ObjectiveTo investigate the effects of migration and expression from chemokine receptor 4 (chemokine receptor-4, CXCR4) of rat bone marrow mesenchymal stem cells (BMSCs) which were pretreated by atorvastatin (ATV) in vitro.MethodsIsolated, cultivated, identified the BMSCs, pretreated P4-P6 of BMSCs with different concentrations of ATV for 12 hours. The experimental group was divided into control group, 0.1 nM/L (group 0.1 nM), 1 nM/L (1 nM group), 10 nM/L (10 nM group), 100 nM/L (100 nM group), 1 000 nM/L (1 000 nM group). The mRNA and protein of CXCR4 were determined by real time-polymerase chain reaction and Western blot. Immunofluoreseence assay were used to detect the expression levels of CXCR4. The migration ability of BMSCs were measured by transwell chamber.ResultsImmunofluoreseence assay showed the protein level of CXCR4 of group 1 nM and 10 nM were significantly higher than the other group. RT-PCR and Western blot showed the protein and mRNA level of CXCR4 in 10 nM was higher than that in group 1 nM. The migration ability of group 10 nM was higher than 1 nM and control group.ConclusionsATV can be dose-dependent promote expression levels of CXCR4 of BMSCs cultivated in vitro.
ObjectiveTo explore the involvement of miR-126 and the role of mammalian target of rapamycin (mTOR)/hypoxia-induced factor 1 α (HIF-1 α) pathway in regulating human umbilical cord mesenchymal stem cells (hUCMSCs) exosomes (Exo) on vascular endothelial growth factor (VEGF)-A levels in high glucose-induced human retinal vascular endothelial cells (HRECs). MethodsThe hREC was cultured in EGM-2-MV endothelial cell culture medium with 30 mmol/L glucose and placed in hypoxic cell incubator by 1% oxygen concentration. The cell model of high glucose and low oxygen was established. After modeling, divided HRECs into Exo group, phosphate buffer saline (PBS) group, PBS+anti-miR126 group, Exo+anti-miR126 group, PBS+anti-mTOR group, and PBS+anti-HIF-1 α group. High-glucose and hypoxia-induced hREC in the PBS and Exo groups were respectively co-cultured with PBS and 100 μg/ml hUCMSC Exo. PBS+anti-mTOR group, PBS+anti-HIF-1 α group: 500 nmol/L mTOR inhibitor ADZ2014, 25 μmol/L HIF-1 α inhibitor YC-1 pretreatment for hREC 12 h, and then co-culture with PBS after High-glucose and hypoxia-induced. PBS+anti-miR126 group, Exo+anti-miR126 group: miR-126 LNA power inhibitor probe was transfected with high glucose, and co-cultured with PBS and hUCMSC Exo 6 h after transfection. Real-time polymerase chain reaction (qPCR) measured miRNA-126 expression levels in PBS, and Exo groups for 0, 8, 16 and 24 h. After 24 hof co-culture, the levels of mTOR and HIF-1 α in the cells of PBS and Exo groups were detected by immunofluorescence, Western blot and qPCR, respectively. Western blot, qPCR detection of VEGF-A expression levels in cells of the PBS+anti-mTOR and PBS+anti-HIF-1 α groups. The expression of VE GF-A, mTOR, and HIF-1 α mRNA was measured in cells of PBS+anti-miR126 group and Exo+anti-miR126 group by qPCR. Comparison between two groups was performed by t-test; one-way ANOVA was used for comparison between multiple groups. ResultsAt 0, 8, 16 and 24 h, the relative mRNA expression of miR-126 gradually increased in the Exo group (F=95.900, P＜0.05). Compared with the PBS group, The mTOR, HIF-1 α protein (t=3.466, 6.804), mRNA in HRECs in the Exo group, VEGF-A mRNA expression (t=8.642, 7.897, 6.099) were all downregulated, the difference was statistically significant (P＜0.05). The relative expression level of VEGF-Aprotein (t=3.337, 7.380) and mRNA (t=8.515, 10.400) was decreased in HRECs of the anti-mTOR+PBS group and anti-HIF-1 α+PBS group, differences were statistically significant (P＜0.05). The relative expression of VEGF-A, mTOR, and HIF-1 α mRNA was significantly increased in the cells of the Exo+anti-miR126 group, the differences were all statistically significant (t=4.664, 6.136, 6.247; P＜0.05). ConclusionsmiR-126 plays a role in regulating the effect of hUCMSCs exosomes on VEGF-A levels in high glucose-induced HRECs via mTOR-HIF-1 α pathway.
