Primary outcome variables were serum sclerostin and serum bone turnover markers (CTx and P1NP), before and after the intervention. Baseline characteristics and variables did not differ between intervention groups. Compared with placebo, prednisolone high‐dose decreased serum sclerostin concentrations , decreased P1NP concentrations  and increased CTx concentrations . Compared with placebo, prednisolone low‐dose did not alter sclerostin concentrations (p = 0.5) or CTx concentrations (p = 0.7), but tended to decrease P1NP concentrations . At baseline concentrations of sclerostin were positively correlated with concentrations of CTx , but not with P1NP. No significant correlations were observed between changes in outcome variables during the interventions. Short‐term high‐dose, but not low‐dose, prednisolone treatment reduces serum sclerostin concentrations in healthy young men. Whether this reflects a counter regulatory mechanism to compensate glucocorticoid‐induced negative effects through other mechanisms remains to be elucidated. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.Glucocorticoid use is the most common cause of osteoporosis in young individuals. In the current study, we investigated the effects of glucocorticoid treatment on circulating sclerostin concentrations and serum bone turnover markers in healthy young men. We performed additional measurements in two combined randomized, placebo‐controlled, double‐blind, dose–response intervention studies: 64 healthy men (age: 22 ± 2 years; BMI: 22.1 ± 1.7 kg/m Glucocorticoid use is the most common cause of secondary osteoporosis.The detrimental effect of glucocorticoids on bone is the net result of hampered new bone formation and an imbalance between bone formation and bone resorption favoring the latter.One of the potential mechanisms contributing to the detrimental effects of glucocorticoid exposure on bone is inhibition of the Wnt/β‐catenin signaling pathway. The Wnt/β‐catenin signaling pathway drives the differentiation of stem cells to the osteoblast lineage and supports differentiation and survival of osteoblasts. Furthermore, the Wnt/β‐catenin signaling pathway inhibits osteoclast function and number through the increased expression of OPG rather than RANKL by the osteoblast.In mice, glucocorticoids increased the expression of Sost, the gene encoding sclerostin production.In accordance with the rodent studies, in patients treated with glucocorticoids for 12 months (7.5 mg/day), serum sclerostin concentrations showed a significant increase. Furthermore, as expected, a decrease in the bone formation marker P1NP and an increase in the bone resorption marker CTx were found.Taken together, human data on the effects of glucocorticoid treatment on circulating sclerostin concentrations and bone turnover markers are scarce and have shown contradictory results. These contradictory results may be explained by the difference in assays used to measure sclerostin, the heterogeneity of the included patients, the different dosages of the used prednisolone, and the difference in follow‐up time. Another main reason for contradictory results is the variability in inflammatory diseases as an indication for glucocorticoid treatment, which may also have directly influenced bone metabolism.Furthermore, the studies were not set‐up in a blinded or randomized fashion. Therefore, the aim of this study was to examine the effects of short‐term low‐ and high‐dose glucocorticoid treatment on circulating sclerostin concentrations and serum bone turnover markers in healthy individuals.n = 64) healthy white men were recruited by local advertisements. Inclusion criteria were age 18 to 35 years, BMI 20.0 to 25.0 kg/m2, and good physical health . Exclusion criteria were smoking, shift work, any current illness, use of any medication, a history of glucocorticoid use, excessive sports activity , and recent changes in weight or physical activity. All participants provided written informed consent before participation. The original studies were approved by an independent ethics committee and were conducted in accordance with the Declaration of Helsinki.For the current study, additional measurements of participants in two combined randomized controlled trials were done.n = 16), prednisolone 7.5 mg once daily (n = 24), or prednisolone 30 mg once daily (n = 24) for 2 weeks using block randomization, as carried out by the Department of Experimental Pharmacology of the VU University Medical Center . Before treatment and on day 13 of treatment, blood was drawn in the fasted state and serum was stored at −80°C for measurement of serum sclerostin concentrations and bone turnover markers, P1NP and CTx.For the current study, data were used from two combined randomized, placebo‐controlled, double‐blind, dose–response intervention studies.Blood pressure (systolic and diastolic) were manually measured  by a single experienced investigator following the 30‐min acclimatization period. The average of three consecutive blood pressure measurements was used.Placebo tablets were obtained from Xendo Drug Development  and prednisolone (7.5 mg and 30 mg) tablets were purchased from Pfizer . To blind the treatment, tablets were encapsulated.Concentrations of circulating sclerostin were measured by an electrochemiluminescence assay  with an intraassay coefficient of variation (CV) ≤6.0%. Interassay CV was 11% and the lower limit of quantitation was 0.040 ng/mL. Electrochemiluminescence immunoassays  were used to measure serum P1NP with an interassay CV of <8.5% and serum CTx with an interassay CV of <5.5%.U test) was performed. Correlations between variables were assessed with Spearman correlations. Statistical analyses were run using IBM SPSS Statistics for Windows version 22.0 ; p < 0.05 was considered statistically significant. Graphs were made using GraphPad Prism for Windows version 8.0.2 .Data are presented as means ± SD or as medians (interquartile range) in case of skewed distribution. Differences in baseline characteristics between treatment groups were evaluated using ANOVA or the Kruskal–Wallis test. Absolute changes from baseline were calculated . To evaluate treatment‐induced differences in serum sclerostin, P1NP, and CTx concentrations, Wilcoxon signed‐rank tests were performed. To analyze differences in treatment‐induced effects between intervention groups, Kruskal–Wallis tests with trend analysis (Jonckheere–Terpstra tests) were performed. In case of relevant differences between intervention groups, post hoc testing using the Wilcoxon rank sum test  and increased serum CTx . However, treatment with prednisolone 7.5 mg once daily decreased serum P1NP . The decrease in serum sclerostin in the prednisolone 30 mg once daily group, but not in the 7.5 mg once daily group, was significantly different from the change in the placebo group (Table A). Furthermore, the absolute changes in serum P1NP were different between treatment groups . The decrease in serum P1NP in the prednisolone 30 mg once daily, but not in the 7.5 mg once daily group, was significantly different from the change in the placebo group (Table B). The increased serum CTx during treatment tended to be different between the intervention groups . The increase in serum CTx in the prednisolone 30 mg once daily group, but not the 7.5 mg once daily group, was significantly different from the increase in the placebo group (Table C).Between‐group changes from baseline were tested by Kruskal–Wallis test with trend analysis (the Jonckheere–Terpstra test). The absolute changes of serum sclerostin during treatment tended to be different between the intervention groups . Moreover, serum CTx concentration was positively correlated with serum P1NP concentration . At baseline, no significant correlation was found between serum P1NP and sclerostin concentrations . No significant correlations were observed between change in P1NP and change in sclerostin concentration (p = 0.629), between change in CTx and change in sclerostin concentration (p = 0.975), or between change in CTx and change in P1NP concentration (p = 0.331) in all treatment groups together. In addition, for each treatment group separately no significant correlations were observed (data not shown).At baseline, the serum CTx concentration was positively correlated with serum sclerostin concentration  prescribed in patients for several indications induced a significant reduction of serum sclerostin concentrations after 96 hours.In agreement with previous studies in humans,In the present study, at baseline a significant positive correlation was found between serum sclerostin with CTx, but not with P1NP concentrations. Results in other studies regarding correlations between sclerostin concentrations and concentrations of bone turnover markers are contrasting.An important strength of our study is that it is the first randomized, controlled, and double‐blinded study on this subject. Another strength is that our study is performed in healthy young men instead of patients with an indication for glucocorticoid treatment. In particular, chronic inflammatory diseases alter osteocyte function and cause osteocyte apoptosis, and may thus interfere with glucocorticoid‐induced effects.An excess of glucocorticoids, either endogenously or iatrogenically induced, leads to loss of bone and is one of the leading causes of increased bone fragility worldwide.


