Understanding of emotions and intentions are key processes in social cognition at which serotonin is an important neuromodulator. Its precursor is the essential amino acid tryptophan (TRP). Reduced TRP availability leads to weaker impulse control ability and higher aggression, while TRP supplementation promotes confidence. In a double-blind placebo-controlled fMRI study with 77 healthy adults, we investigated the influence of a 4 week TRP enriched diet and an acute 5-hydroxytryptophan (5-HTP) intake on two social-cognitive tasks, a moral evaluation and an emotion recognition task. With 5-HTP, immoral behavior without negative consequences was rated as more reprehensible. Additionally, during story reading, activation in insula and supramarginal gyrus was increased after TRP intake. No significant effects of TRP on emotion recognition were identified for the whole sample. Importantly, emotion recognition ability decreased with age which was for positive emotions compensated by TRP. Since the supramarginal gyrus is associated with empathy, pain and related information integration results could be interpreted as reflecting stricter evaluation of negative behavior due to better integration of information. Improved recognition of positive emotions with TRP in older participants supports the use of a TRP-rich diet to compensate for age related decline in social-cognitive processes. Especially in morally challenging situations, the ability of integrating relevant information, dealing with own affective reactions, perspective-taking, and feeling empathy is crucial3.Emotion recognition and understanding of emotions and intentions are key factors in social cognition4 the precursor of which is the essential amino acid tryptophan (TRP). TRP is the scarce amino acid in many foods, however, abundant in e.g. soy, cashew, cacao, and whey products . TRP and other large neutral amino acids (LNAA) like tyrosine compete at the blood brain barrier5 which leads to lower brain TRP levels when more other LNAAs are consumed. As serotonin itself cannot cross the blood brain barrier, it is necessary that a sufficient amount of TRP or 5-hydroxytryptophan (5-HTP) is available in the brain. 5-HTP is the direct precursor of serotonin and is almost completely metabolized to serotonin showing a plasma half-life of about 4–6 h7. 5-HTP can be found in food, e.g. the bean Griffonia simplicifolia8, or can be synthesized from TRP by the enzyme tryptophan hydroxylase, which is the initial and rate-limiting step in the synthesis of serotonin. TRP is metabolized to about 95% via the kynurenine pathway and to about 5% via the serotonin pathway9 mainly in the gut. Serotonin can be converted into melatonin during darkness or it is excreted in the urine in the form of 5-hydroxyindoleacetic acid (5-HIAA). A daily intake of about 4–6 mg TRP/kg is recommended for adults10. Supplementation of TRP increases plasma TRP11 and improves the release of serotonin in the rat brain12.A major neuromodulator in regulating affective and social-cognitive processes is serotonin13 and the recognition of happiness in remitted depression14, others found no effects in participants with a family history of depression15. Further, TRP depletion leads to lower impulse control ability and higher aggression levels16 whereas TRP supplementation seems to reduce aggression and to promote prosocial behavior including interpersonal trust and charitable donating19. It has been argued that the effect of TRP depletion and supplementation acts via serotonin availability on a dimension from agreeability to quarrelsomeness and via that influence mood and social interactive behavior20. An important facet of prosocial behavior which is negatively related to aggression and quarrelsomeness is empathy21.So far, the effect of TRP on social cognition has been mainly investigated in the context of TRP depletion revealing heterogeneous results. While some studies found TRP depletion to impair the recognition of fear in healthy individuals22. While empathizing with the sensations of another person is called affective empathy, cognitive empathy is characterized by perspective-taking24. Cognitive as well as emotional aspects of empathy have been associated to moral decision-making25 supporting the idea that these processes are partly overlapping or that cognitive and emotional empathy, that themselves build on the ability to recognize the emotions of others, are a prerequisite for moral decisions. Furthermore, feeling similar to a person suffering might reduce emotion recognition rather than enhancing it26. These findings are particularly important in the context of moral dilemmas, as perspective-taking increased condemnation when unkind intentions were attributed to a protagonist, whereas (affective) empathy did not increase disciplinary responses3.Empathy supports our ability to understand the intentions, beliefs, and needs of others. Usually, research discriminates affective and cognitive empathy28. A meta-analysis on brain activation during moral decision making, empathy and theory of mind, a concept closely related to cognitive empathy29, found overlapping brain regions between these aspects of social cognition30. Particularly the dorsomedial prefrontal cortex, the bilateral TPJ and the right middle temporal gyrus were found to be activated under all three conditions, reflecting the interconnectivity of empathy, mentalizing and moral decision-making. Further brain areas related to empathy, specifically empathy for pain, were anterior insula and anterior and midcingulate cortex31. These regions were involved in integrating harmfulness information in judgements of the behavior in morally crucial situations2. The response of the right TPJ was highest for attempted harm when protagonists were condemned for their actions32. Regarding TRP on a neural level, Williams and colleagues found in a rather small sample that TRP depletion reduced activity in left posterior superior temporal sulcus and anterior cingulate cortex during the evaluation of the emotionality of faces33. These results might indicate that serotonin, decreased via TRP depletion, plays a role in modulating activity in brain areas associated with social cognition.Brain regions associated with social cognition are the medial prefrontal cortex and bilateral temporo-parietal junction (TPJ)36. Similar associations have been reported in ageing individuals for whom an age related decline of serotonin availability and function was hypothesized38. Consequently, TRP availability and its impact on social cognition are important to human well-being and might be a target for interventions for a broad spectrum of individuals.Thus, it is not surprising that mental disorders which are associated with altered social cognition also show altered serotonin levels32, and the reading the mind in the eyes task (RMET) to assess emotion recognition24 were applied. In addition, on the basis of food diaries completed over one month and urine samples, we analyzed effects of dietary TRP and basic serotonin availability in the body (inferred by urinary 5-HIAA). We expected positive effects of TRP and 5-HTP on emotion recognition and the evaluation of morally relevant behavior, as well as increased activity in areas related to social cognition. These effects should be associated to age, 5-HIAA excretion and the natural diet as we expect greater effects when people have an altered TRP availability or serotonin metabolism, e.g. in older age.The aim of this double-blinded placebo-controlled randomized controlled trial was to further elucidate effects of TRP and 5-HTP supplementation on neural and behavioral measures of social cognition in healthy participants. To allow conclusions on the long-term effects of an adapted diet, we used a 4-week L-TRP enriched diet. To find acute effects more easily, we additionally applied 5-HTP, which is almost completely metabolized into serotonin. For this, during fMRI, a task using short stories to assess moral evaluation39, as well as contraindications for fMRI (including hypertension). Further, food allergies or other illnesses with possible negative influence were assessed and if necessary affected individuals were excluded.82 healthy participants who were randomized assigned  to the intervention groups took part in the study . The last 8 participants were allocated group-wise to match sex and age rates. Exclusion criteria were mental disorders assessed by the screening questions from the Structured Clinical Interview for DSM-IVOne participant was excluded due to a high 5-HIAA baseline value, one quit the study during the first session (comprehension problems), and 3 quit the study after t1 . Demographics of the remaining 77 participants are presented in Table The study was approved by the ethics committee of the University of Heidelberg (2016-518N-MA) and conformed to the Declaration of Helsinki. It was preregistered at the German Clinical Trials Register . All participants gave written informed consent. Data acquisition was from September 2016 until March 2019 in Mannheim, Germany.The whole study was conducted as a double-blinded placebo-controlled trial. The primary outcomes were behavioral and neural measures of social cognition. Participants had three appointments, each comprising laboratory testing and an MRI session. All measures of each participant were planned on the same weekday and at the same time to reduce possible influences of circadian rhythms, working hours and recreational activities.Griffonia simplicifolia) or placebo (cellulose and mannitol) with a 200 ml mixture of fruit juice (15–20%) and water. The capsule was given 1.5 h before placing the participant inside the scanner (mean time capsule taken until first task started: 110 ± 11 min for both conditions). To avoid possible influences of 5-HTP on baseline 5-HIAA, participants gave a urine sample before taking the capsule. 