Changes in Motor and Sensory Cortical Neurotransmitters Following Traumatic Brachial Plexus Injuries in Adults Measured with Magnetic Resonance Spectroscopy

Changes in Motor and Sensory Cortical Neurotransmitters Following Traumatic Brachial Plexus Injuries in Adults Measured with Magnetic Resonance Spectroscopy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Changes in Motor and Sensory Cortical Neurotransmitters Following Traumatic Brachial Plexus Injuries in Adults Measured with Magnetic Resonance Spectroscopy Ryckie G Wade, Gráinne Bourke, Alexandra M Olaru, Steve R Williams, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3910773/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the brain. In response to injury within the central nervous system, GABA promotes cortical plasticity and represents a potential pharmacological target to improve functional recovery. However, it is unclear how GABA changes in the brain after traumatic brachial plexus injuries (tBPI). Therefore, neurometabolite concentrations were serially quantified in 7 males with acute tBPI, up to 19 months post-injury, using magnetic resonance spectroscopy (MRS). We acquired J-difference editing MRS using a MAGNETOM Prisma 3T (Siemens Healthcare, Erlangen, Germany). Data were analysed in jMRUI blind to clinical information to quantify GABA, GABA, creatine (Cr) and N-acetylaspartate (NAA) concentrations. Interhemispheric means were compared using linear methods. Confidence intervals (CI) were generated to the 95% level. Within weeks of injury, the hemisphere representing the injured upper limb had a significantly lower GABA:NAA ratio (mean difference 0.23 [CI 0.06–0.40]) and GABA:Cr ratio (mean difference 0.75 [CI 0.24–1.25]) than the uninjured side, which equalised 12 months post-injury. After BPI, there are interhemispheric differences in GABA concentrations within the sensory and motor cortex. Pharmacological modulation of these metabolites may enhance cortical plasticity, prolong the available time for reconstructive surgery and ultimately, improve function. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cortical representations within the sensory and motor cortices have been shown to ‘remap’ following learning 1–3 or after injury 4 over a time course of minutes to hours. The speed of cortical reorganisation indicates that the mechanism underlying this phenomenon is likely to be via the ‘unmasking’ of existing connections rather than synaptogenesis. Animal studies have demonstrated that a reduction in GABA (γ-aminobutyric acid), the brain’s major inhibitory neurotransmitter, is crucial to this process 5 . Following injury to the brain (e.g., traumatic brain injury 6 , transient ischaemic attack 7 and stroke 8 ) GABA levels reduce in the sensory and motor cortices of the symptomatic hemisphere. Downregulation of GABA (reducing GABAergic inhibition) promotes plasticity analogous to long-term-potentiation (LTP), allowing latent connections to strengthen and lost function to be recovered. In humans and animals, pharmacological modulation of GABA either prevents or facilitates LTP-like plasticity depending on the direction of modulation 9,10 . For example, reducing GABAergic tone has been shown to promote plasticity and functional remapping in the motor cortex after stroke 11 . Consequently, GABA modulation represents a potential pharmacological target which may improve central plasticity following injury and therefore, functional recovery. The brachial plexus is the network of nerves which innervate the upper limb. In England, there are approximately 20,000 cases of major trauma annually 12 , and 1% sustain traumatic injuries to their brachial plexus 13 . Traumatic brachial plexus injuries (BPI) are associated with disability 14,15 , chronic pain 16 , psychological morbidity 17 and reduced quality of life 14,15 . These life changing injuries have been estimated to directly cost health services in the USA $ 38,318 per patient 18 with an indirect cost of $ 1.1 million per patient 19 . Consequently, one in three patients with BPIs are at risk of catastrophic health expenditure, whereby out-of-pocket health spending exceeds 40% of their post subsistence income 20 . Surgical reconstruction of injured nerves is the mainstay of treatment; however, there are tens of thousands of axons in each nerve within the brachial plexus 21 and when surgically repaired, perfect topographical alignment is impossible. This means that regenerating motor neurons may synapse with different muscles and sensory neurons may innervate different cutaneous targets, necessitating cortical reorganisation (Fig. 1 ). Alleviation of GABA-driven synaptic inhibition is essential to facilitate to learning and plasticity mechanisms, but no studies have explored GABA modulation after peripheral nerve injury. Measurement of neurometabolites in vivo in the brain is possible using proton ( 1 H) magnetic resonance spectroscopy ( 1 H-MRS). The most studied neurometabolites include glutamate, N-acetylaspartate (NAA), creatine plus phosphocreatine (Cr) and choline-containing compounds. Although GABA is present in the spectra, the relatively low concentration (1–2mM 22 ) and spectral overlap with other neurometabolites (e.g. creatine at 3ppm) 23,24 means that J-difference editing (as implemented in MEscher–GArwood Point RESolved Spectroscopy, MEGA-PRESS) 25 is necessary to separate the GABA signal contaminated by other molecules. We aimed to quantify the concentrations of GABA, Cr and N-acetylaspartate in the brain and explore how they change in adults with acute BPI, using 1 H-MRS. Results Ultimately, 7 males were included of mean age 42 years (SD 19, range 21–76). No patients had a head injury. Given the severity of the injuries sustained by patients, the nationwide COVID-19 measures in-place during the study and the need to accommodate other surgeries, patients were scanned at different timepoints (Table 1 ) and there was variable attrition. Patient characteristics Five adults were involved in motor vehicle collisions, 1 sustained a Rugby “stinger” injury and 1 sustained a traction injury from horse reigns. At the time of scanning, patients were consuming daily a mean of 1g of paracetamol, 171mg of NSAIDs, 24mg of opioids (in morphine-equivalent units) and 21mg of pregabalin. None were in receipt of benzodiazepines or other neuromodulating agents. Using the EQ-5D-5L, patients reported a median overall quality of life of 85% (IQR 70–89). However, the summary scores were considerably lower for upper-limb specific tools, such as the I-HaND (27%, IQR 11–46), Quick-DASH (42%, IQR 21–75) and BRaT (62%, 7–84). The median VAS scores for pain within the neck, arm, forearm and hand were 20% (IQR 7.5–29), 9% (IQR 0–31), 21% (IQR 5–34) and 20% (IQR 4–36), respectively. Patients had worse motor deficits than sensory deficits, as shown in Table 2 whereby strength in the injured limb was approximately half that of the normal limb, whilst the objective sensory tests, including the static 2-point discrimination (median 2, IQR 2–4) and Semmes-Weinstein Monofilament (median 0.07, IQR 0.07-4) were within the normal range. Table 2 Patient characteristics at the time of their first scan Uninjured side Injured limb Mean difference (95% CI) Mean grip strength in kg (SD) 44 (31–51) 22 ( 19 – 42 ) 18 ( 6 , 31 ) Mean key pinch in kg (SD) 8.4 (1.8) 6.0 (2.8) 2.0 (-1.1, 2.05) Mean Index-to-thumb pinch in kg (SD) 5.0 (2.50) 3.6 (1.87) 1.3 (-0.5, 3.1) MR Spectroscopy 20 spectra were collected from the 7 subjects. Figure 2 shows all 20 edited spectra aligned and overlaid. The spectra are reproducible and of very similar quality except for two