High frequency alternating current (HFAC) stimulation have been shown to produce a peripheral nerve conduction block. Currently, all the studies applying HFAC stimulation in clinical studies, have employed frequencies below 10 kHz. The main aim of this work was to investigate the neuromodulatory effect of transcutaneous 20 kHz stimulation on somatosensory and pain thresholds, and maximal handgrip strength.A randomized, crossover, single-blinded, placebo-controlled trial was conducted following recruitment of fourteen healthy volunteers. Transcutaneous stimulation at 20 kHz and sham stimulation were applied over the ulnar and median nerves of fourteen healthy volunteers for 20 min. Maximal handgrip strength (MHS), mechanical detection threshold (MDT) and pressure pain threshold (PPT) were registered prior to, during (15 min), immediately after the end (20 min), and 10 min following stimulation.p < 0.001 and 34.2 kgs, SE 3.4; p = 0.03, respectively). The 20 kHz stimulation resulted in a slight increase in MDT at 15 min  when compared to the sham stimulation , and no effects were showed for PPT.The 20 kHz stimulation showed a lower MHS during the stimulation at the 15 min (30.1 kgs SE 2.8) and at 20 min  when compared to sham stimulation (35.1 kgs, SE 3.4; High-frequency stimulation at 20 kHz suggests a partial block of nerve activity. Studies in subjects with neurological disorders characterized by nerve hyperactivity are needed to confirm the clinical impact of this non-invasive electrical stimulation technique.NCT02837458. Registered on 12 April 2017.NCT,  Many neurological pathologies lead to neuromuscular dysfunction characterized by an abnormal increase in neural activity, such as the spasticity syndrome or chronic evoked pain . ExaggerTo date only four published studies have reported the effects of non-invasive transcutaneous HFAC stimulation over peripheral nerves , 11, 14.Altogether, these clinical experimental studies support the potential effect of HFAC stimulation on nerve conduction block, even though the optimal stimulation frequency remains unknown. Currently, all the studies applying HFAC stimulation in clinical studies , using eTo date no clinical study has employed transcutaneous HFAC at frequencies greater than 10 kHz. Therefore, the main objective of this study was to investigate the neuromodulatory effect of transcutaneous HFAC stimulation applied on the median and ulnar nerve at a frequency of 20 kHz on somatosensory and pain thresholds and maximal handgrip strength in healthy participants, compared to sham stimulation.ClinicalTrials.gov Protocol Registration System (NCT02837458). Participants received both interventions (20 kHz and sham stimulation), and the order of these interventions was randomized using a web page tool (www.randomizer.org). The duration of the active or sham intervention was 20 min. The evaluation included measures A, B and C (see section D. Outcome measures) assessed throughout the experiment at four time points: i) before stimulation (0 min), ii) during stimulation at 15 min (15 min), iii) at the end of the stimulation (20 min) and iv), at 10 min after the end of the stimulation (30 min). A washout period of 24 h was used between the application of the two interventions [A randomized, crossover, single-blinded, placebo-controlled trial was conducted following recruitment of fourteen healthy volunteers. All volunteers signed the informed consent form approved by the Local Ethics Committee . The present study was registered in the ventions .Healthy volunteers between 18 and 65 years old, without any peripheral or central nervous system pathology, were recruited using non-probabilistic convenience sampling. The exclusion criteria included musculoskeletal pathology in the upper limb, inability to tolerate electrical current stimulation, allergy to the stimulating electrode material, pacemaker or any other implanted device, epilepsy, neurotrauma, recent surgical procedures, diabetes, pregnancy, and cancer.Subjects were seated with their dominant elbow flexed at 90°. Two surface self-adhesive electrodes 5 cm × 5 cm  were used. The proximal electrode was fixed to the skin over the path of the ulnar nerve at the epitrochlea and the distal electrode was placed on the median nerve over the carpal tunnel . The samA charge-balanced, symmetric, biphasic sinusoidal current without modulation was presented at a frequency of 20 kHz for the active intervention. The stimulation intensity was defined as that sufficient to produce a “strong but comfortable” sensation, just below motor threshold , 22.Sham stimulation was applied with the same device and electrode placement, but the intensity was adjusted by simulating to progressively increase the intensity of a non-connected channel. Participants were also blinded to the hypothesis of the study by being informed that in some cases the perceived sensation might be of different as the stimulus intensity could be adjusted to below their sensory threshold, with the possibility that the participant may or may not feel the stimulus current , 20.Maximal voluntary handgrip muscle strength was measured using a handgrip dynamometer . Average maximal handgrip muscle strength was calculated as mean of three measurements and used for statistical analysis  , using the following calibrated filaments to apply forces of 0.25, 0.5, 2, 4, 8, 16, 32, 128, 256 y 512 mN [Mechanical sensory threshold was examined at the palmar surface of the hand, over a 1 cmy 512 mN . ParticiThe PPT was assessed with the application of a digital algometer  applied on the triquetral bone. The stimulus was presented as a circular stimulus tip of 1 cm in diameter, measured on a digital interval scale of 0.1 N. Mechanical pressure was applied at a rate of 5 N/s  and the n = 14). The Gaussian distribution of the data was confirmed with the Shapiro-Walk test, and parametric statistical tests were adopted. A two-way repeated-measures ANOVA, with the “time” factor  and the “intervention” factor (20 kHz and sham stimulation), was performed to compare differences in MHS and PPT. Post-hoc analysis test was assessed with the Bonferroni test. Because MDT is a non-parametric measure, Friedman test was performed to analyse differences within time points . U-Mann Whitney test was used to compare both interventions (20 kHz and sham stimulation). Post-hoc analysis test was assessed with the Tukey test. A p value of < 0.05 was considered statistically significant.Descriptive statistics are reported in mean and standard error or median and interquartile range according to the data distribution . Statistical analysis was performed with a commercial software package Sigmaplot 12.0 for Windows . The estimated sample size for the study was calculated using MHS as the main variable . Taking p < 0.001).Fourteen healthy volunteers completed the study 7 (50%) males, mean age 33.3 years (SD 6.8) ranged between 23 and 44 years. No adverse effects were reported by any participant. The mean current intensity applied at the onset of the stimulus was 44.2 mA (SD 12.8), and the mean final intensity was 85.0 mA  = 4.09; p = 0.013), the “intervention” factor  = 12.14; p = 0.004) and the “time-intervention” interaction  = 4.09; p = 0.013) were detected. Specifically, 20 kHz achieved a decrease in MHS at 15 min during the intervention  when compared with baseline  and to the 10 min post-stimulation time point . Sham stimulation did not show a significant change compared to baseline. A comparison of the both interventions revealed that the 20 kHz stimulus produced a lower MHS  when compared to sham stimulation  during the stimulation at the 15 min test period. Immediately after the intervention at 20 min with 20 kHz stimulation, MHS was statistically lower  when compared to the same measure with sham stimulation . , however the post-hoc test used did not reveal any significant difference. No statistical change in MDT was detected within sham stimulation. When the effect of active or sham stimulation were compared, the 20 kHz stimulus revealed a slight increase in MDT  at 15 min during the stimulation when compared to the sham stimulation   = 0.54; p = 0.66), the “intervention” factor  = 0.26; p = 0.62), or the “intervention-time” intersection  = 0.84; p = 0.48) . With regards to blinding procedures for subjects, the specific method used in this study has been used before , 20, altHigh frequency alternating current stimulation at a frequency of 20 kHz applied over the ulnar and median nerves of healthy volunteers produces a decrease in muscle handgrip strength during the stimulation and a slight change in mechanical detection threshold, when compared to sham stimulation. HFAC at 20 kHz has potential for clinical applications where a selective stimulation protocol is required to partially block alpha-motoneurons without producing large changes in the sensory pathways.