Objective To observe the morphological changes of macular capillary in type 2 diabetic mellitus (DM) patients without clinical features of diabetic retinopathy (DR) by optical coherence tomography angiography (OCTA). Methods This is a prospective clinical case-control study. Forty-three eyes of 22 patients with DM without clinical features of DR (case group) and 40 control eyes of 20 age- and sex-matched healthy physical examination subjects (control group) were enrolled in this study. All subjects underwent OCTA examination with mode of retinal blood flow imaging, macular 3 mm×3 mm and 6 mm×6 mm area, signal strength ＞45. Foveal avascular zone (FAZ) area, foveal capillary density, parafovea capillary non-perfusion, and micro-aneurysm in shallow capillary vessel layer were evaluated. Results In case group, the mean FAZ area was (0.397±0.141) mm2 and the mean foveal capillary density was (44.6±0.62) %. In control group, the mean FAZ area was (0.253±0.112) mm2 and the mean foveal capillary density was (48.6±0.58) %. FAZ area of eyes in case group was larger than that in control group (t=1.017,P＜0.05). There was no difference of foveal capillary density between two groups (t=1.499,P＞0.05). The spider web-like FAZ and normal foveolar avascular zone were observed in eyes of control group. The parafovea capillary non-perfusion, abnormal foveolar avascular zone, micro-aneurysm and tortuosity of vessels were observed in eyes of case group. Parafovea capillary non-perfusion (χ2=4.542), micro-aneurysms (χ2=5.183) were seen more often in case group than control group (P＜0.05). Conclusion Type 2 DM patients have abnormal retinal vascular microcirculation before DR using OCTA, including larger FAZ area, parafovea capillary non-perfusion, abnormal foveolar avascular zone, micro-aneurysm and tortuosity of vessels.
ObjectiveTo observe the effect of exosomes secreted by retinal pigment epithelial (RPE) cells which damaged by blue light to Nod-like receptor protein (NLRP3).MethodsCultured ARPE-19 cells were divided into 2 groups; one group of RPE cells were exposed to blue light irradiation for 6 hours, the other group was cultured in routine environment. Total exosomes were extracted from the two groups by differential ultracentrifugation in low-temperature, and examined by transmission electron microscope to identify their forms. The exosomes were then incubated with normal ARPE-19 cells. The expression level of CD63, interleukin (IL)-1β, IL-18 and caspase-1 on the exosome surface were measured by Western blotting. The expressions of NLRP3 mRNA in RPE cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR).ResultsBlue light damaged the cellular morphology. Transmission electron microscopy showed that the exosomes were 50-200nm in diameter and like double-concave disks. Blue light damaged cell-derived exosomes had significantly higher expression of IL-1β (t=18.04), IL-18 (t=12.55) and caspase-1 (t=14.70) than the control group (P＜0.001). ARPE-19 cells cultured with blue light damaged cell-derived exosomes also had significantly higher expression of IL-1β (t=18.59), IL-18 (t=23.95) and caspase-1 (t=35.27) than control exosomes (P＜0.001). RT-PCR showed that the relative expression of NLRP3 mRNA of PRE cells in experimental group and control group were 1.000±0.069 and 0.2±0.01, respectively, the difference was significant (t=12.20, P＜0.001).ConclusionThe expression IL-1β, IL-18 and caspase-1 and NLRP3 mRNA were upregulated by exosomes secreted by blue light damaged-RPE cells.
