MALAT1 gene polymorphism was associated with cardiac and cerebrovascular diseases. However, the prognostic role of the MALAT1 polymorphism in major adverse cardiac and cerebrovascular events (MACCEs) remains unknown. Therefore, this study intends to explore the association between the MALAT1 rs3200401 polymorphism and MACCEs.Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) participates in the occurrence and development of cardiovascular and cerebrovascular diseases such as stroke and coronary heart disease by regulating inflammatory reactions, programmed cell death, and other pathological processes. Previous studies revealed that the MALAT1 rs3200401 genotype. During the follow-up, MACCEs were recorded. Kaplan–Meier curves and univariate and multivariate Cox survival analyses were used to explore the correlation between MALAT1 gene polymorphisms and the occurrence of MACCEs.We enrolled 617 myocardial infarction (MI) patients and 1125 control participants who attended the First Affiliated Hospital of Xinjiang Medical University from January 2010 to 2018. SNPscan™ typing assays were used to detect the P = 0.047, MI patients 20.7% vs. 14.1%, P = 0.014) and CT + TT genotypes  were significantly higher in subjects with MACCEs than in subjects without MACCEs. However, in control participants, the frequencies of the T allele  and CT + TT genotypes  were not higher in subjects with MACCEs than in subjects without MACCEs. In addition, among the total participants and MI patients, the Kaplan–Meier curve analysis indicated that the subjects with rs3200401 CT + TT genotypes had a higher incidence of MACCEs than CC genotype carriers . Nevertheless, similar results were not observed in the control participants (P = 0.790). Multivariate Cox regression indicated that compared with patients with the CC genotype, patients with CT + TT genotypes had a 1.554-fold increase in MACCE risk .Among the total participants and MI patients, the frequencies of the T allele (total Participants 19.5% vs. 15.3%, MALAT1 rs3200401 CT + TT genotypes could be a risk factor for MACCEs in MI patients, suggesting that the MALAT1 gene may become a biomarker for poor prognosis in MI patients.The  Currently, with the aging of Chinese society and the development of urbanization, cardiac and cerebrovascular diseases, such as stroke, hypertension, and coronary heart disease, have become the leading killer diseases in China. Myocardial infarction (MI) is the main cause of mortality and disability among cardiac and cerebrovascular diseases. The death rate of MI increased by 3 times from 2010 to 2018 .In MI patients, compared with the nonMACCE group, patients in the MACCE group were older  and had lower levels of ApoA .For the control participants, compared with the nonMACCE group, subjects in the MACCE group were also older (59.76 ± 9.80 vs. 54.53 ± 9.96, P = 0.047, CT + TT genotypes, 37.4% vs. 28.1%, P = 0.013, Table Among the total participants, the MACCE group had higher frequencies of the rs3200401 T allele and CT + TT genotypes than the nonMACCE group (T allele, 19.5% vs. 15.3%, P = 0.014, CT + TT genotypes, 39.5% vs. 25.8%, P = 0.003, Table Similarly, in the MI patients, the MACCE group had higher frequencies of the rs3200401 T allele and CT + TT genotypes than the nonMACCE group (T allele, 20.7% vs. 14.1%, P = 0.860, CT + TT genotypes, 31.4% vs. 29.4%, P = 0.760, Table However, for the control participants, we did not observe a prominent difference in the distribution of the rs3200401 T allele or CT + TT genotypes between the MACCE group and the nonMACCE group (T allele, 16.7% vs. 16.0%, P = 0.381, Table P = 0.732, P = 0.076, Table P = 0.567, Table Among the MI patients carrying the CT + TT genotypes or the CC genotype, there was no significant difference in the number of lesion coronary arteries (P = 0.015, Fig. P = 0.001, Fig. P = 0.790, Fig. The Kaplan–Meier analysis revealed that among all participants and MI patients, the MACCE-free cumulative survival rate in the CT + TT genotype group was obviously lower than that in the CC genotype group (P = 0.031, Table Further, univariate Cox survival analysis was used to determine the risk factors for MACCEs among the total participants. The analysis showed that the rs3200401 CT + TT genotype, history of MI, male sex, age, diabetes, Gensini score, number of lesion coronary arteries, revascularization, admission blood glucose, TG, HDL-C, and ApoA were all MACCE risk factors. The multivariate Cox regression analyses suggested that the CT + TT genotypes of rs3200401 were associated with a risk for MACCEs (HR: 1.441, 95% CI 1.035–2.084, P = 0.024, Table In addition, we studied the risk factors for MACCEs among MI patients with univariate Cox survival analysis. It indicated that the CT + TT genotypes of rs3200401, age, smoker, drinker, hypertension, Gensini score, number of lesion coronary arteries and TG were all risk factors for MACCE. After adjustment by multivariate Cox regression analyses, patients with the CT + TT genotypes of rs3200401 were associated with a higher risk of MACCEs than those carrying the CC genotype (HR: 1.554, 95% CI 1.060–2.277, MALAT1 gene polymorphism and MACCE and found that subjects carrying the CT + TT genotypes of rs3200401 were susceptible to MACCE, especially MI patients.In the present study, we investigated the relationship between the Human genome sequencing and the GENCODE project launched in 2003 have revealed that the minority of the human genome can be translated into proteins and that the majority of the genome is primarily transcribed to produce noncoding RNAs , proteinMALAT1 gene rs3200401 CT + TT genotypes were associated with better survival in patients with advanced lung adenocarcinoma [MALAT1 rs3200401 polymorphism and MACCEs occurring among MI patients and control participants and found the rs3200401 CT + TT genotypes were independent factors of MACCEs among MI patients. Nevine Fathy et al. reported that the CT + TT genotypes of rs3200401 were the independent predictors of cerebral ischemic stroke in Egypt [MALAT1 gene rs3200401 polymorphism may be a potential functional mutation that deserves further research and may be a genetic biomarker of MACCEs susceptibility among MI patients.Jian-Zhong Wang et al. first discovered that the arcinoma . In our in Egypt . Moreovein Egypt . Furtherin Egypt , we specin Egypt , and thein Egypt , while Hin Egypt . Taken tMALAT1 gene rs3200401 CT + TT genotypes and MACCE. However, we did not measure the level of lncRNA MALAT1 in plasma, resulting in the linkage between the expression of lncRNA MALAT1 in plasma and MACCE being undetected, which deserves further research.In the present study, there were several limitations. First, this was not a multicenter and large-sample study, which would provide less robust statistical estimations. Second, compared with the MACCE group, the participants were a little older than the participants in the nonMACCE group and it may leave the possibility that the predictive power of the MALAT1 gene rs3200401 polymorphism for MACCE could be influenced. After adjusting the confounders via multivariate Cox regression analyses, the MALAT1 gene rs3200401 CT + TT genotypes remained the risk factor for MACCEs in MI patients. However, age was not the risk factor for MACCEs so the age was not the confounder of MACCEs in this study which did not significantly influence the power of MALAT1 gene rs3200401 polymorphism for MACCEs. Third, we only analyzed the association between MALAT1 gene played an important role in the prognosis of MI patients and that the MALAT1 gene rs3200401 CT + TT genotypes are risk factors for the poor prognosis of MI patients, suggesting that the MALAT1 gene can be used as a biomarker susceptible to MACCE for MI patients. Further gene sequencing for individuals with traditional risk factors could help to identify whether they carry MALAT1 rs3200401 CT + TT genotypes, which may help clinicians distinguish high-risk MACCE patients with a history of MI. This is also the real purpose of precision medicine.In summary, these results indicated that the MALAT1 rs3200401 CT + TT genotypes could be a risk factor for MACCEs in MI patients, suggesting that the MALAT1 gene may become a biomarker for poor prognosis in MI patients.In conclusion, our study revealed that


Gastrointestinal motility disorder is a common gastrointestinal disease, which seriously affects life quality. Traditional Chinese medicine (TCM) has been widely used as an alternative therapy for gastrointestinal motility disorder. Acacetin is a natural flavonoid compound that has antioxidant and anti-inflammatory, antidepressant, and anticancer properties. However, the efficacy of Acacetin in the treatment of gastrointestinal motility disorders has not been studied. Our aim was to investigate the mechanism of Acacetin-alleviated gastrointestinal motility disorder and its efficacy based on network pharmacology. In vivo, we investigated the effects of Acacetin in the gastrointestinal motility disorder model.  We performed network pharmacology to predict the active components, match Weishu decoction (WSD) targets in gastrointestinal motility disorders, and investigate its potential pharmacological mechanisms. We performed the GO and KEGG enrichment analysis.  Based on network pharmacological method, the key active ingredient of WSD was identified as Acacetin, and the enrichment signaling pathway was the PI3K-AKT signaling pathway. Acacetin and Mosapride accelerated gastric emptying time, reduced gastric remnant rate, and increased small intestinal propulsion rate. The levels of GAS and MTL were increased after using Acacetin. These results indicated that Acacetin could improve gastrointestinal motility disorders. Among them, high-dose Acacetin showed a better effect. Acacetin could regulate protein and lipid metabolism in mice with gastrointestinal motility disorder. Furthermore, Acacetin could modulate gastrointestinal inflammation and apoptosis. The detection of the PI3K-AKT signaling pathway-related proteins showed that Acacetin improved gastrointestinal motility disorder by inhibiting the activation of the PI3K-AKT signaling pathway.  