Scleral growth stunting via sub-Tenon injection of cross-linking solutions in live rabbits.

BACKGROUND: Scleral cross-linking is a potential method to inhibit axial elongation of the eye, preventing the progression of pathological myopia. Formaldehyde releasers, which are common preservatives found in cosmetics and ophthalmic solutions, have been shown to be not only effective in cross-linking corneal collagen in vitro and in vivo, but also have minimal toxicity effects on the eye. The present study aims to evaluate the efficacy of scleral cross-linking using sodium hydroxymethylglycinate (SMG) to inhibit eye growth using an in vivo rabbit model. METHODS: A cross-linking solution containing 40 mM SMG was delivered to the sub-Tenon's space behind the equator. The application regimen included a two-quadrant injection performed five times over 2 weeks on New Zealand White rabbits (n=5, group 1), and one-time injection followed for up to 5 days on Dutch-Belted rabbits (n=6, group 2). Group 1 was monitored serially for axial length changes using B-scan ultrasound for 5-6 weeks. Group 2 was injected with a higher viscosity solution formulation. Both groups were evaluated for thermal denaturation temperature changes of the sclera postmortem. RESULTS: Axial growth was limited by 10%-20% following SMG treatment as compared with the untreated eye. Thermal denaturation analysis showed increased heat resistance of the treated eyes in the areas of injection. Overall, the SMG treatment inhibited eye growth with few side effects from the injections. CONCLUSIONS: Cross-linking solutions delivered via sub-Tenon injection provide a potential method for limiting axial length growth in progressive myopia and could be used as a potential treatment for myopia.

Topical Corneal Cross-Linking Solution Delivered Via Corneal Reservoir in Dutch-Belted Rabbits.

Purpose: A topical corneal cross-linking solution that can be used as an adjunct or replacement to standard photochemical cross-linking (UV-riboflavin) methods remain an attractive possibility. Optimal concentration and delivery method for such topical corneal stabilization in the living rabbit eye were developed. Methods: A series of experiments were carried out using Dutch-belted rabbits (3 months old, weighing 1.0-1.5 kg) and topical cross-linking solutions (sodium hydroxymethylglycinate) (10-250 mM) delivered via corneal reservoir. The application regimen included a one-time 30-minute application (10-40 mM sodium hydroxymethylglycinate) as well as a once per week 5-minute application (250 mM sodium hydroxymethylglycinate) for 7 weeks. Animals were evaluated serially for changes in IOP, pachymetry, epithelial integrity, and endothelial cell counts. Keratocyte changes were identified using intravital laser scanning confocal microscopy. Post mortem efficacy was evaluated by mechanical inflation testing. Results: Overall, there were very few differences observed in right eye treated versus left eye controls with respect to intraocular pressure, pachymetry, and endothelial cell counts, although 30-minute cross-linking techniques did cause transient increases in thickness resolving within 7 days. Epithelial damage was noted in all of the 30-minute applications and fully resolved within 72 hours. Keratocyte changes were significant, showing a wound healing pattern similar to that after riboflavin UVA photochemical cross-linking in rabbits and humans. Surprisingly, post mortem inflation testing showed that the lower concentration of 20 mM delivered over 30 minutes showed the most profound stiffening/strengthening effect. Conclusions: Topical cross-linking conditions that are safe and can increase corneal stiffness/strength in the living rabbit eye have been identified. Translational Relevance: A topical corneal cross-linking solution delivered via corneal reservoir is shown to be both safe and effective at increasing tissue strength in living rabbit eyes and could now be tested in patients suffering from keratoconus and other conditions marked by corneal tissue weakness.

Ex vivo anti-microbial efficacy of various formaldehyde releasers against antibiotic resistant and antibiotic sensitive microorganisms involved in infectious keratitis.

