Enucleated Porcine Eyes Research Articles

Intraocular pressure before and after capsulorhexis using two viscoelastic substances and two surgical approaches in enucleated porcine eyes.

To investigate the influence of ophthalmic viscoelastic devices (OVDs) and different surgical approaches on the intraocular pressure (IOP) before and after creation of the curvilinear circular capsulorhexis (CCC) as a measure for anterior chamber stability during this maneuver. Prospective experimental WetLab study carried out on enucleated porcine eyes. IOP was measured before and after CCC with the iCare Rebound tonometer (iCare ic200; iCare Finland Oy, Vantaa, Finland). The OVDs used were a cohesive one [Z-Hyalin, Carl Zeiss Meditec AG, Germany; hyaluronic acid (HA)] and a dispersive [Z-Celcoat, Carl Zeiss Meditec AG, Germany; hydroxy propylmethylcellulosis (HPMC)]. The CCC was created using Utrata forceps or 23 g microforceps in different combinations with the OVDs. Using the Utrata forceps the IOP dropped from 63.65±6.44 to 11.25±3.63 mm Hg during the CCC. The use of different OVDs made no difference. Using the 23 g microforceps the IOP dropped from 65.35±8.15 to 36.55±6.09 mm Hg. The difference between IOP drop using either Utrata forceps or 23 g microforceps was highly significant regardless of the OVD used. Using the sideport for the creation of the capsulorhexis leads to a lesser drop in IOP during this maneuver compared to the main incision in enucleated porcine eyes. The use of different OVD has no significant influence on IOP drop.

Droplet Laplace valve‐enabled glaucoma implant for intraocular pressure management

AbstractGlaucoma, the leading cause of irreversible blindness worldwide, is closely linked to aqueous overaccumulation and elevated intraocular pressure (IOP). For refractory glaucoma, aqueous shunts with valves are commonly implanted for effective aqueous drainage control and IOP stabilization. However, existing valved glaucoma implants have the disadvantages of inconsistent valve opening/closing pressures, poor long‐term repeatability due to their reliance on moving parts, and complex architectures and fabrication processes. Here, we propose a novel valving concept, the droplet Laplace valve (DLV), a three‐dimensional printable moving‐parts‐free microvalve with customizable and consistent threshold valving pressures. The DLV uses a flow discretization unit governed by capillarity, comprising a droplet‐forming nozzle, and a separated reservoir to digitize continuous flow into quantifiable droplets. Unlike the classic one‐time‐use Laplace valves, the DLV's unique design allows for its reusability. The opening pressure is adjustable by varying the nozzle size, like the classic Laplace valves (following the Young–Laplace equation), while the closing pressure can be modified by tuning the separation distance and the reservoir size. Various DLVs with customizable opening pressures from 5 to 11 mmHg have been demonstrated, with opening/closing pressure differences suppressed down to <0.5 mmHg (<0.15 mmHg under the best conditions). Thanks to its moving‐parts‐free nature and digitized flow properties, the DLV shows a highly repeatable valving performance (<1.7%, 1000 cycles) and a predictable linear flow rate–pressure correlation (R2 > 0.99). Preliminary ex vivo validation in an enucleated porcine eye confirms the DLV's efficiency in aqueous shunting and prompt IOP stabilization. The DLV technology holds great promise in glaucoma implants for IOP management and various microsystems for flow control.

Real-Time Measurement of Antiglaucoma Drugs in Porcine Eyes Using Boron-Doped Diamond Microelectrodes.

The primary treatment for glaucoma, the most common cause of intermediate vision impairment, involves administering ocular hypotensive drugs in the form of topical eye drops. Observing real-time changes in the drugs that pass through the cornea and reach the anterior chamber of the eye is crucial for improving and developing safe, reliable, and effective medical treatments. Traditional methods for measuring temporal changes in drug concentrations in the aqueous humor employ separation analyzers such as LC-MS/MS. However, this technique requires multiple measurements on the eyes of various test subjects to track changes over time with a high temporal resolution. To address this issue, we have developed a measurement method that employs boron-doped diamond (BDD) microelectrodes to monitor real-time drug concentrations in the anterior chamber of the eye. First, we confirmed the electrochemical reactivity of 13 antiglaucoma drugs in a phosphate buffer solution with a pH of 7.4. Next, we optimized the method for continuous measurement of timolol maleate (TIM), a sympathetic beta-receptor antagonist, and generated calibration curves for each BDD microelectrode using aqueous humor collected from enucleated porcine eyes. We successfully demonstrated the continuous ex vivo monitoring of TIM concentrations in the anterior chambers of these enucleated porcine eyes. The results indicate that changes in intracameral TIM concentrations can be monitored through electrochemical measurements using BDD microelectrodes. This technique holds promise for future advancements in optimizing glaucoma treatment and drug administration strategies.

