Meningoencephalitis of unknown origin (MUO) describes a group of meningoencephalitides in dogs with a hitherto unknown trigger. An infectious agent has been suggested as one possible trigger of MUO but has not been proven so far. A relatively new method to screen for viral RNA or DNA is next-generation sequencing (NGS) or deep sequencing. In this study, a metagenomics analysis of the virome in a sample is analyzed and scanned for known or unknown viruses. We examined fresh-frozen CSF of 6 dogs with MUO via NGS using a modified sequence-independent, single-primer amplification protocol to detect a possible infectious trigger. Analysis of sequencing reads obtained from the six CSF samples showed no evidence of a virus infection. The inability to detect a viral trigger which could be implicated in the development of MUO in the examined population of European dogs, suggests that the current techniques are not sufficiently sensitive to identify a possible virus infection, that the virus is already eliminated at the time-point of disease outbreak, the trigger might be non-infectious or that there is no external trigger responsible for initiating MUO in dogs. Meningoencephalomyelitis without detectable infectious etiology is a well-known disease entity in dogs , 2. SeveThe hypothesis should be examined that an infectious agent triggers MUO. Therefore, we report in this study a metagenomics analysis of the virome present in cerebrospinal fluid (CSF) samples from a European subpopulation of dogs with clinically suspected MUO in an acute stage of the disease or with acute relapse of clinical signs. In this early stage of the disease the possibility is enhanced that the etiological agent of this condition might be detectable. Identification of virus specifically associated with the development of MUO would greatly enhance our understanding of the pathogenesis of this disease, which could facilitate the development of a specific therapy or strategies to prevent the onset of MUO.Dogs included in this study were client owned patients of the Department for Small Animal Medicine and Surgery of the University of Veterinary Medicine, Hannover, Germany. All dogs were clinically investigated by at least one Resident or Diplomate of the European College of Veterinary Neurology (ECVN). Further diagnostic examinations were performed with informed written owner's consent. MRI examination  and suboccipital CSF collection was performed in general anesthesia (premedication: diazepam 0.5 mg/kg intravenously (i.v.), levomethadone with fenpipramide 0.1 mg/kg i.v. , induction of anesthesia: propofol dose to effect 1–4 mg/kg i.v., orotracheally intubation and connection to a semiclosed circle absorber system .MUO was diagnosed based on criteria outlined by Granger et al.  includinhttps://idseq.net), a cloud-based, open-source bioinformatics platform designed for rapid identification of pathogens in metagenomics data, as described previously , necrotizing leukencephalitis (NLE) and necrotizing meningoencephalitis (NME) are the subtypes most frequently described –6. AlthoDistinct triggers might be involved in the pathogenesis of the different MUO subtypes. Searching such a trigger in a homogenous population of MUO subtypes might be helpful, but was not successful as previously published , 4, 8. TIn the CSF samples, a high percentage of non-host reads were found. It is known that in clinical samples such as CSF which contain very little biomass, the primers over-amplify very small quantities of contaminants and therefore increase the proportion of irrelevant contaminates in the final dataset . To reduAlthough the CSF samples in the current study did not contain viral RNA or DNA which could be associated with MUO, negative NGS results cannot exclude an infectious etiology. An infectious pathogen, which was potentially involved in initiating the inflammation, could have been largely eliminated by the immune system without impeding the inflammatory host response . NGSummarizing, we could not confirm the hypothesis that MUO might be caused by an infectious trigger. Nevertheless, our current state of knowledge of MUO is suggestive of a multifactorial etiology, including an underlying genetic susceptibility and involving an additional unknown external trigger . The poshttps://www.ncbi.nlm.nih.gov/sra/PRJNA698629.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: JN collected clinical data, examined cases, collected CSF samples, wrote, and finalized the report. WJ analyzed CSF samples and NGS data. AO designed the study and finalized the report. ML analyzed the NGS data, supervised the CSF analysis, and wrote the report. AT designed the study, collected CSF samples, and finalized the report. All authors contributed to the article and approved the submitted version.This study was supported by a grant from the Niedersachsen-Research Network on Neuroinfectiology (N-RENNT) from the Ministry of Science and Culture of Lower Saxony, Germany, and by Deutsche Forschungsgemeinschaft and University of Veterinary Medicine Hannover, Foundation within the funding programme Open Access Publishing.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


ObjectiveIn this study, we aimed to assess the topographic anatomy and clinical impact of the vascular foramen on the trochlear groove of adult human dry femora.Materials and methodsThe incidence, shape, size, and location of the foramen present on the trochlear groove of the distal femur were studied using 33 adult human dry femora of unknown age and gender at the Department of Anatomy at Nowshera Medical College, Nowshera, Pakistan. Using a divider and scale, the incidence and structure of the vascular foramen in the trochlear groove were examined and its position in relation to the trochlear groove's upper and lower articulating edges was recorded.ResultsOut of 33 femora, 20 (60.6%) were right-sided and 13 (39.4%) were left-sided. All the foramina had round morphometry. In each of the 33 adult human dry femora, 15 (45.5%) had a single trochlear vascular foramen. One solitary trochlear vascular foramen was found in eight (53.3%) of the left femora and seven (46.7%) of the right femora. The diameter of each trochlear vascular foramina was about 2.5-4.5 mm with a mean diameter of 3.5 mm. Trochlear groove upper margin and foramen were 1.75-2.5 cm apart, whereas the lower margin was 0.5-01 cm apart. On average, foramen was 2.2 cm away from the upper margin, and 0.8 cm away from the lower margin. In the midline, nine (60%) trochlear vascular foramina were found, with five (55.5%) resting on the trochlear groove lower margin, and four (44.5%) lying mid-way at varying places from the upper and lower margins. The left of the centerline had six (40%) of the trochlear vascular foramen, whereas no trochlear vascular foramen was seen on the right side of the midline.ConclusionsBased on our findings, a significant segment of our population has trochlear vascular foramen present on the trochlear groove as the rate of occurrence of this was found to be 45.5%. The trochlear groove center has the most foramen, accounting for 60% of all the foramen. Typical long bones have an extremely vascular structure, which is supplied by four artery systems: periosteal, metaphyseal, diaphyseal, and epiphyseal arteries, which are commonly known as nutrient arteries . The femStudy design and settingThis was a descriptive cross-sectional study conducted at the Department of Anatomy at the Nowshera Medical College, Nowshera, Pakistan.Study duration, sample size, and sampling techniqueAfter obtaining ethical approval from the Institutional Ethical Review Board (IERB) of the Nowshera Medical College vide letter No. 25/NMC/IERB/Sec dated 15/02/2021, this study was conducted in September 2021. A total of 33 adult human dry femora of unknown age and gender were included and studied. Non-probability convenience sampling was employed in the analysis.Sample selectionOnly those femora with undamaged lower ends were examined in this study, whereas those that had injured lower ends were excluded.Data collectionData were collected and entered into a Microsoft Excel 2010 spreadsheet. The incidence, structure, size, and position of vascular foramen found on the patellar surface of the femur, commonly recognized as the trochlear groove, were examined. Because the foramina were found in a variety of places, they were divided into four groups to aid orthopedic surgeons undertaking surgery on the distal femur. The first group consisted of the trochlear groove to the left of the midline. The second group comprised those in the midline, varying in distance from the top and lower trochlear groove edges. Those on the trochlear groove bottom edge, in the midline, were placed in the third group. The fourth group was made up of those on the trochlear groove to the right of the midline. The structure and size were examined for each foramen, and a divider and scale were used to measure trochlear groove diameter and distance from the top as well as bottom edges.Data analysisForamen sizes and distances from the top and bottom borders of the trochlear groove were measured, and the incidence, range, and mean were calculated using SPSS Statistics version 24 .Out of 33 femora, 20 (60.6%) were right-sided and 13 (39.4%) were left-sided. All the foramina had round morphometry. In each of the 33 adult human dry femora, 15 (45.5%) femora had a single trochlear vascular foramen. One solitary trochlear foramen was detected in eight (53.3%) of the left femora and seven (46.7%) of the right femora. The diameter of each trochlear vascular foramina was about 2.5-4.5 mm with a mean diameter of 3.5 mm. Trochlear groove upper margin and foramen were 1.75-2.5 cm apart, whereas the lower margin was 0.5-01 cm apart. On average, foramen was 2.2 cm away from the upper margin, and 0.8 cm away from the lower margin. In the midline, nine (60%) trochlear vascular foramina were found, with five (55.5%) resting on the trochlear groove lower margin as shown in Figure Previous studies have extensively described vascular foramina in the supra, medial, lateral, and intercondylar fossa of the distal femur. However, despite a thorough literature search, we did not find a sufficient number of studies on the incidence, shape, size, and topographic anatomy of vascular foramen situated in the trochlear groove of the distal femur. In this study, 15 out of the 33 adult human dry femora of unknown age and gender had the trochlear vascular foramen, indicating a 45.5% occurrence rate.According to Singh , trochleThe distal femur is important from a morphological, functional, and clinical perspective because of its location, structure, and weight-bearing role, which exposes it to substantial morbidity . The joiAccording to Su et al. and Pereira et al., the developing bone may drag and burst the artery; the nutrition artery goes far from the growing end. As a result, vascular foramina are relocated away from the plant's growing edge. Two variables influence the positioning of vascular foramina: rates of growth at both bones and shaft end remodeling. The rates of bone growth and remodeling in persons of various ages should be investigated. Furthermore, fetal bones may be useful in deciphering this idea. These parameters could not be examined in the current investigation since it involved adult dry bones. The majority of trochlear foramina (64%) were found in the mid-way of the trochlear groove, whereas 28% were found on the left condyle, and 8% were detected along the bottom margin of the trochlear groove, somewhat to the left of the midline. Since there is no previous work in the literature that details the position of the trochlear foramen, there was no way to make a comparison ,16.The irregular orientation of vascular foramina might be caused by the stress of muscle attachments on the periosteum. The foramen can be a source of weakness in certain people, and it can fracture when strained, whether through greater physical activity or deterioration of bone quality. The position of the rupture in relation to the vascular foramen of elongated bone and the morphologies of edema are secondary symptoms in the diagnosis of this kind of fracture. Hence, orthopedic surgeons must be aware of the morphometry, morphology, and topographic anatomy of the trochlear vascular foramen in the trochlear groove of the distal femur.Our findings concluded that a significant proportion of our population has trochlear vascular foramen present on the trochlear groove since the occurrence of this was found to be 45.5%. The trochlear groove center has the most foramen, accounting for 60% of all the foramen. The morphologic, morphometrical, and topographical understanding of this foramen is indispensable for orthopedic and trauma surgeons. Sufficient knowledge is mandatory for the purpose of bone grafting, tumor resections, traumas, congenital skeletal deformities, and equipment and fixation, as well as graft techniques utilized in orthopedics and other procedural processes in the distal femur and knee region to maintain the normal blood flow. Furthermore, knowing where this foramen is located is critical for bone ossification, bone curing, and microvascular bone transplantation.


