Porcine Sclera Research Articles

Microincision vitrectomy surgery: experimental visualization and quantification of vitreous contamination

BackgroundTo visualize and quantify vitreous contamination following microincision vitrectomy surgery (MIVS) using an experimental vitreous contamination model (EVCM).MethodsEnucleated porcine eyes with fluoresbrite carboxylate microspheres applied to the conjunctival surface were used as a type 1 EVCM. Twenty-five- or 27-gauge (G) trocar cannulas were inserted through the conjunctiva and sclera, followed by the placing and opening of an infusion cannula. These procedures were monitored by an intraocular fiber catheter. Secondly, condensed microspheres were applied to an excised sheet of porcine sclera to serve as type 2 EVCM. Twenty-five- or 27-G trocar cannulas were inserted perpendicularly through the top of the sclera where the condensed microspheres were applied, an infusion cannula was inserted, 0.1 mL of saline solution injected through the infusion cannula, and samples collected. The fluorescence strength of samples was then measured using fluorophotometry.ResultsWe visually detected fluorescent microspheres in 10/10 eyes with 25-G and 10/10 with 27-G MIVS. In the experimental quantification study, each MIVS gauge value was significantly higher than the control (P < 0.01). However, there was no significant difference between 25-G and 27-G MIVS.ConclusionsMIVS carries the risk of introducing contamination directly into the eyes when the trocar cannula is inserted and infusion cannula is opened, even when a 27-G MIVS is used. Our study has shown it is essential that the surgeon be aware of the possibility of introducing contamination from the conjunctiva at all times during MIVS.

Scleral defect repair using decellularized porcine sclera in a rabbit model.

The sclera is one of the most commonly used repair materials in ophthalmic plastic surgery and is often used for supporting, wrapping, filling, and pressing during surgery. Although the sclera plays an irreplaceable role in ophthalmology applications, there are many restrictive factors, such as high costs and limited sources. Here, we report the use of a decellularized porcine sclera (DPS) for scleral reconstruction in rabbit models. The DPS generated by a hybrid decellularization protocol was characterized in respect of histological observation, DNA, α-gal, GAG, and collagen content. The mechanical properties were evaluated by uniaxial tensile testing. LIVE/DEAD and Cell Counting Kit (CCK)-8 assays were performed to assess its in vitro cytocompatibility and cytotoxicity. In vivo biocompatibility and biointegration of the DPS for repairing scleral defect in rabbit were measured by slit-lamp and histological analyses. Immunohistochemical (IHC) staining was used to detect the expression of CD4, CD8, CD45, CD68, and vimentin. Through decellularization, the major xenoantigen DNA and α-gal are efficiently removed while abundant matrix components and mechanical properties are well preserved in the DPS. Extracts of the DPS and DPS samples had no inhibitory effects on the proliferation of HFSFs. Moreover, there was no sign that an immune reaction occurred in or around the transplanted DPS grafts within 28days of animal implantation. The decellularization strategy we developed is feasible and effective. The prepared DPS holds great potential for the repair of scleral injury.

Effect of enzyme-induced collagen crosslinking on porcine sclera

This study aims to evaluate the effect of a new type of collagen crosslinking (CXL) mediated by microbial transglutaminases (Tgases) on sclera. Porcine eyes were divided into two groups according to the different crosslinking procedures used: the double-sided CXL group (D-CXL group) and the single-sided CXL group (S-CXL group). In the D-CXL group, 4.0 × 14.0 mm scleral strips harvested from 40 porcine eyeballs were incubated with 1 U/ml Tgases for 30 min at 37 °C. Parallel scleral strips from the same eyeball were incubated with PBS under the same conditions as the controls. In the S-CXL group, 80 whole globes were directly incubated with 1 U/ml Tgases and PBS as the controls for 30 min at 37 °C. After incubation, 4.0 × 14.0 mm scleral strips were cut from each eyeball. Biomechanical testing and light microscopy were used. In the D-CXL group, the general elastic modulus of the Tgases-treated scleral strips was 14.89 ± 6.05 MPa, and the controls was 6.72 ± 2.58 MPa, indicating an increase of 121% with Tgases treatment. In the S-CXL group, the general elastic modulus of the Tgases-treated scleral strips was 12.88 ± 4.29 MPa, and the controls was 7.00 ± 2.45 MPa, indicating an increase of 84% with Tgases treatment. In both the D-CXL and S-CXL groups, significant increases in scleral rigidity were observed compared to that of the respective controls (P < 0.05). The histology indicated increased collagen bundle density, decreased interfibrillar spaces and increased interlamellar spaces after CXL. In conclusion, scleral collagen crosslinking mediated by Tgases produced a significant increase in biomechanical strength.

