Chronic obstructive pulmonary disease (COPD) is associated with high clinical and economic burden. Optimal pharmacological therapy for COPD aims to reduce symptoms and the frequency and severity of exacerbations. Umeclidinium/vilanterol (UMEC/VI) is an approved combination therapy for once-daily maintenance treatment of patients with COPD. This study evaluated the impact of delaying UMEC/VI initiation on medical costs and exacerbation risk.A retrospective analysis of patients with COPD who initiated UMEC/VI between 4/28/2014 and 7/31/2016 was conducted using the Optum Research Database. The index date was the first COPD visit after UMEC/VI available on US formulary . Patients were followed for 12 months post-index, and categorized into 12 cohorts corresponding to month (30-day period) of UMEC/VI initiation (i.e. Months 1–12) post-index. The outcomes studied during the follow up period included COPD-related and all-cause medical costs, and risk of COPD exacerbations. Marginal structural models (MSM) were used to control for time-varying confounding due to changes in treatment and severity during follow up.p = 0.044) for each monthly delay in UMEC/VI initiation, with a 37.4% higher adjusted cost for patients initiating UMEC/VI in Month 12 versus Month 1 . The 12-month adjusted all-cause medical costs increased by 2.8%  for each monthly delay, with a 36.1% higher adjusted cost for patients initiating UMEC/VI at Month 12 versus Month 1 . The monthly risk of severe exacerbation was significantly higher in patients who had not yet initiated UMEC/VI than those who had .2,200 patients initiating UMEC/VI were included in the study sample. Patients’ average age was 69.3 years, 49.9% were female and 69.7% were Medicare insured. Following MSM analysis, 12-month adjusted COPD-related medical costs increased by 2.9%  contains supplementary material, which is available to authorized users. Chronic obstructive pulmonary disease (COPD) is a chronic and disabling respiratory disease characterized by airflow obstruction and persistent breathing difficulties . In the Although COPD is a progressive, irreversible disease, pharmacological treatment can help to control symptoms , improve lung function, and reduce exacerbations, as well as improve exercise tolerance and health status , 8. Trea2-agonist (LABA), or a combination of the two, depending on the symptom burden and exacerbation history  period ran from April 28, 2014  to July 31, 2016 (the most recent date when fully adjudicated pharmacy information was available for patients within the database) to identify patients treated with UMEC/VI Patient data were obtained from the Optum Research Database (ORD), which contains medical data, pharmacy data, and enrollment information for a geographically diverse and representative patient population across the US .th edition, clinical modification [ICD-9-CM] codes: 491.xx, 492.x, 493.2×, 496 or International Classification of Disease, 10th edition, clinical modification [ICD-10-CM] codes: J41*, J42, J43*, J44*) in any position on the visit claim. Patients must also have had ≥1 prescription fill for UMEC/VI within 12 months following the index date. Continuous enrollment with both medical and pharmacy coverage from the start of formulary availability to the index date, for ≥6 months prior to the index date, and for ≥12 months on and following the index date was required. Patients were excluded if they had used UMEC/VI or any other LAMA/LABA fixed dose combination therapy during the 6-month baseline period.Eligible patients were ≥ 40 years old in the index year with ≥1 prescription fill for UMEC/VI during the patient ID period and ≥ 1 COPD-related visit with a potential prescriber during the index date assignment period. Visits were considered COPD-related if they had a diagnosis code for COPD  the index date. Medical costs were considered attributable to COPD if the claim had a diagnosis code for COPD in any position. COPD-attributable pharmacy costs were identified by any pharmacy claim for a COPD-related treatment  analyses, exacerbations were captured in monthly indicators during the 12 months following the index date.Multivariable analyses were conducted using MSMs , 22 to eTreatment selection weighting models for the cost analyses contained potential confounders as independent variables, including severity indicators such as exacerbations, rescue medication prescription fills, respiratory infections, emphysema and Charlson comorbidity index. The complete list of variables used in the weighting models are described in Additional file th percentile [th percentile. If patients had an exacerbation and initiated treatment in the same month, and the exacerbation occurred prior to treatment initiation, treatment initiation was assigned to the following month. All analyses were conducted using SAS version 9.4 .The 12-month COPD-related and all-cause medical cost outcome analyses were estimated for one observation per subject, using weighted GEE with a gamma distribution, a log link, and sandwich variance estimation. The covariates included the primary exposure of interest, month of treatment initiation, as well as the baseline variables used in the weighting models. The analysis was performed using the same weighting scheme for COPD-related and all-cause medical cost outcomes, with weights truncated at the 99.9rcentile . The risrcentile , GEE witSensitivity analyses were conducted to examine the impact of various modifications to the MSM assumptions and covariates on the outcomes. Details of the sensitivity analyses are presented in Additional file A total of 8,317,017 patients aged 40 years and older  had at least one day of enrollment during the index date assignment periods. Out of these, a total of 10,691 patients initiated treatment with UMEC/VI and 2,200 patients met all eligibility criteria and were included in the analytic population  for each month of delay in initiation of UMEC/VI. Patients initiating UMEC/VI therapy in Month 1 had a 12-month adjusted cost of $9,524 versus $13,087 for those initiating UMEC/VI therapy in Month 12. Overall, these results demonstrated that patients initiating treatment with UMEC/VI in Month 12 had 37.4% higher COPD-related medical costs than patients initiating within Month 1  for each month of delay in initiation of UMEC/VI. Patients who initiated UMEC/VI in Month 1 had first-year adjusted costs of $16,727 compared with $22,766 for patients who initiated UMEC/VI in Month 12. Overall, these results demonstrated that patients initiating treatment with UMEC/VI in Month 12 had 36.1% higher all-cause medical costs than patients initiating treatment within Month 1  number of exacerbations during the follow up period was 1.4 (1.7), while the mean (SD) numbers of moderate and severe exacerbations were 1.1 (1.4) and 0.3 (0.7), respectively.p < 0.001; HR for the untreated/month interaction, term 2: 1.08; 95% CI: 1.00–1.17; p = 0.067; combined p-value p < 0.001). Inclusion of the time interaction term in the final model demonstrated a 20% increased risk of a first severe exacerbation for patients not receiving UMEC/VI in the first 30 days relative to patients receiving UMEC/VI in the first 30 days post-index; for each additional month of treatment delay, the risk of a first severe exacerbation increased by 8%   Fig. .Fig. 4CoThis large, retrospective observational cohort study has demonstrated that, among patients initiating treatment with the LAMA/LABA therapy, UMEC/VI, within a year of their first eligible COPD-related visit, consistently lower COPD-related and all-cause medical costs were observed when the delay in initiating UMEC/VI was minimized. A reduced risk of a first severe exacerbation was also observed in these patients. After adjusting for baseline covariates and time-varying confounders using MSM, both COPD-related and all-cause medical costs during the year were found to be nearly 3% higher for each month that treatment initiation of UMEC/VI was delayed. This analysis also found a clear benefit of not delaying treatment with UMEC/VI, with a higher risk of a first severe exacerbation each month that treatment was delayed. These findings suggest that preventing a delay in treatment with UMEC/VI may result in lower medical costs than for similar patients who delay initiation. As exacerbations of COPD are an important cost driver , the obsThe results of this study suggest that healthcare providers may wish to consider the initiation of UMEC/VI earlier in the course of the disease. In the 2017 GOLD report the preferred first initiation of maintenance therapy is based on symptoms and exacerbation risk at presentation. In patients with particularly high symptom burden or who are symptomatic with a history of multiple exacerbations, initial LAMA + LABA therapy is recommended (GOLD Group B/D), while escalation from bronchodilator monotherapy to LAMA/LABA therapy is recommended for patients with a lower exacerbation risk but still experiencing persistent symptoms or those with a lower symptom burden who experience further exacerbations.This study has demonstrated that reducing the delay in UMEC/VI initiation results in a reduced risk of severe exacerbations. Severe breathlessness, severe lung function impairment or a prior severe exacerbation are important predictors of future exacerbations –31, therObservational data have been used to examine the potential benefits of initiating ICS therapy along with regular inhaled bronchodilators in patients with COPD earlier in the course of their disease, rather than using a step-wise approach to intensification. Interestingly, earlier treatment with ICS/LABA reduced severe COPD exacerbations and COPD-related healthcare expenditures , supportTo the best of our knowledge, this is the first outcomes study to assess the benefits of early use of a LAMA/LABA in a real-world setting. In addition, the use of MSM analysis to adjust for differences in multiple measurable factors, including time-varying confounders, between cohorts provides for more robust adjusting for confounding.Claims data are a rich source for examining real-world patterns of healthcare outcomes and for assessing patterns of medication use, and are able to capture information from all providers caring for a patient. However, there can be limitations due to the lack of information relating to clinical measures (e.g. spirometry data and factors relating to exacerbations such as oxygen saturation) and patient characteristics, such as socioeconomic status and frequency of tobacco use. Furthermore, a claim for a filled prescription does not necessarily indicate that a medication has been used or used correctly; and, although a diagnosis code is included on the claim, it may not constitute evidence of the disease due to incorrect coding. The inability to measure and control for unobservable factors using administrative claims data will impact the interpretation of findings in this and similar studies. The population in this study included patients with commercial or MAPD coverage with a minimum of 18 months of continuous enrolment, the generalizability of results to other populations may be limited. As similar analyses using MSM have not been performed for other COPD medication classes (e.g. LAMA or ICS/LABA) we are not able to conclude whether these results are applicable to only UMEC/VI or are a consequence of a delay in initiation in general.This study demonstrates the economic and clinical benefits of preventing a delay in initiation of UMEC/VI dual therapy, with earlier initiation resulting in lower subsequent all-cause and COPD-related medical costs after initiation. This economic benefit may be explained, in part, by the lower risk of severe COPD exacerbation when UMEC/VI initiation is not delayed; however, additional research is needed to confirm this hypothesis, and to assess the relative benefits of initiation of UMEC/VI as a first-line therapy compared with escalation to UMEC/VI from bronchodilator monotherapies.Additional file 1:COPD-related medications. COPD-related treatments identified by pharmacy claims in the baseline period. (DOCX 27 kb)Additional file 2:Independent variables in treatment selection weighting models for cost analyses. Description of independent variables in treatment selection weighting models for the cost analyses. (DOCX 17 kb)Additional file 3:N = 2200). Additional figure illustrating the number of patients initiating UMEC/VI each month. (DOCX 100 kb)Cohort counts by month of UMEC/VI initiation


