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Objective To demonstrate the existence of lymphatics in the canine anterior uvea using lymphatic-specific markers Lyve-1, Prox-1, and podoplanin, the endothelial cell marker CD31, and basement membrane matrix marker collagen IV. Design Prospective Study. Animals Eight normal globes from animals euthanized for unrelated health problems. Procedures Sagittally cut serial sections of six normal canine eyes were immunofluorescence double-stained with Lyve-1 and CD31 and single-stained with colorimetric Prox-1 and collagen IV. Three serial sections from 2 additional eyes were cut in the coronal plane at the level of the ciliary body and immunofluorescence double-stained with Lyve-1 and CD31 to map lymphatic channel distribution. Lymphatics from normal canine lymph nodes were used for validation of podoplanin. Results Four of 6 of the sagitally sectioned eyes had Lyve-1-positive lymphatic-like structures that were distinct from CD31-positive blood vessels in the iris base and ciliary body. Both of the coronally sectioned globes had Lyve-1-positive lymphatic-like structures in the ciliary body. The location of these structures was evaluated and found to be diffusely present circumferentially around the ciliary body. Conclusion and clinical relevance These results support the existence of lymphatic channels in the anterior uveal tract of the canine eye. This could indicate the presence of a novel uveolymphatic outflow pathway, which may play a role in aqueous humor outflow. Future studies are needed to confirm the existence and elucidate the role of this proposed uveolymphatic outflow pathway and potentially develop novel treatment options for managing glaucoma.

Purpose To evaluate the tissue-level effects of goniotomy techniques on human trabecular meshwork (TM). Design Laboratory investigation. Methods The TM from human cadaveric corneal rim tissue was treated using 4 techniques: (1) microvitreoretinal (MVR) blade; (2) 360° trabeculotomy with 5-0 prolene suture; (3) the Kahook Dual Blade (KDB) Glide® device; (4) TrabEx™ device; tissue samples underwent standard histologic processing with H&E stain followed by comparative analyses. Results The MVR blade exhibited incision of TM extending into the scleral wall. The TrabEx device removed a small portion of TM with large leaflet tissue remnants in all treated areas. 360° suture trabeculotomy resulted in incision of the TM proximate to Schwalbe's line with no excised tissue evident in all treated areas. Areas treated with the KDB Glide device resulted in nearly complete excision of TM without injury to surrounding tissues. Conclusion The various methods used for performing goniotomy or trabeculotomy resulted in varying degrees of incision or excision of TM. Only the KDB Glide device resulted in reliable excision of TM with the other devices producing incision or variable excision of tissue. Clinical correlation is required to better understand the implications of the current findings when using these methods to lower intraocular pressure in eyes with glaucoma.

Chloropicrin (CP), a warfare agent now majorly used as a soil pesticide, is a strong irritating and lacrimating compound with devastating toxic effects. To elucidate the mechanism of its ocular toxicity, toxic effects of CP (0-100 μM) were studied in primary human corneal epithelial (HCE) cells. CP exposure resulted in reduced HCE cell viability and increased apoptotic cell death with an up-regulation of cleaved caspase-3 and poly ADP ribose polymerase indicating their contribution in CP-induced apoptotic cell death. Following CP exposure, cells exhibited increased expression of heme oxygenase-1, and phosphorylation of H2A.X and p53 as well as 4-hydroxynonenal adduct formation, suggesting oxidative stress, DNA damage and lipid peroxidation. CP also caused increases in mitogen activated protein kinase-c-Jun N-terminal kinase and inflammatory mediator cyclooxygenase-2. Proteomic analysis revealed an increase in the carbonylation of 179 proteins and enrichment of pathways (including proteasome pathway and catabolic process) in HCE cells following CP exposure. CP-induced oxidative stress and lipid peroxidation can enhance protein carbonylation, prompting alterations in corneal epithelial proteins as well as perturbing signaling pathways resulting in toxic effects. Pathways and major processes identified following CP exposure could be lead-hit targets for further biochemical and molecular characterization as well as therapeutic intervention.

Purpose To quantify the effects of modified Ahmed glaucoma valves® (AGV) with anti-fibrotic plate coatings or a plate surface micro-pattern on outflow resistance and tissue response. Methods Twelve New Zealand rabbits were divided into four groups: commercially available AGV implants (n = 3), AGV with hydrophilic coating (n = 3), AGV with heparin coating (n = 3), and AGV with a plate surface micro-pattern (n = 3). After 6 weeks, the anterior chamber silicone tube was cannulated in situ and perfused with 2.5 μL/min of saline. The pressures were recorded with a perfusion system to measure outflow resistance. The rabbits were then euthanized followed by enucleation of all eyes for bleb histological analyses. Results Hydrostatic pressures were significantly lower in AGVs with the hydrophilic plate coating (mean difference −9.6 mm Hg; p

IntroductionRoughly 13% of all battlefield injuries include some form of ocular trauma. Ocular tissue preservation is critical for wound healing for warfighters with ocular injuries. Our team hypothesized that oxygen plays a vital role in ocular tissue preservation and wound healing and has developed a supersaturated oxygen emulsion (SOE) for the topical treatment of ocular trauma.Materials and MethodsThe partial pressure of oxygen (PO2) was measured in the SOE. Safety and efficacy studies were carried out in primary human corneal epithelial (HCE) cells, as the outermost layer is the first barrier to chemical and mechanical injury. Western blot, scratch assay, and MTT assays were conducted to determine the effect of the SOE on various molecular markers, the rate of scratch closure, and cellular viability, respectively.ResultsData indicate that the SOE releases oxygen in a time-dependent manner, reaching a partial pressure within the emulsion over four times atmospheric levels. Studies in HCE cells indicate that application of the SOE does not lead to DNA damage, promote cell death, or hinder the rate of scratch closure and enhances cellular viability. Preliminary studies were carried out with chloropicrin (CP; developed as a chemical warfare agent and now a commonly used pesticide) as a chemical agent to induce ocular injury in HCE cells. CP exposures showed that SOE treatment reverses CP-induced DNA damage, apoptotic cell death, and oxidative stress markers.ConclusionsMaintaining adequate tissue oxygenation is critical for tissue preservation and wound repair, especially in avascular tissues like the cornea. Further studies examining the application of the SOE in corneal injury models are warranted.

To compare the histological effects of trans-scleral cyclophotocoagulation (TCP) performed with two different probes, the G-probe (IRIDEX Medical Instruments, Mountain View, CA, USA) and the Ciliprobe (Katalyst Surgical, Chesterfield, MO, USA). TCP was performed on two human cadaver eyes from the same corpse. The vertical meridian was marked and opposite sides were treated using either the G-probe or Ciliprobe. The first eye was treated with each probe at 2000ms/2000 mW and the second eye at 3000ms/1500 mW. Histological examination revealed separation and loss of the non-pigmented ciliary epithelium as well as vacuolization in all sections for both probes and settings. Changes to the non-pigmented ciliary epithelium treated at 3000ms/1500 mW were similar between the two probes. A slightly more complete separation of the non-pigmented epithelium was noted on the Ciliprobe treated sections as compared to the G-probe treated sections in the eye treated at 2000ms/2000 mW. Therefore, in human cadaver eyes, both the G-probe and Ciliprobe produced separation, vacuolization, and loss of the non-pigmented ciliary epithelium at two different, clinically utilized settings.