BACKGROUND: Corneal infections with antibiotic-resistant microorganisms are an increasingly difficult management challenge and chemically or photochemically cross-linking the cornea for therapy presents a unique approach to managing such infections since both direct microbial pathogens killing and matrix stabilization can occur simultaneously. The present study was undertaken in order to compare the anti-microbial efficacy, in vitro, of 5 candidate cross-linking solutions against 5 different microbial pathogens with relevance to infectious keratitis. METHODS: In vitro bactericidal efficacy studies were carried out using 5 different FARs [diazolidinyl urea (DAU), 1,3-bis(hydroxymethyl)-5,5-dimethylimidazolidine-2,4-dione (DMDM), sodium hydroxymethylglycinate (SMG), 2-(hydroxymethyl)-2-nitro-1,3-propanediol (NT = nitrotriol), 2-nitro-1-propanol (NP)] against 5 different microbial pathogens including two antibiotic-resistant species [methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), Pseudomonas aeruginosa (PA), and Candida albicans (CA)]. Standard in vitro antimicrobial testing methods were used. RESULTS: The results for MSSA were similar to those for MRSA. DAU, DMDM, and SMG all showed effectiveness with greater effects generally observed with longer incubation times and higher concentrations. Against MRSA, 40 mM SMG at 120 min showed a > 95% kill rate, p < 0.02. Against VRE, 40 mM DAU for 120 min showed a > 94% kill rate, p < 0.001. All FARs showed bactericidal effect against Pseudomonas aeruginosa, making PA the most susceptible of the strains tested. Candida showed relative resistance to these compounds, requiring high concentrations (100 mM) to achieve kill rates greater than 50%. CONCLUSION: Our results show that each FAR compound has different effects against different cultures. Our antimicrobial armamentarium could potentially be broadened by DAU, DMDM, SMG and other FARs for antibiotic-resistant keratitis. Further testing in live animal models are indicated.

Corneal collagen crosslinking in patients treated with dextran versus isotonic hydroxypropyl methylcellulose (HPMC) riboflavin solution: a retrospective analysis.

BACKGROUND: Corneal collagen crosslinking (CXL) is a widely used treatment for halting the progression of keratoconus. Although initial studies of CXL were performed with a riboflavin solution containing dextran, recent protocols for CXL have indicated the use of a riboflavin solution containing isotonic hydroxypropyl methylcellulose (HPMC). This study was performed to investigate differences in visual outcomes and Scheimpflug (Pentacam) analysis in patients who have undergone epithelium-off CXL with riboflavin solution containing either 20% dextran versus 1.1% HPMC. METHODS: All patients in this non-randomized, non-masked, retrospective cohort analysis were treated at Edward S. Harkness Eye Institute, Columbia University Medical Center, New York, NY, USA. Thirty-seven eyes of 33 patients were crosslinked with a dextran solution and 19 eyes of 19 patients crosslinked with an isotonic HPMC solution, both using an epithelium-off 30-min, 3 mW/cm2 protocol. All patients had a diagnosis of keratoconus or post-refractive surgery ectasia. Best spectacle corrected visual acuity (BSCVA) and Pentacam parameters were compared at all follow up visits (1, 6, 12, and 24 months). Differences between groups treated with HPMC and dextran were compared using student's t-test. Differences between treated eye and fellow eye were calculated and compared between HPMC and dextran groups using paired t-test. RESULTS: Patients treated with a dextran solution had significantly greater improvement in BSCVA at 1, 6, and 24 months (p < 0.05) compared to the isotonic HPMC-treated group. Kmax increased in both groups at 1 month; however, HPMC-treated patients had a greater increase compared to dextran-treated patients (p = 0.01). Kmax decreased in both groups at 6 and 12 months, although this finding was only significant in the HPMC-treated group at 12 months. CONCLUSIONS: Our data suggest that crosslinking with the dextran solution may result in significantly better visual outcomes (demonstrated by visual acuity) compared to the isotonic HPMC riboflavin solution. Dextran solutions may have other potential advantages intrinsic to its biochemical properties facilitating more efficient crosslinking. Further research and long-term evidence regarding the use of dextran versus HPMC riboflavin solutions in collagen crosslinking is necessary.

Correlation of Scheimpflug densitometry changes with clinical outcomes after corneal crosslinking.

