Rabbit Sclera Research Articles

Poster Session: Experimental assessment of scleral anisotropy using multi-meridian air-coupled ultrasonic optical coherence elastography.

Scleral biomechanics plays a key role in the understanding of myopia progression. In this study, we characterized the elastic properties of sclera using an air-coupled ultrasonic (ACUS) optical coherence elastography (OCE) system. New Zealand rabbit eyes (n=7) were measured (<24hr postmortem) in four scleral locations: superior/inferior temporal (ST, IT), and superior/inferior nasal (SN, IN) maintaining an intraocular pressure of 15 mmHg. Elastic waves were induced in the sclera, and wave propagation velocity and shear modulus were measured along two directions: circumferential (superior-inferior) and meridional (nasal-temporal). Wave velocity in scleral tissue ranged from 6 to 24 m/s and shear modulus from 11 to 150 kPa. Velocity was significantly higher (p<.001) in the circumferential vs. meridional directions in the following locations: ST:15.83±2.85 vs 9.43±1.68 m/s, IT:15.00±3.98 vs 8.93±1.53 m/s; SN:16.79±4.30 vs 9.27±1.47 m/s; and IN:13.92±3.85 vs 8.57±1.46 m/s. The average shear modulus in the circumferential was also significantly higher (p<.001) than in the meridional direction for all locations: 65.37±6.04 vs 22.55±1.36 kPa. These results show that the rabbit sclera is mechanically anisotropic with higher rigidity in the circumferential direction compared to the meridional direction. ACUS-OCE is a promising non-invasive method to quantify the biomechanical changes in scleral tissue for future studies involving myopia treatments.

Feasibility of Therapeutic Ultrasound Application in Topical Scleral Delivery of Avastin.

PurposeMacromolecules have been shown to be effective in vision-saving treatments for various ocular diseases, such as age-related macular degeneration and diabetic retinopathy. The current delivery of macromolecules requires frequent intraocular injections and carries a risk of serious adverse effects.MethodsWe tested the application of therapeutic ultrasound as a minimally invasive approach for the delivery of Avastin into the diseased regions of the eye. Avastin (bevacizumab) is an anti-vascular endothelial growth factor (VEGF) antibody with a molecular weight of 149 kDa. We tested the effectiveness and safety of Avastin delivery through rabbit sclera in vitro using a standard diffusion cell model. Ultrasound at frequencies of 400 kHz or 3 MHz with an intensity of 1 W/cm2 was applied for the first 5 minutes of 1-hour drug exposure. Sham treatments mimicked the ultrasound treatments, but ultrasound was not turned on. Absorbance measurements of the receiver compartment solution were performed at 280 nm using a spectrophotometer.ResultsAbsorbance measurements indicated no statistical difference between the sham (n = 13) and 400 kHz ultrasound group (n = 15) in the delivery of Avastin through the sclera. However, the absorbance values were statistically different (P < 0.01) between the 3 MHz ultrasound group (0.004, n = 8) and the matched sham group (0.002, n = 7). There was 2.3 times increase in drug delivery in the 3 MHz ultrasound when compared to the corresponding sham group. Histological studies indicated no significant damage in the ultrasound-treated sclera due to ultrasound application.ConclusionsOur preliminary results provided support that therapeutic ultrasound may be effective in the delivery of Avastin through the sclera.Translational RelevanceOur study offers clinical potential for a minimally invasive retinopathy treatment.

Development of a topical tissue cross-linking solution using sodium hydroxymethylglycinate (SMG): viscosity effect.

