When controlling for SUBFAT, the partial correlations demonstrated a consistent negative correlation between GM activation and IMAT  but no relationship with HDL. The current results are important for helping to interpret the results from sEMG by accounting for IMAT. In conclusion, the neuromuscular activation of the GM may be reduced by the quantity of IMAT.The amount of tissue between the muscle and surface electromyography (sEMG) electrode influences the sEMG signals. Increased intramuscular adipose tissue (IMAT) of the hip abductor muscles negatively impacts balance in older individuals, but it is unknown if this is related to the ability to activate the muscles. The aim of this preliminary study was to investigate the influence of gluteus medius (GM) IMAT on sEMG amplitude during maximal voluntary isometric contractions (MVIC) of the hip abductors in older adults. We recruited 12 healthy community-dwelling older adults that underwent a spiral computerized tomography scan. High density lean (HDL), IMAT, and subcutaneous adipose tissue (SUBFAT) cross-sectional area of the GM were assessed. sEMG signal from the GM was recorded while participants performed an MVIC of the hip abductors. There was a negative correlation between GM activation and IMAT ( Surface electromyography (sEMG) is widely used to assess neuromuscular activation across multiple populations, including healthy individuals and those with neurological disorders . MultiplIntramuscular adipose tissue (IMAT), or the amount of adipose tissue within the fascia of a muscle, increases with age and inactivity , and is The ability to activate the hip abductor muscles is important to recover balance and avoid falls . For ins2. Exclusion criteria included: cognitive impairment , sedative use, depression, or a clinically significant functional impairment related to a musculoskeletal, neurological, cardiopulmonary, or metabolic disorder. All inclusion and exclusion criteria were verified by a medical exam with a licensed physician.Healthy community-dwelling older adults were recruited for this study. All participants provided written informed consent, and the study was approved by the Institutional Review Board of the University of Maryland, Baltimore. As previously described , inclusiFAT) cross-sectional area  of the GM and minimus together  between subcutaneous fat thickness and CSA from the CT scans in 50% of the participants; hence, to keep consistency among the study measurements, we used SUBFAT CSA. HDL was defined as 30–100 Hounsfield units (HU), low density lean tissue was representative of IMAT and defined as 0–29 HU and −190 to −30 HU for SUBfat. As previously published, LDL and HDL were normalized for the GM muscle size by calculating a percentage of each measure relative to the total muscle CSA  exam have been published previously . Brieflytogether  were detscle CSA .MVT) at the highest MVIC.Participants performed a standing unilateral hip abduction MVIC on a BIODEX System 4 dynamometer . The torque signal was recorded at a frequency of 1,500 Hz. The test limb was strapped to the Biodex arm with the thigh pad proximal to the knee joint. Support was provided through the arms by a custom stabilizing frame. Three trials were performed at 30° of hip abduction. Each participant was asked to “push” abduction as fast and hard as possible for ∼5 s with 90 s rest between trials. During the MVIC, sEMG signal was recorded using a wireless sEMG system  at a frequency of 1,500 Hz. sEMG electrodes arranged in a bipolar configuration (2 cm inter-electrode distance) were placed on the GM in accordance with SENIAM guidelines . The sEMFAT) was performed. Considering the influence of SUBFAT on sEMG (FAT as a covariate. There was no difference in the correlation between absolute (cm2) or relative (% CSA) values; hence, all correlations are presented as cm2. Additionally, considering possible sex differences, an independent t-test was performed for all variables, and no differences was found (P ≤ 0.081). Thus, results were presented for both sexes as one group. Statistical analysis was performed using SPSS version 25 ; the significance level was set at P < 0.05.A Shapiro–Wilk test was used to assess the normality of the data. Pearson’s product-moment bivariate correlations between sEMG amplitude and each predictor variable  and five females  were tested. There was a significant bivariate negative correlation between GM sEMG amplitude and IMAT , FAT, no significant correlation was found between GM sEMG amplitude and HDL , Seven males . Thus, we provide evidence that GM neuromuscular activation is potentially altered by the amount of adipose tissue inside the muscle. Moreover, although lean muscle tissue positively influenced sEMG, this association disappeared after accounting for subcutaneous fat. Overall, these results bring important information which may be important in the interpretation of sEMG signals in different populations, especially those of disuse and muscle atrophy who may have increased IMAT.Previous work demonstrated that the knee extensor neuromuscular activation, measured by CAR, was partially explained by the amount of adipose tissue inside the muscle of older adults ∼26%; . While wThe present study was not designed to explore the mechanism by which IMAT influences neuromuscular activation, but some factors might help explain the present results. Given that, IMAT represents the deposit of lipids within and between muscle fibers , the incFAT, there was no significant association between variables, albeit did present a moderate correlation (r = 0.503). Thus, although HDL did not directly affect muscle activation in the present study, the small number of participants used here may be a limiting factor, and likely, a larger sample size may be required to produce a significant correlation. It could be hypothesized that higher HDL would provide more motor units to be recruited and result in a greater neuromuscular activation. However, it was previously demonstrated that a decrease in lean muscle mass in older adults does not directly translate into a decline in muscle function  of the neuromuscular activation in older adults. However, after controlling for SUBfunction , which mfunction .Because the present study had a small sample size, we may have been underpowered to detect some correlations. Note that, conclusions for bivariate correlations should be taken with caution. Nonetheless, it is important to highlight that there are several other extrinsic  and intrinsic actors  that also influence sEMG amplitude . The imaIn conclusion, the neuromuscular activation of the hip abductor muscles may be reduced by the quantity of IMAT, while the lean tissue after accounting for subcutaneous fat, may not influence the activation of the GM muscle. Therefore, to better interpret neuromuscular activation from gluteus medius in older adults during a maximal isometric contraction, accounting for the amount of intramuscular fat would provide a more accurate representation of the signal.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 the Institutional Review Board of the University of Maryland, Baltimore. The patients/participants provided their written informed consent to participate in this study.AR, VG, and OA conceived and designed research. ML and WP perform data processing and analysis. All authors contributed to write, read, and approved 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.


Our aim was to assess associations between CSF p-tau with [18F]flortaucipir PET and the associations of both tau biomarkers with cognition and atrophy.In vivo Alzheimer’s disease (AD) biomarkers for tau pathology are cerebrospinal fluid (CSF) phosphorylated tau (p-tau) and [n = 8) and AD dementia (n = 45) and 25 cognitively normal subjects with subjective cognitive decline (SCD) (40% amyloid-positive)). Dynamic 130 min [18F]flortaucipir PET scans were acquired to generate binding potential (BPND) images using receptor parametric mapping and standardized uptake values ratios of 80–100 min (SUVr80-100min) post injection. We obtained regional BPND and SUVr from entorhinal, limbic, and neocortical regions-of-interest (ROIs), closely aligning to the neuropathological tau staging schemes. Cognition was assessed using MMSE and composite scores of four cognitive domains, and atrophy was measured using gray matter volume covering the major brain lobes. First, we used linear regressions to investigate associations between CSF p-tau (independent variable) and tau PET (dependent variable). Second, we used linear regressions to investigate associations between CSF p-tau, tau PET , and cognition (dependent variable). We then assessed the independent effects of CSF p-tau and tau PET on cognition by simultaneously adding the other tau biomarker as a predictor (model 2). Finally, we performed the same procedure for model 1 and 2, but replaced cognition with atrophy. Models were adjusted for age, sex, time lag between assessments, education (cognition only), and total intracranial volume (atrophy only).We included 78 amyloid positive cognitively impaired patients  across ROIs, 0.43–0.46; all p < 0.01). [18F]flortaucipir BPND was more strongly associated with cognition and atrophy than CSF p-tau. When [18F]flortaucipir BPND and CSF p-tau were entered simultaneously, [18F]flortaucipir BPND  was strongly associated with multiple cognitive domains and atrophy regions. SUVr showed comparable results to BPND.Higher [18F]flortaucipir BPND correlated stronger with cognition and neurodegeneration than CSF p-tau, suggesting that tau PET more accurately reflects disease severity in AD.Regional [The online version of this article (10.1007/s00259-020-04758-2) contains supplementary material, which is available to authorized users. Postmortem studies have revealed that tau pathology is highly associated with the degree of cognitive impairment –3 and thau in AD –7.18F]flortaucipir PET provide interchangeable or complementary information. So far, a number of studies have investigated associations between [18F]flortaucipir PET and CSF p-tau. Some studies found moderate to strong correlations across groups which consisted mainly of cognitively normal individuals [18F]flortaucipir uptake values continued to increase with progression of AD [18F]flortaucipir may depend on disease severity.Although both are in vivo biomarkers for tau pathology, it remains unclear whether CSF p-tau and 1Fflortaucividuals , while oividuals . Across ividuals . In addion of AD . Taken t18F]flortaucipir [18F]flortaucipir PET is able to capture regional uptake patterns and mirrors established neuropathological staging schemes of tau [18F]flortaucipir binding with proxies of disease severity, and it remains to be established whether [18F]flortaucipir PET and p-tau can be viewed as equivalent markers for AD staging.Both CSF p-tau , 14 and taucipir –21 have taucipir , 15–19 as of tau , while cs of tau , 24. Hows of tau , 25, 26.18F]flortaucipir binding in cognitively normal and impaired subjects on the AD pathophysiological continuum [18F]flortaucipir PET provide complementary information regarding disease severity, we investigated associations of both CSF p-tau and [18F]flortaucipir PET with multiple cognitive domains and regional atrophy.Therefore, we aimed to compare CSF and PET markers of tau pathology and to assess their relationship with cognitive impairment and atrophy as proxies of disease severity. We first examined regional associations between CSF p-tau and quantitative florbetapir Aß-PET scans PiB or [18F]florbetaben) scan by visual assessment flortaucipir PET scan for SCD subjects  and a maximum of 6 months from the [18F]flortaucipir PET scan for subjects with MCI/AD .All subjects underwent a 3D-T1 weighted sequence on a 3.0 Tesla MRI scanner  volumes using voxel-based morphometry (VBM) implemented in Statistical Parametric Mapping (SPM) version 12 software  as described previously . StructuTotal intracranial volume (TIV) was calculated by combining the native space segmentations  for each subject. We used gray matter density  as a proxy of atrophy in the analyses.18F]flortaucipir PET scans were acquired on a Philips Ingenuity TF-64 PET/CT scanner. The scanning protocol consisted of two dynamic PET scans of 60 and 50 min, respectively, with a 20-min break in between [18F]flortaucipir (injected mass 1.17 ± 0.78 μg). The second PET scan was co-registered to the first dynamic PET