To evaluate the efficacy and safety of pegylated liposomal doxorubicin (PLD) in patients with human epidermal growth factor receptor 2 (HER2)-negative metastatic breast cancer (MBC) heavily pretreated with anthracycline and taxanes.®, generic doxorubicin hydrochloride liposome) 40 mg/m2 every 4 weeks until disease progression, unacceptable toxicity, or completion of six cycles. Primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival (OS), objective response rate (ORR), disease control rate (DCR), clinical benefit rate (CBR), and safety.In this single-arm, phase II study, patients with HER2-negative MBC previously treated with anthracycline and taxanes as second- to fifth chemotherapy received PLD , 41 and 36 were evaluable for safety and efficacy, respectively. In total, 59.1% (26/44) of patients had ≥ 3 metastatic sites, 86.4% (38/44) had visceral disease, and 63.6% (28/44) had liver metastases. Median PFS was 3.7 months  and median OS was 15.0 months (95% CI 12.1–17.9). ORR, DCR, and CBR were 16.7%, 63.9%, and 36.1%, respectively. The most common adverse events (AEs) were leukopenia (53.7%), fatigue (46.3%), and neutropenia (41.5%), with no grade 4/5 AEs. The most common grade 3 AEs were neutropenia (7.3%) and fatigue (4.9%). Patients experienced palmar-plantar-erythrodysesthesia , stomatitis , and alopecia (7.3%). One patient displayed a left ventricular ejection fraction decline of 11.4% from baseline after five cycles of PLD therapy.®) 40 mg/m2 every 4 weeks was effective and well-tolerated in patients with HER2-negative MBC heavily pretreated with anthracycline and taxanes, revealing a potentially viable treatment option for this population.PLD  contains supplementary material, which is available to authorized users. Breast cancer has become the most commonly diagnosed cancer globally in 2020, surpassing lung cancer 11.4%), with an estimated 2,261,419 new cases (11.7%), and the fourth leading cause (6.9%) of cancer-related deaths worldwide, with an estimated 684,996 new deaths in 2020 1.4%, wit. Among pDuring the past 70 years, the highest number of new drugs for breast cancer was approved , 8–12. HPegylated liposomal doxorubicin (PLD), developed in an attempt to reduce the toxicity profile of conventional doxorubicin while preserving its anti-tumor efficacy, became the first United States Food and Drug Administration -approved cancer nanomedicine in 1994, and it is currently indicated for advanced ovarian cancer, acquired immune deficiency syndrome (AIDS)-related Kaposi’s sarcoma, and multiple myeloma in the United States . Various2 day 1 every 4 weeks as one of the preferred regimens for stage IV/recurrent metastatic HER2-negative breast cancer by the National Comprehensive Cancer Network clinical guidelines for breast cancer ) and renal function (serum creatinine ≤ 1.5 × ULN); and negativity on a serum or urine pregnancy test and willingness to use highly effective methods of contraception to prevent pregnancy for potential child-bearing patients during and 3 months following treatment cessation. Bisphosphonate therapy was permitted when entering the study.Between July 2017 and July 2021, female patients with histologically confirmed HER2-negative MBC previously treated with conventional anthracyclines and taxanes were enrolled in this study. HER2 negativity was defined as a 0 or 1 + on immunohistochemistry and/or negativity by fluorescence in situ hybridization in a primary or metastatic tumor sample. Other eligibility criteria included age ≥ 18 and ≤ 70 years; at least one but no more than four lines of prior systemic chemotherapy for metastatic disease; at least one measurable lesion according to Response Evaluation Criteria for Solid Tumors (RECIST) version 1.1; Eastern2 doxorubicin or 550 mg/m2 epirubicin or equivalent, or heart disease due to the use of anthracyclines; history of other malignancies  within the last 5 years; receipt of other anti-tumor therapies or other experimental drugs within 28 days.Exclusion criteria were as follows: congestive heart failure (New York Heart Association class II or higher); untreated or uncontrolled brain metastases; severe systemic infection; allergy to PLD or other related supplementary materials; prior anthracycline exceeding a total cumulative dose of 300 mg/mThis study was approved by the Ethics Committee of Peking University Cancer Hospital  in Beijing, China, and was registered in the Chinese Clinical Trial Registry (ChiCTR1900022568). This study was conducted according to the Declaration of Helsinki and Good Clinical Practice Guidelines. All participants provided written informed consent.2 every 4 weeks monotherapy in patients with HER2-negative MBC pretreated with conventional anthracycline and taxanes. Patients received PLD (Duomeisu®) 40 mg/m2 diluted in 250 mL of 5% dextrose intravenous infusion for 1 h on day 1 of each 28-day cycle. The detailed dose adjustment schemes were presented in Supplemental Methods. Treatment was continually administered until disease progression, unacceptable toxicity, treatment delay of > 3 weeks owing to toxicity, completion of 6 cycles, or patient’s decision to withdraw from the study.This was a prospective, single-center, open-label, single-arm, phase II study designed to evaluate the efficacy and safety of PLD 40 mg/mThe primary objective of the study was to evaluate progression-free survival (PFS) in the intent-to-treat (ITT) population. PFS was defined as the time from the first dose of PLD to disease progression per the RECIST 1.1 criteria, death from any cause without progression, or the last follow-up visit without progression. The secondary objectives included overall survival (OS), objective response rate (ORR), disease control rate (DCR), clinical benefit rate (CBR), and safety. OS was defined as the time from the first dose of PLD to death from any cause, and it was censored at the date of the last follow-up. ORR was defined as the percentage of patients with the best overall response of complete response (CR) or partial response (PR) per the RECIST 1.1 criteria (without confirmation), DCR was defined as the percentage of patients with CR, PR, or stable disease (SD), and CBR was defined as the percentage of CR, PR, or SD for at least 24 weeks. Tumor responses were accessed by computer tomography/magnetic resonance imaging every two cycles (every 8 weeks) during treatment and every 3 months after discontinuation until disease progression according to RECIST version 1.1, as assek = 1, target hazard ratio of 0.667, two-sided alpha level of 5%, and power of 80%, the planned enrollment and follow-up periods were both 12 months. Considering these parameters, a sample size of the least 43 patients was necessary. Therefore, we aimed to recruit 45 patients.The sample size was calculated using the one-sample log-rank test based on the primary endpoint of PFS. According to Al-Batran’s study , median p values were reported and p values less than 0.05 were considered significant. All analyses were performed using SPSS version 20.0 .Patients who received at least two doses of PLD and completed at least one efficacy evaluation were included in the efficacy analysis. Safety was analyzed by descriptive statistics in all patients who received at least one dose of PLD and underwent at least one safety evaluation. PFS and OS analyses were estimated using the Kaplan–Meier method, and 95% confidence intervals (CIs) were also calculated. ORR, DCR, and CBR were summarized with their 95% CIs. Descriptive quantitative data were expressed as median and range according to the data distribution, and qualitative data were expressed as counts and percentages. Two-sided Between July 2017 and July 2021, a total of 45 patients were recruited for this study. One patient withdrew from this study before receiving treatment. Three patients received only one dose of PLD and withdrew from this study without a safety assessment. Therefore, 41 patients were included in the safety analysis. One patient died 21 days after the first dose because of disease progression, and four patients experienced disease progression during the first cycle. Finally, 36 patients who completed at least two cycles of PLD were included in the efficacy analyses. At the data cut-off date of November 2021, the median follow-up time was 13.2 months (range 2.4–48.0 months). Figure N = 44). The median age was 53.5 years . 11 (25.0%) patients were premenopausal, and 32 (75.0%) were postmenopausal. Notably, 25.0% (11/44) of patients had hormone receptor (HR)-negative tumors. 