Objective To investigate the protective effect of Niacin on blood-retina barrier (BRB) in diabetic rats and related mechanism. Methods The male Wistar rats (60) were divided into control (CON) group, diabetes (DM) group and Niacin-treated (NA) group, 20 rats in each group. Rats diabetes models were induced with streptozotocin injection. Niacin (40 mg/kg·d) was administrated orally everyday in Niacin-treated group until sacrificed after 3 months. Pathological outcomes, total cholesterol (TC) and high-density lipoprotein (HDL) were evaluated at month 3. Optical microscopy was used to observe the retinal structure. The integrity of BRB and the vascular permeability was quantified by analyzing albumin leakage using Evans blue (EB) method. The relative expressions of Claudin-5, Occludin, zonula occluden (ZO)-1 and GPR109A mRNA in rat retinas were detected by reverse transcription PCR (RT-PCR) and relative expression of GPR109A, tumor necrosis factor (TNF)-α and interleukin (IL)-6 by Western blot. Results Compared to CON group, the TC content was increased and HDL content was decreased in DM group (t=4.034, 5.831; P < 0.05). Compared to DM group, the TC content was decreased and HDL content was increased in NA group (t=6.868, 3.369; P < 0.05). The retinal structure of CON group was normal. Pathological changes were found in the DM group, such as tumescent nuclei and disorganized structures. The retinal structure of NA group was similar to the control group. Evans blue dye that the microvascular leakage in DM group was increased compared with CON group (t=24.712, P < 0.05), while in NA group was decreased compared with DM group (t=16.414, P < 0.05). The mRNA expression of Occludin, Claudin-5, ZO-1 in DM group were decreased compared with CON group (t=11.422, 12.638, 12.060; P < 0.05), while in NA group were increased compared with DM group (t=5.278, 3.952, 8.030; P < 0.05). The mRNA expression of GPR109A in NA group were increased compared with DM group (t=5.053, P < 0.05). The protein expression of GPR109A, IL-6, TNF-αin DM group were increased compared with CON group (t=4.915, 11.106, 6.582; P < 0.05). Compared to DM group, the protein expression of GPR109A was increased (t=5.806, P < 0.05), while the protein expression of IL-6 and TNF-α were decreased (t=10.131, 5.017; P < 0.05). Conclusion Niacin has the protective effect for BRB by up-regulating GPR109A expression which may suppress inflammation.
Objective To observe the effect of different concentration netrin-1 on retinal vascular permeability in diabetes mellitus (DM) rats. Methods Eighty adult Sprague-Dawley rats were randomly divided into 8 groups, 10 rats in each group, including normal control group (group A), normal＋balanced salt solution (BSS) group (group B), normal＋netrin-1 (500 μg/ml) group (group C) and DM group (50 rats in 5 sub-groups). DM rats were induced by intraperitoneal injection of streptozocin. Three months after intraperitoneal injection, 10 DM rats in the control group were injected with BSS (group D). Forty DM rats were injected with 5 μl of different concentrate netrin-1, and were divided into DM+netrin-1 10 μg/ml group (group E), DM+netrin-1 50 μg/ml group (group F), DM＋netrin-1 100 μg/ml group (group G), DM＋netrin-1 500 μg/ml group (group H) according to the different concentration. Non-DM rats in group C were injected with netrin-1 500 μg/ml. The expression of occludin was determined by immunohistochemistry for protein, and by real-time fluorescence quantitative reverse transcription polymerase chain reaction for mRNA level. Retinal vascular permeability was measured by Evans blue infusion. Results The expression of occludin protein and mRNA in group D were less than group A (t=27.71, 8.59;P=0.00, 0.00). However, the retinal vascular permeability increased in group D (t=−42.72,P=0.00). The expression of occluding protein, occludin mRNA and retinal vascular permeability showed significant differences between group D, E, F, G and H (F=146.31, 16.54, 67.77;P=0.00, 0.00, 0.00). Compared the group B with group C, there was no significant differences between the expression of occludin protein, occludin mRNA and the retinal vascular permeability (t=−1.13, 0.93, 1.04;P=0.27, 0.36, 0.31). The concentrate of netrin-1 showed a significant positive correlation to the expression level of occludin and occludin mRNA (r=0.73, 0.81;P=0.00, 0.00), but negative correlation to the vascular permeability (r=−0.61,P=0.00). Conclusion Netrin-1 can reduce the DM rats' retinal vascular permeability, which depended on the concentration of netrin-1.
