Neonatal mortality from tetanus can be reduced by 94% when pregnant women receive at least two doses of tetanus toxoid. In Ethiopia, immunization programs are suboptimal despite their importance. Therefore, the aim of this study was to examine the geographic distribution and associated factors of poor tetanus toxoid (TT) immunization among pregnant women in Ethiopia.Secondary data analysis was used using the 2016 Ethiopian Demographic and Health Survey (EDHS). ArcGIS version 10.8 statistical software was used to explore the spatial distribution of poor TT immunization and SaTScan version 9.6 software was used to identify significant hotspot areas of poor TT immunization. For associated factors, a multilevel binary logistic regression model was fitted using STATA version 14 software. In the multivariable multilevel analysis, adjusted OR (AOR) with 95% CI was reported to reveal significantly associated factors of poor TT immunization.I = 0.59 at p-value of <0.0001. The highest poor TT immunization clusters were observed in the East and South Tigray, the central part of Amhara, West Afar, East Somali, and West Gambella. Pregnant women with no Antenatal care (ANC) visits , pregnant women with 1–3 ANC visits , media exposure , poor wealth index , middle wealth index , family planning use  and community level education  were significantly associated with poor tetanus toxoid immunization.In Ethiopia, the spatial distribution of poor tetanus toxoid immunization was clustered with Global Moran's Poor tetanus toxoid immunization among pregnant women varies in Ethiopia. It was highest in East and South Tigray, the central part of Amhara, West Afar, East Somali, and West Gambella. Therefore, public health programs should design targeted interventions in identified hot spots to improve tetanus toxoid immunization. Health programmers should be promoting optimal ANC visits, women's education, and family planning use. Maternal and Neonatal Tetanus (MNT) has been among the most common life-threatening consequences of unclean deliveries and umbilical cord care practices, and are indicators of inequity in access to immunization and other maternal, newborns, and child health services , 2. EverThe primary strategies for preventing MNT at birth were immunization of mothers with a protective dose of tetanus toxoid (TT) and clean delivery , 7. TetaDuring antenatal care appointments, pregnant women can receive at least two doses of the TT vaccine in different countries. But among expectant mothers, the coverage of TT2+ vaccinations ranged between 27% to 71% , 10. It Age of the mothers, educational status of the women, marital status, occupational status, joint health decision with husband, distance from health facilities, wealth index, fear of side effects, fear of sterility, ANC follow-up, parity, lack of information about TT vaccination, knowledge, attitude, and low awareness of mothers were significant factors leads to poor TT immunization countries , 13–15. Various studies have been conducted in Ethiopia to identify associated factors for TT immunization. The spatial distribution of TT immunization was unclear in regions of Ethiopia. Understanding the level and geographical variation of TT immunization in Ethiopia can help health planners, programmers, partners in the health sector, and policymakers formulate appropriate strategies and interventions and provide quality reproductive health services to increase TT immunization. Hence, this study aimed to assess the spatial distributions of TT immunization and associated factors in Ethiopia using Ethiopian Demographic and Health Survey (EDHS) datasets.In Ethiopia, there are nine regions  and two administrative cities (Addis Ababa and Dire Dawa) . Based owww.measuredhs.com. Using the 2007 Population and Housing Census (PHC) as a sampling frame, a cross-sectional study design was used with stratified cluster sampling. A stratification was carried out by separating Ethiopia's nine regional states and its two city administrations, then by dividing it into urban and rural areas , which was a nationwide representative sample that was carried out from January 18 to June 27, 2016. Once permission had been obtained by securing an online request after explaining the purpose of the study, the EDHS 2016 was accessed from the DHS official database, al areas .. Additionally, latitude and longitude coordinates were taken from selected EAs (clusters). Full details of the EDHS sampling system were presented in the report   proportional to EA size were selected proportionally to the EA size in the first stage. In the second stage, 28 homes from each cluster (EA) were chosen systematically with an equal chance . For thie report , 19.This study's outcome variable was poor TT immunization, which is defined as poor TT immunization if pregnant women did not receive enough immunization (did not receive two and above tetanus toxoid vaccine) and otherwise no. Depending on different literature reviews, individual and community-level variables were included in the analysis. Age of the women , women's education , occupation , modern family planning use , parity  and number of antenatal care (ANC) visits , person who decides on respondents health care  were considered as individual-level variables –27. MediOf the community level factors, distance to the health facilities , residence , and in accordance with a previous study conducted in Ethiopia and based on its geopolitical features, region was divided into three regions: small peripheral regions , large central regions , and metropolitan  were directly accessed from EDHS data set –31. HoweFor data analysis, we used STATA 14, ArcGIS 10.8, and SaTScan 9.6 software. For the analysis, sample weights were applied to adjust for the non-proportional sampling of strata and regions during the survey process and to restore representativeness. Text, figures, and tables were used to present descriptive statistics and summary statistics , 33.p < 0.05), indicating the presence of spatial autocorrelation and rejecting the null hypothesis (poor TT immunization is randomly distributed). Hotspot analysis was conducted using the Getis-Ord Gi* statistic . A Moran's I value close to −1 indicates that disease/events are dispersed, whereas a Moran's I value close to +1 indicates that they are clustered, and a Moran's I value of zero indicates that they are distributed randomly. There was a significant Moran's I  and Empirical Bayesian Kriging (EBK) were done since they statistically optimized the weight to predict the prevalence of poor immunization in the unobserved areas based on the observed measurement. The ordinary Kriging spatial interpolation method was selected for this study for predictions of poor immunization coverage since it had a smaller residual and Root Mean Square Error (RMSE) than EBK.p-value compared to areas outside the window.Spatial scan statistics applied using Kulldorff's SaTScan software identified statistically significant primary (most likely) and secondary clusters of poor TT immunization. In SaTScan™ works, a window moves across the study areas and the window size needs to be fixed. As the outcome variable was Bernoulli distribution, Kulldorff's method was applied to use a Bernoulli model for a purely spatial analysis. In order to fit the Bernoulli model, respondents with poor TT immunization were considered case, and those with high TT immunization were considered control. Using the default maximum spatial cluster size of 50% of the population as an upper limit, both small and large clusters were detected, and clusters with more than the maximum level were ignored. Poor TT immunization was considered in areas with a high Log Likelihood Ratio and significant Descriptive statistics were described using frequency and percentage. A variance inflation factor (VIF) was used to test for multicollinearity, and a VIF of less than five was obtained for each independent variable, with a mean VIF of 1.47, indicating there was no significant multicollinearity between independent variables. In the EDHS data, there was a hierarchical structure, which violates the independent observations and equal variance assumptions of a traditional logistic regression model. Therefore, women were nested within households, and households were nested within clusters. Within the cluster, they may have similar characteristics. Hence, multilevel binary logistic regression analysis must take into account the variability between clusters. Since the models were nested. Intra-class correlation coefficient (ICC) and Proportional Change in Variance (PCV) were computed to measure the variation between clusters. The ICC reveals that the variation of poor TT immunization between clusters is calculated as;. Moreover, the PCV reveals the variation in the prevalence of poor TT immunization among pregnant women explained by factors and calculated as; where; Vnull = variance of the initial model, and VA = area/cluster level variance . Model fp < 0.2 were considered for multivariable analysis. Finally, multilevel binary logistic regression analysis was done to assess the association between TT immunization and individual and community-level factors. Four models were constructed for multivariable multilevel analysis; the null model (without independent variables), mode I , mode II (containing only community-level factors), and model III  were fitted. In the multivariable model, variables with an adjusted odds ratio (aOR) with a 95% confidence interval (CI) and a p-value of <0.05 were considered significantly associated factors of poor TT immunization.First, bivariable multilevel logistic regression analysis was conducted and those variables with a A total weighted sample of 7,397 pregnant women were included in this analysis. The median age of the respondents was 28 years (IQR: 24–34) and 87.3% of the women were from rural residence. Of the study participants, 63.1% of the pregnant women had no formal education. More than half (53.9%) of the study participants were not employed and most of (65.6%) the study participants had no media exposure. The majority (90.8%) of the pregnant women were from large central regions. More than two fifths (43.7%) of the pregnant women were from households with poor wealth quantiles .The prevalence of poor TT immunization during pregnancy varies across the country. The highest and lowest prevalence of