The aim of this cross-sectional analysis is to investigate the associations between the adherence to the Mediterranean Diet (MD) and semen quality parameters. To assess the adherence to the MD, the Trichopoulou score was used. Semen parameters were assessed as described in the 2010 WHO’s report and the results are showed across tertiles of MD adherence. A total of 106 participants were included. Compared to those in the lowest MD adherence tertile, participants in the top tertile had statistically significant higher BMI and waist circumference and consumed more energy, and also had statistically significant higher semen pH, and total sperm motility and progressive sperm motility percentages, and lower sperm immotility percentages. Moreover, percentage of total and progressive motility were significantly higher among those subjects in the higher adherence to MD in comparison with those in low-medium adherence category. The multivariable linear regression models evaluating the relationship between the sperm quality parameters and tertiles of MD adherence adjusted by age, energy and BMI showed that compared with the lowest tertile, men in the highest tertile had a higher percentage of total sperm motility [β non-standardized coefficient = 12.785]. These findings suggest that adherence to the MD was positively associated with sperm motility. Nowadays infertility affects ∼15% of the world’s population and male factors are responsible for 40–50% of these cases3. Although it has been shown to be a problem worldwide, this decrease is stronger in certain geographic regions of the world, specifically in the developed and industrialized countries5. Therefore, enhancing research on causes of this decline is urgently needed. There are multiple possible causes of this decline, however, the most plausible seems to be related to environmental and lifestyle factors such as pollution6, smoking7, alcohol consumption8, lack of physical activity9, psychological stress10, overweight or obesity11, and unhealthy diets12.Male reproductive health is declining year by year raising serious concerns and implications about human fertility15 supports the hypothesis that specific foods can have beneficial  or deleterious  effects on semen quality. Also, a recent meta-analysis of randomized clinical trials (RCTs)16 proposes that some nutrients and dietary supplements  could beneficially modulate sperm quality parameters and affect male fertility, even though, these last results must be cautiously interpreted due to the limited sample size of the meta-analyzed studies and the considerable observed inter-study heterogeneity. However, there are few studies focused on assessing the possible role of dietary patterns on semen quality parameters, especially in healthy and young population.Accumulating observational literature19, is characterized by a high consumption of olive oil, fruit, nuts, legumes, vegetables and whole cereals; a moderate intake of fish, poultry and wine; and a low consumption of dairy products, red meat, processed meats, and sweets. Because this dietary pattern has been proved to have beneficial effects in several intermediate metabolic outcomes, such as inflammation, oxidative stress and insulin resistance, all of which are related to sperm function, we can hypothesize that adherence to the MD can have benefits in terms of semen quality parameters.One of the healthiest dietary patterns, described as the Mediterranean diet (MD)Therefore, the aim of the present study was to investigate the associations between the adherence to the MD pattern and several recognized semen quality parameters  in healthy and reproductive-age males, using a validated MD score.A total of 244 subjects were assessed for eligibility, 119 were enrolled in the study and finally, 106 participants (89.1%) were included in the present analysis because a completed seminogram and dietary measures were obtained at baseline.6 spz. (27.9 × 106−125.5 × 106) for total sperm count, 24.1 × 106 spz./ml (10.8 × 106−43.1 × 106) for sperm concentration, 79.2% (72.3–84–8%) for sperm vitality, 66.3% (47.6–75.0%) form sperm total motility, 46.5% (28.7–57.8%) for sperm progressive motility and 6.4% (5.2–7.9%) for normal sperm morphology. Most men had at least one major semen analysis parameter  below the WHO 2010 reference values (58.5%).The mean age (±SD) of the study population (n = 106) was 24.7 (±4.7) years old. Most men had a normal BMI , one participant was underweight (0.9%), 26 were overweight (24.5%), and 5 were obese (4.7%). The median and interquartile range (IQR) values for the semen parameters were: 73.8 × 10The baseline characteristics of the participants according to the tertiles of MD are shown in Table Participants with higher adherence to MD score had a higher consumption of vegetables, legumes, fruits and nuts, cereals, fish and seafood. No significant differences across tertiles of MD adherence were detected in dairy products, meat, MUFA/SFA ratio or alcohol intake Table .Table 2GAs shown in Fig. Table In this cross-sectional study conducted in healthy and young population we found that higher adherence to a Mediterranean diet, measured by the validated Trichopolou’s score, was associated with an increased sperm motility. Beyond the potential confounding factors considered in advance for the present study , this association was not only independent of the age and BMI of the participants, but also for the total energy consumption.12 that have reported a positive association between the consumption of individual components of a traditional Mediterranean diet such as fish and seafood20, poultry21, whole cereals22, vegetables21 and fruits22, low-fat dairy23, and the improvement of several sperm quality parameters. The results of the present study are also in line with other studies demonstrating that typical foods highly consumed in western dietary patterns, such us processed meat24, potatoes25, full-fat dairy and total dairy products26, alcohol27, sugar-sweetened beverages28 and sweets21, are detrimentally associated with some quality parameters of semen.Our findings are consistent with previous epidemiologic observational studies29. Unfortunately, research assessing possible associations between dietary patterns and sperm quality outcomes is scarce and needs to be promoted in the future.Our study has been based on the analysis of dietary pattern. Dietary pattern analysis has emerged as an alternative and complementary approach to examining the relationship between diet and the risk of health outcomes. Instead of looking at individual nutrients or foods, dietary pattern analysis examines the effects of overall diet. This approach takes into account possible food or nutrient interactions, and positive or negative synergistic effects35 assessing the association between some a posteriori dietary patterns defined by factor analysis, and semen quality concluded that healthy eating patterns might have a positive association with sperm concentration, but not with other classical sperm quality parameters14. However, due to the small sample size and heterogeneity of the studies, the authors recommended caution in the interpretation of the results.A recent systematic review and meta-analysis of six observational studies36. The MD has consistently been associated with broad healthy benefits on human health, especially in relation to protection against CVD and mortality37. Because this dietary pattern has also demonstrated beneficial effects on inflammation38, oxidation39, insulin sensitivity40, and endothelial function41 among others, we can hypothesize that it can have also benefits in terms of semen quality parameters and male fertility. However, whether adherence to the MD is associated with better semen profile remains largely unexplored.The traditional MD pattern has been considered one of the healthiest dietary patterns existinga priori, and the quality of sperm42. In this cross-sectional study of 225 men from couples attending a fertility clinic in Athens, that has not been included in the aforementioned meta-analysis, men in the lowest tertile of the MD adherence score had higher likelihood of having abnormal sperm concentration, total sperm count and motility, compared to men in the highest tertile of the score. Therefore, our results confirm these previous reported results but only in relation to total and progressive sperm motility, an important parameter related to fertility.To our knowledge, there was only one previously published study analyzing the association between MD adherence, defined 48 adding biological plausibility to the reported association. Only one study has demonstrated that adherence to a healthy dietary pattern is associated to sperm motility in a posteriori analysis. In the context of the Rochester Young Men’s cohort, adherence to a Prudent diet identified by factor analysis rich in fish, chicken, fruit, vegetables, legumes and whole grains was positively associated with progressive sperm motility33.In fact, healthy dietary patterns and specially the MD are rich in several nutrients that have been proven to have benefits in terms of sperm motility18.The principal strength of the present study is the originality of the work, because this is the first study exploring the association between the MD pattern and sperm quality parameters in a young and healthy population. The use of FFQs, as a comprehensive method to obtain detailed information about the habitual intake of foods allowed us to use a validated and widely used MD score measuring adherence to the MD49. Unfortunately, we did not measure physical activity in our participants; therefore, we have not have controlled our models for this variable with the limitations that this entails in the interpretation of the findings. Finally, although an association between adherence to MD and sperm quality was detected, the highest probability of fecundability translation needs to be proven in the future.The main limitation of the study is the cross-sectional nature. Cross-sectional design could not determine whether MD adherence and sperm motility improvements are causally related. Future well-designed observational prospective studies and clinical trials on the current topic are therefore recommended. Although in our study we have excluded several potential confounding factors, these results should be interpreted with caution because of the multifactorial etiology of the sperm quality parameters. Moreover, we cannot discount other possible associations between the adherence to the MD and other sperm parameters by increasing the sample size or by repeating the semen analysis in order to decrease the biological variability. We cannot also discount some analytical error when measuring the sperm parameters, however we have minimized it through strong adherence to standardized protocols. Another limitation it that this study focuses on healthy men and therefore, the results cannot be extrapolated to other populations. Also, in some studies it has been shown that physical activity is associated with an improvement of sperm qualityIn summary, in this healthy and young population, and using a cross-sectional analysis, we demonstrated that adherence to a MD was positively associated with sperm motility parameters. These findings suggest that compliance to a healthy diet may help to improve some semen quality parameters, especially those related to the sperm motility, one of the most important parameter related to fertility. Prospective studies and randomized clinical trials are warranted in the future to increase the scientific evidence in relation to the possible effect