Umbilical Cord blood is an intuitively attractive stem cell source, but its use has declined since it is associated with an increased procedure-related morbidity and transplant related mortality. Some of this reflects that cord blood transplants are more often HLA-mismatched compared to other unrelated donor transplants. The ability to transplant in such a setting, indeed without high rates of chronic Graft versus Host Disease (GVHD), constitutes an advantage compared to other unrelated donor cell sources and there are other advantages specifically associated with cord blood as a donor cell source. These advantages must be weighed against its disadvantage, and we have utilised cord blood preferentially as a donor cell source in certain clinical situations in paediatric medicine. In non-malignant diseases, outcomes in metabolic disease are critically dependent on age at transplant and the enzyme delivered by that transplant, and in cord blood transplantation then the time to transplant can be minimised and the engrafted recipients have higher chimerism that delivers higher enzyme levels. In malignant diseases, studies have described reduced relapse rate and better GVHD-free survival, and so we have prioritised cord as a donor cell source where the risk of relapse is highest, and the effects of higher transplant related mortality is most clearly offset by the reduced relapse rates. Cord blood use is declining. Its decline as a stem cell source in alternate donor haematopoietic stem cell transplant—when there is no matched family donor—has been mirrored by the rise in haplo-identical donor transplant .in vitro graft manipulation, with either positive selection of CD34 stem cells or negative selection of CD3 T-cells, including specifically αβT-cells, or in vivo with post-transplant cyclophosphamide. In cord blood transplant then these techniques are not employed, and allo-reactive, often HLA-mismatched, T-cells are infused with the graft, with higher rates of acute graft versus host disease (GVHD).The reason for this is clear—haplo-identical donor transplant is easier, with lower procedure-related morbidity and mortality than cord blood transplant. This is principally due to the different graft composition in the two clinical settings. The stem cell dose is significantly higher in haplo-identical transplants, ensuring engraftment and transfusion independence earlier than in cord blood transplant, in which the cell dose is relatively small and limiting. Additionally, in haplo-identical transplant allo-reactive donor T-cells are removed from the graft, either during In transplant, however, there is no such thing as a free lunch, and losing this allogeneic effect of transplant may become disadvantageous in certain clinical circumstances. The retention of T-cells that are often mismatched to the recipient in the cord blood graft is likely important in reducing graft immunologic rejection and leukaemia relapse after transplant. Additionally, family donors, including haplo-identical ones, may be carriers of genetic illnesses and constitute inferior donors to wild-type, including cord blood, donors in certain genetic diseases, including metabolic illnesses.This review will examine the properties of cord blood transplant that generates continuing utility as a stem cell source in transplant in children. Cord could conceivably be prioritised as the source of choice to deliver the best achievable long-term outcome, especially in children with high-risk disease. The cord unit donor selection algorithm of our institution will be presented, and the practice of our institution in cord blood transplantation in malignant and non-malignant disease will be reviewed.1) Reduced time to transplantThe time to transplant is critically important in many paediatric conditions. The window of opportunity for transplant success is especially small in high-risk leukaemia and in rapidly progressive neurological, metabolic conditions. Cord blood units are available for immediate shipment, and this is attractive in these conditions requiring urgent transplant and compares favourably with unrelated donor transplant, where there is obligate delay in finding the donor, and scheduling the donor medicals and stem cell collection.Cord blood is often used in metabolic diseases, and this partly is because of this reduced time between diagnosis and recognition of transplant need, and actual transplant with allogeneic stem cell infusion. We have shown that the age at transplant clearly affects both somatic and neurological outcomes in Hurler syndrome, MPSIH . Age at in vivo or ex vivo T-cell depletion—have been used. There is no direct comparison. In HLH there is registry data that outcomes are poor with mismatched cord blood donors in this disease, and many would prefer haplo-identical donors (In Primary Immune deficiency (PID), cord blood is acceptable as a family donor, just as a matched sibling donor. In matched unrelated donor then matched cord is considered comparable to matched adult donor. Where there is no matched donor, then there is no direct large study comparing mismatched cord to mismatched family or unrelated donor. Studies in specific diseases such as Severe Combined Immune deficiency (SCID) and Wiskott-Aldrich Syndrome (WAS) have described that cord blood has similar outcomes to other cell sources. Where there is no matched donor then haplo-identical donors—with either l donors . The rell donors . There aIn haemoglobinopathy then matched sibling donor cord blood gives comparable outcomes to matched sibling donor marrow transplant . The useIn thalassaemia there is good survival with continuing medical care with red cell transfusion and iron chelation and transplant is usually not offered where is not a genotypically identical family donor. In exceptional circumstances such as intolerance of chelation or return to a country with unsafe blood transfusion then alternate donor transplant may be offered. Haplo-identical or cord blood transplant might be considered but cord outcomes will be affected significantly by the degree of HLA mismatch, and haplo-identical approaches might be preferred where there is no well-matched cord blood donor .2) Cord blood increases the donor pool, even for those for whom donors are difficult to findIn sickle cell disease then published results with cord blood transplant are not good enough for it to be a validated approach, and we would prefer haplo-identical transplant in this setting where there is no matched family donor, and where there is a clear disease indication for transplant. The conditioning in cord blood approaches has often been of reduced intensity, and this is likely not to predict for sustained donor cell engraftment. There is greater experience of haploidentical donors in this setting, and this is therefore our preferred cell source in this situation .2) Cord 3) Cord blood transplants can be more safely mismatched at HLA antigensThe unrelated donor cord blood donor pool might be thought to better represent the ethnic diversity of the current population than the unrelated donor registry, since units are taken from women delivering in areas of high ethnic diversity, rather than from adults who have volunteered to donate. This is relevant to the many paediatric patients without a matched family donor and are from an ethnic background that is underrepresented on adult donor panels .3) Cord The utility of cord blood for those children in whom it is difficult to find fully matched family or unrelated donors is further emphasised by the better ability to utilise HLA-mismatch without compromising clinical efficacy or transplant outcomes. The algorithm used at our centre for the selection of matched cord blood unit donors is summarised in We contributed to the study published by Eapen et al., of 1199 paediatric cord blood transplants in children with non-malignant diseases and were reported to either the European Group for Blood and Marrow Transplant or to the Center for International Blood and Marrow Transplant Research (CIBMTR) . There wWe compared outcomes in children with Hurler Syndrome with various graft sources in an EBMT and IBMTR study. This paper was from an earlier era when matching was less exacting and performed at antigen level at class I (HLA-A and -B only) and at high resolution only at HLA-DRB1. The transplant survival was similar between matched sibling donors and 6/6 matched cord blood unit recipients, and superior to that of 5/6 cord blood and 8/8 allele-level matched unrelated donor recipients which were similar to each other. Outcomes were least good in mismatched unrelated donor recipients .Many studies similarly report the relationship between transplant outcomes and HLA-mismatch in malignant diseases in children and adults. The interpretation of such data is more complex, since HLA-mismatch adversely impacts transplant related mortality but is reported in many series—particularly in adults to positively impact leukaemia relapse risk after transplant . For patEapen reported 502 children with acute leukaemia and transplanted with cord blood who were reported to the CIBMTR , and com4) There is less disease relapse after cord blood transplant for malignant disease than after matched sibling or unrelated donor or haplo-identical donor transplantSanz et al. have reported improved leukaemia-free survival rates in 79 adults with AML are actually significantly better in their institution when the cord blood units are mismatched to the patient . YokoyamRelapse is reduced after cord blood transplant compared to other cell sources. This has been reported in many single institution and registry studies. This does not always translate to improved OS or leukaemia-free survival since there is confounding higher TRM in those same cord blood transplant recipients.Milano compared outcomes in Seattle of 140 cord blood transplant recipients with 344 HLA matched and 98 HLA-mismatched unrelated donors. There was reduced relapse in the cord blood group, particularly striking in the MRD positive group, and this translated to an improved OS, particularly against the mismatched unrelated donor transplant recipients .In Eapen’s malignant disease IBMTR cord blood transplant survey then there was reduced relapse in the mismatched cord blood transplant recipients compared with the marrow recipients . A recen5) Engrafted cord blood transplant recipients are more likely to be fully donor engrafted than marrow donor recipientsThis likely reflects an ontogeny difference between cord blood cells, including cord blood T-cells, and adult cells, including adult T-cells. Hiwarkar demonstrated that cord blood T-cells were superior to adult T-cells in controlling disease in a xenograft human B-cell lymphoma model . Cord bl6) Even in the T-cell replete and mismatched donor transplant, the rates of chronic graft versus host disease are lowAlthough the risk of primary graft rejection is increased in cord blood transplant recipients, we have reported that mixed donor chimerism is much reduced in engrafted Hurler Syndrome patients that have a cord blood transplant than a matched sibling or unrelated donor marrow or peripheral blood transplant . Since eThe aim of paediatric transplant is disease-free, graft versus host disease (GVHD) free survival. Chronic GVHD is particularly disabling and limits the utility of transplant in the non-malignant setting where its presence has no benefit at all to disease outcome. The rates of chronic GVHD are reduced after cord blood transplant, and even in the T-cell replete and HLA-mismatched donor setting.Barker reported rates of cGVHD that were less than 10%, even in the double cord setting, without serotherapy and where the median match was 5/8, and none were fully matched . Wagner We reported in a multi-institutional study of 317 children receiving T-cell replete single cord blood transplants for acute myeloid leukaemia that GVHD-free, leukaemia-free survival was similar in matched sibling and unrelated donor cord transplant recipients, and significantly higher than in unrelated donor recipients, even though the cord units were very significantly mismatched, and the unrelated donors were largely matched .7) Cord blood transplantation has higher transplant-related mortality with acute GVHD, increased graft failure, immune cytopenia and respiratory failureMehta RS et al. recently reported that the incidence of GVHD-free, relapse-free survival