PURPOSE: To characterize changes in densitometry after corneal crosslinking (CXL) and correlate it with visual outcomes. SETTING: Tertiary referral academic medical center, New York, New York, USA. DESIGN: Retrospective case series. METHODS: Patients with progressive keratoconus or post-laser in situ keratomileusis ectasia had CXL following the Dresden protocol. The corrected distance visual acuity (CDVA) and Pentacam imaging were obtained at baseline and follow-up visits. RESULTS: Fifty-seven patients were followed for a mean of 15 months (range 1 to 24 months) after CXL. The CDVA improved significantly from baseline to 6, 12, 18, and 24 months postoperatively. The change in densitometry of the mid-stromal layer, 2.0 to 6.0 mm annulus, at 6 months was correlated with the improvement in CDVA at 6, 12, and 24 months (all P < .10). The increase in densitometry of the mid-stromal layer, centermost 0.0 to 2.0 mm annulus, at 6 months was significantly associated with the decrease in maximum keratometry (K) at 6 and 12 months (both P < .05). Last, the change in densitometry at 6 months was significantly correlated with the decrease in specific higher-order aberrations (HOAs) (P < .05). CONCLUSIONS: Although the greatest and most durable post-CXL densitometry change was in the anterior layer, the degree of increased densitometry haze in the mid-stromal layer was most associated with and possibly predictive of improvement in CDVA, maximum K, and HOAs. The persistence of corneal haze at 6 months, measured by increased densitometry, might be a prognostic marker for CXL effectiveness.

Antimicrobial Studies Using the Therapeutic Tissue Cross-Linking Agent, Sodium Hydroxymethylglycinate: Implication for Treating Infectious Keratitis.

Purpose: Our recent studies raise the possibility of using sodium hydroxymethylglycinate (SMG), for pharmacologic therapeutic tissue cross-linking (TXL) of the cornea. The present study was performed to evaluate the antimicrobial effects of SMG for potential use in treating infectious keratitis. Methods: In initial (group 1) experiments, methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa (PA) were treated with SMG (10-40 mM) for 10 to 120 minutes. In group 2 experiments, MRSA, PA, Candida albicans (CA), and vancomycin-resistant Enterococcus (VRE) were treated with SMG (20-200 mM) for 30 minutes. In group 2 experiments, BSA and neutralizing buffer were added to provide a proteinaceous medium, and to ensure precise control of SMG exposure times, respectively. SMG effectiveness was quantitated based on pathogen growth following a 24- to 48-hour incubation period. Results: In group 1 experiments, as expected, time- and concentration-dependent bactericidal effects were noted using MSSA. In addition, the effect of SMG (40 mM) was greatest against MSSA (99.3%), MRSA (96.0%), and PA (97.4%) following a 2-hour exposure with lesser effects following 30- and 10-minute exposures. In group 2 experiments, concentration-dependent bactericidal effects were confirmed for MRSA (91%), PA (99%), and VRE (55%) for 200-mM SMG with 30-minute treatment. SMG was not as effective against CA, with a maximum kill rate of 37% at 80 mM SMG. Conclusions: SMG solution exhibits a dose-dependent bactericidal effect on MSSA, MRSA, and PA, with milder effects on VRE and CA. These studies raise the possibility of using SMG TXL for the treatment of infectious keratitis.

Evaluation of Therapeutic Tissue Crosslinking (TXL) for Myopia Using Second Harmonic Generation Signal Microscopy in Rabbit Sclera.

Purpose: Second harmonic generation signals (SHG) are emitted preferentially from collagenous tissue structures and have been used to evaluate photochemically-induced (CXL) crosslinking changes in the cornea. Since therapeutic tissue crosslinking (TXL) using sodium hydroxymethylglycinate (SMG) of the sclera is a potential treatment for high myopia, we explored the use of SHG microscopy to evaluate the effects. Methods: Single sub-Tenon's (sT) injections (400 µL) using SMG (40-400 mM) were made at the equatorial 12 o'clock position of the right eye of cadaveric rabbit heads (n = 16 pairs). After 3.5 hours, confocal microscopy (CM) was performed using 860 nm two-photon excitation and 400 to 450 nm emission. Pixel density and fiber bundle "waviness" analyses were performed on the images. Crosslinking effects were confirmed using thermal denaturation (Tm) temperature. Comparison experiments with riboflavin photochemical crosslinking were done. Results: Therapeutic tissue crosslinking localization studies indicated that crosslinking changes occurred at the site of injection and in adjacent sectors. Second harmonic generation signals revealed large fibrous collagenous bundled structures that displayed various degrees of waviness. Histogram analysis showed a nearly 6-fold signal increase in 400 mM SMG over 40 mM. This corresponded to a ΔTm = 13°C for 400 mM versus ΔTm = 4°C for 40 mM. Waviness analysis indicated increased fiber straightening as a result of SMG CXL. Conclusions: Second harmonic generation signal intensity and fiber bundle waviness is altered by scleral tissue crosslinking using SMG. These changes provide insights into the macromolecular changes that are induced by therapeutic crosslinking technology and may provide a method to evaluate connective tissue protein changes induced by scleral crosslinking therapies.