of diet on the sperm quality and fecundability.50. Eligible participants were healthy and young men (aged 18–35 years old) reporting a western-style dietary pattern. Exclusion criteria included severe chronic illness, alcohol or drug abuse, frequent consumption or allergy of nuts, use of supplements , history of reproductive disorders or vasectomy, current smoking, or use of drugs for chronic diseases.This cross-sectional analysis was conducted with baseline data of participants recruited between December 2015 and February 2017 in the FERTINUTS study, a parallel randomized clinical trial aimed to assess the effects of nut supplementation on sperm quality parameters. The design of the trial has been reported in detail elsewhere (ISRCTN12857940)The protocol was approved by the Institutional Review Board of the Hospital Universitari Sant Joan de Reus in October 2015. The study was done according to the Declaration of Helsinki for Medical Research involving Human Subjects and all the participants provided a written informed consent.51 over the past year and the mean of 3-day dietary records, including 2 workdays and a weekend day. In the FFQ, dietitians asked the participants about the frequency with which they consumed the different items: never, one to three times per month, once per week, two to four times per week, five to six times per week, once per day, two to three times per day, four to six times per day or more than six times per day. The responses were transformed to grams per day. Energy, nutrient intake, and food groups were estimated using Spanish food composition tables52.Dietary intake was quantified by trained dietitians with a face-to-face 143-item, semi-quantitative, validated food frequency questionnaire (FFQ)18. Subjects were assigned a value of 0 if the consumption of components considered beneficial  was below the median, whereas individuals were assigned a value of 1 if they had a consumption of beneficial food at or above the median. Otherwise, people with consumption of components considered harmful (meat and dairy products) below the median were assigned a value of 1, whereas people with consumption at or above the median were assigned a value of 0. With regards to alcohol, a value of 1 was assigned to subjects consuming a moderate amount (i.e. between 10 and 30 g per day for men) and a value of 0 otherwise. Therefore, the total Trichopoulou score had a potential range from 0  to 9 .In order to assess the adherence to the MD the Trichopoulou score was fitted from FFQ information2), normal weight (18.5 to 25 kg/m2), overweight (25 to 30 kg/m2) and obese (>30 kg/m2). Waist circumference was measured to the nearest 0.5 cm midway between the lowest rib and the iliac crest with an anthropometric tape. Blood pressure was measured in duplicate with a 5 minutes interval by using a semiautomatic oscillometer . Blood samples following 12 h fasting conditions were collected in order to measure the following parameters: plasma glucose, total serum cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very-low-density lipoprotein (VLDL) cholesterol, triglycerides, insulin, C-reactive protein, and folate .General, anthropometrical variables and blood pressure were determined by trained staff at baseline. Briefly, body weight and height were measured using an electronic scale , and BMI was calculated as weight in kilograms divided by squared height in meters. The commonly accepted BMI ranges are: underweight , at ×400 magnification counting 200 spermatozoa. The lower reference limit for sperm concentration was 15 × 106 spermatozoa per ml and for sperm count was 39 × 106 spermatozoa per ejaculate. Sperm motility was assessed also at ×400 magnification (counting 200 spermatozoa) and permits to classify the spermatozoa as: a) Progressive motility (PR), b) Non-progressive motility (NP); and c) Immotility (IM). Total motility was expressed as PR + NP. The lower reference limit for total motility and progressive motility were 40% and 32%, respectively. Sperm vitality was estimated using eosin-nigrosine at ×1,000 magnification . The lower reference limit for vitality (membrane-intact spermatozoa) was 58%. Sperm morphology was assessed with Hemacolor®  staining at ×1,000 magnification (assessing 200 spermatozoa), and expressed as percentage of normal forms. The lower reference limit for normal forms was 4%.Semen parameters were assessed as described in the 2010 World Health Organization’s report: “WHO laboratory manual for the examination and processing of human semen”2). The goodness of fit was expressed as: R square multiply per 100 (R2x100) and the variance analysis of the model and its significance . All P-values are two-tailed at the <0.05 level. Statistical analyses were conducted with the IBM-SPSS statistical package .The results are shown across tertiles of MD adherence. Continuous variables were presented as means and standard deviation (±SD) or medians [25th-75th percentiles (Pc)] based on the normal or non-normal distribution. Missing blood values for the patients who have all the seminogram and dietary information were imputed using the mean of the variables measured. Normal distribution and homogeneity of variances were evaluated using Kolmogorov-Smirnov and Levene’s test, respectively. Categorical variables were presented as number (n) and percentages (%). Differences between groups were assessed with ANOVA test when the variables were normally distributed and Kruskal-Wallis or Mann-Whitney U tests when they were not. A multiple comparison post-hoc Bonferroni test were used to perform pairwise comparisons between groups. The Pearson chi-squared test was used to compare categorical variables. Multivariate linear regression models (enter method) were fitted to assess the relationship between tertiles of MD adherence and the principal sperm quality parameters. Estimates with β coefficient and standard error were shown. The multivariate linear regression models included the following potential confounders: age (years), energy  and BMI (kg/m


Navigated transcranial magnetic stimulation (nTMS) is increasingly applied to map human language functions. However, studies on protocol comparisons are mostly lacking. In this study, 20 healthy volunteers  underwent left-hemispheric language mapping by nTMS, combined with an object-naming task, over a cortical spot with reproducible naming errors within the triangular or opercular part of the inferior frontal gyrus  and the angular gyrus or posterior part of the superior temporal gyrus , respectively. Various stimulation intensities , frequencies , and coil orientations (in steps of 45°) were evaluated, and the adjustments leading to the highest error rates (ERs), combined with low occurrences of errors due to muscle stimulation, were considered optimal. Regarding anterior stimulation, 100% rMT, 5 Hz, and a coil orientation of 90° or 270° in relation to the respective stimulated gyrus resulted in optimal results. For posterior stimulation, 100% rMT, 10 Hz, and coil orientations of 90° or 270° were considered optimal. Errors due to facial muscle stimulation only played a considerable role during analyses of high-intensity (120% rMT) or high-frequency stimulation (20 Hz). In conclusion, this is one of the first studies to systematically investigate different stimulation protocols for nTMS language mapping, including detailed analyses of the distribution of ERs in relation to various coil orientations considered during neuronavigated stimulation. Mapping with 100% rMT, combined with 5 Hz (anterior stimulation) or 10 Hz (posterior stimulation) and a coil orientation perpendicular to the respective stimulated gyrus can be recommended as optimal adjustments. Transcranial magnetic stimulation (TMS), a non-invasive method that creates a magnetic field inducing a transient electric field capable of modulating neurons in their activity, is available for neuroscientific and clinical use in human subjects since the middle eighties . Among iWhile first studies on language mapping by TMS were mostly conducted without simultaneous neuronavigation, the combination of TMS with precise electric-field neuronavigation systems further expanded the role of this evolving technique since we are now able to confirm the cortical area to which TMS is applied very accurately . Thus, nHowever, language mapping by nTMS in patients with brain lesions has repeatedly shown to have a comparably high sensitivity and low specificity when compared to intraoperative direct electrical stimulation (DES) in most studies, which is regarded non-optimal . To imprThe present study was performed in healthy volunteers. Inclusion criteria were German as mother tongue, age of at least 18 years, and right-handedness according to the Edinburgh Handedness Inventory (EHI) . SubjectFigures 1A,B). The mappings were conducted with different stimulation intensities , stimulation frequencies , and coil orientations (in steps of 45°) during performance of an object-naming task. The different adjustments were applied in randomized order, and nTMS-induced errors were counted and categorized during post hoc analyses.Prior to language mapping, anatomical MRI was performed in all subjects. On a separate day, each volunteer underwent repetitive nTMS (rTMS) to the triangular or opercular part of the inferior frontal gyrus  and the angular gyrus or posterior part of the superior temporal gyrus  of the left hemisphere  was performed with a 3-Tesla magnetic resonance scanner , and the obtained sequence was used to be able to apply neuronavigation during language mapping. The acquired three-dimensional (3-D), T1-weighted gradient echo sequence had the following scanning parameters: repetition time/echo time: 9/4 ms, 1 mmpost hoc analyses.Subsequent to the upload of the 3-D T1-weighted gradient echo sequence, the respective subject underwent language mapping by rTMS using a Nexstim eXimia NBS system, version 4.3, with a NexSpeech module . We used a focal, figure-8-shaped stimulation coil with an upward handle position and automatic overheating protection. The used coil applies biphasic pulses (pulse length: 230 μs) and induces an electric field with a maximum field strength of 172 V/m ± 2% . Maximum stimulator output can be approximately 2.5 × rMT of a hand muscle of a healthy adult by default. The stimulation system enables the visualization of all stimulation areas and simultaneous tracking of the coil due to the combination of the stimulation coil with a neuronavigation unit  . Pulse aPrior to language mapping by nTMS, the individual rMT was determined. Pregelled surface electrodes  for electromyography recording were placed over the abductor pollicis brevis and abductor digiti minimi muscles, and single stimulation pulses were applied over the left-hemispheric motor cortex at the area of the anatomical hand knob . After iWe used an object-naming task during baseline assessment and language mapping by nTMS, which was displayed on a screen located approximately 60 cm in front of the volunteers . The taspost hoc analyses of nTMS-induced errors (Two consecutive rounds of baseline assessment (object naming without simultaneous nTMS) were conducted to assess the individual naming skills of each volunteer with respect to the assortment of objects . The subd errors . The basd errors .Subsequent to baseline assessments, language mapping by nTMS was performed with the set of remaining, correctly named objects. The IPI and DT were the same as during baseline assessment; thus, the interval between two consecutive stimulation trains was 3.0 s , with one stimulation train being applied per every single object presentation. Furthermore, the PTI  was set at 0 s, indicating that stimulation started simultaneously with object presentation . Pulse aFigures 1A,B), were mapped in steps of 5–10 mm to identify a cortical spot for each of both cortical regions where nTMS led to reproducible and clear naming errors during task performance (The left-hemispheric trIFG/opIFG (anterior stimulation) and anG/pSTG (posterior stimulation), which were anatomically identified on the reconstructed 3-D head model , 10 consecutive stimulation trains per intensity at the optimal spot  within the trIFG/opIFG and anG/pSTG .