Nitrogen Mustard-Induced Ex Vivo Human Cornea Injury Model and Therapeutic Intervention by Dexamethasone.

Sulfur mustard (SM), a vesicating agent first used during World War I, remains a potent threat as a chemical weapon to cause intentional/accidental chemical emergencies. Eyes are extremely susceptible to SM toxicity. Nitrogen mustard (NM), a bifunctional alkylating agent and potent analog of SM, is used in laboratories to study mustard vesicant-induced ocular toxicity. Previously, we showed that SM-/NM-induced injuries (in vivo and ex vivo rabbit corneas) are reversed upon treatment with dexamethasone (DEX), a US Food and Drug Administration-approved, steroidal anti-inflammatory drug. Here, we optimized NM injuries in ex vivo human corneas and assessed DEX efficacy. For injury optimization, one cornea (randomly selected from paired eyes) was exposed to NM: 100 nmoles for 2 hours or 4 hours, and 200 nmoles for 2 hours, and the other cornea served as a control. Injuries were assessed 24 hours post NM-exposure. NM 100 nmoles exposure for 2 hours was found to cause optimal corneal injury (epithelial thinning [∼69%]; epithelial-stromal separation [6-fold increase]). In protein arrays studies, 24 proteins displayed ≥40% change in their expression in NM exposed corneas compared with controls. DEX administration initiated 2 hours post NM exposure and every 8 hours thereafter until 24 hours post-exposure reversed NM-induced corneal epithelial-stromal separation [2-fold decrease]). Of the 24 proteins dysregulated upon NM exposure, six proteins (delta-like canonical Notch ligand 1, FGFbasic, CD54, CCL7, endostatin, receptor tyrosine-protein kinase erbB-4) associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, showed significant reversal upon DEX treatment (Student's t test; P ≤ 0.05). Complementing our animal model studies, DEX was shown to mitigate vesicant-induced toxicities in ex vivo human corneas. SIGNIFICANCE STATEMENT: Nitrogen mustard (NM) exposure-induced injuries were optimized in an ex vivo human cornea culture model and studies were carried out at 24 h post 100 nmoles NM exposure. Dexamethasone (DEX) administration (started 2 h post NM exposure and every 8 h thereafter) reversed NM-induced corneal injuries. Molecular mediators of DEX action were associated with angiogenesis, immune/inflammatory responses, and cell differentiation/proliferation, indicating DEX aids wound healing via reversing vesicant-induced neovascularization (delta-like canonical Notch ligand 1 and FGF basic) and leukocyte infiltration (CD54 and CCL7).

Patterns of Gene Expression, Splicing, and Allele-Specific Expression Vary among Macular Tissues and Clinical Stages of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness, and elucidating its underlying disease mechanisms is vital to the development of appropriate therapeutics. We identified differentially expressed genes (DEGs) and differentially spliced genes (DSGs) across the clinical stages of AMD in disease-affected tissue, the macular retina pigment epithelium (RPE)/choroid and the macular neural retina within the same eye. We utilized 27 deeply phenotyped donor eyes (recovered within a 6 h postmortem interval time) from Caucasian donors (60-94 years) using a standardized published protocol. Significant findings were then validated in an independent set of well-characterized donor eyes (n = 85). There was limited overlap between DEGs and DSGs, suggesting distinct mechanisms at play in AMD pathophysiology. A greater number of previously reported AMD loci overlapped with DSGs compared to DEGs between disease states, and no DEG overlap with previously reported loci was found in the macular retina between disease states. Additionally, we explored allele-specific expression (ASE) in coding regions of previously reported AMD risk loci, uncovering a significant imbalance in C3 rs2230199 and CFH rs1061170 in the macular RPE/choroid for normal eyes and intermediate AMD (iAMD), and for CFH rs1061147 in the macular RPE/choroid for normal eyes and iAMD, and separately neovascular AMD (NEO). Only significant DEGs/DSGs from the macular RPE/choroid were found to overlap between disease states. STAT1, validated between the iAMD vs. normal comparison, and AGTPBP1, BBS5, CERKL, FGFBP2, KIFC3, RORα, and ZNF292, validated between the NEO vs. normal comparison, revealed an intricate regulatory network with transcription factors and miRNAs identifying potential upstream and downstream regulators. Findings regarding the complement genes C3 and CFH suggest that coding variants at these loci may influence AMD development via an imbalance of gene expression in a tissue-specific manner. Our study provides crucial insights into the multifaceted genomic underpinnings of AMD (i.e., tissue-specific gene expression changes, potential splice variation, and allelic imbalance), which may open new avenues for AMD diagnostics and therapies specific to iAMD and NEO.