Bicyclol, the most commonly-used liver hepatoprotective drug in China, is often selected to control disease progression in CHB patients who refuse anti-viral treatment. However, data on histological changes after bicyclol treatment in these patients are scarce. Therefore, this study has been conducted to find out whether bicyclol has good benefits of histological improvement in CHB patients who refuse anti-viral agents.The demographic, clinical and pathological data were collected from CHB patients who received bicyclol from January 2010 to June 2016. Improvement in liver inflammation or fibrosis is defined as at least one-grade or one-stage decrease as measured by the Scheuer scoring system. Thirty patients treated with ETV for 48 weeks were chosen as a control group to compare the histological improvement between bicyclol and entecavir (ETV) after 48-week treatment.A total of 123 patients with CHB treated with bicyclol were included in this study. Paired liver biopsies were performed in 70 patients. Inter-biopsy interval was 17.44 ± 8.90 months (12–60 months). As shown by facts, 41.4% patients achieved liver inflammation improvement, while only 10.0% patients showed liver inflammation progression after bicyclol treatment. In regarding to liver fibrosis, as shown by facts, 28.6% patients achieved fibrosis improvement. More importantly, It was found that the proportions of patients with liver inflammation and fibrosis improvement were both not significantly lower than those in ETV group (53.3% vs 63.3 and 36.7% vs 43.4%). Most of patients (82.4%) with elevated baseline ALT became normal after bicyclol treatment. More importantly, as shown by the multi-variate analysis, the treatment course of bicyclol was an independent factor for liver inflammation improvement. With the HBeAg status adjusted, ALT and HBV-DNA quantity, the odds ratio  of patients with ≥48-week treatment was 5.756  when compared with patients via < 48-week treatment.Bicyclol can improve liver inflammation and the ALT normalization rate of CHB patients, especially when the treatment course is prolonged. This has confirmed that bicyclol could control hepatitis activity, which might be a good choice for CHB patients who refuse anti-viral treatments. Chronic hepatitis B (CHB) is caused by hepatitis B virus (HBV) infection, while 15–40% of patients with CHB can develop liver cirrhosis and hepatocellular carcinoma (HCC)  suggest P < 0.01, P < 0.05) [Liver cells are constantly attacked by HBV, and continuous liver inflammation, the basic pathologic feature of CHB progression, might induce liver fibrosis or cirrhosis . Therefo < 0.05) . Thirty- < 0.05) . A retro < 0.05) . HoweverEligible patients were selected from patients with CHB hospitalized in Guangdong Provincial Hospital of Chinese Medicine between January 2010 and June 2016 according to the following inclusion and exclusion criteria.G ≥ 2) shown by the first liver biopsy or ALT ≥2 × upper limit of normal, (4) having refused antiviral treatment but received bicyclol treatment instead according to personal wishes of patients, (5) written informed consents obtained from all patients before enrollment. An interbiopsy interval duration of liver biopsy should be at least 1 year in the patient who has received paired liver biopsy. The progression of liver disease was assessed based on the findings of the second liver biopsy. All eligible patients were followed up in the outpatient department integrated with email and telephone until the second liver biopsy for patients maintained on bicyclol treatment, or the beginning of anti-viral therapy for the patients who discontinued bicyclol treatment due to the withdrawal or failure of treatment.The inclusion criteria are as follows: (1) those aged from 18 to 65 years, (2) with a diagnosis of CHB according to the criteria described in the Guideline of Prevention and Treatment for CHB (2010 version) , (3) witThe exclusion criteria are as follows: (1) co-infection with hepatitis A virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, or human immunodeficiency virus; (2) concomitant liver and gallbladder diseases including evident advanced primary biliary cirrhosis, autoimmune hepatitis, decompensated cirrhosis, severe hepatitis, or hepatic carcinoma and a history of excessive alcohol consumption ; (3) serious uncontrollable heart, kidney, lung, endocrine, blood, metabolic, or gastrointestinal primary diseases, or mental illness; and (4) having received any other hepatoprotective drug or anti-viral treatment within 6 months prior to the enrollment.In order to compare the histological improvement between bicyclol and entecavir (ETV) after 48-week treatment, 30 patients treated with ETV in a study registered in 2012 were chosen as a control group to match the the treatment group in a 1:1 ratio by age, gender, HBeAg status and HBV-DNA level.This study has been approved by the Ethics Committee of the Guangdong Provincial Hospital of Chinese Medicine and carried out in accordance with the Declaration of Helsinki. Written informed consents were obtained from all patients during the outpatient follow-up or by telephone and letter follow-up before the enrollment.The demographic, clinical, and laboratory data at the time of liver biopsy were collected from medical records, including the age, gender, levels of ALT, aspartate aminotransferase (AST), and viral parameters. The levels of serum hepatitis B surface antigen (HBsAg) and HBeAg were measured by using electrochemical immunoassay , and the serum HBV-DNA level with a lower limit of detection of 500 IU/mL was measured by using ABI 7300 . The findings of abdominal B ultrasound or upper abdominal computed tomography or magnetic resonance imaging were also recorded.A liver biopsy was performed by using 18G MAXCO needles . Specimens were fixed in 10% formalin, embedded in paraffin, and stained with hematoxylin and eosin. A minimum of 15-mm length of liver tissue and at least 10–15 portal tracts were required for the diagnosis. Pathologic specimens were analized by two experienced histopathologists of our hospital in double-blind manner. The liver necroinflammatory activity and fibrosis were each scored on a 0–4 scale by using the Scheuer scoring system . ImproveAll the enrolled patients received 75 mg  of bicyclol  daily for at least 12 weeks. Patients with other anti-viral drugs or hepatoprotective drugs during the therapy should be excluded.t test or nonparametric test (Wilcoxon) as being appropriate. Categorical variables were presented as counts and percentages. Categorical parameters among groups were compared by using the chi-square test. The causative factors of improvement in liver inflammation were determined by making analysis of univariate and multivariate logistic regression. All P values were two tailed. A P value of less than 0.05 was considered statistically significant. All statistical analyses were made by using the SPSS software version 19.0 .Continuous variables were expressed as the mean and standard deviation, or median and interquartile range, and compared by using the Student A total of 232 CHB patients treated with bicyclol were initially screened in this study. However, 109 patients were excluded for some reasons, such as the combination with other antiviral drugs or hepatoprotective drugs, or incomplete data, etc. Finally, 123 patients with CHB treated with bicyclol tablets were included in this study. The median duration of bicyclol therapy lasted 48 weeks (12-232 weeks). The median age of the patients was 35 years and most of them were males (69.1%). Paired liver biopsies were performed in 70 patients. Inter-biopsy interval was 17.44 ± 8.90 months (12–60 months). Of these 70 patients, 40 patients were treated with < 48-week bicyclol treatment, while 30 patients treated with ≥48-week bicyclol treatment. Table P < 0.001) in patients with elevated base-line ALT level.As shown in Table P = 0.001). Figure After bicyclol treatment, 41.4% patients achieved liver inflammation improvement, while only 10.0% patients showed liver inflammation progression after bicyclol treatment . Meanwhile, 36.7% (11/30) patients showed liver fibrosis improvement, which was no significantly lower than that  in the control group (P = 0.598). Only 6.7% (2/30) patients showed inflammation progression in bicyclol group, which was also no significantly higher than that in ETV group . More detailed information between these two groups can be seen in Table Moreover, there was no significantly difference in histological changes between bicyclol group and ETV group after 48-week treatment. The proportion of patients showed liver inflammation improvement in bicyclol group  was not significantly lower than that in ETV group  of patients achieved inflammation improvement, while 10.0% (7/70) of patients showed inflammation progression. At the same time, ALT changes are similar to the results of previous studies , 15. MosAs for related factors of liver inflammation improvement, the multivariate analysis showed that the treatment course of bicyclol was an independent factor for liver inflammation improvement. The longer of the treatment course, the more significant the improvement in liver inflammation. With the HBeAg status adjusted, ALT and HBV-DNA quantity, the odds ratio  of patients with ≥48-week treatment was 5.756  when compared with patients with < 48-week treatment. Therefore, some famous experts  recommenIn safety analysis, no severe adverse event occurred during the treatment process, suggesting the good safety profile of bicyclol. However, it is worth mentioning that seven patients with elevated ALT showed abnormal ALT after irregular withdrawal of bicyclol during the therapy. Therefore, bicyclol should be withdrawn gradually. Also, it is quite important to increase the patient compliance.However, a potential limitation of this study is that it was a single-center study and the number of enrolled patients was rather limited, although it was merely for the proof of real-world experience. Therefore, further large and prospective studies with a long-term follow-up should be conducted to confirm the histological improvement after bicyclol treatment.In summary, bicyclol can improve liver inflammation and the ALT normalization rate of CHB patients, especially when the treatment course is prolonged. This has confirmed that bicyclol can control hepatitis activity, which might be a good choice for CHB patients who refuse anti-viral treatment. We also suggest that don’t interrupt the treatment casually. Because of the limited number of patients, more clinical trials are still needed to verify the histological improvement of bicyclol in patients with CHB who refuse anti-viral treatment.


