Plants are movers, but the nature of their movement differs dramatically from that of creatures that move their whole body from point A to point B. Plants grow to where they are going. Bio-inspired robotics sometimes emulates plants' growth-based movement; but growing is part of a broader system of movement guidance and control. We argue that ecological psychology's conception of “information” and “control” can simultaneously make sense of what it means for a plant to navigate its environment and provide a control scheme for the design of ecological plant-inspired robotics. In this effort, we will outline several control laws and give special consideration to the class of control laws identified by tau theory, such as time to contact. Bioinspired robotics and artificial intelligence has taken various forms, including genetic algorithms, artificial life, and evolutionary robotics Langton, ; behavioMuch plant-inspiration remains to be discovered  to be informed by the very environment as to where to go about. A plant-inspired ecological robot is a robot that can tune to the structure that the surrounding energy media provides. To do so, we suggest, robots could be engineered to exploit the same type of control laws that plants exploit. It is our hope that a plant-inspired ecological robotics will allow researchers to pay due consideration to some new challenges, and opportunities, for robotics and artificial intelligence.move  exhibits a high degree of metabolism-based tolerance to extreme heat, lack of Nitrogen, or to a salty environment  information” is that the various ambient energy media surrounding an organism are structured by the dynamics of its environment. At a given point of observation (PO), a set of relations exists between the PO and the field, as well as between the sub-fields defined by local distributions of ambient energy. As either the location of a PO or that of an object in the environment is transformed, so too is the set of relations. An organism differs from a generic PO in that it can only access so much of the field—it has a perspective. Nevertheless, the story is similar to that of the PO. As the organism's perspective (or position of an object in the environment) changes, so too do relations within the subfield. Some relations change systematically, while others remain constant . Most importantly, a relationship between transformations of an organism's perspective are directly related to transformations of the relations between distributions of energy in the sub-field . The latter constitute “lawful information” about the former. Processes that generate transformations of the organism's perspective constitute the “control structure.” Mathematically, control and information are duals, akin to the relationship between points and lines, where two points define a line, and two intersecting lines define a point  of the ball on the screen at a given time t isThe Chapman strategy Chapman,  for catcXball, Yball) and  are the position of the ball and outfielder (respectively) in an inertial coordinate system  is ḧ, the second derivative. Control laws, in this situation, arise from the following  is positioned to catch the ball, (2) needs to run backwards, or (3) needs to run forwards. If moving at constant velocity in the same direction as the ball, the outfielder (1) is running at the right speed to catch the ball, (2) needs to run at a faster constant velocity, and (3) needs to run at a slower constant velocity. In both cases, the strategy is cancellation of the optical acceleration . The same strategy can be applied more generally, where constant velocity  of the PO is no longer assumed, so that Ẍoptic field consists of a packed nesting of reflected-light optical cones extending out from a single PO to all the edges and textures around it (see optic flow field (OFF). Note that, as with catching a flyball, the PO need not be occupied by an eyeball or sensor on a creature or robot—it could just as well be a particle of dust. Nevertheless, an observer's perspective is limited by their embodied constitution, including their ocular apparatus, to only a portion of the OFF, the visible optic flow field (VOFF). The VOFF, embedded as it is in the OFF and defined as it is as a relation between the organism (its PO) and the objects in its environment, manifests a number of control laws. To adumbrate a few of them: moving forward expands the VOFF; moving backward contracts it; moving sideways translates it. The distribution of optical cones for an object moving toward the PO (at rest) will expand while the rest of the field remains the same; or, if the PO is also in motion, the object's optical cones will expand more rapidly, and it will occlude some of the others. Objects hidden behind others will introduce accrete or delete cones as it comes in and out of view. And so on  = X(t)/Ẋ(t), which describes the time to close gap X, given the current rate of closure. In the case of control systems, the gap is between the current PO and a final  PO. Rate just at or before colliding with an object requires stabilizing X and its rate of closure Ẋ are constituted in the relationship between a PO and an object. Unlike OA and OFF, X and Ẋ cannot be detected. On the other hand, ρ(t) = 1/τ(t) = Ẋ(t)/X(t), a proportion of the rate of change to the current size of the gap, can be detected  = λX, Yρ, where X and Y are the two gaps, and λX, Y is a scaling factor setting the relative velocity curve. In the case of action guided by a gap closure with constant acceleration from rest, the rho ρ = λX,GρG, where TG is the time it takes to close the guiding gap G, and Gprescribes the value of λx, G. In the case of a guiding gap D with constant deceleration, ρD = 2/(t−TD)  Lee, .power (its magnitude), whether found in ambient energy media, stimulation of sensory organs, or flows of electrochemical energy in a nervous or phytonervous system  moves, it transforms stimulation at the sensors, generating gaps and taus/rhos. If the tau/rho in the environment prescribes tau/rho of a movement, then by picking up source power, stimulus power, then neural/phytoneural power, each tau/rho acts as a prescription for the next, an ensemble of coordinating degrees of freedom.The reader may be willing to admit that between organism or robot and environment, the control law is king, but once getting re Raja, . The schThe foregoing provides a sampling of the richness available to a robotics and artificial intelligence for future research via ecological psychology and an emerging dialogue with neuroscience. Further models of “higher level” psychology exist within and around ecological psychology, such as direct learning (Jacobs and Michaels, PC conceived the original idea. PC and PF devised the project, the main conceptual ideas, and proof outline. PF worked out the technical details and wrote the manuscript in consultation with PC, LJ, and KA. All authors provided feedback and helped shape 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.


CSF levels of total-α-synuclein and phosphorylated-α-synuclein, neurofilament light chain, amyloid-β42, and total-tau and phosphorylated-tau were measured in 26 PD patients (disease duration ≤5 years and Hoehn and Yahr stage 1–2.5). Multivariable linear regression models, adjusted for age, gender, and educational level, were used to assess the relationship between CSF biomarker levels and memory, attention, executive and visuospatial function, and language performance scores. In 26 early-stage PD patients, attention and memory were the most commonly affected domains. A higher CSF phosphorylated-α-synuclein/total-α-synuclein ratio was associated with better executive functioning (sβ = 0.40). Higher CSF neurofilament light was associated with worse memory (sβ = −0.59), attentional (sβ = −0.32), and executive functioning (sβ = −0.35). Reduced CSF amyloid-β42 levels were associated with poorer attentional functioning (sβ = 0.35). Higher CSF phosphorylated-tau was associated with worse language functioning (sβ = −0.33). Thus, CSF biomarker levels, in particular neurofilament light, were related to the most commonly affected cognitive domains in early-stage PD. This indicates that CSF biomarker levels may identify early-stage PD patients who are at an increased risk of developing cognitive impairment.In early-stage Parkinson′s disease (PD), cognitive impairment is common, and a variety of cognitive domains including memory, attention, and executive functioning may be affected. Cerebrospinal fluid (CSF) biomarkers are potential markers of cognitive functioning. We aimed to explore whether CSF α-synuclein species, neurofilament light chain, amyloid-β Cognitive impairment is a common non-motor symptom in early-stage Parkinson′s disease (PD), and can have a major negative impact on the patient′s quality of life . At the 42 (Aβ42), total tau (t-tau), and tau phosphorylated at threonine 181 (p-tau) have been extensively studied in different cohorts [To identify patients who are at an increased risk to develop cognitive impairment, sensitive and specific biofluid biomarkers that reflect underlying pathophysiological mechanisms of cognitive dysfunction are needed. A variety of mechanisms are proposed to contribute to cognitive dysfunction in PD, such as misfolding of the proteins α-synuclein, amyloid-β, and tau, axonal degeneration, synaptic dysfunction, neuro-inflammation, and abnormalities of brain connectivity . In sear cohorts ,10,11,12 cohorts ,11,12. H cohorts ,11,13. I cohorts ,11. Like cohorts ,11,12,13 cohorts . CSF neu cohorts . In PD,  cohorts . Since a cohorts . HoweverThe aim of this study was to explore whether CSF levels of t-α-syn, p-α-syn, p-α-syn/t-α-syn, NfL, Aβ42, t-tau, and p-tau are associated with cognitive performance in early-stage PD patients. First, we determined which of the five cognitive domains, involving memory, attention, executive functioning, visuospatial functions, and language, were most commonly impaired in an early-stage PD cohort. Next, we investigated whether CSF t-α-syn, p-α-syn, p-α-syn/t-α-syn, NfL, Aβ42, t-tau, and p-tau were associated with cognitive functioning of the previously mentioned domains in our cohort.Twenty-six early-stage PD patients with neuropsychological test scores and CSF biomarker levels were included in the study. n = 10, 38.5%), followed by memory functioning . Performance on executive function tests was relatively good with impairment in only one patient (3.8%). The frequency of impairment of visuospatial functions was 7.7% (n = 2). None of the patients had language difficulties.PD-MCI criteria were met by 3 out of 26 patients (11.5%) [To assess relationships between CSF biomarker levels and performance on the five cognitive domains, we performed univariable and multivariable regression analyses. Associations of CSF biomarker levels with cognitive domain scores are presented in p = 0.03, multivariable: sβ = 0.41, p = 0.01) (p = 0.03), also in the model in which we adjusted for age, gender, and level of education  (We found no significant association of single CSF t-α-syn levels with cognitive functioning in univariable and multivariable models . CSF p-α = 0.01) . Similar = 0.02) . Individp = 0.001, and sβ = −0.59, p = 0.002, respectively)  and executive functioning   . In addi = 0.06) .p = 0.01, multivariable: sβ = 0.35, p = 0.04)   . The ass = 0.25) .p = 0.09, sβ = −0.42, p = 0.04, and sβ = −0.40, p = 0.06, respectively) (p = 0.12). However, the associations between CSF p-tau and executive and visuospatial functioning were strongly attenuated   . After ictively) .In this cross-sectional study, we aimed to explore whether CSF biomarkers reflecting protein pathology and axonal degeneration are associated with cognitive performance in early-stage PD patients. Particularly, higher CSF NfL was strongly associated with worse performance on memory, and also associated with worse attentional and executive functioning. Furthermore, we found a moderate association between CSF p-α-syn/t-α-syn and executive functioning, between CSF Aβ42 and attentional functioning and between CSF p-tau and language.In this study of early-stage PD patients, attention and memory were the