Purpose: To test whether correcting for unspecific signal from the cerebral white matter increases the sensitivity of amyloid-PET for early stages of cerebral amyloidosis.Methods: We analyzed 18F-Florbetapir-PET and cerebrospinal fluid (CSF) Aβ42 data from 600 older individuals enrolled in the Alzheimer’s Disease Neuroimaging Initiative (ADNI), including people with normal cognition, mild cognitive impairment (MCI), and Alzheimer’s disease (AD) dementia. We determined whether three compartmental partial volume correction (PVC-3), explicitly modeling signal spill-in from white matter, significantly improved the association of CSF Aβ42 levels with global 18F-Florbetapir-PET values compared with standard processing without PVC (non-PVC) and a widely used two-compartmental PVC method (PVC-2). In additional voxel-wise analyses, we determined the sensitivity of PVC-3 compared with non-PVC and PVC-2 for detecting early regional amyloid build-up as modeled by decreasing CSF Aβ42 levels. For replication, we included an independent sample of 43 older individuals with subjective memory complaints from the INveStIGation of AlzHeimer’s PredicTors cohort (INSIGHT-preAD study).Results: In the ADNI sample, PVC-3 18F-Florbetapir-PET values normalized to whole cerebellum signal showed significantly stronger associations with CSF Aβ42 levels than non-PVC or PVC-2, particularly in the lower range of amyloid levels. These effects were replicated in the INSIGHT-preAD sample. PVC-3 18F-Florbetapir-PET data detected regional amyloid build-up already at higher  CSF Aβ42 levels than non-PVC or PVC-2 data.Conclusion: A PVC approach that explicitly models unspecific white matter binding improves the sensitivity of amyloid-PET for identifying the earliest stages of cerebral amyloid pathology which has implications for future primary prevention trials. Cerebrospinal fluid (CSF) concentrations of the 42-amino acid-long amyloid-β peptide (Aβ42)  and amylHowever, the sensitivity of amyloid-PET may critically depend on the way the PET scans are being analyzed. A particular concern for the quantification of amyloid-PET signal is the so called partial volume effect (PVE). The PVE results from two related mechanisms reflecting the poor spatial resolution of PET scans relative to the thickness of the cerebral gray matter . First, Partial volume effect correction (PVC) methods are designed to decrease the contribution of unspecific signal, and would thus be expected to increase the sensitivity of amyloid-PET for lower levels of amyloid accumulation, particularly when explicitly accounting for spill-in of white matter signal. Indeed, PVC using a 3-compartment approach  was founHere, we explored the effect of different image processing strategies on the association of 18F-Florbetapir-PET signal with CSF Aβ42 levels as reference measure of overall amyloid burden, using combined PET and CSF data of 600 older individuals enrolled in the Alzheimer’s Disease Neuroimaging Initiative (ADNI), including individuals with normal cognition, mild cognitive impairment (MCI), and AD dementia. To test the replicability of our findings in an independent cohort, we used data from 43 cognitively normal individuals with subjective memory complaints (SMC) from the monocentric INveStIGation of AlzHeimer’s PredicTors in subjective memory complainers (INSIGHT-preAD) cohort . Our pri1 The ADNI was launched in 2003 by the National Institute on Aging, the National Institute of Biomedical Imaging and Bioengineering, the Food and Drug Administration, private pharmaceutical companies and non-profit organizations, with the primary goal of testing whether neuroimaging, neuropsychological, and other biologic measurements can be used as reliable in vivo markers of AD pathogenesis. A fuller description of ADNI and up-to-date information is available at www.adni-info.org.Data used in the preparation of this article were obtained from the ADNI database.To test if key findings could be replicated in an independent sample, we used data from the INSIGHT-preAD study . This st2From the ADNI cohort, we retrieved 600 cases with available 18F-Florbetapir-PET data as well as CSF Aβ42 levels in close temporal proximity to the PET acquisition. Mean temporal distance between 18F-Florbetapir-PET acquisition and lumbar puncture was 13 days (SD 34 days). These cases included data of 152 cognitively normal older individuals, 241 cases with early MCI, 129 subjects with late MCI and 78 subjects with AD dementia. Detailed inclusion criteria for the diagnostic categories are available at the ADNI web site.The INSIGHT-preAD study includes 318 cognitively normal Caucasian individuals, recruited from the community in the wider Paris area, aged 70–85 at baseline, with SMC and defined brain amyloid status . Forty-tAs described in the ethics declaration section below, written informed consent was obtained from all participants and/or authorized representatives before any protocol-specific procedures were carried out.ADNI and INSIGHT-preAD cases underwent comprehensive neuropsychological examinations. In the present study, the Mini Mental State Examination (MMSE)  was usedADNI CSF values in the current study were derived from electrochemiluminescence immunoassays for Aβ (1-42) on an automated Elecsys cobas e 601 instrument. The upper technical limit for this assay is 1,700 pg/ml and the values that are higher than 1,700 pg/ml are based on an extrapolation of the calibration curve. Therefore, analyses were repeated with CSF Aβ42 values from the previously used multiplex xMAP Luminex platform  with INNOBIA AlzBio3 kit  .®, Courtaboeuf, France).INSIGHT-preAD CSF Aβ42 values were determined using a double antibody sandwich ELISA method (Innotest-FujirebioDetailed acquisition and standardized pre-processing steps of ADNI imaging data are available at the ADNI website (see text footnote 2). 18F-Florbetapir-PET data was collected during a 50- to 70-min interval following a 370 MBq bolus injection of 18F-Florbetapir. To account for the multicentric acquisition of the data across different scanners and sites, all PET scans undergo standardized pre-processing steps within ADNI.PET data acquisition in the INSIGHT-preAD cohort was performed according to a method previously described . All amyFor anatomical reference and pre-processing of the PET scans we used the corresponding structural MRI scan that was closest in time to the 18F-Florbetapir-PET scan. In ADNI, MRI scans were acquired on multiple 3T MRI scanners using scanner-specific T1-weighted sagittal 3D MPRAGE sequences. Similar to the PET data, MRI scans undergo standardized preprocessing steps aimed at increasing data uniformity across the multicenter scanner platforms (see text footnote 2 for detailed information on multicentric MRI acquisition and preprocessing in ADNI). In INSIGHT-preAD, MRI scans were acquired on a Siemens Verio 3T scanner at Pitié-Salpêtrière Hospital, Paris. A T1-weighted image was acquired using a fast three dimensional gradient echo pulse sequence using a magnetization preparation pulse (Turbo FLASH) .Images were preprocessed using Statistical Parametric Mapping software version 8 (SPM8)  implemented in Matlab 2013. The pre-processing pipeline followed a routine described previously . First, The composition of the cortical SUV mask has been described in WC maps were masked to exclude non-gray matter voxels and smoothed using a Gaussian kernel of 10 mm3 × 10 mm3 × 10 mm3. Due to the low number of cases in the replication cohort, the complementary voxel-wise analyses were only conducted in the ADNI sample.For additional voxel-wise analyses, spatially normalized 18F-Florbetapir-PET scans  were converted to voxel-wise SUVRs by scaling each voxel to the whole cerebellum (WC) reference signal (in uncorrected data). 18F-Florbetapir-PET SUVRa priori to use two knots fixed at the first and second tertile of the range of SUVR values. This decision was made based on the notion that we wanted to analyze three levels of amyloid: low, intermediate and high. In a sensitivity analysis focused on the cases with low amyloid levels, we iterated linear spline estimates for the first knot sliding in 100 steps from the 16.6% quantile to the first tertile of the SUVR values. Of note, for this analysis the overall number of knots of the spline regression was unimportant, as this analysis took only the first knot into account. To render spline estimates comparable across PVC approaches and employed reference regions, the SUVR values and CSF Aβ42 levels were scaled to be mean-centered and have a standard deviation of one before spline regression. Secondly, we determined Pearson’s product moment correlations between SUVR values and CSF Aβ42 levels across the entire sample as well as within tertiles of SUVR values. Correlation coefficients from different PVC methods and reference regions were compared with the correlation coefficients from the standard processing  using Steiger’s Z-test. Again, for sensitivity analysis, we determined the distribution of correlation coefficients when cases were selected sliding in 100 steps from the 16.6% quantile to the first tertile of SUVR values.Associations between global 18F-Florbetapir-PET SUVR values and CSF Aβ42 levels were determined using two complementary approaches. First, we used spline regression to model non-linear associations between SUVR values and CSF Aβ42 levels. We decided http://cran.r-project.org/web/packages.These analyses were performed with RStudio, version 1.1.463, a user interface of R Project for Statistical Computing Analyses. Spline regression