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Purpose: To investigate light-enhanced molecular movement as a potential technology for drug delivery. To do this, we developed an in vitro eye model while representing similar concentration gradient conditions and compositions found in the eye.Methods: The eye model unit was fabricated by inserting a cross-linked type I collagen membrane in a spectrophotometer cuvette with 1% hyaluronic acid as the drug recipient medium. Photokinetic delivery was studied by illuminating 1 mg/mL methotrexate (MTX) placed in the drug donor compartment on top of the membrane, with noncoherent 450 nm light at 8.2 mW from an LED source pulsed at 25 cycles per second, placed in contact with the solution. A modified UV-visual spectrophotometer was employed to rapidly determine the concentration of MTX, at progressive 1 mm distances away from the membrane, within the viscous recipient medium of the model eye after 1 h.Results: A defined, progressive concentration gradient was observed within the nonagitated drug recipient media, diminishing with greater distances from the membrane. Transport of MTX through the membrane was significantly enhanced (ranging from 2 to 3 times, P < 0.05 to P ≤ 0.001) by photokinetic methods compared with control conditions by determining drug concentrations at 4 defined distances from the membrane. According to scanning electron microscopy images, no structural damage or shunts were created on the surface of the cross-linked gelatin membrane.Conclusion: The application of pulsed noncoherent visible light significantly enhances the permeation of MTX through a cross-linked collagen membrane and hyaluronic acid recipient medium without causing structural damage to the membrane. [ABSTRACT FROM AUTHOR]

Exposure of biological chromophores to ultraviolet radiation can lead to photochemical damage. However, the role of visible light, particularly in the blue region of the spectrum, has been largely ignored. To test the hypothesis that blue light is toxic to non-pigmented epithelial cells, confluent cultures of human primary retinal epithelial cells were exposed to visible light (390-550 nm at 2.8 milliwatts/cm²) for up to 6 h. A small loss of mitochondrial respiratory activity was observed at 6 h compared with dark-maintained cells, and this loss became greater with increasing time. To investigate the mechanism of cell loss, the damage to mitochondrial and nuclear genes was assessed using the quantitative PCR. Light exposure significantly damaged mitochondrial DNA at 3 h (0.7 lesion/10 kb DNA) compared with dark-maintained controls. However, by 6 h of light exposure, the number of lesions was decreased in the surviving cells, indicating DNA repair. Isolated mitochondria exposed to light generated singlet oxygen, superoxide anion, and the hydroxyl radical. Antioxidants confirmed the superoxide anion to be the primary species responsible for the mitochondrial DNA lesions. The effect of lipofuscin, a photoinducible intracellular generator of reactive oxygen intermediates, was investigated for comparison. Exposure of lipofuscin-containing cells to visible light caused an increase in both mitochondrial and nuclear DNA lesions compared with non-pigmented cells. We conclude that visible light can cause cell dysfunction through the action of reactive oxygen species on DNA and that this may contribute to cellular aging, age-related pathologies, and tumorigenesis. [ABSTRACT FROM AUTHOR]

Oxidative stress is believed to contribute to the pathogenesis of many diseases, including age-related macular degeneration (AMD). Although the vision loss of AMD results from photoreceptor damage in the central retina, the initial pathogenesis involves degeneration of RPE cells. Evidence from a variety of studies suggests that RPE cells are susceptible to oxidative damage. Mitochondrial DNA (mtDNA) is particularly prone to oxidative damage compared to nuclear DNA (nDNA). Using the quantitative PCR assay, a powerful tool to measure oxidative DNA damage and repair, we have shown that human RPE cells treated with H2O2 or rod outer segments resulted in preferential damage to mtDNA, but not nDNA; and damaged mtDNA is not efficiently repaired, leading to compromised mitochondrial redox function as indicated by the MTT assay. Thus, the susceptibility of mtDNA to oxidative damage in human RPE cells, together with the age-related decrease of cellular anti-oxidant system, provides the rationale for a mitochondria-based model of AMD. [Copyright &y& Elsevier]

