Porcine Eye Globes Research Articles

Investigating dexpanthenol transfer to the anterior segment from coated contact lenses and aqueous solution

Aims/Purpose: Dexpanthenol (Dxp) is a common excipient in topical formulations used in dermatology and ophthalmology. However, little is known about the absorption of Dxp into biological tissues. In this study we investigated the transfer of Dxp into the anterior segment – i.e. cornea and aqueous humor – first, from contact lenses (CL) following treatment with SOLOCARE AQUA® care solution and second, after administration of Bepanthen® and Siccaprotect® eye drops. All products contained approximately equivalent amounts of Dxp (2–3%).Methods: A bioanalytical method for the quantification of Dxp by UHPLC MS/MS was validated. The initial experiments evaluated the ad/absorption of Dxp from the SOLOCARE AQUA® care solution onto FDA class representative CL. Then, ex vivo porcine eye globes were mounted in an eye globe holder developed inhouse, with simulated tear flow, and Dxp uptake into the anterior segment from SOLOCARE AQUA® treated CL and eye drops was investigated.Results: Dxp ad/absorption in CL was correlated to exposure time and CL material. Following exposure to Dxp‐soaked CL and eye drops, transport of Dxp through porcine cornea to the aqueous humor was found in all experimental conditions. However, there was a statistically significant difference in the concentrations measured after CL application and eye drop instillation. Direct application of Dxp containing eye drops resulted in higher Dxp deposition in the anterior segment than the SOLOCARE AQUA® treated CL.Conclusions: In this benchmark study we investigated the deposition of Dxp in the anterior segment of excised porcine eyes from CL treated with a care solution and commercially available eye drops. Despite the excipient Dxp being classified as Generally Recognized as Safe (GRAS), the inferior absorption of the substance from SOLOCARE AQUA® washed CL compared to approved Dxp containing eye drops is considered preferable with regard to medical device safety.

Experimental Evaluation of Ocular Rigidity and Pressure-Volume Relationship After Ex-Vivo Scleral Cross-Linking With Riboflavin and Ultraviolet A in Porcine Eyes.

Scleral cross-linking (SXL) with ultraviolet A (UVA) and riboflavin has already been used in laboratory studies for scleral stiffness increase as a potential treatment for progressive myopia and scleral ectasia. This study aims to investigate whether the regional application of scleral cross-linking (SXL) with ultraviolet A (UVA) and riboflavin in fresh porcine eye globes affects the ocular rigidity as well as its impact on intraocular pressure after an induced acute increase in the volume of intraocular fluid. The study included two groups of fresh porcine eyes: an experimental group (n=20) that underwent scleral cross-linking (SXL) with riboflavin and UVA applied to the posterior sclera and a control group (n=20) that did not receive SXL treatment. Subsequently, a balanced salt solution (volumes 50, 100, 150, and 200 μL) was administered into porcine globes via a syringe, and, at the same time, the intraocular pressure (IOP) was continuously monitored by a pressure sensor that was cannulated to the vitreous chamber. The relationship between volume and pressure was obtained, and the ocular rigidity coefficient (K) was calculated according to Friedenwald's law. Finally, scleral strips were dissected from the globes and were examined macroscopically. In the control group, the mean IOP observed entails gradual, statistically significant increases for higher volumes. Specifically, the mean IOP at 0 μL equals 10 mmHg (SD=0), whereas at 200 μL the mean IOP equals 33.83 mmHg (SD=4.060). The differences were statistically significant with p-values <0.001 in all cases. Similarly, the observed gradual IOP increases in the SXL group were statistically significant with p < 0.001 in all cases except for the comparison of volume 0 μL measurements to volume 50 μL, where the p-value equaled 0.003. Specifically in the SXL group, the mean IOP at 0 μL equals 10.00 mmHg (SD=0.000), the mean IOP at 50 μL equals 13.31 mmHg (SD=2.011), whereas the mean IOP at 200 μL equals 32.06 mmHg (SD=3.078). At no additional injected volume, the differences between the control and the SXL groups were statistically significant. The analysis regarding ocular rigidity indicated significantly higher scores in the control group (K50=0.00812, SD=0.03) compared to the SXL group (K50=0.00552, SD=0.027), t=2.844; p=0.007. The difference regards measures of volumes 0 to 50 μL, while all other rigidity measures were found to be non-significant. Interestingly, the ocular rigidity coefficient in the SXL-treated group did not show changes with an increase in IOP. The macroscopic appearance of the scleral strips showed a significantly increased stiffness of the SXL scleras against the control ones. This study showed that stiffened scleras did not induce substantial change in ocular rigidity and significant IOP elevations. Studying the biomechanical ocular response of laboratory scleral crosslinking applications supports the development of next-generation crosslinking procedures that may constitute potential therapeutic options for severe ophthalmic diseases likepathologic myopia.