Measurement of vitreous humor pressure in vivo using an optic fiber pressure sensor

We conducted a study to assess the pressure difference between the aqueous and vitreous humors in rabbit eyes using a direct intraocular pressure (IOP) measurement method. A micro-optic-fiber pressure sensor was utilized for this purpose. Preliminary experiments with enucleated porcine eyes confirmed the sensor's accuracy in measuring both aqueous and vitreous humor pressure. The main study involved six healthy albino rabbits, where the sensor measured the pressure in the anterior chamber (aIOP) and posterior vitreous-cavity (pIOP). These measurements were compared to aIOP values obtained through rebound tonometry. Additionally, pre- and postoperative pressure comparisons were made after performing a vitrectomy. Results revealed a significant disparity between aqueous and vitreous humor pressures. Prior to vitrectomy, pIOP was 22.8 mmHg, over twice as high as aIOP (11.0 mmHg), but decreased to a similar level following the procedure. Comparison between the sensor measurements and rebound tonometry showed agreement in aIOP values. In conclusion, our study demonstrates that vitreous humor pressure is consistently higher than aqueous humor pressure, reaching the upper limit of normal IOP. Furthermore, vitrectomy effectively reduces pIOP, aligning it with aIOP. These findings contribute valuable insights into intraocular pressure dynamics and have implications for clinical interventions targeting ocular pressure regulation.

Explosives-based vole traps-A novel injury pattern in the eye region

Anovel method for trapping voles is the use of pistol-like explosive tools loaded with bolt-action ammunition. When triggered the vole is killed by the very high gas pressure created. Accidental releases can result in facial and/or eye injuries. The aim of this work was to describe the injury pattern in the patient and to experimentally verify whether there is arisk of penetrating eye injuries. Two emergency patients presented to our eye clinic with eye injuries after unintentional triggering of the explosive trap. Based on the new pattern of injury noted, experiments were performed on enucleated porcine eyes to determine the possible severity. For this purpose, avole trap was clamped in aholder and loaded with aCal. 9 × 17 mm cartridge in each case. In front of the muzzle opening, 3pig eyes each were fixed on Styrofoam at adistance of 20, 40, 60 and 80 cm. The foreign body indentations in the cornea were visualized and measured by spectral domain optical coherence tomography (SD-OCT). The pig eyes were then dissected and searched for foreign bodies using microscopy. The SD-OCT images of an injured patient were also included for human comparison. On patient examination, in addition to the usual fine gunshot marks on the face and conjunctiva/eye area, wax-like, larger and heavier particles of approximately 0.1-0.2 mm in size were found, which originated from the cartridge end cap. Removal of these foreign bodies, some of which were injected more deeply into the cornea, conjunctiva, and tenon, is much more difficult and extensive than in usual blast trauma. There was no evidence of intraocular foreign bodies in either patient. Likewise, no intraocular foreign bodies could be detected experimentally in any pig eyeball (n = 12). Remnants of the wax-like cartridge end were found deeply penetrating into the corneal stroma. The maximum penetration depth measured against the total corneal thickness was 46% at 20 cm device distance and decreased with greater distance to the vole trap (penetration depth at 40 cm at 37%, at 60 cm at 28% and at 80 cm at 19%). For comparisons on the human eye, apenetration depth of 54% was measured at adistance of about 40 cm. In pig eyes the number of foreign bodies per cm2 decreased with increasing distance from the vole trap (mean: n = 174 foreign bodies, FB, at 20 cm distance, n = 46FB at 40 cm, n = 23FB at 60 cm, and n = 9FB at 80 cm). The largest penetrating foreign bodies measured amean of 383 ± 43 μm with amaximum of 451 μm. New vole traps with gas-powered mechanisms result in larger deeply penetrating wax-like foreign bodies in the cornea, conjunctiva, and tenon of the eye, which are difficult to remove and only surgically possible. Despite the significant explosion during triggering, there was no evidence of penetrating ocular injury from the foreign bodies either in the patient or experimentally in the pig eyes. Safety goggles should be worn when handling the traps to avoid penetration of foreign bodies into the eye.

Atomic force microscopy for the evaluation of corneal surface roughness after femtosecond laser flap creation and excimer ablation.

