Neuroinflammation has gained increasing attention as a potential contributing factor in the onset and progression of Alzheimer’s disease (AD). The objective of this study was to examine the association of selected cerebrospinal fluid (CSF) inflammatory and neuronal degeneration markers with signature CSF AD profile and cognitive functions among subjects at the symptomatic pre- and early dementia stages.n = 28, age = 70, 39% female, Mini-Mental State Examination [MMSE] = 27) or non-AD  profile based on the ratio between CSF total-tau (T-tau) and amyloid-β1–42 (Aβ42) values (cut-off point chosen as 0.52). Novel CSF biomarkers included neurofilament light (NFL), YKL-40, S100 calcium-binding protein B (S100B) and glial fibrillary acidic protein (GFAP), measured with enzyme-linked immunosorbent assays (ELISAs). Subjects underwent neuropsychological assessment for evaluation of different cognitive domains, including verbal episodic memory, non-verbal episodic memory, language, processing speed, and executive functions.In this cross-sectional study, 52 subjects were selected from an Icelandic memory clinic cohort. Subjects were classified as having AD  overall, while GFAP correlated with processing speed  and NFL with verbal episodic memory  among subjects with a CSF AD profile.Accuracy coefficient for distinguishing between the two CSF profiles was calculated for each CSF marker and test. Novel CSF markers performed poorly (area under curve [AUC] coefficients ranging from 0.61 to 0.64) compared to tests reflecting verbal episodic memory, which all performed fair (AUC > 70). LASSO regression with a stability approach was applied for the selection of CSF markers and demographic variables predicting performance on each cognitive domain, both among all subjects and only those with a CSF AD profile. Relationships between CSF markers and cognitive domains, where the CSF marker reached stability selection criteria of > 75%, were visualized with scatter plots. Before calculations of corresponding Pearson’s correlations coefficients, composite scores for cognitive domains were adjusted for age and education. GFAP correlated with executive functions (The novel CSF markers NFL and GFAP show potential as markers for cognitive decline among individuals with core AD pathology at the symptomatic pre- and early stages of dementia. In recent years, a paradigm shift in the research criteria of Alzheimer’s disease (AD) has occurred as the primary focus has shifted from clinical to biological criteria. The emphasis is now on the pathology , which iIncreasing evidence shows that inflammation is a contributing factor in the pathogenesis and development of AD and other neurodegenerative diseases , 9. A nuThe glial proteins YKL-40 , S100 calcium-binding protein B (S100B), and glial fibrillary acidic protein (GFAP) have previously been associated with AD pathology . All are42 [42 among elective surgery patients free from dementia and delirium. Ishiki et al. [Inflammation in the brain and its role in AD can be studied indirectly through the analysis of CSF proteins. Increased levels of CSF YKL-40, S100B, and GFAP have been observed in AD patients compared to healthy controls, although results have not been consistent . The rel42 , 27–29. 42 , further42 , 31. Few42  found ani et al.  did not Loss of memory is typically among the first clinical symptoms of AD, marking the beginning of cognitive decline. The medial temporal lobe is an early site of tau accumulation, and its dysfunction may underlie episodic memory decline . Other c1–42 and elevated CSF tau). It is essential for the evaluation of novel biomarkers to examine their relationship with both entities separately, independent of diagnosis. That type of approach could enhance both understanding of the underlying pathology of AD and the sequence of events leading to cognitive impairment. The first aim of this study was to assess the ability of glial  and neurodegeneration (NFL) markers in CSF to discriminate between different CSF profiles (AD and non-AD) among subjects at the symptomatic pre- and early stages of dementia. In addition, the results were compared to the discrimination ability of neuropsychological tests, which are commonly used to aid AD diagnosis. The second aim was to investigate the relationship between the CSF markers with neuropsychological tests reflecting different cognitive domains.In the most recent research criteria from the International Working Group for the diagnosis of AD published in 2014 , the dian = 218). The exclusion criteria were (1) cognitive impairment that, without a doubt, could be explained by a condition other than dementia; (2) difficulties participating due to health or social issues; and (3) residency outside the Reykjavík Capital Area. In entering the study, each subject underwent various assessments, including a standard clinical and neuropsychological assessment and brain magnetic resonance imaging (MRI) for evaluation of medial temporal lobe atrophy (MTA). Lumbar puncture for collection of CSF, which was optional by the requirement of the National Bioethics Committee, was also carried out. For this particular study . The final sample included 52 subjects as four were removed due to excessively high value on CSF GFAP (n = 1) or blood-contamination in the CSF sample (n = 3). Clinical diagnosis of AD was based on the criteria for probable AD dementia defined by the National Institute on Aging-Alzheimer’s Association (NIA-AA) [Individuals, referred to The National University Hospital of Iceland Memory Clinic during a 4-year period which had 1) a score between 24 and 30 on the Mini-Mental State Examination (MMSE) and (2) a score of 4.0 or less on the Informant Questionnaire on Cognitive Decline in the Elderly (IQCODE) , were in a score (NIA-AA) , with ev(NIA-AA) . MCI dia(NIA-AA) , with th42  were carried out in the ISO 15189 accredited medical laboratory MVZ Labor P.D. Dr. Volkmann und Kollegen GbR . Assays for novel markers NFL , YKL-40 , S100B , and GFAP  were performed in technical duplicates and according to the manufacturer’s instructions in a laboratory at the University of Iceland. The mean Intra-assay CV was < 10% and mean Inter-assay CV < 15% for all assays.CSF was collected via lumbar puncture with a 22-gauge spinal needle at the L3/4 or L4/5 interspace. Uncentrifuged samples were frozen in 2-ml polypropylene tubes and stored at − 80 °C. Commercially available sandwich enzyme-linked immunosorbent assays (ELISAs) were used for measurements of all proteins. Analyses of core AD markers T-tau  and Aβ42 values. T-tau/Aβ42 ratio cut-off of 0.52 was chosen based on results from a large memory clinic cohort study [42 ratio > 0.52. The same ratio was also used as a part of the clinical diagnosis of AD, explaining full concordance with CSF AD profile.Each subject was classified independently of clinical diagnosis on the basis of CSF T-tau and Aβrt study , giving All subjects underwent a detailed neuropsychological assessment performed by licensed psychologists. Five cognitive domains, commonly affected by aging and AD, were assessed using seven tests Table . For theU tests and chi-square tests for continuous and categorical variables, respectively. Raw values of CSF measures and selected neuropsychological tests  were naturally log-transformed to account for a non-normal distribution. Composite scores for each cognitive domain were calculated by averaging neuropsychological test z-scores and subsequently converting those scores into z-scores. Before the computation of composite scores, z-scores for tests measuring reaction time were reversed  for the purpose of test consistency . Receiver operating characteristic (ROC) curves were constructed for the differentiation between CSF AD and non-AD profiles. The discrimination abilities of each CSF marker and cognitive domain were compared using the area under the curve (AUC) method, according to DeLong et al. [z-score for each cognitive domain. Two LASSO regressions with a stability selection were performed for each cognitive domain, one which included all subjects and the other, which only included those with a CSF AD profile. Scatter plots were used for visualization of the selected relationships between CSF markers and cognitive domains. Cognitive domain measures were adjusted for age and education before the calculations of corresponding Pearson’s correlations coefficients. For the adjustment, linear regression models were created with each composite test z-score as the dependent variable and age and education as independent variables. The residual for each subject was subsequently calculated (observed minus predicted score). Significance values were not adjusted for multiple comparisons, as this study was viewed as explorative with emphasis on discovering relationships. All statistical analyses were performed using R .Descriptive group comparisons were performed using Mann-Whitney g et al. . The AUCg et al. . LASSO ig et al. , 51. Cutp < 0.05).Table r = 0.62, p < 0.001). GFAP did only significantly correlate with the CSF marker S100B . No CSF markers correlated significantly with Aβ42. All the CSF markers, except for Aβ42, correlated positively with age. Length of education correlated weakly and negatively with T-tau .Pearson’s correlations between the CSF markers, age, and length of education are presented in Fig. n = 19) compared to men (n = 33). LASSO logistic regression with stability selection was performed for the selection of variables distinguishing between the CSF profile groups with the highest consistency. Nine possible predictors could be selected, the four novel CSF markers and the five composite tests presenting each cognitive domain. Only the test reflecting verbal episodic memory was selected as a predictor, with selection frequency (96%) above the cut-off value. All other possible predictors had a much lower selection frequency (≤ 20%).Accuracies for distinguishing between CSF AD and non-AD profiles were based on univariable ROC analyses Table . AUCs foFigure n = 52) and the other only among those with a CSF AD profile (n = 28). Variables with stability selection above 75% were considered reliable predictors. GFAP (78%) was selected as a predictor for executive functions  predicting cognitive scores with the highest consistency Fig. . Two anaons Fig. a and ageons Fig. b within ons Fig. c and NFLons Fig. d. No varons Fig. e.Fig. 4z-scores were therefore adjusted for age and education prior to Pearson’s correlations calculations.Relationships between CSF measures and cognitive domains, as selected with LASSO regression—stability selection analyses Fig. , were vir = − 0.26, p = 0.06, Fig. r = − 0.43, p = 0.02) compared to none among those without . Correlations between the NFL levels and individual neuropsychological tests reflecting verbal episodic memory are presented in Additional file r = − 0.28, p < 0.04, Fig. r = − 0.15, p = 0.45) when analyzed by CSF profile  and inflammation  in relation to core CSF AD markers and cognitive functions in a cohort of subjects at the pre- and early symptomatic dementia stages. While our results indicated that these CSF markers did not accurately distinguish between AD and non-AD CSF profiles, they exhibited different patterns of association with certain cognitive domains, as evaluated by various