Laser-Induced Photic Injury Phenocopies Macular Dystrophy.

OBJECTIVE: To describe the phenotypes associated with laser-induced retinal damage in children. METHODS: Five patients with maculopathy and reduced visual acuity associated with laser pointer use were evaluated. Best-corrected visual acuity, retinal structure, and function were monitored with color fundus, infrared (IR), and red-free images, fundus autofluorescence (AF), spectral domain-optical coherence tomography (SD-OCT), and full-field electroretinography (ERG). RESULTS: All five laser pointer injury patients had retinal lesions resembling a macular dystrophy (one bilateral and four unilateral). These lesions were irregular in shape but all had a characteristic dendritic appearance with linear streaks radiating from the lesion. Photoreceptor damage was present in all patients, but serial OCT monitoring showed that subsequent photoreceptor recovery occurred over time in the eyes of at least four patients. One patient also had bilateral pigment epithelial detachments (PED). Both hyper- and hypoautofluorecence were observed in the laser damage area. CONCLUSIONS: In general, OCT and IR images are quite useful to diagnose laser damage, but AF is not as sensitive. Laser pointer damage in children can occasionally be misdiagnosed as a macular dystrophy disease, but the distinctive lesions and OCT features are helpful for differentiating laser damage from other conditions.

An Evaluation of Lysyl Oxidase-Derived Cross-Linking in Keratoconus by Liquid Chromatography/Mass Spectrometry.

PURPOSE: Current literature contains scant information regarding the extent of enzymatic collagen cross-linking in the keratoconus (KC) cornea. The aim of the present study was to examine levels of enzymatic lysyl oxidase-derived cross-links in stromal collagen in KC tissue, and to correlate the cross-link levels with collagen fibril stability as determined by thermal denaturation temperature (Tm). METHODS: Surgical KC samples (n = 17) and Eye-Bank control (n = 11) corneas of age 18 to 68 years were analyzed. The samples were defatted, reduced (NaBH4), hydrolyzed (6N HCl at 110°C for 18 hours), and cellulose enriched before analysis by C8 high-performance liquid chromatography equipped with parallel fluorescent and mass detectors in selective ion monitoring mode (20 mM heptafluorobutyric acid/methanol 70:30 isocratic at 1 mL/min). Nine different cross-links were measured, and the cross-link density was determined relative to collagen content (determined colorimetrically). The Tm was determined by differential scanning calorimetry. RESULTS: Cross-links detected were dihydroxylysinonorleucine (DHLNL), hydroxylysinonorleucine, lysinonorleucine (LNL), and histidinohydroxylysinonorleucine in both control and KC samples. Higher DHLNL levels were detected in KC, whereas the dominant cross-link, LNL, was decreased in KC samples. Decreased LNL levels were observed among KC ≤ 40 corneas. There was no difference in total cross-link density between KC samples and the controls. Pyridinolines, desmosines, and pentosidine were not detected. There was no notable correlation between cross-link levels with fibril instability as determined by Tm. CONCLUSIONS: Lower levels of LNL in the KC cornea suggest that there might be a cross-linking defect either in fibrillar collagen or the microfibrillar elastic network composed of fibrillin.

Evaluating the Toxicity/Fixation Balance for Corneal Cross-Linking With Sodium Hydroxymethylglycinate (SMG) and Riboflavin-UVA (CXL) in an Ex Vivo Rabbit Model Using Confocal Laser Scanning Fluorescence Microscopy.