Experimental evaluation of stiffening effect induced by UVA/Riboflavin corneal cross-linking using intact porcine eye globes.

UVA/riboflavin corneal cross-linking (CXL) is a common used approach to treat progressive keratoconus. This study aims to investigate the alteration of corneal stiffness following CXL by mimicking the inflation of the eye under the in vivo loading conditions. Seven paired porcine eye globes were involved in the inflation test to examine the corneal behaviour. Cornea-only model was constructed using the finite element method, without considering the deformation contribution from sclera and limbus. Inverse analysis was conducted to calibrate the non-linear material behaviours in order to reproduce the inflation test. The corneal stress and strain values were then extracted from the finite element models and tangent modulus was calculated under stress level at 0.03 MPa. UVA/riboflavin cross-linked corneas displayed a significant increase in the material stiffness. At the IOP of 27.25 mmHg, the average displacements of corneal apex were 307 ± 65 μm and 437 ± 63 μm (p = 0.02) in CXL and PBS corneas, respectively. Comparisons performed on tangent modulus ratios at a stress of 0.03 MPa, the tangent modulus measured in the corneas treated with the CXL was 2.48 ± 0.69, with a 43±24% increase comparing to its PBS control. The data supported that corneal material properties can be well-described using this inflation methods following CXL. The inflation test is valuable for investigating the mechanical response of the intact human cornea within physiological IOP ranges, providing benchmarks against which the numerical developments can be translated to clinic.

Optical Coherence Elastography-Based Corneal Strain Imaging During Low-Amplitude Intraocular Pressure Modulation.

Purpose: Optical coherence elastography (OCE) is a promising technique for high-resolution strain imaging in ocular tissues. A major strain-inducing factor in the eye is intraocular pressure (IOP), with diurnal physiological fluctuations reaching up to 5 mmHg. We study herein low-amplitude IOP modulation to assess local corneal strain patterns.Methods: Ex vivo porcine eye globes were adjusted to an initial IOP of 15 mmHg and subsequently 25 mmHg. Corneal strain was induced by two subsequent pressure cycles, in which IOP was first increased and then decreased, each by a total of 5 mmHg. Two-dimensional optical coherence tomography (2D-OCT) B-scans were recorded after each loading step. Axial strain maps were obtained from magnitude and phase changes and supra-pixel displacements from cross-correlation. The strain detection sensitivity was evaluated in an isotropic material.Results: Deformations arising from a single 1-mmHg step could be resolved. The largest strain amplitudes (5.11·10−3) were observed in the posterior stroma at a low initial IOP. Strain amplitude was 1.34 times higher at 15 mmHg than at 25 mmHg (p = 0.003). Upon IOP increase, the anterior cornea was compressed, whereas the posterior cornea showed axial expansion. Both morphological images and strain maps were sensitive to postmortem time. Strains that are larger than 2.44·10−5 could be reliably measured.Conclusions: Low-amplitude IOP modulation, similar to diurnal physiological changes, induced measurable deformations in corneal tissue. Axial strain maps permit a localized comparison of the corneal biomechanical response. Small-strain OCE can likely be extended to other domains.

Towards Automatically Controlled Dosing for Selective Laser Trabeculoplasty.