neuropsychological tests. This pattern was mainly observed among subjects with a CSF AD profile. Within that group, levels of the neurodegeneration marker NFL associated with verbal episodic memory while inflammatory marker GFAP associated with processing speed. In addition, GFAP associated weakly with executive functions within the whole cohort. Overall, these results indicate that CSF NFL and GFAP levels do relate to cognitive functions, specifically among those with a CSF AD profile.42, in accordance with previous studies [42 levels. In contrast, GFAP did not correlate with the CSF neurodegeneration markers nor with CSF Aβ42 levels. Neither CSF S100B nor GFAP have been much studied in terms of correlation with CSF core AD markers. Hov et al. [42 in CSF. Ishiki et al. [Both CSF NFL and YKL-40 levels correlated with T-tau but not with Aβ studies –56; ther studies , 57, 58.v et al.  found sii et al.  did not The neuropsychological tests reflecting verbal episodic memory did show the best accuracy in differentiating between the CSF profiles out of all the evaluated cognitive measures and the novel CSF markers. The accuracy was good for the composite score of verbal episodic memory and RAVLT delayed recall test (80%), but fair for all the other verbal episodic memory tests (between 70 and 80%). A recent meta-analysis  based onn = 34) were at the SCI or MCI stages, with 23 of those without a CSF AD profile.CSF markers related in different ways to cognitive measures. Both CSF NFL , 60 and 42 and T-tau appear to associate with memory and executive functions in some studies [Knowledge regarding the relationship between core CSF biomarkers and cognition remains incomplete. Overall, Aβ studies , 62, alt studies , 63, 64. studies .Increased CSF levels of inflammatory marker GFAP was found weakly associated with worse performance on tests reflecting executive functions, both within the whole cohort and among subjects with CSF AD profile. Few studies have examined the relationship between CSF GFAP levels and cognitive functions. Ishiki et al.  did not Associations between selected CSF markers and cognitive domains were also examined within each CSF profile. CSF NFL levels moderately related to verbal episodic memory among those with CSF AD profile but not among those without. Higher levels of CSF GFAP also moderately associated with worse performance on processing speed only within the CSF AD profile group. This is of interest because the CSF markers did not directly relate to the CSF AD profile (ability in discriminating between CSF profiles was poor). This outcome could possibly be explained by the additive effects of distinctive processes on cognitive functions. A previous study  showed aThis study has several limitations. First, the sample was relatively small, and hence, present findings need to be validated in a larger study. The sample did not include healthy controls, which could underestimate associations between the studied variables. Another limitation of the study is the lack of information about the ApoE genotype. However, it is unlikely that the ApoE genotype affects the outcome as previous studies have suggested that ApoE ε4 status does not influence CSF NFL or YKL-40 levels , 68, 69.Our findings suggest that levels of CSF markers NFL and GFAP relate to different cognitive profiles at the symptomatic pre- and early dementia stages. The relationships between the levels of NFL with verbal episodic memory and GFAP with processing speed were only observed among those with CSF AD profile, although the CSF markers did not directly relate to the CSF AD profile. These CSF markers could be of potential use as progression markers, monitoring subtle cognitive changes at the earliest symptomatic stages of dementia among those with AD pathology. Further studies with bigger group sizes are needed to validate these results and to evaluate their potential in tracking changes in the more advanced stages of AD and other types of dementia.Additional file 1: Figure 1. Levels of CSF NFL, YKL-40, S100B and GFAP by CSF profile. Table 1. Univariable ROC analysis for distinguishing between CSF profile groups stratified by gender. Figure 2. Pearson’s correlations between levels of CSF NFL with neuropsychological tests reflecting verbal episodic memory by CSF profile. Figure 3. Pearson’s correlations between levels of CSF GFAP with neuropsychological tests reflecting processing speed and executive functions by CSF profile. Figure 4. Pearson’s correlations between CSF levels of NFL and T-tau with verbal episodic memory and GFAP with processing speed and executive functions, within the whole cohort and by CSF profile. Table 2. Pearson’s correlations between CSF markers, age, education and composite z-scores reflecting cognitive domains.


Busulfan is an alkylating agent routinely used in conditioning regimens prior to allogeneic hematopoietic cell transplantation (HCT) for various nonmalignant disorders, including inborn errors of metabolism. The combination of model-based dosing and therapeutic drug monitoring (TDM) of busulfan pharmacokinetics (PK) to a lower exposure target has the potential to reduce the regimen-related toxicity while opening marrow niches sufficient for engraftment in diseases such as mucopolysaccharidosis type I (MPS I). We present four cases of the severe form of MPS I or Hurler syndrome, demonstrating successful and stable CD14/15 donor chimerism following the prospective application of model-based dosing and TDM aimed to achieve lower busulfan exposure. All patients received a busulfan-based conditioning regimen with a median cumulative area-under-the-curve (cAUC) target of 63.7 mg h/L  in protocol-specific combination of chemotherapeutic regimen. The donor source was unrelated umbilical cord blood for three patients and matched sibling donor bone marrow for one patient. The observed median busulfan cAUC was 66.1 mg h/L  and was within 10% of the intended target. Stable, full donor myeloid chimerism was achieved for three patients, while one patient achieved a stable mixed chimerism (76% donor CD14/15 at 53 months) without a recurring need for enzyme replacement. The normalization of α-L-iduronidase enzyme levels followed the attainment of successful donor myeloid chimerism in all patients. Regimen-related toxicity remained low with no evidence of acute graft-versus-host disease (GVHD) grades II to IV and chronic GVHD. The Hurler syndrome is considered the more severe type of MPS I ,2,3,4,5. of life ,2,3,4,5.l-iduronidase enzyme levels.Combination pretransplant conditioning regimen prior to allogeneic HCT with variable degrees of myeloablation have been used in the treatment of MPS I, with the intensity of conditioning associated with both HCT success and regimen-related toxicity ,5. One ol-iduronidase enzyme, and confirmation of pathogenic mutations (Four pediatric patients (2 boys and 2 girls) with newly diagnosed severe MPS I who met the inclusion criteria were enrolled . Diagnosutations . Three putations . All patThe patient-specific conditioning protocols are depicted in l iduronidase levels was followed to assess engraftment and correction of enzyme deficiency following HCT, respectively. Patients 1, 2, and 3 achieved early and stable engraftment as indicated by 99% donor myeloid chimerism both at 1-month and last recorded .l-iduronidase activity. At present, all patients are receiving long-term multi-disciplinary care with close follow-up of donor chimerism and routine monitoring of α-l iduronidase enzyme levels.As of the last follow-up evaluation, all four patients continued to have stable donor chimerism and normal peripheral-blood α-l-iduronidase enzyme levels, the key enzyme deficient in MPS I. None of the patients experienced severe regimen-related toxicity as well as no acute or chronic GVHD. To our knowledge, this is the first report demonstrating that prospective targeting of a lower busulfan cAUC of 65–70 mg h/L is sufficient to achieve a stable donor chimerism and the normalization of α-l-iduronidase enzyme levels in severe MPS I patients undergoing HCT. While these patients did not experience unexpected acute regimen-related toxicity at the time of preparing this report, the need for follow-up to assess long-term regimen-related toxicity and late graft failure [We describe the successful application of model-based dosing and PK-guided low-exposure busulfan-based conditioning in four pediatric patients who underwent allogeneic HCT for newly diagnosed severe MPS I. This approach led to stable CD14/15 donor chimerism, which was associated with normalization of the α- failure  requiresl-iduronidase enzyme levels are three of the most critical factors contributing to the overall long-term success following allogeneic HCT for MPS I [The baseline developmental status before transplantation, age at transplantation, and α-or MPS I ,8,11,12.or MPS I ,6,11,12.or MPS I ,4. ConsiRetrospective studies of large patient cohorts with MPS-1 showed preference for HLA-matched unrelated UCB units based on a relatively faster availability of UCB, better donor chimerism than those engrafted with bone marrow ,8,11 andAutoimmune cytopenias are a well described complication of UCB HCT ,15. A reThe long-term toxicities of myeloablative busulfan exposure in pediatric patients are well established ,19. Whill iduronidase enzyme levels early following HCT, a lower busulfan exposure appears sufficient to effectively facilitate engraftment of donor hematopoietic stem cells. Limiting busulfan exposure to decrease toxicity while maintaining the efficacy of the regimen is the ideal goal in transplanting children with inherited metabolic disorders. Our results in these four cases seem to support the notion that a lower busulfan exposure used in combination with fludarabine may be a suitable alternative to higher busulfan exposure HCT for children with MPS I.As demonstrated by high levels of donor CD14/15 chimerism and normalization of α-l-iduronidase enzyme level is a good predictor of outcomes following allogeneic HCT [The normalization of α-neic HCT ,7,12,24,neic HCT ,25. Factneic HCT ,12,24,26neic HCT .l-iduronidase in peripheral-blood leukocytes, genetic testing for pathogenic mutation, and the clinical phenotype. All patients and/or guardians provided written informed consent to participate in the routine TDM of busulfan as part of their specific transplant protocol. Consent for participation in the PK analysis was waived as part of the University of California San Francisco Committee on Human Subjects’ Research approval process. Eligibility criteria for the inclusion of individual busulfan PK data in this study included (1) subjects 1–26 years of age at the time of HCT; (2) met institutional and protocol specific eligibility criteria for allogeneic HCT that included intravenous busulfan therapy and; (3) busulfan plasma time-concentration data were available for analysis.This was a single-center retrospective study comprised of 4 children affected by MPS I who underwent allogeneic HCT at the University of California San Francisco Benioff Children’s Hospital between January 2012 and August 2018. The diagnosis of Hurler’s syndrome was confirmed on the basis of the activity of α-7 per kilogram of body weight) and with high-normal leukocyte α-l-iduronidase activity that matched