Nonvalvular atrial fibrillation (AF) is the most common cardiac arrhythmia, and it is associated with the prothrombotic state. Circulating microparticles (cMPs) are membrane vesicles that are shed from many cell types in response to cell activation and cell apoptosis. Several studies reported that cMPs may play a role in the hypercoagulable state that can be observed in patients with AF. The aim of this study was to determine the levels of total cMPs and characterize their cellular origins in AF patients.Atotal of 66 AF patients and 33 healthy controls were enrolled. This study investigated total cMP levels and their cellular origin in AF patients using polychromatic flow cytometry.5 counts/mL vs median 15.21, IQR 9.91‐30.86 × 105 counts/mL; P = 0.004), platelet‐derived MPs (PMPs) , and endothelial‐derived MPs (EMPs CD31+ CD41−)  than healthy controls after adjusting for potential confounders. Phosphatidylserine positive MP (PS + MP) levels were similar compared between AF patients and healthy controls.AF patients had significantly higher levels of total cMPs  in the pathophysiology of and the complications associated with AF. MPs are defined by their size, ranging from 100 to 1000 nm in diameter.22.1This study was conducted at the Division of Cardiology, Department of Medicine, Faculty of Medicine Siriraj Hospital, Bangkok, Thailand. The protocol for this study was approved by the Siriraj Institutional Review Board (SIRB) (COA no. Si 199/2017), and complied with the principles set forth in the Declaration of Helsinki (1964). All study participants provided written informed consent prior to inclusion.Sixty‐six AF patients  and 33 healthy individuals were recruited. All subjects underwent thorough historical investigation and 12‐lead electrocardiography to confirm cardiac rhythm. The exclusion criteria were refusal of provide consent, history of myeloproliferative disorders, thrombocytopenia, ischemic stroke within the previous 3 months, malignancy, rheumatic mitral valve disease, acute infectious or inflammatory disease, and pregnancy. Control subjects had no cardiovascular risk factors, no history of atrial arrhythmias, no clinical evidence of disease, and no current cardiovascular treatment. Controls also underwent electrocardiography to document sinus rhythm.2.2Serum was used to analyze creatinine levels using an automatic biochemistry analyzer .2.3g for 15 minutes to remove all cells. We modified the method from Choudhury et al study which determined microparticle levels by flow cytometry from one centrifugation step plasma.Serum was obtained from clotted whole blood, and then centrifuged at 15002.4U test. Kruskal‐Wallis test was used to compare independent samples from more than two groups. cMP levels were expressed as log‐transformed counts per mL (log MPs/mL). Unpaired t test was used to compare normally distributed continuous variables. Proportion comparisons were performed using χ2 test. Analysis of covariance (ancova) was used to adjust for potential confounders. MP levels were transformed on a logarithmic scale before entering ancova analysis. Data are presented at number and percentage or mean ± SD. All statistical tests were two‐tailed, and a P‐value less than 0.05 was considered statistically significant.All data analyses were performed using PASW Statistics version 18 . Kolmogorov‐Smirnov test was used to evaluate the distribution of data. Since circulating MP levels were not normally distributed, comparisons between groups were performed using Mann‐Whitney 33.1P < 0.0001), gender (P = 0.001), and body mass index (BMI) (P < 0.0001) between AF patients and controls. Worsening of renal function was presented by increasing creatinine levels in AF patients. Baseline characteristics of AF patients are shown in Table The baseline characteristics of 66 AF patients and 33 control subjects are summarized in Table 3.2P < 0.001), PMPs (P = 0.002), EMPs (CD31+ CD41−) (P < 0.001), red blood cell‐derived microparticle (RMPs) (P = 0.020), and tissue factor bearing microparticle (TF‐MPs) (P = 0.014) than healthy controls. There were no significant differences in the levels of EMPs (CD144+), leukocyte‐derived microparticle (LMPs), or phosphatidylserine expressing microparticle (PS + MPs) between AF patients and healthy controls  levels were still higher in the AF group than in the control group. Comparisons of total cMP, PMP, and EMP (CD31+ CD41−) levels between AF patients and control subjects are shown in Figure The cellular origins of cMPs in peripheral blood were identified by staining cMPs with cell‐specific surface markers. PMPs were the most abundant population of cMPs in both AF patients and healthy controls. AF patients had significantly higher levels of total cMPs  levels in AF patients, we evaluated each of these parameters in AF patients compared between the presence or absence of each cardiovascular risk factor and antithrombotic drugs. Our results revealed no significant differences in total cMP, PMP, or EMP (CD31+ CD41−) levels relative to any of the evaluated risk factors or antithrombotic drugs .4Our polychromatic flow cytometric analysis showed circulating MPs levels in nonvalvular AF patients. To the best of our knowledge, this is the first study in AF patients to demonstrate increased levels of total cMPs. PS+ is used as a marker for apoptotic cells, and is frequently used in the analysis of cMPs; however, not all cMPs express PS. As such, the study of only PS + MPs may lead to a misunderstanding of the role of cMPs in disease. cMPs also express markers from their parental cells, such as CD41 for platelets, CD235a for erythrocytes, and CD45 for leukocytes. The study of cMP cellular origin may provide insight into the pathophysiology of AF.In this study, total cMP levels were significantly higher in AF patients than in healthy controls even after adjusting for potential confounders. Moreover, total cMP levels did not differ among AF patients relative to cardiovascular risk factors or the type of antithrombotic treatment. Of note, most cMPs (90%) in healthy individuals were characterized using our antibody panel, whereas about 45% of cMPs in the AF group were not able to be characterized. This implies that there are other unknown cellular origins of cMPs in the blood of AF patients. We postulate that increased total cMP levels may result from the pathophysiology of AF, as the atrium is composed of many cell lineages, including endothelial cells, fibroblasts, and cardiomyocytes. Evidence of cellular apoptosis was found in animal and human hearts with AF, including abnormal myocytes, a high degree of DNA breakage, strong caspase‐3 (CASP‐3) expression, and decreased anti‐apoptotic BCL‐2 proteins.We found higher EMP levels (CD31+ CD41−) in AF patients than in healthy controls. It is known that EMPs play a pivotal role in vascular homeostasis. Several stimuli, such as shear stress, inflammation, proapoptotic stimulation, and vascular damage, have been described as promoting EMP secretion.The levels of CD31+ EMPs and CD144+ EMPs appear to increase in apoptotic endothelial cells and have reported as the potential biomarkers for risk assessment of endothelial dysfunction. In our study, we found the increased levels of CD31+ CD41− EMP in AF patients, whereas there was no difference in the levels of CD144+ EMP between AF patients and controls. This contrast may be because of different sites of endothelial injury, noting that PECAM‐1 (CD31) is located mainly outside of the endothelial cell adherens junction, whereas VE‐cadherin (CD144) is localized at the adherens junction.High levels of PMPs were observed in AF patients with no observed differences among the different types of AF, which is consistent with previous finding.PMPs are generated from activated platelets. Platelet activation is observed in AF as represented by increase β‐thromboglobulin levels.In normal conditions, cells release MPs to maintain homeostasis, and this contributes to the regulation of several physiological processes. Relative to the pathophysiology of disease, changes in the number, composition, and function of MPs are thought to promote hypercoagulability, proinflammation, and endothelial dysfunction as part of the development of cardiovascular diseases.Phosphatidylserine‐expressing MPs play an important role in thrombosis, because they provide a catalytic surface to support the assembly of blood coagulation factors. Ederhy et al reported increased procoagulant MP (PS+MP) levels in AF, which suggests the presence of AF as a strong predictor of procoagulant MPs. They suggested that PS+MP may reflect a hypercoagulable state in AF.AF patients were receiving warfarin (78.8%) and aspirin (31.8%) to prevent the ischemic stroke. The effect of warfarin and aspirin on MP levels have demonstrated in AF in which there was no difference in PMP levels between receiving therapy with aspirin and warfarin.Although the reduction of protein C and protein S reflects the high consumption of activated protein C (APC) and was associated with an increase in MP levels in some disease state,Moreover, we found RMP and TF‐MP levels to be significantly higher in AF patients than in controls, but these differences did not persist after adjusting for potential confounders. These results suggest that RMP and TF‐MP levels are more related to age, gender, and BMI than to the AF itself. The number of RMPs was reported to correlate positively with age in women, but not in men.5This is the first report that found a marked increase in total cMP levels in AF patients. The evidence of elevated endothelial damage and platelet activation were demonstrated by increased PMP and EMP levels in AF patients. Additional study is needed to further elucidate the role of cMPs (PMPs and EMPs) in the pathophysiology and the complications associated with AF.The authors declare no potential conflict of interests.FIGURE S1 Flow cytometric gating strategy for the analysis of circulating microparticles. R1 is the gate for all events. (A) The cMP gate was established based on size, using standard calibration beads sized 220, 580, 750, and 1300 nm. (B) cMP are circulating microparticle gate. TC represents TruCOUNT beads gate. The cellular origins of microparticles were identified using cell type‐specific surface markers. (C) PS + MPs were determined by FITC‐Annexin V positive population. (D) PMPs were identified using BV421‐CD41 positive population. Click here for additional data file.FIGURE S2 Effect of cardiovascular risk factors on total cMP, PMP, and EMP (CD31+ CD41−) levels among AF patientsClick here for additional data file.FIGURE S3 Effect of age gender and antithrombotic drugs on total cMP, PMP, and EMP (CD31+ CD41−) levels among AF patientsClick here for additional data file.