was better in the CB group compared to 7/8 BM. In this cohort, only 16% of CB were fully matched at HLA A, B and DRB1 which shows that CB recipients can tolerate HLA mismatch better with lesser incidence of Acute and chronic GVHD compared to BM recipients .7) Cord Cord blood transplantation has higher morbidity and mortality than some other transplant types. Of course, some is comparison of apples with pears. Mismatched unrelated cord blood is often compared with matched unrelated or matched family donors, and for any individual patient then no such donor was available. Milano reports a higher death rate in mismatched unrelated compared to  in mismatched unrelated cord blood transplants , and we Engraftment is slower with cord blood than with marrow, or haplo-identical transplant. Graft failure is the principal course of mortality after cord blood transplant for non-malignant disease , and we Intestinal failure prolongs hospital admission after cord blood transplant. This may be because of GVHD, and Barker reported that the GI tract was the organ most commonly involved in acute GVHD after such transplant . HoweverCB recipients have delayed immune reconstitution post HSCT. Studies suggest that there is delayed viral-specific immune reconstitution leading to higher incidence of viral reactivation in this cohort . This isTaking measures to identify a donor which would offer lifelong immunological accommodation is key to a successful programme. Selection of the optimal cord blood unit involves consideration of HLA and non-HLA factors outlined in Simultaneous national and international donor searches are initiated using targeted filters to optimize the potential number of donors for review. Useful search filters include applying the≤4 mismatches on any locus filter, setting a minimum TNC threshold and restricting choice to frequently used or United Kingdom registries. Other factors that should be considered include the accreditation status of the stem cell bank, the age of the donation, as more recently processed donors use improved methods of isolation, red cell reduction and have higher post thaw viability. A suggested upper limit of 2000 units for review should be retrieved during a search.allelefrequencies.net   without a locus bias, if possible, avoiding mismatching both specificities at any one locus. Extended matching is essential for patients with non-malignant disorders. Where HLA data are missing and an unusual association between loci exists, it is helpful to use HLA haplotype searches in websites such as za 2020)  to selecza 2020) .From the final shortlist of donor units, up to five cord blood units are selected and extended HLA typing requested to confirm the high-resolution match with the patient. Where a choice exists, HLA mismatches in the donor must be selected with consideration of the patient’s HLA antibody profile at the time of transplant, avoiding high titre specificities. The final selection from the shortlist will prioritize the best HLA matched unit with the largest viable cell dose.We prioritize cord blood in difficult-to-cure myeloid disease. We recognize that there is a higher procedure related risk using mismatched unrelated cord blood as a donor to be using matched unrelated donor. However, we further recognize that there is a reduced risk of relapse using the cord blood in a T-cell replete setting, and that the risk of chronic GVHD is low. In children therefore with relapse after a previous transplant or who are MRD positive after chemotherapy, then we will use a cord blood donor, as a platform to immunotherapy of refractory disease. In children with higher risk disease the better disease outcomes overcome the higher risk of such a transplant.In better risk myeloid disease, then we will follow our usual donor hierarchies of matched sibling donor, before matched unrelated donor  or matched cord blood donor , and if there is no matched donor then we would consider mismatched cord blood donor or haplo-identical donor, before mismatched unrelated donor.−4) we use chimeric antigen receptor (CAR) T-cell therapy, and we consider refractory T or bilineage, including infant leukaemias, as similar to refractory myeloid malignancy.In ALL we use the same donor hierarchy but in B-lineage ALL that is refractory  cord blood donor) before a mismatched donor. For a family donor, we would consider a directed cord blood donation from a matched sibling to be equivalent to the bone marrow donation, if the cell count is satisfactory. We will use a mismatched cord blood donor in preference to a mismatched unrelated donor, and in all such diseases the conditioning regimen includes T-cell depleting serotherapy.We will prefer to use cord blood where there is urgency to get to transplant such as in neurological, metabolic diseases and where full donor chimerism is associated with a superior clinical outcome, including metabolic diseases.In other situations, in non-malignant diseases, then judgment is required in selection of alternate donors. Where there is disease that has acceptable non-transplant therapy, such as transfusion-dependent thalassaemia, then no transplant at all might be the best option.However, discretion and judgement are required before turning down a matched sibling donor in favour of a poorly matched unrelated cord blood donor, and transplant survival must clearly be a priority of transplant.In malignant disease then transplant reduces relapse but its utility in both the malignant and the non-malignant setting is impacted by increased treatment-related toxicity. Strategies for cord transplant therefore and its maintained use as a donor cell source in HCT might address both these aspects.ex vivo CD34 stem cell expansion technology and such technologies will likely reduce the risk of graft failure and reduce therefore the risk of cord transplant. Such expansion technologies have been mainly trialed however in malignant diseases. Any expansion must allow preservation of graft T-cells, to permit the graft versus leukaemia effect  and transplant morbidity and mortality .We consider that cord blood transplant has a very significant place in paediatric transplantation, and that the change from cord to haploidentical donors is likely in many clinical circumstances to be associated with adverse outcomes for children.


There are few studies on predictive biomarkers for hyperuricemia, and the predictive value of these biomarkers tends to be poor. Additionally, no reports have described the predictive value of retinol binding protein 4 (RBP4) for hyperuricemia.This study was performed to evaluate the value of RBP4 for predicting the risk of hyperuricemia in a general population, determine whether RBP4 could be used alone or in combination with other factors to predict the risk of hyperuricemia in the general population, and establish an optimum predictive model.We conducted a population-based cross-sectional survey in 2018, involving a questionnaire, physical examination, and laboratory testing. We enrolled 2303 individuals by stratified random sampling, and 2075 were included in the data analysis after applying the eligibility criteria.P<0.001). After adjusting for potential confounders, logistic regression indicated that the risk of hyperuricemia was highest in the highest RBP4 quartile . The area under the receiver operating characteristic (ROC) curve (AUC) for RBP4 was 0.749 , which was higher than that for all the other predictors assessed. The optimum model for predicting hyperuricemia in the general population consisted of RBP4, sex , body mass index, serum creatinine, high-sensitivity C-reactive protein, fasting blood glucose, insulin, and alcohol consumption. The AUC was 0.804 .Serum RBP4 level had a highly significant association with hyperuricemia (RBP4 is strongly associated with hyperuricemia, and its predictive value was higher than that of traditional predictors. The global prevalence of hyperuricemia has increased rapidly in the past few decades –3; it isUnfortunately, studies on the risk and predictors of hyperuricemia are rare. Although some groups have reported on factors  , 17 thatRetinol binding protein-4 (RBP4) was recently recognized as a type of adipokine . SeveralThis large-scale study aimed to explore the association between RBP4 and hyperuricemia in the general population, and to evaluate whether RBP4 can predict hyperuricemia. We also hope that in the absence of serum uric acid results, abnormally elevated levels of RBP4 may suggest the importance of further serum uric acid testing.A cross-sectional survey of the general population in Wanzai town in Zhuhai city, China, was conducted in 2018. Using stratified random sampling, 2303 adults who were permanent residents of Wanzai town were initially enrolled in the study [1054 in the first visit in 2018 and another 1249 later in 2018 ]. We followed the same sampling procedures described in previous reports on our 2012 epidemiological survey –30.The exclusion criteria were as follows: (1) treatment with uric acid-lowering drugs or drugs that affect uric acid in the last 6 months, (2) severe liver or kidney damage, (3) acute cardiovascular or cerebrovascular diseases, and (4) pregnancy or breastfeeding.After applying the eligibility criteria, 2075 individuals were included in the final analysis.The study was approved by the Ethics Committee of the Third Affiliated Hospital of Southern Medical University and was conducted in accordance with the Declaration of Helsinki. All subjects provided written informed consent at recruitment.Data on sociodemographic factors , lifestyle , medical history , and medication use were collected using a questionnaire. The detailed design of this study (including the urine specimen collection) was the same as in our previous epidemiological survey conducted in 2012 –30.2. Obesity was defined as BMI >=28 kg/m2, BMI 24-28 kg/m2 was overweight.Physical examinations were performed to collect data on blood pressure (systolic blood pressure [SBP] and diastolic blood pressure [DBP]), weight and height , and WC. BMI was used to define obesity according to Chinese obesity criteria. Normal weight was defined as BMI<24 kg/mBlood specimens were collected after overnight fasting, stored at 2–8°C immediately after collection, and transported to the Central Laboratory of the Third Affiliated Hospital of Southern Medical University within 3 hours . RBP4 le2 or Fisher’s exact tests.Continuous variables with a normal distribution are presented as the mean and standard deviation, while those with a non-normal distribution are presented as the median and interquartile range. Categorical variables are presented as frequencies and percentages. Continuous variables were compared between groups using independent-samples t tests or analyses of variance  or Mann-Whitney U tests . Categorical variables were compared between groups using χSix binary logistic regression models , with hyperuricemia as the independent variable and RBP4 quartile as the independent variable, were used to calculate odds ratios (ORs) and 95% confidence intervals (CIs). The regression models adjusted for the following covariates: (1) age and sex; (2) model 1 covariates plus hypertension, diabetes, CHD, education of high school or above, physical inactivity, current smoking, and current alcohol consumption; (3) model 2 covariates plus SBP, DBP, log TG, LDL, HDL, BMI, eGFR, FBG, and log insulin; (4) model 2 covariates plus SBP, DBP, LDL, HDL, BMI, eGFR, and log HOMA-IR; (5) model 4 covariates plus log hs-CRP, and log IL-6; and (6) model 5 covariates plus log NAG and log ACR. SCr is strongly correlated with eGFR, so it was not included in the regression models.The area under the receiver operating characteristic (ROC) curve (AUC) was used to assess the predictive value of RBP4, other predictors, and the following three predictive models for hyperuricemia: (1) RBP4, sex, BMI, SCr, log hs-CRP, log insulin, log HOMA-IR, and current alcohol consumption; (2) RBP4, sex, BMI, SCr, log hs-CRP, and FBG; and (3) RBP4, sex, BMI, SCr, log hs-CRP, FBG, log insulin, and current alcohol consumption. Youden’s index (sensitivity + specificity – 1) was used to select the optimum cutoff value of RBP4. The reciprocals of HDL and eGFR were used due to their negative associations with serum uric acid level.P value <0.05 was considered statistically significant.Statistical analyses were performed in SPSS software version 20.0 . A two-sided P<0.05). In addition, there were higher values of SBP, DBP, WC, BMI, TG, LDL, FBG, insulin, HOMA-IR, SCr, cystatin C, NAG, hs-CRP, IL-6, and RBP4 in the hyperuricemia group compared to the non-hyperuricemia group (P<0.05), but lower values of HDL, eGFR, and serum uric acid (P<0.001).The baseline characteristics of the participants are shown in P<0.05). The values of SBP, DBP, WC, BMI, TG, LDL, FBG, insulin, HOMA-IR, serum uric acid, SCr, cystatin C, NAG, hs-CRP, IL-6, and ACR were higher and the values of HDL and eGFR were lower in the fourth quartile compared to the first quartile  .The associations between RBP4 and hyperuricemia, according to six multivariate binary logistic regression analyses, are shown in P<0.001) and Youden’s index was 0.36, with an optimum cutoff of 54.5 mg/L. Its predictive performance was better than the performances of SCr, cystatin C, eGFR, TG, WC, BMI, insulin, HOMA-IR, HDL, FBG, hs-CRP, and SBP . The predictive power of model 1 (AUC: 0.803) and model 2 (AUC: 0.797) were close to that of model 3 (P<0.001). The best predictive model for females was composed of RBP4, SCr, hypertension, log insulin, and log hs-CRP .The best prediction model was model 3, which involved RBP4 and other variables related to uric acid  in a binary logistic regression model . More importantly, RBP4 was a good predictor of hyperuricemia; indeed, it performed better than traditional predictors. We also explored the predictive value of RBP4 combined with routinely assessed clinical factors that are related to hyperuricemia. To our knowledge, this is the first study to investigate the predictive value of RBP4 for hyperuricemia.Using population-based data on southern Chinese adults collected in a single-center cross-sectional epidemiological survey, we found that RBP4 had a highly significant association with hyperuricemia  in regression model 6, the regression results showed that the independent association between RBP4 and hyperuricemia remained significant. We speculate that besides insulin resistance, RBP4 may have additional unique non-renal function mechanisms such as pro-inflammatory effects and direct effects on vascular smooth muscle and uric acid , 39, 40.Our results should be considered in the context of several limitations. First, the participants were all Han Chinese adults from Zhuhai city, and the results may not be generalizable to other ethnicities. In addition, this was a single-center study and therefore inevitably limited regarding the sample size; large multicenter studies are needed to verify the conclusions. Furthermore, the study was cross-sectional, and the underlying mechanisms were not explored in depth. Cohort or case-control studies and basic research on the underlying mechanisms should be performed to verify our findings.This study found that RBP4 is significantly positively associated with hyperuricemia in adults and has good predictive value for the condition. Clinically, it can be used alone or in combination with other traditional indicators to predict the risk of hyperuricemia.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of the Third Affiliated Hospital of Southern Medical University. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.G-BH, HZ, and C-XX participated in the research design, performance of the research, data analysis, and manuscript writing. X-FS, QZ, and X-SK participated in the research design and data analysis. P-CG, X-LL, JN, H-SC, and HX participated in the performance of the research and data analysis. All authors were involved in revising the manuscript and approved the final version.This study was supported by the following sources, (1) Clinical Research Startup Program of Southern Medical University by High-level University Construction Funding of Guangdong Provincial Department of Education , (2) Self-financing Science and Technology Projects of Foshan City , (3) Science and Technique Program of Guangzhou , (4) South Wisdom Valley Innovative Research Team Program , (5) Guangdong Provincial Science and Technique Program , (6) ISN Research Committee grant (2007), and (7) EU FP7 Program .Author P-CG was employed by Nanjing CR Medicon Pharmaceutical Technology Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The etiology of idiopathic normal pressure hydrocephalus (iNPH) is currently unknown. With no visible obstructions, altered cerebrospinal fluid (CSF) dynamics may explain the accumulation of ventricular fluid. We hypothesized that elevated osmolality in the CSF of iNPH patients could potentiate formation of ventricular fluid and thereby cause the disease progression and/or predict the surgical outcome. To address this hypothesis, we determined the lumbar and ventricular CSF osmolality of iNPH patients at different disease stages and compared with lumbar CSF samples obtained from control subjects.The osmolality of CSF was determined on a total of 35 iNPH patients at diagnosis and at the subsequent treatment with shunt surgery (n = 20) and compared with the CSF osmolality from 20 control subjects. Simultaneously collected lumbar and ventricular CSF samples from experimental pigs were used to evaluate the compatibility between CSF from different compartments.We found no evidence of increased osmolality in the CSF of iNPH patients upon diagnosis or at the time of shunt treatment months after the diagnosis, compared with control individuals. CSF tapped from the lumbar space could be used as a read-out for ventricular CSF osmolality, as these were similar in both the patient group and in experimental pigs. We further observed no correlation between the CSF osmolality in iNPH patients and their responsiveness to shunt surgeries.The osmolality of lumbar CSF is a reliable reflection of the ventricular CSF osmolality, and is not elevated in iNPH patients. iNPH therefore does not appear to arise as a function of osmotic imbalances in the CSF system and CSF osmolality cannot serve as a biomarker for iNPH or as a predictive tool for shunt responsiveness. CSF surrounds the mammalian brain and fills the central ventricular cavities. Abnormal expansion of the ventricular space signifies the pathological condition known as hydrocephalus. Although some forms of hydrocephalus arise following blockage of CSF circulation within the ventricular system and/or the CSF drainage routes, clinical hydrocephalus often manifests in the absence of a detectable blockage of these pathways. Idiopathic normal pressure hydrocephalus (iNPH) patients belong to the latter group, as these present with enlarged ventricles in the absence of a discernible blockage of the CSF pathways , 2. In aThe shunt surgery commonly used to treat iNPH patients diverts excessive ventricular fluid into the peritoneal cavity. Even though 80% of the iNPH patients appear to respond to these shunts with clinical improvement of their symptoms , complicWe here assessed the CSF osmolality in iNPH patients versus that obtained in a control group, evaluated the resemblance between lumbar and ventricular CSF osmolality, and determined whether CSF osmolality could serve as a predictive tool to select patients benefitting from surgical management.out). An abnormally high Rout increases the diagnostic accuracy, but a normal Rout does not preclude iNPH . Additional 0.07 ml acepromazine  and 0.5 ml methadone  were administrated intramuscularly at the time of premedication. Upon shaving of all puncture sites and cleaning with 70% ethanol, 1 ml CSF was collected from cisterna magna and from the lumbar cistern (between L7 and S1) with 90 mm and 75 mm spinal needles, respectively. Both CSF samples were obtained with < 5 min interval approximately 3 h after induction of anaesthesia. Animals were excluded if puncturing failed at one site. To avoid bias, the order of CSF collection was randomly controlled for each animal. All animal experiments were performed on pigs employed for veterinary student training of abdominal surgical procedures and according to the legislation for animal protection and care, animal permission no. 2016-15-0201-00957 approved by the Danish Animal Experiments Inspectorate.Danish mixed breeds of Yorkshire, Danish Landrace and Duroc pigs (n = 17) with a mean weight of 24.02 ± 4.37 kg and an estimated age of 10–13 weeks were included. CSF collection was performed with the pigs placed in lateral recumbency under general anaesthesia. Anaesthesia was induced with an intravenous injection of propofol  or Mann–Whitney test (unpaired data) to evaluate statistically significant differences between mean values of groups, as indicated in figure legends. Pearson’s correlation analysis was performed to determine correlations between two variables. P < 0.05 was considered statistically significant. Data are shown as mean values ± standard error of mean (SEM). GraphPad Prism  was applied for statistical analyses.Data were tested for normal distribution using the Shapiro–Wilk test. Data obtained from experimental pigs were normally distributed and were analyzed with paired two-tailed out, Pearson’s correlation coefficient (r) = − 0.28, n = 25, P = 0.17), Fig. To determine whether the enlarged ventricles observed in iNPH patients could develop subsequently to elevated osmotic pressure in the ventricular compartment, we compared CSF osmolality in patients with iNPH to that obtained from control subjects Fig. . The lumTo determine if CSF osmolality could be employed as a biomarker for disease severity and therefore for shunt eligibility, we evaluated the CSF osmolality in iNPH patients enrolled for shunt surgery versus those that were not  or no shunt  patients, Fig. The CSF outflow resistance in the iNPH shunt referral group  was significantly higher than that of the patient group not referred to shunt surgery  reflecting that a high RTo determine the osmolality in paired lumbar and ventricular CSF samples from individual iNPH patients, we employed the lumbar CSF sample taken upon the diagnostic workup  patient samples and ii) samples taken with  ~ 7 months interval representing the time between the diagnostic workup (lumbar samples) and the shunt surgery (ventricular samples). To obtain an experimental scenario in which we could determine the osmolality in both CSF compartments simultaneously in healthy organisms, we employed experimental pigs. Lumbar and ventricular CSF was collected from anaesthetized pigs at time points with only minutes in between. The osmolality of lumbar pig CSF was 289 ± 1 mOsm, which was not statistically different from the osmolality in ventricular CSF of 290 ± 1 mOsm (n = 17, P = 0.05, Fig. To determine whether the responsiveness to shunt surgeries correlates with the CSF osmolality of iNPH patients, the patients were divided into either a shunt responsive or a non-responsive group at a follow-up evaluation of the treatment performed 3–12 months after the ventriculo-peritoneal shunt operation see Fig. a or the We here demonstrate that the enlarged ventricles characteristic of iNPH patients do not appear to arise due to an elevated osmolality of the patient CSF and that the shunt responsiveness does not hinge on the CSF osmolality.Diagnostic workup of various patient groups employ CSF biomarker analysis. Although disturbed brain function is anticipated best reflected in the ventricular CSF bordering the affected brain regions, the CSF samples are generally obtained from the lumbar region. The lumbar sampling is swifter and less invasive than its ventricular counterpart and thus employed for ethical reasons and general feasibility. However, it remains unresolved to what extent the CSF composition at the base of the spine resembles that of the ventricular compartments. Blood-derived proteins, such as albumin, appear elevated in lumbar CSF , 19, 21,The osmolality of bulk CSF is similar to that of the plasma , and CSFMany iNPH patients, regrettably, do not benefit from shunt insertion . To sparIn conclusion, the CSF osmolality appears to be stable throughout the CSF system, as revealed by samples obtained in iNPH patients and in healthy pigs. Such stability allows lumbar CSF sampling to obtain osmolality determinations. iNPH patients do not present with elevated CSF osmolality and their ventriculomegaly, therefore, does not appear to arise as a function of osmotic imbalances in the CSF system. CSF osmolality thus cannot be employed as a clinical marker for disease progression or as a predictive tool for shunt responsiveness.