Rapidly Detachable Microneedles Using Porous Water‐Soluble Layer for Ocular Drug Delivery

AbstractDetachable microneedle (MN) technology is highly desired for treatment of ocular diseases such as keratitis or glaucoma because of its minimally invasiveness and sustained drug delivery. However, shortening of administration time during insertion into target tissues still remains an important issue, although various approaches using mechanical and chemical separation mechanisms have been attempted. Here, a rapidly detachable microneedle pen (RD‐MNP) containing a porous dissolvable sacrificial layer is fabricated for instant separation of the tip. The porous sacrificial layer is fabricated between a drug‐loaded tip and a base by freeze drying. The sacrificial layer is comprised of water soluble polymers: poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP). Porous structure can accelerate dissolution rate compared to solid structure due to its capillary effect that relies on pore size and distribution. The pore size and distribution of the sacrificial layer are controlled and detachment of an RD‐MNP with the optimum pore structures is immediate upon dipping the MNP in PBS. Furthermore, the insertion characteristics of the system are investigated using the gelatin phantom and porcine eye sclera. With the optimized sacrificial layer, a MN tip is almost immediately detached and embedded to the porcine sclera with the aid of impact insertion.

The contribution of sGAGs to stress-controlled tensile response of posterior porcine sclera.

Despite the significant progress in characterizing mechanical functions of individual scleral extracellular matrix (ECM) components, the biomechanical contribution of sulfated glycosaminoglycans (sGAGs) is still poorly understood. The primary purpose of this study was to determine the possible function of sGAGs in scleral mechanical response by characterizing the tensile behavior of normal and sGAG-depleted samples. We used chondroitinase ABC solution to remove sGAGs from scleral samples that were dissected from posterior porcine eyes. We performed biochemical analyses for assessing the efficacy of sGAG removal protocol. Furthermore, we conducted stress-controlled uniaxial tensile tests to characterize the influence of sGAG removal on mechanical properties of sclera. The tensile behavior of scleral strips right after dissection and after being soaked in buffer was also determined. Biochemical analyses confirmed that 18 hour incubation in 0.125 U/ml Chondroitinase ABC solution removed over 90% of chondroitin and dermatan sGAGs. No significant difference was observed in the thickness/hydration of samples because of enzyme- and buffer-treated samples. Furthermore, it was found that sGAG depletion did not significantly alter the tangent modulus, energy dissipation, and peak strain of posterior scleral strips. It was concluded that sGAGs did not influence the stress-controlled viscoelastic tensile response of sclera.

Hollow Microcapsules as Periocular Drug Depot for Sustained Release of Anti-VEGF Protein.

Diseases affecting the posterior segment of the eye such as age-related macular degeneration and diabetic retinopathy are leading causes of blindness all over the world. The current treatment regimen for such diseases involves repeated intravitreal injections of anti- Vascular Endothelial Growth Factor (VEGF) proteins. This method is highly invasive and can lead to severe complications. In an attempt to develop less invasive alternatives, we propose the use of a controlled release system consisting of anti-VEGF loaded hollow microcapsules that can be administered periocularly to form drug eluting depots on the episcleral surface. The microcapsules with either positive or negative surface charge were prepared by a layer by layer approach and showed pH responsive permeability switching. An ex vivo experiment using porcine sclera indicated positively charged microcapsules remained on the episcleral surface over four days while the negatively charged microcapsules were washed away. These positively charged microcapsules were then loaded with anti-VEGF protein ranibizumab using pH dependent permeability switching and protein release from the microcapsules were studied using an in vitro setup. An ex vivo experiment utilizing porcine sclera demonstrated sustained release of ranibizumab over seven days with zero-order kinetics.

