Retinal Neuropathy in IGT Stage of OLETF Rats: Another Characteristic Change of Diabetic Retinopathy.

AIMS: We investigated the changes of retinal structure in normal glucose tolerance (NGT), impaired glucose tolerance (IGT), diabetes mellitus (DM), and diabetic kidney disease (DKD) stages in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. METHODS: We assigned OLETF rats to four groups based on their OGTT results and 24 h urinary microalbumin (24 h UMA) levels: NGT, IGT, DM, and DKD groups. We observed the structural and the corresponding pathological changes and quantified the expression of HIF-1α, iNOS, NF-κB, VEGF, ICAM-1, and occludin in the retina. RESULTS: Significant damage to the retinal structure, especially in retinal ganglion cells (RGCs), was observed in the IGT stage. The expression of HIF-1α, iNOS, NF-κB, VEGF, and ICAM-1 was significantly upregulated, while that of occludin was downregulated. CONCLUSION: Significant retinal neuropathy occurs in the IGT stage. Inflammation and hypoxia may damage the blood retina barrier (BRB), leading to diabetic retinopathy.

Light and electron microscopic studies on the pecten oculi showing blood-retina barrier properties in Turkey's native Gerze chicken.

The pecten oculi is a highly vascularized and pigmented organ that projects from the optic disc into the vitreous body in the avian eye. In this study, the pecten oculi of Turkey's native Gerze chicken was examined by light and scanning electron microscopy. Furthermore, the localization of some adherens junction components (E-cadherin and pan-cadherin) in intact vessels of the blood-retina barrier was investigated by immunohistochemistry. In the Gerze chicken, the pecten oculi was a thin structure, which was located over the head of the discus nervi optici and projected from the retina into the corpus vitreum. The pecten oculi consisted of 18-21 highly vascularized pleats, joined apically by a bridge and resembled an accordion in appearance. Hyalocytes and melanocytes were observed around the small and large vessels. The morphometric data of the pecten oculi showed that there were no statistical differences in terms of sex. The immunohistochemical analysis of the pecten oculi, which is used as a model for the investigation of the formation and maturation of the barrier properties in the central nervous system, revealed cytoplasmic E-cadherin and pan-cadherin immunoreactivity in the endothelial cells of the small, large and capillary vessels. These observations suggest that while the morphological and histological structure of the Gerze chicken's pecten oculi was generally similar to that of other diurnal domestic birds, the pecten oculi, a model system for vascular differentiation and the blood-retina barrier, expressed different cadherins.

Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in mice.

Insight into the molecular and cellular processes in blood-retinal barrier (BRB) development, including the contribution of paracellular and transcellular pathways, is still incomplete but may help to understand the inverse process of BRB loss in pathologic eye conditions. In this comprehensive observational study, we describe in detail the formation of the BRB at the molecular level in physiologic conditions, using mice from postnatal day (P)3 to P25. Our data indicate that immature blood vessels already have tight junctions at P5, before the formation of a functional BRB. Expression of the endothelial cell-specific protein plasmalemma vesicle-associated protein (PLVAP), which is known to be involved in transcellular transport and associated with BRB permeability, decreased during development and was absent when a functional barrier was formed. Moreover, we show that PLVAP deficiency causes a transient delay in retinal vascular development and changes in mRNA expression levels of endothelial permeability pathway proteins.-Van der Wijk, A.-E., Wisniewska-Kruk, J., Vogels, I. M. C., van Veen, H. A., Ip, W. F., van der Wel, N. N., van Noorden, C. J. F., Schlingemann, R. O., Klaassen, I. Expression patterns of endothelial permeability pathways in the development of the blood-retinal barrier in mice.

Gradual Suppression of Transcytosis Governs Functional Blood-Retinal Barrier Formation.

Blood-central nervous system (CNS) barriers partition neural tissues from the blood, providing a homeostatic environment for proper neural function. The endothelial cells that form blood-CNS barriers have specialized tight junctions and low rates of transcytosis to limit the flux of substances between blood and CNS. However, the relative contributions of these properties to CNS barrier permeability are unknown. Here, by studying functional blood-retinal barrier (BRB) formation in mice, we found that immature vessel leakage occurs entirely through transcytosis, as specialized tight junctions are functional as early as vessel entry into the CNS. A functional barrier forms only when transcytosis is gradually suppressed during development. Mutant mice with elevated or reduced levels of transcytosis have delayed or precocious sealing of the BRB, respectively. Therefore, the temporal regulation of transcytosis governs the development of a functional BRB, and suppression of transcytosis is a principal contributor for functional barrier formation.