Hyperviscosity agents are commonly used in ophthalmic formulations for improving corneal drug penetration by increasing tissue contact time. One such viscosity agent is hydroxypropyl methylcellulose (HPMC). HPMC has been used in riboflavin solutions for photochemical UVA cross-linking (CXL). Sodium hydroxymethylglycinate (SMG) is a small molecule formaldehyde releaser that can function as a therapeutic tissue cross-linker for corneal and scleral applications. The present study was undertaken in order to study formulation factors using HPMC and SMG that could positively influence the cross-linking effect in these ocular tissues. Formulations containing 10 mM SMG and 100 mM sodium bicarbonate were prepared with varying HPMC concentrations from 0 to 4.4%. Their cross-linking effects on porcine and rabbit eyes were measured using differential scanning calorimetry (DSC), expressed as the change/difference in melting temperature (ΔTm) compared with the control. SMG in 4.4% HPMC solution resulted in ΔTm of 6.3 ± 1.21, while other concentration showed no differences in Tm shift on porcine cornea. In ex vivo rabbit cornea, there was a trend toward an increasing cross-linking effect with higher viscosity albeit mild differences. While a significant Tm shift was observed in porcine and rabbit sclera, there was no difference in effect of cross-linking between four HPMC concentrations. Increasing the HPMC concentration does not negatively affect the cross-linking efficacy attributed by SMG and could still be a positive cross-linking enhancer by virtue of increasing tissue contact time in a dynamic biological system. This information will be useful for planning further animal and human studies.

Effects of scleral collagen crosslinking with different carbohydrate on chemical bond and ultrastructure of rabbit sclera: Future treatment for myopia progression.

BackgroundMyopia is the most common ocular disorder and is mainly caused by axial elongation of the sclera. If the stiffness of sclera increased, it can inhibit myopia progression. The aim of this study is to compare the effect of the collagen crosslinking with different types and concentrations of carbohydrates on chemical bond and ultrastructural change of rabbit sclera.MethodsNine New Zealand white rabbits were treated with five, sequential sub-Tenon injections of 0.15 mL solutions of ribose, sucrose, and glycogen of 0.1, 0.2 and 0.4 M concentration at the right eye over 14 days. Ten weeks after the last injection, the rabbits were sacrificed and chemical bond and ultrastructural changes were compared with those of the untreated left sclera using Raman spectroscopy, atomic force microscopy (AFM), and histology.ResultsRaman spectroscopy of the control and cross-linked rabbit sclera tissue revealed different types of collagen interactions. Raman shift of 919 cm-1 (C-C stretching and vibration of the proline ring in collagen) was the highest in ribose, followed by sucrose and glycogen. Total energy intensity was also highest in ribose, followed by sucrose and glycogen, and showed a tendency to increase at higher concentrations. AFM revealed interlocking arrangements of collagen fibrils. The collagen fibril diameter was 105.6 ± 21.2 nm, 109.4 ± 28.8 nm, 113.1 ± 30.8 nm and 137.6 ± 25.3 nm for control group, 0.4 M glycogen, sucrose, and ribose, respectively. Histology indicated increased density of the collagen bundle and no increase in inflammatory cell recruitment compared to control at high concentrations of ribose.ConclusionsScleral crosslinking using glycation increased the scleral biomechanical rigidity and these results were particularly pronounced in ribose. Scleral crosslinking using glycation may be a promising method for inhibiting high myopia progression.

Melt-Cast Films Significantly Enhance Triamcinolone Acetonide Delivery to the Deeper Ocular Tissues.

Delivering an effective drug load to the posterior section of the ocular tissues, while using a non-invasive technique, has always been a challenge. In this regard, the goal of the present study was to develop sustained release triamcinolone acetonide (TA) loaded polymeric matrix films for ocular delivery. The TA-films were prepared in two different polymer matrices, with drug loadings of 10% and 20% w/w, and they were evaluated for ocular distribution in vivo in a conscious rabbit model. A 4% w/v TA suspension (TA-C) was used as a control for in vitro and in vivo studies. The TA-films, prepared with melt-cast technology, used polyethylene oxide (PEO) and Soluplus® as the polymer matrix. The films were evaluated with respect to assay, content uniformity, excipient interaction, and permeability across isolated rabbit sclera. The distribution of TA in the ocular tissues, post topical administration, was determined in New Zealand male albino rabbits as a function of dose, and was compared against TA-C. The assay of the 10% and 20% w/w film was in the range from 70–79% and 92–94% for the Soluplus® and PEO films, respectively, and content uniformity was in the range of 95–103% for both the films. The assay of the TA from Soluplus® films was less compared with the PEO films and showed an interaction with TA, as revealed by Differential Scanning Calorimetry (DSC). Hence, Soluplus® films were not selected for further studies. No interaction was observed between the drug and PEO polymer matrix. The enhancement of trans-scleral flux and permeability of TA was about 1.16 and 1.33-folds, respectively, from the 10% w/w PEO and 3.5 and 2.12-folds, respectively, from the 20% w/w PEO films, as compared with TA-C formulations. The in vivo studies demonstrate that significantly higher TA levels were observed in the anterior and posterior segments of the eye at the end of 6h with the PEO films. Therefore, the PEO based polymeric films were able to deliver TA into the back of the eye efficiently and for prolonged periods.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia.