PurposeSelective laser trabeculoplasty (SLT) is a treatment option for open-angle glaucoma; however, it lacks an instant evidence for successful irradiation. So far ophthalmologists use the visible appearance of permanent champagnelike bubbles (macro bubbles) as an indicator for appropriate pulse energy. We hypothesize that micro bubbles, which start energetically far below the appearance of macro bubbles, already trigger the therapeutic benefit. Here we present two methods to capture the onset of these micro bubbles.MethodsThe trabecular meshwork of freshly enucleated porcine eye globes was irradiated with a series of 15 pulses with a pulse duration of 1.7 μs and with increasing energy at a repetition rate of 100 Hz per each spot of 200 μm in diameter. An optical and an optoacoustic method have been developed and appropriate algorithms investigated towards the real-time detection of the onset of micro bubbles.ResultsBoth observation methods are capable of detecting micro bubble nucleation. Threshold radiant exposures were found at 310 ± 137 mJ/cm2. By combination of both methods a sensitivity and specificity of 0.96 was reached.ConclusionsIn case that the therapeutically demanded pressure reduction is already achieved with these micro bubbles, which needs to be proven clinically, then the methods presented here can be used in an automatic feedback loop controlling the laser irradiation. This will unburden the clinicians from any dosing during SLT.Translational RelevanceAutomatic real-time pulse energy dosing based on the formation of micro bubbles in SLT significantly improves and facilitates the treatment for the physician.

Short-duration ocular iontophoresis of ionizable aciclovir prodrugs: A new approach to treat herpes simplex infections in the anterior and posterior segments of the eye

The objective was to investigate (trans)corneal and transscleral iontophoresis of biolabile amino acid ester prodrugs of aciclovir (ACV-X, X = Arg, Gly and Trp) as a means to increase ocular bioavailability of ACV. Prodrugs displayed tissue-dependent susceptibility to hydrolysis. Iontophoresis of ACV-Arg, ACV-Gly and ACV-Trp (5 mM, 0.5 mA/cm2) for 5 min followed by 55 min passive diffusion resulted in appreciable corneal deposition (21.5 ± 5.1, 14.1 ± 2.0 and 5.3 ± 0.6 nmol/cm2, respectively) and transcorneal permeation (13.9 ± 1.6, 10.9 ± 1.8 and 5.7 ± 0.5 nmol/cm2, respectively) of ACV species. In contrast, passive delivery of ACV across porcine cornea after 1 h was < LOQ (i.e. <0.125 nmol/cm2). Transscleral permeation of ACV-Arg, ACV-Gly and ACV-Trp (9 mM, 1.25 mA/cm2) after iontophoresis for 5 min was 20.4 ± 3.8, 12.3 ± 0.3 and 8.4 ± 0.4 nmol/cm2, respectively – far superior to passive delivery which was again < LOQ. Using intact porcine eye globes, 5 min transscleral iontophoresis of ACV-Gly at 3.75 mA/cm2 resulted in considerable delivery of ACV species to the choroid/retina and vitreous humour (5.7 ± 2.3 and 11.7 ± 3.7 nmol/cm2, respectively). Furthermore, the average concentration of ACV species in the whole eyeball (4.5 ± 1.6 nmol/cm3) was significantly higher than the IC50 of ACV against HSV-1 (<0.22 nmol/cm3), demonstrating the potential application for the treatment of ocular HSV infections.

Visualization of porcine eye anatomy by X-ray microtomography

The aim of our study is to obtain, as accurately as possible, porcine ocular tissue visualization using microtomography (micro-CT) method. We propose image contrast enhancement by different staining procedures with combination of micro-CT scanning. Porcine eye globes were investigated with Bruker-SkyScan 1172 micro-CT. We used 4F1G and Bouin's as sample fixation solutions and tincture of iodine, 100% Lugol, phosphotungstic acid and 1% osmium tetroxide solutions for staining. Quantitative and qualitative analysis was performed based on micro-CT reconstruction images histograms and 3D volume rendering models of investigated samples. This investigation showed that staining methods improved micro-CT image quality in case of ocular anatomy visualization. Characteristic profiles of the grey level distributions and quality of the cross-section and 3D volume rendering images confirmed the staining effect. Most significant contrast enhancement was obtained after 96 h staining in osmium tetroxide and Lugol solutions. The images of eye anatomical structures were characterized: cornea, lens, iris, ciliary body, vitreous, retina, choroid and sclera, vasculature and optic nerve. Staining of porcine eye globes used in this work leads to quality improvement of the micro-CT imaging. The most contrast images were obtained for Lugol and osmium tetroxide solutions. Different affinity of staining solutions to eye anatomical structures has been observed in the obtained images. Osmium tetroxide provides sharper image of conjunctiva, sclera, choroid, retina, iris and ciliary body structure. Lugol staining leads to more accurate vessels, cornea and optic nerve imagining.