–stimulation frequency: stimulation with 2 Hz/4 pulses, 5 Hz/10 pulses, 10 Hz/20 pulses, and 20 Hz/40 pulses , 10 consecutive stimulation trains per frequency at the optimal spot within the trIFG/opIFG and anG/pSTG .–coil orientation: stimulation with variations in steps of 45° , 10 consecutive stimulation trains per orientation at the optimal spot within the trIFG/opIFG and anG/pSTG .During mapping, the optimal stimulation intensity and frequency to conduct further stimulation according to the protocol were determined during the ongoing stimulation procedure . This waThe course of mappings applying the different stimulation parameters was randomized for each subject using cards for each adjustment. Prior to language mapping, the first region to map was determined by drawing a card . Then, the different parameters for testing stimulation intensity  and frequency  were individually determined to define the chronological order in which the various settings were tested. Regarding coil orientation, only the initial orientation in relation to a-p coil orientation was drawn , and the other orientations were tested clockwise (in steps of 45°) with respect to the initially used orientation. Like for the baseline assessments, the whole procedure was recorded . After tPost hoc video analyses, which were performed by the investigator who had already conducted the mappings , were based on video and audio recordings acquired during the individual baseline assessments and mapping procedures. These data were streamed and each naming performance was compared with the individual baseline to detect disruption of object naming  were not considered during analyses because we did not use objective measurements of voice reaction times. Furthermore, errors that presumably occurred due to stimulation-induced facial muscle activity according to video analysis or reporting of the volunteers were not included in one of these categories, but were registered separately. For further analyses, we considered all naming errors without hesitations or muscle stimulations , no responses, performance errors, others , and errors due to muscle stimulation.p-value of <0.05 indicated statistical significance for all statistical tests.For all statistical analyses, we used GraphPad Prism . A Errors of the different categories were counted to form absolute and relative frequencies for the different adjustments applied during anterior and posterior stimulation, respectively. Descriptive statistics including mean, standard deviation (SD), median, minimum and maximum values were calculated for subject- and mapping-related characteristics and naming errors of the different categories. Discomfort perceived according to the VAS and the electric field strength during nTMS were compared between anterior and posterior stimulation spots using Wilcoxon matched-pairs signed rank tests.Furthermore, we calculated ERs by dividing the number of errors induced by nTMS by the number of stimulations, which was done for anterior and posterior stimulation as well as for the different adjustments and error categories, respectively . We thenFor the optimal adjustments, we calculated coefficients of variation (CVs) as measures for dispersion regarding error numbers of all errors without hesitations or muscle stimulations. This was again achieved separately for anterior and posterior stimulation and for all subjects together as well as for female and male subjects, respectively. Additionally, were performed Mann–Whitney tests to compare error numbers of this error category between genders.p = 0.0001). There was no statistically significant difference in electric field strengths when comparing anterior and posterior stimulation delivered with optimal adjustments (p = 0.4980). Table 1 displays cohort and mapping characteristics.Twenty healthy volunteers  took part in this study. Overall, the stimulation procedure was tolerated well by all volunteers and no adverse events were observed. Stimulation within the trIFG/opIFG was significantly more discomfortable when compared to nTMS to the anG/pSTG . Overall, 3,000 nTMS trains were applied to 20 subjects, leading to 295 naming errors of the different categories  and 453 errors presumably due to muscle stimulation . Out of the 295 naming errors elicited, 37 were no responses (12.6%), 210 were performance errors (71.2%), 34 were phonological paraphasias (11.5%), 13 were semantic paraphasias (4.4%), and 1 was categorized as neologism (0.3%).We were able to identify a cortical spot with reproducible naming errors during nTMS in all subjects within the trIFG/opIFG at the beginning of language mapping . For all errors without hesitations or muscle stimulations, no responses, and performance errors, 100% rMT led to the highest ERs most frequently when compared to stimulation with 80% rMT or 120% rMT .There was no statistically significant difference between adjustments considering the different error categories except for muscle stimulations . Highest ERs due to muscle stimulation occurred most frequently in response to stimulation with 20 Hz .Stimulation with 5 Hz led to the highest ER for all errors without hesitations or muscle stimulations and performance errors most frequently, with a statistically significant difference in comparison to the other adjustments . However, a statistically significant difference between adjustments for different orientations in relation to a-p coil orientation was only observed for performance errors, with 0°/360° in relation to a-p coil orientation being considered optimal .In relation to a-p coil orientation as the most common adjustment in nTMS language mapping, the highest ERs occurred most frequently for stimulation with 0°/360° , 135°, and 180° when considering all errors without hesitations or muscle stimulations . For this category, the comparison between coil orientations was statistically significant .Regarding coil orientations in relation to the respective stimulated gyrus, 90° and 270° led to the highest ERs most frequently for all errors without hesitations or muscle stimulations (p = 0.8797).For all errors without hesitations or muscle stimulations elicited during nTMS with optimal adjustments, a CV of 55.9% was observed for all subjects together . However, there was no statistically significant difference in error numbers between genders . Application of 3,000 nTMS trains in total resulted in 264 naming errors of the different categories  and 93 errors presumably due to muscle stimulation . Out of the 264 naming errors elicited, 30 were no responses (11.4%), 182 were performance errors (68.9%), 33 were phonological paraphasias (12.5%), 17 were semantic paraphasias (6.4%), and 2 were classified as neologism (0.8%).We identified a cortical spot with reproducible naming errors during stimulation within the anG/pSTG in all enrolled subjects . Furthermore, mapping conducted with 100% rMT also resulted in the highest ER most frequently for all errors without hesitations or muscle stimulations, but without a statistical significance when compared to the other intensities . For no responses, 80% rMT led to the highest ERs most frequently, but without statistical significance .Stimulation with 100% rMT led to the highest ER most frequently for performance errors . Concerning no responses, 5 Hz resulted in the highest ER most frequently, but without showing statistical significance .Statistically significant differences were found between adjustments for all errors without hesitations or muscle stimulations and for performance errors, with 10 Hz leading to the highest ER most frequently . However, a statistically significant difference between adjustments for different coil orientations with respect to a-p coil orientation was only observed for muscle stimulations .Regarding orientations in relation to the a-p coil orientation, the highest ERs occurred most frequently for stimulation with 90° and 180° when considering all errors without hesitations or muscle stimulations . Only for muscle stimulations there was a statistically significant difference between coil orientations , but errors due to this were generally very rare in terms of posterior stimulation.Concerning the coil orientation with respect to the stimulated gyrus, the highest ERs were observed most frequently for stimulation with 90° and 270° when considering all errors without hesitations or muscle stimulations (p = 0.2143).When considering all errors without hesitations or muscle stimulations that were elicited during nTMS with optimal adjustments, a CV of 48.2% was registered for all subjects together . There was no statistically significant difference in error numbers between genders , which seems to be in contrast to these earlier findings. This was not only true for no responses regarding anterior stimulation , but also for all errors together without hesitations or muscle stimulations or performance errors , thus categories not considered in most of the aforementioned earlier studies. The discrepancy between our present and earlier findings might be associated with the use of neuronavigation, which has not yet been available in Epstein’s studies . Of note, the category of no responses preferentially occurred at 20 Hz (anterior stimulation) and 5 Hz (posterior stimulation), thus probably supporting the findings of higher frequencies being more capable of leading to speech arrests  . In this context, muscle stimulation can lead to difficulties during error categorization and discomfort, both potentially hampering proper evaluation of stimulation sessions in terms of language errors, which has been observed also in the studies that preferred higher over lower stimulation frequencies  and 10 Hz (posterior stimulation) showed optimal results during nTMS in the present study  . Discrepes 3A,B) . Furtherquencies . Howeverquencies . AlthougTo the authors’ knowledge, this is one of the first studies to systematically evaluate coil orientation in the context of nTMS language mapping. Here we evaluated both coil orientations in relation to the respective stimulated gyrus and in relation to a-p coil orientation as the most common setting in recent studies on nTMS language mapping . PreviouTable 2 and Figures 4A,B). However, when considering the individual gyral anatomy at the cortical spot stimulated as considered in evaluations related to the respective stimulated gyrus, a clear finding with optimal adjustments of 90° and 270° to this gyrus for both anterior and posterior stimulation was revealed . This finding seems to correspond to the well-known observations during TMS motor mapping where strongest motor-evoked potentials occur when the stimulating coil is oriented perpendicular to the central sulcus during stimulation of the precentral gyrus . For posterior stimulation, 100% rMT, 10 Hz, and coil orientations of 90° or 270° should be considered to achieve optimal results .The present investigation and previous studies suggest that small changes in stimulation adjustments can already have a considerable impact on