The relationship between altered default mode network (DMN) connectivity and abnormal serotonin function in major depressive disorder (MDD) has not been investigated. Using intravenous citalopram and resting-state fMRI, we investigated DMN intra-network connectivity and serotonin function in 77 healthy controls and patients with MDD. There were no significant main effects of MDD or citalopram on DMN intra-network connectivity; however, significant interactions indicated that group differences under saline were modified by citalopram. In MDD patients during saline infusion, in contrast with controls, the DMN (i) did not include the precuneus that was instead part of an anti-correlated network but (ii) did include amygdala that was part of the anti-correlated network in controls. Citalopram infusion in MDD patients restored the pattern seen in controls under saline. In healthy controls, citalopram infusion disengaged the precuneus from the DMN and engaged the amygdala, partially reproducing the abnormalities seen under saline in MDD. In exploratory analyses within the MDD group, greater rumination self-ratings were associated with greater intra-network connectivity of the anterior cingulate cortex with the DMN. We hypothesise that, in MDD, disengagement of the precuneus from the DMN relates to overgeneral memory bias in rumination. The opposite effect, with greater engagement of the amygdala in the DMN, reflects the negative valence of rumination. Reversal of these abnormalities by citalopram suggests that they may be related to impaired serotonin function. That citalopram engaged the amygdala in the DMN in controls may relate to the paradoxical effects on aversive processing seen with acute SSRIs in healthy subjects. We recently found that ketamine evoked blood-oxygen-level-dependent (BOLD) responses in ACC that predicted improvement in mood 1 and 7 days later in major depressive disorder (MDD) patients6. However, the ACC does not function in isolation and it is a component in several neural systems relevant to depression8.Structural and functional magnetic resonance imaging (fMRI) studies have implicated abnormal anterior cingulate cortex (ACC) structure and function in the pathogenesis of depression and as a site of action of antidepressant action10. The network can be defined by an abstracted BOLD waveform (independent component) with which the regional BOLD fluctuations correlate )11. The degree to which regions correlate with the independent component is taken as a measure of their connectivity or integration with the whole network—intra-network connectivity12. Direct connectivity between components of a network are often assessed using regional correlations with the waveform from one or several seed regions to define the network14. The ACC is prominent in the default mode network (DMN), which includes posterior cingulate cortex , precuneus, medial prefrontal cortex (mPFC), ventral ACC and lateral and inferior parietal cortex15.Functional networks involving ACC have been inferred from correlated patterns in the slow oscillations of BOLD signal detected in resting-state fMRI (rsfMRI). A resting-state network consists of a set of regions whose BOLD fluctuations inter-correlate17 in view of its role in visual imagery18 associated with autobiographical memory. The self-reflective role of the DMN has led to much interest in the possibility that excessive rumination in depression involves excessive DMN activity and an inability to switch out of it in response to external demands19. Rumination involves excessive negative inner preoccupation about the personal past, present and future. There is evidence that a tendency to ruminate is a personality trait that predisposes to depression and depressive relapse21. However, few studies have related DMN changes to questionnaire measures of rumination, the most widely used of which is the Ruminative Response Scale (RRS)22. RRS subscales cover negative thoughts associated with depression: brooding rumination and reflective rumination, of which the last may be a constructive form of rumination that is associated with better outcomes23. One study reported that dominance of the DMN over the task positive network (TPN) correlated with brooding rumination but not with reflective rumination; however, DMN/TPN dominance was no greater in MDD patients than in controls23. In an ICA study, Zhu et al.24 reported rumination scores correlated with greater ACC-DMN connectivity while impaired PCC-DMN connectivity correlated more with overgeneral memory recall in MDD patients. Seed-based studies found PCC–subgenual cingulate (SgACC) and SgACC–medial frontal cortex connectivity correlated with RRS scores26. An earlier literature found that high ratings of trait rumination were associated with prolonged amygdala responses to aversive stimuli28. One aim of the present study was to determine whether citalopram-induced 5-HT release modulates brain circuits associated with rumination as predicted by some theories of 5-HT and resilience29.Several lines of evidence suggest that the DMN is involved in self-reflection, autobiographical memory and social cognition. The posterior cingulate/precuneus is one of the most metabolically active regions of the brain at rest and may be particularly important in self-reflective thinking11. In a review of more recent ICA and seed-based studies, Mulders et al.30 conclude that a number of studies are compatible with an increase in connectivity within the anterior DMN connectivity  but that connectivity with the posterior network shows a variety of changes in MDD.Several studies have investigated whether excessive DMN connectivity and other measures of DMN function are associated with MDD but many different methods have been employed and the results are inconsistent. In our review of fMRI resting-state networks, 13 studies reported increased resting-state activity or connectivity of the PCC/precuneus in MDD but 10 reported no changeThe present analysis sought to determine whether acute modulation of serotonin function using intravenous (i.v.) citalopram would modify ACC-DMN abnormalities in depression that are implicated in mechanisms of antidepressant action. We hypothesised that networks associated with the ACC would be abnormal in depression and associated with rumination and that acute i.v. citalopram challenge would normalise the abnormality.Two studies, Remission Mechanisms in Depression (REMEDi) and New Molecules for Mood Disorders (NewMood) were performed at the University of Manchester in accordance with UK ethical procedures and the Declaration of Helsinki, with participants recruited via advertisement. The participants had no previous treatment with a selective serotonin reuptake inhibitor (SSRI) and were unmedicated.The effects of acute citalopram infusion in currently depressed unmedicated MDD and non-MDD control participants were compared using data from the REMEDi study in which participants were randomised to receive 7.5 mg citalopram or saline i.v. over 7.5 min. Thirty-six unmedicated MDD participants were recruited to REMEDi  together with 24 never-depressed (non-MDD) participants . Non-MDD numbers were augmented using 14 participants from the NewMood study all of whom had received 7.5 mg citalopram i.v. in an identical imaging and infusion protocol to REMEDi, carried out in the same scanner by the same research group. Thus a total of 51 participants received citalopram  and 23 received saline infusion .31. Patients met criteria for a DSM-IV major depressive episode. Healthy controls were required to have no personal or family history of psychiatric disorder. Patients’ illness severity was established using the Montgomery Åsberg Depression Rating Scale (MADRS)32 and the seven item Clinical Anxiety Scale adapted from the Hamilton Anxiety Rating Scale33. Currently depressed patients were required to have a MADRS score of ≥20 and psychotropic drug free for at least 2 weeks. The RRS was completed by all participants to measure rumination prior to infusion22.Participants were assessed using the Structured Interview for Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV)Exclusion criteria were: depressive episode >2 years, depression superimposed on dysthymia, failure to respond to adequate treatment trial of 2 antidepressants or to citalopram/escitalopram in the current episode, allergy or intolerance to citalopram or escitalopram, contraindications to SSRI treatment, electroconvulsive therapy or lithium treatment in the current episode, significant suicidal risk, concurrent psychotropic medications, co-morbid Axis 1 psychiatric disorders except anxiety disorders (those with obsessive compulsive disorder were excluded), or personality disorder. Also excluded were participants with any unstable medical condition, pregnancy, neurological disorders, history of significant head trauma, lifetime history of substance or alcohol abuse, current alcohol use over 14 units for women or 21 units for men (these were UK government recommendations at the time the data were collected), caffeine usage >6 cups of coffee per day, smoking >10 cigarettes per day and any contraindication to MRI scanning. For the REMEDi study, the research pharmacy generated and held the randomisation codes and dispensed the citalopram or saline on the day of the experiment. The infusion was made up by an independent research nurse and labelled only with the participant code to maintain the blindness of the research team. Informed consent was obtained from all participants. Ethical approval was gained from the local research ethics committee before each study.34 run under MATLAB 2013a35.Images were acquired on a Philips Intera 1.5 Tesla MR scanner housed within the NIHR/Wellcome Trust Clinical Research Facility, Manchester, UK. The acquisition parameters were single-shot echo-planar imaging (EPI) sequence with an ascending and sequential slice order to give full brain coverage with isotropic (3.5 mm) voxels  = 40 ms, TR (time to repetition) = 2 s). Participants were asked to lie still. Total scan duration was 25 min during which citalopram 7.5 mg or matching saline were infused i.v. over 7.5 min after a 5-min baseline period. Scanning was continued for a further 12½ min and this second half of the scan following infusion was used for analysis. A qualified radiographer performed all MR scans. Images were preprocessed using Statistical Parametric Mapping (SPM)Images were realigned using the first volume as a reference and a six-parameter rigid body transformation. Co-registration of the structural image and EPI images from realignment was performed. This was followed by segmentation of the structural image into grey and white matter. The grey matter segmented image was normalised to the Montreal Neurological Institute (MNI) grey matter probability map supplied with SPM. The normalisation transform matrix was applied to the realigned functional images. The normalised functional images were then smoothed using a 10-mm full-width half maximum isotopic Gaussian kernel.38. To address this, we examined two motion parameters suggested by Power et al.36: framewise displacement (FD) and DVARS . FD measures the movement of any frame relative to the previous frame. DVARS quantifies volume-to-volume BOLD signal change ) that account for patterns of inter-correlation. The preprocessed images were entered into Group ICA of fMRI Toolbox (GIFT) software run under MATLAB 2013a40. The group ICA produces a map of the correlation between regional BOLD signals and each of 20 ICs based on the mean data length. The regional correlations with the independent component are considered to be measures of connectivity showing a region’s connectivity with the network or ‘intra-network connectivity’. The IC map with greatest spatial correlation with an ACC template (Wake Forest University PickAtlas)44 was objectively selected as the ACC-containing network using a multiple regression model. The independent component with the strongest anatomical correlation with the ACC mask proved to be the DMN identified in the GIFT toolbox; thus ‘ACC-containing network’ and DMN are one and the same in this study. The group ICA also identifies regional BOLD responses that are anti-correlated with the IC, in this case, anti-correlated with the DMN component. From the group ICA, individual maps of regional correlation with the DMN (ACC-containing network) were back-reconstructed using GIFT. Parametric cluster-forming thresholds and family-wise error correction at the cluster level leads to an unacceptable amount of type 1 errors45. Therefore, the analysis was performed using non-parametric statistical tests using Multivariate and repeated measures (MRM) toolbox46. MRM is a MATLAB-based toolbox used to analyse group models of neuroimaging data using the multivariate general linear model. The intra-network connectivity maps of the ACC-containing network from each individual were entered into a 2 × 2 factorial model with group (MDD vs control) and