The key ingredient Acacetin in WSD could alleviate gastrointestinal motility disorder by inhibiting the activation of the PI3K-AKT signaling pathway based on network pharmacology analysis. The efficacy and safety of Acacetin treatment provide strong experimental support for the clinical treatment of gastrointestinal motility disorder. Gastrointestinal motility disorder is a common gastrointestinal disease that seriously affects the quality of life and socioeconomic costs . It is cTraditional Chinese medicine (TCM) is the precious treasure of the Chinese nation. Thousands of years of practical experience has made significant contributions to preventing and treating human diseases . At presWith the vigorous development of network pharmacology in recent years, a new approach to finding therapeutic drugs has emerged . Networkhttps://tcmspw.com/tcmsp.php) [http://www.swisstargetprediction.ch/) [https://www.genecards.org/) [https://www.ncbi.nlm.nih.gov/) [https://www.omim.org/) [https://string-db.org/cgi/input.pl) to construct the protein-protein interaction (PPI) network [Homo sapiens,” and the credibility was set as >0.9. In order to better understand the complex interaction relationship between components, diseases, and corresponding targets, a component-disease-target network diagram was constructed based on the included components, therapeutic diseases, and targets and was imported into Cytoscape 3.8.0 to draw the network diagram. Then, the topology analysis was carried out by NetworkAnalyzer [The TCMSP online database (msp.php)  was usedmsp.php)  were incion.ch/) , and theds.org/) , NCBI geih.gov/) , and OMIim.org/) . Among t network . The speAnalyzer . Genes wAnalyzer .P ≤ 0.05 were screened using the String database. The ClusterProfiler, enrichplot, and ggplot2 packages were installed, and the R 3.6.3 software was used to make histograms and bubble plots. KEGG pathway enrichment analysis was carried out on common targets of drug diseases, and the items with corrected P ≤ 0.05 were also screened using the String database.GO biological process (BP), molecular function (MF), and cell component (CC) enrichment were carried out on the common targets of drug diseases, and the items with corrected The adult SPF C57BL/6 male mice were raised in a temperature- and humidity-controlled animal facility on a 12-hour light/dark cycle. The mice were randomly divided into 9 groups with 12 mice in each group. They were divided into the control group (CG), the model group (MG), the low-dose Acacetin group (LAG), the middle-dose Acacetin group (MAG), the high-dose Acacetin group (HAG), the Mosapride group (MPG) (positive control). Acacetin was purchased from Selleck Chemicals , dissolved in dimethyl sulfoxide (DMSO), and stored at -20°C. The model of gastrointestinal motility disorder induced by atropine . AcacetiThe mice were orally administered 0.5 mL of semisolid paste and were killed 30 min later. The difference between total gastric weight and net gastric weight was the residual weight in the stomach, and the percentage of residual weight in the stomach and the weight of semisolid paste was the gastric remnant rate (%). The small intestine of mouse was put on the plane without traction, and the total length from the pylorus to the ileocecal area and the distance from the pylorus to the front end of carbon were measured. The percentage of the advancing length of semisolid paste and the total length of the small intestine was the small intestinal propulsion rate .α, IL-1β, and IL-6 levels. The GAS, MTL, PAB, CER, TRF, TNF-α, IL-1β, and IL-6 were detected by GAS , MTL , PAB , CER , TRF , TNF-α , IL-1β , and IL-6  ELISA kit according to the instructions. The concentration of GAS, MTL, PAB, CER, TRF, TNF-α, IL-1β, and IL-6 was calculated using the Bio-Tek microplate analyzer  by forming a standard curve based on the provided values. The concentration of TG, LDL-C, HDL-C, and TC were detected by the TG , LDL-C , HDL-C , and TC  kit according to the instructions.ELISA was performed to detect serum gastrin (GAS) and motilin (MTL), serum protein metabolism indexes prealbumin (PAB), ceruloplasmin (CER) and transferrin (TRF), and lipid metabolism indexes triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total cholesterol (TC), and inflammatory factors TNF-α, IL-1β, and IL-6 in the gastric antrum tissues were detected by qRT-PCR. Total RNA was extracted by Trizol methods; RNA was reversely transcribed into cDNAs in accordance with the instruction of a reverse transcription kit . SYBR Green qPCR mix (Invitrogen) was performed to test gene relative expression in ABI 7900 system. The relative level of the gene was calculated by 2−ΔΔCt method with β-actin as the internal gene. The primer sequences used in this study are shown in The relative expression levels of Bcl-2, Bax, Caspase-12, TNF-α , IL-1β , IL-6 , Bax , Caspase-12 , Bcl-2 , PI3K , p-PI3K, AKT , p-AKT , and β-actin  at room temperature for 90 min. The secondary antibody HRP goat anti-mouse IgG  or HRP goat anti-Rabbit IgG  was incubated with the membrane at room temperature for 90 min. The protein bands were detected by the Chemiscope6100 system . The integrated density of protein bands was determined by the Quantity One 4.6.2 software and corrected by subtracting the measured integrated density with the background integrated density. β-Actin was used as the internal reference for detecting relative expression levels.RIPA lysis buffer  was applied to extract the total protein from the gastric antrum tissues. The protein was mixed with the SDS-PAGE loading buffer  for 5 min in boiling water at 100°C. The proteins were separated by gel electrophoresis and transferred to the PVDF membrane. Then, they were sealed with 5% skim milk solution for 2 h at room temperature, and incubated with diluted primary antibodies TNF-The expressions of PI3K, p-PI3K, AKT, and p-AKT were detected by IHC in the gastric antrum tissues of different groups. The slices were roasted at 60°C for 12 h. Then, the slices were dewaxed to water and heated to repair the antigen. 1% periodic acid was added, and the endogenous enzyme was inactivated for 10 min at room temperature. The PI3K , p-PI3K , AKT , and p-AKT  primary antibodies were incubated overnight at 4°C. The secondary antibody was incubated at 37°C for 30 min. DAB was used for color development, hematoxylin was restained for 10 min, washed with distilled water, and PBS returned to blue. All levels of alcohol were dehydrated for 5 min. After removal, it was placed in xylene for 10 min. Then, it was sealed with neutral gum and observed under the microscope. We selected the location of the gastric antrum, taking a 400-fold field of view. Image-pro-plus 6.0 analysis software was chosen for IOD analysis. The average optical density (positive area IOD under the field of view/tissue area under the field of view) was performed to indicate the relative expression of PI3K, p-PI3K, AKT, and p-AKT.T-test was used between the two groups conforming to the normal distribution. The one-way analysis of variance (ANOVA) was conducted among multiple groups, followed by Tukey's post hoc test. P < 0.05 was considered statistically significant.Statistical analysis was performed using GraphPad 8.0 software, and three independent experimental data were expressed as mean ± standard deviation (SD). The unpaired https://tcmspw.com/tcmsp.php) [http://www.swisstargetprediction.ch/) [https://www.genecards.org/) [https://www.ncbi.nlm.nih.gov/) [https://www.omim.org/) [In this study, the TCMSP online database (msp.php)  was usedmsp.php)  were incion.ch/) , and theds.org/) , NCBI geih.gov/) , and OMIim.org/) . Among thttps://string-db.org/cgi/input.pl) [Homo sapiens,” and the credibility was set as >0.9. There were 228 nodes and 1017 edges in the PPI network, and the average degree was 8.92. In the R software analysis, all drug-protein targets and disease-related proteins were classified into the two independent groups. The screened drug targets and disease targets were input into the Venny 2.1. As shown in nput.pl)  to constin vivo experiments to test whether Acacetin could inhibit gastrointestinal inflammation and apoptosis from alleviating gastrointestinal motility disorders.The R package provided for GO annotation enrichment analysis, including the CC, MF, and BP analyses. GO annotation showed that the drug-disease crossover proteins were mainly related to the cellular response to drug, peptidyl-tyrosine phosphorylation, peptidyl-tyrosine modification of BP, the national cell body, synaptic membrane of CC, the protein serine/threonine kinase activity, and protein tyrosine kinase activity of MF . In addiBy constructing the gastrointestinal motility disorder model, we found that compared with the CG, gastric emptying was delayed, gastric remnant rate was increased, and small intestinal propulsion rate was decreased in the MG. Compared with the MG, Acacetin and Mosapride accelerated gastric emptying time, reduced gastric remnant rate, and increased small intestinal propulsion rate. These results revealed that Acacetin and Mosapride could improve gastrointestinal motility, and high-dose Acacetin had the better effect . The resTo study whether the alleviating effect of Acacetin on gastrointestinal motility disorder was related to protein and lipid metabolism, we used ELISA to detect indicators related to protein metabolism and lipid metabolism. ELISA results showed that the levels of PAB, CER, and TRF were decreased in the MG compared with the CG. Compared to the MG, PAB, CER, and TRF levels were significantly increased after Acacetin and Mosapride treatment . Compareα, IL-1β, and IL-6 were significantly increased in the MG. Compared with the MG, the levels of TNF-α, IL-1β, and IL-6 were decreased after Acacetin and Mosapride treatment (α, IL-1β, and IL-6 was also increased in the MG. Compared with the MG, the expression of Bcl-2 was increased, the expression of Bax and Caspase-12 was decreased, and the expression of proinflammatory cytokines TNF-α, IL-1β, and IL-6 was also decreased after the treatment of Acacetin and Mosapride (Figures To investigate the effects of Acacetin on gastrointestinal inflammation and apoptosis, we first detected the levels of inflammation-related indicators. Compared with the CG, serum levels of TNF-reatment . The eff Figures . These rBased on the results of KEGG enrichment analysis, we analyzed the role of Acacetin in the regulation of the PI3K-AKT signaling pathway. Western blot and IHC were performed to detect the expression of PI3K-AKT signaling pathway-related proteins PI3K, p-PI3K, AKT, and p-AKT. The results showed that Acacetin and Mosapride improved the expression of p-PI3K and p-AKT. That was, by downregulating PI3K and AKT, the gastrointestinal motility disorders were improved, and the effect of high-dose Acacetin was better Figures . OverallGastrointestinal motility disorder is a common gastrointestinal disease that seriously affects life quality. Based on network pharmacological methods, we conducted many experiments. The results showed that high-dose Acacetin could regulate the PI3K-AKT signaling pathway to inhibit gastrointestinal inflammation and apoptosis and regulate protein metabolism and lipid metabolism. It is helpful to regulate the contents of GAS and MTL in mice with gastrointestinal motility disorders and improve gastric emptying ability, which plays an essential role in alleviating gastrointestinal motility disorders.A variety of hormones play a regulatory role in gastric motility, among which GAS and MTL are the main hormones associated with gastrointestinal motility disorders. GAS is a crucial gastrointestinal hormone that is mainly secreted by G cells . GAS canSerum PAB is an effective tool for evaluating malnourished patients . It has α, IL-1β, and IL-6 was significantly decreased after Acacetin treatment compared with the middle cerebral artery occlusion group [α, IL-6, and IL-1β in the lipopolysaccharide-stimulated human periodontal ligament cells in a dose-dependent manner and inhibit cell apoptosis [α, IL-1β, and IL-6 were decreased after using Acacetin, while the expression of Bcl-2 was increased, and the expression of Bax and Caspase-12 was decreased. These results suggested that Acacetin could regulate gastrointestinal inflammation and apoptosis, and high-dose Acacetin has the best effect. As a classical signaling pathway, the PI3K-AKT signaling pathway plays a vital role in the diseases. Caulis Sargentodoxae could regulate the PI3K-AKT signaling pathway to exert anti-inflammatory effects [α, activate AKT, and affect NF-κB to produce antiapoptotic and proinflammatory effects [During the occurrence of gastrointestinal motility disorders, cytokines mediating the inflammatory response in the injury area can secrete inflammatory factors. Acacetin has antioxidant and anti-inflammatory effects . In the on group . Acacetipoptosis . In our  effects . PI3K co effects . Our stuHowever, our study has some limitations. As shown in In conclusion, our findings suggest that high-dose Acacetin regulates the PI3K-AKT signaling pathway to inhibit gastrointestinal inflammation and apoptosis, regulate protein metabolism and lipid metabolism, and help regulate the levels of GAS and MTL. Acacetin treatment has high efficacy and safety, which could significantly improve the gastrointestinal motility disorder mice, improve the ultrastructure of gastrointestinal tissue, and inhibit the inflammatory response. Our study provides strong experimental support for the clinical treatment of gastrointestinal motility disorders with Acacetin.


