In this area, most cells are spared although their oxidative metabolism is impaired. area penumbra is routinely detected by immunostaining of a molecule named Heat Shock Protein 70 (HSP70). Within the area penumbra, autophagy-related proteins also increase. Therefore, in the present study, the autophagy-related microtubule-associated protein I/II-Light Chain 3 (LC3) was investigated within the area penumbra along with HSP70. In C57 black mice, ischemia was induced by permanent occlusion of the distal part of the middle cerebral artery. Immunofluorescence and electron microscopy show that LC3 and HSP70 are overexpressed and co-localize within the area penumbra in the same cells and within similar subcellular compartments. In the area penumbra, marked loss of co-localization of HSP70 and LC3-positive autophagy vacuoles, with lysosomal-associated membrane protein 1 (LAMP1) or cathepsin-D-positive lysosome vacuoles occurs. This study indicates that, within the area penumbra, a failure of autophagolysosomes depends on defective compartmentalization of LC3, LAMP1 and cathepsin-D and a defect in merging between autophagosomes and lysosomes. Such a deleterious effect is likely to induce a depletion of autophagolysosomes and cell clearing systems, which needs to be rescued in the process of improving neuronal survival.The brain area which surrounds the frankly ischemic region is named the  This region represents the border between ischemia-induced frank cell loss (ischemic core) and healthy brain tissue. Thus, the area penumbra corresponds to a peri-infarct region .area penumbra, autophagy-related proteins are consistently overexpressed [area penumbra as much as HSP70. However, no study so far compared, in the same experimental settings, the increase in LC3 in combination with HSP70.Recent studies indicate that, within the xpressed ,8,9,10. xpressed  or protexpressed ,13. In fxpressed . The incarea penumbra. Similarly, the occurrence of lysosome-specific markers was analyzed. This allows us to establish whether a primary failure of both autophagosome- and lysosome-competent proteins occurs. This is questioned by scoring immunofluorescence at light microscopy and by counting each authentic molecule in situ, within its specific organelle. Again, the merging between markers specific for autophagosomes and lysosomes allows us to answer the question as to whether merging between these compartments is altered. Moreover, assessing the placement of HSP70 within autophagosome and its merging with lysosome extends our vision about specific roles played by HSP70 as a chaperone within specific compartments of ischemic neurons. In fact, the merging of LC3 and HSP70 with LAMP1 and cathepsin-D indicates a potential common fate of these molecules, which mark the area penumbra.Therefore, the present study analyzes the co-expression of HSP70 and LC3 within the penumbra region, its cells and its specific cell compartments. This is carried out following persistent ischemia, which was experimentally produced by occluding the distal middle cerebral artery (MCAO). To measure concomitant expression of HSP70 and LC3, immunofluorescence was joined with stoichiometric measurements of each molecule by transmission electron microscopy (TEM). The measurement of sub-cellular placement of LC3 in combination with the marker HSP70 allows us to establish whether these proteins share similar sub-cellular compartments, where they may synergize. In this effort, autophagy vacuoles need to be primarily analyzed. In fact, previous studies show that up-regulation of cytosol LC3 within ischemic penumbra copes with a failure of LC3 to merge with lysosomal compartment . This waarea penumbra. Therefore, the cortical areas, which are selected for the anatomical study, correspond to those delimited by red squares of Following distal (Sylvian) occlusion of the middle cerebral artery (MCA), a wide permanent ischemic damage is produced in the ipsilateral hemisphere. This involves both cortical and sub-cortical regions . As shown in Ischemic damage extends rostro-caudally from the rostral pole of the striatum to the anterior thalamic nuclei, while the cortical reference spans from the primary motor cortex (M1) to the auditory cortex. Within this region, the red squared areas were dissected both ventrally and dorsally to obtain small blocks from analogous regions in the ischemic side, contralateral side and from homologous areas of sham-operated mice.area penumbra neurons compared with non-ischemic neurons is reported in area penumbra, the number of non-stained vacuoles is not modified. It is rather the specific staining which is modified, as we shall see.Sub-cellular structures such as vacuoles are evident in each group . Direct area penumbra similarly to HSP70 immunofluorescence, which is the classic marker of the area penumbra. The present investigation provides the first evidence for co-expression of both antigens within the area penumbra. In detail, as reported in the graphs of area penumbra compared with analogous regions from sham-operated mice. Such an increase mimics what occurs for HSP70  and in homologous regions contralateral to the area penumbra (694.4 ± 0.054 nm) compared with the area penumbra (683.2 ± 0.049 nm). The range of variability (95%) spans from 200 nm to 1520 nm. The distance is homogenously distributed, with no clustering of co-localization being detectable at any distance. In contrast, when an interaction is expected (as in the case of aggregates of LC3 molecules) stoichiometry reveals a distance approaching zero  double-immuno-gold stoichiometry was carried out. This is shown in representative pictures of area penumbra, the amount and placement of cathepsin-D molecules were quantified at ultrastructural morphometry. The stoichiometry count of cathepsin-D occurring in the cytosol and vacuoles of neurons from the area penumbra was compared with analogous regions from the contralateral side and sham-operated mice. Contrary to LAMP1, the amount of cathepsin-D detected in the cytosol of the area penumbra neurons increases compared with analogous contralateral regions and sham-operated mice  does not increase within neuronal vacuoles. It is rather the opposite, since the vacuolar placement of LC3 (as well as HSP70) is drastically reduced. Thus, the increase in LC3 (and HSP70) in the whole cell occurs within cytosol only, where LC3 may aggregate to produce hot fluorescent spots which had been interpreted as big stagnant autophagy vacuoles. The present study rules out an overexpression of LC3-immuno-positive vacuoles within area penumbra neurons. These findings strongly challenge the hypothesis, based on light microscopy, that within the area penumbra an excess of stagnant autophagosomes occurs, since here LC3-positive vacuoles are markedly reduced within the area penumbra.In the present study, such an investigation was carried out by using a model of permanent occlusion of MCA . Within area penumbra neurons may not correspond to autophagy vacuoles was already formulated [area penumbra compared with the control tissue. In fact, LC3 increases in the cell but it is massively reduced in the vacuoles, where it is needed to induce autophagy clearance. Similarly, the present findings lead to the re-interpretation of previous data based on TEM observations of giant autophagy-like vacuoles [area penumbra, the spots of LC3 immunofluorescence may be due to cytosolic non-vacuolar LC3 protein aggregates [The increase in LC3-positive hot spots is likely dependent on the clustering of LC3 in the cytosol, which may generate spots of immunofluorescence within ischemic penumbra contrasting with the mild fluorescence of controls. The hypothesis that LC3 fluorescent spots in rmulated ,10,15. Ivacuoles ,24,25. Igregates ,27. In farea penumbra is considered to be protective [area penumbra [area penumbra cells. The sole morphological evidence of a remarkable merging between HSP70 and LC3 within the area penumbra neurons needs to be implemented by functional data to explore whether a synergism takes place. A major finding of the present study concerns the merging of LC3 and HSP70 immuno-staining concerning zonal, cellular and sub-cellular compartments. In fact, the very same cortical zones and neurons increase the staining for both antigens. Since the expression of HSP70 within the otective ,29, thispenumbra . Similararea penumbra consists of establishing the fate/progression of LC3-positive autophagy vacuoles. Similarly, due to the co-localization of HSP70, an additional question concerns the fate of HSP70-positive vacuoles. The merging of autophagosomes with lysosomes is key to understand the cell clearance. Zhang et al. (2021) [A key point to interpret the autophagy status in the . (2021)  postulat. (2021) . The pre. (2021) , who indThese findings indicate the lysosomal compartment as a kernel to solve the issue. area penumbra neurons fail to merge with cathepsin-D- or LAMP1-positive vacuoles, which is consistent with the occurrence of a failure in the occurrence of autophagolysosomes. When specific stoichiometry for LAMP1 and cathepsin-D is carried out, a primary defect of both proteins is detected within vacuoles. These findings lead to the hypothesis that within the peri-infarct region a failure of the whole autophagolysosome machinery takes place. This depends on multiple phenomena which are reported in the scheme of In fact, in the second part of the present study, evidence is provided which shows that both LC3 and HSP70 within area penumbra neurons. In fact, in the area penumbra, the merging of vacuolar HSP70 within LAMP1-positive vacuoles is defective. The loss of such a synergism is likely to induce deleterious effects. In fact, the severe depletion of both lysosome and autophagosome is replicated by a loss of their chaperone (HSP70) component. This loss of compartmentalization may be in need to be rescued to improve neuronal survival following ischemia.Remarkably, in the present study, evidence emerges which entraps HSP70 in such a dysfunctional cell clearance within The present data provide direct evidence for a defective compartmentalization of chaperone, autophagy and lysosomal molecules, which could not be detected by using light microscopy alone. In fact, a confounding point, when assessing autophagosomes and lysosomes by light microscopy, is the potential bias of interpreting puncta as a witness of vacuoles. This applies to LC3 spots of immunofluorescence (as discussed above) as well as to the measurement of lysosomal proteins. In fact, this is also the case of the lysosome-associated membrane protein LAMP1 or lysosomal enzymes such as cathepsins. All these proteins may not necessarily occur site-specifically within autophagosome or lysosome compartments. area penumbra, HSP70 is no longer able to merge with lysosome proteins. The loss of interaction between these proteins, though selective protein and organelle degradation, is a massive impairment, which is required for cell clearing machinery to be effective. In line with this, specific studies are needed to address whether compounds, such as rapamycin which promotes LC3 vacuolar placement and protects against ischemia [In fact, we measured that here, in ischemic conditions, the amount of both LC3 compartmentalized within autophagosome and cathepsin-D compartmentalized within lysosomes diminishes, while these proteins dramatically increase in the cytosol. In the case of cathepsin-D, this may lead to non-controlled digestion of cell substrates, which may produce autophagy-dependent cell death. Indeed, it is a loss of enzyme compartmentalization which generates the damage. The issue of preserved autophagosomes and lysosomes include the facet of compartmentalization of specific molecules. The loss of compartmentalization appears a key point. In fact, during ischemia a dissociation may occurs, where the increase in specific proteins does not cope with their placement within appropriate cell compartments. Instead, compartmentalization of molecular pathways is essential in