scan using Vinci software flortaucipir BPND (as dependent variable) and CSF p-tau (as independent variable), adjusted for age, sex, and time lag between LP and tau PET. We performed the analyses across all subjects and additionally stratified for diagnosis  and amyloid status for SCD subjects only. We did not stratify for amyloid status for the rest of the analyses because of the limited amount of amyloid positive SCD subjects (n  = 10).Linear regression models were performed to assess the relationship between entorhinal, limbic, and neocortical [18F]flortaucipir BPND (or SUVr) (dependent variable), we performed a voxel-wise multiple regression analysis for CSF p-tau and [18F]flortaucipir PET, adjusted for age, sex, and time lag between LP and [18F]flortaucipir PET scan. All resulting t-maps were thresholded with p < 0.001 at the voxel-level. Data are shown with and without correction for family-wise error p < 0.05.In order to investigate spatial associations between CSF p-tau (independent variable) and [18F]flortaucipir ROIs on PET with cognition. First, we assessed the predictive effects of CSF p-tau and entorhinal, limbic, and neocortical [18F]flortaucipir separately (model 1), followed by a model including both predictors . Additionally, both models were adjusted for age, sex, education, and time lag between PET/CSF assessments. Finally, we repeated the aforementioned procedure, but replaced cognition with gray matter atrophy (dependent variable) additionally adjusted for TIV.Linear regression models were performed to assess the relationships between CSF p-tau and 3 a priori defined flortaucipir BPND within all brain regions were higher in MCI/AD patients compared to SCD subjects .Subject characteristics are presented in Table 18F]flortaucipir BPND in the entorhinal cortex (sβ = 0.46), limbic (sβ = 0.45), and neocortical region (sβ = 0.43), all p < 0.01. Within-group correlations were stronger for SCD subjects than for MCI/AD patients, with strongest correlations seen in the limbic region  and CSF p-tau. In SCD subjects, associations between tau PET and CSF p-tau were mainly observed in temporoparietal regions, whereas in MCI/AD this association was observed in the fronto-temporo-parietal areas . Contrary, CSF p-tau showed only associations with MMSE and memory , associations between flortaucipir ROIs were related to the different atrophy ROIs , even after adjusting for CSF p-tau . After stratification, these results appeared largely attributable to MCI/AD participants, no significant associations were seen for the SCD subjects. Comparable results for t-tau are presented in Supplementary Table Associations between CSF p-tau and tau PET and atrophy are presented in Table ND values , limbic (sβ = 0.47), and neocortical (sβ = 0.43) [18F]flortaucipir SUVr, all p < 0.01  1Fflortauc18F]flortaucipir PET and their associations with cognition and atrophy. First, we found that higher CSF p-tau was only moderately correlated to higher [18F]flortaucipir binding across the total group. Both tau markers were associated with disease severity, but we found stronger associations for [18F]flortaucipir compared to CSF p-tau in relation to cognition and atrophy, particularly for the MCI/AD group. Taken together, our findings suggest that tau PET may be a better biomarker for tracking Alzheimer’s disease severity.In this study, we examined cross-sectional associations between CSF p-tau and quantitative [18F]flortaucipir ROIs, recapitulating the neuropathological tau stages, and attention, memory, language, and executive functioning as well as with brain atrophy. Associations were most pronounced for MCI/AD dementia patients, especially with regard to limbic and neocortical tau deposition and widespread latrophy, which suggest that later neuropathological tau stages measured by [18F]flortaucipir BPND are tightly related to neurodegeneration in clinical AD. In line with the previous studies [18F]flortaucipir with atrophy [18F]flortaucipir PET). Therefore, CSF may show greater disparity than tau PET with later markers of disease severity, such as atrophy and cognition flortaucipir binding continues to increase over time [18F]flortaucipir PET to dynamically track neurodegeneration and/or (changes in) cognition [18F]flortaucipir PET values were higher for MCI/AD patients than for SCD subject with similar p-tau values  which overestimates true tracer binding as shown in this study.In addition, we examined these associations between the different tau biomarkers in SCD subjects instead of cognitively unimpaired individuals without cognitive complaints. By not using controls from a populations study, these results may be more generalizable to a memory clinic cohort.We acknowledge that there was a time lag in our study between CSF tau sampling and performing of the tau PET scan. Ideally, CSF would be collected on the day of the PET scan, and although our results were comparable if we adjusted the time lag between CSF p-tau and tau PET to a maximum of 1 year and we entered the time lag as a covariate in our main analysis, this time lag may have affected our results. Since p-tau levels may rise before tau PET , 26, 52,18F]flortaucipir PET, more than CSF p-tau, relates to important clinical parameters of disease severity of AD, i.e., cognition and neurodegeneration. As such, tau PET may more accurately reflect disease severity in AD than p-tau.Regional [ESM 1(DOCX 18 kb)ESM 2(DOCX 14 kb)ESM 3(DOCX 16 kb)ESM 4(DOCX 14 kb)ESM 5(DOCX 18 kb)ESM 6(DOCX 18 kb)ESM 7(DOCX 18 kb)ESM 8(DOCX 18 kb)ESM 918F]flortaucipir BPND. Each symbol represents one subject. The fitted lines are stratified over AD (blue) and SCD subjects (red); closed circles are Aβ positive, open circles are Aβ negative. Correlations were adjusted for age, sex, and time lag between LP and [18F]flortaucipir PET scan. (PNG 12830 kb)Scatterplots of the observed relationship between CSF p-tau with entorhinal, limbic, and neocortical [High resolution image (TIF 13503 kb)ESM 1018F]flortaucipir BPND. Voxel-wise associations are shown using a threshold puncorrected < 0.001(red) and pFWEcorrected < 0.05 (blue) at the voxel level. Contrasts were adjusted for age, sex, and time lag between LP and [18F]flortaucipir PET scan. The associations were assessed in the total sample, within SCD subjects only and within MCI/AD subjects only. (PNG 2198 kb)Voxel-wise associations between CSF t-tau and [High resolution image (TIF 20022 kb)


Malocclusion during childhood may affect both morphology and masticatory function and could greatly affect the subsequent growth and development of the jaws and face. The purpose of this study was to evaluate the efficiency of surface electromyography in describing the effects of the rapid palatal expansion (RPE) on Masseter (M) and Temporalis Anterior (T) muscles’ activity in 53 children with different types of malocclusion: bilateral posterior crossbite (BPcb), underdeveloped maxillary complex without crossbite (NOcb) and unilateral posterior crossbite on the right (UPCBr) and on the left (UPCBl). The muscular activities during chewing tasks were assessed bilaterally before and after RPE application and three months after removal. Both the envelope’s peak (µV) and its occurrence (% of chewing task) were extracted from the surface electromyography signal. Our results showed the presence of statistically significant differences (p < 0.05) on temporomandibular joint muscles, across different assessments, in all the tested populations of subjects. Surface electromyography demonstrated a relationship between the correction of a maxillary transverse discrepancy and the restoration of a muscle’s activation patterns comparable to healthy subjects for both T and M. Understanding the characteristic features of primary dentition, as well as the changes that take place in the transitional stage from primary to permanent dentition for a particular population, is essential for dentists involved in planning early preventive and interceptive orthodontic treatment ,2,3,4. PSurface electromyography (sEMG) analysis provides important information on muscular conditions, both in dynamic and static contraction. In the context of MTD, several authors have analyzed sEMG’s characteristics during different dynamic tasks both in subjects with and without malocclusion. SEMG is considered the most objective and reliable diagnostic tool for assessing changes in the electrical activity of the masticatory muscles ,29,30,31Our aim is twofold: firstly, to provide a methodology to verify the ability of RPE to promote selective activity of the temporomandibular muscles and for assessing the possible compensatory mechanisms adopted. Secondly, to highlight differences in sEMG activity as a response to RPE application across three populations of children with different malocclusions: bilateral posterior crossbite (BPcb), underdeveloped maxillary complex without crossbite (NOcb), unilateral posterior crossbite on the right (UPCBr) and on the left (UPCBl), and a control group of healthy subjects (C).53 children (26 girls and 27 boys) were enrolled in the study . They were divided into two groups: 43 patients with different types of malocclusion and 10 healthy controls (C) . The fir6-camera motion capture system 60–120 Hz An 8-channel Free EMG system 1000 Hz Two web cameras Acquisition sessions took place at the BioMov - Lab (Department of Information Engineering of the University of Padua) using the following instrumentation:All instruments were synchronized. The sEMG activity was collected bilaterally on the following muscles: Masseter ((M), right (RM) and left (LM)), Temporalis Anterior ((T) right (RT) and left (LT)), 10-mm diameter pre-gelled sEMG sensors were positioned on the muscles in accordance with Castroflorio et al. . T0. Before the start of the treatment every subject underwent the sEMG analysisStart of treatment with RPE according to individual clinical conditionT1. Stopping of the expansion (between 4 and 6 weeks after treatment start)Removal of RPE 6 months after T1.T2. Three months after the RPE removal we performed a check-up visit to evaluate changes and the sEMG analysis was repeated to identify any further change in masticatory strategy.After signing an Informed Consent form, the following treatment and acquisitions protocol took place :T0. BefoDuring sEMG acquisition at T0, T1 and T2, children were sitting on a chair and were asked to chew the same kind of biscuit. The registrations of muscle activity lasted the time necessary to swallow the biscuit. The sEMG recorded signals were bandpass filtered between 10 and 450 Hz with a 5th order Butterworth filter and wave rectified. The envelope was computed (Matlab R2013a) by low-pass filtering the signals with 4th order Butterworth filter and a cut-off frequency of 5 Hz as in (30), hence the Peak of the Envelope (PoE in µV) was normalized on the mean value of the total sEMG signal, meanwhile, the Position of the Peak of the Envelope (PPoE) was calculated as a percentage of the chewing (0–100%). Ten chewing cycles were extracted from each trial through a Matlab custom code (v. R 2013a) by considering markers trajectories and video sequences.One-way ANOVA or Kruskal Wallis tests were performed after evidencing a normal distribution (Levene’s Test) using SPSS (v. 19.0) and sEMG activity was compared across all subject groups. Paired sample T tests were performed to compare results of the different acquisition’s sessions within each group of subjects. Comparisons were made before RPE application, after RPE application and after the removal. A significance level of p < 0.05 was adopted for all statistical analysis, using a Bonferroni correction when appropriateAll patients concluded the study. No significant differences were observed in term of participants’ age, BMI and sex across groups. All results in terms of PoE and PPoE between the different groups are collected in In On the x-axis of all graphs the names of the muscles are reported: RT , RM (Right Masseter), LT  and LM (Left Masseter).The significant results of one-way ANOVA (p < 0.05) between groups are shown with different symbols as in In On the x-axis of all the graphs, the names of the muscles are reported: RT , RM (Right Masseter), LT  and LM (Left Masseter) and for each muscle, the acquisition time is reported .The significant results of paired T Test (p < 0.05) between different acquisitions  are shown with different symbols as follows: T0 vs. T1, p < 0.05 = *; T1 vs. T2, p < 0.05 = **; T0 vs. T2, p < 0.05= #.In On the x-axis of all graphs the names of the muscles are reported: RT , RM (Right Masseter), LT  and LM (Left Masseter) and for each muscle the acquisition time is reported . The significant results of Paired T-Test (p < 0.05) between different acquisitions  are shown with different symbols as follows: T0 vs. T1, p < 0.05 = *; T1 vs. T2, p < 0.05 = **; T0 vs. T2, p < 0.05 = #.T0 measurements highlighted significant differences in the muscular activities in terms of mean activation and occurrence within the chewing tasks across all the malocclusions and in comparison with the control group. These results demonstrated how the skeletal framework can influence masticatory function in children. In particular, symmetrical malocclusions (BPcb and NOcb) presented symmetrical and similar muscular function while the patients affected by unilateral crossbite presented a lower activity in terms of PoE and earlier activation in terms of PPoE on the contralateral side. Furthermore, BPcb and NOcb presented a slightly increased activity when compared to the control group. At T1 (4/6 weeks) sEMG showed the highest variability in comparison with T0 and T2. The authors expected this result since the muscular activity generally needs a longer time to adapt to the new skeletal framework. Furthermore, other parameters can influence the muscular activity at this time such as altered or earlier occlusal contacts, dental and skeletal pain due to the active maxillary expansion and the presence of the appliance bonded in the mouth. At T2  some differences in sEMG parameters emerged between the different malocclusions. In terms of PoE, all the muscles reduced their mean activity except for the RM in UPCBl patients. The authors expected a similar or lower activity at T2 in all the malocclusions due to the improvement determined by the orthodontic treatment. In terms of PPoE, NOcb had the best improvement showing a significant reduction in RM, LT, and LM and a non-significant reduction in RT. The chewing task does not significantly change in the other malocclusions. The authors expected this result for two main reasons. First of all, NOcb is the easiest malocclusion to correct and generally requires fewer RPE activations. Secondly, the correction often determines earlier contacts on first molars due to the transversal dental compensation present at T0.The posterior crossbite is one of the most frequent malocclusions in children  and doesThe results of our study highlighted two main points: first of all, sEMG is an efficient tool to evaluate and compare masticatory muscle strategies in children affected by maxillary transversal discrepancy. Furthermore, we have demonstrated a positive effect of RPE treatment on the muscular activity in all the malocclusions, particularly in BPcb and NOcb. Other improvements in these populations have been demonstrated in our previous study . Indeed,Alterations in sEMG activity were already recorded by Troelstrup et al, 1970  in the gDespite the good results concerning the use of the RPE in children with different types of occlusions, our study presents some limits. First of all, the small number of patients and their distribution in the four groups, which were not homogeneous. Secondly, the relationship between chewing tasks and maximum voluntary contraction will be the subject of future developments. There is a wide literature on the clinical applications of sEMG and its relevance to evaluate the activity of masticatory muscles ,25,27,38To the authors’ knowledge, our study analyzed for the first time the sEMG of masticatory muscles in BPcb, UPCB and NOcb patients with a control group of healthy children.Besides the observation of sEMG activity in these subjects, while performing a functional task such as chewing, our study highlighted the positive impact of the orthodontic treatment with RPE in correcting the observed sEMG alterations. For this reason, the positive effects of the RPE treatment are not only on the dentition, skeletal framework, and nasal airways but also on the functional activities of the masticatory muscles. Hence sEMG could be used to assess improvements of the masticatory muscles and for personalizing RPE dental treatment plans. In conclusion, when considering the improvement both in the timing (PPoE) and the amount of activity (PoE) registered in each group of subjects, a relationship existed between the correction of a maxillary transverse discrepancy and the restoration of a muscle’s activation patterns comparable to healthy subjects for both T and M. In agreement with Nishi et al.  sEMG rep


Tribulus terrestris L. , the high content of active ingredients , and its frequent uses in folk medicine, and as food supplements highlight the importance of evaluating its phytopharmacological properties. There are miscellaneous hypotheses that the species could have a high potential for the prevention and improvement of various human conditions such as infertility, low sexual desire, diabetes, and inflammatory diseases. Worldwide, numerous herbal supplements are commercialized with indications mostly to improve libido, sexual performance in both sexes, and athletic performance. Phytochemical studies have shown great disparities in the content of active substances . Thus, studies of experimental pharmacology  and clinical pharmacology  have sometimes led to divergent results; moreover, the presumed pharmacodynamic mechanisms have yet to be confirmed by molecular biology studies. Given the differences observed in the composition, the plant organ used to obtain the extract, the need for selective extraction methods which are targeted at the class of phytocompounds, and the standardization of T. terrestris extracts is an absolute necessity. This review aims to highlight the phytochemical, pharmacological, and toxicological properties of T. terrestris, with a focus on the contradictory results obtained by the studies conducted worldwide.The general spread of  Tribulus terrestris (TT) is a plant that grows especially in South Africa, Australia, India, and Europe. It is part of the Zygophyllaceae family, a widespread family with 25 genera and about 250 species. TT is a crawling herbal plant that generally grows in arid climates and sandy soils and grows up to one meter high. The name Tribulus comes from the Greek name “tribolos” which means spike fruit. The fruits are used in traditional Chinese medicine (TCM), in Ayurvedic medicine in India, and traditional medicine in Bulgaria for the treatment of different conditions [http://mpns.kew.org/mpns-portal/). Many compounds with a variety of biological properties and chemical structures have been identified in TT extract, especially steroidal saponins, flavonoids, tannins, terpenoids, polyphenol carboxylic acids, and alkaloids. The composition of TT extract depends on various factors such as the extraction method and whether roots, leaves, or fruits have been used. nditions . In addiFurthermore, the composition and biological activity of TT depends on growth conditions, including soil quality, but also the harvesting period . As showSeveral reviews have been published in recent years. This review presents the most important phytochemical and pharmacological data with an emphasis on the prominent information related to the chemical composition, pharmacological studies, mechanisms of action, and toxicological data. Tribulus terrestris” alone or in combination with “chemistry”, “pharmacology”, “effects”, and “toxicity”. For this review, only full-text articles written in the English language were taken into consideration. Unpublished results or grey literature were not included and only pharmacological actions that demonstrated effects both in vitro and in vivo were discussed in the present review.The information on TT was compiled via an electronic search of the following major scientific databases: Science Direct, PubMed, Web of Science, and Scopus, from 2000 to 2020. Whenever the published data were relevant for the present review, the search was extended to 1982 . The query was supplemented by searching the reference lists of papers included in the first selection. The search terms were as follows: “TT fruits contain important secondary metabolites such as saponins, polyphenolic compounds, and alkaloids. The steroidal saponins are mainly furostanol and spirostanol type . The furStudies have revealed that the composition is strictly linked with the origin of the plant, and hence with climatic conditions.Geographical regions significantly influence the composition of herbal drugs. Dinchev et al.  detectedDioscorea, the pharmacokinetic profile of protodioscin revealed good bioavailability [Dioscorea sp. extracts [Protodioscin is the dominant component in TT fruits and is considered to be the main pharmacologically active steroidal saponin . Studieslability . Despiteextracts ,54. SapoProduction of reactive oxygen species (ROS) in the body and their correlation with the incidence of chronic diseases has been largely described in the scientific literature and is already a fact. •), peroxyl (RO2•), and superoxide (O2•−) radicals [TT extracts contain flavonoids and polyphenol carboxylic acids. The antioxidant activity of these compounds has been convincingly confirmed, based on their ability to donate hydrogen. Polyphenols are capable of scavenging hydroxyl  to water and oxygen, and it has an essential role in the protection of cells from ROS. Superoxide dismutase catalyzes the transformation of superoxide anion free radical (O2−) into oxygen (O2) and H2O2 [Dutt-Roy et al.  observedand H2O2 . MDA is and H2O2 .Studies have shown that STZ-induced diabetes increased oxidative stress, and apparently, TT extracts  were capable of modulating oxidative stress markers (MDA and GSH) .On the basis of the widespread societal presumption that natural compounds are active in erectile dysfunction, but lack the side effects specific to compounds obtained by chemical synthesis , they are often preferred and used for extended periods. Various products containing TT extracts are widely utilized for this purpose, mainly due to the advertising of supplements for professional athletes, based on the alleged effect of testosterone boosting. Existing data in the literature, resulting from in vitro experiments, by analyzing animal models  and evaluating endpoints from clinical trials on subjects with erectile dysfunction are presented below.The main objective of in vitro studies was to evaluate the quality of semen (morphology and viability). In vitro incubation of human spermatozoa with TT extract  had a beneficial effect on motility and viability. These findings suggest that TT extracts could be further used in the preparation of spermatozoa before in vitro fertilization . An orgaPreclinical studies have focused on animal models of human diseases that affect spermatogenesis and androgen secretion ; the effect of TT extracts on spermatogenesis and gonadal steroidogenesis in healthy male subjects, whether or not subjected to standardized physical exertion has also been evaluated.In adult male Swiss albino mice with reproductive damage induced by cyclophosphamide, TT showed an improvement of epididymal sperm characteristics (motility) and an increase in testosterone levels as compared with the control group . ExtractThe analysis of available clinical trials on the effectiveness of TT extracts in men highlights two categories of primary endpoints as follows: Some studies set as their main goal the evaluation of efficacy in erectile dysfunction  and others evaluated the change in the basal secretion of testosterone at the end of the study with the initial values of the subjects serving as the control. However, the available studies did not shed light on the controversy regarding the real efficacy of TT. On the one hand, due to the divergent results ; on the other hand, due to the subjective evaluation , Questionnaire and Global Efficacy Question (GEQ).