Objective To evaluate the accuracy of the biometry using immersion B scan and partial coherence interferometry (Lenstar LS900) for the axial length (AL) of silicone oil-filled eyes respectively. Methods Thirty-five silicone oil-filled eyes (38 patients) were included in the study. All of these eyes underwent silicone oil removal, cataract extraction and intraocular lenses implantation. The AL of all the silicone oil-filled eyes was measured with A/B-scan ultrasound and Lenstar LS900 before operation and with Lenstar LS900 after operation. The measured distance was compared respectively. The method of immersion B-scan guided with respective sonic velocity. AL was the sum of corneal thickness, anterior chamber depth, lens thickness, the apparent length of oil bubble (velocity values 996 m/s), the depth of the water layer beneath the oil bubble. Results Thirty-one eyes were measured with Lenstar LS900 before silicone oil removal, and the mean AL was (24.12±1.70) mm, 7 eyes failed to get the results before the operation; 36 eyes were measured with Lenstar LS900 after silicone oil removal, and the mean AL was (24.45±1.89) mm. All eyes were measured with B-scan before silicone oil removal, and the mean AL was (24.87±2.52) mm. The difference (31 eyes) of AL measurement before silicone oil removal by two methods was (−0.00±0.09) mm; the difference (31 eyes) between pre- and post-surgical AL measurement with Lenstar LS900 was (0.02±0.07) mm; the difference (36 eyes) between pre-surgical AL measured with B-scan and post-surgical AL measured with Lenstar LS900 was (−0.02±0.11) mm. All the differences were not statistically significant (t=−0.205, 1.752, −1.280; P＞0.05). The consistency of the results measured by two methods was well in Bland-Ahamn analysis. Conclusions Measurement results of AL between immersion B-scan guided with respective sonic velocity and Lenstar LS900 are high repeatability on silicone oil-filled eyes. The AL of silicone oil-filled eyes can be measured reliably by immersion B-scan guided with respective sonic velocity.
ObjectiveTo observe the effect and mechanism of human umbilical cord blood mesenchymal stem cells-derived microvesicles (hUMSCs-MVs) on the injury of the primary rat retinal ganglion cells (RGCs) by high glucose environment. Methods The primary RGCs of Sprague Dawley rats were cultured in vitro, hUMSCs-MVs were isolated and extracted by ultra-centrifugation. hUMSCs-MVs were internalized with RGCs. The RGCs were divided into 4 groups under the conditions below: normal control group (group A), high glucose condition group (group B, RGCs+glucose 33 mmol/L), normal RGCs co-cultured with hUMSCs-MVs group (group C, RGCs+hUMSCs-MVs), and RGCs co-cultured with hUMSCs-MVs in high glucose condition group (group D, RGCs+hUMSCs-MVs+glucose 33 mmol/L). The cell activity was detected by CCK-8 test. Annexin Ⅴ/PI staining detected the cell apoptosis rate by flow cytometry. And the relative expression levels of the genes such as Bcl-2, Bax and Caspase-3 were detected by fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Statistical analysis was performed by using One-way analysis of variance and SNK-q test was used for the comparison between groups. Results The hUMSCs-MVs were extracted by ultra-centrifugation, which were characterized as single or cluster of circular membranous vesicle-like structure with diameter ranging from 100 nm to 1000 nm. The flow cytometry analysis showed that hUMSCS-MVs were highly positived by the surface markers of CD44, CD29, CD73, and CD105 whereas been poorly expressed the integrin (CD49f), HLA class Ⅱ, CD34, CD45. There were significant differences in the cell activity and the apoptosis rate among 4 groups, the cell apoptosis rate of group B was higher significantly than that of group A and group D (F=107.92, P=0.000), the cell activity of group B was lower than that of group A and group D (F=382.11, P=0.000). The results of RT-PCR and Western blot showed that the relative mRNA (F=219.79, 339.198, 1 071.21; P=0.000, 0.000, 0.000) and protein (F=544.28, 295.79, 224.75; P=0.000, 0.000, 0.000) expression of Bcl-2, Bax, Caspase-3 and the protein expression of cleaved Capspase-3 (F=533.18, P=0.000) in group B and D were higher significantly than those in group A and C. The relative expression of Bcl-2 mRNA and protein in group B was significantly lower than that of in group D (P＜0.05). The relative expression of Bax, Caspase-3 mRNA and protein in group B was higher than that in group D (P＜0.05). The relative expression of cleaved Caspase-3 protein in group B was higher significantly than that in group D (P＜0.05). Conclusion The hUMSCs-MVs can protect the cultured rat RGCs from the damage of the high glucose condition through increasing the cell activity and reducing the apoptosis rate of RGCs by promoting the Bcl-2 expression, decreasing the expression of Bax and Caspase-3 and inhibiting the Caspase-3 into the activity form of cleaved Caspase-3.