poor TT immunization during pregnancy were observed in the Afar (71.7%) and Dire Dawa (34.2%) regions, respectively .p < 0.0001)  . A highe 0.0001) .According to the Getis OrdGi statistics analysis, the majority of Tigray and Amhara regions had significant hotspots (areas with poor TT immunization). In addition, it is located in the west of Afar and northeast of the Somali region. Cold spots (areas with high TT immunization) were found in Addis Ababa, Dire Dawa, Hareri, the northern and central parts of Oromia, and the north-eastern part of SNNPR .In the Kriging interpolation; the predicted poor TT immunization coverage were identified in the Eastern and North-eastern part of Somali, northern part of Afar, central and south western part of Amhara, and Northern part of SNNPR regions whereas, the predicted high coverage of TT immunization were identified in the Northeastern part of Gambella, some part of eastern, western and central Oromia, Addis Ababa, Dire Dawa, and northwest Somali regions .p = 0.001. According to the study, pregnant women within the spatial window were 1.3 times more likely to have poor TT immunization than pregnant women outside it . As a result of the survey, the primary clusters were found in the Tigray, Amhara, North Eastern Somali region, Benishangul region, and Oromia region. They were located at 14.122895 N, 38.621010 E of geographic location, with a radius of 561.28 km, with a Relative Risk (RR) of 1.3 and Log-Likelihood ratio (LLR) of 77.36, with tside it   in the final model indicates that about 31.63% of the variation of poor TT immunization among pregnant women was attributable to both individual level and community-level factors .Number of ANC visits, media exposure, modern family planning use, wealth index, and community-level education were significantly associated with poor TT immunization in the final model (Model III).Accordingly, pregnant women who had no and 1–3 ANC visits had 10.46  and 1.51  times higher odds of poor TT immunization than those who had recommended ANC visits, respectively. Pregnant women who had no media exposure had 1.45  times more odds to have poor TT immunization than those who had media exposure. Women who did not use modern family planning had 1.28  fold higher odds of poor TT immunization as compared with their counterparts. Compared to study participants in the rich households, pregnant women from households classified as poor and moderate status had 1.22  and 1.23  fold higher odds of poor TT immunization. Moreover, pregnant women who lived in a community with low education had 1.43  times higher odds of poor TT immunization as compared with those who lived in a community with high education .According to the present study, the spatial distribution of poor TT immunization among pregnant women in Ethiopia was clustered. In most parts of Tigray and Amhara regions, there were significant hotspots of poor TT immunization. It is also found in western Afar and north-eastern Somali region. Whereas the significant “cold spot” areas with high rates of TT immunization were located in Addis Ababa, Dire Dawa, Harari, central and North-eastern parts of Oromia, and north-eastern part of the SNNPR regions. The possible justification for this difference could be due to the different socio-economic and obstetric-related factors of the study participants. For instance, the majority of the respondents in Addis Ababa, Dire Dawa, and Harari were educated, rich, and had the recommended ANC visits. In addition, most participants who lived in these regions were exposed to the media. Which in turn enhances the pregnant woman's understanding of TT immunization , 35.Poor TT immunization coverage was higher among mothers who did not attend ANC visits compared to those who attended the recommended ANC visits. This finding was supported by studies conducted in Ethiopia , Kenya , and PakSimilarly, poor TT immunization was more likely to occur among pregnant women from lower-income households. This is in line with studies conducted in Ethiopia  and BangSimilarly, in this study, community-level education was also significantly associated with poor TT immunization. When compared with their counterparts, people who live in a community with low levels of community education are more likely to have poor TT immunization. Scholars showed that maternal health service utilization is low in a community with low community-level education , 40.Poor TT immunization among those who had no media exposure was higher than for those who had media exposure. This is in line with a study conducted in Ethiopia . These fPregnant women who did not use modern family planning methods were more likely to have poor TT immunization as compared with those who used modern family planning. This finding was supported by studies in Ethiopia  and BangThe current study's key strength was the use of weighted data that were nationally representative.As a result, the results of the current study can be applied nationally. In addition, we also identify similar and statistically significant areas with a high cluster of TT immunization by using both ArcGIS and Sat Scan statistical tests. Due to the cross-sectional nature of the study, it does not show the cause-and-effect relationship between the outcome and independent variables. Because the current study relied on secondary data, some essential variables, such as respondents' knowledge of TT vaccines and sociocultural factors, were missing.Poor tetanus toxoid immunization among pregnant women varies in Ethiopia. It was highest in the East and South Tigray, the central part of Amhara, West Afar, East Somali, and West Gambella. Number of ANC visits, media exposure, wealth index, family planning use, and community-level education were significantly associated with poor TT immunization. Therefore, TT immunization should be improved in the identified hotspot areas by designing locally targeted public health interventions. Public health interventions like promoting the dissemination of information related to TT immunization in the media, women's education, and modern family planning use have the potential to improve women's awareness of TT vaccines, which in turn will improve TT immunization.


CSF clearance and paravertebral lymph nodes were quantified after injection and during the stationary phase. Stationary phase of the next day followed the initial perturbed state by injection of 6 ul  and CSF clearance half-time from the subarachnoid space was 93.4 ± 19.7 and 123.3 ± 15.6 min in adult and aged mice (p = 0.01), respectively. While the % injected dose of CSF space were higher, the activity of the paravertebral lymph nodes were lower in the aged mice on the next day. [64Cu]Cu-albumin PET enabled us to quantify CSF-lymphatic drainage across all levels of brain spinal cords and to visualize and quantify lymph node activity due to CSF drainage. [64Cu]Cu-albumin PET revealed the age-related decrease of the lymphatic drainage of CSF due to this decreased drainage from the subarachnoid space, especially during the stationary phase, in aged mice.Age-related cognitive decline is associated with dysfunctional lymphatic drainage of cerebrospinal fluid (CSF) through meningeal lymphatic vessels. In this study, intrathecal [ This pathway is also the route whereby leukocytes enter the CSF and consequently brain parenchyma and spinal cord via pia or ependyma lining the brain and spinal cord4. Mice and rats use their dual pathway of drainage via the nasal cavity and meningeal lymphatics for waste disposal7, whereas humans mainly use the dura lymphatics, not the nasal cavity11. ISF passes through glia limitans to the perivascular space, which communicates with the CSF space. Thus, any waste in ISF is drained to the dura meningeal lymphatics of the brain and spinal cord12. This function and dysfunction of glymphatic drainage13, has been investigated in rodents using fluorescent macromolecules14 and in humans using gadolinium contrast magnetic resonance imaging (MRI)8.Since the discovery of the meningeal lymphatics in 201517 or using L4/5 intervertebral intrathecal administration and MRI in humans11. Both approaches enable us to visualize the reflux of CSF to the perivascular space to evaluate glymphatic function and lymphatic drainage of CSF. In our preliminary study, we found that intra-cisterna magna administration of [64Cu]Cu-albumin was too invasive to unveil the subtle changes in the kinetics of lymphatic drainage of CSF, requiring optimizing the quantity and volume of tracer18. Unlike the initial reports that revealed glymphatic function using fluorescence microscopy in rodents19, recent reports have primarily used intrathecal MRI contrast agents, showing minute differences in the reflux from the submeningeal to the perivascular space22 and thus preventing the use of intrathecal contrast MRI to evaluate glymphatic function. In contrast, the lymphatic drainage of CSF could be studied using intrathecal contrast MRI and intrathecal cisternography positron emission tomography (PET)18, with high contrast and without background, thus demonstrating the superior performance in imaging the primary/secondary sentinel lymph nodes down the skull and along the vertebral columns in rodents23.Glymphatic/lymphatic imaging has been performed using contrast administration via cisterna magna and fluorescent microscopy or MRI in rodents24), which has incremental value further to the in situ clearance of the debris/waste by the glial cells. Dysfunctions beyond the physiologic range of variation associated with sleep, tend to be subtle and minute as observed in age-related changes in the lymphatic drainage of CSF31. Thus, the volume of intrathecal contrast must have been minimized to avoid disturbing the physiological stationarity, but large enough for CSF circulation to deliver the administered tracers along the CSF flow and finally to the outside of the CNS. CSF starts flowing from the ventricular choroid plexus, continuing through the aqueduct/spinal canal and cisterns, rounding and soaking perivascular spaces via the entire subarachnoid space. Then, CSF passes through the arachnoid barrier cell layer of the brain and spinal cord to reach the meningeal extracellular space, eventually entering the lymphatic channels of the cranial/vertebral dura via various pathways such as perineural and dural gaps32. Several radionuclide/radiopharmaceuticals, which