Pure laparoscopic donor right hepatectomy (PLDRH) can provide better operative outcomes for the donor than conventional open donor right hepatectomy (CODRH). However, the complexity of the procedure typically makes transplant teams reluctant to perform it, especially in low-volume transplant centers. We compared the outcomes of PLDRH and CODRH to demonstrate the feasibility of PLDRH in a low-volume transplant program.We carried out a retrospective study of adult living donor liver transplantation in Chiang Mai University Hospital from January 2015 to March 2021. The patients were divided into a PLDRH group and a CODRH group. Baseline characteristics, operative parameters, and postoperative complications of donors and recipients were compared between the two groups.Thirty patients underwent donor hepatectomy between the dates selected (9 PLDRH patients and 21 CODRH patients). The baseline characteristics of the 2 groups were not significantly different. The median graft volume of the PLDRH group was 693.8 mL, which was not significantly different from that of the CODRH group (726.5 mL) The PLDRH group had a longer operative time than the CODRH group, but the difference was not statistically significant . The overall complication rate was not significantly different between the two groups . Additionally, for the recipients, the incidence of major complications was not significantly different between the groups .Even in the context of this low-volume transplant program, whose staff have a high level of experience in minimally invasive hepatobiliary surgery, PLDRH showed similar results to CODRH in terms of perioperative outcomes for donors and recipients.The online version contains supplementary material available at 10.1186/s12893-022-01507-0. Laparoscopic liver resection has become increasingly popular worldwide. The existing literature shows several significant advantages of laparoscopic liver resection over open liver resection (OLR), such as minimized blood loss and shorter hospital stays for patients . RegardiThe first pure laparoscopic donor hepatectomy was left lateral sectionectomy for pediatric liver transplantation, reported by Cherqui et al.  FollowinChiang Mai University Hospital contains a high-volume hepatobiliary and pancreatic surgery center that has been serving the northern part of Thailand since 2005 and began its LDLT program and a minimally invasive liver resection program in early 2015. Right hepatectomy was performed on the donors using the conventional open technique with an intraoperative conventional cholangiogram until the end of 2019, when one of the transplant surgeons received training from a high-volume laparoscopic donor hepatectomy center . Since early 2020, the department has used minimally invasive liver resection, including liver mobilization, parenchymal transection and hilar structure dissection; has started using real-time indocyanine green (ICG) cholangiograms to facilitate bile duct division instead of using the more conventional cholangiogram; and has started the first case of pure laparoscopic donor right hepatectomy in March 2020. To date, 9 cases of PLDRH have been performed in our center.The aim of this study is to present the institutional experience of developing a safe and efficient pure laparoscopic donor right hepatectomy process for adult living donor liver transplantation without transition from hand- or laparoscopic-assisted donor hepatectomy. To the best of our knowledge, this study is the first report of PLDRH from a Southeast Asian transplant center.This study was approved by the Research Ethics Committee of the Faculty of Medicine, Chiang Mai University . Data were collected from all living liver donors who underwent donor right hepatectomy in the department between 1 January 2015 and 31 March 2021. Twenty-one donors underwent CODRH, and 9 donors underwent PLDRH by a single surgeon. There was no hand-assisted or laparoscopic-assisted donor right hepatectomy during this time period. There was also no ABO-incompatible liver transplant. Postoperative complications were classified and reported according to the Clavien–Dindo classification. The criteria for donors were an age of 18–60 years and an underlying medical conditions that are contraindications for surgery and liver donation.Liver volume and vascular anatomy including the hepatic artery, portal vein and hepatic vein were evaluated using multiphase contrast-enhanced computed tomography. The future liver remnant was calculated by liver volumetry software  and must exceed 25% of the total liver volume and be sufficient for the recipient by keeping the graft-to-recipient weight ratio (GRWR) more than 0.8. Biliary anatomy and degree of fatty liver were evaluated by magnetic resonance cholangiopancreatography (MRCP).For the first few cases of CODRH, only donors who had no variations in their liver vasculature or biliary tract were selected; however, these limitations were abandoned after the experience of the team developed. The first 8 liver donors who underwent PLDRH had single right portal veins longer than 1 cm, a single right hepatic artery, a single right bile duct longer than 0.5 cm, and less than 2 inferior hepatic veins. There was no graft size limitation for PLDRH. However, the last case of PLDRH had a right posterior portal vein graft from the main portal vein.The donor was placed supine in a reverse Trendelenburg position with their legs apart. The surgeon, assistant and scope operator were positioned as demonstrated in Fig. The liver was completely mobilized, except for Makuuchi’s ligament. The anterior and lateral planes between the liver and inferior vena cava (IVC) were dissected, and the short hepatic veins were ligated. If there was an inferior hepatic vein, it was left in place to prevent liver congestion and ligated at the last step. No hanging maneuver was performed.The next step was the hilar dissection. To acquire the best exposure of the hilar structure, the gallbladder and round ligament were retracted upward and outward by the assistant. Hilar dissection started with dissection of Calot’s triangle to safely double-ligate the cystic duct and ligate the cystic artery. The gallbladder was left in place to provide traction and exposure.The right hepatic artery (RHA) was dissected laterally to the common hepatic duct (CHD) and wrapped with elastic tape. The right portal vein (RPV) was then dissected until the portal vein bifurcation was clearly identified. The RPV was then wrapped with elastic tape, and the gallbladder was completely removed. After that, the RHA and RPV were temporarily clamped with an endo-bulldog clamp, and 2.5 mg of indocyanine green was injected intravenously. The transection line was clearly visualized and marked, and then the endo-bulldog clamps were removed . The Pringle maneuver was not routinely used. Parenchymal transection was performed following the middle hepatic vein (MHV). V5, V8 or the distal part of MHV was ligated as part of the preoperative planning Fig. . ParenchA Pfannenstiel incision was created and covered with a large wound protector and sealing cap. Pneumoperitoneum was re-established, and the right liver graft was covered with a plastic bag. Heparin was administered intravenously (70 units per kg), and following a 5-min wait, the RHA was double ligated with a titanium clip, the RPV was ligated with an endovascular staple, and the right hepatic vein (RHV) and inferior hepatic vein were also ligated with endovascular staples. Finally, the Makuuchi ligament was divided with an endovascular stapler. Then, the liver graft was removed via a plastic bag through a Pfannenstiel incision.Finally, the heparinized effect was routinely reversed by intravenous protamine sulfate. The Pfannenstiel incision was closed layer by layer, and the pneumoperitoneum was re-established. Any bleeding was secured, and the left lobe of the liver was fixed to the abdominal wall using a suture. One 10 mm Jackson-Pratt drain was routinely placed in the right upper quadrant.. Normally distributed continuous data are reported using the mean ± standard deviation and were compared using t-tests. Non-normally distributed continuous data are reported using the median ± interquartile range and were compared by the Mann–Whitney U test. Categorical data are reported as numbers (percentages) and were compared using Fisher’s exact test. For all analyses, a P-value of less than 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 23.0.0, IBM.The baseline characteristics and surgical outcomes of the donors and recipients between the CODRH and PLDRH groups of this study were compared. The distribution of normality was tested by the Kolmogorov–Smirnov testFrom 2015 to 2021, there were 56 cases of liver transplantation in the department. Thirty of those patients underwent adult LDLT and were operated on using a modified right lobe graft. There were 21 cases of CODRH and 9 cases of PLDRH. The baseline characteristics of the liver donors and donor complications are shown in Table Perioperative data and laboratory investigations of donors are shown in Table E. coli septicemia. The detail of recipients who had mortality were showed in Table Perioperative data and outcomes of recipients are shown in Table The aim of this article is to report the initial experience and outcomes of adult LDLT in a low-volume liver transplant center in a developing country and propose a strategy to develop PLDRH in these circumstances. The donor characteristics, perioperative data, donor outcomes and recipient outcomes were compared between CODRH and PLDRH.The baseline characteristics of donors were not different between the 2 groups. The median graft volume in the PLDRH group was 693.8 mL. which is not different from the CODRH group. Previously, there were donor selection criteria proposed by Kim et al. , 11. SomThere was no bile duct variation from preoperative MRCP to multiple bile duct opening in the CODRH group. However, there were 3 patients in the PLDRH group who had multiple bile duct openings to reconstruct, while all donors had a single short right hepatic duct (< 5 mm) from preoperative MRCP. This may have resulted from the bile duct division and closure technique in donor operation. Our technique used a double 10 mm titanium clip to close the right hepatic duct stump in the PLDRH group, causing an inevitable loss of approximately 3–4 mm in length, in contrast to the CODRH group, which used a polydioxanone suture (PDS) 6/0 closure and resulted in a loss of only 1–2 mm. of length. Our result correlated with a study from a high-volume PLDRH center, which also showed an increased frequency of multiple bile duct openings in the PLDRH group [The operation time in the PLDRH group was longer than that in the CODRH group. Many studies have reported the same result –15. The Regarding postoperative outcomes and donor complications, there were 2 donors in the CODRH group who experienced major complications. Donor number 3 required percutaneous drainage due to intra-abdominal collection, and donor number 7 required immediate biliary reconstruction due to a hepatic duct confluence injury during bile duct transection. There were no major or minor complications in the CODRH group after donor 12. The team started using an ICG cholangiogram in CODRH instead of conventional cholangiograms in donor number 21. After that, we started using ICG cholangiograms to facilitate real-time bile duct division in PLDRH. There were 2 donors in the PLDRH group who had major complications: intra-abdominal collection required percutaneous drainage, and postoperative bleeding required repeat laparoscopy to stop