treatment  as between-subject factors.The multivariate test statistic used was Pillai’s trace. Family-wise error cluster level inference (pFWEc) was used at p < 0.05 with a cluster-forming height threshold of p = 0.002 (two tailed) was used for all analyses with the number of permutations set at 10,000. In subsidiary analyses, MADRS depression ratings and RRS total score and subscale ratings were entered separately as covariates in the complete model and also in the MDD group alone.ICA is a data-driven model-free methodt tests to evaluate group differences.Demographic data, MADRS and RRS total and subscale scores were analysed using two sample Table r = 0.454. Apart from ACC, the regions showing significant intra-network connectivity included most elements of the DMN, including posterior cingulate, precuneus and angular gyrus  with the ACC network, whereas in MDD patients, amygdala showed positive connectivity (positive correlations) with the DMN Fig. . During Severity of depression as measured by MADRS scores did not correlate with intra-network connectivity of any region (in keeping with the lack of a main effect of group) or in any subgroup defined by interaction with group and/or drug. In contrast to the MADRS, total RRS scores correlated with intra-network connectivity of the rostral ACC in the MDD group but not in controls Fig.  producinWithin the MDD group, ACC-DMN connectivity showed a significant total RRS-by-drug interaction Fig. ; Table 3The key findings of the study are (i) that controls and MDD participants differed in how two key structures, implicated in mechanisms of rumination, are integrated with the DMN, and (ii) that the inference that these changes were modulated by acute citalopram-induced 5-HT release. We consider first the effect of MDD in saline-infused participants and second the effect of citalopram. In left precuneus, BOLD fluctuations in saline-treated controls were, as expected, correlated with the DMN network but they were anti-correlated in the MDD patients. In other words, the precuneus was not merely dissociated from the DMN in the depressed patients but instead showed aberrant connectivity with the anti-correlated network. In contrast, the amygdala, part of the anti-correlated network in controls, was integrated with the DMN in MDD participants Fig. . It must49 and the salience network in phases when the DMN is active23.The anti-correlated network to the DMN in the overall group ICA is summarised in 7 reported that in MDD the DMN and emotion networks overlap in a dorsomedial frontal cortical ‘nexus’ in a seed-based study. Hamilton et al.’s50 review and synthesis proposed that aberrant connectivity with an overactive SgACC mediates the affective tone of DMN self-reflection in MDD. The intrusion of left amygdala into the DMN IC in our MDD group is compatible with these ideas and could be a route by which rumination acquires negative emotional valence and becomes maladaptive. Other ICA studies have not reported amygdala correlation with the DMN in MDD. However, the masks used in several studies may have excluded the possibility of detecting such aberrant correlations. A study using bilateral amygdala seeds made the relevant observation of positive connectivity between amygdala and left precuneus in MDD compared to their negative connectivity seen in controls51. This suggests that abnormal amygdala–precuneus connectivity specifically could mediate the negative emotional valency of memory imagery in MDD. In our study, left amygdala and precuneus correlated with opposed DMNs in the healthy controls in keeping with Cullen et al.51; that they switched networks in the MDD group does not imply that the entirety of precuneus and amygdala anatomy and function remained segregated in different networks in our MDD group. Rather, the crossing-over of parts of amygdala and precuneus into anti-correlated networks probably represents a flexible merging of self-reflection and of emotion-processing elements of different networks in MDD. The functional significance of amygdala correlations with the DMN in our MDD group, however, remains unclear, since there was no correlation with depressive symptoms or with RRS scores.An emerging concept is that DMN-mediated self-reflection becomes maladaptive when other networks become integrated with the DMN. For example, Sheline et al.24 found decreased precuneus–DMN connectivity in MDD, but this correlated with an overgeneral memory recall measure rather than their measure of rumination, the 4-item rumination subscale from the Cognitive Emotion Regulation Questionnaire (CERQ-R)52. The CERQ-R scale focusses on cognitive responses to life events and differs considerably from the RRS and its subscales. Given the importance of rumination as a mechanism of overgeneral memory recall53, Zhu et al.’s findings on the functional correlates of precuneus–DMN disconnection may not be far removed from our own. A recent experiment attests to the importance of precuneus in overgeneral recall in depression. MDD patients were able to recruit the DMN in constructing experimental autobiographical memories but their impaired performance related to impaired activation of precuneus and of its seed-based connectivity with hippocampus54. Since some precuneus connectivity was shifted to the anti-correlated DMN in our MDD group rather than merely dissociated from the DMN, we speculate that it may be directly involved in repetitive rehearsal of widely represented depressive semantic self-knowledge, i.e. rumination, that interferes with specific recall of autobiographical memories.In contrast with the amygdala–DMN changes, the shift in precuneus correlation away from DMN to the anti-correlated network in our MDD group correlated with greater RRS scores. Three other studies have reported correlation between RRS scores and RS connectivity but they are not immediately germane to our findings because they probed connections other than precuneus (see Introduction). A fourth study by Zhu et al.6, one motivation for the present analysis was to test whether ACC connectivity abnormalities in MDD would be affected by a single dose of an SSRI. However, we found no group differences in ACC-DMN correlation and no main effects of drug or drug-by-group interaction. Several studies have reported increased ACC DMN connectivity in MDD30, but this is a large region and it is not clear that a particular ACC subregion is consistently implicated except perhaps sgACC55. We observed a group interaction with rumination such that lesser rostral  ACC DMN connectivity was correlated with RRS scores in MDD as found in precuneus but without evidence of association with the anti-correlated network or of modulation by citalopram. A possible inference is that MDD may involve some loss of ACC control of resting-state modes that might contribute to the intrusion of amygdala into the DMN and the dissociation of precuneus. However, caution is required since our finding is in apparent conflict with Zhu et al.’s observation that increased rather than decreased rostral ACC DMN connectivity in MDD correlated with their CERQ measure of rumination.Following our observations on the role of rostral ACC in mediating the antidepressant effects of acute ketamine infusion2C receptors56 or to a deficit of synaptic 5-HT through stimulation of cell-body auto-receptors57. More generally, the engagement of the amygdala in the DMN by citalopram in controls may be another example of many paradoxical findings that acute SSRI treatment enhances aversive processing and symptoms in animals and humans yet work in depression by increasing serotonin function.Citalopram infusion appeared to normalise the abnormalities of precuneus and amygdala integration with the DMN seen under saline infusion: the precuneus was integrated with the DMN and amygdala was anti-correlated as in saline-infused controls. One interpretation of the findings is that the loss of precuneus integration with the DMN and intrusion of the amygdala reflects an impairment of 5-HT neurotransmission in depression that is corrected by i.v. citalopram-induced 5-HT release. A difficulty for this idea is that citalopram had the opposite effects in the healthy controls in whom citalopram shifted an amygdala cluster towards the DMN and shifted out a precuneus cluster  thus mimicking the effect of MDD on the DMN. A possible explanation is the recognised effect of acute SSRIs to induce anxiety variously attributed to stimulation of 5-HT50. There are no other studies of acute 5-HT modulation of the DMN, so the findings are preliminary, although Klaassens et al.58 reported comparable effects of oral sertraline on precuneus connectivity in a single-dose study in 12 volunteers. Given the preliminary nature of the results, it is difficult to comment on whether similar results would have been observed using clinically effective dosing of oral citalopram.This study has several limitations. The smaller numbers of saline- than citalopram-infused participants may have reduced the power to detect regions with hypothesised increase in DMN connectivity. A cross-over design was not considered practical but would have provided more power to detect group and drug effects. No filtering was applied to the data set to compensate for physiological noise contributing to increased variance in the data; however, this should have been removed by the spatial ICA. In addition, our analyses showed a potential effect of head movement in the depressed group compared to healthy controls regardless of treatment. Susceptibility artefact may have prevented replication of altered sgACC connectivity in MDDWe conclude that the DMN is likely to be abnormally configured in MDD with intrusion of amygdala connectivity and loss of some precuneus connectivity, rather than showing a general increase in connectivity; the quality of self-reflection rather than the amount may be the more important change in depression. The DMN abnormalities in MDD may involve altered serotonin modulation of connectivity because they were not seen during acute serotonin release evoked by i.v. citalopram. DMN connectivity in healthy volunteers and patient groups appears to be sensitive to experimental drug challenge and this could be an important approach to understanding neurotransmitter function in depression and in detecting effective new drugs.Supplementary MaterialSupplementary material


Compared with conventional top down costing, micro-costing may provide a more accurate method of resource-use assessment in economic analyses of surgical interventions, but little is known about its current use. The aim of this study was to systematically-review the use of micro-costing in surgery.Comprehensive searches identified complete papers, published in English reporting micro-costing of surgical interventions up to and including 22nd June 2018. Studies were critically appraised using a modified version of the Consensus on Health Economic Criteria (CHEC) Checklist. Study demographics and details of resources identified; methods for measuring and valuing identified resources and any cost-drivers identified in each study were summarised.n = 42, 49.4%) with few conducted in the context of a randomised trial . The majority of studies were single-centre  and almost half  collected data retrospectively. Only half  self-identified as being ‘micro-costing’ studies. Rationale for the use of micro-costing was most commonly to compare procedures/techniques/processes but over a third were conducted specifically to accurately assess costs and/or identify cost-drivers. The most commonly included resources were personnel costs ; materials/disposables  and operating-room costs . No single resource was included in all studies. Most studies  identified key cost-drivers for their interventions.A total of 85 papers were identified. Included studies were mainly observational comparative studies (There is lack of consistency regarding the current use of micro-costing in surgery. Standardising terminology and focusing on identifying and accurately costing key cost-drivers may improve the quality and value of micro-costing in future studies.CRD42018099604.PROSPERO registration  Accurate information regarding the cost-effectiveness of existing and novel surgical interventions is vital to inform policy and ensure scarce resources are used to optimal benefit. Central to the meaningful assessment of cost-effectiveness is the accurate evaluation of resource use . This isThe potential value of this methodology has already been identified and a framework has been developed for applying micro-costing methodology to cost evaluations of surgical technologies . This frDespite the potential benefits of micro-costing, it is time and resource intensive and this may limit its utility in clinical trials. Work is therefore needed to explore whether a more targeted approach may be possible. It is hypothesised that surgical interventions will have ‘cost-drivers’ which create or drive the majority of the cost of the