was conducted using the commands “lspline” and “qlspline” from library “lspline,” and Steiger’s Z-test was conducted using the command “r.test” from library “psych.” The libraries used are available at n = 23) with the highest CSF Aβ42 levels as the reference group. We then compared a series of subsequent groupings of the remaining cases with this reference group as follows. After selecting the 23 cases that comprised the reference group, we took the 23 subjects with the next lower CSF Aβ42 levels and performed voxel-wise contrasts between this group of interest and the reference group. We then dropped the three cases with the highest CSF Aβ42 levels from the group of interest and added the three subjects with the next lowest values and again performed a voxel-wise comparison with the reference group. We continued this process, iteratively creating groups of interest of 23 subjects by dropping the three cases with the highest CSF Aβ42 levels and adding the three cases with the next lowest levels, such that each new group of interest differed from the previous group by only six subjects. In this way, groups of interest gradually moved down the scale of CSF Aβ42 levels, always using the same initial reference group of 23 cases for comparison. This procedure was repeated 36 times until the three healthy controls with the lowest CSF Aβ42 levels were included. For each comparison, voxel-wise two-sample t-tests were performed using SPM12 software with the spatially normalized 18F-Florbetapir-PET SUVR maps from the different PVE correction methods. All voxel-wise analyses were corrected for multiple comparisons using a family-wise error (FWE) at p < 0.05 and cluster size k = 150. With this approach we addressed two questions: First, does the PVC method affect the sensitivity of 18F-Florbetapir-PET for early signs of cerebral amyloidosis as indicated by CSF Aβ42 levels? Second, where does regional amyloid signal occur first in people with low levels of amyloidosis?To detect regional 18F-Florbetapir-PET SUVR increases in early amyloidosis, we used an approach inspired by N = 30; with a slide of 5 cases) and 10% quantiles , respectively. In addition, we applied this approach to all 600 cases, irrespective of diagnosis, with a 15% quantile cut-off (resulting in a group size of 90 cases) and a slide of 5 cases per window.Since the selection of the upper 15% quantile of CSF Aβ42 levels for the reference group was purely arbitrarily , in sensitivity analyses we used other cut-offs, specifically the 20% . By contrast, correlation coefficients for PVC-2 did not differ from the values obtained by the standard processing. For the white matter reference, correlations were significantly more negative than for the standard processing, except for the middle tertile, and correlation coefficients were comparable across PVC methods in this data  = −0.62, r(CSF/no PVC) = −0.48, t(43) = 2.5, p < 0.02]. Numerically, the correlation coefficient for PVC-3 was also markedly more negative than for no PVC data within the first tertile of data, but this effect did not reach statistical significance in this small subsample .In the o no PVC . CorrespWC maps were detected at a group mean CSF Aβ42 level of 840 pg/ml, and localized to the inferior temporal and temporo-occipital cortex, precuneus, superior parietal lobule, and medial prefrontal cortical areas  as for the standard processing. These results were replicated using the 10th and the 20th percentiles for creating the reference samples  than for no PVC and PVC-2  .WC values, taking spill-in of white matter signal into account, but not 2-compartmental PVC , including CSF Aβ levels and amyloid PET scans. Processed partial volume corrected PET scans are available upon request through the corresponding authors of this manuscript. Data from the INSIGHT-preAD cohort are only available upon written request and after approval by the INSIGHT-preAD scientific review board.Data used for the current analysis from the ADNI cohort are freely available from the ADNI website  In vitro Procedure for Diagnosis and Early Diagnosis of Neurodegenerative Diseases Patent Number: 8298784; (3) Neurodegenerative Markers for Psychiatric Conditions Publication Number: 20120196300; (4) In vitro Multiparameter Determination Method for The Diagnosis and Early Diagnosis of Neurodegenerative Disorders Publication Number: 20100062463; (5) In vitro Method for The Diagnosis and Early Diagnosis of Neurodegenerative Disorders Publication Number: 20100035286; (6) In vitro Procedure for Diagnosis and Early Diagnosis of Neurodegenerative Diseases Publication Number: 20090263822; (7) In vitro Method for The Diagnosis of Neurodegenerative Diseases Patent Number: 7547553; (8) CSF Diagnostic in vitro Method for Diagnosis of Dementias and Neuroinflammatory Diseases Publication Number: 20080206797; (9) In vitro Method for The Diagnosis of Neurodegenerative Diseases Publication Number: 20080199966; (10) Neurodegenerative Markers for Psychiatric Conditions Publication Number: 20080131921. MOH received honoraria as a consultant from Blue Earth company. The remaining 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. The reviewer GS declared a past co-authorship with one of the authors, MJG, to the handling editor.SJT participated in scientific advisory boards of Roche Pharma AG, Biogen, and MSD, and received lecture fees from Roche and MSD. AV was an employee of Eisai Inc. [November 2019 – Jun 2021]. This work has been performed during his previous position at Sorbonne University, Paris, France. Before November 2019 he had received lecture honoraria from Roche, MagQu LLC, and Servier. SL received lecture honoraria from Roche and Servier. BD received consultant fees from Lilly, Boehringer Ingelheim and has received grants from Roche for his institution. HH is an employee of Eisai Inc. This work has been performed during his previous position at Sorbonne University, Paris, France. He serves as Senior Associate Editor for the Journal Alzheimer’s & Dementia and does not receive any fees or honoraria since May 2019. Before May 2019 he was supported by the AXA Research Fund, the “Fondation partenariale Sorbonne Université” and the “Fondation pour la Recherche sur Alzheimer,” Paris, France. He had received lecture fees from Servier, Biogen and Roche, research grants from Pfizer, Avid, and MSD Avenir (paid to the institution), travel funding from Functional Neuromodulation, Axovant, Eli Lilly and company, Takeda and Zinfandel, GE-Healthcare and Oryzon Genomics, consultancy fees from Qynapse, Jung Diagnostics, Cytox Ltd., Axovant, Anavex, Takeda and Zinfandel, GE Healthcare and Oryzon Genomics, and Functional Neuromodulation, and participated in scientific advisory boards of Functional Neuromodulation, Axovant, Eisai, Eli Lilly and company, Cytox Ltd., GE Healthcare, Takeda and Zinfandel, Oryzon Genomics and Roche Diagnostics. He is co-inventor in the following patents as a scientific expert and has received no royalties: (1) All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


It was unraveled that RBC osmotic fragility and caspase-like proteasomal activity can link both donor cohorts to post-storage states. In the case of heterozygotes, the geometry, size and intrinsic low RBC fragility might be lying behind their higher post-storage resistance to lysis and recovery in mice. Moreover, energy-related molecules  and purine metabolism factors  were specifically linked to lower post-storage hemolysis and phosphatidylserine exposure. The latter was also ameliorated by antioxidants, such as urate. Finally, higher proteasomal conservation across the transfusion chain was observed in heterozygotes compared to control donors. The proposed “triangularity model” can be (a) expanded to additional donor/recipient backgrounds, (b) enriched by big data, especially in the post-transfusion state and (c) fuel targeted experiments in order to discover new quality biomarkers and design more personalized transfusion medicine schemes.The clarification of donor variation effects upon red blood cell (RBC) storage lesion and transfusion efficacy may open new ways for donor–recipient matching optimization. We hereby propose a “triangular” strategy for studying the links comprising the transfusion chain—donor, blood product, recipient—as exemplified in two cohorts of control and beta-thalassemia minor (βThal While originally suggested by the middle of the 1960s ,3, has oThe unique physiology and RBC storability of these  distinctive donor groups may be linked to differential post-transfusion phenotypes. There is an increasing number of studies discussing the importance of linking specific physiological and metabolic characteristics of freshly drawn or stored RBCs with post-transfusion parameters of individual events ,12,13 to+) differ from those of the general population, especially regarding their osmotic fragility and energy metabolism [+ RBCs are resistant to lysis and to membrane/cytoskeleton disruptions and are also able to counteract oxidative and proteotoxic insults [+ RBCs maintain their resilience against rupture and augmented proteasome activity in an in vitro model of transfusion and they also exhibit a slightly superior recovery in mouse recipients [+ RBCs by bypassing the blood unit or passing through it /saline–adenine–glucose–mannitol (SAGM) from 18 control and 18 beta-thalassemia heterozygotes were analyzed as previously reported ,17,18,19n = 10 per group) in a RBC/plasma volume ratio corresponding to transfusion of two RBC units. Then, the reconstituted samples were incubated for 24 h at body temperature before measuring several hemolysis and redox parameters, as previously reported [Stored RBCs from 10 units per group were reconstituted in plasma from healthy and transfusion-dependent beta-thalassemia major subjects  and significance was accepted at + donor groups. The triangularity analysis highlighted two physiological parameters in both cohorts studied: osmotic fragility and caspase-like (CASP-like) proteasomal activity. Regarding osmotic hemolysis, the pre-storage levels were proportional to those of storage and post-storage in vitro states, with an interesting strong intra-correlation linkage present between the stored and reconstituted samples  or “partial” (two connections) transfusion triangles in both the average control and βThalInitially, we focused on transfusion triangles targeting post-storage hemolysis profiles .