With a possibility for the use of chemical weapons in battlefield or in terrorist activities, effective therapies against the devastating ocular injuries, from their exposure, are needed. Oxygen plays a vital role in ocular tissue preservation and wound repair. We tested the efficacy of supersaturated oxygen emulsion (SSOE) in reducing ex vivo corneal and keratocyte injury from chloropicrin (CP). CP, currently used as a pesticide, is a chemical threat agent like the vesicating mustard agents and causes severe corneal injury. Since our previous study in human corneal epithelial cells showed the treatment potential of SSOE (55 %), we further tested its efficacy in an ex vivo CP-induced rabbit corneal injury model. Corneas were exposed to CP (700 nmol) for 2 h, washed and cultured with or without SSOE for 24 h or 96 h. At 96 h post CP exposure, SSOE treatment presented a healing tendency of the corneal epithelial layer, and abrogated the CP-induced epithelial apoptotic cell death. SSOE treatment also reduced the CP induced DNA damage (H2A.X phosphorylation) and inflammatory markers (e.g. MMP9, IL-21, MIP-1β, TNFα). Further examination of the treatment efficacy of SSOE alone or in combination with other therapies in in vivo cornea injury models for CP and vesicants, is warranted.

Nitrogen mustard (NM) is an analogue of the potent vesicating agent sulfur mustard, with well-established ocular injury models in rabbit eyes to study vesicant-induced ocular toxicity. The effects of NM-exposure to eyes may include irritation, redness, inflammation, fibrosis, epithelial degradation, blurred vision, partial/complete blindness, which may be temporary or permanent, depending on the route, duration, and dosage of exposure. Effective countermeasures against vesicant exposure are presently not available and are warranted in case of any terrorist activity or accidental leakage from stockpiles. Herein, our focus was to evaluate whether dexamethasone (DEX), an FDA approved potent corticosteroid with documented anti-inflammatory activities, could be an effective treatment modality. Accordingly, utilizing NM-induced corneal injuries in rabbit ocular in vivo model, we examined and compared the efficacy of DEX treatments when administration was started at early (2 h), intermediate (4 h), and late (6 h) therapeutic windows of intervention after NM-exposure and administered every 8 h thereafter. The effects of NM-exposure and DEX treatments were evaluated on clinical (corneal opacity, ulceration, and neovascularization), biological (epithelial thickness, epithelial-stromal separation, blood vessels density, and inflammatory cell and keratocyte counts) and molecular (COX-2 and VEGF expression) parameters, at day 1, 3, 7 and 14. Results indicated that DEX treatment markedly and effectively reversed the NM-induced injury markers in rabbit corneas. Early administration of DEX at 2 h was found to be most effective in reversing NM-induced corneal injuries, followed by DEX 4 h and DEX 6 h administration initiation, indicating that DEX has best efficacy at the early therapeutic window in our study model.

Purpose To evaluate the tissue-level effects of goniotomy techniques on human trabecular meshwork (TM). Design Laboratory investigation. Methods The TM from human cadaveric corneal rim tissue was treated using 4 techniques: (1) microvitreoretinal (MVR) blade; (2) 360° trabeculotomy with 5-0 prolene suture; (3) the Kahook Dual Blade (KDB) Glide® device; (4) TrabEx™ device; tissue samples underwent standard histologic processing with H&E stain followed by comparative analyses. Results The MVR blade exhibited incision of TM extending into the scleral wall. The TrabEx device removed a small portion of TM with large leaflet tissue remnants in all treated areas. 360° suture trabeculotomy resulted in incision of the TM proximate to Schwalbe’s line with no excised tissue evident in all treated areas. Areas treated with the KDB Glide device resulted in nearly complete excision of TM without injury to surrounding tissues. Conclusion The various methods used for performing goniotomy or trabeculotomy resulted in varying degrees of incision or excision of TM. Only the KDB Glide device resulted in reliable excision of TM with the other devices producing incision or variable excision of tissue. Clinical correlation is required to better understand the implications of the current findings when using these methods to lower intraocular pressure in eyes with glaucoma.

In general, neurons belonging to the central nervous system (CNS), such as retinal ganglion cells (RGCs), do not regenerate. Due to this, strategies have emerged aimed at protecting and regenerating these cells. Neurotrophic factor (NTF) supplementation has been a promising approach but is limited by length of delivery and delivery vehicle. For this study, we tested a polymeric delivery system (sulfonated reverse thermal gel or SRTG) engineered to deliver cilliary neurotrophic factor (CNTF), while also being injectable. A rat optic nerve crush (ONC) model was used to determine the neuroprotective and regenerative capacity of our system. The results demonstrate that one single intravitreal injection of SRTG-CNTF following ONC showed significant protection of RGC survival at both 1 and 2 week time points, when compared to the control groups. Furthermore, there was no significant difference in the RGC count between the eyes that received the SRTG-CNTF following ONC and a healthy control eye. Intravitreal injection of the polymer system also induced noticeable axon regeneration 500 μm downstream from the lesion site compared to all other control groups. There was a significant increase in Müller cell response in groups that received the SRTG-CNTF injection following optic nerve crush also indicative of a regenerative response. Finally, higher concentrations of CNTF released from SRTG-CNTF showed a protective effect on RGCs and Müller cell response at a longer time point (4 weeks). In conclusion, we were able to show a neuroprotective and regenerative effect of this polymer SRTG-CNTF delivery system and the viability for treatment of neurodegenerations.

Lewisite (LEW), a potent arsenical vesicating chemical warfare agent, poses a continuous risk of accidental exposure in addition to its feared use as a terrorist weapon. Ocular tissue is exquisitely sensitive to LEW and exposure can cause devastating corneal lesions. However, detailed pathogenesis of corneal injury and related mechanisms from LEW exposure that could help identify targeted therapies are not available. Using an established consistent and efficient exposure system, we evaluated the pathophysiology of the corneal injury in New Zealand white rabbits following LEW vapor exposure (at 0.2 mg/L dose) for 2.5 and 7.5 min, for up to 28 day post-exposure. LEW led to an increase in total corneal thickness starting at day 1 post-exposure and epithelial degradation starting at day 3 post-exposure, with maximal effect at day 7 postexposure followed by recovery at later time points. LEW also led to an increase in the number of blood vessels and inflammatory cells but a decrease in keratocytes with optimal effects at day 7 postexposure. A significant increase in epithelial-stromal separation was observed at days 7 and 14 post 7.5 min LEW exposure. LEW also caused an increase in the expression levels of cyclooxygenase-2, IL-8, vascular endothelial growth factor, and matrix metalloproteinase-9 at all the study time points indicating their involvement in LEW-induced inflammation, vesication, and neovascularization. The outcomes here provide valuable LEW-induced corneal injury endpoints at both lower and higher exposure durations in a relevant model system, which will be helpful to identify and screen therapies against LEW-induced corneal injury.