most commonly impaired cognitive domains. Worse performance on memory tasks may be secondary to attentional deficits, even though a previous study in PD patients showed that memory remained impaired after controlling for the effect of impaired attention . Taken tIn particular, domains that predominantly affected our early-stage PD group were associated with CSF NfL levels. Our analyses revealed an inverse association of CSF NfL levels with performance in the memory domain, and, to a lesser extent, with attentional and executive functioning. Since CSF NfL is considered a marker of axonal injury, our results suggest that axonal degeneration may lead to loss of connectivity and play an important role in impairment in multiple cognitive domains in early stage PD. To the best of our knowledge, there have not been any previous cross-sectional studies investigating CSF NfL in relation to cognitive functioning in early-stage PD patients. However, associations of CSF NfL with impairments in various cognitive domains have been reported in Alzheimer′s and frontotemporal dementia, and multiple sclerosis ,20.positive association between CSF p-α-syn/t-α-syn and executive functioning. Additional cross-sectional and longitudinal studies including patients with a wider range of executive performance are needed to validate the potential association of CSF p-α-syn/t-α-syn levels with executive functioning.Although test scores in the executive functioning domain were generally high with little variability, we did find an association of higher CSF p-α-syn/t-α-syn levels with better executive functioning in our patients. Since we observed a very weak association between single CSF t-α-syn levels and executive functioning and a stronger association of CSF p-α-syn levels with executive functioning, the association between the CSF p-α-syn/t-α-syn ratio and executive functioning is mostly due to CSF p-α-syn levels. We previously observed higher CSF p-α-syn/t-α-syn levels in early-stage PD patients compared to healthy controls , suggestThis study showed an association between lower CSF Aβ42 and worse performance on attentional functioning. A similar relation between CSF Aβ42 levels and attentional functioning has been reported previously in a cohort of PD patients with a longer disease duration compared to our cohort . Since rAlthough none of the patients had language difficulties, we found a weak inverse association between CSF p-tau and language. The language domain was assessed in only two previous early-stage PD studies, but no significant association with p-tau was found ,13. ThisData were selected from a cross-sectional cohort of PD patients recruited between 2008 and 2010 at the movement disorders outpatient clinic of Amsterdam UMC, location VUmc, previously described in detail by Van Dijk et al. . All patComprehensive neuropsychological testing was performed by a board-certified neuropsychologist. The testing battery covered five cognitive domains: memory functioning, attention, executive functioning, visuospatial functioning, and language. Aspects of memory were tested using the modified version of the Rey Auditory Verbal Learning Test (RAVLT) to assess delayed recall , and thePatients were classified as PD-MCI if performance on at least two neuropsychological test outcomes in one or multiple cognitive domains was impaired, according to the Movement Disorder Society (MDS) level II criteria . Since tIn order to generate composite domain scores, raw neuropsychological test scores not corrected for age, gender, and level of education, were standardized to z-scores. TMT and Stroop test scores were linearly converted so that lower scores reflect worse performance. Five composite domain scores were formed by calculating mean z-scores from different test outcomes within the same domain. These domain z-scores were used to investigate associations with CSF biomarkers.g for 10 min at room temperature and, thereafter, aliquoted in 0.5 mL polypropylene tubes, and stored at −80 °C [CSF was obtained by lumbar puncture between the 3rd and 4th or the 4th and 5th lumbar vertebrae, as previously described . CSF wast −80 °C . Concentt −80 °C ,23,30.For statistical analyses, SPSS 24 for Windows  was used. We first determined the frequency of PD-MCI and PD dementia, and the frequency of impairment across different cognitive domains. Then, to explore the relationship between CSF biomarkers and cognitive functioning, a linear regression analysis was used with the composite domain scores as dependent variables and CSF biomarker levels as independent variables. For each variable, first univariable linear regression analyses were performed. Then, multivariable linear regression analyses including the potential confounders age, gender, and level of education were used .In conclusion, in our cohort of early-stage PD patients, attention and memory are among the most frequently affected cognitive domains. Our results show that CSF p-α-syn/t-α-syn, CSF Aβ42, p-tau, and, in particular, CSF NfL levels are associated with cognitive performance in early-stage PD patients. This indicates that CSF biomarkers reflecting protein pathology and axonal degeneration may identify early-stage PD patients who are at an increased risk of developing cognitive impairment.


Corynebacterium striatum is common contaminant in clinical specimens. Here, we report a rare case of pyogenic tenosynovitis of the wrist caused by C striatum in a dermatomyositis patient taking oral immunosuppressants.C striatum was detected in the cultures of three samples of synovial fluid taken from the dorsal hand.A 67-year-old Japanese woman with dermatomyositis had a history of multiple intraarticular injections of corticosteroids to the right wrist joint for the treatment of osteoarthritis. She was admitted to our hospital with a painful lump on the right dorsal wrist lasting for three months. MRI revealed cellulitis of the dorsum of the right wrist and hand and fluid collection in the extensor tendon sheath. C striatum.Pyogenic tenosynovitis of the wrist due to The infection was successfully controlled with synovectomy and adjuvant antibiotic therapy.There has been no sign of recurrence for 12-months after the surgical treatment.C striatum in a patient with dermatomyositis. Clinicians should be aware that patients undergoing immunosuppressive therapy have a risk of C striatum infection.This is the first reported case of pyogenic tenosynovitis due to  Except for Corynebacterium diphtheriae, all Corynebacterium spp. are ubiquitous skin commensals. However, some reports have highlighted the importance of C striatum as causes of catheter-related bloodstream infections and various other infections, such as arthritis and endocarditis, in immunosuppressed patients.–5 Here, we report a rare case of pyogenic tenosynovitis of the wrist caused by C striatum in a patient with dermatomyositis who was taking oral immunosuppressants. The infection was successfully controlled with synovectomy and adjuvant antibiotic therapy.2Mycobacterium abscessus and mycotic pneumonia. Her dermatomyositis was controlled with prednisolone 5 mg/day and azathioprine 75 mg/day for 3 years. The patient had received clarithromycin (CAM) (400 mg/day) for the treatment of an M abscessus infection and itraconazole (100 mg/day) for the treatment of mycotic pneumonia for 1 year, in addition to trimethoprim/sulfamethoxazole (160/800 mg twice weekly) as a prophylaxis for opportunistic infections for 4 years. She had a history of multiple injections of corticosteroids into the right wrist for arthralgia due to osteoarthritis for 6 months. Prior to admission, the patient had a 3-month history of a painful lump on the dorsal area of the right wrist, that had gradually increased in size and finally measured approximately 3 × 5 cm  and a normal white blood cell count (WBC: 7200 /mm3) , symmetric proximal muscle weakness, elevated serum skeletal muscle enzymes, and interstitial lung disease. She had a past history of pleurisy caused by conazole 00 mg/dayconazole 00 mg/dayconazole 00 mg/daynt WBC: 700 /mm3 (nt WBC: 700 /mm3 (3Staphylococcus aureus and Streptococcus species are found to be the causative organism in most cases. Atypical organisms including gram-negative rods and mixed flora have been found in immunocompromised patients.Pyogenic tenosynovitis frequently occurs subsequent to a puncture wound. Therefore, skin flora such as C striatum in a patient with dermatomyositis who was taking oral immunosuppressants. There have been few reports of pyogenic arthritis and osteomyelitis due to C striatum.,7,8 To the best of our knowledge, no case of pyogenic tenosynovitis of wrist due to C striatum has been reported.We presented a case of pyogenic tenosynovitis of the wrist due to Corynebacterium species are opportunistic human pathogens that are distributed in the skin and mucous membranes of asymptomatic individuals. These organisms are often considered contaminants when isolated in culture because of their low virulence. However, when isolated repeatedly in pure growth from a normally sterile body site, such as the synovial fluid or blood, in a clinical context consistent with infection, they should be considered clinically relevant, and identification at the species level and antimicrobial susceptibility testing are recommended. In our case, the first bacterial culture of the fluid was thought to indicate skin contamination; however, C striatum was repeatedly detected in the cultures of aspirated material. Therefore, we considered C striatum the pathogenic microorganism. Saglam et al reported an intratendinous septic abscess of the Achilles tendon after multiple local steroid injections and considered that the abovementioned effect may increase the possibility of infection at the site of local corticosteroid injection. Even in our case, this effect may have been the basis for the infection.Corticosteroids results in decreased fibrosis and reduced vascular proliferation in peritendinous tissues, along with anti-inflammatory effects that include decreased leukocyte migration and phagocytosis.C striatum infection include a history of collagen disease, diabetes mellitus and immunosuppressants therapy.,7,12,13 In this case, the patient received prednisolone and an immunosuppressant for approximately 3 years for the treatment of dermatomyositis. Additionally, we concluded that the main cause of the infection was the iatrogenic inoculation of C striatum from the bacterial flora of the skin into the extensor tendon sheath.Previously reported risk factors for C striatum infection includes initiation of appropriate antibiotic therapy and involve possible removal of infectious lesion to ensure complete eradication of the nidus of infection. There is growing concern over resistance of C striatum to penicillins, and cephalosporines, as seen in our case, even though previously reported susceptibility to penicillins. Repeated isolation of C striatum from a sterile body site should prompt early laboratory request for drug-susceptibility testing. Susceptibility testing is not routinely performed otherwise by most laboratories as C striatum is considered a contaminant in most clinical situations. Early detection and appropriate treatment of this infection is important especially in immunosuppressed patients and likely to lead to improved clinical outcomes.Treatment of C striatum with dermatomyositis who was treated with the immunosuppressant azathioprine. Surgical radical debridement and multidrug chemotherapy were useful. Meticulous attention should be paid to aseptic techniques whenever local injections of corticosteroids are administered to patients taking immunosuppressants, and patients should be closely observed during the postinjection period.In summary, we reported the case of a patient with pyogenic tenosynovitis due to Conceptualization: Yohei Kawaguchi.Data curation: Yohei Kawaguchi.Investigation: Yohei Kawaguchi, Hideki Okamoto, Kojiro Endo, Hidetoshi Iwata, Yuji Joyo, Masahiro Nozaki, Shinya Tamechika.Supervision: Yuko Waguri-Nagaya, Hideki Murakami.Writing – original draft: Yohei Kawaguchi.Writing – review & editing: Yohei Kawaguchi, Yuko Waguri-Nagaya.


