Spasticity is a key motor impairment that affects many hemispheric stroke survivors. Intramuscular botulinum toxin (BT) injections are used widely to clinically manage spasticity-related symptoms in stroke survivors by chemically denervating muscle fibers from their associated motor neurons. In this study, we sought to understand how BT affects muscle activation, motor unit composition and voluntary force generating capacity over a time period of 3 months. Our purpose was to characterize the time course of functional changes in voluntary muscle activity in stroke survivors who are undergoing BT therapy as part of their physician-prescribed clinical plan.Our assessment of the effects of BT was based on the quantification of surface electromyogram (sEMG) recordings in the biceps brachii (BB), an upper arm muscle and of voluntary contraction force. We report here on voluntary force and sEMG responses during isometric elbow contractions across consecutive recording sessions, spread over 12 weeks in three segments, starting with a preliminary session performed just prior to the BT injection. At predetermined time points, we conducted additional clinical assessments and we also recorded from the contralateral limbs of our stroke cohort. Eight subjects were studied for approximately 86 experimental recording sessions on both stroke-affected and contralateral sides.We recorded an initial reduction in force and sEMG in all subjects, followed by a trajectory with a progressive return to baseline over a maximum of 12 weeks, although the minimum sEMG and minimum force were not always recorded at the same time point. Three participants were able to complete only one to two segments. Slope values of the sEMG-force relations were also found to vary across the different time segments. While sEMG-force slopes provide assessments of force generation capacity of the BT injected muscle, amplitude histograms from novel sEMG recordings during the voluntary tasks provide additional insights about differential actions of BT on the overall motor unit (MU) population over time.The results of our study indicate that there are potential short term as well as long term decrements in muscle control and activation properties after BT administration on the affected side of chronic stroke survivors. Muscle activation levels as recorded using sEMG, did not routinely return to baseline even at three months’ post injection. The concurrent clinical measures also did not follow the same time course, nor did they provide the same resolution as our experimental measures. It follows that even 12 weeks after intramuscular BT injections muscle recovery may not be complete, and may thereby contribute to pre-existing paresis. Spasticity is a characteristic feature of upper motor neuron lesions, often induced by a hemispheric stroke. The physical signs include hypertonia  as well as hyper-reflexia, an increase in response to muscle stretch or tendon tap. A potential contributor to hyper-reflexia in post-stroke spasticity is hyper-excitability of the associated spinal motor neurons . SpasticEvaluations of BT mediated alterations in spasticity levels have largely been based on clinical assessments  which arAccordingly, our global objective here is to use quantitative measures to accurately characterize the time course of changes in voluntary activation capacity of the biceps brachii on both sides of hemispheric stroke survivors who have received intramuscular BT on their affected side, as a part of their routine clinical care plan for spasticity. This assessment was accomplished in the usual time period between successive BT injections, 3–4 months, using force and sEMG recordings taken during varying voluntary isometric contractions. Based on the aforementioned studies, long term deficits in voluntary capacity could further weaken stroke survivors who routinely are diagnosed with paresis in addition to spasticity.Furthermore, in related animal studies, several researchers reported BT chemo-denervation induced fiber atrophy and also a reduction in total alpha motor neuron numbers, in rats at two weeks post-injection , 19. SubSpecifically, we assessed the initial (expected) decline and subsequent return of both joint torque and sEMG values, and we determined whether both variables varied concurrently. In order to assess possible central effects, i.e., changes in muscle control in addition to peripheral alterations, we computed the slope of the relationship between the generated surface EMG and force during voluntary muscle activation, which has been shown to be disrupted in paretic stroke survivors . We alsoTo summarize our results, we found that there were systematic alterations of force and sEMG induced by BT even at 12 weeks post-injection, and we recorded consistent maximal reductions of the force and the sEMG between 2 and 4 weeks, consistent with results from animal studies. A part of this work was presented at Society for Neuroscience annual meeting, 2017.We have recruited and tested 8 hemiplegic stroke survivors with spasticity who were to receive botulinum toxin as part of their clinical care plan. The initial timing, dosage, and location of the injection were determined by their managing physician. All participants had sustained a single hemispheric stroke at least six months prior to experimental testing. Spasticity was assessed by a physical therapist using the Modified Ashworth Score (MAS) Subjects had to exhibit a score ≥ 1 to be included in the study. The subjects were included only if they were able to perform the required experimental tasks, attain the testing position of the hand, sit continuously during the experiments, and follow the instructions with intact auditory and visual perceptions. All participants gave informed consent via protocols approved by the Institutional Review Board under the Office for the Protection of Human Subjects at Northwestern University.sensor array electrodes were mounted and aligned with the bipolar electrodes at a proximal location on the belly of the biceps muscle on both heads. The locations of electrodes were determined after muscle palpation on the belly of the muscle of interest, based on the recommendation of SENIAM [Participants were seated in a Biodex, Inc. chair with the forearm cast from elbow to the wrist and fixated to a ring-mount interface. The wrist mount was attached to a six-degrees-of-freedom force-torque sensor . After skin preparation, including a light skin abrasion and cleaning with alcohol pads, bipolar sEMG electrodes were placed on the medial and lateral heads of biceps brachii, triceps brachii and brachioradialis muscle (for online monitoring purposes). A pair of Delsys Inc., f SENIAM . The locSpike 2® and the CED-1401®, and dEMG (Delsys Inc.) system decreasing force levels back to resting level. The force and the sEMG signal was sampled at a rate of 2 kHz.A depiction of the experimental setup is shown in Fig. Clinical assessments of spasticity, the Modified Ashworth Score (MAS), and motor impairment, the Fugl-Meyer assessment (FM) were performed by a research occupational therapist at each recording session on the upper extremity on both sides of our participants. After completing the experimental recording sessions on the affected side, patients were asked to return for testing on their contralateral limbs using the same protocol.Our protocol was designed to test the participants in one session before their respective BT injections Pre-S1) and then 6 times after the injection (Post-S1- Post-S6), approximately every two weeks on both the affected and contralateral limbs. We have separated the observations in three segments across the time, initial segment (Post-S1-Post-S2), mid segment (Post-S3-Post-S4), and the final segment (Post-S5-PostS6) as shown in Fig.  and then0 elbow flexion, 450 shoulder flexion, 160 shoulder abduction, and the forearm pronated to 450. These angles were determined by a research therapist, to ensure maximum biceps activation, with the forearm in a mid-supine position. For consistency, we maintained the same joint angles across all recording sessions.Subjects were seated in an upright posture on with arm set at 1200 angle between the two measured force components (Force X and Force Z). A strap was used to maintain trunk and the shoulder position while subjects were performing force-producing tasks.All subjects were instructed to produce isometric elbow flexion force in this position using visual force feedback provided by a display screen, throughout the various sessions. Two-dimensional force feedback was provided to ensure that the subjects generated pure elbow flexion force, which in the seated position results in a 45Matlab®) was used.All subjects were asked to perform a Maximum Voluntary Contraction (MVC) at the beginning of each session. Two MVC trials were performed with 5 min of rest in between. Verbal cues were provided to the participants during the MVC trial. The higher contraction force level of the two trials was selected as the MVC value for that recording session. Subsequent force levels were set at several submaximal levels i.e. 30, 40 and 50%/ 60% of the recorded MVC. Multiple trials at each force level were performed in random order by all of our participants. A gap of two minutes between each trial was used to minimize muscle fatigue. Subjects were queried continuously regarding systemic as well as muscle fatigue. Preprocessing of force and sEMG data was performed before further analysis of RMS-sEMG (RsE)-force relations for all the force-torque levels. A commercial computational package  was calculated from the recorded force data from two principal axes  as:To increase the accuracy of the performance, a visual 2-D display of a trapezoidal force trajectory guide was provided to the subjects along with auditory feedback at the beginning and end of each force trajectory. The instructed force trajectory was programmed to control the rate of force rise. The trapezoidal force trajectory is shown in Fig. x is the component of force in shoulder abduction direction and Fz is the component in the elbow flexion direction. The average resultant force Fr during steady-state force production with minimum variation (5 s) was designated as the mean force value calculation. Identical time windows of steady-state force were selected for further analysis of both force and sEMG. In the case of MVC trials, the peak value of the force was designated for further analysis and other submaximal contraction level mean force values were calculated.FAll channels of single differential sEMG signals were initially amplified with a constant gain of 1000. Each channel data has then been offset nullified and filtered with a Butterworth FIR filter with a passband of 5 Hz to 500 Hz. Five seconds of stationary sEMG and force data were selected during the stable region of the steady-state force trace (less than 10% variation). The average signal power of the sEMG can be calculated by root mean square value of the sEMG (RsE) representing a value proportional to muscle activation capacity. The RsE was calculated within the steady-state force window as:n is the absolute voltage amplitude of the nth sample. These values were used for further processing as described in forthcoming sections. The percentage reduction of contraction force or RsE (Di) at the ith session of the measurements was calculated as:pre is the measurement before the injection and Mi is the measurement at ith session after the BT injections. The correlation of the force and the RsE were analyzed with a linear regression model and all the data points were sampled at the same frequency. Figure Where N is the number of samples within the steady-state force window and vETH was defined as:Surface EMG signal peaks were detected from the raw signals recorded from both heads of the biceps brachii muscle, using novel sEMG sensor array electrodes . The peaks were derived from a 5-s window in the sEMG signal during the steady-state period