Sensory-somatic nervous system neurons, such as retinal ganglion cells (RGCs), are typically thought to be incapable of regenerating. However, it is now known that these cells may be stimulated to regenerate by providing them with a growth permissive environment. We have engineered an injectable microenvironment designed to provide growth-stimulating cues for RGC culture. Upon gelation, this injectable material not only self-assembles into laminar sheets, similar to retinal organization, but also possesses a storage modulus comparable to that of retinal tissue. Primary rat RGCs were grown, stained, and imaged in this three-dimensional scaffold. We were able to show that RGCs grown in this retina-like structure exhibited characteristic long, prominent axons. In addition, RGCs showed a consistent increase in average axon length and neurite-bearing ratio over the 7 day culture period, indicating this scaffold is capable of supporting substantial RGC axon extension.

Purpose Cataract surgery is a procedure by which the lens fiber cell mass is removed from its capsular bag and replaced with a synthetic intraocular lens. Postoperatively, remnant lens epithelial cells can undergo an aberrant wound healing response characterized by an epithelial-to-mesenchymal transition (EMT), leading to posterior capsular opacification (PCO). Aldose reductase (AR) inhibition has been shown to decrease EMT markers in cell culture models. In this study, we aim to demonstrate that AR inhibition can attenuate induction of EMT markers in an in vivo model of cataract surgery. Methods A modified extracapsular lens extraction (ECLE) was performed on C57BL/6 wildtype, AR overexpression (AR-Tg), and AR knockout mice. Immunofluorescent staining for the myofibroblast marker α-smooth muscle actin (α-SMA), epithelial marker E-cadherin, and lens fiber cell markers αA-crystallin and Aquaporin 0 was used to characterize postoperative PCO. Quantitative reverse transcription PCR (qRT-PCR) was employed to quantify postoperative changes in α-SMA, vimentin, fibronectin, and E-cadherin. In a separate experiment, the AR inhibitor Sorbinil was applied postoperatively and qRT-PCR was used to assess changes in EMT markers. Results Genetic AR knockout reduced ECLE-induced upregulation of α-SMA and downregulation of E-cadherin. These immunofluorescent changes were mirrored quantitatively in changes in mRNA levels. Similarly, Sorbinil blocked characteristic postoperative EMT changes in AR-Tg mice. Interestingly, genetic AR knockout did not prevent postoperative induction of the lens fiber cell markers αA-crystallin and Aquaporin 0. Conclusions AR inhibition prevents the postoperative changes in EMT markers characteristic of PCO yet preserves the postoperative induction of lens fiber cell markers.

Purpose The purpose of this article was to evaluate how human trabecular meshwork (TM) is influenced by the chronic presence of trabecular bypass implants. Methods Human TM samples were obtained intraoperatively from 3 patients who had previously undergone implantation of a trabecular micro-bypass stent. Trabecular strips were obtained with a goniotomy blade from areas directly adjacent to the stent after stent removal. Tissue samples were preserved, processed, cut, and stained according to standardized laboratory protocol. Harvested samples were compared with human cadaveric TM from an eye without ocular disease as well as TM obtained from a glaucomatous eye without prior stent placement. Results In all samples, a significant increase in the amount of fibrous material compared with cellular material was noted when compared with controls. In a single strip, a basement membrane-like structure was noted, which correlated with a semiopaque membrane noted intraoperatively overlying the stent and adjacent TM. Further, TM cells were absent from areas adjacent to the stent implantation site with related collapse of collagen beams. Conclusions These findings indicate that inflammatory and fibrotic changes are present surrounding the device with clear differences noted when compared with both healthy and glaucomatous controls. These changes suggest a possible etiology for device failure over time. Further studies are necessary to tease out differences in TM tissue reaction to various implant materials as well as to make comparisons to procedures that excise TM.

Aldose reductase (AR) is thought to play a role in the pathogenesis of diabetic eye diseases, including cataract and retinopathy. However, not all diabetics develop ocular complications. Paradoxically, some diabetics with poor metabolic control appear to be protected against retinopathy, while others with a history of excellent metabolic control develop severe complications. These observations indicate that one or more risk factors may influence the likelihood that an individual with diabetes will develop cataracts and/or retinopathy. We hypothesize that an elevated level of AR gene expression could confer higher risk for development of diabetic eye disease. To investigate this hypothesis, we examined the onset and severity of diabetes-induced cataract in transgenic mice, designated AR-TG, that were either heterozygous or homozygous for the human AR (AKR1B1) transgene construct. AR-TG mice homozygous for the transgene demonstrated a conditional cataract phenotype, whereby they developed lens vacuoles and cataract-associated structural changes only after induction of experimental diabetes; no such changes were observed in AR-TG heterozygotes or nontransgenic mice with or without experimental diabetes induction. We observed that nondiabetic AR-TG mice did not show lens structural changes even though they had lenticular sorbitol levels almost as high as the diabetic AR-TG lenses that showed early signs of cataract. Over-expression of AR led to increases in the ratio of activated to total levels of extracellular signal-regulated kinase (ERK1/2) and c-Jun N-terminal (JNK1/2), which are known to be involved in cell growth and apoptosis, respectively. After diabetes induction, AR-TG but not WT controls had decreased levels of phosphorylated as well as total ERK1/2 and JNK1/2 compared to their nondiabetic counterparts. These results indicate that high AR expression in the context of hyperglycemia and insulin deficiency may constitute a risk factor that could predispose the lens to disturbances in signaling through the ERK and JNK pathways and thereby alter the balance of cell growth and apoptosis that is critical to lens transparency and homeostasis.

Sulfur mustard (SM), a potent vesicating chemical warfare agent, and its analog nitrogen mustard (NM), are both strong bi-functional alkylating agents. Eyes, skin, and the respiratory system are the main targets of SM and NM exposure; however, ocular tissue is most sensitive, resulting in severe ocular injury. The mechanism of ocular injury from vesicating agents' exposure is not completely understood. To understand the injury mechanism from exposure to vesicating agents, NM has been previously employed in our toxicity studies on primary human corneal epithelial cells and ex vivo rabbit cornea organ culture model. In the current study, corneal toxicity from NM ocular exposure (1%) was analyzed for up to 28 days post-exposure in New Zealand White male rabbits to develop an acute corneal injury model. NM exposure led to conjunctival and eyelid swelling within a few hours after exposure, in addition to significant corneal opacity and ulceration. An increase in total corneal thickness and epithelial degradation was observed starting at day 3 post-NM exposure, which was maximal at day 14 post-exposure and did not resolve until 28 days post-exposure. There was an NM-induced increase in the number of blood vessels and inflammatory cells, and a decrease in keratocytes in the corneal stroma. NM exposure resulted in increased expression levels of cyclooxygenase-2, Interleukin-8, vascular endothelial growth factor and Matrix Metallopeptidase 9 indicating their involvement in NM-induced corneal injury. These clinical, biological, and molecular markers could be useful for the evaluation of acute corneal injury and to screen for therapies against NM- and SM-induced ocular injury.