Preclinical Investigation of Ab Interno Goniotomy Using Three Different Techniques.

Purpose: To evaluate incisional or excisional tissue-level effects of ab interno goniotomy techniques on human trabecular meshwork (TM). Methods: The TM from human cadaveric corneal rim tissue was treated using three devices: (1) Kahook Dual Blade (KDB) GLIDE, (2) iAccess, and (3) SION. Two human corneal rims were used for each of the iAccess and SION devices and one with the KDB GLIDE, with 360 degrees of TM treated in each case. Sections were then prepared for analysis and comparison between devices. Tissue samples underwent standard histologic processing with H&E stain, followed by comparative analyses. Results: Areas treated with the KDB GLIDE device resulted in nearly complete excision of TM overlying the canal of Schlemm without injury to surrounding tissues. The iAccess device can be used as a focal trephine to create holes or dragged for TM disruption. When used to create holes, iAccess punched through the full thickness of the TM and also disrupted the anterior scleral tissue. It caused some incisional openings through the TM but with significant leaflets remaining and minimal true "hole-punch" effect. When the device tip was dragged, iAccess incised the TM and left debris behind with little, if any, excision of tissue. SION led to both incision and excision of TM with incision predominating over excision. Conclusion: The various methods evaluated to perform ab interno goniotomy resulted in varying degrees of TM incision or excision. Only the KDB GLIDE device resulted in reliable excision of TM, while the other devices produced incision or minimal excision of tissue with residual leaflets and debris. Use of iAccess resulted in focal disruption of the anterior scleral wall. Because incisional approaches that leave longer residual leaflets may be more prone to fibrosis and closure compared to excisional treatments, clinical correlation will be necessary to better understand the significance of these findings with respect to relative effectiveness of intraocular pressure lowering in eyes with glaucoma.

Effect of dexamethasone treatment at variable therapeutic windows in reversing nitrogen mustard-induced corneal injuries in rabbit ocular in vivo model.

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.

Diabetes-Independent Retinal Phenotypes in an Aldose Reductase Transgenic Mouse Model.

Aldose reductase (AR), the first and rate-limiting enzyme of the polyol pathway, has been implicated in the onset and development of the ocular complications of diabetes, including cataracts and retinopathy. Despite decades of research conducted to address possible mechanisms, questions still persist in understanding if or how AR contributes to imbalances leading to diabetic eye disease. To address these questions, we created a strain of transgenic mice engineered for the overexpression of human AR (AR-Tg). In the course of monitoring these animals for age-related retinal phenotypes, we observed signs of Müller cell gliosis characterized by strong immunostaining for glial fibrillary acidic protein. In addition, we observed increased staining for Iba1, consistent with an increase in the number of retinal microglia, a marker of retinal inflammation. Compared to age-matched nontransgenic controls, AR-Tg mice showed an age-dependent loss of Brn3a-positive retinal ganglion cells and an associated decrease in PERG amplitude. Both RGC-related phenotypes were rescued in animals treated with Sorbinil in drinking water. These results support the hypothesis that increased levels of AR may be a risk factor for structural and functional changes known to accompany retinopathy in humans.

Effect of supersaturated oxygen emulsion treatment on chloropicrin-induced chemical injury in ex vivo rabbit cornea.

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-1ß, 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.

Pilot study supporting the existence of novel lymphatic channels within the canine anterior uveal tract using Lyve-1 and CD31.

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.

