Diet may affect bile acid (BA) metabolism and signaling. In turn, BA concentrations may be associated with cancer risk. We investigated (i) associations of BA concentrations with adenoma recurrence and (ii) the effect of a high-fiber, high-fruit and vegetable, and low-fat dietary intervention on serum BA concentrations.The Polyp Prevention Trial is a 4-year randomized, controlled trial that investigated the effect of a high-fiber, high-fruit and vegetable, and low-fat diet on colorectal adenoma recurrence. Among 170 participants who reported adhering to the intervention and 198 comparable control arm participants, we measured 15 BAs in baseline, year 2, and year 3 serum using targeted, quantitative liquid chromatography-tandem mass spectrometry. We estimated associations of BAs with adenoma recurrence using multivariable logistic regression and the effect of the dietary intervention on BA concentrations using repeated-measures linear mixed-effects models. In a subset (N = 65), we investigated associations of BAs with 16S rRNA gene sequenced rectal tissue microbiome characteristics.Q3 vs Q1 = 2.17; 95% confidence interval = 1.19–4.04; Ptrend = 0.03). Although we found no effect of the dietary intervention on BA concentrations, pretrial dietary fiber intake was inversely associated with total baseline BAs . BA concentrations were associated with potential colorectal neoplasm-related microbiome features .Baseline total BA concentrations were positively associated with adenoma recurrence (odds ratioBaseline circulating BAs were positively associated with adenoma recurrence. Although the dietary intervention did not modify BA concentrations, long-term fiber intake may be associated with lower concentrations of BAs that are associated with higher risk of adenoma recurrence. Multiple circulating bile acids (BAs), particularly conjugated BAs, have been associated with higher risk of colorectal cancer (CRC) ,2, whichin vitro studies support the possible roles of fat and fiber intake in regulating BA synthesis and excretion, respectively  and the alpha-tocopherol, beta-carotene cancer prevention (ATBC) study   . In ATBCIn this study, among men and women with a history of colorectal adenomas, we investigated the associations of circulating BAs with colorectal adenoma recurrence and with the rectal tissue microbiome. We also investigated the effect of adherence to a high-fiber, high-fruit and vegetable, low-fat dietary intervention on circulating BA concentrations and temporal variability of BAs.The Polyp Prevention Trial (PPT)  is a 4-yA colonoscopy was conducted 180 days to 2 years after randomization (around year 1) to remove any lesions missed by the baseline colonoscopy. Any colorectal adenoma identified thereafter was considered a recurrent colorectal adenoma. Those without a recurrent adenoma were considered a noncase. For our main analyses, those with hyperplastic polyps were included in the noncase group. For the secondary analyses by adenoma/polyp characteristics, we included hyperplastic polyps as a separate histology, combined advanced  or ≥2 adenomas into 1 category, and categorized only individuals without colorectal adenomas or hyperplastic polyps as noncases.The specific goals of the dietary intervention included (i) limiting fat intake to 20% of total kcal/d, (ii) consuming ≥ 18 g of fiber per 1,000 kcal/d, and (iii) consuming ≥3.5 servings of fruits and vegetables per 1,000 kcal/d. Dietary goals were calculated based on each participant's total energy intake as determined by the baseline food frequency questionnaire (FFQ), and each participant received a communication with their individual dietary goals and behavior-modification techniques. The intervention group continued to receive dietary counseling by a nutritionist throughout the trial.At each annual follow-up visit, participants answered questionnaires collecting demographic, behavioral, and medical history information and had dietary goal achievement determined by (i) nutritionist assessment and (ii) their responses to a 4-day food record, followed by the Block/National Cancer Institute FFQ modified to assess more detailed intakes of low-fat and high-fiber foods. Each year the investigators also administered unscheduled 24-hour dietary-recall questionnaires to a newly selected 10% random sample. The FFQ ascertained dietary intakes over the past year and average serving sizes. Compared with the 4-day food record and the 24-hour recall, the FFQ slightly overestimated fat and underestimated fiber, fruit, and vegetable intake .We included those who self-reported adhering to the dietary intervention (super compliers) (n = 170) and comparable goal-achieving control arm participants (n = 198), among whom the strongest effects of the dietary intervention on adenoma recurrence were observed . ComposiFasted, baseline venous blood samples were collected after study enrollment and at the end of each year of the 4-year trial and stored in a central repository at −80 °C. We measured concentrations of 15 primary and secondary BAs and their conjugates in serum samples from baseline, year 2, and year 3 using the Metabolon fully quantitative liquid chromatography-tandem mass spectrometry platform. At baseline, serum samples were collected after colonoscopy, and at year 2 and year 3, no colonoscopy was conducted. In brief, serum samples were spiked with a solution of corresponding labeled internal standards for each of the BAs and were subjected to protein precipitation with acidified methanol. Samples were then centrifuged, and a portion of the clear supernatant evaporated to dryness in a gentle stream of nitrogen at 40 °C. The dried extract was then reconstituted, and an aliquot injected onto an Agilent 1290/Sciex QTRAP 6500 mass spectrometer liquid chromatography-tandem mass spectrometry system equipped with a C18 reverse-phase high-performance liquid chromatography column with acquisition in negative-ion mode. The peak area of each parent or product ion was then measured against the peak area of the respective internal standard parent or product ion. Quantitation was performed using least squares regression analysis generated from fortified calibration standards prepared immediately before each run.We included 27 replicates each of 2 pooled, blinded, serum quality control samples randomly distributed throughout each batch, which were used to estimate interbatch coefficients of variation. Thirteen of the 15 BAs were quantifiable in ≥95% of quality control samples. Average interbatch coefficients of variation were ≤20%, except for taurolithocholic acid (26%) and tauroursodeoxycholic acid (45%), which had a high percentage of metabolites below the limit of detection and were excluded from further analyses. Samples from the same participant, collected at different times, were ordered adjacent in the same batch but in a random order.2 transformation was used to improve normality. Thus, a 1-unit increase in the log2 BA variable can be interpreted as a doubling in the concentration . We created summary BA measures, including total, primary, and secondary. To do this, we summed the individual BA concentrations and log2-transformed the sum. Primary BAs included chenodeoxycholic acid, cholic acid, glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, and taurochenodeoxycholic acid while secondary BAs included deoxycholic acid, lithocholic acid, glycodeoxycholic acid, glycolithocholic acid, and taurodeoxycholic acid.Values of BA concentrations (ng/mL) below the limit of detection were replaced by half of the lowest detected value for that metabolite. For analyses treating BA concentrations as continuous variables, a logBriefly, rectal biopsies from baseline, year 1, and year 4 from 455 participants were lysed using an enzymatic cocktail, homogenized in a Bead Ruptor , and centrifuged. The Animal Tissue DNA Extraction Kit  was used for DNA extraction. The V4 region of the 16S rRNA gene was polymerase chain reaction-amplified for 30 cycles, and 2 × 250 bp paired-end sequencing was performed on the Illumina MiSeq v2 using the 500-cycle kit .Using the DADA2 pipeline 1.2.1 , sequenca priori based on prior associations with CRC including Bacteroides, Fusobacterium, Porphyromonas, Parvimonas, Peptostreptococcus, Gemella, Prevotella, Solobacterium, Dialister, and order Clostridiales  and as tertiles and used multivariable logistic regression. We repeated the analysis stratified by sex, given prior evidence for sex differences in BA-colorectal neoplasm associations  using linear mixed-effects models with a random effect for subject clustered by center, a fixed effect for year, and a covariate for randomization group. To investigate associations of circulating BA concentrations with adenoma recurrence, we modeled BA concentrations continuously  that were previously associated with adenoma recurrence in the PPT study and in prior literature.Mean BA concentrations were calculated for each intervention group for baseline, year 2, and year 3. We then conducted analyses estimating the effect of the dietary intervention on mean year 2 and year 3 concentrations of BAs using repeated-measures linear mixed-effects models. The model included a random effect for the subject, the intercept, indicators for the intervention group and follow-up time (baseline or follow-up), and an intervention × follow-up interaction term. We also explored including covariates  in the model that may be associated with systemic BA concentrations based on previous literature but found that their inclusion did not meaningfully change our findings. We repeated the above mixed-effects model analyses stratified by participant characteristics, including age; sex; baseline body mass index (BMI); smoking status; baseline adenoma characteristics; and usual pretrial fat, fiber, and fruit/vegetable intakes. We also conducted analyses stratifying by completion of (i) all-fiber goals at T1, T2, and T3; (ii) all-fat goals at T1, T2, and T3; and (iii) all-fruit and vegetable goals at T1, T2, and T3. To assess whether habitual pretrial diet was associated with baseline BA concentrations, we calculated partial Spearman correlations among pretrial fat, fiber, and fruit and vegetable intakes and BA concentrations. Pretrial dietary intakes were energy-adjusted using the nutrient density method. Finally, we calculated Spearman correlations between circulating BAs and baseline microbiome metrics  correction using the Benjamin-Hochberg method.Institutional review boards from the National Cancer Institute and all participating centers approved of this study .P < 0.001), as were most glycine-conjugated BAs with their taurine-conjugated counterparts . BAs were moderately stable over the 3 time points. The lowest ICC was for glycolithocholic acid  while the highest ICC was for taurocholic acid  and 2-fold  higher odds of adenoma recurrence, respectively, with associations being stronger among men. Associations of