providing appropriate activity. In ischemic cells, the LC3 and cathepsin-D level are increased, but their compartmentalization is suppressed  via drill-operated craniotomy; thus, MCA was identified and exposed using stereomicroscopy [Adult C57BL/6 male mice  weighing 25 g were housed under controlled conditions  on a 12 h light–dark cycle with food and water ad libitum. The experimental protocol was approved by the Ethical Committee of Neuromed Institute  and further supervised by The Italian Ministry of Health (Authorization number 1194/2020-PR). Mice were anaesthetized with chloral hydrate . An incision was made between the outer canthus of the eye and the external auditory meatus and the temporal muscle was bisected and retracted to expose the external temporal aspect of the skull to produce a small hole , acetic acid (10%) and chloroform (30%) for histochemistry and immunofluorescence. Twenty-four hours later, brains are placed in 70% ethanol to be wax-embedded. The brains were cut into 10 μm-thick coronal slices at microtome . Each slice was regularly spaced 550 µm. Slices were used for histochemical and immunofluorescence analysis.For histochemical analysis, slices were de-waxed and processed for staining with thionin (a kind of Nissl staining) to frankly assess infarct and peri-infarct regions. area penumbra, the cortex being analyzed for morphology is restricted to the area delimited by the red squares of As shown in representative area penumbra. Three serial coronal slices were selected from the ventral border at the middle of the rostro-caudal extent of the ischemic region (ranging from bregma +1.94 mm back to bregma +0.14 mm). These specimens were used for staining with rabbit polyclonal anti-LC3B , monoclonal mouse anti-LAMP1  and monoclonal rabbit anti-HSP70 or monoclonal mouse anti-HSP70 . Slices were treated with normal sera for 1 h (10% in TBS). Then, they were incubated overnight (4 °C) with primary antibody anti-LAMP1, anti-HSP70 and anti-LC3B (for details see Paraffin-embedded mouse brain sections (10 μm) were used for immunofluorescence, which was quantified by measuring the number of immunofluorescent cells from three slices per mouse . Slices were obtained from the peri-infarct cortical regions to encompass the 0 (μ1 = μ2) was rejected when p ≤ 0.05.Immunofluorescence was assessed considering the ratio between positive cells and the background fluorescence measured in the corpus callosum by using a Zeiss AxioPhot2 microscope equipped with a digital video camera. Images were acquired at low magnification (10×). Results are given as percentage of the mean ± S.E.M. of 12 values for each group (n = 3 slices × N = 4 mice). Unpaired, two-tailed T-test was used for statistical comparison of collected data. Hypothesis Harea penumbra corresponds to 2  ipsilateral for each mouse (N = 6) undergoing ischemia for a total of 6 × 2 = 12 area penumbra. Similarly, 12 corresponding regions were analyzed from the contralateral side of the same mice, which brings the number of regions from ischemic mice to 24. In addition, since 6 sham-operated mice were used, each one carrying a bilateral dorsal and ventral region, a total of 24 regions were also examined in sham-operated mice. This corresponds to 48 regions owing the very same placement, which were counted from ischemic and sham-operated mice. Corresponding regions from the two hemispheres of sham-operated mice were overlapping concerning all measurements (pilot experiments). This allowed us to pool left and right specimens from sham-operated mice in a total of 6 ventral and 6 dorsal areas to homogenize the number of specimens from each group.Blocks from the cerebral cortex (1 mm × 1.3 mm × 1 mm) were dissected from the ipsilateral and contralateral cortex from ischemic mice (N = 6) and from both hemispheres from sham-operated mice (N = 6). From each side of each mouse, both the ventral border  and the dorsal border  of the pIn fact, the use of sham-operated mice in addition to the contralateral (non-ischemic side) works as an additional control to rule out systemic and widespread compensatory changes altering protein expression  all over the brain following focal ischemia. In fact, this may indirectly influence the contralateral hemisphere through systemic effects or widespread subcortical compensatory innervation. When such a phenomenon occurs, even slightly, on the contralateral side, this may reduce the analytical power of measuring ischemia-induced antigen expression compared with the control tissue.4) for 1 h at 4 °C.For TEM analysis, tissue samples were fixed in a solution containing paraformaldehyde 2.0%, and glutaraldehyde 0.1%, in 0.1 M PBS, pH 7.4. Then, specimens were dissected and immersed in the same solution overnight at 4 °C. After washing in PBS (0.1 M), samples were post-fixed in 1% osmium tetroxide  to be embedded in epoxy resin. The concentration of the fixing and post-fixing solutions along with the use of the epoxy embedding resin were validated in previous studies using immuno-gold at ultrastructural morphometry ,33. In fitecture ,35,36. Tarea penumbra and allowing the identification of specific cell types to be analyzed at TEM.Ultra-thin slices were used both for plain and post-embedding immuno-gold electron microscopy. Ultra-thin slices were stained with uranyl acetate and lead citrate to be visualized at a Jeol JEM SX100 electron-microscope . Before cutting ultra-thin sections at ultra-microtome, semi-thin slices were carried out from blocks embedded in epoxy resin by using the same ultra-microtome, which was set at a width of 700 nm per slice. This was carried out in order to orient the ultra-structural scanning of the Ultra-thin sections, width = 90 nm, collected on nickel grids were processed for proteins detection by using primary antibodies detailed in 4) for 30 min at 22 °C to remove OsO4 for protein unmasking, while keeping optimal preservation of cell ultrastructure [Ultra-thin slices were layered on droplets of aqueous sodium metaperiodate , leading to 120 cells for each region per group.2) were counted from brain slices stained with LC3 and/or HSP70, LC3 and/or LAMP1 and LAMP1 and/or HPS70. Slices spaced 550 µm apart were calculated for each region (area penumbra or homologous regions from sham-operated mice). Three slices were counted for each animal. Values for neuronal density (number of neurons/mm2) were calculated within an area of 82,500 μm2. Results are given as percentage of positive cells compared with the control (sham-operated mice). Results are given as percentage of the mean ± S.E.M per group. Inferential statistics to compare groups was carried out by using a Student’s t-test .Immunofluorescent cells  . For ultEach count was carried out by two blind observers. Data are given as the mean ± S.E.M.0) was rejected when p ≤ 0.05.Data were compared by using one-way ANOVA with Fisher’s Test. Null Hypothesis (Harea penumbra neurons and mark the autophagosome and lysosome compartments (The present manuscript reports the amount and placement of specific molecules which characterize artments .area penumbra, protein amount and compartmentalization varies according to the arrows reported in the scheme of area penumbra neurons, the autophagy-related molecule LC3 increases. This overall increase is specifically due to the cytosolic amount of LC3, while the amount placed within vacuoles is decreased (area penumbra cells due to a massive increase in the cytosol, while its sub-cellular placement within vacuoles is reduced (Within the ecreased . Similar reduced B. These  reduced .area penumbra neurons (area penumbra due to a massive increase in the cytosol, while its specific lysosome compartmentalization is reduced (A similar decrease in vacuolar compartmentalization occurs for the lysosome molecule LAMP1, which is decreased both in lysosome vacuoles and cytosol of  neurons . The lys reduced .This indicates that ischemia produces a dissipation of specific molecules from their own specific physiological compartments. Altogether, these findings generate the concept that in ischemia, lysosomal function and autophagosome formation are impaired .


Background and objectives® are therefore used to support the watertight closure of the dura mater. Recently, we published the results of a large registry documenting the effectiveness and safety of Hemopatch® in various surgical specialties, including neurosurgery. Here we aimed to analyze the outcomes from the neurological/spinal cohort of this registry in more detail.Cerebrospinal fluid (CSF) leakage is a significant complication in cranial and spinal interventions. Hemostatic patches such as HemopatchMethods® registry was designed as a prospective, multicenter, single-arm observational study. All surgeons were familiar with the application of Hemopatch® and it was used at the discretion of the responsible surgeon. The neurological/spinal cohort was open for patients of any age if they had received Hemopatch® during an open or minimally invasive cranial or spinal procedure. Patients with known hypersensitivity to bovine proteins or brilliant blue, intraoperative pulsatile severe bleeding, or an active infection at the potential target application site (TAS) were excluded from the registry. For the posthoc evaluation, we stratified the patients of the neurological/spinal cohort into two sub-cohorts: cranial and spinal. We collected information about the TAS, intraoperative achievement of watertight closure of the dura, and occurrence of postoperative CSF leaks.Based on the data from the original registry, we performed a post hoc analysis for the neurological/spinal cohort. The HemopatchResults® in 147 patients , of which 123 underwent a cranial procedure. Twenty-four patients underwent a spinal procedure. Intraoperatively, watertight closure was achieved in 130 patients . Postoperative CSF leakage occurred in 11 patients . We observed no serious adverse events related to Hemopatch®.The registry comprised 148 patients in the neurological/spinal cohort when enrolment was stopped. The dura was the application site for HemopatchConclusion® in neurosurgery, including cranial and spinal procedures, as also observed in some case series.Our post hoc analysis of real-world data from a European registry confirms the safe and effective use of Hemopatch In crang tissue ,10.® is composed of a thin, pliable collagen pad coated with a protein-reactive polyethylene glycol monomer . The collagen pad has a high liquid absorption capacity, while the covalent binding of the NHS-PEG coating to proteins results in strong adherence to the underlying tissue. The polymerized NHS-PEG builds a hydrogel that allows rapid adhesion to the target site, where the patch forms a liquid-tight barrier. Additionally, blood clotting is promoted by the collagen matrix of the pad. Crosslinking is facilitated by blood and other body fluids or can be achieved with sodium bicarbonate solution if the Hemopatch® is applied on a dry target application site [Hemopatchion site .