for the force trajectory. This electrode consists of five pins and generates four channels of differential sEMG signals. The channel with the largest peak to peak value of the raw sEMG signal was selected for the peak analysis, this was not found consistently in the same channel for every subject. The Delsys sensor array allows a higher bandwidth with reduced chances of superposition of MU spikes, making it a stronger candidate for sEMG peak detection application. The high recording sampling rates of the sensor array further support precise detection of peaks representing the MU firing instances. Figure p < 0.05.Where μ and δ are the mean and the standard deviation of the baseline sEMG signals before the start of the voluntary activation . The totWe report here on eight subjects who were studied before (Pre-S1) and after (Post-S1) the BT injection to assess the short term effect. Six of the eight tested subjects were able to continue testing to the mid segment (Post-S3-Post-S4), and 5 of the tested subjects continued to the final segment (until Post-S6) of the study. The subjects who could not complete the full span of the study  were incidentally random among our cohort and were not systematically different from the participants who had continued with the total course of the study, these three participants could not complete the desired course of the study because of personal reasons .We collected experimental and clinical data biweekly from stroke survivors, across a maximum of 14 weeks per subject, for a total of approximately over 85 experimental recording sessions in both stroke-affected and contralateral arm muscles. Our subject cohort had not undergone physical/occupational therapy sessions, during the course of testing sessions of this study; to our knowledge, this was also not a part of their prescribed care plan. Our subject cohort did not report any pain during the sessions. The results of our experimental data collection and analysis, as well as the clinical assessments, are summarized below.The subject details, injection dosages and clinical measures of the tested subjects are summarized in Table p < 0.001) than the impaired elbow flexion force. Along with the force, the RsE values of the biceps brachii muscles in the contralateral side were also routinely higher (p < 0.05) than on the affected side.For all our subjects, examined across pre-injection and post-injection sessions, recorded contralateral elbow joint flexion force was considerably greater , compared to the Pre-S1 session. Correspondingly, the RsE for biceps medial and biceps lateral declined for all 8 subjects starting at the first post session (Post-S1), except for the biceps lateral recordings in B3.However, the maximal force reduction occurred at different post-BT session. For subjects B1 and B4, the maximal force reduction of 65 and 73% (referenced to MVC force in the pre-injection) was recorded at 4 weeks and 2 weeks’ post sessions (respectively). Whereas maximal force reductions of 54, 38, and 32% were obtained for B2, B3, and B5 respectively at Post-S6, Post-S5 and Post-S3 sessions. B6, B7, and B8 also showed above 25% reduction of contraction force by Post-S1. The initial sharp reduction of MVC force (from pre-BT) values was followed by a general upward trend of MVC force values in majority of participants . In contrast, a return to baseline, i.e., pre-BT values, was not recorded even at 12 weeks’ post-injection. Even at the final segment of the study (Post-S6), approximately 3 months after the injection, the MVC force was found to reach just above 60% of the force produced before the BT injection for the respective subjects. The MVC RsE of the biceps brachii muscle also showed similar trends as the force values, across sessions, however, the maximal reductions of the RsE and force did not occur at the same post-session. Reductions in the force and the RsE were calculated as a percentage difference with respect to their corresponding Pre-S1 values (baseline) using the eq. .We performed similar analyses on data from the p value> 0.8) with respect to the pre-BT values within a subject, however, there was variation in (absolute) values across subjects. The dotted values in Fig. Force and sEMG  data analyzed on the contralateral side also did not show significant systematic variation  MVC at the same session. The submaximal values were determined on the basis of the MVC recorded for that particular session. We used these data to assess the relationship between RsE and force across testing sessions, as well to assess if there were similar trends in the time course of force and sEMG at submaximal levels, as were observed at maximal levels. In Fig. p < 0.05) compared to the pre-BT session at the second session (Post-S2). For subject B1-B5 the value remained low until the final segment (Post-S5).The short term sharp drop of RsE was observed within the initial segment of our study. When compared individually, seven subjects (except B3) show a reduction of the RsE of the injected biceps muscle by Post-S2. A pairwise test of the consecutive recording session reveals that the values of the RsE in both medial and lateral head recording for all the subjects were significantly reduced (p > 0.1). Contrary to the injected biceps muscle, no significant variation in sEMG activity was observed in triceps muscle, to within 10% across all the sessions for the subjects.Unlike the injected side, the RsE values in the contralateral side did not show any similar pattern with any statistical significance  followed by an incomplete return to baseline  in both sEMG and force by the final segment. We observed an average 50% (for B1-B8) force reduction in the initial segment subsequent to the injection and during the final segment at the end of 12 weeks, it remained 35% lower (for B1-B5) compared to their respective baseline force values.The sEMG during the maximal and the submaximal contraction showed a greater reduction compared to baseline (average maximum reduction > 70%) as compared to the respective force values. The reductions of the sEMG also persisted till the final segment after the injection and the value remained lower than baseline values at a statistically significant level  at the end of 12 weeks. An average reduction of 22% of RsE was observed for three subjects  at their last recording session.p values (<<.05).We quantitatively analyzed the correlation between recorded activity in the medial and lateral heads of the affected biceps muscle of each participant. The RsE values of the biceps medial and lateral were found to be substantially correlated (r > 0.85) in the impaired side for four subjects  with the acceptably low We quantified the relationship between the RsE values, for BM and BL separately, and recorded joint force values at submaximal contraction levels during the steady-state portion of the force trajectory (over 5 s duration) at each session for each participant. The objective was to understand if there were significant alterations in muscle control with acute chemical muscle denervation. To quantify this relationship, we regressed the computed RsE with the generated (absolute) force values with a first-order linear relationship. The correlation of the force and the RsE is summarized in Table The computed slope values of the regression analysis across various recording sessions were further analyzed to assess any consistent trends as a function of time after BT injection. A representation of the slope of RsE-force fit during various submaximal contraction levels across all recording sessions for a subject is shown in Fig. Summary plots of the Biceps RsE-force slopes for each subject across all experimental sessions are shown in Fig. contralateral slope values were steady within 8% of the baseline across the sessions, as depicted by the blue dashed line in Fig. The RsE-force slope of the contralateral was found to be significantly higher than the slope on the affected side (p < < 0.05) for all subjects across all sessions, the average The slope value for the triceps RsE-force relation was found to be similar for both the affected and the contralateral side and the slope remained consistently flat (within 2%) across all participants, and unchanged across all sessions.Contralateral biceps: The respective slope values on the contralateral side remained relatively unchanged (within 5%) across all experimental sessions.The slope values of the normalized RsE  and the force were also analyzed. This analysis was performed to better understand the percentage of the available MU pool that was utilized to generate the respective force. During the final segment (at the Post-S6 session), all the remaining subjects (except B5) showed higher slope values at the Post-S6 session compared to the respective pre-injection values in their biceps medial  and lateral . B6, B7, and B8 slope were found to be lower (< 70%) than the baseline at their last recording. Despite variability in the time course, there was an increasing trend of the values with time in the stroke-affected side. In summary, the slope values exhibited several general trends. After attaining global minima at 2–4 weeks following the BT injection, the slope values increased in successive sessions until Post-S6, where the values were higher on average, by 165% in the biceps medial and 108% in the biceps lateral as compared to baseline i.e., pre-BT slope values in four of our five subjects that were tested at the final segment.An sEMG peak analysis from seven of our tested participants was performed (subject B4 was excluded as the sensor array recording was found corrupted by noise during recording). To provide insight as to the variations in RsE-force slope across time and as well to understand potential alterations in motor unit size distribution as a function of time post-BT, this analysis involved replotting the data as an amplitude histogram. All the peaks of the sEMG were identified and fit with a kernel distribution to preserve the versatility of the distribution shape as attributed by individual peak values. Figure p < 0.001) towards the larger values in weeks 2–4 post  despite the fact that during these weeks, the maximum voluntary force and maximum sEMG values were at their lowest recorded values.The median peak values (MPV) during the 50%MVC contraction level were compared across all recorded sessions for all 7 out of the 8 participants. In six out of the seven subjects, , the (median) peak distribution shifted (l side (p ≈ 0.7).A similar shift of the peak amplitude distribution was not observed and the distribution (range and shape) remained unchanged throughout all the recording sessions on the contralateraIn Fig. In summary, larger sEMG peak values were evident in the injected muscle for all the subjects within two/four weeks after the BT injection. The peak amplitude histogram median values subsequently return to baseline values within two sessions in all subjects and remain within 20% of the pre-injection baseline values. The variability of the median values on the contralateral side of the tested cohort remained under 10% across all tested sessions.Modified Ashworth scores (MAS) and Fugl-Meyer scores (FMS) for the upper extremity were recorded at similar time points  to assess both the level of spasticity and the degree of impairment of the affected limbs. The values for MAS and the FMS are plotted in Fig. The primary objective of this study was to characterize the time course of the effects of botulinum toxin on voluntary muscle activation capacity in stroke survivors with spasticity. We have studied the changes over three segments across the 12 weeks to precisely quantify the effect of BT on the voluntary capacity and the muscle control. Our analysis was based jointly on the myoelectric activity of the BB muscle and the associated joint force during voluntary isometric contractions after a single intramuscular BT injection, as prescribed by the participant’s clinician as part of the