activity. If these are consistent across surgical interventions or can be easily identified, it may be possible to develop a simplified micro-costing framework which could be used in future surgical trials.The aim of this systematic review was therefore to identify and critically appraise published studies reporting the use of micro-costing in economic analyses of surgical interventions to explore the resources included in each study; the methods used for measuring and valuing resource use and reported cost-drivers of surgical interventions to inform recommendations for the future use of micro-costing in surgical interventions.The protocol for this systematic review was registered on the PROSPERO International prospective register of systematic reviews (reference number CRD42018099604) before data extraction commenced.The OVID SP versions of MEDLINE, Embase, EconLit, the Cochrane Database and the NHS Economic Evaluation Database (NHS EED) were searched using published micro-costing search strategies –8 and apThe search was limited to human studies, published in English from database inception up to and including 22nd June 2018. Abstracts and conference reports were excluded due to difficulty assessing incomplete information.Duplicate records were excluded, and titles and abstracts of remaining citations were screened for eligibility using prespecified inclusion criteria (see below). The reference lists of identified studies and existing reviews were also manually searched to identify any additional relevant papers.Full papers reporting the use of micro-costing as a method of resource-use assessment in economic analyses of surgical interventions were eligible for inclusion.For the purposes of the review, a surgical intervention was defined as any ‘procedure involving an incision with instruments usually performed in an operating theatre and normally involving anesthesia and/or respiratory assistance’ . Micro-cThe definition of a ‘sufficiently detailed’ micro-costing study was iteratively modified during the pilot abstract screening and data extraction phase which included approximately 100 abstracts and 10 papers respectively. The piloting and iterative modification of the abstract screening and data extraction pro-formas were completed by 2 reviewers (CD/SP) following discussion with experienced health economists (WH/CR). For the purposes of this review, a ‘sufficiently-detailed micro-costing study’ was agreed to be one in which i) the elements of the surgical procedure were sufficiently disaggregated and at the very least two separate elements of the procedure (e.g surgeon costs and consumables) costs presented separately AND ii) the unit cost of at least one element of the procedure  had been considered. Any studies not meeting both of these criteria were excluded.Economic analyses using ‘top down’ or gross-costing approaches (e.g. HRGs), economic models, conceptual papers, reviews, commentaries, letters, abstracts, editorials and studies not evaluating surgery were excluded. Studies comparing surgery with a non-surgical comparator were included provided that micro-costing of the surgical intervention had been performed and met the inclusion criteria.All papers were screened for inclusion by two reviewers (CD and SP/GD) using a standardised two-stage screening proforma ; prospective or retrospective accrual of data; author-reported type of economic analysis undertaken ; number of participating centres (in the micro-costing study separately); surgical specialty with details of the intervention(s) assessed; whether implants were used (yes or no); type of hospital stay ; type of anaesthetic ; details of the study population and stated aims and objectives of the study which were extracted verbatim.In the absence of a specific quality assessment tool for micro-costing studies, a modified version of the Consensus on Health Economic Criteria (CHEC) Checklist  was usedItems 7–9 which assess the degree to which all relevant costs were appropriately identified, measured and valued were the focus of the review and were expanded to include additional details (see below).Details of the micro-costing undertaken in each included study were extracted. This included assessing whether the authors reported performing a ‘micro-costing’ study or if alternative terms were used; the purpose of the study ; methods of resource identification  and the scope of the costing exercise .Resources included in each micro-costing study were recorded (yes/no) using the categories proposed in the existing framework  expandedData extraction was performed by one reviewer (CD) with a proportion (10%) checked by a second reviewer (SP/GD). Discrepancies were resolved by discussion with an experienced health economist (CR/WH).Descriptive statistics were used to summarise study details; compliance with items in the CHEC checklist and details of the micro-costing reported in each study. Simple content analysis  was usedOf the 1009 abstracts identified from the electronic searches, 243 full papers were obtained for further evaluation. Of these, 79 met the inclusion criteria and were retained. A further six papers were identified from the hand-search. A total of 85 papers were therefore included in the review  and the majority originated from Europe  or North America . Included studies were commonly single-centre , retrospective , observational studies with  or without  a comparison group. Authors’ most frequently described their studies as being ‘cost analyses’  and the median number of procedures micro-costed per study was 100 (interquartile range 24–233) although this ranged widely , general  and plastic surgical  procedures were micro-costed most frequently but a comprehensive range of surgical specialities were represented and almost a third of studies micro-costed a surgical procedure involving an implant . Most procedures were performed under general anesthesia  and required an inpatient hospital stay  but this information was often not clearly reported  in a clearly defined population  and were using an appropriate economic study design to achieve their stated objectives . The study perspective was less well-reported  and only a quarter of studies  performed any form of sensitivity analysis. Most studies discussed the generalisability of the results to other settings and patient/client groups . A quarter of studies  did not report any conflicts of interest; only half  reported obtaining ethical approval for the project.The majority of studies scored highly according to the CHEC checklist Table . Includen = 46, 54.1%) describing their methodology as ‘micro-costing’ while a third  used the term (time-driven) activity-based costing (ABC) and a smaller number referred to using a ‘bottom-up’ approach .Table n = 43, 50.6%) but over a third of studies  were specifically undertaken to identify cost-drivers for surgical procedures and a quarter  were performed to compare costing methodologies; most commonly gross or ‘top-down’ methods, such as HRGs or insurance reimbursement with micro-costing or ‘bottom-up’ approaches. In addition to micro-costing the actual surgical procedure, most studies  also micro-costed the patients’ hospital stay. Pre-operative investigations required prior to surgery were included in over half the studies  and a third micro-costed surgical complications  and/or follow-up required after discharge . Most studies reported including either direct costs only  or both direct and indirect costs  but over a third  did not state what types of costs had been considered.Micro-costing was most commonly performed to compare procedures, techniques or processes ; materials and disposables ; operating room costs  and/or the costs of any drugs or medications  in their micro-costing, there was no single type of resource included in all studies and categorisation of resources into pre-defined categories was often difficult due to different studies aggregating and reporting resources in different ways. There was also a lack of consistency in the ways in which relevant resources were identified, measured and valued. Hospital information systems or administrative databases were used in over 40%  of studies with approximately a third  using patient pathway mapping to identify resource-use. Interviews with surgeons and/or patients  and direct observation  were less commonly used  reported having identified cost-drivers but these differed according to whether the authors had micro-costed the surgical procedure alone or the whole episode of care  ; theatre personnel ; operative equipment , implants  and theatre consumables . For studies micro-costing the full episode of care, the main cost-drivers identified were the inpatient stay  and personnel/labour costs . Use of intensive care ; consumables  and overheads  were also identified as potential cost-drivers in these studies . Although routinely available data sources may be accurate, especially in modern ‘fee-for-service’ settings, other reported methods for resource use identification such as case-note review or operating room logs may be less robust –15, poteThere was significant variation in which aspects of the patient pathway researchers elected to micro-cost. Most studies costed both the surgical procedure and the associated inpatient stay, but others also included pre-operative planning/investigations, complications and follow-up required following discharge. To an extent, this variation may appropriately reflect the diversity in the aims of micro-costing studies. For economic evaluations comparing two different surgical interventions researchers may legitimately focus on the incremental cost differences between the two procedures and exclude costs that are common to both procedures (e.g. pre-operative visits). Furthermore, for economic evaluation, methodological guidelines emphasise that the level of resource use detail required is lower for items with a small relative impact on total costs (e.g. volatile anaesthetic agents) . In contThis review has demonstrated the need for specific guidance for researchers undertaking micro-costing. Work to develop a checklist for the conduct, reporting and appraisal of micro-costing studies in healthcare is ongoing . This chThis is, to our knowledge, the first study to systematically identify and critically-appraise the use of micro-costing as a method of resource-use assessment in surgery. We used published search strategies for micro-costing , 7, howeMicro-costing produces an accurate assessment of resource-use but the methodology is time consuming and resource intensive and universal application alongside surgical trials would not be practical , 21. A mAs health systems and technologies become more advanced, routinely-available data may have increasing utility for resource-use assessment and may make future large-scale micro-costing studies possible. Healthcare provider cost-accounting systems that capture details on all resources used in an episode of care combined with electronic theatre systems that could provide proxy time and motion data may represent a time and cost-efficient method for detailed resource-use assessment. Such systems and methodology, however, would need to be consistent across centres if the costs generated were to be comparable. This may be challenging, especially across different healthcare settings and geographical locations.Micro-costing has the potential to improve the accuracy of economic analyses of surgical interventions by providing more accurate assessments of resource-use but the overall quality of existing studies is poor. There is a need to improve the consistency and efficiency of micro-costing in surgery if the potential value of the methodology is to be realised. This review highlights a number of ways in which this could be achieved. Using standardised terminology for micro-costing studies; focusing on identifying and accurately costing cost-drivers relevant to the specific research question and transparently reporting disaggregated costs for each included resource may represent a simple strategy for improving the design and delivery of future studies.Micro-costing may provide a more accurate method of resource-use assessment in economic analyses of surgical interventions and could improve the value of economic evaluations conducted alongside surgical trials, but this systematic review suggests that current use of micro-costing in surgery is inconsistent and lacking in methodological rigor. Using standardised terminology for micro-costing studies; focusing on identifying and accurately costing cost-drivers relevant to the specific research question and transparently reporting disaggregated costs for each included resource may represent a simple strategy for the optimal design and delivery of future studies.Additional file 1. Search strategy for Ovid Medline (1946 to present) .Additional file 2. Screening log (Phase 1 and 2).Additional file 3. Data Extraction Form.Additional file 4 Papers included in the systematic review (n = 85).