Ultra‐high‐performance liquid chromatography–Q–Exactive Orbitrap–mass spectrometry (MS) and gas chromatography (GC)–MS were performed for the qualitative and quantitative analyses of Citri Sarcodactylis Fructus (CSF) from different origins. The contents of eight major CSF components, namely 5,7‐dimethoxycoumarin, scopoletin, hesperidin, tangeretin, nobiletin, limonin, nomilin, and stachydrine, were quantitatively analyzed. Clustering analysis and principal component analysis (PCA) were, respectively, performed to classify and compare the 10 CSF batches. One hundred and two volatile components were identified accordingly by comparing retention times, reference standards, parent peaks, fragment peaks, and findings from relevant literature. Moreover, high content of 5,7‐dimethoxycoumarin and stachydrine was detected in all the CSFs, especially in CSF‐Zhe. Therefore, the high content component coumarin “5,7‐dimethoxycoumarin” was suggested to be quality analysis component rather than hesperidin. Additionally, characteristic compounds were found to distinguish different CSFs. This work was a comprehensive study about the components of various CSF. It distinguished the basic differences in the compositions of CSF from different origins. Eventually, it provided experimental and systematic bases for the quality control analysis of CSF, which has potential application in the further research. Ultra‐high‐performance liquid chromatography–Q–Exactive Orbitrap–mass spectrometry (MS) and gas chromatography (GC)–MS were performed for the qualitative and quantitative analyses of CSF from different origins. The contents of eight major CSF components, namely 5,7‐dimethoxycoumarin, scopoletin, hesperidin, tangeretin, nobiletin, limonin, nomilin, and stachydrine, were quantitatively analyzed. Principal component analysis (PCA) was performed to classify the 10 CSF batches. In addition, CSF is an ornamental plant and its essential oil is used as a raw material for high‐grade spices, which has great economic prospects. CSF is divided into the following four groups according to the origin: CSF‐Guang (from Guangdong or Guangxi Province), CSF‐Zhe (from Zhejiang Province), CSF‐Chuan (from Sichuan Province), and CSF‐Yun (from Yunnan Province). CSF‐Guang and CSF‐Zhe have superior quality and are applied for the treatment of cough with phlegm, stomach distension, vomiting, and other diseases , the dry fruit of Pharmacopoeia of the People's Republic of China (ChP), hesperidin is the only CSF component with a content >0.030% and is therefore used in quality control. Considering that the hesperidin content of CSF is slightly low, hesperidin as a single indicator is not sufficient to distinguish CSFs from different origins. Therefore, scientific index components should be urgently explored via quantitative analysis.Based on Some CSF components have medicinal and commercial values –Q–Exactive Orbitrap–mass spectrometry (MS) , Zhejiang Province (CSF‐Zhe), Sichuan Province or Chongqing municipality (CSF‐Chuan), and Yunnan Province (CSF‐Yun), China in 2019. The CSF samples were stored for 1 year and then used in the experiment. The detailed information of the CSF samples is shown in Table n‐hexane were obtained from Merck (Germany). Deionized water was prepared by a Milli‐Q system .The reference standards of stachydrine, scopoletin, hesperidin, 5,7‐dimethoxycoumarin, limonin, nomilin, nobiletin, and tangeretin were obtained from Weikeqi . The compounds had purities higher than 98%. High‐performance liquid chromatography (HPLC)‐grade formic acid was purchased from Thermo Fisher Scientific (USA). HPLC‐grade acetonitrile and 2.2For UPLC–Q–Exactive Orbitrap–MS analysis, the samples were crushed and passed through a 40‐mesh sieve to obtain a small powder. Each powdered sample (0.1 g) was precisely weighed, soaked with 50 ml of methanol, and ultrasonicated for 30 min . After the samples were cooled to room temperature, methanol was added to the CSF extracts until the weight was the same as its initial weight. The filtrate was prepared and then condensed to 5 ml by a XHRE‐2000C rotary evaporator . After filtering through a 0.22‐μm microporous membrane, the obtained sample solution was injected into the UPLC–Q–Exactive Orbitrap–MS equipment. The standards of stachydrine, scopoletin, hesperidin, 5,7‐dimethoxycoumarin, limonin, nomilin, nobiletin, and tangeretin were accurately weighed and dissolved in 50 ml of methanol. Then, methanol was used to dilute the mixture solution to obtain the concentrations of the tested compounds, which were filtered by a 0.22‐μm microporous membrane for UPLC–Q–Exactive Orbitrap–MS. Then, method validation was performed to determine the repeatability, stability, precision, recovery, and linear correlation of the method.n‐hexane, filtered through a 0.22‐μm microporous membrane, and then injected into the GC–MS system.For GC–MS analysis, each sample was smashed into coarse powder, and approximately 60 g of the crushed powder was weighed in a round‐bottom flask. The mixture was added with 600 ml of deionized water, fully soaked for 12 hr, and heated for 5 hr. Then, volatile oil was flowed back into the extractor with water vapor and then collected at room temperature. The obtained volatile oil was stored at 4°C in the dark to prevent degradation. The weight of the extracted volatile oil and tested compounds were used to calculate the extraction rate in volatile oil. The extracted volatile oil (50 μL) was diluted 20 times with 2.318 column  at 35 ℃. The mobile phase was composed of 0.05% (v/v) water–formic acid (A) and acetonitrile (B) with the following gradient elution conditions: 0–5 min, 10% B; 5–10 min, 10%–20% B; 10–20 min, 20%–50% B; 20–30 min, 50%–85% B; 30–40 min, 85%–100% B . Chromatographic separation was performed on a ZORBAX Eclipse Plus CMS identification was integrated on a Q‐Exactive Orbitrap–tandem MS instrument . Positive ion mode with 3500 V ion spray voltage was used. Nitrogen was applied as sheath gas (30 units), auxiliary gas (10 units), and sweep gas (5 units). Ion source temperature was sustained at 320 ℃, and desolvation temperature was sustained at 300 ℃. Full mass scan was set in the range of m/z 100–1000 Da. All the chemical compounds were discriminated by contrasting the mass spectra with the standard reference, reported literature, and Orbitrap Chinese Traditional Medicine Library (OCTML).2.4‒1 for 3 min, further increased to 250 ℃ at 10 ℃·min‒1, and finally increased to 300 ℃ at a rate of 25 ℃·min‒1 for 5 min. High‐purity helium, the carrier gas, was operated at a flow rate of 1.0 ml·min‒1, and 1 μl of the sample solution was injected in a splitting ratio at 50:1.GC analysis was performed on a TRACE DSQ GC instrument . TG‐5SILMS GC capillary column  was applied, and the temperature program was set as follows: initially, the temperature was set at 60 ℃ for 3 min, then increased to 80 ℃ at 1 ℃·minMS identification was operated on a TRACE DSQ mass spectrometer . Ionization energy was maintained at 70 eV, and temperature in the electron‐impact ion source was set to 270 ℃. The mass scan ranged from m/z 35 to 500 amu, and solvent delay time was set to 4 min. All the constituents in volatile oil were identified based on the comparison of the mass spectra and ion fragmentation with reported literature and MS library (NIST 2008).2.5All the data of CSF from different origins were statistically analyzed using SPSS and GraphPad Prism software. Clustering analysis and principal component analysis were applied to observe the similarities and differences of the CSFs from different origins.33.1Based on the previous study, the methanol extracts of 10 batches of CSFs from various geographical origins were separated and analyzed by UPLC–Q–Exactive Orbitrap–MS. The total ion chromatograms (TICs) of the methanol extract and mixed reference substances in positive ion mode are shown in Figure 3.2R2>0.995 (Table Good linear correlations were obtained for the eight compounds using this method with 95 Table . The lim3.3−1), stachydrine (1.261–1.555 mg·g−1), and nomilin (0.261–0.769 mg·g−1) and extremely low levels of hesperidin (0.044–0.232 mg·g−1), scopoletin (0.003–0.018 mg·g−1), limonin (0.025–0.084 mg·g−1), tangeretin (0.004–0.083 mg·g−1), and nobiletin (0.003–0.050 mg·g−1). Compared with major CSF cultivars, CSF‐Zhe contained the most abundant content of 5,7‐dimethoxycoumarin at 2.730–3.3613 mg·g−1. Additionally, hesperidin concentration in CSF‐Zhe was higher than those in the three other CSFs. The tangeretin contents of CSF‐Zhe and CSF‐Guang were relatively higher than those CSF‐Chuan and CSF‐Yun. However, nomilin was relatively higher in CSF‐Yun than in other CSFs.The contents of the eight principal components in the 10 batches of CSF Table  were obtHesperidin is a flavonoid substance that is widely found in citrus and contains substantial pharmacological activities, such as anti‐neoplastic, anti‐oxidation, and anti‐inflammatory activities; its content has been detected as a biomarker for quality control in CSF as described in ChP (2020). The present study indicated that hesperidin had extraordinarily low content and low response value compared with the other main components; therefore, it is not efficient and suitable as an index for the estimation of CSF quality. In comparison, 5,7‐dimethoxycoumarin has remarkable pharmacological activity  and D‐limonene contents (37.31%–54.33%), which possess excellent antifungal  was performed on 35 major volatiles in the CSF samples. Consequently, five principal components (PCs) were extracted in total, with a cumulative contribution rate of 95.48% Table . These f4In this study, a systemic qualitative and quantitative analysis of nonvolatile and volatile components was conducted. A particularly high content of stachydrine was detected in CSF, and it was the first time to performed stachydrine to quantitative analysis. A new referential index “5,7‐dimethoxycoumarin” was demonstrated to assess the quality and differences among CSF samples from various origins. CSF‐Zhe contained the most abundant 5,7‐dimethoxycoumarin and volatile oil in the four main origins. In addition, some important alkenes in CSF‐Zhe were significantly high, such as D‐limonene, γ‐terpinene, ɑ‐pinene, and β‐pinene, which indicated that there were certain chemical composition advantages in CSF‐Zhe. From the result, tetratetracontane was found to be detected in CSF‐Guang (S1), and terpinolene may be one of the characteristic compounds to distinguish CSF‐Zhe from other CSFs in this study. In summary, this work provided a comprehensive analysis method in CSFs, which conveyed a better understanding and sufficient reference for their further utilization.The authors declare no conflicts of interest.Guodong Zheng: Methodology ; Software ; Supervision ; Validation ; Writing – review & editing . Jinrong Ning: Conceptualization ; Investigation ; Visualization ; Writing – original draft . Kanghui Wang: Conceptualization ; Investigation ; Visualization ; Writing – original draft . Wanling Yang: Data curation ; Methodology ; Software . Mengshi Liu: Data curation ; Methodology ; Software . Jingyuan Tian: Data curation ; Methodology ; Software . Minyan Wei: Methodology ; Software ; Supervision ; Validation ; Writing – review & editing .This study does not involve any human or animal testing.Written informed consent was obtained from all study participants.


