reported changes consistent with a deterioration in her mental state. Fucose-α(1–2)-glycans Fuc 1, Fuc 4, Fuc 5 and Fuc 6 were all repopulating after treatment over the 12-week period. In contrast fucose-α(1–2)-glycan “Fuc 7” was depleted post treatment. This finding differs from that reported in a study of subjects with PTSD(15), where the spectral region encompassing the α-L-fucose substrates and the fucose-α (1–2)-glycans was increased by 30% and the two fucosylated glycans affected “Fuc 4” and “Fuc 6” were increased by 48% (P= 0.002), and 41% (P= 0.02). The PTSD study also did not display any differences in phenylalanine levels. From rat neuronal cultures and immunocytochemistry, the fucosylated glycans are seen at the synapse of the neurons(19) and are implicated in the signalling process of neuron to neuron. Future research should examine cognitive sequelae of CPTSD and MDD, such as confusion in relation to fucosylated glycan activity. The results recorded for this patient are consistent those described by Lener (13) who noted “network dysfunction in association with altered brain levels of glutamate and GABA in both animal and human studies of depression”. The fucose substrate pool was depleted by 20% such that these glycans could not be repopulated adequately prior to treatment. This case report adds to the understanding of this complex situation by demonstrating the marked difference between the patient’s fucose-α(1–2)-glycans and the findings reported in a study of healthy subjects. At the synapse, glutamate, is considered the most abundant neurotransmitter and it plays an important role in synaptic plasticity, learning, memory, and modulation of function within the limbic system. While acute stress can transiently increase glutamate output and transmission,chronic stress is associated with similar findings to those seen in the hyperphenylalaninemic state, with reduced synaptic plasticity and attenuated glutamatergic neurotransmission(28). The Hsieh-Wilson model suggests proteins on neuron “cell A’s” side of synapse contain fucose, which binds to proteins on the surface of neuron “cell B”, acting as a chemical bridge across synapse. The model hypothesises that this activates the cellular machinery in neuron “cell B” and instructs the cell to increase synthesis through a positive feedback loop. Collectively their data identify potentially important roles for fucose-α(1-2)Gal sugars in the regulation of the neurons and morphological changes that may underlie synaptic plasticity. These results support previous reports that the amino-acids tryptophan and phenylalanine play an important role in the pathophysiology of depressive disorders(29). One study suggested that the availability of phenylalanine as a neurotransmitter precursor amino acid in the human brain may play an important role in the pathophysiology of depressive disorders. The relationship between the serotonin system and other systems also requires greater clarity(30), including the glutamatergic neurotransmission. Reduced phenylalanine hydroxylase activity results in significant hyperphenylalaninemia, which leads to alterations in cerebral myelin and reduced levels of serotonin, dopamine, and noradrenaline in the brain(25). It is now clear that due to the complexity of glutamatergic neurotransmission(13, 30) and the realisation that there is a co-location of the fucosylated glycans and glutamine on the neuron, each plays a role in optimal operation of the synapse. Untangling the various altered biochemical pathways is challenging, but the capacity to measure these neurochemicals in the human brain will allow a more detailed understanding of these mechanisms. These results suggest that glutamate and signalling from the fucose-α (1–2)-glycans from one neuron to another are likely to be integrally linked in a dysfunctional brain. In an earlier 2D COSY study of civilian PTSD(15) in first responders being treated while still in employment there was no glutamatergic dysfunction nor GABA dysfunction recorded. There was however a significant effect on the fucose-α(1–2)-glycans and lipid unsaturation. There was no change in the phenylalanine levels. The serendipitous findings in this case study suggest that it is the carbohydrate changes at the synapse may be the first to alter with PTSD and or MDD conditions followed by glutamatergic, GABA dysfunction and phenylalanine dysfunction. The findings in this one patient suggest the need for further studies that evaluate the neurochemistry of patients with either PTSD or CPTSD and comorbid MDD and that further evaluation of the influence of standardised evidenced-based treatment studies that monitor the neurochemistry pre and post treatment should be undertaken. Future studies are needed to determine the specificity of neurochemical bio-markers for PTSD, CPTSD and co-morbid psychological conditions.

Limitations 

While case studies may provide findings not previously identified, it is important to recognise that ‘one off case study’ findings cannot be generalised and must be followed up with well-designed studies. The fortuitous findings in this case highlight the importance of case studies, but also the gaps that may arise. In retrospect it would have been helpful if prior to each scan measures of PTSD or CPTSD symptoms were recorded and that assessment of comorbid MDD and other psychological phenomena such as suicide risk were assessed prior to the second and third scan. 

Conclusion

 In conclusion the patient was originally diagnosed with PTSD but subsequent evaluation via standardised measures (SCID-5 and CAPS-5) the patient was considered to have DSM-5 diagnoses of PTSD and MDD and clinical diagnosis of CPTSD (ICD-11). Prior to therapy when acutely suicidal the neurochemicals, located at the tip of the neuron, fucose-α(1–2)-glycans, glutamine and glutamate, were seriously impacted. When most psychologically well the patient recorded a significant reduction in phenylalanine levels and all except one of the fucose-α(1–2)-glycans were repopulating due to the generation of the substrate α L-fucose. Glutathione was decreasing significantly. Collectively these results suggest that inhibition of neurotransmitter signalling mechanisms plays a critical role in depression in the context of CPTSD. The 2D COSY method offers potential to monitor these neurochemicals in a personalised approach to provide an objective evaluation of response to therapy. This study indicates that technology can facilitate the identification of specific neurochemical changes associated with CPTSD and MDD and the response to treatment. The neurochemical changes provide an opportunity to enhance our understanding of the neurochemistry of mental disorders and address the taxonomic issues related to the harmonisation of DSM-5 and ICD-11.

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