200 mg 5-HTP were chosen as similar doses were reported previously to have an effect in depressed individuals .At t1 and t2, all participants got either 5-HTP or placebo in a randomized cross-over design (double-blinded), age and sex were counterbalanced between groups  the second part of the study using a mixed between-subject design started using the placebo condition from the first part as a baseline. Participants filled in food diaries and ate protein bars over one month. Half of them received placebo/normal protein bars (0.39 mg L-TRP) and the other half got the same protein bars injected with additional 500 mg L-TRP  and creatinine by a commercial laboratory . Creatinine was measured with a double determination to relate 5-HIAA values to urine concentration as this depends highly on e.g. fluid intake and activities. Therefore, 5-HIAA raw values were related to individual creatinine values and afterwards standardized to group mean creatinine (1045 mg/l). For collecting further anthropometric data a body composition scale (Omron body composition monitor BF511) was used.24 is designed to assess mentalizing and emotion recognition. It involves inferring the mental state of a person from photographs of 36 eye regions. As a contrast baseline we used 36 greyscale pictures of plants . Stimuli were presented on a grey background in the center of the screen with four mental state terms or four plant names below a picture  was used to assess self-rated empathy including three subscaleswww.neurobs.com) for stimulus presentation. Participants responded with a response device . Stimuli were presented for 7 s during the RMET  and for 22 s during the moral task  with additional 4 s for response. Trials in the moral task were separated with a variable inter stimulus interval (ISI) of 1 s on average (jitter 0.5 s–1.5 s). During the RMET an ISI of 1 s was used with the presentation of a fixation cross in the position where the next picture would show  and the Presentation software  were recorded with two identical 3T Trio Tim scanners with 12 channel head coils . Further, we collected high-resolution three-dimensional T1 weighted anatomical images  with 192 slices . Heart rate and respiration rate were sampled at 50 Hz with scanner built-in equipment . Half of the sample was scanned on one scanner and the other half on the second .304 (RMET) and 359  T2* weighted EPI images  with 33 slices  running on MATLAB R2017a . Preprocessing included segmentation of the MPRAGE and registration to the SPM12 TPM templates, coregistration of the functional images to the individual MPRAGE, motion correction, slice time correction, normalization of the functional images with parameters derived during MPRAGE segmentation, and smoothing with a 9 mm Gaussian kernel. For first level analyses, a GLM was set up with the onsets of the stimuli  as regressors of interest and the 6 movement parameters  derived from realignment and the response clicks as regressors of no interest. Afterwards, maps were corrected for the baseline measurements which were the corresponding placebo conditions (X − baseline) to get change values for each individual. For the second level analyses on group level (5-HTP vs 0 or L-TRP vs placebo) we used these change values and included age, sex, baseline 5-HIAA, and the order of 5-HTP and placebo administration as covariates. To account for possible effects of the two scanners, we did an additional analysis with the scanner number as covariate. Further, we conducted a parametric modulation for the NP stories with the behavioral data of those.The first 4 images of each task were discarded. Data were corrected for physiological artefacts including a high-pass filter of 1/512 Hz using the Aztec software toolhttps://ndb.nal.usda.gov/ndb/search/list via GitHub 2/2016) was used as a basis and pre-selected for foods for which all relevant amino acids and macronutrients were reported and which contain at least 200 mg of TRP per 100 g. Both TRP/LNAA and TRP/tyrosine ratios were computed and used as sorting criteria. For both sorting criteria, the 25 highest were selected and due to their great overlap, the final list consists of 29 TRP-rich foods (see supplement).A list of foods rich in TRP was used to extract a natural TRP score from the food diaries. The Food Composition Database SR-26 of the United States Department of Agriculture . The natural TRP score is calculated as sum of units over all 28 days. In addition, all food items were grouped into categories, e.g. meat or legumes, and summed up.44. We used age as a covariate as we were especially interested in the effect of age in this research project. The other four covariates were mainly used to control for possible influencing effects .We conducted rmANCOVAs with age, sex, 5-HIAA baseline values, BMI, and order (5-HTP at t1 or t2) as covariates, regressions and correlations with IBM SPSS 25 . Effect sizes were calculated with G*Power 3.1.9.2rmANCOVAs detecting a possible 5-HTP effect were conducted with values from t1 and t2 . rmANCOVAs detecting a possible TRP effect were conducted with t3 values and the placebo condition values from t1 or t2 as a baseline . Please see also supplemental Table Further, we extracted the first eigenvariates (corrected for covariates) of the brain regions significant in MRI results as marker of change in brain activity. Food diary data was used in correlational and 3 regression analyses  where baseline values  were included as additional predictors.1,70 = 0.18, p = 0.670, f = 0.05), but there was a significant effect of the covariate age  was not related to 5-HTP intake . The ratings of the other story categories showed no significant substance effect .With respect to the moral judgement task, even after controlling for covariates there was a main effect of substance showing that the behavior of the protagonist in NP stories was rated as more reprehensible with 5-HTP . Importantly, with respect to the TRP-enriched diet there was a significant effect of the covariate age on emotion recognition  which disappeared for positive emotions with TRP . In addition, a small but non-significant effect of natural TRP regarding the recognition of positive emotions was identified .Regarding the RMET, no significant effects of 4-week TRP supplementation on emotion recognition were found . As for the moral task behavioral effects were only found for NP stories , all following analyses were only applied to these. A significant effect of natural TRP on the rating of NP stories could not be found , whereupon baseline NP ratings , 5-HIAA  and order  were significant predictors.Regarding the moral task, individuals who had received TRP rated the protagonist’s behavior in NP stories not significantly more reprehensible than those who received placebo  but the effects especially in the TPJ were much smaller (peak voxel t = 2.34).For the RMET, the main effect (eyes > plants) found at punc. < 0.001 with a minimum cluster size of 100 voxel. 4-week TRP supplementation was also not related to any neural activation differences compared to placebo in the RMET . For the moral task, a substance effect for NP stories was found. Reading these stories was associated with more activity in posterior insula and supramarginal gyrus in the TRP group. This pattern of results did not change when including the scanner as covariate .The parametric modulation of fMRI time series during reading NP stories and the NP ratings did not show any significantly active brain areas for TRP compared to placebo. The correlation of the activity changes in the two significant clusters and the changes in reprehensibility ratings did not show significant relationships .After 5-HTP, better recognition of positive emotions and worse recognition of negative emotions were associated with higher reprehensibility ratings . Further, a higher BMI was associated with less dietary/natural TRP  and lower excretion of 5-HIAA . Meat consumption (different types were not considered separately) correlated negatively with urinary 5-HIAA , whereas legumes showed a positive, but insignificant correlation .An additional analysis of the effect of TRP on emotion recognition in higher age (40 +) group showed a significant positive effect  or sub scores . There seemed to be no effect of time for the total EQ scale . Cronbach’s α of the EQ at t3 was 0.72 for the total scale, 0.93 for cognitive empathy, 0.86 for emotional reactivity, and 0.63 for social skills.Regarding the empathy quotient (EQ), TRP had no effects on the total scale  with the reprehensibility ratings after receiving 5-HTP give us reason to speculate that the effect of TRP might depend on the specific cognition related empathy-processes at hand. This would fit to the findings of other authors, who propose perspective-taking in moral situations as an important factor in their evaluation3 or report that the feeling of guilt was specially elevated in highly empathic participants under TRP depletion when completing a task on social situations including unjust harm46.The most prominent differences were found for the moral stories in which the protagonist behaved badly although knowing that it might have a negative outcome but this failed to materialize. Normally, people are more outcome oriented47. Brain regions related to empathy for pain were also previously found to be involved in integrating harmfulness information2. For that reason, TRP or 5-HTP intake might improve serotonin availability in the brain and the integration of all available information and therefore accentuate the relevance of the behavior and belief of the protagonist in contrast to the outcome. Curiously, both, the parametric modulation of the ratings with fMRI time series and the correlation between brain activation and the ratings revealed no significant associations. This could be related to a power  or dosage problem but might also indicate that the better integration of information on e.g. beliefs does not automatically lead to the use of this information during evaluation. In short, we speculate that our fMRI results point more to an altered information integration by TRP but further studies are needed to get deeper insights here. Importantly, brain activation was corrected for covariates like 5-HIAA which might has reduced possible associations as 5-HIAA levels explained a significant part in the found behavioral effects.However, in contrast, the subscales of the empathy questionnaire did not show significant changes with TRP either. This might be a problem of the self-rating procedure or suggest that the effects are not primarily related to empathy or perspective-taking. Other authors found the TPJ to be active during moral judgement when encoding beliefs and integrating information49. Additionally, the TRP requirement in the body can vary with some diseases or BMI51 so that the availability in the brain is reduced when more TRP is metabolized in other parts of the body e.g. due to baseline deficiencies. Also in our sample, a higher BMI was associated with lower excretion of 5-HIAA. Therefore, even in healthy populations weight might have effects on TRP availability. All participants got the same dosage, which was necessitated by the double-blind protocol, and considered appropriate, given that all participants were healthy and of normal weight. Nevertheless, it might have been suboptimal for some of the participants and on average especially for TRP the dosage might be too low. Furthermore, participant’s natural diet showed that people with higher BMI consumed less TRP. This could be an additional reason why 5-HIAA levels are lower or this even intensifies a possible lack of TRP in these individuals. In addition, as the placebo bars contained also protein but only a very small amount of TRP, the effects might partly be additionally triggered by other LNAAs. The influence of meat consumption (often tyrosine rich) versus legume consumption (often TRP rich) on 5-HIAA levels may hint to this point, too. But of course several other dietary components like carbohydrates play important roles in this metabolism as well and should be taken more into account. The same is valid for including e.g. urinary kynurenine values to better account for other serotonin metabolites. As some authors found a shift in the tryptophan metabolism from serotonin to kynurenine for different mental disorders52, this might be also of importance in a healthy population especially for vulnerable individuals. As these values were not available in this study, this can be seen as a limitation, which should be addressed in the future.As urinary 5-HIAA is a serotonin metabolite, its excretion indicates that there was a sufficient amount of TRP or 5-HTP in the body to metabolize serotonin. Several processes can influence 5-HIAA levels e.g. the reuptake of serotonin, melatonin production or external variables like alcohol or caffeine53 but for the present whole sample, the positive effect of TRP on emotion recognition was not significant. In a larger sample habitual TRP consumption was related to emotion recognition54. Importantly, in