Components of physical frailty cluster into subtypes, but it remains unknown how these might be associated with age‐related functional declines and multimorbidities. This study aims to investigated associations of physical frailty subtypes with functional declines and multimorbidity in a 10 year longitudinal cohort survey.Complementary longitudinal cohort study used group‐based multitrajectory modelling to verify whether frailty subtypes discovered in Taiwan are presented in another aging cohort, then investigated associations of these subtypes with cognitive decline and multimorbidity. Participants aged ≥50 years were recruited from the third to sixth waves (May 2002 to July 2010) of the National Institute for Longevity Sciences‐Longitudinal Study of Aging, in Japan. People with incomplete data, pre‐frail/frail status before their index wave, and those with incomplete data or who died during follow‐up, were excluded. Group‐based trajectory analysis denoted five established physical frailty criteria as time‐varying binary variables in each wave during follow‐up. Incident frailty was classified as mobility subtype (weakness/slowness), non‐mobility subtype (weight loss/exhaustion), or low physical activity subtype. General linear modelling investigated associations of these frailty subtypes with activities of daily living, digit symbol substitution test (DSST) and Charlson Comorbidity Index (CCI) at 2 year follow‐up.P = 0.03) and higher CCI  than the other groups.We identified four longitudinal trajectories of physical frailty, which corroborated the distinct subtypes we discovered previously. Among 940 eligible participants, 38.0% were robust, 18.4% had mobility subtype frailty, 20.7% non‐mobility subtype, and 20.1% low physical activity subtype. People with mobility subtype frailty were older than those with other frailty subtypes or robust status and had higher prevalence of hypertension, diabetes, and heart failure. In the multivariable‐adjusted general linear models, mobility‐subtype frailty was associated with a significantly lower DSST score (point estimate −2.28, Mobility‐subtype frailty was associated with functional declines and progression of multimorbidity; the long‐term effects of physical frailty subtypes deserve further investigation. Frailty is a prevalent geriatric syndrome, characterized by functional declines and depletion of physiologic reserves, which independently predicts adverse health outcomes and mortality in vulnerable older adults.This retrospective cohort study excerpted data from the National Institute for Longevity Sciences‐Longitudinal Study of Aging (NILS‐LSA), a prospective longitudinal cohort study of community‐dwelling middle‐aged and elderly Japanese.FigureWe analysed data acquired from NILS‐LSA participants aged ≥50 years during the second (April 2000 to May 2002) to sweventh waves, in two complementary studies Figure; the anaThe National Center for Geriatrics and Gerontology Committee on Ethics of Human Research approved the NILS‐LSA protocol. All NILS‐LSA participants gave written informed consent before any study‐related procedure ensued.Physical frailty was defined based on Cardiovascular Health Study (CHS) criteria, comprising exhaustion, weakness, slowness, low physical activity, and weight loss,Physical frailty subtypes were defined based on the inceptive study:FigureIn the first phase, assuming that physical frailty status may change over time, we used group‐based multiple trajectory modellingFigureIn the second phase, having validated that the mobility, non‐mobility and low physical activity frailty subtypes were also present in Japan, and we identified subjects with incidence of these three physical frailty subtypes between the third to sixth waves, and compared them with robust people without any CHS criteria to investigate associations between incident physical frailty and declines in physical and cognitive function, and progression of multimorbidity. People with multiple physical frailty components who could not be categorized into any of these frailty subtype groups were classified as mixed subtype and excluded from subsequent analyses. Follow‐up for each subject started at their index wave and ended on completing the next wave, yielding one‐wave (2 years) follow‐up period for all subjects Figure.Outcomes of interest in the study of associations between frailty subtypes and physical and cognitive decline included activities of daily living (ADL), digit symbol substitution test (DSST) performance, and the modified Charlson Comorbidity Index (CCI), which is a validated weighted multimorbidity score derived from self‐reported medical history.2). Health status and multimorbidity  were assessed based on self‐completed questionnaires and confirmed by subsequent medical examinations. Trained interviewers used a questionnaire to assess the Mini‐Mental State Examination score and the CES‐D score.Demographic data included subjects' age, sex, smoking status, alcohol consumption, education level, and income. Height and weight, measured using digital scales with participants lightly clothed, were used to calculate body mass index . The first study phase classified trajectories of physical frailty subtypes using the group‐based multitrajectory model,All analyses were performed using P values (α = 0.05).Categorical variables were compared by Chi‐square tests and continuous variables by analysis of variance (ANOVA); however, Fisher exact test was used to compare categorical variables if any data value in 2 × 2 Chi‐square tables was <5. General linear models were used to examine associations of physical frailty subtypes with ADL, DSST, and CCI, with multivariable adjustment; associations were presented as point estimates, with FigureGroup‐based multitrajectory modelling in phase 1 involved 728 eligible subjects  selected from among 3135 NILS‐LSA third to sixth wave participants. Although group‐based multitrajectory model analysis and selection identified a model with five physical frailty trajectory patterns over longitudinal follow‐up as being the best based on BIC values , it was too complex to be clinically meaningful and yielded a trajectory with only 5% of participants; therefore, based on clinical expert opinion, the four‐trajectory model was chosen for the final model , non‐mobility frailty subtype , low physical activity frailty subtype, mixed subtype , and robust  based on incident physical frailty in the index wave , with the highest prevalence rates of hypertension (P < 0.01), diabetes (P < 0.01), and ischemic heart disease (P = 0.03). People with the non‐mobility frailty subtype had the highest CES‐D score (P < 0.01) and prevalence of hyperlipidaemia (P = 0.05).In phase 2 study, 940/3135NILS‐LSA third to sixth wave participants who met the inclusion criteria were classified into mobility frailty subtype  and higher CCI scores  than robust people , Taiwan. C.T., R.O., Y.N., L.N.P., M.T., H.S., and H.A. declare that they have no conflict of interest.This research was supported by the Ministry of Science and Technology, Executive Yuan of Taiwan (MOST 107‐2634‐F‐010‐001).All the authors designed the research, drafted the article, revised it critically for important intellectual content, and approved the final version to be published. Huang ST and Chen LK wrote the paper. Huang ST performed the literature search and analysed data. Tange C, Arai H, and Chen LK provided critical methodological inputs. Tange C, Otsuka R, Nishita Y, Peng LN, Hsiao FY, Tomida M and Hiroshi S provided methodological and statistical inputs. Chen LK and Arai H contributed to the clinical interpretation. Chen LK is guarantor.


SQSTM1 gene have been recently identified as a rare cause of progressive childhood neurodegenerative disorder. So far, only 25 patients from 10 unrelated families were reported.Mutations in SQSTM1 gene (GenBank: NM_003900.4).We report on the first Tunisian case of an 11‐year‐old girl with cerebellar ataxia, chorea and ophthalmoparesis. Brain MRI was normal. Whole‐exome sequencing revealed a homozygous mutation c.823_824del(p.Ser275Phefs*17) in By pooling our data to the data of literature, we delineated the phenotypic spectrum and stressed on genetic heterogeneity of this rare neurodegenerative disease. SQSTM1 gene have been recently identified as a rare cause of progressive childhood neurodegenerative disorder. We report on the first Tunisian case of an 11‐year‐old girl with cerebellar ataxia, chorea and ophthalmoparesis. Mutations in  Brain and spine MRI were normal. Laboratory tests, including hemogram, creatine kinase, IgA, and thyroid function tests were unremarkable. Electromyography and muscle biopsy were normal.A written informed consent was obtained from the parents and whole‐exome sequencing was performed using a SureSelect Human All Exon 38 Mb enrichment kit. Co‐segregation analysis revealed homozygous frameshift variant c.823_824del(p.Ser275Phefs*17) in SQSTM1 (GenBank: NM_003900.4) as the likely candidate and confirmed that the mutation was inherited from heterozygous carrier parents. No other mutations were detected. The analysis of the gene dosage using exome depth did not indicate any copy‐number variation. Our patient has been treated with coenzyme Q10 (100 mg daily), L‐carnitine (1 g daily), vitamin E (200 mg twice daily), vitamin C (500 mg per day), and Piracetam (600 mg twice daily). She also underwent occupational and speech therapies.3SQSTM1. Sequestosome 1 (SQSTM1), encoding for p62 protein, is an adaptor protein involved in a variety of key cellular processes including oxidative stress response, apoptosis, and cell differentiation . Dystonia and chorea were noted in 15 and five cases, respectively. Dysautonomic features such excessive sweating and orthostatic hypotension with additional features like iridoplegia and anisocoria were reported by Zuniga‐Ramirez et al (two patients) , two are insertions (c.875_876insT and c.712_713insTCCTCCGAGTGTGAATTTCCTGA), four are substitutions  , amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTLD). Furthermore, causal relationships have been found between mutations of SQSTM1 and the occurrence of these diseases (Le Ber et al., In contrast to PDB‐associated SQSTM1 mutations predominantly affecting the Ubiquitin‐Associated domain, the coding mutations in ALS/FTLD patients are widespread, affecting the regions essential for p62's functions such as the promoter regions (Rubino et al., 4SQSTM1 mutation is a rare cause of neurodegenerative disease characterized by progressive ataxia movement disorders and gaze palsy. Through our study, we highlight the importance of whole‐exome sequencing in the diagnosis of rare neurodegenerative disorders. Description of further cases, will allow to better understand the disease and to develop therapeutic trials.The authors declare no conflict of interest.All authors contributed equally to this work.