Elaeagnus rhamnoides (L.) A. Nelson) is a small tree or bush. It belongs to the Elaeagnaceae family, and has been used for many years in traditional medicine in both Europe and Asia. However, there is no data on the effect of sea buckthorn leaves and twigs on the properties of blood platelets. The aim of the study was to analyze the biological activity of phenolic extracts from leaves and twigs of sea buckthorn in blood platelets in vitro. Two sets of extracts were used: (1) phenolic compounds from twigs and (2) phenolic compounds from leaves. Their biological effects on human blood platelets were studied by blood platelet adhesion, platelet aggregation, arachidonic acid metabolism and the generation of superoxide anion. Cytotoxicity was also evaluated against platelets. The action of extracts from sea buckthorn twigs and leaves was compared to activities of the phenolic extract  with antioxidative and antiplatelet properties. This study is the first to demonstrate that extracts from sea buckthorn leaves and twigs are a source of bioactive compounds which may be used for the prophylaxis and treatment of cardiovascular pathologies associated with blood platelet hyperactivity. Both leaf and twig extracts were found to display anti-platelet activity in vitro. Moreover, the twig extract (rich in proanthocyanidins) displayed better anti-platelet potential than the leaf extract or aronia extract. Sea buckthorn ( Platelets are highly reactive cells activated through various specific membrane receptors by physiological agonists, such as adenosine diphosphate (ADP), thrombin and collagen, as well as non-physiological agonists. They also play an important role in hemostasis, this being the regulation of the flowing properties of blood. In the presence of agonists, blood platelets respond by adhering to various adhesive proteins, including collagen, forming platelet aggregates and secreting various compounds from granules. Moreover, various biochemical processes such as phosphoinositide hydrolysis, arachidonic metabolism and eicosanoid biosynthesis, and reactive oxygen species (ROS) generation, are involved in platelet activation ,2. HowevAronia melanocarpa), blueberries (Vacconium myrtillus) and grapes (Vitis) have been found to possess antioxidant and antiplatelet activities [The most widely-known and popular anti-platelet drug is acetylsalicylic acid (aspirin), which acts as a cyclooxygenase inhibitor, an enzyme involved in eicosanoid synthesis. The most common side effects of aspirin are indigestion, stomach aches and bleeding ,2. Hencetivities ,7,8. Stutivities . In additivities ,15,16,17®) may inhibit platelet activation, by reducing platelet aggregation or eicosanoid synthesis [Elaeagnus rhamnoides (L.) A. Nelson) berries and their products may have therapeutic and protective properties against cardiovascular diseases [2−) production. In addition, the cellular safety of tested extracts was evaluated in vitro using a cytotoxicity test against human blood platelets, measuring extracellular lactate dehydrogenase (LDH) activity. The action of extracts from sea buckthorn twigs and leaves was compared to activities of the phenolic extract, a commercial extract from the berries of Aronia melanocarpa (Aronox®) with antioxidative and antiplatelet properties [Dietary supplements, including commercial products made from aronia berries  inhibition of adhesion to collagen and 62.6 ± 9.0% (p < 0.02) inhibition of adhesion to fibrinogen.Our results show significantly lower adhesion to collagen of resting blood platelets and thrombin-activated platelets following preincubation with 0.5–50 µg/mL twig and leaf extract ; Table 1p > 0.05). However, both 10 and 50 µg/mL leaf extract inhibited thrombin-stimulated platelet aggregation, as did the twig extract at the higher concentration of 50 µg/mL (p < 0.05) (p < 0.05) for twig extract and 29.9 ± 8.9% (p < 0.05) for leaf extract at a concentration of 50 µg/mL  to reduce platelet aggregation stimulated by different agonists, i.e., ADP, collagen and thrombin. The tested extracts were not found to display any anti-aggregatory properties when ADP and collagen were used as agonists ( < 0.05) . For exa50 µg/mL .p > 0.05) (No change was observed in platelet GSH concentration or thiol group number in platelet proteins following exposure to the two tested sea buckthorn extracts at concentrations between 1–50 µg/mL ( > 0.05) .p > 0.05). On the other hand, all used concentrations of twig extract  significantly reduced lipid peroxidation in both the resting and the thrombin-activated platelets (p < 0.05) (As demonstrated in  < 0.05) A,B. At t < 0.05) A,B. In a2− production in resting platelets and activated platelets   .p < 0.05) (In comparative experiments (for blood platelet adhesion), the extract of sea buckthorn twigs  turned out to be more effective than 10 µg/mL aronia extract ( < 0.05) . In addition to multivitamin and multimineral compounds, demand is growing for supplements based on plant sources. One such plant is sea buckthorn, which offers great promise as a supplement, mainly due to its high concentrations of vitamin C, tocopherols and carotenoids, as well as its unique profile of lipids  and various bioactive compounds, including phenolics, believed to be good for human health ,21,22, iSea buckthorn berries are therefore good candidates for functional food production. Recently, other studies have indicated that sea buckthorn leaves and twigs are also good sources of phenolic compounds with various biological activities, including antioxidant and anticoagulant properties ,26,27,28A significant new finding is that these extracts demonstrate antiadhesive activity in the tested system of isolated washed human blood platelets, with the tested extracts reducing blood platelet adhesion to collagen and fibrinogen. In addition, both tested extracts inhibited the aggregation of platelets following thrombin stimulation; interestingly, this inhibition was not observed in the platelets activated by collagen or ADP. It is possible that that tested extracts might interact with the plasma proteins present in platelet-rich plasma (PRP), thus preventing anti-aggregatory activity.Although blood platelet function is known to involve thiol groups ,30, our 2 (TXA2) are produced. Thromboxane A2 is an unstable compound, which is metabolized to inactive thromboxane B2 after about 30 s. In the present experiments, TBARS concentration was used as an indicator of enzymatic peroxidation of arachidonic acid in the thrombin-stimulated platelets. The twig extract was found to reduce the thrombin-induced enzymatic cascade of arachidonic acid metabolism in blood platelets. It is possible that this extract may restore the level of platelet response by helping maintain the redox balance in thrombin-activated blood platelets.Blood platelet activation is associated with arachidonic acid metabolism, in which different intermediate products, including pro-thrombotic thromboxane A2− generation is associated with the enzymatic pathway of arachidonic acid metabolism. A reduction in O2− production was observed in thrombin-activated blood platelets treated with the twig extract, which was accompanied by a decrease in TBARS production. This inhibition of TBARS production and O2− generation by the twig extract suggests that it may also inhibit the thrombin-activated arachidonic acid pathway. It is also possible that the tested leaves and twig extracts may influence platelet reactivity by modifying other signal pathways, not only ROS level, e.g., through the inhibition of enzymatic peroxidation of lipids (TXA2 biosynthesis), or modifying the expression of platelet receptors.It is known that ROS, which may behave as secondary signaling molecules, are generated both in resting platelets and those activated by various agonists, including thrombin. For example, OThe differences in blood platelet activation displayed by the extracts may be accounted for by differences in their phenolic profiles. For example, the greater potency of the twig extract may be associated with its higher proanthocyanidin concentration compared to the leaf extract; for example, it demonstrated greater ability to encourage the inhibiting of platelet adhesion (stimulated by thrombin) to type I collagen. Type I is the most prevalent form of collagen in the arterial vessels changed by atherosclerosis. Two important aspects of the use of natural compounds as drugs or supplements are their toxicity and bioavailability; these parameters are often determined for phenolics intended for use as ingredients of supplements or drugs in in vitro and in vivo models. Our present results demonstrate that none of the tested sea buckthorn extracts induced damage to human blood platelets within the whole tested concentration range. As the concentrations of sea buckthorn extracts used in the study may be achievable in blood during their oral supplementation ,32,33, wA novel finding of our present study is that sea buckthorn twig extract , similar to well-known aronia berry extract, has anti-platelet potential. It is also an interesting that sea buckthorn twig extract had stronger anti-adhesive activity than aronia berry extract. The bioavailability of phenolic compounds varies according to food source. Moreover, it may also depend on the presence of various other compounds in the food matrix, including those with anti-platelet properties . Tormano®, which has a high concentration of anthocyanidins) have been found to be more effective anti-platelet factors than pure phenolic compounds in both in vitro and in vivo models [Chong et al.  indicateo models ,38,40,41o models  report tOur present findings shed new light on the anti-platelet potential of sea buckthorn twig and leaf extracts, particularly those of the twig extract. In future, both may be recommended in the prevention and treatment of cardiovascular diseases associated with hyperactivation of platelets. In addition, our findings may assist the development of further potential anti-platelet supplements or potent drugs against cardiovascular diseases as alternatives to classical drugs such as aspirin, which often induce side-effects. It is important that fruits are harvested together with leaves and twigs; these by products represent a rich source of additional safe phenolic compounds with anti-platelet potential, which would otherwise be regarded as production waste.ADP was obtained from Chrono-Log Corporation . Thrombin was purchased from BioMed Lublin, Poland. Collagen type I, bovine serum albumin (BSA), cytochrome C, 5,5′-dithio-bis(2-nitro-benzoic acid), and dimethylsulfoxide (DMSO) were purchased from Sigma . Fibrinogen was isolated from pooled citrated human plasma by cold ethanol precipitation followed by ammonium sulphate fractionation at 26% saturation at 4 ˚C, according to Doolittle ; its con® by Agropharm Ltd., Poland; batch No. 020/2007k) amounted to 309.6 mg/g of extract, including phenolic acids (isomers of chlorogenic acid), 149.2 mg/g of extract, anthocyanins , 110.7 mg/g, and flavonoids (quercetin glycosides), 49.7 mg/g of extract. The high-performance liquid chromatography (HPLC) determination of this extract was described previously [The content of phenolics in the phenolic-rich powder aronia berr extract , the largest Polish producer of sea buckthorn fruits. The plant material was identified by Mr. Stanislaw Trzonkowski, the owner of the farm. Voucher specimens have been deposited at the Institute of Soil Science and Plant Cultivation, Sate Research Institute, Pulawy, Poland (IUNG/HRH/2015/2).v/v), for 48 h, at room temperature; the extraction was assisted by ultrasonic treatment (6 × 10 min.). After filtration, the methanol extracts were defatted with n-hexane. The defatted extract was concentrated under reduced pressure, the residue was resuspended in Milli-Q water, acidified with formic acid and subjected to n-butanol extraction. The butanol extracts obtained were rotary evaporated, and the residue was suspended in Milli-Q water and freeze-dried. Their composition was determined by reverse-phase ultra high-performance liquid chromatography–mass spectrometry (UHPLC–MS), using the ACQUITY UPLCTM system , coupled with and ACQUITY TQD (Waters) triple quadrupole mass detector. Samples were chromatographed using an ACQUITY BEH C18  column. MS analyses were performed using negative and positive ion mode. More details of the applied analytical method are presented elsewhere [The extracts from leaves and twigs of sea buckthorn were prepared as previously described . Briefly, freeze-dried leaves and air-dried twigs were milled in a laboratory mill . The powdered plant material (140 g of the leaves or 200 g of the twigs) was extracted with 3 L (in three batches) of 80% methanol . The total flavonoid content was 74.7 ± 0.7 mg/g. Catechin and proanthocyanidins were also detected, their total content was 7.2 ± 0.2 mg/g The sea buckthorn twig extract consisted mainly of B–type proanthocyanidins and catechin . Ellagic acid and its glycosides were also present . Flavonoids were present in trace amounts . Major phenolic constituents of the sea buckthorn leaf extract and the twig extract are shown in 2O.Stock solutions of the twig extract and leaf extract were made in 50% DMSO. The final concentration of DMSO in samples was lower than 0.05% and its effect was determined in all experiments. A stock solution of aronia berry extract was made in Hv/v blood/CPD). The samples were not pooled. To obtain platelet-rich plasma, whole blood was centrifuged . The platelet titer was determined spectrophotometrically using a Helios α spectrophotometer at a wavelength λ = 800 nm [8 cells/mL. Analysis of the blood samples was performed under the guidelines of the Helsinki Declaration for Human Research, and approved by the Committee on the Ethics of Research in Human Experimentation of the University of Lodz (resolution No. 3/KBBN-UŁ/II/2016). The first, participants provided verbal consent to the researchers, and later participants provided written the documents.Fresh human blood was obtained from 9 healthy volunteers  in the Lodz Medical Center ; the samples were collected in CPD solution  following the addition of p-nitrophenylphosphate, the phosphatase substrate. The color reaction is created by the addition of 2 M NaOH. The absorbance of control blood platelets (without tested extracts) was expressed as 100% [Platelet adhesion was evaluated by measuring the activity of the platelet exoenzyme (acid phosphatase). Platelets were dissolved with Triton X-100. The formation of  as 100% ,49.Platelet aggregation was measured by turbidimetry in platelet-rich plasma or in a platelet suspended in Barber’s buffer using the optical Chrono-Log aggregometer  .Samples were prepared with 594 μL platelet-rich plasma (PRP) plus 6 μL extracts, or platelets suspended in Barber buffer and 6 μL tested extracts. In addition, a control sample without extract was prepared. Such prepared samples (with extract and without extract) were incubated at 37 °C for 30 min.After incubation, 5 μL ADP  or 5 μL collagen  was added to the platelet rich plasma (PRP) for 10 min. The aggregometer was calibrated against the poor platelet plasma (100% aggregation).After incubation, 5 μL thrombin  was added for 10 min to the platelets suspended in Barber’s buffer. The aggregometer was calibrated against Barber’s buffer (100% aggregation).In these experiments, the blood platelets or PRP not treated with plant extract were used as control samples (positive control), and the rate of agonist-induced aggregation for the control sample (in the absence of plant extract) was 100%.−1cm−1) [The concentrations of thiol groups in platelet proteins and the glutathione concentration was measured spectrophotometrically using a SPECTROstar Nano Microplate Reader  at λ = 412 nm with Ellman’s reagent: 5,5′-dithio-bis- ,52.Lipid peroxidation was determined by measuring TBARS concentration. First, 15% trichloroacetic acid and 0.37% thiobarbituric acid were added to the test samples. The samples were heated in a heating block at 100 °C for 10 min. After cooling, the test samples were centrifuged . The absorbance of the supernatant was measured at λ = 535 nm using a SPECTROstar Nano Microplate Reader  ,18,53,54g). The absorbance of the supernatant was measured at λ = 550 nm using a SPECTROstar Nano Microplate Reader . The O2− determination uses the molar absorption coefficient for cytochrome C, which is 18,700 M−1cm−1 [The superoxide anion level in blood platelets was determined by spectrophotometric measurement of the reduction of ferricytochrome c to ferrocytochrome. Cytochrome c (160 μM) was added to two platelet suspensions: one stimulated by thrombin and another that was unstimulated (resting). The samples were centrifuged  released from platelets is a measure of the toxicity of the tested extracts against platelets. The test samples were centrifuged . The microtiter plate was loaded with 270 μL of 0.1 M phosphate buffer, 10 μL of supernatant and 10 μL of NADH. After a 20-min incubation at room temperature, 10 μL of pyruvate (5 mg) was added and the absorbance was measured. The reading was repeated for 10 min in every minute using a SPECTROstar Nano Microplate Reader  at λ = 340 nm .Several tests were used to perform the statistical analysis. In order to eliminate uncertain data, the Q-Dixon test was performed. All the values in this study were expressed as mean ± SE; n–number of blood donors. Statistical analysis was performed with one-way analysis of variance (ANOVA) for repeated measurements.