traces which show an increased (more negative) and decreased NAA signals. The statistical analyses were run both with and without these 2 spectra and no meaningful differences were noted with a sensitivity analysis excluding the two outlier datasets, so a complete case analysis is presented. Quality assurance metrics (NAA and edited GABA linewidth, CRLB and signal-to-noise ratio) are presented in Table 2 . The quality assurance metrics (eTable 1) show tight distributions of the parameters, consistent with the visual appearance of the overlaid spectra. Both GABA linewidth and CRLBremain within 3 median absolute deviations of the median value, while NAA linewidth lies outside this range in two spectra. In these spectra the linewidth is still well within expected values for human brain at 3T, so does not justify exclusion of the data. An example of the AMARES fit to an edited spectrum is shown in Fig. 3 and Fig. 4 shows an example of an unedited (‘Edit-Off’) spectra. In those who had MRS within 3 months of their injury, the hemisphere representing the injured upper limb had a significantly lower GABA:NAA ratio as compared to the unaffected hemisphere (mean difference 0.23 [CI 0.06–0.40]). After approximately 6-months, the GABA:NAA difference had reduced to a mean 0.14 (CI 0.03–0.25). Beyond a year, there was no detectable interhemispheric difference in GABA:NAA (Fig. 5 ). A similar pattern was observed for GABA:Cr ratio, whereby patients who were scanned early (within 3-months of their injury) had significantly lower GABA:Cr ratios in the hemisphere representing the injured limb (mean difference 0.75 [CI 0.24–1.25]). At approximately 6-months, the interhemispheric difference in GABA:Cr had reduced (mean difference 0.26 [CI -0.14, 0.66]) and by 1-year it had equalised (Fig. 6 ). In contrast, we detected no significant interhemispheric differences in NAA:Cr at baseline or over time (Fig. 7 ). Equally, there were no differences in the proportion of grey matter, white matter and CSF between the injured and uninjured hemispheres (Fig. 8 ). There were no appreciable linear associations between any neurotransmitter ratios and the I-HaND, EQ-5D-5L, Quick-DASH or BrAT scores. Discussion We show prolonged reductions in GABA concentrations within the sensorimotor cortex representing the injured limb, in patients with acute traumatic brachial plexus injuries. This window of GABA reduction (facilitating improved plasticity and cortical remapping) appears to last for several months but not beyond 1 year, meaning that surgeons should aim for their reconstructive procedures to be completed as soon as possible, in the knowledge that reinnervation of target organs make take months and the plastic potential of the brain declines over time. Recent work has shown that following traumatic brain injury 6 , transient ischaemic attack 7 , stroke 8 and ischaemic deafferentation of the upper limb 26 , GABA concentrations fall in the symptomatic sensory and motor cortices. Levy et al (2002) 26 showed that GABA concentrations fall within minutes of ischaemic deafferentation of human limbs. The understanding of temporal changes in GABA were extended by Yasen et al (2018) 6 in their study of mild traumatic brain injury, whereby cortical GABA concentrations were low at 3 days post-injury (compared to controls) and remained low 2 weeks later. Similar patterns were seen in patients with an established stroke (mean 7 months old) whereby patients had lower cortical GABA levels than controls 8 . In the longer term, Tremblay and colleagues (2014) 27 showed that (independent of cortical atrophy) GABA levels normalise 3-years after brain injury. These studies agree with our observations in traumatic BPI, whereby GABA concentrations fall rapidly after injury, remain low months later and normalise approximately 1-year post-injury. It is important to clarify the temporal changes in GABAergic inhibition (whether active or incidental) because GABA levels are known to modulate plasticity. Lower levels of GABA have been shown to promote plasticity and improve perceptual learning 28 . By reducing GABAergic inhibition, vacant neuronal connections are ‘‘unmasked’’ which enables them to form new synapses within minutes-to-hours, such that function can be recovered or new connections fortified. Given the severity of BPI, its wider impact on both patients and their families, and the extraordinary costs to the health services, the potential to pharmacologically modulate GABA (to maintain and extend the window of ‘improved central plasticity’ for surgical reconstruction) might benefit of patients and surgeon alike. In both animals 29,30 and humans 31 early nerve repair after BPI yields better outcomes. Recent work has shown that the time from injury to reconstruction in adults with BPI is linearly associated with functional outcomes, whereby each month of delay to neurotisation reduces the odds of meaningful recovery by 7% 31 . Moreover, the choice of donor nerve(s) used for reconstruction directly influences the chance of useful recovery 31 . This observation cannot be completely explained by local anatomical factors (such as donor-recipient axon counts) and we speculate that the plastic potential of the cortex for some donor nerves may be greater than others. For example, the most prevalent donor nerves for restoring elbow flexion via neurotisation of the musculocutaneous nerve are the Oberlin transfers 32,33 (utilising fascicles from the median and/or ulnar nerves which natively perform wrist flexion) or intercostal nerve transfer (using nerves which provide somatic control of breathing). Although the median/ulnar fascicles (with a mean of 1318 and 1860 axons 21 ) and several intercostals nerves (with axon counts of 520–1353 per nerve, depending on the level 34 ) are well matched for size and axon counts to the musculocutaneous nerve (which has 1600 efferent axons producing elbow flexion 21 ), there are systematic differences in the outcomes between these two transfers which implies that other factors are at play. In this paradigm, we hypothesise that the cortical areas controlling wrist flexion may be more amenable to adaption to controlling elbow flexion (after nerve transfer) than cortical areas designated for voluntary control of respiration. Whatever the mechanisms governing this change in cortical representation, the potential to modulate the plastic potential through GABA represents an important avenue for future research. We were required to exclude some patients with more severe BPI or concurrent injuries. Equally, this population is difficult to access for research for many reasons: at best, patients are young, working age adults with dependents and so have limited time to engage in research; at worst, they are polytraumatised, disabled 14,15 , suffering with chronic pain 16 and psychopathology 17 and so cannot engage. Consequently, our data collection timepoints were inconsistent which could introduce time-related biases and the sample we ultimately recruited may not reflect the population. It is widely known that GABA:NAA ratios are inhomogeneous in the brain 35 so our findings may not be generalisable to other regions. Within the sensorimotor cortex, the hemisphere responsible for the dominant hand exhibits higher GABA concentrations 36 so without controlling for this confound (due to insufficient data), our results may not be precise. Conclusions Our data suggests that there may be a fading window of time (lasting approximately 12 months) where the brain has plastic potential and thus, is receptive to cortical reorganisation following peripheral nerve repair. Future research should explore