clinical plan of care. Clinical scores along with the elbow contraction force and the sEMG of the BB muscle have also been analyzed to understand the variation in the functional deficit of the voluntary force production capacity. Our objective was to understand if voluntary capacity and muscle function exhibited an initial (expected) reduction, followed by a sustained reduction during the final segment, and whether force and sEMG returned to baseline levels within the tracking period. We expected that the return to pre-BT levels would occur within 3 months and that it would be back at least within 10–15% of the pre-injection values.Based on the experimental results we have found that the decrement and subsequent increases in our measured parameters, after the injection across the heterogeneous stroke cohort, are indeed different in some aspects, however, there are also similarities. As expected, the clinical assessments were lacking the necessary resolution to capture the same characteristics across time.One of novel results of our study is that the time course of changes in the clinical scores for spasticity, i.e., the MA score, and motor impairment, i.e., the Fugl-Meyer score differs from the time course of changes in their respective force and the RsE values. Though MAS is a well-accepted measure for spasticity estimation, a measure used to determine the necessity for repeated BT injections in stroke survivors, the rating suffers major reliability issues, as addressed earlier , 27. DurThere was a lack of significant correlation between the max RsE value and the MAS values for all tested subjects. A decrement of the MAS values after BT injection has been reported earlier by other research groups and attributed to the unavailability of the fibers with active NMJ innervation , 31. DesThe FMS also exhibited large variability across time. We expect that BT alone cannot greatly improve functional recovery but recovery may improve with the associated occupational therapy , howeverIn all tested participants, recorded sEMG and voluntary force signals exhibited a systematic decrement in magnitude, as expected within the initial segment of our study in the first few weeks post-BT, with respect to baseline levels . Subjects B1-B6 exhibited increasing trend back towards baseline levels in the subsequent trials. However, there was a disproportionate change in contraction force levels and RsE magnitude after the injection. As well, RsE values did not return back to pre-injection baseline values at the end of 12 weeks for the tested subjects. Submaximal force testing was performed to understand whether similar effects were observed over the successive testing sessions and to be able to compute the relationship between sEMG and force. Reduction of contractile force and of the RsE after the intramuscular injection is assumed to be primarily attributable to the reduced active muscle fiber population, which can be explained by the BT induced presynaptic blockade of the cholinergic transmission . Faster Another possible reason is the potential retrograde transportation of BT to other fibers of the muscle and to other muscles through adjacent motor neurons , that maThe heterogeneity within and between patients was evident in both the affected and the contralateral side as recorded during the experiment. These could potentially be associated with multiple factors i.e. altered contracture in the muscle, architectural changes in the muscle (and varying levels of the injected toxin). Some of these effects have peripheral origin but some of the factors are mediated from upper motor neurons that may have produce ipsilateral deficiencies also .Besides analyzing the deficit in voluntary force production, another focus of this study was to investigate changes in motoneuronal control of muscle force production subsequent to the BT injection. Previous studies have reported that in chronic stroke survivors, the slope of the relation between the RsE and force was higher on the stroke affected side compared to the contralateral side potentially due to reductions in motor unit firing rates and thereby increase in the recruited motor units at a given force level in both upper limb and hand muscle . SimilarOur data shows an increased slope value during the final segment of the study (Post-S6) while the force and the sEMG were lower than the pre-injection baseline for subjects B1-B5. This suggests an increased drive to generate a similar amount of force potentially due to decremental changes in muscle structure and function.It is possible that the slightly higher slopes are indicative of inefficient force production due to an increase in connective tissue deposits, alterations in muscle fiber size or alterations in control due to reinnervation, as reported in animal studies –21. As mThe slope variations reported in our tested subjects cannot be attributed to varying levels of triceps co-contraction, as the triceps activation levels remained relatively constant across the testing sessions. Antagonist recordings during isometric force generation in stroke subjects in other studies  have alsHere, we use the amplitude distribution of the sEMG peak values as a surrogate for the amplitude distribution of the MU pool in the muscle, due to the use of sensor array electrodes, which are very selective electrodes designed to record MU action potential shapes. Our reported shift of the peak values after the BT injection is potentially a reflection of the selective action of BT on the overall MU pool. The median amplitude of the sEMG peak values was increased (in six of seven subjects) despite the reduction in the values of both the RsE and force. The highest median value of the sEMG peak amplitude was found within the initial segment at the second post-injection recording session.The shift in the amplitude of the peaks also can provide an idea about the functional properties of the detectable active motor unit population. The appearance of larger peaks may be attributed by the early terminal and nodal sprouting of motor axons in nonfunctional end plates that begin to form within days after the injection . There iWith time, the sprouting is inhibited but this process is comparatively slower , appearaThere is ample evidence that BT induces progressive loss of contractile fibers  and induThis study provides a quantitative characterization of functional deficits and motor outflow changes after intramuscular BT injections in stroke survivors, which allows us to more clearly understand the effects of BT on voluntary motor capacity. The results suggest the potential need to weigh the benefits of BT therapy for reduction of spasticity with the potential risk of an increase in muscle weakness. The results of this study also show that quantitative measures of force and sEMG do not track with clinical assessments, suggesting that greater measurement precision could provide improved guidance regarding BT injections for spasticity management in stroke survivors.The broad applicability of our results are limited due to the modest number of subjects that we report on. As a function of the variable nature of the chemical denervation and subsequent reinnervation process, the time course of changes in voluntary capacity differed amongst the participants. Part of this variability could also be due to factors that were not under our control, including the dose of the injected BT and the prior history of BT injections in each participant. Thus, it is noted that to utilize these results to guide practice in a clinical setting will require a bigger participant cohort.The variable nature of the denervation/reinnervation process combined with the numerous testing time points employed in this study could contribute to some of the differences reported across participants, i.e., the more frequent testing time points revealed greater variability across the participants. Indeed, there are striking similarities in the sEMG and force parameters observed amongst the majority of the subjects at the very early weeks post-BT injection, when most of the denervation process would have occurred and at the later weeks, when a majority of the reinnervation process may have completed. These similarities include the dramatic reduction of RsE and force in the first 2–4 weeks, with only partial recovery of RsE values at 10–12 weeks, higher force-sEMG slopes after 12 weeks and the appearance of large sEMG peaks in the peak amplitude distribution within 2–4 weeks. It is possible that in the intervening weeks, there was a fluid set of denervated /reinnervated neuromuscular junctions, based on the data reported in this study. These variations of the sEMG values were statistically tested to be significant (we have achieved an effect size of 1.4 at a confidence level of 0.05 with our test cohort), thus our preference was not to report our results as a series of case reports.


In electroantennographic assays, the antennal response of males to thyme oil, thymol, and carvacrol was altered at high doses (103–104 µg), shifting the signal waveform into a biphasic negative–positive potential that caused a decay in the response. In wind tunnel assays, pheromone-mediated attraction of males of both species was interrupted in presence of thyme oil. Further trials demonstrated that thymol alone reduced the number of G. molesta and S. littoralis males landing on the pheromone source. This effect did not differ from that of thyme oil, although the latter provoked a significant reduction on downwind behavior steps in S. littoralis. Overall, our findings provide a preliminary basis for delving into the effect of thyme oil, and especially of its major constituent thymol, as potential mating disruptants of both species.Plant essential oils are considered as important bio-sources for the development of natural and environmentally safe pest control tools due to their multiple modes of action on insects. In this paper we have evaluated the activity of commercially available thyme oil and its constituents thymol, carvacrol, and  Essential oils are complex mixtures of secondary volatile compounds from plant metabolism, mainly composed of terpenes and terpenoids, and, to a lesser extent, by aliphatic and aromatic constituents of low molecular weight 2. Their structural diversity has promoted a vast research on the bioactivity of EOs on insects, as they exert feeding4 and oviposition deterrence6, repellency7, and toxicity in eggs8, immature stages11, and adults12.One of the main challenges of IPM programs is to continuously seek and develop novel eco-friendly and biorational strategies to minimize the application of synthetic pesticides for their side effects. In this context, the use of plant essential oils (EOs) for the development of sustainable and environmentally safe control strategies has gained great attention during the last two decades13. Among Lamiaceae, common thyme Thymus vulgaris has been proved effective against pest insects. Thyme is an aromatic evergreen shrub native to the Mediterranean region, which has been used in traditional medicine since ancient times14. Some of the thyme components are the isomers thymol (2-isopropyl-5-methylphenol) and carvacrol (5-isopropyl-2-methylphenol), both deriving from the precursor p-cymene15, another constituent of T. vulgaris. In terms of repellency, several authors have reported the effect of thyme on different insect orders, including Diptera18, Coleoptera19, Hemiptera21, and Thysanoptera22. In this regard, the two phenolic monoterpenes thymol and carvacrol have been suggested to be repellent for different insect species25. In mosquitoes (Diptera: Culicidae), for instance, the repellency evoked by thyme on Culex pipiens pallens Coquillet is attributable to both phenolic monoterpenes and α-terpinene18, while the repellent effect of three Lamiaceae species against Aedes albopictus Skuse is associated to carvacrol25.Within the broad spectrum of aromatic plant EOs studied, most of them belong to