® in neurosurgery to reduce the incidence of CSF leakage [Several small series previously described the effective and reliable use of Hemopatch leakage ,10,12-14® was published [® in achieving hemostasis and sealing in various tissues, not only in open procedures but also in minimally invasive surgery [Recently, data from a registry documenting the real-world clinical application of Hemopatchublished . This re surgery . Here weStudy design and participants® and decided whether to use it or not according to the institutional standards.Ethical approval for this study was obtained from the Ethics Commission of the University of Cologne (Nr. 17-344 dated 11/8/2017). Details of the study design and selection of participants were reported previously . In brie® during an open or minimally invasive cranial or spinal procedure. Known hypersensitivity to bovine proteins or brilliant blue, intraoperative pulsatile or severe bleeding at the potential target application site (TAS), or an active infection at the TAS were reasons for excluding the patient from the registry. If Hemopatch® was applied, it was held in place for two minutes and, if needed according to the instructions for use, bicarbonate solution was used to moisten the TAS before placement of the pad [Patients of any age were eligible for the neurological/spinal cohort if they had received Hemopatch the pad . The pad® as well as the potential use of bicarbonate was recorded with an electronic case report form (eCRF) [Perioperative data on the effectiveness and safety of Hemopatchm (eCRF) .Effectiveness endpoints and safety outcomesEffectiveness endpoints as well as safety outcomes of the registry have already been reported .Briefly, the primary endpoints for intraoperative effectiveness were the percentage of patients achieving hemostasis within two minutes and the percentage of patients achieving hemostasis without re-bleeding at the time of surgical closure. In the neurological/spinal cohort presented here, an additional intraoperative effectiveness endpoint in terms of leakage control was defined as the achievement of a watertight closure, i.e. no CSF leakage after inspection according to the local standard of care such as the Valsalva maneuver. The primary postoperative effectiveness endpoint for the neurological/spinal cohort was defined as the incidence of a postoperative CSF leakage presenting as an external or internal accumulation of CSF, including pseudo meningocele during the four-week follow-up period .®, the incidence of adverse events of special interest was defined as primary safety endpoints. These were as follows: allergic reaction to Hemopatch®, re-bleeding at the TAS, hematoma at the TAS, and/or local infections at the TAS [To assess the safety of Hemopatch the TAS .® units applied, use of bicarbonate in combination with Hemopatch®, need for intraoperative surgical revisions due to bleeding, air, or other body fluid leakage, intraoperative transfusions , surgery duration, postoperative transfusions up to 72 hours after surgery , days in the intensive care unit, length of hospital stay as well as the surgeon's product assessment and satisfaction, assessed by the surgeon´s questionnaire [Secondary safety endpoints included the number of Hemopatchionnaire .Statistical methods and data sets® registry was mainly descriptive and included sample size (n), mean, standard deviation (SD), median, minimum, and maximum for continuous variables and frequency counts, percentages, and exact 95% binomial confidence intervals (Clopper-Pearson) for proportions for categorical variables. Likewise, analyzed data sets of the registry have been described before [As described previously in detail, statistical analysis of the Hemopatchd before .® were included in the safety analysis set and all patients with a postoperative hemostasis assessment were included in the full analysis set.In the neurological/spinal cohort, all patients who received HemopatchPost hoc evaluation of the neurological/spinal cohort® application  whether Hemopatch® was primarily used to achieve hemostasis or sealing, i.e. in combination with other documented parameters such as achievement of watertight intraoperative closure.Documentation of the TAS provided information on (1) whether HemopatchAs per definition, watertight intraoperative closure of the dura was achieved if no CSF leakage was detected after inspection according to the local standard of care . Intraoperative watertight closure was documented in the eCRF as yes, no, or not applicable (N/A).In the neurological/spinal cohort, the incidence of postoperative CSF leakages was documented during four weeks of postoperative follow-up . The incidence of CSF leakages was therefore determined for the defined sub-cohorts.Characteristics of the neurological/spinal cohortFour centers from two countries contributed cases to the neurological/spinal cohort: two from the Czech Republic and two from Spain, see Appendices. The number of cases per center ranged from 1 to 59.The neurological/spinal cohort comprised 148 patients, adults and children; the mean age was 44.1 years  and within three minutes in the remaining 39 patients (26.4%). Hemostasis was achieved before closure in all cases and no rebleeding was documented. CSF leakage was reported in 11 cases (7.4%). In most procedures  one patch was used to support a watertight closure of the dura . In one ® for all cohorts in this registry have been reported earlier [Detailed results of intra- and postoperative effectiveness of Hemopatch earlier .Post hoc evaluation of the neurological/spinal cohort® was used solely for hemostasis on an extended wound surface after the resection of a sarcoma. Hence, in this case, the TAS was not the dura. Due to the large size of the wound, six patches were used. No other patient received more patches.Of the 148 patients in the neurological/spinal cohort, 123 patients had cranial procedures and 24 underwent spinal procedures , and HemopatchOne patient in the infratentorial sub-cohort was operated on because of a schwannoma. In this case, watertight closure of the dura was not assumed as it was documented in the eCRF as “unknown”. This patient suffered from a CSF leak during the four weeks of postoperative follow-up Table .Watertight closure of the dura was achieved in 119 patients of the cranial sub-cohort . During the four-week follow-up period, a postoperative CSF leak was documented in eight of these 119 patients. For one patient from the infratentorial sub-cohort, the occurrence of a postoperative CSF leak was stated as “unknown”, since the patient died due to malignant brain edema less than a week after the index surgery Table . The inc® application in cranial procedures was the prevention of CSF leakage. Overall, the postoperative CSF leakage rate within four weeks in patients where the product was used as a dural sealant was 7.6% (nine out of 119).The main reason for Hemopatch® in spinal proceduresHemopatchThe spinal cohort comprised 24 patients with spinal dura closure, who were recruited from three different neurosurgical departments. In 13 out of 24 patients, intraoperative watertight closure of the dura could not be achieved with a suture. In the remaining 11 patients, primary watertight closure was documented. Of these 24 patients, two (8.3%) developed postoperative CSF leakage during the four-week follow-up. Remarkably, in both patients, an intraoperative watertight closure was achieved during the index surgery; both patients underwent revision surgery due to a pre-existing CSF fistula  [Schebesch and Brawanski  reported® was used to cover a suture line or a biological graft  to achieve watertight dural closure. In their follow-up, they observed a CSF fistula rate of 5.9% (2/34 patients) [Nowak et al. published a series of 34 prospectively analyzed patients, in which Hemopatchatients) .® and ensure optimal dural sealing [Montano et al. described a small series of eight patients receiving a cranial procedure. During the follow-up, no CSF leakage was observed. However, additional fibrin sealant was used in all cases to seal the edges of Hemopatch sealing .® in dural closure.The additional use of fibrin sealant by Montano and colleagues as well as the smaller number of cases in all three studies might contribute to the lower rates of postoperative CSF leaks observed. Nevertheless, the observations of Schebesch and Brawanski , MontanoTwo other studies describe postoperative CSF leakage rates following cranial interventions that are comparable to the rate of 7.3% reported here Table .® as an adjunct during reconstruction or other products such as glues, dural substitutes, etc. The repair procedure employing Hemopatch® led to a CSF leakage rate of 7.8% vs. 30.4% for the procedures that did not employ Hemopatch® [® in preventing CSF leakage.Sánchez Fernández and Rodríguez-Arias reported complex dural reconstructions including the posterior fossa. In their cohort study, they compared procedures in which the surgeon decided to use either Hemopatchmopatch® . This su® as a dural sealant in posterior fossa neurosurgery to Tachosil®. They used Hemopatch® in 40% of the cases for complex reconstructions and in 60% for sealing the dura suture. Overall, their CSF leakage rate in the cohort with Hemopatch® was 10.3%; (four out of 39). In the Tachosil® cohort, this product was only used in 9.4% for complex reconstructions and in more than 90% for sealing dura suture lines. The CSF leak rate was 8.0% in this cohort (18 out of 224) [Nowak et al. also compared the effectiveness of Hemopatch of 224) .Post hoc evaluation of CSF leakage rates after spinal proceduresIn the 24 spinal procedures of our registry, a postoperative CSF leak was observed in two cases , or additional suture holes after suture closure of the dura following resection of intradural tumors (five cases). In none of the 14 patients undergoing spinal surgery was a CSF leak observed during the postoperative follow-up. However, as described above for all the patients of Montano et al., fibrin sealant was used in addition to seal the edges of Hemopatch® [®. The consistent additional use of fibrin sealant and the low number of cases might explain the lower CSF leakage rate observed in the series by Montano et al.So far, spinal procedures with HemopatchThe results of other studies reported a range from 3% to 20% after removal of an intradural spinal tumor -21 and 1Role of pre-existing CSF fistula® was initially achieved during the index surgery. The repair of existing CSF fistulas can be challenging due to various factors such as scarring, low vascularity of the surrounding tissues, shortage of dura to close the leak, and the difficulty of identifying the exact position of the leak in an inflammatory sub-acute wound [In our registry, two documented cases of postoperative spinal CSF fistula appeared in patients who underwent repair of a pre-existing CSF fistula. It is worth noting that in both patients, watertight closure of the dural repair with Hemopatchte wound . Alexandte wound .Limitations® registry have already been stated in detail [®. In addition, documentation of the amount of CSF entering the drainage system at postoperative follow-up would be useful to further define effectiveness. Furthermore, due to its dual properties as a “sealing hemostat” and the fact that bleeding grades were not recorded before Hemopatch® application, it was sometimes impossible to determine whether the main purpose of applying the product was hemostasis or sealing. Moreover, the conditions under which Hemopatch® was applied may have varied widely because of the multicenter design and large sample size for the entire spine and the fact that supra- and infratentorial regions were included. However, the purpose of this registry was to investigate general efficacy and safety under real-world conditions.Limitations of the Hemopatchn detail . Brieflyn detail  before t® use in neurosurgery in a relatively large cohort including 148 patients. Watertight closure of the dura was virtually the exclusive reason for the application of Hemopatch®. This objective was accomplished in the majority of patients, and we observed no serious adverse events related to the use of Hemopatch®. The data obtained from the neurological/spinal cohort of our registry, therefore, confirm that the clinical use of Hemopatch® as a dural sealant is safe and effective.In this registry, we had the opportunity to analyze the outcomes of Hemopatch