changes in GABA in other peripheral nerve disorders and whether modulation of GABA improves outcomes for patients. Methods This cohort study was designed and reported in accordance with the STROBE and STARD guidance, taking into account the domains of the QUADAS-2 37 and PRISMA-DTA 38 tools. Approval was provided by the United Kingdom National Health Research Authority (ID 19/NW/0324) and conducted in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all participants. The raw MRS data are available open-source at https://osf.io/un24g/ . Recruitment Between January 2019 and July 2020, we screened 22 consecutive adults with acute traumatic BPI admitted to three Major Trauma Centres within England. Fifteen were excluded for the following reasons: 4 declined to participate or were uncontactable, 4 required immediate surgery which precluded preoperative MRI, 3 were unable to meet COVID-related isolation requirements, 2 were claustrophobic, 1 had learning difficulties and so could not provide informed consent, and 1 patient had a c-spine injury requiring extended immobilisation in a collar. Image Acquisition Data were acquired at a field strength of 3 Tesla (T) using a MAGNETOM Prisma (Siemens Healthcare, Erlangen, Germany) with a 64-channel head coil. Firstly, we acquired an MPRAGE (T1-weighted) dataset with the following parameters: TI 900 ms, TE 2.98 ms, TR 2300 ms, flip angle 9 o , 176 sagittal slices, field of view 256x248, 1mm isotropic resolution, GRAPPA factor 4, TrueForm B1 shim, no partial Fourier, AP phase encoding and bandwidth 240 Hz/Px. Thereafter, we acquired J-edited spectra using MEscher–GArwood Point RESolved Spectroscopy (MEGA-PRESS) 25 from 2 cm isotropic voxels centred on the right and left “hand knob” areas (using the ‘delta sign’ on axial slices), respectively, with the following parameters: TE 68 ms, TR 2000 ms, 64 averages, water suppression bandwidth 50 Hz, automated field-map-based B0 shim, TrueForm B1 shim, 4 preparation scans, edit pulse frequency 1.9 ppm, edit pulse bandwidth 50 Hz, centre frequency 4.7 ppm, receiver bandwidth 1200 Hz for an acquisition time of 853 ms. Spectra were interleaved every acquisition between ‘edit-on’ and ‘edit-off’. Real time frequency adjustment was performed automatically on the scanner to prevent field drift affecting the difference spectra. The total scan time was 4 minutes 24 seconds per hemisphere. Data were stored as averaged fids for the ‘edit-on’ and ‘edit-off’ subspectra and exported from the scanner as .RDA files. Spectroscopic processing After acquisition, a custom-made processing script was used to generate phase-corrected spectra for the ‘edit-on’ and ‘edit-off’ conditions as well as the edited (difference spectrum) itself. Anonymised data were imported into jMRUI 39 v5.2 ( http://www.mrui.uab.es/mrui/ ) for analysis by SW and CLC, who were blind to clinical information. Prior to analysis using AMARES 40 , residual water was removed from the spectra using the HLSVD routine. The edited spectra were fitted with the following prior knowledge: NAA: relative phase 180 o , lorentzian line shape, frequency, linewidth and amplitude unconstrained; GABA: relative phase 0 o , Gaussian line shape, frequency and amplitude unconstrained, linewidth constrained between 15 and 25Hz. Co-edited glutamate + glutamine: fitted as 2 peaks of equal amplitude separated by 10.25Hz, relative phase 0 o , lorentzian line shape, each linewidth constrained to equal that of NAA, frequency unconstrained. The prior knowledge for the edited GABA signal takes account of the contribution from co-edited macromolecules 24 , hence a gaussian line shape was assumed with constraints on the linewidth to force the fit to be meaningful. GABA measured from non-macromolecule-suppressed spectra is often referred to as GABA + , but we do not use this extra abbreviation for simplicity. The ‘edit-off’ spectra were also quantified by AMARES with the following prior knowledge for NAA, Cr at 3.03 and 3.96 ppm and choline-containing compounds (Cho): amplitudes and frequencies were all unconstrained, lorentzian line shape was stipulated and linewidths were all constrained to equal that of Cr at 3.03 ppm. Example spectra, together with the AMARE fit for an edited spectrum are shown in Figs. 2 – 4 . To quantify GABA, Cr and NAA are assumed to be of quasi-constant concentration (both within and between subjects) such that GABA:Cr and GABA:NAA ratios may be computed as a proxy for GABA concentration. The edited signal for NAA was used to construct the GABA ratio, since any slight missetting or drift of the amplitude or phase of the pulse would affect both the edited GABA and NAA signals similarly. The quality of the spectra was assessed by visual inspection and by measuring the linewidth and Cramer-Rao lower bound (CRLB) of the AMARES 40 fit for GABA in the edited spectra. The signal-to-noise ratio of NAA was also recorded for edited and spectra. Additionally, grey matter and white matter proportions were assessed using SPECTRIM 41 . This provides a graphical user interface to enable segmentation of the T1-weighted anatomical DICOM images, with extraction of the grey matter, white matter and CSF content of the spectroscopic voxels. This is a necessary step to interpret any changes in GABA, as it is known that the GABA content of grey matter is approxiamtely double that of white matter 42 . Functional outcomes assessment Grip strength was assessed using a digital grip dynamometer. Key (lateral) and tip (index pulp to thumb pulp) pinch were assessed using the Jamar Plus Digital Pinch Dynamometer For all assessment, 3 measurements were taken the best (strongest) was used. Analysis Data were analysed using Stata v16/MP (StataCorp LLC, Texas). Scaled variables approximating the normal distribution are represented by the mean (and standard deviation, SD). To estimate the interhemispheric differences in the concentrations of neurotransmitters, means were compared by linear regression. As the dataset is small, we used resampling with replacement (bootstrapping) with 1000 iterations to improve estimates of the variance. Age may affect GABA levels 43,44 but as there is no conceptual association between these variables and interhemispheric differences after nerve injury, adjustment was not required. Although the patients in this series were exposed to analgesics which modulate GABA and Glutamate (Gabapentin or Pregabalin), as we were directly comparing interhemispheric differences, the effect of these drugs will be neutralised, and so further adjustment is unnecessary. Confidence intervals (CI) were generated to the 95% level. Declarations Funding RGW is funded by the National Institute for Health Research (NIHR, DRF-2018-11-ST2-028 and CL-2021-02-002). SP is funded by a British Heart Foundation Chair (CH/16/2/32089). CLC is funded by the Medical Research Council (MRC) Grant MR/PO14445/1. This research is also supported by the University of Leeds Advanced Imaging Centre, which is funded by the Medical Research Council (MR/M008991/1) with support from the British Heart Foundation (BHF-SP/14/7/31351) and Arthritis Research UK (ARUK-21078). The views expressed are those of the author(s) and not necessarily those of the United Kingdom’s National Health Service, NIHR or Department of Health. Competing Interests The authors have declared that no competing interests exist References Kami A, Meyer G, Jezzard P, et al. Functional MRI evidence for adult motor cortex plasticity during motor skill learning. Nature . 1995;377(6545):155–158. Lea-Carnall CA, Trujillo-Barreto NJ, Montemurro MA, et al. Evidence for frequency-dependent cortical plasticity in the human brain. Proc. Natl. Acad. Sci. 2017;114(33):8871–8876. Vidyasagar R, Folger SE, Parkes LM. 