Ptyodactylus guttatus) ranging in body mass between 4.2–11.5 g. We then  used this calibrated  DXA measurements to determine the best linear measurement calculated CI for this species. Condition indices (CIs) are used in ecological studies as a way of measuring an individual animal’s health and fitness. Noninvasive CIs are estimations of a relative score of fat content or rely on a ratio of body mass compared to some measure of size, usually a linear dimension such as tarsus or snout-vent length. CIs are generally validated invasively by lethal fat extraction as in a seasonal sample of individuals in a population. Many alternatives to lethal fat extraction are costly or time consuming. As an alternative, dual-energy X-ray absorptiometry (DXA) allows for non-destructive analysis of body composition and enables multiple measurements during an animal’s life time. DXA has never been used for ecological studies in a small, free-ranging lizard before, therefore we calibrated this method against a chemical extraction of fat from a sample of 6 geckos . Fat percentage regressed with body mass significantly predicted the DXA fat percentage . Live wet mass was significantly correlated with predicted fat mass  for specimens more than 4.8 g. Among the five calculated non-invasive CIs that we tested, the best was mass/SVL.We found that fat mass measured with DXA was significantly correlated with the mass of chemically extracted fat for specimens more than 4.8 g  for both sexes. Branta bernicla bernicla, [Neochmia phaeton, [Odocoileus virginianus, [Suricata suricatta, [Callospermophilus lateralis, [Chelonia mydas, [Zootoca vivipara, [Body condition is a term used by ecologists to rank the ‘quality’ of an individual animal, usually in relation to the amount of energy reserves  an animal has available . Many anernicla, ), reprodphaeton, ), foragiinianus, ; meerkatricatta, ), survivteralis, ), diseasa mydas, ) and disivipara, ). It is ivipara, . In popuivipara, , 34.Body fat, due to its high energy content, is the best measure of body condition of an animal . Body faBioelectrical Impedance Analysis (BIA) appears to be a better predictor of body fat than body condition estimates calculated from mass and SVL . HoweverMacaca mulatta ; measuridation, ), identiidation, ), determviduals, ) and chaviduals, ). In repviduals, , 37 whicviduals, , 28). Inviduals, ). The usPtyodactylus guttatus inhabited the premises [The study was conducted at Midreshet Ben-Gurion in the northern Negev desert, Israel (30°51′8.27″N 34°47′0.24″E) from summer 2003 until autumn 2004. The study site was a complex of guest rooms surrounded by a two-meter high wall over an area 13x150m. A dense population of the Israeli fan-toed gecko premises . The Isrpremises , 45 Zlotpremises  in the fpremises , 47, 201premises , 46).Ohaus digital scale to 0.1 g precision, snout-vent length (SVL), using digital calipers, and the width at the base of the tail. Six additional individuals were captured and euthanized for the calibration necessary for this study  The smallest gecko (55.6 mm) with body mass 4.2 g was excluded to improve accuracy from 55 to 8.5% error. The snout vent length (SVL) of these geckos was 61.5–91.7 mm and their body mass 4.8–11.5 g. We killed only six geckos in order to minimize destructive sampling as much as possible.Fifty-five geckos were hand captured, measured (morphometrics) and scanned (DXA) and released at the site of capture. Of these, 30 gecko’s scan data were included in the comparison, since their body mass was above the lowest possible accurate reading with minimal body mass (> 4.8 g) as indicated in the results Table . The snoGeckos were anesthetized using cotton balls wet with Isofluran which was inserted with the gecko in a sealed jar for a minute or less until the gecko stopped moving. To obtain the DXA measurements, each gecko was scanned twice using a Lunar PIXImus® 2 densitometer . Each sThe DXA machine protocol was designed for 10–50 g. laboratory mice , but it R2 and P values. For statistical analysis, we used SPSS (version 20).We used backward selection mode of a step-wise regression to test the relation between DXA values of body mass and the values determined from the chemical analysis of body composition. Single and multivariate regression models were then constructed to predict the fat mass from DXA readings. We report the results of our statistical analysis in terms of their N = 6, R2 = 0.834, P = 0.011; Fig. N = 5, R2 = 0.995, P < 0.001; Fig. R2 = 0.995) and a lack of significant difference between fat content by chemical extraction and by DXA estimation .Six geckos were used for the validation portion of this study Table . The valN = 30, R2 = 0.984, P < 0.001; Fig. N = 5, R2 = 0.995, P < 0.001.To determine the accuracy of the DXA in calculating mass compared to actual weighed mass, we compared all lizards above 4.8 g. The DXA body fat readings were highly correlated to live wet mass (linear regression: P = 0.045) but the percentage of explained variation was low (R2 = 0.136). We regressed a multivariate regression model, constructed with mass and DXA machine fat percentage  as variants to predict the correct fat percentage: N = 30, R2 = 0.847, R2adj. = 0.836, P < 0.001; y = − 30.412 ± 3.669 + 1.523 ± 0.157(xFat%) + 1.289 ± 0.151(xMass)  + 1.903 ± 0.179(xMass).A simple linear regression of calculated  vs. DXA machine’s predicted fat percentage was significant ( R2 = 0.16. We regN = 29), and did not significantly depart from a normal distribution . Females tended to have a higher percentage of their body mass as fat  compared to males (7.94 ± 4.19); however, this difference was not significant .Average total body wet mass was 11.1 ± 7.0% body fat  mass/SVL3.We applied five different morphological condition indices that are commonly used on reptiles to the 30 geckos in the dataset in order to determine their accuracy. The indices tested were 1) width of tail base; 2) ordinary least squares (OLS) linear regression of body mass (g) against SVL (mm); 3) mass/SVL; 4) mass/SVLP < 0.05). The correlations between calibrated body fat percentage and body condition indices was largest for the mass/SVL index. The correlation coefficient was highest for the Mass/SVL index > mass/SVL2 > width of tail base > Mass/SVL3 > OLS  and not significant for females.All body indices were highly correlated with each other , a non-destructive method that accurately measures fat mass in humans and other mammals, could be used to determine the fat mass in the small, common Israeli fan-toed gecko. We found that the lowest value for body mass calculation by the DXA machine and that was still accurate was 4.8 g. Thus, when we exclude the smallest gecko (4.2 g), we found that DXA and chemical extraction of fat mass were significantly correlated. It should be stressed that the validation sample size was small and there were not enough individuals to explore sex differences, so further exploration of the technique is warranted. Regression equations using DXA values were able to predict the fat mass and total gravimetric body mass accurately with an average error of 8.55 and 3.5% respectively. Using the predicted DXA values, we were able to test common body condition indices against both actual and predicted DXA values and show that mass/SVL was the most accurate method for estimating the condition index of these small lizards. Measuring the tail base width was not correlated with females body percentage; however for males, it was more accurate than mass/SVL. Females and males may differ in their patterns of fats storage, which might explain why the tail base width was not correlated with fat percentage in females, while it was the best indicator for fat percentage in males.2) predict the fat mass of diamondback water snakes (Nerodia rhombifer) with significantly different values from the DXA and chemical analysis results (mean error ranging from 20 to 41%). Weatherhead & Brown [N. sipedon). Other methods for the estimation of fat percentage  have been fairly successful in predicting body condition, but again they lack accuracy  and DXA (this study). The mean error percentage for fat mass we calculated for using DXA was 8.55% when the animal body mass was more than 4.8 g. This percentage designated the difference between the fat mass predicted by the model and the fat mass that was extracted chemically. The mean error of the wet body mass was even lower, at 3.5%. Thus, the body index method we used—measurement of body fat% by DXA— was proved to be most reliable and highly correlated to chemically extracted body fat.Previous work on reptile body condition indices and non-destructive sampling have been notoriously inaccurate. Secor and Nagy  found th & Brown  found anccuracy . Total ote that  found thWe found that DXA was a reliable rapid and accurate means to predict fat mass and body mass in live small lizards. It enables a researcher to accurately track seasonal and ontogenetic changes in an individual and to correlate these changes to fitness traits. The DXA machine and software were easy to use, and such machines are widely available due to DXA use in human medicine. The cost for such a machine solely for use on wildlife would be prohibitive , but as a secondary use for machines already purchased for medical or veterinary laboratories, DXA becomes a cost-effective option. While cost is always a drawback for DXA, we suggest its primary benefit is to select the most accurate CI of the non-invasive methods. A validation study is required to generate the predictive models necessary to know which CI is the most accurate; the sacrifice of study animals becomes then a serious liability for studies on rare species. Another disadvantage is the necessity that subjects remain absolutely still, which requires anesthesia. In a pilot trial, we attempted to place the lizards in the freezer prior to the trial to prevent them from moving during the scanning phase, but this proved unreliable and had to be substituted with an anesthesia which can prove fatal in overdose.r = 0.961). This result concerning tail width should be explored in other species of lizards in order to understand if this specific measurement is a general basic physiological difference between the sexes.As a final note on practicalities, when comparing five common traditional CI’s, we found that mass/SVL was the index with the highest correlation coefficient to the DXA estimates. We conclude that when DXA is not available, mass/SVL is the most reliable alternative index for this species and should be used unless actual fat mass is required. In situations where more accuracy is needed, DXA is a suitable method for small lizards. An interesting question raised here is the insignificant correlation between females tail base width and fat percentage, while in males it was the best predictor (