+ RBCs positively correlated with the mechanically induced one in the unit, which was in turn linked to osmotic fragility post storage , which varied proportionally to the in vivo levels B. An intThe levels of spontaneous hemolysis post storage in the same donor group were positively related to the oxidatively induced hemolysis levels of both freshly drawn and stored RBCs C. A “com+ plasma intra- and inter-correlated with the respective levels in the supernatant, as well as with the intracellular levels of urate in stored RBCs. Moreover, all three storage parameters were negatively associated with the externalization of phosphatidylserine (PS) and the production of PS+ extracellular vesicles (EVs) under recipient-mimicking conditions  and uric acid-dependent antioxidant capacities (UAdAC) of fresh βThalnditions A. Urate  storage A.In the case of proteasome, the (mainly cytosolic) levels of chymotrypsin-like (CH-like) activity were linked with each other in a “complete transfusion triangle” B. Notabl+ RBCs with thiol-oxidizing agents , was proportional to the in vivo and storage levels (The accumulation of reactive oxygen species (ROS) post stimulation of reconstituted βThale levels C. On thee levels C.+ RBCs, as shown in Finally, some interesting correlations were evident between freshly drawn and reconstituted/transfused βThal+ EVs of the same RBC states. Of note, the in vivo levels of 2,3-bisphosphoglycerate negatively correlated with the magnitude of lipid peroxidation in the reconstituted samples’ membrane , in vivo levels of 2,3-BPG and lipid peroxidation post storage ) presented the same trend in the control group, too. However, the smaller number of paired control samples compared to the βThal+ pairs rendered the statistical analysis highly unsound in this cohort. Last but not least, osmotic and mechanical fragilities of freshly drawn βThal+ RBCs exhibited an inverse link with their 24 h recovery in mouse recipients .The levels of spontaneous hemolysis in the reconstituted samples were negatively associated with those of ATP, pyridoxamine and phosphoethanolamine of freshly drawn RBCs A. Regardmembrane A. Some ocipients B. In thi+ donor groups: osmotic fragility and CASP-like proteasome activity, which have the potential to link donors to post-storage RBC features, at least in vitro. Furthermore, we provide evidence regarding a well-characterized and homogeneous  donor group, in which a variety of fragility and redox parameters, along with metabolites of purine and energy pathways, seem to be linked directly or indirectly to their overall superior post-storage phenotypes.We hereby propose a paired “triangular” model for studying the transfusion chain, presenting it through a correlation analysis of paired donor–storage–post-storage data. Interestingly, out of hundreds of variables tested in freshly drawn blood and stored RBCs, two parameters stood out, fulfilling the triangularity criteria in both the average and genetically distinct βThal+ RBCs and (b) direct linking between donor attributes and transfusion outcomes, which presuppose analyses in freshly drawn blood. Regarding the first point, the correlation profile of sublethal defects versus the typical hemolysis metric stands as a representative example. It seems that the multiparametric nature of spontaneous hemolysis [The currently proposed triangular approach of transfusion research has the potential to provide reliable and statistically solid links between donors, blood units and post-storage/post-transfusion metrics . It also highlights the significance of (a) cellular indices that may affect RBC performance, as with the case of sub-lethal storage lesions in βThalemolysis  does notemolysis ,8. Howevemolysis ,22, as wemolysis ,19.Paired analyses, like the one presented in our study, can serve as the first step in elucidating biomarkers of storage and transfusion quality out of a large number of metrics that arise through the implementation of high- or low-throughput techniques. Such studies can feed more targeted experimental approaches in larger cohorts and different donor groups to validate, expand or reject hypotheses that lean on initial observations. Whatever the result of the latter approaches might be, the search for donor/recipient signatures in specific transfusion events will narrow, step by step, the distance between where we currently stand and the much-anticipated future of precise transfusion medicine. Such information can also guide blood logistics strategies in periods of inventory shortage, such as the one imposed worldwide by the COVID-19 pandemic.+ RBCs to both osmotic and mechanical lysis benefits them in the circulation of transfused mice mainly due to their unique geometry and membrane/cytoskeleton proteome [+ RBCs is also hinted at by the presence of hematological indices, such as MCV and RDW in the respective transfusion triangles. The lower volume , expressed (directly or indirectly) by the above-mentioned variables, probably protects the cells from stress-induced rupture, a hypothesis consistent with the previously observed positive correlation between RDW and osmotic stability in diabetic samples [The application of the proposed model expanded the previously reported  biomarkeproteome ,23 in co samples . The siz samples ) upon re+ cells was found to be tightly related to several physiological features. Breakdown intermediates of pyrimidines and purines were found to be “connected” positively or negatively, respectively, to cell fragility and the exposure of removal signals. Focusing on purine metabolism, the negative association between in vivo levels of IMP and mechanical hemolysis post storage came as no surprise in the light of the formerly revealed positive correlations between IMP and storage hemolysis in control samples [+ and control (though as a trend). This regular emergence of energy-related molecules in such analyses, both ex vivo and in vivo, is consistent with the abundance of volume-regulating channels, membrane-asymmetry maintaining molecules and antioxidants that rely on the energy reservoir of the cells [+ osmotic hemolysis with maltose/sucrose, oligosaccharides known to decrease the membrane lesions on frozen RBCs [The metabolism of βThal samples . In the  samples . Of note samples . High-en samples ,25,26,27he cells . An intezen RBCs . Anotherzen RBCs , as prev+ RBCs. Pyridoxamine, which has been found to be elevated in these RBCs [+/K+-ATPase activity [+ RBCs to plasma and body temperature. Indeed, hemoglobin oxidation is associated with cellular aging, loss of integrity and, consequently, hemolysis during cryopreservation [As in the case of phosphocreatine, the donor levels of antioxidant pyridoxamine—the storage levels of which also anticorrelate with post-storage hemolysis —similarlese RBCs , plays aactivity . On the ervation . In the ervation , and the+ RBCs. The mitigation of oxidative stress inside and outside the cell, mediated by non-enzymatic antioxidants, such as urate, appears to be associated with a decrease in PS exposure. The slightly elevated, though within normal range, baseline levels of Ca2+ in βThal+ cells [+ membranes [+-specific feature in RBCs previously reported to exhibit unique proteo-vigilance abilities during storage [The most integrated transfusion triangle in our study was the one connecting the antioxidant powers (extra- and intra-cellular) with the post-storage exposure of PS in βThall+ cells  seemed tl+ cells  nor calpl+ cells  but, on l+ cells , includil+ cells  and the embranes ) that maembranes . Interes storage .2+, pyridoxamine, extracellular antioxidant capacity) that are linked—directly or indirectly—to post-storage physiology or performance differ between βThal+ and control donors from the time of donation or during storage, too [+ donors, indicating a unique, βThal+-related variability. Although it is tempting to hypothesize the presence of thresholds producing differential correlation profiles in distinct genetic subgroups  the small size of our cohort cannot allow us to draw strict conclusions for such cut-off values.Overall, the application of the currently proposed approach to our donor cohorts provided interesting  information and unraveled variables as potential biomarkers of RBC performance. The abundance of transfusion triangles in the group of βThal minor donors reflects the “compactness” of this cohort in regard to several of the currently measured parameters. It may also be associated with the fact that a significant part of the freshly drawn blood variables  corresponding to RBCs, plasma and extracellular vesicles may lead to the discovery of a significant number of candidate biomarkers for assessing the transfusion quality of RBCs.