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon's space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

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

PurposeSecond 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.MethodsSingle 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.ResultsTherapeutic 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.ConclusionsSecond 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.

Biomechanics of sclera crosslinked using genipin in rabbit.

To strengthen the biomechanics of collagen by crosslinking rabbit scleral collagen with genipin to develop a new therapy for preventing myopic progression. Ten New Zealand rabbits were treated with 0.5 mmol/L genipin injected into the sub-Tenon's capsule in the right eyes. Untreated contralateral eyes served as the control. The treated area was cut into scleral strips measuring 4.0 mm×10.0 mm for stress-strain measurements (n=5). The remaining five treated eyes were prepared for histological examination. Compared to the untreated scleral strips, the genipin-crosslinked scleral strips showed that the ultimate stress and Young's modulus at 10% strain were increased by the amplitude of 130% and 303% respectively, ultimate strain was decreased by 24%. There had no α-smooth muscle actin (α-SMA) positive cells in control and treated sclera. Histologically, there was no sign of apoptosis in the sclera, choroid, and retina; and no side effects were found in the peripheral cornea and optic nerve adjacent to the treatment area. Genipin induced crosslinking of collagen can increase its biomechanical behavior by direct strengthening of the extracellular matrix in rabbit sclera, with no α-SMA expression seen in the myofibroblasts. As there is no evidence of cytotoxicity in the scleral, choroidal, and retinal cells, genipin is likely a promising agent to strengthen the weakened sclera to prevent myopic progression.

Ultrasound-enhanced penetration through sclera depends on frequency of sonication and size of macromolecules

We previously employed ultrasound as a needleless approach to deliver macromolecules via the transscleral route to the back of the eye in live animals (Suen et al., 2013). Here, we investigated the nature of the ultrasound-enhanced transport through sclera, the outermost barrier in the transscleral route. Thus, the possible role of cavitation from ultrasound was explored; its effect during and after sonication on scleral penetration was measured; and the dependence on the size of macromolecules was determined. We applied ultrasound frequency from 40kHz to 3MHz at ISATA (spatial-average-temporal-average intensity) of 0.05W/cm2 to fresh rabbit sclera ex vivo. Fluorescent dextran of size 20kDa to 150kDa was used as macromolecular probes. We measured the distance of penetration of the probes through the sclera over 30s during sonication and over 15min after sonication from cryosectioned tissue images. Deeper penetration in the sclera was observed with decreasing frequency. The presence of stable cavitation was further verified by passive acoustic detection. The effect during sonication increased penetration distance up to 20 fold and was limited to macromolecular probes ≤70kDa. The effect post sonication increased penetration distance up to 3 fold and attributed to the improved intrasscleral transport of macromolecules ≥70kDa. Post-sonication enhancement diminished gradually in 3h. As the extent of cavitation increased with decreasing frequency, the trend observed supports the contribution of (stable) cavitation to enhancing transport through sclera. Effect during sonication was attributed to flow associated with acoustic microstreaming. Effect post sonication was attributed to the temporary increase in scleral permeability. Flow-associated effect was more pronounced but only applied to smaller macromolecules.