59.1% (26/44) of patients had ≥ 3 metastatic sites, 86.4% (38/44) had visceral disease, and 63.6% (28/44) had liver metastases. All patients had received conventional anthracycline and taxane previously in the neo/adjuvant setting , metastatic setting , or both settings . Seventeen patients (38.7%) received ≥ 3 prior systemic chemotherapies for metastatic disease, and the median number of prior chemotherapy regimens in the metastatic setting was two (range 1–4). The median anthracycline-free interval  and median PFS of the last therapy were 59.5 months (range 10.0–212.0) and 3.0 months (range 1.0–18.0), respectively.The demographics and baseline characteristics of the patients are presented in Table n = 1; two cycles, n = 1), one who experienced disease progression after receiving two months of toremifene maintenance therapy, and one patient underwent modified radical mastectomy for the primary breast lesion after CR of distant metastases following six cycles of PLD.Thirty-six patients were assessable for clinical response. Ten patients (27.8%) completed the planned six cycles, and 26 patients (72.2%) discontinued treatment earlier than planned: 22 patients 61.1%) because of disease progression, 2 patients (5.6%) because of AEs, and 2 patients due to patient choices  Fig. a, and mep > 0.05). The median PFS times were 3.7, 3.7, and 4.0 months in the second, third, and fourth/fifth lines, respectively , liver metastasis, HR status, and the treatment line . The median dose intensity of PLD was 10 mg/m2/week . Dose reduction occurred in four patients (9.8%) because of grade 2/3 PPE (4.9%), grade 3 fatigue (4.9%), and grade 2 palpitation (2.4%). Dose delay occurred in two patients (4.9%) because of grade 3 neutropenia. Two patients (4.9%) discontinued treatment after the fifth cycle of PLD due to grade 3 fatigue (2.4%), and grade 2 dyspnea with an 11.4% LVEF decline compared with baseline (2.4%).Of 44 patients enrolled, 41 who received at least one dose of PLD were evaluable for safety. PLD was administered for a mean of 3.4 cycles . The mean cumulative dose of PLD was 134 mg/mForty (97.6%) patients reported AEs. The most common all-grade AEs included leukopenia 53.7%), fatigue (46.3%), neutropenia (41.5%), nausea (31.7%), and aspartate aminotransferase elevation (29.3%). Overall, treatment was well-tolerated with no grade 4/5 AEs. Nine patients (22.0%) experienced grade 3 AEs, including neutropenia (7.3%), fatigue (4.9%), PPE (2.4%), anemia (2.4%), vomiting (2.4%), and constipation (2.4%). Patients reported PPE , stomatitis , and alopecia (7.3%) during treatment. There was no evidence of marked LVEF decreases in 40 patients (97.6%) during treatment. Only one patient observed LVEF declined from 70% at baseline to 62% after cycle 5 of PLD . No other cardiac AEs were observed. AEs are listed in Table .7%, fati® monotherapy at 40 mg/m2 every 4 weeks in Chinese women with HER2-negative MBC heavily pretreated with conventional anthracyclines and taxanes. The results of this study revealed that median PFS was 3.7 months (95% CI 3.3–4.1) with an ORR of 16.7% and CBR of 36.1%, whereas median OS was 15.0 months (95% CI 12.1–17.9). Most AEs were mild or moderate, with no grade 4/5 AEs. Grade 3 AEs were infrequent, with 7.35% neutropenia, 4.9% fatigue, and 2.4% PPE.This study first evaluated the efficacy and safety of Duomeisu2 every 4 weeks as a second- or third-line treatment by Al-Batran et al. [2 every 4 weeks showed that the PFS and OS were 3.6 months (95% CI 2.7–6.4) and 12.3 months (95% CI 7.7–16.3), respectively [2 every 4 weeks achieved median PFS of 2.9 months and median OS of 11.0 months [2 every 4 weeks in our study was not associated with worse survival outcomes. Conversely, numerically longer OS was observed in this study. This discrepancy might be explained by patients with advanced breast cancer in earlier studies including the HER2-positive subtype and the likely superiority of Duomeisu® to DOXIL®  or Caelyx®  though Duomeisu® was demonstrated to be bioequivalent to Caelyx® [An unmet need exists for patients with heavily pretreated HER2-negative MBC. After anthracycline and taxane failure, there is no optimal chemotherapy regimen established for such patients . Single-n et al.  recordedectively . Similar0 months . In our 0 months . Further Caelyx® . Nowaday Caelyx® , 28, 45.More recently, eribulin, a novel non-taxane microtubule inhibitor, was approved as monotherapy for the treatment of MBC previously treated with anthracyclines and taxanes based on results of EMBRACE  and StudCurrently, novel antibody–drug conjugates (ADCs) are rapidly evolving therapies for heavily pretreated HER2-negative MBC, including trastuzumab deruxtecan (T-DXd)  and saci2 every week [The overall safety profile was good in this study. No grade 4/5 AEs were observed. The most frequently reported AEs were leukopenia, fatigue, and neutropenia. The most common grade 3 AEs were neutropenia (7.3%) and fatigue (4.9%). All grades of PPE were 24.4% with 2.4% grade 3, and all grades of stomatitis 9.5% with no grade 3. Only three patients (7.3%) experienced grade 1 alopecia. Regarding cardiotoxicity, most patients (97.6%) had no significant decrease in LVEF during treatment, and only one patient experienced a decrease in LVEF from 70% at baseline to 62% after five cycles of PLD. These findings were consistent with those in the previous studies of PLD 10 mg/mery week , 43.This study had several limitations. First, this study had a single-center, open-label design with no control arm. Second, the implementation of the study took longer than originally planned. The slow enrollment of patients was aggravated by the COVID-19 pandemic. Third, this was an exploratory trial with small sample sizes.®) monotherapy at 40 mg/m2 every 4 weeks was effective and safe in patients with HER2-negative MBC heavily pretreated with conventional anthracycline and taxanes, indicating that this treatment is a viable treatment option for this population.In summary, our study demonstrated that PLD Below is the link to the electronic supplementary material.


These markers were measured in facial mid-sagittal section through ViewPoint 6 software. The diagnostic value of these markers for abnormal fetuses was assessed by receiver operating characteristic (ROC) curves.p = 0.013; p = 0.003). The mean MNM angle of fetuses with trisomy 18 and CLP were 6.98° (SD 2.61) and 9.41° (SD 2.57), respectively, which were significantly greater than the normal fetuses . The mean FMA values of trisomy 18 fetuses were 63.95° (SD 4.77), which was significantly smaller than the normal fetuses (p < 0.001). The mean FS distance of CLP fetuses was -0.22 mm (SD 1.38), which was significantly smaller than the normal fetuses (p < 0.001). The mean PL distance of trisomy 21, trisomy 18 and CLP fetuses were 2.89 mm (SD 0.41), 2.91 mm (SD 0.56) and 2.71 mm (SD 0.37), respectively. The difference with the normal fetuses had no statistical significance .According to the first-trimester scanning (FTS) and follow-up, 31 fetuses were enrolled in the abnormal group, including 14 cases of trisomy 21, 7 cases of trisomy 18, 10 cases with cleft lip and palate (CLP), and 1000 normal fetuses were selected. Among the normal fetuses, the IFA, FS distance and PL distance had negative correlations with CRL. The MNM angle and FMA had positive correlations with CRL. The mean IFA values for fetuses with trisomy 21 and trisomy 18 were 74.11° (standard deviation (SD) 7.48) and 69.88° (SD 7.08), respectively, which were significantly smaller than the normal fetuses (Fetal facial profile markers had excellent correlations with CRL during the first trimester. IFA had certain clinical significance in detecting trisomy 21. FMA, IFA and MNM angle were reliable indicators for screening trisomy 18. The abnormal MNM angle and FS distance could be used as sensitive indicators for CLP. However, PL distance was not the best markers for trisomy 21, trisomy 18 and CLP.The online version contains supplementary material available at 10.1186/s12884-022-05028-9. Facial malformations are the most common fetal surface malformations, including cleft lip and palate (CLP), micrognathia and so on. It not only brings great adverse effect to the children and their family, but also aggravates the social burden , 2. WithThe fetal facial profile contains abundant information. The fetal facial mid-sagittal section is an important section to evaluate the facial profile in first-trimester scanning (FTS). In this section, not only the NT thickness can be measured, but also such facial structures as the forehead, nasal bone, palate, upper lip, lower lip and mandible can be observed from top to bottom. In recent years, some scholars have converted these complex facial structures into objective ultrasonographic measured markers, such as inferior facial angle (IFA), maxilla-nasion-mandible (MNM) angle, facial maxillary angle (FMA), frontal space (FS) distance and profile line (PL) distance, et al. Some studies have reported that the abnormal values of facial markers are closely related to fetal chromosomal abnormalities  and facial deformities  –10. HoweThe normal fetuses had normal ultrasound and normal follow-up outcomes. Fetuses diagnosed as chromosomal abnormalities by invasive genetic diagnosis or those with facial structural abnormalities suspected by ultrasound and confirmed after induced abortion or birth were categorized as the abnormal group. Ultrasound images of FTS, performed in the Affiliated Suzhou Hospital of Nanjing Medical University during August 2017 to December 2020, were selected. We have included all the cases in the selected time frame without a priori calculation of the sample size.Inclusion criteria: (1) both parents of the fetus were Chinese; (2) singleton pregnancy; (3) Two-dimensional (2D) stored images were the fetal facial mid-sagittal section, which strictly followed the guidelines of Fetal Medicine Foundation (FMF). The forehead, nasal bone, palate, mandible, upper lip and lower lip of the fetuses were clearly distinguishable; (4) There was no umbilical cord or limb in front of the fetal face.Exclusion criteria: (1) pregnancies with significant maternal complications; (2) fetuses were lost to follow-up.This study was approved by the Ethics Committee of the Affiliated Suzhou Hospital of Nanjing Medical University (K2016038). Pregnant women voluntarily participated in the follow-up and signed informed consent forms.A GE Voluson E8 Expert and a Philips Affiniti70 four-dimensional (4D) color ultrasound diagnostic instruments were used in this study. The ultrasound examinations were performed transabdominally, using convex probe C5-1 (frequency 1 ~ 5 MHz) and C9-2 (frequency 2 ~ 9 MHz). The image was stored in DICOM  format and the measurement software was ViewPoint 6 ultrasound workstation.1) IFA , 5, 11:  IFA , 5,2) MNM angle , 11: the MNM angl3) FMA , 11: the FMA , 114) FS distance , 9: the  FS dista5) PL distance , 11: the PL distaThese facial markers were measured through ViewPoint 6 software by two experienced sonographers, who had obtained