at least six of eight HLA loci was selected. Standard clinical laboratory procedure was used to prepare UCB cells for transplantation, including thawing, quantifying, and assessing the viability of the nucleated cells, clonal hematopoietic progenitor cells, and CD34+ cells; bone marrow was processed as per the cell therapeutic laboratory standard operating procedure.If a HLA-matched sibling donor (MSD) who was not a carrier of the disease was available, this was the donor of choice. Otherwise, a unit of cord blood (UCB) with the highest number of nucleated cells  or 24 h (patient 3 and 4) as outlined in the predefined protocol-specific combination pre-transplant conditioning regimen. The timing for collection of busulfan PK samples was based on the dose interval and methodology for AUC estimation as previously described . Brieflyobs = new dose in mg/desired AUCtarget.Dose administered in mg/AUCFor patients 3 and patient 4, the first dose and cAUC was estimated using a validated population pharmacokinetic model and nonlinear mixed effects modeling using model-informed precision dosing platform ,28,29. Cl-iduronidase enzyme level post-HCT. Chimerism of peripheral blood (PB) samples was performed for all patients at regular intervals following HCT using Short Tandem Repeat (STR) markers, as previously described [The primary outcome of interest was a donor CD14/15 chimerism and normalization of the α-escribed . The cell-iduronidase enzyme levels in patient’s blood was performed by the lysosomal diseases testing laboratory at the Thomas Jefferson University, Philadelphia, and activity is expressed in nmol/h/mg protein.Neutrophil engraftment was defined as the first of three consecutive days with an absolute neutrophil count (ANC) ≥ 500/μL. Hepatic sinusoidal obstruction syndrome (SOS) was scored according to EBMT Criteria . MucositGraphPad Prism  software was used to perform descriptive statistics and generate graphs. l-iduronidase enzyme and with no significant regimen-related acute toxicity in four pediatric patients with newly diagnosed severe MPS I. Long-term follow-up will be required to assess the durability of the donor chimerism and the persistence of the normal levels of α-l-iduronidase enzyme and to monitor potential late effects of busulfan conditioning.In conclusion, lower exposure busulfan in combination with fludarabine provided stable multi-lineage engraftment, with prompt and durable normalization of the α-


Infertility is one of the major medical problems nowadays. Couples who opt for In Vitro Fertilization (IVF) face a great deal of stress which certainly affects the outcome of the procedure. Therefore, we aimed to reduce the stress during the oocyte retrieval procedure by applying midazolam. Total oxidant (TOC) and antioxidant (TAC) capacities of serum, as well as glutathione (GSH) content and catalase activity, were measured in both control and midazolam groups. Follicular fluid was also tested for oxidant capacity and IL1β. Results implied that the midazolam group increased TAC at the end of the procedure. At the same time, the control group decreased GSH at the beginning of the procedure, and both groups decreased catalase activity at the end of the procedure. The results imply that stress during the procedure affects oxidative and antioxidative parameters of the patients, but did not affect the frequency of the pregnancy at the end of this pilot study. Yet, the results imply that oxidative and antioxidative mechanisms during IVF should be investigated in detail as they could affect the outcome of IVF. Today, infertility is recognized as a growing medical problem, affecting 9% to 15% of the world’s fertile population, with the number of affected couples in Europe reaching up to 25 million . The IVFOxidative stress is a disturbed balance between oxidative and reductive processes in cell, organ and organism . This oxThe main objective of this study was to determine whether premedication with midazolam prior to oocyte retrieval affects the oxidative stress in subjects undergoing IVF by measuring oxidative stress markers in paired samples of blood serum and follicular fluid at different time points during the procedure.This research was conducted in University Hospital Center “Sestre milosrdnice”. The study was approved by the Ethics Committee of UHC “Sestre milosrdnice” (EP-2258/18-3). The pilot study is registered at DRKS – German Clinical Trials Register, as a primary registry in the WHO Registry Network (Registration ID: DRKS00021657) and the ClinicalTrials.gov, ID number: NCT0416061. The procedure was explained in detail to the patients, after which the patients signed written informed consent. The total of 60 patients who underwent IVF procedure were randomized into two groups (30 patients per group): the control group and the group that received midazolam orally before the oocyte retrieval procedure. The two groups had no significant differences in clinical parameters (described in detail in the results). Blood samples from each patient were collected on admission to the hospital, before midazolam administration (time point A), 40 ± 5 min after midazolam administration, immediately before intravenous anesthesia for oocyte retrieval, (time point B), and 2 h after the procedure (time point C). Blood was then stored as whole blood, or serum at −80 °C until analyses. Retrieval A sample of follicular fluid was also collected during the oocyte and stored at −80 °C until analyses. Blood sampling was adjusted to the pharmacokinetics of midazolam and was incorporated into the medical protocol for oocyte retrieval. By comparing the groups at the first time point (A), we controlled for the uniformity of the stress response. The second time point (B) indicated the way midazolam affected the stress response at its maximum effective concentration. The third time point (C) indicated whether there was a residual effect on the stress response in the subjects’ plasma after midazolam ceased to be active.Total oxidative capacity (TOC) is a colorimetric assay based on the cascade reaction of peroxides in the sample (serum and follicular fluid) with peroxidase, which produces oxygen, which in turn oxidizes tetramethyl-benzidine  . The lasTotal antioxidant capacity (TAC) is measured by a similar reaction to TOC, the serum samples are incubated with hydrogen peroxide which is removed by endogenous antioxidants. This is followed by the reaction of the remaining hydrogen peroxide with peroxidase and addition of TMB . The rea2PO4, 5 mM EDTA pH 7.4). The total GSH content was detected as yellow product, 2-nitro-5-thiobenzoic acid, and measured on the plate reader at 405 nm . GSH levels were measured in whole blood lysate by the modified method by Tietze . The rea2O2 is added to start the reaction. The reaction is left for 5 min and then stopped by adding 100 μL of 200 mM ammonium molybdate and the colour development was measured spectrophotometrically in a plate reader at 450 nm . The amount of degraded H2O2 is calculated from the standard curve (0 to 75 mM H2O2). One unit of catalase activity is defined as the amount of enzyme needed for degradation of 1 μmol of H2O2/min at 25 °C. Catalase activity was expressed as units per milligram of proteins in the cell lysate (U*mg−1).Catalase activity was measured from the whole blood lysate according to the modified method by Goth . The asst-test for significance between the two groups and Fisher’s exact test was used to analyze the differences between hormonal status and positive outcome of IVF. Values of p lower than 0.05 were considered as statistically different.Statistical analyses were performed using Prism GraphPad 8.0 software , and the methods used were Student’s As patients were randomly assigned to each group, the two groups were analysed for possible differences in age, hormonal status , and body mass index (BMI). The two groups showed no significant difference in any of the monitored parameters .Total oxidant capacity (TOC) was measured in serum and in follicular fluid at three-time points in both groups. There were no significant differences between the control group and the midazolam group at any of the observed time points in the serum and follicular fluid samples .p = 0.0072). However, there was no significant difference between control and midazolam groups in serum.Total antioxidant capacity (TAC) was measured in serum at three time points in both groups , whereasp = 0.04). GSH levels at all time points in the control group were not significantly different compared to the midazolam group . Similar to GSH, catalase activities at all time points in the control group were not significantly different from those in the midazolam group , 8/30 (27%) were in the control group and 3/30 (10%) were in the midazolam group. When the distribution frequencies were analysed, the exact Fischer’s test showed no difference between control and midazolam groups. Again, we have analysed the differences in hormonal parameters between pregnancies in the control and midazolam groups and found no significant difference .In order to attribute the observed differences to midazolam administration, we first eliminated possible sources of bias by ensuring that midazolam administration was the only different intervention between the groups studied. The random selection revealed no statistically significant differences between the two groups in factors shown to influence the outcome of the procedure. We hypothesized that the anxiolytic and sedative effects of midazolam would reduce systemic stress during oocyte retrieval in IVF. A reduction in systemic stress would then produce a measurable difference in markers of oxidative stress in patients’ serum and follicular fluid, and thus have a potential beneficial effect on the outcome of IVF. To date, no studies have investigated the effect of premedication in IVF procedures, making a comparative analysis of our results difficult.Oxidative stress is a shift of equilibrium in oxidation-reduction processes toward oxidation . To prevMidazolam, as a benzodiazepine drug, reduces neuronal excitability by increasing the efficiency of the brain neurotransmitter gamma-aminobutyric acid (GABA) and thereby reducing anxiety and sedation . Our hypData suggest that the immune status contributes greatly to fertility and that, in IVF, follicular fluid immune status contributes significantly to the success of the procedure . TherefoThe results showed a consistent trend of stress markers reduction in serum, whole blood, and follicular fluid, but are not equivocal and do not confirm either positive or negative effects of midazolam at the biochemical level or at the level of a positive outcome of the IVF procedure. Our study is a pilot study conducted on a small number of patients, with an even smaller number of positive outcomes. We aimed here to compare the standard treatment with a novel approach based on a possible additive effect of midazolam premedication. In addition, due to a limited number of patients, it was difficult to confirm with certainty the statistical trends observed. This study points to the need to investigate the effects of axiolysis during IVF on the outcome of this procedure. This is of importance, especially since patients may use anxiolytics either by themselves or by advice of their MD.