Simultaneous spatial temporal focusing (SSTF) is used to deliver microjoule femtosecond pulses with low numerical aperture geometries (

Purpose To visualize and quantify the three-dimensional (3D) spatial relationships of the structures of the aqueous outflow system (AOS) within intact enucleated mouse eyes using spectral two-photon microscopy (TPM) techniques. Methods Spectral TPM, including two-photon autofluorescence (TPAF) and second-harmonic generation (SHG), were used to image the small structures of the AOS within the limbal region of freshly enucleated mouse eyes. Long infrared excitation wavelengths (930 nm) were used to reduce optical scattering and autofluorescent background. Image stacks were collected for 3D image rendering and structural segmentation. For anatomical reference, vascular perfusion with fluorescent-conjugated dextran (150 KDa) and trans-corneal perfusion with 0.1 μm fluorescent polystyrene beads were separately performed to identify the episcleral veins (EV) and the trabecular meshwork (TM) and Schlemm's canal (SC), respectively. Results Three-dimensional rendering and segmentation of spectral two-photon images revealed detailed structures of the AOS, including SC, collector channels (CC), and aqueous veins (AV). The collagen of the TM was detected proximal to SC. The long and short axes of the SC were 82.2 ± 22.2 μm and 6.7 ± 1.4 μm. The diameters of the CC averaged 25.6 ± 7.9 μm where they originated from the SC (ostia), enlarged to 34.1 ± 13.1 μm at the midpoint, and narrowed to 18.3 ± 4.8 μm at the junction of the AV. The diameter of the AV averaged 12.5 ± 3.4 μm. Conclusions Spectral TPM can be used to reconstruct and measure the spatial relationships of both large and small AOS structures, which will allow for better understanding of distal aqueous outflow dynamics.

Aldose reductase (AR) in the lens plays an important role in the pathogenesis of diabetic cataract (DC) by contributing to osmotic and oxidative stress associated with accelerated glucose metabolism through the polyol pathway. Therefore, inhibition of AR in the lens may hold the key to prevent DC formation. Emodin, a bioactive compound isolated from plants, has been implicated as a therapy for diabetes. However, its inhibitory activity against AR remains unclear. Our results showed that emodin has good selectively inhibitory activity against AR (IC50 = 2.69 ± 0.90 μM) but not other aldo-keto reductases and is stable at 37 °C for at least 7 days. Enzyme kinetic studies demonstrated an uncompetitive inhibition against AR with a corresponding inhibition constant of 2.113 ± 0.095 μM. In in vivo studies, oral administration of emodin reduced the incidence and severity of morphological markers of cataract in lenses of AR transgenic mice. Computational modeling of the AR-NADP(+)-emodin ternary complex indicated that the 3-hydroxy group of emodin plays an essential role by interacting with Ser302 through hydrogen bonding in the specificity pocket of AR. All the findings above provide encouraging evidence for emodin as a potential therapeutic agent to prevent cataract in diabetic patients.

Purpose To evaluate the toxic effects and associated mechanisms in corneal tissue exposed to the vesicating agent, nitrogen mustard (NM), a bifunctional alkylating analog of the chemical warfare agent sulfur mustard. Methods Toxic effects and associated mechanisms were examined in maximally affected corneal tissue using corneal cultures and human corneal epithelial (HCE) cells exposed to NM. Results Analysis of ex vivo rabbit corneas showed that NM exposure increased apoptotic cell death, epithelial thickness, epithelial-stromal separation, and levels of vascular endothelial growth factor, cyclooxygenase 2, and matrix metalloproteinase-9. In HCE cells, NM exposure resulted in a dose-dependent decrease in cell viability and proliferation, which was associated with DNA damage in terms of an increase in p53 ser15, total p53, and H2A.X ser139 levels. NM exposure also induced caspase-3 and poly ADP ribose polymerase cleavage, suggesting their involvement in NM-induced apoptotic death in the rabbit cornea and HCE cells. Similar to rabbit cornea, NM exposure caused an increase in cyclooxygenase 2, matrix metalloproteinase-9, and vascular endothelial growth factor levels in HCE cells, indicating a role of these molecules and related pathways in NM-induced corneal inflammation, epithelial-stromal separation, and neovascularization. NM exposure also induced activation of activator protein 1 transcription factor proteins and upstream signaling pathways including mitogen-activated protein kinases and Akt protein kinase, suggesting that these could be key factors involved in NM-induced corneal injury. Conclusions Results from this study provide insight into the molecular targets and pathways that could be involved in NM-induced corneal injuries laying the background for further investigation of these pathways in vesicant-induced ocular injuries, which could be helpful in the development of targeted therapies.

There are no effective and approved therapies against devastating ocular injuries caused by vesicating chemical agents sulfur mustard (SM) and nitrogen mustard (NM). Herein, studies were carried out in rabbit corneal cultures to establish relevant ocular injury biomarkers with NM for screening potential efficacious agents in laboratory settings. NM (100 nmol) exposure of the corneas for 2 h (cultured for 24 h), showed increases in epithelial thickness, ulceration, apoptotic cell death, epithelial detachment microbullae formation, and the levels of VEGF, cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9). Employing these biomarkers, efficacy studies were performed with agent treatments 2 h and every 4 h thereafter, for 24 h following NM exposure. Three agents were evaluated, including prescription drugs dexamethasone (0.1%; anti-inflammatory steroid) and doxycycline (100 nmol; antibiotic and MMP inhibitor) that have been studied earlier for treating vesicant-induced eye injuries. We also examined silibinin (100 µg), a non-toxic natural flavanone found to be effective in treating SM analog-induced skin injuries in our earlier studies. Treatments of doxycycline + dexamethasone, and silibinin were more effective than doxycycline or dexamethasone alone in reversing NM-induced epithelial thickening, microbullae formation, apoptotic cell death, and MMP-9 elevation. However, dexamethasone and silibinin alone were more effective in reversing NM-induced VEGF levels. Doxycycline, dexamethasone and silibinin were all effective in reversing NM-induced COX-2 levels. Apart from therapeutic efficacy of doxycycline and dexamethasone, these results show strong multifunctional efficacy of silibinin in reversing NM-induced ocular injuries, which could help develop effective and safe therapeutics against ocular injuries by vesicants.