Early detection of psychosocial problems post-injury may prevent them from becoming chronic. Currently, there is no psychosocial screening instrument that can be used in patients surviving a physical trauma or injury. Therefore, we recently developed a psychosocial screening instrument for adult physical trauma patients, the PSIT. The aim of this study was to finalize and psychometrically examine the PSIT.n = 1448) received the PSIT, Impact of Events Scale-Revised (IES-R), Patient Health Questionnaire-9 (PHQ-9), Rosenberg Self-Esteem Scale (RSES), State-Trait Anxiety Inventory-State (STAI-S), and the World Health Organization Quality of Life-Abbreviated version (WHOQOL-Bref). After 2 weeks, a subgroup of responding participants received the PSIT a second time. The internal structure , internal consistency , test-retest reliability , construct validity (Spearman’s rho correlations), diagnostic accuracy , and potential cut-off values (sensitivity and specificity) were examined.All adult (≥ 18 years) trauma patients admitted to a Dutch level I trauma center from October 2016 through September 2017 without severe cognitive disorders  patients participated, of whom 128 completed the PSIT again after 19.5 ± 6.8 days. Test-retest reliability was good (ICC = 0.86). Based on PCA, five items were removed because of cross-loadings ≥ 0.3. Three subscales were identified: (1) Negative affect ; (2) Anxiety and Post-Traumatic Stress Symptoms ; and (3) Social and self-image . CFA supported this structure . Four of the five a priori formulated hypotheses regarding construct validity were confirmed. The following cut-off values represent maximum sensitivity and specificity: 7 on subscale 1 (89.6% and 83.4%), 3 on subscale 2 (94.4% and 90.3%), and 4 on subscale 3 (85.7% and 90.7%).The final PSIT has good psychometric properties in adult trauma patients. Each year, injuries resulting from physical trauma cause worldwide over five million deaths . Tens ofRecently, the Psychosocial Screening Instrument for physical Trauma patients (PSIT) was developed, a self-report instrument which screens for several psychosocial problems after injury. To develop the PSIT, first a systematic review was conducted to generate a comprehensive list of psychosocial problems following physical trauma (submitted). Second, focus groups with trauma patients and HCPs were organized to ask patients which psychosocial problems they experienced and to ask patients and HCPs feedback on the problems list resulting from the review and which problems they perceived as most important (submitted). Whereas studies most frequently have assessed symptoms of depression, post-traumatic stress, and anxiety –24, our Patients were eligible if they were 18 years or older and admitted to a ward or the Intensive Care Unit (ICU) of the ETZ Hospital, a level I trauma center in the Netherlands, from October 2016 to September 2017. Patients were invited using the Brabant Trauma Registry (BTR) database. Exclusion criteria were (i) severe cognitive impairment  and (ii) insufficient knowledge of the Dutch language. The Medical Ethical Committee Brabant approved the study. The data were collected between October 2017 and March 2018.Eligible participants received written explanation about the study and contact details of one of the researchers. When a patient was willing to participate, he/she was asked to sign an informed consent form, complete the questionnaires, and return all documents together in a return envelope. Patients who did not return the questionnaires were called to remind them of the study and, if they were unreachable, they received a reminder by post. After approximately 2 weeks, patients who completed the first set of questionnaires were sent the PSIT again, with a request to complete this instrument a second time to establish test-retest reliability. This approach was chosen because a smaller sample size is needed to examine test-retest reliability compared with other psychometric properties . ParticiThe following variables were derived from the patient database: sex, date of birth, date of hospital admission, injury cause, injury mechanism, injury severity score (ISS), and whether they were admitted to the ICU. Patients were asked to provide the following demographic information: level of education, living situation , and whether they currently had a paid job (yes/no). Furthermore, to gain insight in pre-existing psychosocial problems, patients were asked whether they experienced psychological problems before the trauma (yes/no) and if they could briefly describe those problems (if applicable), and if they ever received counseling for psychological problems (yes/no). In addition, patients were asked if they currently received counseling for psychological problems (yes/no).not at all) to 3 (very much). After completion of the PSIT, patients were asked whether they found one or more items confusing or difficult , whether they missed a topic , and whether they had any remarks about the PSIT.The PSIT is a recently developed Dutch psychosocial screening instrument for adult trauma patients. The preliminary PSIT consists of 20 items and covers the following topics: anxiety symptoms (2 items), mood disturbances (2 items), sexual problems (1 item), impaired body image (1 item), loneliness (1 item), feeling burdensome to others (1 item), inadequate social support (1 item), decreased self-confidence (1 item), employment-related problems (1 item), post-traumatic stress symptoms (3 items), impairments in social activities/leisure time (1 item), frustration (1 item), disappointment (1 item), powerlessness (1 item), anger (1 item), and relationship issues (1 item). This preliminary version of the PSIT ended with an open-ended question to provide patients the opportunity to indicate any other psychosocial problem or problems that they experienced. Each item can be answered on a 4-point Likert scale from 0  to 3 (nearly every day) [The PHQ-9 is a 9-item measure to assess depressive symptoms. It is considered a suitable questionnaire to screen for depressive symptoms following injury . Each syery day) . The totery day) –33. The ery day) , 34, 35.not at all) to 4 (extremely). Scores can range from 0 to 88 and a score of 33 or higher represents the most appropriate cut-off value of PTSS [The IES-R consists of 22 items and measures three symptom clusters of PTSS, namely intrusive, avoidance and hyperarousal symptoms . Each sy of PTSS . Studies of PTSS , 37, 38.almost never) to 4 . Despite limited research on useful cut-off values, a score of 40 or higher has been reported to reflect anxiety symptoms [The STAI-S is a 20-item questionnaire which measures state anxiety . Each itsymptoms . Studiessymptoms , 41. strongly disagree) to 4 (strongly agree). Although it has been stated that scores should preferably be analyzed in a continuous manner, scores below 15 reflect low self-esteem [The RSES has 10 items and is a self-report instrument to assess global self-esteem . Responsf-esteem . The RSEf-esteem .The WHOQOL-Bref consists of 26 items and is the short form of the WHOQOL-100 which is developed to assess QoL . Scores Several recommendations exist regarding the minimum sample size needed to assess psychometric properties of an instrument , 49, 50.To compare responders and non-responders on demographic and clinical characteristics, chi-squared and Mann-Whitney U tests were calculated. Descriptive statistics were used to create an overview of the sample characteristics. The distribution of item scores on the PSIT was explored with regard to kurtosis and skewness and by performing frequency analyses. Moreover, the presence of floor and ceiling effects was assessed using frequency analyses. Next, principal components analysis (PCA) was used to examine the internal structure of the PSIT. Appropriateness of PCA was checked using the Kaiser-Meyer-Olkin measure (KMO), which should be at least 0.06, and by Bartlett’s test of sphericity, which should be statistically significant . ObliqueReliability was measured by examining internal consistency and test-retest reliability. Internal consistency was assessed using Cronbach’s alpha coefficients (α) and values of at least 0.70 reflect satisfactory internal consistency . Test-rer < 0.50, and unrelated constructs should show r < 0.30 [To examine construct validity, Spearman’s rho correlation coefficients were calculated between the PSIT subscales and the additional questionnaires. A priori, five hypotheses were formulated Table . Instrumr < 0.30 , 56. Conr < 0.30 .Table 1Receiver operating characteristics (ROC) analyses were performed to evaluate the ability of the PSIT to detect patients with psychosocial problems . The aren = 78), had insufficient knowledge of the Dutch language (n = 63), had severe cognitive disorders such as dementia (n = 116), did not have an injury after all according to the electronical medical file (n = 5), or if their address was unknown or incomplete (n = 6). Furthermore, 13 patients were registered twice in the BTR database. In total, 1448 eligible patients were invited to participate of which 364 returned the questionnaires (response rate: 25.1%). The PSIT was completed a second time by 128 patients , gender  = 0.43, p = 0.50), injury cause  = 9.25, p = 0.24), and ICU admission  = 1.20, p = 0.27; Table p = 0.02) but this was a small effect (r = 0.06). In addition, patients with penetrating injury were less likely to respond, although the effect size was small  = 5.95, p = 0.02, phi = −0.06). Table The BTR database contained 1729 trauma patients admitted to the ETZ from October 2016 through September 2017. Patients were excluded if they had died  Negative affect, (2) Anxiety and PTSS, and (3) Social and self-image (Table 2 (87) = 240.55, CFI = 0.95, RMSEA = 0.07, and SRMR = 0.05). To improve the model fit, two correlations of two error terms were added to the model . This resulted in an excellent model fit (χ2 (85) = 191.58, CFI = 0.96, RMSEA = 0.06, and SRMR = 0.04) , subscale 1 , subscale 2 , and subscale 3  , 0.88 for Anxiety and PTSS , and 0.92 for Social and self-image . Table Figures n = 7) and that some of the experienced problems were not related to the trauma (n = 5). In other words, only three patients had difficulty with interpreting one or more items of the PSIT. Therefore, it was decided that it was not needed to change the wording of the items or the response options.Thirty-four patients (9.3%) reported that they found one of the questions in the PSIT confusing or difficult. One patient required assistance to complete the PIST, another patient found the item regarding re-experiencing symptoms ambiguous, and a third patient was confused regarding the difference between ‘frustration’ and ‘disappointment’. The most common remarks were that patients found the questions confronting (n = 11) and less often to psychosocial problems . Since the goal of the PSIT is to screen for psychosocial problems, the suggested topics were not included in the final PSIT. The optional open-ended question was retained to provide patients the opportunity to write an experienced problem not listed in the PSIT.Thirty patients (8.2%) stated that they missed a topic in the PSIT, most often