The extended endoscopic approach provides unimpaired visualization and direct access to ventral skull base pathology, but is associated with cerebrospinal fluid (CSF) leak in up to 25% of patients. To evaluate the impact of improved surgical techniques and devices to better repair skull base defects, we assessed published surgical outcomes of the extended endoscopic endonasal approach in the last two decades for a well-defined homogenous group of tuberculum sellae and olfactory groove meningioma patients.Random-effects meta-analyses were performed for studies published between 2004 (first publications) and April 2020. We evaluated CSF leak as primary outcome. Secondary outcomes were gross total resection, improvement in visual outcomes in those presenting with a deficit, intraoperative arterial injury, and 30-day mortality. For the main analyses, publications were pragmatically grouped based on publication year in three categories: 2004–2010, 2011–2015, and 2016–2020.We included 29 studies describing 540 patients with tuberculum sellae and 115 with olfactory groove meningioma. The percentage patients with CSF leak dropped over time from 22% (95% CI: 6–43%) in studies published between 2004 and 2010, to 16% (95% CI: 11–23%) between 2011 and 2015, and 4% (95% CI: 1–9%) between 2016 and 2020. Outcomes of gross total resection, visual improvement, intraoperative arterial injury, and 30-day mortality remained stable over timeWe report a noticeable decrease in CSF leak over time, which might be attributed to the development and improvement of new closure techniques , refined multilayer repair protocols, and lumbar drain usage.The online version contains supplementary material available at 10.1007/s00701-020-04641-x. In the last two decades, the limits of endoscopic endonasal skull base surgery have been investigated. Current extended approaches allow exposure of the area between the olfactory groove and the odontoid process for resection of different pathologies  , 31. OriWith the addition of the extended endoscopic approach to the arsenal of the surgeon, certain tumors can be approached from below with unimpaired visualization and direct access to the pathology, with minimal exposure and manipulation of unaffected critical neurovascular structures . In patiTo evaluate the impact of these modifications and a possible learning curve, we evaluated outcomes of the extended endoscopic endonasal approach in the last two decades for a well-defined homogenous group of patients with tuberculum sellae and olfactory groove meningioma in terms of CSF leak and other surgical outcomes using a meta-analyses approach.A previously published literature search in Pubmed and Embase considering publications after 2004 (first paper) on outcomes of tuberculum sellae and olfactory groove meningioma patients operated with the extended endoscopic and transcranial approach was updated on 19 April 2020 . DetailsWe have adapted the New-Castle Ottawa Scale for risk of bias assessment. This scale is scored out of 6 and assesses sample selection, outcome reporting, and comparability between treatment arms. As no comparative studies were assessed in our study, we omitted the latter domain.For the main analyses, we pragmatically grouped publications based on publication year in three categories . As in earlier years, fewer publications were published; we chose the first category to span a year longer than the other categories. We evaluated the percentage patients with a CSF leak as primary outcome. Secondary outcomes were the percentage patients with a gross total meningioma resection, improvement in visual outcomes in those with preoperative deficits, intraoperative arterial injury, and 30-day mortality.We also performed multiple sensitivity analyses to assess the robustness of the results and the possible effects of information bias, classification bias, and selection bias.First, as publications from the same year might cover different study periods, we categorized studies in three categories based on the median calendar year of the described study period: 2000–2005, 2006–2010, and 2011–2015.Second, we performed analyses separately for patients with tuberculum sellae meningioma and olfactory groove meningioma. Although the analyses with only patients with olfactory groove meningioma should be interpreted with caution, as the number of studies and patients within some analyses is very small.. No other comparisons were made concerning closure techniques, due to paucity of data on other well-defined techniques.Third, we compared publications that specifically described routine use of pedicled nasoseptal flaps  with those that did not describe routine use of these flaps.Fourth, we compared publications that specifically described the routine use of lumbar drains to prevent CSF leaks with those that did not describe routine use of lumbar drainsI2 statistics were used for quantification of between-study heterogeneity. If multiple patient groups  were described separately within one publication, each group was entered separately in the analyses to account for the heterogeneity between the groups with the use of the random-effects model. No formal statistics were assessed to obtain p values for the performed comparisons, as none of the comparisons were described in the original studies. Comparison of different patient groups could be strongly affected by differences in patient and tumor characteristics, which are often confounders for the comparisons. Instead results are reported for each group, including 95% confidence intervals (95%CI), describing the accuracy of the aggregated results within the group [Random-effect estimates with 95% confidence intervals were calculated using the DerSimonian and Laird method . A Freemhe group . PublicaA total of 2285 articles were screened for title and abstract, and of 241 articles, the full text was read to assess eligibility. We eventually included 29 studies describing 36 groups of patients consisting of 540 patients with tuberculum sellae meningioma patients and 115 with olfactory groove meningioma  in studies published between 2004 and 2010, to 16% (95% CI: 11–23%) between 2011 and 2015, and 4% (95% CI: 1–9%) between 2016 and 2020  of patients from studies conducted between 2000 and 2005, compared with 13% (95%CI: 8–22%) between 2006 and 2010, and 3% (95%CI: 0–8%) between 2011 and 2015 Fig. , or olfa0 of patiIn articles clearly describing the routine use of a pedicled nasoseptal flap, CSF leak was reported in 3% 95%CI: 0–8%) of patients, compared with 12% (95%: 6–19%) in those articles that did not describe routine use of a pedicled flap , further decreases CSF rhinorrhea rates (21% vs 8%) without an increased risk of complications, such as infections [Standard use of lumbar drains to prevent CSF leaks is controversial as complications such as pneumocephalus and infections might not outweigh the potential benefit, especially as the percentage patients with a CSF leak has reduced with the development and improvement of closure techniques , 29. Howfections . Direct fections . These rfections , 33, 34.The decrease in CSF leak might also be attributed to a surgical learning curve. However, no clear improvement in the percentage patients with a gross total resection or improvement in visual outcomes was observed. This is in contrast with studies on the learning curve within a single large referral center, which showed improvement of both outcomes , 23. SubA limitation of this study is the small number of published studies with small, possibly highly selected, patient groups, and therefore selection bias and publication bias cannot be ruled out. However, to address selection bias to the best of our ability, we performed multiple sensitivity analyses which generally showed results in line with the main analyses, adding to the robustness of the results. Furthermore, we expect the possible impact of publication bias to be limited, as heterogeneity was seen in the reported outcomes, and asymmetry in the funnel plots was limited without any major changes in effect estimates using the trim and fill method. Nevertheless, the bar to submit and publish outcomes worse than the first reports, might have affected our outcomes. While we were able to perform analyses with studies routinely using a vascularized pedicled nasoseptal flap, we did not perform a separate analysis with studies routinely using and not using gasket seal closure techniques, as these studies were almost all from the same surgical group , 2, 27. We report a noticeable decrease in CSF leak over time, which might be attributed to the use of lumbar drains, development and improvement of new closure techniques  and integration of these techniques within multilayer and graded repair protocols Fig. . No imprESM 1(DOCX 607 kb)


This exploratory study investigated the time-course of lectin complement pathway (LCP) initiators in cerebrospinal fluid (CSF) and plasma in patients with subarachnoid hemorrhage (SAH), as well as their relationship to delayed cerebral ischemia (DCI) and functional outcome.PATIENTS = 63, n_SAMPLES = 399) and day 8 , respectively. Twelve neurologically healthy patients undergoing spinal anesthesia and 12 healthy blood donors served as controls. The development of DCI during hospitalization and functional outcome at 3 months  were registered for patients.Concentrations of ficolin-1, ficolin-2, ficolin-3, and mannose-binding lectin (MBL) were analyzed in CSF and plasma from patients with SAH. Samples were collected daily from admission until day 9  and multiple testing . In patients who developed DCI compared with those without DCI, CSF levels of ficolin-1 and MBL tended to increase slightly more over time ; however, no association was found after adjustments for confounders and multiple testing . Plasma ficolin-1 and ficolin-3 were lower in SAH patients compared with healthy controls on all days. DCI and functional outcome were not associated with LCP initiator levels in plasma.Patients with SAH displayed elevated CSF levels of ficolin-1, ficolin-2, ficolin-3, and MBL. Increased CSF levels of ficolin-1 and MBL were associated with a poor functional outcome.NCT01791257, February 13, 2013, and NCT02320539, December 19, 2014).This study was a retrospective analysis of samples, which had been prospectively sampled and stored in a biobank. Registered at clinicaltrials.gov (The online version contains supplementary