The glymphatic system is a network of perivascular spaces that promotes movement of cerebrospinal fluid (CSF) into the brain and clearance of metabolic waste. This fluid transport system is supported by the water channel aquaporin-4 (AQP4) localized to vascular endfeet of astrocytes. The glymphatic system is more effective during sleep, but whether sleep timing promotes glymphatic function remains unknown. We here show glymphatic influx and clearance exhibit endogenous, circadian rhythms peaking during the mid-rest phase of mice. Drainage of CSF from the cisterna magna to the lymph nodes exhibits daily variation opposite to glymphatic influx, suggesting distribution of CSF throughout the animal depends on time-of-day. The perivascular polarization of AQP4 is highest during the rest phase and loss of AQP4 eliminates the day-night difference in both glymphatic influx and drainage to the lymph nodes. We conclude that CSF distribution is under circadian control and that AQP4 supports this rhythm. Glymphatic function is increased during the rest phase while more cerebrospinal fluid (CSF) drains directly to the lymphatic system during the active phase. The water channel aquaporin-4 supports these endogenous, circadian rhythms in CSF distribution. Acute sleep deprivation impairs cognitive function4, and sleep disruption is often an early correlate of neurodegenerative disease5. Sleep quality is highly dependent on both sleep deficit and sleep timing controlled by circadian rhythms, 24 hour cycles in gene transcription, cell signaling, physiology, and behavior6. Understanding how circadian rhythms contribute to sleep quality is necessary to promote long-term brain health.Sleep is an evolutionarily conserved biological function, clearing the brain of harmful metabolites such as amyloid beta that build up during wakefulness1. The glymphatic system is a network of perivascular tunnels wrapped by astrocyte endfeet7. The localization of the water channel aquaporin-4 (AQP4) to astrocyte vascular endfeet promotes CSF movement through the brain8. This fluid movement is also dependent upon arterial pulsation10, and correlated to decreased heart rate11. Although the cardiovascular system and sleep, two main influencers of the glymphatic system, are tightly regulated by circadian rhythms12, it remains unknown whether glymphatic fluid movement is under circadian control.During sleep, there is increased cerebrospinal fluid (CSF) movement into the glymphatic system, promoting waste clearance14. In fact, lymphatic drainage may prevail when the animals are awake and glymphatic influx is low15. The immune system is also regulated by circadian rhythms17, yet whether lymphatic drainage of CSF to the lymph nodes is under clock control has to be determined.CSF moves not only into the brain and spinal cord via the glymphatic system, but also drains out of the central nervous system to the lymphatic system of the bodyHere, we test the hypothesis that glymphatic influx of CSF tracer from the cisterna magna (CM) into the brain, and clearance of solute from the brain varies across the day independent of arousal state. We also test whether AQP4 may have a role in rhythmic glymphatic function. We show that glymphatic influx and clearance of a small tracer from the parenchyma is increased during the day compared with night in mice independent of the type of anesthesia or light/dark cycle, corresponding to day/night changes in AQP4 localization. These rhythms in influx, clearance, and AQP4 persist after 10 days in constant light, indicating that the glymphatic system is under circadian control. AQP4 knockout (AQP4 KO) animals exhibit no day/night difference in glymphatic influx. In fully anesthetized animals, in vivo imaging of mandibular lymph nodes showed increased tracer outflow during the night compared with day, when entry of CSF to the brain is low. This day/night difference in lymph node filling persists in constant conditions, and is absent in animals without AQP4.n = 6 mice, night: 24.56 ± 7.1 MPI, n = 6 mice; t test: t(10) = 2.617, p = 0.0257). Because there is also increased CSF tracer in areas outside of cortex, we re-analyzed the images using an ROI more tightly confined to the MCA and confirmed similar results  of the brain. Mice were anesthetized with ketamine/xylazine (KX) at mid-day or mid-night . In order to measure glymphatic function, fluorescent tracer was injected into the CSF pool in the CM and allowed to circulate for 30 min prior to brain collection. During circulation, fluorescent tracer movement was imaged through the skull of anesthetized mice Fig. . We founn = 10–12 mice per time point; Supplementary Fig. p = 0.004; R2 = 0.151). The difference in influx between the day and night was ~22% . Because these experiments were all performed under KX anesthesia, and some anesthetics have potential to shift circadian timing18, we generated full time courses under two additional anesthetics, pentobarbital and 2,2,2-tribromoethanol  . Anesthesia impacted overall tracer influx, as reported previously11, where KX had significantly more influx (average influx under KX: 7.08 ± 0.3 MPI) when compared with pentobarbital (4.42 ± 0.1 MPI) or tribromoethanol  = 105.2, p < 0.0001). More importantly, we show significant rhythms under both pentobarbital  = 5.527, p = 0.0069; R2 = 0.175) and 2,2,2-tribromoethanol  = 6.479, p = 0.0035; R2 = 0.209) with acrophases around mid-day  and similar amplitudes , a measure of phase and mesor of the data sets, and found similar results to our 95% confidence intervals above, where anesthetic changed the mesor but each phase estimate was approximately mid-day  and night  in total influx, anterior to posterior distribution, or in distinct subregions  and night (n = 6 mice). We chose this method because although we cannot measure where in the vasculature EB enters and binds to albumin, any fluorescence detected in the femoral vein had to be cleared from the brain parenchyma. Thus, this approach enabled us to test the hypothesis that net clearance of EB from the brain is under diurnal control. Mice were implanted with a microdialysis cannula into the striatum 24–36 h prior to experiments. At the time of experiment, the microdialysis probe was inserted in the cannula, the femoral vein exposed, and placed under the macroscope . EB was significantly (t test: t(13) = 2.605, p = 0.0218) higher in the femoral vein as early as 15 min post infusion . By 90 min there was 37% more EB during the day than the night (t test: t(13) = 2.519, p = 0.0256; Fig. U = 9, p = 0.6095; Fig. Glymphatic influx does not reflect function of the whole glymphatic system, therefore we measured clearance of Evans blue (EB), a small molecule (960 Da) that freely diffuses through the brain and binds tightly to albumin in the blood, from the brain to the femoral vein in anesthetized animals during the day  or ZT18 , we found increased polarization of AQP4 around vascular structures across the cortex during the day  = 4.173, p = 0.0004), with clear trends persisting in both the hypothalamus  = 2.342, p = 0.0577) and CA1 of the hippocampus  = 2.139, p = 0.1188) , or in higher molecular weight bands of AQP4 complexes  =  1.912, p = 0.0688; Fig. Influx of CSF into the brain parenchyma is highly dependent upon localization of the water channel AQP4 to the endfeet of astrocytes that surround brain vasculature29, components of which exhibit highly enriched gene expression in astrocytes  according to a cell-specific transcriptome data base30. We hypothesized that day/night changes in gene expression for components of this complex may support rhythmic localization of AQP4 to perivascular endfeet of astrocytes. Brains were harvested at ZT6 (n = 5 mice) and ZT18 (n = 5 mice), then reverse transcription PCR (RT-qPCR) was used to measure gene expression. First, we measured the clock gene Arntl1 and the non-clock gene homolog Arnt31. As expected, there was a significant day/night difference in Arntl1  = 2.504, p = 0.037; Fig. Arnt. Next, we measured gene expression of key components of the DAC: Dag1, Dmd, Dtna, Snta1, and Aqp4  = 2.335, p = 0.0483; Dtna: t(6.272) = 2.837, p = 0.0283; Aqp4: t(7.973) = 0.038). Both Dmd and Snta1 had similar trends of expression, but the day/night differences were not significant , cation channels (Mlc1), and bicarbonate transporters (Slc4a4) are upregulated in astrocytes30, and putatively associated with the DAC27. We found only Gja1 exhibited day/night differences in gene expression, with a reduction during the night  = 2.43, p = 0.0445). This may indicate that temporal localization of connexin-43 to astrocytic endfeet is regulated by the DAC. Increased gene expression of key components of the DAC, including AQP4, match the increased localization of AQP4 and increased glymphatic influx of CSF into the brain during the day.AQP4 localization to the vascular endfeet of astrocytes is dependent upon the dystrophin-associated complex (DAC)qp4 Fig. . We founant Fig. . In addi33. Mice were housed in constant light (LL) for 10 days with continuous activity monitoring to ensure chi-squared periodogram estimates of free-running period were accurate34. Because long-term (>30 days) LL induces behavioral arrhythmicity and altered SCN organization36, we wanted to ensure that general cage activity was unchanged in LL (n = 6 cages of 2 mice) compared with DD (n = 10 cages of two mice). Using chi-squared periodogram analysis we found animals in DD and LL had similar variability in periodicity, and every cage had significant Qp values from a chi-squared periodogram analysis  = 2.477, p = 0.0325). Similar to previous reports, mice in LL had an average period of 25.35 ± 0.1 h. Using average activity profiles for 10 days in LL and DD, we found no evidence of splitting in LL  = 2.089, p = 0.0555; Supplementary Fig. t test: t(14) = 1.727, p = 0.1062). Thus, 10 days in LL does not drastically alter circadian behavior, indicating the circadian system is intact.Next, we tested the hypothesis that diurnal changes in glymphatic CSF tracer influx are not driven by cycles of light and dark (LD). We chose constant light (LL) over constant dark (DD) to avoid effects of light pulses that are inevitable during live-animal surgery. The surgery takes ~10 min, more than enough to phase shift mice in constant darknessn = 13–14 mice per group; Mann–Whitney test: U = 32, p = 0.0033). In addition, AQP4 polarization to vascular endfeet was increased during rest phase of the animal  = 2.552, p = 0.0288; Fig. n = 5 mice) or active phase . Similar to LD, clearance was significantly increased by 55% during the rest phase compared with the active phase at 90 min (t test: t(8) = 3.823, p = 0.0051), with no significant differences in percent coverage of EB in the brain  or mid-active phase (CT 16–20) of the animal’s behavior Fig. . Influx ain Fig. . These d1 and drainage of CSF to the lymph nodes is increased when awake15. Because we observed a strong underlying circadian rhythm in glymphatic influx, our next question was whether CSF outflow via the cervical lymphatic system showed circadian variation. Mice received CM injections of tracer under KX anesthesia, as discussed above, during the day (n = 12 mice) or night (n = 15 mice). The mandibular lymph nodes were then exposed and imaged for 50 min post CM. We found that filling of the submandibular lymph nodes was 46% lower during the day compared with the night  = 2.25, p = 0.0335). This effect persisted after 10 days in constant light  = 2.603, p = 0.0231). Appearance of tracer in the lymph node occurs as early as the first frame of recording  with a trend for this pattern to persist in constant conditions , indicating there are diurnal timing mechanisms for how fast the CSF can distribute into different compartments. We conclude that CSF drainage to the cervical lymph nodes is under circadian control with a peak during the active phase that temporally is in antiphase to CSF entry into the glymphatic system of the brain  and in 10 days LL  we found no significant differences in contractions per minute between the rest and active phases , main effect of phase: F = 0.603, p = 0.4457; interaction light*phase: F = 0.173, p = 0.6815). We conclude that intrinsic contraction rate of the mandibular lymph node vessels is not responsible for circadian drainage of CSF to the lymph nodes.Lymphatic vessels contract to draw fluid into the lymph nodesate Fig. . KX anesn = 10 mice) or night  to test whether this day/night difference in AQP4 is necessary for day/night differences in glymphatic influx. AQP4 KO animals did not have a day/night difference in glymphatic influx, as measured by average slice intensity (t test: t(18) = 1.511, p = 0.1481; Fig. n = 6 mice) and night (n = 7 mice). There was no difference in lymph node drainage after 50 min between day and night (t test: t(11) = 1.103, p = 0.2937; n = 6–7 mice), nor in the slope of drainage calculated from the first 20 min of recording (t test: t(11) = 0.9202, p = 0.3772; Fig. We performed CM injections of CSF tracer (BSA647) into AQP4 KO mice during the day  = 0.2208, p = 0.8284) between LM (n = 3 mice) and KO animals  and KO influx in the brain (t test: t(48) = 1.962, p = 0.0556).The reduction in day-time influx in AQP4 KO animals compared with littermate (LM) controls has already been publishedt test: t4 = 0.220n = 3 mice per time point) and KO , time  = 0.191, p = 0.667), or the interaction between the two factors  = 0.902, p = 0.353). Because our AQP4 KO line is backcrossed to C57, and the majority of LM fell within the two median quartiles of drainage distribution, we next pooled LM with C57 values at 30 min post pump start . Overall, drainage to the mandibular lymph nodes was different between groups  = 8.464, p = 0.037; Fig. p = 0.020 and p = 0.011, respectively) supporting increased drainage only during the night in WT mice, though this effect was gone after correction.To measure lymph node drainage, we compared fluorescence in the mandibular lymph nodes after 30 min of CSF circulation during the day and night in LM , and an increase in clearance of EB from the brain parenchyma to the periphery indicate that glymphatic system function peaks during the day. Conversely, drainage of CSF tracer to the mandibular lymph nodes is highest in anesthetized mice during the night, when animal activity will be at its peak. Day/night differences in glymphatic influx, solute clearance, and CSF drainage to the lymph nodes persists under constant light, supporting the hypothesis that these are endogenous circadian oscillations. Circadian glymphatic function is supported by circadian regulation of AQP4 polarization in astrocytes, and genetic deletion of AQP4 effectively eliminates the circadian regulation of CSF distribution detected as an absence of day/night differences both in cortical tracer influx and in drainage of tracer to the lymph node.38. In addition, restoring molecular clock rhythms in SCN astrocytes is sufficient to drive rhythmic sleep/wake behavior in animals that are genetically arrhythmic39. Our data provide evidence supporting the idea that astrocytic regulation of glymphatic rhythms occurs at least in part via AQP4 vascular polarization in the cortex, which is supported by rhythmic gene expression of key components in the DAC. Deletion of the molecular clock in astrocytes induces mis-regulation of AQP4 gene expression, supporting our hypothesis that the DAC is under molecular clock control40. Circadian regulation of proteins in the DAC beyond AQP4 may provide insight into conserved astrocyte clock function controlling the release and buffering of extracellular signals such as glutamate, as in the SCN41, or regulating sleep- and wake-promoting extracellular ion concentrations and fluid transport throughout the cortex42. Our data show that expression of connexin-43  is diurnally regulated in a pattern similar to genes in the DAC, consistent with Cx43 being highly enriched in the vascular endfeet of astrocytes43, and provide additional mechanisms for clock-controlled astrocytic signaling. These observations support the notion that astrocytes stabilize and perhaps drive circadian behavior, in part by regulating bulk fluid movement through and CSF/ISF exchange across the brain to entrain circadian rhythms.Recent work has provided evidence that astrocytes in the clock center of the brain, the suprachiasmatic nucleus (SCN), may be responsible for setting periodicity of circadian behavior44. In addition, CSF production in humans may be rhythmic with peak CSF production during the night46, though results may be inconclusive47. This poses at least two potential paths the CP might regulate glymphatic rhythms: first, by regulating concentration of signaling molecules in the CSF and second by mechanically supporting glymphatic influx by providing a larger volume of CSF during the rest phase. When improved methodology for CSF production in rodents becomes available49, it should be investigated whether CSF production regulates glymphatic CSF influx along the perivascular spaces of the brain.CSF is produced in the choroid plexus (CP) of the brain, an epithelial layer of cells located within the ventricles. The CP exhibits robust cycling of the molecular clock in vitro, and can tune periodicity of molecular rhythms in SCN co-cultures and in behavior15. This was based on two main observations: no tracer enters perivascular spaces of penetrating arterioles prior to death based on macroscopic imaging, and lymphatic outflow is increased in wakefulness, whereas glymphatic influx is decreased along the MCA. Our laboratory has already demonstrated CSF tracer alongside penetrating arterioles in live animals using two-photon microscopy9, which has considerably higher spatial resolution than macroscopic imaging50. In addition, we have used rapid decapitation followed by drop fixation to process all of our brain samples since 2017 to reduce the death15 and perfusion artifact10 of CSF tracer localization in the brain. Here, we collected both in vivo and ex vivo data in fully anesthetized mice and observe changes in glymphatic and lymphatic CSF distribution dependent upon circadian timing, with underlying molecular rhythms in the DAC and AQP4 polarization. Based on these data, we propose that the lack of glymphatic influx is not simply a downstream effect of the lymphatic system drainage of CSF before it reaches the brain. Instead, the awake brain actively suppresses periarterial influx10, has less perivascular AQP4, and prevents CSF/ISF exchange by reducing the interstitial space volume1. This would cause CSF to follow alternative, pre-existing routes out of the subarachnoid space around the brain and spinal cord, such as into the mandibular lymph nodes, meningeal lymphatics, and/or the deep cervical lymph nodes51. In short, CSF distribution will change based on fluid changes and space availability within the brain.Proulx and colleagues recently proposed a model whereby rapid CSF turnover through lymphatics precludes significant bulk flow into the brain11. One hypothesis is that the synchronous, slow wave firing characteristics of non-REM sleep drive consolidated movement of extracellular ions across cortex, supporting pulsating inflow of CSF into the neuropil. This process would not be compatible with the asynchronous patterns of neuronal activity that characterize the awake brain.The mechanisms behind brain-regulated suppression of glymphatic activity during wakefulness remain unknown. EEG activity tightly correlates to glymphatic influx and the patterns of EEG activity that resemble wakefulness strongly suppress CSF influx52. Cortical neuronal activity as measured by EEG has strong circadian components in humans54. The cardiovascular system as a whole is under tight circadian control55, and arterial pulsatility is a key driving factor in transport of CSF along the penetrating arteries10. In flies, there is evidence of a circadian-clock in the equivalent of the blood–brain barrier57. Finally, immune system functionality is regulated by circadian timing58 and we describe a clear interaction of CSF entry to the brain and lymphatic clearance of CSF. Although glymphatic function has yet to be studied in models of circadian disruption, such as in shiftwork, it has been established that shift workers are at increased risk for neurodegenerative disorders, cardiovascular disease, and exhibit increased markers of systemic inflammation61. Understanding how these rhythms, all with different timing and biological functions, interact to affect glymphatic function and lymphatic drainage may help prevent morbidity associated with circadian misalignment.There are multiple circadian rhythms that may interact to promote rhythmic glymphatic function. Recent work hypothesized increased glymphatic clearance during the sleep phase, driving rhythmic fluid flow in the brain62 were bred in the University of Rochester vivarium, and backcrossed to C57BL/6 mice for 20+ generations before use. A minimum of five mice were used in each group, with exact animal numbers stated in the results and figure legends. Mice were group-housed either in a 12:12 light/dark