related to physical or cognitive problems . It is plausible that patients did not answer this item because they did not have a romantic relationship, since several patients had written down that they were single. Nevertheless, this missing rate (3.8%) is still far below the threshold of a problematic missing rate of 15% or more .All subscales of the PSIT had floor effects. A disadvantage of floor effects is that discrimination between patients without psychosocial problems is not possible . HoweverAs expected, strong correlations were found between the subscales of the PSIT. Research shows that psychosocial problems can be related or co-existing –62. ConsConcerning the construct validity, only one hypothesis for the third subscale of the PSIT  could not be confirmed. Moderate correlations were found between this subscale and the RSES and domain 3 of the WHOQOL-Bref, while high correlations were expected. This could be explained by the fact that this PSIT subscale contains items related to self-confidence and social problems and therefore measures a slightly broader construct than the other two instruments, which are focused on either self-esteem (the RSES ) or sociThe current study has some limitations. First, response bias might have occurred as only 25.1% of the eligible trauma patients responded to the questionnaire. Analyses revealed that younger age and penetrating injury were associated with being a non-responder, although effect sizes for these variables were small. Responders and non-responders were comparable on other characteristics . The majority of eligible patients were not reachable. Patients declining participation and willing to provide the reason often indicated that they were not interested because they were participants in other studies, they did not experience any psychosocial problems, or they found the questionnaire too long and/or burdensome. The response rate for the second PSIT (to assess test-retest reliability) was higher, namely 78.5%. This group completed the first questionnaire and was therefore already willing to participate in this study. Second, 63 patients were excluded based on their insufficient knowledge of the Dutch language. Yet, this is only 3.6% of the total trauma population, implying a relatively low risk for language or cultural bias.Future research should explore whether the established cut-off values are useful in clinical practice and how the referral system could be organized. For instance, to whom should referral occur ? Another relevant research area is appropriate timing of psychosocial screening . Moreover, future studies might consider exploring how the PSIT can be best implemented in trauma care. Once these questions are addressed, the PSIT could be translated in different languages to assess its cross-cultural validity.This study also has a number of clinical implications. While various questionnaires and screening instruments are available, these mainly assess depressive and anxiety symptoms , or PTSIn conclusion, this study showed that the PSIT is a reliable, valid, and easy to complete psychosocial screening instrument. It appears to be a useful instrument to screen for psychosocial problems after injury.Additional file 1: Table S1. Items of the PSIT, missing scores, distribution of responses, kurtosis, and skewness.Additional file 2. The PSIT and its scoring instructions.


NGF-TrkA is well known to play a key role in propagating and sustaining pruritogenic signals, which form the pathology of chronic pruritus. Inhibition of NGF-TrkA is a known strategy for the treatment of pruritus. In the present paper, we describe the identification, in vitro characterization, structure–activity analysis, and inhibitory evaluation of a novel TrkA inhibitory scaffold exemplified by Cucurbitacins (Cus).Cus were identified as TrkA inhibitors in a large-scale kinase library screen. To obtain structural models of Cus as TrkA inhibitors, AutoDock was used to explore their binding to TrkA. Furthermore, PC12 cell culture systems have been used to study the effects of Cus and traditional Chinese medicinal plants (Tian Gua Di and bitter gourd leaf) extracts on the kinase activity of TrkA.Cus block the phosphorylation of TrkA on several tyrosine sites, including Tyr490, Tyr674/675, and Tyr785, and inhibit downstream Akt and MAPK phosphorylation in response to NGF in PC12 cell model systems. Furthermore, traditional Chinese medicinal plants (Tian Gua Di and bitter gourd leaf) containing Cu extracts were shown to inhibit the phosphorylation of TrkA and Akt. These data reveal mechanisms, at least partly, of the anti-pruritus bioactivity of Cus.Taken together, with the recent discovery of the important role of TrkA as a therapeutic target, Cus could be the basis for the design of improved TrkA kinase inhibitors, which could someday help treat pruritus. Cucurbitacins (Cus) are tetracyclic triterpenes, consisting of at least five cucurbitacin compounds, named Cucurbitacin B (CuB), Cucurbitacin D (CuD), Cucurbitacin E (CuE), Cucurbitacin I (CuI), and Cucurbitacin Q (CuQ). Among them, CuB, CuE, and CuI Fig. a have beHigh-throughput kinase screening provides a powerful platform to support profiling a compound against hundreds of pharmaceutical targets in a single screen . KinasesThe PC12 cells used in the present study were obtained from the American Type Culture Collection . The cells were maintained in Dulbecco’s modified Eagle’s medium  containing 10% (v/v) fetal bovine serum (FBS) and 5% horse serum, supplemented with penicillin/streptomycin . Cus were obtained from Sigma-Aldrich  and were dissolved in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM. The TrkA kinase inhibitor, GW441756, was purchased from Selleck Chemicals . The drugs were dissolved in DMSO to an initial stock concentration of 10 mM, and aliquots were made and stored at − 80 °C until use.Cucumis melo fruit peduncles) were collected from Wulian County , Rizhao City, Shandong Province, China. Bitter gourd (Momordica charantia) leaves were collected from Xindu District , Chengdu City, Sichuan Province, China in July 2016. Voucher specimens  have been deposited in the School of Pharmacy, Chengdu Medical College, Chengdu, China. These plants were identified by Professor Dr. Yuhang chen, Department of Pharmacy, The Chengdu Medical College.The Gua Di (Gua Di and Bitter gourd leaves were separately pulverized into fine powder using a stainless-steel blender and mixed with ethanol in a 1:10 ratio (w/v). The material was then extracted by boiling in a flask under reflux twice (1 h per cycle). Then, the extracts were concentrated under vacuum in a rotary evaporator. The decoction was lyophilized and stored at 4 °C. The lyophilized powder was dissolved in DMSO and filtered through a 0.22 μm syringe filter to create a stock solution. GDE and BGLE denote Gua Di and Bitter gourd leaf ethanol extract, respectively. GDE and BGLE were diluted with culture medium to the final concentration indicated for each experiment.The HPLC analysis was performed on a Waters 2695 apparatus coupled to a Waters 2996 photodiode array detector, assisted by Waters Empower 2 software. A Kromasil 100-5C18  column was used for analytical HPLC , with the absorbance detection at 230 nm.http://www.kinase-screen.mrc.ac.uk/) as described previously [33P] ATP (∼800 cpm/pmol) was used at 5 μM for protein kinases. The assays were stopped by adding 5 μL of 0.5 M (3%) orthophosphoric acid, and were then harvested onto Whatman® P81 UNIFILTER® plates with a washing buffer containing 50 mM orthophosphoric acid.All assays were carried out by the International Centre for Protein Kinase Profiling (eviously . Briefly33P] ATP (50–1000 cpm/pmole), and was incubated for 30 min at room temperature. The assays were stopped by adding 5 μL of 0.5 M orthophosphoric acid, and were then harvested onto plates with a wash buffer of 50 mM orthophosphoric acid. Controls for solvents and background phosphorylation levels were included in all assays, and the non-specific 33P incorporation obtained was subtracted from all values. IC50 values were generated from log dose-response curves using Prism 5 software .The assays to determine the 50% inhibitory concentration (IC50) values for each enzyme were performed as described previously . In brieTrkA (PDB code: 4PMM) was obtained from the Protein Data Bank. The molecular structures of all compounds were obtained from PubChem. All water atoms were removed, and hydrogen atoms were added to the structures using the pyMol. The nonbonded interactions were calculated using all atomic parameters of AutoDock based on the AMBER force field, since they best reproduced crystal ligand conformations. Implicit solvation parameters were added from the AutoDock Tools package. Ligand nonpolar hydrogen atoms were marked as atom type ‘X’. Docking grids for carbon, oxygen, and polar and nonpolar hydrogen atoms were calculated by AutoGrid, also part of AutoDock. Grid maps contained points for TrkA and all of the compounds to constrain them within the docking cavity. Docking cavities were detected with AutoDockTools, and the candidate cavity was chosen referencing the inhibitor in the crystal structure, 4PMM. The binding conformation was screened with the Genetic Algorithm with Local Search docking method. The docking results were analyzed using pyMol.6 cells) were serum-starved in DMEM in the presence of CuB, CuE, CuI, GDE, BGLE, and GW441756 for 8 h. NGF  was added to the culture medium at a concentration of 50 ng/mL. After 5 min, the cells were lysed, and the protein concentration of all cell lysate samples was measured using the bicinchoninic acid (BCA) protein assay kit . The cell lysates with equal amounts of protein were mixed with SDS loading buffer containing 10% SDS, 50 mM dithiothreitol, 312 mM Tris-HCl (pH = 6.8), and 50% glycerin, boiled for 5 min, and separated by SDS-PAGE electrophoresis. The proteins were then transferred to polyvinylidene difluoride membranes . The membranes were blocked in PBS containing 0.05% (v/v) Tween®20 and 5% (w/v) nonfat dry milk at room temperature for 1 h. The primary antibody incubations were performed in TBS with 0.1% Tween®20 (TBST) with 5% nonfat dry milk overnight. After washing, horseradish peroxidase (HRP)-linked secondary antibodies (1:4000 dilution) were added and incubated for 1 h. Subsequently, the membranes were washed and incubated in ECL detection reagent . The following primary antibodies were used: rabbit anti-Phospho-TrkA 490 (1:2000), anti-Phospho-TrkA 674/675 (1:2000), rabbit anti-Phospho-TrkA 785 (1:2000), rabbit anti-Phospho-Akt 473(1:2000), rabbit anti-TrkA (1:2000), rabbit anti-Akt (1:2000) and rabbit anti-GAPDH (1:4000). All antibodies were purchased from Cell Signaling Technology, Danvers, MA, USA.PC12 cells  . CuB inhTo obtain structural models of Cus as TrkA inhibitors, AutoDock was used to explore their binding to TrkA. The model was generated using the coordinates of known TrkA crystal structures, particularly those of the complex with known TrkA inhibitor (PDB code 4PMM), without crystal water and coligands. Two reasonable binding modes of Cus in the pocket of TrkA were found Fig. , in whicIn this regard, it is noteworthy that pheochromocytoma 