However, current cardiovascular magnetic resonance (CMR) methods are insensitive, require a coalesced mass of lipid core, and rely on multicontrast acquisition with contrast media and extensive post-processing.2 maps and compared directly to plaque lipid defined by histology.Patients scheduled for carotid endarterectomy were recruited for 3-T carotid CMR before surgery. Lipid area was derived from segmented T2 mapping and histology showed excellent correlation, both by individual slices  and plaque average . Lipid area (%) on T2 maps was significantly higher in symptomatic compared with asymptomatic plaques  despite similar degrees of carotid stenosis and only modest difference in plaque volume . Receiver-operating characteristic analysis showed that T2 mapping has a good ability to discriminate between symptomatic and asymptomatic plaques with 67% sensitivity and 91% specificity .Lipid area (%) on T2 mapping distinguishes different plaque components and accurately quantifies plaque lipid content noninvasively. Compared with asymptomatic plaques, greater lipid content was found in symptomatic plaques despite similar degree of luminal stenosis and only modest difference in plaque volumes. This new technique may find a role in determining optimum treatment .CMR T  Lipid accumulation in the subendothelial space, following deposition and retention of apolipoprotein B–containing plasma lipoproteins, is a key process in the initiation and progression of atherosclerosis Although intensive lipid-lowering therapy can reduce total carotid vessel wall area Multicontrast cardiovascular magnetic resonance (CMR) is an established technique in plaque characterization 2 relaxation times of plaque components on a voxel-by-voxel basis 2 mapping addresses the need for an absolute physical parameter that can be standardized among different CMR systems and widely adopted in multicenter studies. Accordingly, here we sought: 1) to validate lipid quantification on carotid T2 maps using histology gold standard; and 2) to evaluate its potential clinical application in relation to identifying recently unstable plaques.We recently reported a quantitative CMR method to map TEthical approval was obtained from National Research Ethics Services and local R&D committee. Forty patients awaiting carotid endarterectomy at Oxford University Hospitals NHS Trust were recruited from November 2011 to June 2014. Patients were scanned at the Oxford Acute Vascular Imaging Centre ≤24 h before surgery. Carotid plaques were collected at operation. Patients either had recently symptomatic (median time from index event 2 weeks) or asymptomatic carotid disease, with 50% to 99% stenosis according to NASCET , or 70% to 99% according to ESCT  criteria. Plaques were defined as culprit plaques where they were deemed to have given rise to either a minor cerebrovascular accident or a transient ischemic attack as assessed clinically and supported, where available, by brain magnetic resonance imaging or computed tomography imaging. Asymptomatic carotid plaques were those that had no documented clinical symptoms, but with an indication for carotid endarterectomy on the basis of percent stenosis.2 maps were generated from 14 images with echo time of 9 to 127 ms and repetition time of 2,000 ms, acquired using the multislice delay alternating with nutation for tailored excitation multiecho spin-echo (DANTE-MESE) sequence that combined black-blood preparation on the basis of nonselective DANTE pulse trains Patients were imaged on a Verio 3-T scanner  with a 4-channel phased-array carotid coil . Bright-blood time-of-flight (TOF) angiography of the carotid arteries was acquired to localize carotid bifurcation and lumen stenosis. Two-dimensional carotid TDANTE-MESE image quality was assessed by an experienced reviewer (L.B.) who inspected the visibility of the carotid wall boundaries and rated them from 1 (poor) to 5 (excellent) according to standard procedures 2 maps of the carotid arteries were generated voxel by voxel using monoexponential nonlinear fitting 2 than normal vessel wall and fibrous tissue, whereas with recent IPH into the core, T2 was longer than all other plaque components 2 thresholds, a lower one (T2L) indicating the maximum T2 value associated with lipid alone and a higher one (T2H) above which IPH was recorded inside the plaque lipid core. Voxels with T2 values below T2L or above T2H were thus classified as LRNC. Significant IPH (T2 > T2H) was defined as >5% of the total cross-sectional area. We also reported LRNC values on the basis of T2L alone, taking no account of hemorrhage.T2L (range 30 to 50 ms) and T2H (range 70 to 120 ms) to find the pair of threshold values that achieved the optimal lipid segmentation on T2 maps . All algorithms were implemented in Matlab . Plaque type was also classified on the basis of T2 mapping and TOF data  using the modified American Heart Association (AHA) scheme To use the data most efficiently, segmentation performance was estimated by leave-1-out cross-validation. Lipid area (%) was computed for all possible combinations of TCarotid plaques were collected at endarterectomy and divided into 2 halves at the point of maximal stenosis: one half was snap-frozen and the other half processed for paraffin embedding. Paraffin sections (5 μm) were obtained at 1-mm intervals for histological examination using hematoxylin and eosin  and Masson’s trichrome , which identified collagen in the fibrous tissue (green), as well as fibrin and erythrocytes from IPH (bright red). Lipid area without previous IPH appeared transparent or unstained in both hematoxylin and eosin and Masson’s trichrome stain. Stained slides were scanned using the NanoZoomer  virtual pathology system. Frozen sections (20 μm) at the mirror plane of division on 20 randomly selected plaques were obtained for Oil Red O (Sigma-Aldrich) staining to confirm lipid presence. Because IPH occurred exclusively inside, and as part of, the LRNC, the boundary of IPH against LRNC was often impossible to define on histology . The tot2 map locations and provided the corresponding T1-weighted images to a second investigator (J.T.C.) who obtained histology on the basis of the specific distance from bifurcation, and fine-tuned the exact matching location using local vessel morphology from the T1-weighted images provided.One investigator (L.B.) blinded to histology measured the distance from carotid bifurcation of T2 mapping was studied in 10 additional patients recruited on an unselected sequential basis. They were taken out of the scanner after the first scan and left sitting for 2 min before the second scan. Both scans acquired 10 contiguous slices to cover the entire plaque and assess the reproducibility of lipid area (%) per plaque.Scan-rescan reproducibility of lipid quantification by Tt tests and chi-square tests were performed. Lipid area correlation was calculated using both slice locations and plaques as the unit of analysis. Leave-1-out cross-validation was performed on the slice-by-slice dataset of independent lipid area measurements from T2 maps and histology.All statistical analyses are reported as mean ± SEM, unless otherwise stated; Student CMR scan quality ≥3 was achieved for 26 of 40 plaques, 15 symptomatic and 11 asymptomatic. Patient characteristics are summarized in 2 maps. To evaluate how accurately T2 mapping can determine plaque types, we used the modified AHA plaque classification system and compared this directly against histological classification. 2 map (+ TOF) against histology, which showed good agreement (80.8%) between the 2 methods (Cohen’s κ = 0.73).2 maps to quantify plaque lipid content  against lipid area (%) measured on histology  and Bland-Altman analysis confirmed a high degree of agreement between T2 maps and histology, with only 0.03% underestimation on T2 maps   .Figure 42 mapping whether LRNC size varied between different clinical presentations. Symptomatic plaques were found to have significantly higher lipid area (%), compared to asymptomatic plaques  . In addition, using an established definition of large LRNC as ≥25% of cross-sectional area 2 mapping alone (p = 0.003)  (We then tested using Te 99.4%) . This wa= 0.003) . Finallyy = 91%) .Figure 52 threshold had influenced our results, we examined the prevalence of IPH and its contribution to the total lipid area (%). Sixteen of the total 60 slices contained significant (>5%) IPH. Of these, only 7 had IPH infiltrating >50% of the lipid core. Thus IPH made a relatively minor contribution to the total lipid measurement. 2 threshold of <42 ms for segmentation and lipid area calculation, by so doing effectively excluded IPH (long T2) as part of the LRNC in T2 map segmentation, yet the strength of correlation was similar to our dual T2 threshold approach (R = 0.79 vs. 0.85). In fact, if we excluded those 7 slices with large (>50%) IPH from the cohort, the correlation of the remaining 53 slices using a single threshold (<42 ms) approach went back to R = 0.85.To clarify whether the inclusion of IPH in our calculation with a second TOne of the 10 carotid plaques scanned to evaluate reproducibility was rejected due to low image quality during rescan. For the remaining 9 plaques, lipid core measurements ranged from 5.7% to 36.6% with a mean of 20.3%. The scan-rescan reproducibility was excellent .2 mapping, on a voxel-by-voxel basis, and at high resolution, enables accurate and highly reproducible quantification of plaque lipid, validated with histology. Second, lipid quantification by T2 mapping can distinguish recently symptomatic plaques with high sensitivity and specificity.This work makes 2 principal findings. First, in vivo TDeposition and retention of cholesterol-rich lipoproteins is a cardinal feature of atherosclerosis, occurring from the earliest foam cell lesions through to the complex plaques of advanced disease. Where the rate of deposition exceeds the rate of clearance, cholesterol and cholesterol derivatives can accumulate within the plaque. The presence of a large LRNC  has been associated with high risk of rupture and atherothrombotic complications 2 mapping and multicontrast CMR. The main advantage of T2 mapping is to provide quantitative information, on a per-voxel basis, using direct measurement of an absolute physical property of plaque components. Our approach to lipid quantification is therefore more objective and less dependent on acquisition parameters than multicontrast CMR, which relies on relative signal intensities for plaque segmentation. In addition, T2 mapping does not suffer from blurring artifacts intrinsic to fast spin echo acquisition Since the first description of Toussaint et al. Atherosclerosis regression is a term used to denote favorable remodeling of plaque, for instance in response to aggressive reduction in plasma level of atherogenic lipoproteins 2 mapping in our different clinical cohorts, we found that despite having similar degree of carotid stenosis, and only a modest 21.2% difference in terms of plaque volume, symptomatic plaques had 99.4% larger LRNC than asymptomatic plaques. ROC analysis confirmed our technique having good ability to distinguish between symptomatic and asymptomatic plaques with optimal LRNC cutoff at 25% of the cross-sectional area, in agreement with large-scale histological series in both carotid 2 mapping data may assist interventional procedural planning to minimize such risks . This demonstrated empirically that T2 mapping does track plaque lipid content accurately, whether or not IPH is considered as part of the LRNC calculation. Finally, this histological validation study has a relatively small number of subjects and CMR slices. To utilize the available data most efficiently, we employed leave-1-out cross-validation to test the performance of our T2 segmentation model on data that were not used to build it.The main limitation of our method is the sensitivity to motion artifacts, a fact reflected in the scan rejection rate (35%), which is comparable to that of multicontrast CMR (30%) 2 mapping technique for quantification of plaque lipid content. Not only can T2 mapping distinguish between symptomatic and asymptomatic plaques on the basis of their lipid content, but we showed that despite similar degree of luminal stenosis and only modest difference in plaque volume, symptomatic plaques contained nearly double the lipid content compared with asymptomatic plaques on T2 maps. This technique may be of value in patient selection and evaluating response to new antiatherosclerotic treatment and interventions.PerspectivesCOMPETENCY IN MEDICAL KNOWLEDGE: Quantitative T2 mapping is a novel technique in characterization of carotid atherosclerotic plaques. Using histological validation, this study demonstrated its ability to differentiate various plaque components, with an emphasis on lipid quantification. Symptom-related plaques were shown to contain significantly more lipid despite similar degree of luminal stenosis and only modest difference in plaque volume compared with asymptomatic plaques. T2 mapping is accurate and reproducible; it is an alternative technique to the conventional multicontrast approach, which may be of value in future lipid intervention trials and cardiovascular risk stratification.TRANSLATIONAL OUTLOOK: Technical improvements are actively pursued, to reduce imaging time by compressed sensing, increase spatial resolution coverage and signal-to-noise ratios by better multichannel coils, and reduce motion artifacts by the development of image navigator–based approaches. How the changes in the size of lipid-rich core in vulnerable plaques, as an imaging biomarker, translate to the modification of cardiovascular risks remain to be elucidated.We provided histological validation of a new in vivo multislice T