Effect of Toxoplasma gondii infection on the junctional complex of retinal pigment epithelial cells.

Ocular toxoplasmosis is the most frequent cause of uveitis, leading to partial or total loss of vision, with the retina the main affected structure. The cells of the retinal pigment epithelium (RPE) play an important role in the physiology of the retina and formation of the blood-retinal barrier. Several pathogens induce barrier dysfunction by altering tight junction (TJ) integrity. Here, we analysed the effect of infection by Toxoplasma gondii on TJ integrity in ARPE-19 cells. Loss of TJ integrity was demonstrated in T. gondii-infected ARPE-19 cells, causing increase in paracellular permeability and disturbance of the barrier function of the RPE. Confocal microscopy also revealed alteration in the TJ protein occludin induced by T. gondii infection. Disruption of junctional complex was also evidenced by scanning and transmission electron microscopy. Cell-cell contact loss was noticed in the early stages of infection by T. gondii with the visualization of small to moderate intercellular spaces. Large gaps were mostly observed with the progression of the infection. Thus, our data suggest that the alterations induced by T. gondii in the structural organization of the RPE may contribute to retinal injury evidenced by ocular toxoplasmosis.

Imaging pericytes and capillary diameter in brain slices and isolated retinae.

The cerebral circulation is highly specialized, both structurally and functionally, and it provides a fine-tuned supply of oxygen and nutrients to active regions of the brain. Our understanding of blood flow regulation by cerebral arterioles has evolved rapidly. Recent work has opened new avenues in microvascular research; for example, it has been demonstrated that contractile pericytes found on capillary walls induce capillary diameter changes in response to neurotransmitters, suggesting that pericytes could have a role in neurovascular coupling. This concept is at odds with traditional models of brain blood flow regulation, which assume that only arterioles control cerebral blood flow. The investigation of mechanisms underlying neurovascular coupling at the capillary level requires a range of approaches, which involve unique technical challenges. Here we provide detailed protocols for the successful physiological and immunohistochemical study of pericytes and capillaries in brain slices and isolated retinae, allowing investigators to probe the role of capillaries in neurovascular coupling. This protocol can be completed within 6-8 h; however, immunohistochemical experiments may take 3-6 d.

[The estimation of age-related sensitivity of the retinal pigment epithelium of Japanese quail (Coturnix japonica) to the light damage].

The effect of blue light damage (445-455 nm, 4 J/cm2) to retinal pigment epithelium (RPE) subcellular structures was investigated in 4 age risk groups (9, 25, 40 and 52 weeks) of Japanese quail Coturnix japonica by light and electron microscopy. The indicator of biological aging of RPE was age-related accumulation of lipofuscin granules: 5-6-fold increase in their quantity increasing by 5-6 times in quails at 52 weeks. The main photo-induced changes observed after 24 h of the photo radiation were located in the blood-retinal barrier, such as loss of homogeneity of Bruch's membrane, disorganization of basal processes, deformations of the nuclei and mitochondria shapes. Those effects ofphotobleaching were more expressed in young birds. But for the older 52-week age birds it was not so noticeable, because their retinal pigment epithelium structures had disorders which were similar to those in younger birds after photodamage.

Loss of caveolin-1 causes blood-retinal barrier breakdown, venous enlargement, and mural cell alteration.

Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma.

Effect of blood-retinal barrier development on formation of selenite nuclear cataract in rat.

Selenite cataract, as an experimental animal model of nuclear cataract to mimic human senile cataract, is produced only when overdose selenite is injected to neonatal rats before eyelid opening. To clarify the cause of age differences on selenite cataract formation in rats, mRNA expression of GPx1, MsrA and MsrB1, as well as GPx activity in Wistar rat lens at different ages were assayed, level of lipid peroxidation, extent of lens damage induced by sodium selenite and barricade function of blood-retinal barrier (BRB) were investigated. The results showed that mRNA expressions and activity of antioxidant enzymes in neonatal rat lens before eyelid opening were the highest and then decreased with age, and revealed by transmission electron microscopy (TEM) using lanthanum hydroxide as tracer that higher selenite content entering eyes injured lens and resulted in cataract formation for immature BRB before eyelid opening, moreover, a little selenite content entering eyes was not enough to induce cataract formation after eyelid opening because of mature BRB.