have been clinically used for radioisotope cisternography for decades, could be used for imaging the lymphatic drainage of CSF. In our preliminary study, we chose [64Cu]Cu-albumin and established [64Cu]Cu-albumin PET protocols for imaging lymphatic drainage of CSF in mice18. This approach also visualized primary/secondary sentinel lymph nodes down the skull along the vertebral column in mice by [64Cu]Cu-albumin PET18.The contribution of the lymphatic drainage of CSF is now known to be mandatory for central nervous system (CNS) health because waste disposal is crucial for maintaining the brain/spinal cord function. This lymphatic drainage is particularly important during slow wave sleep and anesthesia  of [64Cu]Cu-albumin within 9 min. After resting for a day, the mice were imaged again for assessing the stationary CSF-lymphatic drainage and lymph node activity under continuous influx/drainage of its own.In this study, we corroborated the findings of our previous study, using the aforementioned protocol in a simple mouse aging model and assessed the variations in the lymphatic drainage of CSF with aging using intrathecal Cu-albumin PET protocol for evaluating lymphatic drainage of CSF in adult mice. Animal experiment was approved by Institutional Animal Care and Use Committee (IACUC) of Seoul National University (SNU-200513-8-6) and all methods were carried out in accordance with relevant IACUC guidelines and regulations of Seoul National University and ARRIVE guidelines (https://arriveguidelines.org). Relevant details are in Supplemental Notes.Male C57BL/6 mice at 2–9 months of age were used as the adult, whereas those at 15 to 25 months of age were used as the aged miceBehavioural test was performed in a Y-shaped maze under dim lighting. For adaptation of this test, mice were introduced into the center of the maze and allowed to visit all three arms the day before testing. For Y-maze test, a mouse was introduced into the center of the maze and allowed to freely explore the maze for 8 min. To calculate the alternation, visits of each arm were recorded when the limb and tail were within an arm. The alternation percentage was calculated by dividing the number of sequential alternations of the three arms by the total number of alternations possible.64Cu]Cu  using a click chemistry-based method34. Briefly, (1) 64Cu in HCl solution was dried with a nitrogen flow of 5–10%, (2) 1 M sodium acetate buffer was added to adjust the pH to 5.3, (3) p-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA)-N3-1300 nmol/mL, 10 μL was added, and the mixture was placed in a heat block at 60–70 °C for 10 min, (4) labeled NOTA-N3 of [64Cu]Cu NOTA-N3 was added to 1 mg/mL of azadibenzocyclooctyne (ADIBO)-albumin  and placed in an orbital shaker for 10 min, (5) the mixture was filtered and then centrifuged at 15,000 rpm for 5 min. The labeling efficiency of [64Cu]Cu-albumin was 99%, as confirmed by instant thin layer chromatography (ITLC), with 0.1 M citric acid solution as a developing solvent.Human serum albumin, with a molecular weight of 66.5 kDa, was radiolabeled with Cu-albumin injection was performed using a syringe pump (Harvard Apparatus) at an infusion rate of 700 nl/min, injecting a total volume of 6 μL with an amount of radioactivity ranging from 0.74 to 1.85 MBq. The needle was kept in place until the first imaging session was over. Then, the needle was removed, and the wound was closed.Intrathecal administration followed the procedure used for preclinical or clinical radioisotope cisternographyGenisys PET box (Sofie Biosciences) was used for whole-body PET acquisition of static PET in list mode for 6 min at each of the image acquisition time points, which were 9 min, 2, 4, 6, 12, and 24 h post-injection for adult mice, and 9 min, 2, 4, 6, 17, and 24 h post-injection for aged mice. Injection and image acquisition were performed under isoflurane anesthesia, with 3% for induction, and 2.8% for maintenance with 500 ml/min oxygen inhalation.0) were used as a surrogate of the injected dose for normalization of the counts of all other ROIs at any given time point (hn), which were expressed as %ID. The %ID of each time point (hn/h0) was plotted against the corresponding time points to generate the time-%ID curves. Using the one-compartment exponential model of GraphPad Prism, the half-life (t1/2) was calculated for each mouse. The coefficient of determination R2 > 0.95 showed a good fit. Unpaired t-test with Welch’s correction was used to compare half-lives between groups.Using the MIM software, 3D regions of interest (ROI) were drawn, and counts were measured for the entire subarachnoid space. Subarachnoid space  in the spatial memory among aged mice in comparison with adult mice , the animation image of a mouse , revealing that [64Cu]Cu-albumin was in intact form in the subarachnoid space at 1 and 4 h after intrathecal injection. Intact [64Cu]Cu-albumin of blood and urine samples peaked at the origin, and the corresponding degraded fragments moved to the solvent-front at 1 and 4 h, with the amount increasing over time lapse  and the central nervous system  regionally4. However, it remains to be proven whether the flow from CSF to meningeal lymphatics is segmental, that is, whether vertebral segmental routes are taken separately by lymphatic channels in vertebral segmental divisions. According to two recent reports, two possibilities are there. In one suggestion, brain lymphatics flow from the nasal cavity to the deep cervical lymph nodes (as a secondary stop) via superficial cervical lymph nodes and brain meningeal lymphatics directly flow to deep cervical lymph nodes (as primary stop), whereby vertebral lymphatics exit the vertebral segmental subarachnoid space, with the sacral lymph node as a primary stop and the iliac lymph node as secondary stop27. In another suggestion, as every vertebra has its epidural lymphatics to the smaller paravertebral lymph nodes, as was indicated by a mouse whole-body vDISCO study with transparent mounting/imaging30. At least a long column of many vertebral segments with epidural lymphatics could have connection with the dura outlets and make primary lymph nodes in their vicinity. This distributive drainage paths might also take the role of gates for adaptive immune surveillance against brain/spinal cord-derived waste and debris38. A recent report surprisingly showed that meningeal lymphatics were the route for senescent astrocytes to be deployed to the systemic immune system39.Brain waste materials contribute to brain aging both physiologically or pathologically . Obviously, they are first cleared from the brain parenchyma into CSF within cerebral ventricles and subarachnoid space40, which could act as a good connection path between these two central systems, neuro and immune, of brain/spinal cord parenchyma and bone marrow of skull/vertebrae. Myeloid/lymphoid cells are known to commute between bone marrows of the skull and vertebrae and brain parenchyma via CSF4. Lymph nodes are the intermediary station of the information contained in the macromolecules or cells from CSF in every level of the brain and spinal cord segments. Kwon et al.16 and Ma et al.27 explicitly reported the sacral drainage path in animals, and Jakob et al.38 visualized the epidural lymphatic channels of vertebrae, which drain CSF solutes to regional lymph nodes39. Thus, the paravertebral epidural lymphatic channels to the nearby lymph nodes are now added for every vertebral segment32 in addition to the nasal routes and meningeal lymphatics to the superficial and deep cervical lymph nodes. In our study of [64Cu]Cu-albumin using PET, segmental CSF-lymphatic drainage was assessed in a snapshot on serial visual reading of the MIP images  was used as a surrogate of extracellular (CSF) tau (ca. 60 kDa) or amyloid beta (100–150 kDa), the sustained radioactivity observed, even at 24 h, in the image of the lymph nodes suggests that pathologic proteins flown out from the CSF to the CNS-sentinel lymph nodes would be exposed to systemic immune surveillance, i.e., antigen-processing/presenting myeloid cells and adaptive immune cells in the lymph nodes.PET using Cu-albumin, and volume and injection rate for intrathecal administration.Various diseases have been associated with dysfunctional lymphatic drainage of CSF in the literature14 used ex vivo fluorescent microscopy imaging to visualize/quantify paravascular flux/reflux of intra-cisterna magna injected with fluorescent dyes. The researchers found the size dependence of lymphatic drainage of CSF15, as well as influx through perivascular spaces from the subarachnoid space19, which reportedly differ between young, middle age, and old mice. As expected, young mice had deeper perivascular penetration of intra-cisterna magna-injected materials such as sizable fluorescent dye-labelled dextran (3 kDa) or ovalbumin (45 kDa)19. These findings were reproduced in a multicenter study authored by Mestre et al.53 but challenged by Smith et al. for their mechanistic interpretation of interstitial fluid space drainage54. Notwithstanding these competing ideas of convection/advection through a glymphatic mechanism or diffusion across any interstitial fluid space, the perivascular space between basement membranes of glia limitans and capillary endothelium is crucial under physiological conditions for flowing waste/debris from the brain/spinal cord parenchymal interstitial fluid space to the CSF. Studies with intrathecal gadolinium contrasts, mostly gadobutrol (450 Da), conducted in mice or rats, have even highlighted subtle differences in influx to the perivascular space, showing effects on this glymphatic flow in anesthesia or sleep58. However, unlike initial fluorescent microscopic studies, the perivascular reflux found in these MRI experiments was not so profound, and only shallow infiltration was observed, especially in rats58.For the same purpose of understanding the lymphatic drainage of CSF, pioneering studies99mTc -DTPA , in a trace amount, was chosen to be the optimal candidate for most physiological examinations. In our study, we were not able to observe the brain parenchymal perivascular influx on [64Cu]Cu-albumin PET, so we gave up on the glymphatic evaluation, especially on the age-related decline of glymphatic or interstitial lymphatic drainage of CSF, which had been studied previously by reversing the