language performance . Hence, Figures 5). Thus, few subjects may show optimal results for other than the recommended parameters, which underlines the importance of individualization of stimulation parameters in each subject, if possible. In neuroscientific use among healthy subjects or for spatially circumscribed nTMS mappings, this may be achievable and should still be aimed for, with the current recommendations serving as a good starting point for further individual adjustments in single subjects. In clinical use and for spatially widespread nTMS mappings especially among patients suffering from brain tumors, the present recommendations may replace current procedures that commonly apply partially different protocols, especially in terms of coil orientations  or 10 Hz (posterior stimulation within the anG/pSTG) and a coil orientation perpendicular to the stimulated gyrus, are recommended to achieve optimal results. As the next step, these adjustments should be applied in the preoperative setting among patients with language-eloquent brain tumors, followed by intraoperative DES to evaluate whether improved sensitivity and specificity of nTMS language mapping can be achieved with these settings.All data used for analysis are presented in the manuscript. The discussion and conclusions only rely on the data presented. Raw mapping data can be provided upon request.The experimental setup was approved by the local ethics committee  and was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects prior to the mapping procedures.NS designed the experiments, acquired, handled, analyzed (including statistics), and interpreted the data, performed the literature research, drafted the manuscript, and read and approved the final version. SF-R acquired, handled, analyzed, and interpreted the data, performed the literature research, drafted the manuscript, and read and approved the final version. CZ and BM designed the experiments, acquired and handled the data, supervised the study, and read and approved the final version. SK designed the experiments, acquired, handled, and interpreted the data, performed the literature research, drafted the manuscript, supervised the study, and read and approved the final version.SK is a consultant for Brainlab AG  and Nexstim Plc. . The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In this study we present the porous silica-based material that can be used for in situ drug delivery, offering effective supply of active compounds regardless its water solubility. To demonstrate usability of this new material, three silica-based materials with different pore size distribution as a matrix for doping with Photolon (Ph) and Protoporphyrin IX (PPIX) photosensitizers, were prepared. These matrices can be used for coating cardiovascular stents used for treatment of the coronary artery disease and enable intravascular photodynamic therapy (PDT), which can modulate the vascular response to injury caused by stent implantation—procedure that should be thought as an alternative for drug eluting stent. The FTIR spectroscopic analysis confirmed that all studied matrices have been successfully functionalized with the target photosensitizers. Atomic force microscopy revealed that resulting photoactive matrices were very smooth, which can limit the implantation damage and reduce the risk of restenosis. No viability loss of human peripheral blood lymphocytes and no erythrocyte hemolysis upon prolonged incubations on matrices indicated good biocompatibility of designed materials. The suitability of photoactive surfaces for PDT was tested in two cell lines relevant to stent implantation: vascular endothelial cells (HUVECs) and vascular smooth muscle cells (VSMC). It was demonstrated that 2 h incubation on the silica matrices was sufficient for uptake of the encapsulated photosensitizers. Moreover, the amount of the absorbed photosensitizer was sufficient for induction of a phototoxic reaction as shown by a rise of the reactive oxygen species in photosensitized VSMC. On the other hand, limited reactive oxygen species (ROS) induction in HUVECs in our experimental set up suggests that the proposed method of PDT may be less harmful for the endothelial cells and may decrease a risk of the restenosis. Presented data clearly demonstrate that porous silica-based matrices are capable of in situ delivery of photosensitizer for PDT of VSMC. Percutaneous coronary interventions (PCI) involving stent implantation are routinely used for the treatment of coronary artery disease. Proliferation and migration of tunica media layer (middle coat) smooth muscle cells (SMC) in response to the vascular wall injury, are essential events leading to the subsequent neointimal thickening, which eventually causes vessel narrowing . TherefoPhotodynamic therapy (PDT) was shown to modulate the vascular response to injury caused by PCI ,6. PDT iDelivery of the soluble form of PS and its good selectivity are crucial for successful PDT. Usually, for intravascular PDT administration of PS is performed via intravenous injections. PS is taken up and retained in the lesion and administered prior to exposure to irradiation. However, there are some well-known disadvantages of the systemic infusion of PS due to poor tissue selectivity . It has For our study we have chosen two widely used photosensitizers: Photolon (Ph) and Protoporphyrin IX (PPIX). Both drugs were already successively used in the cardiovascular PDT ,26,27,28Our goal was to create a material suited for use as coating of cardiovascular stent. Such a material can serve as a protective layer as well as a source of PS. In contrast to DES, which is designed for very slow releases of drug, in this study we propose delivery of PS directly to cardiovascular plaque. Such an approach eliminates the need for systemic administration of a photo-sensitizer, which is the main source of side effects in PDT.The sol–gel materials were prepared from silica precursor named TEOS  mixed with 96% ethyl alcohol  with addition of 36% HCl  as a catalyst and Triton X-100 (Sigma-Aldrich). The mixture was stirred for 4 h with the speed of 400 rot/min at the room temperature. Diverse material samples were produced with the various ratio of number of TEOS moles to the number of ethyl alcohol moles. The suitable amount of solvent was used, thus to obtain the required materials, named as M1, M2, and M3. These materials were stored for one month in controlled temperature and humidity conditions . Then the morphology of solid samples was studied (pore size distribution and X-ray micrograph distribution).For atomic force microscopy (AFM) studies as well as Attenuated Total Reflectance with Fourier Transform Infrared Spectroscopy (FTIR-ATR) and UV-vis spectroscopy, Photolon (18-carboxy-20-(carboxymethyl)-8-ethenyl-13-ethyl-2,3-dihydro-3,3,12,17-tetramethyl-21H, 23H-porphin-2-propionicacid)  and Protoporphyrin IX dimethylester   were used as photosensitizers (PS). Stock solutions 0.5% of photosensitive dyes were prepared by dissolving the photosensitizer in ethyl alcohol. The adequate amounts of stock solutions were added to 1 mL of the fresh prepared silica sol in order to get the final concentrations of PS depicted by Examination of the porous structure of solid samples was carried out quantitatively by mercury porosimetry. Thermo Scientific Pascal 440 porosimeter  provides information about pore size distribution, particle size distribution, bulk density, and specific surface for most porous solids. For this study the structural parameters, such as relative pore volume and pore radius distribution, were determined. Each sample M1, M2, and M3 mass was 0.15 g .2 statistic, the degrees of freedom adjusted-R2 statistic and RMSE (root mean squared error).The obtained results of the mercury porosimetry measurements in the form of dependence of the relative pore volume on the pore size, were used to characterize the pore size distribution in each analyzed material . A non-standard estimation method was developed and applied. The raw data produced by Pascal 440 device were fitted by Gaussian functions to evaluate the pore size distribution. For estimation of the goodness of fit the following measures were used: R2 statistic measures with what accuracy the applied fit has in explaining the variation of the analyzed data. It is defined as the square of the correlation between the observed values and the predicted values from the fit. It can take values from 0 to 1, where values closer to 1 are indicating that a greater proportion of variance is accounted for by the applied fitting model.The R2 statistic, but without the practical improvement of the fit. To omit this problem, the additional degrees of freedom adjusted-R2 statistic, were applied. This approach uses the R2 statistic and adjusts it basing on the residual degrees of freedom, which are defined as the difference between the number of the observation value and the fitted coefficients estimated from the observation values. As R2 statistic, adjusted-R2 statistic takes values from 0 to 1 and increases only when the additional fitted coefficients will improve the prediction model. Additionally, the RMSE measuring differences between observation values predicted by a fitting model and observed values, were applied. This parameter is estimating the standard deviation of the random component in the data. When RMSE takes values closer to 0, it indicates that the fit will be more useful for prediction of the observation’s values.In some cases, the increase of the number of fitted coefficients in the model can increase the value of the RThe energy density H and power density E were set up experimentally because the producer suggestions were tissues established only . Power dThe used irradiation setup included laser diode (λ = 655 nm) with adjustable power control unit coupled into the optical fiber system  .The structure of coatings was examined by scanning electron microscopy Hitachi S-4000  equipped with energy dispersive X-ray system. Prior to the examination, the samples were stored in a desiccator. The spectra were recorded by the following conditions: accelerating voltage 20.0 kV, beam current 100,000 nA, magnification 1000, live time 77 s, preset time 0, Nb channels 2048, Ev/Channel 10, offset 0 keV, width 20 keV, whereas the geometry was as follows: tilt angle 0, azimuth angle 0, take-off angle 33 deg.AFM images were acquired in the tapping mode using a Nanoscope IIId scanning probe microscope with Extender Module  in the dynamic modus. An active vibration isolation platform was applied. Olympus etched silicon cantilevers were used with a typical resonance frequency in the range of 200–400 kHz and a spring constant of 42 N/m. The set-point amplitude of the cantilever was maintained by the feedback circuitry to 80% of the free oscillation amplitude of the cantilever. All samples were measured at room temperature in air. The sample was first adjusted with an optical light microscope .For this study, mica plates (Nanowords) were coated by undoped and Photolon doped sol–gel matrices, as well as undoped and Protoporphyrin IX (PPIX) doped sol–gel layers. The distribution of PS molecules on the coating surface was analyzed.All absorption spectra were recorded in wavelength range 200–1100 nm by the means of the AVA-Spec 3648 spectrophotometer  equipped with deuterium-halogen lamp  as the light source. As the excitation source in luminescence measurement the continuous wave semiconductor laser λ = 405 nm  was used. For spectroscopic studies the PSs doped M1, M2, and M3 materials were measured in standard UV cuvette. After that materials were deposited on a round coverslip glass with a diameter of 12 mm (20 µL per slide). An important step in this process was the deposition of a thin and uniform layer of silica material in which the PSs are immobilized.