Resistance associated mutations were shown in six CSF samples from discordant patients with LLV (one had insufficient sample for testing), which affected antiretroviral therapy at sampling in five. Overall discordance was present in 20/153 (13 %) and was associated with nadir CD4 but not antiretroviral concentrations in plasma or CSF. CSF/plasma discordance is observed in patients with LLV and is associated with antiretroviral resistance associated mutations in CSF. The implications for clinical practice require further investigation.The central nervous system has been proposed as a sanctuary site where HIV can escape antiretroviral control and develop drug resistance. HIV-1 RNA can be at higher levels in CSF than plasma, termed CSF/plasma discordance. We aimed to examine whether discordance in CSF is associated with low level viraemia (LLV) in blood. In this MRC-funded multicentre study, we prospectively recruited patients with LLV, defined as one or more episode of unexplained plasma HIV-1 RNA within 12 months, and undertook CSF examination. Separately, we prospectively collected CSF from patients undergoing lumbar puncture for a clinical indication. Patients with durable suppression of viraemia and no evidence of CNS infection were identified as controls from this group. Factors associated with CSF/plasma HIV-1 discordance overall were examined. One hundred fifty-three patients were recruited across 13 sites; 40 with LLV and 113 undergoing clinical lumbar puncture. Seven of the 40 (18 %) patients with LLV had CSF/plasma discordance, which was significantly more than 0/43 (0 %) with durable suppression in blood from the clinical group ( The central nervous system (CNS) has been proposed as a sanctuary site where HIV-1 replication may continue during stable antiretroviral therapy (ART). Concentrations of many antiretrovirals (ARVs) in cerebrospinal fluid (CSF) fall below the minimum inhibitory concentration for wild type virus, and there is large inter-individual variability in CSF penetration  in plasma is a common clinical problem, occurring in up to a quarter of ART-treated patients (Doyle and Geretti We prospectively recruited HIV-1 positive adults (≥18 years) receiving ART from 13 centres in the UK. Written informed consent was obtained from all participants. The study was approved by the North Wales Research Ethics Committee .Patients were recruited in two groups. In the first group, patients with LLV underwent lumbar puncture (LP) for research purposes. LLV was defined as at least one episode of unexplained plasma HIV-1 RNA viraemia above detection threshold (40 copies/ml) within the previous 12 months. No upper limit was specified. We included patients with blips and persistent LLV. We excluded those with an alternative explanation for virological non-suppression such as adherence <95 % on self-report or recent commencement of ART. Recent modification of ART was not an exclusion.It was not considered ethically justifiable to perform research LP on aviraemic controls without neurological symptoms; therefore, we prospectively recruited patients undergoing LP for a clinical indication and collected extra CSF at the time of LP. From this group, we identified patients with durable suppression of HIV-1 RNA detection limits (40 copies/ml) over the past year and no evidence of CNS infection to act as controls for those with LLV.In a separate analysis, we examined factors associated with discordance in the two groups combined, including patients in the clinical group that were not durably suppressed or had evidence of CNS infection.Virology testing and quantification of drug concentrations were performed centrally at the University of Liverpool. CSF cell count, protein and glucose measurements and microbiological investigations were performed by routine methods at the centres of care. In the LLV group, CSF cell count was performed locally at the discretion of the treating physician.10 higher than those in plasma based on criteria from other studies  with a lower limit of quantification of 40 copies/ml, as previously described . Drugs were extracted by protein precipitation (plasma) or directly injected (CSF). Standard curves were made up in artificial CSF , and matrix effects assessed by post column injection. Assays were validated against repeated runs of quality controls (inter and intra-assay variability) and external quality assurance standards.The revised CNS penetration effectiveness (CPE) score (2010) was calculated by assigning a predefined number of points to each component of the ART regimen, as proposed by Letendre et al.  in the previous 12 months. HIV-1 RNA had been detected >50 copies/ml on 117 of 198 occasions; median HIV-1 RNA levels during viraemic episodes was 92 copies/ml . Eight patients (20 %) reported cognitive symptoms on questionnaire. One patient (3 %) had a score below ten on the International HIV Dementia scale and one other patient had a score below eight on the Instrumental Activities of Daily Living scale. Ten patients (25 %) were anxious and two patients (6 %) were depressed according to the Hospital Anxiety and Depression scale.n = 50), headache (n = 20), suspected CNS infection or malignancy (n = 18), follow-up of a previous CNS infection (n = 8), abnormal neurological examination (n = 7), seizure (n = 5), neuropathy/radiculopathy (n = 2) and others (n = 3). On the basis of clinical findings and results of LP CNS infection was excluded in 92/113 patients (81 %). Where CNS infection was proven or probable, the diagnoses were neurosyphilis (n = 4), cryptococcal meningitis (n = 2), cryptococcal immune reconstitution inflammatory syndrome (n = 2), herpes simplex virus type-2 meningitis (n = 1), viral radiculopathy (n = 1), Ebstein-Barr virus in CSF of uncertain significance (n = 4) and no diagnosis (n = 7). Details of alternative diagnoses in those without CNS infection were not recorded; symptoms were cognitive (n = 43), headache (n = 16), suspected CNS infection or malignancy (n = 12), follow-up of a previous CNS infection (n = 4), abnormal neurological examination (n = 7), seizure (n = 5), neuropathy/radiculopathy (n = 2) and others (n = 3). In this group, 43/113 (38 %) patients had durable HIV-1 RNA suppression for 12 months and no CNS infection. The remaining 70 either had CNS infection (n = 21), were not suppressed (n = 26) or were not durably suppressed (n = 23).In the clinical group, 113 patients underwent LP to investigate cognitive symptoms  and those without (n = 33) discordance. There were no significant differences in the rate cognitive symptoms, International HIV Dementia Scale results, functional impairment on the Instrumental Activities of Daily Living scale, or tests of anxiety and depression. There was a trend to more frequent raltegravir use in patients with LLV compared to durable suppression , which likely reflects use of integrase inhibitors to intensify ART in patients with viral persistence.CSF/plasma discordance was found in 7/40 18 %) patients with LLV. None of the 43 patients in the clinical group with durable suppression had discordance. The difference in rate of discordance between patients with LLV (18 %) and durable suppression (0 %) was statistically significant, 8 % patier = 0.51; p < 0.001). HIV-1 RNA levels were <50 copies/ml in 113/153 (74 %) plasma samples and 115/153 (75 %) CSF samples. In discordant patients median HIV-1 RNA levels were 501 copies/ml  in CSF versus <40 copies/ml  in plasma. Differences in measured variables among patients with and without CSF/plasma discordance are shown in Table 3, p = 0.029).In the clinical group, discordance was found in 13/113 (12 %) patients. All discordant patients in this group were either not suppressed in plasma, not durably suppressed in plasma or had CNS infection. When combined with the LLV group, the rate of discordance overall was 20/153 (13 %). Overall HIV-1 RNA levels in plasma and CSF were directly correlated (Spearman CSF samples from 6/7 discordant patients with LLV underwent sequencing for the detection of HIV-1 RAMs (one patient had insufficient CSF for resistance testing). All six samples tested showed RAMs; this affected one or more of the ARVs taken at the time of sampling in 5/6 patients Table . At leasIn the clinical group, 12/13 CSF samples from discordant patients underwent sequencing (one patient had insufficient CSF for resistance testing). Five CSF samples did not amplify despite repeated attempts and one sample only amplified for the protease gene. Of the six CSF samples successfully tested, all showed RAMs; this affected one or more of the ARVs taken at the time of sampling in all six patients (Table Resistance testing of plasma virus was attempted in patients 7, 10 and 11. Plasma virus from patients 7 and 10 did not amplify. Plasma from patient 11 showed no NRTI or NNRTI RAMs; the integrase gene did not amplify.ARV concentrations in plasma and CSF are shown in Table We observed CSF/plasma discordance in 18 % of patients undergoing a research-based LP for intermittent or continuous LLV. No discordance was observed in patients with durably suppressed plasma viral load undergoing clinically indicated LP. This suggests that a significant proportion of HIV-positive patients with LLV despite ART may have suboptimal control of virus replication within the CNS. CSF examination of discordant patients with LLV revealed RAMs in all samples tested, which related to ART taken at the time of sampling in all but one. Of note, in these patients, plasma viraemia was generally too low to perform resistance testing in blood.One interpretation of these findings could be that ongoing replication of HIV in a CNS sanctuary site results in seeding to plasma leading to LLV. Alternatively, the observation of LLV and CSF/plasma discordance may represent two coinciding aspects of overall suboptimal virological suppression. It is of interest that in all cases at least part of the resistance detected in CSF virus was consistent with the previous history of failure and older resistance tests in plasma. This, combined with the observed association of discordance with low nadir CD4, suggests that advanced immune suppression may allow the establishment of a viral reservoir in the CNS that may be only partially responsive to ART. This virus may then continue to evolve and develop resistance to some ARVs, for example raltegravir, and thus become progressively less susceptible to suppression than virus in peripheral blood. LLV with a genetically different virus may originate from a similar process of incomplete suppression at other sites such as lymphoid tissue, where drug penetration and activity may also vary (Fletcher et al. Screening for cognitive impairment in the LLV group with a cognitive symptoms questionnaire and International HIV-Dementia Scale did not reveal associations with CSF/plasma discordance. The numbers are small and the screening methods used are insensitive for milder cognitive disorders (Haddow et al. CSF/plasma discordance was present in 12 % of patients undergoing clinically indicated LP. This rate of discordance is similar to other reports from clinical settings (Eden et al. There are a number of limitations to our study. Selecting controls from patients undergoing clinically indicated LP was not ideal; however, as clinical cohorts have shown higher rates of discordance than those undergoing LP for research purposes, this risked producing a false negative rather than false positive result. We assessed adherence by self-report which may not be reliable in all patients. Of note, there were no observed differences in measured plasma ARV concentrations between those with and without discordance suggesting that adherence was similar between groups, at least in terms of very recent ART use. Differences in the rate of viral decay between compartments in those recently starting or changing ART could have led us to identify some with discordance that did not have true compartmentalisation (Schnell et al.