®, Sopharma AD-coated tablets containing 250 mg of dry extract equivalent to furostanol saponins not less than 112.5 mg) for the treatment of men with mild to moderate erectile dysfunction and with or without hypoactive sexual desire disorder in a prospective, phase IV, randomized, double-blind, placebo controlled clinical trial in parallel groups. The characteristics of the study can be summarized as follows: dose of three coated tablets per day; sample size of 90 subjects in each group (treated vs. placebo); duration of 12 weeks; primary endpoint, the change in IIEF score at the end of the treatment. The authors showed a significant improvement in erection, libido, and orgasmic function in the treated group, in the absence of any difference in the profile of side effects as compared with the placebo [Recently, Kamenov et al.  evaluate placebo .Santos et al.  conducteTribulus”, anaerobic muscle power and serum testosterone increased significantly in young men [Tradamixin”, consisted of TT, Alga Eckonia, D-glucosamine, and N-acetyl-glucosamine, was given daily for two months, and improved libido in elderly men and increased testosterone. It should be noted, however, that in both experiments, there was no certainty that a particular component would have caused those biological benefits or if TT contributed to those effects [In contrast to these data, two studies confirmed the beneficial effects after treatment with pharmaceutical products containing TT and other components. The first study showed that after 20 days of supplementation with the dietary supplement “oung men . The oth effects .In a study conducted on male boxers, the administration of a TT supplement (with >40% saponins) produced no effect on plasma testosterone and dihydrotestosterone. Although the results were inconclusive, the authors suggested that a possible mechanism of action for TT compounds could be related to insulin-like growth factor (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3). IGF-1 is a growth hormone that showed the capacity of elevating skeletal muscles and preventing age-related loss of muscle mass . Additionally, IGF improves insulin signaling, which could explain the beneficial effects obtained with TT extracts in diabetes, but the exact mechanism of action is not fully known .The booster effects of TT extracts have been confirmed by some authors, both in experimental research and in clinical studies, as shown above, but are questioned by others. The available data on the mechanisms underlying the use in sexual disorders can be summarized as follows : steroidGiven the testosterone boosting action of the extract, research has been performed to evaluate if the consumption of TT extracts could influence the doping tests of athletes regarding the urinary testosterone/epitestosterone TS/ET ratio limit of 4:1 (World Anti-Doping Agency) .The in vitro and in vivo studies are briefly presented in Staphylococcus aureus, Streptoccocus mutans, Streptococcus sanguinis, Actinomyces viscosus, Enteroccocus faecalis, and Bacillus subtilis were susceptible and among the Gram-negative bacteria Escherichia coli, Salmonella typhi, Proteus mirabilis, and Klebsiella pneumoniae were susceptible [E. coli, Salmonella, Staphylococcus aureus, and Streptococcus [There are several in vitro studies that have revealed the antibacterial potency of TT total or fractionated extracts on Gram-negative and Gram-positive bacterial strains. Among the Gram-positive bacteria, facultative anaerobe strains such as ceptible ,90,91,92ceptible . The antceptible ,94. Kiantococcus ,40.Chromobacterium violaceum, Serratia marcescens, and Pseudomonas aeruginosa strains. The main compound was found to be ß-1, 5-O-dibenzoyl ribofuranose [A recently published paper demonstrated the quorum quenching activity of TT (origin India) root extracts on furanose .Studies conducted with extracts from TT have been shown to inhibit the activity of alpha-glucosidase and alpha-amylase in vitro. Alpha-glucosidase and alpha-amylase are enzymes involved in the hydrolysis of carbohydrates. Alpha-amylase breaks down the oligosaccharides into disaccharides and alpha-glucosidase breaks down the disaccharides into absorbable monosaccharides. Inhibition of the activity of these enzymes has been proven to reduce postprandial hyperglycemia in diabetic patients. The TT extracts exhibited a relatively higher inhibition capacity on alpha-amylase than on that of alpha-glucosidase . The actIn vivo animal studies are in concordance with the in vitro studies, as it was shown that the saponins from TT administered to rats were able to delay the postprandial hyperglycemia by inhibiting alpha-glucosidase . StudiesAlkaloids could act synergistically with the steroidal saponins, as it was shown that imidazolidine derivatives stimulate insulin secretion by activation of imidazoline receptor type 3 binding sites in the pancreatic beta cells .Samani et al.  conducteRecent research suggests that there is a correlation between testosterone levels and type 2 diabetes and that low testosterone levels in men predict a high risk of type 2 diabetes . The dirSeveral studies have demonstrated that extracts of TT have anti-inflammatory activities. The primary mechanisms involved are thought to be downregulation of inflammatory pathway protein NFκB . The extAnimal experiments have confirmed the anti-inflammatory effects demonstrated in vitro. Mohammed et al.  showed tThe β-Carboline indole alkaloids are known to be monoamine oxidase inhibitors (MAOIs), primarily MAO-A, as they prevent biogenic amine from binding to the active site of the MAO molecule and undergoing deamination. Consequently, their presence in TT is thought to have been responsible for the unusual locomotory disturbance in sheep that grazed in areas with TT ,113. If In several studies, the neuroprotective effect has been demonstrated and several mechanisms of action have been proposed. Chaudary et al.  demonstrPart of the previously mentioned effects of TT extracts is mediated through the central nervous system, and therefore are not included in the present section. These include the modulation of pituitary gonadotropin secretion. The toxic effects observed in sheep also involve the modulation of the GABAergic and dopaminergic system and are further presented.Evaluation of toxicological effects in vitro has demonstrated that TT extracts  have estrogenic and genotoxic effects .Medicago sativa). The symptoms included depression, jaundice, weight loss, conjunctivitis, and also the reddening of the muzzle, nose, ears, and eyelids [Hepatogenous photosensitivity appeared after 11 days in sheep fed with a mixture of TT and alfalfa ( eyelids . The stu eyelids , on diab eyelids . Bourke  eyelids  reported eyelids .Acute and subacute toxicity tests were performed by Hemalatha and Hari  with butTalasaz et al.  reportedThe toxicity of TT extracts has not been fully evaluated, and the toxic compounds have not been properly identified. With respect to the reported cases of toxicity, no clinical trial in which TT-based products were administered, have reported these side effects. Particular attention should be given to the herbal supplements, and an elaborate analysis should be performed in order to identify the toxic compounds. There is a constant risk of adulteration of food supplements, primarily when these are used for their anabolic effects. There is also the possibility of trace metal accumulation in herbal drugs. A single research article was found that analyzed the content of some essential and trace elements in TT organs . AlthougConsidering all of the above information, a complete analysis of the supplements should be performed when a toxicity case is reported.Tribulus extracts. Although TT has been extensively researched, further studies are needed in order to clarify important aspects such as a more accurate correlation between the phytochemical and pharmacological profiles, pharmacokinetic studies of the most important compounds, as well as the evaluation of possible pharmacokinetic and pharmacodynamic interactions with other compounds. Researchers should provide full information on the plant origin and the tested organ. Methods for standardization are necessary in order to achieve reproducible results. To date, it seems that the chemical compounds found in TT are capable of activating multiple pathways, hence, the various effects. Different phytochemical profiles of the herbal drugs from TT, highlighted both in the concentration of the main active compounds and in the absence of some active compounds, explain the major differences in the therapeutic effects reported over the years in the literature. The main pharmacological research on TT has been focused on sexual disorders, but other important effects have been demonstrated in vitro and in vivo studies, i.e., anti-hyperglycaemic, anti-inflammatory, antioxidant, and antibacterial. Toxicological studies, although limited, have highlighted the risk of nephrotoxicity following the administration of TT supplements. However, additional studies are needed to determine some of the still unknown molecular mechanisms of action of the therapeutic active compounds found in


Retinol binding protein 4 (RBP4) is a member of the lipocalin family and a vitamin A carrier in the blood. More recently, RBP4 has been described as an adipokine that is involved in insulin resistance and metabolic syndrome (MetS). As obesity, MetS and some adipokines contribute to the pathogenesis of osteoarthritis (OA), we investigated RBP4 in patients with OA.Cartilage, synovial fluid and blood samples were collected from 100 OA patients undergoing total knee replacement surgery. Primary chondrocytes and cartilage tissue were cultured to measure the RBP4 expression. The concentrations of RBP4, other adipokines  and biomarkers of OA  were measured by immunoassay, and gene expression was measured by next-generation RNA sequencing.The OA cartilage samples released RBP4 into the culture medium, and the levels correlated positively with the expression of the adipokines adipsin, adiponectin, leptin and resistin. RBP4 was the most prominently expressed of these adipokines in the OA chondrocytes, and the expression of the RBP4 receptors STRA6 (stimulated by retinoic acid gene homologue 6) and TLR4 (Toll-like receptor 4) was also detected. Within the cartilage culture medium, RBP4 showed a positive correlation with MMP-1, MMP-3 and YKL-40. RBP4 was also present in the synovial fluid from the OA patients and correlated positively with the concentrations of RBP4 found in the plasma and the cartilage culture medium. Plasma RBP4 concentrations also showed a positive correlation with MMP-3 and adipsin.We show here, for the first time, that RBP4 is produced within OA joints and that it is associated with increased levels of adipokines and MMPs. The results suggest a role for RBP4 in the pathogenesis of OA and as a possible target for the disease-modifying drugs for the treatment of OA. Osteoarthritis (OA) is the most prevalent joint disease and a leading cause of disability that affects an estimated 10% of the world’s population over the age of 60 years . It is aAdipose tissue produces cytokine-like hormones known as ‘adipokines’. Adipokines not only regulate energy metabolism and appetite but also other functions in the human body. Several studies have reported an important role for adipokines in cartilage and bone homeostasis, metabolism and inflammation and suggest that these molecules serve as a link between obesity and OA . RetinolRBP4 acts by binding to the receptor ‘stimulated by retinoic acid gene homologue 6′ (STRA6) , 18. In 2; age 72 (14) years, median ; 62/38 females/males] undergoing total knee replacement surgery at Coxa Hospital for Joint Replacement, Tampere, Finland, participated in the study. All patients fulfilled the American College of Rheumatology classification criteria for OA  undergoing knee replacement surgery at Coxa Hospital for Joint Replacement, Tampere, Finland. The cells were isolated as described [Analysis of mRNA expression was performed using chondrocytes isolated from the knee cartilages from ten additional OA patients whose samples were not used in the experiments described above [escribed  and cultn = 10).The sequencing depth was 20 million paired-end reads of 100 bp. The read quality was first assessed using FastQC , and ther)  and t tests . A p value less than 0.05 was considered significant.The data are reported as the mean ± SEM, unless stated otherwise. The statistical analysis was performed using Spearman's rank correlation coefficient  were higher than those in synovial fluid , resistin , leptin  and adipsin . Interestingly, RBP4 also showed a positive correlation with the levels of MMP-1 , MMP-3  and YKL-40  released into the culture medium but did not correlate with BMI.The