It is well known that the femtosecond laser lamellar cut induces some degree of surface roughness. Nevertheless, as in femtosecond laser-assisted LASIK (FS-LASIK), an excimer LASIK ablation is performed, and the post-ablation stromal bed should show some degree of smoothening. We decided to compare, using atomic force microscopy (AFM), the roughness of the corneal stromal bed, after a femtosecond lasers device flap was created with or without an excimer myopic ablation. Using 6 freshly enucleated porcine eyes, we created in every eye a flap using a femtosecond laser. Additionally, in 3 eyes, an excimer laser ablation to correct-3 diopters (D) was made. AFM imaging of the remaining corneal stroma was performed. Ten different square areas of 20μm x 20μm at the central area of the stroma of each corneal sample were studied. The roughness parameters used were the root-mean-square deviation from a perfectly flat surface. The RMS deviation was 360 ± 120nm in femtosecond laser only, and 110 ± 20nm in those cases where excimer is also involved (p < 0.0001). Our results show that the roughness of the surface treated with excimer is clearly lower than in the group with no excimer ablation; thus, the application of laser excimer after a flap created by femtosecond laser seems to soften the nano-irregularities created by this technique.

OCT based corneal densitometry: the confounding effect of epithelial speckle.

Corneal densitometry is a clinically validated method for objectively assessing the transparency of stroma. The technique is currently dominated by Scheimpflug technology. Still, optical coherence tomography (OCT), in which examination of the statistical properties of corneal speckle is undertaken, has also been considered to assess corneal densitometry. In-vivo, the stroma is observed via the epithelium. However, the effect of this external layer on stromal densitometry has not been considered as yet. This study aims to quantify the influence of epithelium integrity on corneal OCT densitometry. OCT images from eleven freshly enucleated porcine eyes before and after epithelial debridement were used. OCT densitometry was investigated at different stromal depths using four metrics of speckle statistics. Results indicate that there exist statistically significant differences in speckle statistics for a given stromal depth depending on the presence or absence of the epithelium. The estimation error in speckle statistics can reach over 20% depending on the stromal depth. The anterior stroma densitometry values are the ones most affected by epithelial integrity. In conclusion, if OCT densitometry stromal parameters are to be considered in absolute terms, it is essential to consider the confounding effect of the epithelial layer in the analysis.

Analysis of Biomechanical Response After Corneal Crosslinking with Different Fluence Levels in Porcine Corneas

Purpose To evaluate corneal stiffening of porcine corneas induced by corneal crosslinking (CXL) with constant irradiance as a function of total fluence. Methods Ninety corneas from freshly enucleated porcine eyes were divided into five groups of 18 eyes. Groups 1–4 underwent epi-off CXL using a dextran-based riboflavin solution and an irradiance of 18 mW/cm2, group 5 served as the control group. Groups 1 to 4 were treated with a total fluence of 20, 15, 10.8, and 5.4 J/cm2, respectively. Thereafter, biomechanical measurements were performed on 5 mm wide and 6 mm long strips using an uniaxial material tester. Pachymetry measurements were performed on each cornea. Results At 10% strain, the stress was 76, 56, 52, and 31% higher in groups 1–4, respectively compared to the control group. The Young’s modulus was 2.85 MPa for group 1, 2.53 MPa for group 2, 2.46 MPa for group 3, 2.12 MPa for group 4, and 1.62 MPa for the control group. The difference between groups 1 to 4 and the control group 5 were statistically significant (p = <0.001; p = <0.001; p = <0.001; p = 0.021). In addition, group 1 showed significantly more stiffening than group 4 (p = <0.001), no other significant differences were found. Pachymetry measurements revealed no statistically significant differences among the five groups. Conclusion Additional mechanical stiffening can be achieved by increasing the fluence of the CXL. There was no threshold detected up to 20 J/cm2. A higher fluence could compensate the weaker effect of accelerated or epi-on CXL procedures.

Region-related and layer-specific permeability of the iris vasculature with morphological mechanism: A novel understanding of blood-aqueous barrier

The permeability of iris blood vessels has an important role in maintaining aqueous humor (AH) homeostasis, contributing to variation in iris volume and probably the pathogenesis of angle closure glaucoma. This study investigates the permeability of the iris microvasculature to plasma-derived protein and correspond it with the morphologic characteristics of vascular mural cells (MCs).Twenty-two enucleated porcine eyes were used in this study. 12 eyes were micro-perfused with vehicle alone as control or with FITC-albumin as a marker of protein leakage and histological sections subsequently made to examine for FITC-albumin presence. The other 10 eyes were immunolabeled via micro-perfusion for αSMA and VE-cadherin to investigate their topographic distribution in the porcine iris vasculature, and to cross correspond with the locations of FITC-albumin deposits.Distribution of FITC-signals exhibited a site-dependent pattern and time-dependent change in the iris. Fluorescence was initially detected around capillaries in the superficial and deep layer of the iris microvascular network. The pupillary region and the iris root retained more fluorescent signal than the iridal ciliary region. At low magnification, αSMA labelling displayed a regional variation which was inversely correlated with vascular permeability. At the cellular level, αSMA labeling corresponded with vascular MCs distribution in the iris vascular network.The correspondence between iris microvascular permeability to FITC-albumin and the pattern of αSMA distribution and MCs coverage adds to the understanding of the elements comprising the blood-aqueous barrier with implications for the bio-mechanics of iris volume change.