The trabecular meshwork (TM) region of the eye is exposed to a constant low-level of oxidative insult. The cumulative damage may be the reason behind age-dependent risk for developing primary open angle glaucoma. Chronic and acute effects of hydrogen peroxide (H(2)O(2)) on TM endothelial cells include changes in viability, protein synthesis, and cellular adhesion. However, little if anything is known about the immediate effect of H(2)O(2) on the biochemistry of the TM cells and the initial response to oxidative stress. In this report, we have used two-photon excitation autofluorescence (2PAF) to monitor changes to TM cell nicotinamide adenine dinucleotide (NADPH). 2PAF allows non-destructive, real-time analysis of concentration of intracellular NADPH. Coupled to reduced glutathione, NADPH, is a major component in the anti-oxidant defense of TM cells. Cultured human TM cells were monitored for over 30 min in control and H(2)O(2)-containing solutions. Peroxide caused both a dose- and time-dependent decrease in NADPH signal. NADPH fluorescence in control and in 4 mM H(2)O(2) solutions showed little attenuation of NADPH signal (4% and 9% respectively). TM cell NADPH fluorescence showed a linear decrease with exposure to 20 mM H(2)O(2) (-29%) and 100 mM H(2)O(2) (37%) after a 30 min exposure. Exposure of TM cells to 500 mM H(2)O(2) caused an exponential decrease in NADPH fluorescence to a final attenuation of 46% of starting intensity. Analysis of individual TM cells indicates that cells with higher initial NADPH fluorescence are more refractive to the apparent loss of viability caused by H(2)O(2) than weakly fluorescing TM cells. We conclude that 2PAF of intracellular NADPH is a valuable tool for studying TM cell metabolism in response to oxidative insult.

We investigated the potential cytotoxicity of various topical ophthalmic glaucoma formulations containing different preservatives in cultured human trabecular meshwork (TM) and non-pigmented ciliary epithelial (NPCE) cell lines.We tested 0.004% travoprost preserved with either 0.015% benzalkonium chloride (BAK), sofZia or 0.001% Polyquad (PQ); and 0.005% latanoprost preserved with 0.020% BAK. We also tested a range of BAK concentrations in balanced salt solution (BSS). TM cells were treated for 10 min at 37°C with solutions diluted 1:10 to mimic the reduced penetration of topical preparations to the anterior chamber. Viability was determined by the uptake of the fluorescent vital dye calcein-AM (n = 6).BAK solutions (diluted 1:10) demonstrated a dose-dependent reduction in cell viability in both cell types (TM and NPCE). With a 1:10 dilution of 0.020% BAK, there were significantly more living NPCE cells (89 ± 6%) than TM cells (57 ± 6%; p0.001). In TM cells, travoprost + BAK had statistically fewer live cells (83 ± 5%) than both travoprost + sofZia (97 ± 5%) and travoprost + PQ (97 ± 6%; p0.05). Compared with BSS-treated NPCE cells, travoprost had statistically fewer live cells (p0.05) when preserved with BAK (85 ± 16%), sofZia (91 ± 6%) or PQ (94 ± 2%).These results demonstrate that substitution of BAK from topical ophthalmic drugs results in greater viability of cultured TM cells, the cells involved in the conventional outflow pathway. Cultured NPCE, responsible for aqueous inflow, appear more resilient to BAK.

To determine the effect of 4 formulations of commercially available prostaglandin analogs (PGAs) on human corneal epithelial cells in vitro.The test solutions (PGAs) examined were tafluprost 0.005% with 0.010% benzalkonium chloride (BAK), travoprost 0.004% with 0.015% BAK, travoprost 0.004% with sofZia, and latanoprost 0.005% with 0.020% BAK. Also tested independently were the 4 respective BAK or sofZia concentrations related to each PGA. Balanced salt solution (BSS) was used as the live control, and a fixative solution containing 70% methanol and 0.2% saponin was used as the dead control. Immortalized human corneal epithelial cells were exposed to test or control solution for 25 min at 37 degrees C and 5% CO(2). A live/dead assay was used to measure the toxicity of the PGAs.The percentage of live cells in the PGA groups ranged from 2% to 72% of the BSS group (live control). The PGA with the highest relative live cell percentage, at 72% of the live control, was travoprost with sofZia. The next highest PGA, exhibiting 14% live cells, was the formulation of travoprost containing BAK. The other 2 PGAs, tafluprost and latanoprost, had few surviving cells, with 3% and 2% live cells, respectively. The BAK concentrations exhibited 4%, 3%, and 3% for the 0.01%, 0.015%, and 0.02% concentrations, respectively. The stand-alone sofZia cell survival was 68% of the live control.All 4 PGA formulations tested demonstrated significantly more toxicity in human corneal epithelial cells than the live control, but there were significant differences among the PGAs. Travoprost with sofZia exhibited the least toxicity, followed by travoprost with BAK, and then tafluprost and latanoprost. The stand-alone preservative systems were also tested and showed similar survival percentages to each respective PGA. The true clinical implications of these findings require further investigation.

The purpose of this study was to determine the effects of two commercially available combination topical ophthalmic medications on human ocular surface cells in vitro.Tissue culture plates (96-well) containing immortalized human corneal and conjunctival epithelial cells were divided into five groups. The test solutions examined were: timolol 0.5%+brimonidine 0.2%, containing 0.0050% benzalkonium chloride (BAK); timolol 0.5%+dorzolamide 2%, containing 0.0075% BAK; and preservative-free artificial tears. Balanced salt solution (BSS) was used as the live control, and a fixative solution containing 70% methanol and 0.2% saponin was used as the dead control. Cells were exposed to 100 microL of test or control solution for 25 minutes at 37 degrees C and 5% carbon dioxide (CO(2)). A live cell assay was used to measure the toxicity of combination treatments compared with BSS controls.Exposure to timolol 0.5%+brimonidine 0.2% resulted in a significantly higher percentage of living conjunctival cells (48%+/-12%) as compared with timolol 0.5%+dorzolamide 2% (10%+/-5%, P0.00001). In corneal cells, testing revealed 12%+/-3% live cells after timolol 0.5%+brimonidine 0.2% exposure compared with 2%+/-3% after timolol 0.5%+dorzolamide 2% (P0.001).Both combination medications demonstrated a significant reduction in the percentage of live corneal and conjunctival epithelial cells compared with control. However, cell cultures exposed to timolol 0.5%+dorzolamide 2% had significantly fewer live cells compared with cell cultures exposed to timolol 0.5%+brimonidine 0.2%. Further studies are needed to better understand the clinical significance of these findings in patients using these medications for chronic treatment of glaucoma and ocular hypertension.

Vacuolar proton-translocating ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although V-ATPases are involved in a number of cellular processes, how the proton pumps are regulated under physiological conditions is not well understood. We have reported that the glycolytic enzyme aldolase mediates V-ATPase assembly and activity by physical association with the proton pump (Lu, M., Holliday, L. S., Zhang, L., Dunn, W. A., and Gluck, S. L. (2001) J. Biol. Chem. 276, 30407-30413 and Lu, M., Sautin, Y., Holliday, L. S., and Gluck, S. L. (2004) J. Biol. Chem. 279, 8732-8739). In this study, we generate aldolase mutants that lack binding to the B subunit of V-ATPase but retain normal catalytic activities. Functional analysis of the aldolase mutants shows that disruption of binding between aldolase and the B subunit of V-ATPase results in disassembly and malfunction of V-ATPase. In contrast, aldolase enzymatic activity is not required for V-ATPase assembly. Taken together, these findings strongly suggest an important role for physical association between aldolase and V-ATPase in the regulation of the proton pump.