cartilage samples from the OA patients released RBP4 protein into the culture medium (11.9 ± 0.5 ng/10 mg cartilage), and these levels correlated positively with the other adipokines measured: adiponectin  and MMP-3 .For the synovial fluid, no correlations between RBP4 and adipokines, MMP-1, MMP-3 or YKL-40 were found. The plasma RBP4 concentrations positively correlated with adipsin . As shown in Table RBP4 is a retinol transport protein in blood that is prevalently expressed in the liver but is also highly present in adipose tissue , 7. BecaTo date, there have been no studies on the RBP4 production in joint tissues. Here, we showed that cultured cartilage released RBP4. Furthermore, these RBP4 levels correlated positively with the other adipokines implicated in OA pathogenesis, i.e., adiponectin , 32–34, RBP4 acts as an immunomodulatory adipocytokine. Kahn et al. reported that RBP4 induces the secretion of proinflammatory cytokines in mouse dendritic cells and macrophages, causing Th1 polarization and proliferation in vivo . RBP4 alIn the present study, we show that OA synovial fluid contains RBP4 and that there is a significant positive correlation between the RBP4 levels in the OA synovial fluid and the cartilage culture medium from the same patients. Regarding the synovial fluid, we did not find any correlations between the RBP4 levels and the OA biomarkers. This finding is likely due to the contribution by other joint tissues than cartilage to the content of the synovial fluid, but this possibility was not explored in the present study. Instead, the findings point to cartilage as a significant target tissue that bears the destructive effects of RBP4. OA chondrocytes express STRA6 and TLR4, receptors known to be activated by RBP4 –21. The We also measured the levels of RBP4 in the plasma from the OA patients. These levels correlated positively with adipsin, an adipokine recently demonstrated as a factor that contributes to cartilage degradation in osteoarthritis , 42. MorSeveral studies reported an increased concentration of RBP4 in obesity , 55–57 aIn conclusion, our data demonstrated, for the first time, that RBP4 is a prominently expressed adipokine in OA chondrocytes and is present in synovial fluid and plasma from OA patients at considerable μg/ml levels. RBP4 was also found to be associated with increased levels of adipokines and matrix metalloproteinases MMP-1 and MMP-3.The results suggest a role for RBP4 in the pathogenesis of OA and as a potential target for disease-modifying drugs for the treatment of OA, which encourages additional studies to reveal the more detailed role of RBP4 in the pathogenesis and pathology of OA. Further studies in large cohorts of patients and in cells and joint tissues are needed to confirm the implications of RBP4 in OA.


Xenopus with optical coherence tomography (OCT) imaging to investigate in vivo ventricular and neural development during the onset of CSF circulation.Hydrocephalus, the pathological expansion of the cerebrospinal fluid (CSF)-filled cerebral ventricles, is a common, deadly disease. In the adult, cardiac and respiratory forces are the main drivers of CSF flow within the brain ventricular system to remove waste and deliver nutrients. In contrast, the mechanics and functions of CSF circulation in the embryonic brain are poorly understood. This is primarily due to the lack of model systems and imaging technology to study these early time points. Here, we studied embryos of the vertebrate Xenopus tadpole brains and to dynamically detect in vivo ventricular morphology and CSF circulation in real-time, at micrometer resolution. The effects of immobilizing cilia and cardiac ablation were investigated.Optical coherence tomography (OCT), a cross-sectional imaging modality, was used to study developing Xenopus, using OCT imaging, we demonstrated that ventriculogenesis can be tracked throughout development until the beginning of metamorphosis. We found that during Xenopus embryogenesis, initially, CSF fills the primitive ventricular space and remains static, followed by the initiation of the cilia driven CSF circulation where ependymal cilia create a polarized CSF flow. No pulsatile flow was detected throughout these tailbud and early tadpole stages. As development progressed, despite the emergence of the choroid plexus in Xenopus, cardiac forces did not contribute to the CSF circulation, and ciliary flow remained the driver of the intercompartmental bidirectional flow as well as the near-wall flow. We finally showed that cilia driven flow is crucial for proper rostral development and regulated the spatial neural cell organization.In Xenopus embryonic ventriculogenesis and rostral brain development are critically dependent on ependymal cilia-driven CSF flow currents that are generated independently of cardiac pulsatile forces. Our work suggests that the Xenopus ventricular system forms a complex cilia-driven CSF flow network which regulates neural cell organization. This work will redirect efforts to understand the molecular regulators of embryonic CSF flow by focusing attention on motile cilia rather than other forces relevant only to the adult.Our data support a paradigm in which  Cerebrospinal fluid (CSF) provides hydromechanical protection to the central nervous system (CNS) and delivers nutrients and active metabolites, while also eliminating catabolites. Hydrocephalus develops when cerebral ventricles pathologically expand due to CSF accumulation within the ventricles, leading to a group of diseases with high mortality and morbidity , 2. In aThere are two significant drivers of the CSF circulation, the heart, and the ependymal cilia. The heart is known to drive the bulk CSF circulation. Flow, in this case, is generated by the expulsion of the CSF via dilated intracranial arteries during cardiac systole. Based on the magnetic resonance imaging and radionuclide cisternography studies in adult human brains, this bulk flow is driven mainly by the choroid plexus pulsations, and additionally by the ventricular wall motion . When coXenopus  staging [We serially imaged  staging .CSF flow velocity analysis and particle tracking:Particle tracking was performed using TrackMate particle tracker plugin built in ImageJ. The plugin allows two-dimensional tracking and analysis of particle trajectories. The region of interest (ROI), the ventricle, was segmented manually. We obtained a velocity color map and a vector map that shows all five flow fields. We also obtained median velocities (described below in detail) from each flow field , and we compared experimental velocities with the corresponding ventricle velocities in controls.Gaussian process regression:We used a Gaussian process regression (GPR)—based post-processing ensemble method, reported in , to accuStage 1 uses the LAP simple tracking algorithm provided by TrackMate –47 to obXenopus) to compute statistics for the subregion. To calculate the median speed in a subregion, for example, the software collects all of the vectors in the subregion and computes the median norm of the collected vectors, thus giving the median speed of movement in the subregion. The code for our GUI, including the entire three-stage GPR estimation pipeline, is available at https://github.com/tommymtang/PTVProcessorA custom-made GUI, written in Matlab, visualizes the calculated dense fluid velocities, their standard deviations, and other statistics. The estimated velocity vector at each pixel is viewed as a quiver-map overlaid on top of an OCT image. The GUI also displays the speed (magnitude of the estimated velocity) and the standard deviation of the estimate as color-maps. Finally, the GUI allows selections of subregions -Glucose) from the original suspensions to a final concentration of 0.25% solids (0.81 µm beads) or 0.5% solids (4.61 µm beads). After embedding stage 30, and 49 tadpoles in 1.5% low melt agarose, they were injected with the bead suspension into the ventricular space at the hindbrain (stage 30) or into the third ventricle  using the microinjection apparatus. Injection volumes were 4 nL for stage 30 tadpoles and 25 nL for stage 47, 48, and 49 tadpoles.0.81 µm or 4.61 µm diameter polystyrene microsphere suspensions (Bangs Laboratories) were diluted in artificial CSF  chloride hexahydrate (50 mM) (Sigma Aldrich) and sodium orthovanadate (10 mM) (Sigma Aldrich). After the injection of beads into the third ventricle of agarose embedded stage 49 tadpoles and recording pre-treatment OCT movies, we injected 25 nL of one of the above solutions into the same location as the beads injection, then recorded OCT movies at 5 min time intervals.Xenopus tropicalis, stage 46 tadpoles were fixed in 4% paraformaldehyde in PBS for 2 h at room temperature. The tadpoles were washed 3 × 15 min with PBS, and then the brains were dissected out. The brains were incubated for 1 h at room temperature in blocking buffer , then incubated overnight at 4 °C in primary antibody against glutamylated tubulin  diluted 1:1000 in blocking buffer [For g buffer . SamplesXenopus laevis, stage 46 tadpoles were stained whole-mount according to Willsey et al. 2018 [For al. 2018  using th4, 3.7% (v/v) formaldehyde) for 2 h at room temperature. The tadpoles were then washed multiple times with 100% ethanol, then stored at − 20 °C. c21orf59 morphants were imaged using OCT at stage 46 and tadpoles with aqueductal stenosis were recorded and selected to be fixed as above with MEMFA.Wildtype tadpoles were imaged using OCT at stage 46 to confirm typical ventricle size, shape, and CSF flow, then fixed with MEMFA (100 mM MOPS (pH 7.4), 2 mM EGTA, 1 mM MgSOIn situ hybridization was performed according to the standard protocols . Here wec21orf59 morphants we utilized Mann–Whitney test  and used violin plot graph to show the distribution of the data. Significance was determined when the p value is lower than 0.01.Data was analyzed using Prism7 statistical software. For comparison between pre and post cardiac ablation we utilized the Wilcoxon matched-pairs  and used before-after graph including standard error of the mean. Significance was determined when the p value is lower than 0.01. For comparison between controls and Xenopus Optical Coherence Tomography platform, in a non-destructive fashion, we can detect global CSF flow mapped to CNS structures in the Xenopus tadpole [Xenopus embryonic CSF circulation, we again capitalized on the power of OCT imaging. Despite the optically dense yolk, unexpectedly, OCT imaging allowed us to serially examine each developmental stage of the Xenopus embryo, starting from stage 19  until stage 49 (tadpole) (Nieuwkoop—Faber staging. ~ post-fertilization day 4 at 25 °C) . Using Xenopus embryonic ventricular development from as early as post neurulation. The ventricular space enlarges in a rostrocaudal fashion throughout the early tailbud stages (Stage 22–28). Then, five flow fields emerge, circulating and mixing CSF between compartments throughout the late tailbud stages, where we show a caudal to ostral decreasing CSF flow rate (Stage 30–44). We further expanded the interrogation of the earlier (before stage 32) and later stages (after stage 46) by introducing intraventricular polystyrene beads into the ventricular space  with intraventricular bead injection, we noted an intricate CSF circulatory pattern  chloride hexahydrate or sodium orthovanadate , 57 injeXenopus embryonic CSF circulation, we first examined the distribution of the cilia along the ventricular surface and the impact of cilia-driven flow on CNS development. Previous reports have described that frog ventricles are densely populated with ciliated cells [Xenopus tropicalis and laevis species, where 4th and 5th flow fields generate the fastest CSF circulation, we observed translationally polarized multiciliated ependymal cells, where cilia are confined to the center of the apical surface  to knockdown c21orf59 (cilia and flagella associated protein 298). c21of59 is required for ciliary dynein arm assembly, which is known to regulate motile cilia function [c21orf59 is lost, motile cilia morphologically form correctly but are unable to beat, remaining paralyzed. We had previously shown that when we knocked down c21orf59  and 3rd ventricle (500 µm2) were much smaller than controls  whereas the midbrain ventricle area (1410 µm2), anterior 4th (1060 µm2) and posterior 4th ventricles (3020 µm2) were much closer to control  is known to regulate