12 (PC12) cells express TrkA receptor that mediates neurite outgrowth and anti-apoptotic signaling by binding to NGF . Hence, M. charantia L) leaf helps to prevent or counteract pruritus, and that the plants of the genera Momordica contain a special group of Cus [‘Gua Di’ (in Chinese), a traditional Chinese herb prescribed for liver disorders by native Chinese physicians, is rich in CuB and CuE . It is np of Cus . Thus, wAberrant kinase function or regulation can contribute to the rise of many diseases . While pUnlike other protein kinase inhibitors currently in use, there have been relatively few reports of TrkA inhibitors. Most of these molecules share a similar structure with staurosporine. For example, both CEP-701 and K252a are TrkA inhibitors that are structurally related to staurosporine. Thus, characterization of novel classes of potent and specific TrkA inhibitors is still a challenge. Due to their important therapeutic promise, significant efforts to identify novel TrkA inhibitors have been made in recent years. The natural product, wrightiadione, was discovered as a new template for the development of TrkA inhibitors. The wrightiadione derivative, 2 h, showed a potent inhibitory activity (IC50 = 6.6 μM) toward TrkA at the molecular level [The discovery of artemisinin came from an intensive search for plant natural products representing the wisdom of Chinese medicine , 27. TheThere is evidence that the NGF-TrkA pathway plays a key role in the pathogenesis of psoriasis and associated pruritus. Human mast cells express functional TrkA, are a source of NGF, and NGF-induced histamine secretion from mast cells induces itch or pruritus , 29. KerIn conclusion, Cus block the phosphorylation of TrkA on several tyrosine sites, including Tyr490, Tyr674/675, and Tyr785, and inhibit downstream Akt and MAPK phosphorylation in response to NGF in PC12 cell model systems. Furthermore, traditional Chinese medicinal plants (Tian Gua Di and bitter gourd leaf) containing Cu extracts were shown to inhibit the phosphorylation of TrkA and Akt. These data reveal mechanisms, at least partly, of the anti-pruritus bioactivity of Cus. Considering the development of this TrkA inhibitor, together with the recent discovery about the important role of TrkA as a therapeutic target, Cu could act as a precursor for the development of improved TrkA kinase inhibitors with enhanced activity, which might one day contribute to the treatment of pruritus due to psoriasis.


To determine the effective dose of SI, the right eyes of 19 male New Zealand white rabbits received an intravitreal injection of SI or sham. Based on the dose-dependence results, 0.4 mg of SI in 0.05 mL of total volume was injected into the right eyes of 10 rabbits at two weeks after vitrectomy. In the dose-dependence study, localized retinal atrophy was observed with 0.3- and 0.4-mg SI injections without vitrectomy. Severe and diffuse retinal atrophy was identified by spectral-domain optical coherence tomography (SD-OCT) at one month after a 0.5-mg SI injection following vitrectomy. In the second experiment, 0.4 mg of SI in 0.05 mL was injected, and the severity of outer retinal degeneration was graded as one of two types according to electroretinography (ERG) response change. There was no response on ERG in complete retinal degeneration, 30% of all 10 rabbits. Intravitreal injection of 0.4 mg of SI into vitrectomized rabbit eyes induces diffuse outer retinal degeneration, and the degree of retinal degeneration can be evaluated through  Visual prosthetics such as retinal implants have been developed for treatment of retinal degeneration due to advances in electronic device technology and biomaterials5. Recently, implantation of visual prosthetics has been performed in humans. Therefore, to further develop and refine such medical devices, larger experimental animal models  with specific loss of photoreceptors are inevitably needed.Retinal degeneration, which includes conditions such as retinitis pigmentosa (RP), choroideremia, and geographic atrophy (GA) of age-related macular degeneration (ARMD), is the main cause of irreversible vision loss and greatly affects quality of life. RP is the most common inherited retinal dystrophy and leads to irreversible vision loss. Initial degeneration due to RP occurs in the photoreceptors, and inner retinal thickness is gradually decreased in advanced-stage RP6. Specifically, SI primarily induces necrosis in RPE cells8, which is followed by choriocapillaris atrophy9 and panretinal degeneration10. In addition to these effects on RPE cells and photoreceptors, SI also provokes necrosis of the inner retina11. SI induces the production of reactive oxygen species, which contribute to damage in the RPE cells12. SI retinal toxicity has been demonstrated in many different mammalian species, including sheep7, rabbits14, rats15, and mice16, with varying doses and routes of administration. Most studies have used relatively high doses of SI (50–100 mg/kg) and have reported rapid RPE damage characterized by defragmentation and loss of RPE cell nuclei. Systemic application of SI leads not only to bilateral retinal degeneration, but also to reduced general health of the experimental animals. Systemic intoxication of SI after systemic administration includes gastrointestinal problems such as diarrhea, general weakness, and convulsion18. A high dose of SI was found to be lethal in experimental animals18. Therefore, local administration of SI is required to avoid its systemic effects.The retinotoxin sodium iodate (SI) is an oxidizing compound toxic to retinal pigment epithelial (RPE) cells, with secondary effects on photoreceptors and the choriocapillarisIn the present research, we attempted to induce unilateral diffuse homogeneous outer retinal degeneration of the whole retina by intravitreal administration of SI in rabbits. We hypothesized that this approach would avoid the known systemic side effects. The primary objective of this study was to elucidate the necessary effects of vitrectomy and the proper intravitreal SI dose following vitrectomy to induce diffuse homogeneous outer retinal degeneration in rabbits. Secondarily, we evaluated the ability of the determined dose of intravitreally injected SI to induce diffuse outer retinal degeneration.At one month after SI injection, no significant changes were observed in fundus photography (FP), fundus autofluorescence (AF), histology with hematoxylin and eosin (H&E) staining, or spectral-domain optical coherence tomography (SD-OCT) images of rabbit eyes injected with 0.1 mg of SI Fig. . LocalizAt one month after SI injection, no localized hyper- or hypo autofluorescence was observed by ultra-wide-field color FP or AF Fig. .Figure 2Additionally, no significant retinal changes were observed with SD-OCT in eyes injected with sham at one month, and 0.1 mg of SI at one week and one month Fig. . HoweverAn example of histological examination by H&E staining at one month is presented in Fig. Based on the dose-dependence study, 0.4 mg of SI in 0.05 mL of total volume was selected for further evaluation. Six weeks after the 0.4-mg SI injection, the severity of outer retinal degeneration was graded into two types according to change in OCT images and electroretinography (ERG) response Fig. . For aniHistology and immunohistochemistry findings demonstrated differences between the control eyes  and SI-injected eyes with incomplete and complete changes in retinal degeneration Fig. .Figure 4In the control eyes, all layers of the retina were easily distinguished by histology Fig. . AdditioCataracts caused by contact with the instrumental lens during vitrectomy were observed in four out of 10 eyes (40.0%) during examination at six weeks after injection. We observed no signs of systemic toxicity such as weight loss or death in any rabbit.We compared the effects of a 0.4-mg SI injection depending on vitrectomy. In non-vitrectomized rabbit eyes, total retinal thickness was significantly decreased after 0.4 mg SI injection 20. However, in this experiment, RPE staining remained normal despite retinal degeneration compared with the control. There was no previous report about RPE staining after intravitreal injection of SI. A previous study reported morphologic changes of RPE with immunohistochemistry staining after subretinal injection of SI in other species21. In this report, the RPE nuclei became bigger and more rounded after subretinal injection of SI, but RPE remained relatively stable and appeared to be more resistant than the photoreceptors. The previous and present studies suggest that systemic administration of SI could induce toxicity of RPE, but localized administration of SI such as subretinal and especially intravitreal injection induce less damage to the RPE. We did find that intravitreal injection of SI after vitrectomy induced direct damage to the photoreceptor, not the level of RPE. Previous studies reported that, after SI was administered, Müller glial cells proliferated on the third day, and the ratio of GFAP-positive cells increased markedly for 28 days22. In this study, GFAP staining was also increased in all the eyes that received an intravitreal injection of SI, and it is likely that the SI injection itself caused fibrosis and proliferation of glial cells, regardless of degeneration. The TUNEL assay was positive in eyes with outer retinal degeneration, even though the degree of staining was not extensive. This could be due to a lack of apoptotic cells. SI induced the death of photoreceptors and may have triggered apoptosis. TUNEL-positive cells were restricted to the ONL, where photoreceptor nuclei are located12.When ERG response was partially decreased, greater damage to the IS/OS with an intact ONL was observed on SD-OCT. Finally, when all responses were lost on ERG, ONL and the photoreceptor layer were indistinguishable in SD-OCT images. Additionally, eyes with incomplete changes in retinal degeneration had fewer cone cells based on PNA staining. Eyes with complete changes in retinal degeneration, including those with flat ERG responses, also had less staining with PNA and rhodopsin. Immunohistochemistry demonstrated fewer bipolar and ganglion cells in eyes with complete changes in retinal degeneration. SI not only affects RPE cells and photoreceptors, but can also lead to toxicity and necrosis of the inner retina when intravenously injectedet al.23 reported a transgenic (Tg) rabbit model of progressive retinal degeneration. That rabbit model showed decreased rod ERG response in 5% of animals at 48 weeks of age. In that study, 15% of newborn rabbits were transgene-positive, and some died. In general, it is difficult to develop a genetically modified animal model, and it takes a relatively long time for disease to fully manifest. Because of these limits, intravitreal injection of compounds to induce retinal degeneration has been tried with various drugs in several different kinds of animals31. The main advantages of localized application of SI through intravitreal injection are that it spares the second eye as a control and avoids unwanted systemic side effects. With a known effective drug dose, many adequate animal models for retinal degeneration could