Influence of optical clearing on collagen crosslinking of sclera

The dynamics of the change of dimensions (cross-section area) of porcine sclera specimens in the process of optical clearing was measured using optical coherence tomography (OCT). Based on the obtained data, the optimal (corresponding to maximum tissue contraction) time interval between the application of the immersion agent and UV/riboflavin photo-crosslinking was found. The ability of multiphoton microscopy (tomography) to detect morphological changes in scleral tissue subjected to UV/riboflavin crosslinking in optically cleared state (with subsequent rehydration) was discussed based on measured BSHG images of UV/riboflavin treated porcine sclera ex vivo.

Nanostructural and mechanical changes in the sclera following proteoglycan depletion

The mechanical properties of ocular tissues, such as the sclera, have a major impact on healthy eye function, and are governed by the properties and composition of the microstructural components. For example, biomechanical degradation associated with myopia occurs alongside a reduction of proteoglycans (PGs). In this study, the role of PG degradation in the nanomechanical properties of the porcine sclera is explored. In-vitro enzymatic degradation of PGs was conducted with α-amylase and chondroitinase ABC enzymes. Collagen fibril morphology and nanomechanical stiffness were measured with atomic force microscopy (AFM). The elastic modulus of the tissue was reduced in all enzyme-treated samples relative to controls. In addition, collagen fibril organization was disrupted by PG depletion. Our data demonstrate that PGs play an important role in determining not only the mechanical properties at these length scales, but also collagen fibril arrangement.

THE PERMEABILITY OF THE SCLERAL TISSUE FOR ULTRAVIOLET A IN THE EXPERIMENT

Purpose : to evaluate the permeability of the scleral tissue for ultraviolet A (UVA). Materials and Methods. The study was conducted on 15 cadaver porcine eyes. Rectangular scleral flap 12*15 mm was cut out from each eyeball. The thickness of the sample was measured with a digital caliper. The outer surface of the scleral flap was irradiated with ultraviolet A. A sensor of the UV-radiometer was placed under the sample. It measured the UVA irradiance passing through the sclera. The absorption coefficient of ultraviolet A by the sclera was calculated from the formula derived from the Bouguer-Lambert Beer law: , where E 0 — the absorption coefficient, mm -1 ; — irradiance of the transmitted light, mW/cm 2 ; I 0 — irradiance of the received light, mW/cm 2 ; — thickness of the sclera, mm. Results . The absorption coefficient of ultraviolet A by the sclera was 4,91±0,43 mm -1 . According literature the threshold damaging radiation for retina is 7.7 J/cm 2 . Substituting these values in the converted formula of the Bouguer-Lambert-Beer law, we can derive an equation for calculating the threshold UVA irradiation dose of the sclera: , where 0 — the threshold UVA irradiation dose of the sclera, J/cm 2 ; — the base of the natural logarithm; — thickness of the sclera, mm. Conclusion. The absorption coefficient of ultraviolet A by the cadaver porcine sclera is 4,91±0,43 mm -1 . The derived formula of the threshold UVA irradiation dose of the sclera calculating allows to avoid the threatening for the retina procedure parameters in the performance of the scleral crosslinking with riboflavin/UVA.

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques.

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera’s collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.

EXPERIMENTAL VISUALIZATION AND QUANTIFICATION OF VITREOUS CONTAMINATION FOLLOWING INTRAVITREAL INJECTIONS

To detect and quantify vitreous contamination after intravitreal injection using an experimental vitreous contamination model. Enucleated porcine eyes served as a Type 1 experimental vitreous contamination model with fluoresbrite carboxylate microspheres applied to the conjunctival surface. Saline solution (0.05 mL) was injected using a 27-, 30-, or 32-gauge (G) needle. Injection procedures were monitored using an intraocular fiber catheter. Condensed microspheres were applied to an excised sheet of porcine sclera (Type 2 experimental vitreous contamination model). Saline solution (0.05 mL) was injected from the top of an applied condensed microsphere through the sclera using a needle of one of the aforementioned gauges, and samples were then collected. The fluorescence strength of samples was measured using fluorophotometry. We visually detected fluorescent microspheres in 10/10, 9/10, and 9/10 eyes injected with 27-G, 30-G, and 32-G needles, respectively. In the experimental quantification study, values at all needle gauges were significantly higher than those of controls (P < 0.01). Fluorescence strength was significantly higher in the 27-G group than in the 30- (P < 0.01) and 32-G (P < 0.01) groups. Intravitreal injection carries the risk of introducing contamination directly into the eyes even when a 32-G needle is used. Furthermore, the 27-G needle carries the highest contamination risk.