material available at 10.1186/s12974-020-01979-y. Aneurismal subarachnoid hemorrhage (SAH) is a life-threatening condition accounting for ~5% of all stroke cases. Despite low annual incidence rates , the totDelayed cerebral ischemia (DCI) is a main contributor to a poor functional outcome in patients surviving SAH , 5. The The complement system is part of the innate immune system, which comprises a first-line defense against invading microorganisms, enabling clearance of dying cells, and amplifying adaptive and inflammatory immune responses upon stimulation . Three pRecent studies have reported an association between LCP initiators in plasma and functional outcome in ischemic stroke patients –16. LCP We aimed to investigate the time-course of LCP initiator levels in CSF and plasma after SAH and to describe their potential relationship to DCI and functional outcome.clinicaltrials.gov (NCT01791257 and NCT02320539). Informed consent was obtained from the next of kin, the general practitioner and, when possible, from the patient.This study was a retrospective analysis of samples from CSF and plasma, which had been prospectively sampled and stored in a biobank, and some of which were included in a prospective observational cohort study of microRNAs . ProtocoThis manuscript adheres to requirements set by the STrengthening the Reporting of OBservational studies in Epidemiology (STROBE) statement.Patients with spontaneous aneurysmal subarachnoid hemorrhage (SAH) as diagnosed by computed tomography (CT) and CT angiography who were admitted to the Neurointensive Care Unit at Rigshospitalet  were assessed for eligibility. Inclusion criteria were >18 years of age. Exclusion criteria were more than 24 h uncertainty of the time of ictus, as well as the inability to speak and understand Danish.Neurologically healthy patients >18 years of age with an American Society of Anesthesiologists physical status classification ≤2, who had spinal anesthesia before lower limb orthopedic surgery at Frederiksberg Hospital, were included and served as the control group for the CSF analyses.Neurologically healthy blood donors >18 years of age were included from the National Donor Registry  and served as the control group for the plasma analyses.The clinical severity of SAH was assessed using the World Federation of Neurosurgical Societies (WFNS) grading scale, which categorizes patients from 1 to 5; 1 indicating the lowest and 5 indicating the highest disease severity . The extClinical deterioration due to DCI was defined according to Vergouwen et al. as the development of a new neurological deficit or a decrease of at least 2 points in GCS, persisting for at least 1 h, and which was not explained by any other potential cause . Two cliFunctional outcome at 3-months follow-up was based on the modified ranking scale (mRS) , which mFrom patients with an external ventricular drain (EVD), CSF was collected immediately after placement with a sterile procedure, and once daily using an aseptic procedure the following 7 days. From neurologically healthy patients, one CSF sample was collected using a sterile procedure immediately before spinal anesthesia for orthopedic lower limb surgery. CSF samples were immediately put on ice and spun within 15 min at 500 g for 10 min, and the supernatant was stored at −80 °C.Blood was collected from an arterial catheter from SAH patients upon admission and once daily the following 7 days. If no arterial catheter was present, sampling was done from a central venous catheter. From healthy volunteers, blood was sampled once during a blood donation. Blood samples were immediately put on ice and spun within 15 min at 2000 g for 10 min, and the plasma samples were stored at −80 °C. Both plasma and CSF samples were collected at the same time of the morning each day.2SO4. Assays were optimized for automated analysis in a 384-well format using the Biomek FX platform  for pipetting followed by a DTX880 Multimode detector system where assays were analyzed at 490 nm.The concentration of LCP initiators  in CSF and plasma was determined using sandwich enzyme-linked immunosorbent assay (ELISA) developed in the Laboratory of Molecular Medicine, Rigshospitalet, according to previously described procedures –28. BrieThe lower detection limit was as follows in plasma: 5 ng/ml for ficolin-1, 0.005 μg/ml (5 ng/ml) for ficolin-2, 1 ng/ml for ficolin-3, and 20 ng/ml for MBL; it was 0.4 ng/ml in CSF for all four biomarkers. We obtained the following coefficients of variation as calculated from the raw triplicate measurements: Ficolin-1: CSF 9.3%, plasma 7.1%; ficolin-2: CSF 20.2%, plasma 10.6%; ficolin-3: CSF 18.1%, plasma 9.6%, MBL: CSF 9.2%, plasma 7.7%.P values were corrected for multiple testing using the Benjamini-Hochberg procedure.Plasma and CSF levels of LCP initiators on admission and specific days after the initial bleeding in SAH patients were compared with those of neurologically healthy individuals using Wilcoxon’s rank-sum test. Within SAH patients, levels were compared between the groups, ‘definitely DCI’ vs ‘definitely not DCI’ and ‘poor functional outcome’ vs ‘good functional outcome’, using a linear mixed model with subgroup  and day as fixed effects, and patients as a random effect. Crude comparison was done without including any further covariates , whereas adjusted analyses included sex, age, and WFNS and Fisher scores as covariates . The linear mixed model was applied twice: (1) Assuming no interaction  thereby evaluating the overall difference in concentrations (p), and (2) including the group*day interaction to explore potentially different time developments between groups (p_interaction). Goodness of fit was assessed by residual plots. Due to substantial skewness, CSF levels were log-transformed prior to analysis and results were expressed accordingly . All statistical analysis was performed using Statistical Analysis System v.9.4 .N = 32), the plasma study alone (N = 19), or both (N = 31) , although insignificant. However, after adjustment for multiple testing, the association was no longer found (p-adj = 0.20). In multivariate analysis adjusted for sex, age, WFNS, and Fisher score no association was found.In univariate analysis, the CSF level of ficolin-1 seemed elevated in patients with DCI versus patients without DCI . Adjusted for confounders , the increase over time for ficolin-1 and MBL remained significant, but with further adjustment for multiple testing, the increase over time became non-significant  and 1.69 ng/mL higher in average, 95% CI, 1.09 to 2.63, p = 0.040).In univariate analyses, CSF levels of ficolin-1, ficolin-2, ficolin-3, and MBL were higher in patients with a poor functional outcome compared with those with a good functional outcome. In multivariate analyses adjusting for sex, age, WFNS, and Fisher score as well as for multiple testing, ficolin-1 and MBL levels remained higher in patients with a poor functional outcome .In contrast, LCP initiators in plasma did not differ between patients with good and those with poor functional outcomes either overall or by time Fig. .We investigated the day-by-day changes of LCP initiators in both CSF and plasma after SAH. Our study revealed three major findings. First, CSF concentrations of LCP initiators were increased throughout the first week following SAH, followed by a gradual decline over time. Second, increased CSF levels of all investigated LCP initiators, especially ficolin-1 and MBL, were associated with a poor functional outcome in SAH patients. Third, in univariate analysis, patients with DCI compared with those without DCI, no association was found between DCI and overall LCP initiator levels in CSF. However, ficolin-1 tended to increase slightly more over time in patients with DCI than without , but the increase was not significant after adjustment for multiple testing  dysfunction after SAH. In favor of spill-over from plasma, invasion of complement and inflammatory cells over the disrupted BBB has been proposed as a major contributor to the BBB pathology in other neuroinflammatory central nervous system (CNS) disorders, such as traumatic brain injury, bacterial meningitis, and cerebral malaria . HoweverOne other study has looked at LCP initiator levels in CSF after SAH . The autA study assessing the involvement of the LCP initiators in contused human brain tissue from patients with head trauma found that the LCP initiators were present inside and outside of brain vessels in all contusions examined . Only fiAnother study examining brain samples in patients with acute ischemic stroke showed that certain complement proteins, including MBL, were deposited in ischemic lesions in the brain . The autPlasma levels of all investigated LCP initiators were lower or comparable in SAH patients compared with healthy individuals, and ficolin-1 and ficolin-3 were lower in both the hyperacute, acute, and the intermediate phase after SAH. Also, circulating LCP initiators were associated neither to DCI nor to functional outcome.Few research groups have examined the changes of LCP initiators in plasma after SAH, and conflicting results have been reported , 18, 20.This study is the first to provide information about the day-by-day changes of LCP initiators after SAH. The high number of consecutive samples of both CSF and plasma from each patient yielded a higher resolution than previous studies, conferring improved robustness and precision of the natural course of LCP initiators after SAH.Furthermore, our study complies with the recent reporting recommendations in cellular and molecular SAH studies using recommended methods of sample acquisition, use of collection tubes, biospecimen processing and storage, inclusion of a neurologically healthy control group for both CSF and plasma, as well as using functional outcome and DCI as outcome measures . This waHowever, this study also has noteworthy limitations. First, we evaluated multiple outcome measures  which increase the risk of chance findings. We reduced this risk by correcting for multiple testing.Due to the post hoc nature of this study, we cannot exclude a potential risk of selection bias as well as a risk of analytical variation due to the chosen method to detect LCP initiators. Furthermore, the retrospective design of the study rendered us unable to measure the actual activity of the LCP system and establish any potential causal relationship with the levels of LCP