Misfolded protein aggregation, including cataract, cause a significant amount of blindness worldwide. α-Crystallin is reported to bind misfolded proteins and prevent their aggregation. We hypothesize that supplementing retina and lens with α-crystallin may help to delay disease onset. The purpose of this study was to determine if αB-crystallin subunits containing a cell penetration peptide (gC-tagged αB-crystallin) facilitate the uptake of wild type αA-crystallin (WT-αA) in lens and retina. Recombinant human αB-crystallin was modified by the addition of a novel cell penetration peptide derived from the gC gene product of herpes simplex virus (gC-αB). Recombinant gC-αB and wild-type αA-crystallin (WT-αA) were purified from E. coli over-expression cultures. After Alexa-labeling of WT-αA, these proteins were mixed at ratios of 1:2, 1:5 and 1:10, respectively, and incubated at 37°C for 4 hours to allow for subunit exchange. Mixed oligomers were subsequently incubated with tissue culture cells or mouse organ cultures. Similarly, crystallin mixtures were injected into the vitreous of rat eyes. At various times after exposure, tissues were harvested and analyzed for protein uptake by confocal microscopy or flow cytometry. Chaperone-like activity assays were performed on α-crystallins ratios showing optimal uptake using chemically-induced or heat induced substrate aggregation assays. As determined by flow cytometry, a ratio of 1:5 for gC-αB to WT-αA was found to be optimal for uptake into retinal pigmented epithelial cells (ARPE-19). Chaperone-like activity assays demonstrated that hetero-oligomeric complex of gC-αB to WT-αA (in 1:5 ratio) retained protein aggregation protection. We observed a significant increase in protein uptake when optimized (gC-αB to WT-αA (1:5 ratio)) hetero-oligomers were used in mouse lens and retinal organ cultures. Increased levels of α-crystallin were found in lens and retina following intravitreal injection of homo- and hetero-oligomers in rats.

Background The findings that α-crystallins are multi-functional proteins with diverse biological functions have generated considerable interest in understanding their role in health and disease. Recent studies have shown that chaperone peptides of α-crystallin could be delivered into cultured cells and in experimental animals with beneficial effects against protein aggregation, oxidation, inflammation and apoptosis. Scope of review In this review, we will summarize the latest developments on the therapeutic potential of α-crystallins and their functional peptides. Major conclusions α-Crystallins and their functional peptides have shown significant favorable effects against several diseases. Their targeted delivery to tissues would be of great therapeutic benefit. However, α-crystallins can also function as disease-causing proteins. These seemingly contradictory functions must be carefully considered prior to their therapeutic use. General significance αA and αB-Crystallin are members of the small heat shock protein family. These proteins exhibit molecular chaperone and anti-apoptotic activities. The core crystallin domain within these proteins is largely responsible for these prosperities. Recent studies have identified peptides within the crystallin domain of both α- and αB-crystallins with remarkable chaperone and anti-apoptotic activities. Administration of α-crystallin or their functional peptides has shown substantial inhibition of pathologies in several diseases. However, α-crystallins have been shown to promote disease-causing pathways. These two sides of the proteins are discussed in this review. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

Purpose. To compare morphologic changes in human trabecular meshwork (TM) after selective laser trabeculoplasty (SLT) and argon laser trabeculoplasty (ALT).Design. Laboratory evaluation of ex vivo human eye TM after laser trabeculoplasty.Methods. Corneoscleral rims from human cadaver eyes were sectioned and treated with varying powers of either SLT or ALT. Specimens were examined using light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM).Results. TEM of SLT at all powers resulted in disrupted TM cells with cracked and extracellular pigment granules. SEM of SLT samples treated at high power revealed tissue destruction with scrolling of trabecular beams. SEM of ALT-treated tissue showed increasing destruction with exposure to higher power. The presence or absence of “champagne” bubbles during SLT did not alter the histologic findings.Conclusions. SLT-treated human TM revealed disruption of TM cells with cracked, extracellular pigment granules, particularly at higher treatment powers. Tissue scrolling was noted at very high SLT energy levels. ALT-treated tissue showed significant damage to both the superficial and deeper TM tissues in a dose-dependent fashion. Further studies are needed to guide titration of treatment power to maximize the IOP-lowering effect while minimizing both energy delivered and damage to target tissues.

Simultaneous spatiotemporal focusing (SSTF) is applied to lens tissue and compared directly with standard femtosecond micromachining of the tissue at the same numerical aperture. Third harmonic generation imaging is used for spatio-temporal characterization of the processing conditions obtained with both a standard and SSTF focus.

PURPOSE The aim of this study was to nondestructively monitor morphological changes to the lipid membranes of primary cultures of living human trabecular meshwork cells (hTMC) without the application of exogenous label. METHODS Live hTMC were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). The hTMC were treated with a commercial formulation of latanoprost (0.5 μg/mL) for 24 hours before imaging. Untreated cells and cells treated with vehicle containing the preservative benzalkonium chloride (BAK; 2 μg/mL) were imaged as controls. After CARS/TPAF imaging, hTMC were fixed, stained with the fluorescent lipid dye Nile Red, and imaged by conventional confocal microscopy to verify lipid membrane structures. RESULTS Analysis of CARS/TPAF images of hTMC treated with latanoprost revealed multiple intracellular lipid membranes absent from untreated or BAK-treated hTMC. Treatment of hTMC with sodium fluoride or ouabain, agents shown to cause morphological changes to hTMC, also did not induce formation of intracellular lipid membranes. CONCLUSIONS CARS microscopy detected changes in living hTMC morphology that were validated by subsequent histological stain. Prostaglandin-induced changes to hTMC involved rearrangement of lipid membranes within these cells. These in vitro results identify a novel biological response to a class of antiglaucoma drugs, and further experiments are needed to establish how this effect is involved in the hypotensive action of prostaglandin analogues in vivo.

PURPOSE To image the cellular and noncellular structures of the retina in an intact mouse eye without the application of exogenous fluorescent labels using noninvasive, nondestructive techniques. METHODS Freshly enucleated mouse eyes were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). Cross sectional transverse sections and sequential flat (en face) sagittal sections were collected from a region of sclera approximately midway between the limbus and optic nerve. Imaging proceeded from the surface of the sclera to a depth of ∼60 μm. RESULTS The fluorescent signal from collagen fibers within the sclera was evident in the TPAF channel; the scleral collagen fibers showed no organization and appeared randomly packed. The sclera contained regions lacking TPAF and CARS fluorescence of ∼3 to 15 μm in diameter that could represent small vessels or scleral fibroblasts. Intense punctate CARS signals from the retinal pigment epithelial layer were of a size and shape of retinyl storage esters. Rod outer segments could be identified by the CARS signal from their lipid-rich plasma membranes. CONCLUSIONS CARS microscopy can be used to image the outer regions of the mammalian retina without the use of a fluorescent dye or exogenously expressed recombinant protein. With technical advancements, CARS/TPAF may represent a new avenue for noninvasively imaging the retina and might complement modalities currently used in clinical practice.

