Andrew Tsin

The Role of Microglia in Diabetic Retinopathy

There is growing evidence that chronic inflammation plays a role in both the development and progression of diabetic retinopathy. There is also evidence that molecules produced as a result of hyperglycemia can activate microglia. However the exact contribution of microglia, the resident immune cells of the central nervous system, to retinal tissue damage during diabetes remains unclear. Current data suggest that dysregulated microglial responses are linked to their deleterious effects in several neurological diseases associated with chronic inflammation. As inflammatory cytokines and hyperglycemia disseminate through the diabetic retina, microglia can change to an activated state, increase in number, translocate through the retina, and themselves become the producers of inflammatory and apoptotic molecules or alternatively exert anti-inflammatory effects. In addition, microglial genetic variations may account for some of the individual differences commonly seen in patients susceptibility to diabetic retinopathy.

Streptozotocin-induced diabetes in rats is associated with impaired metabolic availability of vitamin A (retinol)

Using streptozotocin-induced diabetic Wistar rats, studies were carried out to examine the metabolic availability of vitamin A in the plasma, liver and the retina of the eye. Control and diabetic rats were fed ad lib. on a semi-purified diet either with or without (basal) vitamin A supplementation, or pair-fed on the basal diet for 4 weeks. Despite the fact that diabetic rats consumed 48% more feed, they had lower plasma concentrations of retinol (P < 0.003). The decrease in plasma retinol concentration was a response to diabetes (or diabetes-induced trauma), since neither pair-feeding (P < 0.01) nor vitamin A supplementation altered this effect (P < 0.05). Furthermore, the hepatic concentrations of the vitamin in these animals remained elevated and this increase was greater in the supplemented diabetic group (P < 0.001). Decreases in 11-cis retinal (a component of rhodopsin) concentrations in the retina were also observed in diabetic animals. The increased hepatic and the decreased plasma and retina vitamin A levels suggest a defect in the transport of the vitamin from the liver.

Bone morphogenetic protein‐4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non‐diabetic human donors and from adult retinal pigment epithelium‐19 (ARPE‐19) cells expressed significant bone morphogenetic protein‐4 (BMP‐4) message. In addition, ARPE‐19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP‐4 into the conditioned media (CM). However, the effect of BMP‐4 on the release of VEGF by ARPE‐19 cells has not been studied. Accordingly, ARPE‐19 cells were treated with BMP‐4 to determine VEGF secretion. BMP‐4 and VEGF levels in the CM and cell lysates were measured by enzyme‐linked immunosorbent assay (ELISA). Cells treated with exogenous BMP‐4 had higher VEGF in the CM and this treatment effect was dose‐ and time‐dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin‐D (ACT) significantly reduced VEGF secretion from cells treated with BMP‐4, suggesting that the BMP‐4‐induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP‐4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells. J. Cell. Biochem. 98: 1196–1202, 2006.

Distribution of ascorbate in the retina, subretinal fluid and pigment epithelium

The posterior segment of the eye was divided into four compartments: retinal cytosol (R), subretinal fluid on the retinal surface (S/R), retinal pigment epithelial (RPE) cytosol, and subretinal fluid on the RPE surface (S/RPE). The volume of each compartment was estimated from the dilution of creatinine (in the extraction buffer) by the endogenous tissue fluid. The ascorbate concentrations in R, S/R, S/RPE, and RPE were 20.6, 12.3, 3.7, and 5.8 mg/dl respectively. Dehydroascorbate was observed only in the RPE and S/RPE. The decreasing ascorbate concentration from the retina to RPE, and the distribution of dehydroascorbate suggest a movement of ascorbate from the vitreous cavity into the subretinal space. The permeability of retinal cell layers to ascorbate was confirmed by the high radioactivity observed in the subretinal space after an intravitreal injection of C14-ascorbate. The occurrence of dehydroascorbate in the RPE and the S/RPE indicates the presence of oxidative reaction of ascorbate in these compartments, where light induced free radicals are located.

Optical scattering, absorption, and polarization of healthy and neovascularized human retinal tissues

The optical scattering, absorption, and polarization properties of human retinal tissues are investigated for a number of laser wavelengths in the visible range. The indices of refraction of these tissues are determined by applying Brewsters law. The inverse adding doubling method based on the diffusion approximation and radiative transport theory is applied to the measured values of total diffuse transmission, total diffuse reflection, and index of refraction to determine the optical absorption, scattering, and scattering anisotropy coefficients of the intact retinal tissues from healthy and diseased (neovascularized) human eyes. The polarization studies show that the retinal tissues possess significant intrinsic polarization characteristics, that are more pronounced in diseased tissues than in healthy tissues.