physiologic flux upon administration intra-cisterna magna with a larger volume (10 to 15 μL) at a tolerable, but high rate (1 to 5 μL/min)  versus intrathecal injection and the larger fractional amount of contrast (relatively in mice) versus the smaller fractional amount of contrast (in rats) and larger quantity of fluorescent contrast in ex vivo/ in vivo fluorescent microscopy versus the relatively smaller quantity of contrast (gadobutrol) in MRI. Among these issues, we hypothesized that the volume was the primary confounder. Because we did not observe any activity in the lymph nodes upon 27 that sacral spine CSF flows out to the sacral lymph nodes as a first stop and then to the iliac lymph nodes which is collecting hub, on our study it seemed that the sacral lymph nodes drained the CSF from sacral epidural lymphatics and the iliac lymph nodes drained the CSF from lumbar epidural lymphatics. We suspect that the iliac lymph nodes work as the collecting site of the vertebral segmental lymphatic drainage from the adjacent spines and also the efferent lymph flows from the sacral lymph nodes, as a local leakage of albumin into the lumbosacral tissues. If recapitulated with the following studies on list-mode PET studies on the earlier time points (immediate to 1 h post-injection), this finding will propose the hierarchical structure of pre/paravertebral lymph nodes covering the entire length of cranium and vertebrae with the possible bidirectional communication between the systemic immune system and central nervous system. Hosang et al. recently reported that infectious inflammation of the lungs affected the states of microglia of the thoracic spinal cord59. Likewise, the compartmentalized composition between immune structures (lymph nodes) and spinal cord segments and the brain may be the general platform of the periphery to CNS interaction, especially gut with microbiome and brain/spinal cord61. [64Cu]Cu-albumin PET will help to scrutinize, as noninvasively as possible, the kinetics of the lymphatic drainage of CSF until 24 h after intrathecal administration, without further manipulation or with repeated imaging as this method revealed that aging-related decline was there in normal aging mice without much variation between individual mice per lymph nodes activity over %ID of CSF space activity at 24 h imaging  and sagittal sinus vein of cranial dura67. At least in mouse, we assume that CSF fluid and/or macromolecules pass through arachnoid barrier cell layer gaps to reach dura lymphatic vessels. As pigs were already shown to have possible pass through arachnoid granulation-like gaps, monkey/primate/human studies remain. Age-related decline of meningeal lymphatic vessels would also be accompanied by the well-known pathology of systemic lymphatic vessels such as (1) decreased lymphatic vessel contractility, (2) basal hyperpermeability and thus reduced inflammatory responsiveness and (3) delayed immune response68. Meningeal lymphatic vessels and their mechanistic contribution to age-decline of CSF-lymphatic drainage should be elucidated further.Limitation of our current PET study is that it did not show direct evidence that CSF cross arachnoid barrier cells layer to reach dura lymphatic vessels. Instead, it did show intrathecally laden exogenous albumin reach paravertebral lymph nodes. Ma et al.64Cu]Cu-albumin PET is recommended when seeking to understand the physiological and pathological mechanisms of the lymphatic drainage of CSF as the producer of major candidate parameter of a disease course and/or intervention outcomes. The clearance half-life and the surrogate of CSF retention, %IDs at 4, 6 or 24-h post injection are proposed as a marker of CSF drainage abnormality in line with evidence of such an age-related phenotypic change suggesting a dysfunctional clearance of brain waste25. Lymph node activities on [64Cu]Cu-albumin PET from earlier and late stationary phase is another interesting finding ensuring that [64Cu]Cu-albumin PET is showing lymphatic drainage at every level of skull and vertebrae. These biomarkers and the corresponding protocols can be applied to assess the response to novel therapeutic strategies known to enhance the lymphatic drainage of CSF69.In the future, cause and effect relationships and their therapeutic significance should be tested by repeatedly imaging small animals under physiological conditions using sophisticated PET/ computed tomography (CT) or PET/MRI. These methods use small quantities of radiopharmaceutical, avoiding any concern about toxicity or perturbation of the stationary state of animals. Animal models of traumatic brain injury, intracerebral/subarachnoid hemorrhage or even aging/sleep/circadian cycles should be accompanied at various levels, degrees of a severity or time points to model disease course in individual animals. Neuroinflammation or dementia models should also be observed repeatedly during their course of disease even at 3/ 7/ 12/ 18 months of age. For these studies at multiple time points, [Aged mice show a significantly higher clearance half-life and tracer retention within the subarachnoid space than adult mice. The imaging parameters of the PET protocol used in this study can be applied to evaluate lymphatic dysfunction and the response after therapeutic enhancement of lymphatic function.Supplementary Video 1.Supplementary Video 2.Supplementary Information.


Child sexual abuse (CSA) has become a focal point for lawmakers, law enforcement, and mental health professionals. With high prevalence rates around the world and far-reaching, often chronic, individual, and societal implications, CSA and its leading risk factor, pedophilia, have been well investigated. This has led to a wide range of clinical tools and actuarial instruments for diagnosis and risk assessment regarding CSA. However, the neurobiological underpinnings of pedosexual behavior, specifically regarding hands-on pedophilic offenders (PO), remain elusive. Such biomarkers for PO individuals could potentially improve the early detection of high-risk PO individuals and enhance efforts to prevent future CSA.To use machine learning and MRI data to identify PO individuals.Post hoc, we investigated the PO model decision scores with respect to sociodemographic  and forensic characteristics  in the PO subpopulation. We assessed model specificity in an external cohort of 53 HC individuals.From a single-center male cohort of 14 PO individuals and 15 matched healthy control (HC) individuals, we acquired diffusion tensor imaging data  in literature-based regions of interest . We trained a linear support vector machine to discriminate between PO and HC individuals using these WM microstructure data. P5000 = 0.018) and an out-of-sample specificity to correctly identify HC individuals of 94.3%. The predictive brain pattern contained bilateral fractional anisotropy in the anterior cingulate cortex, diffusivity in the left amygdala, and structural prefrontal cortex-amygdala connectivity in both hemispheres. This brain pattern was associated with the number of previous child victims, the current stance on sexuality, and the professionally assessed risk of future sexual violent reoffending.The classifier discriminated PO from HC individuals with a balanced accuracy of 75.5% (sensitivity = 64.3%, specificity = 86.7%, Aberrant white matter microstructure in the prefronto-temporo-limbic circuit could be a potential neurobiological correlate for PO individuals at high-risk of reoffending with CSA. Although preliminary and exploratory at this point, our findings highlight the general potential of MRI-based biomarkers and particularly WM microstructure patterns for future CSA risk assessment and preventive efforts. Child sexual abuse (CSA) is defined as any completed or attempted sexual act or contact with a child, as well as any form of sexual exploitation of a child . CSA is To effectively allocate preventive resources, it is crucial to identify and stratify individuals who belong to pedophilic offending risk groups. Pedophilic offending includes hands-on crimes, that is, actual sexual contact with a child, as well as hands-off crimes, namely the use of media depicting CSA –9. In thFor that reason, we decided to explore brain structural correlates of PO individuals using advanced machine learning techniques, which have proven to be highly valuable in tackling the biological elusiveness of complex disorders and phenotypes . We presμ = 23.77, SD = 3.02), which were scanned in the same MRI unit as part of a different study cohort  . A speci3. The sum of all five ROIs  amounts to 8.912 cm3. The CC ROIs were located midsagittally and had a left-right extension of 11 mm (from −5 to +5 mm).MRI data from the main study sample and the external HC sample were acquired on a General Electric Signa LX 1.5-T scanner located at the University of Magdeburg, Germany. Data for diffusion tensor calculations were collected with 12 non-collinear gradient orientations, each additionally measured with the opposite diffusion gradient polarity . The ori1 The regions were co-registered with the diffusion data using the FSL tools flirt and fnirt . Due to this approach, the amygdala region used for fiber tracking slightly differed from the one used for acquisition of the DTI anisotropy and diffusivity parameters. This approach also led to individual, non-spherical amygdala and PFC regions, for which MNI coordinate description is not suggested. A total of 68 anisotropy/diffusivity features  and 4 SC features  entered the analysis , defined as the mean of sensitivity and specificity. Furthermore, we used the sensitivity and specificity of the model to calculate the number needed to diagnose (NND), which is the inverse of Youden’s index given by [1/(sensitivity + specificity – 1)]. The NND displays how many individuals have to be examined in order to correctly detect one individual with the phenotype of interest . HoweverIn our case, a binary classification (−1 vs. 1) was carried out, whereas PO individuals were defined as positive label , i.e., the phenotype of interest, while HC individuals were defined as negative label . SVM models place weights on all input features, ranging from −1 to 1. SVM models then predict the labels by multiplying an individual’s feature values  with the model feature weights. In our case this leads to 72 multiplication products which are then