the families Myrtaceae, Lauraceae, Lamiaceae, and Asteraceae29, and naturally-occurring compounds, such as volatiles and EO components from non-host plants35. Based on this, we wonder whether thyme would interfere in the pheromone-mediated attraction of the major pest moths Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) and Grapholita molesta (Busck) (Lepidoptera: Tortricidae). The Egyptian cotton leafworm, S. littoralis, is a serious pest of cotton and many other crops36. In turn, the oriental fruit moth G. molesta is a primary pest of stone and pome fruits worldwide37. Traditionally, pheromone-based communication has been one major target for the management of both species, including attract-and-kill, mass trapping and mating disruption39. In this context, mating disruption has become a widespread control approach during the last decades. Therefore, the development of new natural mating disruptants could open promising avenues for environmentally-friendly management of both species. In this paper, we have tested commercially available thyme oil, and three of its major components, i.e., thymol and carvacrol, for their proven repellent properties, and their precursor p-cymene. The effect induced by these chemicals on the male chemoreception system of both species was assessed by electroantennography (EAG), and the possible antagonism to the pheromone response of thyme oil, single compounds and binary/ternary mixtures was evaluated in a wind tunnel.Despite the considerable literature on the repellent effect of thyme oil or constituents, to the best of our knowledge no research has been focused on a possible effect of this EO on insect pheromone communication. It is well known that the male attraction to sex pheromones can be disrupted by synthetic pheromone analogsp-cymene (30%), followed by three minor compounds: γ-terpinene (5%), linalool (4%), and carvacrol (3%)  and G. molesta, χ2 = 17.726, df = 3, p = 0.001; S. littoralis, χ2 = 15.175, df = 2, p = 0.001). Males exhibited a dose-dependent response at low doses (1–102 µg), and reached the maximum EAG amplitude at 102 µg , with less than 60% of the antennae of both species responding at 1 µg. However, a clear drop in the antennal response was observed at 103 and 104 µg. Indeed, a remarkable change in the shape of the response was observed at the highest dose in both species, with a small depolarization followed by a more pronounced positive polarization. Representative shape changes of S. littoralis males antennae to different treatments are shown in Fig. in S. littoralis. In the case of G. molesta, no significant differences were detected among doses higher than 10 µg, and, even though the EAG response decayed at the highest dosages (103–104 µg), the percentage of antennae responding to 104 µg was higher (81%) than in S. littoralis.The EAG amplitudes in response to thyme oil and three of the constituents are displayed in Figs. p-cymene, a dose-dependent effect was detected in both species   responded at the lowest doses (1–102 µg). This trend shifted at the highest dose (104 µg) with only 40% of the antennae responding (0.81 ± 0.23 mV), in contrast to the 100% of G. molesta (1.62 ± 0.20 mV).With regard to p-cymene. Males of both species only responded to low doses (≤ 102 µg), and the number of antennae sensitive to both stimuli was also relatively low, especially for thymol (less than 60%) almost all doses. Regarding carvacrol, no significant differences were detected between the active doses (1–102 µg) in either species . Here again, depolarization recordings of both phenolic compounds changed dramatically at 103 and 104 µg in both species . In G. molesta, a similar change of the electroantennographic potential was noticed at the two highest doses of carvacrol and thymol, although the antennal recovery after the positive polarization did not return to the baseline, as similarly occurred with the thyme oil stimulus  in presence of thyme oil at 1:1 and 1:10 ratios was not affected for any of the behaviors except in landing, which significantly decreased from 93 to 69% at 1:10 ratio  .Flight response of p-cymene, 15 µg). Thymol significantly reduced to 54% the percentage of landings on the pheromone source , albeit this effect did not statistically differ from that elicited by the combination of thyme oil (50 µg) and pheromone (5 µg)   with single compounds in quantities present in 50 µg of thyme oil , although this effect did not differ from that of thymol . No effect was observed when binary blends containing p-cymene and carvacrol or thymol were tested  :thyme oil ratios inhibited the number of landing males, displaying significantly lower percentages than that elicited by 10 µg of the major component of the pheromone (84%) . Thus, while the 1:1 ratio reduced the landing percentage to 60% , the 1:10 resulted in a four-fold decrease (23%) . Indeed, the 1:10 proportion significantly disrupted the remaining behavioral steps in response to the pheromone  . Among the single components, thymol was the unique chemical displaying a disruptive effect, reducing the approach and landing scores to 67%  and 43% , respectively. Other downwind behavioral steps, i.e. take-off and halfway, were unaffected . Approach to the source, in contrast, differed significantly between the whole EO mixture (37%) and thymol . The percentage of landing males was also decreased to 50% by the binary mixture of thymol and carvacrol , and the ternary blend (60%) , although neither treatment exerted the disruptive effect of thyme oil (50 µg) when mixed with the pheromone component. The presence of p-cymene in the tests either when singly co-released with the pheromone or in binary mixtures with thymol or carvacrol and the pheromone did not evoke any appreciable disruptant effect on any behavioral step. Indeed, it interrupted the activity of thymol alone, with 80% of males contacting the pheromone source when p-cymene was mixed with the former   Fig. a. Among 01) Fig.  online. see Fig.  online. see Fig.  online.FS. littoralis that the attractant effect of the pheromone in a wind tunnel is interrupted by volatiles of two non-host plants, namely Picea abies and Adhatoda vasica40. Likewise, other authors have reported that significantly fewer G. molesta males orient upwind towards a virgin calling female when the monoterpenoid citral is simultaneously released31. Similar activity of citral has also been reported for Lobesia botrana (Denis & Schiffermüller) (Lepidoptera: Tortricidae)32. In Plutella xylostella (L.) (Lepidoptera: Plutellidae), the terpenoids eucalyptol and α-terpineol suppress the attraction to the sex pheromone in wind tunnel and field trapping experiments30.In most moth species, sexual communication is primarily ruled by female-released species-specific pheromones that provoke a stereotyped behavior on males. Even though insect olfactory systems are highly tuned for sensing mating chemical cues in fluctuating and complex odor landscapes, attraction to sex pheromone may be interrupted in presence of non-host volatiles. For example, it has been noticed in G. molesta and S. littoralis males. Thymol is abundantly found in EOs of Lamiaceae41, Verbenaceae24, and Apiaceae23, among other plant families. Toxic and repellent activity of thymol on arthropod pests of agricultural and medical-veterinary importance has been well documented42. For example, thymol is strongly repellent to the bean bug Riptortus clavatus (Thunberg) (Hemiptera: Alydidae)23, and Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae)24. In S. littoralis, thymol is an acute toxic for larvae43, and also affects the behavior of adult females. In fact, a six-component blend containing thymol, carvacrol, benzaldehyde and three additional terpenes  significantly deterred oviposition44. So far, this is the first time in which thymol is reported as an inhibitor of sex pheromone attraction of moths.Here we provide evidence of the activity of thyme oil and its major component thymol as disruptors of the sex pheromone attractiveness on S. littoralis and G. molesta. The number of the noctuid males approaching to the source was also diminished, while no effect was observed for this parameter in G. molesta. It should be pointed out that in both insects thymol was only evaluated at the amount contained in the corresponding pheromone:thyme oil ratio (1:10), and, therefore, additional research would be necessary to elucidate whether the effect of the chemical is dose-dependent. It is also remarkable that in S. littoralis thyme oil has a stronger effect than thymol in all behavioral steps considered. In addition, since binary and ternary blends of thymol with p-cymene and carvacrol did not improve the inhibitory response of thymol alone, the effect of thyme oil could be the result of a possible synergism of thymol with another minor constituent of the EO not considered in our bioassays. In this context, linalool, a monoterpene alcohol present in thyme oil -4,8-dimethyl-1,3,7-nonatriene (DMNT), another herbivore-induced volatile, has been reported to exert disruption of S. littoralis behavior in response to pheromone and other attractant odors47. Physiological studies have revealed that these volatiles interfere with pheromone perception at peripheral level47. These results denote that volatiles from injured cotton plants act as ecologically significant signals interfering the sensory perception and, subsequently, modulating the behavior of both sexes. In addition and as mentioned before, volatiles from unsuitable or non-host plants may act as disruptive cues to avoid maladaptive choices and favor an efficient localization of conspecifics and hosts. In this sense, perception of the sex pheromone and host/non-host volatile plumes is a concomitant phenomenon that simultaneously converges into the olfactory system of the insect, with the volatiles affecting the sensitivity of pheromone olfactory receptor neurons (PHE-ORNs)49. Although plant volatile odorants are detected by other ORNs, it has been reported that some PHE-ORNs are activated by plant signals, possibly in a competing way50, and a concrete compound may differently act from one species to other. For instance, heptanal is known to activate PHE-ORNs in Agrotis epsilon (Hufnagel) (Lepidoptera: Noctuidae) males, while no activation is reported for S. littoralis50. Apart from direct activation, coding activity of the sex pheromone may be affected by the suppressive action of some volatiles. In the case of DMNT, the response of PHE-ORNs is reduced due to the suppression of the calcium activity47. As a consequence of these multimodal interactions of plant odorants in the pheromone detection process, antagonistic effects on the behavior are occasionally reported4751. Considering our results, it is possible that thymol, a non-host volatile, may play a disruptive role on the pheromone detection at the peripheral level, leading to relevant changes in the sex pheromone-guided behavior. Nevertheless, the ecological significance of these interactions at PHE-ORNs level is still far from being fully understood.Herein, we demonstrate that in wind tunnel thymol inhibited landing on the pheromone source in p-cymene and carvacrol, did not exert any disruptant effect in either species at the doses present in the EO. Crude EO extracts have been proved to be more effective than individual components alone, owing to additive and/or synergistic interactions among constituents52. In our case, carvacrol did not induce any synergistic effect to the mixtures containing thymol. In contrast, p-cymene exerted an antagonistic effect when mixed with thymol, overriding the disruptive effect of the latter in both species. As previously stated, carvacrol by its own exerts repellency on insects25. Nonetheless, it must be taken into consideration that relatively low dosages (1.5–3 µg) were tested, and thus additional trials would be necessary to state consistent conclusions. Given the structural similarity between thymol