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Wade","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYHACZoYEEHWA+QCQlJAhRQsbiJLgIU4LGBzgMQBRhLWYz0h+bPCg4rA83/Gez69u1FjwMLAfProBnxaZG2nGCQlnDhvOPHN2m3XOMaDDeNLSbuDTIiGRw3wgse0244YbuduMc9iAWiR4zIjQ8u+2/Yb7b54Z5/wjUktCYsPtxA03eJgf57YRo4XnmbFBwrH/yTPPpJkx5/ZJ8LAR9At78mPJHzVptn3HDz/+nPOtTo6f/fAxvFoYBBLgTDYJMIlXOQjwH4AzmT8QVD0KRsEoGAUjEgAADetLjAyZKZgAAAAASUVORK5CYII=","orcid":"","institution":"Leeds Teaching Hospitals Trust","correspondingAuthor":true,"prefix":"","firstName":"Ryckie","middleName":"G","lastName":"Wade","suffix":""},{"id":274285033,"identity":"65f4dbcf-b31f-4fc7-92bf-d7c7c3a4eacb","order_by":1,"name":"Gráinne Bourke","email":"","orcid":"","institution":"Leeds Teaching Hospitals Trust","correspondingAuthor":false,"prefix":"","firstName":"Gráinne","middleName":"","lastName":"Bourke","suffix":""},{"id":274285034,"identity":"7f31d20e-2418-44a5-96ca-877f17085019","order_by":2,"name":"Alexandra M Olaru","email":"","orcid":"","institution":"Siemens Healthcare Ltd","correspondingAuthor":false,"prefix":"","firstName":"Alexandra","middleName":"M","lastName":"Olaru","suffix":""},{"id":274285035,"identity":"25f7e268-3a34-42c4-8e00-f768d9b90412","order_by":3,"name":"Steve R Williams","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Steve","middleName":"R","lastName":"Williams","suffix":""},{"id":274285036,"identity":"c7a3d36f-4b70-4b14-af19-42a63a60b308","order_by":4,"name":"David Shelley","email":"","orcid":"","institution":"University of Leeds","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Shelley","suffix":""},{"id":274285037,"identity":"c4e9a563-0243-489a-8cae-a606d491cb90","order_by":5,"name":"Sven Plein","email":"","orcid":"","institution":"University of Leeds","correspondingAuthor":false,"prefix":"","firstName":"Sven","middleName":"","lastName":"Plein","suffix":""},{"id":274285038,"identity":"3ccd0082-ae36-4451-9b7d-a3f371b92447","order_by":6,"name":"Robert D Bains","email":"","orcid":"","institution":"Leeds Teaching Hospitals Trust","correspondingAuthor":false,"prefix":"","firstName":"Robert","middleName":"D","lastName":"Bains","suffix":""},{"id":274285039,"identity":"8d22e263-d4e0-42ff-8832-48e4b7e2bd03","order_by":7,"name":"James D Bedford","email":"","orcid":"","institution":"Manchester University NHS Foundation Trust","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"D","lastName":"Bedford","suffix":""},{"id":274285040,"identity":"dca53f49-862a-40af-81d0-af77350b9588","order_by":8,"name":"Lucy Homer Newton","email":"","orcid":"","institution":"Manchester University NHS Foundation Trust","correspondingAuthor":false,"prefix":"","firstName":"Lucy","middleName":"Homer","lastName":"Newton","suffix":""},{"id":274285041,"identity":"a02086af-9903-494f-8275-9056b6bb2c3e","order_by":9,"name":"Chye Yew Ng","email":"","orcid":"","institution":"Wrightington, Wigan and Leigh NHS Foundation Trust","correspondingAuthor":false,"prefix":"","firstName":"Chye","middleName":"Yew","lastName":"Ng","suffix":""},{"id":274285042,"identity":"8201c356-8a17-4660-855f-4aca2d7e7b55","order_by":10,"name":"Laura Parkes","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Laura","middleName":"","lastName":"Parkes","suffix":""},{"id":274285043,"identity":"c0d1b25a-3b48-4ab3-9890-54f19ad742c7","order_by":11,"name":"Caroline Lea-Carnall","email":"","orcid":"","institution":"University of Manchester","correspondingAuthor":false,"prefix":"","firstName":"Caroline","middleName":"","lastName":"Lea-Carnall","suffix":""}],"badges":[],"createdAt":"2024-01-30 13:52:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3910773/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3910773/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51565520,"identity":"e6f10153-340b-467f-b901-1d4369dd975a","added_by":"auto","created_at":"2024-02-23 19:05:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":440493,"visible":true,"origin":"","legend":"\u003cp\u003eAfter division of a peripheral nerve (top) and surgical repair (bottom), the topography of the repair site is imperfect, so the cortical representation must change.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/1caf7a86e19e96a456a5777e.png"},{"id":51565526,"identity":"35912b10-15d2-4835-9a5f-090c1e319128","added_by":"auto","created_at":"2024-02-23 19:05:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":62013,"visible":true,"origin":"","legend":"\u003cp\u003eOverlay of 20 edited spectra. Peaks are assigned to NAA, GABA + macromolecules (GABA+) and glutamate + glutamine (Glx). The two outlier spectra are indicated by *. The spectra have had 6.0 Hz line-broadening applied for display purposes.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/1267a9bb4100d7416f28fa2f.png"},{"id":51565523,"identity":"5c43a29e-9e10-4249-9ed3-e3476e1740a7","added_by":"auto","created_at":"2024-02-23 19:05:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":43139,"visible":true,"origin":"","legend":"\u003cp\u003eExample of AMARES fit to an edited spectrum. A) residual, B) individual fits and C) model fit overlaid onto original data with NAA, GABA + macromolecules and glutamate + glutamine (Glx) indicated. The spectra have had 6.0 Hz line-broadening applied for display purposes.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/8a800027328852f8ee149649.png"},{"id":51565525,"identity":"afd57860-f39e-4e06-bf67-c515c7c4912c","added_by":"auto","created_at":"2024-02-23 19:05:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":75091,"visible":true,"origin":"","legend":"\u003cp\u003eExample unedited (‘Edit-Off’) spectra from the right and left hand knob areas of the motor cortex, with the quantified resonances indicated: NAA – N-acetylaspartate; Crn – creatine + phosphocreatine methyl; Cho – choline-containing compounds. The spectra have had 6.0 Hz line-broadening applied for display purposes.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/aa2d5379fc8814041c93b60c.png"},{"id":51565528,"identity":"bb76ee39-99e5-469b-987c-c76f8e0986b3","added_by":"auto","created_at":"2024-02-23 19:05:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":46979,"visible":true,"origin":"","legend":"\u003cp\u003eGABA:NAA ratios in the hemispheres representing the injured and uninjured limbs of adults with traumatic brachial plexus injuries. The linear fit indicates that GABA:NAA ratios fall in the hemisphere representing the injured limb (relative to the normal hemisphere) immediately after injury and normalise over months.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/8e6f361225ac58dd49584e81.png"},{"id":51565527,"identity":"335a9222-25c8-45c3-912b-5ed739cb3220","added_by":"auto","created_at":"2024-02-23 19:05:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":45656,"visible":true,"origin":"","legend":"\u003cp\u003eGABA:Cr ratios in the hemispheres representing the injured and uninjured limbs of adults with traumatic brachial plexus injuries. The linear fit indicates that GABA:Cr ratios fall in the hemisphere representing the injured limb (relative to the normal hemisphere) immediately after injury and normalise over months.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/fbb401d6ca1529533ed4591f.png"},{"id":51565524,"identity":"dd7db344-15ec-4161-82d8-cdd61026488c","added_by":"auto","created_at":"2024-02-23 19:05:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":45403,"visible":true,"origin":"","legend":"\u003cp\u003eChange in NAA:Cr ratio in the hemispheres representing the injured and uninjured limbs of adults with traumatic brachial plexus injuries. The linear fit of the injured and normal hemispheres was not different.