initiators. However, another study found plasma ficolin-3 and ficolin-3-mediated functional LCP activity to be well correlated . SimilarCSF samples were collected from patients treated with an EVD; thus, our CSF findings mainly is related to this selected subgroup of SAH patients. In addition, SAH patients with intraventricular blood (Fisher grade of IV) could conceivably experience variation in the CSF composition depending on positioning and other manipulations affecting cerebrospinal fluid compartments.All included patients were treated with tranexamic acid (TXA) (an antifibrinolytic) based on the Danish National Guidelines, which might have an impact on complement activity. Many fibrinolytic compounds work as natural C3 and C5 convertases, thus generating C3a and C5a (active anaphylatoxins) . The useIn our study, we did not control for the presence of intercurrent infection; neither did we measure other markers of inflammation in plasma or CSF. Although SAH is associated with a marked systemic inflammatory response , 45, infp values for individual days are less important than the visualization of the dynamic changes over time.Additionally, a longitudinal analytical approach results in a high-resolution due to frequent repeated measures, which eases the process of visualizing on which time point (day) potential differences between groups occur. However, this analytical approach limits the likelihood of finding significant differences between groups on a specific day. Thus, in our study Finally, this is still a novel investigation field in which our exploratory and retrospective study design was not able to include all potential laboratory and clinical confounders, which may alter LCP profiles in plasma and CSF. Future studies should investigate comorbidities, in particular on-going inflammatory/infectious diseases, neoplastic, paraneoplastic and demyelinating conditions, premorbid hypertension as stated in recent recommendations as well as other potential confounders , 47.Our study revealed that SAH patients treated with external ventricular drain had elevated levels of LCP initiators in CSF compared with neurologically healthy patients. Intrathecal ficolin-1 and MBL were increased in SAH patients with a poor functional outcome. No association was found between DCI and overall CSF levels of LCP initiators. Future studies should clarify the pathophysiological mechanism behind ficolin-1 in the central nervous system and determine the potential of ficolin-1 as a prognostic marker of functional outcome.Additional file 1. Supplementary Table 1Additional file 2. Supplementary Fig. 1Additional file 3. Supplementary Fig. 2


Brachycephalic dogs remain popular, despite the knowledge that this head conformation is associated with health problems, including airway compromise, ocular disorders, neurological disease, and other co-morbidities. There is increasing evidence that brachycephaly disrupts cerebrospinal fluid movement and absorption, predisposing ventriculomegaly, hydrocephalus, quadrigeminal cistern expansion, Chiari-like malformation, and syringomyelia. In this review, we focus on cerebrospinal fluid physiology and how this is impacted by brachycephaly, airorhynchy, and associated craniosynostosis. Brachycephalic dogs and cats have proved to be increasingly popular since they were introduced to Europe in Victorian times. This rising popularity defies a high prevalence of conformation-related morbidity, including breathing, ocular, and neurological disorders ,2. BrachThe skull develops in response to the expanding brain mass. The ultimate shape of the skull is determined by the growth of the cranial sutures. The sutures are fibrous joints between calvarial bones, which develop at the site of dural reflections, where the inner dural layer descends into the brain, forming septa, which divide the brain into compartments  ,10. SutuRecently, understanding of CSF physiology has shifted from a choroid, plexus, arachnoid, and granulation-centric model of production and absorption to an interstitial, fluid, lymphatic, and vessel-centric model . In cats and dogs, approximately 40% of CSF is secreted by the choroid plexus into the ventricular spaces; the remainder originates from the brain and spinal interstitial fluid, leptomeningeal and parenchymal capillaries, and ependyma ,16. The As the majority of CSF is absorbed through lymphatics in the skull base and via the olfactory bulbs, the balance between CSF production and absorption may be impacted by craniofacial hypoplasia and cranial base shortening.There is direct continuity between the subarachnoid space and the perineurial spaces of the olfactory nerve fibers penetrating the cribriform plate. These nerve fibers are encircled by lymphatics, providing one of the main drainage routes of CSF ,29. CSF In addition to olfactory perineural drainage, basal meningeal lymphatics and cranial nerve perineural lymphatics are important for CSF drainage ,36,37,38The skull base foramina allow passage of the cranial venous sinuses. Reduction of the skull base and stenosis could therefore impede venous drainage and increase intracranial venous pressure, reducing venous resorption of cerebrospinal fluid. For example, stenosis of the jugular foramen will impede drainage through the sigmoid sinus and therefore the transverse sinus ,41.However, craniosynostosis may be associated with a primary venous abnormality. Cavalier King Charles spaniels with syringomyelia associated with Chiari malformation have reduced volume caudal cranial fossa dorsal sinuses . Mouse mBrachycephalic dogs with short muzzles are predisposed to the conformation-related respiratory disorder brachycephalic obstructive airway syndrome (BOAS) . Soft tiRespiration drives CSF movement through the ventricular system . The sysThe oropharynx, larynx, and hyoid are suspended from the cranial base. Skull base shortening may displace these tissues and the tongue, caudally affecting swallowing and respiration, especially if there are other conformation problems reducing airway space. In rats and humans, these conformational changes are associated with obstructive sleep apnea and sleep-disordered breathing ,58.Animals with severe BOAS have increased thoracic effort with increased inflation of the lungs and use of accessory abdominal musculature . This deThe skull base forms the lower part of the foramen magnum, which accommodates the craniospinal junction. Premature closure of the spheno-occipital synchondrosis alters the angulation between the skull base and the cervical vertebrae ,60,61. TCSF passes from the ventricular to the subarachnoid space via the lateral apertures of the fourth ventricle . Primates have an additional foramen of Megendie in the median plane between the fourth ventricle and cisterna magnum . In specHydrocephalus is defined as an active distension of the ventricular system of the brain, resulting from inadequate passage of CSF from its point of production within the cerebral ventricles to its point of absorption into the systemic circulation . VentricThe authors propose that the tendency for brachycephalic animals to have ventriculomegaly, and in more extreme cases hydrocephalus, is because of a combination of reduced absorption of CSF through lymphatics and altered neuroparenchymal compliance and pulsatility. Contributary factors may include reduced movement of CSF due to apnea and interruption of sleep, cranial venous stenosis, constricted CSF pathways through the lateral apertures and craniocervical junction, and intrathoracic pressure gradients. However, if there is expansion of the lateral and third ventricles only  with aqueduct stenosis, then mesencephalic developmental anomalies, including fusion of the colliculi, are also likely .The quadrigeminal cistern is a midline dilatation of the subarachnoid space, which is connected to the peri-cerebellar subarachnoid space caudally and medial cerebral hemisphere subarachnoid space laterally. It is dorsal to the third ventricle and mesencephalic aqueduct, but separated from the ventricular system by a thin membrane of pia mater and ependyma . ExpansiPathogenesis of quadrigeminal cistern expansion is unproven. Although this subarachnoid cistern is anatomically separated from the ventricular system, there is evidence that the quadrigeminal cistern is connected to the third ventricle via the velum interpositum and to the fourth ventricle via the dorsal medullary velum . These vChiari-like malformation associated pain in dogs describes a syndrome of pain associated with brachycephaly and hindbrain herniation. It is often compared to Chiari type I and 0 malformation in humans. However, it is more like the hindbrain herniation seen with syndromic and complex craniosynostosis in humans, for example, Crouzon’s and Pfeiffer syndrome . CraniofMany small breed dogs are predisposed to Chiari-like malformation, and may have magnetic resonance imaging (MRI) evidence of brachycephaly with hindbrain herniation. However, dogs with signs of pain have more extreme brachycephaly with craniofacial and occipital bony tissue reduction ,75. The Syringomyelia describes spinal cord cavitation (syrinx) with fluid similar to CSF . SyringoIn comparison to dogs with Chiari-like malformation only, dogs with syringomyelia have more extreme brachycephaly with craniocervical junction deformation, including cervical flexure, change in angulation of the odontoid peg, increased proximity of the atlas to the skull , kinking or elevation of the craniospinal junction, and loss of the cisterna magna ,82,83,84Dogs and cats with neonatal characteristics of a reduced muzzle and brachycephaly have impaired CSF circulation, which predisposes ventriculomegaly, hydrocephalus, quadrigeminal cistern expansion, Chiari-like malformation associated pain, and syringomyelia. Reduction of the lymphatic absorption of CSF through the nasal and skull base lymphatics, in conjunction with restriction of CSF movement through the lateral apertures and craniocervical junction, are hypothesized to be key features. Cranial venous stenosis, spinal canal conformation, chronic intermittent hypoxia, and thoracic cavity pressure gradients may contribute. The ethics of choosing to breed animals with this predisposition is questionable, despite their popularity as companion animals. Education of the pet-buying public, avoiding breeding extreme animals, and screening at-risk breeding stock is recommended.