Preclinical Investigation of Goniotomy Using Four Different Techniques.

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.

Effect of Novel Design Modifications on Fibrotic Encapsulation: An In Vivo Glaucoma Drainage Device Study in a Rabbit Model.

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 < 0.001), heparin-coated plates (mean difference -4.4 mm Hg; p < 0.001), and micro-patterned plates (mean difference -18.6 mm Hg, p < 0.001), indicating lower outflow resistance compared to control AGV models. Fibrotic encapsulation was lower in hydrophilic plate coating (84.2 µm; mean difference -6.2 µm, p = 0.425), micro-patterned surface (63.7 µm; mean difference -26.7 µm, p = 0.003), and heparin plate coating (49.3 µm; mean difference -41.1 µm, p = 0.006) when compared to control AGV models. CONCLUSIONS: Modified AGVs with plate coatings and AGVs with micro-patterned plates both appear to reduce postoperative fibrotic encapsulation and aqueous outflow resistance by altering the tissue response to implanted materials. Further studies are needed to characterize the safety and role of plate surface modifications on glaucoma drainage devices.

Toxic consequences and oxidative protein carbonylation from chloropicrin exposure in human corneal epithelial cells.

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.

Acute corneal injury in rabbits following nitrogen mustard ocular exposure.

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 Metalloproteinase 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.

Trans-scleral cyclophotocoagulation: a tale of two probes.

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.

Aldose Reductase Inhibition Prevents Development of Posterior Capsular Opacification in an In Vivo Model of Cataract Surgery.

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.

Histopathologic Examination of Trabecular Meshwork Changes After Trabecular Bypass Stent Implantation.

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.

Injectable Neurotrophic Factor Delivery System Supporting Retinal Ganglion Cell Survival and Regeneration Following Optic Nerve Crush.

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.

Histopathological and Molecular Changes in the Rabbit Cornea From Arsenical Vesicant Lewisite Exposure.

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.

A Self-Assembling Injectable Biomimetic Microenvironment Encourages Retinal Ganglion Cell Axon Extension in Vitro.

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.

Three-Dimensional Segmentation and Quantitative Measurement of the Aqueous Outflow System of Intact Mouse Eyes Based on Spectral Two-Photon Microscopy Techniques.

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.

Clinical progression of ocular injury following arsenical vesicant lewisite exposure.

Ocular injury by lewisite (LEW), a potential chemical warfare and terrorist agent, results in edema of eyelids, inflammation, massive corneal necrosis and blindness. To enable screening of effective therapeutics to treat ocular injury from LEW, useful clinically-relevant endpoints are essential. Hence, we designed an efficient exposure system capable of exposing up to six New-Zealand white rabbits at one time, and assessed LEW vapor-induced progression of clinical ocular lesions mainly in the cornea. The right eye of each rabbit was exposed to LEW (0.2 mg/L) vapor for 2.5, 5.0, 7.5 and 10.0 min and clinical progression of injury was observed for 28 days post-exposure (dose-response study), or exposed to same LEW dose for 2.5 and 7.5 min and clinical progression of injury was observed for up to 56 days post-exposure (time-response study); left eye served as an unexposed control. Increasing LEW exposure caused corneal opacity within 6 h post-exposure, which increased up to 3 days, slightly reduced thereafter till 3 weeks, and again increased thereafter. LEW-induced corneal ulceration peaked at 1 day post-exposure and its increase thereafter was observed in phases. LEW exposure induced neovascularization starting at 7 days which peaked at 22-35 days post-exposure, and remained persistent thereafter. In addition, LEW exposure caused corneal thickness, iris redness, and redness and swelling of the conjunctiva. Together, these findings provide clinical sequelae of ocular injury following LEW exposure and for the first time establish clinically-relevant quantitative endpoints, to enable the further identification of histopathological and molecular events involved in LEW-induced ocular injury.

Nitrogen Mustard-Induced Corneal Injury Involves DNA Damage and Pathways Related to Inflammation, Epithelial-Stromal Separation, and Neovascularization.

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.