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/d750ec96d2f80beeb95eecf6.png"},{"id":51565530,"identity":"cf45b95a-9a56-41ff-939e-136fff7337d2","added_by":"auto","created_at":"2024-02-23 19:05:04","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":97010,"visible":true,"origin":"","legend":"\u003cp\u003eThe volume of the grey matter, white matter and cerebrospinal fluid (CSF) in the hemispheres representing the injured and uninjured limbs\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/b845497024373c6e9364a1aa.png"},{"id":52578931,"identity":"54e453a6-2f93-432f-a2c1-068a0a085338","added_by":"auto","created_at":"2024-03-13 07:18:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1098445,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/e9aa350e-eb7f-4d3f-8087-771e53a0fade.pdf"},{"id":51565522,"identity":"f954fd16-4a43-4c41-9dee-e537522a8da4","added_by":"auto","created_at":"2024-02-23 19:05:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14977,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3910773/v1/05945a6398f4fc69883d1b9c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Changes in Motor and Sensory Cortical Neurotransmitters Following Traumatic Brachial Plexus Injuries in Adults Measured with Magnetic Resonance Spectroscopy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCortical representations within the sensory and motor cortices have been shown to \u0026lsquo;remap\u0026rsquo; following learning\u003csup\u003e1\u0026ndash;3\u003c/sup\u003e or after injury\u003csup\u003e4\u003c/sup\u003e over a time course of minutes to hours. The speed of cortical reorganisation indicates that the mechanism underlying this phenomenon is likely to be via the \u0026lsquo;unmasking\u0026rsquo; of existing connections rather than synaptogenesis. Animal studies have demonstrated that a reduction in GABA (γ-aminobutyric acid), the brain\u0026rsquo;s major inhibitory neurotransmitter, is crucial to this process\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFollowing injury to the brain (e.g., traumatic brain injury\u003csup\u003e6\u003c/sup\u003e, transient ischaemic attack\u003csup\u003e7\u003c/sup\u003e and stroke\u003csup\u003e8\u003c/sup\u003e) GABA levels reduce in the sensory and motor cortices of the symptomatic hemisphere. Downregulation of GABA (reducing GABAergic inhibition) promotes plasticity analogous to long-term-potentiation (LTP), allowing latent connections to strengthen and lost function to be recovered. In humans and animals, pharmacological modulation of GABA either prevents or facilitates LTP-like plasticity depending on the direction of modulation\u003csup\u003e9,10\u003c/sup\u003e. For example, reducing GABAergic tone has been shown to promote plasticity and functional remapping in the motor cortex after stroke\u003csup\u003e11\u003c/sup\u003e. Consequently, GABA modulation represents a potential pharmacological target which may improve central plasticity following injury and therefore, functional recovery.\u003c/p\u003e \u003cp\u003eThe brachial plexus is the network of nerves which innervate the upper limb. In England, there are approximately 20,000 cases of major trauma annually\u003csup\u003e12\u003c/sup\u003e, and 1% sustain traumatic injuries to their brachial plexus\u003csup\u003e13\u003c/sup\u003e. Traumatic brachial plexus injuries (BPI) are associated with disability\u003csup\u003e14,15\u003c/sup\u003e, chronic pain\u003csup\u003e16\u003c/sup\u003e, psychological morbidity\u003csup\u003e17\u003c/sup\u003e and reduced quality of life\u003csup\u003e14,15\u003c/sup\u003e. These life changing injuries have been estimated to directly cost health services in the USA \u003cspan\u003e$\u003c/span\u003e38,318 per patient\u003csup\u003e18\u003c/sup\u003e with an indirect cost of \u003cspan\u003e$\u003c/span\u003e1.1\u0026nbsp;million per patient\u003csup\u003e19\u003c/sup\u003e. Consequently, one in three patients with BPIs are at risk of catastrophic health expenditure, whereby out-of-pocket health spending exceeds 40% of their post subsistence income\u003csup\u003e20\u003c/sup\u003e. Surgical reconstruction of injured nerves is the mainstay of treatment; however, there are tens of thousands of axons in each nerve within the brachial plexus\u003csup\u003e21\u003c/sup\u003e and when surgically repaired, perfect topographical alignment is impossible. This means that regenerating motor neurons may synapse with different muscles and sensory neurons may innervate different cutaneous targets, necessitating cortical reorganisation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Alleviation of GABA-driven synaptic inhibition is essential to facilitate to learning and plasticity mechanisms, but no studies have explored GABA modulation after peripheral nerve injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMeasurement of neurometabolites in vivo in the brain is possible using proton (\u003csup\u003e1\u003c/sup\u003eH) magnetic resonance spectroscopy (\u003csup\u003e1\u003c/sup\u003eH-MRS). The most studied neurometabolites include glutamate, N-acetylaspartate (NAA), creatine plus phosphocreatine (Cr) and choline-containing compounds. Although GABA is present in the spectra, the relatively low concentration (1\u0026ndash;2mM\u003csup\u003e22\u003c/sup\u003e) and spectral overlap with other neurometabolites (e.g. creatine at 3ppm)\u003csup\u003e23,24\u003c/sup\u003e means that J-difference editing (as implemented in MEscher\u0026ndash;GArwood Point RESolved Spectroscopy, MEGA-PRESS)\u003csup\u003e25\u003c/sup\u003e is necessary to separate the GABA signal contaminated by other molecules.\u003c/p\u003e \u003cp\u003eWe aimed to quantify the concentrations of GABA, Cr and N-acetylaspartate in the brain and explore how they change in adults with acute BPI, using \u003csup\u003e1\u003c/sup\u003eH-MRS.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eUltimately, 7 males were included of mean age 42 years (SD 19, range 21\u0026ndash;76). No patients had a head injury. Given the severity of the injuries sustained by patients, the nationwide COVID-19 measures in-place during the study and the need to accommodate other surgeries, patients were scanned at different timepoints (Table\u0026nbsp;\u003cspan\u003e1\u003c/span\u003e) and there was variable attrition.\u003c/p\u003e\n\u003cdiv\u003e\n \u003cdiv align=\"left\"\u003e\u003cbr\u003e\u003cimg src=\"https://myfiles.space/user_files/122228_c8a1650c59388082/122228_custom_files/img1708676443.png\"\u003e\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec3\"\u003e\n \u003ch2\u003ePatient characteristics\u003c/h2\u003e\n \u003cp\u003eFive adults were involved in motor vehicle collisions, 1 sustained a Rugby \u0026ldquo;stinger\u0026rdquo; injury and 1 sustained a traction injury from horse reigns. At the time of scanning, patients were consuming daily a mean of 1g of paracetamol, 171mg of NSAIDs, 24mg of opioids (in morphine-equivalent units) and 21mg of pregabalin. None were in receipt of benzodiazepines or other neuromodulating agents.\u003c/p\u003e\n \u003cp\u003eUsing the EQ-5D-5L, patients reported a median overall quality of life of 85% (IQR 70\u0026ndash;89). However, the summary scores were considerably lower for upper-limb specific tools, such as the I-HaND (27%, IQR 11\u0026ndash;46), Quick-DASH (42%, IQR 21\u0026ndash;75) and BRaT (62%, 7\u0026ndash;84). The median VAS scores for pain within the neck, arm, forearm and hand were 20% (IQR 7.5\u0026ndash;29), 9% (IQR 0\u0026ndash;31), 21% (IQR 5\u0026ndash;34) and 20% (IQR 4\u0026ndash;36), respectively. Patients had worse motor deficits than sensory deficits, as shown in Table\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e whereby strength in the injured limb was approximately half that of the normal limb, whilst the objective sensory tests, including the static 2-point discrimination (median 2, IQR 2\u0026ndash;4) and Semmes-Weinstein Monofilament (median 0.07, IQR 0.07-4) were within the normal range.