the NT screening qualification certificate issued by the FMF.Neither of the two surveyors knew the fetal karyotype, and the mean value of each marker was taken after three measurements. It took 5 to 6 min for three measurements of all 5 markers.p < 0.05, the differences were considered as statistically significant. The diagnostic value of these markers for abnormal fetuses was assessed by receiver operating characteristic (ROC) curves.All statistical analysis was performed by SPSS21.0  and Graphpad Prism8.0. Continuous variables with a Gaussian distribution were expressed as mean . Continuous variables without a Gaussian distribution were expressed by Median . Spearman’s rank correlation test was used to analyze the correlations between fetal facial markers and CRL. The Student’s T test or Mann–whitney U test were used to analyze the differences among the groups. When One thousand normal fetuses whose image was clear and typical for measurement were selected. The median maternal age was 28 years (IQR 26–31). The median CRL was 66 mm (IQR 62–71), and the median NT thickness was 1.6 mm (IQR 1.3–1.9).s = -0.614, p < 0.001). MNM angle was distributed from 1.38° to 10.7°, and the median was 4.07° (IQR 3.21–5.29). MNM angle depended significantly on CRL during the first trimester . FMA was distributed from 56.29° to 91.87°, and the mean was 73.02° (SD 5.01). FMA had significant positive correlation to CRL . FS distance was distributed from 0 to 4.3 mm, and the median was 1.97 mm (IQR 1.53–2.37). FS distance decreased with CRL . PL distance was distributed from 1.1 to 4.67 mm, and the mean was 2.81 mm (SD 0.53). PL distance decreased with CRL .IFA was distributed from 55.9° to 107.89°, and the median was 78.25° (IQR 74.75–82.98). Spearman’s rank correlation analysis showed that IFA was negatively correlated with CRL during the first trimester . The mean CRL was 65.65 mm (SD 6.23), and the mean NT thickness was 3.13 mm (SD 1.84). Ultrasonographic measurements of each facial marker of trisomy 21, trisomy 18 and CLP fetuses were shown in Figs. p = 0.013; Z = -3.018, p = 0.003). The mean IFA values of CLP fetuses were 82.03° (SD 11.52). The difference with the normal fetuses had no statistical significance .The mean IFA values of trisomy 21 and trisomy 18 fetuses were 74.11° (SD 7.48) and 69.88° (SD 7.08), respectively, which were significantly smaller than the normal fetuses . The mean MNM angle of trisomy 21 were 4.59° (SD 1.71). The difference with the normal fetuses had no statistical significance .The mean MNM angle of trisomy 18 and CLP fetuses were 6.98° (SD 2.61) and 9.41° (SD 2.57), respectively, which was significantly greater than the normal fetuses . The mean FMA values of trisomy 21 and CLP were 74.33° (SD 6.25) and 74.42° (SD 7.62), respectively, which had no statistically significant difference .The mean FMA values of trisomy 18 fetuses were 63.95° (SD 4.77), which was significantly smaller than the normal fetuses . The mean FS distance of trisomy 21 and trisomy 18 fetuses were 2.26 mm (SD 0.85) and 1.70 mm (SD 1.81), respectively, which had no statistically significant difference .The mean FS distance of CLP fetuses was -0.22 mm (SD 1.38), which was significantly smaller than the normal fetuses .The mean PL distance of trisomy 21, trisomy 18 and CLP fetuses were 2.89 mm (SD 0.41), 2.91 mm (SD 0.56) and 2.71 mm (SD 0.37), respectively. The difference with the normal fetuses had no statistical significance  ultrasound could obtain a real standard mid-sagittal section through multi-plane mode . HoweverThere was a good correlation between fetal facial profile markers and CRL during the first trimester in a Chinese population. IFA had a certain value in the diagnosis for trisomy 21 and trisomy 18, with the cut-off of 76.34° and 75.37°, respectively. The advantage of MNM angle in detecting CLP was superior to trisomy 18. When the MNM angle was above 6.83° or FS distance below 0.35 mm, the possibility of CLP should be considered. Among FMA, IFA and MNM angle, FMA had the highest accuracy (91.4%) in detecting trisomy 18, with a cut-off of 67.96°. These cut-off need to be confirmed by a large number of prospective studies. While PL distance was not the best ultrasound markers for trisomy 21, trisomy 18 and CLP during first trimester.Additional file 1.


Similarly, SCP vessel density significantly increased from 15.14 ± 3.41 mm−1 to 18.14 ± 2.57 mm−1 after surgery (p < 0.001). The mean preoperative FAZ area significantly increased from 0.27 ± 0.12 mm to 0.24 ± 0.11 mm seven days postoperatively (p = 0.008). When comparing softer and harder cataracts, no significant variations in SCP vessel density, as well as SCP perfusion density parameters and the FAZ area, perimeter, and circularity index, were noted before and after surgery. Conclusions: Macular SPC vessel density and macular SCP perfusion density increase after uncomplicated cataract surgery regardless of the cataract severity.Background: To investigate macular microvascular changes after uncomplicated phacoemulsification surgery according to the cataract severity grade. Methods: Retrospective, cross-sectional study involving 23 eyes of 23 patients who underwent elective cataract extraction. All patients underwent routine ophthalmologic examination, including optical coherence tomography angiography (OCTA) at baseline  and seven days postoperatively (T7). OCTA scans were obtained with the spectral domain system Cirrus 5000 , and 3 mm × 3 mm raster fovea-centered scans were obtained to evaluate the superficial capillary plexus (SCP) vessel density, perfusion density, and foveal avascular zone (FAZ) parameters. Results: SCP perfusion density significantly increased from 28.3 ± 5.73% to 33.74 ± 4.13% after the surgery ( Cataract remains the leading cause of blindness in middle-income and low-income countries, with an estimated 16 million people worldwide . Today, As demonstrated by Zhao et al., cataract surgery induces a significant increase in vessel density at the parafoveal and perifovea regions, along with a decrease in the foveal avascular zone (FAZ) .Various methods such as Doppler ultrasound and fundus fluorescein angiography (FFA) can observe retinal blood flow. However, these clinical applications have some limitations, as Doppler ultrasound can only see large vessels with low resolution, and FFA is invasive and difficult to quantify . OpticalOCTA has been used to assess the FAZ area and macular vascular flow density in healthy and pathologic eyes . StartinIn this retrospective cross-sectional study, we collected pre-and post-operative data from 23 people who underwent elective cataract extraction performed between 1 June 2020 and 1 November 2020 at our ophthalmology unit . Data from patients who underwent phacoemulsification surgery with intraocular lens (IOL) implantation were retrieved and collected.This study was approved by the Institutional Review Board and followed the tenets of the Declaration of Helsinki. The inclusion criteria for the study were the presence of a nuclear or cortical cataract with no concomitant intraocular disease and intraocular pressure (IOP) lower than 21 mmHg. Eyes with IOP higher than 21 mmHg, with a diagnosis of posterior subscapular or posterior polar cataract, history of ocular trauma or intraocular surgery, axial length > 26, or any abnormal intraocular findings, were excluded. Patients diagnosed with glaucoma or any retinopathies that might result in the abnormal microvascular network  or previously treated by laser or photodynamic therapy were also excluded. In addition, patients with either a previous or current history of systemic diseases such as diabetes and inflammatory or cardiovascular diseases that may interfere with retinal vascular abnormalities were excluded. Furthermore, poor OCT images due to severe cataracts, unstable fixation, or any signs of intraoperative or postoperative complications were also grounds for exclusion.All patients underwent routine examinations prior to cataract surgery, including the measurement of corrected distance visual acuity (CDVA) in the logarithm of the minimum angle of resolution (logMAR), the measurement of intraocular pressure (IOP) with a non-contact tonometer, the measurement of axial length (AL) with partial coherence interferometry , a slit lamp biomicroscopy, cataract grading with the Lens Opacities Classification System III  between the preoperative and postoperative SCP perfusion density, vessel density, and FAZ parameters in patients with different cataract severity grades was assessed by linear regression analysis. A p-value < 0.05 was considered statistically significant.Between-group differences for each parameter considered , effective phacoemulsification time (EPT), phacoemulsification energy, and postoperative follow-up examination interval) were assessed, as for qualitative variables, either by the Fisher Exact test or the Chi-square test, with Yates’ correction as appropriate. Quantitative variables were evaluated by Student t-test if normally distributed; otherwise, the Mann—Whitney U test was applied. Specifically, “Intra-group” comparative analyses, based on preoperative and postoperative quantitative variables, were conducted on both groups. Specifically, the pre-and post-intervention measures of quantitative variables were compared separately for each group using Student’s Twenty-three eyes of twenty-three patients with senile cataracts fulfilled the assessment visits at baseline and after surgery. All patients did not develop any surgical complications during and after surgery.p < 0.01).The mean correct distance visual acuity (CDVA) before surgery was 0.55 ± 0.23 LogMar (range 0.10–1.00), whereas the mean CDVA after surgery was 0.07 ± 0.08 (range 0.00–0.2). Therefore, the CDVA was significantly improved (The patients’ mean age was 75.86 ± 8.85 years (range 55–98 years), the mean effective phacoemulsification time was 91.56 + 25.56 s, and the mean phacoemulsification energy was 11.83 + 5.05 µJ. The mean interval of the postoperative follow-up examination was 7.70 ± 0.97 days.p < 0.05).All parameters of perfusion density and vessel density significantly increased after the cataract surgery when comparing baseline (T0) to 1-week follow-up (T7). . SCP vessel density in the inner ring was 16.04 ± 3.62 mm−1 at baseline, and 19.02 ± 2.64 mm−1 at T7, showing a statistically significant increase compared to baseline (p < 0.001). At last, SCP vessel density in the full ring increased from T0 to T7, with a significant variation when comparing baseline to 1-week follow-up .The preoperative SCP vessel density in the central ring was 8.20 ± 3.30 mmp = 0.003), 35.59 ± 4.17% for the inner ring (p = 0.001), and 33.74 ± 4.13% for the full ring (p < 0.001)  .2 to 0.24 ± 0.11 mm2 seven days after surgery (p = 0.008). The mean preoperative FAZ Perimeter decreased from 2.31 ± 0.54 mm to 2.17 ± 0.58 mm after the surgery in a non-significant way (p = 0.057), and the mean preoperative circularity index also did not show any statistical variation at T7   .Fourteen eyes belonged to group 1 (less severe cataracts), whereas nine eyes belonged to group 2 (severe cataracts). Group 1’s mean CDVA before surgery was 0.44 ± 0.10 LogMar (range 0.3–0.7), whereas the mean CDVA after surgery was 0.03 ± 0.06 (range 0.00–0.2).Group 2’s mean CDVA before surgery was 0.71 ± 0.29 LogMar (range 0.1–1), whereas the mean CDVA after surgery was 0.12 ± 0.08 (range 0.00–0.2).Group 1’s phacoemulsification cumulative dissipated energy (CDE)  of the phacoemulsification was 8.70 ± 2.13 µJ, whereas Group 2’s CDE was 16.71 ± 4.35 µJ. Group 1’s effective phacoemulsification time (in seconds) was 75.60 ± 7.8 s, whereas group 2’s effective phacoemulsification time was 116.38 ± 23.59 s.p < 0.05)  .p = 0.03, p = 0.007, and p = 0.005, respectively).Group 1’s mean preoperative SCP perfusion density in the central ring, inner ring, and full ring was 14.87 ± 6.33%, 31.20 ± 4.98%, and 29.35 ± 5.01%, respectively, which significantly increased to 18.22 ± 7.11%, 35.89 ± 4.38%, and 33.75 ± 4.61% after the surgery .Group 2’s mean preoperative SCP perfusion density in the central ring, inner ring, and full ring was 15.06 ± 5.96%, 28.06 ± 7.62%, and 26.66 ± 6.68%, respectively, which significantly increased to 22.72 ± 11.11%, 35.13 ± 4.04%, and 33.73 ± 3.52% after the surgery . In addition, there was no significant mean increase difference [Δ (T7-T1)] between the preoperative and postoperative SCP perfusion density in the central ring, inner ring, and full ring between groups . Linear regression was calculated to predict the mean increase difference [Δ (T7-T1)] between the preoperative and postoperative SCP perfusion density in the central ring, inner ring, and full ring in both groups from the cataract severity grade. A non-significant linear regression was found .There was no significant difference between the mean preoperative and postoperative SCP perfusion density in the central ring, inner ring, and full ring between groups . Group 1’s mean preoperative SCP vessel density in the central ring, inner ring, and full ring was 8.17 ± 3.34 mm−1, 15.04 ± 4.37 mm−1, and 14.26 ± 3.96 mm−1, respectively, which significantly increased to 12.74 ± 5.89 mm−1, 18.63 ± 2.36 mm−1, and 17.97 ± 2.93 mm−1 after the surgery .Group 2’s mean preoperative SCP vessel density in the central ring, inner ring, and full ring was 8.25 ± 3.25 mmp = 0.95, p = 0.22, p = 0.98, and p = 0.24, p = 0.58, and p = 0.60, respectively). In addition, there was no significant mean increase difference [Δ (T7-T1)] between the mean preoperative and postoperative SCP vessel density in the central ring, inner ring, and full ring between groups . Linear regression was calculated to predict the mean increase difference [Δ (T7-T1)] between the preoperative and postoperative SCP vessel density in the central ring, inner ring, and full ring in both groups from the cataract severity grade. A non-significant linear regression was found .There was no significant difference between the mean preoperative and postoperative SCP vessel density in the central ring, inner ring, and full ring between groups . The mean preoperative FAZ area in group 2 was 0.25 ± 0.14 mm2, which decreased to 0.22 ± 0.12 mm2 after the surgery in a non-significant way (p = 0.08). There was no significant difference between the mean preoperative and postoperative FAZ area between the groups . In addition, there was no significant mean increase difference [Δ (T7-T1)] between the mean preoperative and postoperative FAZ area between groups (p = 0.5).The FAZ decreased significantly seven days after surgery in both groups. The mean preoperative FAZ area in group 1 was 0.27 ± 0.12 mmp = 0.23). The mean preoperative FAZ perimeter in group 2 was 2.25 ± 0.63 mm, which decreased to 2.06 ± 0.61 mm after the surgery) in a non-significant way (p = 0.14). There was no significant difference between the mean preoperative and postoperative FAZ perimeter between groups . In addition, there was no significant mean increase difference [Δ (T7-T1)] between the mean preoperative and postoperative FAZ perimeter between groups (p = 0.65).The mean preoperative FAZ perimeter in group 1 was 2.35 ± 0.49 mm, which decreased to 2.23 ± 0.57 mm after the surgery in a non-significant way (p = 0.05). The mean preoperative circularity index in group 2 was 0.60 ± 0.03, which remained stable after the surgery (0.60 ± 0.09) in a non-significant way (p = 0.9). There was no significant difference between the mean preoperative and postoperative FAZ circularity index between groups . In addition, there was no significant mean increase difference [Δ (T7-T1)] between the mean preoperative and postoperative FAZ circularity index between groups (p = 0.66). The mean preoperative circularity index in group 1 was 0.59 ± 0.06, which increased to 0.62 ± 0.09 after the surgery in a non-significant way  ] between the preoperative and postoperative FAZ area, perimeter, and circularity index in both groups from the cataract severity grade. A non-significant linear regression was found  .Nowadays, OCTA is a reliable noninvasive tool that evaluates the retinal vascular pattern and circulation. OCTA is a reliable tool for detecting these parameters with a high degree of repeatability . Many stIn our study, we aimed to evaluate the retinal vasculature changes in terms of perfusion density as well as vessel density at the macular area after uncomplicated phacoemulsification in patients with neither ocular nor systemic diseases. Our findings are consistent with those reported in previous research held by Zhao et al. and Yu et al. ,14.Zhao et al. assessed that the increased vascular density was related to three factors: (1) increased pulsatile ocular blood flow after cataract surgery due to the postoperative decrease in IOP  ; (2) an To further investigate whether the lens density and thus the lower quality image may be related to the VD and PD’s increase after lens extraction, we split our sample into two subgroups according to lens opacity grade.p > 0.05). Therefore, we can assess that cataract severity does not significantly affect OCTA-calculated parameters. In addition, as the baseline (T0) VD and PD values were quite similar between the two groups (p > 0.05), the media opacity density (lens density) did not significantly alter the PD and VD.Analyzing the mean increase difference [Δ (T7-T1)] between the preoperative and postoperative SCP perfusion density and vessel density, we did not find any substantial variations between the two groups  (p > 0.05) To further establish this hypothesis, we run a linear regression to evaluate if the cataract severity could be related to the mean increase difference [Δ (T7-T1)] between the preoperative and postoperative SCP perfusion density, vessel density, and FAZ parameters in both groups. It showed us that the lens density does not statistically significantly predict the mean increase difference [Δ (T7-T1)] between the preoperative and postoperative SCP perfusion density, vessel density, and FAZ parameters in both groups. (Thus, in our small sample, the lens density impact on image quality in terms of artifacts and signal strength did not affect the mean increase of VD and PD in the higher density group. These findings show that the enhanced retinal vascular density and perfusion along with the FAZ area reduction are not strictly related to the image quality.However, this study has several limitations. First, the results may not be generalizable due to the limited sample size. Second, we did not deeply analyze the peripheral retinal vascular abnormalities. Third, the increased macular retinal vessel density and perfusion were assessed just one week after surgery when the postoperative inflammation is in its thriving phase. Finally, we included only patients with neither systemic nor ocular disease.Therefore, the assessment of confounding factors such as medications, medication adherence, early-stage diseases, cardiovascular diseases, or inflammatory disorders that may be related to vessel changes could not be assessed in the study’s population. Therefore, further investigations with a longer follow-up should be considered due to the known links between diabetes, cataract, and blood vessel changes. In addition, evaluating the underlying mechanisms will be of great importance and interest.In conclusion, after uncomplicated lens extraction, the macular perfusion density and the vessel density significantly increased regardless of the cataract severity grade. Studies with a bigger sample size are required to investigate the underlying mechanisms further.


