Protective effect of asiatic acid on blood-retinal barrier in diabetic rats / 中华实验眼科杂志

Objective:To investigate the protective effect of asiatic acid (AA) on blood-retinal barrier (BRB) in diabetic rats and its possible mechanism.Methods:Ninety-six healthy 8-week-old male SD rats were randomly divided into normal control group, diabetes group, low-dose AA group and high-dose AA group, with 24 rats in each group.Intraperitoneal injection of streptozocin (STZ) was used to establish diabetes model.One month after the establishment of the model, the low-dose AA group and the high-dose AA group were given intragastrical administration of 37.5 mg/kg AA and 75.0 mg/kg AA, respectively, once a day according to grouping.The normal control group and the diabetes group were administrated with the same amount of 0.5% sodium carboxymethyl cellulose.The body weight of the rats were weighted at week 0, 1, 2, 3, 4 after intragastrical administration.Blood was taken from the tail vein and the blood glucose level was measured.The retina was obtained one month following the administration.Pathological changes of the rats retina were detected by hematoxylin-eosin (HE) staining.Evan's blue quantitative method was used to detect the damage of blood-retinal barrier (BRB). Immunofluorescence staining was performed to detect the distribution of Occludin, Notch1, Jagged canonical Notch ligand 1 (JAG1) and Delta like canonical Notch ligand 4 (DLL4) in retina.The mRNA and protein expressive levels of Occludin, Notch1, JAG1 and DLL4 were detected by Real-time PCR and Western blot.The study protocol was approved by a Scientific Research and Clinical Trial Ethics Committee of The First Affiliated Hospital of Zhengzhou University (No.2020-KY-228). The use and care of animals complied with the Guide for the Care and Use of Laboratory Animals of National Institutes of Health and the 3R rules.Results:At 4 weeks after intragastrical administration, the body weight of the high-dose AA group was significantly higher than that of the diabetes group, and the blood glucose values were significantly lower in the high-dose AA group and the low-dose AA group in comparison with the diabetes group (all at P<0.05). The cells were arranged orderly with clear layered structure in the normal control group.In the diabetes group, the retina was thicker than that of the normal control group, with a thicker outer nuclear layer, disordered cell arrangement and unclear layered structure.Compared with the diabetes group, the total retinal thickness and structure were obviously improved in the low-dose AA group and the high-dose AA group.Evan's blue leakage in retina was (3.07±1.30), (13.73±3.88), (9.57±2.69) and (6.55±1.61)ng/mg in the normal control group, the diabetes group, the low-dose AA group and the high-dose AA group, respectively.There was a significant difference in leakage of Evan's blue among the four groups ( F=18.50, P<0.01), among which the leakage of Evan's blue dye in the high-dose AA group was significantly lower than that of the diabetes group ( P<0.01). Compared with the diabetes group, there was significantly higher relative expression level of Occludin protein and significantly lower relative expression levels of Notch1, JAG1 and DLL4 proteins in the other three groups (all at P<0.05). The relative expression level of Occludin protein was significantly higher and the relative expression levels of Notch1, JAG1 and DLL4 proteins were significantly lower in the high-dose AA group than those in the low-dose AA group (all at P<0.05). Compared with the normal control group, the Occludin mRNA expression level was significantly decreased and the expression levels of Notch1, JAG1 and DLL4 mRNA were significantly increased in the diabetes group and low-dose AA group (all at P<0.01). The Occludin mRNA expression level was higher and the Notch1 mRNA expression level was lower in the high-dose AA group than those in the diabetes group and the low-dose AA group, and the expression levels of JAG1 and DLL4 mRNA were lower in the high-dose AA group in comparison with the diabetes group, and the differences were statistically significant (all at P<0.05). Conclusions:Asiatic acid might play a protective role on BRB in diabetic rats by inhibiting Notch1 signaling pathway.

Identificação de um novo motivo peptídico específico para a vasculatura cerebral e que diferencia as barreiras hematoenfálica e hematoretiniana / Identification of a new specific peptide motif to the brain vasculature that differentiates between the blood brain barrier and the bloodretinal barrier

O conceito de heterogeneidade vascular é bem aceito pela comunidade cientifica, desempenhando papel essencial em processos fisiológicos e patológicos. Uma vez que os vasos sanguíneos são importantes na organogênese, diferenciação e morfogênese de tecidos e órgãos, torna-se interessante desvendar a diversidade vascular cerebral, identificando novos marcadores moleculares para este órgão tão importante. Utilizando tecnologia combinatorial de phage display in vivo, identificamos um novo motivo peptídico, na qual os aminoácidos FenilalaninaArginina-Triptofano (Phe-Arg-Trp; FRW) predominam. Este motivo peptídico é um ligante seletivo para vasos sanguíneos cerebrais e não se acumula em outros órgãos, incluíndo tecidos como intestinos e gônadas, que também apresentam barreiras endoteliais especificas. No entanto, mais surpreendente foi a observação de que o motivo FRW não se liga aos vasos sanguíneos da retina, o que implica em uma diferença até então desconhecida entre duas barreiras: a barreira hematoencefálica e a barreira hematoretiniana. Combinando phage display in vivo e microscopia eletrônica de transmissão, observamos a presença de partículas de fago ligadas à vasculatura cerebral em um nível supramolecular: aglomerados de fagos filamentosos expressando o motivo FRW foram visualizados ligados às regiões de contato entre as células endoteliais. Por fim, a utilização do peptídeo CFFWKFRWMC permite imageamento in vivo, demonstrando que novas ferramentas para estudar e visualizar o cérebro podem surgir deste motivo