secondary BAs with adenoma recurrence were weaker and not statistically significant. Associations of BAs at year 2 and year 3 with adenoma recurrence were in similar directions but were weaker compared with baseline. At baseline, glycochenodeoxycholic acid and glycocholic acid were most strongly, positively associated with adenoma recurrence, particularly among men . For example, comparing men in the highest relative to lowest tertile of glycochenodeoxycholic acid and glycocholic acid, there were 2.24-fold  and 2.51-fold  higher odds of adenoma recurrence, respectively. In our analyses according to adenoma characteristics, baseline BA concentrations were generally inversely associated with hyperplastic polyps and were more strongly, positively associated with adenomas in the left vs right colon .Associations of summary BAs at baseline, year 2, and year 3 with adenoma recurrence around year 3–4 of the trial are summarized in Table https://links.lww.com/CTG/A877 for changes in individual BA concentrations). We found no appreciable or statistically significant treatment effects of the dietary intervention on BAs, alone or in combination. The effects were similarly null among categories of age; sex; BMI; smoking status; baseline adenoma characteristics; usual pretrial fat/fiber/fruit and vegetable intakes; and adherence to fiber, fat, or fruit and vegetable intervention goals at year 1, year 2, and year 3 .Changes in summary BA concentrations for super compliers relative to goal-achieving control arm participants are summarized in Table s = −0.15; PFDR = 0.02), primary BAs , and secondary BAs . Of the individual BAs, fiber was most strongly inversely associated with glycochenodeoxycholic acid . Fat and fruit/vegetable intakes were not statistically significantly associated with the BAs after FDR correction.To assess whether habitual diet was more strongly associated with BA concentrations, we calculated partial Spearman correlations for associations of baseline fat, fiber, and fruit and vegetable intakes with baseline BA concentrations Table . Fiber wa priori selected rectal tissue microbiome measurements, multiple BAs were inversely associated with alpha diversity . For example, the Spearman correlation for the association of secondary BAs with observed ASVs (species richness) was −0.39 (P = 0.001) and the Spearman correlation for the association of taurochenodeoxycholic acid with Bacteroides abundance was 0.30 (P = 0.02).Among 65 individuals with both baseline BA concentration and Bacteroides, two microbiome metrics that have been associated with colorectal neoplasms generally  and with adenoma prevalence in PPT (under review).Among men and women with a history of colorectal adenoma, we found that baseline BA concentrations were positively associated with adenoma recurrence, particularly primary BAs among men. We also found that adhering to a high-fiber, high-fruit and vegetable, and low-fat diet did not alter circulating BA concentrations over 3 years. However, we found that longer term, pretrial fiber intake was inversely associated with BAs at baseline. BA concentrations, predominantly secondary BAs, were inversely associated with rectal microbial alpha diversity and positively associated with abundance of We found that BAs, particularly glycine-conjugated primary BAs, were positively associated with colorectal adenoma recurrence. Our findings were mostly limited to baseline, possibly reflecting the moderate temporal stability of BAs potentially due to underlying changes in adenoma status across the follow-up period. There is accumulating evidence that dysregulation of BA metabolism may be associated with higher risk of colorectal neoplasms. Human studies have found positive associations of circulating BA concentrations with colorectal adenomas –34. A caQ4 v Q1 = 2.85, 95% CI = 1.45–5.60; Ptrend = 0.011) . There were similar positive findings for other glycine-conjugated and taurine-conjugated primary and secondary BAs (.52–3.26.= 0.011) .Bacteroides, a bacterium which has been positively associated with adenomas and CRC . We also found positive associations of BAs with  and CRC . Given tin vitro and animal studies demonstrates that dietary fat stimulates hepatocyte secretion of BAs into the bile canaliculi, a necessary function for solubilization and absorption of lipids in the gut  Sequence Read Archive . This analysis was not preregistered in an independent, institutional registry.✓ Circulating bile acids (BAs) have been associated with higher risk of colorectal cancer.✓ Few human studies have investigated interrelations of diet, BA concentrations, and colorectal adenoma recurrence.✓ BA concentrations at baseline were strongly, positively associated with adenoma recurrence.✓ Although a high-fiber, high-fruit and vegetable, and low-fat diet was not associated with circulating BAs, pretrial fiber intake was inversely associated with total baseline BA concentrations.✓ BAs may be a potentially intervenable biomarker for colorectal neoplasms.✓ Additional prospective studies are needed to better elucidate interrelationships among diet, BAs, and colorectal neoplasia.


WHO handbook for guideline development. The guideline was initially registered in the International Practice Guidelines Registry Platform (IPGRP-2021CN096). The clinical questions included in the guideline were identified through a three-round Delphi vote. Databases search was performed in PubMed, Embase, the Cochrane Library, ClinicalTrials.gov, Chinese National Knowledge Infrastructure, and WanFang Database from their inception to 31 March 2021. Systematic reviews and meta-analyses for each clinical question were conducted individually to synthesize available scientific evidence. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used to assess the quality of evidence and grade the recommendations’ strengths. The multidisciplinary guideline groups were set up, including ophthalmologists, pharmacists, methodology experts, pharmacologists, pharmacoeconomists, and lawyers. The guideline identified 25 clinical questions included. A total of 32 systematic reviews, including 24 conducted by the systematic review group and eight high-relevance published within 2 years, were referred to address these questions. Finally, the guideline presented 32 recommendations addressing 25 clinical questions, involving five strong recommendations and 27 weak recommendations for the treatment of ocular fundus, corneal disease, glaucoma, and endophthalmitis. Current evidence from clinical studies supports the off-label drugs used in ophthalmology. We developed an evidence-based guideline using a rigorous multidisciplinary approach to guide these usages in route clinical practice.Off-label drugs use is widespread in ophthalmology due to the delay in drug approvals and package inserts update. It has been found to vary among different medical institutions in China, leading to safety problems since inappropriate use. Guidance is urgently needed regarding how best to use the drugs for unapproved indications and routes of administration. We aimed to develop an evidence-based guideline to guide off-label drugs used in ophthalmology in China. The practice guideline was developed by the Hospital Pharmacy Professional Committee, Chinese Pharmaceutical Association, following the  Vision loss contributes to a substantial public health burden worldwide, causing significant economic impact, reducing the quality of life, impacting educational opportunities, and increasing the risk of premature death . World HOff-label drugs refer to pharmaceutical drugs used for an unapproved indication or in an unapproved age group, dosage, or route of administration. Off-label drug use is not prohibited in the United States and the European Union, in which physicians could prescribe off-label drugs with the support of extensive scientific evidence . China’sThe use of drugs for unapproved indications and routes of administration in ophthalmology has been widespread due to the delay in drug approvals and package inserts update in China . PreviouAt present, there are only scattered studies to systematically evaluate the efficacy and safety of ophthalmic drugs used in new unapproved indications or non-recommended routes. Furthermore, no guidelines or consensuses regarding off-label drug use in ophthalmology have been developed. Therefore, we aimed to investigate the medications used in ophthalmology that have not yet been approved in China. Additionally, this study aimed to develop Chinese evidence-based guideline for off-label drugs in ophthalmology to standardize the ophthalmic prescriptions and promote their rational use among different medical institutions.Handbook for guideline development and the Appraisal of Guidelines for Research and Evaluation (AGREE II)  statement (The guideline was developed following the WHO GREE II) . The guitatement . The floThe project was led by the Hospital Pharmacy Professional Committee of the Chinese Pharmaceutical Society and implemented by the Department of Pharmacy, Beijing Tongren Hospital, Capital Medical University. Methodological support was obtained from the Chinese GRADE Center. The guideline was initially registered in the International Practice Guidelines Registry Platform (IPGRP-2021CN096).The guideline focused on off-label drugs used in ophthalmology in China, including medications in the treatment of ocular fundus, corneal disease, glaucoma, and endophthalmitis. Medications including antagonists of vascular endothelial growth factor (anti-VEGF) agents, immunosuppressive drugs, glucocorticoids, and antimicrobial agents were comprehensively evaluated to treat the above-mentioned disorders. Several drug usages approved in the United States and the European Union, not in China, were also included. The primary target populations were ophthalmologists, pharmacists, and nurses. Nevertheless, other health care professionals and policymakers involved in medication regulation can also benefit from this guideline.Handbook for guideline development, the steering group, guideline development group (GDG), external review group, and systematic review (SR) group were established , and three outstanding ophthalmologists . The steering group’s role was to provide administrative support for the guideline development, make decisions at every stage, and control the entire process. Finally, they led the writing of the final draft of the guideline.The GDG consisted of 21 multidisciplinary specialists: 11 clinical pharmacy specialists, five ophthalmology specialists, two methodology experts, one pharmacoeconomics consultant, one pharmacy-administration expert, and one health lawyer. They were responsible for determining the clinical questions, formulating recommendations, and reviewing and approving the final guideline document.The SR group consisted of 16 qualified pharmacists or students trained in evidence-based medicine. They were responsible