The integrity of the retinal pigment epithelium, especially that of the macula is essential for the preservation of vision into old age. The chronic exposure to sunlight and peroxidized lipids from phagocytized photoreceptor outer segments imposes a high level of oxidative stress on the retinal tissues, which increases with age as antioxidant protection declines and therefore could accelerate apoptosis. Bcl-2 known to facilitate mitochondrial DNA repair and cellular survival in other tissues was overexpressed in a single clone of human retinal pigment epithelium cells after stable transfection with humanbcl-2 in ρSFV-neoexpression factor. Near confluent cells (2nd–4th generation permanently bcl-2 transfected) were protected from mitochondrial dysfunction after exposure to H2O2 up to 150 μM. With 200 μM H2O2, function in transfected cells declined by only 25% control activity as determined by MTT reduction assays, compared to wild type and vector only transfected cells expressing normal bcl-2 levels. Similarly the bcl-2 -transfected cells were more resistant to mitochondrial DNA damage after H2O2 treatment than the other groups and suffered 50% less damage after exposure to 200 μM H2O2, as assayed by quantitative polymerase chain reaction assays. These data suggest that bcl-2 overexpression protects human RPE cells from mitochondrial respiratory dysfuction, mitochondrial DNA damage and promotes cellular survival in response to oxidative stress induced by H2O2. [Copyright &y& Elsevier]

Abstract: Mitochondria are central to retinal cell function and survival. There is increasing evidence to support an association between mitochondrial dysfunction and a number of retinal pathologies including age-related macular degeneration (AMD), diabetic retinopathy and glaucoma. The past decade has highlighted mitochondrial genomic instability as an important factor in mitochondrial impairment culminating in age-related changes and age-related pathology. This represents a combination of the susceptibility of mitochondrial DNA (mtDNA) to oxidative damage and a limited base excision repair pathway. This random cumulative mtDNA damage leads to cellular heteroplasmy and, if the damage affects a sufficient proportion of mitochondria within a given cell, results in loss of cell function and greater susceptibility to stress. mtDNA damage is increased in the neural retina and RPE with ageing and appears to be greatest in AMD. It thus appears that the mitochondrial genome is a weak link in the antioxidant defenses of retinal cells and that deficits in mitochondrial DNA (mtDNA) repair pathways are important contributors to the pathogenesis of retinal degeneration. Specifically targeting mitochondria with pharmacological agents able to protect against oxidative stress or promote repair of mtDNA damage may offer potential alternatives for the treatment of retinal degenerations such as AMD. [Copyright &y& Elsevier]

Purpose . To investigate the interrelationships between DNA damage,mitochondrial activity, and apoptosis in retinal pigment epithelial cells(RPE) after exposure to rod outer segments (ROS). Methods . After incubation of cultured human RPE with ROS, mitochondrialredox function was evaluated from MTT reduction. Mitochondrial (mt) and nuclear(n) DNA damage were determined by quantitative polymerase chain reactions(QPCR). Apoptotic RPE cells were detected by binding of annexin V to phosphatidylserine (PS) using fluorescence microscopy. The expression of the pro-apoptoticproteins, p53 and p21[sup waf-1] , and DNA repair enzymes, apurinic/apyrimidinicendonuclease (APE[sup ref-1] ) and DNA polymerase β (β-pol) werequantitatively determined by Western blotting analysis. Results . Mitochondrial function decreased by 20 ± 5% andannexin V immunofluorscent binding was enhanced after exposure of cells tophysiological levels of ROS (3.8 × 10[sup 6] cm[sup -2] )for 4 h. MtDNA was preferentially damaged after exposure to ROS with increasedlesion frequencies of 1.49 ± 0.37 and 2.2 ± 0.14 per 10 kb basepairs (bp), respectively after 5 and 7 h contact, compared to untreated controls(zero class damage). APE[sup ref-1] expression increased more than 340%above controls after exposure to ROS for 7 and 24 h. The expression of β-polin cultures increased 110% above controls after 24 h contact with the ROS.The expression of p53 and p21 in cells increased 100 and 38% above controlsafter 24 h exposure to the ROS. Conclusions . Exposure of ROS to ROS induced mtDNA damage and dysfunctionand activated nDNA repair pathways, which did not prevent apoptosis. [ABSTRACT FROM AUTHOR]