Investigation of barrier characteristics in the hyaloid-retinal vessel of zebrafish.

The blood-retinal barrier (BRB) is essential for the physiological integrity of the retinal vessels. In particular, ocular pathologies of retinal neovascularization could be causally related to the BRB breakdown. Zebrafish have emerged as an advantageous model for studying vascular development and characteristics. Here we investigated for the first time the barrier characteristics of the hyaloid-retinal vessel using fli1-EGFP transgenic zebrafish. By 7 dpf, the hyaloid-retinal vessel was formed between lens and retina, where intercellular junctional complexes were already present between endothelial cells. Interestingly, NG-2 expression, but not GFAP, was colocalized with EGFP-positive cells of the hyaloid-retinal vessel. Among endothelial tight junction proteins, claudin-5 was expressed on EGFP-positive cells of the hyaloid-retinal vessel, whereas occludin and ZO-1 were not observed on the vessel. In addition, the hyaloid-retinal vessel was so leaky that a mixture of fluorescein tracers (2,000-kDa FITC-dextran, 10-kDa rhodamine-dextran, and 350-Da DAPI) diffusely infiltrated into all retinal layers. Our results suggest that, unlike retinal vessels of higher vertebrates, the hyaloid-retinal vessel of zebrafish shows insufficient characteristics to meet a functional endothelium-based CNS barrier. Therefore, it might be not suitable to use the hyaloid-retinal vessel of zebrafish for studying BRB biogenesis.

Huperzine A activates Wnt/ß-catenin signaling and enhances the nonamyloidogenic pathway in an Alzheimer transgenic mouse model.

Huperzine A (HupA) is a reversible and selective inhibitor of acetylcholinesterase (AChE), and it has multiple targets when used for Alzheimer's disease (AD) therapy. In this study, we searched for new mechanisms by which HupA could activate Wnt signaling and reduce amyloidosis in AD brain. A nasal gel containing HupA was prepared. No obvious toxicity of intranasal administration of HupA was found in mice. HupA was administered intranasally to ß-amyloid (Aß) precursor protein and presenilin-1 double-transgenic mice for 4 months. We observed an increase in ADAM10 and a decrease in BACE1 and APP695 protein levels and, subsequently, a reduction in Aß levels and Aß burden were present in HupA-treated mouse brain, suggesting that HupA enhances the nonamyloidogenic APP cleavage pathway. Importantly, our results further showed that HupA inhibited GSK3α/ß activity, and enhanced the ß-catenin level in the transgenic mouse brain and in SH-SY5Y cells overexpressing Swedish mutation APP, suggesting that the neuroprotective effect of HupA is not related simply to its AChE inhibition and antioxidation, but also involves other mechanisms, including targeting of the Wnt/ß-catenin signaling pathway in AD brain.

The blood-retinal barrier: structure and functional significance.

Formation and maintenance of the blood-retinal barrier is required for proper vision and loss of this barrier contributes to the pathology of a wide number of retinal diseases. The retina is responsible for converting visible light into the electrochemical signal interpreted by the brain as vision. Multiple cell types are required for this function, which are organized into eight distinct cell layers. These neural and glial cells gain metabolic support from a unique vascular structure that provides the necessary nutrients while minimizing interference with light sensing. In addition to the vascular contribution, the retina also possesses an epithelial barrier, the retinal pigment epithelium, which is located at the posterior of the eye and controls exchange of nutrients with the choroidal vessels. Together the vascular and epithelial components of the blood-retinal barrier maintain the specialized environment of the neural retina. Both the vascular endothelium and pigment epithelium possess a well-developed junctional complex that includes both adherens and tight junctions conferring a high degree of control of solute and fluid permeability. Understanding induction and regulation of the blood-retinal barrier will allow the development of therapies aimed at restoring the barrier when compromised in disease or allowing the specific transport of therapies across this barrier when needed. This chapter will highlight the anatomical structure of the blood-retinal barrier and explore the molecular structure of the tight junctions that provide the unique barrier properties of the blood--retinal barrier.

Intravenously administered gold nanoparticles pass through the blood-retinal barrier depending on the particle size, and induce no retinal toxicity.