for collecting clinical questions, retrieving, evaluating, synthesizing, and grading evidence, conducting a systematic review, and formulating the summary of findings tables and the GRADE evidence profiles.The external review group consisted of 29 first-line health care professionals . They reviewed the final guideline to identify errors or missing data and commented on clarity, feasibility, setting-specific issues, and implementation implications.In the early stage, the clinical questions about off-label drugs used in ophthalmology were collected from several hospitals by retrieval records and individual interviews with experienced ophthalmologists and pharmacists. Subsequently, a three-round Delphi vote was performed among the steering group and GDG to select the most important clinical questions that needed to answer. Finally, a total of 25 clinical questions were included in the guideline.Cochrane Handbook for Systematic Reviews of Interventions to answer the clinical questions selected  framework in the selected . DatabasThe quality of systematic reviews/meta-analyses was assessed using the Assessment of Systematic Reviews (AMSTAR) tool , the quaThe certainty of quantitative and qualitative evidence was assessed using the GRADE and GRADE-CERQual approaches, respectively . FurtherTwenty-five clinical questions about off-label drugs used in ophthalmology in China were included. Seventeen drug uses were off-label in the United States and the European Union. A total of 32 systematic reviews, including 24 conducted by the SR group and eight high-relevance published within 2 years, were referred to address these questions. Finally, the guideline developed 32 recommendations, including five strong and 27 weak recommendations for the treatment of ocular fundus, corneal disease, glaucoma, and endophthalmitis. Drug approval status, GRADE evidence profiles, and summaries of findings are shown in the Supplementary Appendix. The characteristics of the systematic reviews included for recommendations formulation are presented in Recommendation 1. Anti-VEGF drugs alone or in combination with panretinal laser photocoagulation (PRP), are recommended for the treatment of proliferative diabetic retinopathy (PDR), which can improve best-corrected visual acuity (BCVA), reduce vitrectomy and vitreous bleeding rates (strong recommendation with high certainty evidence).n = 632, five RCTs, high certainty evidence, Evidence. A systematic review and meta-analysis article .n = 880 eyes, 11 RCTs, moderate certainty evidence, Evidence. A systematic review and meta-analysis article (Recommendation. The efficacy and safety of anti-VEGF drugs in treating type I retinopathy of prematurity (ROP) are comparable to those of PRP. Anti-VEGF drugs delay disease recurrence and improve refraction (weak recommendation with low certainty evidence).n = 3701 eyes, 24 controlled studies involving 8 RCTs, low certainty evidence, Evidence. A systematic review and meta-analysis article (Recommendation. Anti-VEGF drugs in neovascular glaucoma are suggested to improve BCVA and reduce the incidence of surgery-related adverse events (weak recommendation with low certainty evidence).n = 1189, 14 RCTs, low certainty evidence, Evidence. The systematic review conducted by the SR group  (weak recommendation with moderate certainty evidence).n = 720, 7 RCTs, moderate certainty evidence, Evidence. The systematic review conducted by the SR group .n = 310, 16 RCTs, high certainty evidence, Evidence. The systematic review conducted by the SR group .n = 310, 16 RCTs, high certainty evidence, n = 1631, 8 RCTs, moderate certainty evidence) showed that anti-VEGF drugs were more effective in improving visual acuity and reducing macular edema in patients with branch retinal vein occlusion than placebo, laser photocoagulation, and local ocular injection of glucocorticoids .n = 378, 24 case series, very low certainty evidence, n = 35), followed by cataract formation (n = 13), and traction retinal detachment (n = 12), with no systemic adverse events.Evidence. The systematic review conducted by the SR group .n = 839, 2 RCTs, 1 cohort study, 3 case-control studies, 12 case series, low certainty evidence, Evidence. The systematic review conducted by the SR group .n = 3329, 33 RCTs, high certainty evidence, Evidence. The systematic review conducted by the SR group .n = 281 eyes, 1 RCT plus 3 cohort studies, very low certainty evidence, versus oral glucocorticoids. One retrospective comparative study found that the frequency of uveitis occurrence in the IV pulse group was significantly decreased (number/year 0.23 ± 0.21 vs. 0.40 ± 0.35). Five days after treatment, the maximum retinal detachment height was also considerably lower than that in the oral treatment group. No significant difference was observed in ocular complications such as anterior retinal membrane, neovascularization, and subretinal fibrosis.Evidence. The systematic review conducted by the SR group .n = 144 eyes, 3 RCTs, very low certainty evidence, Evidence. The systematic review conducted by the SR group (Recommendation 1. It is suggested to combine cyclosporine with glucocorticoid for the treatment of VKH disease when the effect of glucocorticoid is not satisfactory (weak recommendation with very low certainty evidence).n = 177, 3 RCT plus one cohort study, very low certainty evidence, n = 60) found that cyclosporine therapy plus oral glucocorticoid gained better visual acuity than glucocorticoid therapy. In both studies , cataracts, glaucoma, and systemic adverse events were reported sporadically in both groups. Two RCTs evaluated the efficacy and safety of cyclosporine plus oral glucocorticoid therapy, compared with intravenous pulse followed by oral glucocorticoid administration. One RCT showed no significant difference in the 12-months recurrence risk between the two groups. In both RCTs, inconsistent visual acuity results were reported, while a significant improvement in fundus lesions was observed in the cyclosporine group. One RCT found that the incidence of cataracts was lower in the cyclosporine group, but there were 3 cases of drug withdrawal due to adverse events, which was higher than in the control group.Evidence. The systematic review conducted by the SR group .Evidence. One RCT .n = 862, 1 RCT plus 4 case series, low certainty evidence, Evidence. The systematic review conducted by the SR group .n = 693, 6 RCTs, low certainty evidence, Evidence. The systematic review article  .n = 321, 1 cohort study plus 5 case series, very low certainty evidence, Evidence. The SR group conducted the systematic review .n = 1328, 19 RCTs, moderate certainty evidence, Evidence. The SR group updated the systematic review  .n = 437, 1 RCT plus five observational studies, low certainty evidence, Evidence. A systematic review article  .n = 221, 5 case series, very low certainty evidence, Evidence. The systematic reviews conducted by the SR group  glaucoma to relieve glaucoma-related symptoms (weak recommendation with very low certainty evidence).n = 351, 13 case series, low certainty evidence, Evidence. The SR group conducted the systematic review .n = 370, 6 RCTs, high certainty evidence, Evidence. The systematic review conducted by the SR group .n = 1085, 11 RCTs, moderate certainty evidence, Evidence. Studies retrieved did not differentiate between various levels of dry eye severity. The latest systematic review  .n = 130 eyes, 3 RCTs, low certainty evidence, Evidence. Studies retrieved did not differentiate between various levels of dry eye severity. The SR group conducted the systematic review .n = 380, 1 cohort study plus 6 case series, very low certainty evidence, Evidence. The SR group conducted the systematic review .n = 145, 2 RCTs plus 1 cohort study, low certainty evidence, Evidence. The systematic review conducted by the SR group .n = 141, 2 RCTs, moderate certainty evidence, Evidence. The SR group conducted the systematic review .n = 165, 1 quasi-experimental study plus 4 case series, very low certainty evidence, Evidence. The SR group conducted the systematic review .n = 115, 5 case series, very low certainty evidence, Evidence. The systematic review conducted by the SR group .n = 329, 1 cohort study plus 4 case series, very low certainty evidence, Evidence. The SR group conducted the systematic review .n = 76, 1 RCT plus 6 case series, very low certainty evidence, Evidence. The systematic review was conducted by the SR group .n = 297, 1 RCT plus 12 case controls, very low certainty evidence, Evidence. The systematic review was conducted by the SR group  controls the commercial availability of novel therapeutics but does not restrict physician prescriptions. Consequently, the physician may legally prescribe approved agents for unapproved indications  based on their best clinical judgement . China pTo our knowledge, this was the first evidence-based guideline for off-label drugs used in ophthalmology in China. The guideline has several strengths. Firstly, we followed the internationally recognized guideline development methodology, ensuring the guideline’s validity, reliability, and reproducibility. For example, we registered the guideline online, performed a systematic review for each clinical question, used the GRADE approach and Delphi vote to generate recommendations, conducted an external peer review on the recommendations, and reported the guideline according to the RIGHT statement. Secondly, we emphasized the management of conflicts of interest and involvement with frontline medical workers in the guideline development, which enhanced the transparency and applicability of our guideline. Thirdly, our guideline provided a practical recommendation regarding the off-label drugs used for many refractory eye diseases, including anti-VEGF agents, immunosuppressive drugs, glucocorticoids, and antimicrobial agents, to promote the rational use of drugs.The guideline also has limitations. Firstly, off-label drug usages in the guideline are mainly for China, which might have been approved in other countries. For example, only 17 usages in the guideline are off-label in the United States and the European Union. However, this does not affect applying the guideline as a reference for medical professionals in these countries. Secondly, some recommendations for off-label drug use are only supported with low-quality evidence and formulated more based on expert opinions, which may bring some bias. However, we had adopted the Delphi method of voting to minimize the bias.In conclusion, we developed an evidence-based guideline concerning off-label drug use in ophthalmology using a rigorous and multidisciplinary approach. The guideline provides more comprehensive recommendations for informing clinical care, which is of greater applicability. However, considering the lack of robust evidence for some off-label usages, more high-quality extensive observational studies and RCTs are still urgently needed.