PURPOSE. To determine whether hydrogen peroxide (H2O2), a physiological mediator of oxidative stress induces apoptosis in retinal pigment epithelial (RPE) cells. METHODS. To demonstrate that oxidatively stressed retinal pigment epithelial cells undergo apoptosis consequential to mitochondrial dysfunction, biochemical parameters of apoptosis were determined in cultured cells after treatment with 50–200 mM H2O2 for different times. Caspase-3 protease activity was determined from hydrolysis of DEVD-ρ-nitroanilide. Expression of the anti-apoptotic protein, bcl-2 and the pro-apoptotic proteins p53 and p21 were analyzed by western blotting. RESULTS. Caspase-3 activity significantly increased in cells exposed to H2O2. Also, the expression of bcl-2 in cells treated with 200 μM H2O2 was diminished, whereas expression of p53 and p21waf-1 was increased compared to the controls. CONCLUSIONS. Exposure of retinal pigment epithelial cells to concentrations of H2O2 that cause in vitro mitochondrial DNA damage also promotes apoptosis. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the effects of nicotine on retinal alterations in early-stage diabetes in an established rodent model. Materials and Methods: Sprague-Dawley rats were examined using a combination of confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography to determine changes in retinal structure in response to nicotine exposure, diabetes and the combined effects of nicotine and diabetes. Diabetes was induced by a single injection of 65 mg/kg streptozotocin and nicotine injections were administered subcutaneously daily. Retinal thickness in the superior, inferior, nasal and temporal quadrants were determined based on the spectral domain optical coherence tomography (SD-OCT) volume scans (20° × 20°) centered on the optic disc. Segmentation of discrete retinal layers was performed on a subset of SD-OCT cross-sections to further examine changes in each treatment group. Survival of neurons within the ganglion cell layer (GCL) was assessed by confocal morphometric imaging. Results: The control group did not experience any significant change throughout the study. The nicotine treatment group experienced an average decrease in total retinal thickness (TRT) of 9.4 µm with the majority of the loss localized within the outer nuclear layer (ONL) as determined by segmentation analysis ( p < 0.05). The diabetic group exhibited a trend toward decreased TRT while segmentation analysis of the diabetic retinopathy (DR) group revealed significant thinning within the ONL ( p < 0.05). The combination of nicotine and diabetes revealed a significant increase of 8.9 µm in the TRT ( p < 0.05) accompanied by a decrease in the number of GCL neurons. Conclusions: We demonstrated significant temporal changes in retinal morphology in response to nicotine exposure, diabetes and with the combined effects of nicotine and diabetes. These findings may have implications in determining treatment strategies for diabetic patients using products containing nicotine, such as cigarettes, smokeless tobacco, electronic cigarettes or smoking cessation products. [ABSTRACT FROM AUTHOR]

Objective The protease HTRA-1 is closely associated with rheumatoid arthritis (RA). The molecular mechanisms that control HTRA-1 expression are currently unknown. This study was undertaken to determine the regulatory role of Toll-like receptors (TLRs) on HTRA-1 expression in mice with collagen-induced arthritis (CIA) and in synovial cells from RA patients. Methods HTRA-1 messenger RNA and protein production in mouse fibroblasts, mouse macrophages, and freshly isolated RA patient synovial cells treated with TLR ligands were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Arthritis incidence and severity were determined using clinical scores and histopathologic analysis. Involvement of HTRA-1 in lipopolysaccharide (LPS)-increased arthritis incidence and severity in mice was determined using anti-HTRA-1 monoclonal antibody. The signal pathways involved in HTRA-1 expression were accessed by specific inhibitors, RNA interference, dual-luciferase reporter, and chromatin immunoprecipitation methods. Results LPS and tenascin-C, but not the other TLR ligands tested, strongly induced HTRA-1 expression. LPS significantly increased HTRA-1 expression in the joint tissue as well as arthritis incidence and severity in mice with CIA. Blocking HTRA-1 by antibody significantly decreased LPS-promoted CIA severity. Inhibiting NF-κB significantly decreased LPS-induced HTRA-1 expression in mouse and human cells. Dual-luciferase reporter assay and ChIP analysis showed that p65 directly binds to HTRA-1 promoter (amino acid 347). Conclusion Our findings indicate that TLR-4 activation increases HTRA-1 expression through the NF-κB pathway in fibroblasts and macrophages. HTRA-1 expression is involved in the enhancing effects of LPS on CIA. This study offers new insights into the regulation of HTRA-1 expression via LPS/TLR-4 and the role of HTRA-1 in RA pathogenesis. [ABSTRACT FROM AUTHOR]