Photodynamic therapy of tumors requires the topical, systemic or oral administration of a photosensitizing compound, illumination of the tumor area by light of a specific wavelength and the presence of oxygen. Light activation of the photosensitizer transfers energy to molecular oxygen creating singlet oxygen, a highly reactive and toxic species that rapidly reacts with cellular components causing oxidative damage, ultimately leading to cell death. Tumor destruction caused by photodynamic therapy is not only a result of direct tumor cell toxicity via the generation of reactive oxygen species but there is also an immunological and vascular component involved. The immune response to photodynamic therapy has been demonstrated to significantly enhance its efficacy. Depending on a number of factors, including type of photosensitizer, light dose and dose rate, photodynamic therapy has been shown to induce cell death via apoptosis, necrosis, autophagy and in particular immunogenic cell death. It is the purpose of this review to focus mainly on the role photodynamic therapy could play in the generation of specific anti-tumor immunity and vaccines for the treatment of brain tumors. The therapeutic goal following gross surgical resection of brain tumors therefore is to prevent recurrence by eliminating the infiltrating tumor cells both remaining in the margins of the resection cavity as well as remote from the tumor site. With this goal in mind anti-cancer immunotherapy is being actively researched as an important therapeutic modality for the treatment of HGG. Of the several methods to induce effective anti-tumor immune response, photodynamic therapy (PDT) has some potentially unique properties. PDT can induce combinations of apoptosis, autophagy and necrosis and immunogenic cell death, depending on a number of factors including type of photosensitizer, light dose and dose rate. It is the purpose of this review to focus mainly on the studies related to PDT-generated anti-tumor immunity and vaccines for gliomas.Tumor resection is the primary treatment employed in the treatment of high grade gliomas (HGG). The main functions of surgery are decompression of the brain, obtaining a histopathological and molecular classification of the tumor and reducing tumor load, allowing maximum effect of postoperative therapy. Despite employing modern imaging and surgical techniques, that increase the incidence of gross tumor resection, the tumor invariably recurs, usually in the vicinity of the surgical resection cavity-8. The photon energy activates the photosensitizer and initiates a complex photochemical reaction that generates cytotoxic reactive oxygen species (ROS) as shown in .PDT of tumors requires the topical, systemic or oral administration of a photosensitizing compound, illumination of the tumor area by light of a specific wavelength and the presence of oxygenDifferent photosensitizers react with specific intracellular organelles, resulting in cell death via several varied mechanisms .. Additionally, PDT is a highly site-specific form of treatment, since its effect is restricted to the immediate vicinity of the region of illumination.Unlike ionizing radiation and many chemotherapeutic agents, PDT does not exert its effects via DNA damage-14.Tumor destruction caused by PDT is not only a result of direct tumor cell toxicity via the generation of ROS but it is well established that there is also a significant immunological component involved. The great majorities of experimental studies have been done on extra-cranial tumors, and are reviewed in several extensive recent publications while immunosuppressed counterparts were not able to resist the re-challenge. Induction of systemic and memory immunity following PDT treatment has been verified in numerous studies. Systemic immunity following PDT treatment has been demonstrated by the ability of a locally induced immune response to affect distant non-treated areas-19. Treatment of subcutaneous (s.c) primary tumors that led to 90%-100% of tumor ablation after PDT treatment showed a significant reduction of metastasized lung tumors compared to non-treated controls. In particular, a study using s.c colon carcinoma treated with hypericin-PDT yielded 100% of tumor cures, and i.v. re challenge with viable tumor cells showed no development of new tumors.The first evidence for induction of a tumor-specific immune response came by the demonstration that normal mice cured by PDT were able to resist a re-challenge with tumor cells in a tumor-specific manner. ICD is a cell death mode where the dead and dying cancer cells expose and/or release damage associated molecular patterns (DAMPs). Although DAMPs are present in cells under normal conditions, they are exposed on the cell surface or released from cells upon the damage caused by the ROS generated by PDT. DAMPs reported to be necessary for the generation of antitumor immunity and induced upon PDT include surface calreticulin (CRT), heat shock protein (HSP) 70, HSP90, secreted adenosine triphosphate (ATP), and high-mobility group box 1 protein (HMGB1)-25. Importantly, DAMPs cause maturation, activation and antigen processing/presentation of APCs, leading to their migration and proliferation in local lymph nodes. The mature APCs in the lymph nodes present the tumor antigens to a specific subset of CDs+ T cells.PDT has been shown to induce apoptosis, necrosis, autophagy and immunogenic cell death (ICD)-28. This strategy induces in situ DC activation which enhances antigen acquisition and processing as well as migration of the DCs to draining lymph nodes.In addition, several studies have shown that PDT-treatment of extra cranial tumors followed by direct intra-tumoral injection of immature DCs, leads to an enhanced anti-tumor immune response compared to PDT treatment as single therapy-33. PCI is based on the use of specially designed photosensitizers, such as AlPcS2a, TPPS2a, TPcS2a that localize preferentially in the membranes of endocytic and lysosomal intracellular vesicles. Upon exposure to light of appropriate wave lengths, the photosensitizers induce the formation of short range singlet molecular oxygen, destroying the intracellular vesicles membranes, thus leading to the release of the contents of these vesicles into the cell cytosol. The released macromolecules can now exert their full biological activity instead of being degraded by lysosomal hydrolases.Photochemical internalization (PCI), a derivative of PDT, has been shown to increase the efficacy of drugs, gene transfection as well as a variety of other anti-cancer agents that are taken up into cells by endocytosiset al. (2017) has examined the efficacy of PCI delivery of bleomycin (BLM-PCI) and its impact on systemic anti-tumor immunity in an extra-cranial mouse model. Their results showed that both PDT and BLM-PCI were incapable of inducing a curative effect in athymic mice at the light dose tested. In contrast, 50% of the light dose of that used in athymic mice resulted in a curative effect in 90% of the animals after BLM-PCI and 70% after PDT in normal mice. Inhibition of tumor cell growth was observed when combined with co-injection of splenic T cells from mice treated and cured with BLM-PCI. The anti-tumor immunity induced by BLM-PCI was equal to that obtained with PDT treatment, but at a lower light dose. Furthermore, the induced immune response after BLM-PCI was sufficient to reject tumor re-challenge immediately after PCI and lasted for at least two months.Norum et al.  (2018) utilizing PCI to promote the escape of trapped endocytosed peptides into the cytosol of APCs. Their results showed that PCI caused a 30-fold increase in MHC class I/peptide complex formation and surface presentation on APCs, and a subsequent 30- to 100-fold more efficient activation of antigen-specific CTLs compared to using the peptide alone. These in vitro effects of PCI were translatable in vivo by the successful induction of antigen-specific CTL responses to cancer antigens in C57BL/6 mice following intradermal peptide vaccination and local light treatment. It is noteworthy that both macrophages and DC were used as APCs with approximately equal efficacy in these experiments. If these promising PCI strategies might be translatable to post-operative HGG treatment by the use of indwelling balloon light applicators, as has been proposed and tried for both radiation and PDT treatment, remains to be determined-39.An additional and novel method for enhancing the efficacy of peptide vaccines in extra cranial studies has been explored by Haug -43. Although numerous clinical trials have been initiated, the vast majority have consisted of uncontrolled phase I/II studies containing small numbers of patients. For example, in the single center phase III trial reported by Eljamel et al. using both flourecent guided resction combined with ALA and Photofrin repetitive PDT, a mean overall survival (OS) of the treatment group was 52.8 weeks compared to 24.6 weeks in the control group. In a phase II uncontrolled trial of 22 patients reported by Muragaki et al., using talaporfin sodium as a PS, a median of survival of 99 weeks was observed. This compared favorably to the 54-64 weeks OS obtained from previous trials employing standard post operative treatment consisting of radiation and TMZ. Due to differences in methodology and types of malignant brain tumors treated, it has been very difficult to evaluate PDT efficacy from these limited trials. For a more detailed account of the results of a number of PDT clinical trials for HGG, Bechet et al. and Quirk et al. give an excellent overview. Additionally, none of these clinical trials have included an evaluation of the effects of PDT on the immune response to treatment. Overall, the results of PDT trials for malignant gliomas have been relatively modest, thus providing the rationale for alternative PDT mediated treatment approaches such as PDT induced immunotherapy.PDT has been investigated as an adjuvant for the treatment of malignant gliomas for approximately 35 yearset al. showed that PDT in vivo generated regional and systemic anti-tumor immunity in mice with G422 gliomas in the brain. The infiltration of immune cells and the release of inflammatory factors, such as TNF-α and IFN-γ, were increased in animals with G422 gliomas following PDT, compared to non-treated controls. Splenic lymphocytes, isolated from PDT-treated mice, were able to induce anti-tumor immunity in nude mice. These workers could also demonstrate that PDT induced anti-glioma immunity was significantly reduced in tumor bearing complement C3 knockout as well as in nude mice.There have been few experimental studies exploring the effects of direct PDT of brain tumors. Li . It would therefore not affect the glioma cells in more distant infiltration zones in the resection cavity wall, which can be measured in cm. In addition, the tumor cells infiltrating normal brain that lead to tumor recurrence are protected by the blood brain barrier, so uptake of PS can be inadequate. To overcome the difficulties of in situ light delivery and dosimetry in postoperative brain tumor resection cavities, PDT produced anti glioma vaccines are a related approach that takes advantage of the immune stimulatory effects of ex vivo PDT of tumor cell cultures.Although PDT/PCI has clearly demonstrated the induction of a significant anti-tumor immune response, light based therapies are limited by the rapid absorption of light in tissue. For this reason the therapeutic efficacy of PDT, using presently available PSs, has been clinically confined mainly to superficial