Urinary incontinence is a highly prevalent condition that impacts self-esteem and overall quality of life. Many non-surgical treatment options are available, ranging from pharmacological approaches to pelvic exercises. We aimed to summarize the available evidence regarding these non-surgical interventions. Review of systematic reviews, conducted in the Discipline of Evidence-Based Medicine, Escola Paulista de Medicina, Universidade Federal de São Paulo (EPM-UNIFESP).  A sensitive search was conducted to identify all Cochrane systematic reviews that fulfilled the inclusion criteria. Titles and abstracts were screened by two authors.  We included 20 Cochrane systematic reviews: 4 assessing methods of vesical training, 3 evaluating pharmacological interventions, 4 studying pelvic floor muscle training approaches and 9 aimed at other alternatives . The reviews found that the evidence regarding the benefits of these diverse interventions ranged in quality from low to high.  This review included 20 Cochrane systematic reviews that provided evidence  for non-pharmacological interventions for patients with urinary incontinence. Moderate to high quality of evidence was found favoring the use of pelvic floor muscle training among women with urinary incontinence. To establish solid conclusions for all the other comparisons, further studies of good methodological quality are needed. Urinary incontinence (UI) can affect up to 50% of women worldwide.,,Urinary incontinence can be classified into three main types: stress, urge and overflow incontinence. Many women have mixed characteristics of more than one type.Women presenting stress incontinence (the most common type in younger women) have involuntary leakage of urine relating to increased intra-abdominal pressure  in the absence of a bladder contraction.,It is important to point out that despite the considerable prevalence of UI, it continues to be an underdiagnosed and an undertreated disease: only about a quarter of women with complaints pursue medical care and, of those, less than 50% receive some type of treatment.Because UI is a highly prevalent condition, with high impact on quality of life, several treatment options for it are currently available. These options range from pelvic exercises to surgical techniques, including pharmacological therapies and use of local devices.The aim of the present study was to identify and summarize the evidence from Cochrane systematic reviews (SRs) regarding non-surgical therapeutic options for preventing urinary incontinence and for treating women with this condition.A review of Cochrane SRs was conducted within the Discipline of Evidence-based Medicine of Escola Paulista de Medicina, Universidade Federal de São Paulo (EPM-UNIFESP). This article was specifically developed for the section Cochrane Highlights, which is an initiative for disseminating Cochrane reviews. This initiative results from a formal partnership between the São Paulo Medical Journal and Cochrane, and it is supported by Cochrane Brazil.We included full Cochrane SRs published in the Cochrane Database of Systematic Reviews (CDSR). Protocols for SRs and withdrawn or outdated versions of SRs were not included. There was no limit on publication dates.The participants considered were women who had been diagnosed with any type of urinary incontinence. We did not take into consideration reviews that included participants of both sexes but did not have any subgroup analyses that considered women alone.We considered SRs assessing any non-surgical therapeutic option, whether used in isolation or in combination with each other.We considered any clinical or laboratory outcomes, as evaluated by the authors of the SRs included.We conducted a systematic search in the Cochrane Database of Systematic Reviews (CDSR) (via Wiley) on October 18, 2017, sensitively using the MeSH term “urinary incontinence” and all related variants, in titles, abstracts and keywords. The detailed search strategy is presented in The titles and abstracts were screened by two authors  independently. The SRs that met the inclusion criteria were selected. Any disagreement was resolved by consulting a senior author (RR).The results from the search and the SRs included were presented through a descriptive approach .Our search strategy retrieved 106 references and, after screening the titles and abstracts, 31 SRs were preselected. After assessing full texts, 20 reviews were found to fulfill our inclusion criteria and were included for qualitative synthesis.In the following section, we present a brief individual summary of each review included, according to the intervention assessed. For details about the characteristics of the interventions, comparisons, outcomes and quality of evidence,This reviewFor further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD001842.pub2/abstract.This reviewNumber of participants with no improvement of urinary incontinence: use of oral systemic estrogens gave rise to a higher number of participants with no improvement , while use of local estrogens gave rise to a lower number of participants with no improvement .Episodes of urinary incontinence over 24 hours: no difference between the groups . Women with urge incontinence: no difference between use of systemic estrogens and placebo , but lower rate with local estrogens than with placebo .Women with adverse events: higher rate with use of systemic estrogens . No difference in adverse events was found with use of local estrogens, compared with placebo .The authors concluded that local estrogen treatment may improve UI, but that there was only limited evidence regarding its effects in the post-treatment period and over the long term. However, it was observed that systemic estrogen treatment might worsen incontinence. The data were insufficient to identify the optimal estrogen type and dose, and there was no direct evidence for comparing routes of administration. The risk of endometrial and breast cancer after long-term use of systemic estrogen suggested that this treatment should only be administered for limited periods, especially among women with an intact uterus. For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD001405.pub3/abstract.This reviewNumber of participants with urinary incontinence at the end of the follow-up: no benefits from use of duloxetine 80 mg/day , 40 mg/day  and 20 mg/day .Number of participants without improvement during treatment: lower through use of duloxetine 80 mg/day , 40 mg/day  and 20 mg/day .Adverse events: more frequent through use of duloxetine 80 mg/day .These results showed that duloxetine could improve symptoms but failed to show any objective improvements in cure rate. The number of participants presenting adverse events was higher in the duloxetine group. Further studies comparing duloxetine and other SNRIs with other treatments are needed in order to reach additional conclusions.For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD004742.pub2/abstract.This reviewPelvic floor muscle training (PFMT) with biofeedback versus PFMT alone: PFMT with biofeedback was statistically significantly better for improving women’s perception of change in incontinence  and women’s satisfaction . There was also a marginally significant result regarding reduction of the number of leakage episodes , but this result might not be clinically relevant.PFMT with feedback versus PFMT alone: The group that received feedback had better perception of change in incontinence  and greater satisfaction .Other comparisons were made, but the effects of biofeedback/feedback interventions could not be individually interpreted. The authors of this SR concluded that feedback or biofeedback might provide benefits when added to PFMT in treating urinary incontinence. However, no assessment of quality of evidence was made and no data for safety assessment were provided in the RCTs included. For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD009252/abstract.This reviewBladder training versus oxybutynin: Patient’s perception of cure at six months: better in bladder training group .Quality of life : better in bladder training group .Bladder training versus imipramine plus flavoxate:Patient’s perception of cure immediately after treatment: better in bladder training group .Bladder training plus pelvic exercises plus biofeedback versus pelvic exercises plus biofeedback:Patient’s perception of improvement: better with bladder training .All the results of this systematic review were based on poorly reported low-quality RCTs with small sample sizes. The main outcomes were subjective and not pre-specified. Therefore, conclusions regarding the effectiveness and safety of bladder training must await further studies with good methodological and reporting qualities. For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD001308.pub2/abstract.This reviewFor further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD002801.pub2/full.This reviewdietary changes weight losssmoking cessationphysical activity andfluid intake in relation to management of UI. Eleven RCTs were included .The following results were found regarding comparison of weight loss programs versus any other intervention:Patient’s subjective impression of improvement: better in the weight loss program group .Cure rates: no difference between groups .Frequency of incontinence episodes per week: no difference between groups .All the trials included were judged to present high risk of bias. Taking into account the quality of the body of evidence relating to weight loss programs and the lack of high quality RCTs, no solid conclusion can be drawn until new studies are available. For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD003505.pub5/abstract.This reviewReduction of the proportion of hourly checks that found wetness (mean difference (MD) 12%; 95% CI -18.79% to -5.21%; one RCT; 148 participants).Fewer episodes of incontinence over 24 hours . No statistical difference between the groups were found regarding the number of people with no improvement in wet episodes . Many of the RCTs included did not provided minimum statistical data for analysis. Also, the clinical outcomes assessed in these RCTs were too diverse and poorly reported. The small sample size and poor methodological quality of the RCTs were an obstacle to drawing any recommendations for practice. Further studies investigating prompted voiding are needed in order to evaluate the safety and effectiveness of this intervention. For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD002113/abstract.This reviewFor further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD002802.pub2/abstract.This reviewFor further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD007408/abstract.This reviewImprovement of symptoms (subjective assessment): better with acupuncture . Cure rate: no difference between groups .Because this review included only one small head-to-head study with low methodological quality and poor reporting, no conclusions can be reached regarding the efficacy and safety of acupuncture among UI patients. For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD009408.pub2/abstract.The objective of this reviewFor further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD001756.pub6/abstract.This reviewQuality of life: no difference between groups Adverse events: no difference between groups Urinary tract infection: no difference between groups .The authors concluded that there was insufficient evidence to be able to state that TRCD improves quality of life among patients with UI. For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD010217.pub2/abstract.This reviewMeclofenoxate versus placebo: reduction in the perception of urinary symptoms through use of meclofenoxate .Acupuncture versus usual care/no intervention: reduction in the number of participants with incontinence after treatment in the acupuncture group .The authors concluded that there was insufficient evidence to guide UI management after stroke. For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD004462.pub3/abstract.This reviewInjection versus conservative treatment: the injection treatment group had a higher number of participants with improvement of continence than did the conservative treatment group  and it achieved a statistically significant improvement of quality of life .Injection versus surgical management: the surgical group had higher rates of symptom improvement in two trials: RR 4.77; 95% CI 1.96 to 11.64; one RCT; 45 participants in one study; and RR 1.69; 95% CI 1.02 to 2.79; one RCT; 54 participants in the other study.The results from the remaining RCTs included were poorly reported and no data could be pooled because of the lack of relevant outcome results