aggregated into a final decision score. If the decision score is positive, then the positive label (in this case: PO) is predicted, while a negative decision score leads to the prediction of the negative label (in this case: HC). Before entering the analysis pipeline, all input features are rescaled from 0 to 1, so that beyond zero only positive features enter the analysis. Therefore, multiplication of a positive feature weight with the corresponding feature value of an individual always yields a positive multiplication product, while a negative feature weight always yields a negative multiplication product. Consequently, a positive feature weight indicates that the corresponding feature increases the decision score and therefore pushes the model toward prediction of the positive label, while a negative feature weight decreases the decision score and pushes the model toward prediction of the negative label. The absolute value of the feature weight further indicates how strongly a certain feature contributes to either a positive or a negative decision score. Finally, the distance between the decision score and the decision boundary  reflects the prediction certainty of the model. Higher positive decision scores or lower negative decision scores reflect that the model is more certain of either the positive or negative label prediction. The closer the decision score gets to zero, the more uncertain a model is of its prediction. If a feature weight is zero, the corresponding feature does not influence the decision score and therefore also does not impact the prediction, regardless of the input feature value of the individual.To obtain a stable visualization of such an SVM model and its feature weights, we used measures of pattern element stability  and pattern element significance (sign-based consistency) . The CVRP-value can be computed for each feature, determining whether the rate at which that feature received either a positive or negative sign  exceeded chance level. Therefore, this method quantifies how consistently a feature contributed to the prediction of a certain target and whether that rate is significant beyond chance level.Complementary to the CVR measure, we implemented a sign-based consistency method, which is based on an approach proposed by Gómez-Verdejo et al.  toward wPost-hoc investigation of the PO model was performed using all available sociodemographic data  and forensic information  and years of education . Compared to a German norm sample of 230 PO individuals  and its subdomains “fantasy” , “searching/sneaking around and persuasion tactics”  and “sexual assault/attack” . Furthermore, elevated levels were found in the “paraphilia” domain , which were driven by significantly higher scores in the “voyeurism” sub-domain . PCL-R scores of PO individuals  were below the 95% confidence interval of non-standardized samples of incarcerated violent offenders  were below the 95% confidence interval of non-standardized samples of incarcerated violent and sexual offenders . When applied to the external HC sample, the PO model correctly identified 50 of 53 as HC individuals (specificity = 94.3%).The classifier correctly identified 9 of 14 PO individuals (sensitivity = 64.3%) and 13 of 15 HC individuals (specificity = 86.7%), with a cross-validated BAC of 75.5%, a positive predictive value (PPV) of 82.0% and a negative predictive value (NPV) of 72.0% . The PO When given assumed prevalence rates of PO individuals between 1 and 75%, the model yielded robust PPV, NPV, PSI, and NNP values . SpecifiUsing a wrapper-based feature selection, 22 of 72 input features were included in the final PO model . The CV Sign-based consistency analysis determined four features to be the most reliable predictors: FA pgACC left/right and SC amygdala to PFC left/right .F = 26.1, P = 5.75E-05, df = 9). The prediction was based only on three features: The final SVR-20 assessment score , the number of previous child victims  and the MSI subscale “sexual knowledge and beliefs” . Furthermore, this regression model explained 88.5% of the variance in the decision scores (adjusted R2= 0.885).Given the complete set of sociodemographic and forensic information on PO individuals, the stepwise linear regression analysis successfully predicted the brain-based PO model decision scores . We combined the efficacy of linear SVM at low sample sizes with a literature-based region of interest approach, focusing on key brain areas in the prefronto-temporo-limbic circuit that have previously been implicated in pedophilia, sexual behavior, and pedophilic offending , 20, 21.The DTI parameters used in this study are sensitive to certain aspects of white matter (WM) microarchitecture . SpecifiThe development of WM microstructure follows a distinct trajectory throughout the life of an individual and is strongly linked to cognitive abilities, social behavior, language skills, and motor function –66. InitThis development of WM microstructure is a multifactorial process . The genThis is supported by a large body of evidence reporting particularly higher loads of hormonal and environmental risk factors in pedophilic men, but even more so in PO individuals , 73–75. Detecting this signature, particularly in the microstructure of WM, is of great forensic relevance due to the strong involvement of WM maturation and development in the context of higher-order cognitive abilities and behaviors, which could all play a role in the enactment of pedophilic impulses and the commission of pedosexual crimes –66. WM mHowever, the regions of interest chosen in this study are also highly involved in the generation and control of sexual arousal . While tIn summary, our findings, specifically the increase in FA in the pgACC and the increased structural amygdala to PFC connectivity, support a pathophysiological model of disinhibited aberrant sexual impulses and deficient higher-order cognitive control in PO individuals. In this model, dysfunctional ACC and amygdala signaling could lead to pedophilic arousal and the generation of pedosexual behavioral impulses, which are not sufficiently controlled, as the amygdala overrides prefrontal control in a bottom-up manner. However, our current knowledge on WM suggests that this aberrant pattern of WM microstructure could potentially be influenced by learning and/or intense activity even in adulthood . Therefopost hoc investigation pointed to the possible clinical and forensic utility of such a brain-based PO biomarker. Using stepwise linear regression, the PO classifier scores were predicted by the number of previous child victims, the MSI subscale “sexual knowledge and beliefs,” as well as the final assessment score in the SVR-20, with the latter reflecting the assessment by a mental health professional of the risk of future sexual violent reoffences. We therefore assume that the model indeed captured a WM microstructure pattern which is potentially modifiable and appears to be closely related to past child sexual offending, current stance on sexuality, and risk of future sexual violent reoffending.The Since this model was developed in an evenly distributed sample of PO and HC individuals, we assessed the added value of using such predictive tools in possible real-world scenarios, where the rate of PO individuals is highly dependent on the selection of individuals, i.e., the context in which such a tool is used . While tThe most important limitation of this study is the small sample size of 29 subjects in the main study sample and 53 subjects in the external HC sample . MachineBeyond these more methodical elaborations, further limitations of our study are related to the intricacies of the study populations. Our HC populations from both samples were non-offending, non-pedophilic individuals, whereas non-pedophilic child sexual offenders or non-offending pedophilic individuals would have been more specific control groups. Our PO population was an inpatient cohort of hands-on pedophilic offenders without any comorbid psychiatric disorders, which carries additional limitations. First, the inpatient status could mean that certain effects of deprivation or incarceration might have influenced the results. Second, our study sample did not include hands-off pedophilic offenders, namely consumers of media depicting child sexual abuse, which is an important mode of child sexual offending leading to the victimization of a great number of children , 112. ThTo our knowledge, this is the first study to successfully train a supervised machine learning classifier on WM microstructure patterns to distinguish between PO and HC individuals. The brain-based PO model produced a BAC of 75.5%, an out-of-sample specificity of 94.3% and was related to the individual’s previous number of child victims, current stance on sexuality, and future risk of sexual violent reoffending. We hypothesize that the discriminatory pattern of WM in the amygdala, ACC, and PFC could reflect a high-risk interaction between genetic predisposition, hormonal influence, and environmental factors throughout an individual’s life. We propose this WM microstructure pattern in the prefronto-temporo-limbic circuitry as a template for further research on brain-based biomarkers for pedophilic offenders and we hope that such biomarkers will improve secondary and tertiary prevention of child sexual abuse.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the local ethics advisory board of the Medical School at Otto-von-Guericke University, Magdeburg. The patients/participants provided their written informed consent to participate in this study.DP and KS had full access to all the data in the study, took responsibility for the integrity of the data and the accuracy of the data analysis, and contributed to the concept and design of the study. DP, MWe, CG, JK, KS, MWa, and JW contributed to the acquisition, analysis, or interpretation of data. DP, MWe, CG, and JK drafted the manuscript. DP, MWe, ML, JL, NK, KS, PF, BB, and MWe contributed to the critical revision of the manuscript for important intellectual content. DP, MWe, JK, and NK contributed to the statistical analysis. DP, KS, JW, and BB obtained the funding. PF, JK, KS, BB, JW, and NK contributed to the administrative, technical, or material support. PF, NK, and KS contributed to the supervision. All authors contributed to the article and approved the submitted version.