Content of Lipid Nutrients in the Milk of Fulani Women

Little is known about the nutrition of the infants of the Fulani, migratory nomads of the western Sahel of Africa. Milk was collected from 18 Fulani women 10 to 30 days postpartum and the fatty acid compositions of the triacylglycerol and phospholipid fractions were determined by capillary gas-liquid chromatography. De novo fatty acids (10:0-14:0) comprised 36.3 12.7% of fatty acids of the triacylglycerols. Compared to the milk of various populations worldwide, the milk of the Fulani women contained adequate proportions of oxlinolenic acid (0.50 ± 0.16%) and arachidonic acid (0.42 ± 0.22%), but relatively low amounts of linoleic acid (9.95 ± 2.13%) and docosahexaenoic acid (DHA) (0.15± 0.08%). In addition, the milk of the Fulani women contained adequate concentrations of P-carotene (1.58 ± 0.69 Rtgfdl) and vitamin A (42.7 ± 40.3,ug/dl), but very low levels of vitamin E (0.1 1 ± 0.10 mg/dl). These data indicate that exclusively breastfed infants of Fulani women were receiving relatively low amounts of critical fatty acids and vitamin E.

Distribution of 11-cis LRAT, 11-cis RD and 11-cis REH in bovine retinal pigment epithelium membranes

Our recent finding of the co-localization of 11-cis retinyl esters and 11-cis retinyl ester hydrolase (11-cis REH) activity in bovine retinal pigment epithelium (RPE) plasma membrane (PM) has led us to explore the possibility that the PM may provide 11-cis retinal for rhodopsin regeneration. In the RPE, visual chromophore is synthesized via a membrane associated 11-cis retinol dehydrogenase (11-cis RD). Accordingly, bovine RPE membranes enriched with either endoplasmic reticulum (ER) or plasma membrane (PM) enzyme markers were prepared and assayed for visual cycle enzyme activities. Pronounced 11-cis RD activity was associated with both ER- and PM-enriched membrane fractions. In contrast, 11-cis REH activity was mostly recovered in PM-enriched fractions while LRAT activity was found only in ER-enriched membranes. The finding that both 11-cis retinol and 11-cis retinal can be produced at the PM of the bovine RPE strongly suggests that 11-cis retinyl esters at this subcellular locale serve as a precursor of visual chromophore for pigment regeneration.

Glucose and TGFβ2 modulate the viability of cultured human retinal pericytes and their VEGF release

Purpose: Determine the effects of glucose and exogenous TGFβ2 on viability and VEGF release by human retinal pericytes (HRP). Methods: Human retinal pericytes (HRP) were cultured in 5 mM (physiologic) or high (18 mM) glucose with or without added TGFβ2. Viable cells were counted; TGFβ2 and VEGF in the conditioned media (CM) were measured by ELISA. Results: High glucose significantly reduced viable cell number and increased the levels of TGFβ2 and VEGF. TGFβ2 caused a significant dose-dependent effect on viable cell number and on the level of VEGF secreted into the CM by HRP in physiologic glucose, decreasing viable cell number, and increasing VEGF release per 1000 cells at a low concentration (0.1 ng/ml) and increasing viable cell number and decreasing VEGF release per 1000 cells at higher concentrations (1.0 and 10 ng/ml). TGFβ2 affected neither parameter in high glucose. Conclusions: Elevated glucose decreased HRP viability and modulated changes in TGFβ2 and VEGF release. This suggests a novel mechanism for HRP dropout in diabetic retinopathy.

Distribution of retinoids in different compartments of the posterior segment of the rabbit eye

Using high-performance liquid chromatography, the amounts of all-trans retinol, retinal and retinyl palmitate were measured in the following ocular tissues and fluid of the light (LA) and dark adapted (DA) rabbit: cytosol and membrane fractions of the retina (R/C and R/M), cytosol and membrane fractions of the retinal pigment epithelium (RPE/C and RPE/M), subretinal fluid collected from the inter-photoreceptor matrix (S/R) and the matrix between apical microvilli of the RPE (S/RPE). The total amount of all-trans retinol extracted from LA eyes, 2.74 nmol per eye, was ten times greater than the amount extracted from DA eyes. In the LA eye, most of the all-trans retinol was extracted from the membrane fraction of the retina (67%); in the DA eye, most of the retinol was extracted from the cytosol fraction of the retina (58%). In contrast, the DA eye yielded more all-trans retinal (9.84 nmol) than the LA eye (5.80 nmol) and most of this retinoid was recovered from the cytosol and membrane fractions of the retina. A higher amount of all-trans retinyl palmitate was recovered from the LA eye (5.88 nmol) than the DA eye (2.02 nmol). Although most of this retinyl palmitate was extracted from the cytosol fraction of the RPE (45%, LA eye), appreciable amounts were found in all other ocular compartments. The amount of retinyl palmitate in the LA eye exceeded that of the DA eye in every compartment examined in the present study, suggesting a possible important role of retinyl esters in the visual cycle.

Enhancement of nonlinear optical properties of BaTiO3 nanoparticles by the addition of silver seeds.

Barium titanate (BaTiO3) is a technologically important material because of its nonlinear properties, such as its strong second harmonic generation and high third order susceptibility. While many nonlinear effects have been extensively studied on the bulk scale, there are still questions regarding the strength of nonlinear effects in nanoparticles. The nonlinear properties of BaTiO3 nanoparticles and nanorods have been studied using the closed aperture z-scan technique. Silver was then grown photochemically on the surface of the BaTiO3 nanoparticles, and it was found that the third order susceptibility increases dramatically.