−1 using the OMNIC software  at the standard analysis field. Further, spectral analysis and processing was carried out with Origin software.316 L stainless steel functionalized surfaces (10 mm in diameter) were characterized with FTIR-ATR spectroscopy. Spectra were recorded using attenuated total reflection method with Nicolet 6700 FTIR spectrometer  and Diamond Top-Plate of the Golden Gate MK II ATR Accessory . Samples were placed directly on the diamond at steady temperature of 21 ± 1 °C. Spectra were obtained as a mean of 32 scans with the resolution of 4 cm2.Human peripheral blood mononuclear cells (PBMCs) were cultured in RPMI medium supplemented with 10% fetal bovine serum . Blood samples were collected from healthy consent, volunteers, with agreement with local regulations and Ethical Board permit no. KB-158/2010. Human umbilical vein endothelial cells (HUVEC) and human aorta/smooth muscle cells (T/G HA-VSMC) were maintained in F12 K medium supplemented with 2 mM l-glutamate (Gibco) and 0.1 mg/mL heparin (Sigma-Aldrich), 0.03 mg/mL endothelial cell growth supplement (Sigma-Aldrich), and 10% FCS. HUVEC, T/G HA-VSMC cells and PBMCs were cultured on flasks and 24, 48, or 96-well plates (Corning Costar) at 37 °C in a humidified atmosphere of 5% CO3 cells per well) coated with photosensitizer-loaded silica sol–gel carriers. After 4 h incubation the probes were illuminated with light wavelength 655 nm  and incubated 1 h before fixation. Uptake of PS by PBMCs was examined after 45 min incubation with test compound surfaces. Briefly after isolation, lymphocytes were seeded on 24 well PS-loaded silica sol–gel carriers plate. Next some of the probes were illuminated with light wavelength 655 nm . After each treatment cells were washed twice with PBS  and fixed with 4% paraformaldehyde  in PBS, at 37 °C for 30 min. Then cells were washed three times with PBS. The confocal images of cells were collected on confocal laser microscope Leica TCS SPE using a 63× high numerical-aperture oil immersion objective. Image size was set to 512 pixels × 512 pixels. For excitation of PS laser line at 405 nm was operated at 20% of maximum power. Optimal gain settings 1260 V was used.For PS uptake HUVEC cells were seeded on a 96 well plate  in 15 mL conical tubes  and washed in 0.9% NaCl or PBS pH 7.4. Cells were extracted in 15 mL conical tubes (Sarstedt) and diluted in 50 mL conical tube .For hemolysis assay 8 mL of peripheral blood was obtain from anonymous human donor and drawn into K2-EDTA-coated Vacutainer tubes . Erythrocytes were prepared as described by Evans et al. , with miv/v) in PBS pH 7.4 and 190 μL was added to each well. After 1 h incubation at 37 °C, the cells were centrifuged at 450 × g for 10 min at 14 °C and 100 μL of supernatant was carefully transferred to a transparent 96 well plate (Corning Costar Inc.). The absorbance at 450 nm corresponding to hemoglobin release from lysed erythrocytes was measured by Thermo LabSystems Multiskan RC Microplate Reader (Thermo Fisher Scientific). Erythrocytes incubated with 1% Triton X-100 (Sigma-Aldrich) served as positive control (100% hemolysis). Result below 5% was considered as negative (no hemolysis).Hemolysis assay was performed at black 96 well plates previously coated with the test compounds. Red blood cells were diluted 1:49 (6 were seeded on 24-well plate (Corning Costar Inc.) on coated surfaces and incubated . For assessment photo-toxicity cells were illuminated  and removed to fresh plates and cultured for 48 h. Next, the apoptosis was evaluated using the method of Nicoletti et al. [Peripheral blood lymphocytes were isolated from blood using Lymphocyte Separation Medium 1077  according to the supplier’s instruction. Lymphocytes at density 1 × 10i et al. , basing 2DCFDA ; Molecular Probes, Thermo Fisher Scientific, Inc., Waltham, MA, USA. Briefly, cells were stained with 10 μM of H2DCFDA for 30 min in PBS with 10% FBS and washed twice with warm PBS with 2.5% FBS. Then HUVEC and T/G HA-VSMC cells were seeded on photoactive surfaces on 48-well plate at density 60 × 103 cells per well, after 2 h the basal fluorescence was measured. Next, the cells were illuminated  and then fluorescence was measured again. Basal fluorescence and fluorescence collected from matrices without photosensitizer values were used as the background and was subtracted from all the samples after illumination.Reactive oxygen species (ROS) concentration was assessed by measuring the 485-/535-nm fluorescence of HThe matrices with controlled porosity make silica materials highly attractive as the structural basis for a wide variety of technological applications, e.g., medical devices, therefore we report the effects of synthesis parameters on pores size distribution and the elemental composition.On The energy dispersive X-ray spectroscopy (EDS) was used to verify the chemical composition of the materials. The EDS results on 2 statistic it was shown that the used fitting models enabled the explanation of the 99.66%, 92.41%, and 93.66% of the total variation in the data obtained for M1, M2, and M3, respectively.The raw data from mercury porosimetry was fitted by two Gaussian functions on a decaying exponential background using the nonlinear model, to estimate the pore size distribution of investigated materials. Obtained values of the measures used to evaluate the goodness of the fit are presented in The fitted pore size distributions in each analyzed material are shown on The surface morphology of silica sol–gel materials was characterized by atomic force microscopy. The AFM images of M1, M2, and M3 sol–gel layers revealed that the different porosity of materials had limited impact on the surface composition and surfaces of all coatings were smooth and homogeneous  with rouPS’s absorption and emission bands should not overlap with absorption or fluorescence bands of endogenous chromophores occurring in human body. Therefore, optical properties of PSs and PS-doped sol–gel carriers were determined by spectrophotometry. The absorption spectrum of Photolon (Ph) shows a strong band at 410 nm (Soret band) and weaker Q-bands at 528 nm, 595 nm, and 645 nm a. For PrThe changes related to the type of material were firmly visualized by luminescence intensity in colloids c,d. Uponex = 405 nm) the strongest luminescence for M2-Ph, and lower for M3-Ph and M1-Ph, was observed  favored the monomeric form of PSs. Aggregated PS are less photochemically active because of nonradiative deactivation of excited states, which shortens the fluorescence lifetime .In the case of silica coatings doped with PP IX f all ban−1 is the most differentiating and full of various signals, most of which are very likely to be coming from bending vibrations of groups, such as (C–H), (C=C), and (N–H). Spectral region between 1000 cm−1 and 1300 cm−1, marked as II is filled with peaks attributed to stretching vibrations of amines (C–N), alcohols (C–O), carboxylic species (C–OH), some skeletal vibrations from (C–C), and, finally, of ester groups (C–O–C). The last described area is the least differentiating, however the dissimilarities are also easy to spot. In both spectra, slightly shifted signals of 1375 cm−1 and 1422 cm−1 are assigned to the bending vibrations of (C–H) groups and to skeletal stretches of (C=C). These signals are followed by an amine doublet, with maxima located at 1566 cm−1 and 1652 cm−1 , that seems to differ in each spectrum regarding the signal intensity, most likely due to the exposure of (N–H) groups. Peaks located at 1669 cm−1 and 1712 cm−1 are present only in Protoporphyrin IX spectrum, they are most likely coming from stretching vibrations of imine groups (C=N) or skeletal (C=C) and (C=O) from ketonic species, respectively. Next in line are the signals located between 2700 and 3100 cm−1 assigned to stretches of (C–H) groups coming from various kinds of alkanic and alkenic species. This area is definitely more diverse in the case of Protoporphyrin IX spectrum, because there are at least six different local maxima, as opposed to a simple triplet in case of Ph. The last and perhaps the most prominent difference between the two spectra are the peaks located at 3271 cm−1 for Ph and at 3309 cm−1 for PPIX. Broad nature of this signal in Ph spectrum suggests that it is correlated with the stretching vibrations of hydroxyl (O–H), which is not the case for PPIX, because this compound simply lacks those groups. However, the sharp character of the PPIX 3309 cm−1 signal leads to a conclusion that it corresponds to amine stretches of (N–H) groups.FTIR spectroscopy was used to determine the structural characteristics of functionalized 316 L samples. Initially, spectra of Ph , whereas in M3 it noticeably differentiated into two maxima at 1110 cm−1 for (Si–O–Si) and at 1157 cm−1 for carboxylic stretches of (C–OH). Otherwise, the signals looked very similar in all three regions. However, slight spectral changes became visible after the addition of Ph, when a small doublet signal emerged at 1620 cm−1 suggesting the presence of bending vibrations of amine (N–H), most likely coming from the addition of photosensitizers. The most visible difference seemed to be the flattening of a broad 3300 cm−1 signal, coming from stretching vibrations of hydroxyl (O–H) groups. At the same time a steep slope-like signal was formed at 3350 cm−1, which corresponded to amine stretches of (N–H) bonds, again confirming the presence of Ph. The other difference, noticeable after the addition of this porphyrin was the slight shift and change of shape of the signal of the highest intensity, located in II region at 1100–1200 cm−1. In the case of M1 it emerged as a doublet similar to M3 before the addition of porphyrins, whereas in M2 and M3 it shifted a little to the lower wavelength values. The addition of Ph also seemed to smoothen the signals located in the area ranging from 1250 cm−1 to 1510 cm−1, which was mostly responsible for bending vibrations of (C–H) groups. When it came to photoactive surfaces, the most visible spectral differences appeared in the slight shifts of the 1100 cm−1 signal and the appearance of the amine doublet at 1620 cm−1, also coming from stretching vibrations of (N–H) groups. In the case of Protoporphyrin IX addition, the regions I and III stayed relatively unchanged.The spectra of surfaces modified with M1, M2, and M3 were evaluated  groups of PPIX were closing in on each other due to the deformation or aggregation of porphyrins located in pores of the matrices. This effect was also observable, and thus confirmed, in luminescence spectra.Interestingly, all matrices doped with PPIX seemed to exhibit the “red shift” effect of bending vibrations of (N–H) groups, meaning that the signals responsible for those groups tended to shift slightly towards the lower wavenumbers  with some silica-based materials may result in disruption of RBC membrane due to hydrophobic  or electWith the lack of dark cytotoxic effect, we used the same assay for testing phototoxic properties of silica-based photoactive matrices in PBMCs. As in the previous studies cells were preincubated on photosensitizing surfaces for as long as 24 h . All theFor assessment of biological activity two cell lines relevant to the proposed application were selected: human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMC). Cellular uptake of photosensitizers encapsulated in silica matrix carriers was studied using confocal microscopy