Seeds diluted in cerebrospinal fluid (CSF) accelerated the seeding reaction and slightly increased the sensitivity without affecting specificity. Repeated freeze–thaw cycles decreased the apparent lag times of seeds diluted in ddH2O but did not alter the seeding activity of seeds diluted in CSF. High levels of artificial contamination with blood resulted in prolonged apparent lag times, while sensitivity and specificity were unaffected. Altogether, qRT‐QuIC with aSyn seems to be robust concerning sensitivity and specificity in our model system, but quantitative interpretation might be limited under certain conditions.Parkinson’s disease (PD) is a progressive neurodegenerative disease which is histologically characterized by loss of dopaminergic neurons in the substantia nigra and deposition of aggregated alpha‐synuclein (aSyn) in the brain. The detection of aSyn in well accessible fluids has been one of the central approaches in the development of biomarkers for PD. Recently, real‐time quaking‐induced conversion (RT‐QuIC) has been successfully adapted for use with aSyn seeds. Here, we systematically analysed parameters potentially impacting the reliability of this assay by using quantitative real‐time quaking‐induced conversion (qRT‐QuIC) with  RT‐QuIC with alpha‐synuclein represents a promising tool for the early diagnosis of synucleinopathies from cerebrospinal fluid. We tested typical conditions like repeated freeze–thaw cycles or contamination with blood which may impair the functionality of the assay. While freeze–thaw cycles do not seem to have a major impact, blood prolonged the apparent lag phases but did not change sensitivity or specificity of the assay. First clinical symptoms occur, when 50–70% of the nigral dopaminergic neurons are already lost , 2, 3, win vitro‐formed aSyn seeds and substrate to systematically analyse parameters which in daily practice potentially interfere with the reliability of the assay.For the present study, we have used our qRT‐QuIC assay with Escherichia coli . Protein expression was induced with 1 m IPTG  for 4 h at 37 °C. Cells were lysed by boiling after heat inactivation of proteases. After centrifugation, the supernatant was filtered through a Filtropur S 0.2 filter , loaded onto a 5‐mL HiTrap Q HP anion exchange column  and eluted over a linear NaCl gradient (25–500 mm). The fractions containing aSyn were pooled, concentrated and gel filtrated via a Superdex 75 prep grade column . After adjusting the protein concentration to 1 mg·mL−1, aliquots were frozen in liquid nitrogen and stored at −80 °C until use.Expression and purification were performed as previously described , 20. Brim tris pH 7.0 was incubated at a concentration of 50 µm along with 100 mm NaCl and 0.02% NaN3 in a total volume of 1.4 mL at 37 °C for 96 h under vigorous orbital shaking (1400 r.p.m.) using an Eppendorf Thermomixer Comfort   were loaded on the top of the sucrose gradient. Ultracentrifugation was performed with 40 000 r.p.m. (163 900 g) at 4 °C for 1 h using a Sw60Ti rotor (Beckman Coulter). Samples were harvested after centrifugation from the top to the bottom of the sucrose gradient in 200‐µL fractions . 28 µL of each fraction was used for western blot analysis. aSyn was detected using an antibody against full‐length aSyn .For sucrose‐gradient centrifugation, solutions containing 50 mg for 10 min at room temperature, subsequently transferred to new tubes and frozen at −80 °C within 30 min.All patients provided written informed consent to clinical assessment and lumbar puncture (LP) in order to collect CSF according to protocols approved by the local ethics committees . All procedures of this study were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Diagnostic/therapeutic high‐volume LP was performed in patients with normal pressure hydrocephalus (NPH). After the first 2–3 drops were discarded, 30–40 mL CSF was withdrawn and collected into 15‐mL polypropylene tubes. A sample was sent for routine analysis . The remaining CSF was centrifuged at 2500 et al. [−1 monomeric aSyn and 10 µm ThT were pipetted into a black 96‐well plate  in the presence of 1× QuIC buffer . Seeds were diluted in ddH2O or CSF in 0.5‐mL Protein LoBind Tubes (Eppendorf AG) and added at final concentrations of 500 nm–500 fm , corresponding to 7 × 10−7–7 × 10−13 g of aSyn seeds, to the seeding reaction. The plate was immediately sealed with transparent tape  in order to avoid cross‐contamination and aerosol formation. Reactions were performed on FLUOstar OPTIMA and FLUOStar Omega multiwell plate readers  at 50 °C for at least 90 h with cycles of 1 min double‐orbital shaking at 600 r.p.m. followed by 1 min of stationary incubation. ThT fluorescence was recorded every hour using an excitation wavelength of 440 nm and an emission wavelength of 480 nm. Apparent lag times were determined in a blinded manner and defined by the last time point before the ThT signal increased compared to the baseline. At least three independent experiments with duplicates or triplicates per seed concentration were performed for all different conditions.The qRT‐QuIC protocol is based on the protocol previously described by Shi et al. : for a fm–5 pm) of seeds in ddH2O or CSF were prepared at a time and 0, 1 or 5 times frozen in liquid nitrogen and subsequently thawed at room temperature. Of note, always the whole dilution series with all subsequent dilutions were subjected to the number of freeze–thaw cycles indicated.For repeated freeze–thaw cycles, three dilution series . Subsequently, the whole blood sample was diluted in CSF to the number of RBC indicated and the prepared CSF samples were spiked with aSyn seeds as specified above. All procedures of this study were in accordance with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.To mimic contamination with blood, blood by courtesy of two healthy volunteers [volunteer 1: sex: F, age: 30–35, red blood cell count (RBC): 4.6 × 10in vitro‐aggregated aSyn as seeds to provide standardized conditions with low interexperimental variation and to save precious and irretrievable patient samples. aSyn seeds were prepared by incubating monomeric aSyn at a concentration of 50 µm for 96 h at 37 °C under vigorous orbital shaking at 1400 r.p.m. ThT fluorescence measurements of the seed preparations obtained showed an increased fluorescence intensity compared to buffer (baseline) or aSyn monomer   Fig.  without nt) Fig. . Shaking. et al. : 1 min dg) at 20 °C for 30 min to possibly increase the concentration of active seeds or where the seed preparation was sonicated to receive a more homogeneous seed population  was diluted to final concentrations of 1000, 100 and 10 RBC per µL in the CSF sample to be analysed. While with 1000 RBC per µL a discrete reddish discolouration was still visible, admixtures of 100 or 10 RBC per µL could not be detected by eye anymore. Increasing blood concentrations led to a nearly parallel increase of the apparent lag times for all amounts of seeds tested, with an average increase of 2.3 h at 10 RBC per µL Fig. , 5.3 h aNeurodegenerative diseases like Alzheimer’s and Parkinson's disease have become a growing problem in our ageing population, as no causal and only limited symptomatic therapy is available to date. To provide effective and causative treatment strategies, an early and reliable diagnosis of specific neurodegenerative disease entities is required. Various approaches to develop sensitive and specific biomarkers to diagnose synucleinopathies or other neurodegenerative diseases have been followed, of which qRT‐QuIC seems currently to be one of the most promising approaches. We and others have adapted qRT‐QuIC for the use with aSyn seeds and substrate. In this study, we have systematically characterized factors of daily routine, which potentially interfere with the reliability of the assay.in vitro‐formed aSyn seeds, which we characterized by ThT fluorescence measurements, continuous sucrose‐gradient centrifugation, and EM. ThT fluorescence measurements revealed strong fluorescence intensities for the seed preparations compared to buffer or monomeric protein, indicating the presence of amyloid fibrils, which could be confirmed by EM. Using sucrose‐gradient centrifugation, we could demonstrate a continuous size distribution suggesting that the seed preparation contains a variety of different‐sized aggregates. To address the question, if it was possible to enrich the fraction of active seeds or to retrieve a more homogenous seed population, single experiments were performed, where the seed preparation was ultracentrifuged or sonicated. However, we could not find obvious differences of the seeding activity compared to unprocessed seed preparations. Moreover, as it is unclear which particular aggregate species in fact trigger the seeding reaction and as there possibly exists a spectrum of different aggregate species in patient brains and CSF, we decided to proceed without further processing of the seed preparations.To adapt qRT‐QuIC for use with aSyn seeds and substrate, we used 2O, thus, reaching absolute amounts of 7 × 10−7–7 × 10−13 g of aSyn seeds, corresponding to concentrations of 500 nm–500 fm of aSyn seeds in the final reaction. A reaction temperature of 50 °C was chosen, as the seeding reaction seems to be reasonably fast. Moreover, at this temperature, microbial growth is virtually avoided, while at the same time there is no risk of protein denaturation yet. However, homogeneous heating of the multiwell plate should be ensured, and plates should be densely sealed to prevent evaporation. Decreasing seed concentrations led to increasing apparent lag times, which are correlated by an exponential relationship. This was used to establish a standard calibration curve.To adapt qRT‐QuIC for use with aSyn seeds and substrate, we diluted the seed preparations by factors of ten in ddHper se does not impair the seeding reaction. Therefore, we spiked CSF with in vitro‐formed seeds and performed qRT‐QuIC under the same conditions as described before. In contrast to seeds diluted in ddH2O, we reliably observed seeding activity at 7 × 10−13 g aSyn seed. The lag times were generally shorter, especially at lower seed concentrations. Specificity was not affected. A possible explanation for the more sensitive and faster reaction could be that the functional seed concentration is increased, as the additional proteins from CSF might prevent the adsorption of seeds to the walls of the wells. Assuming a normal CSF protein concentration of 1.5–4.5 mg·mL−1, the concentration of protein in the reaction well would be at least doubled compared to our experiments with seeds diluted in ddH2O, where the aSyn substrate displays the only considerable protein source.As CSF is a body fluid of complex composition, we wanted to ensure that the CSF matrix in vitro‐formed seeds after one and five freeze–thaw cycles. While there was no obvious difference for CSF spiked with seeds, already one freeze–thaw cycle of seeds diluted in ddH2O was associated with a remarkable reduction of the apparent lag times, especially at lower seed concentrations. A potential explanation for this could be an increased fractionation of seeds diluted in ddH2O leading to a higher functional concentration of seeds (while the protein concentration remains the same) and thus to a reduction of the apparent lag phases. In