PURPOSE: To develop methods to delineate the relationship between endothelial cell toxicity and tissue fixation (toxicity/fixation) using sodium hydroxymethylglycinate (SMG), a formaldehyde releaser, and riboflavin-UVA photochemical corneal cross-linking (CXL) for therapeutic tissue cross-linking of the cornea. METHODS: Eleven fresh cadaveric rabbit heads were used for ex vivo corneal cross-linking simulation. After epithelial debridement, the tissue was exposed to 1/4 max (9.8 mM) or 1/3 max (13 mM) SMG at pH 8.5 for 30 minutes or riboflavin-UVA (CXL). The contralateral cornea served as a paired control. Postexposure, cross-linking efficacy was determined by thermal denaturation temperature (Tm) and endothelial damage was assessed using calcein AM and ethidium homodimer staining (The Live/Dead Kit). Confocal laser scanning fluorescence microscopy was used to generate live/dead cell counts using a standardized algorithm. RESULTS: The ΔTm after CXL, 1/3 SMG, and 1/4 SMG was 2.2 ± 0.9°C, 1.3 ± 0.5°C, and 1.1 ± 0.5°C, respectively. Endothelial cell damage was expressed as the percent of dead cells/live + dead cells counted per high-power field. The values were 3 ± 1.7% (control) and 8.9 ± 11.1% (CXL) (P = 0.390); 1 ± 0.2% (control) and 19.5 ± 32.2% (1/3 max SMG) (P = 0.426); and 2.7 ± 2.4% (control) and 2.8 ± 2.2% (1/4 max SMG) (P = 0.938). The values for endothelial toxicity were then indexed over the shift in Tm to yield a toxicity/fixation index. The values were as follows: 2.7 for CXL, 14 for 1/3 max, and 0.1 for 1/4 max. CONCLUSIONS: Quarter max (1/4 max = 9.8 mM) SMG effectively cross-linked tissue and was nontoxic to endothelial cells. Thus, SMG is potentially a compound that could achieve both desired effects.

Vitrectomy for macular hole following Nd:YAG laser injury.

The Q-switched Nd:YAG laser can cause significant ocular injury, because it can emit an invisible and powerful load of energy in a short period of time. One of these injuries is macular hole formation. We report the case of a 21-year-old woman who presented with acute floaters in her left eye after exposure to a Q-switched Nd:YAG laser. Her initial best-corrected visual acuity (BCVA) in the left eye was 20/80. Examination demonstrated an acute vitreous hemorrhage, and spectral-domain optical coherence tomography (SD-OCT) confirmed a full-thickness macular hole (FTMH). Four months after her injury, her BCVA deteriorated to 20/400, and she underwent vitrectomy, internal limiting membrane peeling, and gas injection. Three weeks following the procedure, her FTMH was closed, however, her BCVA remained 20/150. At her last office visit, 19 months after the surgery, the examination was unchanged. There are few reports of vitrectomy to close an FTMH after Nd:YAG laser exposure, and the factors that contribute to visual recovery remain unclear. The aim of this case report is to emphasize the importance of early diagnosis and surgical repair, and to review the literature and surgical outcomes of cases of Nd:YAG laser-induced macular holes. Imaging with SD-OCT was essential in evaluating the visual outcome, as it was dependent on the degree of photoreceptor and retinal pigment epithelium injury.

Cosmetic preservatives as therapeutic corneal and scleral tissue cross-linking agents.

PURPOSE: Previously, aliphatic ß-nitroalcohols (BNAs) have been studied as a means to chemically induce tissue cross-linking (TXL) of cornea and sclera. There are a number of related and possibly more potent agents, known as formaldehyde releasers (FARs), that are in commercial use as preservatives in cosmetics and other personal care products. The present study was undertaken in order to screen such compounds for potential clinical utility as therapeutic TXL agents. METHODS: A chemical registry of 62 FARs was created from a literature review and included characteristics relevant to TXL such as molecular weight, carcinogenicity/mutagenicity, toxicity, hydrophobicity, and commercial availability. From this registry, five compounds [diazolidinyl urea (DAU), imidazolidinyl urea (IMU), sodium hydroxymethylglycinate (SMG), DMDM hydantoin (DMDM), 5-Ethyl-3,7-dioxa-1-azabicyclo [3.3.0] octane (OCT)] were selected for efficacy screening using two independent systems, an ex vivo rabbit corneal cross-linking simulation setup and incubation of cut scleral tissue pieces. Treatments were conducted at pH 7.4 or 8.5 for 30 minutes. Efficacy was evaluated using thermal denaturation temperature (Tm), and cell toxicity was studied using the trypan blue exclusion method. RESULTS: Cross-linking effects in the five selected FARs were pH and concentration dependent. Overall, the Tm shifts were in agreement with both cornea and sclera. By comparison with BNAs previously reported upon, the FARs identified in this study were significantly more potent but with similar or better cytotoxicity. CONCLUSIONS: The FARs, a class of compounds well known to the cosmetic industry, may have utility as therapeutic TXL agents. The compounds studied thus far show promise and will be further tested.