after incubation of HUVECs and VSMC on Ph- or PPIX-loaded M1, M2, and M3 photoactive materials. Fluorescence of Ph or PPIX was observed in all probes, showing that both photosensitizers could be efficiently taken up by the cells from M1, M2, and M3 surfaces in 2 h . Inside The direct outcome of photodecomposition is the generation of ROS inside the cells. This event usually starts a cascade of reactions leading to programmed cell death. In this regard, we checked the sufficiency of cellular uptake of PSs to induce ROS production upon illumination. Both the HUVECs and the VSMC were incubated on the M1, M2, and M3 materials loaded with Ph or PPIX and illuminated with 655 nm light beam corresponding to PDT window of Ph and PPIX. The fluorescence collected from cells incubated on matrices without a photosensitizer, was negligible and ranged from 154 to 225 RLU. In the case of matrices with photosensitizers, the M1 material was most effective for induction of ROS in the both cell lines, regardless the photosensitizer. Two other materials were less efficient, especially in case of PPIX. Importantly, ROS production was markedly higher in vascular smooth muscle cells than in endothelial cells . To furtAs cardiovascular stents are commonly used during PCI procedures, PS release could be accomplished from the surface of the stent coating. At the same time immobilization of PS may reduce its systemic toxicity by limited release from the matrix. This approach combines encapsulation of PS into the interconnected porous matrix with procedure of the catheter accessible lesion illumination. We show that cardiovascular stents can be upgraded into PDT devices by deposition of PS loaded layer onto the inner surface. Our paper presented in vitro studies concerning PSs contained coatings that might be deposited on the medical device. During the PDT, the reactive oxygen species could be generated not from the surface of the layer but from interconnected pores network of deposited coating still containing entrapped PS molecules.The preparation of silica-based materials via sol–gel technology was affected by variations in the synthesis conditions. The materials were properly obtained if a catalyst (hydrochloride acid) was used, otherwise a non-durable or opaque material, indicating the formation of the internal stresses, was obtained. It was found that the gelling time increased as the dilution increased , and the gel formed was transparent and rigid . During We observed the best optical properties  for M1 material. Using a mercury porosimetry it was stated that this particular material had monomodal pores distribution. Materials M1 prepared with TEOS to ethyl alcohol molar ratio 20  was usedIt is worth to mention that we were able to prepare photoactive materials regardless of differences in chemical properties of photosensitizers. Both, well water soluble Ph and low water soluble PPIX were successfully encapsulated in silica based porous matrices.Low material roughness results in reduced friction between the rough implant surface and the blood vessel wall during stent placement . This liThe biological studies show that even short incubation of the SMCs on the photoactive matrices could result in PS uptake sufficient for induction of phototoxic reaction upon illumination measured as ROS induction. The uptake was detectable in both smooth muscle cells and endothelial cells. The photosensitizing surfaces varied in the activity, but the M1 material was the most efficient in induction of ROS. As this material had the smallest pores and lowest pore volume ratio it strongly suggests that the presence of small pores and/or low pore volume ratio was important for overall biological activity of obtained materials. It clearly indicates that M1 material was best for in situ delivery of a photosensitizer for PDT using Ph or PPIX. It can be expected that different pore size or pore volume ratio would be required by different drugs. It seems to be contradictory in view of a poor release of various drugs from porous matrixes with small pores ,46, but Although, it is known that different cell subsets can differently response to PDT , in thisIn clinical conditions, at the site of angioplasty, EC cells were absent , while SMC became activated and proliferated at a high rate. Thus, targeting the SMC proliferation is important for hindering intimal hyperplasia. On the other hand, proper reendothelialization is important for successful vascular repair decreasing the risk of in-stent thrombosis. Here, we show that PDT was able to discriminate between the different cellular subsets and could be more effective in smooth muscle cells than in the endothelial cells. It made the in situ PDT even more valuable therapeutic option for treatment of cardiovascular disease.We designed porous and smooth silica-based matrices that could be successfully used for delivery of Photolon and PPIX photosensitizers. Differences in physiochemical properties of these two photosensitizers revealed the extent of the universality designed material. All tested materials were biocompatible and capable of in situ delivery of PS for PDT of VSMC.


IAA19 promoter fusion with a destabilized luciferase reporter, written by Kotaro T. Yamamoto, Masaaki K. Watahiki, Jun Matsuzaki, Soichirou Satoh and Hisayo Shimizu, was originally published electronically on the publisher’s internet portal (currently SpringerLink) on 10 April 2017 without open access.The article Space–time analysis of gravitropism in etiolated Arabidopsis hypocotyls using bioluminescence imaging of the http://creativecommons.org/licenses/by/4.0/), which permits use, duplication, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.With the author(s)’ decision to opt for Open Choice the copyright of the article changed on 16 July 2018 to © The Author(s) 2018 and the article is forthwith distributed under the terms of the Creative Commons Attribution 4.0 International License (


RC), emission from water erosion (Ewat), and HC, which can be obtained from public data sources. Then the revised CSF was applied to the Liaoning province of China. The estimated carbon input of Liaoning province during 2000–2014 was 114.77 ± 8.41 TgC yr−1, while the carbon output was 110.48 ± 8.38 TgC yr−1. The difference between input and output induced a carbon sink of 4.30 ± 2.20 TgC yr−1, accounting for 3.75% of total carbon input. The carbon sink spatially decreased from northeast to southwest, which was highly correlated with that of GPP. However, though its forming fluxes significantly increased from 2000 to 2014, the carbon sink showed a decreasing trend. In addition, the revised scheme only needed published and public data, which made it serve as an alternative approach for regional carbon budget assessment.Assessing the regional carbon sink sets the basis of regional carbon management, which involves many measures but has large uncertainties. Carbon sink assessment scheme based on its forming processes (CSF) is a recently proposed measure but repeatly calculates emission from water erosion and ignored human inducing carbon inputs. Therefore, we revised the CSF by calculating the direct outputs from land surface and adding human returned carbon (HC) to the input. The revised CSF thus involved gross primary productivity (GPP), ecosystem respiration (ER), carbon removal from cropland (CRC), emission from reactive carbon (E Increasing the carbon sink of terrestrial ecosystems has been regarded as an important approach in mitigating climate change, which needs fully assess the spatial distributions of carbon sink and its increasing potential3.Carbon sink, defined as the residue of organic matter sequestrated by vegetation photosynthesis after natural and human disturbance4, process models6, and atmospheric inversion8, were involved in assessing the regional carbon sink. Resource inventory assessed the regional carbon sink as the changes of carbon stocks during different periods, which was much accurate and was deemed as the base of other measures9. However, resource inventory had a coarse temporal resolution and only accounted the value of carbon sink, which can’t directly reveal the processes how carbon sink was formed. Process models quantified regional carbon sink by simulating the carbon cycles with climatic and biotic drivers, which can simulate the carbon sink at different time-scale and can address the forming processes of carbon sink, while its parameters had some uncertainties11. Atmosphere inversion calculated the regional carbon sink by analysing the changes of atmosphere CO2 concentration and other carbon emissions from natural and human activities, which was much simple but strongly relied on the accuracy of other carbon emissions such as land use changes12. In addition, most of those measures differed obviously as the definitions of carbon sink varied among approaches. For example, resource inventory defined carbon sink as the changes of carbon stock during a period, which ignored the carbon stored in human-beings13, while carbon sink in atmospheric inversion reflected the net carbon fluxes including not only the terrestrial ecosystems but also the human beings14. It is thus necessary to build an assessment scheme fully accounting the involved carbon fluxes, which would improve the clarity of carbon sink they assessed.Many approaches, such as the resource inventory15. However, the current version of CSF had some shortages, which can be ascribed into two aspects. First, it involved many fluxes that may be repeatedly calculated. For example, the carbon emission from water erosion (Ewat) may be recalculated by the carbon flowing to the ocean. Second, gross primary productivity (GPP) was regarded as the sole input, while there were other input fluxes such as the manure, the straw return and industrial fertilizer (IF) introducing carbon to the ecosystems. Therefore, it was urgent to clearly define the carbon sink and fully considering the involved carbon inputs, which would be helpful for accurately assessing the regional carbon budget.Carbon sink assessment scheme based on its forming processes (CSF), a recently proposed approach, involved various carbon fluxes during the forming processes of carbon sink and served as an alternative approach in quantifying the regional carbon sink5.In this study, selecting Liaoning province as the target, we revised the CSF by calculating the direct outputs from land surface and adding the carbon input by human returned carbon (HC). Then the revised scheme was applied to assess the spatial distributions of carbon sink and its components. Our results will provide a reference for assessing the regional carbon sink in other regions, which will be helpful for reducing the uncertainties in regional carbon sink assessments. Our results will also provide a data basis for carbon management in Liaoning province, whose carbon sink had substantial uncertainties and was sensitive to climate change−1. The carbon input were primarily comprised of GPP, which was 110.78 ± 7.87 TgC yr−1 and accounted for 95.62% of total carbon input  also occupied a large portion of output carbon flux, with a value of 19.98 ± 3.72 TgC yr−1 accounting for 17.41% of carbon input. However, the amounts of emission from reactive carbon (ERC) and emission from water erosion (Ewat) were relative small, which were 0.97 TgC yr−1 and 2.57 TgC yr−1, respectively, and accounted for 0.85% and 2.23% of carbon input, respectively , while the returning coefficient may differ among regions. If we assumed the returning coefficient randomly varied from 0 to 0.5 in each prefectural level city, which was randomly repeated 