Conclusions: Our results demonstrate a strong correlation between POCUS TT localization measurements and traditional measurements via fluoroscopy. This study further supports the utility of POCUS for pediatric care.Introduction: A non-radiographic technique to measure the location of the tracheal tube (TT) in children is of value given the risk of inappropriate TT placement along with concerns about radiation exposure. Airway point-of-care ultrasound (POCUS) has demonstrated utility in children, but the examinations vary by age and may require non-traditional techniques or utilize less common probes. This study evaluated the performance of measuring the tracheal location of the cuffed TT using a novel, linear probe-based POCUS examination over a wide age range of children. After adjusting for the subjects’ height and TT size, ultrasound measurements of the TT cuff location were compared with fluoroscopy measurements of the TT tip location. Methods: Perioperative pediatric patients (<10 years) requiring a cuffed TT were enrolled. After routine TT placement, ultrasound and fluoroscopy images were obtained. Measurements from the TT cuff to the cricoid cartilage were obtained from the POCUS examination. Chest fluoroscopy was reviewed to measure the TT’s distance from the carina. Both measurements were then compared after scaling for patient height. The duration of the ultrasound examination and image quality scores were also recorded. Results: Forty-one patients were enrolled, with a median age of 3 (25th/75th percentile: 1.50/7.00) years. The POCUS examination identified the TT cuff in all cases with the highest image quality score. The median POCUS exam time was 112 (25th/75th percentile: 80.00/156.00) seconds. There was a strong correlation between the POCUS measurements and the fluoroscopy measurements, r = −0.7575, 95% CI ,  Tracheal intubation is a critical skill for acute care providers working with children. One of the most important aspects of tracheal intubation is the proper positioning of the tracheal tube (TT). Previous studies have demonstrated the incidence of TT malposition to be between 15% and 38% ,3. AdditMalposition of the TT can result in serious consequences such as: (1) hypoxemia, (2) inadequate ventilation, (3) pneumothorax, (4) barotrauma, (5) atelectasis, and (6) the potential for inappropriate interventions such as chest tube placement. In neonates and children, these concerns are magnified, due to the smaller margin of error when positioning the TT. Additionally, flexion and extension have shown to impact TT positioning in this population .While not commonly used in the operating room, fluoroscopy has been used for confirmation of the correct position of the TT and has demonstrated benefits for the identification of TT malposition by allowing measurements of the placement of the TT within the trachea. . HoweverPoint-of-care ultrasound (POCUS) is a new modality which has demonstrated promise for the evaluation of appropriate TT positioning. For the perioperative setting, the principal investigator for this manuscript performed a randomized control trial in which adult patients were selected to have the TT placed either in the trachea or the R/L bronchi. This study compared the efficacy of detecting tracheal vs. bronchi TT position between stethoscope auscultation and a POCUS examination, termed PLUS (pulmonary tree and lung expansion ultrasound study). The results from this study demonstrate a significantly greater sensitivity and specificity in the PLUS exam vs. stethoscope auscultation . This stIn regard to children, the utility of POCUS for identifying the appropriate location of the TT has been demonstrated ,11,12. TThis study sought to evaluate a novel perioperative POCUS exam for pediatrics. The exam consisted of a modified PLUS exam  that included the evaluation of the dilation of the trachea with TT cuff inflation along with the evaluation of bilateral pleural sliding. The theoretical benefit of this exam is that it may provide both the identification of a mainstem intubation  and a method of measuring the location of the TT within the trachea . To evaluate the potential utility of this POCUS exam, TT location measurements obtained from a PLUS exam were compared with TT location measurements obtained from chest fluoroscopy over a wide age range of children under 10 years old. Our hypothesis was that a POCUS exam could be an alternative to chest fluoroscopy by demonstrating a strong degree of correlation in TT location measurements between the two modalities. Specifically, after adjusting for the subjects’ height and TT size, the authors evaluated the correlation between measurements from the cricoid cartilage to the TT cuff, obtained by POCUS, and measurements from the TT tip to the carina, obtained by fluoroscopy.This article is presented following the Standards for the Reporting of Diagnostic Accuracy (STARD) checklist methodology for the reporting of studies of diagnostic accuracy.The study was performed at Loma Linda University Medical Center after Institutional Review Board (IRB #5160372) approval from February 2017 to June 2018. Written informed consent and information release were obtained from parents or legal guardians prior to study participation. Assent was obtained from children aged 7 years or older who were able to read and understand a simplified description of the study. Patients scheduled for procedures in the cardiac catheterization laboratory were screened for this study. This patient population was selected for screening secondary to a current clinical protocol at the study institution for obtaining a chest fluoroscopy for the verification of appropriate TT placement prior to procedure initiation. Under this protocol, the fluoroscopic measurement of the TT tip’s distance from the carina was used to evaluate appropriate positioning. Additional inclusion criteria were children aged from birth to 10 years and general anesthesia with a cuffed TT. The exclusion criteria were emergency procedures, patients with known airway anomalies, and parents/guardians who were non-English- or non-Spanish-speaking.Ultrasound training: A total of four physician examiners  performed the POCUS examinations. Each examiner received one-on-one instruction with the lead author of the original PLUS examination (D.R.) and each examiner performed 25 examinations prior to performing the study exams.2O. The final component of the examination consisted of the placement of the probe on the left and right anterior chest walls, at the third rib space along the midclavicular line, to ascertain the presence of the pleural sliding sign. A graphic of the modified PLUS examination used for this study is shown in All patients underwent tracheal intubation via direct laryngoscopy. The TT was positioned by the anesthesiologists using traditional methods including capnography verification, standard American Society of Anesthesiology recommended monitors, and stethoscope auscultation. After appropriate TT placement was confirmed via these techniques, the POCUS and fluoroscopy images were obtained sequentially. All patients received ShileyTM cuffed tracheal tubes from Covidien . The trained physician examiners performed the POCUS examination using an institutionally purchased Sonosite X-Porte  ultrasound machine with a high-frequency linear probe 13–6 MHz). The examination consisted of the placement of the probe transversely on the anterior neck approximately 2 cm from the sternal notch. The probe was adjusted in the cephalad or caudad direction to identify the second tracheal ring and the esophagus location was then identified. The trachea was then placed in the middle of the ultrasound footprint and the probe was then rotated 90 degrees to achieve a long-axis cross-section of the airway anatomy . The probe was adjusted in either the cephalad or caudad direction such that the cricoid cartilage was identified on the end of the ultrasound image. The pilot balloon was inflated and deflated to identify the position of the TT cuff in the airway, as previously described , < 0.001) . SecondaThis study further supports the utility of POCUS for evaluating TT position and providing a method of measuring the location of the TT within the trachea over a wide age range of pediatric patients. Specifically, our results demonstrate a statistically significant correlation between a proven technique of measuring TT localization (chest fluoroscopy) and a more novel technique (point-of-care ultrasound). Additionally, this study demonstrates that after receiving structured training, users can perform the described POCUS examination (PLUS exam) within 3 min, 85% percent of the time, yielding high image quality. Future studies could directly evaluate the performance of POCUS as compared to conventional radiologic methods as a primary means for measuring the location of the TT within the trachea.The utility of POCUS for identifying the appropriate location of the TT in children has been demonstrated. In infants, the majority of these studies evaluated the distance between the tip of the TT, visualized with a curved low-frequency transducer, and the maximal curvature of the aortic arch . While aHowever, despite being a growing area of interest, the use of point-of-care ultrasound is not mainstream in the perioperative environment. This was nicely summarized in a recent position paper by a consensus group of international experts in the fields of cardiothoracic, general, and critical care, pain management, and regional anesthesiology. This group reported a “call to action” on this topic and emphasized the importance for our specialty’s societies to develop standards of training such that proficiency in POCUS is expected for our specialty in the United States . FurtherThis study has several limitations. First, it is important to acknowledge that this study was an observational study designed to assess the efficacy of TT location measurements obtained from a novel POCUS examination for children. The goal of this study was to evaluate whether POCUS could be used as another tool to measure the location of the TT, which has the primary benefit of not requiring radiation exposure. This concept should be applied to scenarios in which TT location measurements are useful. There was no component of this study that evaluated the utility of applying POCUS routinely for intubations. Secondly, all patients in this study demonstrated the appropriate positioning of their TT with primary placement, and so no direct evaluation of inappropriate TT placement was able to be performed. It is the authors’ hope that future studies can evaluate the utility of POCUS in this manner. Thirdly, as with most diagnostic tools, there is a possibility for error in the ability to both perform and interpret the exam. While this study demonstrated a high image quality for all POCUS examinations, only a few providers performed the ultrasound exams. Further studies are needed to evaluate the ability to train a larger group of providers in the PLUS exam. Also, the TTs used during this study were all from the same manufacturer; however, the authors feel that this should minimally impact the ultrasound and fluoroscopy measurements. Finally, the small sample size, wide age range of children enrolled, and inclusion of patients only undergoing cardiac catherization procedures are additional limitations. For this reason, we did not separate our analysis by any subgroup, such as age or gender. Indeed, the goal of this study was to demonstrate the feasibility of applying a single POCUS examination across a wide age range of children. Future studies with larger sample sizes are needed to evaluate the utility of applying the PLUS exam in these subgroups.This study supports the utility of using ultrasound as an accurate method of measuring the location of the tracheal tube within the trachea in a wide age range of children. Future studies could directly evaluate the performance of POCUS as compared to conventional radiologic methods as a primary means for measuring the location of the TT within the trachea.