ObjectiveTo explore the effect and mechanism of netrin-1 on blood-retinal barrier permeability in diabetes mellitus (DM) rats. MethodsEighty Sprague-Dawley rats were randomly divided into the normal control group, DM+balanced salt solution (BSS) group, DM+netrin-1 low dose group and DM+netrin-1 high dose group, with 20 rats in each group. DM rats were induced by intraperitoneal injection of streptozocin (STZ). These rats were feed with high sugar and fat for 3 months after STZ injection. All rats were sacrificed at 1 month after intravitreal injection. Retinal vascular permeability was measured by Evans blue. The expression level of occludin was determined by immunohistochemistry. Hematoxylin-eosin (HE) staining of retina was used to observe the pathological change of DM and the level of occludin mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). Five rats of each group. ResultsHE staining of retina showed that the degree of edema and vascularization in DM+netrin-1 high dose group was better than DM+BSS group. Staining of occludin in retina was limited to nerve fiber layer, ganglion cells, inner plexiform layer and inner nuclear layer in normal rats, but in DM+BSS group, the color of staining positive of occludin was lighter and more reduced. However, DM+ netrin-1 group occludin staining was deepen and enlarged. The result of RT-PCR showed that the expression of occludin mRNA in other three groups was less than normal control group (P < 0.05). The significant difference during DM+BSS group, low dose group and DM+netrin-1 high dose group (F=177.13, P=0.00), and the more concentrate of netrin-1 the higher expression of occluding. Compared the DM+netrin-1 low dose group with DM+BSS group, there was significant difference expression of occludin (t=-13.98, P=0.00). There was significant difference between the DM+netrin-1 high dose group and normal control group (t=12.87, P=0.00). There was statistically significant difference in DM+BSS group, DM+netrin-1 low dose group and DM+netrin-1 high dose group (F=179.69, P=0.00). Compared the two group of different concentration netrin-1, the quantification of vascular permeability in DM+netrin-1 high dose group reduced more (t=12.73, P=0.00). ConclusionsNetrin-1 can protect the blood-retinal barrier in DM rats. Netrin-1 may decrease BRB leakage in DM rats by protecting the expression of occludin.
Objective To investigate effects of neural retina on development of the structure of outer blood retinal barrier in embryogenesis. Methods The retinal neural epithelium (RNE) and pigment epithelium (RPE) layers of 150, 120 and 90 embryonic chicken eyes incubated for 7,10, and 14 days were peeled off. RNE was used to prepare the culture medium with different conditions (7drcSF3, 10drcSF3, 14drcSF3). RPE cells of 7- and 14-incubated chicken embryos were cultured on laminin-coated transwell filter. The SF3, 7drcSF3, 10drcSF3 , 14drcSF3 medium were used respectively in the apical chamber and SF2 was used in basolateral chamber. After the formation of monolayer, the transepithelial electrical resistance of the RPE was detected. After the fixation of RPE cells, the condition of the tight junction among the cells was observed by immunohis tochemistry and transmission electron microscopy. Results For the RPE cells of 7-and 14-day incubated embryonic eyes, the difference of TER in various medium of SF3/SF2, 7drcSF3/SF2, 10drcSF3/SF2, 14drcSF3/SF2 was statistically significant (P＜0.01). The polarity of RPE cells was induced and the netlike tight junctional strands was urged in the retina-conditioned medium. Conclusion The neural retina may actively promote the formation of the structure of outer blood retinal barrier. (Chin J Ocul Fundus Dis，2004，20：237-240)
In vivo and in vitro tracer studies, e. g., fundus fluorescein angiography, fluorescein and lanthanum tracer procedures were carried out on mild and severe blunt ocular trauma in rabbits to investigate pathological changes of the blood retinal barrier. Noo difusion of the tracers was found in the retinal after mild blunt trauma. However, severe disorganization of the retinal pigment epithelial cells and breakdown of the outer blood retinal barrier with permeation of tracers in the interphotoreceptor space were evident after severe blunt trauma. These results suggest that contusional retinal edema is mainly due to disruption of cells in the outer retinal layer barrier may, in part, play a role in pathogenesis of the retinal edema. （Chin J Ocul Fundus Dis,1992,8:130-132）
Objective To investingate the ultrastructural changes of retinal pigment epithelium(RPE) and its permeability in spontaneously hypertensive rats(SHR)and explore the relation between these changes and hypertensive retinopathy.MethodsThe ultrastructure of RPE cells in the SHR aged five,six,seven months wasobserved with transmission electronmicroscope and compared to its normotensive control strain(WKY) with the same age.Then,lanthanum tracer procedures were carried out to investigate pathological changes of the blood-retinal barrier.Results (1)In SHR the main pathological changes involved swelling of mitochondria,enlargement of endoplasmic reticula,decrease of RPE cell infolding,and sparseness of microvilli.These degenerations were more serious in older rats with higher blood pressure.(2)The breakdown of outer blood-retinal barrier with permeation of lanthanum tracers were evident in SHR aged six or seven month,however,in WKY and five-month SHR the traces were prevented from passing by tight junctions.ConclusionThe degeneration of RPE owing to ischemia and anoxia arises in early periosd of hypertensive retinopathy.The pathological changes of ultrastructure and permeability might interact with the damage of visual cells and play a main role in the hypertensive retinopathy.