Pharmacologic alternatives to riboflavin photochemical corneal cross-linking: a comparison study of cell toxicity thresholds.

PURPOSE: The efficacy of therapeutic cross-linking of the cornea using riboflavin photochemistry (commonly abbreviated as CXL) has caused its use to become widespread. Because there are known chemical agents that cross-link collagenous tissues, it may be possible to cross-link tissue pharmacologically. The present study was undertaken to compare the cell toxicity of such agents. METHODS: Nine topical cross-linking agents (five nitroalcohols, glyceraldehyde [GLYC], genipin [GP], paraformaldehyde [FA], and glutaraldehyde [GLUT]) were tested with four different cell lines (immortalized human corneal epithelial cells, human skin fibroblasts, primary bovine corneal endothelial cells, and immortalized human retinal pigment epithelial cells [ARPE-19]). The cells were grown in planar culture and exposed to each agent in a range of concentrations (0.001 mM to 10 mM) for 24 hours followed by a 48-hour recovery phase. Toxicity thresholds were determined by using the trypan blue exclusion method. RESULTS: A semiquantitative analysis using five categories of toxicity/fixation was carried out, based on plate attachment, uptake of trypan blue stain, and cellular fixation. The toxicity levels varied by a factor of 10(3) with the least toxic being mononitroalcohols and GLYC, intermediate toxicity for a nitrodiol and nitrotriol, and the most toxic being GLUT, FA, GP, and bronopol, a brominated nitrodiol. When comparing toxicity between different cell lines, the levels were generally in agreement. CONCLUSIONS: There are significant differences in cell toxicity among potential topical cross-linking compounds. The balance between cross-linking of tissue and cell toxicity should be borne in mind as compounds and strategies to improve mechanical tissue properties through therapeutic tissue cross-linking continue to develop.

Aliphatic ß-nitroalcohols for therapeutic corneoscleral cross-linking: chemical stability studies using 1H-NMR spectroscopy.

Recent studies suggest that aliphatic ß-nitro alcohols may represent a useful class of compounds for use as in vivo therapeutic corneoscleral cross-linking agents with higher order nitroalcohols (HONAs) showing enhanced efficacy over the mono-nitroalcohols. The current study was undertaken in order to evaluate the chemical stability of these compounds during storage conditions. Two mono-nitroalcohols (2-nitroethanol=2ne and 2-nitro-1-propanol = 2 nprop) and two HONAs, a nitrodiol (2-methyl-2-nitro-1,3-propanediol = MNPD), and a nitrotriol (2-hydroxymethyl-2-nitro-1,3-propanediol = HNPD) were monitored for chemical stability by (1)H-NMR for up to 7 months. Each compound was studied at two concentrations (1% and 10%) either in unbuffered H2O or 0.2 m NaH2 PO4/Na2 HPO4 (pH = 5), and at 0 °C and room temperature (RT) for a total of eight conditions for each compound. The (1)H-NMR spectra for the starting material were compared to subsequent spectra. Under all four of the conditions studied, both the nitrodiol (MNPD) and nitrotriol (HNPD) were stable for the duration of 7 months. 2nprop became unstable under all conditions at 3 months. 2ne was the most unstable of all the compounds tested. HONAs exhibit excellent chemical stability under long-term storage conditions. In contrast, the nitromonols tested are significantly less stable. These findings are relevant to the translation of this technology into clinical use.

Aliphatic ß-nitroalcohols for therapeutic corneoscleral cross-linking: corneal permeability considerations.