Social interaction plays an essential role in species survival for socialized animals. Previous studies have shown that a lack of social interaction such as social isolation, especially in the early-life phase, increases the risk of developing mental diseases in adulthood. Chronic social stress alters blood-brain barrier (BBB) integrity and increases peripheral cytokines to infiltrate the brain, which is linked to the development of depressive-like behaviors in mice, suggesting that BBB function is crucial in environmental stimuli-driven mood disorders via increased neuroinflammation in the brain. However, the precise mechanisms of inflammation and BBB integrity underlying the behavioral profiles induced by social isolation remain poorly understood. Here we showed that chronic childhood social isolation from post-weaning for consecutive 8 weeks in female but not male C57BL/6J mice induces anxiety-like behaviors. The levels of peripheral inflammatory cytokines including interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the plasma of socially isolated female mice were increased. Importantly, we found decreased expression of the endothelial cell tight junction protein Claudin-5, increased BBB breakdown and microglial activation in the amygdala of isolated but not group-housed female mice. Moreover, the neuronal activity in the amygdala was increased as evidenced by c-fos positive cells, and the levels of IL-1β in the amygdala, a critical brain region for regulating social processing and interaction, were also higher in female mice exposed to social isolation. Finally, down-regulation of Claudin-5 induced anxiety-like behaviors in group-housed females and overexpression of Claudin-5 with adeno-associated virus in the amygdala to restore BBB integrity decreased subsequent anxiety-like behaviors. Together, these findings suggest that chronic childhood social isolation impaired BBB permeability and caused neuroinflammation in the amygdala by recruiting peripheral cytokines into the brain and activating microglia, consequently triggering the development of anxiety-like behaviors in female mice. Social behavior, like eating and sleeping, is an essential function for both socialized animals and human beings survival and development . Lack ofExposure to inflammation is associated with several neuropsychiatric disorders, including depression and anxiety . Among tThis study aims to clarify the mechanisms underlying sex-specific influence of behavioral outcomes induced by chronic social isolation. We exposed mice from post-weaning for 8 weeks of social isolation and measured anxiety-, depressive-like behaviors, social interaction and cognitive functions. In addition, peripheral inflammation, neuroinflammation and BBB function were measured. Finally, we regulated the tight junction protein Claudin-5 using virus-mediated manipulation to reveal the role of the BBB in behavioral deficits induced by social isolation.Both male and female C57BL/6J mice  were used in this study and maintained in standard temperature (22 ± 2°C), humidity (50% ± 10%), and 12 h circadian rhythm cycle (8:00–20:00 is the light cycle) with free food and water access. Mice were 3 weeks old at the start of the experiments (weighing 7–9 g). Behavioral tests were carried out during the dark cycle. All mouse experiments met the ethical requirements of experimental animals and complied with the regulations issued by the state and the Animal Use and Protection Committee of the Peking University Health Science Center .On post-natal day 21 (PND21), mice were housed in isolation (1 mouse per cage) or groups (4–6 mice per cage). Mice were placed in the same rooms, but baffles were arranged between adjacent cages for isolated mice. Isolated mice can hear and smell other mice but cannot see and interact physically with other mice. According to previous study, social isolation procedure was maintained for 8 weeks .The novelty suppressed feeding test (NSFT) was performed in an opaque acrylic plate box with an open top (42 cm × 42 cm × 42 cm). Mice were tested after 24 h of food deprivation. Four food pellets from home cage were placed in the central area of the test box. Mice were gently put into the test box from one corner. The latency to start eating food pellets was used to evaluate the anxiety-like behaviors. During the test, mouse was allowed to explore freely in the box for 10 min, and the longest latency was recorded as 600 s .The elevated plus maze (EPM) apparatus consists of two open arms (30 cm × 5 cm), two closed arms , and a central area (5 cm × 5 cm); the open arms were elevated 75 cm from the ground. During testing, mice were gently placed in the central area with their head facing the open arm, and the test lasted for 6 min. The EPM test is a widely accepted approach to reflect the behavioral changes following psychosocial stress. The time that mice stayed in open arms and the numbers entered open arms were recorded as indicators to evaluate anxiety-like behaviors .The open field test (OFT) was performed in an opaque acrylic plate box with an open top (42 cm × 42 cm × 42 cm). Mice were placed in the center of the apparatus in the beginning of the test, and were allowed to explore freely for 5 min. Total travel distance (cm) was recorded as the measure of spontaneous locomotion. The time of mice in the central area (24 cm × 24 cm) was recorded as a measure of anxiety-like behaviors. At the end of the test, mice were put back to their home cages. During the test interval of two mice, the test apparatus was wiped with 75% alcohol to remove the residual odor and excreta .The sucrose preference test (SPT) is a useful parameter of anhedonia simulating a core symptom of depression in humans based on a two-bottle free-choice paradigm in rodents. Briefly, the sucrose preference test consists of three stages: 1) Adaptation stage: 1% sucrose solution was prepared with daily drinking water of mice, 1% sucrose and drinking water were put into two bottles of the same size and appearance, respectively, and then two bottles were placed on the cages of mice. Mice can freely choose to drink each liquid from the two bottles for 48 h, and the positions of the two bottles were exchanged at the 24th hour. 2) Fasting stage: mice were deprived of water and food for 24 h before testing. 3) Sucrose preference test: 1% sucrose bottles and drinking water bottles were weighed and carefully placed on the cage simultaneously. Mice can freely choose to drink either liquid from the two bottles for 24 h. The positions of the two bottles were exchanged at the 12th h. After testing, two bottles were weighed again, and the intakes of sucrose and drinking water within 24 h were calculated. The sucrose preference value was used as the indicator of anhedonia . SucroseThe forced swim test (FST) was conducted in a plexiglass cylinder with a diameter of 18 cm and a height of 27 cm. The water depth was 15 cm, and the water temperature was kept at 25 ± 1°C. When testing, mouse cannot be supported by their limbs or tail. Baffles were placed between adjacent cylinders to prevent mice from learning from each other. The water in the cylinder was replaced every three mice to avoid the interference of excreta. The forced swim test was divided into two stages:1) Adaptation stage: 1 day before the test, mice were forced to swim for 15 min. 2) Forced swim test: 24 h later, mice were subjected to the swimming test for 6 min, and the floating immobility time of the last 4 min after recording was used as an indicator of depressive-like and despair behavior .For the tail suspension test (TST), mouse was hung up by the tape (3/4 of the distance from the bottom of tail) and were suspended about 35 cm away from the ground after hanging. A total of 6 min of testing was carried out and the time that mice spent immobility in the last 4 min of each 6-min test was recorded as an indicator of despair behavior .The social interaction (SI) test equipment consists with an open top (42 cm × 42 cm × 42 cm), and the social interaction area was a 12 cm × 24 cm rectangle on one side within a 7 cm × 11 cm × 30 cm transparent plastic box. This box separated the test mouse from the other mouse used for interaction. The top of this box was open, and there were some holes at its walls so that the test mouse could smell the odor of the mouse used for interaction . The tesThe novel object recognition test (NORT) was performed in a 40 cm × 27 cm × 18 cm box. The test was divided into two sessions, each session was 3 min, and the interval between two sessions was 15 min. In the first session, mice were placed in the test device from the corner. There were two same objects A in the center of the test box. In the second session, there were two objects: familiar object A and novel object B. A and B were different in character, material, and color. Mice were placed in the test device from its corner, and the time of mice exploring A and B was recorded, respectively. The cognitive function of mice was evaluated with the novel object recognition index, which is determined by a ratio of the time of exploring object B to (the time of exploring object A + the time of exploring object B) .Mice were sacrificed, and brain tissues were frozen in n-hexane cooled with dry ice and ethanol (−65°C) and were stored in a −80°C refrigerator until use. The positions of the mPFC, NAc, amygdala (Amy), hypothalamus (Hypo) and hippocampus (Hip) were determined according to the second edition of Mouse Brain in Stereotaxic Coordinates and brain tissues were removed with a freezing microtome . The collected tissues were homogenized in RIPA lysis buffer  supplemented with protease inhibitors cocktail  and phosphatase inhibitors cocktail  and were homogenized by bead rotor homogenizer (Omini). After centrifugation , the supernatant was collected, and protein concentration was quantified with BCA Protein Assay Kits . Protein samples were adjusted to an equal concentration and were mixed with 5 × loading buffer  and heated at 100°C for 5 min to be denaturalized. Protein samples were separated by SDS-PAGE electrophoresis  at 120 V (for about 1 h), subsequently were electrotransferred onto polyvinylidene difluoride membranes (Millipore) via wet transfer (250 mA at 4°C for 1 h). Protein membranes were blocked using 5% defatted milk powder in TBST buffer for 1 h at room temperature. The membranes were incubated overnight with primary antibodies against Claudin-5 , IL-1β , TNF-α , IL-6 , and β-actin  overnight at 4°C. The membranes were washed using TBST for 3 × 10 min and were incubated with appropriate secondary antibody conjugated to horseradish peroxidase  for 1 h. Chemiluminescence was performed with a hypersensitive ECL Chemiluminescence kit (Millipore) and visualized using a chemiluminescence Imager (GelDoc XR). β-actin was set as a loading control, and the signal of immunoblot was analyzed by Image J software.2 EP tube, blood sample was centrifuged . The supernatant was the blood plasma and was stored at −20°C until use. According to mouse IL-1β, IL-6, TNF-α, S100 Enzyme-linked immunosorbent assay (ELISA) Kit instructions , the kit was placed at room temperature for 30 min in advance, and 50 μl of different concentrations of standard samples, 40 μl of sample diluent, and 10 μl of sample + 40 μl of sample diluent were added to the standard wells, blank wells, and sample wells respectively. 