the bleeding. Fortunately, all donors recovered well, and there were no consequent complications during the long-term follow-up period. We believe that the ICG cholangiogram is a gamechanger that facilitates the smooth transition from CODRH to PLDRH and obviously beneficial to the donor. In our study, there were no biliary complications among donors in the PLDRH cohort. However, there were four donors (14.4%) in the CODRH cohort who had biliary complications, and one of them (4.8%) had an injury affecting the confluence of the bile ducts, which was corrected with Roux-en-Y hepaticojejunostomy. Our hypothesis is that, while using a real-time ICG cholangiogram, the surgeon can simultaneously perform bile duct dissection, which will result in precise division of the bile ducts. For conventional cholangiography, the fluoroscope needs to move in and out of the operative field, which may cause some error during bile duct division. We strongly believe that dynamic ICG cholangiography is a transformative technology for donor surgery using both laparoscopic and open approaches.As the small volume liver transplant center, we try our best to build the safe and sustainable LDLT program. Back to 2010, Our hospital was first started our adult LDLT and five cases were performed but unfortunately the mortality rate was high as 60% which resulted from early allograft dysfunction, vasculo-biliary complication or even chronic allograft rejection. The leader transplant surgeon at that time who was not HB surgeon was then departed which resulted in the shutting down of transplant program. The many abdominal transplant programs might lead by transplant surgeon who has proficiency in liver, pancreas and kidney transplantation. The vascular surgeon can also lead the transplant program due to familiarity with the vascular technique. However, the adult LDLT is one of overlapping area of transplant and HB surgery. The comprehensive knowledge which includes liver biology, anatomy and complex hepatectomy skill are necessary for building the sustainable and successful LDLT team. We decided to send the young HB surgeon (SJ) who had the extensive experience in complex hepatobiliary surgery which included major hepatectomy and vascular reconstruction in cholangiocarcinoma surgery to United states and South Korea to obtain the experience in both deceased donor and living donor liver transplantation. After he came back, the first DDLT was performed in November 2014 and after the successful of 3 cases of DDLT, the first adult LDLT was performed in July 2015. Afterwards, the LDLT is a majority of transplant volume in our center , who had extensive experience in both open and laparoscopic complex hepatobiliary surgery and CODRH received adequate training from a high-volume minimally invasive liver transplantation center that used the reproducible technique –21. UnliThe strategy to convert from CODRH to PLDRH in small liver transplant centers in developing countries has several steps, which are summarized in Fig. The type of laparoscope is also very important for PLDRH starters. Many high-volume centers favor a 3-dimensional (3D) flexible endoscope over a rigid endoscope due to the flexibility of the camera angle and depth of perception . HoweverMany studies have shown that PLDRH has several advantages over CODRH, such as reduced donor recovery times, better cosmetic results, and improved quality of life without compromising the quality of the liver graft and donor safety , 5, 16. 4. So, we could not confidentially conclude that the complication in both groups were not significantly difference due to shorter follow up time and lower volume of LDLT. Several studies mention that the number of cases of LDLT affects the result of the transplantation program [The major complications and 30-day and 90-day mortality of recipients in his study were relatively high compared to the results from high-volume centers. A majority of the patients died from infection, which may be related to surgical technique. Four recipients in the CODRH group who underwent surgery during the initial phase of the program died, two of them from unpreventable causes (aortic dissection and cardiac arrythmia). The learning curve of the team, which included patient selection, surgical technique, and perioperative care, was the main influence on the short- and long-term outcomes of transplant patients. We believe that posttransplant survival will improved when the program is able to maintain a median transplant volume (more than 10 cases/year) ,which wi program –27. We sThis study had several limitations. First, this report had a small number of cases in both groups; thus, the sample size was not sufficient to obtain a statistically significant result. Second, there is a potential bias in the study due to the retrospective nature of this study and the selection bias in the selection criteria. Finally, the long-term outcomes of donors and recipients of PLDRH groups were not examined in this study, despite their critical importance.The results of our initial experience with PLDRH showed that PLDRH is safe and beneficial for donors without compromising graft function. PLDRH can be attempted in centers with high levels of experience in minimally invasive hepatobiliary surgery and well-established adult LDLT programs. However, starting a PLDRH program without changing from a hand-assisted or laparoscopic-assisted program has limited evidence and should be done cautiously.Additional file 1. The total liver transplant volume from 2009 to 2021.Additional file 2. The case number and 90 day mortality.


Cerebrospinal fluid (CSF) has recently experienced a revival in diagnostics and research. However, little progress has been made regarding CSF cell analysis. For almost a century, CSF cell count and cytomorphological examination have been central diagnostic parameters, with CSF pleocytosis as a hallmark finding of neuroinflammation and cytology offering valuable clues regarding infectious, autoimmune, and malignant aetiologies. A great deal of information, however, remains unattended as modern immune phenotyping technologies have not yet been broadly incorporated into routine CSF analysis. This is a serious deficit considering the central role of CSF cells as effectors in central nervous system (CNS) immune defence and autoimmune CNS processes, and the diagnostic challenges posed by clinically overlapping infectious and immune-mediated CNS diseases. Here, we summarize historical, specimen-intrinsic, methodological, and technical issues determining the state-of-the-art diagnostics of CSF cells and outline future perspectives for this underutilized window into meningeal and CNS immunity. Cerebrospinal fluid (CSF) is a clear colourless liquid that surrounds the brain and spinal cord and plays a vital role in central nervous system (CNS) homeostasis . As far Speaking about CSF usually refers to the fluid itself. However, cerebrospinal fluid also contains cells, namely a special selection of blood-derived lymphocytes and monocytes. These cells are capable of crossing the blood–CSF barrier (BCSFB) of the choroid plexus, which allows minimal entry of immune cells and macromolecules, such as antibodies, in steady-state . Immune The subarachnoid space is part of the leptomeninges and contains immune sentinels and non-fenestrated blood vessels, which cover and traverse the brain surface, encased in pial sheaths with CSF flowing along in the perivascular spaces . The funDue to its CNS-ensheathing proximity, accessibility, and obvious role in CNS immune surveillance, much attention is paid to CSF in laboratory diagnostics for neurological disorders—a development largely driven by German pathologists, clinicians, and researchers during the past century . Viral iWhile advances in laboratory techniques have broadened the indications and significance of CSF diagnostics by allowing investigating, for instance, an increasing repertoire of neuronal autoantibodies in suspected autoimmune encephalitis (AIE) and biomarkers of neurodegenerative diseases , little Why is this so? Here, in the quest for an answer, we outline the historical and state-of-the art methods of investigating CSF cells for diagnostic purposes and summarize technologically available yet underutilized opportunities for enhancing our understanding of meningeal CNS immunity.Lumbar punctures were first performed in the 1890’s to reduce intracranial pressure in patients with hydrocephalus and tubercular meningitis ,24. At aWith the methodological issues of cell preparation resolved, the time from bedside to the lab remained the most critical factor for investigating CSF cells. Ex vivo cerebrospinal fluid quickly turns toxic to its cells, thus severely limiting their lifespan . MoreoveWith the crucial role of timing in preanalytics established, reducing manual processing and speeding up analytics by introducing modern hematoanalysers capable of special fluidic acquisition modes was pursued. Although alluring and intensely investigated for CSF cells, automation reached no breakthrough in fully replacing the manual counting chamber method or cytospin preparations . Again, This brings us to the central role and relevance of CSF cell count and May–Grünwald stained cytology in the CSF diagnostic work-up of neurological disorders. Absolute CSF cell numbers frequently earn the most attention from clinicians, as they are available faster than cytology. In addition, if elevated, they are highly informative via the degree of elevation. However, only combined interpretation with cytomorphology allows accurate diagnostic clues about the underlying pathology . For insMycobacterium tuberculosis, Listeria monocytogenes, Treponema Pallidum, or Borrelia burgdorferi. In these cases, the cell picture is typically more varied, featuring numerous plasma cells, activated lympho- and monocytes and, more or less frequently, granulocytes [Normal CSF cell counts are below 5 cells/µL, consisting of mononuclear white blood cells with a majority of lymphocytes and fewer monocytes . Cell coulocytes ,36. Plasulocytes . In suspulocytes .Thus, the informative value of examining CSF cells is evident. However, dependent on the individual patient’s immune system, age, comorbidities, disease stage, and (pre-)medication, cytological findings may be less characteristic or even overlap between aetiologies. Aside from bacterial infection, granulocytes may also occur in the early stages of viral infections . A lymphThe main limitation of conventional CSF cytology relates to it being confined to cell morphology, enabling no further differentiation of lymphocytes and monocytes than their activation state, and the occurrence of plasma cells as evidence of intrathecal B cell activity. Fortunately, CSF diagnostics is not built on CSF cytology alone; it also combines and integrates CSF cell count, protein analysis, antibody tests and indices, and microbiological and genome analysis.Nevertheless, the clinical need for a broader understanding of the inflammatory CSF cell phenotype is underlined by an estimated 10–20% of unresolved meningoencephalitis cases that are believed to be non-infectious and immune-mediated, relating to AIE ,39. KnowFlow cytometry was quickly recognized as a highly promising method to close the knowledge gap regarding the inflammatory immune cell phenotype. Complicating factors, such as the need for immediate processing combined with a limited availability of cells, did not dampen the rising enthusiasm for CSF immune cell phenotyping in the 1990’s, when early flow cytometers allowed 2–4 colour experiments.Those years produced pivotal studies that