Dual pathology of Alzheimer's disease (AD) and vascular cognitive impairment and dementia (VCID) commonly are found together at autopsy, but mixed dementia (MX) is difficult to diagnose during life. Biological criteria to diagnose AD have been defined, but are not available for vascular disease. We used the biological criteria for AD and white matter injury based on MRI to diagnose MX. Then we measured multiple biomarkers in CSF and blood with multiplex biomarker kits for proteases, angiogenic factors, and cytokines to explore pathophysiology in each group. Finally, we used machine learning with the Random forest algorithm to select the biomarkers of maximal importance; that analysis identified three proteases, matrix metalloproteinase-10 (MMP-10), MMP-3 and MMP-1; three angiogenic factors, VEGF-C, Tie-2 and PLGF, and three cytokines interleukin-2 (IL-2), IL-6, IL-13. To confirm the clinical importance of the variables, we showed that they correlated with results of neuropsychological testing. Alzheimer's disease (AD) and vascular cognitive impairment and dementia (VCID) are the major causes of dementia  or trained research coordinators and scored according to standard procedures. Standardized (T) scores were calculated for each test. Averaged composite T-scores were calculated for separate cognitive domains: memory , executive function , attention , language , and processing speed . An overall cognitive composite score was derived as the mean of individual domain T-scores. Control participants for the MRI studies underwent the same neuropsychological test battery.A number of biomarkers were measured in CSF and blood plasma. CSF biomarkers were obtained by lumbar puncture performed in the morning after fasting by one of the authors (JCA). Blood draws were performed during the same patient visit. Samples were centrifuged, aliquoted, and stored at −80°C for later analysis.1−42 (Aβ1−42) and β-amyloid1−40 (Aβ1−40) to calculate the Aβ1−42/Aβ1−40 ratio . The output data were used to quantify the concentrations based on the 2-fold sample dilation and the supplied in-assay standard curve. All data were expressed as pg/mL, though the ratio is unitless.Levels of CSF Tau protein phosphorylated at threonine position 181 (pTau) were measured using the Innotest Phospho-Tau (181P) ELISA . Prior to analysis, all CSF underwent one freeze-thaw cycle. Assays were performed according to manufacturer protocols and were read with a Bio-Tek multimodal plate reader with absorbance at 450 nm. The output data were used to quantify the concentrations based on the supplied in-assay standard curve. We measured β-amyloidThe biomarkers we selected were based on the MesoScale Discovery (MSD) multiplex assay kits. These have been adapted for use by the MarkVCID consortium. Matrix metalloproteinases  were measured with two ELISA kits . Angiogenic growth factors were measured by ELISA . Similarly, multiple proinflammatory factors were measured with the Proinflammatory Panel 1 (MSD). For these assays, all CSF samples were run undiluted while all plasma samples were diluted 2-fold except for the MMP 3-Plex, in which case the plasma samples were diluted 10-fold. All data were expressed as pg/mL.Assays were performed using established protocols on an MSD Quickplex SQ 120 plate reader, followed by analysis performed in the MSD Discovery Workbench 4.0 software that was used to quantify analyte concentrations and all data were expressed as pg/mL. Protein markers measured with MSD assays were subjected to intra-plate variability tests which calculated the coefficient of variation (CV), as determined by duplicate runs for each sample. Samples with a CV ≥ 15% were removed from further analysis. Another assessment involved two CSF and two plasma pooled control samples run in duplicate on the same plate in all assays. These control samples were held to the same intra-plate CV (≥15%) and were also assessed for plate-to-plate variability.2 with 30 volumes collected with different gradient directions and five volumes with b = 0. The acquisition time was 6.5 min. The experiments done on the 32-channel coil used a CMRR multi-band sequence, which enabled us to collect more gradient directions. On the 32-channel coil, we collected three shells with a maximum b-value = 3000 s/mm2, 155 volumes with different gradient directions, and eight volumes with b = 0. The acquisition time was 12.5 min.To obtain information on the integrity of the white matter, we used MRI scans that were performed on a Siemens 3T scanner. Initial scans were performed on a 12-channel radio frequency (RF) coil and later scans were acquired with a 32-channel RF coil. The imaging parameters with the two RF coils were closely matched. The 3D MPRAGE sequence had TR = 2530 ms, four echoes, and TI = 1200 ms with an acquisition time of 6.5 min. The 3D FLAIR sequence had a TR = 6000 ms, TE = 427 ms, and TI = 2000 ms. The diffusion data were collected with a FOV = 224, 2 mm isotropic resolution, and 72 slices for both RF coils. On the 12-channel coil, the diffusion protocol had a single-shell of b-value = 800 s/mmwww.xinapse.com). The diffusion images were corrected for motion, distortion, and mean diffusivity (MD), and fractional anisotropy (FA) was calculated (www.fmrib.ox.ac.uk).White matter hyperintensity (WMH) volume was calculated from FLAIR images based on JIM software  if it was likely due to measurement error by outlying from the majority of points by roughly greater than twice the range of the majority of points on the variable's original scale. This resulted in removing roughly one or two values from about half of the features, a total of 54 values over 55 features. Observations were filtered to include patients who did not have missing values for more than 30% of the features, retaining 86 observations for our three primary diagnosis groups and controls. Missing values were imputed using the method “Multivariate Imputation by Chained Equations” via the mice R package , a supervised ensemble machine learning algorithm that is based on classification trees Breiman,  in whichThe three neurologists arrived at a consensus clinical diagnosis based on clinical history, neuropsychological tests and MRI FLAIR results. Initially, the results of the diffusion tensor MRI and some results of the CSF and blood studies were not available: AD CSF biomarkers were done initially, and the subsequent biomarkers in CSF and plasma were from the proteases, angiogenic factors and cytokines. Since VCID includes a number of forms of vascular disease, we focused on the small vessel form, subcortical ischemic vascular disease (SIVD), which can be detected by MRI and has a progressive course, making it more amenable to clinical trials Pantoni, . The diap = 0.010) (p < 0.001). There were no significant between-group differences for other cognitive domains  and composite cognitive score .Eighty-six (86) subjects had complete data permitting a full analysis; the numbers in each category are shown in = 0.010) . Control1−42/Aβ1−40 ratio and pTau. In addition, CSF values for MMP-1, MMP-9, and MMP-10, VEGF-D, Flt-1, PlGF, IL-8, IL-10, and IL-13 were significantly different from controls , while pTau was higher in the MX than in SIVD or AD (p = 0.001)  . The Aβ1ng speed .p = 0.001) (p < 0.054). Plasma MMP-2 negatively correlated with most of the cognitive measures , and plasma MMP-3 positively correlated with age (CSF MMP-10 was highest in MX and AD relative to SIVD (= 0.001) . No othewith age . CSF MMPwith age .p = 0.018) and CSF VEGF-C was lower in AD relative to SIVD and MX (p = 0.008)  was elevated in MX relative to AD (= 0.008) . No othe= 0.008) . Plasma = 0.008) .p = 0.043) . Plasma IL-13 was lower in SIVD relative to MX (= 0.043) . No otheCSF IL-1β was positively correlated with executive function, language, and overall cognition, CSF IL-2 was positively correlated with memory, TNF-α was positively correlated with language, memory, and overall cognition, and CSF IL-13 was negatively correlated with attention, executive function, and processing speed . Plasma We performed supervised classification using Random Forests with subsets of features from CSF and plasma to classify diagnosis groups in three ways . We considered three broad scenarios. First, we considered “All Factors” of CSF and plasma together, as well as CSF and plasma features separately. Second, we considered the separate “CSF Factors” of AD Proteins, Proteases, Angiogenesis, and Cytokines. Third, we considered the separate “Plasma Factors” of Proteases, Angiogenesis, and Cytokines. To improve classification accuracy, each model is first fit using the complete set of features and then we perform manual stepwise backward selection based on variable importance (VIMP) until all remaining variables have reliably positive VIMP values. The classification accuracy results for all scenarios are summarized in 1−42/Aβ1−40 ratio and pTau from the modeling; accuracies were similar for both All features  and CSF features . The ROC Curves indicate the optimal threshold  and CSF alone features have similar accuracies of roughly 77%, 88%, and 67% for the three diagnosis groups, while Plasma alone features had much lower accuracies . Therefore, the Plasma features do not add additional classification benefits to the CSF features , top rowhreshold , circle hreshold . AdditioVariable importance (VIMP) values for the “All Factors” scenario for the three diagnosis group definitions are given in In the “CSF Factors” scenario, separate models were considered for each set of features. The classification accuracy indicates that AD biomarkers and Angiogenesis factors are more predictive of diagnosis category than Proteases, with the Cytokines being the least predictive , 4. In tFeatures that did not improve classification  because Using the biological diagnosis to diagnose AD and MRI white matter injury to indicate vascular disease, we identified during life a group of cognitively impaired patients with dual pathology. Having separated patients into AD, VCID, and MX, we then used a large number of biomarkers determined in CSF and plasma with multiplex assay kits to determine the biomarkers of maximal importance. Finally, we used neuropsychological testing to validate the biomarkers identified. An important part of the study was the use of a statistical machine learning method to determine the relative importance of the biomarkers. In this manner, our study was a step in the realization of precision medicine for dementia studies.We studied the variable importance of biomarkers in a diverse group of cognitively impaired patients classified into AD, SIVD, and MX. We included the MX group by expanding the AD biological research criteria to include a vascular factor to identify dual pathology patients (Jack et al., The classes of biomarkers that we studied had inflammation and repair in common. We found that those with dual pathology had the highest values for the biomarkers, which is consistent with studies that show an acceleration of cognitive decline suspected to be due to the cumulative effects of the different pathological processes (Snowdon et al., Angiogenic factors have been identified in a number of studies in AD, but it is unclear whether they participate in injury or repair. It is possible to conceptualize a pathological scenario in which the growth of blood vessels begins with the proteolytic disruption of the extracellular matrix by one or more of the MMPs, which is analogous to vessel growth in tumors where the proteases remove pericytes and breakdown extracellular matrix proteins to prepare the vessels for sprouting under the control of angiogenic factors (Rundhaug, Correlating biomarkers with neuropsychological testing was important in that it showed their clinical relevance. The relationship between fluid biomarkers and cognition is complex and, given modest correlations and small sample size, our data should be considered as hypothesis-generating rather than instructive. Positive correlations between cognitive performance and CSF levels of inflammatory cytokines pose a paradox if inflammation precedes injury to brain structure. Scatterplots in Our results reveal the role of the angiogenic factors. It is interesting that Flt-1 besides being the receptor for VEGF, is a signaling factor for microglia (Ryu et al., reg cells that are linked to chemokines, CCL1 and CCL20, which suppress astrocytosis, contributing to repair (Ito et al., Random Forests, a machine learning method, selected several of the cytokines as variables of importance for distinguishing patient groups, including IL-2, IL-6, and IL-13. These may influence the inflammatory response: IL-2 amplifies TThere are several caveats with our data. First, patients were from a single center and only a subset had complete CSF/plasma and MRI datasets, reducing the numbers available for statistical analysis. Second, biomarkers selected were those available from MesoScale Discovery and had been used by the MarkVCID consortium, which included our group; other biomarkers and platforms with different biomarkers could have been used. Furthermore, the study was cross-sectional rather than longitudinal, precluding inferences about the temporal dynamics of analyte levels. A major caveat is the small sample size, which was further hindered by forming an additional MX group. However, despite the small numbers, the results were statistically significant. A follow-up study on a larger population is necessary to further validate the results of this present study.In conclusion, we expanded the biological definition of AD by adding vascular factors, allowing the identification of patients with dual pathology prior to autopsy. Using Random Forests, a machine learning method, we have determined the major proteases, angiogenic factors, and cytokines of importance in classification in a diverse group of dementia patients. Following an initial classification into diagnostic groups, we identified the proteases, MMP-1, MMP-3 and MMP-10, the angiogenic factors, VEGF-C, PlGF, Flt-1, Tie-2, and the cytokines, IL-2, IL-6, and IL-13. Our results suggest that the combined action of proteases and angiogenic growth factors may be important in dementia with cytokines fueling the inflammatory processes. Further studies in larger numbers of patients will be needed to confirm these results.