Angiostrongylus vasorum has different freshwater aquatic and terrestrial gastropod molluscs as an intermediate host, e.g. Arion spp. The mollusc Achatina fulica is a danger to public health, given the large diversity of nematodes utilizing it as an intermediate host, such as the parasites of the genus Angiostrongylus, of importance in human and veterinary medicine. Achatina fulica has been shown to have an excellent capacity for maintaining outbreaks and natural infections with A. cantonensis in Asia. Within the mollusc, the nematode parasites activate haemocytes and/or haemolymph factors and in some invertebrates, phenoloxidase (PO), that induces the release of toxic elements and eliminates the parasites. Despite the importance of A. fulica in the life-cycle of nematodes, little is known regarding the defence mechanisms involving PO in molluscs infected with nematodes. Here, the presence of PO and nitric oxide (NO) in the haemolymph and haemocytes of A. fulica infected with first-stage (L1) larvae of Angiostrongylus vasorum was evaluated, together with the presence of melanin in the cephalopod mollusc tissue.F = 6.73, P = 0.00006), L-DOPA  = 22.67, P = 0.02) and p-phenylenediamine (PPD)  = 27.58, P = 0.0019), was observed. PO increase coincided with the presence of melanin in the cephalopodal tissue. At 8 dpi, PO activity, compared to L-DOPA  = 22.67, P = 0.00002) and PPD  = 27.58, P = 0.079) decreased, while melanin increased. At 13 dpi, PO decreased with PPD  = 27.58, P = 0.000015) and also the amount of melanin observed in histology. At 30 dpi, PO increased along with the substrates L-DOPA and PPD, while melanin decreased. NO levels increased until 8 dpi, and decreased after 13 dpi.An increase in PO at one day post infection (dpi), in comparison with the control using the substrates L-tyrosine (A. fulica and provides the first observation of an L-tyrosine dependent PO activity in molluscs infected with A. vasorum. This work suggests that PO pathway may help to control A. vasorum infection in A. fulica.To our knowledge, this is the first study that illustrates PO activity in a helminth-infected  Angiostrongylus vasorum parasitizes pulmonary arteries in domestic and wild Canidae and has terrestrial molluscs as intermediate hosts [te hosts –6. The mte hosts .The mollusc intermediate host is capable of mounting a cellular immune response to parasite infection via factors soluble in the haemolymph that activate haemocytes. In addition, elements of the acellular fraction of the haemolymph can also damage the parasites’ structures , 9. In sPO exists in the haemolymph in its inactive form (proPO) and is activated by external components of microorganisms such as β-1,3-glucans  and lipoPOs are copper-binding proteins including tyrosinases EC 1.14.18.1), catecholases (EC 1.10.3.1), and laccases (EC 1.10.3.2), and play a vital role in the immunological defence mechanism of Pacific oyster . All the.1, catecMelanin deposition around the pathogens, or in the pathogens themselves, is formed during the oxidation and polymerisation of phenols. The cytotoxic molecules generated in this process include reactive oxygen (ROS) and nitrogen (RNS) species and quinoids, which are intermediates of melanin. Additionally, an increase in the production of nitric oxide (NO), an effector molecule very effective against invasive organisms, occurs in immunoreactive hosts , 20.Angiostrongylus cantonensis in areas endemic for angiostrongylosis such as Asia and South America [A. fulica. The role of the PO pathway in controlling infection by nematode parasites is still poorly understood in molluscs and elucidating this process is crucial for understanding the defence mechanisms present in this intermediary host. Achatina fulica is less susceptible to A. vasorum in comparison with other molluscs and can be infected in laboratory conditions [A. fulica infected with A. vasorum to measure NO levels and characterise PO activity in the soluble fractions of the haemolymph using the substrates L-tyrosine, L-DOPA, and PPD. Additionally, we verified the occurrence of melanin in the cephalopodal tissue of A. fulica.Despite its importance as an intermediate host of  America –26, littA. fulica with an average shell length of 30 mm bred in the Veterinary Helminthology Laboratory of the Federal University of Minas Gerais (LabHelVet-ICB-UFMG) were individually infected with 1000 L1 of A. vasorum obtained from dog faeces using the Baermann technique [A total of 240 specimens of echnique .L1 were added to 2 ml of tap water and placed in a polystyrene container (4 × 5 cm). The molluscs were individually immersed in the suspension for 24 h. The container was sealed with gauze and adhesive tape. Afterwards, the molluscs were transferred to a maintenance tank containing lettuce, water, and autoclaved soil. The L1 that remained in the polystyrene containers were counted to estimate the infection rate .A. vasorum in A. fulica; these stages changes are described according to [A. vasorum larval stages were 1, 8, 13 and 30 dpi, for recovering L1, L1 to L2, L2 to L3 and L3 stage, respectively. The larval stages were identified using the descriptions of Bessa et al. [The haemolymph was collected according to the changes of stages of rding to . The daya et al. , Barçanta et al. .For removal of haemolymph, the shell was disinfected with 70% alcohol and dried on absorbent paper. Then, in the heart region, the shell was pierced with a mini drill , and the haemolymph was collected by cardiac puncture with a 1000 μl insulin syringe (Manoject 50 unit) and transferred for a 12 ml polypropylene plastic tube in an ice bath. Approximately 200 μl of haemolymph was collected per mollusc and added to the pool of the group, keeping the control and infected pools separated. Samples were collected from 20 molluscs per group, 10 controls and 10 infected, for each day analyzed. The experiments were repeated three times with other snails of the same lineage.A. fulica haemolymph in the control group were evaluated to analyze the best haemolymph-like buffer solution, which would allow the study of the internal defence system in A. fulica. It was noted that pH and osmolarity of haemolymph were pH 7.2 and 160 mOsm/kg, respectively. Thereafter, PBS solution was standardised with the osmolarity and pH of A. fulica .Initially, pH and osmolarity of g for 10 min at 4 °C in a 15 ml polystyrene vial. The supernatant (acellular fraction) was used for the analysis of PO and NO concentration. The haemocytes (cellular fraction) were washed 4× with PBS by centrifugation at 80× g for 10 min at 4 °C. The viability of the cells was observed by counting in a hemocytometer after a 1:10 dilution in 0.4% trypan blue (Sigma-Aldrich) [g for 10 min at 4 °C, and the supernatant kept on ice for analysis of PO.The total haemolymph of each group was centrifuged at 80× Aldrich) . The haeBoth the acellular fraction and the haemocyte lysate were subjected to a similar analysis of PO. Three substrates specific for PO enzymes were tested: (i) L-tyrosine  metabolized by tyrosinase only; (ii) L-DOPA  metabolized by the enzymes catecholase, tyrosinase and laccase; and (iii) p-phenylenediamine  metabolized by laccase only .We used 10 mM of L-DOPA  , L-tyrosine , and PPD  diluted in PBS immediately before the experiment to avoid oxidation.One-hundred microlitres of cell-free haemolymph, 50 μl of substrate solution, and 50 μl of PBS  were added to each well into a 96-well microtiter plate. As controls, we used 50 μl of substrate solution with 150 μl PBS. For each treatment, we tested samples, controls, and blanks (200 μl of PBS) in triplicate.The reaction kinetics were measured for 2 h. Readings were performed every 20 s at 28 °C in a microplate reader , using λ = 490 nm for L-tyrosine and L-DOPA, and λ = 465 nm for PPD. The results were plotted using the maximum reaction speed (i.e. the increase in the absorbance/min), according to Alves et al. .A. fulica was determined by measuring the concentration of nitrite (NO2) using the Griess test. Fifty microliter aliquots of the samples were allocated to a 96-well plate  and 50 μl of Griess reagent were added to each well. Following 10 min of reaction at room temperature, the reading was performed in a spectrophotometer at λ = 570 nm.NO dosage in the acellular fraction of A. fulica specimen used for haemolymph extraction. A 2 cm tissue fragment was separated from the shell and fixed in Milloning [Histological techniques also analyzed each illoning . Tissuesilloning . Histoloilloning  and Fontilloning  and thenilloning .post-hoc test. Differences at P < 0.05 were considered statistically significant.PO activity and NO production were analyzed by a one-way ANOVA, followed by the Tukey’s A. fulica was observed.The average proportion of L1 remaining in the containers used for infection was 25%. Thus, the average number of L1 that penetrated the molluscs was 750. Following the collection of haemolymph and digestion with 1% HCl-pepsin, we recovered larvae from all the molluscs. Percentages varied from 8 to 25% for L1, 7–19% for L2, and 12–30% for L3. Throughout the experiment, no mortality of A. fulica infected with A. vasorum and controls.We observed PO activity with the substrates L-DOPA, L-tyrosine, and PPD. Figure F = 22.67, P = 0.02), L-tyrosine  = 6.73, P = 0.00006) and PPD  = 27.58, P = 0.0019) was observed  = 22.67, P = 0.00002) decreased, while in PPD  = 27.58, P = 0.079) increased  = 22.67, P = 0.00029) and PPD  = 27.58, P = 0.000015) in relation to control  = 94.59, P = 0.003687). The amount of NO started to decrease at 13 dpi but was still significantly higher than the control  = 94.59, P = 0.001). At 30 dpi, NO levels were 15 times lower than the levels observed at 8 dpi (peak).NO levels were indirectly estimated in the soluble fraction of A. fulica in molluscs of the control  and tyrosinase (L-tyrosine).In the present study, we demonstrated that the soluble fractions of the haemolymph of Drosophila melanogaster [A. fulica was not detected inside the circulating haemocytes, nor in the cells infiltrated in the tissues analyzed.Very few studies have explored PO activity in molluscs, and most models involving molluscs do not have PO activity with the substrate L-tyrosine , 34. Actnogaster , 35, 36.nogaster , 37, 38.PO activity in the haemolymph at 1 dpi, detected with the substrates L-DOPA, L-tyrosine and PPD, coincided with the beginning of the detection of melanin in the epithelium. However, a lower amount of melanin was observed in the connective tissue in the surroundings of L1, where cells migrated.At 8 dpi, the levels of PO detected in the haemolymph decreased with L-DOPA and L-tyrosine substrates, in this day the moult from L1 to L2 may modulate PO activity through the antigens produced during the moult. The melanin and the cellular infiltrate increased in the connective tissue surrounding L2, suggesting that this tissue could lead to a mechanical contention of the parasite.A. fulica presented