Leptomeningeal metastases (LM) occur in patients with breast cancer (BC) and lung cancer (LC) showing exceptionally poor prognosis. The cerebrospinal fluid (CSF) tumour microenvironment (TME) of LM patients is not well defined at a single‐cell level. Based on the 10× genomics single‐cell RNA sequencing (scRNA‐seq) data from GEO database including five patient‐derived CSF samples of BC‐LM and LC‐LM, and four patient‐derived CSF samples of idiopathic intracranial hypertension (IIH) as controls, we analysed single‐cell transcriptome characteristics of CSF TME in LM patients compared to controls simultaneously and comprehensively. In addition, we performed 10× genomics scRNA‐seq on CSF cells derived from a BC‐LM patient to help generate a solid conclusion. The CSF macrophages in LM patients showing M2‐subtype signature and the emergence of regulatory T cells in LM confirmed the direction of tumour immunity toward immunosuppression. Then, the characteristics of CSF circulating tumour cells (CTCs) of breast cancer LM (BC‐LM) patients were classified into five molecular subtypes by PAM50 model. The communication between macrophages and five subtype‐specific CSF‐CTCs showed largest number of ligand‐receptor interactions. The five subtypes‐specific CSF‐CTCs showed great heterogeneities which were manifested in cell proliferation and cancer‐testis antigens expression. Gene regulatory networks (GRNs) analysis revealed that transcription factor SREBF2 was universally activated in the five subtypes‐specific CSF‐CTCs. Our results will provide inspiration on new directions of the mechanism research, diagnosis and therapy of LM. Integrated analysis of characteristics of CSF tumour environment of LM patients.Five subtype‐specific CSF‐CTCs of breast cancer showed great heterogeneities on cell proliferation and cancer‐testis antigens expression.Gene regulatory networks (GRNs) highlighted transcription factor SREBF2 activated in the breast cancer CSF‐CTCs Breast cancer (BC) is the most common women's malignant tumours occurring brain metastasis.BC‐BM included parenchymal brain metastases (PBM) and leptomeningeal metastases (LM), which can occur either separately or simultaneously. PBM refer to multiple intracranial space‐occupying lesions shown on CT or magnetic resonance imaging (MRI). LM refers to dissemination of cancer cells to the pia mater, arachnoid and subarachnoid space. A positive cerebrospinal fluid (CSF) cytology result is the gold standard for LM diagnosis. BC‐LM can present as a late‐stage complication of systemic progression or the first sign of metastatic disease.Interests always abound in understanding the mechanisms that drive LM, which are thought to originate from the seeding of circulating tumour cells (CTCs) into leptomeninges. Single‐cell RNA sequencing (scRNA‐seq) technology make us available to obtain the single‐cell transcriptome data of CSF cells. For instance, scRNA‐seq methods have fuelled the investigations of CSF‐CTCs of LCIn this study, we intended to analyse the characteristics of CSF tumour microenvironment (TME) of LM patients. We enrolled scRNA‐seq data of nine CSF samples from Gene Expression Omnibus (GEO) database, including five CSF samples from three breast cancer LM patients (BC‐LM) and two lung cancer LM (LC‐LM) patients.22.1https://www.ncbi.nlm.nih.gov/geo/) database (GSE150681), including three BC LM patients  and two LC LM patients .The 10× genomics scRNA‐seq data of five LM patient‐derived CSF samples were from GEO  at Huashan Hospital, Fudan University in this study is GEO: GSE202501. The consent forms and the proposed studies were approved by Institutional Review Board of Huashan Hospital .The single cell date of BC blood‐CTCs were obtained, including 35 blood‐CTCs sequenced by smart‐seq2 method2.2https://figshare.com, https://doi.org/10.6084/m9.figshare.19130036).Cells had less than 1000 counts and 300 genes were removed for following analyses (Seurat package version 4.0.4). After filtration, gene expression matrix were merged and the batch effect was removed with the R package harmony v1.0 . The merged expression matrix was prepared for clustering using the Seurat 4.0.4, following the common pipeline. Uniform Manifold Approximation and Projection (UMAP) dimensionality reduction was used to project cells in two dimensions. An R project including counts and celltypes of five LM patient‐derived CSF samples and four control CSF samples are provided on figshare website (The feature plots based on R package Seurat were used to demonstrate the expression marker genes. Marker genes: T cells (T), CD3E and TRAC; CD4+ T cells (CD4): CD4 and IL7R; CD8+ T cells (CD8): CD8A, CD8B; regulatory T cells (Treg): FOXP3 and CTLA4; NK cells (NK): GNLY, PRF1, and XCL1; myeloid lineage cells: LYZ; monocyte cells (Mono): FCGR3A, S100A8; Macrophage cells (Mac): CD14, PLTP, MRC1, NLRP3 and IL1B; myeliod DC type 1 cells (mDC1): XCR1; myeliod DC type 2 cells (mDC2): FCER1A, CD1C; plasmacytoid DC cells (pDC): TNFRSF21; B cells (B): CD79A; tumour cells: EPCAM, CDH1, KRT18 and KRT19.2.32FoldChange ≥ 0.25, adjusted P‐value < 0.01.We applied the FindAllMarkers function in R package Seurat (4.0.4) to identify differentially expressed genes (DEGs) for each cell clusters. The Wilcoxon Rank‐Sum test was applied to determine the significance of the difference. The DEGs in the cell state over the trajectory were selected for subsequent analysis by average Log2FoldChange ≥ 1, adjusted P‐value < 0.05), so that their expression is significantly higher in CSF‐CTCs.The DEGs between CSF‐CTCs and blood‐CTCs were filtered based on the statistical threshold . Signature genes need to be expressed in > 25% of cells in a cell cluster and < 5% in other cell clusters.FindAllMarkers function was also performed to select signature genes in different cell clusters according to the statistical threshold (log2.4AddModuleScore function in R package Seurat (4.0.4) was applied to the calculation of the gene expression modular scores per cell. The conventional classic activated macrophages (M1‐subtype) modular included genes IL1B, IL1A, TNF, IL6, CXCL9, CXCL10, IL12A, IL12B, IL23A, FCGR1A, FCGR1B, FCGR1C, CCR7, IL8, CCL5, HLA‐DRA, IRF5, IRF1. The alternative activated macrophage (M2‐subtype) modular contained genes IL10, CD163, MARCO, MRC1, MSR1, ARG1, STAB1, TGM2, MMP7, MMP9, MMP19, TGFB1, TGFB2, TGFB3, VEGFA, FN1, CCL4, CCL22, CCL17, CCL18, IL4R, IL7R, IRF4.2.52Foldchange ≥ 0.25, adjusted P‐value < 0.01, Wilcoxon Rank‐Sum test.Pseudotime analysis was performed using R package Monocle2 (v2.18.0) to determine the dramatic translational relationships among different cells. FindAllMarkers function in R package Seurat (4.0.4) was taken to identify specific genes in each state with average log2.6https://www.cellphonedb.org/) package was applied to investigate the cell–cell communication, ligand–receptor interaction with P‐value < 0.05 were considered as significant.The python CellPhoneDB , basal‐like , human epidermal growth factor receptor‐2 positive (Her2), Luminal A (LumA) and Luminal B (LumB), according to the expression of 50 signature genes2.8https://www.gsea‐msigdb.org/gsea/msigdb).The gene set variation analysis (GSVA) made use of the R package GSVA (1.40.1)2.9The R package SCENIC (v1.1.0) was applied to analysing the enrichment of TF‐binding motifs per cell.The hub gene in the 5 GRNs was identified by CytoHubba in Cytoscape (3.8.2).2.10t‐test, or Wilcoxon Rank‐Sum test. Difference among multiple pairwise comparisons was analysed by one‐way ANOVA test or R Package Dunn.test (v1.3.5) using Kruskal–Wallis test. P‐value < 0.05 was considered to be significant.Differential analysis between two groups was performed using two‐sided Student's 33.1Immune components have been reported to exert crucial influence on tumorigenesis and progression,P‐value <  0.01). Signature genes need to be expressed in > 25% of cells in a cell cluster and < 5% in other immune cell clusters. As expected, the macrophage cluster expressed markers of microglia  were divided into CD4+ T cells (IL7R and CD4) and CD8+ T cells (CD8B) Figures  and S1B. Figures  and S1D.3.2Myeloid cells play a critical role in tumour inflammation regulation. Tumour‐associated macrophages (TAMs) are commonly characterised as M2‐polarised phenotype macrophages, which contribute to tumour aggressiveness.CCL3, CCL4, CCL4L2, CCL3L3 CCL8, CXCL8).An unsupervised trajectory analysis was conducted to understand the transcriptional transition from monocytes to macrophages in LM CSF samples.3.3CD8A and CD8B) displayed cytotoxic  or exhausted state   mainly showed naïve state  in LM compared to controls , delivered a direction of tumour immunosuppression  expression and a higher variable of copy number variations (CNVs) than normal immune cells  in the five subtype‐specific CSF‐CTCs. BC CSF‐CTCs had higher expression of 11 CTAs  family is necessary for the synthesis of fatty acids and cholesterol.3.6https://github.com/ddiez/DoubletFinder). Six thousand nine hundred forty‐four CSF single‐cell transcriptomes data in total were obtained and classified into eight final cell clusters, no obvious pDC cluster and mDC1 cluster were discovered . We removed cells which had less than 300 genes and 1000 counts, more than 20% mitochondrial‐gene counts, and computed doublet detection by DoubletFinder , which providing a crucial energy source for M2 polarization.In this study, we concentrated on the CSF TME of BC‐LM and LC‐LM patients, and the characteristics of BC‐LM CSF‐CTCs based on the scRNA‐seq data. We identified nine different immune cell types, and revealed monocytes and macrophages enriched in CSF of LM patients Figure . M2‐polaThe paracrine interactions between malignant cells and TME immune cells promote the immune surveillance escape and tumour progression. Interactions between myeloid cells  were the most prominent in the network of immune cells Figure . The macBC CSF‐CTCs had great heterogeneity shown on the cell‐cycle state and CTAs expression Figure , same asWe enrolled one BC‐LM patient to help generate a solid conclusion and 6944 CSF single‐cell transcriptomes data in total were obtained for analysis Figure . The TMEP‐value < 0.05, FoldChange > 1.5, Figure CTCs in blood of BCs have been reported into two subpopulations: epithelial  cells and mesenchymal  cells.In summary, our study is the first one to define the single‐cell transcriptome characteristics of CSF TME in LM patients of BC and LC. The emergence of M2‐subtype macrophages and regulatory T cells in LM confirmed the direction of tumour immunosuppression. The communication network in CSF TME showed most significant interactions between BC five subtype‐specific CSF‐CTCs and macrophages/monocytes, especially the interactions of MDK‐SORL1 and MDK‐LRP1 ligand–receptor pairs. The great heterogeneities of BC CSF‐CTCs were manifested in cell proliferation, CTAs expression and GRNs. GRNs revealed that SREBF2 was critical for the BC‐LM progression. Our study provided an important reference point for CSF microenvironmental research of LM and revealed transcriptional plasticity of BC CSF‐CTCs, which facilitated the understanding of LM mechanism research. Further, we intend to enlarge specimen to further support and enrich research results. It is interesting to analyse the association between survival and CSF immune cell composition.The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.Supporting MaterialClick here for additional data file.