and carvacrol, and considering that synergism depends not only on the suitable combination but also on the ratio of the components, a putative synergism between both phenolic compounds cannot be excluded.The other constituents of thyme oil considered, i.e., p-cymene than to any other stimulus tested. While p-cymene induced electroantennographic activity in a dose-dependent manner, thyme oil, and particularly carvacrol and thymol, exhibited a notorious decrease in the antennal response at the highest doses. Indeed, the phenolic compounds (thymol and carvacrol) induced in both species moderate-to-low response at doses below 103 µg, whereas no measureable EAG response was recorded at the highest stimuli. In a previous work, thymol evoked no EAG response in S. littoralis females at any of the doses tested (1–104 µg), whereas the response to carvacrol decreased at the highest doses (102–104 µg)44. In our case, the drastic drop in the EAG response could be attributed to the change in the EAG waveform at the highest doses, characterized by a small negative potential followed by a large positive response. This effect was more acute when single compounds were puffed, since the effective dose of these stimuli represents a higher amount of compound than that contained in its equivalent thyme oil dose. Electroantennographic responses in insects are typically considered as single negative depolarizations, albeit the chemical structure of the odorants can alter the EAG shape53. This negative–positive response was firstly detected during the early development of the EAG technique54, while recording the effect of the exposure to chloroform on Bombyx mori (Lepidoptera: Bombycidae). Similarly, Light et al.55 recorded the same biphasic EAG shape on Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) in response to short-chain acids. Some authors have suggested that the EAG response pattern can be correlated with behavioral activity of a particular compound. First evidence was reported for Periplaneta americana (L.) (Blattodea: Blattidae), in which those compounds generating positive polarizations were demonstrated to act as repellents56. Similar results were obtained by Ramachandran et al.57 in Cnaphalocroeis medinalis (Guenee) and Marasmia patnalis Bradley . A stimulus of 102 µg of either thymol or carvacrol evoked a negative–positive response in males and females of both species, and both compounds resulted to be an antifeedant for immature stages. Recently, Lee et al.23 obtained reversed EAG profiles from R. clavatus in response to 103 µg of thymol and carvacrol, and both compounds were strongly repellent to females. These findings are partially in accordance with ours. Here, we suggest that the particular EAG shape at high doses may be correlated with an interference of the sexual communication in both species by reducing the attraction of males to the pheromone, specially in the case of thymol, which was proven to interrupt the behavior of males towards sex pheromone. Thus, we consider that thymol should be referred here as a disruptor of the pheromone attraction rather than a repellent. Nevertheless, as cited above, whether or not pheromone perception is affected by thymol at the peripheral level is unknown. In a similar vein, why a background of thyme oil constituents alters male innate orientation within the pheromone plume remains unanswered. In-depth electrophysiological recordings at the sensillum level combining pheromone, thymol, or other constituents, would help answer these questions. As outlined earlier, S. littoralis females did not respond to thymol in EAG assays, but single-sensillum recordings revealed the presence of two receptor neurons, one responding specifically to carvacrol, and another being sensitive to carvacrol, eugenol and thymol44.Our EAG results showed that males of both species are capable of perceiving thyme oil and the three single compounds, although the response profile is notably different among compounds. Thus, the antennae of both species were more sensitive to 59. In our case, no deleterious sign on neurone membrane integrity was detected, since posterior stimuli to sex pheromone (1 µg) elicited a characteristic V-shape response in both species after the highest doses of thymol and carvacrol  level via tyramine receptors60, we suggest as a possible explanation that binding of thymol and carvacrol to receptors linked to the signal transduction pathway might provoke an alteration in second messenger cascade, which in turn would alter the EAG signal profile.The mechanisms underlying this shift in the EAG response profile are unclear. Thymol and carvacrol are known to affect the surface electrostatics and integrity of the cell membrane, where they can be incorporated and provoke membrane lesion and loss of permeability, leading to intracellular content leakageS. littoralis and G. molesta. This result represents a preliminary contribution for the development of novel practical approaches targeting pheromone communication of these two pest species. Further studies would be required to assess the potential of thyme oil or thymol as effective behavior manipulation tools under field conditions. In addition and to disclose the mechanism of the disruptive action of these chemicals, new electrophysiological experiments on specifically-tuned pheromone receptors would be necessary.In conclusion, our study demonstrates that thyme oil disrupts the sex pheromone attraction in G. molesta, a first batch of pupae was provided by the School of Agrifood and Forestry Science and Engineering—ETSEA , to establish a new colony in our laboratory based on a previously described rearing methodology61.For S. littoralis, males were obtained from a long-term colony reared at the Institute of Advanced Chemistry of Catalonia—CSIC . Rearing methodology and maintenance have been described elsewhere62.In the case of G. molesta (Z)-8-dodecenyl acetate (Z8-12:OAc) (≥ 97%) and (E)-8-dodecenyl acetate (E8-12:OAc) (≥ 96%) were prepared in our laboratory63, while (Z)-8-dodecen-1-ol (Z8-12:OH) (≥ 99%) was purchased from Pherobank . The major sex pheromone component of S. littoralis -9,11-tetradecadienyl acetate  was purchased from Bedoukian Research, Inc. . Thyme oil and carvacrol (98%) were obtained from Merck/Sigma-Aldrich . p-Cymene (> 97%) and thymol (> 98%) were acquired from Alfa Aesar .The pheromone components of p-cymene, carvacrol and thymol was estimated by peak area normalization. Additional compounds with an abundance of ≥ 3% were considered for identification by comparison of their mass spectra with those of reference compounds from NIST mass spectra library , and synthetic standards.Prior to behavioral assays, the chemical composition of thyme oil was analyzed using a GC (Thermo Finnigan Trace 2000 GC) coupled to a Trace MS quadrupole mass spectrometer  equipped with a TR-5MS capillary column  (ThermoFisher Scientific). The percentage of 64. For sample preparation, each antenna was excised under a binocular stereo microscope, and the first 2–3 antennomeres of the tip were cut off. Then, the antenna was mounted on a forked microelectrode holder , and both ends were fixed to the corresponding electrode with a drop of conductive gel . The holder was connected to a MP-15 micromanipulator (Syntech), and placed 1.0 cm below the main branch of a glass tube (7 cm long × 5 mm diameter) that continuously blew humidified pure air (ca. 750 mL/min) over the antenna to keep it clean and prevent desiccation. The output signals were amplified (10×), filtered (DC to 1 kHz) with an IDAC-2 interface (Syntech), further amplified (10×), digitized on a PC and analyzed with the EAG Pro software Version 2.0 (Syntech).EAG assays were conducted following previous standardized proceduresviz. thyme oil, p-cymene, carvacrol, and thymol, were obtained by testing five doses (from 1 to 104 µg) on antennae of 1–2 day old virgin males . To obtain the desired doses, decimal serial dilutions in analytical n-hexane were prepared from a stock solution (500 mg/mL) of each chemical, and 20 µL of the corresponding dilution were loaded on a filter paper strip . For delivering the stimulus, the filter paper was placed into a disposable glass Pasteur pipette. Stimuli were carried out by giving air puffs (ca. 200 mL/min and 200 ms duration) with the aid of a stimulus controller CS-01 (Syntech). Doses were tested from the lowest to the highest, and for each treatment two stimulations were applied on the antenna at 60 s intervals. Control puffs with a filter paper disc containing 20 µL of solvent that had been previously evaporated were intercalated between two consecutive stimuli to determine the mechanosensory response. The net EAG amplitudes (mV) were calculated by subtracting the mean response to the solvent before and after each stimulus from the mean response of the corresponding dose. Doses which did not exert a response on at least 60% of the antennae were excluded from statistical analysis. In the same way, when solvent subtraction from chemical response resulted in negative values, the corresponding recordings were also discarded.Dose–response profiles for each treatment, 62. An incoming filtered air speed of 30 cm/s was set. For G. molesta, appropriate illumination was obtained using a 58 W fluorescent white lamp located 16 cm above the tunnel, which provided ca. 100 lx inside the tunnel. For S. littoralis assays, the tunnel was illuminated by a similar 58 W red fluorescent light. All tests were conducted 2 h into the scotophase under a relative humidity of 30 ± 10%. The tunnel was thoroughly cleansed with 70% ethanol prior and after a set of assays.Flight response of virgin males of first and second scotophase to sex pheromone alone or in combination with thyme oil and constituents was evaluated in a glass wind-tunnel (180 cm long × 55 cm wide × 50 cm high), as previously describedG. molesta, n = 60; S. littoralis, n = 56), and for each trial including pheromone plus thyme oil or constituents in single/binary/ternary combinations, 30 males were tested. Prior to the beginning of each test, insects were individually isolated in Petri dishes (9 cm diameter), and acclimatized to the room conditions for 1 h. In all assays each male was used only once. Sex pheromone was co-released with thyme oil in 1:1 and 1:10 ratio, while the dosages for single constituents and binary/ternary mixtures were defined according to their percent composition in the EO. The pheromone and mixtures of the chemicals were loaded on Whatman filter paper discs (2.5 cm diameter) separated by 2 cm, approximately. For G. molesta, 5 µg of the sex pheromone blend of Z8-12:OAc, E8-12:OAc, and Z8-12:OH in 100:6:10 ratio61 was used. On the other hand, 10 µg of Z9E11-14:OAc were used as attractant for S. littoralis, since we reported earlier that the major component alone is capable of eliciting the full behavioral sequence in wind tunnel assays62. Filter papers were replaced every day. Grapholita molesta males were allowed to respond for 2 min61, and S. littoralis males for 5 min62. Four responding behaviors were scored: (1) take-off from the release platform at 110 cm downwind from the odor source, (2) upwind flight up to half-way of the tunnel, (3) approach to the source, and (4) landing. Landing was positively annotated when the insect remained more than 5 s in contact with the source.The response to the sex pheromone alone in both species was daily assessed (U test. Bonferroni correction (α/number of comparisons) was applied to adjust probability values. Behavioral steps in the wind tunnel were analyzed by Chi-square 2 × 2 test of independence with Yates' correction. All tests were performed using SPSS Statistics 17.0.Differences in absolute EAG amplitudes within a treatment were analyzed by the non-parametric Kruskal–Wallis test, at a significance level of α = 0.05. When significant differences were detected, pairwise comparisons among doses were subjected to the Mann–Whitney Supplementary Information.