the highest levels of NO in the haemolymph at 8 dpi.Melanin circulates in the tissue infiltrate through the haemolymph spaces and, during the activation of PO pathway in insects, cytotoxic elements such as quinones and oxygen and nitrogen derivates are produced to boost the destruction of the pathogens’ tegument , 39, 40.The transformation from L2 to L3 seems to also negatively interfere with PO activity at 13 dpi for all the three substrates tested, and particularly PPD. At this interval, the levels of NO and circulating melanin in the epithelial and connective tissues and the cellular infiltrate next to the parasite started to decrease.A. vasorum penetrates and exits the A. fulica tissues [Diatraea flavipennella infected with the parasitoid Cotesia flavipes, modulation was observed in NO and PO levels throughout parasite infection [Finally, at 30 dpi, the modulation of the L-tyrosine-dependent PO pathway continued at the same level as the previous days of infection, whereas PO levels increased with L-DOPA and PPD. The PO oscillations observed during larval development may occur due to the detection of products released by L1 and L3 when  tissues , 42. Ano tissues . In insenfection . HoweverAngiostrongylus spp. that exhibits motility and allows its active output from the granuloma in molluscs tissues [Biomphalaria tenagophila infected with A. vasorum at 30 dpi, a decrease in NO level was observed in relation to the control group [The decrease of NO at 30 dpi in comparison to the control may be due to the association of biological characteristics of L3 of  tissues , 45. Als tissues , 46. In ol group .A. fulica presents an innate immune defence mechanism based on PO pathway. According to Allam & Raftos [A. vasorum may be taking place. Le Clec’h et al. [Biomphalaria glabrata infected with Schistosoma mansoni presents laccase-dependent PO activity but did not associate the activation of this pathway with the susceptibility to the parasite. The oscillations in the levels of PO, tissue melanin, and NO observed in our model may contribute to the control of the infection. Indeed, Coaglio et al. [A. fulica infected with A. vasorum, in contrast with the over 80% recovery of L3 reported by Mozzer et al. [Omalonyx matheroni and B. glabrata.Here, we suggest that & Raftos , this meh et al.  observedo et al.  recoverer et al.  in OmaloA. fulica during the infection with A. vasorum, similar to previous observations in B. glabrata [Lymnaea stagnalis infected with Plagiorchis sp. [D. melanogaster, PO is located in the crystal cells and lamellocytes [Unlike the arthropods (insects and crustaceans) that have PO located mainly in the haemocytes, we did not detect a response of PO in the populations of haemocytes of glabrata  and in hchis sp. . In insellocytes , 48.A. fulica, which may explain the lower susceptibility of A. fulica to Angiostrongylus spp. [L-tyrosine-dependent PO activity is frequent in arthropods , 49. To lus spp. , 50, 51.We suggest that the cytotoxic products, such as NO, released in the PO pathway, may play a role in controlling the infection inside the mollusc. Further studies will be needed to understand the PO-dependent defence mechanisms in this terrestrial mollusc.


To have the participants look at each face carefully, an identity judgement task was used: they were asked to judge whether the two faces presented in succession were of the same person or not. The ERP waveforms in response to the first faces were analyzed. In two experiments with different stimulus probabilities, the amplitudes of N170 and vertex positive potential (VPP) were smaller for faces with light makeup than for faces with heavy makeup or no makeup. The P1 amplitude did not differ between facial types. In a subsequent rating phase, faces with light makeup were rated as more attractive than faces with heavy makeup and no makeup. The results suggest that the processing fluency of faces with light makeup is one of the reasons why light makeup is preferred to heavy makeup and no makeup in daily life.The effects of makeup on attractiveness have been evaluated using mainly subjective measures. In this study, event-related brain potentials (ERPs) were recorded from a total of 45 Japanese women ( Facial makeup, which is intended to accentuate one’s attractiveness to others, might be crucial for many women in their daily lives ,2. When For women, makeup manipulates the typical femininity of the face by modifying the bilaterally symmetric balance, the configurations of parts of the eyes and mouth, and skin color and texture . Women wThe effects of makeup on attractiveness have been evaluated using mainly subjective measures. For example, Mulhern et al. (2003) reported that both men and women judged Caucasian female faces with full makeup more attractive than faces with lip-only makeup or no makeup . Nash etThe purpose of this study is to illustrate the effect of makeup on interpersonal cognition by examining event-related brain potential (ERP) responses to faces with no makeup, light makeup, and heavy makeup. ERP measures have been used to examine the differences in processing between attractive and unattractive faces –15. To oAnother line of research suggests that unnatural faces elicited a larger N170 response. Minami et al. (2011) reported that N170 amplitude was larger and N170 latency was longer for a bluish-colored  face than for a natural-colored face . Using fOn the other hand, Halit et al. (2000) reported no N170 differences between the original faces and the unnaturally stretched faces of which the eyes were moved upward and the mouth was moved downward . Wiese eAlthough there are some exceptions, the previous findings generally suggest that N170 amplitude is modulated by the atypicality or unnaturalness of the face and that fluent processing of a face is associated with a smaller N170 response. As processing fluency leads to positive affect , 21, facIn the present study, we investigated the influence of light makeup and heavy makeup on facial perception by means of ERP and ratings of attractiveness. We hypothesized that makeup influences the early stages of facial processing that are reflected in the N170 amplitude and its polarity-inversed counterpart, the vertex positive potential (VPP). If the N170 amplitude is inversely related to the processing fluency of the face, as proposed by Trujillo et al. (2013), a smaller N170 amplitude would be expected for faces with makeup than for faces with no makeup, because makeup would conceal the negative features of individual faces and make them closer to the average . On the In addition to the N170, the P1 component, which precedes the N170, was analyzed to see whether any physical differences in the faces and/or attention modulation affect ERP responses to faces with makeup. Halit et al. (2000) showed that the P1 amplitude was larger for unattractive faces than attractive faces . They alTo have the participants look at each face carefully, an identity judgment task similar to that in Ueda and Koyama (2014) was used . In thisSD = 7.0, range = 20–33).Thirty Japanese women were recruited by a research firm and paid for participation. The conditions of recruitment were (1) physically and mentally unimpaired, (2) being right-handed determined according to self-report, (3) having reported normal or corrected to normal vision determined according to self-report, and (4) wearing makeup with a mid-priced lipstick and foundation three times a week or more. We considered that women who wear makeup regularly in daily life are aware of the effects of makeup and can make use of the advantage it provides. This was also the reason why only female participants were recruited. Participants gave written informed consent before testing. The Ethical Committee of the Shiseido Global Innovation Center approved this study. Participants who showed a large number of eye blinks during electroencephalogram (EEG) recording and who thus had an insufficient number of artifact-free EEG trials were excluded from the analysis . The following analysis was conducted on the data from 23 women  = 2.30, p = .217, ηp2 = .54.The stimuli were 18 Japanese female faces that encompassed three types of facial images  of six female models (25–35 years old). All the styles of makeup were manipulated by a professional makeup artist. Participants were seated in a comfortable chair in a dimly lit room. The stimuli were presented on a computer screen  using Inquisit 4.0 . All the faces were displayed against a black background and subtended 8° in width and 10° in height at a viewing distance of 70 cm.makeup sequence) or from makeup to no makeup (no-makeup sequence). Each sequence consisted of 144 trials. The first face and the second face were of the same person in 72 trials and of different persons in 72 trials. The faces of the six models were presented equiprobably. In the makeup sequence, the first face was always a no-makeup face and the second face was one of the three types . In the no-makeup sequence, the first face was one of the three types and the second face was always a face with no makeup. The faces were changed either from no makeup to makeup  using a 100-point visual analog scale on paper for their impressions of the faces of 10 models with no makeup, light makeup, and heavy makeup. Each participant rated only one makeup type of each model, that is, 10 faces in total. The makeup types of each model were counterbalanced across participants.Electroencephalogram (EEG) data were recorded from 19 scalp sites  according to the 10–20 system using BIOSEMI active electrodes . Horizontal and vertical electrooculograms (EOGs) were recorded from the outer anti of both eyes and from above and below them. The sampling rate was 256 Hz.The EEG data were converted to the average reference, and an offline filter of 0.1–30 Hz was applied. Ocular artifacts were corrected by the independent component analysis (ICA) function implemented in the Brain Vision Analyzer 2.1.0 . ERPs in response to the first faces were calculated for 400 ms (from 100 ms before and 300 ms after stimulus onset). Because a visual mask was presented 300 ms after stimulus onset, we only analyzed the epoch before the mask that might evoke other ERP responses. The periods containing artifacts exceeding ±80 μV in amplitude (after the ocular correction) were rejected from the average. The participants who had an insufficient number of artifact-free trials  were excluded from the analysis. Each ERP waveform was aligned to the 100-ms pre-stimulus baseline by subtracting the mean amplitude of this period from each point of the waveform. ERPs were quantified using the mean amplitudes of P1 (80–110 ms) components for O1 and O2 and N170 (120–170 ms) components for T5, T6, and Cz.t tests to a stepwise fashion and increases the probability of detecting differences without changing the control of the Type I error [The response accuracy was analyzed by a multivariate analysis of variance (MANOVA) with