The cerebrospinal fluid (CSF) plays an important role in delivering nutrients and eliminating the metabolic wastes of the central nervous system. An interrupted CSF flow could cause disorders of the brain and eyes such as Alzheimer's disease and glaucoma. This review provides an overview of the anatomy and flow pathways of the CSF system with an emphasis on the optic nerve. Imaging technologies used for visualizing the CSF dynamics and the anatomic structures associated with CSF circulation have been highlighted. Recent advances in the use of computational models to predict CSF flow patterns have been introduced. Open questions and potential mechanisms underlying CSF circulation at the optic nerves have also been discussed. The cerebrospinal fluid (CSF) is primarily composed of water (99%)  and occuIn the ON, interrupted CSF circulation, also referred to as compartment syndrome of the ON , 8, causIn this review, the anatomy and circulation route of the intracranial and ON CSF system have been introduced with an emphasis on the ON. Interstitial fluid (ISF) in parenchyma can be rapidly transported by AQP4 at the apex of the astrocytes , and theVarious tissues and structures are involved in the generation, circulation, and absorption of CSF, including the choroid plexus, ependyma, cisterna magna, subarachnoid spaces, Virchow–Robin space (VRS), brain parenchyma, the hypothalamus in the intracranial space, dura, ON subarachnoid space, and ON parenchyma . In thisMost of the CSF is produced in the choroid plexus. The choroid plexus is located in the ventricular system, which is a set of interconnected cavities composed of two lateral ventricles, the third ventricle, the cerebral aqueduct, and the fourth ventricle. Capillaries, enfolded by fenestrated endothelial cells, form the core of the choroid plexus . The epeCSF flowing from cisterna magna enters the subarachnoid space. The subarachnoid space is the space between the arachnoid mater and the pia mater, which is filled with CSF. It is occupied by delicate connective trabeculae and intercommunicating channels as well as branches of the arteries and veins of the brain .The VRS  are regaThere are three layers of meninges covering the central nervous system. The pia mater directly enfolds the parenchyma. Dura mater is the exterior most layer of the brain meninges. Between the dura and the pia is the arachnoid layer. Studies have shown that a network of lymphatic vessels exists in the dura, which is capable of draining CSF out of the brain , 24. How18F) fluoro-D-glucose ([18F]FDG) secreted by the hypothalamus takes part in the regulation of metabolic processes and sleep processes . Du. Du40). It is speculated that ON dural sheath is involved in CSF transport through the lymphatic vessels in the dura . HoweverInside the ON parenchyma, a perivascular transport system exists to allow CSF communications between the ON subarachnoid space and the ON parenchyma . Cells, Human eyes exhibit frequent movements in daily activities with approximately 170,000 saccades per day . The angInteractions between the eye globe and the ON –53 durinOf note, the eye rotates frequently during rapid eye movement (REM) sleep, which is a sleep stage, despite the eyes being closed. This state persists for 10–30 min at a time . ThroughFluid in the lymphatic systems  plays a Microscopically, the metabolic waste of brain tissues is cleared by a brain-wide perivascular pathway along which there is an exchange of CSF and ISF through AQP water channels , which iThe glymphatic system provides novel insights into the pathology of AD. The deposition of β-amyloid plaques  in AD reThe ON is part of the central nervous system. Therefore, the glymphatic system should also exist in the ON to assist in the clearance of metabolic wastes. The ON glymphatic system can assist in understanding the development of glaucoma . In WangA higher trans-lamina cribrosa pressure difference (TLCPD) will cause deformation of the intraocular portion of the ON and distort the pathways of the ON glymphatic system, which could hinder the metabolic clearance mechanism and result in glaucoma . The intOwing to the limitations of currently used imaging technologies and anatomical findings, whether the glymphatic system has an impact on those diseases is still unknown. Further research is thus required.Various imaging technologies with corresponding indicator elements or substances can be utilized to visualize the pathways of CSF to understand its properties and functions. These techniques include magnetic resonance imaging (MRI), two-photon imaging, computer-assisted cisternography (CT cisternography), and scanning electron microscopy (SEM). Moreover, phase contrast techniques are also widely used in the CSF study .Phase-contrast MRI (PCMRI) is a commonly used method in studying CSF dynamics . PCMRI iBesides PCMRI, time-spatial labeling inversion pulse (Time-SLIP) is another technique that can visualize the CSF movement . It is uEcho planar imaging (EPI) is another technique frequently used in MRI. EPI is capable of acquiring an image in milliseconds, allowing it to image dynamic physiologic processes such as CSF movement. In 1990, Stehling and associates showed that EPI with a snap-shot imaging time of 128 ms allows detailed demonstration of transient intraventricular CSF flow patterns . It alsoBecause a contrast agent is not necessary for MRI, potential artifacts induced by contrast agent injection, such as the increase in the CSF volume, can be avoided. However, in a complex CSF flow pattern, because anterograde and retrograde flows mix, they may not be detectable with fMRI as a result of spatial overlap.in vivo two-photon imaging to detect the perivascular CSF pathway through the brain parenchyma. In this study, it is found that CSF enters the parenchyma along perivascular spaces, which surround the penetrating arteries and the ISF is cleared along perivenous pathways.Two-photon imaging is a type of fluorescence imaging with a limited imaging depth of approximately 300 μm . Becausein vivo. The study conducted by Wang used this technique and found that the ocular β-amyloid clearance was affected by glial water channel AQP4  is a branch of fluid mechanics that utilizes equations governing a fluid motion to simulate complex fluid flows. Generally, four steps are required in a CFD simulation that are as follows: 1) constructing the geometry of the problem, usually through computer-aided design or reconstruction from medical images. 2) dividing the volume of fluid and the boundaries into discrete cells (elements). 3) defining the physical modeling such as the equations of fluid motion. 4) defining the boundary conditions . All comComputational models are versatile in terms of predicting the CSF properties in perivascular spaces , CSF volIntracranial CSF circulation has been investigated to a considerable extent. However, the corresponding flow in the ON is largely unexplored . The narAccording to Killer's research, the subarachnoid space in the ONs is divided into different cavities by trabeculae and septae . HoweverMoreover, the relationship between eye movements and the CSF circulation in the ON deserves further investigation. The functions of eye movements in human REM sleep are still unknown. Eye movements have been hypothesized to drive the CSF flow in the ONs.This review described the anatomy and CSF flow in the ON and cranium, as well as research tools for CSF dynamics. The role of the glymphatic system in the brain and the relationship between CSF flow and ophthalmic and neurological diseases were emphasized. Potential mechanisms and driving forces of CSF circulation in the ON are still uncertain. Several questions about ON CSF dynamics remain to be answered. The eye movement was speculated to be the driving force of CSF exchange between the ON and cranial subarachnoid spaces. We are interested in analyzing the pressure gradients in the ON and their impact on the visual pathway.XW and JS contributed to the conception of the manuscript and wrote the first draft of the manuscript. QL and TL wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This study was funded by the National Natural Science Foundation of China (12002025 and U20A20390) and the 111 Project (B13003).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The early diagnosis and treatment of bacterial meningitis (BM) in young infants was very critical. But, it was difficult to make a definite diagnosis in the early stage due to nonspecific clinical symptoms. Our objectives were to find the risk factors associated with BM and develop a prediction model of BM especially for young infants.We retrospectively reviewed the clinical data of young infants with meningitis between January 2011 and December 2020 in Children’s Hospital of Soochow University. The independent risk factors of young infants with BM were screened using univariate and multivariate logistic regression analyses. The independent risk factors were used to construct a new scoring model and compared with Bacterial Meningitis Score (BMS) and Meningitis Score for Emergencies (MSE) models.Streptococcus  and Escherichia coli  were the main pathogens of BM in the young infants. Multivariate logistic regression analysis identified procalcitonin (PCT), cerebrospinal fluid (CSF) glucose, CSF protein as independent risk factors for young infants with BM. We assigned one point for CSF glucose ≤ 1.86 mmol/L, two points were assigned for PCT ≥ 3.80 ng/ml and CSF protein ≥ 1269 mg/L. Using the not low risk criterion (score ≥ 1) with our new prediction model, we identified the young infantile BM with 100% (95% CI 91.9%-100%) sensitivity and 60.3% (95% CI 46.4%-72.9%) specificity. Compared with BMS and MSE model, our prediction model had larger area under receiver operating characteristic curve and higher specificity, the differences were statistically significant.Among the 102 young infants included, there were 44 cases of BM and 58 of aseptic meningitis. Group B Our new scoring model for young infants can facilitate early identification