ObjectiveTo observe the changes of macular structure and choroidal capillary blood flow density in patients with acute central serous chorioretinopathy (CSC).MethodsProspective cross-sectional study. A total of 24 eyes of 24 patients with monocular acute CSC (case group) diagnosed by clinical examination from Shanxi Eye Hospital during January and March 2018 were included in the study. The eyes (24 eyes) and contralateral eyes (24 eyes) of the patients in the case group were set to CSC group and contralateral eye group, respectively. Twenty-one eyes of 21 healthy volunteers with age and gender matching were selected as normal control group. The macular structure of the eyes were observed by OCT and OCT angiography (OCTA), and the blood vessel density of choroidal capillary layer in the circular area of the macular area with a radius of 1 mm was measured. The paired t-test was used to compare the differences in blood flow density in the choroidal capillaries between the three groups.ResultsThe results of OCT showed that the serous neuroepithelial detachment in the macular area was observed in all eyes of the CSC group, with or without RPE detachment being 20 or 4 eyes, respectively. Of the 24 eyes in the contralateral eye group, 13 eyes (54.2%) had thick choroidal RPE lesions (PPE). There was no abnormality in the retina and choroidal structure in the macular area of the normal control group. The results of OCTA showed that the blood flow density of choroidal capillaries in the CSC group, the contralateral eye group and the normal control group were 1.759±0.132, 1.924±0.463, and 1.940±0.033, respectively. Compared with the eyes of the contralateral eye group and the normal control group, the blood flow density of choroidal capillaries in the CSC group was significantly lower (t=6.611, 6.474; P=0.000, 0.000). There was no significant difference in the blood flow density of choroidal capillary layer between the contralateral eye group and the normal control group (t=1.328, P＞0.05). In the contralateral eye group, there was no significant difference in the blood flow density of choroidal capillary layer between PPE eyes and no RPE eyes (t=0.806, P＞0.05).ConclusionsThere is 54.2% of the contralateral eyes in the monocular acute CSC patients with PPE. The choroidal capillary layer blood flow density is lower than that of the contralateral and normal eyes.
ObjectiveTo observe the changes of macular microvessels in patients with retinal vein occlusion (RVO) and macular edema (ME) after intravitreal injection of aflibercept (IVA), and analyze its correlation with best corrected visual acuity (BCVA).MethodsA retrospective case study. Thirty patients (30 eyes) with monocular RVO with ME (RVO-ME) who were diagnosed in the clinical examination of Tianjin Eye Hospital from April 2019 to February 2020 were included in the study. Among them, there were 12 males (12 eyes) and 18 females(18 eyes); the average age was 54.30±13.17 years. The average course of disease was 3.43±1.97 months. Both eyes were examined by BCVA and optical coherence tomography (OCTA). The on-demand injection was adopted after the first injection in IVA treatment regimen. The macular area 6 mm×6 mm in both eyes was scanned with an OCTA instrument, and the area of the foveal avascular area (FAZ), FAZ circumference (PERIM), and out-of-roundness were measured at baseline and 1, 3, and 6 months after treatment. Index (AI), blood flow density within 300 μm width of FAZ (FD-300), foveal retinal thickness (CMT), superficial retinal capillary plexus (SCP), deep retinal capillary plexus (DCP) blood flow density. The paired t test was used to compare the quantitative parameters of the affected eye and the contralateral healthy eye at baseline; the changes of the quantitative parameters at baseline and 1, 3, and 6 months after treatment were analyzed by repeated measures analysis of variance. Pearson correlation analysis was used to analyze the correlation