Background: The role of cerebrospinal fluid (CSF) alpha-synuclein as a potential biomarker has been challenged mainly due to variable preanalytical measures between laboratories. To evaluate the impact of the preanalytical factors contributing to such variability, the different subforms of alpha-synuclein need to be studied individually.Method: We investigated the effect of exposing CSF samples to several preanalytical sources of variability: (1) different polypropylene (PP) storage tubes; (2) use of non-ionic detergents; (3) multiple tube transfers; (4) multiple freeze-thaw cycles; and (5) delayed storage. CSF oligomeric- and total-alpha-synuclein levels were estimated using our in-house sandwich-based enzyme-linked immunosorbent assays.Results: Siliconized tubes provided the optimal preservation of CSF alpha-synuclein proteins among other tested polypropylene tubes. The use of tween-20 detergent significantly improved the recovery of oligomeric-alpha-synuclein, while multiple freeze-thaw cycles significantly lowered oligomeric-alpha-synuclein in CSF. Interestingly, oligomeric-alpha-synuclein levels remained relatively stable over multiple tube transfers and upon delayed storage.Conclusion: Our study showed for the first-time distinct impact of preanalytical factors on the different forms of CSF alpha-synuclein. These findings highlight the need for special considerations for the different forms of alpha-synuclein during CSF samples’ collection and processing. A vast majority of studies done on neurological disorders use cerebrospinal fluid (CSF) as the biofluid of choice to explore and assess biomarkers as CSF protein levels most closely reflect the pathophysiology of the brain   and University Medical Center . The samples were pooled and aliquoted as 500 μl aliquots. Samples collected from the Biobank at VUmc were used in α-syn stability and adsorption experiments while those from University Medical Center Göttingen were used in investigations done on adsorption of α-syn. Aliquots were stored at −80°C in 1.5 ml polypropylene tubes with screw caps . All samples were blinded, and donors gave written informed consent at study entry for the use of clinical information and CSF material for scientific research purposes. The study was conducted according to the revised Declaration of Helsinki and Good Clinical Practice guidelines and approved by the local ethics committee of the VU University Medical Center.To assess the extent of adsorption of α-syn to the surface walls of storage tubes, three experimental approaches were followed: (1) we tested the difference in α-syn levels when using six different polypropylene (PP) types from different vendors (see n = 3). The first five tubes of each set were subjected to 1–7 freeze-thaw cycles (reference sample-1 freeze-thaw cycle). The remaining tubes were split into two sets which were then exposed to delayed storage times: 1, 2, 4, 24, 72, or 168 h at either 4°C or room temperature (RT) before finally storing them at −80°C .CSF pools were aliquoted into 19 (Sarstedt-0.5 ml) tubes per set  at 50 μl/well in 200 mM NaHCO3, pH 9.6 and incubated overnight at 4°C. Plates were washed thrice in phosphate-buffered saline with 0.05% Tween-20 (PBST) then blocked with 100 μl/well of blocking buffer (PBST with 2.5% gelatin) for 2 h at 37°C. After washing plates again, CSF samples and assay standards prepared as serial dilutions of recombinant human α-syn and o-α-syn in artificial CSF were added to the plates in duplicates at 50 μl/well and incubated at 37°C for 2.5 h. Detection antibodies, 11D12  against t-α-syn and FL-140  against o-α-syn were used at the diluted concentration of 1:5,000 and 1:1,000, respectively. Following a washing step, 50 μl/well of species-appropriate secondary antibodies: donkey anti-mouse IgG HRP or goat anti-rabbit IgG HRP , was added to the respective plates at the diluted concentration of 1:20,000 and incubate at 37°C for 2 h. After a final wash, the plates were incubated with an enhanced chemiluminescent substrate . The chemiluminescence (relative light units) was immediately measured using a PerkinElmer Envision multilabel plate reader .CSF total and oligomeric α-syn levels were measured using our previously described ELISA assays , and CSF α-syn concentrations were calculated by interpolating from the curves. The generated figures and as well as the calculations used to establish the curves generated and determine α-syn concentrations were carried out using GraphPad Prism 7.0. The variations in the α-syn concentration due to the type of storage tube used, repeated freeze-thaw cycles and tube transfers, and delayed storage are expressed as mean percentage change compared to the values of the reference samples. Normality tests were not productive due to the small sample size hence the statistical significance of observed variations was assessed through non-parametric analysis of variance (ANOVA) in repeated measures (Friedman’s test) followed by post-test Dunn’s multiple comparison tests compared to one control reference value using GraphPad Prism. A To investigate the preanalytical variations due to non-specific binding of α-syn species to storage tubes, the effect of different types of polypropylene tubes, use of non-ionic detergents, and multiple tube transfers on measured t- and o- α-syn concentrations was tested. The stability of the α-syn species (t- and o-α-syn) against multiple freeze-thaw cycles and delayed storage was also tested.p = 0.003) was observed. For measurements of both t- and o-α-syn levels, the use of Eppendorf Safe Lock, and Extragene tubes showed a significant reduction in the measured levels by 23 ± 4.2% (p = 0.025) and 35 ± 2.8% (p = 0.042) for t-α-syn and by 34 ± 13.4% (p = 0.049) and 51.5 ± 0.7% (p = 0.004) for o-α-syn levels, respectively.Three CSF pools were distributed and incubated in six types of polypropylene tubes from different vendors  then meap = 0.046) while levels Triton X-100 treated samples increased by 39.2 ± 3.2% although statistical significance was not reached   or Real-time quaking-induced conversion (RT-QuIC). Additionally, application studies assessing the correlation between the stability of α-syn forms and their impact on biomarkers studies would also be of great benefit to the research community.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.NM and OE-A: study design and supervision. IA, NM and EW: conducting of the experiments. IA and NM: data analysis. IA: writing the first draft of the manuscript and incorporating revisions from other authors. IA, NM, WB, BM, CT and OE-A: data interpretation and critical revision of the manuscript for important intellectual content. OE-A: final review of the draft and approval to submit for publication. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The permeability of the blood-spinal cord barrier (BSCB) is mainly determined by junction complexes between adjacent endothelial cells (ECs), including tight junctions (TJs) and adherens junctions (AJs), which can be severely damaged after spinal cord injury (SCI). Exercise training is a recognized method for the treatment of SCI. The destruction of the BSCB mediated by matrix metalloproteinases (MMPs) leads to inflammation, neurotoxin production, and neuronal apoptosis. The failure of new blood vessels to effectively regenerate is also an important cause of delayed recovery after SCI. For the first time, we introduced water treadmill training (TT) to help SCI rats successfully exercise and measured the effects of TT in promoting recovery after SCI and the possible mechanisms involved.Sprague-Dawley (200–250 g) rats were randomly divided into the following three groups: sham operated, SCI, and SCI + TT. Animals were sacrificed at 7 or 14 days post-surgery. The degree of neurological deficit, tissue morphology and BSCB permeability were assessed by the Basso-Beattie-Bresnahan (BBB) motor function scale and appropriate staining protocols, and apoptosis, protein expression and vascular EC ultrastructure were assessed by TUNEL staining, Western blotting, immunofluorescence and transmission electron microscopy (TEM).Our experiments showed that TT reduced permeability of the BSCB and decreased structural tissue damage. TT significantly improved functional recovery when compared with that in the SCI group; TJ and AJ proteins expression increased significantly after TT, and training reduced apoptosis induced by SCI. TT could promote angiogenesis, and MMP-2 and MMP-9 expression was significantly inhibited by TT.The results of this study indicate that TT promotes functional recovery for the following reasons: TT (1) protects residual BSCB structure from further damage, (2) promotes vascular regeneration, and (3) inhibits MMP-2/9 expression to mitigate BSCB damage. Spinal cord injury (SCI) is responsible for heavy societal and economic burdens . CurrentMatrix metalloproteinases (MMPs), a family of zinc-containing peptidases that degrade and reshape the extracellular matrix and other extracellular proteins, play a key role in barrier function , 8. StudPrevious studies on this topic have mainly focused on drug treatment after SCI , 19. HowAnti-MMP-9, anti-MMP-2, and anti-Tubulin antibodies were purchased from Proteintech . Anti-VEGF, anti-BrdU, anti-Laminin and anti-β-Actin antibodies were purchased from Abcam . Anti-p120-Catenin, anti-β-Catenin, anti-ZO-1, anti-Occludin, anti-Claudin-5 and anti-CD31 antibodies were purchased from Affinity . An in situ cell death detection kit was purchased from Roche Molecular Biochemicals.A total of 123 adult male Sprague-Dawley rats (200–250 g) were purchased from the Shanghai Laboratory Animal Center. The animal-related protocols had been approved by the Animal Research Committee of Wenzhou Medical University. All animals were housed in a controlled environment and regularly fed food and water. Rats were randomly divided into the following three groups: sham-operated , SCI , and SCI + TT .Rats were anaesthetized with 2% pentobarbital sodium (30 mg/kg) and shaved, after which a 2-cm incision was made to expose the T10 segment of the spinal cord . The expOur research group provided the initial design for the water treadmill  and submitted it for patenting. The rats were given adaptive training for three days before SCI. The surface of the water was adjusted to the xiphoid process of the rat sternum, the water temperature was set at 30 °C, and the speed of the water treadmill was maintained at 10–15 m per minute. Training started one day after SCI. Rats in the training group(TM) began training that lasted for 7 or 14 days   motor functional scale test in an open field. The maximum total BBB score was 21 points, and the higher the score was, the closer the animal’s motor function was to normal .n = 5), after which the rats underwent cardiac perfusion with 0.9% normal saline and 0.5 cm of the T10 spinal cord segment was removed. The degree of oedema in this segment was assessed by the dry and wet weight method as previously reported [At 7 or 14 d after SCI, 2% sodium pentobarbital was intraperitoneally injected to anaesthetize the animals  were injected with EB dye (4 ml/kg) by the tail vein at 7 or 14 d after SCI to assess BSCB permeability, followed by treatment with 2% sodium pentobarbital anaesthesia 2 h later and 0.9% saline perfusion. Tissues containing T10 were soaked in N,N′-dimethylformamide at 50 °C for 72 h. The concentration of EB dye in the samples was determined based on a standard curve (μg/g). Tissues were cut into 15-μm thick sections with a freezing microtome at − 20 °C, and then analysed. Quantitative data analysis was performed with ImageJ software.According to previously reported methods , 8, ratsBriefly, T9–T11 spinal cord tissue was removed from the rats at 7 or 14 d after SCI, and stored in 4% paraformaldehyde for 24 h (4 °C). The spinal cord tissue was immersed in a 0.1 M phosphate buffer solution and 30% sucrose solution overnight (4 °C). Successive sections (15-μm thick) were frozen and stored for subsequent HE staining.Tissues containing T10 segments were put into a collection tube containing a mixture of phenylmethanesulfonyl fluoride (PMSF) and RIPA lysis buffer (100:1) and then microcentrifuged at 12,000 rpm for 5 min at 4 °C. We extracted the supernatant and calculated the protein concentration with a BCA protein assay kit. The mixed solution was heated to 100 °C for 10 min. After electrophoretic transfer to membranes, they were incubated with the appropriate primary  and secondary antibodies, and the signal was digitally