of BM and has a better performance than BMS and MSE models. Bacterial meningitis (BM) is a life-threatening bacterial infection, with the highest incidence reported in young infants . Due to Physicians have been trained to administer antibiotics for infants suspected bacterial infection as soon as possible. If antibiotics need to be given before the lumbar puncture (LP) is performed, the antibiotics can sterilize the cerebrospinal fluid (CSF) making it less likely that bacteria will grow . This ca6 /L). In our study, young infants suspected of meningitis and who have completed LP must have the following examinations: peripheral blood cell count, peripheral blood inflammatory markers , CSF cell count, CSF glucose and protein, CSF Gram stain, CSF and peripheral blood culture, cranial magnetic resonance imaging or computerized tomography. It was also suggested to conduct CSF virus detection or CSF metagenomics next generation sequencing if conditions permit. We excluded those patients who were not previously healthy , underwent a traumatic LP (> 1000 × 106 /L red blood cells in the CSF), diagnosed with a definite viral meningitis , or treated with antibiotics within 72 h before the diagnostic LP. We also excluded the cases with incomplete clinical data. Then, we selected eligible patients who were finally diagnosed as BM (the ICD code was G00.900) or Aseptic meningitis . The eligible patients were from Children’s Hospital of Soochow University between January 2011 and December 2020. The study was approved by the Ethics Committee of Children’s Hospital of Soochow University.We retrospectively reviewed the clinical records of young infants aged from 29 to 90 days with suspicions of meningitis , and patients in whom a LP was accepted and with CSF pleocytosis , Escherichia coli, Staphylococcus aureus, Neisseria meningitidis, Haemophilus influenza, etc.); or (2) the presence of CSF pleocytosis and with a positive blood culture. Organisms  cultured in previously healthy patients were considered to be contaminants. AM was defined as CSF pleocytosis with negative bacterial cultures of the CSF and blood. In our study, AM also include the presence of negative viral tests if performed. Complications of acute meningitis included seizure, subdural effusion, hydrocephalus, brain abscess, suspected ventriculitis and cerebral infarcts.Infantile BM was diagnosed according to either one of the following two criteria: (1) the CSF culture was positive for a bacterial pathogen . The presence of seizures and increased AFP were determined by two treating physicians. The laboratory values were obtained closest before the time to the LP, the data included peripheral white blood cell (WBC) count, peripheral absolute neutrophil count (ANC), CRP, PCT measurements, blood culture. LP results included CSF WBC count and CSF ANC, CSF glucose, CSF protein, CSF Gram stain, CSF culture.P < 0.05 indicated that the difference was statistically significant.Statistical analyses were performed using SPSS 27.0. Our analysis showed that the measurement data in this study were not normally distributed. Therefore, the measurement data were expressed as medians (quartiles), and the count data were expressed as frequencies (percentages). Mann–Whitney test for measurement date and χ2 test for count date were used to compare variables between groups. We conducted a receiver operating characteristic (ROC) curve analysis including significant continuous variables selected by univariate analysis. Continuous variables were converted to dichotomous variables according to the optimal cutoff points used by the Youden index. Risk factor analysis was performed using univariate and multivariate logistic regression analyses. In multivariate logistic regression analysis, the forward stepwise method was used to select independent risk factors for young infants with BM. Hosmer–Lemeshow test was used to identify the fitness of the regression model. The score point of each predictor was determined by the value of logistic coefficient. Area under the ROC curve was calculated to evaluate capacity of the models. The sensitivity and specificity for each scoring models were calculated. Two-tailed analysis with Escherichia coli , Streptococcus pneumonia , Klebsiella pneumonia , Enterococcus species , and Staphylococcus aureus . In the BM group, there were 6 infants with positive CSF Gram stain, and all of them were eventually cultured pathogenic bacteria. The bacterial pathogen was identified in both CSF and blood culture in 18 patients (40.9%), CSF culture alone in 16 patients (36.4%), and blood culture alone in 10 patients (22.7%). All the complications identified (37.9% of the patients) were seizures in AM group. Infants with BM could be combined with multiple complications rather than a single complication, the incidence of one or more complications was 59.1% in BM group. The main characteristics of the patients with BM and AM are shown in Table There were 133 infants aged from 29 to 90 days who had CSF pleocytosis and were initially diagnosed with meningitis, 27 cases with incomplete data were excluded. We also excluded 4 infants who were finally diagnosed with enteroviral meningitis. Eventually, the remaining 102 young infants including 44 (43.1%) with BM and 58 (56.9%) with AM were enrolled in our study. In the AM group, 32.8% (19/58) of the cases received Enterovirus testing, 77.6% (45/58) received Herpes simplex virus testing, and none of them had positive results. These children without positive pathogens were finally diagnosed as AM. Cases of BM were caused by the following pathogens: GBS , P < 0.05) were demonstrated in peripheral WBC count, CRP, PCT, CSF WBC count, CSF ANC, CSF glucose, CSF protein and positive Gram stain between the BM and AM . We evaluated the performance of the three models in predicting young infants at not low risk of BM in terms of specificity and sensitivity. Thus, we got 100% (95% CI 91.9%-100%) sensitivity and 60.3% (95% CI 46.6%-72.9%) specificity in our new model, 90.9% (95% CI 78.3%-97.4%) and 10.3% (95% CI 3.9%-21.2%) in the BMS model, 100% (95% CI 91.9%-100%) and 19.0% (95% CI 9.9%-31.4%) in the MSE model, respectively. Our prediction model had a higher specificity than the other two models in our study patients . The results are shown in Table We also tested BMS model and MSE model in our study, the area under ROC curve was 0.64 (95% CI 0.53–0.75) for BMS model and 0.82 (95% CI 0.74–0.90) for MSE model. Compared with BMS and MSE model, our prediction model had a larger area under ROC curve, and the differences were statistically significant . Using the not low risk criterion (score ≥ 1) with our new prediction model, we identified complication with 77.1% (95% CI 62.7%-88.0%) sensitivity and 38.9% (95% CI 25.9%-53.1%) specificity.Finally, we calculated sensitivity and specificity of our new scoring model to identify BM complication. The incidence rate of complication of the BM was 59.1%, which was significantly higher than that of the AM patients  with the 1 month old of the median age [Escherichia coli remain the most common causes of BM in the young infants.GBS accounted for a half of cases of BM in young infants in our study, and om Japan , 38% of om Japan , and 32%om Japan . GBS proom Japan , 16. So iagnosis . In Japadian age . UnfortuGenerally, it is difficult to distinguish between bacterial and aseptic meningitis at the early stage of the disease, especially in young infants. The majority of infants with CSF pleocytosis receive broad-spectrum antibiotics while awaiting the results of culture tests. Due to the high mortality and morbidity rates of BM , 4, it iSeveral diagnostic prediction models have been developed to assess the likelihood of BM in patients presented with suspected central nervous system infection. Up to now, the frequently reported predictors of the BM included seizures; higher level of peripheral WBC count, peripheral ANC, CSF ANC, CSF protein, CRP; lower lever of CSF glucose or CSF/blood glucose ratio –9. HowevStreptococcus pneumonia and Haemophilus influenza in older infants and children, leukopenia was most common in young infants with Escherichia coli and GBS [s obtained earlier than in others’ studies. We reviewed the clinical data and found that 76.5%(78/102)of the infants had peripheral blood routine examination within 24 h of fever. CSF protein concentrations were higher in healthy infants than in older infants and children [Our new scoring model included PCT ≥ 3.80 ng/ml, CSF glucose ≤ 1.86 mmol/L and CSF protein ≥ 1269 mg/L. PCT as a predictor of BM was also proposed by Dubos and Mintegi , 20. In  and GBS , 24. Anochildren , that wachildren . We alsoCSF Gram staining is fast, convenient, and well validated for detecting bacteria. In the BM group, there were 6 infants with the positive Gram-stain, who got the risk score ≥ 4 using our prediction model. Therefore, we recommend that broad spectrum antibiotics should be used to infants with CSF pleocytosis and positive Gram-stain until culture results are available. According to the not low risk criterion (score ≥ 1) of our new model, we identified the young infantile BM with 100% (95% CI 91.9%-100%) sensitivity and 60.3% (95% CI 46.4%-72.9%) specificity. Thus, we also recommend that physicians should give antibiotics to young infants with CSF pleocytosis and risk score ≥ 1.We also assessed the role of BMS and MSE scoring models in the present study. Their areas under the ROC curve were 0.64 and 0.82, respectively, which were smaller than our new scoring model of 0.93. Most prediction models were developed to accurately identify patients with BM. Because missing BM will have devastating consequences, only 100% sensitivity seems good enough. But, the sensitivity of the BMS model was 90.9%, lower than 100% sensitivity of MSE model and our model. Meanwhile, BMS results were found to be negative for a few children with BM in other studies , 27. ThaSeveral limitations of our analyses should be considered. First, this was only a retrospective study and was not evaluated in a prospective study. Second, blood glucose before lumbar puncture was not always timely registered, leading to the lack of a more appropriate predictor of CSF/blood glucose ratio. Furthermore, Distribution of main pathogens of BM varies in other regions and would alter the performance of the prediction models. Since our study was realized in a single hospital in an east China population, one may question the generalizability of the findings.We presented a new scoring model, appearing sufficiently accurate to permit the timely diagnosis of BM in young infants with CSF pleocytosis. This simple prediction model is more appropriate for young infants and it has a better performance than BMS and MSE models.