cycle or under constant light with ad libitum access to food and water. All experiments were approved by the University of Rochester Medical Center Committee on Animal Resources. All of the University of Rochester’s animal holding rooms are maintained within temperature (18–26 degrees Celsius) and humidity ranges (30–70%) described in the ILAR Guide for the Care and Use of Laboratory Animals (1996). All efforts were made to keep animal usage to a minimum.Male and female C57BL/6 mice  were acquired from Charles River Laboratories  in equal numbers for each experimental group to control for any potential sex differences. Male and female AQP4 KO mice63. Activity was monitored continuously in 5 min bins via the Comprehensive Lab Animal Monitoring System (Columbus Instruments). Circadian behavioral analysis was completed with ActogramJ. Free-running period of each cage was determined using at least 10 days of activity and a χ2 periodogram, which was used to confirm behavioral rhythmicity and estimate experimental times in conjunction with activity onset of the cage.For constant light experiments, animals were housed two per cage to reduce risk of hypothermia−1 racemic ketamine and 20 mg kg−1 xylazine kg i.p. (KX); pentobarbital 60 mg kg−1 i.p.; 2,2,2-tribromoethanol  120 mg kg−1 i.p.. Depth of anesthesia was determined by the pedal reflex test; if the mouse responded to toe pinch, an additional one-tenth the initial dosage was given and the tracer experiment delayed until full unconsciousness was obtained. Directly prior to CM infusion, the animal received an additional one-tenth the initial dosage, and the pedal reflex was tested every 5–10 min during the tracer circulation time to ensure proper anesthesia throughout the study. Animals under avertin anesthesia were most likely to need supplemental dosing.Anesthesia was administered as follows: 100 mg kgTM 647 conjugate; 66 kDa; Invitrogen, Life Technologies, Eugene, OR) was formulated in artificial CSF at a concentration of 0.5% weight by volume. Anesthetized mice were fixed in a stereotaxic frame, the CM surgically exposed, and a 30 gauge needle connected to PE10 tubing filled with the tracer was inserted into the CM. Ten microliters of CSF tracer was infused at a rate of 2 μl min−1 for 5 min with a syringe pump (Harvard Apparatus)10. For the awake animal cohort: mice were anesthetized with 1–2% isoflurane, and 0.25% bupivicane was applied topically to the surgery site at the beginning of and after recovery from the procedure. Mice were allowed to resurface from anesthesia before pump start. Total volume of CSF tracer was increased to 12 µL to compensate for decreased glymphatic function, pump speed remained the same (2 μl min−1).Fluorescent CSF tracer . The brain was fixed overnight by immersion in 4% paraformaldehyde in phosphate-buffered saline (PBS). Coronal vibratome slices (100 μm) were cut and mounted. Tracer influx into the brain was imaged ex vivo by macroscopic whole-brain and whole-slice conventional fluorescence microscopy .11. The cerebral cortex in each slice was manually outlined, and the mean fluorescence intensity within the cortical ROIs was measured. An average of fluorescence intensity was calculated between six slices for a single animal, resulting in a single biological replicate . A CM injection was then performed as described above, with imaging beginning at pump start. Image acquisition was once per minute on the Cy5 channel, 30 min total .Animals were anesthetized with KX at ZT6 or ZT18 and were prepared for transcranial imaging−1). Images of the femoral vein were taken under a macroscope  using the Cy5 filter set, for EB visualization, and GFP, for venous structure visualization, once every 15 min for 90 min  beginning at the start of EB injection. Brains were collected upon experiment end, drop-fixed overnight in paraformaldehyde (PFA) and sliced the next day in the same manner as the CSF influx experiments. Slices were used to ensure parenchymal injections did not hit the ventricle and volume of injection, as measured by % area, was similar between animals. All images were analyzed in ImageJ. Mean pixel intensity in the femoral vein was quantified on the Cy5 channel, with the GFP channel used to ensure placement of the intra-venous region of interest.All experiments occurred between CT 2–7 or CT 14–19 depending on if it was the rest or active phase of the animal, respectively. Cannulas  were implanted into mice 24–36 h prior to experimentation into striatum . At the appropriate experimental time points, mice in either LD or 10 days LL were anesthetized with KX and fitted with a microdialysis probe , had skin over the femoral vein resected to better visualize EB in the blood stream, and EB was injected into the brain  in PBS with 0.03% Triton-X-100 and incubated with primary antibody overnight, followed by three washes in PBS and incubation with the fluorophore-linked secondary antibodies (Invitrogen) for 2 hours. Stained slices were mounted with Fluoromount G (Thermofisher Scientific). Primary antibody used was rabbit anti-AQP4 , secondary antibody used was Alexa 594 donkey anti-rabbit , cell nuclei were identified using DAPI . A subset of animals were cardiac-perfused with wheat germ agglutinin conjugated with Alexa Fluor 488 (Thermofisher Scientific) at a working concentration of 15 µg mLAll analyses were done with investigators blinded to time-of-day, as images were taken by a separate investigator who randomized file names. Equivalent images of dorsal cortex, lateral cortex, ventral cortex, were imaged for each slice (three images per slice). For a subset of animals, additional images were taken of CA1 in hippocampus and hypothalamus. The mean immunofluorescence intensity was quantified using ImageJ. For quantification of AQP4 polarization, 50 μm segments centered on blood vessels  were analyzed using the line-plot tool in ImageJ. Vessels chosen were ~6 µm wide (range of 5–8 µm) and 32 µm long (range of 20–55 µm). Three vessels that matched this criteria were chosen per image.For polarity calculation, the baseline was defined as the average intensity over 10 µm, −20 µm to −10 µm from peak fluorescence. Baseline fluorescence was subtracted from the peak intensity of the segment. This subtraction method is ideal because it looks at changes beyond background fluorescence localized to vascular endfeet. For each dataset (LD and LL), the adjusted peak fluorescence values were normalized to the highest value out of all groups compared in the statistical analysis, one value per graph for ease of comparison and visualization, giving a range from 0 to 1.m Tris•HCl pH 7.6, 150 mm NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS)) with a protease inhibitor cocktail  using a Fisherbrand 150 homogenizer. Samples were kept at 4 °C or on ice, to avoid freeze thaw cycles. Samples were sonicated 3 × 10 s (Branson Sonifier 150D) and centrifuged at 13,000 × g for 5 min at 4 °C. Supernatant was transferred to a new tube and diluted 1:10 in lysis buffer. Protein concentration was determined by a Pierce BCA assay (Thermofisher). Samples were prepared for loading in sample buffer  at 20 µg protein per well. Dual Color Ladder (Bio-rad) was loaded to identify molecular weights. The gel was 4–15% Bis-acrylamide (Bio-rad). Gels were transferred to PVDF membranes (Bio-rad), then membranes were blocked in: Tris-buffered saline (Bio-rad), 0.1% Tween-20 (Sigma), and 5% fat free milk (Bio-rad), for 1 h on an orbital rocker. Membranes were incubated with primary Rabbit anti-AQP4 antibody  in TBST 5% milk overnight at 4 °C. Then, incubated 1 h at room temperature with Donkey anti-Rabbit-HRP secondary antibody . Membranes were developed using Femto kit  and imaged on a Chemidoc MP imaging system (Bio-rad). Membranes were stripped using stripping buffer  at 50 °C for 10 min. Membranes were blocked again, then incubated with mouse anti-βactin primary antibody  in TBST 5% milk at 4 °C overnight. Donkey anti-Mouse-HRP secondary antibody  was applied for 1 h and the membranes developed and imaged.Mice were killed at ZT6 and ZT18, mice at ZT18 were killed in a dark room using red light lamps to avoid light pulses. Brains were extracted and cut in half along the sagittal plane with the cerebellum removed, one half was used for protein extraction. Brain tissue was homogenized in lysis buffer (25 mn = 5 per group). RNA was isolated using Trizol®, cleaned via RNeasy Mini kit  following manufacturer’s instructions and quantified on a nanodrop spectrophotometer. First-strand cDNA was synthetized using TaqMan Reverse Transcription Reagents . Real-time PCR samples were prepared in triplicate with 5 ng of RNA in FastStart Universal SybrGreen Mastermix  and amplified on a CFX Connect Real-Time System Thermocycler . Primer sequences are listed in Supplementary Table 66.Brains were collected at ZT6 or ZT18, cut in half along the midline with the cerebellum removed, and flash-frozen on dry ice then stored in a −80 °C freezer until RNA isolation (Mice were anesthetized with KX between CT 4–8 or CT 16–20 and received a CM injection as described above. Upon completion of the tracer infusion (5 min) the mice were placed supine under the macroscope  with all skin over the neck resected to reveal the superficial lymph nodes. Images were taken on the Cy5 channel for 50 minutes at a rate of one image per minute, 51 images total . The speed of the lymph node filling (fluorescent intensity per min) was evaluated by the slope of a linear regression on the first 20 minutes of each recording (20 images).37) was calculated from each lymph vessel, and averaged for each animal.Mice were anesthetized with KX between CT 4–8 or CT 16–20. Mice had superficial lymph nodes exposed and were placed under the macroscope, as described above. FITC-conjugated 3 kD dextran  was injected into each cheek of the mouse. Imaging acquisition began following lymph vessel filling (<2 min post cheek injection) at a rate of 2.5 hz for 5 min . Images were analyzed in ImageJ. A horizontal line segment centered on the lymph vessel was drawn and fluorescence over time was calculated using this region of interest. Contractions per minute , an unpaired t test was used for two groups. For more than two groups an ANOVA was used for comparisons between means, followed by Bonferroni p value correction for multiple comparisons. In cases of a non-normal distribution (as indicated by a Shapiro–Wilk test) or unequal variances (Levene’s test), a nonparametric Mann–Whitney test or a Welch’s t test was used for comparisons between two means, and a nonparametric Kruskal–Wallis test was used for comparisons between more than two means, followed by Bonferroni p value correction for multiple comparisons. Significance was ascribed at p < 0.05.All statistical analysis was performed in Graphpad Prism version 7.0, with the exception of the cosinor analyses. Cosinor analysesFurther information on research design is available in the Nature Research Reporting Summary linked to this article.Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Movie 1Supplementary Movie 2Reporting Summary