500 times, the uncertainty represented by the standard deviation of HC also spatially varied Fig. . The uncRC and Ewat were not available. Though we attempted to included ERC and Ewat in the revised CSF, there was little effort in quantifying their spatial and inter-annual variations in this region, which made the derived carbon sink have some uncertainties.The spatial and inter-annual variations of EFurthermore, owning to the simplicity of the revised CSF, there were some uncertainties for the forming fluxes thus the carbon sink, which needed to be addressed in the future.1. Furthermore, we also found that the amount of HC (4.00 TgC yr−1) was comparable with the sum of ERC (0.97 TgC yr−1) and Ewat (2.57 TgC yr−1) , and emissions from water erosion (Ewat), which relied on public and published data. The estimated input and output were 114.77 ± 8.41 TgC yr−1 and 110.48 ± 8.38 TgC yr−1, which made a carbon sink of 4.30 ± 2.20 TgC yr−1. Carbon sink spatially decreased from northeast to southwest and positively correlated with GPP. Though its forming fluxes significantly increased, carbon sink decreased from 2000 to 2014. The revised scheme was very simple thus provided an alternative approach to assess the regional carbon budget.In this study, we revised the carbon sink assessment scheme and applied it to Liaoning province of China. The revised scheme only involved gross primary productivity (GPP), ecosystem respiration (ER), carbon removal from cropland (CRC), human returned carbon (HC), emissions from reactive carbon . Then the remained organic carbon, which was deemed as the net primary productivity (NPP), was further consumed by animal and microbes through heterotrophic respiration (Rh). Ra and Rh constructed to ER. Furthermore, the sequestrated carbon was consumed by creature ingestion, which can be deemed as the emission from creature ingestion (ECI). During the stages that ecosystem released carbon through CO2, the sequestrated carbon was also released by reactive carbon such as CH4, CO, and so on, which was deemed as the emission from reactive carbon (ERC). Besides the natural releases, the sequestrated carbon also suffered from natural disturbances and human disturbances. Emissions from natural disturbances (END) was caused by fire (EF) and soil erosion, where END from soil erosion can be further separated into wind erosion (Ewin) and water erosion (Ewat). Emissions from anthropogenic disturbances (EAD) were comprised by the carbon removal from cropland (CRC), grassland (CRG), and forest (CRF). After human disturbances, some removed carbon can be returned to the ecosystems as human returned carbon (HC). In addition, industrial fertilizer (IF) such as ammonium bicarbonate (NH4HCO3), urea (CO(NH2)2) also took some carbon into the ecosystems.The theoretical basis of carbon sink assessment scheme was the carbon cycle processes in terrestrial ecosystems Fig. 15,34. Ca34. Therefore, the carbon sink assessment scheme based on its forming processes involved carbon fluxes occurred between the ecosystems and the surrounding environments. According to the theoretical basis of carbon sink assessment scheme, the input carbon fluxes were GPP, HC, and IF, while the output carbon fluxes included ER, ERCCI, EAD, and END , urea (CO(NH2)2), we only considered GPP and HC as the input carbon fluxes. In addition, given ECI occupied a small proportion of GPP15 and Liaoning province was dominated by cropland, whose ECI can be neglected, we set ECI to 0 in this region. Furthermore, EAD was primarily comprised by cropland carbon transfer in this region and the carbon removal from grassland and forest were small but were hard to obtain35, we just had CRC as EAD. Given fire seldom occurred and soil erosion by wind was hardly quantified in this province, we only considered emissions from water erosion (Ewat) as the emissions of natural disturbance (END).Given the low carbon content of IF, which can be calculated from their chemical formula such as ammonium bicarbonate . Given GPP and ER were positively spatially coupled36, ER, whose spatial resolution was same as GPP, was calculated from GPP as:The magnitudes of some carbon fluxes, including GPP and ER, which had detailed spatial distributions, were calculated by each grid. The magnitude of GPP was set to the Moderate Resolution Imaging Spectroradiometer data (MOD17A2) having a spatial resolution of 30 arc second from University of Montana , the crop harvest index (HIi), the water content (Cwi) and the carbon fraction of dry matter (CCi), which can be found in our previous work30. The amount of HC was set to 20% of CRC.The magnitudes of some carbon fluxes, including CRC and HC having limited information on their spatial distribution, were calculated by each prefectural level city from statistical yearbooksRC and Ewat, whose spatial distributions were seldom available, were calculated as the ratio of their total amounts to the regional area. The amounts of these carbon fluxes can be calculated from the following subsection.While the magnitudes of other carbon fluxes, such as EThe magnitude of carbon sink was calculated from the input and output fluxes, with a spatial resolution of 30 arc second.30.The amounts of GPP and ER, were summed from the magnitude of carbon fluxes by each grid, while these of CRC and HC were calculated as the sum of their corresponding fluxes in each prefectural level city, which can be found in our previous work4), nonmethane volatile organic compounds (NMVOC) and carbon monoxide (CO)15. Emission from CH4 (Em) occurred at rice paddies, lakes, and natural wetlands52. From a recently published paper52, we can get the values of Em of Liaoning province from rice paddies and natural lake. In addition, Em from natural wetland was comparable with that from natural lake52. Therefore, Em of Liaoning province was estimated from previous work52 by summarizing the Em from rice paddies, natural lake, and natural wetland. Given little attention was paid to the spatial variation of NMVOC and CO emissions in China, we had no choice but calculated the emissions of NMVOC (ENM) and CO (ECO) of Liaoning province from the total emissions of China15 and the proportion of Liaoning land area in Chinese area. ERC was the sum of Em, ENM, and ECO.Emission from reactive carbon included that from methane  and the deposition of eroded carbon (F2), while the vertical transportation included the replacement of atmosphere CO2 (F3), the decomposed carbon from buried and transported carbon to atmosphere (F4 and F5). The net lateral and vertical transportations of eroded carbon were F1-F2 and F3-F4-F5, respectively. Ewat was the sum of net lateral and vertical transportations with a positive value indicating the carbon emission. In a recent paper, Ewat of northeast China was reported by Yue, et al.16. After integrating the area of three northeast provinces and Ewat of northeast China, we estimated emissions of soil water erosion in LiaoninNg province.Water erosion caused the lateral and vertical transportations of soil carbonRC and Ewat were smaller and were hardly obtained, their inter-annual variations were neglected.In this study, we analysed the spatiotemporal variations of carbon sink and its components during 2000–2014. The data of GPP, ER, CRC, and HC were calculated from 2000 to 2014 by each year. However, given the values of Ehttp://www.esri.com/software/arcgis), with α of 0.05.Based on data from 2000 to 2014, we calculated their mean values during the study period to investigate their spatial distributions. The spatial correlations between fluxes were conducted with ArcGIS 10.0 software (Using the Manna-Kendall trend analysis, we detected the trend of each carbon fluxes from 2000 to 2014, which was conducted with Matlab 2014.


The purpose of this article is to compare optical coherence tomography angiography (OCTA) and indocyanine green angiography (ICGA) in patients with central serous chorioretinopathy (CSC). OCTA, ICGA and fluorescein angiography (FA) images of all enrolled patients were collected and compared. Abnormal areas were annotated on en face choriocapillaris OCTA and ICGA images and compared with each other. We found three main types of anomalies in choriocapillaris OCTA images: type A, coarse granulated high reflective area (61 eyes [92.4%]); type B, roundish dark halo around Type A (54 eyes [81.8%]); and type C, coarse granulated low reflective area (66 eyes [100%]). There were 54 eyes (81.8%) that exhibited all three types abnormalities, 7 (10.6%) had only type A and C abnormalities, and 5 (7.6%) had only type C abnormalities. The Mean JI of type A on OCTA and hyperfluorescence area on ICGA was 0.84 ± 0.15 and 0.82 ± 0.23 for grader 1 and 2, respectively. Type A area on OCTA had a statistically larger area than hyperfluorescence on ICGA (P = 0.01 [paired t-test]). In summary, abnormalities were found on OCTA images of CSC. Coarse granulated high reflective area in OCTA corresponded well with the hyper-permeability area in ICGA in most of the eyes. CSC incidence varies among ethnic groups, and a higher frequency has been suspected among Asians, Caucasians, and Hispanics compared with African Americans1. CSC patients report symptoms including blurred vision, relative central scotoma, metamorphopsia, moderate dyschromatopsia, hypermetropization, and micropsia, and reduced contrast sensitivity2. Bilateral, multifocal cases of CSC, with worsened visual acuity, have been reported more frequently in Asians3. In most cases, acute CSC (disease duration ≤6 months) is self-limiting with favorable outcome; however, 30% to 50%4 of acute CSC may be associated with permanent vision loss or recur within one year. Chronic CSC (disease duration >6 months) is characterized by diffuse leakage on fluorescein angiography (FA), widespread RPE alterations and photoreceptor defects, resulting in progressive and inevitable vision loss5. Despite observations, CSC with paramacular leakage points can be treated with laser photocoagulation. For individuals with CSC and leakage points directly on the macula, half-dose photodynamic therapy (HD-PDT) is the mainstay treatment choice6.Central serous chorioretinopathy (CSC) is a disorder characterized by serous retinal detachment with or without retinal pigment epithelial (RPE) detachment, and associated with hyper-permeability of the choriocapillary layer. A higher prevalence of CSC among men in their fourth and fifth decades of life has been observed, with the male: female ratio ranging from 2.7:1 to 7:17. OCT reveals subretinal fluid (SRF), with or without serous pigment epithelial detachment (PED), and the thickening of choroid. Intraretinal fluid (IRF) and macular cysts may be encountered in some patients with chronic CSC. A double-layer sign (DLS), defined as an undulated RPE layer and intact underlying Bruch’s membrane, appears in 87% of chronic and 63% of acute CSC patients8. FA reveals one or multiple leakage points and/or diffuse RPE alterations. ICGA reveals choriocapillary hyperpermeability areas with hyperfluorescence. ICGA often reveals a more diffuse hyperfluorescent area compared with FA. In some cases, ICGA may reveal choriocapillary ischemia with hypofluorescence in the early stages9.In addition to a full clinical examination, FA, indocyanine green angiography (ICGA), and optical coherence tomography (OCT) can help diagnose CSC11. More importantly, OCTA detects the movement of red blood cells, which facilitates blood flow imaging and provides the ability to analyze blood flow in different layers12. Because abnormal choroidal blood flow is considered to be important in the pathogenesis of CSC, OCTA may