PURPOSE: Our recent tissue cross-linking studies have raised the possibility of using aliphatic ß-nitroalcohols (BNAs) for pharmacologic, therapeutic corneal cross-linking. The present study was performed to determine the permeability of BNAs and to explore the use of permeability-enhancing agents. METHODS: Ex vivo rabbit corneas were mounted in a typical Franz diffusion chamber. BNA permeability was determined by assaying the recipient chamber over time using a modification of the Griess nitrite colorimetric assay. The apparent permeability coefficient (Ptot) was determined for 2 mono-nitroalcohols [2-nitroethanol (2ne) and 2-nitro-1-propanol (2nprop)], a nitrodiol [2-methyl-2-nitro-1,3-propanediol (MNPD)], and a nitrotriol [2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD)]. Permeability-enhancing effects using benzalkonium chloride (BAC) (0.01% and 0.02%), ethylenediaminetetraacetic acid (0.05%), and a combination of 0.01% BAC + 0.5% tetracaine were also studied. RESULTS: The Ptot (±SE) values (in centimeters per second) were as follows: 4.33 × 10 (±9.82 × 10) for 2ne [molecular weight (MW) = 91 Da], 9.34 × 10 (±2.16 × 10) for 2nprop (MW = 105 Da), 4.37 × 10 (±1.86 × 10) for MNPD (MW = 135 Da), and 8.95 × 10 (±1.93 × 10) for HNPD (MW = 151 Da). Using the nitrodiol, permeability increased approximately 2-fold using 0.01% BAC, 5-fold using 0.02% BAC, and 5-fold using the combination of 0.01% BAC + 0.5% tetracaine. No effect was observed using 0.05% ethylenediaminetetraacetic acid. CONCLUSIONS: The results indicate that the corneal epithelium is permeable to BNAs, with the apparent permeability corresponding to MW. The findings are consistent with previous literature indicating that the small size of these compounds (<10Å) favors their passage through the corneal epithelium via the paracellular route. This information will help to guide dosing regimens for in vivo topical cross-linking studies.

Photoactivated riboflavin treatment of infectious keratitis using collagen cross-linking technology.

PURPOSE: To evaluate riboflavin/ultraviolet-A (UVA) as an adjunct treatment for infectious keratitis. METHODS: This prospective, dual-center, interventional case series included cases of infectious keratitis that were treated by instilling riboflavin 0.1% solution for 30 minutes to saturate the cornea, followed by exposure to 365-nm UVA light (3 mW/cm(2)) for 15 to 45 minutes, with continued instillation of riboflavin. Eyes continued on standard antibiotic treatment. The primary outcome measures were the times to resolution of the infiltrate and the epithelial defect. RESULTS: Forty patients aged 14 to 86 years were enrolled. Seven (18%) eyes had a previous keratoplasty. Bacterial species were identified in 24 eyes, fungal in 7, protozoan in 2, viral in 1, and no organism in 6. The maximum infiltrate diameter ranged from 1 to 12 mm and the epithelial defect diameter was 0 to 8 mm before treatment. In 6 cases (2 bacterial, 3 fungal, and 1 without growth), the keratitis did not resolve successfully and the eye received a penetrating keratoplasty (PK). In 1 eye with prior PK, the infection resolved following treatment, but a regraft was required to address perforation of the PK incision. CONCLUSIONS: Riboflavin/UVA should be avoided in eyes with prior herpes simplex but otherwise posed no obvious safety risk in this series and appeared to be most effective when the infection depth was limited. The success rate was higher for bacterial infections than fungal infections. Randomized studies against antibiotics alone are needed to further evaluate efficacy.

Correlation between extraocular muscle size and motility restriction in thyroid eye disease.

PURPOSE: Evaluate the relationship between extraocular muscle (EOM) size, measured by computed tomography, and ocular motility in thyroid eye disease (TED). METHODS: This case series is based on a retrospective review of the records of 54 patients (108 orbits) with TED. Main outcome measures included EOM size and degree of motility restriction. The average diameter of each rectus muscle was compared with published norms. Four subpopulations based on Age (< 40 or ≥ 40 years) and State of thyroid eye disease (active or stable) were studied. Versions were measured by the corneal light reflex method. The trend of muscle diameter versus motility restriction was evaluated. RESULTS: The average EOM diameter was greater than the norm in the study cohort and 4 subpopulations. The average diameter was largest in the Older and Active TED subpopulations. The inferior rectus and medial rectus were most frequently restricted in the study cohort and 4 subpopulations. The medial rectus had the strongest trend between increasing diameter and motility restriction, followed by the inferior rectus and the superior muscle group (comprised of the superior rectus and levator palpebrae superioris). However, there was a general lack of strong correlation between the diameter of the rectus muscles and their respective motility, especially in the Younger subpopulation. CONCLUSION: EOM diameters are larger and have more restricted motility in the Older and Active TED subpopulations. Contrary to prior publications, the correlation between EOM diameters and motility was weak, especially in the Younger subpopulation. These findings suggest that the pathophysiology of EOM enlargement is different based upon the age of the patient and the activity of the orbitopathy.