dorsal telencephalon development [emx1 in humans, mice, and Xenopus is spatially restricted to the cerebral cortex [c21orf59 with morpholino and confirmed the paralysis of motile cilia by the loss of CSF circulation by OCT imaging, we observed that emx1 dorsal telencephalic expression was mis-patterned in these tadpoles. Expression of emx1 was not lost or diminished but instead, was no longer limited to the dorsal telencephalon. In morphants, we were able to see ectopically-expressed emx1 within the diencephalon, extending towards the mesencephalon  and the midbrain-hindbrain (isthmic organizer) transition zones harbor regions that act as secondary organizers that regulate patterning following neural tube formation [zli is an evolutionarily conserved structure that demarcates the boundary between pre-thalamus and thalamus  and is referred as the mid-diencephalic organizer [zli, we investigated LIM homeobox 1 (lhx1), a transcription factor known to regulate differentiation and morphogenetic tissue movement in head development [Xenopus, lhx1 depletion leads to loss of anterior head structures [zli. As shown in Fig. lhx1 marks the thalamic region in Xenopus stage 46 tadpole as well as the midbrain region. This pattern was explicitly disrupted at the telencephalic region and was relatively conserved elsewhere when the cilia were paralyzed, and the CSF circulation ceased in c21orf59 morphants  without a heart [Xenopus developing central nervous and ventricular systems during the genesis and maturation of the embryonic CSF circulation. This examination has uncovered multiple novel observations in the Xenopus embryonic nervous system  We analyze the CSF movement using particle tracking, which relies on the endogenous particles that are readily present, suspended within the ventricular space. Therefore, the unmanipulated, endogenous CSF movement`s detection is limited by the OCT resolution and the availability of these particles. The earliest time point where we were able to detect CSF movement by OCT imaging was at stage 32—tailbud stage. (2) As the density of the intraventricular particles diminishes over time, particle tracking improves. Currently, available particle detection algorithms are more sensitive and able to detect and track more efficiently when particle density is low enough to discriminate a particle from the background. We can quickly qualitatively assess CSF movement at the earliest stages of CSF circulation by following these particles. However, we cannot precisely outline polarity or reliably quantify particle speeds at the very early stages. As OCT imaging technology advances, we anticipate an improvement in particle tracking when the density is high.Xenopus. When we quantified the median CSF speed, interestingly, we observed that the speed also is markedly different along the caudal-rostral axis. The presence of such a difference in the magnitude of ~ tenfold between the forebrain and hindbrain hints at potential compartmental differences in the CSF circulation's regulatory effects. When we couple the different ependymal cilia populations with our CSF polarity map, we see that the discrete motile mono and multicilia polarize circulation even within the same compartment. As we know, near-wall CSF dynamics do regulate neuroprogenitor migration, proliferation, and axon pathfinding [We showed that during the rostrocaudal ventricular expansion, CSF circulation is compartmentalized in a retrograde caudal-rostral direction. Circulation initiates caudally and eventually forms five mature flow fields, and this can be easily tracked in hfinding , yet thetnnt2a-/- mutants (that lack a heartbeat), maximal CSF flow (Vmax) was lower from the diencephalic and mesencephalic ventricles to the rhombencephalic ventricle. The authors concluded that the heart indeed contributed to the embryonic CSF movement. The following work by Olstad et al. demonstrated that the zebrafish embryonic CSF circulation had two major components: a highly pulsatile flow located in the aqueducts and a directional flow along the ventricular wall [New animal models, especially zebrafish, paired with in vivo imaging, revealed much about the embryonic CSF circulation. Fame et al. showed that CSF's directional movement in the zebrafish embryo, which preferentially moved from the diencephalic to the rhombencephalic ventricle, reversed between the early and late larval stages, and one of the key results was that CSF flow was partially dependent on the heartbeat . In tnntlar wall . The pullar wall , 22.Xenopus, our findings, although agreed with the cilia's importance as the main driver of the embryonic CSF circulation, were fundamentally different. We observed no cardiac forces' contributions, no pulsatile flow, in contrast to the larval zebrafish model during early embryogenesis. Instead, intra and interventricular mixing across the entire ventricular system was solely driven by Xenopus's ciliary network. Until the active feeding stages (Stage 45), Xenopus remains mostly dormant; therefore, the contribution of bodily movement is minimal during the polarization of CSF circulation. On average, over 5 min, stage-37/38 animals swim at 0.4%, stage-43 3.11% and stage-45 36% of the time [Xenopus tadpoles swim with a relatively constant frequency, and the amplitude of the head oscillations is rather variable and decreases as tadpoles grow [Xenopus' head and liberalized bodily movements , where Xenopus . When weInterestingly, despite the changes in the prosencephalon, the impact of the absence of CSF flow on the midbrain and hindbrain was minimal. However, CSF flow speed is highest in these regions, suggesting that CSF circulation regionally may be interpreted differently. Together, these observations are in line with the long-standing notion that a connection exists between CSF circulation and neurogenesis. However, here, we establish another link and suggest that compartmental flow driven by the cilia contributes to “molecular boundaries.” The frog, as well as other semi-transparent animal model systems , in which CSF fluid dynamics can be easily coupled with molecular profiling using OCT imaging, will enable future studies to understand this complex interaction.Altogether, our study demonstrates that proper embryonic CSF circulation and subsequently, CNS development goes through stages of development where the ciliary flow is the sole driver before the emergence of pulsatile forces. At this critical phase, CSF flow is compartmentalized and sets molecular boundaries to regulate proper prosencephalic regionalization, and when lost, can lead to aqueductal stenosis, a hallmark of human hydrocephalus. Further studies are needed to investigate the links between cilia, CSF circulation, and human disease states in which these processes may be compromised, such as infantile hydrocephalus. A better understanding of embryonic CSF homeostasis could uncover targets that could be modulated to ameliorate hydrocephalus and other conditions.Additional file 1: Figure S1. Xenopus tropicalis ventricular development map by OCT imaging. (A)Xenopus tropicalis tadpole ventricular system developmental map shown between the stages 18 to 46. Stereomicroscopy image presented with the corresponding mid-sagittal OCT imaging. (B) OCT images of the stage 49 tadpole from coronal and midsagittal plane. Anterior choroid plexus outlined with red circles and magnified view in red square. Posterior choroid plexus outlined with green circles and magnified view in green square. a: anterior; p: posterior; d: dorsal; v: ventral.Additional file 2: Movie S1. Video showing OCT movies of the ventricular space in stage 18, 32, 39, 46 Xenopus tropicalis tadpoles. The initial CSF movement is at stage 32. Polarization and bulk movement of the intraventricular particles and CSF is visible at stage 39. Five mature and distinctly polarized flow fields are visible at stage 46.Additional file 3: Movie S2. Video shows a stage 30 tadpole mid-sagittal plane before and after the injection of the polystyrene microbeads. Beads remain static, no CSF movement visible (30fps).Additional file 4: Movie S3. Video shows a stage 49 tadpole mid-sagittal plane before and after the injection of the polystyrene microbeads. Within the compartmental and intercompartmental space, the CSF movement is polarized. Intercompartmental CSF movement is bidirectional, dorsal flow directed to the rostral region, whereas ventral flow is directed caudally (30fps).Additional file 5: Movie S4. Video is focused at the lateral and 3rd ventricle of the stage 49 tadpole. Intercompartmental CSF movement is bidirectional. Dorsal—rostral CSF flow from the third ventricle is opposite to the dorsal- caudal flow generated from the lateral ventricle. Similarly, ventral flow is also polarized in the opposite direction, 3rd ventricle flow is now caudal, whereas the lateral ventricle flow is directed rostrally (30fps).Additional file 6: Movie S5. Video shows a stage 49 tadpole mid-sagittal plane focused at the 3rd, midbrain, and fourth ventricles. CSF movement is bidirectional and polarized along the rostral-caudal axis (30fps).Additional file 7: Movie S6. Video shows a stage 49 tadpole mid-sagittal plane focused on 4th ventricle choroid plexus before and after NiCl2 injection (30fps).Additional file 8: Movie S7. Video shows a stage 49 tadpole mid-sagittal plane focused on 4th ventricle choroid plexus after microbead injection followed by before and after NiCl2 injections.Additional file 9: Figure S3. Ventricular Volume Measurements at Stage 46 tadpole: Control, Heart Ablation, c21orf49 morphant. Data was analyzed using Prism8 statistical software. Significance was determined when the p value is lower than 0.01. For comparison between controls, heart ablated tadpoles and c21orf59 morphants we utilized Mann–Whitney test  and used scatter plot graph where we show the mean with SEM. Significance was determined when the p value is lower than 0.01. .Additional file 10: Movie S8. Video shows a stage 46 tadpole ventricular system, CSF circulation, and the cardinal vein with the temporal color map at pre (left) and post (right) cardiac ablation states. Systemic cardiovascular circulation abolishes post cardiac ablation, whereas CSF circulation, ventricular morphology, and size remain unchanged.Additional file 11: Movie S9. Video shows a stage 49 tadpole mid-sagittal plane focused on the hypothalamic region following NiCl2 injection. Pulsatile flow pointed with the white arrow.Additional file 12: Movie S10. Video shows a stage 49 tadpole before and after Na2VO4 injection. The last movie is following multiple washes where ciliary movement is partially restored. The right column shows the corresponding temporal heat maps of 100frames (30fps).Additional file 13: Movie S11. Video shows a stage 49 tadpole before and after nickel chloride hexahydrate injection. The last movie is following multiple washes where ciliary movement is partially restored (30fps).Additional file 14: Figure S2. Stage 46—Xenopus laevis ventricular cilia distribution in whole brain. Fluorescence images of (A) the most lateral and (A’) mid-sagittal view of the whole brain. Cilia (green) marked with anti-GT335 antibody. (B) Fluorescence image of the dorsal view the whole brain shows clusters of MCCs along the dorsal lateral, third and fourth ventricles. (B1) Anterior choroid plexus, (B2) pineal gland region, (B3) posterior choroid plexus, magnified views of the representative regions. R: right, L: left, a: anterior; p: posterior; d: dorsal; v: ventral.Additional file 15: Figure S4.emx1, lhx1 and en2 expression in controls, c21orf59 morphants and heartless tadpoles. Quantification of emx1, lhx1, en2 mRNA expression in stage 46 control vs. c21orf59 morphant tadpoles (A-C) and control vs. heart ablated tadpoles (D-F).emx1 expression was categorized as dorsal telencephalon only or dorsal telencephalon + diencephalon extension. lhx1 expression was categorized as telencephalon positive or negative. en2 expression was categorized as mid-brain hindbrain border positive or negative. UIC: un-injected control, HA: heart ablated.Additional file 16: Movie S12. Left video shows freely swimming stage46 tadpole, where the distal tail mostly drives bodily movements. Similarly, the middle video shows the partially immobilized stage 46 tadpole, where the tail remains free. The OCT video is obtained from the partially immobilized tadpole during free tail movements (30fps).