The aqueous outflow system (AOS), including the trabecular meshwork (TM), the collector channels (CC) and the Schlemm’s canal (SC), regulates intraocular pressure (IOP) through the drainage of the aqueous humor (AH). Abnormal IOP elevation leads to increased pressure stress to retinal ganglion cells, resulting in cell loss that can ultimately lead to complete loss of eyesight. Therefore, development of imaging tools to detect abnormal structural and functional changes of the AOS is important in early diagnosis and prevention of glaucoma. Multiphoton microscopy (MPM), including twophoton autofluorescence (TPAF) and second harmonic generation (SHG), is a label-free microscopic technique that allows molecular specific imaging of biological tissues like the TM. Since the TM and other AOS structures are located behind the highly scattering scleral tissue, transscleral imaging of the TM does not provide enough optical resolution. In this work, a gonioscopic lens is used to allow direct optical access of the TM through the cornea for MPM imaging. Compared to transscleral imaging, the acquired MPM images show improved resolution as individual collagen fiber bundles of the TM can be observed. MPM gonioscopy may have the potential to be developed as a future clinical imaging tool for glaucoma diagnostics.

Intraocular Biocompatibility of Gold-Nanoparticles Background: The present study is to assess the in vitro biocompatibility of colloidal gold nanoparticles (CGN) and to describe the effect of CGN over the normal electrical activity of the retina on an in vivo rat model. Material and Methods: Retinal pigment epithelium cells (ARPE- 19) were cultured to confluence. Three wells were incubated with a mixture of CGN conjugated to goat anti-mouse IgG and three were incubated with culture media as a control. After 2 days, media was aspirated and incubated again with 4,5-dimethylthiazol- 2-yl-2,5-diphenyltetrazolium bromide for 2 hrs. The absorbance of each well was measured by spectrophotometry at a 540 nm wavelength, subtracting the background measurement at 670 nm. A total of 16 eyes of eight brown Norway rats were used, being divided in two groups of eight eyes each; Right eyes received an intravitreal injection of 1μM/5μL CGN suspension at baseline. An electroretinogram (ERG) was done at baseline and then again after six weeks. A two-sample t-test was used as statistical method. Results: Absorbance signal for the CGN Group was 89.06% with respect to the reference values of 100% for untreated controls. There was no statistical difference between groups (p=0.1). The ERG yielded no statistically significant difference between groups at any time point for any of the five steps of the ERG. Conclusion: The in vitro viability of ARPE-19 cells seems not to be affected by addition of 5 nm CGN to the culture, with no significant morphological changes. The normal electrical activity of the retina seems not be disturbed by the intravitreal injection of CNG.

We have used a homology based approach to identify G protein-coupled receptors preferentially expressed in retinal and taste cells. Rat and bovine sequences encoding a novel G protein-coupled receptor have been isolated. Analysis indicates that while the protein sequence is most similar to the receptors for somatostatin and opiates, it is unlikely to be a subtype of these receptors. Northern and RNase protection analysis indicates that the gene is preferentially expressed in neural and sensory tissues.

To determine and compare the short-term, in vitro effect of prostaglandin analogues (PGAs), timolol, and benzalkonium chloride (BAK) on pre-adipocyte proliferation and adipocyte cytotoxicity.For the proliferation assay, human primary subcutaneous pre-adipocytes were incubated in the presence of either bimatoprost (0.015%, 0.03%, 0.06%) travoprost (0.002%, 0.004%, 0.008%), latanoprost (0.0025%, 0.005%, 0.010%), tafluprost (0.00075%, 0.0015%, 0.003%), timolol maleate (0.25%, 0.5%, 1.0%), BAK (0.005%, 0.010%, 0.015%, 0.020%, 0.040%), or control of BAK vehicle. After 72 h in culture, cell numbers were determined by fluorescent assay with CellTiter-Blue(®) reagent. For the cytotoxity assay, mature human adipocytes were cultured for 72 h with the same test compounds and controls. Cell numbers were again determined in the same manner through fluorescence.In the proliferation assay, all four PGA medications failed to show a statistically significant difference from BAK vehicle alone (p = 0.065-0.751). Both timolol and BAK alone demonstrated near complete inhibition of pre-adipocyte proliferation at all concentrations tested (p0.01 for all). In the cytotoxicity assay, all four PGAs again exerted no significant effect when compared to control (p = 0.211-0.901). Timolol and BAK again displayed near complete toxicity of adipocytes at all concentrations (p0.01 for all).All four PGAs had similar and negligible short-term effects on pre-adipocyte proliferation and adipocyte toxicity in vitro. Timolol and BAK had profound cytotoxic effects on mature adipocytes and antiproliferative effects on pre-adipocytes in vitro. Further testing is needed to determine whether similar effects are present in vivo.

To evaluate the effects of a novel ab interno trabeculectomy device on human trabecular meshwork (TM).Laboratory evaluation.The TM from human cadaveric corneal rim tissue was incised using 3 instruments: (1) novel dual-blade device; (2) microvitreoretinal (MVR) blade; and (3) Trabectome. Tissue samples underwent histologic processing and comparative analyses. Subsequently, human eye perfusion studies were performed to evaluate intraocular pressure (IOP)-lowering effects of each device. Main outcome measures were degree of TM removal by histology and IOP in a perfusion model.The MVR blade exhibited minimal removal of TM and obvious injury to the adjacent sclera. The Trabectome removed a large portion of the central TM, but leaflets of residual tissue remained and thermal injury was noted in all samples. The dual-blade device achieved a more complete removal of TM without injury to surrounding tissues. All devices resulted in statistically significant lowering of IOP during perfusion model studies. MVR blade treatment across 170.0 ± 14.1 degrees of TM resulted in a decrease of IOP from 18.5 ± 1.9 mm Hg to 12.8 ± 2.2 mm Hg (P.01). Trabectome treatment across 117.5 ± 12.6 degrees resulted in a decrease of IOP from 18.8 ± 1.7 mm Hg to 11.3 ± 1.0 mm Hg (P.01). Dual-blade device treatment across 157.5 ± 26.3 degrees resulted in a decrease of IOP from 18.3 ± 3.0 mm Hg to 11.0 ± 2.2 mm Hg (P.01).The novel dual-blade device demonstrated a more complete removal of TM without residual TM leaflets or damage to surrounding tissues and significantly reduced IOP in a human eye perfusion model.