Increasing evidence suggests that retinal hyper-reflecting foci (HRF) might be clusters of activated and proliferating microglia. Since microglia are widespread activated in multiple sclerosis (MS) brain, its evaluation in retina may help to understand and monitor MS-related pathology.This study aims at investigating the association of HRF with cerebrospinal fluid (CSF) cytokines and MRI parameters in relapsing–remitting MS (RRMS).Nineteen RRMS at clinical onset and 15 non-inflammatory neurological disorders (NIND) underwent brain 3T MRI and CSF examination. Optical coherence tomography (OCT) analysis, including HRF count, was performed on RRMS patients. Sixty-nine cytokines/chemokines were analyzed in the CSF by multiplex technology.2: 0.47) with both CXCL-2  and CXCL-13 . This latter cytokine increased in RRMS with high HRF count compared with NIND and RRMS with low HRF count. Finally, the CXCL-13/CXCL-2 ratio strongly associated with HRF count  and cortical lesion volume .In RRMS, HRF count in the ganglion cell layer (GCL) was associated with IL-1Ra, IL-9, IL-15, IFN-γ, and G-CSF. Moreover, in RRMS patients CSF concentrations of IL-1Ra and G-CSF associated with global cortical thickness. The HRF count in the inner nuclear layer (INL) correlated with IL-22, IL-34, IL-35, CXCL-2, CXCL-10, and CXCL-13, and multivariate analysis confirmed a strong association (rThe association of HRF with intrathecally produced monocyte/microglia-derived cytokines confirms their microglial origin and indicates they are worth further evaluating as markers of activated microglia. Retinal hyper-reflecting foci (HRF) have been observed by optical coherence tomography (OCT) in a wide range of neurological and ophthalmological diseases –4. Howevin vivo evaluation of activated microglia in the brain, OCT with a single linear scan through the macula proved to identify and correlate the HRF with different parameters of local inflammation and damage  cytokines/chemokines and magnetic resonance imaging (MRI) parameters of both gray and white matter inflammation and degeneration.2-glycoprotein I, anti-cardiolipin, and LAC); CSF examination; and brain and spinal cord MRI to exclude neurological disorders, as previously described , CSF analysis, OCT, and brain and spinal cord MRI at the time of the diagnosis. Inclusion criteria were 1) diagnosis of RRMS achieved according to the revised 2017 McDonald criteria ; 2) inteescribed . Even ifNo difference in demographic and clinical variables was observed between NIND and RRMS Table 1.CSF specimens were collected by non-traumatic lumbar puncture between 8.00 and 9.00 a.m., as previously reported . FollowiAll MS patients underwent spectral domain OCT  examination of both eyes, in a dark room without the injection of any mydriatic agent. In line with recent publications , 6, 14, 3; (ii) 3D-FLAIR: RT = 4,800 ms, ET = 310 ms, inversion time  3D-DIR: RT= 55,000 ms, ET = 284 ms, inversion time (IT) = 2,550 ms; contiguous axial slices with thickness of 1.0 mm; matrix size 212 × 212; and FOV = 256 × 256 × 182 mm3.MRI was achieved on a 3.0-T scanner  with 33-mT/m power gradient and a 32-channel head coil. No major hardware upgrades occurred during the study, and bimonthly quality assurance sessions were done for measurement stability. The MRI protocol included the following sequences: (i) three-dimensional (3D) T1 MPRAGE: repetition time (RT) = 7.8 ms, echo time (ET) = 3.6 ms; 180 contiguous axial slices with the off-center positioned on zero with thickness of 1.0 mm; flip angle = 8°; matrix size = 220 × 220; FOV 220 × 220 × 180 mmT1-weighted images were processed following these procedures: correction for magnetic field inhomogeneity, performed with ANTs N4 Bias Field Correction tool,  brain exData were reported as mean (± standard deviation) or median (range) for continuous variables. Group differences between RRMS patients and NIND were tested by the chi-square test for sex and by the T-test for age. Non-parametric Spearman correlation was applied to correlate HRF count and cytokines. Multiple linear regression analysis considered HRF count as a dependent variable and cytokines as an independent variable. Differences between ONIND and RRMS subgroups were tested by the Kruskal–Wallis test corrected with multiple-comparison Dunn’s test. A p-value lower than 0.05 was considered statistically significant. Prism 9.2.0 was used for all the analyses.high) and low GCP HRF count (G-HRFlow). Interestingly, significantly lower levels of IL-1Ra, IL-9, and G-CSF were found in G-HRFhigh compared to G-HRFlow , IL-9 , IL-15 , G-CSF , and IFN-γ   (high (I-HRFhigh) and INL HRFlow (I-HRFlow). I-HRFhigh had a CXCL-13/CXCL-2 ratio (4.05 ± 2.67) than both I-HRFlow  and NIND  , IL-34 , IL-35 , and CXCL-2  and directly with CXCL-10  and CXCL-13  , the CXCL-13/CXCL-2 ratio correlated with gray matter lesion volume (GMLV) .in vivo keeps the question on the origin and pathologic significance of HRF still open. Nevertheless, indirect evidence, accumulated over the last decade, supports the hypothesis that these nodules are constituted by clusters of activated retinal microglia that migrate close to the blood-retinal barrier probably in response to detrimental triggers.The impossibility of obtaining histological specimens of the human retina Inasmuch as a widespread microglial activation has been demonstrated by histological and neuroimaging (advanced MRI and PET techniques) studies in MS , this diThus, we investigated the possible correlation of HRF with a wide range of inflammatory soluble molecules  in the CSF of RRMS patients at clinical onset and untreated. The most significant and interesting finding was the inverse correlation between GCL HRF counts and G-CSF values. This cytokine showed a neuroprotective effect in a rat model of anterior ischemic optic neuropathy (rAION model)  and has Among several monocyte/microglia-derived cytokines/chemokines, CXCL-13, whose role in MS has been repeatedly ascribed , 34, seeTaken altogether, our data strongly support the hypothesis that HRF might be constituted, at least partially, by activated microglia. HRF should be interpreted in the context of the widespread microglial activation and proliferation that take place in MS CNS. The different concentrations of macrophage/microglia-derived cytokines/chemokines strongly indicate that these pro-inflammatory factors may also be produced in the retina, thus explaining the morphological changes observed here. Indeed, microglial activation in GCP and INL could induce a cytokine release that leads to BRB dysfunction and increases permeability determining an increase in the INL volume. This immunopathological hypothesis agrees with a recent observation on blood–brain barrier dysfunction associated with specific patterns of microglia-derived pro-inflammatory cytokines in the CSF  . The michigh had higher CSF CXCL-13 than NIND and I-HRFlow, while the CXCL-2 concentration was slightly increased in I-HRFlow and then dropped in I-HRFhigh. This behavior may reflect phenotypical changes of retinal microglia. Indeed, I- or G-HRFlow patients have CSF cytokine concentrations similar to those observed in NIND, suggesting a “physiological” low-level release of soluble mediators by microglia, in line with the homeostatic role of these cells. Furthermore, the association of increased HRF count with different cytokine profiles in I- or G-HRFhigh suggests a microglial shift from a homeostatic to an activated profile, characterized by proliferation and production of higher levels of pro-inflammatory mediators. However, since no histological or cytological evaluation is here provided and considering that these data derive from a limited cohort of MS patients, our findings need to be handled with caution and need to be further confirmed. Longitudinal studies will reveal whether the association of CSF cytokines with HRF counts is worthy of further experimental development and will help to better weight our preliminary findings. Moreover, to further explore the origin of HRF, studies evaluating the parallel behavior of retinal and brain microglia should be performed [as mentioned above, PET allowed the observation of a widespread microglial activation in the gray matter of a small cohort of MS patients . The patients/participants provided their written informed consent to participate in this study.MP: study concept and design, collection of clinical, OCT, and immunological data, writing and critical revision of the manuscript, statistical analysis of the data. TT: collection of OCT data, writing and critical revision of the manuscript. SF collection of clinical and immunological data, writing and critical revision of the manuscript. AM: collection of immunological data, writing and critical revision of the manuscript. FN: collection of clinical and immunological data. VM: collection of clinical and OCT data. MS: study concept and design, collection of clinical and OCT data. EP: study concept and design, writing and critical revision of the manuscript. EM: study concept and design, writing and critical revision of the manuscript. PG: study concept and design, writing and critical revision of the manuscript. All authors contributed to the article and approved the submitted version.This project was funded by “Progetto di Eccellenza 2020”, Department of Neuroscience, Università degli Studi di Padova, and “Roche per la Ricerca 2016”.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.


Daily exposure to bisphenols can affect reproductive functions due to their pseudo-estrogenic and/or anti-androgenic effects. Testicular lipids contain high levels of polyunsaturated fatty acids necessary for sperm maturity, motility, and spermatogenesis. Whether prenatal exposure to bisphenols alters testicular fatty acid metabolism in adult offspring is unknown. Pregnant Wistar rats were gavaged from gestational day 4 to 21 with BPA and BPS . Despite increased body and testis weight, the total testicular cholesterol, triglyceride, and plasma fatty acids were unaffected in the offspring. Lipogenesis was upregulated by increased SCD-1, SCD-2, and expression of lipid storage (ADRP) and trafficking protein (FABP4). The arachidonic acid, 20:4 n-6 (ARA) and docosapentaenoic acid, 22:5 n-6 (DPA) levels were decreased in the BPA-exposed testis, while BPS exposure had no effects. The expression of PPARα, PPARγ proteins, and CATSPER2 mRNA were decreased, which are important for energy dissipation and the motility of the sperm in the testis. The endogenous conversion of linoleic acid,18:2 n-6 (LA), to ARA was impaired by a reduced ARA/LA ratio and decreased FADS1 expression in BPA-exposed testis. Collectively, fetal BPA exposure affected endogenous long-chain fatty acid metabolism and steroidogenesis in the adult testis, which might dysregulate sperm maturation and quality. Global decline in fertility rate and increasing male infertility could originate from the fetus. Testicular dysgenesis syndrome is suspected due to adverse environmental exposure to fetal life gonadal programming . ExposurLipids are essential in sperm membrane formation since their compositional changes during testis maturity determine sperm motility and affect spermatogenesis . SpermatIn addition to the liver and other fatty acid-synthesizing tissues, the testis is the site for the active biosynthesis of polyunsaturated fatty acids (PUFAs). The activities of desaturases and elongases regulate it. The testicular fatty acids profile affects their cells and influences sperm maturity, capacitation, acrosome reactions, and sperm–oocyte fusion, contributing to male fertility . The metPUFAs influence male reproductive functions in several ways. They are the precursors of prostaglandin (PG) synthesis and modulate the expression of key enzymes involved in steroid and fat metabolism of the testis. Spermatozoa need plasma membrane PUFA enrichment to support membrane fluidity during fertilization. However, it also makes spermatozoa susceptible to attack by reactive oxygen species. BPA exposure promotes oxidative stress in the testis, thereby establishing a potential association with reduced fertility. Eicosanoid biosynthesis involves testicular development by supporting PG synthesis, which is needed for germ cell development and steroidogenesis. Arachidonic acid, 20:4 n-6 (ARA), is a precursor for the PG-2 series during testicular steroidogenesis in spermatogenesis . AlthougFatty acid composition is influenced by diet and endogenous desaturation activities in the testis. The LCPUFAs, such as docosapentaenoic acid  and docosahexaenoic acid  are essential for sperm quality. The local accumulation of these LCPUFAs