100 μl of enzyme labeling reagent was added to each well (excluding blank wells), and the plate was sealed with a sealing film. After incubation at 37°C for 1 h, each well was washed with washing solution 5 × 30 s, and then patted dry. Each well was added 50 μl of chromogenic reagent and 50 μl of chromogenic reagent B. After incubation at 37°C for 15 min in the dark, 50 μl of stop solution was added to each well. Absorbance was detected in microplate photometer at 450 nm . Then the absorbance was calculated as concentration from the working curve. Furthermore, the value was normalized with the average of the control group for subsequent statistical analysis.Mice were anesthetized with 5% chloral hydrate, and blood was collected from the inferior vena cava. After anticoagulation by EDTA-KTotal RNA was extracted from mouse amygdalar tissues with Trizol reagent (Invitrogen) and was quantified by AceQ Universal SYBR qPCR Master Mix Kit  under the following conditions: . Primers sequences (Sangon Biotech) for amplification were follows CLDN5 , GAPDH .We anesthetized mice with 5% chloral hydrate, perfused with 0.1 M PBS and 4% paraformaldehyde (PFA) successively. Subsequently, mice brains were removed and postfixed in 4% PFA for 24 h, dehydrated in 30% sucrose three times, and frozen by n-hexane on dry ice. The brains were then sliced into 20 μm thick coronal sections on the freezing microtome . All of the sections were soaked in blocking solution  in 0.1 M PBS) for 1 h at 37°C. Sections were incubated with primary antibodies in a blocking solution for 36 h at 4°C . After washing 3 × 10 min in 0.1 M PBS, sections were incubated with goat anti-rabbit alexa Fluor 488 secondary antibodies  for 3 h at room temperature. The sections were rewashed 3 × 10 min with 0.1 M PBS. Images of amygdala were acquired with fluorescence microscope (Olympus VS120) and analyzed by Image J software referring to previous report .2 anticoagulant tube (IDXX Vet Collect™). Within 6 h after collecting, the blood cell analysis was performed with a five-classification automatic blood cell analyzer (IDEXX procyte DX™). Peripheral inflammation markers including leukocytes counts, neutrophils counts, neutrophils percentage, neutrophil/lymphocyte ratio (NLR), monocytes counts, monocytes percentage, lymphocytes counts and lymphocytes percentage were recorded.Mice were anesthetized with 5% chloral hydrate. The blood was collected from the inferior vena cava and were quickly placed into the EDTA-KMice were anesthetized with isoflurane, and the head of mice were fixed in stereotaxic instrument (RWD Life Science). AAV particles were injected bilaterally into the amygdala of the mouse brain  using hamilton syringes at a rate of 2 nl/min, needles were left in the place for additional 10 min to allow the virus to diffuse from the injection site .Cldn5-shRNA-mCherry and its negative control virus AAV9-CAG-shRNA-mCherry were used for down-regulation of Cldn5, and their titers were 5.03 × 1012 and 5.58 × 1012 vg/ml, respectively. The virus construction was referred to previous report . Short-hairpin RNA (shRNA) AAV: AAV9-CAG-s report . The ove3 blocks, and postfixed in 3% glutaraldehyde overnight, dehydrated in 30% sucrose for three times. Next, the tissue blocks were fixed in 1% osmium tetroxide for 2 h at 4°C and were dehydrated using graded acetone and then immersed in acetone/epoxy resin (1:1) for 2 h, acetone/epoxy resin (1:2) for 4 h. The blocks were embedded by epoxy resin at 37°C for 24 h, 45°C for 24 h, 60°C for 24 h. The embedded blocks were sliced into ultrathin sections (50–70 nm) using an ultramicrotome . Finally, the micrographs were captured under the transmission electron microscope .Mice were anesthetized with 5% chloral hydrate and perfused with 0.1 M PB and 3% glutaraldehyde. The amygdala tissues were dissected into 1 mmp < 0.05 was used as the standard to measure the significant difference, and the results were expressed as mean ± SEM.All statistical analyses were performed with Graphpad Prism 8.0 software. D’Agostino & Pearson normality was used to test the normal distribution, and then Brown-Forsythe was used to test the homogeneity of variance. If the data conforms to the normal distribution and homogeneity of variance, the two-tailed unpaired T-tests were used to analyze the univariate data in experiments with two groups. If the variance of univariate data was unequal, the Mann-Whitney test was used to analyze. Two-way ANOVA was used for bivariate data, and then a post hoc comparison was carried out. Bonferroni’s test was used to test the difference between groups. p = 0.0005, Group median: 99.00; Isolation median: 417.5, Mann-Whitney test). The total traveled distance in OFT (an index of locomotor) was not changed (t(19)= 0.952, p = 0.35) but time in the center area was decreased (t(19)= 2.822, p = 0.01). In the EPM test, time in open arms was decreased (t(22)= 3.147, p = 0.005) with no changes of open arms entries (t(22)= 2.039, p = 0.05). These data revealed that social isolation induced anxiety-like behaviors in female mice. Additionally, social isolation decreased the SI index of female mice in the social interaction test (t(18)= 3.386, p = 0.003), indicating that isolation induced social anxiety in female mice. Moreover, sucrose preference (t(22)= 0.655, p = 0.519), immobility in FST (t(22)= 0.677, p = 0.51), and immobility in TST (t(22)= 1.107, p = 0.280) were not changed, suggesting that social isolation did not affect depressive-like behaviors in female mice. Novel object recognition index in isolated female mice was not changed compared with group rearing mice (t(10)= 0.263, p = 0.79), which showed that social isolation did not alter the cognitive performance of female mice. In order to determine the sex differences in mental disorders induced by social isolation, we used a balanced design for male mice. Behavioral tests of male mice showed that the latency to feeding (t(10)= 0.951, p = 0.36), the total traveled distance in OFT (t(14)= 0.530, p = 0.60), the time spent in the center area (t(14)= 1.171, p = 0.16), time in open arms (t(10)= 1.494, p = 0.16) and open arms entries (t(10)= 0.639, p = 0.54) in EPM were not affected by social isolation, indicating that chronic social isolation did not induce anxiety-like behaviors in male mice. In addition, social isolation did not change the SI index , sucrose preference (t(10)= 1.679, p = 0.12), immobility in FST (t(10)= 1.832, p = 0.09), and immobility in TST (t(10)= 0.179, p = 0.86), showing that social isolation had no effects on social anxiety and depressive-like behaviors in male mice. Notably, isolated male mice showed an increased recognition index measured in NORT = 1.312, p = 0.21), neutrophils/lymphocytes ratio (t(17)= 0.985, p = 0.34), number of monocytes (t(18)= 0.908, p = 0.38), percentage of monocytes (t(18)= 0.153, p = 0.88), number of lymphocytes (t(18)= 1.380, p = 0.18), percentage of lymphocytes (t(18)= 0.871, p = 0.39), number of neutrophils (t(18)= 0.238, p = 0.98), and the percentage of neutrophils (t(18)= 0.845, p = 0.41). It has been verified by t-test that isolation did not affect the WBCs of female mice. We next examined the pro-inflammatory cytokines and found that the levels of plasma IL-1β (t(9)= 2.944, p = 0.02), IL-6 (t(9)= 5.654, p = 0.0003) and TNF-α (t(9)= 5.010, p = 0.0007) were significantly higher in female mice undergoing isolation than group rearing mice. These data indicate that activated peripheral inflammation is possibly linked to female sensitivity to social isolation.Peripheral inflammation levels are elevated after social isolation, mediating anxiety- and depressive-like behaviors . To invet(10)= 2.184, p = 0.05) and neutrophils/lymphocytes ratio (t(10)= 0.729, p = 0.48) were not significantly changed in male mice. While the number of monocytes (t(10)= 2.449, p = 0.03) was decreased significantly. Moreover, the percentage of monocytes (t(10)= 1.764, p = 0.11), number of lymphocytes (t(10)= 2.066, p = 0.07), percentage of lymphocytes (t(10)= 0.846, p = 0.42), number of neutrophils (t(10)= 1.362, p = 0.20) and the percentage of neutrophils (t(10)= 0.607, p = 0.56) were not significantly changed. The levels of IL-1β (t(10)= 0.356, p = 0.73), and IL-6 (t(10)= 0.739, p = 0.48) were not changed in isolated male mice, while the level of TNF-α (t(10)= 4.264, p = 0.002) was higher in isolation than group rearing mice.We also performed WBCs analysis and pro-inflammatory cytokines assay in male mice to uncover the potential sex difference of social isolation on peripheral inflammation. We found that after social isolation, the number of WBCs (t(10)= 0.731, p = 0.48; t(10)= 2.134, p = 0.06; t(10)= 0.366, p = 0.72, respectively) IL-1β expression levels were unchanged in social isolation. However, in the NAc and Amy, IL-1β expression levels were increased (t(18) = 3.058, p = 0.007; t(10)= 2.862, p = 0.02, respectively). The levels of IL-1β were increased by 91.4% in Amy after isolation and increased by 24.9% in NAc. The expressions of IL-6 in the mPFC, NAc, Amy, Hypo and Hip were not significantly changed after isolation (t(10)= 1.489, p = 0.17; t(10)= 1.676, p = 0.12; t(10)= 0.148, p = 0.89; t(10)= 1.486, p = 0.17; t(10)= 0.203, p = 0.84, respectively). TNF-α expressions in these regions were not significantly changed after social isolation (t(10)= 0.737, p = 0.48; p = 0.31, Group median: 1.000; Isolation median: 2.042, Mann-Whitney test; t(10)= 0.781, p = 0.45; t(10)= 0.135, p = 0.89; t(10)= 0.45, p = 0.66, respectively).The above results showed that the development of anxiety-like behaviors was influenced by the activation of peripheral inflammation during chronic social isolation in female mice. To further examine how neuroinflammation affects the susceptible response to social isolation in female mice, we detected the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α levels in mPFC, NAc, Amy, Hypo and Hip . In femat(8)= 0.291, p = 0.78; t(8)= 0.054, p = 0.96; t(8)= 0.509, p = 0.62; t(8)= 0.408, p = 0.69; t(8)= 0.376, p = 0.72, respectively) were unchanged in isolated mice. The IL-6 expressions in the NAc, Amy, Hypo and Hip were also not significantly changed after isolation (t(8)= 0.122, p = 0.91; t(8)= 0.420, p = 0.69; t(8)= 1.120, p = 0.29; t(8)= 0.119, p = 0.91, respectively), whereas IL-6 expression in the mPFC was decreased after isolation (t(8)= 2.525, p = 0.04). TNF-α expressions in the mPFC, NAc, Amy, Hypo and Hip were not significantly changed after isolation (t(8)= 0.044, p = 0.97; t(8)= 0.6217, p = 0.55; t(8)= 0.149, p = 0.89; t(8)= 0.119, p = 0.91; t(8)= 0.048, p = 0.96, respectively). These data suggested that socially isolated female mice exhibited increased levels of neuroinflammation in the amygdala compared with the group rearing mice, as manifested by significantly elevated levels of the pro-inflammatory cytokine IL-1β. Therefore, we further detected microglia in the amygdala by immunofluorescence staining (t(12)= 3.112, p = 0.009), although the difference of Iba-1-positive cells in the central amygdala (CeA) was not statistically significant (t(12)= 1.890, p = 0.08), indicating that the microglia in the BLA was activated. Taken together, social isolation induces BLA-specific neuroinflammation as manifested by markedly elevated levels of IL-1β and increased numbers of microglia in female mice.In males, IL-1β expression levels in the mPFC, NAc, Amy, Hypo and Hip (staining . The rest(10)= 1.156, p = 0.27; t(10)= 0.256, p = 0.80; t(10)= 1.623, p = 0.14; t(9)= 0.172, p = 0.87) were not significantly changed. In contrast, the Claudin-5 in Amy was significantly decreased (t(9)= 3.384, p = 0.008). In male mice, social isolation did not affect Claudin-5 expression in the mPFC, NAc, Amy, Hypo and Hip (t(8)= 1.498, p = 0.09, t(8)= 1.498, p = 0.17; t(8)= 0.048, p = 0.96; t(8)= 1.799, p = 0.11; t(8)= 0.319, p = 