Abstract: Ocular structural and functional changes, including optic disk edema and reduction of near visual acuity, have been recently described in some astronauts returning from long-duration space travels. It is hypothesized that ocular changes related to spaceflight may occur, in predisposed individuals, as a result of cephalad shift of body fluids, possibly leading to elevated intracranial pressure (ICP). Results from head-down bed-rest studies (used to simulate the effects of microgravity) and from parabolic flight experiments (used to produce transient periods of microgravity) indicate that ocular blood flow and intraocular pressure (IOP) may undergo changes in a low-gravity environment. Recent studies suggest that changes in translaminar pressure (i.e., IOP minus ICP) may be implicated in the pathophysiology of optic disk neuropathies. Because postural changes exert an effect on both IOP and ICP, the head-down bed-rest analog may also be used as a platform to characterize the relationship between IOP and ICP, and their reciprocal influence in the pathophysiology of conditions such as optic disk edema or glaucoma. [Copyright &y& Elsevier]

Oxidative stress is involved in the pathogenesis of age-related macular degeneration (AMD). Administration of conventional antioxidants has been shown to slow the progression of AMD and vision loss. Melatonin, an endogenous neurohormone produced by the pineal gland and retina, has been reported to be a potent antioxidant and free radical scavenger. In this study we tested whether melatonin can protect retinal pigment epithelial (RPE) cells against hydrogen peroxide (H2O2)-induced cell death. Since mitochondrial DNA (mtDNA) is preferentially susceptible to oxidative damage, we tested whether melatonin can reduce H2O2-induced mtDNA lesions. A human RPE cell line (ARPE-19) was cultured and exposed to H2O2 (100 and 200 μm) for 1 hr to induce cell death. Prior to H2O2 treatment, cells were treated with various concentrations (0·1–200 μm) of melatonin for 2, 24 or 72 hr. Control cells received either melatonin or ethanol alone. Cell viability, as determined by MTT assay, showed no significant (P>0·05) protection against H2O2 toxicity in cells receiving 2- and 24-hr pretreatment of melatonin at either concentration. However, when melatonin was administered diurnally for 3 consecutive days, this prolonged treatment markedly reduced H2O2-induced cell death (P<0·05). MtDNA damage, as assessed with quantitative PCR, was significantly decreased (P=0·031) in RPE cells pretreated with melatonin as compared to those without melatonin treatment. These results suggest that melatonin may play a role in protecting RPE cells from oxidative stress. [Copyright &y& Elsevier]

: PurposeTo examine the factors affecting visual outcome after phacoemulsification and evaluate the use of preoperative visual potential in assessing the visual prognosis in diabetic patients.: SettingDepartment of Ophthalmology, University of Texas Medical Branch, Galveston, Texas, USA.: MethodsIn a retrospective chart review of 1345 consecutive patients who had uneventful small-incision phacoemulsification, operated eyes from 106 diabetic and 55 nondiabetic control patients were selected. Data on demographics, level of retinopathy, perioperative glycosylated hemoglobin (HbAIc), surgical duration, preoperative best corrected visual acuity (BCVA), and visual potential were collected.: ResultsThe age, sex, preoperative BCVA, and visual potential in the diabetic and control eyes were comparable. Throughout the postoperative period, BCVA was worse of the diabetic group. At 1 year, BCVA was 20/40 in 82.1% of the diabetic group and 94.7% of the control group (P = .01). The most important factors affecting postoperative BCVA included coexisting diabetes and preoperative level of retinopathy. No correlation was found between perioperative and postoperative BCVA. Diabetic patients were less likely than control patients to achieve a BCVA better than or equal to the preoperative visual potential at 4 years (hazard ratio 0.6; 95% confidence interval, 0.4-0.9; P = .011). Patients with nonproliferative diabetic retinopathy were nearly 5 times less likely (P = .023) and patients with proliferative diabetic retinopathy 30 times less likely (P < .0001) to achieve a postoperative BCVA of 20/40 than diabetic patients without retinopathy.: ConclusionsAlthough uneventful small-incision phacoemulsification improved visual acuity in diabetic patients, this group had an overall worse visual outcome than nondiabetic patients. The most important predictors of visual outcome were coexisting diabetes and the extent of preoperative retinopathy. Methods used to assess preoperative visual potential provided a reasonable estimate of postoperative BCVA in diabetic patients. Given the inverse association between the level of retinopathy and visual outcome, it may be better to perform cataract extraction in diabetic patients during earlier stages of retinopathy. [Copyright &y& Elsevier]