and because of the heterogeneity between studies. Until further high-quality studies are available, the benefits of urethral injection therapy in patients with urinary incontinence will remain a matter that needs to be resolved.For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD003881.pub4/abstract.This reviewWeighted vaginal cones versus no treatment: the intervention group had a lower number of patients with urinary incontinence at the end of the follow-up .Weighted vaginal cones versus pelvic exercises: no difference between the groups regarding subjective assessment of cure .Weighted vaginal cones versus electrostimulation: no difference between the groups regarding subjective assessment of cure .The authors concluded that these findings suggested that cones were a better option than no treatment. However, no assessment of the quality of the body of evidence was made. More high-quality trials are necessary in order to fully evaluate the possible benefits and adverse events of use of weighted cones for treatment of urinary incontinence, preferably with larger sample sizes and using standard relevant outcomes.For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD002114.pub2/abstract.,,,Four reviewsOne reviewPFMT plus electrical stimulation versus electrical stimulation alone: no difference between groups regarding cure or improvement of symptoms .versus heat and steam-generating sheet alone: the group that received PFMT had higher likelihood of participants presenting cure or symptom improvement .PFMT plus heat and steam-generating sheet PFMT plus vaginal cones versus vaginal cones alone: no difference between groups .The adverse events were only evaluated in one RCT in which PFMT in combination with drug therapy was assessed, and this did not find any statistical difference . In the light of these findings, the authors concluded that there was insufficient evidence to state what the effects of addition of PFMT to treatments for urinary incontinence were.For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD010551.pub3/abstract.The second reviewCure at the end of the follow-up: higher likelihood in the PFMT group .Symptom improvement: higher likelihood in the PFMT group .The authors concluded that these findings supported a recommendation to use PFMT as first-line conservative therapy for women with urinary incontinence. For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD005654.pub3/abstract.Other comparisons were made, but the data were sparse and based on low-quality RCTs. Moreover, there was no assessment of the quality of the body of evidence for any comparison. The authors concluded that currently there was insufficient evidence to make any strong recommendations about the best approach to use for PFMT.For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD009508/abstract.The fourth reviewIncidence of incontinence over the first six months after delivery (prevention): lower likelihood among pregnant women with intensive antenatal PFMT than among those with postnatal PFMT .Rate of incontinence 12 months after delivery (treatment): no difference in likelihood between pregnant women with intensive postnatal PFMT and those who received intensive antenatal PFMT .The authors concluded that there was some evidence to support the use of PFMT among women who were having their first baby, to prevent UI over the first six months after delivery. However, there was no appraisal of the quality of the evidence, and the clinical and methodological heterogeneity among the studies included prevented other pooled analyses.For further details, refer to the original abstract, available from: http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD007471.pub2/abstract.This review included 20 Cochrane systematic reviews that assessed non-surgical interventions for women with urinary incontinence. The studies included evaluated pharmacological interventions (three SRs), behavioral interventions (six SRs), miscellaneous interventions (seven SRs) and pelvic floor muscle training interventions (four SRs).,,,,Despite the considerable numbers of studies included in many of these SRs, most of them were poorly reported with low methodological quality, which compromised confidence in the data synthesis. However, the quality of the body of evidence was only assessed in five SRs.Regarding the clinical implications, the only systematic reviews that presented moderate to high quality of evidence were those relating to use of pelvic floor muscle training among women with urinary incontinence. On the other hand, the quality of evidence reported was very low to moderate in relation to use of estrogen for treating and preventing urinary incontinence. Moreover, the outcomes reported from all other interventions presented very low to low quality of evidence. The results presented in Regarding the implications for further research, our results suggest that it is important to plan and conduct randomized controlled trials assessing non-pharmacological interventions for women with urinary incontinence strictly in accordance with rigorous protocols. The trial protocols need to be published before the study begins and the results need to be presented in accordance with the CONSORT guidelines.This review included 20 Cochrane systematic reviews that provided evidence ranging in quality from unknown to high, in relation to non-pharmacological interventions for patients with urinary incontinence. Moderate to high quality of evidence favoring use of pelvic floor muscle training among women with urinary incontinence was found. To establish solid conclusions for all the other comparisons, further studies of good methodological quality are needed.


This paper explores how changing digital literacy practices in educational contexts require that we continually revisit conceptualisations of digital literacy education. We begin by analysing the positions taken by stakeholders who contribute to digital literacy discourses in Australia, exploring how competing interests produce effects which manifest in ways that differently consecrate social and cultural practice in the digital age. We advocate the need for pedagogic frameworks that support digital literacy education. Existing approaches tend to privilege the operationalisation of digital technology. By contrast, teaching is needed which focusses on meaning-making and creating. However, the ‘datafication of everyday life’ Barassi, , p.170 h It has become a truism to observe that the world has been radically changed by the digital and to note the pervasiveness of digital platforms and technologies in everyday lives, and the effects of these technologies on users. Prior to COVID-19, the importance of digital literacy, and the need for young people to become effective and agential users and makers of digital forms, texts and practices was already widely recognised . At other times technology is seen as the cause of all the social ills of today’s youth. A two-dimensional view of youth interactions with and through digital technologies has continued throughout the pandemic, with digital technologies lauded for their ability to keep students learning during times of school closures, but also criticised for distracting young people from more worthwhile pursuits.Framed within a mythologisation of a golden age not hindered by ‘all-consuming’ digital technologies, a discourse emerges which holds digital devices, the Internet, and video games as obstacles to the growth of a generation ready for the literacy challenges of life. The confusion regarding the relationship between digital practices and literacy is understandable given the public has been positioned to think about ‘the literacy crisis’ in terms of digital technologies as both a cause, and simultaneously the tool to mitigate the crisis.illusio, captures their investment in the game and is tied to beliefs about the field and the investments which should produce distinction in such a field. Digital literacy education has come to represent many forms of knowledge, skills and practices because of the distance between those positions discussed above, as they struggle to consecrate particular cultural dispositions.Each of the above positions represents a stake in the game. We refer here to Bourdieu’s metaphorical use of ‘the game’, which he draws on to examine the games of culture which are inherently tied to education systems and their imposition of cultural practices (1993). These practices matter greatly as they become internalised as dispositions for future action. The different stakeholders discussed above, as well as many others that space does not allow us to explore here, bring with them a specific interest in the game, implied by their participation. This interest, Our concern is that despite the incongruent nature of many of these positions, there is an expectation that literacy educators can navigate this landscape unproblematically with their students. This expectation ignores what sociology has taught us about the origins of people’s dispositions and their ability to perceive the field as a field of possibilities. All dispositions are associated with certain social and cultural origins . Well beyond the use of apps to regulate a wide range of services that were once offered ‘in-person’, the intrusion of the digital now includes ‘the datafication of childhood’ (p.171), where children become datafied citizens as they are coerced into participating in society through digital means which leave digital traces. Lupton and Williamson  refer toPlatformatisation is the process of using digital platforms to offer digital services and encourage online participation. In the context of formal schooling, concerns have been raised about the extent to which such platforms shroud what they do with data collected about users’ activities. Kumar et al.’s  investigAnother example of educational technology reshaping the digital literacies of schooling is the ubiquitous edu-app. While critical approaches to the study of educational apps have received only modest attention (Decuypere, We offer these examples as catalysts for asking questions about what teachers and students need to know, and be able to do, in response to the widespread incorporation of such technologies into formal schooling. Operationalising these technologies does not appear to be a challenge for most users. For the most part, platforms and apps are designed in ways that make their operation almost seamless. However, the risk is that this only contributes to their uncritical use. Approaches to digital literacy education that are limited to pedagogies supporting students to be digital makers and designers are not enough.Importantly, work is already underway which considers digital citizenship in light of the issues caused by the expansion of digital literacies into almost every aspect of contemporary life. Emejulu and McGregor  articulaThis paper raises core questions about what constitutes digital literacy education. What, if anything, is the relationship between using digital texts and possessing the knowledge to critique such texts? Should students be aware that they are generating data as they use edu-apps and social media, and of the ways in which platforms and technologies make use of these? Should data literacy be included as part of critical digital literacy conceptualised as an expansion of traditional literacy terms? What are the implications for pedagogy and curriculum?Some of the issues we raise reprise longstanding debates within the field. One set concerns conceptions of curriculum, and of the organisation of school learning, and what Green calls ‘the representation problem’, led by questions: what knowledge is of most worth? What should the schools teach? Green, , p.21.Another issue relates to the great need for teachers to have access to specific knowledge about digital technologies, both hardware and software, and their features, and about how they might teach and work with them. Building knowledge in this field, and providing preservice and practising teachers with information, strategies and resources is patently a clear task for literacy educators, as for the profession as a whole.As always, actions and possibilities are complex, often contradictory. Digital technologies and communication have massive roles, amongst them disseminating information and misinformation on the one hand, and enabling social cohesion and creative forms of meeting and relationships on the other; threatening or supporting mental health; facilitating workplaces and practices of some kinds, while limiting and risking others. One thing is sure. Now, more than ever, young people — and all of us — need to be critically and digitally literate, astute, and informed.