The concept of vascular heterogeneity is well accepted by the scientific community, playing an essential role in physiological and pathological processes. Since blood vessels are important in organogenesis, differentiation, and morphogenesis of tissues and organs, it becomes interesting to unveil the cerebral vascular diversity, identifying new molecular markers for such important organ. Using in vivo phage display, we show that a new peptide motif that emerged from our combinatorial screening of the vasculature binds selectively to blood vessels in the brain in vivo but not to vessels in other organs. Peptides containing a conserved motif in which amino acids Phenylalanine-Arginine-Tryptophan (Phe-Arg-Trp; FRW) predominate could be visualized by transmission electron microscopy bound to the junctions between endothelial in all areas of the brain, including the optic nerve but not in other barrier containing tissues, such as intestines and testis. Remarkably, peptides containing the motif do not bind to vessels in the retina, implying an important molecular difference between these two vascular barriers. Furthermore, the peptide allows for in vivo imaging, demonstrating that new tools for studying and imaging the brain are likely to emerge from this motif

Pathophysiology of Diabetic Retinopathy: The Old and the New

Vision loss in diabetic retinopathy (DR) is ascribed primarily to retinal vascular abnormalities—including hyperpermeability, hypoperfusion, and neoangiogenesis—that eventually lead to anatomical and functional alterations in retinal neurons and glial cells. Recent advances in retinal imaging systems using optical coherence tomography technologies and pharmacological treatments using anti-vascular endothelial growth factor drugs and corticosteroids have revolutionized the clinical management of DR. However, the cellular and molecular mechanisms underlying the pathophysiology of DR are not fully determined, largely because hyperglycemic animal models only reproduce limited aspects of subclinical and early DR. Conversely, non-diabetic mouse models that represent the hallmark vascular disorders in DR, such as pericyte deficiency and retinal ischemia, have provided clues toward an understanding of the sequential events that are responsible for vision-impairing conditions. In this review, we summarize the clinical manifestations and treatment modalities of DR, discuss current and emerging concepts with regard to the pathophysiology of DR, and introduce perspectives on the development of new drugs, emphasizing the breakdown of the blood-retina barrier and retinal neovascularization.

Effect of light damage on the function of blood-retinal barrier in rats / 国际眼科杂志(Guoji Yanke Zazhi)

Abstract?AIM: To investigate the influence on blood-retina barrier after intense light exposure in rats.?METHODS: The rats were randomly divided into light exposure group and control group. Rats in light exposure group were exposed in white light (10000lux, 12h on-off, continuing 1-14d) .Rats in control group were only exposed in natural light.The eyes of the rats in the two groups were removed when the rats in light exposure group acceptted intense light after 1, 3, 7 and 14d.We observed the change of retinal structure using hematoxylin-eosin ( HE ) staining, and observed the change of retinal ultrastructure using electron microscope.We quantified the change of retinal vascular permeability using laser scanning confocal fluorescence microscope and spectrophotometry after perfusion of Evans-blue, to evaluate the change of blood-retinal barrier.?RESULTS: At 1d after intense light exposure, the retinal ultrastructure of rats changed, such as denaturation of photoreceptor cells and falling of membranous disc outer segment and thinning of the outer nuclear layer thickness, and so on;and the longer the rats exposure to intense light, the more serious change of the retinal ultrastructure were found.At 3d later, photoreceptor cells began apoptosis.At 14d later, the outer nuclear layer became thinner obviously, and the number of cells reduce obviously.At 1d after intense light exposure, EB leaked from the retinal vascular, and at 14d later the leaking of EB was more obvious.?CONCLUSION: The photoreceptor cell of the outer nuclear layer of retina will degenerate and apoptosis, and the outer nuclear layer will be thinner, and the structure and function of blood-retinal barrier will be destroied, if the eyes of rats exposed in intense light.

The effect of Evans blue on determining the breakdown of blood-retina barrier / 中华眼底病杂志

Objective To verify the effect of Evans blue dye on determining the retina blood vessel leakage. Methods Male Sprague Dawley rats were used in this study. The VEGF induced retinal blood vessel leakage was checked with Evans blue dye. Then the blood retina barrier breakdown of 1 week diabe tic animals was quantified with Evans blue. The dye was extracted from retina by formamide and the extraction was checked with spectrophotometer. Evans blue leakage was normalized against wet or dry retina weight. Results The retinal Evans blue content of eyes treated with VEGF was remarkably higher than that of the controls (n=17, P