in spermatids supports spermatogenesis . The fatSpermiogenesis is an energy-intensive process transforming round spermatids into elongated spermatozoa for fertile sperm production. Site-specific metabolic fuel utilization supports reproductive energy requirements during spermatogenesis. Peroxisome proliferator-activated receptor gamma (PPARγ) is a master regulatory switch for the energy metabolism of fats, predominantly expressed in the germ cells and Sertoli cells of the testis, involved in regulating the expression of lipid metabolic genes by forming PPARγ-RXRα heterodimer in Sertoli cells , and itsFatty-acid-binding proteins (FABPs) involve intracellular fatty acid trafficking and metabolism to support the synthesis and remodelling of membrane lipids in the testis . FABP9 iExcess adiposity affects the fatty acid composition of spermatozoa via regulating testis fatty acid metabolism . Fetal BOur recent data demonstrated that prenatal exposure to bisphenols (BPA and BPS) significantly increased body and testis weight and altered testosterone levels, androgen, estrogen receptor expression, hyperplasia, inflammation, and oxidative stress in the offsprings’ testis . We hypop < 0.05), while MUFAs  and PUFA (linoleic acid) increased (p < 0.05) in BPA-exposed (4.0 µg/kg) testis. While in the control testis, LA was converted efficiently to produce ARA and DPA and increased their levels by ~2.9 and ~3.2 folds, but their levels were decreased by ~5.8 and ~9.6 folds, respectively, in BPA-exposed testis , monounsaturated fatty acid (MUFA), PUFA, and LCPUFA levels across the experimental groups involved with various concentrations  of bisphenols  or delta-9-desaturase are involved in the biosynthesis of MUFA from SFAs by introducing a double bond. Therefore, the SCD indexes (SCD-16 and SCD-18) were estimated by the product/precursor ratios of the fatty acids. The increased SCD-16 and SCD-18 in the BPA-exposed testis  reflect p < 0.05). In contrast, the levels of oleic acid, C18:1 (OA), and linoleic acid, C18:2 (LA), were significantly increased (p < 0.05) by ~2.4 and ~4.9 folds, respectively, only in BPA-exposed offspring , adrenic acid (C22:4 n-6), and docosapentaenoic acid, C22:5 n-6 (DPA) were significantly decreased by ~3.4, ~2.0, and ~6.3 folds (ffspring . Howeverffspring . Collectp < 0.05) in BPA-exposed testis . In contrast, the expression of lipoprotein lipase (LPL) was decreased (p > 0.05) in the offspring’s testis (p < 0.05) in the rat testis exposed to BPA (p > 0.05) in BPA-exposed rat testis.To examine the mechanisms associated with lipogenesis, the mRNA expression of the genes that regulate lipogenesis and steroidogenesis was investigated in the adult testis. In utero, BPA exposure significantly upregulated hormone-sensitive lipase (LIPE) expression by ~1.35 fold (s testis . The expd to BPA . The expp < 0.05) in BPA-exposed testis (p < 0.05), but the expression of CATSPER1 had no significant effects compared to the control  in the testis of the BPA-exposed (4.0 µg/kg) rats compared to the control (p > 0.05) in the testes of these rats. However, the mRNA levels of FADS2, ELOVL2, and ELOVL5 remained unaltered due to BPA exposure. Since LCPUFA biosynthesis was severely affected in the testes, the expression of fatty acid desaturase 1 (FADS1 or delta-5 desaturase), fatty acid desaturase 2 (FADS2 or delta-6 desaturase), delta 4 desaturase sphingolipid 1 (DEGS1), the elongation of very long-chain fatty acids-like 2 (ELOVL2), the elongation of very long-chain fatty acids-like 5 (ELOVL5), stearoyl-coenzyme A desaturase 2 (SCD2), and stearoyl-coenzyme A desaturase 1 (SCD1) were screened quantitatively. The mRNA expression of fatty acid desaturase 1 (FADS1) was significantly downregulated . Green dots indicate the expression and localization of FADS1 protein in the testicular tissue obtained from the control and BPA-exposed rats A. The noFor the first time, this study reports that the TDI level (4.0 µg/kg bw/day) of prenatal BPA exposure affected lipogenesis, stearoyl-CoA desaturase (delta-9-desaturase) index, the levels of MUFAs and n-6 LCPUFAs, and fatty acid desaturase 1 (FADS1 or delta-5 desaturase) expression in adult offspring testis, which in turn may be responsible for the dysregulation of sperm maturity. The PUFA composition of the whole testis showed a reduced proportion of n-6 LCPUFAs in the BPA-exposed testis than the controls, where ARA (20:4 n-6) and DPA (22:5 n-6) levels were dramatically decreased. The lower content of these n-6 LCPUFAs can reduce membrane fluidity  in the BDecreased levels of n-6 LCPUFA may affect spermatogenesis by reducing prostaglandin (PG) production. The cyclooxygenase (COX) enzyme regulates gonadal development via PG in the testis . ARA, liWe observed that testicular MUFA levels  were significantly increased in BPA-exposed offspring. A decreased level of oleic acid is associated with normal sperm maturation in rats , indicatSimilar to BPA-exposed testis in this study, a higher level of palmitoleic acid was associated with increased fat synthesis in BPA-exposed rat adipose , and theEstrogen mimics of BPA might affect spermatogenesis in several ways, including a disruption of the blood–testis barrier , wideninThe testicular metabolism observed in BPA-exposed rats exhibits similarities to its obesogenic effects demonstrated by disrupting metabolic activity and making the body prone to overweight and obesity. Excess BMI regulates fatty acid metabolism in the testis . DespiteThe composition of n-6 LCPUFAs (ARA and DPA) in the testis was decreased in BPA-exposed rats . In contSeveral enzymes are involved in LCPUFA biosynthesis by desaturation and elongation activities, such as FADS1, FADS2, ELOVL2, ELOVL5, and others. In addition, SCD1, SCD2, FASN, and Acyl Co-A synthetase 6 are also involved in maintaining fatty acid homeostasis in the testis. Despite higher levels of LA, the precursor of n-6 LCPUFAs, the ARA and DPA levels decreased, indicating that the desaturation and elongation of fatty acids were inhibited by BPA exposure. The increased LA and decreased ARA in BPA-exposed testis indicate that both FADS1 and FADS2 are likely to be affected. Again, due to reduced ARA and DTA, DPA was synthesized much less in BPA-exposed rat testis. Although FADS2 is considered the rate-limiting step as it acts twice in the LCPUFA synthesis pathway, FADS2 mRNA expression did not change in BPA-exposed testis  in our sIn addition to the liver, brain, and adipose, the mammalian testes are also involved in PUFA metabolism. The testis and epididymis also perform specific spermatogenesis roles by modulating LCPUFA metabolism. Viable sperms have a high level of LCPUFAs in their membrane, which maintains sperm fluidity and moves around with a greater velocity. Adequate LCPUFAs are ensured by liver metabolism involving desaturase and elongase enzymes. FADS1 metabolites (such as ARA and their metabolites) are active in Sertoli cells, helping elongate spermatids. FADS1 is involved in the initial step of LCPUFA synthesis, i.e., ARA, by introducing double bonds to LA. Decreased expression of FADS1 in the ARA metabolic pathway of BPA-exposed testis may result in abnormal sperm maturation as the latter depends on this metabolite. The BPA exposure impairs the processes associated with spermatogenesis by modulating fatty acid metabolism in the testis, and the changes were closely similar to HFD-mediated effects .The immunolocalization of FADS1 underlines their active metabolism in the seminiferous tubule. FADS1 is regulated by the transcription factors controlling fatty acid metabolism and lipogenic gene regulation by producing PUFAs. FADS1 is typically detected in elongated spermatids and epididymal cells in animals . A decrePPARα and PPARβ are expressed in interstitial, germ, and Sertoli cells, while PPARγ expression is specific to Sertoli cells . TesticuThe involvement of testicular cells with the available LCPUFAs depends on the expression of the PPARs . The skePPAR signalling affects sperm energy capacitation and motility and thus attracts targets for male infertility. The mechanism involving BPA-induced changes in testicular fatty acid composition and PPAR signalling with sperm energy metabolism is not understood yet. The reduced expression of PPARγ and PPARα could be due to the lower availability of ARA and DPA in the testis. The reduced expression of FADS1 in BPA-exposed rat testis might be mediated by the decreased expression of PPARα in producing lower levels of LCPUFAs observed in our study.Endothelial lipase or hormone-sensitive lipase (LIPE) and lipoprotein lipase (LPL) are expressed in normal testis but perform distinct biological roles. Increased LIPE expression could be responsible for increased testosterone production  in BPA-eFatty-acid-binding protein 9 (FABP9) is required in sperm acrosomal vesicle assembly and its formation. Its absence is associated with increased sperm head abnormalities . In rodeFABP4 regulates lipid trafficking and is involved in lipogenesis. BPA-activated PPARγ disturbs lipid metabolism by modulating the expression of FABP4 . Mutant Testicular cells produce neutral lipids, store them in lipid droplets (LD) and maintain lipid homeostasis by regulating the expression of LD proteins. LDs control the regulation of lipid storage and oxidation in Sertoli cells. ADRP (PLIN2) is expressed during testis development in rodents and helps maintain scrotal temperature for effective spermatogenesis. Increased ADRP A could pThe study is limited to the fact that the PUFA composition in the testis was based on total fatty acids rather than the phospholipid composition. Again, VLC-PUFAs (C ≥ 22) could not be measured in testis due to the unavailability of the standards. Despite changes in the SCD or delta-9-desaturase index, its protein expression could not be measured. While the earlier report suggests that BPA delivery in pregnancy altered fatty acid composition at a concentration of 0.5 µg/kg bw/day ; howeverIn summary, the LA level is increased relatively higher in BPA-exposed testis than in the control rats. Still, their utilization was less in these testes, indicating reduced metabolism and increased lipogenesis as measured by increased SCD-1 and SCD-2, FABP4, and ADRP expression in the testis. Despite higher MUFAs in BPA-exposed testis, the endogenous conversion to ARA was reduced due to decreased desaturase activities in these testes. Decreased FADS1 and PPARγ expression and reduced ARA production in BPA-exposed testis suggest that FADS1 expression is regulated by the dysregulated expression of transcription factors, PPARγ, and the desaturation of fatty acids. Compared to the control testis, BPA-exposed rats exhibited a dispersed FADS1 signal around ST cells, reduced PPARγ expression and decreased n-6 LCPUFA and FADS1 expression levels. The desaturation/elongation rate of PUFAs is differed by species, sex, endocrine status, and diet but is not reported due to BPA exposure in utero. This first report suggests that, in utero, BPA exposure can affect FADS1 expression in the offspring of the mature testis. The Sertoli cells were possibly affected more than germ cells of the testis because Sertoli cells are more active in LCPUFA metabolism, richer in polyunsaturated fatty acids (PUFAs), and correlate a higher expression of Δ5- (FADS1) and Δ6-desaturase (FADS2). The reduced ARA could have produced fewer metabolites supporting steroidogenesis in BPA-exposed testis.Three-month-old females (n = 40) and males (n = 20) Wistar rats were obtained from the animal facility of the National Institute of Nutrition, India. The rats were housed in pairs per cage at a constantly regulated temperature (22 ± 2 °C) under light-controlled housing conditions (~12:12 h light/dark cycles). The rats were allowed to acclimate for a week and provided with a chow diet and water ad libitum throughout the study. The fertile estrous cycle was determined by using methylene blue staining of vaginal smears. The rats were kept for mating overnight (M:F = 2:1) during their fertile period. The