In this work, the hydrodynamic characteristics of spiral groove bearing in centrifugal rotary blood pump is investigated for the cases with different structural parameters. The simulation model is proposed based on the CFD technology and the effectiveness of simulation model is demonstrated by the published data. Then, the pressure, load carrying capacity and friction torque are calculated and the characteristics of pressure distribution are analyzed. It is found that the structural parameters of spiral groove would lead to the complex pressure distribution of blood film and the load carrying capacity also changes at the same time. Moreover, the deep analysis of structural characteristics for spiral groove bearing is conducted based on the orthogonal design method, which could improve the computational efficiency of hydrodynamic behavior of spiral groove bearing. And, the mapping relationship between structural parameter and hydrodynamic performance of bearing preferably is also illustrated. At presThe effects of structural characteristics for spiral groove are attracted researchers . Han andIt is important to note that many investigations focus on the structural design of spiral groove bearing ,16. FesaIn this work, the hydrodynamic analysis model of spiral groove bearing in centrifugal rotary blood pump is established, which considers the structural characteristics of spiral groove and pressure boundary conditions. Then, the effects of structural parameters on the pressure distribution and load carrying capacity are discussed. Moreover, the orthogonal design approach is employed to investigate the hydrodynamic behavior of spiral groove bearing with different structural characteristics.2ω is the rotational speed of spiral groove bearing. Then, the characteristics of spiral groove can be described by logarithmic spiral, that is,The spiral groove bearing of centrifugal rotary blood pump is plotted in The thickness of blood film at the land area  can provide helpful for the analysis of structural parameters for spiral groove bearing, which is given as:In order to improve the accuracy of hydrodynamic analysis for spiral groove bearing, the structural parameters of groove are always chosen detailedly. The more parameters would increase the calculation duration and complexity of analysis. Then, the design approach of spiral groove bearing is established by the orthogonal experiment method. For example, the number of structural parameters is mr times ,29. As tIn addition, the numerical method flowchart of spiral groove bearing is displayed in 3−5. The simulation results and experiment data are listed in In order to demonstrate the effectiveness of the presented method, the simulation results are compared with the published data in Ref. . The strIn addition, the groove shape plays the key role in the hydrodynamic response of spiral groove bearing. Then, the numerical simulation would be conducted considering the effects of structural parameter variation. The mesh model is shown in The pressure and velocity distribution of spiral groove bearing with different structural parameters are plotted in 4In order to reveal the influence of groove shape on the hydrodynamic response of bearing, the spiral angle, groove width, groove number and groove depth are defined as the variables. Then, the numerical simulation can be conducted in detail. Moreover, the orthogonal table is established by the orthogonal design approach and more structural parameters of spiral groove can be considered. The hydrodynamic performance of spiral groove bearing are analyzed by the maximum pressure, load carrying capacity, friction torque and energy loss.4.1hg = 0.06 mm) decreases at the spiral angle 35°, as the increases of maximum pressure. Although the maximum pressure increases, the average value of pressure decreases, which can illustrate this phenomenon. Meanwhile, the increase of groove depth would expand the motion region of blood, and the intermolecular force of blood is reduced, which is the reason of decrease of load carrying capacity. As the growth of spiral angle, Case 1: In this model, the groove number, groove width ratio and inner radius are defined as 16, 1 and 6 mm, respectively. The values of groove depth are 0.04 mm, 0.06 mm, 0.08 mm and 0.1 mm. The spiral angles are chosen to 20°, 25°, 30°and 35°, respectively. And, the simulation results are shown in −3 MPa and 0.5138 N. With the groove depth expands to 0.1 mm, the maximum pressure and load carrying capacity are 8.851 × 10−3 MPa and 0.969 N, respectively. The hydrodynamic characteristics of spiral angle bearing can be explained that the increase of groove width ratio change the blood flow state and the distribution and value of blood-film pressure also change. Moreover, the simulation results shows that the friction torque and energy loss increase as the growth of groove width ratio. Compared with the influence of groove width ratio, the effects of groove depth on the friction torque and energy loss are weak, especially in the condition of a larger groove depth. The increase of groove width ratio could enlarge the contact region area, and the viscosity characteristics of blood would raise the friction force between blood and solid, which would cause the appearance of more energy consumption. As the increase of energy loss, the hydrodynamic performance of blood film would drop and it is not the design objective of spiral groove bearing. In addition, the increase of groove width ratio would reduce the extruded area of blood film, which is the main reason for the decrease of supporting capacity for spiral groove bearing. And then, the increase of clearance between blood molecules would cause the more energy loss during the collision.Case 2: In this model, the groove number is defined as 16, the spiral angel is given as 25° and the inner radius is 6 mm. Case 3: The spiral angle, groove width ratio and inner radius are 25°, 1 and 6 mm, respectively. 4.25 (1024) times, which has a heavy workload. According to the orthogonal design method, the orthogonal table  are listed in 4E3B1C2A4) could provide a better hydrodynamic performance of spiral groove bearing. Based on the orthogonal design method, the efficiency of structural design for spiral groove bearing in centrifugal rotary blood pump has been improved. However, it should be note that the numbers of parameters and variable value should be satisfied the requirement of orthogonal table, which could represent the coupling relationship of design parameters. More structure parameters and variable values could improve the establishment difficulty of orthogonal table.According to the simulation results, the influence of structural parameters on the hydrodynamic behavior of spiral groove bearing is complicated and confused. Then, the simulation results analysis should be conducted and the optimization design object is also proposed (ODO). The average value  [No. CZQM2020054].Dr Cong Xue was supported by Funding from Young Talent Changzhou Science and Technology Planning Project [CJ20210067].Xiuying Wang was supported by Data will be made available on request.The authors declare no conflict of interest.


Evidence-based practice (EBP) is essential in improving the quality of healthcare and of importance for all health care personnel. No study in Norway has investigated attitudes, skills and use related to EBP among chiropractors. The aim of this study was to describe Norwegian chiropractors’ attitudes, skills, and use of EBP, as well as the barriers and facilitators to their use of EBP.A national cross-sectional survey, the online version of the Evidence Based practice Attitudes & Utilisation SurvEy (EBASE), was sent by email to 770 Norwegian practicing chiropractors, all members of the Norwegian Chiropractic Association. Three EBASE sub-scores were generated , and the demographic characteristics of the sample were reported. Linear regression analyses were conducted to examine the association between responses of the three sub-scores and demographic characteristics. Information on main barriers and facilitators of EBP was collected and described.A total of 312 (41%) chiropractors responded to the survey, and 95% agreed that EBP is necessary for chiropractic practice. While overall use of EBP activities was low participants were interested in learning and improving their skills to incorporate EBP into practice. Chiropractors’ attitudes, skills, and use of EBP were positively associated with being female and having spent more than one hour per week on research, but negatively associated with having practiced more than 10 years. Main barriers of EBP were lack of skills to critically evaluate, interpret, and apply research findings to practice. Main facilitators of EBP included access to the internet and free online databases in the workplace.Although chiropractors in Norway reported positive attitudes and moderate skills in EBP, their use of EBP activities was limited. The main barriers and facilitators to EBP were primarily related to perceived skills deficits, whilst enablers of EBP were mostly related to infrastructure requirements. The foundation of evidence-based practice (EBP) is the integration of the best available research evidence into clinical practice to improve both health outcomes and quality of care for individual patients , 2. In aPrevious surveys on EBP readiness of manual therapy professions conducted in Sweden , Canada Surveys of manual therapy professions to date indicate that access to workplace internet and free online databases are frequently favoured facilitators of EBP uptake, whereas lack of time and lack of clinical evidence in the profession have been found main barriers to delivering EBP , 11, 12.Chiropractic is an allied health profession with a scope of practise that largely focuses on musculoskeletal conditions . In NorwWe also know little of EBP uptake within the Norwegian chiropractic profession. Only one previous survey, from 2014, has described Norwegian chiropractors attitudes towards clinical guidelines and research participation , and werThe overall aim of this study was to describe Norwegian chiropractors’ attitudes, skills, and use of evidence-based practice, as well as the barriers and facilitators to the use of EBP in the Norwegian chiropractic context. Specifically, this study aimed to address the following objectives:attitudes toward EBP,levels of perceived skill in EBP,levels of engagement in EBP activities,facilitators of, and barriers to EBP uptake, and.attitudes, skills, and use of EBP, and their association with demographic factors.An online, descriptive cross-sectional survey.2 (0.8) and used mean and standard deviation of outcomes in this study sample. This specified a required sample size of 254. Our total sample size included 312 chiropractors and was within acceptable limits.The target population consisted of 770 Norwegian practising chiropractors registered with the Norwegian Chiropractic association as of June 2021. No exclusion criteria were applied. We used the method by Riley et al. to calculate for the required sample size for multivariable linear regression modeling . In the The Evidence-Based practice Attitude and utilization SurvEy (EBASE)  is an inPart A: attitudes ,Part B: skills ,Part C: education and training (5 multiple-choice items),Part D: use ,Part E: barriers .Part F: facilitators  and.Part G: demographics (14 multiple-choice items and 1 open-text item).Items from three of the survey parts can be generated into sub-scores, as described elsewhere , and sumPart A: attitude sub-score, sum of the first 8 items, ranging from 8 (predominantly strongly disagree) to 40 (predominantly strongly agree).Part B: skills sub-score, sum of all 13 items, ranging from 13 (primarily low-level skill) to 65 (primarily high-level skill), and.Part D: use sub-score, sum of the first 6 items, ranging from 0 (mainly infrequent use) to 24 (mainly frequent use).The survey was translated from the English and Swedish versions to Norwegian by members of the research team who were fluent in these languages. After the translation, the questionnaire was pilot tested on 2 selected chiropractors. Suggestions for minor modification  were integrated into the final version of the survey.Data collection were handled by a digital questionnaire created with Nettskjema.no, survey solution developed and hosted by the University of Oslo, Norway (nettskjema@usit.uio.no). The link to the survey was sent by email to all practicing