provide additional information about blood supply in CSC, and may help to better understand the underlying pathophysiology of the disease. Previous studies16 have described findings of OCTA with CSC. Hence, the aim of the present study was to describe OCTA findings in Asian patients with chronic and acute CSC, and to compare OCTA with traditional angiographic imaging modalities.Although the imaging techniques mentioned above can help to diagnose most cases of CSC and differentiate from other diseases, FA and ICGA are invasive techniques that require a high demand for trained technicians and patient cooperation. Recently, OCT angiography (OCTA) has emerged as a new imaging technology. Dyeless OCTA is a fast, easy, and noninvasive technique, combining Doppler OCT and en face OCTThis study was performed at the Department of Ophthalmology, Peking University People’s Hospital, Beijing, China. All patients diagnosed with symptomatic CSC between February 2016 and January 2017 were included. The inclusion criteria were as follows: presence of active focal or diffuse leakage on FA; presence of SRF with or without serous PED on OCT; and presence of abnormal dilated choroidal vasculature on ICGA. Patients with other ocular conditions commonly associated with SRF, such as choroidal neovascularization, polypoidal choroidal vasculopathy (PCV), diabetic retinopathy, retinal vascular occlusion, Coat’s disease, or any disease that may affect the quality of imaging , such as cataract, high myopia or nystagmus; history of ocular surgeries including retinal laser; pregnancy; any uncontrolled systemic disease; or any condition rendering patients intolerable to image acquisition were excluded. The research protocol adhered to the tenets of the Declaration of Helsinki. Informed consent was obtained from all subjects, and the protocol was approved by Ethics Committee of People’s Hospital of Peking University.17. All studied eyes were scanned using Angio scan 6 × 6 mm section, 3 × 3 mm section, and HD 21 lines. The built-in OCTA software was used to manually alter the automated segmentation if necessary. All OCTA examinations were performed by one well-trained physician (HJ) on the same day when FA and ICGA were performed to maximize the comparability of the different modalities.All patients underwent a comprehensive ophthalmological examination, including best-corrected visual acuity (BCVA), intraocular pressure, slit-lamp biomicroscopy, dilated fundus examination, FA , ICGA  and OCTA . The spectral domain OCT instrument uses an algorithm, known as OCT microangiography-complex, that is capable of scanning at a rate of 68,000 A-scans per second and introduces improved tracking software known as FastTracTM retinal-tracking technologyTwo experienced masked observers (QJF and ZMW) independently read the OCTA and ICGA images. En face choriocapillary OCTA images and representative ICGA images at early (within 30 s post-injection) and mid phase (60–180 s post-injection) were exported in JPG format and imported into ImageJ  for qualitative analysis. The OCTA images and ICGA images were aligned using Photoshop CS6 . After translation, rotation, and rescaling the target images, three user-specified landmarks on both modalities were accurately matched. A representative example of registered images is shown in Fig. Each observer annotated abnormal areas on en face choriocapillary OCTA images, hyperfluorescence area on mid-phase ICGA images, and hypofluorescence area on early-phase ICGA images. Adjacent abnormal areas that appeared confluent to each other were amalgamated into a single area.To determine interobserver agreement of image annotations, spatial overlap of the annotations of type A on en face choriocapillary OCTA and the hyperfluorescence area on mid-phase ICGA was calculated separately according to Jaccard index (JI) as follows:entclass1pt{minimaA qualitative analysis and comparison of the entire imaging data set were conducted. Statistical analysis was performed using a commercially available spreadsheet program . The Student’s t-test for paired samples and Chi-squared test were used for statistical analysis; P < 0.05 was considered to be statistically significant.This study included 72 eyes of 64 patients (17 women and 47 men) with a mean age of 46.3 ± 10.5 years. Mean BCVA was 0.35 ± 0.29logMAR. 56 patients exhibited a unilateral involvement, whereas the disease occurred bilaterally in 8 patients. Of the 72 eyes, 44 (61.1%) were diagnosed with acute CSC and the remainder (38.9%) with chronic CSC.We found 54 eyes (75%) presenting double-layer sign (DLS) in OCT B-scans. 6 of them  were found abnormal vascular network, which could be choroidal neovascularization (CNV) or branching vascular network (BVN), within the DLS on OCTA images after manually adjusting the slab boundary just above and under the DLS. These 6 eyes were excluded for the following analyze of OCTA images and the comparison of OCTA and ICGA images. Patient demographics and clinical characteristics of the remained 66 eyes are listed in Table Abnormal choriocapillaris OCTA images were noticed in all 66 remained eyes. According to the abnormal patterns, three types of abnormalities were defined: type A, coarse granulated high reflective area  . The mean area of type A abnormalities on OCTA was 12.8 ± 4.0 mm2 in acute CSC patients and 13.4 ± 3.7 mm2 in chronic CSC  eyes and larger than the hyperfluorescence area on ICGA in 12 (18.2%) eyes , which was found in all eyes. Since we have known that various factors such as PED, and subretinal fibrin might cause shadow artifacts16, We had used combinations of OCT B scan and color fundus photograph to exclude shadow artifact at choriocapillaris level caused by PED, fibrin and also the migration of pigment. The low signal area caused by shadow effect was not included in our type C area in our study. Shinojima et al., however, high signal intensity on choriocapillary OCTA was observed in all eyes; the different result may be explained by the smaller sample size of their study20. Although it remains unclear what the type A represents, since there was high spatial correspondence between type A abnormality on OCTA and hyperfluorescence area on ICGA (mean JI 0.84 ± 0.15 for grader 1 and 0.82 ± 0.23 for grader 2), we presume it was caused by the dilation and increased blood flow of choriocapillaries in CSC patients. RPE atrophy may also contribute to the type A abnormality in chronic CSC eyes, but by comparing the OCTA images with the FA and OCT B scan images, type A area was much larger than the RPE atrophy area in certain eyes. Furthermore, if type A area was merely artifacts caused by the atrophy of RPE due to long lasting SRF, it should be more frequent in the chronic CSC than the acute CSC since RPE atrophy was more frequent in the chronic CSC, but in fact we found there is no significant difference of type A area between acute and chronic patients (P = 0.72). Automatic segmentation error might also cause artifacts and affect the abnormal finding at choriocapillaris level, one must be cautious in the interpretation of all types of abnormalities at the level of choriocapillaris on OCTA.In our study, the type A abnormality, which was the coarse granulated high reflective area, was present in 61 (92.4%) eyes. In the study by Chan 2 vs 11.9 ± 4.4 mm2; P = 0.01[paired t-test]) in our study. 49 eyes (74.2%) exhibited an equal area of type A abnormality on OCTA and hyperfluorescence area on ICGA, which was consistent with the findings in the study by Teussink et al.13, while 12 eyes exhibited a larger highly reflective area on OCTA than on ICGA, and 5 eyes did not present type A abnormality on OCTA at all. This finding may be explained by the different mechanism of OCTA and ICGA imaging. OCTA depicts blood flow dependent on intravascular cell movement and maybe affected by the velocity of blood flow. If the blood flow is too high or too slow, it may be beyond the test limitations of the OCTA device, and we would not be able to detect the abnormality. Although ICGA depicts blood flow independent of intravascular cell movement, as long as there is plasma, it is visible on ICGA. Different imaging modalities provide blood flow information that is supplementary to one another. Another possible explanation is that the dilation of choriocapillaries and increased blood flow within them may not present in all CSC patients, or not in all stages of CSC.The mean type A area was larger than high permeability area on ICGA . It was caused by the shadow effect of SRF because we found it co-located with SRF on structural OCT in all eyes. Patients with the type B abnormality had a greater mean maximum SRF thickness than patients without type B abnormalities. We postulate the thickness and the composition of the SRF may be an influential factor in the shadow effect.et al. reported13. That was mainly because we were using a circumcircle of the abnormal area instead of the border of the abnormal area on OCTA to calculate the JIs. The reason why we chose to use different methods was as follows. First, the border between the appearance of healthy and affected area on OCTA may not easily be drawn between graders, even though the appearance of these changes was significantly different from the fairly homogeneous appearance of the healthy choriocapillary layer. Second, we used a round laser spot that covered the choriodal lesion related to the active leakage when applying PDT to CSC patients, we believe a circumcircle would be more practical in our clinic. Our study also revealed a higher agreement of type A abnormality on OCTA and hyperfluorescence on ICGA, and this result may provide the opportunity to use OCTA to guide PDT for active CSC patients. Whether OCTA-guided PDT is equally as effective as ICGA-guided PDT still needs further prospective study to draw a definitive conclusion.The inter-observer agreement for type A on OCTA and hyperfluorescence area on ICGA was higher than Teussink Among OCTA studies of CSC, our investigation included the most number of patients; nevertheless, it was still limited by cross-sectional data acquisition. We included treatment-naïve patients only and excluded potential confounders of different interventions on OCTA images. However, the manual segmentation and annotation methods could also be a factor that may have introduced bias in the result. Because of the limited length of each OCTA scan, we only compared the foveal-centered OCTA images with the ICGA images at the corresponding position and did not perform the OCTA image montage because it was time prohibitive. We were using spectral domain OCTA but not swept source OCTA instrument in this study and it is not satisfying for choroidal circulation at this moment because of OCT light attenuation due to RPE. Because of this limitation we only analyzed the choriocapillary layer but not the Sattler and Haller layer. Nevertheless, we believe that with development of OCTA technology, wide-field swept source OCTA images would provide more information.In conclusion, OCTA enables noninvasive visualization of the retinal and choroidal vasculature in CSC patients. Abnormalities were found in all en face choriocapillaris OCTA images of CSC. The most common abnormality was the coarse granulated low reflective area and the coarse granulated high reflective area on en face choriocapillaris OCTA, which suggested choriocapillary ischemia and compensatory dilation were important in the pathogenesis of CSC. Coarse granulated high reflective area in OCTA corresponded well with the hyper-permeability area in ICGA and may guide PDT instead of ICGA in the future.