provided the basis of current knowledge about normal and inflammatory CSF immune cell phenotypes. One of the most intriguing findings was that CSF lymphocytes were not a random sample of blood-derived lymphocytes but were a special selection capable of migrating across brain barriers and proposedly involved in immune surveillance ,41. CompHowever, these early achievements suffered severely from a delay in technical advances for flow cytometry. Too many years with too few colour experiments for too few CSF cells (due to specimen-intrinsic cellular paucity) hampered both the broader translation into clinical diagnostics and further research. The situation was aggravated as lumbar punctures were considered invasive, and technological advances in brain imaging substituted some of the indications for CSF ,44. The Today, flow cytometry has evolved into an indispensable tool for disease and treatment monitoring of blood immune phenotypic markers in both clinical diagnostics and research . New fluThese advances, however, have only partially reached CSF diagnostics. In addition to specimen-intrinsic factors relating to the instability and paucity of CSF cells, clinical implementation of flow cytometry for CSF cells has suffered from: (i) low CSF collection volumes (lumbar punctures were considered invasive), (ii) lack of consensus protocols for preanalytics, processing, antibody panels, and gating strategies, and (iii) outsourcing of CSF diagnostics from specialized neuroimmunological to large central laboratories observable at least in German speaking European countries.Today, the only widely accepted indication for diagnostic flow cytometry and its sole mention in the current S1 guidelines for CSF diagnostics of the German Society ,17 are sConventional cytology has a rate of up to 60% false negative results . ProvideThe situation is even more difficult in primary CNS lymphoma (PCNSL), a highly aggressive form of B-NHL of the diffuse large B cell-type with manifestations exclusively in the CNS and not the periphery . The likInvestigating the CD4/CD8 T cell ratio, however, finds no mention in the current S1 CSF diagnostic guidelines. This may be because earlier findings highlighting the CD4/CD8 T cell ratio, i.e., if decreased, supportive of suspected viral infection and, if increased, supportive of suspected MS, were not confirmed by more recent studies ,53. In cThis does not mean that earlier studies reporting decreased CD4/CD8 T cell ratios associated to viral infection were inaccurate. Rather, it indicates that similar to CSF cytology, immune phenotyping also depends on disease stage and therapy. Thus, expansions of cytotoxic CD8 T cells occurring earlier in the infection may be limited to the initial phase, after which activated CD4+ T-helper cells, B cells, and plasma cells build up the pathogen-specific humoral immune response. This interpretation is supported by the presence of elevated B cell frequencies detected by flow cytometry , activatNevertheless, the strikingly low CD4/CD8 T cell ratio of HIV patients (0.5–1.8) and the fact that it is difficult to clinically differentiate HIV encephalitis from opportunistic infections ,55 is juIn neurosarcoidosis, an increased CD4/CD8 T cell ratio in CSF (>5.0) has been intensely investigated as a surrogate marker, aiding in differential diagnosis for many years with inconclusive results. Of clinical relevance, however, may be recent findings reporting (i) a negative predictive value of 88%, if a CD4/CD8 T cell ratio <5.0 is combined with an absence of pleocytosis , (ii) thFollowing the idea that immune phenotyping is helpful for differential diagnoses in neuroinflammation, also the analysis of other lymphocyte subsets, e.g., B cells and NK cells, could be of help. Patients with HIV, LNB, bacterial meningitis, and MS showed differences in elevated fractions of B cells, with the highest elevations seen in LNB, and patients with viral meningitis showed significantly higher NK cell frequencies than MS patients .To summarize, immune phenotyping of CSF cells for indications other than haematological CNS malignancies appears to be helpful. However, it is mainly restricted to neurological centres equipped with on-site neuroimmunological laboratories, multicolour flow cytometers, and associated research groups, discussed in more detail further below in the text ,62,63,64Cerebrospinal fluid is currently experiencing a renaissance as a window into our CNS. This renewed interest was ignited by the rise of molecular techniques allowing valuable mechanistic insights into disease pathologies for diagnostic, therapeutic, and research purposes. The result was an increasing repertoire of neurodegenerative CSF biomarkers and neuronal antibodies, the detection of previously unknown pathogens, and the opportunity of analysing CSF as liquid biopsy in suspected CNS malignancies, which altogether broadened the indications for lumbar punctures. The concurrent development and application of atraumatic needles reducing complications on the one hand, and the guideline recommendations for improved atraumatic lumbar punctures on the other hand, helped to expand acceptance that lumbar punctures in general are safe procedures ,20. ThisIn short, the combination of technological advances broadening indications for lumbar punctures and the higher CSF collection volumes alleviating limitations regarding the availability of sufficient CSF brought unforeseen opportunities for investigating CSF cells. Single cell high-throughput technologies allowing cell-type specific proteomic, transcriptomic, and genomic analyses appear as particularly promising methods to advance our understanding of intrathecal inflammatory CNS processes and, ultimately, CSF cytodiagnostics . These sMultiparametric flow cytometry, for instance, is broadly accessible and widely used; however, it is limited by spectral overlap of fluorescent-labelled detection antibodies. Hence, it is very useful for phenotyping CSF cell heterogeneity by width rather than depth. Mass cytometry, in contrast, appears perfect for high dimensional, deep immune profiling into CSF cell subset diversity because the use of rare metals for detection obviates the spectral overlap of fluorophores . This teUnforeseen opportunities provide the rapid emergence of single-cell sequencing technologies allowing the production of high-throughput transcriptomic and, latest, surface expression data, which enables the identification of specific immune cell subsets and cell-type specific regulation patterns. First studies already successfully employed single-cell RNA sequencing (scRNAseq) for investigating CSF cells. One reported the compartment-restriction of T follicular helper cells in the CSF of MS patients, and another the occurrence of neurodegenerative disease-associated microglia-like cells in the CSF of patients with HIV ,72.Next-generation sequencing (NGS) is the method of choice when it comes to investigating the B cell receptor (BCR) and T cell receptor (TCR) repertoires. In fact, the intrathecal BCR repertoire has been intensely investigated in CSF and compared to blood, lymph nodes, and meningeal B cell accumulations in patients with MS. These results provided the basis of the concept that class-switched memory B cells and plasmablasts traffic across the blood–brain barrier ,74,75. NThe targeting of meningeal and CNS immunity with single cell technologies is developing at high speed. As an alternative to NGS, an approach combining scRNA-TCRseq of CSF cells with unbiased bioinformatics approach was recently successfully applied to quantify and visualize clonally expanded CSF T cells of patients with neurodegenerative diseases to pin down the intrathecal adaptive immunity in neurodegeneration .It is obvious that these technologies hold great promise for a deeper understanding of intrathecal cellular processes and future cytodiagnostics. However, substantial further efforts and developments towards standardized protocols, multiplexing, and barcoding technologies allowing the pooling of samples from different individuals are necessary in order to gain high quality results despite the limitations of CSF cell paucity. Another important step is the inclusion of CSF investigation in clinical trials. This has been increasingly postulated as a necessity for new knowledge formation that is condensable into diagnostically exploitable formats ,78,79.From all the above mentioned single-cell technologies, immune phenotyping by flow cytometry currently has the greatest potential for advancing CSF cytology by translating new findings into clinical diagnostics. Flow cytometers are widely available and the costs are comparably low. Standardized protocols exist for clinical and trial settings, and shared platform activities help with optimized panel design and panel recommendations to improve data quality and reproducibility in research. Moreover, flow cytometry allows for the analysis of low cell numbers and can reliably identify T-lymphocytes even if the cell count is lower than 5 leukocytes/µL ,67,80,81Accordingly, a great deal of effort has been dedicated to the special considerations and needs of CSF flow cytometry. De Graaf et al. , for insFurthermore, a great deal of effort has been dedicated to identifying cellular biomarkers of neurological diseases and disease activity. Alvermann et al.  compreheCSF immune phenotyping data for MS patients is quite extensive. Though comparably modest in numbers, the knowledge gain obtainable through flow cytometry of CSF cells is increasingly recognized and applied in order to investigate other neurological diseases. These diseases include paraneoplastic disorders ,79,83, cIncreasing efforts further aim at deciphering the intrathecal inflammatory milieu by integrating cellular and humoral parameters, such as cytokines and chemokines ,61,64,92Calculating immune cell ratios was identified early as a useful tool for capturing additional information from immune phenotyping data. A shift of the CD4/CD8 T cell ratio reportedly may help to differentiate between possible CD4+ T cell dysregulation in CNS autoimmunity from a cytotoxic CD8+ T cell anti-viral immune defence ,43 and aToday, more sophisticated algebra in the form of modern computational methods promise profound advances via integrated data analysis of routine CSF diagnostic and multiparameter CSF immune phenotyping data. One of the first reports by Han et al.  investigTo summarize, CSF cells represent an important window into CNS disorders. These cells are intensely investigated but still underutilized in clinical CSF diagnostics. To date, not a single immune phenotyping biomarker, other than suspected lymphomatous malignancy, has been included in the consensus guidelines for aiding in differential diagnosis, individual prognosis, or treatment decisions. Besides the restricted time-points of lumbar punctures in the diagnostic work-up, major hurdles include specimen-intrinsic characteristics, such as low cell count and quick decay. This complicates the validation of findings in controlled settings of multicentre studies and the inclusion of immune phenotyping of CSF cells in clinical trials. In contrast, emerging efforts towards the qualitative cryopreservation of CSF, which, combined with modern molecular and immunologic technologies and computational methods, will accelerate the speed towards unforeseen insights into meningeal and CNS immune processes and further translation into clinical routine. With immune phenotyping by flow cytometry, a routine-qualified diagnostic method at hand, the revival of CSF cell analysis in clinical practice is highly feasible.