chiropractors that were members of the Norwegian Chiropractors’ Association (n = 770) in the fall of 2021. The email contained information on the purpose of the study, survey composition, informed consent considerations, reporting of results, storage of data, and ethics. Verbal advertisement and encouragement to participate in the survey were given during the annual National Norwegian chiropractic Conference. Several participation advertisements, encouragement messages and reminders to complete the EBASE survey were distributed before and during data collection. These were sent by email or as small films and personal reminders posted on relevant social media groups (Facebook and Messenger), using collegial language . Data collection took place between October and November 2021.2 was used to select/choose the best linear regression model. We set the significance level at 5% for all tests and performed all analyses in STATA/SE 16 .Data from the online survey was imported into “Tjenester for Sensitive Data” (TSD), owned by the University of Oslo. The TSD  service is designed for storing and post-processing sensitive data in compliance with the Norwegian “Personal Data Act” and “Health Research Act”. Statistical analyses were performed on the TSD facilities. There were no missing data, as all items were made compulsory. Categorical data were described using frequencies and percentages. Medians and the interquartile range (IQR) were used for non-normally distributed data. Linear regression models explored associations between a priori selected demographic variables , and each of the following: attitudes sub-score, skills sub-score and use sub-score. Previously, these variables have shown to be associated with attitudes, skills, and use of EBP . SelectiStudy participation was anonymous and voluntary, and no ethical approval was needed according to Norwegian law . The stuThe response rate was 41% (312/770 chiropractors). Characteristics of the participating chiropractors are shown in Table Chiropractors’ attitudes toward EBP were positive with a median sub-score of 33, (IQR (30–36); range 8–40; scores ranging between 32.0 and 40.0 are indicative of predominantly agree to strongly agree) , EBP assists clinical decision-making (94%), and EBP improves the quality of patient care (89%), Table . The majMultivariable analysis between demographic characteristics and the attitude sub-score found the best subset model included age, number of years in practice, undertaking research activities, clinical setting, and country of education. The model found that spending one or more hours per week on research were significantly associated with a higher attitude sub-score (i.e. more positive attitude towards EBP) whereas having practiced between 11 and 15 years, and being educated in the USA was significantly associated with a lower attitude sub-score Table .The median skill sub-score (40.5 (IQR 35–47); range 13–65) was indicative of a predominantly moderate to moderate-high skill level of perceived skills in EBP (as defined by scores ranging between 39.1 and 51.9) Table . ChiroprMultivariable analysis between demographic characteristics and the skill sub-score found the best subset model included gender, number of years in practice, country of education and undertaking research activities. The model found that being female was significantly associated with a higher skill sub-score  whereas spending one or more hours per week on research was significantly associated with a lower skill sub-score Table .Chiropractors’ median use sub-score of 8  reflected a moderate-low level of EBP activities in the previous month. A score between 6.1 and 12.0 is suggestive of a moderate-low level of use) Table .Multivariable analysis between demographic characteristics and the use sub-score found the best subset model included gender, age and number of years in practice, educational institution and undertaking research activities. The model found that being female, older, and spending one or more hours per week on research were significantly associated with a higher use sub-score (i.e. greater use of EBP in the previous month) whereas having practiced more than 6–10 years was significantly associated with a lower use sub-score Table .Information sources that were ‘Used a lot’ or ‘Always’ by chiropractors to inform their clinical decision-making included clinical practice guidelines (62%), however, chiropractors also favoured traditional knowledge (56%), and fellow practitioners or experts (51%). Information sources ‘Never used’ or ‘Used a little’ were experimental/laboratory research (87%), trial and error (44%), and textbooks (36%) Table .Of the included chiropractors, about one-half indicated that the following topics were major parts of their chiropractic education: coursework about EBP (55%), applying research evidence to clinical practice (55%), and critical thinking/analysis (56%). Only 14% of chiropractors indicated they never had any critical thinking/analysis included in their chiropractic education. Approximately one-third had never received any education on conducting systematic reviews (31%) or clinical research (27%).not to be a barrier to EBP uptake were lack of collegial support for EBP (69%), lack of profession support (57%), lack of interest for EBP (55%), lack of resources (53%), lack of relevance to chiropractic (49%), and patient preference for a specific treatment (36%). Factors largely reported as a major or moderate barrier to EBP uptake are reported in Table The majority (55–96%) of chiropractors found that 12 of 13 potential barriers were either ‘not a barrier’ or ‘only a minor barrier’. Those factors considered by most chiropractors Most chiropractors (53–89%) found that most of the listed factors were ‘very useful’ facilitators of EBP uptake, as reported in Table This is the first study to examine Norwegian chiropractors’ attitudes, skills and use relative to EBP. The study found that Norwegian chiropractors generally report positive attitudes towards EBP and moderate to high levels of perceived EBP skills. Despite this, chiropractor participation in EBP activities over the previous month was mostly low and infrequent.The results from this survey indicate that participating Norwegian chiropractors were relatively young and graduated recently from a European educational institution. Most worked in private practices together with other healthcare providers, with few working in solo practices. The demographic and work characteristics of the study sample were similar to those reported in the previous national survey of Norwegian chiropractors . This prThe positive attitudes toward EBP among participating chiropractors agree with previous studies of Swedish, Australian, American, and Canadian chiropractors –6, 12, aNorwegian chiropractor attitudes toward EBP were found to be significantly associated with engagement in research activities. These findings concur with those reported among chiropractors in Sweden and Canada , 9, 11, Participating chiropractors reported that their poorest skills in EBP related to conducting clinical research and/or systematic reviews. This is not surprising as such skills are unlikely to be developed in most chiropractic programs. By contrast, most chiropractors were comfortable with identifying clinical problems and knowledge gaps in practice, and generally judged the level of these skills as relatively high. This was also found in studies of Swedish and US chiropractors , 6, and Overall, few chiropractors participated in research activities, and those who did, only engaged in such activities between 1 and 5 h per week. Given that most participating chiropractors worked in private practice, and not in an academic or research institution, this is to be expected as practicing chiropractors may not have the time and/or resources to engage in academic activities. An interesting finding of this study was the statistically significant inverse association between participation in research activities and perceived skill level regarding EBP. While this finding may appear illogical, it can be explained by the Dunning-Kruger effect, which states that people with high levels of skill (such as EBP-related skills) tend to underestimate their skill level, and vice versa . This coMost chiropractors indicated that a moderate to large proportion of their practice was based on clinical research evidence. This is consistent with findings from a study of Swedish chiropractors , yet is When participating chiropractors were specifically asked about the information sources used to support clinical decision-making, the majority reported using clinical practice guidelines, followed by traditional knowledge and fellow practitioners or experts. Similar findings of using clinical guidelines, traditional knowledge and fellow practitioners to inform their clinical decision-making were reported in both the Swedish and the Australian study, however, in these studies the overall use was lower compared to our results , 12. It Understanding barriers and facilitators of EBP may help facilitate EBP uptake and implementation in Norwegian chiropractic practice. In our study, the major facilitators of EBP use were accessibility to the internet and online databases in the workplace. These facilitators of EBP uptake are similar across countries and manual therapy professions –6, 9–11,Further, approaches to overcoming these barriers should be integrated into chiropractic educational programs, as well as continuing education for practicing chiropractors. Appropriate investment in such approaches will be critical to the successful implementation of EBP in Norwegian chiropractic practice.Although our study was novel, and we had employed several participation reminder strategies, the response rate was modest. Still, our response rate was similar to a previous survey of Norwegian chiropractors , and subThe survey was based on both the Swedish and English versions of EBASE , 17, andDue to differences in the year of publication, response rates, and context ), drawing comparisons between our sample, and those of other EBASE studies, was challenging. For instance, if a study was done some time ago, EBP might not have penetrated clinical practice to the level observed today. Another difference is the context in which the chiropractic profession practices , as the former typically will encourage EBP use. For instance, being in solo practice was less common in Norway relative to the other studies , 7, 12. Concerning educational curricula, chiropractic programs are different across and even within countries, schools, and universities , 30. TheNorwegian chiropractors reported positive attitudes toward EBP, and perceived they had a good level of EBP-related skill, yet rarely used EBP. A key enabler of EBP uptake was access to the internet and open-access papers. However, the results also indicated that there was a need for relevant training to enable chiropractors to engage in EBP. Training courses that facilitate EBP skills development and use  have been shown to be a feasible strategy for healthcare professionals . In addiChiropractors’ adoption of EBP should be systematically included within program curricula and be prioritized and provided in a manageable form as continuing education for practicing chiropractors. Currently, there is no chiropractic education in Norway, and thus, there is no academic base for the profession to grow. However, EBP training and access to research findings are part of academicization of a profession. This may be achieved by delivering chiropractic education within a university environment. Until this is achieved, a temporary solution may be to establish a centre for professional excellence to foster the provision of continued professional development related to EBP. Such a centre could also assist in overcoming other barriers identified in this study, such as EBP incentivisation and evidence synthesis.The study provides insight into the attitudes, skills, and use of EBP among Norwegian chiropractors. Chiropractors had positive attitudes and moderate to high perceived EBP skills and believed in the value of EBP to improve outcomes and healthcare quality of individual patients. However, chiropractors had limited time and incentive to find research evidence; accordingly, use of EBP was low. The barriers to EBP were primarily related to perceived skills deficits, whilst facilitators of EBP were mostly related to infrastructure requirements.