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of New Mexico Human Research Review Committee. The patients/participants provided their written informed consent to participate in this study.EE performed the statistical analysis and wrote a draft. JA and JK recruited the patients. AC performed the MRIs and analyzed the MRI data. JT and SH analyzed the CSF and blood. JP performed the neuropsychological testing. DS obtained the control CSF during surgery. GR obtained the funding, recruited patients, and contributed to the writing of 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Six months postoperatively, there were no statistically significant differences in OOM activity compared to preoperative values (p = 0.2). Conclusions: Eyelid sEMG may be a useful diagnostic tool in post-operative OOM recovery monitoring. sEMG parameters of the maximal contraction of the OOM normalize within 6 months after anterior approach LAA. Electrode placement in the mid-pretarsal area of the upper eyelid offers several advantages and therefore may enhance the feasibility of sEMG in clinical practice.Introduction: In this article, we propose a new application for eyelid surface electromyography (sEMG). By placing the electrode in the mid-pretarsal area of the upper eyelid, one can easily perform a fast examination and achieve repeatable results. We believe that this technique may increase the feasibility of eyelid sEMG in clinical practice. Methods: 126 sEMG examinations of the upper eyelid were performed by using the above-described method. Thirty-nine controls and 29 ptotic patients were enrolled. The controls underwent one measurement while the ptotic patients were employed for four sessions: Before anterior approach levator aponeurosis advancement (LAA), 2 weeks, 3 months, and more than 6 months after surgery. The relaxation and maximal contraction of the orbicularis oculi muscle (OOM) using root mean square (RMS) values were measured. Results: The results showed a statistically significant decrease in RMS values of the maximal contraction of the OOM 2 weeks after surgery ( Many of the anterior-approach oculoplastic procedures require an incision or resection of the orbicularis oculi muscle (OOM). Levator aponeurosis advancement (LAA) is one of the most frequently performed methods. LAA can be performed from the anterior or posterior approach ,2. In thSurface EMG (sEMG) is a noninvasive, quantitative, and objective method of assessing muscle activity. It allows the measurement of muscle activity in both eyelids simultaneously. The utility of sEMG in the assessment of OOM, mainly in facial palsy, has been proven in experimental studies ,5,6. HowThe OOM is a striated muscle that lies just below the skin. It is a large muscle, considering eyelid structures. Anatomically, it is divided into four parts: Orbital, preseptal and pretarsal segments, and the muscle of Riolan. The orbital part overlies the bony orbital rim. The palpebral portion is positioned over the mobile eyelid and is divided into preseptal and pretarsal parts. The preseptal part overlies the orbital septum in both upper and lower eyelids. The pretarsal OOM is positioned over the tarsal plates. Between the tarsal plate and the pretarsal OOM runs the muscle of Riolan. Below the OOM lies the orbital septum that separates the preaponeurotic fat pads . The locThe frontalis muscle is a large and strong paired muscle that extends from the coronal suture line towards the supraorbital rim. Its medial fibers interdigitate with those of the procerus muscle. Under the brow, frontalis muscle fibers blend with the corrugator muscle and the depressor supercilii. The sEMG signal from the palpebral or supraorbital area of the OOM is compromised by the activation of the above-mentioned muscles.OOM in the lower eyelid neighbors the zygomaticus major, zygomaticus minor, levator labii superioris, and levator labii superioris alaque nasi . These lTaking into consideration these anatomical conditions, we assumed that the pretarsal area represents less crosstalk than any other part of the OOM.We propose a new application for eyelid sEMG using single-electrode placement in the pretarsal area of the upper eyelid. Using RMS analysis of the maximal contraction of the OOM, the objectives of our study included (1) implementing this technique in a clinical setting in which patients presented with involutional ptosis; (2) comparing results in healthy controls to patients with involutional ptosis; (3) comparing results in patients with ptosis before and after surgery and (4) assessing the feasibility of sEMG in muscle recovery after ptosis surgery.The prospective experimental study was conducted in the Department of Ophthalmology and the Department of Neurology at the Central Clinical Hospital of the Ministry of Interior and Administration in Warsaw. Sixty-eight participants  were included in the study. The exclusion criteria were previous eyelid surgery or injury, eyelid retraction, lagophthalmos, eyelid skin lesions, stroke, myasthenia gravis, myopathies, and lack of consent.The study was implemented in the Electromyography Unit of the Department of Neurology. A Keypoint electromyography device with a 24kHz resolution and the Keypoint-Net Program was applied . For standard BR assessment, the sweep speed of the EMG device was set at a 50–800 ms/division interval according to the interstimulus interval. Amplitude sensitivity was set at s 100–200 uV/division, and upper and lower frequency filters were set between 20 Hz and 10 kHz.During the examination, the subject was in a sitting position with their head in an upright position. The active electrode was placed in the central portion of the upper eyelid horizontally, 5 mm above the lash line, while the reference electrode was placed on the forehead . The groThe sEMG examination was standardized and performed by one technician in the presence of the ophthalmologist (LKW). The procedure was explained to the participants. The real measurement was conducted when a subject fully learned how to perform the movement after preliminary measurement. The controls were committed to one research session lasting approximately 20 min. This included informed consent, setup time, and cleanup time. The ptotic patients were employed for four sessions . A one-time electromyography signal measurement was set at five seconds. Each test was repeated with three measurements. The average value of all three measurements was used as a measurement value. Between each measurement, subjects were required to take a one-minute rest. The measurement was taken while performing maximum eyelid closure  and withAll patients underwent anterior approach LAA. When indicated, blepharoplasty was performed. A strip of OOM was excised in all cases. After LAA, OOM was closed with Vicryl 6.0 interrupted sutures. The skin incision was closed with a running Prolene 5.0 suture.Statistical analysis was performed. The Shapiro–Wilk test was used to test the normal or non-normal distribution of variables. The mean and standard deviations, as central tendency measures, were estimated in the normal distributed variables, and the median and interquartile range were estimated in the not normally distributed variables.p < 0.05 was selected as the threshold for the statistical significance.The nonparametric Mann–Whitney U-test was adopted to evaluate whether there was a statistically significant difference in the average contraction values between controls and patients. Meanwhile, the comparison of the average contraction values between patients before and after surgery was analyzed using the Wilcoxon test. A level of The experiment was well tolerated by all participants. Demographics of the examined groups are summarized in In the control group, 39 individuals had a single sEMG examination. Patients in the ptosis group initially consisted of 29 participants prior to surgery, 28 people 2 weeks after the operation, 13 patients after 3 months, and 17 patients more than 6 months after the surgery. That provided 87 sEMG examinations in the patient group and 126 measurements in both groups. An equal number of left and right eyelids were operated on.p > 0.05). None of the subjects experienced any limitation in the maximal contraction of the eyelid with an electrode attached in the mid-pretarsal area of the upper eyelid. The results of RMS values in men and women and in left and right eyelids were compared  decrease in RMS values of maximal contraction of the OOM 2 weeks after surgery. An improvement in muscle activation within the first 3 months after surgery was observed. However, there were still statistically significant differences between OOM activation patterns before surgery and 3 months after (p = 0.03). We found no statistical differences between RMS values pre-op and post-op more than 6 months after surgery. Future studies can be conducted to detect the onset of OOM recovery during the early follow-up after surgery. Moreover, it would be interesting to investigate the relationship between OOM electrophysiological recovery and the presence of lagophthalmos.Iatrogenic damage of the OOM occurs during many oculoplastic procedures. LAA is a procedure that can be performed through a skin incision or through a conjunctival approach ,2. The aThese preliminary results provide the basis for further research towards a new approach to OOM activity assessment in clinical practice. The presented technique offers several advantages. It is simple and fast, as placing only one electrode reduces the preparation time and costs. It does not require special training or anatomical knowledge by the technician to properly install the electrodes. Therefore, it is repeatable and can be applied by different technicians with no loss to the quality of data collected. All these advantages can increase the utility of eyelid sEMG in a clinical setting.The electrodes attached to the upper eyelid may disconnect during forced eyelid closure since the electrodes are secured with adhesive tape. This problem could be resolved by introducing new materials used for electrode construction, such as soft carbons .The electrode can be attached exclusively to the undamaged skin. This limits the utility of the procedure within the first week post-op.We encountered some difficulties in correctly placing the electrodes in patients with dermatochalasis. Excessive drooping of the skin may be a contraindication in some cases.The disadvantages of this technique are as follows: In conclusion, the results of the present study indicate that the OOM recovers from surgical injury within 6 months after the anterior approach LAA procedure. Electrode placement in the mid-pretarsal region of the upper eyelid is a novel approach introduced to limit crosstalk. sEMG may become a useful diagnostic tool in OOM activation assessment in an objective manner. However, further studies are needed to verify the usefulness of eyelid sEMG with the electrode placed in the mid-pretarsal area.