Hirudo) has begun to unravel, unveiling their diversity in practically all of Europe, except for its westernmost peninsula, Iberia. We discovered for the first time H. verbana in Spain and conducted an integrative approach  to characterize the newly recorded Iberian populations. We found two endemic and geographically separated Iberian lineages of H. verbana. One of them is easily distinguished by its distinctive colour-pattern and is described as H. verbana bilineata ssp. nov. We established its phylogenetic relationships with other European Hirudo spp. and confirmed the presence of a second species in Iberia, H. troctina. The provided distribution pattern of medicinal leeches contributes to a better understanding of the complexity of the Iberian Peninsula as a glacial refugium/cradle for endemisms, sheltering species populations that began to settle throughout the Pleistocene. Finally, we highlight the urgent need for implementing conservation measures to reverse the evident decline of Iberian populations of medicinal leeches.During the last decade, our understanding of the biogeography of Western-Palaearctic leeches , two endemic and geographically separated Iberian lineages have been found. One of them is easily distinguished by its distinctive colour-pattern and is described as H. verbana bilineata ssp. nov. We characterized the new subspecies morphologically, ecologically, and genetically. We also established its phylogenetic relationships with other European Hirudo spp. and confirm the presence of H. troctina in Iberia, occurring as far as 43° lat. N. Iberian H. verbana records constitute its westernmost known distribution to date. The provided distribution pattern of H. verbana contributes to a better understanding of the complexity of Iberia as a glacial refugium/cradle for endemisms, harbouring populations with a high degree of genetic structure that began to settle throughout the Pleistocene. Iberian Hirudo populations are declining in recent decades and there is an urgent need to assess their conservation status and to initiate conservation measures to reverse their decline.Until the beginning of the 21st century, the famous medicinal leech was thought to be represented by only one species,  Medicinal leeches are haematophagous ectoparasites of vertebrates and are widely used in medicine and as model organism in biology and physiology  accessed on 29 June 2020).In relation to the physicochemical parameters, the Bikuña pond presents a marked seasonality. In the summer months it suffers from low-water but does not get completely dry. Its maximum depth does not exceed 1 m. The water temperature varies from 4 °C in the winter months (never reaching the freezing point) to over 20 °C, reaching temperatures above 25 °C on the hottest days of the summer. The pH through the whole annual cycle is always above 7, normally varying in a range between 7.5 and 8.5 Hirudo verbana “typical”: Two specimens, Doniños lake, 4.5 m altitude, 42°29′31″ N, 8°18′44″ W, A Coruña, Galicia, Spain; two specimens, Gándaras de Budiño lake, 20 m altitude, 42°06′43″ N, 8°37′48″ W, Pontevedra, Galicia, Spain; one specimen, Water channel of Antela lake, Antela Valley, 610 m altitude, 42°05′24″ N, 7°45′00″ W, Ourense, Galicia, Spain; four specimens, Xuño lake, 4 m altitude, 42°38′00″ N, 9°02′18″ W, A Coruña, Galicia, Spain; three specimens, Ocelo lake, Pena Trevinca mountains, 1517 m altitude, 42°13′36″ N, 6°52′22″ W, Ourense, Galicia, Spain. For historical examined material see Examined material: Diagnosis: Medium-sized species, maximal length of about 70 mm including suckers; maximum body width 9 mm; pigmentation of dorsum greenish with an orange-pigmented longitudinal reticulum; lateral margins with a longitudinal beige to yellow stripe; venter unicoloured greenish to yellowish, with no (or very few) dark spots and a pair of black marginal stripes d,e; jawsHirudo verbana cf. verbana is only known from the lakes, ponds, and pools of Galicia and the Basque Country from 4 to 1517 m of altitude (NW to N Spain) (Habitat and distribution: N Spain) ; Table 3Hirudo troctina Johnson, 1816Diagnosis: Small to medium-sized species, medium length of about 50 mm including suckers; pigmentation of dorsum greenish to greyish with four or six dorsal metameric black dots; lateral margins with a longitudinal orange stripe; venter unicoloured greenish to yellowish, with dark spots and a pair of black marginal stripes arranged in zigzag f,g; jawsExamined material: See Habitat and distribution: High to medium flow rivers and streams from southern, central and northern Spain. Our last confirmed historical record of the species dates back 1992 from northern Spain . No specH. verbana from the Iberian Peninsula, constituting its westernmost distribution to date.Medicinal leeches were historically popularised for their utility in bloodletting (phlebotomy) in the 18th and 19th centuries. They have experienced a recent prominence again in post-operative treatments for flap and replantation surgeries, and in terms of characterization and isolation of salivary anticoagulants ,24. MediHirudo [H. verbana specimens with “typical” colour (max. 70 mm long/9 mm width) in relation to the other European clades (max. 100–140 mm/10–12 mm width) [H. verbana cf. verbana. These findings suggest that the genetic diversity within H. verbana is considerably higher than it was accepted before. Thus, each of the four provided clades within H. verbana may represent a subspecific status. This hypothesis should be investigated further based on examination of additional material and genetic markers. The taxonomic rank of subspecies remains highly contentious, largely because traditional subspecies boundaries have sometimes been contradicted by molecular data [Hirudo morphotypes is divided into two well-delimited geographical groups with some morphological differences. Hirudo verbana bilineata inhabits the mountain small ponds or pools (of more than 800 m high) of the Cantabrian Mountains (“Cordillera Cantábrica” in Spanish) that stretch for over 300 km across northern Spain   constitute an important component in the evolutionary legacy of the European medicinal leeches, and thus, the populations of H. verbana bilineata and “typical” H. verbana, are here considered as Evolutionarily Significant Units (ESUs). Accordingly, they should be treated as distinct and as a priority for purposes of conservation. This is a pivotal point, because Iberian medicinal leech populations have been declining in recent decades as both direct and indirect results of human activity [Iberian populations correspond to two different morphotypes, which are also supported by molecular data. It is widely accepted that external pigmentation is not only one of the most useful features to distinguish leech species, but also arguably the best character to diagnose species within the genus Hirudo ,3,6,24. lar data . Howeverrn Spain a–c. OtheW Spain) e,f. Usinactivity ,15,17,31H. medicinalis and H. officinalis may actually refer to this new subspecies [H. medicinalis showing the typical bilineated pattern of H. verbana bilineata [H. medicinalis by Ocharan [H. verbana of the “typical” pattern [H. verbana, since they were traditionally marketed as “green leeches” or “Lisbon brand” (common names attributed to H. verbana) [H. medicinalis) were historically known as “Black leeches” or “Hamburg brand” [H. medicinalis as being a parasite on the Iberian brown frog Rana iberica. Although it is difficult to ascertain from the non-diagnostic photograph provided, the location where they found this medicinal leech  falls within the distribution area of the “typical” morph of H. verbana herein reported. Therefore, these leeches may be referred to as H. verbana. On the contrary, in other records of amphibian predation by H. medicinalis from northern Spain [Hirudo, but most likely to the horse-leech Haemopis sanguisuga.Some northern Iberian literature records of bspecies ,29. Howebspecies  in his hilineata . In the  Ocharan  from Kua Ocharan , are ind pattern ,31. Simiverbana) . Conversg brand” . Ayres ag brand”  have reprn Spain , it is eH. troctina was not confined to southern Spain as previously considered. Almost 100 years later, Fernández-Bernaldo-de-Quirós [H. troctina differs in its habitats from H. verbana, preferring moving bodies of water to lentic ecosystems. This statement is also supported by bibliographic data, since, to our knowledge, all the Iberian records come from rivers or streams , which do not overlap in their geographical distribution, support the hypothesis of a distinct postglacial colonization process from Mediterranean peninsulas [H. verbana bilineata in the upper reaches of the Duero, Ebro and northern rivers catchment and H. verbana “typical” in the Miño-Sil watershed) indicates survival in at least two different Pleistocene glacial refugia. Therefore, the Iberian Peninsula is regarded more likely as a cradle for freshwater endemisms rather than a single refugium, as has been previously suggested by Gómez and Lunt [H. verbana ssp. distribution patterns contribute to a better understanding of the internal complexity of the Iberian Peninsula as a source of glacial relics/endemic taxa, harbouring populations with a high degree of genetic structure that began to settle throughout the Pleistocene.It is very likely that before Pleistocenic glaciations ninsulas . The Easand Lunt . CertainHirudo populations are declining in recent decades as both a direct and indirect result of human activity. We have here described H. verbana bilineata, which was only found in two localities in low numbers. Thus, this new subspecies, as well as the typical morph of H. verbana, can be considered as locally rare taxa, being also consistent with the definition of an Evolutionarily Significant Unit. Rarity of species is usually explained by a combination of extensive versus restricted geographic range, broad versus narrow habitat tolerance and large versus small population size. Iberian H. verbana populations combine restricted geographic ranges with narrow habitat and small population size, making them vulnerable to extinction. Therefore, there is an urgent need to assess in detail the conservation status and to initiate conservation measures for this endemic subspecies and its congeners, in order to reverse their decline and recover them.Iberian H. verbana is required. Application of scanning electron microscopy and molecular tools may shed light on the evolutionary history of medicinal leech populations from other Mediterranean peninsulas and the Western European countries, which may preserve unrecognized diversity of Hirudo species. The broader view is that H. verbana subspecies/phylogroups can provide an effective short-cut for estimating patterns of intraspecific genetic diversity, thus constituting a useful tool for the study of evolutionary divergence and conservation in medicinal leeches in Europe.Despite the long history of studies about the taxonomy and phylogeny of medicinal leeches, our knowledge is still incomplete. Undoubtedly, further investigations involving testing of nuclear genetic markers and morphological variation in “Iberian typical”