repeated measures. The same analysis was also conducted on the data after arcsine transformation. Because both analyses yielded similar results, the results without transformation are reported here. The percentage of the choice of faces with light makeup in a forced-choice task was analyzed by a binomial test. Subjective rating scores were analyzed by a MANOVA with a single factor of makeup type . The mean amplitudes for P1 and N170 were analyzed by a MANOVA with two factors: makeup type  and electrode site . Because the present study used repeated-measures designs, we follow the recommendation of Vasey and Thayer (1987) and report MANOVA solutions to compensate for possible type I error inflation by the violation of sphericity . The res I error . The sigp = .67, .17, and .17 for faces with no makeup, light makeup, and heavy makeup, respectively. In Experiment 2, we equalized the presentation rates of the three types of faces (p = .33). Moreover, to cause participants to think of the identity of the faces deliberately, a visual mask was not presented and the interstimulus interval between the first and second faces was extended to 700 ms. Unless otherwise mentioned, the methods were identical to Experiment 1.Because Experiment 1 replicated the protocol of Ueda and Koyama (2014) , the preSD = 2.4, range = 27–35) were analyzed.Thirty Japanese women were recruited and participated in Experiment 2. They had not participated in Experiment 1. Based on the same criteria used in Experiment 1, the data of 22 women  were used. For each model, three makeup types could appear either in the left or right position, which resulted in nine combinations . A set of 54 trials (9 combinations × 6 models) was conducted twice. For each combination, the chosen type was coded as +1 and the unchosen type was coded as -1. The sum of the values was calculated for each makeup type. If a certain makeup type was chosen consistently, the sum would be 48. Subjective ratings using analog visual scales were not administered.p = .001, two-tailed).In the forced-choice task, faces with light makeup were selected as a face with a better impression more frequently than faces with heavy makeup. On average, faces with light makeup were chosen 17.87 times (89.3%) out of 20 pairs  = 3.08, p = .067, ηp2 = .23. Multiple comparisons showed that the P1 amplitude was marginally smaller for heavy makeup than for no makeup (p = .070). The effect of site and the interaction effect were not significant, F = 1.99, p = .172, ηp2 = .08; F = 0.75, p = .486, ηp2 = .07, respectively. On the other hand, the N170 amplitude showed a significant main effect of site and a significant interaction, Makeup type: F = 1.63, p = .220, ηp2 = .13, Site: F = 3.75, p = .041, ηp2 = .26, Makeup type × Site: F = 3.70, p = .022, ηp2 = .44. The significant effect of site reflects the polarity inversion of the N170/VPP. A one-way MANOVA was conducted for each site. The effect of makeup type was significant at T5 and Cz, F = 4.05 and 5.21, p = .032 and .015, ηp2 = .28 and .33, respectively. Multiple comparisons of means showed that the N170 amplitude was smaller (less negative) for no makeup than for heavy makeup at T5 (p = .009) and that its counterpart VPP was smaller (less positive) for light makeup than for heavy makeup at Cz (p = .004). The main effect of makeup type was marginally significant at T6, F = 2.62, p = .097, ηp2 = .20. Multiple comparisons showed that the N170 amplitude was smaller for light makeup than for heavy makeup (p = .037). There were no significant differences between no makeup and light makeup in the N170/VPP amplitude and between no makeup and heavy makeup in the VPP amplitude. In sum, faces with heavy makeup elicited a larger N170/VPP than did faces with no makeup or light makeup.M = .86) as compared with light-makeup pairs (M = .89) and no-makeup pairs (M = .91), F = 9.12, p < .001, ηp2 = .48. Post-hoc pairwise comparisons showed significant differences between heavy makeup and light makeup (p = .002) and between heavy makeup and no makeup (p = .001).M = 35.91 (SD = 10.57), M = -7.82 (SD = 14.29), and M = -28.09 (SD = 17.88) for faces with light makeup, heavy makeup, and no makeup, respectively. A Friedman test showed a significant effect of makeup type . Post-hoc multiple comparisons using Wilcoxon signed-rank tests revealed that the measures of the three makeup types differed from one another (p < .01).In the forced-choice task, faces with light makeup were chosen as faces with a better impression most frequently. Faces with heavy makeup were in second place, and faces with no makeup were the least chosen. The choice measure (varies from 48 to -48) were F = 0.39, p = .685, ηp2 = .04; Site: F = 2.80, p = .109, ηp2 = .12; Makeup type × Site: F = 0.33, p = .723, ηp2 = .03. For the N170 amplitude, the main effects of site and interaction were significant, Makeup type: F = 1.38, p = .274, ηp2 = .12; Site: F = 18.99, p < .001, ηp2 = .66; Makeup type × Site: F = 3.33, p = .033, ηp2 = .43. The significant effect of site reflects the polarity inversion of the N170/VPP. A one-way MANOVA was conducted for each site. The effect of makeup type was significant at T6 and Cz, F = 4.13 and 6.30, p = .031 and .008, ηp2 = 0.29 and .008, respectively. Multiple comparisons of means showed that the N170 amplitude was smaller (less negative) for light makeup than for heavy makeup at T6 (p = .009) and that its counterpart VPP was smaller (less positive) for light makeup than for heavy makeup at Cz (p = .002). In addition, the VPP was smaller for light makeup than for no makeup at Cz (p = .019). The main effect of makeup type was not significant at T5, F = 1.24, p = .311, ηp2 = 0.11. Multiple comparisons did not show any amplitude differences between makeup types.This study examined how makeup influences the early components of ERPs (P1 and N170) in the facial recognition process using faces of the same female models with no makeup, light makeup, and heavy makeup. Two experiments with different stimulus probabilities consistently showed that the amplitude of P1 (80–110 ms) at the occipital sites showed no significant difference between makeup types. In both experiments, the amplitude of N170/VPP (120–170 ms) was smaller for faces with light makeup than for faces with heavy makeup at the central site (Cz). The latter difference was also significant at the right posterior temporal site (T6) in Experiment 2, in which the three types of faces were presented equiprobably. In addition, the N170/VPP amplitude at Cz was smaller for faces with light makeup than for faces with no makeup in Experiment 2. As compared to no makeup and light makeup, heavy makeup deteriorated the performance in the identity judgement task in Experiment 2. In both experiments, faces with light makeup were chosen more frequently as a face with a better impression than faces with heavy makeup. On the other hand, Experiment 2 showed that faces with heavy makeup were chosen more frequently than faces with no makeup.The result that the P1 amplitude did not significantly differ between makeup types suggests that the ERP differences occurred at a level higher than physical characteristics. Although the P1 amplitude was numerically smaller for heavy makeup than for no makeup in Experiment 1, this difference was not found in Experiment 2. In Experiment 1, differences in stimulus probability across makeup types may affect P1 amplitudes. Therefore, the present study did not provide strong evidence that makeup types affect P1 amplitudes. It has been suggested that the P1 effect depends on low-level visual cues of faces , 29, wheM = 18.57 out of 100) was rated to be significantly less natural than light makeup (M = 46.47) and no makeup (M = 72.05). In the present study, the N170 amplitude at the right posterior temporal site (T6) was smaller for light makeup than for heavy makeup. Schultz et al. (2012) reported that the N170 amplitude in the right hemisphere was greater for a spatially exaggerated face than for veridical and non-exaggerated faces [A puzzling finding is that a smaller N170 amplitude was found for faces with light makeup but not for faces with heavy makeup. With heavy makeup, individual faces become less distinctive and more difficult to be recognized correctly . In a seIn both experiments, there were no significant differences in the P1 and N170 amplitudes between faces with heavy makeup and no makeup. Because faces with heavy makeup were rated to be more attractive than faces with no makeup, processing fluency, which is reflected in the reduction of the N170 amplitude, alone does not explain the subjective ratings of attractiveness. Heavy makeup involved larger physical changes in appearance than light makeup. Therefore, the finding that the P1 and N170 amplitudes did not differ between faces with heavy makeup and faces with no makeup supports the idea that the N170 reduction for faces with light makeup  was not due to low-level perceptual differences.The most accurate facial recognition was achieved when the first and second faces were of the same makeup type . This result is reasonable, because the identical pictures were presented twice in half of the trials for these pairs. Interestingly, the identification performance for heavy makeup–heavy makeup pairs was worse than for the other pairs. As Tagai et al. (2016) pointed out, heavy makeup may conceal the idiosyncratic features of the person and thus hinder identity judgement . The ligIn the forced-choice task, light makeup was chosen more frequently than heavy makeup as a face with a better impression. This result is consistent with previous findings in Japan . Light mThere are several limitations in this study. First, we could not determine exactly which features of the light makeup led to the reduction of the N170 response. For example, according to the definition of the present study, the light and heavy styles of makeup used different colors: the light makeup was based on soft red with natural skin tone, whereas the heavy makeup was based on dark brown with matte skin. Minami et al. (2011) and Nakajima et al. (2012) suggested that facial color can affect the N170 amplitude , 17. As In summary, the present study suggests that faces with light makeup are processed more fluently than faces with heavy makeup or faces with no makeup. This may be one of the reasons why light makeup is preferred and rated to be more attractive than heavy makeup in daily life. Together with the findings of Tagai et al. (2016) , light m