relatively flat tumors limited to skin and head and neck surfaces as well as bladder and esophagus. Effective PDT has been shown to extend only up to a depth of approximately 4 mm in cerebral tissueet al. demonstrated that lysates from PDT-treated tumor cells were more effective as preventative vaccines than tumor cells treated with UV, ionizing irradiation or cells subjected to freeze-thaw (F/T) cycles. Other groups have extended these results in several extra cranial models and could demonstrate that PDT-treated tumor cells could act as therapeutic anti-cancer vaccines,49.In earlier studies, using extra cranial tumor models, Gollnick in vitro co-culture of PDT treated glioma cells and DC or macrophages (Ma) acting as APCs. PDT generated vaccines against glioma cells have taken the form of CD activation by the use of crude tumor lysates, acid eluted crude lysates, and in vitro study, employing human glioma spheroids and dendritic cells from human donors, Etminan et al. (2011) showed that ALA-PDT of glioma spheroids in vitro promoted DC attraction, uptake of tumor antigens and maturation of DCs, three important initial steps of the afferent phase of adaptive immunity. Co-cultured DCs with ALA-PDT-treated spheroids promoted the induction of CD83 (a marker for mature DCs) and upregulation of the co-stimulatory molecules CD40, CD80 and CD86. Additionally HSP-70 was upregulated on the spheroids after ALA-PDT treatment.The generation of vaccines in experimental models using PDT has been explored by a number of groups. For example, in an et al. generated DC vaccines using Photofrine-PDT-treated C6 glioma cell to produce antigenic peptides to activate DCs ex vivo. Immune response parameters between DC vaccines from PDT acid-eluted induced supernatants, DC vaccines from PDT-induced C6 supernatants or DCs exposed to antigens generated by direct acid elution only or freeze-thawing. Effects of these adaptively transfer DCs on host immunity were evaluated by measuring cytokine induction, as well as assessing DC-induced cytotoxic T lymphocyte (CTL)-mediated lysis of C6 target. Their results demonstrated that PDT-acid elution resulted in more effective DC differentiation associated with a high expression of CD80 and MHC-II compared with the other vaccine treatment groups. In addition the induction of the highest rat serum levels of IL-12 and TNFα and the lowest IL-10 levels were observed in the PDT acid eluted peptide group. Spleen cells isolated from these animals effectively mediated lysis of C6 target cells. They concluded that PDT of C6 cells significantly enhanced tumor cell immunogenicity compared to freeze-thawed C6 cells.Shixiang . The photosensitizer Hypericin, a naturally occurring photosensitizer, mainly locates to the membranes of the endoplasmic reticulum and Golgi apparatus. Hyp-PDT has been shown to induce major DAMPs characteristic of ICD including surface-exposed calreticulin (CRT), surface exposed HSP 70/90, secreted adenosine triphosphate (ATP), and passively released high-mobility group box 1 (HMGB1) protein-56. ICD induced by Hyp-PDT was more effective in comparison to that induced by chemotherapy or radiotherapy.Reactive oxygen species (ROS) production and endoplasmic reticulum stress are believed to be important factors inducing ICDet al., combined HYP-PDT induced ICD with DC immunotherapy in an orthotopic HGG mouse model involving both prophylactic (immunization before i.c tumor cell implantation) as well as theraputic (immunization after i.c tumor cell implantation) treatment protocols. Both protocols using ICD-based DC vaccines demonstrated a significant anti-HGG survival benefit. In particular using a theraputic protocol, Hyp-PDT induced ICD-based DC vaccines together with chemotherapy (temozolomide) increased survival of HGG-bearing mice by up to 300%, resulting in half of the immunized animals becoming long-term survivors. Noteworthy was the observation that ALA-PDT treated glioma revealed no significant increase of CRT and release of HMGB1, two important DAMPs for the induction of ICD. The different characteristics of the various PSs used for PDT will in all probability determine their impact on subsequent antitumor immunity. Additionally, Hyp-PDT induced ICD-based DC vaccines appeared to induced an immune-stimulatory shift in the brain, from regulatory T cells to TH1/cytotoxic T lymphocyte/TH17 cells. A similar T cell shift has been shown to be associated with good patient prognosis in several tumor types,60.In a recent study Garg . Therefore, DCs it is argued, have no unique adaptation for antigen presentation that is not shared by other Ma and, as such, it is not surprising that both cell types are equally active vis a vis antigen presentation. We have used Ma together with the photosensitizer disulfonated aluminum phthalocyanine (AlPcS2a) mediated PDT of F98 rat glioma cells ex vivo. AlPcS2a is a photosensitizer which enters the cell by endocytosis, and tends to localize in endosomes and lysosomes. PDT at relatively low light dose rates causes partial damage to lysosomes resulting in the release of hydrolases, which trigger both apoptotic and/or autophagy cell death.Although DCs have been used as APCs in the vast majority of immunization studies recent work has shown that DCs are part of the mononuclear phagocyte system and that they are indistinguishable from macrophages (Ma) in several key areas including developmental pathways, markers and efficacy as APCs4C  glioma cells were used in these experiments, in an in vivo brain tumor development model. Co-incubation of naive Ma with AlPcS2a-PDT treated F98 glioma cells led to pronounced morphological changes of the Ma. Naïve Ma were round in shape, approximately 10 to 15 μm in diameter and are composed of an equal population of both adherent and floating cells in vitro. By contrast, activated Ma were significantly larger, irregular in shape with increased intracellular inclusions and all of the cells were adherent in culture. Inoculation of these primed Ma (acting as APC), significantly inhibited but did not completely prevent the growth of F98-induced tumors in the brain. Complete suppression of tumor development though, was obtained via AlPcS2a-PDT-treated tumor cell primed Ma inoculation combined with direct intra-cranial injection of allogeneic glioma cells. Interestingly, allogeneic glioma cells injected into the brain in one hemisphere did not form tumors but surprisingly slowed the growth of syngeneic tumors induced in the contra-lateral hemisphere in the same animal. This appeared to indicate a systemic immune response generated via i.c inoculation by allogeneic glioma cells, though inadequate to prevent tumor development, did have an inhibiting effect.Fischer rats and F98 (syngeneic) and BTet al.,64 in preclinical studies in rats using both allo and syngeneic stimulation. The underlying DAMPs developed by AlPcS2a-PDT, as has been previouslydemonstrated for Hyp-PDT-58, remains to be determined in detail.Allogeneic cells likely contain antigen determinants shared with the syngeneic tumor, leading to the observed reduction in tumor growth. This hypothesis is in agreement with the previous findings of Stathopoulos in vitro. In a subset of brain tumor patients, harboring surgically inaccessible tumors, interstitial PDT (iPDT) has been evaluated. Here light treatment is applied via stereotactically placed implantable fibers directly into the tumor. iPDT could be combined with direct injection of naïve APCs as has been done in several previously described extra-cranial experimental tumor models-28. This protocol translated to intra-cranial tumors is illustrated in In all of the above mentioned studies glioma tumor cells were PDT treated in situ APC activation which enhances antigen acquisition and processing as well as migration of the APCs to draining lymph nodes. This in vivo produced vaccine would potentially enhance the primary PDT effect and prevent tumor regrowth. It would also obviate the time consuming and costly necessity of priming APCs in vitro.This combined iPDT-APC injection strategy would both directly destroy portions of the tumor and additionally induce ex vivo PDT/PCI generated APC or peptide vaccines seems the most attractive approach. A deeper and detailed understanding of the induction of the antitumor immunity induced by light activated therapies would allow in the defining of protocols which would focus and enhance the immune system to recognize and prevent the inevitable post-operative recurrence of the tumor. Combining PDT induced anti-tumor vaccines with other therapeutic modalities including check-point inhibitors, is an exciting field to explore. Although not discussed in this review both PDT and PCI have an effect on the vasculature and have been show to temporarily open the blood brain barrier in a limited site specific region-68. What additional role this would play in the development of an effective and safe anti-HGG patient therapy, remains to be established.Although the experience with PDT/PCI produced anti HGG vaccines is limited and no clinical trials have been done, PDT/PCI appears to be an inducer of immunogenic cancer cell death, an important step in the afferent phase of the immune anti-tumor response. Light activated induced immunotherapy therefore holds the potential to become a complementary therapeutic option for for patients with HGG. Taking into account the penetration limitations of light activated therapies in the brain the further development of


Hyperscanning studies, wherein brain activity is recorded from multiple participants simultaneously, offer an opportunity to investigate interpersonal dynamics during interactive tasks at the neurophysiological level. In this study, we employed a dyadic juggling paradigm and electroencephalography (EEG) hyperscanning to evaluate functional connectivity between EEG sources within and between jugglers’ brains during individual and interactive juggling. We applied graph theoretical measures to identify significant differences in functional connectivity between the individual and interactive juggling conditions. Connectivity was measured in multiple juggler pairs with various skill levels where dyads were either skill-level matched or skill-level unmatched. We observed that global efficiency was reduced during paired juggling for less skilled jugglers and increased for more skilled jugglers. When jugglers were skill-level matched, additional reductions were found in the mean clustering coefficient and small-world topology during interactive juggling. A significant difference in hemispheric brain lateralization was detected between skill-level matched and skill-level unmatched jugglers during interactive juggling: matched jugglers had an increased right hemisphere lateralization while unmatched jugglers had an increased left hemisphere lateralization. These results reveal multiple differences in functional brain networks during individual and interactive juggling and suggest that similarities and disparities in individual skills can impact inter-brain dynamics in the performance and learning of motor tasks. Juggling is a unique and complex skill that depends on multiple perceptual-motor and cognitive abilities and processes including coordination, visuo-spatial attention, motor vigilance, performance related self-monitoring, and sensorimotor learning . AdvanceJuggling can be performed individually and between two or more interactive participants. Thus, it represents an opportunity to explore social factors such as team dynamics, cooperation, and interpersonal interaction . By acquhyperbrain