line with our hypotheses and research proposing an altered serotonin metabolism in higher age37, we found age to be a significant contributing factor to TRP and 5-HTP effects. In older participants, especially the recognition of positive emotions was improved after TRP. This might point to a compensatory effect of TRP in older individuals who could improve social cognitive processing with a TRP-rich diet. Of course, those results should be seen critically and need replication, especially also in samples with older participants. Accordingly, also genetic and epigenetic features seem to play an important role in this pathway55, the monoamine oxidase A high-activity allele together with the degree of methylation at a promoter CpG site on the tryptophan hydroxylase 2 gene explain significant quantities of variance in the RMET performance, pointing to people at risk might profit more from TRP supplementation or a TRP rich diet. This and the possible age-related effect could imply that especially people with a lack of TRP or other metabolic variations which have influence on the serotonin metabolism are those who profit most which is why effects are harder to detect in mixed samples including also individuals with a normal serotonin metabolism.We find first hints for a positive influence of TRP on recognition of positive emotions and of the better recognition on reprehensibility ratings which was even more pronounced for 5-HTP. The opposite effect, worse emotion recognition after TRP depletion, was found before56 and multiple sclerosis patients57. As ratings might be influenced by the wording and form of questions asked when evaluating moral behavior45 this might have had a further influence on learning processes (e.g. asking for reprehensibility vs. permissibility). However, we aimed at reducing this influence as much as possible by structuring the stories following uniform rules and using the same question with a rating scale for all stories.Importantly, analyses of natural TRP on moral stories showed an effect of the time point of 5-HTP administration which could imply related altered learning and memory effects. Such effects could be found before in animalsAn important limitation of the study is that some effects were only significant in one condition and not in both (TRP and 5-HTP). Due to the different designs of the two parts of the study, we had 77 participants for the 5-HTP part but only 36 for the TRP part. This might have led to power problems especially in the latter one. Additionally, for detecting TRP effects the tasks were applied thrice and for the 5-HTP effect only twice which might had an influence too. This could explain the non-finding of behavioral changes with TRP but cannot explain that possible 5-HTP effects were not detected on the chosen threshold with fMRI. It should be a point of future studies if this is also true for larger sample sizes, higher 5-HTP doses or another application of the tasks.In sum, even in healthy individuals who have lower TRP availability or an altered serotonin metabolism , TRP or 5-HTP supplementation seems to have a positive influence on the recognition of positive emotions and the integration of information in moral judgements. Besides above mentioned factors, the 5-HIAA level, indicative of serotonin status, might be an additional rough measure for individualized diet recommendations. Notably, future studies should use not only individualized dosages of TRP, but also include more biological and psychological factors to deepen our knowledge of this important pathway influencing our social behavior, and physical and mental health.Supplementary Information.


Microglia play an important role in the pathogenesis of multiple sclerosis and the mouse model of MS, experimental autoimmune encephalomyelitis (EAE). To more fully understand the role of microglia in EAE we characterized microglial transcriptomes before the onset of motor symptoms (pre-onset) and during symptomatic EAE. We compared the transcriptome in brain, where behavioral changes are initiated, and spinal cord, where damage is revealed as motor and sensory deficits. We used a RiboTag strategy to characterize ribosome-bound mRNA only in microglia without incurring possible transcriptional changes after cell isolation. Brain and spinal cord samples clustered separately at both stages of EAE, indicating regional heterogeneity. Differences in gene expression were observed in the brain and spinal cord of pre-onset and symptomatic animals with most profound effects in the spinal cord of symptomatic animals. Canonical pathway analysis revealed changes in neuroinflammatory pathways, immune functions and enhanced cell division in both pre-onset and symptomatic brain and spinal cord. We also observed a continuum of many pathways at pre-onset stage that continue into the symptomatic stage of EAE. Our results provide additional evidence of regional and temporal heterogeneity in microglial gene expression patterns that may help in understanding mechanisms underlying various symptomology in MS. In healthy and developing brain, microglia interact with neurons and play a significant role in synaptic pruning, neuronal wiring and synaptic plasticity2. These cells are also the “first responders” to changes in homeostasis in the brain parenchyma3. Under physiological conditions, microglia exist in a ramified state and are highly motile as they extend their fine processes to survey their surroundings. When microglia become activated in response to changes in homeostasis, they retract their processes and become more ameboid4. However, this morphological correlate of microglial activation state has been challenged in the past decade5. Transcriptional profiling of microglia has contributed immensely to the understanding of the microglial function6. Microglial activation has been associated with several neurological disorders including Alzheimer’s disease7, amyotrophic lateral sclerosis8 and multiple sclerosis9.Microglia are the resident myeloid cells in the CNS that play an important role in brain function in both homeostatic and disease states10. MS may either be associated with, or occur independently of neuronal loss12 that results in sensory and motor impairments. While MS may exist as more than one subtype14, experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that recapitulates several features of MS including motor and sensory alterations associated with demyelination, synaptic and neuronal loss and inflammation of the CNS18. EAE is induced by provoking an antibody response using a myelin associated antigen given in conjunction with Complete Freund’s adjuvant and pertussis toxin. The latter two compounds provide an early innate immune response to initiate a subsequent T cell invasion and attack of CNS myelin. We and others found that EAE animals develop changes in coping and anxiety-like behaviour prior to the onset of motor symptoms (the pre-onset stage)24. As we found changes in synaptic function at this early stage of EAE, we explored the contribution of microglia because of their known involvement in synaptic pruning25. We found that early, pre-symptomatic EAE was associated with an altered microglia phenotype in the basolateral nucleus of the amygdala26. At the electron microscopic level, we found increased process area, increased perimeter and reduced extracellular digestion in the microglia of EAE animals compared to controls, which was indicative of a de-activated phenotype27. This observation contrasted with the expected, activated microglia phenotype usually found in inflammation1. Even though these animals showed behavioural changes, we did not observe any T cell invasion and demyelination in the brain at this early stage of disease19. In contrast, later stages of the disease, (associated with profound motor impairment) feature the well-known T cell invasion, synaptic impairment and associated demyelination18. This continuum of innate (in early EAE) and acquired (late EAE) immune response provides an opportunity to characterize microglial phenotype under different inflammatory conditions. This is made relevant by the appreciation of significant interactions between the innate and acquired immune responses within the brain in MS28. Furthermore, the progression of the inflammatory immune response in EAE in different parts of the central nervous system allows us to expand on the increasing evidence for phenotypic and functional differences in microglial populations by comparing brain and spinal cord. This is important because disease pathology in the grey matter of central nervous system is quite different from that of the largely white matter of the spinal cord12 and there is also compelling evidence that microglia derived from the cortex and spinal cord display different phenotypes29.Multiple sclerosis (MS) is an inflammatory disease of the nervous system characterized by cerebral and spinal cord white matter demyelination, inflammation and axon injury and degeneration30. Furthermore, it has been suggested that the traditional mechanical dissociation and isolation methodology may not capture the ribosomes that are located in the fine processes of the microglia31. Thus, isolating mRNA bound to ribosomes enabled us to study only the RNA that is ready to be translated rather than the global transcriptome of microglia. Using RiboTag technology, we compared microglial transcriptomic profiles of the brain and spinal cord in both the pre-onset stage of EAE, when behavioral alterations, but not motor difficulties are observed, and in symptomatic animals that display gross motor disability associated with demyelination. While there has been a previous study on microglial transcriptomics in EAE 32—it used pooled spinal cord and CNS tissues, thus not differentiating between microglial functions associated with the motor and sensory changes involving the spinal cord, and those associated with the behavioral changes that would be more evident in higher brain regions. To the best of our knowledge, this is the first study comparing the transcriptomic profile of microglia in the pre-onset and symptomatic brain and spinal cord in EAE.In order to better understand the microglial activation state during pre-onset and symptomatic stages of a disease, we have conducted a transcriptomic profile of the microglia in the brain and spinal cord from EAE animals using RiboTag technology. This technology allowed us to isolate ribosome-bound mRNA from the frozen brains by immunoprecipitation, without tissue dissociation methods, which has been shown to induce microglial activation markers and also has the potential to be associated with cargo contaminations and transcripts that are sequestered from the ribosomes10. Eight-ten week-old C57BL/6 N female mice , housed under specific pathogen free conditions, 4/cage, on a 12:12 h light cycle were used in this study. EAE was induced by subcutaneous immunization with 100 μg myelin oligodendrocyte glycoprotein (MOG35–55) in emulsion of a 1:1 volume with complete Freund’s adjuvant . The mice were also injected with intravenous Bordetella pertussis toxin  in 1X Phosphate Buffered Saline (PBS) at the time of, and two days following, immunization33. In each cohort of mice, subjects were randomly chosen for either the EAE or naive group so that equal numbers of each group were represented. Animals were assessed for motor deficits associated with EAE as previously described19; animals described as “pre-onset” that were used 8 days