The vagus nerve affects innate immune responses by activating spleen-projecting sympathetic neurons, which modulate leukocyte function. Recent basic and clinical research investigating vagus nerve stimulation to engage the cholinergic anti-inflammatory pathway (CAP) has shown promising therapeutic results for a variety of inflammatory diseases. Abundant sympathetic innervation occurs in rodent spleens, and use of these species has dominated mechanistic research investigating the CAP. However, previous neuroanatomical studies of human spleen found a more restricted pattern of innervation compared to rodents. Therefore, our primary goal was to establish the full extent of sympathetic innervation of human spleens using donor tissue with the shortest procurement to fixation time. Parallel studies of porcine spleen, a large animal model, were performed as a positive control and for comparison.Human and porcine spleen tissue were fixed immediately after harvest and prepared for immunohistochemistry. Human heart and porcine spleen were stained in conjunction as positive controls. Several immunohistochemical protocols were compared for best results. Tissue was stained for tyrosine hydroxylase (TH), a noradrenergic marker, using VIP purple chromogen. Consecutive tissue slices were stained for neuropeptide Y (NPY), which often co-localizes with TH, or double-labelled for TH and CD3, a T cell marker. High-magnification images and full scans of the tissue were obtained and analyzed for qualitative differences between species.TH had dominant perivascular localization in human spleen, with negligible innervation of parenchyma, but such nerves were abundant throughout ventricular myocardium. In marked contrast, noradrenergic innervation was abundant in all regions of porcine spleen, with red pulp having more nerves than white pulp. NPY stain results were consistent with this pattern. In human spleen, noradrenergic nerves only ran close to T cells at the boundary of the periarterial lymphatic sheath and arteries. In porcine spleen, noradrenergic nerves were closely associated with T cells in both white and red pulp as well as other leukocytes in red pulp.Sympathetic innervation of the spleen varies between species in both distribution and abundance, with humans and pigs being at opposite extremes. This has important implications for sympathetic regulation of neuroimmune interactions in the spleen of different species and focused targeting of the CAP in humans.The online version contains supplementary material available at 10.1186/s42234-022-00102-1. Efferent neuronal regulation of the immune system occurs through the sympathetic nervous system, which innervates primary and secondary lymphoid tissues including bone marrow (BM), thymus, and the spleen , which provides a novel approach for activating anti-inflammatory mechanisms in the spleen , were evaluated to assure maximum visualization of sympathetic nerves in samples collected from different regions of the spleen. Human heart and porcine spleen samples were evaluated as positive controls. Since neuropeptide Y (NPY) is a recognized co-transmitter in sympathetic nerves, we also evaluated the presence and localization of this marker in spleen samples. Double labeling methods were used to evaluate the proximity of TH + nerves to CD3 + T cells.Use of human tissue for this study was approved by the Institutional Review Board of the University of Alabama Birmingham (UAB) (Protocol# 300001087 and 300005723) and was conducted in accordance with the Declaration of Helsinki. Samples of human spleen were collected from six brain dead donors after organ procurement at the UAB Legacy of Hope. Demographic and patient history information for the donors is listed in Table After removal of donor spleens, tissue was dissected into a 1–2 cm block of about 0.5 -1 cm thickness and was immediately fixed for 3–7 days in 10% neutral buffered formalin at room temperature before embedding in paraffin. Paraffin blocks were then shipped to East Tennessee State University (ETSU) for study. We also received paraffin sections of normal human left ventricle from UAB to use as a positive control for sympathetic nerve staining. Samples of porcine spleen (Yucatan mini-pig) were provided by UCLA for use as a positive control and to evaluate sympathetic innervation of spleen in another large species. Porcine spleens were collected at the end of terminal experiments approved by the UCLA Institutional Animal Care and Use Committee. These samples were dissected into blocks of about the same size as used for human spleen, fixed for 3–7 days in 10% neutral buffered formalin, and transferred to 70% ethanol for shipment to ETSU with ice packs. Porcine samples were embedded in paraffin at ETSU. Two spleens were collected from normal Yucatan mini-pigs and one more from a mini-pig at six weeks after myocardial infarction. Paraffin sections were cut at 5 μm thickness using a Leica RM2135 microtome and collected on SuperFrost® Plus slides (Fisher Scientific).2O2 in PBS, rinsed an additional time with PBS and incubated 10 min in PBS containing 0.4% Triton X-100 and 0.5% BSA. Slides were then placed in an incubation box and covered with blocking buffer . After 2 h, the blocking buffer was replaced with fresh blocking buffer containing the primary antibody  and incubated overnight at room temperature. Sections were washed with PBS and PBS containing 0.5% BSA followed by a two-hour incubation in biotinylated secondary antibody (1:200 dilution) from the kit. Slides were washed again before a 1.5-h incubation with the ABC reagent from the kit. Slides were next washed for 20 min in 50 mM Tris buffer (pH 7.6) before treatment for 1–10 min with the chromogen  to visualize targets (purple reaction product). Slides were washed, dehydrated, and cover glasses were attached using Cytoseal XYL (Thermo Scientific Cat. No. 8312–4).Representative sections from each human spleen were stained with hematoxylin & eosin (H&E) by standard methods to view cellular organization and morphology. Immunohistochemistry was used to stain for TH (noradrenergic marker) or NPY (co-transmitter in sympathetic nerves). Prior to staining, sections were deparaffinized, hydrated, and treated for antigen retrieval (either 1 mM ethylenediaminetetraacetic acid (EDTA), pH 8, 30 min at 92 °C or 1 mM citrate, pH 6.0, 30 min at 92 °C). Staining was done at room temperature using the Avidin-Biotin Complex (ABC) immunohistochemistry method (Rabbit ABC- horseradish peroxidase (HRP) Kit, PK-4001, Vector Labs). Briefly, slides were rinsed with phosphate buffered saline , incubated for 10 min in PBS containing 0.4% Triton X-100 and 0.5% bovine serum albumin (BSA), treated for 15 min with 1.0% HThe TH and NPY stains were repeated using the Vector ImmPRESS Excel Amplified Polymer Kit for Anti-Rabbit IgG, Peroxidase , following the manufacturer-recommended protocol. This kit uses a diaminobenzidine (DAB) chromogen (brown reaction product).To examine the association of TH + nerves with CD3 + T cells, double labeling experiments were performed using the Vector ImmPRESS Duet Double Staining Polymer Kit , with a modified version of the manufacturer-recommended protocol. The kit used 2.5% normal horse serum blocking buffer, and sections were incubated with a PBS solution containing 0.4% Triton X-100, 1% BSA, and two primary antibodies . The ImmPRESS Duet HRP/AP  Polymer Reagent included a mixture of HRP anti-rabbit IgG and AP anti-mouse IgG. The tissue was developed first with DAB EqV Substrate, yielding brown reaction product for TH. This was followed by washing and the addition of Red Substrate, yielding a magenta product for CD3.Stained sections were viewed, and digital images collected using an Olympus BX41 microscope equipped with an Olympus DP74 digital camera and cellSens Dimension software . Additionally, digital images of full sections were collected for a subset of experiments using a Leica Biosystems Aperio CS Console . Magnification for all full scans was set to 20X, and all controls were set to the manufacturer’s default settings.2, selected for consistency across samples of different sizes, were manually counted in three regions  of tissue stained with H&E using Aperio ImageScope 12.4.6. . This was repeated in the same regions on consecutive slides stained for TH, counting only the number of innervated blood vessels within the region of interest. These numbers were used to estimate the percentage of innervated blood vessels in each region. Statistical analysis was done using Prism version 8.4.3 . Values obtained for different regions of spleen were compared using repeated measures analysis of variance (P < 0.05) and pairwise comparisons conducted using Tukey’s multiple comparisons test.To quantify the extent of vasculature innervation by noradrenergic nerves within human spleens, total number of blood vessels within two standardized areas of 26 mmMost previous studies evaluating sympathetic innervation of the human spleen used samples collected during autopsy and fixed at unspecified times after death. Since there is the potential for loss of some immunostaining during this interval, we circumvented this issue by using spleen samples collected after organ retrieval and fixed immediately. To be certain that our immunostaining for TH revealed complete noradrenergic innervation of the human spleen, we evaluated sections from the same samples from each donor using different permutations of the immunohistochemical protocol and different antigen retrieval methods. Staining with the ABC Elite kit and VIP chromogen yielded similar localization of noradrenergic nerves after citrate or EDTA antigen retrieval Fig.  