functional networks, which include the functional connectivity within the brain of each participant (intra-brain connectivity) as well as the functional connectivity between the brains of two or more participants (inter-brain connectivity).In the last several years, hyperscanning techniques have expanded the field of social neuroscience and are yielding new insights into the neurodynamics that underlie interpersonal interaction  juggling . In thisIn the present study, we sought to extend our previous work by examining interactive juggling in multiple pairs of jugglers with diverse levels of expertise. To better assess the influence of skill-level on hyperbrain dynamics, we quantified general properties of hyperbrain functional networks at source level using measures derived from graph theory and measures of regional and hemispheric connectivity. Specifically, the aims of the present study were to (a) quantify differences in functional connectivity during individual juggling versus dyadic juggling tasks, (b) examine how individual juggling skill might affect functional connectivity in both conditions, and (c) explore how similar or dissimilar skill-levels affect functional connectivity in juggling dyads.To meet the aforementioned aims, we acquired EEG data from multiple pairs of jugglers during both individual and interactive juggling, reconstructed hyperbrain functional connectivity maps at the source level, and employed multiple measures of functional brain network topology to typify functional connectivity within and between jugglers’ brains.Thirteen jugglers , recruited from a professional juggling school in Northern Italy, participated in the study. Jugglers had between 3 and 19 years of experience in individual juggling but no prior experience in interactive juggling. All jugglers provided informed written consent prior to study participation according to the policies outlined in the Declaration of Helsinki and approved by the local institutional ethics committee.The experimental task was based on previous research on the juggling paradigm. The juggling paradigm purports that interactive juggling serves as an ideal platform to study social interaction in general, and the notion of team mental models in particular , 2017. Shalf-shower pattern. In the PAIRED trials, the same two jugglers were asked to juggle five balls between them (interactively) in an adapted half-shower pattern ((df) rationale . If the jugglers failed to complete a 20 s trial in either condition, additional trials were completed until a total of 12 successful trials were obtained for each condition. The order of conditions was counter-balanced to control for learning and vigilance confounds.In each session, data were collected from two jugglers in two conditions: an individual juggling (SOLO) condition and a paired juggling (PAIRED) condition. Both conditions consisted of juggling trials of at least 20 s duration with an inter–trial interval of approximately 1 min to allow the jugglers restart when they felt ready. In the SOLO trials, each juggler was asked to juggle three balls continuously in a so-called  pattern . Importaationale  in whichAn overview of the EEG data processing pipeline is provided in Electroencephalography recordings from each session were epoched into trials for each juggler in each condition. Only trials longer than or equal to 20 s  were retained for further processing. Epoched trials were trimmed to 10 s by removing the first and last 5 s from each trial, thus retaining only the 10 s interval of steady juggling. All trials were band-pass filtered between 1 and 100 Hz and a notch filter was applied to remove power line interference at 50 Hz. The EEG time-series from each trial was visually inspected and EEG channels exhibiting excessive noise or absence of signal were removed.To remove artifacts, each EEG trial was decomposed into 20 independent components (ICs) using an extended version of the Infomax ICA algorithm, which has been demonstrated to be more suitable to separate sources which may possess super-Gaussian and sub-Gaussian distributions . The top1. The eLORETA algorithm constructs an inverse (source) solution based on a realistic head model employing the MNI152 template where the solution space is restricted to 6239 voxels of 5 mm3 resolution comprising possible cortical gray matter and hippocampal sources estimated from the probabilistic Talairach atlas  algorithm  and implch atlas . We confch atlas . Since wWe chose to investigate functional connectivity only in the alpha frequency band (8.5–12 Hz) because (1) alpha band frequencies have been shown to be an important marker of human social coordinated behavior  and (2) To estimate functional connectivity within and between the jugglers’ brains, we calculated the phase synchronization (PS) between all ROIs within each juggler’s brain in the SOLO and PAIRED conditions and between jugglers’ brains in the PAIRED condition for each retained trial. PS is a measure of stability of phase difference between two time-series . PS values vary between 0 and 1, where zero indicates random phase differences and unity indicates a constant phase difference between two time-series. Because PS can over-estimate the degree of connectivity between brain sources due to contamination from brain volume conduction effects , we conshttps://doi.org/10.6084/m9.figshare.9384512.To calculate the LPS between ROI pairs, we employed a method described by 2) inter-brain ROI pairs, resulting in a total of 1326 averaged LPS values for each PAIRED condition trial.The preceding procedure was applied between all ROI pairs for each trial. In the SOLO condition, where 26 ROIs were defined, each ROI was compared to the remaining 25 ROIs. Because PS is a bi-directional measure, this resulted in 325 /2]) intra-brain averaged LPS values for each SOLO condition trial. In the PAIRED condition, there were 26 ROIs from each of the two jugglers’ brains. Thus, there were 325 intra-brain ROI pairs for each juggler and an additional 676  where the diagonals were self-connections. Additionally, there was one 52-by-52 ROI adjacency matrix for the PAIRED condition (hyperbrain matrix) where the upper and lower diagonal matrices were intra-brain connectivity maps and the off diagonal matrices were inter-brain connectivity maps . In eachTo synthetically describe the features of the functional networks , we employed multiple measures of functional brain network topology, including global measures derived from Graph Theory, which provides effective means to quantify the degree of integration, segregation, and efficiency in functional brain networks . We alsoGlobal efficiency (G) is a graph theoretic measure which quantifies the ability of a network to exchange information globally and is generally defined as the reciprocal or inverse of the average path length (L) of the network  or totally regular (lattice) structural features, urations . Random G suggested by L calculated using 100 random networks and 100 lattice networks. The random and lattice networks were generated by changing the connectivity values between the ROIs in the original intra-brain or hyperbrain network while preserving the same intra-brain and inter-brain degree distributions as in the original network  configuration  and to the high global efficiency of a totally disordered (random) configuration . Because it is possible for G to occasionally be less than 0 or greater than 1, particularly in highly sparse or dense networks, values less than 0 were set to 0 and values greater than 1 were set to 1  is a measure of the prevalence of clustered connectivity around individual nodes (ROIs) and reflects the tendency of the network to exchange information locally  is a measure of a network’s “small-worldness,” which is a tendency for a functional network to jointly possess a short average path length and a high mean average clustering coefficient as compared to random and lattice networks. Small-world networks therefore have the unique feature of combining high global and local efficiencies. It has been demonstrated that the functional organization of the human brain has small-world features both at rest and during the execution of simple motor tasks  that aims to measure the extent to which the two small-world characteristics of an observed network are jointly maximized, and can be calculated as the product of the normalized global efficiency G and the normalized clustering coefficient C. Given that both G and C can range from 0 to 1, also the product of these two terms  ranges from 0 to 1: when the network displays only one or neither small-world characteristic , SWI equals 0; when the network displays both small-world characteristics , SWI equals 1.The “small-worldness” of a functional network is generally quantified with a metric introduced by L and C and the generation of random and lattice networks were performed using the Brain Connectivity Toolbox  is another global measure from Graph Theory that describes the extent of connectivity in a network. D is defined as the proportion of connections out of all possible connections in graph, and is the simplest estimator of the physical cost  of a network  in the graph as a proportion of the total number of possible edges, which equals 325 for intra-brain networks, and 1326 for hyperbrain networks. network . D is caintra-brain/inter-brain ratio (IIR) is a measure unique to hyperbrain networks that quantifies the asymmetry in the functional brain maps of interacting subjects. IIR is defined as the ratio between the total number of intra-brain connections from both jugglers in the network and the number of inter-brain connections between the jugglers  and regional asymmetry (RA) measures similar to those used in other reports .Based on prior functional mapping studies where intrinsic brain activity lateralization within the attention system in normal subjects  and the  reports  to quantLatintra quantifies the asymmetry between the number of connections within the left and right hemispheres of each juggler. The values of Latintra are bounded between –1 and +1, where +1 indicates that all observed intra-brain connections occur in the right hemisphere, –1 indicates that all observed intra-brain connections occur in the left hemisphere, and 0 indicates that there are an equal number of right and left intra-brain connections . RAintra quantifies asymmetries between fronto-limbic regions and occipito-parietal regions. The values of RAintra are likewise bounded between +1 (only fronto-limbic intra-brain connections) and –1  with a value near 0 indicating lack of regional asymmetry.For intra-brain networks, Lat and RA are calculated for the connections between jugglers’ brains. In this case, Latinter and RAinter are measures specific to each juggler’s brain in the hyperbrain network and describe asymmetries in the distribution of connections to/from the left and right hemispheres and to/from the fronto-limbic and occipito-parietal regions of the brain of each juggler during dyadic juggling. As with the intra-brain connections, values of Latinter and RAinter are bounded between –1 and +1.For inter-brain networks, G, C, SWI, D, Latintra, and RAintra. We then evaluated functional connectivity in the MATCHED only sessions and UNMATCHED only sessions separately by performing two-factor ANOVAs for each skill-level group (MATCHED or UNMATCHED) where skill-level (more vs. less skilled) and condition (SOLO vs. PAIRED) were within-session factors. To determine the direction of effects, follow-up t-tests were performed for all ANOVAs that reached