after EAE induction were verified to lack the motor weakness that typically emerges only after 10–13 days in our hands. Symptomatic animals had a score of 2–3  as previously described19 and showed tail and hindlimb paralysis. For all experiments, we used three naïve animals, three pre-onset animals and three symptomatic animals.All animal protocols were approved by the Health Sciences Animal Care Committee of the University of Calgary, following the guidelines of the Canadian Council on Animal Care. All experiments and use of animals complied with the ARRIVE guidelines. Female mice were used throughout the study as the incidence of MS in females versus males is 3:1Rlp22HA mice 34 were crossed with male homozygote Cx3cr1CreER(B6.129P2(Cg)-Cx3cr1tm2.1(cre/ERT2)Litt/WganJ, Stock # 021,160, Jackson Laboratories) mice to generate Cx3cr1CreER: Rpl22HA mice30. The floxed Rlp22HA allele bearing mice carry three copies of hemagglutinin (HA) epitope that is inserted after a ribosomal protein Rlp22 allele with a floxed C-terminal, and the HA terminal is positioned just before the stop codon34. CX3CR1, a chemokine receptor, is highly expressed in myeloid cells including monocytes and macrophages and has been widely used as a microglial marker36. In the Cx3cr1CreER allele bearing mice, Cre recombinase is expressed under the control of the Cx3cr1 promoter, and tamoxifen injection allows the estrogen receptor fused Cre-recombinase to gain entry into the nucleus and cause loxP dependent recombination. Mice homozygous for Rlp22HA were crossed with Cx3cr1CreER mice to generate Rlp22HA:Cx3cr1CreER mice, which were then treated with tamoxifen to induce Rlp22HA expression in the microglia/macrophages  and the lumbar spinal cord and subcortical regions of the brain anterior to the pons  were microdissected on ice and snap-frozen in dry-ice. RNA was isolated from the fresh frozen samples using the TRAP (translating ribosome affinity purification) approach40 based on a published protocol34 with some modifications. Briefly, the tissue was homogenized in polysome buffer and the supernatant from the lysate was incubated with 50 µl of anti-HA magnetic beads  for 2 h at 4 ˚C. Bead bound RNA was recovered by magnet, washed with high salt buffer three times and purified using RNeasy Micro Kit .The Iba1) as well as non-microglial marker genes  between the RiboTag RNA (immunoprecipitated (IP) RNA) and the RNA from the whole tissue (Input RNA) by qPCR. An equal amount of RNA from the IP RNA and input RNA was used to generate cDNA using the High Capacity cDNA Reverse Transcription Kit . qPCR was performed using TaqMan Gene Expression assay kit (Applied Biosystems) with the probes for the following cell type specific marker genes; Iba1 (Mm00479862), Gfap (Mm01253033), Map2 (Mm00485231), Mbp (Mm01266402) and Gapdh (Mm03303349), which was used as an internal standard. Triplicates of each cDNA were amplified by Step One Real Time PCR system (Applied Biosystems). The results were analyzed using Step One Software (Applied Biosystems) and the enrichment for each marker gene was represented as relative quantification (RQ) value reflecting the relative amount of the transcript in the IP RNA compared to input RNA by ΔΔCT method.In order to verify that the RNA isolated by the RiboTag method is enriched for the transcripts from microglia, we compared the relative amount of mRNA of a microglial marker gene , then perfused with PBS and 4% paraformaldehyde (PFA). Whole brains were dissected, and fixed in 4% PFA overnight at 4 °C. The brains were placed in 20% sucrose + PBS solution overnight, embedded in optimal cutting temperature (OCT) compound, and then flash frozen. Blocks were cut into 20 µm sections on a cryostat. The sections were then incubated with anti-GFP  and goat anti-HA  overnight. The tissues were washed in PBS and incubated with a donkey anti-rabbit-Cy3 antibody  and a donkey anti-goat-Alexa488 antibody , at room temperature for 1 h. The sections were then mounted using glass slides in bicarbonate-buffered glycerol at pH 8.6 containing 1 µg/ml DAPI, visualized with a Zeiss Axioplan fluorescence microscope, and photographed with a digital camera.41, using the Mus musculus GRCm38 cDNA sequences as reference. Kallisto was run with the sequence bias correction and 50 bootstraps. Statistical analysis was done in R v3.3.142. For the parametric analysis, Sleuth43 was used for differential gene expression using a linear model with the day and tissue factors as independent explanatory variable, plus a factor interaction term (synergistic effect). A custom filter function was used that keeps any transcript with at least 5 reads in > 47% of samples from any factor-combination. Transcripts passing the Wald test with Benjamini–Hochberg corrected p values  less than 0.05, and with at least twofold increase or decrease in transcript expression were considered differentially expressed.100 ng of high-quality total RNA was purified using poly-T magnetic beads. The poly-A mRNA was then fragmented to ~ 200 bp and first- and second-strand cDNA synthesis was done, preserving strand specificity. The resulting double stranded cDNA was converted into adapter-ligated libraries which were amplified, quantitated by qPCR, and sequenced on two 75-cycle NextSeq500 sequencing cartridges to generate the data. Transcript abundances were quantified with Kallisto v0.43.0 p < 0.05.For Fig. Cx3cr1CreER: Rlp22HA mice following tamoxifen injection, we carried out immunofluorescence analysis of the brain tissue of these mice (n = 3). The CX3CR1 + cells in the Cx3cr1CreER carried GFP and hence microglia were identified by using antibodies to GFP. Immunolabeling of GFP and hemagglutinin revealed that HA expression was exclusively limited to GFP-expressing cells in the subcortical regions examined, including hippocampus, hypothalamus and amygdala. A sample micrograph of the immunohistochemical labeling in amygdala is shown in Fig. The RiboTag technology allows isolation of ribosome bound mRNA from microglia as described in the schematic Fig. p = 0.0066), and Tukey's post-hoc test revealed significant enrichment (p < 0.05) of Iba1 compared to Gfap, Map2 and Mbp with no other significant group differences. The enrichment level of Iba1 was 135.8 ± 44.34% while those of Gfap, Map2 and Mbp were 2.8%, 7.26% and 2.7% respectively. Thus, we were confident that we were isolating microglia-specific RNA.We further verified the enrichment of cellular markers for astrocytes, microglia, neurons and oligodendrocytes using GFAP, IBA1, MAP2 and MBP respectively in the isolated, microglia-specific IP RNA quantified by qPCR. The enrichment in the microglial IP RNA was compared to the total RNA before IP in 3 animals , using all mapped genes, showed separation of all the groups and samples clustered in a tissue and disease state dependent manner Fig. A. The diFalse Discovery Rate < 0.05, and two-fold up or down regulation) were chosen for further analysis where they will be referred to as differentially expressed genes (DEGs) from hereon. We first examined if there were any changes at the pre-onset time point in the DEG in the microglia and found that 289 genes were enriched and 64 genes were depleted in the brain, while in the spinal cord 287 genes were enriched and 41 genes were depleted showing that there were roughly comparable number of genes altered in the brain and the spinal cord .We ran an analysis to ensure residual macrophages were not influencing differential gene expression, using p value 1.54e−15; Z-score 4.123). Seventeen genes in this pathway were upregulated in the process, with the chemokine Cxcl10 showing the highest expression (Log2 Fold change 2.864) followed by tnf (Log2 Fold change 2.759). Tnf was the most activated gene for the causal network and IPA showed that this cytokine is a master regulator of 169 of these genes in the chemokine/cytokine signaling pathway  and TNF exerts its effect through 12 intermediary factors, including IL1β, IFN-g, NF-kB complex, NF-KB1, IRF-1, STAT1, STAT6, STAT3, RELA, REL, NFkBIA and CDK2A  and G2/M checkpoint signaling was downregulated , which indicates that there is enhanced cell-proliferation  of the tissues at the pre-onset stage showed that both tissues showed upregulation of 24 pathways and downregulation of three pathways . Specific to spinal cord at the pre-onset stage, twelve genes  related to the formation of cellular protrusions were upregulated .C3 was upregulated in both brain and spinal cord at the pre-onset stage. Combining the known enrichment of genes involvement in cell motility from IPA knowledge-base with the observed causal network with our gene expression data at pre-onset stage, we posit that C3 has a regulatory effect on cell motility by its effect on 10 genes, namely p value = 1.82e−29), pathways linked to pattern recognition receptors , hypercytokinemia/hyperchemokinemia , neuroinflammatory signaling pathway  and Toll-like receptor signaling . There was also upregulation of T-cell linked pathways, including Th1 pathway , Th2 pathway  and iCOS-iCOSL signaling in T-helper cells . Of note, there was a strong downregulation of PD1 pathways, some of which were related to self-recognition of immune system at the symptomatic stage. This was especially strong in spinal cord. This was on a background of spinal cord generally having higher PD1 activity .In the symptomatic stage of the disease Fig. , both tiCdkz2a, Birc5, Cdkn1a), but much stronger induction in both tissue types at the symptomatic stage. A few genes were constitutively less expressed in spinal cord compared to the brain , but additional genes were only downregulated in symptomatic spinal cord  which led to opposite p53 pathway activity signals in IPA at symptomatic stage for the two tissues. We also observed changes in IL-17 signaling in EAE, with both tissues strongly affected at the symptomatic stage (Z-score = 5.7) but also an early signal at the pre-onset stage (Z-score = 2.6).Figure This study has revealed a number interesting findings concerning microglial transcriptional regulation throughout various stages and in various tissues in the EAE mouse model of MS. Principal Component Analysis of the RNAseq data revealed that the brain and the spinal cord samples clustered separately at both stages of EAE. When we compared the gene expression profile between pre-onset and symptomatic EAE mice in the brain and the spinal cord, we observed changes both in the pre-onset and symptomatic stages of EAE that differed somewhat between the brain and spinal cord.30 showed that microglial RNA isolated using Ribotag technology was superior to traditional cell-sorting methods in that it avoided incurring artifacts arising from tissue dissociation, cargo contamination and transcripts de-sequestered from ribosomes.In this study, we isolated microglial transcriptomes from the spinal cord and the brain of EAE animals using RiboTag technology. To the best of our knowledge, this is the first study in which this technology has been exploited to study the transcriptome in the brain and spinal cord microglia in the context of EAE. Haimon et al.30 showed that the usage of Cx3cr1CreER mice conferred higher specificity of microglial cell-type expression of the HA-tag compared to Cx3cr1Cre. They found that the HA tag was restricted to microglia when Cx3cr1CreER mice were used and our immunohistological examination suggested a similar conclusion. A problem when attempting to understand microglial function is that in the symptomatic