Background: Early shunt obstruction (SO) remains the most common cause of lumboperitoneal shunt (LPS) failure. Although there is anecdotal evidence that the level of cerebrospinal fluid (CSF) parameters might affect shunt performance, its association with early LPS obstruction in adults with post-hemorrhagic hydrocephalus (PHH) is unclear.Methods: The retrospective study was performed by reviewing the adults with PHH treated by LPS from years 2014 to 2018. We included patients with CSF samples analyzed within 1 week prior to shunt insertion or at the time of shunt insertion. Baseline characteristics of each patient were collected. The primary outcomes were the incidence rate and associated factors of SO occurring within 3 months of shunt placement. The secondary outcomes included scores on the National Institute of Health Stroke Scale (NIHSS) and Evans Index at discharge.Results: A total of 76 eligible patients were analyzed, of whom 61 were obstruction-free and 15 were early SO. The overall rate of early SO was 15.6%. The RBCs count and nucleated cells count in preoperative CSF were actually higher in patients with early SO, compared to patients in the control group. Multivariate analysis identified RBC elevation  as a dependent risk factor for early SO. NIHSS dramatically decreased at discharge while the alteration of ventricular size was not observed.Conclusions: This study suggested that the presence of RBCs in preoperative CSF was associated with early SO in patients with PHH treated by LPS. Intraventricular hemorrhage (IVH) and subarachnoid hemorrhage (SAH) frequently lead to post-hemorrhagic hydrocephalus (PHH), resulting in ventriculomegaly, intracranial hypertension, and damage of the periventricular parenchyma . CerebroDespite immediate alleviation of symptoms after LPS placement, early shunt obstruction (SO) is unacceptable and is the most common cause of shunt failure , 12. TheThe retrospective study was performed by reviewing the adult patients with PHH treated by LPS at West China Hospital from years 2014 to 2018. We included patients with CSF samples analyzed within 1 week prior to shunt insertion or at the time of shunt insertion. Patients aged below 18 years, together with patients who were lost to follow-up, were excluded. Since bleeding at the time of lumbar puncture might distort the RBC count, we excluded patients with confirmed or susceptible bleeding that was related to lumbar puncture. LPS was totally performed by skilled and practiced neurosurgeons, and the shunting system with programmable pressure valve was obtained from either Medtronic (USA) or Sophysa (France).Baseline characteristics of each patient including age, sex, duration of hydrocephalus, history of prior shunt, follow-up time, symptoms, National Institute of Health stroke scale (NIHSS), Glasgow Coma Scale (GCS), and brand of shunt system were collected. CSF parameters including red blood cell (RBC) count, nucleated cell count, protein level, glucose level, and chlorine level were analyzed within 1 week prior to shunt insertion or at the time of shunt implantation.The primary outcomes were the incidence rate and associated factors of SO occurring within 3 months of shunt placement. Based on a previous study, obstruction was defined as a blockage of the catheter with debris, such as blood, proteinaceous fluid, or pieces of choroid plexus, which was confirmed within the process of shunt revision . Patientt-test was used to compare the difference. Other quantitative data  were described as median (range), and Wilcoxon rank sum test was used to compare the difference. Categorical data  were described as number (percentage). In the binary logistic regression analysis, first, variables with a p < 0.2 in univariate analysis were selected for further multivariate analysis. Probability values (p) < 0.05 (two-sided) were considered to have statistical difference.SPSS version 19  was used to perform statistical analysis. The Kolmogorov–Smirnov test was first used to determine the normality for quantitative data. Continuous data following normal distribution  were described as mean ± standard deviation (SD), and The flowchart of the selection of patients is shown in The baseline characteristics are listed in p = 0.981), etiology (p = 0.843), history of prior shunt (p = 0.489), time from onset to shunt insertion (p = 0.358), and type of implanted system (p = 0.945).In order to identify any factors associated with SO, we synchronously compared the baseline data between these two groups. However, factors highly suspected to be related to SO were not of statistical significance, including age (p = 0.032), indicating an immediate improvement of clinical symptoms. In terms of Evans Index, no statistical difference was observed  following LPS. The proportion of patients with positive response was 73.7% (56 patients) within 3 months after surgery. The overall rate of early SO was 15.6%  while 61 patients (control group) were obstruction-free without shunt revisions.NIHSS dramatically decreased at discharge with statistical significance  and nucleated cell count  of preoperative CSF significantly elevated in the study group compared with control group. However, there was no statistical significance between the two groups in the level of protein , CSF glucose , and CSF chlorine .The values for CSF parameters and the comparison of the study group to control group are shown in p < 0.2 in the univariate analysis including elevated CSF nucleated cell counts , elevated CSF RBC counts , and CSF chlorine level  were selected to perform further multivariate analysis. Other CSF parameters were not associated with early SO, including CSF protein level  and CSF glucose level . Multivariate analysis identified the presence of RBCs in preoperative CSF  as a dependent risk factor for early SO while elevated nucleated cells  and CSF chlorine level  were not of statistical significance.In the binary logistic regression analysis as shown in SO is the most common cause of shunt failure, remaining a significant problem with diversion procedures . The incin vitro  showed a statistically significant increase in the risk of early SO , indicating that it might be necessary to wait for blood to clear from the CSF before LPS insertion. Meanwhile, early SO was not statistically associated with CSF protein level, glucose level, and chlorine level. Despite our findings, a further prospective study with a large sample size is essential to draw strong conclusions.Nevertheless, there are surprisingly no studies examining CSF parameters with SO following LPS implantation in adults. It should be noted that results found in VPS cannot be easily translated to the LPS-treated populations. Given that patients with a high level of CSF parameters before shunt insertion might be at a high risk of SO, we analyzed the RBC count, nucleated cell count, protein level, glucose level, and chlorine level of preoperative CSF and assessed whether CSF parameters were associated with early SO following LPS implantation in a group of adult patients with PHH. The RBC count and nucleated cell count in preoperative CSF were actually higher in patients with early SO. Importantly, patients with RBCs in preoperative CSF  showed improvement in NIHSS at the time of discharge while the statistical difference on Evans Index between the two groups was not observed, indicating that the majority of patients obtained favorable outcomes immediately after LPS implantation despite the bare reduction on the ventricular size. Besides, the proportion of patients with positive response (improvement of one point or more on the NIHSS) was 73.7% within 3 months after surgery. Although patients treated by LPS might face some complications, severe adverse events, e.g., subdural hematoma, cerebral infarction, and cerebral hernia, were not observed in the current work (% showed We recognize that the current study still has some limitations. The most critical issue is that the current study is retrospective from a single institution in the presence of possible systematic bias and variation. Second, the evaluation of improvement of symptoms is subjective. There is no scale to assess the symptoms caused by PHH and the commonly used scales describing the severity of INPH, such as INPH grading scale and Keifer's hydrocephalus scale, are not suitable for our cohorts.This study suggested that the presence of RBCs in preoperative CSF was associated with an increased risk of early SO in patients with PHH treated by LPS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The study was conducted according to the guidelines of the Declaration of Helsinki. The patients/participants provided their written informed consent to participate in this study.TS and JG: conceptualization. TS, WC, and SC: formal analysis. TS and XL: methodology. YZ, YY, and HY: investigation. WC and JY: software. CY and JG: funding acquisition and writing—review and editing. JG: project administration. TS and WC: writing—original draft. All authors have agreed to be listed and have seen and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


Recent studies have suggested alternative cerebrospinal fluid (CSF) clearance pathways for brain parenchymal metabolic waste products. One fundamental but relatively under-explored component of these pathways is the anatomic region surrounding the superior sagittal sinus, which has been shown to have relevance to trans-arachnoid molecular passage. This so-called parasagittal dural (PSD) space may play a physiologically significant role as a distal intracranial component of the human glymphatic circuit, yet fundamental gaps persist in our knowledge of how this space changes with normal aging and intracranial bulk fluid transport.T1-weighted and T2-weighted MRI for PSD volumetry. PSD volume was extracted using a recently validated neural networks algorithm. Non-parametric regression models were applied to evaluate how PSD volume related to tissue volume and age cross-sectionally, and separately how PSD volume related to CSF flow (significance criteria: two-sided p < 0.05).We re-parameterized MRI methods to assess CSF circulation in humans using high resolution imaging of the PSD space and phase contrast measures of flow through the cerebral aqueduct to test the hypotheses that volumetric measures of PSD space (1) are directly related to CSF flow (mL/s) through the cerebral aqueduct, and (2) increase with age. Multi-modal 3-Tesla MRI was applied in healthy participants  across the adult lifespan whereby phase contrast assessments of CSF flow through the aqueduct were paired with non-contrasted p < 0.001, Spearman’s-p < 0.001, Spearman’s-p < 0.001) were observed. The elevation in PSD volume was not significantly related to gray or white matter tissue volumes. Findings are consistent with PSD volume increasing with age and bulk CSF flow.A significant PSD volume enlargement in relation to normal aging (Findings highlight the feasibility of quantifying PSD volume non-invasively in vivo in humans using machine learning and non-contrast MRI. Additionally, findings demonstrate that PSD volume increases with age and relates to CSF volume and bi-directional flow. Values reported should provide useful normative ranges for how PSD volume adjusts with age, which will serve as a necessary pre-requisite for comparisons to persons with neurodegenerative disorders.The online version contains supplementary material available at 10.1186/s12987-022-00320-4. Classically, cerebrospinal fluid (CSF) production is recognized to occur in the choroid plexus (ChP) complexes, after which the fluid circulates through the ventricular system and around the brain and spinal cord with subsequent resorption mediated by dural venous sinus arachnoid granulations. However, there is emerging evidence that CSF clearance may also occur in the regions surrounding the dural sinuses . SupportThe primary source of CSF production is the ChP, which is estimated to produce approximately 500 mL of CSF daily at a rate of approximately 20–25 mL CSF per hour in adult humans . While mThe logical extension of this work, particularly given the increased attention to the role of the PSD in effective fluid transport from the subarachnoid space, is to understand how such CSF flow parameters relate to quantitative estimates of the PSD volume. Here, we applied deep learning algorithms to non-contrasted head MR images in healthy adults across the lifespan, for the first time, to test the hypotheses that increases in PSD volume parallel changes in CSF flow through the cerebral aqueduct, and additionally that PSD volume increases with advancing age. Findings are discussed in the context of the growing literature on PSD morphology and CSF clearance.All participants provided informed, written consent in accordance with the local institutional review board (IRB) and consistent with the Declaration of Helsinki and its amendments. All participants were scanned between January 2020 and September 2021 at Vanderbilt University Medical Center. Inclusion criteria for healthy control participants: age = 20–83 years, no history of cerebrovascular disease, anemia, psychosis, or neurological disorder including but not limited to prior overt stroke, sickle cell anemia, schizophrenia, bipolar disorder, existing neurodegenerative disorder, or multiple sclerosis. Presence of non-specific white matter lesions was not an exclusion criterion, as these lesions are extremely prevalent with aging, and we sought our cohort to be generalizable and representative. Clinical history was reviewed by a board-certified Neurologist  and anatomical imaging and angiography by a board-certified neuroradiologist  to ensure that inclusion criteria were met.T1-weighted magnetization-prepared-rapid-gradient-echo (MPRAGE) , 3D T2-weighted volume isotropic-turbo-spin-echo-acquisition (VISTA) , 2D T2-weighted FLAIR , 3D time-of-flight magnetic resonance angiography , and diffusion weighted imaging (DWI) . These scans were primarily used for confirming healthy status and ensuring inclusion criteria; T1-weighted and T2-weighted scans were additionally used for brain parenchymal and PSD volume segmentation as described below.All participants were scanned at 3.0 Tesla  using body coil radiofrequency transmission and phased array 32-channel reception. The scan protocol included standard anatomical imaging consisting of 3D CSF movement was recorded within the aqueduct of Sylvius. To achieve this, four MR-compatible ECG electrodes were placed on the chest to enable retrospective cardiac phase correction. A single slice orthogonal to the aqueduct of Sylvius was placed above the location of the 4th ventricle, where the aqueduct is bound by the tectum posteriorly and mid-brain anteriorly. A velocity encoding gradient of 12 cm/s was applied, and measurements were acquired for five minutes, after which 12 cardiac phases were time-averaged to generate the mean CSF flow profile over the cardiac cycle.CSF flow The directionality of CSF flow was defined as anterograde  or retrograde , whereby anterograde flow occurs