significance (p = 0.05). These follow-up tests were Bonferroni corrected to account for multiple comparisons.In order to accomplish the aims of the study, we first performed a series ANOVAs where we compared the intra-brain networks of each of the two jugglers from each session during the SOLO and PAIRED conditions. In each session, one of the jugglers had at least two more years of juggling experience than the other, allowing us to include juggling skill (defined as years of juggling experience) as a factor in the ANOVAs by comparing the intra-brain network of the more skilled juggler with the intra-brain network of the less skilled juggler. Thus, we performed three-factor ANOVA tests where skill-level (more vs. less skilled) and condition (SOLO vs. PAIRED) were within-session factors, and similarity in jugglers’ skill-levels (MATCHED vs. UNMATCHED) was a between-session factor. We performed six separate ANOVAs to identify significant differences in each of the different measures of functional connectivity applicable to intra-brain networks: Latinter and RAinter) due to skill-level (more vs. less skilled) and similarity of jugglers’ skill-levels (MATCHED vs. UNMATCHED) by performing two-factor ANOVAs in the PAIRED condition.We also investigated differences in inter-brain connectivity  revealed significant differences between the SOLO and PAIRED conditions in global efficiency (G) and hemispheric lateralization (Latintra).At the intra-brain level, the three-factor ANOVA tests performed for the applicable measures of functional connectivity  revealed a significant condition (SOLO vs. PAIRED) by skill-level (more vs. less skilled) interaction such that the less skilled juggler showed a decrease in G whereas the more skilled juggler showed an increase in G during PAIRED juggling compared to the SOLO juggling condition . No other significant contrasts were observed. Results for all comparisons are provided in the The three-factor ANOVA test of global efficiency (Latintra), which revealed a significant condition (SOLO vs. PAIRED) by skill-level groups (MATCHED vs. UNMATCHED) interaction  to model complex brain dynamics during complex “real-world” interactive tasks.To conclude, despite the aforementioned limitations, our study clearly revealed that multiple differences in functional brain networks during individual and interactive juggling exist, thus suggesting that individuals’ skill levels influence inter-brain dynamics in the performance and potentially the learning of complex motor tasks. Perhaps most importantly, our study is among the first to estimate source level connectivity across brains engaged in a naturalistic task and thus advances our neuropsychological understanding of the costs and benefits of teamwork performed by teammates with different skill levels, while also advancing a stepwise methodology [see attached homemade scripts available on FigShare . This protocol was approved by the University G. d’Annunzio of Chieti-Pescara  with Ethical Application Ref. no. 10-21/05/2015. All subjects gave written informed consent in accordance with the Declaration of Helsinki.All co-authors contributed to the study design. EF, SdF, and MB collected the data. DS, GT, and SC designed the procedure for data analysis. DS developed the software and wrote the manuscript. DS and GT analyzed the data. SC supervised all phases of the work. All co-authors evaluated the results and revised the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Introduction: Previous studies have proven the effectiveness of bilateral cochlear implantation compared to unilateral cochlear implantation. In many of these studies the unilateral hearing situation was simulated by switching off one of the cochlear implants in bilateral cochlear implant users. In the current study we assess the accuracy of this test method. Does simulated unilateral hearing (switching off one cochlear implant) result in the same outcomes as real life unilateral hearing with one cochlear implant and a non-implanted contralateral ear?Study design: We assessed the outcomes of one arm of a multicenter randomized controlled trial.Methods: In the original trial, 38 postlingually deafened adults were randomly allocated to either simultaneous bilateral cochlear implantation or sequential bilateral cochlear implantation. In the current study we used the data of the sequentially implanted group (n = 19). The primary outcome was speech perception-in-noise from straight ahead. Secondary outcomes were speech perception-in-silence, speech intelligibility-in-noise from spatially separated sources and localization capabilities. A within-subjects design was used to compare the results of hearing with one cochlear implant and a non-implanted contralateral ear (1- and 2-year follow-up) with the results of switching off one cochlear implant after sequential bilateral implantation (3-year follow-up).Results: We found no significant differences on any of the objective outcomes after 1-, 2-, or 3-year follow-up.Conclusion: This study shows that simulating unilateral hearing by switching off one cochlear implant seems a reliable method to compare unilateral and bilateral hearing in bilaterally implanted patients.Clinical Trial Registration: Dutch Trial Register NTR1722. Cochlear implantation (CI) has become a widely applied intervention in the treatment of patients with severe to profound bilateral sensorineural hearing loss, who obtain limited benefit from conventional hearing aids.Although many patients with a single cochlear implant achieve high levels of speech perception-in-silence, even the most successful cochlear implantees experience difficulty with speech perception-in-noise and localization capabilities , 2.In 2009, our study group started a randomized controlled trial (RCT) concerning the effectiveness of simultaneous bilateral CI (simBiCI) compared with either 1) unilateral CI (UCI)  unilater, or (2) Earlier (cohort) studies showed similar benefits of BiCI compared to UCI, however they used different methods to simulate the unilateral listening situation. In most of these studies, differences between bilateral and unilateral hearing were assessed using a within-subjects study design by switching off one cochlear implant in a group of bilaterally implanted patients and comparing the results with the bilateral listening situation –13.Our hypothesis was that this simulated unilateral listening situation would not be representative for an actual UCI situation. The electrode in a patient with bilateral implants would have diminished residual hearing. On the other hand, patients with bilateral implants are used to listening with two ears in everyday life, while in patients with a unilateral cochlear implant, patients may not have used one ear for an extensive period of time.We performed the current study to assess the reliability of switching off one cochlear implant as a method to simulate unilateral hearing in bilateral cochlear implant users.Data for the current study were collected as part of a multicenter RCT that compared simBiCI to seqBiCI –4.This study was approved by the Human Ethics Committees of all participating centers  (NL2466001808), registered in the Dutch Trial Register (NTR1722) and conducted according to the Declaration of Helsinki. Written informed consent was obtained from all participants.All participants eligible for CI were discussed in our cochlear implant team. Inclusion- and exclusion criteria were verified for each participant . After rIn the current study, we will focus on the first implanted side (CI1) in the seqBiCI group, by using a within-subjects design. Patients in this arm received their second cochlear implant 2-years after their first cochlear implant. Objective outcomes were measured after the first implantation at 1- and 2- years follow-up. After 2-years of follow-up, the patients received their second implant. In order to assess whether simulated unilateral hearing (switching off one cochlear implant) provides the same outcomes as real life unilateral hearing, we compared the data of this group 1- year after unilateral implantation (1-year follow-up) with the situation after bilateral implantation (3-year follow-up), in which we switched off the second cochlear implant (CI2). As a sensitivity analysis, to correct for a possible learning effect with the first implanted ear, we also compared the unilateral 2-year follow-up data with the simulated unilateral 3-year data (switching off the second cochlear implant).The primary outcome was speech perception-in-noise from straight ahead, measured with the Utrecht-Sentence Test with Adaptive Randomized Roving levels resulting in a speech reception threshold in noise (SRTn). A lower threshold value reflects better speech perception. A SRTn of 30 dB was considered speech perception in relative silence and was used as a cut-off point for all scores above 30 dB.The other outcomes were (1) speech perception-in-silence, (2) speech intelligibility-in-noise from spatially separated sources (SISSS), and (3) localization capabilities. All these objective tests were conducted using the AB-York Crescent of Sound set-up. In previous articles of our study group more detailed information was presented about the test procedures and setup.Speech perception in silence was measured using the standard Dutch consonant-nucleus-consonant (CNC) test.In the SISSS, in which the outcome is also expressed as an SRTn, sentences were presented from 60° azimuth to the left of the patient and noise from 60° azimuth to the right of the patient (S-60 N+60) or vice versa (S+60 N-60). When sounds come from different directions, participants usually have a best performing situation and a worst performing situation. In current study, in which we evaluate the unilateral group or situation, speech presented to the cochlear implant side was indicated as the best performing situation. If speech was presented to the contralateral ear and noise to the cochlear implant side we indicated this as the worst performing situation.For the localization test, participants were instructed to face the loudspeaker in front of them during the entire procedure. Thirty phrases were presented randomly at 60, 65, or 70 dB SPL from one of the loudspeakers. The results were percentage correct responses in three localization conditions: 15° angle azimuth between 5 loudspeakers, 30° angle azimuth between 5 loudspeakers, and 60° angle azimuth between 3 loudspeakers.All gathered data were double-checked by two independent persons who did not have any further connections to the otorhinolaryngology department.t-test for numeric normally distributed data, the Wilcoxon signed rank test for non-normally distributed data and the chi-square test for ordinal data.In order to compare baseline characteristics, means or medians were reported depending on normality of data. We used the Student Between December 2009 and September 2012, a total of 19 participants were included in the seqBiCI group. During the second and third year, two participants in the seqBiCI group withdrew because of personal reasons. Another participant was excluded because of poor results with the first implant and low expectations after sequential implantation owing to central deafness caused by rhesus antagonism .We found no significant differences between simulated unilateral hearing (switching off one cochlear implant) 1-year after seqBiCI and real life unilateral hearing 1- and 2-years after UCI. The results of all outcomes separately are presented in In order to assess methodological issues with simulation of cochlear implant use, in