stage of EAE, macrophages enter the CNS from the periphery. In order to mitigate isolating mRNA from this population of cells, we waited for one month prior to EAE induction so that there would be turnover of macrophages containing the RiboTag39. However, we concede that some non-parenchymal brain macrophages may have long half-lives like microglia and hence might still be present with an HA-tag44. We addressed this concern by using Meg3 gene expression level as a proxy for the macrophage and found that its omission or inclusion did not materially affect the DEGs in our existing linear model. Thus, if invading macrophage RNA was in our sample, it was present in insignificant amounts.Haimon et al.45. This study pointed that while there were many genes upregulated at the RNA level, the numbers of upregulated proteins were far less. Thus, we are cautious about our interpretation of the ribosome bound RNA as the translatome. We are unable to isolate only microglial-derived protein in sufficient abundance for quantification of specific proteins, and since the large majority of the RNA we are quantifying from bulk tissue is not microglial-specific, protein analysis by Western blots or ELISAs from whole, multi-cellular tissue would not necessarily apply to microglia; nor would it be able to differentiate between brain/spinal cord produced molecules and those that might arise from the macrophages and other immune cells that cross into the brain through the compromised blood brain barrier in latter stage EAE46. Nonetheless, it should be noted that for a number of the microglial predominant DEGs we found to be altered in EAE, the genes or their protein products have previously been shown to be altered, even in whole brain or spinal cord tissue. Relevant examples include products of the complement cascade49.The RiboTag technology enabled us to isolate the RNA bound to the ribosome and therefore we focused on the genes that were actively translated and contributing to the cellular translatome. However, one study showed that there is dissociation between the levels of ribosomal bound RNA and the protein level in microglia after an LPS challenge50 . We observed many of the same directional changes as well as the specific DEGs identified in this previous study. Below we discuss the changes in different stages of the disease.It is difficult to compare our data with other studies as no one so far has investigated the differences in microglial gene expression between the different stages of EAE in brain and spinal cord separately. However, it has been shown in microglia isolated from intact brain and spinal cord and subjected to FACS sorting that RNA phenotype changes with disease progression19 showed that there was modest increase only in Il-1ß transcripts in the hypothalamus  at the pre-onset stage. However, using the RNAseq strategy, we found that there was increased activation of a number of pathways related to cytokines/chemokines. This indicates that analysis of a specific identifiable cellular population gives a more granular data that may not be apparent in the examination of whole, multi-cellular tissue. Cytokines such as CXCL10 and TNF, that were found to be upregulated in the pathway analysis are secreted by microglia and exert several downstream effects not only on immune regulation, but also, in the case of TNF, on synaptic plasticity, neurogenesis and astrocyte function51. For example, it has been shown that TNF changes AMPA receptor current52 similar to what we saw in our earlier work26. TNF is also an important molecule in cellular immunity, in particular T cell function53 and it is no surprise that we found it to be the most activated gene for the causal network. This is in keeping with extensive evidence for secretion of TNF by microglial cells in EAE54 and an important role for TNF in the pathology of MS and EAE55. CXCL10 is a chemokine that binds to CXCR3 and thus is a chemo-attractant for activated T cells and natural killer cells56. There is evidence its upregulation in MS as well as in EAE and its expression in advance of evidence of T cell invasion in the pre-symptomatic stage would be consistent with its role in helping to initiate subsequent T cell invasion57.Previous data from our lab58, where it has been shown to be causally involved in microglial dependent synaptic pruning59. In the pre-onset EAE amygdala, we previously implicated downregulation of C3 in the increased numbers of dendritic spines and augmented excitatory synaptic activity seen at this early stage. This inference was based upon reduced mRNA expression for C3 mRNA, but this did not seem to be localized specifically to the microglia26. Likewise, in the current study we saw a generalized upregulation of the complement system in microglia. This is in keeping with an extensive body of evidence reporting upregulation of the complement system in MS and EAE60. This was seen in both brain and spinal cord; in the former, it has been implicated in the extensive synaptic pruning and synaptic degeneration seen in these disease states. An upregulation of the complement system at this early stage of EAE has not previously been reported, to the best of our knowledge, but the fact that C3 has a regulatory effect on cell motility, which was a highly upregulated pathway, would be compatible with a role in synaptic pruning. Likewise, the upregulation of numerous genes related to cellular protrusions may point to dynamics of microglial processes that are highly motile both in performing immune surveillance and in engulfing material for phagocytosis.Complement proteins in the brain are membrane expressed proteins found in microglia that interact with receptors on target cells to initiate phagocytosis. There is now extensive evidence that complement plays an important role in normal brain function61. It has been demonstrated that the rate of microglia proliferation significantly increased in the early stage of EAE before T cell or monocyte infiltration in the CNS62. The proliferation of microglia seems to occur specifically early in the pathogenesis and correlates with our finding that genes and pathways regulating cell proliferation were activated during the pre-onset stage of EAE. Although microglia may not play an active role in tissue damage, the proliferation of tissue resident microglia in the early stage of EAE has been shown to be important in subsequent events during the pathogenesis64. Thus, it can be speculated that the initial increase in microglial population may be a mechanism to maintain the microglial population in the tissue during the microglial activation.The pathway analysis revealed that there is upregulation of kinetochore metaphase signaling and downregulation of G2/M damage pathways at the pre-onset stage. These two pathways are related to cell division and the changes in expression of the related genes suggest progression of cell cycle and proliferation of microglia. In resting states, microglia are known to have a low mitotic rate, but their proliferation capacity increases after activation32 where genetically labeled microglia and invading monocytes were flow sorted and RNA expression of a number of inflammation related genes was analyzed at disease onset, there was a general downregulation of a number of metabolic and activation pathways. As noted above, we saw downregulation of pathways linked to cell division (G2/M damage checkpoint regulation) and LXR/RXR signaling, but we did not see a significant alteration of pathways linked to metabolism.In an earlier study47. This may be due to an inability in the previous study to discriminate between cells, for example invading monocytes and microglia that can show divergent expression patterns32 and thus effectively cancel each other out, or perhaps may reflect the agnostic nature of the RNAseq in our study that provides a more extensive survey relative to the more targeted neuropathology panel of the earlier study.Many more pathways were activated in the symptomatic stage compared to pre-onset. Of note is that using the same twofold cutoff, but applied to specific neuropathology panel encompassing only 770 genes, another study reported differentially expressed genes in whole brain and spinal cord tissue that were markedly lower in number than what we describe here65 and it is conceivable that TREM1 may also facilitate phagocytosis in EAE models. Interestingly, TREM1 signaling did not cross the statistical threshold in the pre-onset stage. TLR and pattern recognition signaling receptor pathways were also upregulated. We know that these pathways in microglia mediate innate immunity and also link with the adaptive immune response, providing an important mechanism by which microglia are able to sense both pathogen‐ and host‐derived ligands within the CNS66. In an acute model of EAE, it has been shown that the activated microglia acquire an immature dendritic cell phenotype and may terminate the immune response67. This may be reflected in activation of the pathway linked to dendritic cell maturation. The pathways linked to T-cell signaling may be a reflection of T-cell-microglia interaction that occurs in the EAE. Activated microglia interact with the encephalitogenic T-cells and exacerbate their capacity to induce injury68.TREM1 signaling, which was upregulated in the symptomatic stage, facilitates microglial phagocytosis in some disease conditions69. There is evidence that T cells enter the CNS through the lumbar region (L5) of spinal cord at early stages of EAE70. With that knowledge, we expected that we would see increased number of DEGs in the spinal cord compared to the brain in the pre-onset stage. To our surprise, we found that the changes in the brain and the spinal cord were somewhat comparable.In our study, we could not probe the regional variation within the brain because the ribosome-bound RNA yield from each region using RiboTag technology was very low. We decided to utilize the sub-cortical region of the brain, which includes amygdala, hippocampus, thalamus, basal ganglia and hypothalamus, all being grey matter regions known to be important in behaviour. Concurrently, we analyzed the transcriptome from the spinal cord because demyelination is prominent in the spinal cord of the EAE mouse50. In our dataset, antigen presentation showed up without any activation pattern (Z-score not applicable) and these differences could be a factor of method of microglial isolation.An earlier study compared the pre-onset and symptomatic stages of RNA expression derived from FACS sorted microglial cells and found that oxidative phosphorylation, protein ubiquitination, antigen presentation and mitochondrial dysfunction were all highly enriched in the symptomatic compared to pre-onset groupThis is the first study to our knowledge that has analyzed the microglial transcriptome in EAE from the brain and the spinal cord using the RiboTag technology. The microglial transcriptome analysis showed up- and down-regulation of many pathways in tissue and disease state dependent manner, thereby opening door for future in-depth analysis on each of these pathways. Of particular importance is the fact that we have identified certain unique alterations in gene expression well before onset of classic motor and sensory symptoms emerge. These findings may be instructive in addressing some of the behavioral issues that are associated with MS even in the absence of active disease symptomology.Supplementary Information 1.Supplementary caption.