quantified.After the sections had been dried, they were washed 3 times for 15 min. They were treated with non-immune goat serum for 1 h and then incubated with the following primary antibodies for 50 min at room temperature: rabbit anti-Occludin antibody (1:100), rabbit anti-claudin-5 antibody (1:100), rabbit anti-p120-Catenin (1:200), rabbit anti-β-Catenin (1:100), anti-CD31(1:100), anti-BrdU (1:100) and anti-Laminin (1:100) at 4 °C. This was followed by incubation with Alexa Fluor 488 Affinipure goat anti-rabbit IgG (H + L)  for 50 min at room temperature. Phosphate-buffered saline (PBS) was used in place of the primary antibody as a negative control. We use an in situ cell death detection kit to detect apoptotic cells. The nuclei were coloured by staining with Hoechst or DAPI. The fluorescence signal was observed by laser confocal microscopy. Five fields on each of three slides per animal were randomly selected for visualization and analysis performed using ImageJ software .3 pieces on ice and soaked in 2.5% glutaraldehyde. The tissue was then fixed with a 1% oxidizing fixative for 1 h, stained with 1% uranyl acetate for 2 h, and embedded after dehydration in a gradient acetone solution. After semi-thin sectioning and toluidine blue staining, ultrathin sections were cut and observed using a Hitachi transmission electron microscope.A total of 18 mice were subjected to TEM. Tissue was quickly removed, cut into 1 mmImages were taken under the same conditions, including brightness and contrast, to better compare TJs among different groups. According to standard protocols , 31, thep > 0.05 indicating a normal distribution, Levene’s test was used as a test of homogeneity of variance, with p > 0.05 used to indicate homogeneous variance, and vice versa. A t-test was used to compare two groups. One-way ANOVA and Dunnett’s test were used to evaluate the data when more than two groups were compared. Statistical analyses were performed with SPSS 16 statistical software, and p < 0.05 was used to indicate statistical significance.All experimental data are expressed as the mean ± standard deviation. The Kolmogorov–Smirnov (K–S) test was used as a normality test, with p7 < 0.001, p14 < 0.001; TM vs. M: p7 < 0.01, p14 < 0.001). The amount of EB dye in the tissue increased significantly after SCI compared with that in group S, suggesting BSCB leakage . After TT, infiltration of EB dye was significantly reduced . The fluorescence intensity of the EB dye after SCI was much higher than that in the S group. However, the fluorescence level of the TM group was significantly lower than that of the M group . All these data indicate that TT could reduce BSCB disruption.We used an impactor to generate a model of SCI Fig. a. Subseqp7 < 0.01, p14 < 0.05; sagittal section, p7 < 0.01, p14 < 0.01). The functional recovery of the S, M and TM groups was evaluated by BBB motor functional scoring at 6 h, 1 d, 3 d, 7 d, and 14 d after SCI . We further tested the relationship between apoptosis and TT at 7 d after SCI . These results show that TT could significantly improve functional recovery and preserve tissue.At 7 and 14 d after injury, histological differences in the T9-T11 spinal cord were observed by HE staining Fig. a. The arSCI Fig. c. The BBSCI Fig. d. The nup < 0.001). However, compared to the rats in M group, rats treated with TT showed higher TJ and AJ protein expression at 7 days . Staining for Occludin/CD31/Hoechst , TJ proteins (Claudin-5 and Occludin) and ZO-1 expression was estimated by Western blotting. According to the results Fig. a, TJ andays Fig. b . Furthermore, TT significantly upregulated VEGF expression compared to that in rats that received SCI alone . Then, blood vessels co-labeled with 5-bromo-2-deoxyuridine (BrdU) and laminin in the ischaemic penumbra . Additionally, the neovascularization density of the TM group was increased compared with that in the M group, suggesting that TT could effectively promote angiogenesis (TM vs. M: p < 0.001).We investigated VEGF protein expression in the epicentre at 7 d after SCI Fig. a. Comparp < 0.001; MMP-9, p < 0.01). Moreover, to detect defects in vascular ECs, we analysed them by TEM. In group S, spinal cord vascular ECs showed tight connections with a rivet-like structure, and a series of electron-dense bands between the plasma membranes, forming a relatively closed vascular lumen, were observed. The intercellular TJs were partially opened after SCI (M group), and compared with that in group S under the same conditions, the structure of the junctions was more disordered; for example, the TJ length was shorter and the gap was wider . In contrast, rats in the TM group had more electron-dense bands, and the ultrastructural changes in these rats were between those observed in the S and M groups .MMP-2/9 protein expression levels were detected by Western blotting, which showed that TT could significantly inhibit the upregulation of MMP-2/9 after SCI Fig. a–c  protects the residual BSCB structure from further damage, (2) promotes vascular regeneration, and (3) inhibits MMP-2/9 expression to mitigate BSCB damage.Emerging evidence indicates that exercise therapy can promote recovery after SCI –35. It iFor the first time, mechanisms to mitigate damage after SCI were studied through our experiments in rats. Trauma, infection, tumour growth or obstruction of the blood supply can cause oedema of the central nervous system (CNS). Cytotoxicity and vasogenic edema are interdependent factors involved in the development of CNS oedema. Prolongation of cytotoxic oedema leads to vasogenic oedema, and vice versa , 40. CytThe BSCB protects the spinal cord by restricting the entry of plasma components and blood cells . Followi+/Laminin+ cells increased significantly after TT treatment in rats with SCI, helping to maintain the stability of the BSCB (Fig. To determine the protective mechanism of TT, we performed more in-depth research. VEGF stimulates EC proliferation and survival vascular structure formation, nitric oxide-dependent vasodilatation and vascular leakage . It has SCB Fig. .The activation of MMP-2/9 after SCI plays an essential role in the destruction of the BBB/BSCB , and MMPAs far as we know, this is the first time that TT has been applied in the treatment of SCI in rats. We found that TT could enhance the expression of TJ and AJ proteins after SCI. We also found that TT could promote angiogenesis and inhibit MMP-2/9 expression, which may be an important mechanism by which TT maintains the stability of the BSCB. These experimental results provide a better understanding of the possible mechanisms of TT in the treatment of SCI and a reliable basis for the application of TT in the future.We acknowledge the limitations of our experiments. In this study, we only used male rats, ignoring any possible differences caused by sex. Due to limitations in the experimental conditions, we could not observe changes at the functional level through electrophysiological techniques to understand the role of TT. The training period was relatively short and the specific mechanism by which TT inhibits MMP-2/9 expression remains to be further studied.TT participates in protecting the BSCB after SCI by protecting the residual BSCB structure from further damage. Furthermore, TT also promotes vascular regeneration and inhibits MMP-2/9 expression to mitigate BSCB damage Fig. . HoweverAdditional file 1. The movement of the rats on the water treadmill.


Surface electromyography (sEMG) monitoring has recently inspired new applications in the field of patient diagnose, rehabilitation therapy, man–machine–interface and prosthesis control. However, conventional wet electrodes for sEMG recording cannot fully satisfy the requirements of these applications because they are based on rigid metals and conductive gels that cause signal quality attenuation, motion artifact and skin allergy. In this study, a novel flexible dry electrode is presented for sEMG monitoring. The electrode is fabricated by screen-printing a silver–eutectic gallium–indium system over a transfer tattoo paper, which is then hydrographically printed on 3D surface or human skin. Peano curve in open-network pattern is adopted to enhance the mechanics of the electrode. Hydrographic printing enables the electrode to attach to skin intimately and conformably, meanwhile assures better mechanical and electrical properties and therefore improves the signal quality and long-term wearability of the electrode. By recording sEMG signal of biceps under three kinds of movement with comparison to conventional wet electrode, the feasibility of the presented flexible dry electrode for sEMG monitoring was proved. Flexible devices or epidermal electronics are newly developed wearable electronics, which are much more lightweight, robust and flexible in contrast to traditional devices, therefore allowing them intimately mounted on human skin ,2,3,4,5.EMG signal is generated by the electrical activity of the muscle fibers that are activated almost synchronously by the motor neuron innervating them ,11,12. EThe most common and commercially available electrodes for bioelectrical signals acquisition are wet conductive gel electrodes. While they are characterized by causing irritation and allergy, signal degradation during long-term monitoring, as well as difficulty in adhering skin without adhesive. Dry flexible electrode is a promising alternative as they can attach to skin with intimate attachment, less motion artifact and no skin reaction . While mIn this work, a novel flexible dry electrode for sEMG monitoring is presented. In the following sections, the pattern, structure, fabrication process and characterization of the electrode are introduced. Then the contact impedance and the stretch–resistance characteristics of the electrode are tested. Finally, the feasibility of the electrode for sEMG monitoring is verified through recording sEMG signal of biceps under three kinds of movement and testing the long term wearability in comparison to traditional wet electrode.Considering the generation and location of sEMG, the electrode pattern and size are well designed according to the guideline of SENIAM (surface electromyography for the non-invasive assessment of muscles) project. The electrode pattern consists of three phases, i.e., reference (REF), ground (GND), record (REC) phase, as shown in The electrode is manufactured by hydrographic printing silver–indium–gallium alloy system  on the tattoo paper. It has been found out that ternary alloy system silver–indium–gallium would form when silver is added into eutectic Ga–In at atmosphere condition . Weak acAfter finishing the electrode, scanning electronic microscopy (SEM) and energy dispersive X-ray spectroscopy (XPS) were taken to examine the surface and material characteristics of the traces. As shown in The ability of electrodes to record sEMG depends strongly on the skin–electrode contact impedance. Comparative experiments between traditional wet electrode and the flexible dry electrode are carried out, which demonstrates that the contact impedance declines with the increasing intimacy. To better understand the influence of the mechanical deformation on electrical characteristics, the resistance of an eight-millimeter trace under stretch was examined. Referring to The electrical and mechanical features are summarized above as the fundamental for the sEMG signal measuring. The primary application test is to measure sEMG signal of biceps under voluntary contraction . Three cIn addition to mechanical and electrical property tests, the conformable long-term wearability also takes a critical role in sEMG monitoring. An ultrathin, flexible and skin conformable dry electrode that can be applied without skin preparation was presented. The electrode is fabricated through hydrographic printing which can magnificently lower the cost and simplify the processing. Due to the novel structure and elaborate pattern, the electrode can susceptibly attach to the skin like a tattoo. This unique feature assures better signal quality and long-term wearability of the electrode in sEMG recording. In the comparative tests, the flexible dry electrode performs equally well or even better than traditional wet electrode, especially on aspects of skin–electrode contact impedance and wearability. Even though the ACF connector used in the experiments introduces a baseline noise, the dry electrode performs well under movement. In conclusion, the flexible dry electrode can be expected for sEMG monitoring applications.