between BCVA, retinal perfusion, and macular blood supply parameters at 6 months after IVA treatment.ResultsAt baseline, compared with the contralateral healthy eye, the FAZ area (t=−4.091), PERIM (t=−5.098) and AI (t=−9.093) of the RVO-ME eye were enlarged, and FD-300 (t=7.237) and overall SCP and DCP blood flow density (t=8.735, 9.897) decreased, the difference was statistically significant (P＜0.001). Six months after treatment, the BCVA of RVO-ME eyes was significantly increased, CMT decreased, FAZ area expanded, and AI decreased (t=8.566, 16.739, −6.469, 9.719; P＜0.001), the difference was statistically significant. There was no significant change in the blood flow density of FD-300 and overall SCP and DCP, and the difference was not statistically significant (t=1.017, 1.197, 0.987; P＞0.05). Compared with baseline, the FAZ area of RVO-ME eyes gradually expanded at 3 and 6 months after treatment, and the difference was statistically significant (F=21.979, P＜0.001). Correlation analysis results showed that BCVA at 6 months after treatment was positively correlated with the overall SCP and DCP blood flow density at baseline and 6 months after treatment (r=−0.538, −0.484, −0.879, −0.854; P＜0.05). There was a negative correlation with the area of FAZ 6 months after treatment (r=0.544, P=0.001). The number of ME recurrences was negatively correlated with BCVA and overall SCP and DCP blood flow density 6 months after treatment (r=0.604, −0.462, −0.528; P＜0.05), it was positively correlated with FAZ area (r=0.379, P=0.043).ConclusionWithin 6 months of IVA treatment in RVO-ME eyes, ME is significantly reduced and visual acuity is improved; SCP blood flow density decreases, and FAZ area expands.
Objective To observe the clinical and multimodal imaging characteristics of eyes with acute macular neuroretinopathy (AMN) associated with COVID-19. MethodsA retrospective clinical study. From December 18 to 26, 2022, 16 eyes of 8 patients with AMN associated with COVID-19 were included in the study. There were 4 males and 4 females; all cases were bilateral. The age was (31.5±9.6) years old. The time from fever to decreased vision was (3.75±1.04) days. The best corrected visual acuity (BCVA), intraocular pressure, slit lamp microscopy, indirect fundus microscopy, fundus color photography, and optical coherence tomography (OCT) were performed in all patients. Infrared fundus photography (IR), OCT angiography (OCTA) and fluorescein fundus angiography (FFA) were performed in 14, 6 and 4 eyes respectively. The BCVA examination was performed using the international standard visual acuity chart, which was converted into logarithm of the minimum angle of resolution (logMAR) BCVA for statistics. The clinical data, IR, OCT and OCTA imaging features of the patients were retrospectively analyzed. ResultsThe logMAR BCVA of AMN eyes was 4.21±0.74, intraocular pressure was (14.87±1.50) mm Hg (1 mm Hg=0.133 kPa). Fundus color photography showed that multiple gray-white petal-shaped lesions were arranged around the macular fovea in 2 eyes; no obvious abnormality was found in the macular area in 14 eyes. Of the 14 eyes examined by IR, 6 eyes had irregular weak reflective lesions around the macular fovea. OCT showed strong reflex in the outer nuclear layer and outer plexiform layer of all eyes, including 15 eyes with elliptical zone injury. In 6 eyes examined by OCTA, the blood flow density of the superficial and deep capillary plexus (DCP) of retina decreased, and the blood flow density of DCP decreased significantly. The en-face image of DCP showed the wedge-shaped strong reflective lesion area with the tip pointing to the central fovea in 2 eyes. No abnormal fluorescence was observed in FFA. ConclusionsThe characteristic manifestation of AMN associated with COVID-19 is weak reflex focus in IR; OCT shows strong reflection in outer core layer and outer plexiform layer; OCTA showed that retinal DCP blood flow density decreased.