\u003c/p\u003e\n \u003cdiv\u003e\n \u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv\u003eTable 2\u003c/div\u003e\n \u003cdiv\u003e\n \u003cp\u003ePatient characteristics at the time of their first scan\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eUninjured side\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eInjured limb\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMean difference (95% CI)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean grip strength in kg (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e44 (31\u0026ndash;51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e22 (\u003cspan\u003e19\u003c/span\u003e\u0026ndash;\u003cspan\u003e42\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18 (\u003cspan\u003e6\u003c/span\u003e, \u003cspan\u003e31\u003c/span\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean key pinch in kg (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8.4 (1.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.0 (2.8)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.0 (-1.1, 2.05)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eMean Index-to-thumb pinch in kg (SD)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.0 (2.50)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.6 (1.87)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.3 (-0.5, 3.1)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\"\u003e\n \u003ch2\u003eMR Spectroscopy\u003c/h2\u003e\n \u003cp\u003e20 spectra were collected from the 7 subjects. Figure\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e shows all 20 edited spectra aligned and overlaid. The spectra are reproducible and of very similar quality except for two traces which show an increased (more negative) and decreased NAA signals. The statistical analyses were run both with and without these 2 spectra and no meaningful differences were noted with a sensitivity analysis excluding the two outlier datasets, so a complete case analysis is presented. Quality assurance metrics (NAA and edited GABA linewidth, CRLB and signal-to-noise ratio) are presented in Table\u0026nbsp;\u003cspan\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eThe quality assurance metrics (eTable 1) show tight distributions of the parameters, consistent with the visual appearance of the overlaid spectra. Both GABA linewidth and CRLBremain within 3 median absolute deviations of the median value, while NAA linewidth lies outside this range in two spectra. In these spectra the linewidth is still well within expected values for human brain at 3T, so does not justify exclusion of the data. An example of the AMARES fit to an edited spectrum is shown in Fig.\u0026nbsp;\u003cspan\u003e3\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan\u003e4\u003c/span\u003e shows an example of an unedited (\u0026lsquo;Edit-Off\u0026rsquo;) spectra.\u003c/p\u003e\n \u003cp\u003eIn those who had MRS within 3 months of their injury, the hemisphere representing the injured upper limb had a significantly lower GABA:NAA ratio as compared to the unaffected hemisphere (mean difference 0.23 [CI 0.06\u0026ndash;0.40]). After approximately 6-months, the GABA:NAA difference had reduced to a mean 0.14 (CI 0.03\u0026ndash;0.25). Beyond a year, there was no detectable interhemispheric difference in GABA:NAA (Fig.\u0026nbsp;\u003cspan\u003e5\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eA similar pattern was observed for GABA:Cr ratio, whereby patients who were scanned early (within 3-months of their injury) had significantly lower GABA:Cr ratios in the hemisphere representing the injured limb (mean difference 0.75 [CI 0.24\u0026ndash;1.25]). At approximately 6-months, the interhemispheric difference in GABA:Cr had reduced (mean difference 0.26 [CI -0.14, 0.66]) and by 1-year it had equalised (Fig.\u0026nbsp;\u003cspan\u003e6\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eIn contrast, we detected no significant interhemispheric differences in NAA:Cr at baseline or over time (Fig.\u0026nbsp;\u003cspan\u003e7\u003c/span\u003e). Equally, there were no differences in the proportion of grey matter, white matter and CSF between the injured and uninjured hemispheres (Fig.\u0026nbsp;\u003cspan\u003e8\u003c/span\u003e). There were no appreciable linear associations between any neurotransmitter ratios and the I-HaND, EQ-5D-5L, Quick-DASH or BrAT scores.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe show prolonged reductions in GABA concentrations within the sensorimotor cortex representing the injured limb, in patients with acute traumatic brachial plexus injuries. This window of GABA reduction (facilitating improved plasticity and cortical remapping) appears to last for several months but not beyond 1 year, meaning that surgeons should aim for their reconstructive procedures to be completed as soon as possible, in the knowledge that reinnervation of target organs make take months and the plastic potential of the brain declines over time.\u003c/p\u003e \u003cp\u003eRecent work has shown that following traumatic brain injury\u003csup\u003e6\u003c/sup\u003e, transient ischaemic attack\u003csup\u003e7\u003c/sup\u003e, stroke\u003csup\u003e8\u003c/sup\u003e and ischaemic deafferentation of the upper limb\u003csup\u003e26\u003c/sup\u003e, GABA concentrations fall in the symptomatic sensory and motor cortices. Levy et al (2002)\u003csup\u003e26\u003c/sup\u003e showed that GABA concentrations fall within minutes of ischaemic deafferentation of human limbs. The understanding of temporal changes in GABA were extended by Yasen et al (2018)\u003csup\u003e6\u003c/sup\u003e in their study of mild traumatic brain injury, whereby cortical GABA concentrations were low at 3 days post-injury (compared to controls) and remained low 2 weeks later. Similar patterns were seen in patients with an established stroke (mean 7 months old) whereby patients had lower cortical GABA levels than controls\u003csup\u003e8\u003c/sup\u003e. In the longer term, Tremblay and colleagues (2014)\u003csup\u003e27\u003c/sup\u003e showed that (independent of cortical atrophy) GABA levels normalise 3-years after brain injury. These studies agree with our observations in traumatic BPI, whereby GABA concentrations fall rapidly after injury, remain low months later and normalise approximately 1-year post-injury. It is important to clarify the temporal changes in GABAergic inhibition (whether active or incidental) because GABA levels are known to modulate plasticity. Lower levels of GABA have been shown to promote plasticity and improve perceptual learning\u003csup\u003e28\u003c/sup\u003e. By reducing GABAergic inhibition, vacant neuronal connections are \u0026lsquo;\u0026lsquo;unmasked\u0026rsquo;\u0026rsquo; which enables them to form new synapses within minutes-to-hours, such that function can be recovered or new connections fortified. Given the severity of BPI, its wider impact on both patients and their families, and the extraordinary costs to the health services, the potential to pharmacologically modulate GABA (to maintain and extend the window of \u0026lsquo;improved central plasticity\u0026rsquo; for surgical reconstruction) might benefit of patients and surgeon alike.