Objective To investigate the bloodretinal barrier（BRB）function of porcine retinal pigment epithelial（RPE）cells cultured in vitro. Methods Primary porcine RPE cells were cultured，and the third generation were inoculated in a microporous filter with the filter membrane of polyvinylpyrrolidone（PVP）-free polycarbonate membrane.After 1，2，3 and 4 weeks of culture，the surface of filter membrane was observed by light microscope，and after 2 weeks of culture，the section of filter membrane was observed by light microscope and transmission electron microscope.Transepithelial electrical resistance（TER）was detected and the permeability was measured with fluorescein sodium（FS）and horseradish peroxidase（HRP）. Results Primary porcine RPE cells were cultured successfully.RPE cells converged1week after inoculation; 2 and 3 weeks after inoculation，the density of RPE cells did not changed obviously; 4 weeks after inoculation，the density of RPE cells decreased.The characteristics of polarized growth of monolayer were found in RPE cells on the surface of filter membrane; 2 weeks after inoculation，the TER of RPE cells was（97.44plusmn;11.36）Omega;/cm2 which maintained till the 3rd week after inocubation.After incubated for 30 minutes，only 0.27% of FS and 0.17% HRP reached the inferior filter membrane，and the permeability rate of SF with low molecular weight was higher than which of HRP with high molecular weight. Conclusions The filter with PVPfree polycarbonate membrane may be used to set up the model of RPE cells with polarized growth of monolayer and investigate the barrier function of RPE cells. (Chin J Ocul Fundus Dis, 2006, 22: 188-191)
Objective To investigate the effect of intravitreal injection with dexamethasone on leukocyte accumulation, vascular permeability, and the expression of intercellular adhension molecule (ICAM-1) in rats with diabetes. Methods Seventy-two BN rats were divided into 4 groups: control group, diabetes group, diabetes+ physiologic saline group, and diabetes+ dexamethasone group, with 18 rats in each group. Streptozotocin was injected into the rats to set up the diabetic model. Accumulated leukocytes were counted in vivo by acridine orange leukocyte fluorography, and retinal vascular permeability was measured by Evans blue assay. The expression of mRNA and protein level of ICAM-1 were evaluated by real-time quantitative polymerase chain reaction analysis and enzymelinked immunosorbent assay. Results In the diabetes+ dexamethasone group, accumulated leukocytes were reduced, retinal vascular permeability decreased, and the expression of ICAM-1 decreased. The expression of ICAM-1 mRNA and protein levels in control group, diabetes group, diabetes+ physiologic saline group, and diabetes+ dexamethasone group were 0.43plusmn;0.07，0.76plusmn;0.21，0.74plusmn;0.18，and 0.55plusmn;0.13; (37.90plusmn;4.56), (76.74plusmn;6.68), (74.32plusmn;7.11), and (39.61plusmn;4.47) pg/mg respectively. Conclusions Dexamethasone can reduce accumulated leukocytes and retinal vascular permeability, which may be caused by inhibiting the expression of ICAM-1. (Chin J Ocul Fundus Dis,2007,23:273-276)
Iron death is an alternative to normal cell death and is regulated by a variety of cellular metabolic pathways. Iron death has become a hot topic of research because it can cause damage to various organs and degenerative diseases in the body. Metabolism, signalling pathways, endoplasmic reticulum stress, and immune cells can all affect the occurrence of iron death, and the blood-retina destruction induced by iron death plays an important role in autoimmune uveitis. Exploring the components of the blood-retina regulatory mechanism of iron death in autoimmune uveitis can lead to the search for targeted drug targets, which can provide a new research idea for the subsequent study of the diagnosis and treatment of autoimmune uveitis.