A and B.Three regions of spleen from six human donors were evaluated for the presence and distribution of noradrenergic nerves. With the notable exception of human spleen 002, TH + nerves occurred primarily in the wall of arteries ranging in size from the splenic artery to arterioles within the white pulp Fig. . Larger The spleen from donor 002 differed from the above pattern in that slightly more sympathetic nerves were detected overall, and more TH + nerve fibers penetrated a short distance into the white pulp Fig. A. This sGiven the limited distribution of noradrenergic nerves detected in human spleen sections, we used the same immunostaining methods to evaluate sympathetic innervation of two positive control tissues: human heart and porcine spleen. Sections of left ventricular muscle from normal human heart contained many noradrenergic nerve fibers that were closely associated with arteries and myocytes Fig. . This fiNoradrenergic innervation of porcine spleen was evaluated because the pig is a large animal species often used as a model for human biological systems and pathology  or red pulp (rp). Sympathetic nerve bundles (nb) were occasionally seen in large arteries. Arrows indicate central arterioles. Montage was created by stitching multiple 10X images using an Olympus BX41 microscope equipped with an Olympus DP74 digital camera and cellSens Dimension software.Additional file 2. Montage image showing a large section of porcine spleen stained for TH. TH + nerve fibers were more prevalent in the red pulp (rp) than the white pulp (wp), trabeculae (t), or capsule (c). Sympathetic nerve bundles (nb) are scattered throughout the section. Montage was created by stitching multiple 10X images using an Olympus BX41 microscope equipped with an Olympus DP74 digital camera and cellSens Dimension software.Additional file 3. Montage image showing a large section of porcine spleen stained for NPY. NPY + nerve fibers were found in all regions of pig spleen, with red pulp being the most densely innervated. White pulp (wp), red pulp (rp), and trabeculae (t) are labeled. Montage was created by stitching multiple 10X images using an Olympus BX41 microscope equipped with an Olympus DP74 digital camera and cellSens Dimension software.Additional file 4. Spleen from donor 003 exhibited a unique, robust pattern of NPY staining. (A) Image showing NPY nerve staining around a splenic artery (sa) and staining within surrounding red pulp (rp). Arrow indicates NPY staining of littoral cells. (B) Image showing NPY staining of littoral cells (arrow) in the subcapsular region. Note that the trabeculae (t) and capsule (c) are largely free from NPY staining while the red pulp shows dense staining. (C) Image showing lack of innervation within the white pulp (wp) and limited innervation around a central arteriole (ca.). (D) Image showing intense and abundant staining of littoral cells (arrows) within the red pulp.


The aim of this study was to evaluate the health professionals’ attitudes towards parental presence during their child’s invasive procedure. pediatric healthcare providers  from one of the Spain’s largest hospitals were asked to complete a questionnaire and write free-text comments.  227 responded the survey. Most (72%) participants, in their answers, reported that parents are sometimes present during interventions, although there were differences between professional categories in this respect. The procedures in which the parents were present were those considered “less invasive” (96% of cases), while only 4% were present in those considered “more invasive”. The older the professional, the less necessary parental presence was considered. the attitudes towards parental presence during pediatric invasive procedure are influenced by the professional category, the age of the healthcare provider and the invasiveness of the procedure.  The psychoanalysts René Spitz and John Bowlby strongly contributed to our understanding and knowledge of the psycho-affective consequences of this parental deprivation for young infants, defining it as “hospitalism” and proposing the “attachment theory”, respectively-,-Until the first half of the 20--Invasive pediatric procedures can be painful and frightening experiences for children and their parents. In the European Charter for hospitalized children (1986), it is established that it is a child’s right to be accompanied by their parents throughout the hospitalization process, with parents becoming an active part of hospital lifeHowever, some of the healthcare professionals are less willing to allow such parental presence despite it being the child’s right. The two main reasons expressed by the care providers in this respect include parental anxiety and the greater nervousness on the part of children. Other reasons are related with the time required to explain the procedure to parents and the healthcare providers’ anxiety for the parental presence during the interventionThe aim of this study was to evaluate the health professionals’ attitudes towards parental presence during their child’s invasive procedure.This is a cross-sectional study, guided by the STROBE  guideline, used to report observational studiesrd, 2019 and June 30th, 2020.The data collection period was between October 3. Pediatric population are defined in Spain as children from 0 to 14 years old although this range vary between countriesThe hospital employs 4000 health professionalsWorkers from the pediatricians, nursing and nursing assistant teams  belonging to eleven different pediatric units  in the hospital were invited to the study. All the workers of pediatric units were invited to the study. In order to avoid bias a minimum sample size base on the total eligible population (n=444) was calculated using the statistical software SAS University Edition. To determine the minimum sample size of responders with statistical relevance, a 75% rate of parental presence with a precision of 5% and a confidence level of 95% was estimated based on previous studies in SpainAn anonymous self-administered questionnaire, which consisted of 14 multiple response questions were used. The questionnaire was based on a published questionnaireThe questionnaire contained sociodemographic information , professional category  and pediatric units. The survey covered variables such as parental presence during invasive pediatric procedures , the type of procedures performed which parents were allowed to be present (eleven invasive procedures could be selected), the reasons for any reluctance to allow parental presence  and an evaluation of the existence for protocols and specific training regarding parental presence . Moreover, a Likert scale were used to determine the frequency of the problems derived from the presence of the parents . After completing the questionnaire, participants were invited to write free-text comments, motivated by the sentence: “We would appreciate any comment on the subject”. Eleven invasive procedures were included in the study and divided according to the degree of invasiveness, into less and more invasive .The workers were invited through the institutional email service. After that, printed questionnaires and informed consent form were sent to each pediatric unit. As a strategy during data collection, visits were made to all of the units to invite and instruct the professionals to participate in the research.2 analysis. A multivariate analysis (Fisher logistic regression) was explored considering presence/absence of parents as dependent variable and professional category and age as independent variables. The questionnaires received were processed using Microsoft Excel, and statistical analysis was performed using the statistical software SAS University Edition. The presence/absence of parents during invasive procedures with their children, the age ranges of healthcare professionals , and the proportion of parents present in “more” or “less” invasive procedures were analyzed using Pearson’s χThe free-text comments in the questionnaires were evaluated by a text analyst program KH coderThis study was carried out in accordance with the ethical standards established by the Helsinki Declaration (2000) and Istanbul Declaration (2008).Health care professionals were invited to answer the questionnaire after being informed of the confidentiality of the survey according to the European General Data Protection Regulation 2016/679 and allowed sufficient time to read it.Two hundred and twenty-seven out of 444 questionnaires were received (51.1%) completed by 196 female (86.34%) and 31 male (13.65%) participants from 11 different pediatric services. The respondents’ ages were aggregated and balanced into three groups <40 years old, from 40 to 50 and >50 years . The respondents were also placed into 3 professional categories: 1. Pediatrician and pediatric residents (n=50); 2. Pediatric nurses and pediatric nurse residents (n=109) and 3. Nursing assistants (n=68).In the first part of the questionnaire, the presence of parents in the different units was investigated for the 227 respondents. Most (72%) reported that parents were sometimes present during interventions, 19% never performed procedures in the presence of parents and 9% reported that parents were always present during procedures (p<0.0001) .No differences were found when these results were analyzed taking into account the professional category of the respondents. However, there were differences according to professional category when only the professionals that always or never performed procedures in the presence of parents were analyzed. The percentages in this respect were that pediatricians reached 100% of presence, followed by pediatric nurse residents (50%), pediatric nurses (31%), nursing assistants (27%) and pediatric residents (14%) (p<0.05) .In terms of the type of procedures where parents were allowed to be present, the percentages were 96% in the case of “less invasive” and 4% in the case of “more invasive” (p<0.0001). In regard to any problems derived from parental presence, 61% of the professionals reported occasional problems (n=122), 29% had never had problems (n=58) and 10% said they frequently had problems (n=18). The reported problems related to such presence were: greater nervousness on the part of child , parent’s indisposition (example: dizziness) , interrupting the procedure  and others such as aggression or complaints .Most professionals thought that parental presence was unnecessary and preferred to perform procedures without them (72% n=164), while 28% (n=53) thought such presence was necessary. The