Evaluation of Dimensional and Flow Properties of ExPress Glaucoma Drainage Devices.

ExPress devices are available as P50 and P200 models, the numbers related to their luminal diameters in μm. We compared their Poiseuille's Law-based theoretical resistance values with experimental values and correlated these with their luminal dimensions derived from electron microscopy. Scanning electron microscopy was performed on P50 and P200 devices. Bench-top flow studies were performed to find the resistances of the devices. Devices were also incorporated into a perfused, ex vivo porcine sclera model to test and compare their control of pressure, with and without overlying scleral flaps, and with trabeculectomies. The luminal dimensions of the P200 device were 206.4±3.3 and 204.5±0.9 μm at the subconjunctival space and anterior chamber ends, respectively. Those of the P50 device were 205.0±5.8 and 206.9±3.7 μm, respectively. There were no significant differences between the P200 and P50 devices (all P>0.05). The resistances of the P200 and P50 devices were 0.010±0.001 and 0.054±0.002 mm Hg/μL/min, respectively (P<0.05). Equilibrium pressures with overlying scleral flaps were 17.81±3.30 mm Hg for the P50, 17.31±4.24 mm Hg for the P200, and 16.28±6.67 mm Hg for trabeculectomies (P=0.850). The luminal diameters of both devices are externally similar. The effective luminal diameter of the P50 is much larger than 50 μm. Both devices have low resistance values, making them unlikely to prevent hypotony on their own. They lead to similar equilibrium pressures as the trabeculectomy procedure when inserted under the scleral flap.

Preparation and Evaluation of Chitosan Submicroparticles Containing Pilocarpine for Glaucoma Therapy.

The objective of the present study was to optimize the preparation of chitosan submicroparticles and to assess whether they enhanced ocular permeation of pilocarpine. Submicroparticles were produced by spray drying and characterized to determine process yield, encapsulation efficiency, morphology, size distribution, drug-polymeric matrix interaction, porcine sclera permeation as well as ocular irritancy and drug retention. Quantification of pilocarpine using High Performance Liquid Chromatography was found to be selective, linear, precise, accurate and robust. The spray drying method proved to be simple and reproducibly produced particles with satisfactory yields, thus showing potential for industrial scale applications. The pilocarpine-loaded chitosan particles exhibited adequate morphological characteristics as well as high encapsulation efficiency. The particles produced were on a submicrometric scale and compatible with intraocular administration. In pilocarpine-loaded particles, the interaction between pilocarpine and chitosan polymeric matrix resulted in delayed release of the drug, attributed to formation of a reservoir system. The best fit for drug release was obtained using the Higuchi equation. The chitosan submicroparticles enhanced the permeation effect and increased the passage of pilocarpine through porcine sclera and also demonstrated low irritancy potential. Therefore, the particles produced can be considered a promising system for the ocular delivery of pilocarpine.

Characterization of silver nanoparticle-infused tissue adhesive for ophthalmic use

In this work, we demonstrate the successful enhancement of breaking strength, adhesive strength, and antibacterial efficacy of ophthalmic tissue adhesive (2-octyl cyanoacrylate) by doping with silver nanoparticles, and investigate the effects of nanoparticle size and concentration. Recent work has shown that silver nanoparticles are a viable antibacterial additive to many compounds, but their efficacy in tissue adhesives was heretofore untested. Our results indicate that doping the adhesive with silver nanoparticles reduced bacterial growth by an order of magnitude or more; nanoparticle size and concentration had minimal influence in the range tested. Tensile breaking strength of polymerized adhesive samples and adhesive strength between a T-shaped support and excised porcine sclera were measured using a universal testing machine according to ASTM (formerly American Society for Testing and Materials) standard techniques. Both tests showed significant improvement with the addition of silver nanoparticles. The enhanced mechanical strength and antibacterial efficacy of the doped adhesive supports the use of tissue adhesives as a viable supplement or alternative to sutures.