Anatomical differences of the protein profile in the rabbit sclera during growth

We aimed to investigate the proteome changes in anatomical regions of sclera during growth and development of the rabbit. Sclera from New Zealand white rabbits of three ages (1 month, 2 months and 6 months) was dissected into three segments - anterior, equatorial, and posterior. A total of 36 samples were divided into groups by age and anatomical region. Tryptic digests of total proteins were analyzed by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS). Label-free quantification based on spectral counts was used to determine the relative protein abundance and identify proteins with statistically significant differences between age groups or anatomical regions of the sclera. Western blotting was performed to validate some of the differentially expressed proteins. A total of 840 non-redundant proteins was identified in the sclera at different ages and regions with protein and peptide false discovery rate (FDR) at ≤1.0% and ≤0.1%, respectively. Differentially expressed proteins were identified by comparing age or anatomical region. Among these, periostin showed decreasing abundance with age, while myocilin, latent-transforming growth factor beta-binding protein 2, hyaluronan, proteoglycan link protein 1 and selenbp1 showed increasing abundance with age. In mature rabbits, alcohol dehydrogenase showed region-related differences in the sclera. Periostin showed an age-related decrease while selenbp1 showed an age-related increase in abundance in the anterior region. Vitronectin and extracellular superoxide dismutase had greater expression with age in the equatorial and posterior regions, respectively. The age related differential expression of periostin and selenbp1 was confirmed by western blotting. In conclusion, the protein profile of sclera showed age- and region-related differences. The differential protein profiles provide a baseline for understanding changes in the protein expression in the young and mature rabbit that appears to show regional changes. The changes observed in the present study add to the existing knowledge about regional alterations in biomechanical properties of sclera during growth.

The prospects of using the radiation for the assessment of corneal and scleral hydration

PurposeAn adequate water balance (hydration extent) is one of the basic factors of normal eye function, including its external shells – the cornea and the sclera. THz systems creating images in reflected beams are likely to become ideal instruments of noninvasive testing of corneal and scleral hydration degree as THz radiation is highly sensitive to water content. The paper aims at studying the transmittance and reflectance spectra of the cornea and the sclera of rabbit and human eyes, as well as those of the whole rabbit eye, in the frequency range of 0.13–0.32 THz.MethodsThe experiments were carried out on 3 corneas and 3 rabbit scleras, 2 whole rabbit eyes, and 3 human healthy adult scleras using a specially developed THz system based on reliable and easy‐to‐use continuous wave sources: a backward‐wave oscillator and an avalanche transit‐time diode.ResultsThe transmittance spectra of the cornea and the sclera and the dependence of the reflection coefficient of these tissues in THz range on water percentage content were determined. Comparison of the rabbit cornea hydrated from 73.2% to 76.3% concentration by mass demonstrated an approximately linear relationship between THz reflectivity and water concentration. The decrease of free water concentration by 1% leads to a drop of the reflectance coefficient by 13%. The parameters studied displayed noticeable differences between the sclera and the cornea of rabbits and between rabbit sclera and human sclera.ConclusionsPreliminary results demonstrate that the proposed technique, based on continuous THz radiation, may be used to create a device for noninvasive testing of corneal and scleral hydration, which has good potential of wide‐scale practical application.The work was supported by the Russian Foundation of Basic Research (grant No.15‐29‐03843)

The ultrastructure of rabbit sclera after scleral crosslinking with riboflavin and blue light of different intensities.