As a commonly used anesthetic agent, midazolam has the properties of water-soluble, rapid onset, and short duration of action. With the rapid development in the field of neuroimaging, numerous studies have investigated how midazolam acts on the human brain to induce the alteration of consciousness. However, the neural bases of midazolam-induced sedation or anesthesia remain beginning to be understood in detail. In this review, we summarize findings from neuroimaging studies that have used midazolam to study altered consciousness at different levels and content. We also compare the results to those of neuroimaging studies using diverse anesthetic agents and describe the common neural correlates of anesthetic-induced alteration of consciousness. Midazolam is a widely used intravenous anesthetic agent with rapid onset, short duration of action and relatively rapid plasma clearance. It is mainly used to produce preoperative sedation and the induction of general anesthesia , 2. EverIn the past decades, with the development of the field of neuroimaging, significant progress has been made in understanding functional changes in human brain associated with consciousness. Functional imaging methods are comprehensive, real-time, non-invasive techniques and show in vivo metabolic and functional changes. These methods, especially positron emission tomography (PET) and functional magnetic resonance imaging (fMRI), have been extensively used for exploring neural correlates of anesthetic-induced altered consciousness , 5. As aThis review first summarizes the pharmacology, pharmacodynamics of midazolam and altered cognitive function during midazolam-induced sedation. Then, we review findings from neuroimaging studies that have used midazolam to study changes in brain activity at reduced levels and content of consciousness, both with stimulation task and at resting state. In the following, we compare these results to those of neuroimaging studies using diverse anesthetic agents and describe the common neural correlates of anesthetic-induced alteration of consciousness.Midazolam is a 1,4-imidazole benzodiazepine and the first water-soluble benzodiazepine . MidazolPharmacodynamics of midazolam is similar to those of other benzodiazepine drugs, including sedation, anxiolytic, anterograde amnesia, muscle relaxant, anticonvulsant effect, as well as hypnotic activities , 8–11. AAbsorption of midazolam is rapid regardless of the administration route. After oral doses of midazolam, peak plasma concentrations generally occur between 0.5 and 1 h , 16. In Since midazolam is unique among benzodiazepines with rapid onset of action, good effectiveness and few adverse effects, it is one of the most commonly used sedative medications for a variety of therapeutic and diagnostic procedures . MidazolAnesthesia aims to enhance patient comfort, improve operating conditions , and prevent recall of noxious physical and emotional experiences during the medical procedures. Midazolam has a great advantage in eliminating adverse experiences because of its marked effects on cognitive functions. As mentioned before, midazolam acts on GABA-A receptor to reduce the excitability of neurons, resulting in impairment of multi-domain cognitive functions , 12.Midazolam, in common with other benzodiazepines, has strong anterograde amnesic property . The antFor types of memory, one of the most popular concepts of memory suggests that there are three basic memory types: sensory memory, short-term memory, and long-term memory. Sensory memory refers to the initial process of storing information that is a very brief recall of a sensory experience, such as what we just saw or heard. Short-term memory, also refers to as primary memory, is that brief period of time where you can recall information you were just exposed to. Long-term memory, also calls secondary memory, encompasses memories that range from a few days to decades. In order to learn something successfully, information has to move from the sensory or the short-term memory to the long-term memory . There aAlong with midazolam-induced anterograde amnesia, a previous study also suggested that subjects with intravenous midazolam showed a marked enhancement in implicit transitive inference (TI) performance . TransitIn the past decades, with the development of the field of neuroimaging, significant progress has been made in understanding of the brain mechanisms associated with midazolam-induced sedation. Arterial spin labeling (ASL) methodology is a MRI technique used to assess cerebral blood flow (CBF) noninvasively by magnetically labeling inflowing blood . In contOn the other hand, positron emission tomography (PET), which enables three-dimensional metabolic and flow studies in the human brain, can also detect changes in the regional cerebral blood flow (rCBF) and has been used to investigate the regions subserving conscious brain activity . A previThere are several different ways that we could apply to identify the neural substrate of midazolam-induced sedation. One powerful approach is to examine brain regions whose activity changes when subjects who ingested midazolam are asked to perform different types of tasks . Task-related fMRI will help us identify and characterize functionally distinct nodes in the brain and interpret the neural correlates underlying tasks assessing different neural systems which include primary sensory processes  and different cognitive processes  .A number of studies have investigated the effects of midazolam-induced sedation on brain activity during primary sensory stimulation and high-order cognitive processing –52. MostHowever, mild sedation may impair more complex cognitive processing . For exaTo date, inherent and spontaneous neural activity of the brain can be well characterized by resting-state functional magnetic resonance imaging (rs-fMRI). It is one of the most important technologies for the non-invasive mapping of brain activity pattern and functional interactions between several large-scale networks known as resting-state networks (RSNs) , 63. It By contrast, functional connectivity within high-order networks is significantly disrupted by midazolam-induced light sedation . In geneRecent studies of resting state have revealed that the human brain is intrinsically organized in two brain networks showing a reciprocal pattern of spontaneous activity: an “intrinsic” and an “extrinsic” network , 80, 81.In addition to the altered static functional connectivity within and between the RSNs, recent studies of anesthetic-induced sedation or unconsciousness have focused on functional brain dynamics and connectivity patterns –95. One A receptors like midazolam [In comparison with midazolam-induced sedation, there are other anesthetic agents that have the similar mechanism of action and sites of action for sedation or anesthesia. Like midazolam, propofol is also an intravenous sedative agent with a rapid onset and a short duration of action. It is widely used for initiation and maintenance of anesthesia, combined sedation and regional anesthesia, induction and maintenance of general anesthesia . Propofoidazolam , 97. Dueidazolam , 98, 99.idazolam . These fidazolam , 100–102idazolam . In addiidazolam . Thus, tDespite common neural changes related to anesthesia, there are some specific effects on brain function for other anesthetic drugs. Numerous studies have suggested that anesthetic-induced sedation disturbs cerebral connectivity. However, the drug effects on higher-order brain networks still vary among diverse anesthetic agents . For exaFor decades, anesthetic agents are used to manipulate the global level of consciousness to explore the neural correlates of consciousness in healthy individuals. Combined with various neuroimaging techniques, the studies are consistent with the notion that the cortex and subcortical structures are suppressed sequentially in a dose-dependent manner . BroadlyThere is growing evidence that functional brain dynamics captures important information about cognition and the emergence of momentary neural coalitions forms the basis for complex cognitive functions, such as consciousness , 110. Li2 levels [2) and partial pressure of oxygen (PaO2) than did others [2 and PaO2 during MRI procedures that involve BOLD signaling and take into account when interpreting findings.In addition, previous studies suggested that anesthetic agent can indirectly affect BOLD signaling by altering physiological parameters, such as arterial concentration of carbon dioxide. Since individuals are not intubated but spontaneously ventilate during midazolam-induced sedation, respiratory depression associated with intravenous midazolam may have resulted in hypercapnia and increased arterial CO2 levels . During d others . We expeIn this review, we have summarized neuroimaging mechanisms of midazolam-induced sedation or anesthesia, which is important for understanding how midazolam-induced sedation impacts on various domains of cognition and the patterns of preserved brain activity during sedation. In particular, midazolam-induced alteration of consciousness is specifically associated with a significant decreased CBF in the bilateral medial thalamus and precuneus/PCC. Studies of individuals who are sedated with midazolam suggest that cortical activity is sequentially impaired from higher-order brain cortices to primary cortical areas in a dose-dependent manner. Under midazolam-induced sedation, functional connectivity within lower-level networks is preserved. By contrast, functional connectivity within and between higher-order networks, particularly the anticorrelation between the DAN and the DMN, is seemingly of neuronal origin of consciousness. Nevertheless, although much published work has suggested the effective role of preserved brain activity in the emergence of the conscious awareness, more evidence should still be required to further explore brain mechanisms of midazolam-induced alteration of consciousness.