presence of vaginal plugs confirmed mating. After mating, the males were removed, and the day was considered a gestational day (gD) 0. The pregnant female rats were randomly divided into seven groups. Bisphenols  were dissolved in extra virgin olive oil to obtain the desired concentrations of 0.4, 4.0, and 40.0 µg/kg body weight per day. The control group was only administered extra virgin olive oil. The pregnant rats were administered bisphenols via oral gavage from gD 4 to 21, as described previously [The blood was collected in EDTA-coated tubes from the adult rats (90 d) by puncturing the retro-orbital plexus using a sterile capillary tube. The plasma was separated and stored at −80° C for further analysis. The male adult rats were euthanized by carbon dioxide asphyxiation. The testis was removed and washed in sterile PBS to remove traces of blood. Testis was stored in Bouin’s solution and snap-frozen in liquid nitrogen.The plasma and testis from adult (90 days) rats were used to estimate the total fatty acid composition. As performed before, the lipids were isolated from the plasma and testis tissue (~100 mg) . The totUsing a blade homogenizer, the testis tissue (50–100 mg) was homogenized in 5% aqueous Nonidet P40 (NP-40). The homogenate was heated at 80 °C in a water bath to solubilize the lipids. Testicular triglycerides and cholesterol were assayed using an enzymatic kit .Total RNA isolation was carried out using TRIzol reagent . Briefly, the testis tissue (50 mg) was homogenized in TRIzol solution using zirconia beads  in a mini bead beater (Biospec). The addition of chloroform achieved phase separation. The upper aqueous phase was separated, and RNA was precipitated using chilled isopropanol. The RNA pellet was dissolved in sterile nuclease-free water. The total RNA was purified from genomic DNA by employing a DNase I kit . Subsequently, Nanodrop  was employed to assess the quality and quantity of the RNA. Next, the total RNA (1 µg) was converted to cDNA using a kit . Finally, qRT-PCR was performed using the Stratagene Mx3005p qPCR system  using TB green pre-mix Ex-Taq II  and KiCqStart SYBR green primers . An equal quantity of protein was loaded in each well and resolved onto 12% SDS-PAGE gel. After resolving, the proteins were transferred onto a nitrocellulose membrane  using a wet transfer system. The membrane was blocked with 5% skim milk powder in TBS to avoid non-specific antibody binding. The blots were incubated with primary antibody overnight using corresponding dilutions . The bloTestis tissues were collected from adult rats (90 d) and fixed in Bouins solution overnight. The fixed tissues were embedded in paraffin, and 4 µm sections were obtained with the help of a microtome. The paraffin sections were mounted onto pre-coated slides. The slides were heated at 60 °C for deparaffinization, followed by a couple of xylene washes. The sections were rehydrated using varying ethanol concentrations and washed with distilled water. Antigen retrieval was achieved by boiling the slides in a microwave oven for 5 min using citrate buffer (pH 6.0). The slides were washed with PBS and blocked with 3% horse serum in a moist, humidified chamber. Primary antibody was added and incubated at 4 °C overnight. The slides were washed with PBS twice and incubated with Alexa fluor conjugated secondary antibody  for 1 h, then mounted with fluoroshield in DAPI medium . Images were captured using a confocal microscope (DMi8) with SP8 infrared lasers (Leica microsystems).t-test to compare the two groups. The results were calculated from the data derived from repeated independent experiments mentioned in the text or figure legend. Statistical significance was considered when p < 0.05. All experimental data are expressed as mean ± standard error of the mean (SEM).The data, which are normally distributed, were analyzed by one-way ANOVA followed by post hoc Sidak’s or Tukey’s multiple comparison tests using Graph pad Prism v.8. The data were also calculated by paired In utero BPA exposure could disrupt the fetus’s gonadal programming via dysregulated fatty acid metabolism in the offspring testis. This supports the notion that disrupted testicular development in fetuses could arise due to adverse environmental exposure. Prenatal BPA exposure decreased n-6 LCPUFAs in the testis by modulating the endogenous metabolism of these fatty acids aided with concomitant reduction in the expression of FADS1, which may affect sperm maturity and quality. Thus, fetal BPA exposure reduces testicular long-chain fatty acid availability in adult testis, which may ultimately affect spermatogenesis and pose a risk for male infertility. The development disturbance of the gonads due to prenatal BPA exposure could be linked with fertility impairment due to aberrant changes in the LCPUFA composition of the offspring testis. Thus, lifestyle choices and ubiquitous chemical exposure may affect the reproductive health of adults due to adverse effects on fetal development.Male fertility is declining globally. In addition to depleted LCPUFAs in the diet, daily exposure to endocrine-disrupting substances could negatively affect spermatogenesis at metabolic and gonadal levels. Testicular dysgenesis syndrome causally originates from adverse environmental exposure, which may disrupt fetal gonadal development and programming due to BPA exposure. Furthermore, the interplay of bisphenol exposure and the endogenous lipid metabolism of the testis could dysregulate the functions of male reproductive systems. However, further studies are required to examine the endocrine-metabolic cross-talk to the early life exposure of this most abundant plasticizer on the male reproductive function in humans.


Soluble growth stimulator gene 2 protein (sST2) is associated with heart failure and myocardial infarction; however, the predictive value of plasma sST2 level for coronary slow flow/no-reflow (CSF/NRF) is unclear. This study aimed to explore the predictive value of plasma sST2 levels for CSF/NRF in patients with ST-elevation myocardial infarction (STEMI) who underwent emergency percutaneous coronary intervention (PCI).  A total of 242 STEMI patients who underwent emergency PCI at our hospital between November 2020 and July 2021 were enrolled in this study. According to the postprocedural procedure, these patients were divided into the CSF/NRF and control groups. Clinical data were collected from both groups and were used to explore the predictive value of serum sST2 levels for CSF/NRF. P < 0.05) were observed in age, diabetes mellitus, sST2 level, neutrophil-to-lymphocyte ratio (NLR), fasting blood sugar, preprocedural blood pressure, intraprocedural hypotension, N-terminal pro-B-type natriuretic peptide, MB isoenzyme of creatine kinase (CK-MB), and cardiac troponin I (cTNI). Multivariate analysis showed that the sST2 level, NLR, and intraoperative hypotension were independent risk factors for CSF/NRF. ROC curve analysis showed that the sensitivity and specificity of the sST2 level for predicting CSF/NRF were 68.0% and 75.5%, respectively, when the sST2 level was more than 64.6 ng/mL .  Of the total 242 patients, CSF/NRF was observed in 50 patients (20.7%). Statistically significant differences ( For STEMI patients, preprocedural sST2 levels significantly correlated with CSF/NRF occurring in PCI. sST2 level is a potential predictor for CSF/NRF occurrence. Acute ST-elevation myocardial infarction (STEMI) is a cardiovascular disease with high lethality, and a mortality rate as high as 4–21% has been reported . SolubleThe study was approved by the Institutional Review Board of the People's Hospital of Liaoning Province and complied with the Declaration of Helsinki. All patients signed informed consent before participation.STEMI was diagnosed according to the 2017 European Society of Cardiology guidelines [All patients were administered aspirin (300 mg), clopidogrel (300 mg), or ticagrelor 180 mg) before PCI, and unfractionated heparin was administered during PCI. The operators determined the periprocedural medication and procedural strategies. The procedures were performed through the radial or femoral artery. The coronary flow velocity was determined using the TIMI frame count method (30 frames/s). It was defined as the first frame when the contrast agent touched the two medial walls of the coronary artery and the diameter of the stained vessel was more than 70%, and it was defined as the last frame when the contrast agent reached the end of each branch of the coronary artery 0 mg befo were excIn all patients, 2 ml of arterial blood was collected after successful puncture of the radial or femoral artery, stored at 4–8°C, and was used to determine sST2 levels by fluorescent immunoassay and chromatography with a dry immunity analyzer .x ± s), and comparison between the two groups was made using the t-test. The measurement data with abnormal distribution were expressed as P50 (P25–P75), and interquartile range and comparison between the two groups were made using the nonparametric Mann–Whitney U test. The enumeration data were expressed as percentages, and number of cases and comparison between the two groups was made using χ2 test or Fisher's exact test. Ranked data were analyzed using the nonparametric rank-sum test. Multivariate logistic regression was used to identify the independent risk factors for CSF/NRF. A receiver operating characteristic (ROC) curve was drawn, and the area under the curve (AUC) was used to evaluate the predictive value of sST2 levels for CSF/NRF occurrence. Statistical significance was set at P < 0.05.Statistical analyses were performed using the SPSS 26.0 software. The measurement data with normal distribution were expressed as mean ± standard deviation (P < 0.05) were observed in age, diabetes mellitus, sST2 level, neutrophil-to-lymphocyte ratio (NLR), fasting blood sugar, N-terminal pro-B-type natriuretic peptide (NT-proBNP), MB isoenzyme of creatine kinase (CK-MB), and cardiac troponin I (cTNI). However, the two groups did not differ in sex, smoking, body mass index (BMI), preinfarction angina pectoris, Killip grade, lipoprotein α (Lpa), blood uric acid, creatinine clearance(CCr), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), and ejection fraction (EF) (P > 0.05)  were included in the CSF/NRF group and 192 (79.3%) in the control group. The general and laboratory data of the two groups are given in  > 0.05) .P < 0.05). However, the two groups did not differ in the position of myocardial infarction, infarcted arteries, left main coronary artery involvement, diffuse long coronary lesions, Gensini score, stent length, contrast agent dosage, or operation duration (P > 0.05)  .P < 0.05)  .P=0.009), and the sensitivity and specificity of NLR were 44.0% and 84.9%, respectively . sST2 and NLR can predict values for CSF/NRF, and the predictive value of sST2 was better than that of NLR  and hsCRP, increasing reactive oxygen species in the vascular endothelium. These oxygen radicals can lead to oxidative stress and vascular endothelial damage. The injured endothelium also promotes TNF-α and hsCRP release [Similar to a previous study , this st release , resulti release . NeutropThis study also indicated that intraprocedural hypotension was an independent risk factor for CSF/NRF occurrence in patients with STEMI. Hypotension is associated with ischemia-reperfusion injury. Appropriate perfusion pressure provides the basis for reperfusion. Intraprocedural hypotension changes microvascular blood rheology, and P-selectin and cytokines promote platelet thrombosis, leading to the occurrence of CSF/NRF .This study had some limitations. First, the sST2 levels were measured once. Second, some specific inflammatory factors such as CRP and TNFs were not considered in this study. Finally, it needs to be confirmed with a larger sample size and multicenter studies.For STEMI patients, preprocedural sST2 levels significantly correlated with CSF/NRF occurring in PCI. The sST2 level is a potential predictor for CSF/NRF occurrence.