To generate a Cerenkov scatter function (CSF) for a primary proton beam and to study the dependence of the CSF on the irradiated medium.The MCNP 6.2 code was used to generate the CSF. The CSF was calculated for light‐pigmented, medium‐pigmented, and dark‐pigmented stratified skin, as well as for a homogeneous optical phantom, which mimics the optical properties of human tissue. CSFs were generated by binning all of the Cerenkov photons which escape the back end (end opposite beam incidence) of a 20 × 20 × 20 cm phantom. A 4 × 4 cm, 500 × 500 bin grid was used to create a histogram of the Cerenkov photon flux on the simulated medium’s back surface (surface opposite beam incidence). A triple Gaussian was then used to fit the data.2 value for the fit was calculated to be 0.99. The phantom material was found to have a significant effect  on the CSF amplitude and full width at half maximum . The difference in these parameters for the three skin pigments was found to be small.From the triple Gaussian fit, the coefficients of the CSF for the four phantom materials was generated. The individual CSF fit coefficient errors, with respect to the Gaussian representation, were found to be between 0.92% and 4.11%. The RThe CSF was obtained for a proton beam using the MCNP 6.2 code for a phantom constructed of light, medium, and dark stratified human skin, as well as for an optical phantom. The CSFs were then fit with a triple‐Gaussian function. The coefficients can be used to generate a radially symmetric CSF, which can then be used to deconvolve a measured Cerenkov image to obtain the dose distribution. PT has the advantage of significantly reduced toxicity to non‐malignant tissue due to the absence of exit dose, while providing focused high linear energy transfer radiation to the tumor target. However, real‐time in vivo dosimetry for both electronin vivo dose measurement method using Cerenkov radiation produced by secondary electrons and positrons within the patient volume. This could be helpful for evaluating the accuracy of the prescribed radiation treatment, monitoring hot spots of dose deposition, and improving the therapeutic outcomes of PT due to the ability to accurately monitor dose in real time. However, there is no method that can perform this type of dosimetry in real time with sufficient accuracy.There has been increasing interest in using Cerenkov emissions for quality assurance and Proton therapy beams produce electrons and positrons that are responsible for dose deposition in a medium. Above a certain energy threshold, these charged particles will be superluminal within the medium and emit Cerenkov photons along their path. The Frank‐Tamm formula gives the amount and spectrum of Cerenkov photons emitted.This approach has been used for photon and electron beamsTo date, no CSF has been produced for a primary proton beam. In this work, a CSF is generated for a proton therapy beam for four different tissue equivalent phantom material compositions. The MCNP 6.2 code is used to simulate the interactions in the phantom.22.ACerenkov radiation is electromagnetic radiation which is emitted by charged particles passing through a dielectric material at a speed in excess of the phase velocity of light in that material. When a charged particle moves inside a dielectric material, it excites molecules to higher energy levels and excited states. Upon returning back to their ground state, the molecules re‐emit some photons in the form of electromagnetic radiation. According to Huygens principle, the emitted waves move out spherically at the phase velocity of the medium. If the particle motion is slow, the radiated waves bunch up slightly in the direction of motion, but they do not cross. However, if the particle moves faster than the light speed, the emitted waves add up constructively leading to a coherent radiation at an angle with respect to the particle direction, known as Cherenkov radiation. The result of this effect is a cone of photon emission in the direction of particle motion.2.BFigure 15O) which are responsible for both energy deposition and the creation of Cerenkov photons within the medium. Cerenkov photons created within the tissue volume will undergo scattering and absorption. Some of the Cerenkov photons will be absorbed, but a fraction (about 1%)A modulated proton beam incident on a tissue volume in the Z‐direction is considered. A system capable of imaging Cerenkov photons is placed at some angle θ relative to the beam. The proton beam produces secondary electrons and positrons (mostly from The following convolution equation represents the relationship between the surface flux of the incident proton beam entering the tissue volume and the intensity of the Cerenkov photons reaching the imaging systemI(θ)=J(Ω)The CSF can be written as a convolution of the dose scatter function (DSF) kernel and the Cerenkov dose scatter function (CDSF) kernel as follows:The CDSF kernel represents the production of Cerenkov photons by the secondary electrons and positrons from the incident proton beam and the transport of these Cerenkov photons toward the surface of the tissue volume. Equation 2 indicates that the CDSF can be found by deconvolving the CSF with the DSF.0 can be written as:The dose deposited in a plane at depth dInserting Equation 2 into Equation 0 in the tissue volume; the problem then becomes one of deconvolution of the Cerenkov photon intensity I, which is captured by the camera, with the CDSF, which can be found by deconvolving with the CSF produced herein and the DSF which will be calculated in a later work. The result is the dose profile at depth d0.The objective now is to determine the 2D dose distribution at depth d2.Co beam angle is assumed in the present work.The convolution statements in Equations 33.AThe MCNP 6.2in = 0°, where θin is the angle of entrance vector defined from the normal surface. As aforementioned, this is to ensure shift invariance. The phantom was modeled as a 20 x 20 x 20 cm3 box. The CSF simulations used the spread‐out Bragg peak (SOBP) shown in Table The MCNP model consisted of a pencil beam proton radiation source and the phantom being irradiated, with air surrounding. The radiation source was directed at the phantom with a constant beam angle of θThe CSF is created by the transport of Cerenkov photons created by a pencil beam of ionizing radiation incident on a dielectric material. For PT, a high‐energy proton beam is the primary radiation beam. The primary proton beam produces secondary electrons and positrons, which can exceed the speed of light in the medium, producing Cerenkov photons. Most of these photons will be absorbed in the tissue, but about 1% travel toward the surface and escape the irradiated medium.2 pixels.The initial position and direction at the time of the Cerenkov photon creation and the final position and final direction at which the Cerenkov photon escapes the phantom were recorded. The CSF was calculated by scoring the flux of the Cerenkov photons which escaped from the phantom surface. The positions of the photons leaving the surface were scored in a grid of 0.2 x 0.2 mm3.BFour different phantom materials were used in this study: light‐pigmented, medium‐pigmented, and darkened‐pigmented stratified skin and an optical phantom material. The stratified skin models were included to allow the effect of material properties, in particular the melanin content, on the CSF to be studied. The compositions for all four materials were taken from the National Institute of Standards and Technology soft tissue library.3.C2, 500 x 500 bin grid was used to create a histogram of the Cerenkov photon flux on the simulated medium surface. The histogram was sampled along a line from r = −10 to 10 cm using spoke‐sampling which was centered at the origin of the pencil beam on the medium surface. The sampling angle, θs, was defined on the medium surface as the rotation from the Y‐axis about the Z‐axis. Sampling angles of 0‐180 degrees in one‐degree increments were used. A triple‐Gaussian distribution was used to fit the mean of the data:CSFs were generated by binning all of the Cerenkov photons which escape the back end of the phantom (end opposite beam incidence). A 4 x 4 cmmean is the mean of the CSF cross‐sections over the sampling angle θs. The parameter r is the radial distance from the entrance of the proton pencil beam on the optical phantom surface. The parameters α, β, γ, δ, ε, and ζ are fit coefficients, generated using the Mathworks MATLAB fitnlm function; they were scaled by the number of primary protons incident on the optical phantom. The fitnlm function also produces the error in the fit coefficients. The triple‐Gaussian fit was chose due to it having the best goodness of fit (R2) value of the fits tested, as well as having the lowest error in the fit coefficients. Single‐Gaussian and double‐Gaussian fits were also tested but yielded lower R2 values and larger errors in the fit coefficients. The error in CSFmean was found as the standard deviation at each radial point from the CSF cross sections. The large number of histories and the incorporated variance reduction ensured that the error at each point on CSFmean was <1% of the maximum value for each CSF. The Gaussian fit of the CSF data is shown in Figure CSF of fit R value of42 value was calculated to be 0.99.The coefficients of the CSF for the four phantom materials are shown in Table Table 5In the present work, the CSF was calculated for a proton beam incident on phantoms of light, medium, and dark stratified skin, as well as an optical phantom, which mimics the optical properties of human tissue. The SOBP from Table The material was found to have a significant effect on the CSF amplitude and FWHM. The optical phantom CSF amplitude was found to be 63% greater than the light skin phantom; the FWHM for the optical phantom was found to be 195% greater than the light skin phantom material. The skin pigment was found to have a small impact on the CSF amplitude and almost no effect on the FWHM. The lack of effect on the FWHM is likely due to the fact that the only difference between the three pigments in the melanin content in the epidermis. The other layers of skin have identical scattering and absorption characteristics. As a result, Cerenkov photons have similar behavior underneath the epidermis layer, but have different amounts of absorption between the three skin models for Cerenkov photons leaving the tissue. As a result, the FWHM for the three skin models are similar.As can be observed from Table 6In the present work, the CSF was obtained for a proton beam using the MCNP 6.2 code for a phantom constructed of light, medium, and dark stratified human skin, as well as for an optical phantom. The CSFs were then fit with a triple‐Gaussian function using the MATLAB fitnlm function. The coefficients for this fit are provided in Table 2 value of 0.99.The phantom material was found to have a significant effect on the CSF amplitude and FWHM. The difference in these parameters for the three skin pigments was found to be small, meaning that melanin content has a diminutive effect on the CSF. The % errors for the parameters were found to vary from 0.92% to 4.11%, with an R