The retina maintains homeostasis through the blood-retinal barrier (BRB). Although it is ideal to deliver the drug to the retina via systemic administration, it is still challenging due to the BRB strictly regulating permeation from blood to the retina. Herein, we demonstrated that intravenously administered gold nanoparticles could pass through the BRB and are distributed in all retinal layers without cytotoxicity. After intravenous injection of gold nanoparticles into C57BL/6 mice, 100 nm nanoparticles were not detected in the retina whereas 20 nm nanoparticles passed through the BRB and were distributed in all retinal layers. 20 nm nanoparticles in the retina were observed in neurons (75 +/- 5%), endothelial cells (17 +/- 6%) and peri-endothelial glial cells (8 +/- 3%), where nanoparticles were bound on the membrane. In the retina, cells containing nanoparticles did not show any structural abnormality and increase of cell death compared to cells without nanoparticles. Gold nanoparticles never affected the viability of retinal endothelial cells, astrocytes and retinoblastoma cells. Furthermore, gold nanoparticles never led to any change in expression of representative biological molecules including zonula occludens-1 and glut-1 in retinal endothelial cells, neurofilaments in differentiated retinoblastoma cells and glial fibrillary acidic protein in astrocytes. Therefore, our data suggests that small gold nanoparticles (20 nm) could be an alternative for drug delivery across the BRB, which could be safely applied in vivo.

Effect of fat embolism with triolein emulsion on blood-retinal barrier.

BACKGROUND: Triolein emulsion is under investigation for supplemental use to open the blood-brain barrier during chemotherapy. The effects of triolein emulsion on the blood-retinal barrier (BRB) were investigated. METHODS: Fat emboli were induced in 20 cats by injecting triolein emulsion through the carotid artery. At 30 min, 4, 12 and 48 h after embolization, electroretinography (ERG) and fluorescein angiography (FA) were performed. The eyeballs were enucleated for transmission electron-microscopic study. RESULTS: FA revealed multiple leaking points at 30 min, and prominent diffuse leakage at 4 h when scotopic b-waves showed significant differences between the study and control eyes. Multiple focal disruptions of the blood vessels by fat vacuoles were found with electron microscopic study. ERG improved at 12 and 48 h, and the BRB appeared to be recovered on FA and electron microscopic studies after 48 h. CONCLUSION: An experimental embolism with triolein emulsion disrupted the blood retinal barrier. Delayed maximal change was observed, and it could be implicated in the latent interval of clinical fat embolism syndrome.

Expression of alpha-dystrobrevin in blood-tissue barriers: sub-cellular localisation and molecular characterisation in normal and dystrophic mice.

The alpha- and beta-dystrobrevins (DBs) belong to a family of dystrophin-related and dystrophin-associated proteins that are members of the dystrophin-associated protein complex (DAPC). This complex provides a link between the cytoskeleton and the extracellular matrix or other cells. However, specific functions of the two dystrobrevins remain largely unknown, with alpha-DB being believed to have a role mainly in skeletal muscle. Here, we describe previously unknown expression patterns and the localisation and molecular characteristics of alpha-DB isoforms in non-muscle mouse tissues. We demonstrate a highly specific sub-cellular distribution of alpha-DB in organs forming blood-tissue barriers. We show alpha-DB expression and localisation in testicular Sertoli cells, stomach and respiratory epithelia and provide electron-microscopic evidence for its immunolocalisation in these cells and in the central nervous system. Moreover, we present the molecular characterisation of alpha-DB transcript in these tissues and provide evidence for a distinct heterogeneity of associations between alpha-DB and dystrophins and utrophin in normal and dystrophic non-muscle tissues. Together, our results indicate that alpha-DB, in addition to its role in skeletal muscle, may also be required for the proper function of specific non-muscle tissues and that disruption of DAPC might lead to tissue-blood barrier abnormalities.

Structural changes in components of the blood-retina barrier in rat retina during photodamage in alloxan diabetes and their correction with ascovertin.

The outer part of the blood-retina barrier was most sensitive to light exposure (6000 lx, 6 h) during photodamage. It was manifested in hemodynamic disturbances, endothelial dysfunction, and focal death of the pigment epithelium. The photo effects increased during alloxan diabetes. The specific area of open vessels decreased, while the number of thrombotic vessels in the choroid increased. Administration of ascovertin improved hemodynamic parameters of the eye and decreased the specific area of focal damage.