Oxytocin (OT) concentration in the blood is considered to be a marker of its action in the brain. However, two problems have emerged when measuring OT level in the blood. First, it is unclear whether different methods of assessment lead to similar OT values. Second, it is unclear if plasma OT concentrations is informative on what OT does in the brain. To clarify these issues, we collected cerebrospinal fluid (CSF) from the brain ventricle of 25 patients during surgery to compare with plasma OT after simultaneous blood withdrawal. Additionally, we collected 12 CSF and blood samples from non-human primates while awake or under anaesthesia. We used four methods to assay OT concentrations: Commercial EIA with/without extraction, laboratory developed EIA with filtration and RIA with extraction. Three of these methods showed a positive correlation between plasma and CSF OT, suggesting a link between plasma and central OT, at least under specific testing conditions. However, none of the methods correlated to each other. Our results show major disagreements among methods used here to measure peripheral and brain OT and therefore they call for more caution when plasma OT is taken as a marker of central OT. Notably, OT is implicated in various aspects of social behaviour and can positively or negatively modulate how an individual interacts with conspecifics2. This OT functional property has attracted a lot of attention within the scientific community, hoping to develop new pharmaceutical approaches to treat psychiatric disorders such as autism or schizophrenia3, which are characterized by social dysfunction. In this context, one way to study OT in humans is to measure its concentration in the plasma and to correlate it with a behavioural outcome4. Although widely used this approach suffers of two major flaws. Firstly, it is unclear whether peripheral oxytocin (OTp) is linked to central oxytocin (OTc). To demonstrate such a link, researchers have tried to compare plasma OT concentrations to OT levels in the cerebrospinal fluid (CSF), the latter thought to reflect the global activity of the oxytocinergic system in the brain5. Secondly, there is an important debate regarding how to measure OT levels, since it is unclear whether different assessing methods can generate converging results.Oxytocin (OT) is a neuropeptide synthesized in the hypothalamus which has a wide range of actions both in the brain and in the body5. OT is distributed in extracellular space and CSF mainly through dendritic release7. Axonal and dendritic release of OT seem to be independently regulated8 and do not act on the same time scale: axonal release is instantaneous while dendritic release is slower (OT peaks in CSF dozens of minutes after stimulation)9. Despite evidence against a coordinated action between brain and body OT, yet some animal studies suggest the existence of a link. Wotjak and colleagues found that in chronically stressed rats plasma OT concentrations reflected OT levels in the SON10, thus suggesting that although independent, OTc and OTp can be released simultaneously. Further, Knobloch et al.11 discovered a group of PVN magnocellular neurons projecting both to the amygdala and to the posterior pituitary, releasing OT in these two regions. These results were confirmed recently by studies showing central projections of OT neurons, notably an axonal magnocellular pathway in the forebrain14. Following these results, it is reasonable to conclude that, under certain conditions, there is a coordinated axonal release of OT in specific brain areas and in the blood.Anatomical evidence shows that OTp and OTc are released through independent pathways. The paraventricular (PVN) and the supraoptic (SON) nuclei of the hypothalamus, the centres of OT synthesis, contain magnocellular neurons with axons projecting to the posterior pituitary, where OT is released into the bloodstream16 four others failed to find a significant link20. In all of these experiments, CSF OT was collected by doing a lumbar puncture, a method that extracts CSF in a region far from OT synthesis site. OT collected at this remote place can be degraded compared to brain OT because CSF flow is slower in the spinal cord7. Moreover, lumbar puncture procedure interfere with neurotransmitters levels including OT22, by generating acute stress, a condition that further modulates OT concentrations23. This view finds support in a recent meta-analysis which shows that the relationship between central and peripheral levels of OT is dependent on sampling condition24.Whether similar mechanisms regulate OT release in humans is unknown. Previous attempts to link OTp and OTc have led to divergent results. Although two studies performed in headache patients and in children have found a correlation between OTp and OTc26. Before the advent of commercial Enzyme Linked-Immunosorbent Assay (EIA), the most common approach was the Radioimmunoassay (RIA) technique associated with solid phase extraction. Several laboratories have developed this technique in the eighties and many studies have obtained physiologically plausible results. Thus, RIA has been since considered as the gold standard for assessing peripheral OT. More recently, plasma concentrations of OT have been measured with the immunoassay method combined with an extraction process to remove interfering proteins. With this procedure, baseline plasma OT levels are typically comprised between 1–10 pg/mL25. When several teams started to assay non-extracted samples, their results conducted to extremely high values. For instance OT levels can go from 1.8 pg/mL after extraction up to 358 pg/mL without extraction27. High values obtained without extraction are problematic because they do not match the theoretical concentration when considering OT production rate, release and clearance rate26. Hence, OT pre-processing whether performed with solid phase extraction or filtration is strongly recommended in order to obtain robust results on plasma OT real concentrations. Thus, we believe it is urgent to conduct a comparison between the methods employed to investigate plasma OT.In addition to the lack of knowledge regarding the biological significance of OTp, serious methodological criticisms have recently emerged concerning its measurements28 and RIA18).The aim of this work was to study the correlation between OTc and OTp and to compare different sample pre-processing techniques  and type of assay .In addition, to look at the effects of anaesthesia and to test a fourth method, the most used one , we collected simultaneous CSF and blood samples from awake or anaesthetized macaques.Twenty-five adult patients  requiring brain surgery  participated in this study. Patients with history of severe depression or other major psychiatric disorders, those abusing of drug, alcohol and/or tobacco, and pregnant women were excluded from the study.All patients or their legal guardian gave a written informed consent. The study was approved by the Ethical Committee Lyon Sud-Est IV and by the Biomedical French Agency (ANSM). The experiment and all of the involved procedures were performed in accordance with the relevant guidelines and regulations applied in Europe.Nine patients suffered brain damage because of a tumour . Of the remaining 16 patients, 9 suffered from hydrocephalus, 6 from CSF hydrodynamic abnormalities and 1 from skull base defect. CSF extraction (location was contingent on surgery type) were collected in the subarachnoid space (N = 9), within the ventricle (N = 7) or at the end of a ventriculoperitoneal shunt tap (N = 9).Blood and CSF were simultaneously collected during surgery at the Neurological Hospital in Lyon. Patients fasted overnight. Seven millilitres of blood were collected in EDTA tube and up to five millilitres of CSF were collected in a polypropylene tube, immediately after CSF aspiration by the neurosurgeon. Tubes were immediately placed on ice and transported to the nearby Neurobiotec Center , where samples centrifugation and storage occurred. CSF and blood were centrifuged during 10 minutes at 2000 × g at 4 °C. Note that there is no need for CSF centrifugation, but this was done so all samples undergo the same treatment. Plasma and CSF were then stored in a freezer at −80 °C until assay occurred within an hour after sampling.29 with a head post and a chamber located above the right lateral ventricle were involved in the present experiment. We precisely localized the right lateral ventricle, guided by structural MR images, and sampled 250 µL of CSF with a 23 Gauge needle and a 1 mL syringe. This procedure was done in both awake animals under head restraint conditions (7 samples) and on anesthetized animals (5 samples). For procedure details, see Lefevre and colleagues29. Simultaneously, 1 mL of blood was withdrawn from the saphenous vein. Immediately after collection, samples were centrifuged during 10 minutes at 2000 × g at 4 °C, and stored at −80 °C until assay. The experiment and all of the involved procedures were performed in accordance with the relevant guidelines and regulations  and approved by the animal ethical committee CELYNE n°42 (ref 02075-01) and the French Ministry of agriculture and environment.Three male rhesus macaques (mean age 4.1 years), implanted for another experiment28), OT RIA (the method from Landgraf’s lab18), OT M (macaques).Three different methods were used to measure OT in human samples, and one for macaques’ samples. They will be referred as, respectively, OT ELS (Enzo Life Science), OT Lg  kits . It should be noted that extraction is “strongly recommended” by the manufacturer and that we deliberately chose to skip this step-in order to demonstrate the importance of the procedure on OT values. This EIA is a competitive assay (as opposed to the “sandwich” technique), and is therefore sensitive to the various interfering products in samples. Each sample was tested in duplicate . Prior to assay, plasma samples were diluted to 1:4 . Cross-reactivity with related peptides is according to manufacturer <0.001% and assay sensitivities 11.6 pg/ml. We will refer to this technique as OTc (CSF) ELS and OTp (plasma) ELS.et al.28 and already reported in several studies32. Briefly, this method is a laboratory developed EIA involving a filtration protocol before the assay in order to remove all molecules  that weight more than 3 kDa and with a sensitivity of 1 pg/ml. Thus, samples bellow 1 pg/ml were truncated at 1 pg/ml. We will refer to this technique as OTc Lg and OTp Lg (“Lg” stands for “Liège”).Second, we sent our samples to an independent lab  blind to the goal of the study, where they assayed OT using a method previously described by Péqueux 18. Prior to the assay, samples were extracted as previously described in the literature18. The analysis was performed on codified samples, without knowing which CSF samples matched to plasma samples. We will refer to this technique as OTc RIA and OTp RIA.Third, we sent our samples to another independent lab headed by Pr Rainer Landgraf, who has developed the Radioimmunoassay (RIA) procedure for OT. This method has often been used to assay OT in various species and details can be found in2O) and 250 µL of plasma were centrifuged at 16000 g for 15 minutes at 4 °C. The supernatant was applied to a 200 mg Sep Pak column (previously equilibrated with 1 mL of acetonitrile and 15 mL of 0.1% TFA- H2O) and washed with another 15 mL of 0.1% TFA- H2O. The sample was then eluted with 3 mL of 95% acetonitrile/5% of 0.1% TFA- H2O, and the eluate (kept cold in an ice bath) was evaporated with compressed nitrogen gas and stored at −20 °C until assay. Prior to assay, samples were reconstituted with 250 µL of assay buffer. One sample was spiked with 500 pg of OT and found to have a recovery rate of 102.5%. We will refer to this technique as OTc M and OTp M.Finally, monkey samples were assayed using the same EIA kit (Enzo Life Science) that was used for human samples, but with the addition of a solid phase extraction (SPE). 