\u003c/p\u003e \u003cp\u003eIn both animals\u003csup\u003e29,30\u003c/sup\u003e and humans\u003csup\u003e31\u003c/sup\u003e early nerve repair after BPI yields better outcomes. Recent work has shown that the time from injury to reconstruction in adults with BPI is linearly associated with functional outcomes, whereby each month of delay to neurotisation reduces the odds of meaningful recovery by 7%\u003csup\u003e31\u003c/sup\u003e. Moreover, the choice of donor nerve(s) used for reconstruction directly influences the chance of useful recovery\u003csup\u003e31\u003c/sup\u003e. This observation cannot be completely explained by local anatomical factors (such as donor-recipient axon counts) and we speculate that the plastic potential of the cortex for some donor nerves may be greater than others. For example, the most prevalent donor nerves for restoring elbow flexion via neurotisation of the musculocutaneous nerve are the Oberlin transfers\u003csup\u003e32,33\u003c/sup\u003e (utilising fascicles from the median and/or ulnar nerves which natively perform wrist flexion) or intercostal nerve transfer (using nerves which provide somatic control of breathing). Although the median/ulnar fascicles (with a mean of 1318 and 1860 axons\u003csup\u003e21\u003c/sup\u003e) and several intercostals nerves (with axon counts of 520\u0026ndash;1353 per nerve, depending on the level\u003csup\u003e34\u003c/sup\u003e) are well matched for size and axon counts to the musculocutaneous nerve (which has 1600 efferent axons producing elbow flexion\u003csup\u003e21\u003c/sup\u003e), there are systematic differences in the outcomes between these two transfers which implies that other factors are at play. In this paradigm, we hypothesise that the cortical areas controlling wrist flexion may be more amenable to adaption to controlling elbow flexion (after nerve transfer) than cortical areas designated for voluntary control of respiration. Whatever the mechanisms governing this change in cortical representation, the potential to modulate the plastic potential through GABA represents an important avenue for future research.\u003c/p\u003e \u003cp\u003eWe were required to exclude some patients with more severe BPI or concurrent injuries. Equally, this population is difficult to access for research for many reasons: at best, patients are young, working age adults with dependents and so have limited time to engage in research; at worst, they are polytraumatised, disabled\u003csup\u003e14,15\u003c/sup\u003e, suffering with chronic pain\u003csup\u003e16\u003c/sup\u003e and psychopathology\u003csup\u003e17\u003c/sup\u003e and so cannot engage. Consequently, our data collection timepoints were inconsistent which could introduce time-related biases and the sample we ultimately recruited may not reflect the population. It is widely known that GABA:NAA ratios are inhomogeneous in the brain\u003csup\u003e35\u003c/sup\u003e so our findings may not be generalisable to other regions. Within the sensorimotor cortex, the hemisphere responsible for the dominant hand exhibits higher GABA concentrations\u003csup\u003e36\u003c/sup\u003e so without controlling for this confound (due to insufficient data), our results may not be precise.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur data suggests that there may be a fading window of time (lasting approximately 12 months) where the brain has plastic potential and thus, is receptive to cortical reorganisation following peripheral nerve repair. Future research should explore changes in GABA in other peripheral nerve disorders and whether modulation of GABA improves outcomes for patients.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThis cohort study was designed and reported in accordance with the STROBE and STARD guidance, taking into account the domains of the QUADAS-2\u003csup\u003e37\u003c/sup\u003e and PRISMA-DTA\u003csup\u003e38\u003c/sup\u003e tools. Approval was provided by the United Kingdom National Health Research Authority (ID 19/NW/0324) and conducted in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all participants. The raw MRS data are available open-source at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://osf.io/un24g/\u003c/span\u003e\u003cspan address=\"https://osf.io/un24g/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRecruitment\u003c/h2\u003e \u003cp\u003eBetween January 2019 and July 2020, we screened 22 consecutive adults with acute traumatic BPI admitted to three Major Trauma Centres within England. Fifteen were excluded for the following reasons: 4 declined to participate or were uncontactable, 4 required immediate surgery which precluded preoperative MRI, 3 were unable to meet COVID-related isolation requirements, 2 were claustrophobic, 1 had learning difficulties and so could not provide informed consent, and 1 patient had a c-spine injury requiring extended immobilisation in a collar.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eImage Acquisition\u003c/h2\u003e \u003cp\u003eData were acquired at a field strength of 3 Tesla (T) using a MAGNETOM Prisma (Siemens Healthcare, Erlangen, Germany) with a 64-channel head coil. Firstly, we acquired an MPRAGE (T1-weighted) dataset with the following parameters: TI 900 ms, TE 2.98 ms, TR 2300 ms, flip angle 9\u003csup\u003eo\u003c/sup\u003e, 176 sagittal slices, field of view 256x248, 1mm isotropic resolution, GRAPPA factor 4, TrueForm B1 shim, no partial Fourier, AP phase encoding and bandwidth 240 Hz/Px. Thereafter, we acquired J-edited spectra using MEscher\u0026ndash;GArwood Point RESolved Spectroscopy (MEGA-PRESS)\u003csup\u003e25\u003c/sup\u003e from 2 cm isotropic voxels centred on the right and left \u0026ldquo;hand knob\u0026rdquo; areas (using the \u0026lsquo;delta sign\u0026rsquo; on axial slices), respectively, with the following parameters: TE 68 ms, TR 2000 ms, 64 averages, water suppression bandwidth 50 Hz, automated field-map-based B0 shim, TrueForm B1 shim, 4 preparation scans, edit pulse frequency 1.9 ppm, edit pulse bandwidth 50 Hz, centre frequency 4.7 ppm, receiver bandwidth 1200 Hz for an acquisition time of 853 ms. Spectra were interleaved every acquisition between \u0026lsquo;edit-on\u0026rsquo; and \u0026lsquo;edit-off\u0026rsquo;. Real time frequency adjustment was performed automatically on the scanner to prevent field drift affecting the difference spectra. The total scan time was 4 minutes 24 seconds per hemisphere. Data were stored as averaged fids for the \u0026lsquo;edit-on\u0026rsquo; and \u0026lsquo;edit-off\u0026rsquo; subspectra and exported from the scanner as .RDA files.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eSpectroscopic processing\u003c/h2\u003e \u003cp\u003eAfter acquisition, a custom-made processing script was used to generate phase-corrected spectra for the \u0026lsquo;edit-on\u0026rsquo; and \u0026lsquo;edit-off\u0026rsquo; conditions as well as the edited (difference spectrum) itself. Anonymised data were imported into jMRUI\u003csup\u003e39\u003c/sup\u003e v5.