Study of the spectral features of different biological samples

In the present study we have observed and analyzed the fluorescence changes in the fluorescence spectra of four different samples like brilliant sulphaflanine, quinine bisulphate, coumarine 120 and porcine cornea and sclera including the changes in fluorescence spectrum of cornea are also observed after CO2 laser exposure. The preliminary study clearly explains the proof of concept only.

In vitro scleral lutein distribution by cyclodextrin containing nanoemulsions.

Lutein is a macular pigment that contributes to maintaining eye health. The development of lutein-laden nanocarriers for ocular delivery would have the advantages of user friendliness and cost-effectiveness. Nano-scaled vehicles such as cyclodextrin (CD) and nanoemulsion could overcome the barriers caused by the scleral structure. This study focused on the development of hybrid nanocarriers containing nanoemulsion and CD for scleral lutein accumulation. In the presence of the nanoemulsion, CD forms such as βCD and hydroxyethyl (HE) βCD increased the partition of lutein into the porcine sclera. A combination of nanoemulsion and 2% HEβCD enhanced lutein accumulation to 119±6 µg g(-1) h(-1), which was 9.2-fold higher than that with lutein suspension alone. We explored the dose effect of CD in nanoemulsion on scleral lutein and found that the scleral accumulation of lutein was enhanced by increasing the CD content. The novel nanoemulsion had 95% drug-loading efficiency and low cytotoxicity in retinal cells. The CD-modified nanoemulsion not only improved the stability and entrapment efficacy of lutein in the aqueous system but also enhanced scleral lutein accumulation. An increase in the partition coefficient of lutein in porcine sclera when using the CD-modified nanoemulsion was also confirmed.

Transsclera Drug Delivery by Pulsed High-Intensity Focused Ultrasound (HIFU): An Ex Vivo Study

Purpose/aim of study: Drug delivery to the ocular posterior segment is of importance, but it is a challenge in the treatment of irreversible blindness disease, such as age-related macular degeneration. Although some methods (i.e. intraocular injection, sustained release by polymer and iontophoresis) have been applied, some technical drawbacks, such as slow rate and damage to the eye, need to be overcome for wide use.Materials and methods: In this study, the feasibility of high-intensity focused ultrasound (HIFU) to enhance the transsclera drug delivery was tested for the first time. One-hundred HIFU pulses with the driving frequency of 1.1 MHz, acoustic power of 105.6 W, pulse duration of 10–50 ms and pulse repetition frequency of 1 Hz were delivered to the fresh ex vivo porcine sclera specimen.Results: In comparison to the passive diffusion (control), 50-ms HIFU can increase the penetration depth by 2.0 folds (501.7 ± 126.4 µm versus 252.4 ± 29.2 µm) using bicinchoninic acid assay and Rhodamine 6 G fluorescence intensity by 3.1 folds (22.4 ± 12.3 versus 7.1 ± 4.1) and coverage area by 2.6 folds (40.4 ± 9.1% versus 15.8 ± 2.9%). No morphological changes on the sonicated sclera samples were found using a surface electron microscope.Conclusions: In summary, pulsed-HIFU may be an effective modality in the transsclera drug delivery with a high transporting rate and depth. In vivo studies are necessary to further evaluate its performance, including the drug penetration and its possible side effects.