250 µL of 0.1% trifluoroacetic acid . We applied log transform to all variables in order to restore normal distribution and to perform parametrical statistical tests. Pearson’s correlation tests were used to evaluate correlation between log transformed OT concentration of simultaneous CSF and blood samples. Moreover, we also tested correlations between methods for both CSF and plasma OT values.To complement this analysis, we also used Spearman rank correlation tests on raw values, as non-parametric statistics are not sensitive to biased distribution and outliers.Our sample size of 22–25 patients (depending on the assay) has 80% power (20% chance to fail rejecting the null hypothesis) to detect r = 0.57–0.54. We consider however that correlation coefficient lower than 0.5 would not be strong enough to use plasma OT as a reliable indicator of central OT.All statistical tests were performed with STATISTICA 8 or MATLAB 2013a.33 to determine if we could conclude statistical equivalence for the correlation between methods. We set the equivalence bounds to r = −0.5 and 0.5 as we estimate that lower coefficients of correlation are not indicating equivalent measures of the same samples by the different methods.For cases of non-significant correlations between the methods, we used equivalence tests based on the two-one sided test procedureThe datasets generated and analysed during the current study are available from the corresponding authors on reasonable request.Out of the 25 patients involved, we could not obtain enough CSF for 3 of them to perform every measurement method. Thus only 22 simultaneous CSF and plasma samples were measured with the OT Lg and OT RIA methods. In addition, one CSF sample could not be assessed with the OT ELS methods due to technical issue. Table Our analysis on log transformed concentrations revealed significant positive correlations between OT concentration in the plasma and OT concentration in the CSF for three out of the four methods used, namely, OT ELS , OT Lg , OT M   and plasma (OTp) OT concentrations, none of the correlations we performed revealed an effect, whether we used Pearson ) or Spearman tests on log transformed , ELS and Lg tended to be independent (p = 0.07), and RIA and ELS were not independent (0.1) but the negative correlation of r = −0.25 is highly unexpected and does not suggest in our view an agreement between the two methods.Equivalence tests for measurements in plasma revealed that RIA and Lg led to independent measures (p = 0.04), ELS and RIA were not independent (p = 0.16) but once again negatively correlated (r = −0.31) and ELS and Lg measures were not independent (p = 0.21).To sum up, it can be concluded that we did not found any concordance among the methods tested, excepted for OTp ELS and RIA, for which our data are not sensitive enough to reject the null hypothesis or to support statistical equivalence.For each method, we did not find an effect of sex on OT levels in both CSF and plasma  . No significant effect was found  for hydrocephaly, subarachnoid cavity (n = 7) for tumours, ventriculoperitoneal shunt tap (n = 9) for CSF hydrodynamic disorders), had an impact on OT concentrationWe also tested the influence of different analgesic drugs . We found no effect .Our study provides two main results. First, we found a significant relationship between OTp and OTc with three methods  but the one measure considered to be the gold standard for OT measurement (OT RIA) did not led to similar results. These findings therefore call for more caution when considering what information is inferred from peripheral OT levels. Second, we found no correlations between any of the measures. The lack of agreement between OT ELS without extraction, OT Lg and OT RIA raises doubts on exactly what is measured by these methods, at least in human subjects under stressful conditions.1, OT has also many other physiological roles, such as in pain regulation35, skeletal homeostasis37, muscles maintenance and regeneration38, energy metabolism39, body temperature41, cardiovascular monitoring44. It must be noted that while pain seems to be jointly regulated by OTc and OTp, it is less clear if other peripheral effects of OT are linked to central OT, even though this could still occur through peripheral feedback to the brain46. Thus, even if OTp and OTc are correlated under specific circumstances, the information given by these two measures is unlikely the same. Hence, correlating OTp with behavioural scales seems to be a suboptimal method to investigate links between the OT system and behaviour, as OTp might be a noisy proxy of OTc. Of course, it can be hypothesized that physiological actions of OTp in the body are synchronized to OTc effects on behaviour, for instance to adapt cardiac rhythm to the emotional state47. But this assumption still need to be demonstrated.Given these divergent results on the relation between OTp and OTc, it appears clear that OTp cannot be used as a biomarker of central OT as its biological significance remains unclear. Aside from its well-known action in giving birth and lactationAdding to this problem is the absence of agreement between methods tested here. Importantly, while this issue was expected for OT plasma concentration given the influence of pre-processing procedure , the fact that CSF OT levels assayed with three different specific antibodies were completely unrelated  is surprising, as there are supposedly no interfering proteins in this matrix. One can expect different concentrations values due to dissimilar assay sensitivity, but the order between values should remain identical. This was not the case here, as even non-parametric tests did not lead to significant results.27. A recent finding showing that in the blood OT can bind to a larger molecule48 may explain the differences we observed here, suggesting that after extraction or filtration we might only assay “free OT” but ignore the OT that is bound. This hypothesis leads to new questions regarding the biological significance of “free OT” and “bound OT”, the rate of exchange between the two compartments and if the OT we measure without extraction or filtration corresponds to the combined free and bound OT. One critical step will be the identification of the molecule(s) to which OT binds to, and whether several potential candidates such as albumin or Neurophysin 1 can be considered50. Unfortunately, this idea is still very speculative and deserves confirmation and further research.Thus, more work is necessary to compare OT methods and one important factor is sample pre-processing52. They all reported plasma OT levels, following extraction, within the classical range of 1–10 pg/ml, (but see53). We suggest that it would be interesting to look at the effect of pre-processing on these values, and whether OT levels measured in the CSF with LCMSMS are coherent with those measured by EIA and RIA.We have no recommendations here regarding the optimal way to assess OT levels, as these issues should be clarified by scientists and companies providing these methods. We believe that in depth chemical analysis is required to check and compare the influence of pre-processing methods on endogenous OT and if differences in binding site recognition by the various antibodies could be responsible for such discrepancies. Recently, several teams have used liquid chromatography coupled to tandem mass spectrometry (LCMSMS) to assess OT levels23. Lastly, CSF flow is much slower in spinal cord than in central system7, so CSF OT at lumbar level can be reduced. Altogether, we believe that CSF OT levels obtained after lumbar puncture might be too variable.Our experiment has some limitations. We extracted CSF and blood at least 30 minutes after general anaesthesia and skin incision. We assumed this was an optimal interval to observe a similar modulation in plasma OT and CSF OT concentrations contrary to lumbar puncture procedure, which causes acute stress, and therefore OT release may not have enough time to act on CSF OT concentration. In addition, lumbar puncture generates pain which probably prompts a fast release of oxytocin in some segments of the spinal cordIt should be noted that due to limited volume of samples in humans, we were not able to perform extraction prior to assay with the commercial EIA (ELS). Notably, the manufacturer strongly recommends this step and therefore we must acknowledge that this might have increased the disagreement between the methods tested here. Thus, from the present results we cannot conclude whether ELS with extraction correlates with the other methods.Our patients were suffering from different types of pathologies  but no differences in the hormones’ levels were observed between the three groups. This can be explained by the absence of damage in the hypothalamus, the core area of oxytocin synthesis. Moreover, the wide variety of patients’ aetiology indicates that our results are unlikely due to a specific pathological effect.54. Furthermore, our results in non-human primates also show that anaesthesia does not alter OT levels.The various anaesthetics and analgesic drugs used during surgery did not produce any effect on the neuropeptide concentrations as well, and this result is in accordance with a previous study showing that, during surgery, plasma levels of OT raised after skin incision but not after anaesthesia inductionIn our equivalence analysis, because of our sample size, we had to set equivalence bounds to r = −0.5 and 0.5. Thus, we could not reject smaller correlations. However, correlations lower than 0.5 would still, in our opinion, be insufficient to use plasma OT as a reliable marker of its action in the brain. Future studies are needed to clarify this point.Following assay of plasma with the OT Lg technique, several values (n = 7) had to be truncated to 1 pg/ml. It should be noted that higher sensitivity could have impacted the correlation between plasma OT and CSF OT, and therefore this specific result should be interpreted with additional caution.Finally, although we found a correlation between OTc and OTp with the commercial EIA after extraction, this result was obtained in a limited number of samples (n = 12) from rhesus macaques, and we cannot rule out potential species differences with humans.24 suggesting that OTp and OTc might be correlated under specific stress conditions although this seems to vary with the method employed. Therefore, we believe that plasma OT should not be used to infer central OT levels, unless one can prove that in the sampling conditions, and with the assay method used, plasma OT is correlated to central OT. Last but not least, we call for more inter methods comparisons in both plasma and CSF  samples, as it seems that each method is producing different values of OT concentrations.Altogether, we believe that our protocol produced a release of OT at both peripheral and central level, given the intensity and the length of stress induced by a surgical intervention. Thus, it should be noted that, given the specific context in which we collected our data, we cannot firmly conclude on the link between OTc and OTp at basal state. Our study is in agreement with a recent reviewsupplementary results