Arthroscopic subacromial decompression is one of the most commonly performed shoulder surgeries in the world. It is performed to treat patients with suspected shoulder impingement syndrome, i.e., subacromial pain syndrome. Only few studies have specifically assessed return-to-work rates after subacromial decompression surgery. All existing evidence comes from open, unblinded study designs and this lack of blinding introduces the potential for bias. We assessed return to work and its predictors in patients with shoulder impingement syndrome in a secondary analysis of a placebo-surgery controlled trial.n = 57), diagnostic arthroscopy, a placebo surgical intervention, (n = 59), or exercise therapy (n = 68). We assessed return to work, defined as having returned to work for at least two follow-up visits by the primary 24-month time point, work status at 24 and 60 months, and trajectories of return to work per follow-up time point. Patients and outcome assessors were blinded to the assignment regarding the arthroscopic subacromial decompression vs. diagnostic arthroscopy comparison. We assessed the treatment effect on the full analysis set as the difference between the groups in return-to-work rates and work status at 24 months and at 60 months using Chi-square test and the predictors of return to work with logistic regression analysis.One hundred eighty-four patients in a randomised trial had undergone arthroscopic subacromial decompression .There was no difference in the trajectories of return to work between the study groups. By 24 months, 50 of 57 patients (88%) had returned to work in the arthroscopic subacromial decompression group, while the respective figures were 52 of 59 (88%) in the diagnostic arthroscopy group and 61 of 68 (90%) in the exercise therapy group. No clinically relevant predictors of return to work were found. The proportion of patients at work was 80% (147/184) at 24 months and 73% (124/184) at 60 months, with no difference between the treatment groups (Arthroscopic subacromial decompression provided no benefit over diagnostic arthroscopy or exercise therapy on return to work in patients with shoulder impingement syndrome. We did not find clinically relevant predictors of return to work either.NCT00428870.ClinicalTrials.gov identifier  Subacromial pain, shoulder impingement syndrome and rotator cuff disease describe the most common shoulder conditions. The syndrome is distinctively characterised by pain located subacromially while lifting the arm and has traditionally been attributed to ‘impingement’ of the rotator cuff tendons between the humeral head and the overlying acromion. Following to this logic, a surgical procedure called subacromial decompression was developed. It is a surgical procedure premised on smoothening the undersurface of the acromion, which decompresses the rotator cuff tendons passing through the subacromial space. With the advent of arthroscopy, arthroscopic subacromial decompression (ASD) became a safe, quick and convenient procedure, and thus became one of the most frequently performed orthopaedic surgical procedures in the world .A Cochrane review  and a BMWe conducted this superiority trial at three orthopaedic clinics  in Finland from February 1, 2005 through October 10, 2018. The study was registered at ClinicalTrials.gov . Details of the trial design and conduct have been published elsewhere [The study was conducted in accordance with the Declaration of Helsinki. All patients gave written informed consent. On entering the study, they were unequivocally informed that they might undergo diagnostic arthroscopy and that they would be allowed to consider crossing over to ASD if they did not have adequate relief of symptoms, preferably no sooner than 6 months after randomisation.We enrolled patients 35 to 65 years old who had subacromial pain (for more than 3 months) that was unresponsive to conventional conservative treatment and had clinical findings consistent with shoulder impingement syndrome. Magnetic resonance imaging with intra-articular contrast was performed on all patients to exclude a rotator cuff tear. Detailed inclusion and exclusion criteria are provided in Table In an attempt to obtain three balanced study groups of similar group size, we planned a two-fold, sequential randomization as follows: First, during the baseline appointment, patients were randomized to surgical or conservative treatment  in 2:1 ratio. Those randomised to ET started standardised physiotherapy within 2 weeks of the baseline appointment, while those allocated to surgery were scheduled for surgery aimed to be performed within 12 weeks of this first randomisation. In those allocated to surgery, a diagnostic arthroscopy was first carried out to rule out a rotator cuff tendon tear and other obvious intra-articular pathology requiring surgical treatment. If a rotator cuff tear large enough to require repair according to current clinical practice guidelines [Randomisation was carried out using sequentially numbered sealed opaque envelopes. Separate randomisation lists for each three centres, with blocks varying randomly in size, were prepared by a statistician with no involvement in the clinical care of participants in the trial.Supervised, progressive, individually-designed physiotherapy was started within 2 weeks of randomisation using a standardised protocol that relied primarily on daily home exercises as well as 15 visits to an independent physiotherapist . The detArthroscopic examination of the glenohumeral joint and subacromial space was performed with the patient under general anaesthesia. We performed an intra-articular and subacromial assessment of the rotator cuff integrity. If arthroscopic examination revealed any pathology requiring intervention other than ASD, the patient was excluded from the trial Fig. . Once thAfter arthroscopic examination of the shoulder , the ASD procedure that involved the debridement of the entire subacromial bursa (bursectomy) and resection of the bony spurs and the projecting anterolateral undersurface of the acromion, was carried out with a shaver, burr and/or electrocoagulation .In the ASD and DA groups, the postoperative rehabilitation was identical, consisting of one visit to an independent physiotherapist, blind to the group assignment, for guidance and instructions for home exercises.Questionnaires were administered at baseline and 3, 6, 12, 24, and 60 months after randomisation. Work status was inquired with the following answering four options: 1) At work; 2) On sickness absence; 3) Unemployed; 4) Retired. Work status at each time point was dichotomised (At work / Not at work). The dichotomous RTW (Yes/No) was defined as follows: the study participant was at work at the primary 24-month time point or was at work at least at two of the three earlier time points. We also calculated the trajectories of return to work using dichotomous data (At work/Not at work) per each follow-up time point.We originally powered the study to detect a difference of at least the minimal clinically important difference (15 points [The present study was primarily designed to ascertain whether ASD is superior to DA in RTW with the two outcomes (the primary comparison). We also included a pragmatic comparison of the relative benefits of ASD vs. ET (the secondary comparison).We primarily quantified the treatment effect on full analysis set basis as the difference between the groups in RTW rates up to 24 months and in work status at 24 and 60 months after the primary randomisation. In these analyses, the participants were included as randomized and we analysed all available data, the full analysis set. As a sensitivity analysis, we performed on-treatment analysis of unblinding, in which the participants were included as treated in case the participants in the DA and ET groups had received ASD after unblinding.P-values were calculated. We estimated the prediction model accuracy by calculating a confusion matrix  and calculated the positive and negative predictive values thereof.Categorical variables were analysed using Chi-square test and logistic regression analysis. We compared the rates of RTW between the study groups with Chi-square test. In the logistic regression model RTW was the independent variable and dependent variables were baseline work status (Yes/No), age (dichotomised by median 50.6 years), gender , physical work strain (dichotomised) and study group (three): Odds ratios with 95% confidence intervals and p value of .05 was considered to indicate statistical significance. The estimation of the logistic regression model parameters was conducted using R 3.5.2 statistical software.All analyses are secondary by nature and must be interpreted as exploratory and supportive only. A There were 281 eligible patients, of whom 71 were excluded  during the 24-month follow-up and of 170 patients (88%) at 60 months out of the 193 randomised  or at 60 months (Chi-square p = 0.94).At the primary 24-month time point, 147 patients (80%) were at work, 23 (13%) on sickness absence, 10 (5%) had retired, two (1%) were unemployed or students, and data of two patients (1%) was missing. The respective figures at 60 months were 124 (67%), 26 (14%), 2 (1%), and 14 (8%). Work status at the 24-month and 60-month time points by group on the full analysis set basis are shown in Table p = 0.93). In the ASD group 50 patients out of 57 (88%) returned to work at least at two time points, while the respective figures were 52 out of 59 (88%) in the DA group and 61 out of 68 (90%) in the ET group.Trajectories of RTW were virtually identical by time points between the study groups Fig. A. TrajecParticipation in working life  and age below the median  at baseline predicted RTW in the multivariable logistic regression (Table p = 0.49). In the ASD group, 2 participants underwent a consequent re-operation, manipulation under anaesthesia for one participant while the other first had acromioclavicular resection and then later manipulation under anaesthesia. In the DA group, there were seven ASDs and one ASD coupled with subscapularis tendon repair. In the ET group, the overall incidence of surgery due to persistent symptoms was 15/71. Of these, 14 participants underwent ASD, and one underwent acromioclavicular resection. Further, three participants experienced persisting symptoms after surgery and underwent consequent reoperations.After surgery, the incidence of persisting symptoms sufficiently severe to lead to unblinding of the study-group assignment was similar in the two surgically treated groups .Of the 29 patients who were unblinded, 24 (83%) returned to work while the respective figure for those who remained in their assigned study groups were 139 out of 155 (90%)  was not superior to diagnostic arthroscopy (DA) or exercise therapy (ET) in enabling patients to