Swiss chiropractors have been licensed since 1995 to prescribe from a limited formulary of medications for treating musculoskeletal (MSK) conditions. In January 2018, this formulary was expanded to include additional muscle relaxant, analgesic, and anti-inflammatory medications. Internationally, controversy remains over whether or not medication prescribing should be pursued within the chiropractic profession.The purpose of this study was to assess Swiss chiropractors’ attitudes, beliefs, and practices regarding their existing medication prescription privileges. This information will provide new insights on the topic and help inform research and policy discussions about expanding chiropractic prescription rights in other jurisdictions.A 13-item questionnaire and Q-methodology approach were used to conduct the assessment. Recruitment was conducted by e-mail between December 2019 and February 2020, and all members of the Swiss Chiropractic Association were eligible to participate. Data were analyzed using by-person factor analysis and descriptive statistics.prescribers, non-prescribers, collaborators, and integrators. All except non-prescribers thought medication prescription privileges were advantageous for the chiropractic profession in Switzerland. There was also strong consensus among all four groups that medication prescribing should not replace manual therapy in chiropractic practice.In total, 187 Swiss chiropractors participated in this study (65.4% response rate). Respondents reported prescribing analgesics, anti-inflammatories, and muscle relaxants to a median of 5, 5, and 0% of patients, respectively. Forty-two percent of respondents expressed interest in further expanding the range of current medications available to Swiss chiropractors for treating MSK conditions. Only 15% expressed interest in expanding this range to include medications for treating non-MSK conditions. In the Q-methodology analysis, four salient viewpoints/groups regarding medication prescribing emerged: This was the first national survey on attitudes toward prescribing medications among Swiss chiropractors since the year 2000, and the first using Q-methodology. With this approach, four unique groups of chiropractic prescribers were identified. Even with diversity among clinicians, the findings of this study showed general support for, along with conservative use of, prescribing privileges within the Swiss chiropractic profession. Studies in jurisdictions outside of Switzerland are needed to assess whether chiropractors are interested in expanding their scopes of practice to include similar prescribing privileges. The right to prescribe medications is a controversial topic within the chiropractic profession , 2. At pThe purpose of this study was to assess Swiss chiropractors’ current attitudes toward, the frequency of, and indications for, medication prescribing for MSK conditions in clinical practice. Based on previous literature , 5, 9–12The results of this study are important because they can be used by clinicians, educators, decision-makers, and health policy-makers to inform future directions and research regarding prescribing practices for chiropractors in Switzerland, and possibly in other jurisdictions. For instance, reports indicate that a growing number of chiropractors from countries outside Switzerland are also interested in expanding their scopes of practice to include similar prescribing privileges –17. HoweQ-sort table [www.nowhereroad.com/qsort/]), and administered the demographic questionnaire through SurveyMonkey (www.surveymonkey.com). The instruments were developed in English because the target population was able to communicate in this language.We conducted a cross-sectional study of chiropractors across Switzerland and employed a Q-methodological approach to identify major viewpoints held about medication prescribing for MSK conditions among study participants , 19. Thert table , 19 . A concourse is a list of comprehensive descriptive statements on the topic of interest  = 0 − 11%), 5% (IQR = 1 − 12%), and 0% (IQR = 0–3%) of patients, respectively. Of the 185 participants, 77 (41.6%) were also interested in expanding the range of current medications available to Swiss chiropractors for treating MSK conditions. However, only 28 (15.1%) expressed interest in expanding this range to include medications for treating non-MSK conditions .A summary of the demographic data and scope of practice characteristics for all study participants is displayed in Table p < 0.05) on any of these factors were excluded from further comparative analyses among the four factors  = 9.7) years of clinical experience, eight worked in a multidisciplinary/hospital-based setting, and 25/37This factor was represented by 38 significantly loading Q-sorts (participants). The chiropractors in this group had an average of 20.5  believed that prescribing painkillers and NSAIDs was a useful addition to chiropractic practice, particularly for treating patients who were in severe pain (statement 35). As summarized by one respondent,[With prescription rights], I can better control the healing of the patient, reassure him, prescribe exercises, and finish the case with less costs than the medical doctor. P 122ce Table . They stPrescribers strongly disagreed (− 4) with the statement that “medication prescription should only be performed by general practitioners or pharmacists” (statement 10). In their Q-sort comments, a common theme among many of these respondents was that chiropractors should be allowed to prescribe medications. For example,Why should it be forbidden [for] chiropractors to make use of a treatment tool that can be beneficial to the patient? This makes no sense. P 132Medication, “natural” or “synthetic,” should be an option in the treatment of MSK pain and [should] not be automatically excluded because of purely dogmatic rhetoric. P 23Some prescribers suggested that the chiropractic profession should move on from its historical past to become a more modern and integrated healthcare profession. For instance,We are in a time where we drive cars and airplanes, not buggies and horse chariots. We have to evolve to [be able to provide] the best care possible and [deliver it in] the most efficient way. P 122Some also felt that such privileges should not be denied by the chiropractic profession simply for ideological reasons. As stated by the following respondent,prescribers strongly agreed (+ 3.5) that medication prescription privileges were an advantage for the chiropractic profession in Switzerland  years of clinical experience, eight worked in a multidisciplinary/hospital-based setting, and 13/23This factor was represented by 24 participants. prescribers, non-prescribers were generally opposed to medication prescribing in chiropractic practice (Table non-prescribers strongly (+ 4) believed that it should be part of the definition of the chiropractic profession that chiropractors “assist the body in self-healing WITHOUT the use of drugs or surgery,” a sentiment reflective of the profession’s international history [I don’t think the chiropractic profession should be blending [its scope of practice] with the medical profession to such a degree. We need to stay separate and apart and not give up our roots! P 128Contrary to ce Table . They fe history . This isOne very common reason why patients seek care in my clinic is because they have already tried the medications, injections, cortisone shots, etc. [and these] have not given them the results they [had] hoped for. P 103Some respondents indicated that patients often visit a chiropractor for pain management because medications and other therapies have been ineffective. For instance,Non-prescribers also strongly disagreed (− 4) that adding new drug classes (opioids) to their prescription rights would be useful (statement 2). This particular respondent stated that,Too many other health care providers prescribe medication (including opioids). We should be the ones providing the alternative. P 14non-prescribers compared with prescribers, collaborators, and integrators . Moreover, non-prescribers were the only group out of the four to disagree (− 1.5) with the statement that, “medication prescription privileges are an advantage for the chiropractic profession in Switzerland”  years of clinical experience, two worked in a multidisciplinary/hospital-based setting, and seven (77.8%) collaborated with other medical professionals on a daily basis. The age range was 41–56 years.This factor was represented by nine participants. non-prescribers, collaborators were generally opposed to medication prescribing in chiropractic practice  believed that continuous education concerning medication prescribing should be mandatory (statement 14). They also strongly agreed  with the statements, “I do not like to prescribe because it could interfere with other medical prescriptions ,” and “chiropractors should get more continuing education (CE) about medications and side effects.” These concerns are further expressed by the following respondent:Medical doctors are trained in pharmacology, we had only one course in toxicology, not enough to take [on] that kind of responsibility. P 51Similar to the ce Table . HoweverCollaborators also strongly disagreed  that their knowledge of, and chiropractic training for, prescribing medications to treat MSK or non-MSK conditions was sufficient (statements 1 and 8). As such, many of these chiropractors reflected the sentiment that medication prescribing should be co-managed with their medical colleagues, as elucidated by the following respondent:I personally have [a] very good rapport with local MDs [medical doctors] whom I call and ask … for drug support – which is very beneficial for our interprofessional rapport and also reduces many potential risks . P 8collaborators still agreed (+ 1) that medication prescription privileges were an advantage for the chiropractic profession in Switzerland, albeit to a lesser extent than did prescribers (p = 0.003)  years of clinical experience, one worked in a multidisciplinary/hospital-based setting, and all five (100%) collaborated with medical professionals on a daily basis. The age range was 29–70 years.This factor was represented by five participants. These chiropractors had an average of 24  for the chiropractic profession in Switzerland. They strongly (+ 4) believed that their medication prescription privileges allowed for better integration within the healthcare system (statement 11). In their Q-sort comments, some respondents also suggested that the responsibility of prescribing raised the standard of practice for chiropractors in Switzerland. For example, this respondent stated the following:I find that [because] we can give medication, it’s a privilege and it’s a big difference for our statute between us and the [chiropractic profession in] other countries. P 142Similar to ce Table . In partIntegrators also strongly agreed (+ 4) that medication prescription rights give them better credibility among their medical colleagues and their patients . One respondent also commented on how these privileges are needed for chiropractors to have greater autonomy and cultural authority:…I believe that medication (excluding opioids) is a necessary tool to be able to fully assume the responsibility and independence of a primary care practitioner. P 65integrators strongly disagreed (− 4) with the statement that “medication prescriptions are a burden because they bring added professional responsibility to the chiropractor” (statement 3). One respondent (P 2) quipped that this “is a statement for lazy chiropractors.”Furthermore, integrators’ frequency of medication prescribing was significantly higher than that of their chiropractic colleagues. For instance, compared with prescribers, non-prescribers and collaborators, integrators reported prescribing analgesics to a median of 25% of patients , NSAIDs to a median of 25% of patients , and muscle relaxants to a median of 10% of patients .When comparing factors, There were no statistically significant relationships between the four factors and any of the remaining demographic variables, including age, sex, number of years in practice, chiropractic school of graduation, postgraduate university education, collaboration with other medical professionals, type of practice , and interest in expanding the range of current medications available to prescribe for non-MSK conditions.Consensus statements are statements for which all participants in a Q-methodology study, regardless of factor, generally agree or disagree with to a similar extent  manually because of medication. P 32Medical prescription is a part of our education at the University of Zürich and has to be allowed and also used!  …our main work is and remains the manual therapy, which includes manipulation and should never be replaced by prescribing medications! P 123And,Whoever agrees with [statement] No. 18 should be kicked out of the profession! P 82r extent . In the prescribers, non-prescribers, collaborators, and integrators.This was the first national study of chiropractors in Switzerland on attitudes, beliefs, and practices regarding medication prescribing for MSK conditions conducted since the year 2000 . It was prescribers, collaborators, and integrators) also agreed that the ability to prescribe from a limited formulary of medications was advantageous for the profession. However, these four identified groups also represented four distinct perspectives or viewpoints about medication prescribing for MSK conditions. These viewpoints can be used, at least conceptually, to help explain why the majority of Swiss chiropractors seem to feel that prescription privileges are an advantage for the profession, and why the minority do not. For instance, in the current study, the prescribers and the integrators favoured medication prescribing in chiropractic practice because they felt this was an important tool in the management of patients with MSK conditions. Integrators also believed that such privileges have allowed for better integration of chiropractors within the healthcare system. Indeed, chiropractic is one of five government-recognized medical professions in Switzerland ; chiropractic services are also fully covered under the country’s publicly-funded national health insurance program [non-prescribers were opposed to medication prescribing in chiropractic practice, feeling that chiropractic treatment should remain “drug-free” and be provided to patients as an alternative to medication. Although collaborators were also generally opposed to medication prescribing by themselves, this was based on the viewpoint that chiropractors needed more training in pharmacology and toxicology. Research involving other independent prescribing professionals such as nurses and pharmacists has shown that continuing education in pharmacology can improve practitioners’ confidence in prescribing medications [collaborators might convert to prescribers if given such additional training.In previously conducted quantitative surveys of Swiss chiropractors , 5, majo program , 7, 27. ications , suggestprescribers, collaborators, and integrators) or were at least neutral  with the idea that a review of new medications relevant to chiropractic practice should be organized for the profession every 2–5 years. In New Mexico (USA), chiropractors complete 10 h of continuing education per year in pharmacology, toxicology, or medication administration to maintain their ‘advanced practice certification’ for prescribing medications in that state [In terms of ChiroSuisse members’ beliefs toward the adequacy of their current pharmacology training, respondents in all four groups, at least to some extent, felt that with prescription privileges chiropractors should receive more continuing education about medications and side effects. This is consistent with the results from a study of Swiss chiropractors conducted by Wangler et al. , where nat state . Outsideat state  endorsedat state .The differences identified between the four factors in the current study indicate that other contextual variables might influence chiropractors’ viewpoints about medication prescribing for MSK conditions. For instance, previous surveys of chiropractors in North America –11 have non-prescribers also alluded to this issue in their comments provided for the current study. However, Swiss chiropractors have maintained their profession’s distinct identity despite having limited prescription privileges since 1995 [A concern that is often raised by those who are against chiropractors gaining access to medication prescribing rights relates to the perception that the chiropractic profession might lose its distinct  brand and identity , 2. Somence 1995 , 7. Swisintegrators, who had the highest reported frequency of medication prescribing among the four identified groups, still only prescribed medications to a maximum of 25% of their patients. In comparison, Swiss medical doctors prescribe medications to nearly two-thirds of patients with spine-related / MSK complaints [Using quantitative survey methods, this study queried Swiss chiropractors about the frequency with which they prescribed medications. In line with previously published research conducted in 2009 , 2010 55, and 20mplaints . Similarmplaints .Regarding their existing medication prescription privileges, ChiroSuisse members were also asked about their interest in expanding the range of current medications available to prescribe for MSK or non-MSK conditions. In general, respondents were divided over being able to prescribe opioids, while most were opposed to incorporating medications into their formulary for treating non-MSK conditions. These findings are consistent with the results of several international chiropractic surveys , 9–12. IPrescribers, non-prescribers, and integrators further indicated that they prescribe medications to patients in “extremely acute cases where absolutely no range of motion can be achieved and pain levels are too high.” These findings are consistent with those of a previous pilot study [As for the clinical indications for prescribing by Swiss chiropractors, participants in all four groups also agreed, at least to some extent, that prescribing medications were “useful in helping patients who could not sleep because of pain.” ot study  where thprescribers and non-prescribers both agreed with, and the collaborators were at least neutral to, the notion that medications should be used conservatively in the management of MSK conditions. Similarly, participants in all four groups either agreed with or were neutral to the statement, “I personally take very little to no medication on a yearly basis and think we should encourage this same approach with our patients.” Although we did not specifically ask Swiss chiropractors about their attitudes and practices toward taking patients off previously prescribed MSK medications , several respondents indicated that, with prescription privileges, they were in a position to advise patients against improper usage of these types of medications. For example, in the Q-survey, prescribers indicated that they only prescribe analgesic medications “when they think it would be useful, and not every time the patient asks.” In their Q-sort comments, some non-prescribers indicated that limited prescription privileges allow them to counsel their patients with MSK pain against overusing or over-relying on medications prescribed to treat their condition. This concept of ‘medication counselling’ was also described by Wangler et al. [Because chiropractors share primary care status with general practitioners in Switzerland , 8, the r et al.  in theirr et al. . When coIf practiced across the chiropractic profession globally, such a role could very well have public health implications in light of the growing opioid crisis in numerous countries around the world –37. For This study has several strengths. First, using Q-methodology, this study revealed four distinct viewpoints toward medication prescribing among Swiss chiropractors not identified in previous cross-sectional surveys , 5, 7. AA limitation of this study is that just under two-thirds of all ChiroSuisse members participated, and only half of these respondents also completed the Q-sort table. Although the overall response rate 65.4%) was high compared with many previous chiropractic surveys on medication prescription rights % was hig, 41, 42,In using Q-methodology, this study was able to demonstrate how Swiss chiropractors prioritized their viewpoints on medication prescribing for MSK conditions in chiropractic practice. With this approach, four distinct viewpoints were identified. In response to our demographic survey, participants also reported prescribing MSK medications infrequently to patients in clinical practice. These findings suggest that, even with diversity among chiropractors, limited prescribing rights can be incorporated and conservatively used within the profession. With such privileges, chiropractors internationally would have an important role to play as MSK specialists within their respective healthcare systems. If utilized judiciously, chiropractors, working alongside general practitioners, could also have a positive influence on public health in these countries. Clinicians, associations, and health policy-makers can use the results of the current study to inform the discourse on whether to extend prescribing rights to chiropractors in other jurisdictions.Additional file 1. Final Q-sample used in the Q study instrument.Additional file 2. Demographic comparison of Q-sort study respondents versus Q-sort non-respondents.Additional file 3. Comparison of Q-sort study respondents versus all non-respondent ChiroSuisse members by gender and region of practice.


We also correlated MCP-1 and NLK levels in CSF to several parameters of disease. We showed that MCP-1 levels in CSF positively correlate with PD progression  but could not differentiate between PD, MSA, and controls. NLK levels in CSF neither differentiated between PD, MSA, and controls, nor correlated with disease progression. Our results indicate that MCP-1 levels in CSF cannot distinguish between PD, MSA, and controls but correlate with disease progression in PD patients, suggesting that neuroinflammation is associated with clinical progression in PD. The correlation with disease progression was only moderate, so MCP-1 levels in CSF should be included in a larger battery of prognostic biomarkers that also tackle different pathophysiological processes.Parkinson’s disease (PD) and multiple system atrophy (MSA) have overlapping symptoms, challenging a correct early diagnosis. Prognostic information is needed to predict disease progression and provide appropriate counseling. Neuroinflammation plays a role in the pathology of both disorders, as shown in genetic and postmortem tissue studies. Monocyte chemoattractant protein 1 (MCP-1) and neuroleukin (NLK) are two inflammatory proteins with potential to serve as biomarkers of the neuroinflammatory process. Here, we aimed to study the biomarker potential of both MCP-1 and NLK protein levels in cerebrospinal fluid (CSF) from a longitudinal cohort study , consisting of PD patients ( The exact etiology remains unknown but likely involves both genetic and environmental factors. Classically, neural degeneration is associated with presence of Lewy Bodies, which contain aggregates of the protein α-synuclein (α-syn). However, the pathology of the disease also affects numerous fundamental cellular processes such as neuroinflammation, mitochondrial function, protein trafficking and the proteasome-mediated protein degradation5. The characteristic PD motor symptoms appear when 50–80% of dopaminergic neurons have already died. These motor symptoms include bradykinesia, resting tremor, and rigidity7. Establishing a correct diagnosis of PD in its early stages can be challenging since PD shares many clinical features with multiple system atrophy (MSA). MSA is a sporadic, rare, and aggressive neurodegenerative disease with an average clinical course of 9 years from symptom onset to death9. Pathologically, MSA is associated with cytoplasmic inclusions of abnormally folded α-syn, predominantly in oligodendrocytes. In the parkinsonian form of MSA (MSA-P), neuronal loss mainly occurs in the nigrostriatal pathway9. Clinically, MSA-P is an akinetic-rigid syndrome characterized by autonomic dysfunction, gait disturbance, rigidity, progressive bradykinesia, and a poor response to dopaminergic therapy10.Parkinson’s disease (PD) is the second most common neurodegenerative disorder affecting 1% of the worldwide population older than 65 years, and is expected to affect 12 million people by 204012. Currently, the diagnosis is based on clinical examinations made by movement-disorder specialists, who still reach error rates as high as 24%10. Since the gold standard diagnosis is postmortem brain tissue examination, there is a need to discover good biomarkers for diagnosis at early disease stages, and to establish biomarkers that may predict disease progression.To determine treatment strategies and to counsel patients appropriately, an early and reliable differential diagnosis of PD versus MSA is needed. Ideally, clinicians should also have tools to predict disease progression in these patients13. Thus, we hypothesized that inflammatory proteins present in the cerebrospinal fluid (CSF) may reflect this chronic state of neuroinflammation, and might be good candidate biomarkers to differentiate PD from MSA, and to predict disease progression. Monocyte chemoattractant protein 1 (MCP-1) is a cytokine that recruits monocytes and T cells to the sites of inflammation. Here, we specifically further investigate its diagnostic and prognostic value in PD and MSA. MCP-1 levels in blood are elevated in PD compared to controls and correlate with PD progression14. In other studies, upregulated MCP-1 expression in brain tissue and higher levels in CSF from Alzheimer’s disease patients compared to controls were reported, and this upregulation correlated with disease progression16. Neuroleukin (NLK) is a neurotrophic factor with axonal growth activity and a product of lectin-stimulated