main reasons stated for the reluctance to allow parents to be present were parental anxiety (65%), the belief that the parents are not prepared to stay (41%), the invasiveness of the procedures (39%), reduced physical space (23%), the idea that they would perform less well (20%) and greater nervousness on the part of child (18%).Most of the professionals in group aged >50 perceived as unnecessary the present of the parents showing the highest percentage of negative answers (84%). Although there were differences between the three subgroups  (p=0.004) .When the “Yes” or “No” answers concerning the need for parental presence were compared within the same age range, statistically significant differences were found within the 40 to 50 group and >50 group .Furthermore, 82% of the professionals indicated that there were no written protocols related to parental presence in the different pediatric units, while the need to create them and to receive relevant training was supported by 75% of those interviewed. Statistical differences in terms of the need of training were found between group <40 (83%) and the groups 40 to 50 (66%) and >50 group (66%) p<0.005. Fifty-nine respondents wrote comments in the free-text part of the questionnaire. Analysis of their comments showed that 1435 words were used in a total of 75 sentences. The most frequent words were the verb “be” (101 times), followed by “parent” and “child” , the adverb “not” (35 times), “procedure” (26 times), and the noun “presence” (24 times). The results of the co-occurrence network of words analysis revealed 11 clusters regarding how the professionals evaluated parental presence during invasive pediatric procedures . The most important cluster in terms of frequency of the words used and the thickness of it edges was cluster 7 represented in green in Another important cluster is number 10 (in purple), which matched the words “get” and “nervous” as exemplified by the sentences: “I think that parents should not be present, they get very nervous” or “I do not agree that the parents be present during procedures, they get very nervous”.Cluster 5 (in blue) brought together “prepare”, “hurt”, “understand” and “more”, as in the comments: “Children are more restless (they do not understand that they are hurting them and their parents do not defend them), professionals feel supervised and most families are not prepared”. “I think that in some techniques some parents are not prepared to understand what we do”.Another cluster relates to the performance of professionals  said that they sometimes perform invasive procedures in children with parental presence. Nineteen percent of them claimed that they never perform invasive procedures with parental presence and 9% affirmed that they always performed procedures in the presence of the parents.vs. 31%). Our results agree with those described in other Spanish studies, which showing pediatricians to be more in favor to parental presence than nurses, with those that disagree most being pediatric residents--There were differences between the different professional categories concerning the answers “never” and “always”. Pediatricians performed more procedures with parental presence than pediatric residents . Furthermore, previous studies in Europe showed that residents from various specialties more often prefer paternalistic decision making than their supervisors. They suggest that this might be attributed to differences in the degree of professional confidence and their lack of knowledge about shared decision making-,-e.g., breastfeeding, kangaroo/maternal holding, nonnutritive sucking, white noise in new-born-e.g., clown therapy, distraction--In regard to the wishes of parents to be present during invasive procedures even in worse-case scenarios such as critically ill children or resuscitation, they want to be thereFew problems regarding parental presence during invasive procedures were reported by the respondents. Most of the providers (90%) mentioned occasional (61%) or no problems (29%) had arisen during a given procedure, coinciding with other study, which found a high level of satisfaction (57%) of the professionals when an invasive procedure is performed in the presence of parents. Furthermore, in the same study, 90% of family members and 76% of professionals believed that it had been beneficial for the child--The main problems mentioned by professionals in our study concerning parental presence were, greater nervousness on the part of child (37%), parent´s indisposition (35%), procedure interruption (22%) and aggression or complaints (7%). These problems have been reported in another study, in which the most frequently mentioned problems were dizziness (78.6%), greater nervousness on the part of child (63.3%), interruption of the procedure due to the parents  (46.4%) and finally complaints or reclamations (8%)-,In our study, 65.4% of the 227 professionals did not consider the presence of the parents to be necessary. Regarding that, an innovative free-text analysis was used for the first time to evaluate the comments on parental presence wrote by healthcare providers, although KH Coder have been used in studies of public healthThe present study notes that the reasons given for any reluctance to allow parental presence are mainly parent-related, including parental anxiety (65%), belief that parents are not prepared to stay (41%), the invasiveness itself of the procedure (39%), reduced physical space (23%), poorer care performance (20%) and greater nervousness on the part of child (18%). In the case of parental anxiety, these results were reinforced in the free text analysis, where cluster number 10 associated words like “get” and “nervous”. The belief that parents are not prepared was represented in the free text comments by cluster number 5 with words like: “prepare” and “understand”. Somehow the clusters 10 and 5 can be associated with the theme “Not prepared for the experience” that was reported by some healthcare providers in face-to-face, in-depth, semistructured individual interviews in a qualitative study in South Africae.g., a fear of needles) interfered with their ability to be present during a procedure, making them feel guilty or that they were not fulfilling their parental role. In some cases could be the “important others” (families of parents or friends) the ones who help and support the pediatric invasive procedureParental anxiety was also given for reluctance to allow parental presence in another study carried out in an emergency pediatric service (86% of the respondents), being similar to our results. By contrast, other explanations were more frequent: fear of poorer staff performance (77%), belief that parents are not prepared to stay (76%), the invasiveness of the procedure (70%), and increase child anxiety (39%)-There is a vast quantity of scientific information concerning the benefits of parental presence during invasive pediatric procedures-In reference to the ages of professionals and their views concerning necessity of parent presence, our study found that the older the care provider the less parental presence was considered beneficial . A possible explanation in our context would be the rare negative experiences the professionals had had during their careers and the lack of formal training in pediatrics in the case of nursing assistants and pediatric nurses. Similar results are described in studies from Brazil and South Africa, in which parental presence was more widely accepted by professionals with less experienceParents want to be present during invasive procedures and they recommend being present to support increased coping for their children,The findings of our study could also help to determine which groups need to receive such training in the first place. Defining roles within work teams could speed up not only parental presence during invasive procedures but also decrease children’s pain, stress and negative behavior in general during invasive proceduresThe present study brings us the opportunity to know what is behind parental presence or not within one of the largest hospitals in the south-east of Spain, but this could also be a limitation of our study, since it really reflects the opinion of the professionals of this hospital. Another limitation could be the lack of a pure qualitative study. Future research with individual interviews or even through focal groups may be a future path for a better understanding of the object and scenario studied. The contribution of this study allows us to know the perspectives of healthcare professionals about a very important subject. In most of the countries, where family-centered care in more implemented, exist internal policies to be follow by the professionals that allow parents to be present in all procedures. Thus, in these cases the attitude of practitioner is less important. However, in our context, parental presence is determined by the professional´s attitude. According to our study findings, there is a demand for written protocols or guidelines and specific trainings. This could speed up the acceptance of parental presence during invasive procedures. Moreover, the main reason stated for the reluctance to allow parents to be present were parental anxiety. This can be management with timely and relevant information to make parental interventions an important tool for professionalsThe results showed that the attitudes towards parental presence during invasive pediatric procedures are influenced by the professional category, age and the degree of invasiveness of the procedure. Moreover, more than half of the respondents did not perceive the presence of a parent as being necessary when invasive procedures are being performed. The rare problems reported when parents were present during invasive procedures were mainly related with greater nervousness on the part child, the parent’s indisposition, and interruption of the procedure. The findings show that there is a widely felt demand for written protocols and specific training in the different medical units to clarify how best to include parents in child´s care during invasive procedures.