0.76). The mRNA expression level of Cldn5 in Amy was detected by qPCR, and the data showed that there was no significant change in the mRNA levels of Cldn5 mRNA in female isolated mice compared with the group rearing group (t(10)= 0.448, p = 0.66). We also used ELISA to detect the BBB damage marker S100 in the plasma of female mice, and the results showed that there was no significant difference between the isolation and the group rearing mice (t(18)= 0.28, p = 0.78). The results from transmission electron microscope for assessment of the ultrastructure of tight junctions in amygdala showed that after social isolation, the proportion of discontinuous tight junction (DTJ) was increased significantly (t(6) = 3.249, p = 0.02), while the level of intact tight junction (ITJ) decreased significantly (t(6) = 2.532, p = 0.05). In group housed female mice, intact tight junctions accounted for 88.1% of all observed tight junctions, and discontinuous tight junctions accounted for 11.9%. But in isolated female mice, intact tight junctions accounted for 55% of all observed tight junctions, and discontinuous tight junctions accounted for 45% = 4.361, p = 0.001), confirming increased neuronal activity in this brain region. However, there was no significant difference in c-fos expression in the CeA (t(14)= 1.289, p = 0.22).Neuronal activity is regulated by the inflammatory cytokine IL-1β and microglia, which in turn regulates microglia activation, consequently affecting BBB permeability . TherefoCldn5-shRNA-mCherry or negative control (Control) virus AAV9-CAG-shRNA-mCherry in the amygdala (t(7)= 2.649, p = 0.03). In the experiment on down-regulation of Claudin-5, female mice were group rearing from PND 21, and stereotaxic surgery was performed at 4 weeks of age. The mice were divided into two groups, the Control group, and the KD group, and were injected with the negative control virus AAV9-CAG-shRNA-mCherry or adeno-associated shRNA interference virus AAV-CAG-Cldn5-shRNA-mCherry into the amygdala, respectively. On PND 79, behavioral tests were performed (t(12)= 2.72, p = 0.02). In the EPM test, down-regulation of Claudin-5 did not affect the time in the open arms (t(13)= 0.49, p = 0.63) and entries into the open arm (t(13)= 0.459, p = 0.65). In OFT, down-regulation of Claudin-5 did not alter the locomotor in female mice (t(13)= 0.994, p = 0.34) but caused a decrease of time in the center area . The above results indicated that down-regulation of Claudin-5 expression in the amygdala induced anxiety-like behaviors in female mice. In the social interaction test, down-regulation of Claudin-5 caused a significant decrease in the SI index of female mice (t(18)= 2.539, p = 0.02), indicating that female mice exhibited social anxiety behavior.Given the findings that decreased expression of Claudin-5 in the amygdala is linked to isolation-induced anxiety-like behaviors in females, we further investigated whether down-regulation of Claudin-5 in the amygdala could induce anxiety-like behaviors in group-housed female mice. Firstly, the protein knockdown effect of the adeno-associated virus (AAV) short hairpin RNA (shRNA) was verified. Stereotactic injection of knockdown (KD) virus AAV-CAG-amygdala . After 4erformed . The resCldn5-mCherry or the negative control AAV-CAG-mCherry (Control) into BLA   = 6.877, p = 0.01) and Claudin-5  = 4.615, p = 0.04), but no significant effect of isolation × Claudin-5 interaction  = 4.024, p = 0.08) for the latency to feeding in NSFT. Comparing with Group + Control, the latency to feeding of the Isolation + Control group was significantly increased (p = 0.007) and relative to Isolation + Control group, Isolation + OE group had significantly decreased latency to feeding in novel environment (p = 0.02)  = 5.259, p = 0.03) but no effects of isolation  = 2.360, p = 0.13) and isolation × Claudin-5 interaction  = 4.094, p = 0.05) for the time in the open arms. Compared with Group + Control, the time in open arms of the Isolation + Control group was significantly decreased (p = 0.04), and the time in open arms was significantly increased in the Isolation + OE group compared with Isolation + Control (p = 0.01)  = 0.379, p = 0.06), Claudin-5  = 5.259, p = 0.03), and isolation × Claudin-5 interaction  = 0.717, p = 0.43) for the open arms entries  = 24.10, p < 0.0001), no significant effects of Claudin-5  = 0.000, p = 0.98) and isolation × Claudin-5 interaction  = 3.249, p = 0.08) for the locomotor. The locomotor of the Isolation + OE group was significantly increased compared to the Group + OE group (p < 0.0001)  = 5.487, p = 0.02), no significant effects of Claudin-5  = 0.475, p = 0.49) and isolation × Claudin-5 interaction  = 1.299, p = 0.49) for the time in the center area in the open field test. Compared with the Group + Control group, time in the central area of the open field in the Isolation + Control group was significantly decreased (p = 0.04), but compared with the Group + OE group, there was no significant difference in the time spent in the central area of the open field in the Isolation + OE group (p = 0.80), indicating that the overexpression of Claudin-5 in the amygdala increased time in the central area  = 5.863, p = 0.02) but no significant effects of isolation  = 3.032, p = 0.09) and isolation × Claudin-5 interaction  = 1.405, p = 0.24) for the SI index. Compared with Group + Control, the SI index of Group + OE group increased significantly (p = 0.03)  virus AAV-CAG-into BLA . After 4ey test) . In the peration . Results = 0.02) . EPM tes = 0.01) . There w entries . These d 0.0001) . There wral area . In the  = 0.03) , suggestIn this study, we have demonstrated that after 8 weeks of post-weaning social isolation, female mice show anxiety-like behaviors, while male mice do not demonstrate behavioral alterations. We have also found that the vulnerability to social isolation in female mice is related to the increased peripheral cytokines, neuroinflammation and impaired BBB integrity in the amygdala. In female mice, viral-mediated downregulation of Claudin-5 is sufficient to induce anxiety-like behaviors, while overexpression of Claudin-5 prevents these behavioral deficits induced by social isolation .Social isolation, as psychosocial stress, has been found to induce anxiety-, depressive-like behaviors, and changes in social interaction and cognitive performance . Among sPrevious studies revealed that both female and male mice exhibit depressive-like behaviors after experiencing early-life social isolation, while male mice also exhibit anxiety-like behaviors . In addiRegarding the mechanism of sex differences in social isolation, previous studies have shown that social isolation induced different neural circuit activity changes in both sexes, different effects on neuroplasticity, the hypothalamo-pituitary-adrenal axis, and inflammatory changes in females . ConsistComparing with females, male mice exposed to social isolation had a lower inflammatory response, manifested as only elevated TNF-α but not IL-1β and IL-6. In addition, the percentage of the increase was smaller than that of female mice. After social isolation, male mice showed decreased monocytes, suggesting that males may have lower levels of inflammation. In different studies, the results of monocyte changes in mental disorders were not consistent. Most of the studies showed increased levels of monocytes in patients with anxiety and depression, but it has also been confirmed that monocytes in animal models of depression were decreased . In thisThe findings of current study that social isolation caused a decrease in the expression of Claudin-5 protein in the amygdala of female mice, and the tight junction structure in isolated female mice was also more incomplete than that in the group rearing mice. This was consistent with changes in higher neuroinflammation levels in the amygdala of female mice. Previous evidence have shown that peripheral pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α can actively transport across BBB, inducing microglia, astrocytes and endothelial cells to release local secondary cytokines and triggered neuroinflammation . A recenIt is worth noting that the changing trend of Claudin-5 mRNA expression level and the protein expression level was not consistent. The mRNA level remained unchanged but the protein was decreased. It has also been reported that the changing trends of Claudin-5 mRNA and protein levels were inconsistent or even opposite. For example, the mRNA level of Claudin-5 was increased in the prefrontal cortex of schizophrenia patients, while the protein expression was decreased . It can BBB damage and microglia activity have been shown to correlate with neuronal activation in the brain. We therefore examined neuronal activity in the amygdala of female mice after social isolation. The results showed that the number of c-fos positive cells in the BLA of female mice was significantly increased, indicating that the neurons were activated. There are as high as 80% of neurons in the BLA are glutamatergic, and the high local concentrations of excitatory neurotransmitter glutamate are associated with impaired BBB . PreviouThe results illustrated that BBB damage in the amygdala is a crucial target in susceptibility to isolation-induced behavioral impairments in female mice. Based on previous studies, the presumed mechanism is that social isolation mediates peripheral inflammation transferring to the amygdala, activates local neuroinflammation and neurons, and induces anxiety-like behaviors. However, for this speculation, our study still has some inevitable limitations. First, the present results cannot provide strong evidence to explain the cause of the BBB damage in the amygdala of female mice, and the key mechanisms through which BBB damage induces behavioral deficits, particularly in females. In addition, we proposed that BBB damage may be caused by peripheral inflammation, neuroinflammation, or neuronal activation, and BBB damage may also play a role by promoting neuroinflammation and causing neuronal activation. Our results confirmed that Claudin-5 down-regulation in the amygdala can induce anxiety-like behaviors without social isolation. However, it also cannot explain the role of Claudin-5 in social isolation induced behavioral deficits. For the above speculation, further experiments are needed, such as whether the up- or down-regulation of the BBB affects local neuroinflammation and neuronal activation and their effects on behavioral responses to social isolation. The effects of knockdown of Claudin-5 on anxiety states under the condition of subthreshold social isolation stress should also be evaluated. Moreover, the types of activated neurons after social isolation were not determined, so we cannot provide directional guidance for further modulation of specific neuronal activity in the amygdala. Future studies aiming at characterizing distinct neurons responsible for the emotional behavior encoding will therefore be required to demonstrate how BBB alters neuronal activity by optogenetic or chemogenetic manipulations.In conclusion, our data have revealed that female, but not male, mice exposed to chronic childhood social isolation exhibit increased anxiety-like behaviors with activated peripheral cytokines, neuroinflammation and BBB leakiness in the amygdala. Considering the crucial regulation of Claudin-5 that links BBB integrity and emotional behaviors, the development of selective regulators of tight juction proteins targeting BBB morphology and function is needed in the future particulary for individuals suffered social isolation during their early life.