return to work over the course of up to 60-month follow–up. Participation in working life at baseline and younger age predicted sustainable return to work, but differences in participation in working life by these predictors were minimal at 24-month follow-up and the accuracy of the predictive model was unacceptably poor for clinical practice.There are three previous studies that have reported on RTW after treatment of subacromial pain syndrome, all unblinded, open trials comparing surgery to exercise therapy –18. The The existing literature concerning predictors of RTW among patients with subacromial pain syndrome is very limited. Engebretsen at al  found thAs surgery has been shown to produce a profound placebo response –23, the lack of any difference between the treatment groups in RTW leads us to conclude that our findings are not based on insufficient evidence, as in an underpowered study, but rather on evidence demonstrating the lack of a clinically relevant treatment benefit.Our study had some limitations. The study sample calculations for the trial were based on continuous variables and the present study is apparently underpowered to detect subtle differences in the dichotomous RTW outcome variables between the treatment arms. Moreover, the number of participants with RTW data available who completed the entire 2-year follow-up was 57 and 59 in the ASD and DA groups, below the pre-specified target of 68. However, the Our RTW outcomes were based on self-reports by questionnaires, which may be considered as a limitation. What mitigates this concern is having a study nurse checked the work status records at each follow-up visit, looking for inconsistencies, which likely improved the quality of the data. Also, sustainable RTW is typically defined as a stable full-time or part-time RTW to either original job or a modified job for a pre-defined period of, for example, at least 3 months, without reoccurring sickness absence . Our arbIn addition to our primary sham-surgery controlled efficacy comparison between ASD and DA, our study also included a pragmatic, exploratory secondary comparison between surgical and non-operative care (ASD vs. ET). In apparent contrast to four previous, similar randomised trials that found no benefit of ASD over various exercise therapy regimens , 13, 26,The absence of a clinically relevant benefit of ASD in the 2- and 5-year follow-up reports of this trial  has promThe results of this randomised controlled trial indicate that arthroscopic subacromial decompression provides no benefit over diagnostic arthroscopy or exercise therapy on return to work in patients with shoulder impingement syndrome. We also failed to find predictors for return to work that would be applicable to clinical practice. Along with previous controlled trials on the subject, these findings form a credible body of evidence that refutes the notion that patients suffering from subacromial pain syndrome will return to work faster if they have surgery. Given this body of evidence, patients should be made fully aware of the potential benefits and harms related to the various treatment options for subacromial pain and be invited to agree on a treatment plan as a fully informed participants in the shared decision-making between clinician and patient. At a policy level, we strongly encourage decision makers to consider alternatives to subacromial decompression for managing shoulder impingement symptoms.


There is limited data on pediatric ventilator-associated events (PedVAE) in the neonatal intensive care unit (NICU) setting, since the CDC mandated state reporting of these events in January 2019. This study sought to describe PedVAE rates and characteristics in the NICU population.Single-center case-control study of infants requiring mechanical ventilation in a 39-bed level IV NICU between January 1, 2018 and December 31, 2020. Baseline infant demographic, respiratory support and antibiotic use data was obtained and comparisons were performed between patients with potential PedVAEs and those without events.Two hundred and nine infants were mechanically ventilated. Two of the 126 patients ventilated for ≥4 days met CDC criteria for PedVAEs with a total of 3 events, and 32 (25%) received antibiotics with escalation of respiratory support, primarily for tracheitis.NICU-specific data on PedVAE is limited. Only 2 infants in the study period met the current CDC criteria for PedVAE with a rate of 0.9 events per 1000 ventilator days. The current CDC PedVAE definition might be inadequate to identify actionable VAEs to inform prevention efforts in the NICU population, and alternate indices could better characterize these events. Mechanical ventilation is an essential, life-saving therapy for infants with critical illness and respiratory failure . HoweverTraditionally, surveillance for VAEs was limited to VAP alone, but the most widely accepted VAP definitions are neither sensitive nor specific, due to the subjectivity of clinical and chest radiographic findings of pneumonia, key requirements of the CDC’s VAP definition , 6. The 2) and/or mean airway pressure (MAP) after a period of stability or improvement on the ventilator [VAE surveillance was initially not used in neonatal and pediatric locations based on recommendations of a separate working group the CDC organized in 2012 . Subsequntilator , 9.In a retrospective cohort study involving adult patients, VAEs were associated with more days to extubation, more days to hospital discharge, and higher mortality risk . In addiSt. Christopher’s Hospital for Children’s NICU, a 39-bed Level IV regional perinatal center, is located in Philadelphia, PA. Nearly 250 neonates are admitted annually; all patients are out-born and transferred in from regional NICUs. Admitted infants frequently require advanced surgical and pediatric subspecialty services.A database of admissions to the NICU is maintained by the Division of Neonatology for reporting purposes. All admissions from January 1, 2018 to December 31, 2020 were screened for use of mechanical ventilation. This single center, case-control study was conducted with a detailed retrospective chart review of these mechanically ventilated patients. Inclusion criterion was any patient in the NICU receiving mechanical ventilation via endotracheal tube or tracheostomy. Patients on extracorporeal membrane oxygenation were excluded from PedVAE surveillance for the duration of time when this support was in place, based on the CDC definition of PedVAE.This study was reviewed by the Institutional Review Board at Drexel University College of Medicine and was determined to be exempt (protocol: 190500718). The need for informed consent was waived as this was deemed exempt human subjects research. All methods were carried out in accordance with relevant guidelines, and the information was recorded by the investigator in such a manner that subjects could not be identified, directly or through identifiers linked to the subjects.2 or MAP values. The baseline period is defined as the two calendar days immediately preceding the first day of increased daily minimum MAP or FiO2. The daily minimum FiO2 is defined as the lowest value of FiO2 documented during a calendar day that is maintained for more than 1 h. Daily minimum MAP is the lowest value documented during the calendar day. For patients less than 30 days old, daily minimum MAP values 0–8 cmH2O are considered equal to 8 cmH2O for the purposes of surveillance. For patients ≥30 days old, daily minimum MAP values 0–10 cmH2O are considered equal to 10 cmH2O for the purposes of surveillance.Patients needed to be mechanically ventilated for at least 4 calendar days to fulfill PedVAE criteria. PedVAEs are defined by a 14-day period, starting on the day of onset of worsening oxygenation . A new PedVAE cannot be identified or reported until this 14-day period has elapsed [2 of ≥25% over the daily minimum FiO2 of the first day in the baseline period, sustained for ≥2 calendar days; or 2. Increase in the daily minimum MAP values of ≥4 cmH2O over the daily minimum MAP of the first day in the baseline period, sustained for ≥2 calendar days.A PedVAE is defined if after a period of stablity or improvement on the ventilator, the patient has at least one of the following indicators of worsening oxygenation: 1. Increase in the daily minimum FiOAdditionally, infants who were mechanically ventilated for at least 4 days and had a new antibiotic treatment course initiated during the time of mechanical ventilation were analyzed for worsening oxygenation. We coined these events as potential PedVAEs for the purpose of this study to identify another surrogate marker of events that require quality improvement interventions.n = 32) and the No-Event cohort (n = 94) were conducted using chi-square test of association for categorical variables and group t-test for continuous variables.All statistical analysis for this research was conducted using SPSS version 25.0. Descriptive data were reported for categorical variables using either count or percent for each category, and for continuous data mean with standard deviation. Comparisons between potential PedVAE/PedVAE cases  using greater than 32 as a reference, and birth weight measured categorically with greater than 2500 g as the reference. A reasonable sample size for a logistic regression model is at least 10 events per independent variable. For our study, we had 32 patients with a PedVAE/potential PedVAE. Therefore, three independent variables were included in the logistic regression. Reporting on the logistic regression included beta coefficient, p < 0.05) was used to determine significance for all tests. No corrections were applied to the data for multiple comparisons, and no missing value imputations were performed.Due to the exploratory nature of this analysis and alpha level of 0.05  were excluded because they were ventilated for less than 4 days  based on a sustained increase in her daily minimum MAP by 4 cmH20, and the second event occurred on DOL 181  based on another sustained increase in her daily minimum MAP by 5 cmH20.Of the 126 infants eligible for evaluation, only 2 patients met the CDC criteria for PedVAE during the 3-year study period. Both patients who developed PedVAE based on the CDC criteria were 25-week gestation infants, and Hispanic in origin. The first patient was male and developed a PedVAE on day of life (DOL) 40  based on a sustained increase in the daily minimum FiOEnterococcus faecalis. He ultimately succumbed to the infection within 24 h of the PedVAE. The two events in the second patient were treated for culture-negative sepsis for 7–14 days.In all three of these events, antibiotics were prescribed by the primary medical team. The first patient was 1 day post-operative from an exploratory laparotomy for necrotizing