ST‐elevation myocardial infarction (STEMI) patients with multivessel disease (MVD) are associated with a worse prognosis. However, few comparisons are available according to coronary status in the era of modern reperfusion and optimized secondary prevention.We hypothesized that the difference in prognosis according to number of vessel disease in STEMI patients has reduced.All consecutive STEMI patients undergoing primary percutaneous coronary intervention (PCI) within 24 h of symptoms onset between January 1, 2014 and June 30, 2016 enrolled in the CRAC  France PCI registry were analyzed. Baseline characteristics, management, and outcomes at 1‐year were analyzed according to coronary status .p = .28). In multivariable adjusted Cox proportional hazard regression model, two‐ and three‐VD were not associated with higher rate of MACE compared to patients with single VD .A total of 1886 patients  were included. Patients with MVD (two or three‐VD) represented 53.7%. They were older with higher cardiovascular risk factor profile. At 1 year, the rate of major adverse cardiovascular events  was 10%, 12%, and 12% in one‐, two, and three‐VD respectively (MVD still represents an important proportion of STEMI patients but their prognoses were not associated with worse clinical outcomes at 1‐year compared with one‐VD patients in a modern reperfusion area and secondary medication prevention. It is an observational prospective multicenter registry, which includes all patients undergoing coronary angiography or coronary angioplasty in each participant center. The methods used for this registry have been detailed previously.For the present analysis, we enrolled all consecutive STEMI patients undergoing PCI within 24 h of symptoms onset between January 1, 2014 and June 30, 2016 in the six ICCs which had been part of the CRAC registry (n = 1886). One ICC was excluded because of incomplete data. Non‐culprit lesion was defined as ≥50% diameter stenosis by visual estimate in at least one non‐infarct related vessel. Patient characteristics, management, and outcomes were analyzed according to coronary status . To define CAD extent, all three coronary arteries were assigned one point each and two points for left main coronary artery (LMCA) whatever the status of left anterior descending and left circumflex, resulting in a maximum score of 3  in patients without a history of coronary artery bypass grafting (CABG). Patients with previous CABG were considered as three‐VD (n = 25). Multivessel CAD was defined as 2‐ or 3‐VD. Complete myocardial revascularization was considered in our analyses if the additional procedure was performed before discharge or during the first 3 months after index event. Complete myocardial revascularization was defined by successful PCI of all non‐culprit lesion(s) . The primary endpoint of the study was a composite of MACE at 1‐year defined as all‐cause death, re‐MI, or stroke.2.2The anonymous database includes up to 150 variables per procedure with hospital follow‐up data and at 1 year for any coronary angioplasty and pre‐hospital data for STEMI <24 h.Patient follow‐up was conducted by local research technicians on site at the participating centers. Major complications such as death, intrastent thrombosis, re‐MI, unplanned coronary revisions, major hemorrhage (BARC 3), and stroke were collected at discharge and after 1 year (telephone contact), in the form of antithrombotic therapy and cardiac rehabilitation. The 1‐year telephone call was made with direct access to medical and administrative information, which simplified and accelerated the process. In addition, the data collected was limited, focusing on MACE complications (which were expected to be low). Follow‐up at 1‐year was obtained in 95%. The data are anonymized before being automatically transferred to the central database of CRAC‐France PCI.2.3The study was conducted according to contemporary clinical practice guidelines and French regulations . The French Persons Protection Committee (IRB00003888) approved the study protocol (no. 15–231). Data file collection and storage were approved by the French National Commission for Data Protection and Liberties (no. 2014–073). All patients were informed of the aims of the survey. All included patients gave their informed consent to participate before data collection.2.4SDs) or medians and interquartile ranges (IQR), when appropriate. Discrete variables are described as counts and percentages. Groups were compared by analysis of variance for continuous variables and χ2 (or Fisher exact tests) for discrete variables. Hazard ratios (HR) are presented with their 95% confidence intervals (CI). Survival curves were estimated using the Kaplan Meier estimators and compared using log rank tests. The rates of MACE at 1‐year were analyzed according to number of VD, and the impact of MVD  was compared using a multivariate backward stepwise Cox analysis with a threshold of 0.10 for variable elimination, among the different risk groups. Variables included in the final models were selected ad hoc, based on their physiological relevance and potential to be associated with outcomes; they comprised age, gender, risk factors, comorbidities, year, and management. Two sensibility analyzes were performed in survival population after index hospitalization; and, in MVD patients according to complete myocardial revascularization. In addition, we repeated the multivariate analysis using a composite endpoint focused on cardiac clinical outcomes  to better evaluate the role of MVD on specific cardiac clinical outcomes. Analyses were repeated using forward stepwise analysis to check the consistency of the results. Statistical analyses were performed using IBM SPSS 26.0 (IBM SPSS Inc). For all analyses, 2‐sided p values <.05 were considered significant.Continuous variables are reported as means . The rates of TIMI score 0/1 of the culprit lesion before primary PCI was similar in all groups. Procedural characteristics are detailed in Table All patients had an invasive strategy and were referred to a cardiac catheterization laboratory. Coronary angiogram showed that the site of the culprit lesion differed according to coronary status  Table . PatientAntithrombotic treatment used before admission and medications prescription at discharge are given in Table Finally, duration of dual antiplatelet therapy after AMI was mainly ≥12 months whatever the coronary status Figure .3.3In‐hospital complications  did not differ according to coronary status as the case for the rate of in‐hospital death  Table .p = .28) . The rate of death was higher in patients with MVD compared with one‐VD patients (10% vs. 7%), while the rate of stroke and re‐MI were similar in all groups. Major bleeding was observed similarly in all groups (2%). Finally, the use of urgent myocardial revascularization was higher in MVD patients .The proportion of MACE at 1‐year was 11% in the overall population and was not significantly different in patients with one‐VD (10%), two‐VD (12%), and three‐VD (12%) (p < .001).In patients with MVD, the rate of MACE at 1 year was 5.2% in patients with complete myocardial revascularization and 14.4% in those without complete myocardial revascularization   at 1 year compared with those with culprit lesion alone .4.1During the last 30 years, several registries specific to AMI and large administrative or billing databases have shown a decrease in mortality in patients with STEMI.Improved survival among STEMI patients was observed in all categories of patients over the last 30 years.4.2Primary PCI is the preferred reperfusion strategy in patients with STEMI within 12 h of symptom onset.In addition, our data show that patients with MVD received more aggressive secondary medication prevention at discharge and the proportion of DAPT at 1 year was numerically higher in this population.4.3As in any observational study, there are limitations to our analysis. Only STEMI ≤24H patients admitted to a cardiac catheterization laboratory were included, which represents a selection bias. Several data are missing in the database to better define the study groups such as atrial fibrillation (AF). Recent data have shown that patients with AF have generally less severe CAD compared to non‐AF ones.5MVD in STEMI patients still represents half of the patients despite a substantial change in the patient risk profile. However, the prognosis of patients with two or three VD is not associated with higher rate of MACE  at 1‐year compared to patients with single‐VD.None of the companies had a role in the design and conduct of the study, data collection, and management. Etienne Puymirat has received research grants/consultant fees/lectures fees: Amgen, Astra‐Zeneca, Abbott, Bayer, Biotronik, BMS, Boehringer Ingelheim, Daiichi‐Sankyo, Lilly, MSD, Novartis, Sanofi; Nicolas Danchin has received grants, personal fees, and non‐financial support from Amgen, AstraZeneca, Bayer, Bristol‐Myers Squibb, and Sanofi and personal fees from Boehringer Ingelheim, Intercept, Merck Sharp & Dohme, Novo Nordisk, Pfizer, and UCB Pharmaceuticals. Guillaume Cayla has received research grants/consultant fees/lectures fees from Amgen, AstraZeneca, Abbott, Bayer, Biotronik, Bristol‐Myers Squibb, Pfizer, Sanofi‐Aventis; Gilles Montalescot reports consulting or speaker fees from Abbott, AIM group, Amgen, Actelion, American College of Cardiology Foundation, Astrazeneca, Axis‐Santé, Bayer, Boston‐Scientific, Bristol‐Myers Squibb, Beth Israel Deaconess Medical, Brigham Women's Hospital, Fréquence Médicale, ICOM, Idorsia, Elsevier, Fédération Française de Cardiologie, Fréquence Médicale, ICAN, Lead‐Up, Menarini, Medtronic, MSD, Novo‐Nordisk, Pfizer, Quantum Genomics, Sanofi‐Aventis, SCOR global life, Servier, WebMD; Ariel Nakache, Christophe Saint Etienne, Pierre Marcollet, Olivier Fichaux, Marie‐Pascale Decomis, Stephan Chassaing, Philippe Commeau, Hakim Benamer, Rene Koning, Pascal Motreff, and Grégoire Rangé have no conflict of interest.Table S1. Antithrombotic used during prehospital management and medications prescribed at discharge.Figure S1: Flow chart.Figure S2: Dual antiplatelet therapy duration according to number of vessel disease.Click here for additional data file.