We aimed to determine the ultrastructural changes of collagen fibrils and cells in the rabbit sclera after scleral crosslinking using riboflavin and blue light of different intensities. Scleral crosslinking is known to increase scleral stiffness and may inhibit the axial elongation of progressive myopic eyes. The equatorial parts of the sclera of one eye of six adult albino rabbits were treated with topical riboflavin solution (0.5%) followed by irradiation with blue light (200, 400, 650mW/cm(2)) for 20min. After 3weeks, the ultrastructure of scleral cells and the abundance of small- (10-100nm) and large-diameter (>100nm) collagen fibrils in fibril bundles of different scleral layers were examined with electron microscopy. In the scleral stroma of control eyes, the thickness of collagen fibrils showed a bimodal distribution. The abundance of small-diameter collagen fibrils decreased from the inner towards the outer sclera, while the amount of large-diameter fibrils and the scleral collagen content did not differ between different stroma layers. Treatment with riboflavin and blue light at 200mW/cm(2) did not induce ultrastructural changes of cells and collagen fibrils in the scleral stroma. Treatment with blue light of higher intensities induced scleral cell activation in a scleral layer-dependent manner. In addition, outer scleral layers contained phagocytes that engulfed collagen fibrils and erythrocytes. Blue light of the highest intensity induced a reduction of the scleral collagen content, a decreased abundance of large-diameter collagen fibrils, and an increased amount of small-diameter fibrils in the whole scleral stroma. The data indicate that in rabbits, scleral crosslinking with riboflavin and blue light of 200mW/cm(2) for 20min is relatively safe and does not induce ultrastructural alterations of scleral cells and of the collagen composition of the scleral stroma. Irradiation with blue light of intensities between 200 and 400mW/cm(2) induces scleral cell activation, which may contribute to scleral scarring and stiffening. Higher intensities cause scleritis.

Investigation on the concentration of riboflavin in sclera tissue

To investigate the permeability and saturation characteristics of riboflavin in sclera tissue, in order to explore a reasonable dosage regimen in sclera cross-linking. Experimental study. Five human cadaver eyes were also included in this study, every eye was cut into seven sclera stripes and then 0.1% riboflavin solution was dripped to the sclera stripes for 5, 10, 15, 20, 25, 30 minutes respectively. After that, the stripes were made into frozen sections and observed by confocal microscope. To calculate the mean fluorescence density and compare with each other. In addition, 36 New Zealand rabbits were randomly divided into group A and B groups. Each group was divided into six subgroups. In group A, 0.1% riboflavin solution was dripped to the sclera of right eye for 5, 10, 15, 20, 25 and 30 minutes respectively. In group B, 0.5% ribof lavin solution was dripped to the sclera of right eye for 5, 10, 15, 20, 25 and 30 minutes respectively. The left eye were given with physiological saline as blank check. Executed the rabbits to take out the eye and then the nose upper quadrant of the equator sclera was taken to made into frozen sections which were observed by two-photon microscope. To calculate the mean fluorescence density and compare the six subgroups in every group respectively, and compare the subgroups with the same riboflavin-applicated time between group A and B respectively. The fluorescence intensity of sclera gradually strengthened with the longer riboflavin-applicated time, but there was no statistical difference between 20 minutes and the longer time groups (P > 0.05). With the same riboflavin-applicated time in rabbit sclera, the fluorescence intensity of 0.5% concentration group was higher than 0.1% concentration group (P < 0.05). The riboflavin reached an saturation level in the sclera of human cadaver eyes and rabbit eyes after 20 minutes application. The higher concentration of riboflavin-applicated, the higher concentration of riboflavin in the rabbit sclera.

Novel dexamethasone-loaded nanomicelles for the intermediate and posterior segment uveitis.

Development and characterization of dexamethasone (DEX)-encapsulated polymeric nanomicelles have been reported. A low molecular weight di-block copolymer was synthesized and characterized for its structure, molecular weights, critical micelle concentration (CMC), and cytotoxicity in ocular cells. In order to delineate the effects of drug-polymer interactions on drug solubilization in micelle core, a response surface methodology was generated with the help of SAS 9.02 (exploratory model). The method for preparing micelle was modified based on the results obtained from exploratory model. The formulation was optimized by response surface methodology (optimization model) to achieve DEX solubility of above 1mg/mL. The optimized formulation was characterized for DEX solubility, nanomicelle size, polydispersity index, surface morphology, in vitro transport across conjunctival cell line, and ex vivo transport across excised rabbit sclera. Nanomicelles exhibited average sizes in range of 25-30nm with unimodel size distribution and low polydispersity of 0.125. Nanomicelles increased DEX permeability by 2 times across conjunctival cell line and by 2.5 times across the excised rabbit sclera as compared to DEX suspension. A design of experiment (DOE) strategy was successfully applied to understand the effects of drug-polymer interaction on drug solubility. DOE was also employed to achieve optimal formulation with high DEX solubility. Nanomicellar formulation significantly enhanced DEX permeability across the excised rabbit sclera. Therefore, nanomicellar formulation may provide therapeutic levels in the back of the eye following topical administration.