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.mrui.uab.es/mrui/\u003c/span\u003e\u003cspan address=\"http://www.mrui.uab.es/mrui/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for analysis by SW and CLC, who were blind to clinical information. Prior to analysis using AMARES\u003csup\u003e40\u003c/sup\u003e, residual water was removed from the spectra using the HLSVD routine. The edited spectra were fitted with the following prior knowledge: NAA: relative phase 180\u003csup\u003eo\u003c/sup\u003e, lorentzian line shape, frequency, linewidth and amplitude unconstrained; GABA: relative phase 0\u003csup\u003eo\u003c/sup\u003e, Gaussian line shape, frequency and amplitude unconstrained, linewidth constrained between 15 and 25Hz. Co-edited glutamate\u0026thinsp;+\u0026thinsp;glutamine: fitted as 2 peaks of equal amplitude separated by 10.25Hz, relative phase 0\u003csup\u003eo\u003c/sup\u003e, lorentzian line shape, each linewidth constrained to equal that of NAA, frequency unconstrained. The prior knowledge for the edited GABA signal takes account of the contribution from co-edited macromolecules\u003csup\u003e24\u003c/sup\u003e, hence a gaussian line shape was assumed with constraints on the linewidth to force the fit to be meaningful. GABA measured from non-macromolecule-suppressed spectra is often referred to as GABA\u003csup\u003e+\u003c/sup\u003e, but we do not use this extra abbreviation for simplicity.\u003c/p\u003e \u003cp\u003eThe \u0026lsquo;edit-off\u0026rsquo; spectra were also quantified by AMARES with the following prior knowledge for NAA, Cr at 3.03 and 3.96 ppm and choline-containing compounds (Cho): amplitudes and frequencies were all unconstrained, lorentzian line shape was stipulated and linewidths were all constrained to equal that of Cr at 3.03 ppm. Example spectra, together with the AMARE fit for an edited spectrum are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. To quantify GABA, Cr and NAA are assumed to be of quasi-constant concentration (both within and between subjects) such that GABA:Cr and GABA:NAA ratios may be computed as a proxy for GABA concentration. The edited signal for NAA was used to construct the GABA ratio, since any slight missetting or drift of the amplitude or phase of the pulse would affect both the edited GABA and NAA signals similarly.\u003c/p\u003e \u003cp\u003eThe quality of the spectra was assessed by visual inspection and by measuring the linewidth and Cramer-Rao lower bound (CRLB) of the AMARES\u003csup\u003e40\u003c/sup\u003e fit for GABA in the edited spectra. The signal-to-noise ratio of NAA was also recorded for edited and spectra.\u003c/p\u003e \u003cp\u003eAdditionally, grey matter and white matter proportions were assessed using SPECTRIM\u003csup\u003e41\u003c/sup\u003e. This provides a graphical user interface to enable segmentation of the T1-weighted anatomical DICOM images, with extraction of the grey matter, white matter and CSF content of the spectroscopic voxels. This is a necessary step to interpret any changes in GABA, as it is known that the GABA content of grey matter is approxiamtely double that of white matter\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFunctional outcomes assessment\u003c/h2\u003e \u003cp\u003eGrip strength was assessed using a digital grip dynamometer. Key (lateral) and tip (index pulp to thumb pulp) pinch were assessed using the Jamar Plus Digital Pinch Dynamometer For all assessment, 3 measurements were taken the best (strongest) was used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis\u003c/h2\u003e \u003cp\u003eData were analysed using Stata v16/MP (StataCorp LLC, Texas). Scaled variables approximating the normal distribution are represented by the mean (and standard deviation, SD). To estimate the interhemispheric differences in the concentrations of neurotransmitters, means were compared by linear regression. As the dataset is small, we used resampling with replacement (bootstrapping) with 1000 iterations to improve estimates of the variance. Age may affect GABA levels\u003csup\u003e43,44\u003c/sup\u003e but as there is no conceptual association between these variables and interhemispheric differences after nerve injury, adjustment was not required. Although the patients in this series were exposed to analgesics which modulate GABA and Glutamate (Gabapentin or Pregabalin), as we were directly comparing interhemispheric differences, the effect of these drugs will be neutralised, and so further adjustment is unnecessary. Confidence intervals (CI) were generated to the 95% level.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cu\u003eFunding\u003c/u\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRGW is funded by the National Institute for Health Research (NIHR, DRF-2018-11-ST2-028 and CL-2021-02-002). SP is funded by a British Heart Foundation Chair (CH/16/2/32089). CLC is funded by the Medical Research Council (MRC) Grant MR/PO14445/1. This research is also supported by the University of Leeds Advanced Imaging Centre, which is funded by the Medical Research Council (MR/M008991/1) with support from the British Heart Foundation (BHF-SP/14/7/31351) and Arthritis Research UK (ARUK-21078). The views expressed are those of the author(s) and not necessarily those of the United Kingdom\u0026rsquo;s National Health Service, NIHR or Department of Health.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKami A, Meyer G, Jezzard P, et al. Functional MRI evidence for adult motor cortex plasticity during motor skill learning. \u003cem\u003eNature\u003c/em\u003e. 1995;377(6545):155\u0026ndash;158.\u003c/li\u003e\n\u003cli\u003eLea-Carnall CA, Trujillo-Barreto NJ, Montemurro MA, et al. Evidence for frequency-dependent cortical plasticity in the human brain. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e 2017;114(33):8871\u0026ndash;8876.\u003c/li\u003e\n\u003cli\u003eVidyasagar R, Folger SE, Parkes LM. 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Med.\u003c/em\u003e 2013;69(2):317\u0026ndash;320.\u003c/li\u003e\n\u003c/ol\u003e "}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-3910773/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3910773/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the brain. In response to injury within the central nervous system, GABA promotes cortical plasticity and represents a potential pharmacological target to improve functional recovery. However, it is unclear how GABA changes in the brain after traumatic brachial plexus injuries (tBPI). Therefore, neurometabolite concentrations were serially quantified in 7 males with acute tBPI, up to 19 months post-injury, using magnetic resonance spectroscopy (MRS). We acquired J-difference editing MRS using a MAGNETOM Prisma 3T (Siemens Healthcare, Erlangen, Germany). Data were analysed in jMRUI blind to clinical information to quantify GABA, GABA, creatine (Cr) and N-acetylaspartate (NAA) concentrations. Interhemispheric means were compared using linear methods. Confidence intervals (CI) were generated to the 95% level. Within weeks of injury, the hemisphere representing the injured upper limb had a significantly lower GABA:NAA ratio (mean difference 0.23 [CI 0.06\u0026ndash;0.40]) and GABA:Cr ratio (mean difference 0.75 [CI 0.24\u0026ndash;1.25]) than the uninjured side, which equalised 12 months post-injury. After BPI, there are interhemispheric differences in GABA concentrations within the sensory and motor cortex. Pharmacological modulation of these metabolites may enhance cortical plasticity, prolong the available time for reconstructive surgery and ultimately, improve function.\u003c/p\u003e","manuscriptTitle":"Changes in Motor and Sensory Cortical Neurotransmitters Following Traumatic Brachial Plexus Injuries in Adults Measured with Magnetic Resonance Spectroscopy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-23 19:04:56","doi":"10.21203/rs.3.rs-3910773/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"14c9a43e-328d-4743-a8ff-b38f43a96808","owner":[],"postedDate":"February 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-13T07:18:16+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-23 19:04:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3910773","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3910773","identity":"rs-3910773","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}