Chitinase 3-like 1 (CHI3L1) is an extracellular monomeric single-chain glycoprotein expressed by many types of cells. Its elevated levels were found in cerebrospinal fluid in central nervous system (CNS) inflammatory diseases patients. The aim of the study was 1) to validate the reference interval of cerebrospinal fluid (CSF) CHI3L1 in a control group; 2) to measure the CHI3L1 concentration in different diagnosis groups .including multiple sclerosis (MS); and 3) to correlate those values with other biomarkers of axonal damage or neuroinflammation in different grous.The study included 132 CSF samples sent to the Department of Clinical Biochemistry, Institute of Laboratory Diagnostics, University Hospital Ostrava. Concentrations of CHI3L1, CXCL13 chemokine, neurofilament light chains, and phosphorylated neurofilament heavy chains were determined by enzyme-linked immunosorbent assays. IgG oligoclonal bands were detected by isoelectric focusing in agarose gels followed by immunofixation. IgM and FLC oligoclonal bands were analyzed by IEF followed by affinity immunoblotting. The group consisted of 42 patients with multiple sclerosis, 14 with clinically isolated syndrome, 11 with other central nervous system inflammatory diseases, 46 with non-inflammatory diseases of the central nervous system, 4 with inflammatory diseases of the peripheral nervous system, and 15 controls.-1. Statistically significant differences of CSF CHI3L1 concentrations were found among diagnosis groups (p < 0.0001), after age adjustment (p = 0.002). There was a statistically significant relationship between CHI3L1 and NFL in the MS group , and between CHI3L1 and pNFH in the MS group . No statistically significant difference was found in the categorical comparison of CHI3L1 in the MS group and other diagnostic groups as well as when using the Mann-Whitney U test for CHI3L1 with additional parameters with and without oligoclonal bands present.The estimated reference values of CHI3L1 were 28.6–182.5 μg.LCSF CHI3L1 values differ depending on diagnosis and correlate significantly with concentrations of the axonal damage markers CSF neurofilament light chains, and CSF phosphorylated neurofilament heavy chains, but not with CSF concentrations of the inflammatory marker CXCL13. Thus, CSF CHI3L1 could be another promising prognostic, albeit probably etiologically nonspecific, biomarker of MS. Multiple sclerosis (MS) is a chronic disease affecting the central nervous system. In recent years several biochemical markers in cerebrospinal fluid have been suggested as prognostic tools –3.CHI3L1, also known as YKL-40, belongs to the chitin glycoside hydrolase 18 family. Unlike true chitinases, it lacks enzymatic activity. It is a glycoprotein produced by a wide variety of cells, such as macrophages, chondrocytes, synovial cells, osteoblasts, neutrophils, and astrocytes .CHI3L1 is expressed in astrocytes in the brain tissue of patients with multiple sclerosis, and is associated with reactive gliosis in different neuropathological states, particularly those associated with neuroinflammation. A correlation between the time course of the CHI3L1 concentration and the CSF viral load was shown in lentiviral encephalitis . CHI3L1 MS is a demyelinating disease associated with increasing and decreasing inflammation, gliosis, and variable axonal loss. Therefore, we expect to find increased concentrations of CHI3L1 in MS patients.The aim of the study was 1) to validate the reference interval (RI) of cerebrospinal fluid (CSF) chitinase 3-like 1 (CHI3L1) in a control group; 2) to measure the CHI3L1 concentration in different diagnosis groups, including MS; and 3) to correlate those values with other biomarkers of axonal damage or neuroinflammation in different groups.RIs were estimated on the basis of the guidelines of the Clinical and Laboratory Standards Institute (CLSI C28-A3), which recommends the use of nonparametric tests for statistical data processing and the evaluation of data according to gender and age –11.Our study includes 132 patients of the Moravian-Silesian region of the Czech Republic whose CSF samples were sent for analysis to the Institute of Laboratory Diagnostics, Department of Clinical Biochemistry, University Hospital Ostrava. Informed consent was obtained from all patients at the University Hospital Ostrava who were included in the study. The study was approved by the Ethics Committee of the University Hospital Ostrava as a part of the project ‘CSF biomarkers of multiple sclerosis’ (reference number 400/2017). Patients were subdivided into diagnosis groups: MS , clinically isolated syndrome , other central nervous system inflammatory diseases , inflammatory diseases of the peripheral nervous system , non-inflammatory diseases of the central nervous system , and controls . For diagnosis of multiple sclerosis, we used the 2017 Revisions of the McDonald Criteria [CSF samples were collected into polypropylene tubes . Samples were centrifuged at 390 × g for 10 minutes at room temperature, and the supernatants for determination of CHI3L1, CXCL13, neurofilament light chains (NFL), and phosphorylated neurofilament heavy chains (pNFH) were then aliquoted into at least 3 vials  and stored at -70 °C until analysed.The concentrations of CHI3L1 , CXCL13 , NFL , and pNFH  were determined by ELISA. Undiluted CSF was used for CXCL13 and pNf-H whereas 1/2 and 1/50 dilution was used for NFL and CHI3L1, respectively.-1 for CHI3L1, 10.7 ng.L-1 for CXCL13, 32 ng.L-1 for NFL, and 27 ng.L-1 for pNFH. All samples were measured in duplicate. The mean coefficients of variation for CHI3L1, CXCL13, NFL, and pNFH were 5.8%, 5.2%, 1.9%, and 3.3%, respectively.The quality control samples supplied by the manufacturers of the diagnostic kits were used for accurate and reproducible measurement of CHI3L1, CXCL13, and pNHF; for NFL measurements, the patient sample was used, because the diagnostic kit did not include a quality control sample. Kit manufacturers reported that analytical sensitivity was 3.55 ng.LIgG oligoclonal bands (oIgG) were detected by isoelectric focusing in agarose gels followed by immunofixation, using a commercial kit on a Hydrasys instrument .IgM and FLC oligoclonal bands  were analyzed by IEF focusing followed by affinity immunoblotting as originally described by Sindic and Laterre  and sligThe statistical analysis was rendered using Excel, Stata version 13, MedCal version 17.9.7., R and NCSS 2007 software –17. MedCBasic descriptive statistics, including frequency tables, medians, arithmetic means, standard deviations and percentiles, were used to describe the results. With the Shapiro-Wilk test of normality, the normality of the parameters CHI3L1, CXCL13, NFL, and pNFH was verified.The normality hypothesis was rejected; therefore, non-parametric tests were used, including the Kruskal-Wallis rank test and the two-sample Wilcoxon rank-sum (Mann-Whitney) test.The relationship between the parameters was assessed by Spearman’s correlation coefficient. Data values were classified as positive and negative. Fisher’s exact test was used to test categorized data. Conformity between assay results was measured using the kappa index with confidence intervals of 95%. Statistical tests were evaluated using a significance level of 5%.-1) and the limit of detection (LoD = 5.48 ng.L-1) values were calculated. The average recovery of CHI3L1 obtained using spiked samples of CSF was 101.4% . After age-adjusted analysis according to regression dependence CHI3LI = 51.874 + 1.997 * Age, OIND diagnosis group had higher CHI3L1 levels than other diagnosis groups, (p = 0.002), s = 0.595; n = 105; P < 0.0001) and between CHI3L1 and pNFH concentrations .The regression relationship between studied parameters was evaluated using Passing–Bablok regression . There ws = 0.460; P = 0.002), NIND , and OIND  diagnosis groups, and between CHI3L1 and pNFH in the MS  and NIND diagnosis groups   . We perf0 ng.L-1 ; CSF pNF.L-1 [-1 ; CXCL13  [-1 [-1 , in the The Cohen´s kappa statistic was used to compare the assays based on clinical interpretation (positive and negative results) because the methods had different reference intervals . The higThe Mann-Whitney U test was chosen to compare parameters  with and without the presence of oligoclonal bands . Statistically significant differences were not found with the presence of oligoclonal bands for CHI3L1, but were found with oIgG for pNFH and CXCL13 , with oIgM for CXCL13 (P = 0.0279), with oFLC lambda for NFL and CXCL13 , oFLC kappa and CXCL13 (P <0.0001).In this study we tested CHI3L1 as a marker of multiple sclerosis. The CHI3L1 ELISA diagnostics kit is sensitive enough to measure CHI3L1 in cerebrospinal fluid. We also estimated the normal CSF CHI3L1 values that were show to be independent of gender but associated with age. The CHI3L1 concentration increased with increasing age. This is consistent with published data showing increased levels of CSF CHI3L1 in patients with CNS inflammation compared with healthy individuals, and an increase with increasing age, consistent with the hypothesis that lower-grade inflammatory processes are induced in the aging brain .The dependence of CH3L1 concentration in different diagnostic groups was studied. An elevated level of CSF CHI3L1 was found in patients with MS, but it was much higher in patients with other inflammatory neurological diseases. Modvig et al.  reportedAt the same time, a number of studies dealt with the study of CHI3L1 in relation to CNS tissue damage. Burman et.  found a Concurrently, CSF CHI3L1 concentration correlates significantly with CSF NFL and even more with CSF pNFH concentrations, making pNFH kit more convenient for routine analysis. The correlation between CHI3L1 and NFs varies depending on the diagnosis.We also assessed the correlation between CHI3L1 and CXCL13. CXCL13 is a potent B cell chemoattractant. In cerebrospinal fluid, it is expressed by monocytes and especially macrophages in perivascular inflammatory lesions and scattered parenchymal cells . KhademiThe present study also provides the first results that compare CHI3L1, NFL, pNFH, and CXCL13 with IgG, IgM, and FLC kappa and FLC lambda oligoclonal bands. Our reason for conducting the comparison is the fact that intrathecal synthesis of IgG as well as of free light chains (FLC) is detectable in the majority of patients with multiple sclerosis and less frequently in other, mostly inflammatory, nervous system diseases. In addition, a positive finding of intrathecal FLC synthesis can be a marker of disease progression . The detSince CSF CHI3L1 concentrations correlated much more closely with CSF pNFH, an established biomarker of disease progression than with inflammatory biomarker CSF CXCL13 and oFLC, we hypothesize that CSF CHI3L1 levels might reflect the extent of tissue damage rather than the degree of inflammatory activity. Lowest CSF CHI3L1 level found in the CIS group might further support this hypothesis, although patients with CIS  are probably at lower risk of progression to CDMS than before the implementation of 2017 revision of MS diagnostic criteria.Our study has several weaknesses. Although three CSF and serum aliquots were frozen for each patient in the study, there was not always enough material, especially cerebrospinal fluid, for all tests. At the same time, the patient population was obtained during a 3-year study (n = 356), but in some patients no definitive diagnosis has been established. Therefore, these patients were not included in the final evaluation, which also affected the unequal representation of patients in the individual diagnostic groups. Finally, detailed clinical characteristics of patients within individual diagnostic groups were not taken into account in the analysis.In this study, we tested a diagnostic kit for the determination of CHI3L1 concentrations in biological fluids. The CHI3L1 ELISA assay has adequate sensitivity and is suitable for CSF analysis. The data showed good correlation and moderate conformity between CHI3L1 and pNFH concentrations. When assessing the relationship of CHI3L1 concentrations and diagnosis, correlations were found between the concentrations of CHI3L1 and NFL in the MS, NIND, and OIND groups, and between the concentrations of CHI3L1 and pNFH in the MS and NIND groups. The results support the hypothesis that CSF CHI3L1 could be another promising prognostic, albeit probably etiologically nonspecific, biomarker of MS.S1 Table(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.