Antithrombotic therapy in patients with nonvalvular atrial fibrillation (NVAF) concomitant with the acute coronary syndrome (ACS) or underwent percutaneous coronary intervention (PCI) is challenging and has evolved in recent years. However, real-world data on this issue about antithrombotic regimens at discharge and its evolving trend were relatively scarce, especially in China.n = 674; cohort 2 (2018), n = 793; and cohort 3 (2019), n = 512. Baseline characteristics and antithrombotic therapy at discharge were analyzed by cohort.A total of 2,182 patients with NVAF and ACS/PCI were enrolled from 2017 to 2019. A total of 1,979 patients were finally analyzed and divided in three sequential cohorts: cohort 1 (2017), P trend < 0.001), while triple therapy (TT)/double therapy (DT) increased from 27.2 to 50.0% (P trend < 0.001). This trend was also seen in different subgroups stratified by CHA2DS2-VASc score, HAS-BLED score, coronary artery disease type, or management type, and was validated after multivariate adjustment. Persistent atrial fibrillation and history of congestive heart failure, hypertension, diabetes mellitus, and stroke/transient ischemic attack/systemic embolism were the independent predictors of TT/DT use, while ACS, PCI, or advanced chronic kidney disease was related with more DAPT prescription.In our cross-sectional study, the majority of patients (59.6%) received dual antiplatelet therapy (DAPT). Over the 3 years, DAPT prescription reduced from nearly 70% to <50% (There is a shift of antithrombotic regime at discharge for patients with NVAF with recent ACS/PCI with reducing DAPT prescription and increasing TT/DT prescription. While the appropriate antithrombotic regimen for patients with NVAF having ACS/PCI is still underused in China. Atrial fibrillation (AF) is the most common cardiac arrhythmia with increasing prevalence  and is aPatients with nonvalvular atrial fibrillation (NVAF) having moderate-to-high stroke risk require chronic oral anticoagulation (OAC) for thromboembolism prevention , 8, wherAntithrombotic therapy of patients concomitant NVAF and ACS/PCI has evolved in recent years with new evidence from pivotal clinical trials in this field –14. EvidThis study is a single-center cross-sectional study of adults with AF and concomitant with ACS or who underwent PCI from 2017 to 2019 in Fuwai Hospital, Beijing, China. Men and women aged over 18 years with AF and ACS or who underwent PCI were enrolled to assess eligibility . At leasThe diagnosis of AF was confirmed by reviewing clinical records and electrocardiographic evidence, namely, ECGs, Holter, and rhythm strips. ACS comprised a series of acute coronary diseases, namely, ST-elevation myocardial infarction (STEMI), non-STEMI (NSTEMI), and unstable angina (UA). The diagnosis of ACS was confirmed by clinical physicians based on clinical evaluation, namely, symptoms of cardiac ischemia, 12-lead electrocardiography abnormalities, cardiac biomarkers (cardiac enzyme and cardiac troponin) change, and echocardiography. PCI was defined as percutaneous transluminal coronary revascularization with balloon angioplasty, drug-eluting balloon angioplasty, bare-metal/drug-eluting stent implantation, or unsuccessful coronary intervention attempt.The definitions of comorbidities are listed as follows. Congestive heart failure was defined with prior symptomatic heart failure and/or left ventricular ejection fraction <40%. Hypertension was defined as a documented history of hypertension or blood pressure over 140/90 mm Hg. Vascular diseases include CAD with prior myocardial infarction (MI), acute MI (AMI), and peripheral artery disease with artery stenosis or occlusion. Prior stroke was defined as a combination of ischemic stroke and hemorrhage stroke.The CHA2DS2-VASc scores and HAS-BLED scores were used to evaluate the risk of ischemic stroke and bleeding. CHA2DS2-VASc scores were calculated with 1 point for congestive heart failure, hypertension, diabetes, vascular disease, age 65–74 years, and sex category , and 2 points for age ≥75 years or stroke. HAS-BLED were calculated with 1 point each for uncontrolled hypertension with systolic blood pressure over 160 mm Hg, abnormal renal function, abnormal hepatic function, previous ischemic or hemorrhagic stroke, bleeding history or predisposition, elderly (age > 65 years), concomitant use of antiplatelet or NSAID and excessive alcohol intake per week, and labile international normalized ratio (INR) is applied for patients receiving warfarin with over 3 times INR measurements. The high risk of ischemic thromboembolism was defined as CHA2DS2-VASc score ≥2, and the high risk of bleeding was defined as HAS-BLED score ≥3.The antithrombotic regimens were evaluated by the prescription of antithrombotic drugs at discharge including antiplatelet and oral anticoagulants (OACs). The antiplatelet drugs include aspirin, clopidogrel, and ticagrelor, and OACs comprise both vitamin K antagonist  and nonvitamin K oral anticoagulants .We classified the antithrombotic treatment according to the combination of prescribed drugs into the following six regimens: no treatment, single antiplatelet therapy (SAPT), dual antiplatelet therapy (DAPT), OAC monotherapy, double therapy , and triple therapy . And TT and DT together are called combined therapy. Besides, TT and DT were further classified into NOAC-based TT (NOAC + DAPT), VKA-based TT (VKA + DAPT), NOAC-based DT (NOAC + SAPT), and VKA-based DT (VKA + SAPT).via interviewing the participants, reviewing medical records , and contacting their treating physicians. Individual CHA2DS2-VASc score and HAS-BLED score were calculated by using the information on comorbidities at baseline retrospectively. And source data verification was conducted by the computer-based patient record management system. The study design and protocol have been approved by the Ethics Committee of Fuwai Hospital (Approved No. 2017-923) and conformed to the Declaration of Helsinki. All the patients have signed consent to participate in this study.Data about demographic information, baseline comorbidities, and medication usage were collected by trained research personnel Descriptive statistics were used to summarize and analyze the baseline demographic characteristics, medical history, clinical risk score assessment, and in-hospital invasive treatment by cohort. Besides, the antithrombotic therapy and drug usage were summarized and analyzed by cohort. Then, the temporal trend in antithrombotic therapy was further analyzed by CHA2DS2-VASc score and cohort, by HAS-BLED score and cohort, by CAD type and cohort, and by treatment type and cohort.2], and in-hospital invasive treatment.To investigate factors affecting the prescription of TT or DT after recent ACS/PCI in patients with AF, baseline demographic characteristics, medical history, clinical risk score, and in-hospital invasive treatment were compared between patients with TT/DT and those with only DAPT. Then logistic regression analysis was performed to investigate the difference in the TT/DT or DAPT prescription across different CHA2DS2-VASc scores and HAS-BLED scores. In addition, multivariable logistic regression analysis was performed using the forward stepwise (LR) method to evaluate the independent predictors of favoring TT/DT over DAPT, with variables comprising year cohort, AF type, CAD type, gender, age, congestive heart failure (CHF), hypertension, diabetes, CAD, PAD, stroke/transient ischemic attack (TIA)/systemic embolism (SE), history of bleeding, advanced chronic kidney disease (CKD) [estimated glomerular filtration rate (eGFR) < 30 ml/min/1.73 mThe multivariate logistic regression models were applied to verify the independent effect of time trend on TT/DT use by adjusting for potential confounders. Model 1 was the crude model without adjustment. Model 2 was adjusted for AF type, CAD type, and treatment type. Model 3 was further adjusted factors in the CHA2DS2-VASc score, namely, age, sex, CHF, hypertension, diabetes mellitus, stroke/TIA/SE, prior MI, and peripheral arterial disease.U-test was used to compare the difference among groups. Categorical variables were presented as the frequency with percentage and the chi-squared test was used. The significance of the linear time trend over the study period was assessed using linear regression analysis or logistic regression analysis. Statistical significance was set at p < 0.05. All the analyses were performed using software packages SPSS (version 26.0).Continuous variables were expressed as mean ± SD or median with interquartile ranges as appropriate and one-way ANOVA or the Mann–Whitney n = 674; cohort 2 (2018), n = 793; cohort 3 (2019), n = 512.Between January 2017 and December 2019, 2,182 patients with AF and ACS or who underwent PCI were enrolled to assess eligibility. A total of 1,979 patients were included in the final analysis, and divided into three consecutive sequential cohorts by year: cohort 1 (2017), P trend = 0.014), while the rest variables were similar among the groups.Baseline characteristics and in-hospital treatment strategy were listed in P trend < 0.001). The combined therapy  was increased from cohort 1 (27.2%) to cohort 3 (50.0%) (P trend < 0.001). Both the proportion of TT  and DT  increased over time. The rise was due to the prevalence use of NOAC, as the prescription of both NOAC + DAPT  and NOAC + SAPT  increased over the study period. At the same time, there was a decline in the use of VKA, regardless of VAK + DAPT  or VKA + SAPT . While the proportion of patients not receiving antithrombotic therapy remained unchanged . Considering the drug-specific prescription, there is a decreasing trend in aspirin  and VKA  use. And there is an increasing trend in NOACs  use, especially for rivaroxaban  use. While the prescription rate of clopidogrel, ticagrelor, and dabigatran stayed almost the same.P for trend < 0.001, P for trend = 0.058, The increasing trend of TT/DT prescription was observed regardless of CHA2DS2-VASc score level , HAS-BLEp < 0.001), CHF (p < 0.001), hypertension (p < 0.001), DM (p < 0.001), prior history of stroke/TIA/SE (p < 0.001), and possessed higher CHA2DS2-VASc score (p < 0.001). Besides, patients with TT/DT were more likely treated conservatively without invasive treatment (p < 0.001) and with less stent implantation (p < 0.001).2 was associated with underuse of the combined therapy, the most significant predictor of DAPT was PCI , while age, sex, history of vascular diseases, and previous bleeding events had little effect on the choice of antithrombotic therapy.Then, the multivariate logistic regression analysis was performed to evaluate the independent predictors associated with the prescription of TT/DT or DAPT . The incp < 0.001) and cohort 3  has a higher prescription rate of TT/DT and is increasing over time (P trend < 0.001). The results remained statistically significant after adjustment for confounders in Model 2–3. In the full adjusted model, the association between year cohort and TT/DT remained statistically significant .The multivariate logistic regression model was applied to verify the independent effect of time trend on TT/DT use. The association between yearly trends and TT/DT use is shown in 2 were associated with underuse of the combined therapy; and (5) Temporal trend was the independent factor related to the incremental use of TT/DT.In this study, we investigated the evolving trend in antithrombotic therapy, and our principal findings were as follows: (1) The majority of patients were administered DAPT without OAC; (2) Both the TT and DT prescriptions showed a gradual increment over time, and the total amount of TT/DT prescriptions surpassed 50% in 2019; (3) An incremental relationship between CHA2DS2-VASc score and combined therapy prescription was identified; (4) The predictors of TT/DT use were persistent AF type and history of CHF, hypertension, diabetes mellitus, and stroke/TIA/SE. ACS or PCI and CKD with eGFR <30 ml/min/1.73 mThe choice of antithrombotic therapy for patients concomitant of AF and ACS/PCI presents a great challenge in the real-world clinical scenario. The risk of ischemic events and the risk of bleeding need to be balanced carefully. Plenty of efforts have been made to explore this issue since 2013. Based on the result of WOEST , ISAR-TRn = 6,362) and/or underwent PCI  from 2011 to 2017, finding <1/2 patients were treated with DT/TT . The underprescription of OAC-based TT/DT in our study might be caused by concerns of physicians in China. As TT/DT has been shown to increase the risk of bleeding , rather than the guideline-recommended TT/DT. And there is a small proportion of patients were prescribed with merely OAC, SAPT, or no treatment. These results highlight the undertreated status of patients with NVAF and ACS/PCI in China and are in line with other countries. A Korean study  reportedbleeding , Asians bleeding , 27. Forbleeding . The neeCI n = 2,7 from 20Then, we investigate the difference in the TT/DT or DAPT prescription across different CHA2DS2-VASc scores and HAS-BLED scores by utilizing logistic regression analysis. The CHA2DS2-VASc score was significantly related to the prescription of TT/DT, and the association was more pronounced in higher risk score subgroups. While, the bleeding risk evaluated by HAS-BLED score has little effect on the choice of antithrombotic regimens, as the proportion of patients on TT/DT stayed almost the same across different HAS-BLED scores. Besides, patients with higher bleeding risk (HAS-BLED score ≥3) possessed a borderline increased likelihood of TT/DT prescription. This might due to the fact that bleeding risk factors frequently overlap with thromboembolism risk factors. Those patients with high bleeding risk often possess high thromboembolic risk as well. Besides, clinical guidelines suggest that HAS-BLED scores should be utilized to assess the risk of bleeding, rather than define whether a patient should be treated with OAC . By usinMoreover, we explored the independent predictors of TT/DT prescription and the factors associated with the suboptimal use of combined therapy by using multivariable logistic regression analysis. In addition to the temporal trend of TT/DT use, persistent AF type and history of CHF, hypertension, diabetes mellitus, and stroke/TIA/SE were associated with a higher prescription of TT/DT. This is in line with a previous study utilizing Korea nationwide registry data, which also found traditional thromboembolism risk factors, namely, female gender, DM, prior cerebrovascular accident, and CHF are significant determinants of OAC use . And patAnother important finding in this study was that a pronounced change in the pattern of antithrombotic therapy prescription has been made in China. There is a shift of antithrombotic regime with reducing DAPT prescription and increasing combined therapy (TT/DT) prescription. This trend was also seen in different subgroups stratified by CHA2DS2-VASc score, HAS-BLED score, CAD type (ACS or SCAD), or management type . Moreover, this trend stayed validated after adjusting for confounders, namely, AF type, CAD type, and treatment type, factors in the CHA2DS2-VASc score. This phenomenon demonstrated that clinical practice has been influenced by evolving evidence and guidelines recommendations, which is in line with the result from Taiwan . This paThere are several strengths in this study. First, this study was conducted in the largest medical center for cardiovascular diseases in China, representing the most advanced level of clinical practice in our country. To the best of our knowledge, this is the largest cross-sectional study conducted in China until now in this field. Second, a great deal of clinically important information was collected, namely, patient baseline characteristics, previous history, bleeding profile, and specific treatment strategy. In addition, source data verification was conducted by the computer-based patient record management system to ensure validity.This study also has some limitations. First, since our data were derived from a single medical center, there may be a discrepancy between the drug prescription habit of this single center and the overall population of China. Generalizing the antithrombotic utilization patterns beyond this level of healthcare setting warrants caution. Second, the prescription of TT/DT might be overestimated as some OAC prescription was due to transient purposes like cardioversion or AF ablation. Third, patients with AF may have some specific angiographic characteristics . The sev2 were associated with underuse of the combined therapy. Further studies are warranted to elucidate the clinical outcome with different antithrombotic treatment regimes in Chinese real-world clinical practice.The antithrombotic treatment at discharge for patients with NVAF having recent ACS/PCI has noticeably changed in recent years. There is a shift of antithrombotic regimen with reducing DAPT prescription and increasing combined therapy (TT/DT) prescription. The predictors of TT/DT use ware persistent AF type and history of CHF, hypertension, diabetes mellitus, and stroke/TIA/SE, while ACS or PCI and CKD with eGFR <30 ml/min/1.73 mThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Fuwai Hospital (Approved No. 2017-923) and conformed to the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.NS collected the data, performed the statistical analysis, drafted, and wrote the manuscript. Y-mY and JZ designed the study and revised the manuscript. JW, HZ, X-hS, and SW collected the data. All authors read and approved the final manuscript.This work was supported by Capital's Funds for Research and Application of Clinical Diagnosis and Treatment Technology (Grant/Award Number: Z191100006619121).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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