Finally, the impact of the distribution of and total compliance on CSF pressures and velocities is evaluated. Without respiration effects, compliance of 0.17 ml/mmHg yielded pressure pulsations with an amplitude of 5 mmHg and an average value within the physiological range of 7–15 mmHg. Also, model flow rates were found to be in good agreement with reported values. However, when adding respiration effects, similar pressure amplitudes required an increase of compliance value to 0.51 ml/mmHg, which is within the range of 0.4–1.2 ml/mmHg measured in vivo. Moreover, altering the distribution of compliance over the four different outlets impacted the local flow, including the flow through the foramen magnum. The contribution of compliance to each outlet was directly proportional to the outflow at that outlet. Meanwhile, the value of total compliance impacted intracranial pressure. In conclusion, a computational model of the CSF has been developed that can simulate CSF pressures and velocities by incorporating boundary conditions based on physiological processes. By tuning these boundary conditions, we were able to obtain CSF pressures and flows within the physiological range.Cerebrospinal fluid (CSF) dynamics play an important role in maintaining a stable central nervous system environment and are influenced by different physiological processes. Multiple studies have investigated these processes but the impact of each of them on CSF flow is not well understood. A deeper insight into the CSF dynamics and the processes impacting them is crucial to better understand neurological disorders such as hydrocephalus, Chiari malformation, and intracranial hypertension. This study presents a 3D computational fluid dynamics (CFD) model which incorporates physiological processes as boundary conditions. CSF production and pulsatile arterial and venous volume changes are implemented as inlet boundary conditions. At the outlets, 2-element windkessel models are imposed to simulate CSF compliance and absorption. The total compliance is first tuned using a 0D model to obtain physiological pressure pulsations. Then, simulation results are compared with  Cerebrospinal fluid (CSF) flows freely around the brain and spinal cord in the central nervous system. This water-like fluid has multiple functions including mechanical protection of the neurological tissues and transport and secretion of brain metabolites and fluids . The dynin vivo measurements and assessment of physiological processes and their impact on the CSF pressures and velocities is challenging. Pressure measurements with a ventricular catheter allow real-time CSF pressure monitoring but require invasive implementation of measurement devices and provide no spatial pressure data .The CSF is not easily accessible for ure data . In contsurement  and accusurement , artifacsurement . AlthougCadaver and animal studies are typically performed to investigate CSF turnover, whereby absorption should be equal to production to preserve stable CSF pressures. CSF is primarily produced by the choroid plexus in the cerebral ventricles, whereas multiple sites of absorption have been identified to drain CSF both into the venous and lymphatic system . CSF folComputational fluid dynamics (CFD) models allow for a detailed evaluation of fluid pressures and velocities within complex fluid spaces under different (patho)physiological conditions. Because of the complexity of the CSF space, most CFD studies focused on a small part of the CSF geometry . This alWhile the aforementioned studies provided valuable insights and results, CFD studies only presented spatial pressure differences and thus provided no absolute pressures over time as recorded through intracranial pressure measurements. Also, although there is evidence for fluid exchange with the interstitium and lymphatic absorption , only vein vivo measurements of pressure and flow reported in the literature. We address some limitations of previous studies through the implementation of CSF production, secretion into the lymphatic and venous system, arterial and venous volume changes, and intracranial and spinal compliance.This work aims to build a robust computational model, which can realistically predict CSF pressures and velocities under different physiological conditions. Therefore, a three-dimensional CFD model was developed that allows the simulation of absolute pressures and velocities within the complex CSF space, which are validated against A CFD model of the CSF circulation was constructed from medical images and literature data following four steps: 1) Segmentation of the 3D geometry, 2) generation of computational mesh, 3) implementation of boundary conditions, and 4) set up of numerical solver. These are elucidated in detail in the next paragraphs. The CFD analysis was performed using the numerical software Fluent 2021 R2 .First, the three-dimensional geometry was segmented from clinical T2 MRI images of a patient with Chiari type 1 malformation  using MiA computational mesh was generated using ICEM 2021 R2 . The mesh is composed of tetrahedral and prismatic elements: tetrahedra occupy the largest part of the volume and three prism layers with a total thickness of 0.7 mm were created next to the walls to accurately resolve the laminar velocity profile. The tetrahedral mesh elements have a maximal seed size of 3 mm and a refinement factor of 10 was applied. The thickness of each prism layer is determined by an exponential growth law with height ratio 2 and the total thickness. The filet ratio, maximal prism angle, maximal height over base, and prism height limit factor were set to 1, 180, 0.8, and 1 respectively. A mesh sensitivity study was executed for a stationary flow case with a constant velocity inlet of 0.4 ml/min and zero pressure boundary condition at the interstitial outlet and outflow boundaries for the spinal, lymphatic, and arachnoid villi outlets with 20%, 30%, and 30% of outflow, respectively. Spinal SAS pressure, wall shear stress, maximal velocity, and average pressure were evaluated for 13 meshes ranging from 0.3 to 5 million cells. Based on this study, the mesh with 1.18 million cells was selected which is the coarsest mesh with a maximal deviation of 5% compared to the finest mesh.Boundary conditions are based on our current understanding of physiological processes impacting the CSF flow. An overview of the applied in -and outlet boundary conditions is depicted in 2 to account for constant CSF production of 0.4 ml/min (production) is presented as production in First, at the lateral ventricles  is derived from volumetric flow measurements at the level of the 3rd ventricle (Qv3)  . A valuearterial2 caused by these volume changes is estimated from cervical (Qc) and aqueduct (Qv3) CSF flow measurements and the relative contribution of the spinal compartment to the total CSF compliance . The amplitude of the corresponding volumetric flow Qarterial2 , an avevenous1) was considered 50% of the amplitude of the arterial pulsations based on cervical flow measurements by , spinal (sp), lymphatic (lym), and arachnoid villi (av) absorption pathway . To acco to zero .Resistance and compliance are accounted for by coupling a 2-element windkessel to each outlet of the CFD model. Therefore, the pressure at each outlet i at timestep n is set following the differential equation coupling pressure and flow .Qi,n=Pii,n and Pi,n-1 pressure in the current and previous step, respectively. This results in an expression of the pressure at outlet i in function of the outflow at timestep n and the pressure in the previous timestep n-1i is the compliance and Ri the resistance at outlet i. To couple this equation to the CFD solver for all outlets, the algorithm described in  and the CSF production rate (Qproduction). The average intracranial pressure is assumed 10 mmHg within the normal physiological range of 7–15 mmHg (R–15 mmHg .Rtot=ICi) are placed in parallel, contributing to total resistance asThe four outlet resistances , corresponding to capacitors placed in parallel.The total compliance in the model is the sum of the compliances of each outlet (Ci is the contribution of outlet i (in %) to total compliance. For the spinal outlet, this value is initially set to 66% based on in vivo reports with values ranging from 63% to 73% spinal compliance contribution for humans in lying position  at time step nThe discretized expression  is then n-1 the pressure in the previous timestep and timestep size ∆t set equal to 0.05 s. The compliance value Ctot that leads to intracranial pressure pulsations with an amplitude of 5 mmHg is then selected. This approach is applied for the calculation of compliance for cases A (Qvenous1 is zero) and B.With PFive different cases are set up to validate the CFD simulation outcomes against reported literature data and investigate the impact of respiration and compliance distribution on CFD simulation outcomes.case A, only production and cardiac pulsations are accounted for as inlet conditions, thus discarding the impact of respiration. Outlet boundary conditions are resistances for the arachnoid villi (av) and lymphatic (lym) outlet, and 2-element windkessel models for the interstitial (int) and spinal (sp) outlet with compliance distribution of 33 and 66% respectively. The total compliance is calculated following case B, respiratory effects are added to the inlet boundary conditions described in case A, while outlet boundary conditions do not change. This addition leads to a new value of total compliance, again calculated following case C, inlet boundary conditions described in case B are imposed, while outlet boundary conditions are 2-element windkessel models for all outlets with compliance distribution of 11%, 66%, 11%, and 11% for the interstitial (int), spinal (sp), lymphatic (lym), and arachnoid villi (av) outlet respectively. The total compliance is unchanged compared to case B. In case D, the spinal and cranial compliance are reversed compared to case B. Hence, outlet boundary conditions are resistances for the arachnoid villi (av) and lymphatic (lym) outlet, and 2-element windkessel models for interstitial (int) and spinal (sp) outlet with compliance distribution of 66% and 33% respectively. Total compliance is the same as in cases B and C. Finally, in case E, inlet and outlet boundary conditions are the same as in case B but the total compliance is doubled.In The CSF was modeled as an incompressible Newtonian fluid with properties the same as water (density 998.2 kg/m³ and dynamic viscosity of 0.001003 kg/m.s). The motion of the fluid was governed by the continuity and Navier-Stokes equations.nd order spatial and temporal discretization. An absolute convergence criterion of 1E-9 was imposed for all residuals . All simulations were run using a time step of 0.05 s. The central computing infrastructure of Ghent university (HPC) was used for the simulations using up to 96 processor cores. Simulation of 25 cardiac cycles or a total simulation time of 25 s took about 14 h to complete.With the fluid density ρ [kg/m³], the velocity vector u [m/s], the fluid viscosity μ [kg/m.s], and the pressure field p [Pa]. The flow is considered incompressible, the effects of gravity were neglected, and the flow was considered laminar because of low Reynolds numbers, i.e., maximal order of 100s . The transient simulations were run using a PISO scheme and a finite volume method with 2In In the first five cardiac cycles , the avein vivo and in silico in the order of 0.1 ml/s whereas pulsations through the foramen magnum are more than 10 times larger in the range of 2–5 ml/s.First, we consider the results corresponding with case A, thus without the inclusion of respiration. In Intracranial pressure, corresponding with average pressure at the interstitial outlet (int), is presented over 25 s . ContourCalculated pressures for cases A and B are visualized in The flow through the cerebral aqueduct  is identin vivo measurements (0.4–1.2 ml/mmHg)  , suggestin vivo obtained flow values. Here, fluid flow across a cross-section of the SAS and the cerebral aqueduct showed a good agreement with phase-contrast MRI measurements . This is because the simulations were only run for 25 cardiac cycles to save computational time. The 0D results, presented in In vivo pressure measurements in patients with idiopathic normal pressure hydrocephalus suggest a maximal pressure difference between the subdural and ventricles of 1 mmHg/m or 0.1 mmHg difference between the ventricles and foramen magnum (10 cm)  . Thus, bMultiple windkessel outlets enable modeling a heterogenous CSF compliance and absorption, which are thought to be impaired in different types of hydrocephalus . The incFirst, the simulation results are validated by comparison to literature data, which are recorded at a limited number of points or planes in the CSF space. Also, these are typically obtained through cardiac-gated acquisition only, meaning that the measurements are not real-time and respiration information is not quantified. In that way, they do not account for respiratory effects. Therefore, only simulation results without respiratory influences (case A) were compared to flow measurements. Pressure recordings presented are typically acquired in the context of traumatic brain injury  or hydroFurther, this model includes some important simplifications. First, the currently used 3D geometry is simplified to overcome the limited resolution of the original scan (slice thickness of 3 mm) and reduce complexity to limit computer time. Second, the model geometry was obtained from clinical MRI images of a patient with Chiari type 1 malformation. However, above mentioned simplifications of the CSF geometry resulted in a CSF layer at the level of the foramen magnum of minimally 4 mm, hereby removing the obstruction from the segmented geometry. As such, simulations are representative for a healthy subject, rather than for a patient with Chiari malformation. Third, the locations of the veins and arteries are limited to two locations and the morphology of the boundary surfaces is loosely based on literature. Fourth, the inlet boundary conditions are simplified to sinusoidal time signals with a constant value over the complete inlet surface. Last, CSF volume compensation mechanisms within and beyond the simulated 3D space are simplified by introducing windkessel models including compliance at the outlets. These models are distributed, yet still implemented at a discrete and limited number of sites. It is, without any doubt, more physiologically correct to use an FSI approach to account for compliance effects arising from the deformability of the tissues surrounding the simulated 3D fluid spaces, as demonstrated by among others . As suchDespite these simplifications and assumptions, this model is a proof of concept that by setting proper boundary conditions, thus inlet and windkessel boundary conditions in a 3D model of the CSF, one can obtain pressures and velocities within the physiological range. Now, the model can be stepwise optimized, including more detailed information on the neural anatomy and physiology. Toward the future, we aim to apply this framework to neurological disorders with known disruptions of CSF pressure and flow and to expand validation to extended datasets, including data on the cerebral arteries and veins.A computational model of the 3D CSF space has been developed. Through the implementation of physiological processes and adequate tuning of inlet- and outlet boundary conditions, the model yielded CSF pressures and velocities within the physiological range as indicated by invasive intracranial pressure monitoring and phase-contrast MRI. CSF absorption and compliance are modeled by the implementation of 2-element windkessel models at the outlets. We found that the distribution of compliance has an important impact on CSF flow direction, while total compliance impacts the amplitude of the pressure fluctuations.