The effects of glycosaminoglycan degradation on the mechanical behavior of the posterior porcine sclera

Pathological changes in scleral glycosaminoglycan (GAG) content and in scleral mechanical properties have been observed in eyes with glaucoma and myopia. The purpose of this study is to investigate the effect of GAG removal on the scleral mechanical properties to better understand the impact of GAG content variations in the pathophysiology of glaucoma and myopia. We measured how the removal of sulphated GAG (s-GAG) affected the hydration, thickness and mechanical properties of the posterior sclera in enucleated eyes of 6–9month-old pigs. Measurements were made in 4 regions centered on the optic nerve head (ONH) and evaluated under 3 conditions: no treatment (control), after treatment in buffer solution alone, and after treatment in buffer containing chondroitinase ABC (ChABC) to remove s-GAGs. The specimens were mechanically tested by pressure-controlled inflation with full-field deformation mapping using digital image correlation (DIC). The mechanical outcomes described the tissue tensile and viscoelastic behavior. Treatment with buffer alone increased the hydration of the posterior sclera compared to controls, while s-GAG removal caused a further increase in hydration compared to buffer-treated scleras. Buffer-treatment significantly changed the scleral mechanical behavior compared to the control condition, in a manner consistent with an increase in hydration. Specifically, buffer-treatment led to an increase in low-pressure stiffness, hysteresis, and creep rate, and a decrease in high-pressure stiffness. ChABC-treatment on buffer-treated scleras had opposite mechanical effects than buffer-treatment on controls, leading to a decrease in low-pressure stiffness, hysteresis, and creep rate, and an increase in high-pressure stiffness and transition strain. Furthermore, s-GAG digestion dramatically reduced the differences in the mechanical behavior among the 4 quadrants surrounding the ONH as well as the differences between the circumferential and meridional responses compared to the buffer-treated condition. These findings demonstrate a significant effect of s-GAGs on both the stiffness and time-dependent behavior of the sclera. Alterations in s-GAG content may contribute to the altered creep and stiffness of the sclera of myopic and glaucoma eyes.

Correlation of discoloration and biomechanical properties in porcine sclera induced by genipin.

To study the feasibility of using the discoloration to evaluate the biomechanical properties after treating with genipin. Porcine cadaver eyes were treated for 30min with 1.0% (by w/v) genipin. Untreated samples were used as controls. After treatment, scleral strips of 4.0×10.0-mm(2) were cut. The denaturation temperature (Td) measurement and stress-strain test were performed after taking photograph to analyze the color. Within 24h after treating with genipin, the sclera exhibited a bluish color which became deeper with time. And the denaturation temperature also was increased gradually. Compared with untreated groups, at 1, 6, 12, 24 and 36h after treatment, the ultimate stress were increased by 56%, 153%, 173%, 225% and 211% respectively. The Young's modulus at 10% strain also increased by 170%, 246%, 264%, 389% and 288% respectively. There were strong correlation between the discoloration and the biomechanical properties (ΔE-Ultimate stress: R(2)=0.892, P=0.00; ΔE-Young's modulus: R(2)=0.602, P=0.00). Genipin could be used to strengthen collagen gradually in a relatively short time span. And the biomechanical properties could be reliably evaluated via simple visible discoloration.

Scleral birefringence as measured by polarization-sensitive optical coherence tomography and ocular biometric parameters of human eyes in vivo.

The relationship between scleral birefringence and biometric parameters of human eyes in vivo is investigated. Scleral birefringence near the limbus of 21 healthy human eyes was measured using polarization-sensitive optical coherence tomography. Spherical equivalent refractive error, axial eye length, and intraocular pressure (IOP) were measured in all subjects. IOP and scleral birefringence of human eyes in vivo was found to have statistically significant correlations (r = -0.63, P = 0.002). The slope of linear regression was -2.4 × 10(-2) deg/μm/mmHg. Neither spherical equivalent refractive error nor axial eye length had significant correlations with scleral birefringence. To evaluate the direct influence of IOP to scleral birefringence, scleral birefringence of 16 ex vivo porcine eyes was measured under controlled IOP of 5-60 mmHg. In these ex vivo porcine eyes, the mean linear regression slope between controlled IOP and scleral birefringence was -9.9 × 10(-4) deg/μm/mmHg. In addition, porcine scleral collagen fibers were observed with second-harmonic-generation (SHG) microscopy. SHG images of porcine sclera, measured on the external surface at the superior side to the cornea, showed highly aligned collagen fibers parallel to the limbus. In conclusion, scleral birefringence of healthy human eyes was correlated with IOP, indicating that the ultrastructure of scleral collagen was correlated with IOP. It remains to show whether scleral collagen ultrastructure of human eyes is affected by IOP as a long-term effect.