contrast, seeds diluted in CSF might be protected against fragmentation due to the composition of CSF so that the functional concentration of seeds would not change. This hypothesis is supported by the observation that the ThT fluorescence of seeds diluted in ddH2O is clearly reduced after one or five freeze–thaw cycles, whereas only a slight reduction of the ThT fluorescence was recorded for seeds diluted in CSF , the blood cells are presumably quickly damaged leading to release of intracellular components or membrane fragments, which may contribute to the altered aggregation behaviour. Finally, it is very likely that not only one single factor, but several factors together contribute to the prolonged lag times observed. Based on our observations, we would recommend to carefully interpret the qRT‐QuIC results of CSF samples with visible contamination with blood in terms of quantitative aspects.As clinical diagnostics is typically a sequential, multi‐step process, it is inevitable to store samples refrigerated or frozen over certain time periods and thaw them again for subsequent analyses. Thus, we tested the seeding activity of the CSF Fig. . In addihrocytes , 23, 24  as aSyn , 26, 27.In this study, we show for the first time that the qRT‐QuIC seeding reaction is altered – particularly regarding quantitative aspects – under conditions, which are relevant in handling of clinical samples. The strength of our model with artificial seeds is that we can systematically and easily vary different parameters like freeze–thaw cycles and blood contamination and analyse their effects at different and defined amounts of seeds. However, one must be cautious with transferring the results one‐to‐one to real patient‐derived CSF samples. Nevertheless, the results of this study address an important aspect and represent a reasonable and solid basis for further considerations and investigations with real patient‐derived CSF specimens.With regard to clinical practicability and handling of CSF samples, we have used our model system and systematically characterized parameters which potentially interfere with the seeding reaction and thus lead to false‐positive or false‐negative results. Altogether, qRT‐QuIC turned out to be quite a robust assay in terms of sensitivity and specificity although quantitative interpretation, for example, in terms of disease progression or therapy monitoring might be impaired under certain conditions. Thus, explicit requirements to the quality of CSF samples would need to be defined. Although our findings still need to be confirmed using patient‐derived CSF samples, they provide a good starting point for further investigations.The authors declare no conflict of interest.AG and BM oversaw the project. VCR, SS, FS, DW, GSN and AG designed, performed, analysed and interpreted the experiments. BM and UK provided CSF samples. VCR wrote the manuscript. All authors critically read, edited and approved the manuscript.Fig. S1. Higher temperatures decrease apparent lag times. Compared to 50 °C , the seeding reaction is much slower at 37 °C.Click here for additional data file.Fig. S2. Different pre‐treatments of the seed preparation have no effect on their seeding activity. Ultracentrifugation (A) or sonication (B) of the seed preparation does not alter the seeding activity of the seed preparation. Error bars indicate standard deviation; n = 1.Click here for additional data file.Fig. S3. Multiple freeze–thaw cycles reduce ThT fluorescence of seeds diluted in ddH2O but not of seeds diluted in CSF. Seeds diluted in either ddH2O or CSF were subjected to 0, 1, or 5 freeze–thaw cycles before being added to the seeding reaction. Diagrams depict the baseline fluorescence at the beginning of the seeding reaction at a seed concentration of 500 nm, where a clear decrease of the ThT fluorescence is observed for seeds diluted in ddH2O but only a slight reduction of ThT fluorescence can be seen after five freeze–thaw cycles. Error bars indicate standard error of means; n = 3.Click here for additional data file.


Cultural limitations are identified in the traditional use of qualitative approaches at the expense of quantitative measurement-based monitoring methods to design and assess ergonomic interventions and train operators. To bridge the gap between the return-to-work rehabilitation and other disciplines, several teaching courses, accompanied by further electrodes and instrumentations development, should be designed at all Bachelor, Master and PhD of Science levels to enhance the best skills available among physiotherapists, occupational health and safety technicians and ergonomists.Patients affected by neurological pathologies with motor disorders when they are of working age have to cope with problems related to employability, difficulties in working, and premature work interruption. It has been demonstrated that suitable job accommodation plans play a beneficial role in the overall quality of life of pathological subjects. A well-designed return-to-work program should consider several recent innovations in the clinical and ergonomic fields. One of the instrument-based methods used to monitor the effectiveness of ergonomic interventions is surface electromyography (sEMG), a multi-channel, non-invasive, wireless, wearable tool, which allows in-depth analysis of motor coordination mechanisms. Although the scientific literature in this field is extensive, its use remains significantly underexploited and the state-of-the-art technology lags expectations. This is mainly attributable to technical and methodological  and cultural limitations. The technical  Bipolar and multi-channel (high-density) surface electromyography (sEMG and HDsEMG) represent non-invasive physiological approaches, which enable greater comprehension of the upper limb, lower limb, and trunk muscle behaviors during the execution of movement –4. Diffe° maximum,° averaged rectified value,° root mean square, and- amplitude indices , 6° max- muscle activation timings .Furthermore, sEMG-based algorithms enable greater in-depth comprehension of the muscle coordination mechanisms adopted by the central nervous system (CNS) by estimating the following:- simultaneous activation of several muscles or muscle groups (co-activation), a mechanism adopted by the CNS to stabilize joints, upper and lower limbs, and the spine –11,- myoelectric manifestation of muscle fatigue, estimated by measuring the decrease in fiber conduction velocity , 13, whi- locomotor coordination –26, analHigh-density sEMG recordings are performed using high-density surface grids placed on the skin to evaluate the online spatial distribution of the sEMG activity and estimate the discharge times of several motor units by using decomposition algorithms , 31.Despite the many advantages of sEMG and HDsEMG, these instrumental tools are largely underexploited and their application lags expectations in the fields of ergonomics and occupational medicine. These tools have begun to be applied in the prevention of work-related musculoskeletal disorders, a set of painful inflammatory and degenerative conditions affecting the joints, spinal dizcs, cartilage, muscles, tendons, ligaments and peripheral nerves, caused by manual lifting, pushing and pulling, repetitive movements, and patients handling activities –39. Howeoccupational health and safety technicians and ergonomists which should operate in a multidisciplinary team also constituted by neurologists, occupational physicians, physiatrists, biomedical engineers and movement scientists. We believe that no new professions should be created.People affected by neurological pathologies need to be integrated/reintegrated into their workplaces because their motor disease symptoms appeared when they were of working age, reducing their working capacity , 41 and Occupational health and safety technicians have an academic degree offered at Bachelor of Science level in the same field of physiotherapists . Instead, professionals from different disciplines, for example occupational physicians, who aspire to practice the profession of ergonomist, need a degree that is not conferred by universities. In Europe, the Center for the Registration of the European Ergonomists (CREE) is the professional certification supported by European ergonomics associations and recognized by the International Ergonomics Association (IEA) which provides the title of “Eur.Erg.” and allows to practice the profession of ergonomist in 47 countries.The above premise serves as a rationale for identifying and discussing the barriers to the coherent and widespread use of sEMG in work integration/reintegration.This article represents the perspective, under the aegis of the SOPHIA project, of the Laboratory of Ergonomics and Physiology of the Italian Institute for Insurance against Accidents at Work (INAIL), a public non-profit entity aimed at facilitating the return-to-work of people with motor disorders.Ethical review and approval and written informed consent were not required for the study because no human participants were recruited in the study and in accordance with the local legislation and institutional requirements.Degenerative and acquired neurological diseases, including neuropathies, multiple sclerosis, stroke, spastic paraplegia, cerebellar ataxia, dystonia, traumatic spine and brain lesions, and encephalitis, are disorders, which can affect the motor function during working age and severely limit the autonomy and efficiency of workers , 54–58. Combined with kinematic and kinetic measurements, sEMG is currently widely used in research laboratories by movement scientists and still little in clinical routine by health operators to classify quantitatively the nature and degree of motor dysfunction, analyze the complex relationship between the primary deficit and the adaptive and compensatory mechanisms, categorize patients based on their specific neurological disease, and finally monitor pre-post-treatment. Importantly, as sEMG essentially investigates the final output of motor commands, it can quantify the residual motor function, which can theoretically be monitored continuously in workplace adaptation and integrated into HRC technologies.When a worker affected by a neurological pathology with motor disorders is reintegrated at work, an exhaustive assessment of his/her residual motor function is of primary importance to design and/or adapt his/her workplace well. Additionally, it is necessary to verify and monitor the efficacy of these ergonomic interventions over time.Although no consistent studies have specifically investigated the sEMG of patients with neurological disorders in the workplace context, there are several reports on the residual muscle function assessment of patients with neurological disorders [, for revSome long-lasting degenerative neurological diseases, such as cerebellar ataxia and spastic paraplegia, often begin at a young age and can persist for the entire duration of a subject's working life. This is of interest in the context of work-related rehabilitation because the majority of patients consider themselves capable of working, and approximately 78% of non-working patients seek employment . FurtherThe use of sEMG analysis in patients with degenerative cerebellar ataxia  reveals a series of muscle activation abnormalities . SpecifiIt has been reported that