Microneedle-mediated intrascleral delivery of in situ forming thermoresponsive implants for sustained ocular drug delivery

This paper describes use of minimally invasive hollow microneedle (HMN) to deliver in situ forming thermoresponsive poloxamer-based implants into the scleral tissue to provide sustained drug delivery. In situ forming poloxamer formulations were prepared and investigated for their rheological properties. HMN devices 400, 500 and 600 μm in height were fabricated from hypodermic needles (i.e. 27, 29 and 30 G) and tested for depth of penetration into rabbit sclera. Maximum force and work required to expel different volumes of poloxamer formulations was also investigated. Release of fluorescein sodium (FS) from intrasclerally injected implants was also investigated. Optical coherence tomography (OCT) was used to examine implant localisation and scleral pore-closure. Poloxamer formulations showed Newtonian behaviour at 20°C and pseudoplastic (shear-thinning) behaviour at 37°C. Maximum force and work required to expel different volumes of poloxamer formulations with different needles ranged from 0.158 to 2.021 N and 0.173 to 6.000 N, respectively. OCT showed intrascleral localisation of implants and scleral pore-closure occurred within 2-3 h. Sustain release of FS was noticed over 24 h and varied with depth of implant delivery. This study shows that the minimally invasive HMN device can localise in situ forming implants in the scleral tissue and provide sustained drug delivery.

Comparison of Riboflavin/Ultraviolet-A Cross-Linking in Porcine, Rabbit, and Human Sclera

Purpose. To compare the biomechanical properties of porcine, rabbit, and human sclera before and after riboflavin/ultraviolet-A (UVA) collagen cross-linking (CXL). Methods. Eight rabbits, 8 porcine eyeballs, and 8 human eyeballs were included. One rabbit eye and half of each bisected human and porcine eyeball were treated with riboflavin/UVA CXL. Untreated fellow rabbit eyes and eyeball halves served as controls. A 10 mm × 20 mm scleral band was harvested from each specimen. From this band, two 3.5 mm × 15.0 mm strips were prepared for biomechanical testing. The biomechanical parameters were ultimate stress, stress and Young's modulus. Results. Values of stress, and Young's modulus showed that human sclera was 4 times stiffer than porcine sclera and 3 times stiffer than rabbit sclera. In rabbit sclera, both the stress and Young's modulus were significantly increased by CXL (P < 0.05). In porcine sclera, only the ultimate stress was significantly increased by CXL (P < 0.05). The biomechanical properties of human sclera were not statistically affected by CXL (P > 0.05). Conclusions. Human sclera has higher biomechanical stiffness than porcine and rabbit sclera. With the same irradiation dose, riboflavin/UVA CXL increases the biomechanical stiffness of rabbit sclera but not porcine or human sclera.

Scleral thickness topography of the adult Chinchilla pigmented rabbit with implications for transscleral drug delivery research