Leptomeningeal disease is a feared sequelae of malignant paediatric brain tumours. Current methods for its detection is the combined use of cranio-spinal MRI, and CSF cytology from a post-operative lumbar puncture. In this study, the authors hypothesize that CSF taken at the start of surgery, either from an external ventricular drain or neuroendoscope will have equal sensitivity for positive tumour cells, in comparison to lumbar puncture. Secondary hypotheses include positive correlation between CSF cytology and MRI findings of LMD. From a clinical perspective, the key aim of the study was for affected paediatric patients to avoid an additional procedure of a lumbar puncture, often performed under anaesthesia after neurosurgical intervention.This is single-institution, retrospective study of paediatric patients diagnosed with malignant brain tumours. Its main aim was to compare cytological data from CSF collected at the time of surgery versus data from an interval lumbar puncture. In addition, MRI imaging of the same cohort of patients was examined for leptomeningeal disease and corroborated against CSF tumour cytology findings.Thirty patients are recruited for this study. Data analysis demonstrates a statistically significant association between our intra-operative CSF and LP sampling. Furthermore, our results also show for significant correlation between evidence of leptomeningeal disease on MRI findings versus intra-operative CSF positivity for tumour cells.Although this is a retrospective study with a limited population, our data concurs with potential to avoid an additional procedure for the paediatric patient diagnosed with a malignant brain tumour. Active perquisition of their existence is an important priority in affected patients. At present, the recommended method for LMD detection is the combinatorial use of gadolinium-enhanced magnetic resonance imaging (MRI) of the neuroaxis, and the cytologic examination of cerebrospinal fluid (CSF) specimens obtained within 2 to 3 weeks after surgery. This practice, utilised by our institution, is largely extrapolated from Gajjar et al . As partat the beginning of surgery, versus interval samples taken 2 to 3 weeks post-operatively via lumbar puncture. Given that CSF cytology is hypothesized to be dependent on neoplastic cells being shed from the primary tumour source, a logical assumption will be that at this time-point, a high pickup rate will be more likely—tumour at its largest and most number of cells. Given that false-negative detection of malignant cells on cytology is a common occurrence, higher CSF volumes will theoretically increase positive yield. Some studies have quoted a CSF volume of at least 10.5 ml to have feasible detection of malignant cells [Paediatric brain tumour patients commonly present with symptoms secondary to obstructive hydrocephalus, owing to the frequent midline, peri-circumventricular locations of their lesions. Here, hydrocephalus results from direct mechanical blockade of CSF flow from the primary tumour, often corroboratively with LMD, if the latter is present . Under snt cells , 5. UnfoFor this study, the authors hypothesize that CSF collected during at the start of brain tumour surgery will be reflective of overall LMD in affected patients. The primary objective for this project is to demonstrate that the sensitivity of CSF cytology when sampled intracranially at the beginning will be corroborative with post-operative CSF findings from lumbar puncture. Secondary objectives include the assessment of correlation between pre-operative MRI results with CSF results for LMD in the same cohort of patients.This is an ethics-approved, single-institution, retrospective study of patients aged less than 18 years old under the care of the Neurosurgical Service, KKH. The study is designed for proof-of-concept: focusing on the comparison of cytological data from CSF collected at the time of surgery versus data from an interval LP. Inclusion criteria involved all paediatric patients with malignant brain tumours who had a ventricular drain, or a neuro-endoscope inserted at the time of surgery, prior to tumour resection or biopsy. Following that, the same patient would have undergone LP up to 60 days post-operatively. Cerebrospinal fluid collected at both events were sent for cytology. For the purposes of this manuscript, ‘malignant brain tumours’ is defined as WHO III and IV as per WHO 2016 classification –11. Excl™ Signa on HD23 platform) or 3 Tesla scanner (Siemens™ Skyra on VE 11A platform) at our institution. Post-contrast T1-weighted images depicting leptomeningeal enhancement,  was the primary mode of diagnosis. Findings of contrast-enhancement over cranio-spinal areas in a‘sugar-coated’ manner are also included as positive findings. In addition, the MRI images were screened for presence of lesions along the cranio-spinal sites away from the primary tumour of interest.  point, or Frazier’s  point . For neuBroadly speaking, the patient was placed in lateral decubitus/ foetal position. If the child was too young or uncooperative, appropriate anaesthesia would be administered for the procedure. For very young children or those with difficult spinal anatomy, the lumbar procedure was performed by an interventional radiologist using imaging guidance. Under asepsis, a spinal needle would be usually introduced at the level of the L3/L4, or L4/L5 interspinous space. Up to 40 drops of CSF (this roughly equated to an estimated 2 ml) and thereafter, collected for required investigations once smooth flow was established. Post-procedure, the patient was kept supine for about 6 hours. During this period, there would be close monitoring of vital signs and the symptoms of post-LP headaches.™ preservative fluid , placed in a cytospin sample chamber and centrifuged at 600 rpm for 6 minutes. Alcohol-fixed smears (95% ethanol for at least 60 minutes) were placed in an autostainer machine for Papanicolau staining, and air-dried smears were stained using the Hemacolour® stain. Both cytospin samples and stained smears were then reviewed by our in-house pathologists.  CSF sampling for presence of tumour cells versus the current standard of practice of CSF sampling via LP, the diagnostic odds ratio, sensitivity and specificity of the test were presented. Level of significance was calculated using either chi-square test or Fisher’s Exact test. The sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of CSF cytology from EVD or NED, in comparison to LP, were evaluated against MRI findings of LMD. Statistical significance was set at A total of 30 patients were recruited for this study. This cohort consisted of 21 medulloblastoma, 2 intracranial germinoma, 1 pineoblastoma, 1 embryonal tumour with multi-layered rosettes (ETMR), 4 atypical teratoid rhabdoid tumour (ATRT) and 1 ependymoma. Patient ages ranged from 8 months to 15 years old (median age 7 years 2 months), and there were 20 males and 10 females. Intra-operatively, 5 patients had CSF collected via NED and 25 patients had individual EVDs inserted at the start of the surgery. These patients would be henceforth, be referred to as the ‘EVD/ NED’ group. Interval lumbar punctures were performed up to 30 days post-operatively (median time 17.7 days). . Based oFollowing that, 10 patients had MRI findings positive for LMD at the time of diagnosis. In this group, 4 patients had corroborative positive CSF cytology on both EVD/ NED and LP procedures. One patients with positive MRI LMD results had positive cytology from EVD/ NED sampling, but was negative on LP. A total of 3 patients were negative on cytology for both EVD/ NED and LP, despite their MRI scans depicting LMD. . Followip = 0.0034). The CSF sampling via EVD/ NED as a test for evidence of LMD demonstrates a sensitivity of 71.43% and specificity of 91.3%. Based on this, the results imply if EVD/NED sampling is negative, this will be more likely that the LP sampling will be negative for tumour cytology.  of 26.25 (2.94 to 234)  [Leptomeninges consist of the arachnoid and pia mater and form the boundaries of the subarachnoid space, separated from each other by fine trabeculae and CSF . Tumour  stages) , 15. OwiThe essential goal of CSF diagnostics is to detect neoplastic presence in the CSF for clinicians to react with the most compatible therapy . Early iDiagnostic gold standard for LMD is cytologic examination of the CSF, although this has limited sensitivity, and currently MRI is frequently the initial study for diagnosing LMD , 22. Lepstart of surgery, the previously undisturbed in situ tumour mass will be at its maximum volume. Hence, the yield for circulating daughter cells released by the primary tumour will potentially be higher. Next, majority of published papers had the cranial CSF tapped from either Ommaya reservoir or ventriculo-peritoneal shunt  [already planned operation. If successful, will hence, avoid the need for an additional invasive procedure which may require anaesthesia.Currently, CSF cytology is still considered the gold standard for diagnosis of LMD . While iservoir) , 5, 29. The authors acknowledge that there are limitations that should be highlighted. First and foremost, this is a retrospective study. Next, the patient population is smaller in comparison to larger, prospective series  reportedThis is a study focused on attempting to answer if direct CSF sampling at the time of naive surgery of malignant paediatric tumours will be a feasible option to avoid a post-operative LP in the same patient. The main objective was to firstly, ascertain if our cranial CSF sampling results will be corroborative with post-operative LP CSF cytology in our local cohort; and next, if this proposed method of CSF sampling can be an acceptable surrogate to replace LP for young children under such disease circumstances. Although the authors are aware that this is a retrospective study with a small population, our data concurs with potential to obliterate an additional procedure for the paediatric patient diagnosed with a malignant brain tumour. In meantime, we strongly advocate continued efforts to elucidate mechanistic pathways for better understanding of LMD in individual tumour types.