primarily during systole and retrograde flow occurs primarily during diastole  and converting cubic millimeters to milliliters (1 cm3 = 1 mL). The mean volumetric flow of CSF through the aqueduct of Sylvius was calculated during 12 prescribed phases of the cardiac cycle which leads to computation of the following flow parameters: the maximum anterograde flow, maximum retrograde flow, net flow, absolute flow, and regurgitation fraction  was estimated using the brain mask computed by the advanced normalization tools (ANTs) package [ package , which u package . CSF, gr package .Parasagittal dural (PSD) space quantification PSD volumes were computed using a semi-supervised segmentation method based on a combination of a fully connected neural network (F-CNN) and voxel clustering based on a gaussian mixture model (GMM) to label voxels as PSD or sagittal sinus based on their T2-weighted MRI signal intensities. The first step of our method used an F-CNN to extract parasagittal space; this deep-learning model was trained using 20 T2-weighted MRI scans, as we found T2-weighted contrast to provides the best contrast to the adjacent subarachnoid space (relative to proton density or T1-weighted contrast). This deep-learning model aims to estimate a binary mask of parasagittal space which includes both the PSD, superior sagittal sinus, and contributing veins in the region of the PSD. Once the parasagittal space mask was computed, voxels belonging to the parasagittal mask were labeled as PSD, or superior sagittal sinus/contributing veins.T2-weighted MRI images were aligned to the MNI template [To achieve this, bias field inhomogeneity was reduced using N4 inhomogeneity correction . Next, itemplate  using notemplate  with a cT2-weighted scans. This region of interest contains the superior sagittal sinus volume and PSD space volume along the sinus. Next, manual segmentation maps were used to train an automatic segmentation method based on an F-CNN using a U-Net architecture [T2-weighted MRI signal distribution using Bayes framework.First, a board-certified neuroradiologist (CDM) manually segmented the parasagittal space of 20 itecture . U-Net aT2-weighted scans) and venous structures including the superior sagittal sinus and contributing veins (hypointense in the T2-weighted scans). Resulting segmentation maps were transformed to native space using the inverse transform. Therefore, all subsequent analyses were performed in the native space of the T2-weighted scans. All segmentation maps (PSD and tissue masks) were visually inspected and validated by a neuroradiologist (CDM).Thus, the final maps provide labels for voxels belonging to the PSD space  we defined a model with PSD and maximum anterograde or retrograde flow as separate dependent variables and age, sex, and intracranial volume as independent variables.Demographics and tissue volumes  we defined a model with PSD and maximum anterograde or retrograde flow as separate dependent variables and age, sex, CSF, GM, and WM volumes as independent variables.PSD volume and CSF flow we defined a model with PSD volume as the dependent variable and the CSF flow characteristics including maximum anterograde flow, maximum retrograde flow, net flow , absolute flow , or regurgitant fraction , as well as age, and sex as independent variables.To assess the validity of our hypotheses, we used a generalized linear model (GLM). We defined three multivariate models to assess three different hypotheses.p-values are reported as raw and corrected with FDR with significance fixed to 0.05. All analyses were performed in Matlab  using the statistical toolbox.In addition to these linear models, for completeness we also assessed the correlation of each pair of features using the Spearman’s rank correlation coefficient. This was evaluated using analysis of variance (ANOVA) and corrected for multiple comparison using false discovery rate (FDR) . DemograA summary of participant demographics is provided in Table 3, 3) or sex was observed. The analysis of the relationship between brain tissue volume and age indicates that GM significantly declines with age with p-value < 0.001 and No significant difference in ICV over the different age groups of 20–39 years, 40–59 years, or 60–83 years . Spearman’s rank correlation method indicated a correlation of retrograde CSF flow and CSF volume, with a correlation coefficient mentclass2pt{minimUsing the same method, we assessed the relationship between PSD volume and brain tissue and CSF volumes Figs. , 5. No eTwo linear models were applied to evaluate the relationship between PSD volume and maximum CSF flow through the cerebral aqueduct. The results provide evidence for a significant correlation of PSD volume and CSF flow for both anterograde and retrograde directionality. The model indicates that anterograde CSF flow correlates with PSD volume with a p-value = 0.01 , and a Spearman’s correlation coefficient of 0.36 . Retrograde CSF flow showed stronger correlation, with a p-value = 0.001 , and a Spearman’s correlation coefficient of Figure T2-weighted imaging, and furthermore, that PSD hypertrophy correlates with age and directly relates to CSF flow through the cerebral aqueduct.CSF egress pathways along dural sinuses have recently gained attention given their potential importance to cerebral waste clearance, which has in turn prompted a reevaluation of neuro-fluid circulation and relevance. It has recently been suggested that such fluid clearance may occur along parasagittal dural spaces, which co-localize with the anatomic region surrounding the superior sagittal sinus  and compIt is noteworthy that parasagittal dural space is not the sole potential pathway for CSF egress. Alternative routes have been hypothesized, such as routes involving arachnoid villi and granulations, olfactory pathways through the cribriform plate, and outflow along the spinal canals ; these ren route to the brain parenchymal interstitial space [Increasing animal and human evidence supports the presence of this highly organized system whereby fluid passes from the subarachnoid space to the periarterial space al space –22. ISF al space , 24. Netal space , 26.The PSD spaces, along with other pathways, have been proposed to help clear fluid exiting the interstitial space. For example, Aspelund et al. demonstrated the importance of a dural lymphatic network for the clearance of macromolecules using transgenic mouse models expressing a VEGF-C/D trap and associated aplasia of dural lymphatics . Here, aComplementary findings have been observed by Louveau et al. . Using aT1-weighted imaging detailing contrast progression to the PSD. Following this finding, a more recent study using intrathecal gadobutrol injection assessed the clearance of CSF in humans over an extended time period of 48 h in adults with CSF leakage disorders [Additionally, in vivo measurements in humans demonstrating trans-arachnoid molecular passage in the PSD region were conducted by introducing gadolinium contrast into the subarachnoid space via intrathecal injection , with suisorders . This stisorders .T1-weighted postcontrast after gadobutrol injection; participants were undergoing clinically-indicated scanning for suspected or confirmed cancer and brain metastases screening [A separate study assessed so-called peri-sinus lymphatic space volume in 165 neurologically healthy adults using black blood MRI imaging with creening . ConsistT2-weighted imaging of the brain to assess similar volumes without exogenous contrast agent injection. The technique we used allows for distinction of the PSD from both the adjacent subarachnoid space as well as the superior sagittal sinus and feeding cortical veins. Our delineation of this PSD volume is visibly and topographically similar to the 2D appearance and the 3D volumetric maps as detailed by Ringstad et al. [In our study, we used submillimeter 3D Our results also confirm the previous findings by Park et al. that demonstrated increasing PSD volumes with age . Our stuWe observed that PSD volumes were significantly correlated with maximum anterograde and retrograde CSF flow through the cerebral aqueduct. This correlation presents further evidence of an organized system of CSF circulation and metabolism and suggests of complex physiologic interplay between various anatomic structures. Further work is needed across the human lifespan and in disease to determine the sequence of dysfunction across these components and components of the glymphatic circuit. Investigations which characterize these findings across differing neurodegenerative disease cohorts may shed light on how aberrant CSF flow contributes to various neurodegenerative conditions.We also assessed for a relationship between CSF flow through the cerebral aqueduct with white and gray matter volumes, CSF volume, gender, and age. Our experiments indicate that CSF volumes show significant correlation with maximum retrograde CSF flow in the cerebral aqueduct. This again supports that these findings cannot be fully explained by brain volume loss.In one prior study investigating the relationship of different CSF dynamics at the level of the cerebral aqueduct, CSF movement was shown to be dependent on age and gender . Only paFinally, while our primary analysis focused on maximum anterograde and retrograde flow through the aqueduct, we performed supplementary analyses where we also considered net flow, absolute flow, and regurgitant fraction  were utilized, and also that compressed sensing algorithms led to similarly poorer performance. The proposed non-contrasted scan is 0.8 mm isotropic without compressed sensing and requires a duration of approximately 5 min. We believe that future work to reduce scan times is warranted, however we recommend caution for applying PSD segmentation at lower spatial resolution or with advanced accelerated acquisition or reconstruction algorithms without further refinement.Second, in parallel work, we explored the possibility of faster imaging using compressed sensing and lower spatial resolution  for the T2-weighted signal distribution. Among the many proposed deep-learning architectures, a variant of the well-known U-net architecture showed the best performance in the estimation of the parasagittal mask. Following this initial step, our experiments indicated that techniques based on maximizing a posteriori model using the Bayes framework provide a robust delineation of PSD over superior sinus, afferent veins, and dura mater.Finally, extensive work has been conducted to design an effective algorithm for the automatic delineation of PSD space using non-contrasted anatomical MRI. We believe that the benefit of using such techniques is twofold. First, dedicated computer-aided delineation techniques have demonstrated increased accuracy and enables the study of large-scale datasets. Second, and more importantly, these techniques enable us to substantially improve the reproducibility of studies. Therefore, in our work, we adapted recent advances in machine learning with a fully convolutional neural network that has been combined with non-learning statistical modeling of the Visual quality assurance done over the entire dataset by our expert radiologist suggest that there is no overestimation of the PSD volume due to size of the sinus. However, it is noteworthy that we did notice some areas appearing as arachnoid granulations can mimic partial occlusion of the superior sinus lumen. This has been reported in a few of case studies in the past , 32. ThiThe study findings should also be considered in light of several limitations. First, we evaluated the PSD volume across the lifespan cross-sectionally as is common in neuroimaging studies, rather than longitudinally. Second, while the largest cohort of PSD volume data presented to date, the sample size of 62 was moderate and precluded multiple co-variates from being included in analysis. However, we characterized the health of each participant both radiologically and neurologically as described in the inclusion criteria, and all participants met rigorous healthy volunteer criteria. Given the moderate sample size, we also focused hypotheses on those that could be tested responsibly with the sample size.T2-weighted MRI, that PSD volume increases with age, and that PSD volume relates to bulk CSF volume and maximum bi-directional flow through the cerebral aqueduct. Values reported should provide useful normative ranges for how PSD volume adjusts with age, which will serve as a necessary pre-requisite for comparisons to persons with neurodegenerative disorders. Findings also provide new tools that may help to complement the growing number of studies of CSF egress. These processes may contribute to age-related neurological changes and possible vulnerability to further pathological disruption of CSF clearance processes.Findings highlight the feasibility of quantifying PSD volume non-invasively in vivo in humans using deep learning and non-contrasted submillimeter Additional file 1. Coefficients of regression analyses and results for the analysis of the relationship of additional CSF flow measures  and anatomical measures .