Background: This study aimed to evaluate changes in markers of calcification and of endothelial dysfunction during the development of calcification and instability of atherosclerotic plaques and to identify associations of calcification factors with the formation of unstable plaques. Methods: We analyzed 44 male patients with coronary atherosclerosis who underwent endarterectomy in coronary arteries during coronary bypass surgery. The endarterectomy material (intima/media) was examined using histological and biochemical methods, and the stability and calcification degree of atherosclerotic plaques were assessed. In homogenates of the tissue samples and in blood, concentrations of osteoprotegerin, osteocalcin, osteopontin, osteonectin, monocyte-chemoattractant protein type 1 (MCP-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), and E-selectin were determined by enzyme immunoassays. Results: Unstable atherosclerotic plaques proved to be calcified more frequently (80.4% of plaques) than stable ones (45.0%). Osteonectin, E-selectin, and sVCAM-1 levels were lower in unstable plaques and plaques with large calcification deposits. Osteocalcin content increased with the increasing size of the calcification deposits in plaque. Blood osteocalcin concentration directly correlated with osteocalcin concentration in atherosclerotic plaques and was higher in the blood of patients with calcified plaques in coronary arteries. Conclusions: The results provide the basis for further research on the suitability of osteocalcin as a potential biomarker of an unstable calcified atherosclerotic plaque in a coronary artery. Coronary heart disease (CHD) is one of major causes of high morbidity and mortality worldwide . The patCoronary calcification is linked with the risk of adverse cardiovascular events in patients with coronary atherosclerosis. For 15 years, Shaw et al. observed 9715 patients without clinical manifestations of CHD at baseline. The authors noted that, in patients with even a low degree of arterial calcification, the risk of mortality was almost 70% higher, whereas it was sixfold higher in patients with the largest calcification deposits in arteries than in patients who have no calcification deposits in arteries . AccordiBecause of the abovementioned discrepancy, addressing the question of whether calcification indeed affects the formation of an unstable atherosclerotic plaque was chosen as one of the aims of this study. In addition, we investigated possible associations (including the joint effect) of calcification factors and of endothelial-dysfunction factors with the formation of calcified (including unstable) atherosclerotic plaques.This study was approved by the local Ethics Committee of the Research Institute of Internal and Preventive Medicine—Branch of the Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences . The study involved 108 male patients (mean age 60.6 ± 7.8 years) admitted to the Clinic of the Federal State Budgetary Institution “National Medical Research Center named academician E.N. Meshalkin” of the Ministry of Health of the Russian Federation for coronary bypass surgery. All the patients signed an informed consent form for participation in the study. The inclusion criteria were the absence of myocardial infarction less than 6 months old, the absence of acute chronic infectious and inflammatory diseases and their exacerbations, and the absence of renal failure, liver diseases, cancer, and hyperparathyroidism . All patEach endarterectomy tissue sample was longitudinally and transversely divided symmetrically into several parts for histological and biochemical assays. Under an AxioLab.A1 binocular microscope , an examination of 140 fragments of the coronary arteries was conducted, including the description of the cap, core, and periphery of each atherosclerotic plaque. In accordance with the results of the histological analysis, 89 samples of stable atherosclerotic plaques were identified  59 tissue samples without calcification deposits a, (2) 67Venous blood was collected from the patients before the surgical operation, 12 h after a meal. For biochemical assays, 1% homogenates in PBS  were prepared from intima/media samples of coronary arteries. In the intima/media homogenates, protein was quantified using the Lowry method. In the homogenates of intima/media and in the blood samples, concentrations of osteoprotegerin, osteopontin, monocyte-chemoattractant protein type 1 (MCP-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), E-selectin , osteocalcin, and osteonectin  were determined on a Multiscan EX . All results on the intima/media homogenates were normalized to the protein content of the samples.p < 0.05.The results were statistically analyzed using the SPSS software . The normality of distribution of biomarker levels was determined using the Kolmogorov-Smirnov test. These distributions were not normal; therefore, nonparametric tests were applied. The results are presented as the 25th, 50th, and 75th percentiles. The significance of differences was evaluated using the Mann-Whitney test and chi-square test for categorical variables. Multiple comparisons among the groups were performed with the Kruskal-Wallis method. Univariate correlation (Spearman’s) analysis and multivariate linear regression and logistic regression analyses were carried out to find independent predictors of unstable plaques and calcified plaques. Data were considered statistically significant at Analysis of histological data showed that 80.4% of unstable plaques were calcified , as were 45.0% of stable plaques .Comparative analysis of calcification factors and endothelial-dysfunction factors between stable and unstable plaques revealed significant differences only in three markers . In unstA comparative analysis of the calcification factors and endothelial-dysfunction factors among different stages of plaque calcification  uncovered significant differences in five of the seven studied markers . Levels The opposite result was obtained for the calcification-related substance osteocalcin. Specifically, the level of osteocalcin increased with the progressing plaque calcification and was the highest in plaques with large calcification deposits  .r = 0.388, p = 0.001) and MCP-1  and between osteonectin and sVCAM-1 , MCP-1 , and E-selectin . Multivariate linear regression analysis—where one of the calcification factors served as a dependent variable, and markers of endothelial dysfunction served as independent variables—also yielded significant results. It was found that osteonectin is associated with sVCAM-1  and E-selectin , R2 = 0.596, p = 0.0001, osteopontin is associated with sVCAM-1 , R2 = 0.296, p = 0.0001, and osteoprotegerin with sVCAM-1 , R2 = 0.084, p = 0.014. These results confirmed the unidirectionality of changes in the levels of osteopontin and osteonectin with respect to the changes in the levels of endothelial-dysfunction markers as the calcification of atherosclerotic foci increased.The analysis of relationships between calcification indicators and markers of endothelial dysfunction in the atherosclerotic foci revealed direct moderate correlations between osteopontin and sVCAM-1 (p = 0.047) and directly correlates with the calcification degree of the atherosclerotic focus. For example, in the presence of small calcification deposits, the probability of development of an unstable plaque was 4.4-fold higher (Exp(B) = 4.413, 95% CI 1.545–12.602, p = 0.006) and 39.4-fold higher in the presence of large calcification deposits in it (Exp(B) = 39.443, 95% CI 3.564–436.53, p = 0.003). We also conducted multivariate logistic regression analysis with the construction of models where the parameter “calcification in the plaque is present/absent” was the dependent variable. We noticed that relative risk of calcification formation in atherosclerotic plaques of coronary arteries was associated with osteocalcin (Exp(B) = 1.011, 95% CI 1.004–1.018, p = 0.001).Next, we performed multivariate logistic regression analysis with the construction of models where the parameter “stable/unstable atherosclerotic plaque” was a dependent variable. We found that the probability of unstable plaque presence inversely correlates with the plaque content of E-selectin (Exp(B) = 0.924, 95% confidence interval (CI) 0.854–0.999, r = 0.180, p = 0.038), MCP-1 , and osteocalcin . These results point to the feasibility of these markers’ assays in the blood for evaluating the state of atherosclerotic plaques.To understand the pathogenesis features of an atherosclerotic lesion in the vascular wall that lead to the formation of an unstable plaque, it is necessary to consider not only local but also systemic processes. Therefore, assays performed in parallel on the vessels and on the blood are relevant and important in this context. In our assessment of correlations of the studied parameters between atherosclerotic plaques and blood, significant correlations were found for sVCAM-1  blood samples that corresponded to tissue samples containing stable plaques , and (2) blood samples that corresponded to tissue samples containing unstable plaques . No signLikewise, to identify biomarkers in the blood that are significantly characteristic of atherosclerotic plaque calcification, all blood samples were distributed into two groups: (1) blood samples that corresponded to tissue samples containing calcified plaques , and (2) blood samples that corresponded to tissue samples containing plaques without calcification .Among all the studied parameters, only osteocalcin showed a significant difference between the two groups . In maleThese results also indicate the suitability of osteocalcin assays in the blood for evaluating the state of atherosclerotic plaque calcification.In this study, unstable plaques (which are the cause of adverse cardiovascular events) turned out to be more calcified. In a multivariate logistic regression analysis, we found that the risk of development of an unstable plaque is associated with the degree of calcification of the lesion. Our results are consistent with the finding of Hoffmann et al. that coronary-artery calcium is most strongly associated with CHD, regardless of other cardiovascular risk factors, and that an increase in the arterial calcium level increases the risk of adverse events .When examining markers of endothelial dysfunction, we noted that unstable plaques and plaques with large calcification deposits contain lower levels of sVCAM-1 and E-selectin. Our data are in agreement with several studies. For instance, Oishi et al.  revealedIn our analysis of calcification markers, unstable plaques manifested a lower osteonectin level and insignificant downregulation of osteoprotegerin and osteopontin in comparison with stable plaques. In addition, plaques with large calcification deposits featured lower concentrations of osteonectin and osteopontin and insignificant downregulation of osteoprotegerin. In the discussion of these results, it is important to point out that the findings about these three calcification-related biomolecules are contradictory among different studies. For example, judging by the results of Lin at al., high blood levels of osteoprotegerin and osteopontin are strong predictors of mortality in patients with CHD . TousoulLastly, the results of our study indicate a major role of osteocalcin as a biomolecule that potentiates the development of a calcified unstable plaque. International literature data about the effects of osteocalcin on vascular calcification and stabilization/destabilization of atherosclerotic plaques are contradictory. For instance, the results of Millar et al. imply that osteocalcin is not a mediator of vascular calcification . RashdanIn general, our results suggest that the calcification of an atherosclerotic plaque promotes its transition to an unstable state and that osteocalcin performs an important potentiating function in these processes. Here, we obtained the same results when assaying the concentration of this biomolecule in the blood; specifically, the concentration of osteocalcin in the blood directly correlates with its content in atherosclerotic plaques and is significantly higher in the blood of male patients having calcified plaques in the arteries. Our findings provide the basis for further research on the suitability of osteocalcin as a potential biomarker of an unstable calcified atherosclerotic plaque in a coronary artery.