Using survey data to calculate composite financial literacy (CFL), existed studies do not consider the geographical difference of the means of objectively-measured financial literacy and subjectively-perceived financial literacy, i.e., comparative benchmark.Taking the survey data of National Financial Capability Study (NFCS) for example, we explain why it is more reasonable to use the within-state average rather than the national average of financial literacy as the comparative benchmark to measure CFL. Then we use NFCS 2009, 2012, 2015 and 2018 dataset to comparatively analyze the difference between CFL measured with the two benchmarks.The results of statistical analysis show that there is a great difference among the four groups of CFL measured with the two benchmarks, and 10.7% of respondents are categorized as a particular group of CFL incorrectly for all datasets. Additionally, the findings of spatial distribution analysis unveils that 36, 19, 15, and 6 states have respondents miscategorized in the four groups of CFL for 2009, 2012, 2015, and 2018 respectively, in which the highest proportion of the population miscategorized in a state is up to 49.91%. Finally, we find that several groups of CFL measured with the two benchmarks have significantly different effects on stock market participation behavior.Using the national average as a benchmark to determine all the respondents’ relative financial literacy levels for different states is not meaningful, and will lose the practical appeal to tackle the regional inequalities of financial literacy among the households. Therefore, we suggest that the within-state average of financial literacy, not the national average, should be taken as the comparative benchmark for identifying the more precise groups of CFL in survey. The 2008–2009 global financial crises attach more importance to individual’s financial literacy (FL) and its impact on household wealth. However, the lack of FL may lead to poor financial decisions and then decrease household financial welfare. Many studies have proved that this is evident for many countries . While cTo study the OFL’s interaction with SFL could be beneficial because it can further help us to understand how FL comprehensively affects individuals’ financial behaviors. As an original research, The method provided by Therefore, it is more reasonable to replace national average with within-state average as comparative benchmark to measure CFL. Using the survey data of NFCS 2009, 2012, 2015 and 2018 to comparatively analyze the difference between CFL measured with SA method and NA method,The rest of this paper is organized as follows. Section 2 provides a detailed description of data source and study method. The results of empirical analysis and the discussions are presented in section 3, and section 4 provides the conclusions, limitations and future prospects of this study.This paper uses a representative survey data, i.e., NFCS 2009, 2012, 2015, and 2018.In all the four survey years, NFCS contains five basic questions to provide information about respondents’ OFL covering fundamental financial concepts encountered in daily life: compounding interest, inflation effect on the time value of money, the relationship between bond price and interest rate, interest payment difference on shorter and longer mortgages, and principle relating to diversification and risk. Although the five NFCS test items focus on basic financial knowledge in a simple question form, they have been found to be challenging for respondents and have been served as valuable indictors of OFL in many studies e.g., .Respondents can choose either they do not know the answer or refuse to answer, which can prevent respondents choosing at random. Specifically, the five financial questions in the OFL test include two options, ‘Do not know’ and ‘Prefer not to say.’ In line with previous research , a ‘1’ rThe NFCS questionnaire also contains an alternative measure of overall FL to provide information about how respondents self-assess their own level of FL. In the survey, the self-assessment FL question is answered before the five test items of OFL to ensure that the self-rating is not affected by the objective answers.Specifically, respondents in the NFCS survey are asked to assess their own level of financial knowledge on a 7-point Likert scale, whereby a ‘1’ reflects the lowest self-assessed level of financial knowledge and a ‘7’ reflects the highest level. Those choosing the answer ‘I do not know’ are excluded from our sample. This self-assessment question provides insights into how respondents perceive their own level of financial knowledge, and we label this as SFL. As shown in Similar to the previous studies, we divide respondents in each year into ‘OFL-high’ and ‘OFL-low’ groups using the individual OFL scores compared with OFL average scores (OFL-high: OFL > mean and OFL-low: OFL ≤ mean). The split of the sample in each year into “SFL-high” and “SFL-low” groups is based on the SFL scores and their averages in each year (SFL-high: SFL > mean and SFL-low: SFL ≤ mean). Like As shown in Based on SA method and NA method, we can obtain two types of CFL for each respondent. In addition, the higher proportion of the population above NAOFL than those above SAOFL in each year implies that if we use NAOFL as the comparative benchmark, the relative OFL level of respondents is overestimated, just as shown in Using the NASFL and NAOFL as cutoff points, Y, to represent whether the respondents in the sample are incorrectly categorized. Y equals 1 if the CFL of a respondent calculated by the two methods are different, and equals to 0 otherwise. Y = 1 represents that the respondent is categorized in a wrong group of CFL by NA method. Y, which shows that there are 10.70% of respondents categorized as a particular group of incorrect CFL in all the survey years. In particular, the largest proportion (23.03%) incorrectly categorized occurs in 2009, and the lowest proportion (4.06%) occurs in 2018.We argue that the CFL measured with SA method is more reasonable than NA method, and then create a binary variable, SAOFL, NAOFL) < OFL score < max min  < SFL score < max Taking the region II in Y = 1 in states for the 4 years. Y = 1, while the other states do not have any respondent miscategorized for 2009, 2012, 2015, and 2018, respectively. From 2009 to 2018, the lowest proportion of population miscategorized is 19.27% in Alaska in 2012, whereas the highest proportion is 49.91% in South Carolina in 2009. This finding means that once there are respondents miscategorized in a state, the proportion of population miscategorized is substantial. Therefore, if we use the national average as the comparative benchmark to measure CFL, the results will mislead our judge on the CFL of respondents in many states.We use ArcGIS software to illustrate the distribution of the proportion of respondents with We further provide a comparative analysis of the difference between the effects of the two types of CFL on financial behavior. Stock market participation is one of the essential financial behaviors of households,Φ is the standard normal distribution, β is coefficient vector to be estimated. The datasets of 4 years provide weights to match Census distributions for age by gender, ethnicity, education, and Census division, and then we use the survey commands available in Stata to compute clustered robust standard error. The definitions of variables other than CFL are described in Where For simplicity, This paper provides a new method to measure the CFL and proves its reasonability through the comparative analysis with the NA method. We find the significant difference between the results of CFL of the two methods, and explain the cause of the difference. Our findings imply that if we use NA method to measure the CFL, a particular group of respondents will be miscategorized. Especially, the difference of the effects between the two types of CFL on stock market participation behavior indicates that if we use the imprecise measure of CFL to study the relationship between CFL and financial behaviors, the conclusions could mislead the practitioners and policymakers. Using the national average as a benchmark to determine all the respondents’ relative FL levels for different states is not meaningful, and will lose the practical appeal to tackle the regional inequalities of FL among the households. Therefore, we suggest that the within-state average of OFL and SFL, not the national average, should be taken as the comparative benchmark for the measure of CFL.This study unveils that the comparative benchmark related to geographical factor really plays an important role in the measure of CFL. However, we only explore the different impacts of CFL measured with the two methods on stock market participation behavior. The other finical behaviors, such as retirement planning and investment decision, are also significantly affected by individuals’ CFL e.g., ,b. Therehttps://finrafoundation.org/knowledge-we-gain-share/nfcs/data-and-downloads.Publicly available datasets were analyzed in this study. This data can be found at: FL: acquization of original data, software, formal analysis, methodology, and manuscript revision. HL: funding acquisition, reference management, and manuscript revision critically. WL and QY: conceptualization, writing – review and editing, methodology, and manuscript revision. FL and HL: data calculation and writing. All authors contributed to the article and approved the submitted version.The authors would like to acknowledge funding from the National Social Science Fund of China (Grant No. 21XJY018), the Social Science Fund of Xinjiang (Grant No. 20BJY04), the Major Counselor and Investigation Project of Xinjiang University of Finance and Economics (Grant No. 2022XZZ001), and the Social Science Fund of Jiangsu University of Technology (Grant No. KYY20512).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. 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