The separation distance was at a millimeter level, benefiting the subsequent processing of the specimen. Within only three separating cycles, the mass ratio between particles with diameters of 1.0 μm and 0.1 μm can be effectively increased to 13 times of its initial value. A theoretical analysis indicates that this spontaneous separation is attributed to the size-dependent adsorption between the colloidal particles and the aromatic substrate due to the strong hydrated cation-π interactions.Great efforts have been made to separate micro/nanoparticles in small-volume specimens, but it is a challenge to achieve the simple, maneuverable and low-cost separation of sub-microliter suspension with large separation distances. By simply adding trace amounts of cations (Mg Separating and isolating micro/nanoparticles in suspension, especially in small-volume specimens, is a critical step in various environmental and biomedical applications ,11,12,13The coffee ring effect (CRE), which applies to small droplets, is a good candidate for the separation of small-volume specimens. As a sessile droplet evaporates, an outward capillary flow is generated due to the much faster evaporation rate near the three-phase contact line (TCL) ,29,30,312, CaCl2 or NaCl) in a suspension droplet with a volume down to 0.2 μL. The separation distance observed is at a millimeter level, which even enabled us to manually sample the deposit pattern. A theoretical analysis indicates that this spontaneous separation is attributed to the size-dependent adsorption between the colloidal particles and the aromatic substrate due to the strong hydrated cation-π interactions. These findings have direct implications for the development of simple, maneuverable and low-cost technologies for low-volume sample preparation.In this work, we experimentally achieve the spontaneous separation of fluorescent polystyrene particles by simply adding trace amounts of salt  were mixed in equal volumes to obtain a suspension containing bi-dispersed F-PS particles, followed by thoroughly mixing with MgCl2 solutions of different concentrations. Individual small droplets of these suspensions (0.2~1.5 μL) were then placed on a graphene substrate  microspheres were used as reported previously . The susubstrate a. After  pattern f. In con pattern e.m = M1.0/M0.1, where M1.0 and M0.1 are the total masses of F-PS particles with diameters of 1.0 μm and 0.1 μm in the sampling region  and on other aromatic substrates such as the most common thermoplastic polymer resin of polyethylene terephthalate (PET). 2 on PET substrate. The red color traces the 1.0 μm diameter particles under excitation wavelength λex = 535 nm (or orange color under λex = 365 nm), while the green color traces the 0.2 μm diameter particles under λex = 488 nm and λex = 365 nm. For the MgCl2 concentration of 3.0 mM, a red pan-like pattern is observed under λex = 535 nm  are 2.0 mM and 5.0 mM for particles with diameters of 1.0 μm  and different concentrations of MgCl cations a,d, we of 1.0 μm b and 0.1f 1.0 μm e, respecw/v, red fluorescent with excitation wavelength λex = 535 nm and emission wavelength λex = 610 nm; (II) 0.1 μm in diameter, 1% w/v, green fluorescent with excitation wavelength λex = 488 nm and emission wavelength λex = 525 nm; (III) 0.2 μm in diameter, 1% w/v, green fluorescent with excitation wavelength λex = 488 nm and emission wavelength λex = 525 nm. All the polystyrene microsphere suspensions were centrifuged, washed and redispersed into Milli-Q water 4 times before use. Graphene monolayer on copper foil  were manufactured by Chongqing Graphene Technology Company through the CVD method (2), sodium chloride (NaCl) and calcium chloride (CaCl2) of AR grade were provided by Sinopharm Chemical Reagent Company. Polyethylene terephthalate (PET) films were produced by Shanghai Zicheng Packaging Materials Company. All the substrates and the salts were used as received.Milli-Q water  was used for all experiments. The mono-dispersed fluorescent polystyrene (F-PS) particle suspensions were purchased from ACME microspheres, Inc. The nominated parameters are listed as follow: (I) 1.0 μm in diameter, 1% D method . Magnesi2 was dissolved into 10 mL Milli-Q water to prepare the aqueous solution at a concentration of 0.25 M. Then, twice echelon dilution was used to get a serial of concentrations.0.24 g of MgClTo prepare the suspension containing mono-dispersed F-PS particles, 100 μL of F-PS particle suspension with single particle diameters (1.0 μm or 0.1 μm), 100 μL of Milli-Q water and 200 μL of salt solution with different concentrations were added into an Eppendorf tube, reciprocatingly sucked with a micropipette (Eppendorf), and ultra-sounded for 5 min to thoroughly mix them.To prepare the suspension containing bi-dispersed F-PS particles, 100 μL of 1.0 μm diameter red F-PS particle suspension, 100 μL of 0.1 μm (or 0.2 μm) diameter green F-PS particle suspension and 200 μL of salt solution with different concentrations were added into an Eppendorf tube, reciprocatingly sucked with a micropipette (Eppendorf), and ultra-sounded for 5 min to thoroughly mix them.2 were then immediately deposited onto the substrate in a petri dish, which was loosely covered to avoid air disturbance or contaminants from the environment. The environment temperature was 18 ± 3.5 °C and the relative humidity was 47 ± 3.0%. The deposition of suspension droplets containing CaCl2 and NaCl followed the same procedure.Small droplets (0.2~1.5 μL) of the as-prepared suspensions containing mono/bi-dispersed F-PS particles and different concentrations of MgCl2 on PET substrate were used for every single measurement. The rim part . About 35 independently dried deposits from suspensions containing bi-dispersed F-PS particles and 2.0 mM MgClMorphologies of the deposit patterns were captured by scanning electron microscopy . Fluorescence images of the deposit patterns were captured by optical fluorescence microscopy . All images were acquired in similar illumination conditions and acquisition settings. They are displayed without any post-processing.2 (0 mM and 6.0 mM) were placed on single-layer graphene and PET substrates. The contact angles were immediately examined by a surface tensiometer . The results are shown in Droplets (~0.2 μL) of suspensions containing bi-dispersed F-PS particles (1.0 μm and 0.1 μm in diameters) and different concentrations of MgCl2+, Ca2+ or Na+). This cation-controlled separation method enlarges the separation distance between particles of different sizes in the deposit pattern , which is convenient for subsequent processing such as manually sampling from the deposit pattern and performing multiple separating cycles. The mass ratios between F-PS particles with diameters of 1.0 μm and 0.1 μm were effectively increased from 1.0 ± 0.1 to 12.9 ± 2.2 by merely three separating cycles. A theoretical analysis indicates that this cation-controlled spontaneous separation is attributed to the size-dependent adsorption of particles onto the aromatic substrate due to the strong hydrated cation-π interactions. We note that innovative morphological analysis tools have recently been developed by Lotito et al. [In summary, we experimentally achieved the spontaneous separation of fluorescent polystyrene particles of different sizes in small-volume specimens by only adding trace amounts of cations (i.e., Mgo et al. ,49,50,51