Aliphatic beta-nitroalcohols for therapeutic corneoscleral cross-linking: chemical mechanisms and higher order nitroalcohols.

PURPOSE: The recent tissue cross-linking studies indicate that aliphatic beta-nitroalcohols (BNAs) may be useful as pharmacologic corneoscleral cross-linking agents. The present study was performed to identify the specific chemistry involved under physiologic conditions, with the intent of identifying more effective agents. METHODS: The mechanism of chemical cross-linking at pH 7.4 and 37 degrees C was studied using three techniques. The colorimetric Griess assay was used to follow the release of nitrite from three mono-nitroalcohols (2-nitroethanol [2ne], 2-nitro-1-propanol [2nprop]), and 3-nitro-2-pentanol [3n2pent]). Second, the evolution of 2nprop in 0.2 M NaH(2)PO(4)/Na(2)HPO(4)/D(2)O was studied using (1)H-NMR. Third, thermal shrinkage temperature analysis (T(s)), a measure of tissue cross-linking, was used to support information from (1)the H-NMR studies. RESULTS: A time-dependent release of nitrite was observed for all three mono-nitroalcohols studied. The maximum levels were comparable using either 2ne or 2nprop (approximately 30%). However, much less (approximately 10%) was observed from 3n2pent. Using (1)H-NMR, 2nprop evolved into a unique splitting pattern. No match was observed with reference spectra from three possible products of denitration. In contrast, 2-methyl-2-nitro-1,3-propanediol (MNPD), a nitro-diol, was identified, implying the formation of formaldehyde from a retro-nitroaldol (i.e., reverse Henry) reaction. In support of this mechanism, T(s) shifts induced by the nitro-triol 2-hydroxymethyl-2-nitro-1,3-propanediol (HNPD) were superior to the nitro-diol MNPD which were superior to the mono nitroalcohol 2nprop. CONCLUSIONS: BNAs function as both formaldehyde and nitrite donors under physiologic conditions to cross-link collagenous tissue. Higher order BNAs are more effective than mono nitroalcohols, raising the possibility of using these agents for therapeutic corneoscleral cross-linking.

Initial studies using aliphatic beta-nitro alcohols for therapeutic corneal cross-linking.

PURPOSE: Corneal collagen cross-linking through UVA-riboflavin photochemistry (UVAR) has been shown to be an effective treatment for keratoconus and related keratectasias. In recent studies using sclera, the authors observed that short-chain aliphatic beta-nitro alcohols can cross-link collagenous tissue under physiologic conditions. Thus, this study was undertaken to evaluate these agents as potential pharmacologic alternatives to UVAR. METHODS: Porcine corneal strips (8 x 4 mm) and corneoscleral complexes were cross-linked using 1 to 100 mM 2-nitroethanol (2ne), 2-nitro-1-propanol (2nprop), and 3-nitro-2-pentanol (3n2pent) at pH 7.4, 34 degrees C. Cross-linking by UVAR was carried out for comparison. Thermal shrinkage temperature analysis was used to evaluate cross-linking effects, and changes in corneal light transmission were determined with a fiber-optic spectrophotometer. RESULTS: At 10 and 100 mM for 96 hours, initial shrinkage temperature (T(i)) was shifted by 3.3 degrees C (P < 0.001) and 9.8 degrees C (P < 0.001) for 2ne, 2.9 degrees C (P = 0.008) and 4.9 degrees C (P < 0.001) for 2nprop, and 3.8 degrees C (P = 0.003) and 4.9 degrees C (P < 0.001) for 3n2pent. Reacting at 1 mM through daily exchange of fluid over 7 days shifted T(i) by 3.8 degrees C (P < 0.001), 4.4 degrees C (P = 0.002), and 3.2 degrees C (P = 0.005), for 2ne, 2nprop, and 3n2pent, respectively. These shifts were greater than cross-linking using UVAR (T(i) = 1.9 degrees C; P = 0.012). In the blue light region (400-500 nm), transmission was decreased by 5.6% (P = 0.003), 2.1% (P = 0.260), and 0% (P = 0.428) for 2ne, 2nprop, and 3n2pent, respectively. CONCLUSIONS: beta-Nitro alcohols can induce corneal cross-linking in vitro better than the UVAR technique and can induce negligible effects on light transmission. These early results suggest that such compounds could be used as topical stiffening agents for keratoconus and related disorders.