T cells18. Previous studies showed that NLK is upregulated in the brain in patients with Huntington’s disease19 and promotes axonal growth in spinal cord injury20, indicating a role of NLK in neurodegenerative processes. Therefore, in the present study, we aimed to assess the potential of the inflammatory proteins MCP-1 and NLK in CSF as biomarkers for diagnosis and progression of PD and MSA.Aggregates of α-syn activate microglia and astrocytes, inducing an immune response. Chronic activation of microglia and astrocytes causes a constant release of pro-inflammatory proteins that enhance neuroinflammation and neurotoxicity, leading to cell deathWe analyzed the levels of MCP-1 and NLK in CSF from 46 PD, 17 MSA, and 52 controls. We first compared the MCP-1 and NLK levels in CSF between clinical groups. The levels of both MCP-1 and NLK did not differ between groups  , probably because of the small number of patients. We neither observed correlations between MCP-1 and UPDRS, ICARS or MMSE progression over 3 years in the PD group nor with any of the scales in the MSA group. We did not observe a correlation between NLK CSF levels and any of the disease progression parameters for either PD or MSA (data not shown). We also correlated MCP-1 and NLK levels in CSF with parameters of disease severity at baseline and 3-year follow-up. MCP-1 levels in CSF positively correlated with HY score at 3-year follow-up  in the PD group. NLK levels in CSF positively correlated with baseline UPDRS-III  and baseline ICARS  in the MSA group.We then correlated MCP-1 and NLK levels in CSF with parameters of disease progression. MCP-1 levels in CSF positively correlated with the change in HY score over a period of 3 years in the PD group (43) Fig. . However21 for MCP-1, and multiple sclerosis (MS) and HD for NLK22.Several studies showed that the levels of the inflammatory proteins MCP-1 and NLK are altered in neurodegenerative diseases, such as PD and AD24. Thus, we anticipated that CSF levels of MCP-1 would be increased in PD and/or MSA compared to controls. However, and in agreement with other studies29, we found that CSF levels of MCP-1 are comparable in PD, MSA and controls. In contrast, other studies described increased levels of MCP-1 in CSF of either PD or MSA patients compared to controls31. An explanation for these discrepancies might be differences in clinical features and pathological state across the various cohorts of patients. In addition, in our study, CSF was collected when there was uncertainty of diagnosis, and diagnosis was reviewed after an extended follow-up period of time. In contrast, other studies analyzed CSF of patients with more advanced disease and shorter follow-up. Finally, different assay types were used to quantify MCP-1 levels in the various studies. One study found an association between MCP-1 and more severe non-motor symptoms in PD26. Also, higher MCP-1 expression has been demonstrated in patients with dementia33. Thus, MCP-1 levels might be higher in later stages of the disease and be associated with disease progression, as we also observed previously29. In the present study, we found a weak positive correlation in the PD group between MCP-1 levels in CSF and both final HY score at 3-year follow-up and progression of this score over a 3-year period. This may indicate that a more advanced degree of neuroinflammation may be associated with more advanced motor dysfunction in PD. However, the weak correlation of MCP-1 levels with the HY scores was not supported by UPDRS-III score suggesting that MCP-1 has limited predictive power on its own, but may help as part of a bigger panel that includes a broader range of molecular, imaging, or clinical parameters that also tackle different pathophysiological processes. Unfortunately, the correlation between MCP-1 and HY progression in the PD group was lost after 10-year follow-up. This result might be biased by the lower number of clinical data after 10 years. Alternatively, it might indicate that long-term disease progression may be affected by additional factors.MCP-1 is a chemokine expressed by glial cells and neurons, and involved in the recruitment of astrocytes, microglia and infiltrating cells from the blood. The MCP-1 receptor (CCR2) is constitutively expressed in brain areas associated with dopaminergic and cholinergic neurotransmission, which are the most affected in PD. Preclinical studies in mouse models suggest that MCP-1 causes neuronal loss and that its downregulation is neuroprotective36.We did not observe a difference in UPDRS-III progression between PD and MSA patients, and, moreover, we observed that the UPDRS-III progression in patients with MSA was remarkably low. This could be explained by the following observations: (1) 10 of the initial 17 MSA patients could not complete the UPDRS-III assessment at 3-year follow-up (deceased or too severely disabled); (2) 2 of them had more prominent cerebellar features and less pronounced parkinsonian features; and (3) 2 of the 7 patients with follow-up were scored ‘on’ dopaminergic medication because skipping doses of dopaminergic medication would lead to unacceptable deterioration in these patients. However, further analysis on the progression of the complete cohort of PD and parkinsonism patients at 3- and 12-year follow-up showed that the degree of UPDRS-III progression in our cohort is similar as in other longitudinal studies. Noteworthy, recent observations in the large Parkinson’s Progression Markers Initiative cohort have shown a substantial amount of error variance and individual fluctuations of the UPDRS score and therefore, UPDRS might not be precise enough to assess disease progression18. When located intracellularly, NLK is involved in the insulin-like growth factor 1 (IGF-1)/insulin-like signaling pathway37, which has been implicated in neurodegeneration and human aging. NLK may suppress α-syn accumulation and toxicity, dopamine neurodegeneration and inflammation in Caenorhabditis elegans and Drosophila37. These studies suggest protective effects NLK in neurodegenerative diseases. Despite the possible association of NLK with the pathology of α-synucleinopathies, our data suggest that NLK neither has a role as a biomarker for diagnosis nor for monitoring disease progression of PD and MSA.NLK is neurotrophic factor with axonal growth activityThe uniqueness of our patient cohort is the major strength of our study. Only patients with diagnostic uncertainty at baseline were included in the study and their clinical diagnosis was re-evaluated after 3 and 12 years. Although this may also be regarded as a weakness, i.e., bias toward more complicated cases, this experimental setting exactly reflects the clinical situation when biomarkers are needed most, i.e., when there is diagnostic uncertainty during first visit.14, which reinforce the quality and reliability of our results.Our study also had limitations. First, patients presented heterogeneity with respect to dopaminergic medication at the time of lumbar puncture. The majority of patients were tested drug-naïve or “off” medication. However, for some of the patients medication withdrawal was ethically not possible. Second, our group of MSA patients was relatively small, which may have affected our analyses; especially for the correlation between the biomarkers and disease progression parameters. Third, the final diagnosis was based on clinical evaluation according to international diagnostic criteria but has not been confirmed by postmortem neuropathologic examination. This may have caused potential misdiagnoses, but we have reduced this risk by the very long follow-up of the patients. Some concerns might arose regarding the long storage of the CSF samples previous to analysis (23 years). However, a study has shown the stability of long-term storage of CSF samples in BiobanksIn summary, our results indicate that MCP-1 levels in CSF cannot distinguish between PD, MSA, and controls, but correlate with disease progression in the PD group, suggesting its usefulness as part of a bigger panel to predict motor dysfunction in PD.38. In this study, 156 patients, referred to our center between January 2003 to December 2006 because of parkinsonism and diagnostic uncertainty, were included. Exclusion criteria were age younger than 18 years, history of brain surgery or neurodegenerative disease other than parkinsonism or unstable comorbidity. All patients underwent a structured interview, detailed and standardized neurologic examination, blood collection, lumbar puncture and other ancillary investigations , 123I-iodobenzamide single photon-emission computed tomography, analysis of CSF, and anal sphincter electromyography) within 6 weeks after inclusion. The study design, methods and patient population have been extensively described elsewhere38. These patients were followed up for 3 years and a final clinical diagnosis was established by two expert neurologists in movement disorders based on a repeated structured interview and extensive neurological examination. In 2018, 12 years after inclusion, all diagnoses were re-evaluated and updated by two independent neurologists, according to the most recent clinical criteria42 and based on clinical data collected at time of inclusion and at 3- and 10-year follow-up. The diagnoses of some patients may have changed over time as illustrated in Supplementary Fig. A total of 46 PD and 17 MSA patients were selected based on CSF availability from a prospective cohort study performed at the Radboud University Medical Center The control group consisted of 52 patients aged above 40 years with neither a neurological nor an inflammatory disease and who underwent a lumbar puncture because of a suspected neurological disorder that was subsequently ruled out in the diagnostic process.This study was approved by the Central Committee on Research Involving Human Subjects in the region Arnhem-Nijmegen (2002/188) and all participants provided written informed consent.38. CSF samples had no blood contamination (leukocyte count fewer than 5 cells/μL and erythrocyte count fewer than 200 cells/μL)43. CSF was immediately frozen at −80 °C and only thawed once for aliquoting and analysis previously to measurements. CSF samples from either PD or MSA patients were obtained in the same period and were all treated similarly.Lumbar puncture was performed as described previouslyn = 3, range: 26.6–38.6 pg/mL), the inter-assay variation , the intra-assay variation  and the parallelism (100–120%)44.MCP-1 and NLK levels in CSF were measured in 2019, i.e., 23 years after CSF withdrawal. MCP-1 levels in CSF were measured using a human MCP-1 ELISA Kit . CSF samples were diluted eight times and measurements were performed according to company’s recommendations. We validated the reliability of the ELISA kit for CSF analysis by measuring the limit of detection (LOD = 1.28 pg/mL), the lower limit of quantification (LLOQ = 6.48 pg/mL) the dilutional linearity , the intraplate variation (coefficient of variation (CV): 7.2 ± 5.2%; n = 5).NLK levels in CSF were measured using the human Glucose-6-Phosphate Isomerase ELISA Kit . CSF samples were diluted six times and measurements were performed according to the manufacturer’s recommendations. To validate the reliability of the kit for CSF analysis we measured the LOD (0.06 ng/mL), LLOQ (0.14 ng/mL), the dilutional linearity , the intra-assay variation  and the inter-assay variation  samples were included in duplicate in all measurements to correct for interplate variation. Briefly, a correction factor was calculated per plate using QC concentrations by dividing the concentration of the QC on the reference plate  by the same QC on the other plates. Then, the average of the five QC correction factors was calculated per plate and multiplied by the protein levels of the samples of that specific plate.p value < 0.05 was considered as statistically significant.Statistical analyzes were performed using IBM SPSS Statistics (v.25.0.0.1). Two-sided Kruskal–Wallis test or Mann–Whitney test with Bonferroni correction was performed to assess differences between groups for age, baseline and follow-up parameters, as well as disease progression. Chi-square test was used to assess sex differences. Group comparison of MCP-1 and NLK concentration in CSF was performed by rank analysis of covariance to correct for age. Briefly, the dependent variables and the covariate were ranked. Then, a linear regression of the ranks of the dependent variable on the ranks of the covariate was performed and the unstandardized residuals were saved. Finally, a two-sided ANOVA with Hochberg correction for multiple testing was performed using the unstandardized residuals. Disease progression was calculated using annual change in HY, UPDRS, ICARS, and MMSE scores using the 3-year follow-up and baseline scores. Spearman’s test was used to correlate the levels of biomarkers at baseline with the annual progression scores, as well as disease severity at baseline and at 3-year follow-up. In all cases, a Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.Supplementary figure 1Reporting Summry