be developed quickly. Animal models with monocular drug–induced retinal degeneration could be useful because they do not show binocular blindness. Cho et al.30 reported that intravitreal injection of SI induced monocular retinal degeneration in New Zealand white rabbit eyes. They further reported that retinal damage was reversible at low doses (0.1 and 0.2 mg of SI) but irreversible at higher doses (0.4 and 0.8 mg of SI). In the 0.4-mg SI group in that study, the outer retina was significantly destroyed, whereas the inner retina was relatively preserved. Conversely, in the 0.8-mg SI group, the entire retinal layers were irreversibly destroyed. Those authors reported that monocular intravitreal injection with SI provided an animal model for monocular retinal degeneration. However, Cho et al.30 only tested the performance of intravitreal SI injection and did not perform vitrectomy. Our study showed that intravitreal SI injection without vitrectomy did not induce diffuse retinal degeneration and produced only localized, widely varying degrees of retinal degeneration. In addition, intravitreal SI injection without vitrectomy could induce severe retinal thinning to affect the inner retinal layer. With intravitreal injection after vitrectomy, retinal degeneration was relatively uniform, and the RPE and inner retinal layer remained relatively intact per the results of immunohistochemistry and quantitative outcomes of retinal thickness. Additionally, because only conventional FP without autofluorescence imaging was used instead of ultra-wide-field FP with AF imaging, those authors were limited in their ability to determine the distribution of effects in the peripheral retina. In the present study, we demonstrated that intravitreal injection of SI after vitrectomy was an effective method to produce a globally diffuse outer retinal degeneration animal model with no systemic complications and low cost. Furthermore, if future researchers screen the degree of outer retinal degeneration before experimentation, the two types of outer retinal degeneration models induced here by SI injection can be effectively used for other study purposes.Kondo Notably, there were some limitations in this study. First, the intravitreally injected SI dose was different between the post-vitrectomy group and the without-vitrectomy group. Because we learned that diffuse homogenous retinal degeneration in the whole retina could not be induced with intravitreal SI injection without vitrectomy in the dose-dependence study, we did not try to inject a higher SI dose than 0.4 mg. In the dose-dependent study of SI with vitrectomy, we confirmed that intravitreal injection of SI after vitrectomy affected the whole retina homogeneously based on wide AF and SD-OCT images and focused on detecting the effective dose that could induce diffuse retinal degeneration. We initially could not predict the effect of intravitreal SI injection after vitrectomy and could not rule out the possibility that even lower doses of SI might induce retinal degeneration in the vitrectomized eye. Therefore, sham and a higher dose of SI (0.5 mg) were also injected in vitrectomized eyes. Second, the follow-up periods of the dose-dependent study and the second study were different. In the dose-dependent study, the purpose was to find the effective dose to induce diffuse retinal degeneration, so rabbits were observed for four weeks after intravitreal injection. In the second study, the purpose was to evaluate the retinal change after a 0.4-mg SI intravitreal injection, so rabbits were sacrificed at six weeks after intravitreal injection. Third, the numbers of animals included were relatively small. However, with the rabbits used, we could compare the effects of vitrectomy on the induction of diffuse retinal degeneration and determine the effective dose. Furthermore, in the second study, complete retinal degeneration was induced at 30% among 10 rabbits. So, while minimizing the number of sacrificed animals, the aim of this study was achieved.This SI-induced method provides an animal model that includes incomplete and complete outer retinal degeneration and will be useful for evaluating therapeutic strategies for diseases involving retinal degeneration. After vitrectomy, an animal model induced by intravitreal SI injection showed diffuse and uniform outer retinal degeneration.In conclusion, a vitrectomized rabbit model with an intravitreal a 0.4-mg/0.05-mL SI injection induced diffuse outer retinal degeneration with disruption of photoreceptors.In a dose-dependent study of SI, the right eyes of male New Zealand white rabbits (n = 19), aged five months and weighing between 2.5 and 3.5 kg, received either an intravitreal injection of SI without vitrectomy or an intravitreal injection of SI or sham injection at two weeks after vitrectomy. For all injections, each dose of SI was diluted in 0.05 mL of phosphate-buffered saline (PBS). Two rabbits per concentration received the following intravitreal injections of SI without vitrectomy: 0.4 mg, 0.3 mg, 0.2 mg, and 0.1 mg. Intravitreal injection of SI after vitrectomy was performed as follows: sham  or 0.5 mg, 0.3 mg, or 0.1 mg of SI (n = 3 rabbits per concentration). To identify morphological changes to the retina, ultra-wide-field color FP, fundus AF imaging, and SD-OCT were performed at one month after intravitreal SI injection. Histological examinations after H&E staining were also completed on select rabbit eyes at one month after injection. Additionally, to analyze the change of retinal degeneration over time, SD-OCT was also performed at one week after intravitreal SI injection in the vitrectomized eyes.Based on the results of the dose-dependence study, 0.4 mg of SI in 0.05 mL of PBS was chosen for injection into the right eyes of rabbits (n = 10) at two weeks after vitrectomy. The rabbits in the efficacy experiments had a mean body weight of 3.32 kg and mean axial length according to A-scan ultrasonography of 15.96 mm. In addition, FP, AF imaging, SD-OCT, and histology were performed at six weeks after injection. To identify physiological changes to the retina, ERG was also performed at six weeks after injection.All procedures adhered to the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research . Approval for this study was obtained from the Institutional Animal Care and Use Committee of Korea University College of Medicine in Seoul, Korea.The rabbits were anesthetized by injection of alfaxalone  into the marginal auricular vein and intramuscular injection of xylazine . After anesthesia, 0.5% tropicamide and 0.5% phenylephrine  were administered for pupil dilatation, and then the eye was irrigated with 5% povidone iodide and draped for surgery. Two-port, 23-gauge core vitrectomy  was performed with a direct biconcave lens. The two ports were prepared by inserting a trocar cannula into the sclera at 4 mm from the limbus on the superoventral and superodosal sides. A surgical microscope was used for lighting. The vitreous was removed using a vitreous cutter while continually supplying balanced salt solution .Animals were anesthetized as described above. Immediately before the injections, SI  was dissolved in PBS. The right eye of each rabbit was prepared, and the corresponding dose of SI  was injected intravitreally at 4 mm posterior to the limbus using a 30-gauge needle. No injections were performed in the left eyes.FP and AF images were captured using an ultra-wide-field scanning laser ophthalmoscope . SD-OCT was performed using the Spectralis® OCT system . The area of the visual streak below the optic disc was evaluated. Vertical line scans, horizontal line scans, and raster scans (33 B-scans over a 16.5-mm × 16.5-mm area in a 55-degree image) were performed in high-resolution mode . Up to 100 single images were averaged in automatic real-time mode to obtain a high-quality mean image.34. The rabbits were anesthetized as described above, dark-adapted for 30 minutes, and their pupils were dilated. One eye per animal was studied to avoid accidental contralateral light adaptation. Light stimulation and ERG signal recording were performed with a commercial system  using a contact lens electrode with a built-in light resource . The reference and ground electrodes were platinum subdermal needle electrodes. The reference electrodes were placed in the skin near the lateral canthus of the eyes, while the ground electrode was placed on the forehead between the two eyes.The ERG protocol was based on the international standard for ERG from the International Society for Clinical Electrophysiology of Vision (ISCEV)Immediately after euthanasia, both eyes were enucleated, immersion-fixed in Davidson’s solution for 24 hours, dehydrated, and embedded in paraffin. Sections measuring 4 µm were cut and stained with H&E. The slides were examined for pathological retinal changes using a light microscope  and photographed with a digital camera .Tissue sections were deparaffinized, rehydrated, and microwave-heated in antigen retrieval buffer . Sections were then blocked with 4% horse serum in PBS, followed by primary antibody incubation at 4 °C overnight. Anti-RPE65  staining was performed following the manufacturer’s protocol. For anti-PKCα  and rhodamine-labeled anti-peanut agglutinin (PNA)  coimmunostaining, fluorescent detection was performed with an Alexa Fluor 488–conjugated goat anti-mouse secondary antibody . For anti-PKCα and anti-rhodopsin  coimmunostaining, fluorescence detection was performed with Alexa Fluor 488–conjugated goat anti-mouse and Alexa Fluor 594–conjugated goat anti-mouse secondary antibodies . For anti-Brn3  immunostaining, fluorescence detection was performed with an Alexa Fluor 594–conjugated goat anti-mouse secondary antibody. Lastly, for anti-GFAP  staining, sections were incubated for two hours at room temperature and then for one hour with Alexa Fluor 594–conjugated goat anti-mouse secondary antibody. A deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay  was performed following the manufacturer’s protocol. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) . Cells stained by TUNEL were evaluated using fluorescence microscopy .In each rabbit, we measured total retinal thickness and inner retinal thickness at 10 different inferior retinal sites using linear horizontal SD-OCT imaging performed at baseline and at one month after a 0.4-mg SI/0.05-mL injection in the non-vitrectomized and vitrectomized eyes, respectively. Total retinal layer was defined as from ganglion cell layer (GCL) to RPE layer and inner retinal layer was defined as from GCL to INL.To compare the effects of SI injection on total retinal thickness and inner retinal thickness between baseline and post-injection, statistical analysis was conducted using a Wilcoxon signed-rank test. To compare the effects of vitrectomy on total retinal thickness and inner retinal thickness, statistical analysis was conducted using the Mann–Whitney U test. To compare the type of retinal degeneration after SI injection, statistical analysis was conducted using the Mann–Whitney U test or Kruskal Wallis test. Data are presented as mean ± standard error (SE). Differences were considered statistically significant at p < 0.05.