Cases of preclinical AD were identified using biomarker-guided classification . The prediction of dementia onset (questionable dementia) among CH participants was assessed as the risk of conversion from Clinical Dementia Rating [CDR = 0] to CDR ≥ 0.5 over 6 years. Mixed linear models were used to assess the utility of baseline CSF NfL levels for predicting the rate of cognitive decline among participants with MCI over 4.5 years. Results: Neurofilament light levels were significantly higher in preclinical AD participants  as compared to A-T-N- (p < 0.001). Baseline levels of CSF NfL were higher in CH participants who converted to CDR ≥ 0.5 over 6 years (p = 0.045) and the risk of conversion to CDR ≥ 0.5 was predicted . CH participants with CSF NfL > cut-off were at a higher risk of developing dementia . Participants with MCI and with higher baseline levels of CSF NfL (>median) had a higher rate of decline in cognition over 4.5 years. Conclusion: An assessment of CSF NfL levels can help to predict dementia onset among CH vulnerable individuals and cognitive decline among those with MCI.Background: Biomarkers that are indicative of early biochemical aberrations are needed to predict the risk of dementia onset and progression in Alzheimer’s disease (AD). We assessed the utility of cerebrospinal fluid (CSF) neurofilament light (NfL) chain for screening preclinical AD, predicting dementia onset among cognitively healthy (CH) individuals, and the rate of cognitive decline amongst individuals with mild cognitive impairment (MCI). Methods: Neurofilament light levels were measured in CSF samples of participants (CH,  Alzheimer’s Disease (AD) is a disease of multiple etiologies . The assNeurodegeneration initiates early in the AD continuum, believed to be triggered by several pathological changes, such as amyloidopathy, tauopathy, neuroinflammation and oxidative stress . TherefoIn addition to its diagnostic utility for AD, NfL can be used as a screening biomarker of early neurodegeneration to predict the risk of dementia onset in the vulnerable population, as well as a prognostic biomarker of downstream neurodegenerative changes to identify cognitively impaired individuals who are likely to progress further along the trajectory of cognitive impairment. Studies indicate that elevated levels of CSF NfL predict the risk of conversion to mild cognitive impairment (MCI), among CH participants , and corn = 186, aged ≥ 60 years). Of these, 154 were CH, and 32 had MCI. Participants’ CSF was collected between 2009 and 2016. Participants were clinically classified as MCI or healthy controls/cognitively healthy (CH), using all the available information  = 0.79, CI 0.60–0.98, p = 0.011). CSF NfL levels were also significantly elevated in the preclinical AD group compared to the preclinical AD pathological change group . CSF NfL levels were higher in the preclinical AD group compared to non-AD pathological change, though not significantly.This analysis was carried out in CH participants . CSF NfL levels (Z scores) significantly predicted the risk of conversion from CDR = 0 to CDR ≥ 0.5 over 6 years, with a hazard ratio (HR) of 1.60 , after controlling for age, sex and APOE genotype. Participants (CH) whose CSF NfL levels were above the optimum cut-off  were at a higher risk of dementia onset . The Kaplan–Meier survival curves shown in p < 0.001). The high CSF NfL group had 22 CH participants who converted to CDR ≥ 0.5 or developed questionable dementia over 6 years, while the low CSF NfL group had three such participants  after controlling for covariates age, sex and APOE genotype . The prognostic utility of NfL was assessed using mixed linear models, for predicting the rate of cognitive decline in participants with MCI over 4.5 years, assessed through the annual change in MMSE and CDR-SB scores. For comparison, a disease prognostic analysis was also carried out for core CSF biomarkers . MCI participants (n = 32) were dichotomized into groups (low and high CSF measure) based on the 50th percentile of CSF measures . The annual change in MMSE and CDR-SB scores was assessed in the two groups after controlling for age, sex and APOE genotype  were significantly elevated in participants with MCI vs. CH (genotype .p < 0.001), and 0.19 in the low CSF NfL group (p = 0.003). The annual change in MMSE was −0.99 points in the high CSF NfL group (p = 0.001), and −0.12 in the low CSF NfL group (p = 0.372). p < 0.001), and 0.20 in the low CSF T-tau group (p = 0.002). The annual change in MMSE was −1.03 points in the high CSF T-tau group (p = 0.003), and −0.14 in the low CSF T-tau group (p = 0.214). For CSF P-tau, the annual change in CDR-SB was 0.78 points in the high CSF P-tau group (p < 0.001), and 0.18 in the low CSF P-tau group (p = 0.003). The annual change in MMSE was −1.31 points in the high CSF P-tau group (p < 0.001), and −0.04 in the low CSF P-tau group (p = 0.734). For CSF Aβ42, as expected, the annual change in cognitive scores was higher in the low CSF Aβ42 group. Lower levels of CSF NfL, T-tau, P-tau and higher levels of CSF Aβ42 did not indicate the rate of change in MMSE scores. Therefore, further studies are required to establish the association.For CSF NfL, the annual change in CDR-SB (rate of increase) and annual change in MMSE (rate of decrease) was higher in group 2 (high CSF NfL group). A higher rate of decrease in MMSE and a higher rate of increase in CDR-SB indicates a higher rate of cognitive decline. The annual change in CDR-SB was 0.68 points in the high CSF NfL group (p = 0.00125) compared to A-N- for both groups) and much higher in A+N+ (1.66 times higher) [Cerebrospinal fluid NfL is a biomarker of neurodegeneration whose levels are elevated in AD and associate with central neuropathological changes–tauopathy, amyloidopathy and brain atrophy ,9,10,16.We found significantly elevated baseline levels of CSF NfL in CH individuals who developed questionable dementia or very mild dementia over 6 years. In addition, the elevated levels predicted the risk of developing questionable dementia. We noted that individuals with CSF NfL levels above optimal threshold are at much higher risk of dementia onset. Future studies with a larger sample size should test our findings by classifying CH participants using a specific threshold, or into tertiles or quartiles. Since AD is a neurodegenerative disease, the preclinical stage could be associated with an initial stage of neurodegeneration whereby clinical symptoms are not evident, but neurodegeneration exits, though below a certain threshold. Upon disease development or onset, the evolution of the neurodegenerative process in the brain could be equated to the rate of change in the severity of clinical symptoms or vice versa . As a biOur results indicate that CSF NfL could be used as a pathophysiological biomarker of stage-associated neurodegeneration. The identification of stage-associated changes will help in targeting a focused therapy, which can be applied at an early stage, defined by specific pathological changes. An early and targeted intervention can help in reducing the risk of developing dementia or can postpone development. In addition, assessments of CSF NfL levels can help in enriching the clinical trials, by including individuals who are likely within the early phase of the AD continuum, based on their biomarker profile . MoreoveA small cohort of MCI participants represents the major limitation for our study. Given the small number of participants with MCI, we separated them using the 50th percentile or median value of CSF NfL, for disease prognostic analysis. A relatively small number of CH participants could present another limitation. Therefore, the estimated risk of dementia onset among CH participants (high CSF NfL group vs. low CSF NfL group), and rates of cognitive decline among participants with MCI could have been overestimated. Findings should be tested in larger cohorts by stratifying participants into terciles or quartiles, or dichotomizing them using an optimal cut-off. The utility of CSF NfL to predict the risk of dementia onset should also be assessed in longitudinal studies using CH participants who convert to MCI, and to predict disease progression using MCI participants who progress to AD.In conclusion, CSF NfL can be used as a pathophysiological biomarker which reflects early neurodegeneration, and as a prognostic biomarker which reflects downstream neurodegenerative changes along the AD continuum. As a diagnostic biomarker, it can help to screen preclinical AD and predict the risk of dementia onset. Thereby, it can help to optimize the drug development process by targeting the right drug candidate, at the right biochemical pathway and at the right stage. As a prognostic biomarker, it can be used as a treatment response biomarker for interventions under trial that aim at slowing down disease progression by combating axonopathy. A biomarker-based diagnostic algorithm, which includes CSF NfL, could be developed to screen preclinical AD with high accuracy. To increase the clinical applicability of NfL, future studies should explore the potential of plasma NfL for screening preclinical AD, predicting the risk of dementia onset among CH participants, and the rate of cognitive decline in participants with MCI.