present study we assessed whether simulated unilateral hearing (switching off one cochlear implant) provides the same outcomes as real life unilateral hearing.Binaural hearing has been proven to be superior to unilateral hearing with regard to speech perception in noise and sound localization , 14–17. We assessed if this is a reliable test method.However, the current study demonstrated similar results after UCI and hearing with one cochlear implant switched off after seqBiCI on speech perception-in-silence, speech intelligibility-in-noise and localization tests.This is the first study that reports whether simulated unilateral hearing (switching off one cochlear implant) provides the same results as real life unilateral hearing.A strength of our study is that we used a prospective within-subjects study design. All data were collected at fixed moments. Secondly, by measuring after a follow-up of at least 1-year after implantation, it was safe to assume that patients were used to their implants and that we had corrected for a possible learning effect. Furthermore, at time of inclusion, all patients suffered from profound sensorineural hearing loss [pure-tone average of greater than 90 dB ]. Because of the profound hearing loss in the contralateral ear (second implanted ear) patients were not used to listening with two ears after the first implantation. Therefore, the situation before the second implantation can be considered as actual unilateral hearing.As this study was based on a secondary analysis from a larger RCT, a power analysis for the present study was not performed and the study may be underpowered.We found no significant differences between simulated unilateral hearing (switching off one cochlear implant) and real life unilateral hearing. This study shows that simulating unilateral hearing by switching off one cochlear implant seems a reliable method to compare unilateral and bilateral hearing in bilaterally implanted patients.This study was carried out in accordance with the recommendations of the medical ethics committee (MEC) of the Academic Medical Center (AMC) with written informed consent from all subjects in accordance with the Declaration of Helsinki. The protocol was approved by the MEC AMC. This study was also registered in the Dutch Trial Register (NTR1722).All authors have made substantial contributions to the paper. AvZ collected parts of the data, analyzed the data, and wrote the paper. YS designed the study, recruited and included participants, collected parts of the data, and critically revised the paper. VK collected parts of the data and critically revised the paper. GvZ provided audiometric advice and critically revised the paper. RS recruited and included participants and critically revised the paper. IS provided advice and was involved in the methodology for present study, statistical analysis and co-authored the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Equine laminitis is a complex disease that manifests as pain and lameness in the feet, often with debilitating consequences. There is a paucity of data that accounts for the multifactorial nature of laminitis and considers time-varying covariates that may be associated with disease development; particularly those that are modifiable and present potential interventions. A previous case-control study identified a number of novel, modifiable factors associated with laminitis which warranted further investigation and corroboration. The aim of this study was to identify factors associated with equine laminitis in horses/ponies in Great Britain (GB) using a prospective, web-based cohort study design, with particular interest in evaluating modifiable factors previously identified in the case-control study.Self-selected horse/pony owners in GB submitted initial baseline and follow-up health and management questionnaires for 1070 horses/ponies between August 2014 and December 2016. The enrolled horses/ponies contributed 1068 horse-years at risk with a median of 38 days between questionnaire submissions. Owners reported 123 owner-recognised and/or veterinary-diagnosed episodes of active laminitis using a previously-validated laminitis reporting form. Multivariable Cox regression modelling identified 16 risk/protective factors associated with laminitis development. In keeping with the previous case-control study, a prior history of laminitis (particularly non-veterinary-diagnosed episodes), soreness after shoeing/trimming and weight gain were associated with higher rates of laminitis. There is now strong evidence that these risk factors should be used to guide future recommendations in disease prevention. Factors with some prior evidence of association included breed, steroidal anti-inflammatory administration, transport and worming. The modifiable factors amongst these should be the focus of future laminitis studies. The remainder of the identified factors relating to health, turnout and grazing management and feeding are novel, and require further investigation to explore their relationship with laminitis and their applicability as potential interventions.This study has demonstrated a temporal relationship between a number of horse- and management-level factors and laminitis, identifying potential interventions and important risk groups for which these interventions would be of particular importance. These results serve as a sound evidence-base towards the development of strategic recommendations for the horse/pony-owning population to reduce the rate of laminitis in GB.The online version of this article (10.1186/s12917-019-1798-8) contains supplementary material, which is available to authorized users. Equine laminitis is a complex, multifactorial disease that manifests itself in the foot when a cascade of inflammatory, endocrine and/or vascular reactions cause disruption to the structure and function of the suspensory apparatus of the distal phalanx (SADP) . Pain-inFew robust epidemiological studies have been conducted to ascertain the animal- and management-level factors that contribute to laminitis development . A 2012 The aim of the current study was to identify factors associated with owner-reported equine laminitis  in horses/ponies in GB using a prospective cohort study design, with particular interest in evaluating modifiable factors previously identified in a case-control design .A prospective, dynamic web-based cohort study was conducted in GB between August 2014 and December 2016 (29 months). Recruitment of participants was based on self-selection of individuals who owned or cared full-time for a horse/pony that resided in England, Scotland or Wales. Prior to, and for the duration of, the recruitment period (August 2014 to July 2016), the study was advertised nationally across a variety of equestrian and veterinary media platforms and/or organisations. Owners enrolling only some of their animals were instructed to enrol those whose names appeared first in the alphabet, to avoid bias. A dedicated website hosted the data collection tools and assisted with participant recruitment and retention.Sample size estimates (OpenEpi v3.03)Participants were required to submit a baseline management and health questionnaire (hereafter referred to as ‘the questionnaire’), supplemented with regular consecutive follow-ups and the reporting of incident active equine laminitis episodes. Where internet access was limited, participation was facilitated by posted questionnaires and structured telephone/in-person follow-ups. Retention of participants was aided by automated monthly reminder e-mails and manual personalised e-mails in instances where no follow-ups had been obtained for more than 3 months. Study updates were e-mailed to all participants approximately every 2 months and regular incentives, mainly in the form of prize draws and competitions, were used to attract new participants and encourage follow-up submissions.The questionnaire  for 14 days or more’. An active episode was described as ‘recognition of pain in one or more feet attributed to laminitis, with stance and/or gait abnormalities’. A default laminitis ‘recovery period’ of 44 days was used for episodes where recovery time was missing, based on the median recovery time estimated from historical episodes in the study population that occurred prior to study enrolment ) has previously been associated with higher laminitis risk [Presence of endocrine disease  receiving NSAIDs were older than 17 years, with owners reporting that many were on long-term maintenance doses of phenylbutazone to control arthritis-related pain and promote joint mobility. The effect of chronic NSAID usage, and its contribution to laminitis development, should be considered given the ability of NSAIDs to alter faecal microbiota and contribute to gastrointestinal injury in both horses and people –56. AlteNon-laminitis-related soft tissue injury was associated with laminitis development. Where details were provided (21 cases), 42.9% were described as ligament and 19.0% as tendon injuries. These animals were commonly on restricted turnout or field rest, with only 19.8% on box rest (defined as confinement to stable/shelter for 24 h per day with no exercise) – a previously identified risk factor for laminitis , 58, it An association between laminitis and new access, or abrupt changes in access, to grass , 10 was Multiple environmental factors influence accumulation and storage of NSCs in grass, the rate and storage of which also depend on grass type . Thus thThe only protective factor identified related to animals whose turnout bordered woodland. This association is potentially a proxy measure for another exposure, such as geographical location or pasture characteristics , althougLimitationsThe main challenges with field-based prospective cohort studies are recruiting and retaining a large and representative cohort –67. The This study has contributed to improved knowledge of laminitis epidemiology in GB and has demonstrated a temporal association between a number of animal- and management-level factors and owner-reported laminitis. It has corroborated the association between previously-identified risk factors and laminitis and identified potential management-related interventions and risk group identifiers which should be targeted for future detailed study. There is now consistent evidence that weight gain, a previous history of laminitis, and soreness following routine hoof care are associated with laminitis development, as the same factors were found to be associated with laminitis in a previous case-control study . PrioritAdditional file 1:Online health and management questionnaire used by participants in the cohort study of equine laminitis in Great Britain. (PDF 1040 kb)Additional file 2:The monitoring of weight and condition booklet provided to participants in the cohort study of equine laminitis in Great Britain. (PDF 2645 kb)Additional file 3:Online owner laminitis reporting form used by participants in the cohort study of equine laminitis in Great Britain. (PDF 331 kb)Additional file 4:A table showing the demographics of the cohort population of horses and ponies at baseline presented in descending order of frequency, unless the variable was ordinal in nature. (DOCX 23 kb)Additional file 5:P < 0.25) in a cohort study of horses and ponies in Great Britain. (DOCX 108 kb)A table showing the univariable Cox regression results of variables associated with laminitis  in a cohort study of horses and ponies in Great Britain. (DOCX 37 kb)A table showing the multivariable Cox regression interim models of variables associated with laminitis (