enterocolitis and had blood culture-proven sepsis with Upon further review of the cases where there was an escalation of respiratory support, but the event did not meet CDC criteria for a PedVAE, antibiotics were prescribed often. We next analyzed the 126 patients who were mechanically ventilated for more than 4 days and there was initiation of a new antibiotic course. Using this as a surrogate marker for a clinical deterioration while mechanically ventilated, respiratory support parameters were evaluated when antibiotics were initiated and an additional 30 patients with potential PedVAE were identified , had a lower birth weight (p < 0.001), spent a longer duration on the ventilator (p < 0.001) and had a longer NICU stay (p < 0.001) compared to those who did not have potential PedVAE/PedVAE. They were also more likely to be small for gestational age and have other complications of prematurity including respiratory distress syndrome, bronchopulmonary dysplasia, patent ductus arteriosus and retinopathy of prematurity .To determine the clinical factors with the greatest biologic plausibility in terms of predisposing to PedVAE, we performed a logistic regression. Although there were several clinical characteristics which were more frequent in the potential PedVAE/PedVAE group, these clinical characteristics are also much more common in the extremely preterm population. Based on the number of events in our population 32), we analyzed three variables which were likely to be driving these events: gestational age, birth weight and duration of mechanical ventilation. When birth weight and gestational age were controlled for, there was no longer a significant association with potential PedVAE/PedVAE. However, increased ventilator duration remained significant, with higher duration demonstrating a risk factor with a slightly elevated odds ratio equal to 1.030  were treated for culture-negative sepsis for 7 to 10 days, and in 26 out of 79 (33%) antibiotics were prescribed for 48–72 h for sepsis rule out  were treated for blood culture-proven sepsis. Two patients were treated for Pseudomonas aeruginosa , followed by Klebsiella pneumoniae  and Staphylococcus aureus . In 8 out of 79 events (10%), patients were treated for pneumonia. In addition to chest radiograph findings, tracheal aspirate cultures associated with these 8 events showed the following: cytomegalovirus , Staphylococcus aureus (with a concern for aspiration), Pseudomonas aeruginosa (2 events), methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Serratia marcescens and Klebsiella pneumoniae. These patients did not meet CDC criteria for ventilator-associated pneumonia.The most common indication for antibiotic use was tracheitis Fig.  comprisi2/MAP prior to the event, as well as the sustained increase for two or more calendar days following the event. Subsequently, the thresholds of the daily minimum increase in FiO2 were reduced in 5 % increments from the CDC guideline of 25%  [NICU-specific PedVAE data are limited, with most studies of PedVAE occurring in the PICU or other acute care settings such as the CICU. In this single-center case-control study of ventilated infants, 2 patients met the PedVAE surveillance definition based on the CDC criteria with a total of 3 events. The PedVAE definition is based on an increase in the daily minimum FiOe (PEEP) . PEEP cae (PEEP) .In the adult population, most VAEs are because of pneumonia, pulmonary edema, atelectasis or acute respiratory distress syndrome . The low2 increase on the ventilator, the event did not qualify because the definition is based on the daily minimum value for the day. Other reasons for exclusion included no 2-day period of stability or decreasing FiO2 prior to the increase. This was evidenced by an increase in the number of potential events once the period of stability or decrease was removed. Different thresholds of fraction of inspired oxygen (FiO2) and PEEP can result in increased sensitivity and better correlation between the VAE definition in children and clinical outcomes in ventilated critically ill children [Although many patients had episodes of significant FiOchildren .20 in the NICU population is very unlikely unless the patient is being transitioned from the conventional ventilator to high frequency oscillatory ventilation, which was the case in the patient who developed 2 PedVAEs based on the increase in her MAP. There were 2 potential events when the period of stability or decrease was removed and the threshold for increase in MAP was reduced to 2 cmH20.An increase in MAP of 4 cmHIn the NICU population, infants who have a clinical deterioration and require increased ventilator settings frequently are initiated on empiric antibiotics and a blood culture is obtained to rule out sepsis. Therefore, initiation of a new course of antibiotics while mechanically ventilated was used to identify an additional 30 patients with 79 potential PedVAEs. There is poor correlation between clinician diagnosed ventilator-associated infection in PICUs and the proposed PedVAE definition .Staphylococcus epidermidis in 2 events and Candida albicans in 1 event. The primary indication for antibiotic use in our study population was tracheitis and the most common organism isolated in tracheal aspirate cultures was Pseudomonas aeruginosa. Similarly, 60% of tracheal aspirates in a Canadian PICU grew Haemophilus influenzae and Pseudomonas aeruginosa [Out of these 79 events, only 3 events (4%) had culture-proven sepsis with ruginosa . Antibioruginosa , 17.Patients with potential PedVAE/PedVAE were more likely to be born preterm, and there was a statistically significant increase in complications of prematurity such as respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD) and retinopathy of prematurity in this population. Of note, patients with potential PedVAE/ PedVAE were admitted at an older age compared to those without PedVAE. This study was conducted at a Level IV NICU with all admitted infants transferred from peripheral Level II and III NICUs. Therefore, these patients have usually had complicated hospital courses at the referral sites, which culminates in transfer.Age-specific comorbidities such as RDS, BPD and necrotizing enterocolitis in neonates and infants can independently result in prolonged duration of mechanical ventilation, and complicate clinical and laboratory diagnostic criteria of VAP . To remoThe rate of PedVAE in our study was 0.9 per 1000 ventilator days, and thus difficult to assess the attributable morbidity and mortality in our patient population. Only 2 infants met current PedVAE criteria in the 3-year study period. However, about one-quarter of ventilated infants received multiple sepsis evaluations and antibiotic courses associated with increased ventilator settings. The current PedVAE definition offers little guidance relating to antibiotic use, and might be inadequate to identify actionable VAE events to inform prevention efforts in the NICU population. To the best of our knowledge, there have been no studies on PedVAE in the neonatal population since the implementation of mandatory reporting of these events by the CDC in January 2019. Screening daily ventilator settings is feasible and efficient, however, this study reveals the challenges with the current surveillance definition and the need to identify alternate indices which could better characterize these events in the NICU population and predict adverse outcomes.


Novel coronavirus 2019 (COVID-19) has been one of the largest and most devastating global pandemics of our time. There have been several complications of this disease that have also proven to be debilitating and deadly. While primarily affecting the respiratory system, some cases presented with uncommon complications such as pneumopericardium and spontaneous pneumothorax. We present a case of an elderly female diagnosed with COVID-19 found to have both spontaneous pneumothorax and pneumopericardium. She had a complicated hospital course and ultimately succumbed to her illness. While the pathogenesis of these conditions is not yet fully understood, further studies are needed to help clinicians develop treatment and prevention strategies to improve patient outcomes. Coronavirus 2019 (COVID-19) became a public health international emergency in December 2019 leading to hundreds of thousands of deaths to date -5. Sever2) of 90% on room air. Physical examination was only remarkable for tachypnea and increased work of breathing for which she was placed on high flow oxygen via nasal cannula with a fraction of inspired oxygen (FiO2) of 85% and RR of 35 to maintain an oxygen saturation of 89%-90%. Laboratory findings on admission were unremarkable and she was diagnosed with COVID-19 infection through reverse transcription-polymerase chain reaction (RT-PCR). Chest X-ray was also obtained on admission showing bilateral ground-glass opacities consistent with COVID-19 infection  of 20 breaths per minute, blood pressure of 119/64 mm Hg, the temperature of 100.3 degrees Fahrenheit, and oxygen saturation . She was encouraged to self-pronate but was unable due to discomfort. She was ultimately intubated due to persistent hypoxia and tachypnea and brought to the intensive care unit (ICU) for further management. Post intubation chest X-ray was performed showing both pneumothorax of the right lung and pneumopericardium which were not present prior to intubation  of 10 cm H2O, and FiO2 of 100%.Six days later, she began to desaturate again while on 100% FiOAfter the discovery of the pneumothorax, a right-sided chest tube was emergently placed; however, the following day it was noted on chest X-ray that the right pneumothorax persisted and a second chest tube was placed which helped to resolve the pneumothorax Figure . On day 2O is associated with an increased risk of alveolar rupture during mechanical ventilation [Pneumothorax is defined as the presence of air in the pleural space . Spontantilation . There htilation . Althougtilation -11. In oPneumomediastinum is defined as free air in the mediastinum and is classified as spontaneous pneumomediastinum (SPM) and secondary pneumomediastinum . SPM tenPneumopericardium is defined as the presence of air in the pericardial cavity. It can be the result of blunt chest wall trauma in deceleration injury and as a rare complication of pericardiocentesis or positive pressure ventilation . Lee et Although associated with poor outcomes and uncommon, several published cases and our own experience emphasize the importance of early identification and management of these complications in affected patients. While the pathogenesis of these conditions is not yet fully understood in the setting of COVID-19 infection, further studies are needed to help clinicians develop treatment and prevention algorithms to improve patient outcomes.