Objective To measure the scleral thickness of the rabbit eye at different locations in the globe,providing a theoretical basis for advanced research in transscleral drug delivery.Methods In this experimental study,98 eyes of 49 adult pigmented rabbits were sacrificed after deep anesthesia and the eyeballs were enucleated and fixed in 1％ buffered formaldehyde and 1.25％ glutaraldehyde for a minimum of 36 hours.The eyes were processed for routine paraffin embedding and sectioning.The sagittal sections at the optic nerve level were collected and used for scleral thickness study.The images were acquired at serial locations between the limbus and optic nerve and the thickness was measured with SPOT image software.The data were analyzed with generalized estimating equations (GEE) and Tukey's HSD Post Hoc Test.Results In general,the mean scleral thickness of the inferior eye globe was significantly thinner than that of the superior globe (342.9±91.3 μm versus 400.4±67.6 μm,x2=43.57,P＜0.01,GEE).There were no significant differences in the scleral thickness from the pre-equator vicinity to the posterior pole,with a mean thickness of 366.8±56.3 μm at the superior globe.For the inferior globe,the mean scleral thickness at the pre-equator vicinity and at the equator was similar,340.9±72.5 μm and 340.8±76.3 μm.However,scleral thickness decreased to 293.9±57.4 μm at the post-equator vicinity and to 209.0±51.8 μm at the posterior pole.The thinnest sclera was located immediately around the optic nerve,273.5±90.5 μm for the superior and 187.7±60.1 μm for the inferior,compared to the other locations (P＜0.05,Tukey's HSD Post Hoc Test).Conclusion Rabbit sclera is thinner than human sclera.In the rabbit eye globe,the superior sclera is significantly thicker than the inferior sclera and superior sclera is shorter and more uniform in thickness between the equator and the posterior pole.Scleral thickness has larger variations between the limbus and equator than the variations between the equator and the posterior pole. Key words: Rabbits; Scleral thickness; Transscleral drug delivery

Effect of irradiation time on riboflavin–ultraviolet-A collagen crosslinking in rabbit sclera

To determine the effect of the duration of irradiation on the biomechanical parameters of combined riboflavin-ultraviolet-A (UVA) collagen crosslinking (CXL) in rabbit sclera. Department of Ophthalmology, Provincial Hospital affiliated with Shandong University, Shandong, China. Experimental study. Thirty-six New Zealand rabbits were divided into 6 groups based on the duration of irradiation (10, 20, 30, 40, 50, or 60 minutes). After the application of riboflavin 0.1% drops (without dextran) as a photosensitizer, the animals were irradiated with 3 mW/cm(2) UVA at 365 nm. Only the left eye of each rabbit was treated. All the animals were humanely killed 24 hours postoperatively. One eye in each treated group was used for light microscopy. The other treated eye and all control eyes were prepared for biomechanical testing. The biomechanical parameters were ultimate stress, Young modulus, and the physiological modulus. The eyes irradiated for 10 or 20 minutes did not differ significantly from the control eyes. Stress-strain measurement of scleral strips irradiated for 40 minutes or longer showed a significant increase in the ultimate stress, Young modulus, and the physiological modulus. There was a significant increase in the physiological modulus of scleral strips irradiated for 30 minutes or longer. Eyes that were irradiated for 50 minutes and 60 minutes had retinal damage. Riboflavin-UVA CXL can lead to a noticeable increase in the biomechanical stiffness of the sclera. The physiological modulus is the most sensitive tool to measure stiffness. In this study, the optimum duration of irradiation was 40 minutes.

Collagen crosslinking of porcine sclera using genipin

To evaluate the effect of collagen cross-linking induced by genipin in porcine sclera. Porcine cadaver eyes were treated with genipin at concentrations (by w/v) of 0.01%, 0.03%, 0.1%, 0.3%, 1.0% for 15 and 30 min. Riboflavin/ultraviolet A(UVA)-treated and untreated samples were used as controls. After treatment, scleral strips of 4.0 × 10.0 mm were cut. Twenty-four hours later, the stress-strain parameters of the strips were measured using a biomaterial microtester. The stress and Young's modulus at 8% strain were evaluated. Compared with untreated groups, after treatment with genipin for 15 min, the stress was increased by 66-246%, depending on the concentration of genipin. As for the 30-min groups, the stress was 171-444% higher than that of the control. The difference of the Young's modulus between genipin 15-min groups, except the 0.01% groups (p = 0.095), also had statistical significance (p < 0.05). The Young's modulus had significant difference between the untreated group and the genipin 30-min groups (all p < 0.05). Of 0.3% genipin for 15 min or 0.01% genipin for 30 min had a similar stress-strain curve with those of eyes treated with the riboflavin/UVA group. The sclera exhibited a bluish colour which became deeper with increase concentration and cross-linking time. Collagen cross-linking induced by genipin could increase the biomechanical strength in porcine sclera. The effect depends on the concentration and treatment time of genipin.