patients with degenerative spastic paraplegia  show increased global  and segmHuman-robot collaborative technologies, particularly exoskeletons, have proven significantly useful in the rehabilitation programs of patients affected by neurological diseases. Physiological parameters with sEMG play a key role in monitoring muscle activation amplitude and fatigue in the design of innovative active exoskeleton controller systems and assessment of their effectiveness on motor performance .Recent improvements in the measurement and real-time classification of myoelectric signals have already facilitated the use of sEMG in man-machine interfaces for controlling prostheses and orthoses among other devices –76.Very recently, human-exoskeleton interfaces were developed for the purpose of rehabilitation to support physically weak and disabled people in performing several motor activities of daily living, such as walking. These interfaces were developed based on new technical and mathematical approaches, including new sEMG signal-processing procedures –82.The performance of these man-machine interfaces is quickly improving owing to neuro-musculoskeletal models driven by neural information obtained from the decomposition of HDsEMG .methodological and cultural limitations. The technical issues are attributable to both monitoring and control functions. With regard to monitoring, the most critical technical aspects are strongly associated with the sEMG technique:The critical issues hindering the widespread adoption of sEMG in return-to-work programs are mainly attributable to technical, - electrode-skin impedance, noise, and electrode contact stability;while the methodological aspects are associated to:- - problems linked to electrode location, size, configuration, and distance;- presence of crosstalk signals .- placement of sEMG electrodes for long hours;- selection of the right sensor setup on the base of the neurological pathology and manual handling activity to be investigated;- management the sEMG amplitude normalization;- definition of appropriate sEMG-related outcomes and normative data.and methodological limitations of the sEMG approach can be minimized and do not justify its non-usage in return-to-work rehabilitation plans. In addition, new wearable sensors and electronic smart devices such as smartphones and tablets allow simple monitoring of the worker at the workplace. Wearable sensors do not interfere with the typical movements performed by workers owing to their miniaturization and wireless communication protocols. In addition, multi-channel sEMG systems are available in wireless versions despite their high number of channels and high data rate. The combination of sensor networks and intelligent work environments provides real-time estimation of physiological parameters, enabling direct feedback to workers who are monitored directly and constantly at the workplace. Real-time monitoring is additionally useful for providing acoustic, visual, and vibro-tactile stimuli to workers  with a significant involvement of erector spinae and rectus abdominus muscles, while handling low loads at high frequency imply neck and upper limbs muscles fatigue. For this reason the choice of channels and the rationale for selecting them should be task-guided.The criticisms related to the use of sEMG to control collaborative wearable trunk and upper limb devices designed to assist people with neurological disabilities are attributable to the algorithms used in human-robot interfaces. These algorithms are used for pattern recognition and classification of patients' movement intentions. Only a few years ago, the performance of sEMG-based interfaces had not reached accuracies acceptable for widespread commercial use . AccuracAnother technical limitation of sEMG-based interfaces for use in HRC technology control is the fact that most wearable assistive devices use traditional control tools, such as bipolar sEMG, to record antagonist muscle activities. This low spatial sampling implies that a maximum of one degree of freedom (DoF) can be controlled. Furthermore, managing up to two DoFs requires slow, sequential, and unintuitive control. The related limited functionality in conjunction with the extensive training required of neurological subjects led to the high rejection rates of these technologies . Currenthas so far been disregarded, owing to cultural barriers, which lead to a preference for qualitative approaches.Without doubt, in job integration/reintegration, the sEMG approach has yet to be adopted and the critical issues associated with it are managed with difficulty. In fact, while reasonable workplace accommodation and disability employment issues are being historically and widely addressed by the governments of the industrialized world, the adoption of instrument-based quantitative assessments of ergonomic interventions A clear testimony of the presence of this educational gap is the worldwide social policies for persons with disabilities , 91, sucThe lack of quantitative approaches suggests that, except in rare cases, sEMG is not taught in the Ph.D. programs of universities and occupational medicine specialization schools. Furthermore, the domestic and international chapters of the Human Factors and Ergonomics Society do not actively promote meetings, conferences, and events to address the challenges of sEMG use. Finally, the reference society for sEMG, the International Society of Electrophysiology and Kinesiology, has thus far not dedicated specific training programs regarding job accommodation. All the above mentioned training activities would typically be successful to address methodological issues, as increased knowledge alone cannot overcome technical limitations.The enormous potential of the sEMG and HDsEMG approaches and their very limited use in return-to-work programs represent a real paradox. It is difficult to determine why sEMG is underused. Perhaps, the most likely reason is that the training provided to professionals is based on more qualitative rather than quantitative approaches, and the transition from one approach to the other is evidently difficult. Professionals in the field have to be trained to understand the extremely variable abnormalities of workers suffering from neurological pathologies and to associate an appropriate return-to-work plan with them.Although sEMG is considered the most informative instrument for muscle monitoring when wearable robots are used, its use poses a number of challenges.One such challenge, which we believe is on the verge of being addressed, pertains to the use of exoskeletons for active rehabilitation therapies. It pertains to the optimization of appropriate smart algorithms to detect patients' intentions and allow exoskeletons to act in synergy with them , 94. TheMyocontrol should be increasingly based on HDsEMG to increase spatial resolution with respect to low-density sEMG and to improve the accuracy of motor workers' intentions recognition. Moreover, machine learning approaches such as artificial neural networks should be used to evaluate the capacity of workers with neurological diseases for myocontrol.In a recent study , HDsEMG occupational health and safety technicians, physiotherapists, ergonomists, occupational physicians, rehabilitation engineers and movement scientists) that has always reached the maximum allowed limit (30 in the case of training courses). The authors of this article begin to observe a first positive impact of these initiatives on the need that operators in the field have in using the sEMG approach.To bridge the gap between the return-to-work concept and other disciplines, several educational activities should be developed to enhance and apply the best skills available in rehabilitation engineering, physiotherapy, occupational therapy, and ergonomics. For a few years, INAIL has been promoting a Ph.D. program together with the Sapienza University of Roma titled “Kinematic, Kinetic, and Electromyographic Characterization of Motor Disabilities and Biomechanical Overload Risk Management for Job Reintegration,” a Masters' course titled “New Methodologies for the Evaluation and Management of Biomechanical Risk and Criteria and Methods for the Adaptation of Workplaces,” and several training courses regarding the role of sEMG in occupational medicine and ergonomics. The participation, although the events were not free and not recognized as continuing education, has always been conspicuous with a number of participants  results in altered motor unit recruitment and decreased maximal voluntary motor unit firing rate that can be detected by sEMG monitoring. A specific scenario could be to adapt the workplace and to modify the work-task according to the subjects' abnormal fatiguing performance, by assisting the workers with devices and/or reducing the amount and the duration of the work-related activity. Furthermore, an individualized rehabilitative program could be planned to improve the impact of the work-task on the fatigability in a long-term period.The abnormal muscle co-activation is another example of common problem identifiable by sEMG in several neurological disorders  which can be detected by measuring the simultaneous time-varying sEMG signal in many muscles. It is known that patients with balance disorders increase the muscle co-activation to control their walking instability in the attempt to stiffen the body segments. Unfortunately, this compensatory mechanism has some negative effects, such as increased metabolic cost and risk of cartilage degeneration. A specific scenario for these workers is to plan an appropriate workplace rehabilitation to improve their balance, to stabilize the body segments and to reduce the need to increase the muscle co-activation.In conclusion, sEMG should be used in job integration plans to:- classify the nature and degree of residual motor function in order to design/adapt the workplace;- assess the efficacy of work-task rehabilitation and ergonomic interventions;- control new assistive technologies such as collaborative robots;- evaluate the biomechanical risk during the execution of manual handling activities;- plan a preventive rehabilitation program to prevent injury.Including the sEMG approach in work integration/reintegration offers the possibility of designing programs based on the residual motor abilities of the worker and adapting his/her workplace. This allows to consider a wide range of workers with several neurological pathologies and different levels of severity but without a complete inability to perform activities of daily life.To make this possible, Bachelor, Master and PhD programs should be promoted, or at least, supervised and monitored, by scientific societies in the fields of physiotherapy, ergonomics, occupational medicine, biomechanics, electrophysiology and kinesiology, and should include continuing education courses on the use of sEMG specifically oriented to teachers in these fields.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval and written informed consent were not required for the study as per local legislation and institutional requirements.AR, MS, and FD planned the manuscript, performed the literature searches, wrote the text and revised the manuscript. All authors contributed to the article and approved the submitted version.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.