Histological analysis of a cornea following experimental femtosecond laser ablation.

Corneal photorefractive surgery is currently performed by ablation of corneal stroma under the stromal flap. A stromal flap is created using a femtosecond (FS) laser or mechanical microkeratome, although the FS laser procedure is considered safer and more accurate. This review assesses and compares the use of FS laser versus mechanical microkeratome ablation for corneal stromal characteristics mainly examined by histology and cellular biological responses. Supporting data from our studies, using corneas of enucleated porcine eye globes, are included in this review. Histological analysis and experimental studies of cellular/tissue responses to FS laser irradiation should be further investigated, and the equipment used to perform these techniques should be improved.

The Mechanical Properties of Ex Vivo Bovine and Porcine Crystalline Lenses: Age-Related Changes and Location-Dependent Variations

The mechanical properties of ex vivo animal lenses from three groups were evaluated: old bovine (25–30 mo old, n = 4), young bovine (6 mo old, n = 4) and young porcine (6 mo old, n = 4) eye globes. We measured the dynamics of laser-induced microbubbles created at different locations within the crystalline lenses. An impulsive acoustic radiation force was applied to the microbubble, and the microbubble displacements were measured using a custom-built high pulse repetition frequency ultrasound system. Based on the measured dynamics of the microbubbles, Young's moduli of bovine and porcine lens tissue in the vicinity of the microbubbles were reconstructed. Age-related changes and location-dependent variations in the Young's modulus of the lenses were observed. Near the center, the old bovine lenses had a Young's modulus approximately fivefold higher than that of young bovine and porcine lenses. The gradient of Young's modulus with respect to radial distance was observed in the lenses from three groups.

Ultraviolet light-induced changes in the glucose-6-phosphate dehydrogenase activity of porcine corneas.

Corneas have the high activity of the pentose phosphate pathway. To clarify the relevance of the pentose phosphate pathway to their antioxidant defense, we examined the activity of glucose-6-phosphate dehydrogenase (G6PDH), the rate-determining enzyme of the pathway, in corneas exposed to ultraviolet light (UV). Fresh porcine eye globes were exposed to ultraviolet light A (UVA) or ultraviolet light C (UVC), and the tear film-removed eye globes to UVA. After UV exposure, the corneas were dissected from the eye globes and extracted with a saline solution, and the G6PDH activity in the extract was assayed. The G6PDH activity of unilateral opaque corneas, their paired, transparent corneas, and normal corneas, all of which were obtained from fresh, UV-unexposed eye globes, and the L-lactate dehydrogenase (LDH) activity of these opaque and normal corneas also were assayed. The G6PDH activity of corneas increased with UVA exposure, and decreased with long-term UVC exposure, although it increased with short-term UVC exposure. A UV-blocking contact lens screened corneas from the UVA-induced increase. Removal of the tear film enhanced the UVC-induced decrease. The G6PDH activity of unilateral opaque corneas was lower than that of paired, transparent corneas or normal corneas, all of which were obtained from fresh, UV-unexposed eye globes. The LDH activity of the opaque corneas was much higher than that of the normal corneas. Exposure of corneas to UVA or a small dose of UVC enhances the G6PDH activity, i.e., the pentose phosphate pathway. This activity enhancement may play an important role in corneal antioxidant defense against UV-induced oxidative stress. However, exposure of corneas to large doses of UVC appears to damage the pathway.