Purpose To demonstrate the ability of multiphoton microscopy to obtain full three-dimensional high-resolution images of the intact mouse eye anterior chamber without need for enucleation. Methods A custom multiphoton microscope was constructed and optimized for deep tissue imaging. Simultaneous two-photon autofluorescence (2PAF) and second harmonic generation (SHG) imaging were performed. A mouse holder and stereotaxic platform were designed to access different parts of the eye for imaging. A reservoir for keeping the eye moist was used during imaging sessions. Results Non-invasive multiphoton images deep inside the anterior chamber of the mouse eye were obtained without the need for enucleation. The iris, corneal epithelium and endothelium, trabecular meshwork region and conjunctiva were visualized by the 2PAF and SHG signals. Identification of the anatomy was achieved by the intrinsic properties of the native tissue without any exogenous labeling. Images as deep as 600 microns into the eye were clearly demonstrated. Full three-dimensional image reconstructions of the entire anterior chamber were performed and analyzed using custom software. Conclusions Multiphoton imaging is a highly promising tool for ophthalmic research. We have demonstrated the ability to image the entire anterior chamber of the mouse eye in its native state. These results provide a foundation for future in vivo studies of the eye.

PURPOSE To examine the protective effects of antioxidants in cultured trabecular meshwork (TM) cells exposed to oxidative stress. METHODS Primary porcine TM cells were pretreated with 50 μM resveratrol, 0.2 mM urate, 1 mM ascorbate, 1 mM reduced glutathione (rGSH), or 1 mM ρ-coumarate followed by exposure to hydrogen peroxide (0.5-4 mM). Cell metabolism was determined by mitochondrial enzyme activity and cell viability by uptake of the vital dye calcein, a fluorescent calcium binding dye. Reactive oxygen species (ROS), which may reflex oxidative damage, were determined by 2'7'-dichlorofluorescein diacetate. RESULTS Trabecular meshwork cell metabolism was reduced to 72 ± 5% of control levels with 1 mM hydrogen peroxide (H2O2) treatment. TM cells that co-incubated with ascorbate (85% ± 5%), ρ-coumarate (98 ± 11%) or rGSH (103 ± 17%) had significantly increased metabolism compared to 1 mM H2O2 treatment. Resveratrol significantly increased TM cell metabolism at both 2 mM (102 ± 14% live) and 4 mM H2O2 (27 ± 12% live), with H2O2-treated cultures containing mostly metabolically inactive cells (3% at 2 mM; 2% at 4 mM). Similar results were obtained in cell viability assays. Ascorbate and resveratrol, but not ρ-coumarate or rGSH, decreased ROS levels in TM cells exposed to a sublethal dose of H2O2 (0.5 mM). Urate had no protective effect against H2O2 damage in any of the assays. CONCLUSIONS Increased oxidative damage was demonstrated in the TM of patients with primary open angle glaucoma. The antioxidants (resveratrol, ascorbate, ρ-coumarate) and the antioxidant enzyme cofactor (rGSH) protected TM cells from H2O2-induced damage. TRANSLATIONAL RELEVANCE Future experiments are needed to determine whether addition of antioxidants may maintain TM cell viability in vivo. Antioxidants could be applied either topically or coupled with extended-release vehicles for intraocular injection to reduce free radical formation leading to enhanced therapeutic outcomes. Ultimately, studies using animal models could determine whether application of antioxidants can ameliorate progression in diseases such as glaucoma and macular degeneration.

This work compares the concentration of active ingredients and preservatives in commonly used brand name versus generic glaucoma medications.Active ingredient and benzalkonium chloride (BAK) concentrations in brand name latanoprost and dorzolamide-timolol were each compared to two generic counterparts using liquid chromatography-mass spectrometry at baseline and after exposure to 25°C and 50°C for 30 days. Micro flow imaging was used to quantify particulate material greater than one micron in diameter.Brand name formulations contained active ingredients and BAK in concentrations that were generally in agreement with their package inserts at baseline. The two generic formulations of latanoprost contained baseline levels of active ingredients that were 10% greater than their labeled value. Generic latanoprost formulations had significant loss of active ingredient concentration after exposure to 25°C and 50°C for 30 days. Both generic and brand name dorzolamide-timolol appeared relatively resistant to degradation. BAK concentrations remained stable at 25°C but decreased in some bottles at 50°C. Bottles of both generic medications had higher levels of particulate matter compared to brand name versions.Exposure to temperatures at the high end of the labeled value may lead to a significant decrease in concentration of active ingredients in generic formulations that could influence clinical efficacy. Re-evaluation of intraocular pressure lowering efficacy may be indicated in glaucoma patients switching from brand name to generic formulations.

Purpose To image the human trabecular meshwork (TM) using a non-invasive, non-destructive technique without the application of exogenous label. Methods Flat-mounted TM samples from a human cadaver eye were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). In TPAF, two optical photons are simultaneously absorbed and excite molecules in the sample that then emit a higher energy photon. The signal is predominately from collagen and elastin. The CARS technique uses two laser frequencies to specifically excite carbon-hydrogen bonds, allowing the visualization of lipid-rich cell membranes. Multiple images were taken along an axis perpendicular to the surface of the TM for subsequent analysis. Results Analysis of multiple TPAF images of the TM reveals the characteristic overlapping bundles of collagen of various sizes. Simultaneous CARS imaging revealed elliptical structures of ~7×10 µm in diameter populating the meshwork which were consistent with TM cells. Irregularly shaped objects of ~4 µm diameter appeared in both the TPAF and CARS channels, and are consistent with melanin granules. Conclusions CARS techniques were successful in imaging live TM cells in freshly isolated human TM samples. Similar images have been obtained with standard histological techniques, however the method described here has the advantage of being performed on unprocessed, unfixed tissue free from the potential distortions of the fine tissue morphology that can occur due to infusion of fixatives and treatment with alcohols. CARS imaging of the TM represents a new avenue for exploring details of aqueous outflow and TM cell physiology.

The aim of this study is to investigate potential adverse effects of fixed combination glaucoma medications preserved with either benzalkonium chloride (BAK) or Polyquad® (PQ; Alcon Research Ltd., Fort Worth, TX, USA) on cultured ocular epithelial cells.Confluent cultures of human cornea and conjunctival cell lines were exposed for 25 minutes to different glaucoma medications as well as a range of concentrations of BAK (0.001%-0.050%). Balanced salt solution was used as the "live" control and a solution containing 70% methanol and 0.2% saponin was used as a "dead" control. The number of dead and live cells were determined via ethidium homodimer (Eth-1) and calcein acetoxymethyl ester (AM) fluorescence, respectively.The toxicity of the prostaglandin analog with beta-blocker timolol fixed-combination formulations preserved with BAK was different from that observed in the respective BAK concentrations. Travoprost plus timolol fixed combination with BAK performed better than its respective BAK concentration alone, while the latanoprost plus timolol fixed combination performed worse than its respective BAK concentration. Travoprost plus timolol fixed combination preserved with PQ had greater corneal and conjunctival cell survival than either the travoprost plus timolol fixed combination preserved with BAK or the latanoprost plus timolol fixed combination.Ocular surface side effects have previously been demonstrated with chronic, long-term exposure to intraocular-pressure-lowering medications containing the common preservative BAK. BAK alone has significant in-vitro cytotoxicity to cultured ocular epithelial cells. Substitution of BAK with PQ resulted in significantly higher percentages of live conjunctival and corneal cells. Further studies are needed to understand the clinical implications of these findings.