GDAP1 pathogenic variants cause Charcot-Marie-Tooth (CMT) disease, the most common hereditary motor and sensory neuropathy. CMT-GDAP1 can be axonal or demyelinating, with autosomal dominant or recessive inheritance, leading to phenotypic heterogeneity. Recessive GDAP1 variants cause a severe phenotype, whereas dominant variants are associated with a milder disease course. GDAP1 is an outer mitochondrial membrane protein involved in mitochondrial membrane contact sites (MCSs) with the plasmatic membrane, the endoplasmic reticulum (ER), and lysosomes. In GDAP1-deficient models, the pathophysiology includes morphological defects in mitochondrial network and ER, impaired Ca2+ homeostasis, oxidative stress, and mitochondrial MCSs defects. Nevertheless, the underlying pathophysiology of dominant variants is less understood.GDAP1 clinical variants located in the α-loop interaction domain of the protein. p.Thr157Pro dominant variant causes the increase in these MCSs that correlates with a hyper-fissioned mitochondrial network. In contrast, p.Arg161His recessive variant, which is predicted to significantly change the contact surface of GDAP1, causes decreased contacts with more elongated mitochondria.Here, we study the effect upon mitochondria–lysosome MCSs of two 2+ transfer from the lysosome to mitochondria, our results support that GDAP1 clinical variants have different consequences for Ca2+ handling and that could be primary insults determining differences in severity between dominant and recessive forms of the disease.Given that mitochondria–lysosome MCSs regulate Ca Summary: The relevance of mitochondria-lysosome membrane contact sites dysfunction as a primary event in axonopathies etiology, and as a key signaling hub opening novel therapeutic strategies in peripheral neuropathies, especially in Charcot-Marie-Tooth disease. GDAP1 gene cause Charcot-Marie-Tooth (CMT) disease, a motor and sensory neuropathy, which is the most common hereditary neuromuscular disorder. CMT-GDAP1 can be expressed as an autosomal recessive demyelinating neuropathy  (ne (OMM) , which ine (OMM)  with memne (OMM) . The GDAne (OMM) , a hydroGDAP1 participates in the dynamics of the mitochondrial network, specifically in fission processes , and in GDAP1 dominant variants on the biology of mitochondria-lysosome MCSs is not known.Recently, we described that GDAP1 is located at mitochondria–lysosome MCSs , which hGDAP1 missense pathogenic variants located inside the α-loop domain on the structure and function of mitochondria-lysosome MCSs. We found that GDAP1 recessive and dominant variants have different effects on these mitochondrial MCSs.Here we aimed to study the effect of dominant and recessive GDAP1 missense variants p.Arg161His (recessive)   and p.Thcessive) , which lcessive) . Additioin-silico predictors tools and data mining. The Mutation Taster , while GDAP1p.Arg161His-MYC (recessive) showed a significant reduction   . In the GDAP1 deficient cell models present abnormalities in the mitochondrial network and lysosomes , pCMV-GDAP1-MYC p.Thr157Pro (dominant variant), and pCMV-GDAP1-MYC p.Arg161His (recessive variant) .The following antibodies were used: β-ACTIN mouse monoclonal , LAMP-1 rabbit polyclonal , LAMP-1 mouse monoclonal , and MYC mouse monoclonal .GDAP1 variants was done using VarSome (last accessed & February 2023) according to the American College of Medical Genetics and Genomics (ACMG) standard guidelines. In-silico analyses of the genetic variants were performed using: MutationTaster  . The eleonTaster  and CADDonTaster .2 incubator. HEK293T cells were transfected with FuGENE Transfection Reagent  according to the manufacturer's instructions.HEK293T cells were cultured in Dulbecco's Modified Eagles’ Medium high-glucose  supplemented with 10% fetal bovine serum , 2 mM L-glutamine  and 100 mg/ml penicillin-streptomycin , at 37°C in a 5% COHEK293T cells were seeded onto glass coverslips, washed with PBS, and fixed in pre-warmed 4% paraformaldehyde for 20 min at room temperature. After three PBS washes, cells were permeabilized with 0.2% Triton in PBS for 30 min and they were blocked with 1% BSA and 4% serum in PBS. The specific primary antibodies were incubated overnight at 4°C, and the secondary conjugated antibodies were incubated for 1.5 h at room temperature. The coverslips were mounted with Fluoromount-G with DAPI  .In Situ-Fluorescence, Sigma-Aldrich, DUO92008) and the coverslips were mounted with Duolink In Situ Mounting Medium with DAPI .HEK293T cells were seeded onto glass coverslips, washed with PBS, fixed in pre-warmed 4% paraformaldehyde for 20 min at room temperature, and permeabilized with ice-cold methanol at −20°C for 20 min. After 1 h of incubation with the blocking solution in a pre-heated humidity chamber at 37°C, cells were incubated overnight at 4°C with the specific primary antibodies. Afterwards, we performed the PLA assay according to the manufacturer's instructions  containing a protease inhibitor cocktail . Homogenates were centrifuged at 13,200 rpm (FA-45-30-11 Rotor) for 15 min at 4°C and the protein concentration of the supernatant was quantified by the BCA method , resolved in sodium dodecyl sulfate-polyacrylamide gels (SDS-Page) and transferred onto PVDF Immobilon-P membranes . The membranes were blocked with 5% defatted milk in TBS-T buffer . Afterward, the membranes were blotted with the specific primary antibodies, which were detected using secondary antibodies coupled to horseradish peroxidase. Proteins were processed for chemiluminescence with Amersham ECL Prime Western Blotting Detection Reagent  and visualized by iBrBand CL1000 Imaging System (ThermoFisher Scientific). Band intensity was measured using ImageJ software .HEK293T cells were homogenized in lysis buffer  overnight at 4°C. Beads were softly washed with lysis buffer, resuspended in Laemmli Buffer, heated at 95°C, and analyzed by SDS-Page and western blotting.http://rsb.info.nih.gov/ij).Super-resolution images were acquired with a Leica TCS SP8 X White Light Laser confocal microscope with Hybrid spectral detectors and HyVolution  using the Leica LAS X software (version 3.1.5). Image deconvolution was performed with Huygens Essential software v.4.4 0p6 . Image processing and analysis were performed using the Leica Application Suite X (LAS-X) software  or ImageJ . For LAMP-1 fluorescence intensity ROIs were drawn around representative areas of the cell. Three regions next to the cells without fluorescence were taken as background. The corrected total cell fluorescence (CTCF) formula was used: CTCF = integrated density – (area of selected cell × mean fluorescence of background readings). For lysosome area, maximum intensity projections were generated from Z-stacks followed by automated 8-bit Otsu-thresholding, and the binary images were evaluated to obtain the LAMP-1 total area in each cell.P-values less than 0.05 were considered significant. P-values are indicated by asterisks *P<0.05, **P<0.01, ***P<0.001.Statistical analysis was performed using GraphPad Prism  with a minimum of three independent experiments. Normality was assessed by the Kolmogorov–Smirnov test. The specific test applied in each case is indicated in the figure legend.


Staphylococcus aureus meningitis requires antimicrobial penetration into the CSF. Anti-staphylococcal penicillins, including flucloxacillin, are generally recommended as first-line treatment. However, some evidence suggests that the first-generation cephalosporin, cefazolin, may be an appropriate alternative. We investigated the CSF concentration of cefazolin and flucloxacillin in children.Effective treatment of methicillin-susceptible CSF samples from children aged 0-18 years treated with cefazolin or flucloxacillin were collected when there was sample remaining after diagnostic tests were completed. Unbound antibiotic concentrations of cefazolin and flucloxacillin were measured.6/L) than those without . Notably, only those aged ≤1 year receiving cefazolin  achieved steady state CSF concentrations above the CLSI breakpoint for sensitive S. aureus (MIC ≤2 mg/L).Overall, 222 CSF samples were collected from 163 children, median age 12 months (range 0.0-17.5 years). Of these, 157 were collected while the antibiotic was at steady state. For cefazolin, children ≤1 year had a higher median CSF concentration compared with those >1 year , whereas for flucloxacillin the concentration was similar between the two age groups . In infants aged ≥ 1 month to 1 year, the CSF concentration of both antibiotics was higher in those with pleocytosis (WCC >5 x10S. aureus MIC.CSF penetration of cefazolin was higher in younger infants and for both antibiotics was higher in those with pleocytosis. In most children, the CSF concentrations in the CSF for either cefazolin or flucloxacillin were not above the All Authors: No reported disclosures