Intracorneal Ring Segment Implantation Results in Corneal Mechanical Strengthening Visualized With Optical Coherence Elastography.

To quantify the mechanical impact of intracorneal ring segment (ICRS) implantation of different dimensions in an ex vivo eye model. A total of 30 enucleated porcine eyes were assigned to ICRS implantation (thickness: 300 µm, angle: 120°, 210°, or 325°), tunnel creation only, or virgin control groups. For mechanical evaluation, each globe was mounted on a customized holder and intraocular pressure (IOP) was increased in steps of 0.5 mm Hg from 15 to 17 mm Hg, simulating physiologic diurnal IOP fluctuations. At each step, an optical coherence tomography volume scan was recorded. Deformations between subsequent scans and the locally induced axial strains were analyzed using a vector-based phase difference method. The effective E-modulus was derived from the overall induced strain as a measure of global mechanical impact. ICRS implantation increased the effective E-modulus from 146 and 163 kPa in virgin and tunnel-only eyes to 149, 192, and 330 kPa in eyes that received a 5-mm optical zone ICRS with 120°, 210°, and 325° arc length, respectively; and to 209 kPa in a 6-mm optical zone ICRS with 325° arc length. The most consistent effect was a shift toward positive strains in the posterior stroma by 0.1% to 0.46% (factor 1.15 to 2.15) after ICRS surgery. ICRS implantation reduces the overall tissue strain under the load of the IOP and provokes posterior tissue relaxation. This effect is more prominent the longer the arc length and the smaller the optical zone of the ICRS is. ICRS have not only a geometrical, but also a mechanical impact on corneal tissue. This behavior might have clinical implications when ICRS implantation is performed in biomechanically weakened keratoconic corneas. [J Refract Surg. 2022;38(7):459-464.].

Corneal Strain Induced by Intracorneal Ring Segment Implantation Visualized With Optical Coherence Elastography.

To record the axial strain field in the cornea directly after creating a stromal tunnel and implanting an intracorneal ring segment (ICRS). Freshly enucleated porcine eyes were obtained and assigned to either ICRS implantation, tunnel creation only, or virgin control groups. Immediately after manual tunnel creation and ICRS positioning, the entire eye globe was mounted on a customized holder and intraocular pressure (IOP) was adjusted to 15 mm Hg. Then, IOP was inreased to 20 mm Hg, in steps of 1 mm Hg. At each step, an optical coherence tomography volume scan was recorded. Displacements between subsequent scans were retrieved using a vector-based phase difference method. The induced corneal strain direction was determined by taking the axial gradient. In addition, corneal surface was detected and sagittal curvature maps computed. Corneal tissue presented a localized compressive strain in the direct vicinity of the stromal tunnel, which was independent of IOP change. The central and peripheral (exterior to the ICRS) cornea demonstrated compressive strains on IOP increase, and tensile strains on IOP decrease. ICRS implantation induced an annular-shaped tensile strain at its inner border, particularly during IOP increase. The compressive strains close to the tunnel remained after ICRS implantation. Corneal curvature changes were concentrated on regions where strain was induced. ICRS implantation induces localized strains in the regions subjected to refractive changes, suggesting that corneal strain and curvature are directly related. Studying corneal strain in response to surgical intervention may provide new insights on underlying working principles. [J Refract Surg. 2022;38(3):210-216.].

Combining robot-assisted surgical system and 3D visualization system for teaching minimally invasive vitreoretinal surgery.

To investigate the feasibility of teaching minimally invasive vitreoretinal surgery with a robot-assisted surgical system and a three-dimensional (3D) visualization system. Enucleated porcine eyes were established as an animal model for removing foreign bodies. Forty medical students were recruited to remove foreign bodies to compare the traditional microscope and the 3D system. One junior resident performed the surgical task with manual and robot-assisted operations on 20 porcine eyes for each group. One senior surgeon evaluated the retinal invasion by a graded injury degree. The learning curve for minimally invasive vitreoretinal surgery was described. Compared with the robot-assisted group, the injury degree was higher in the manual group. For the first ten surgeries, the manual and robot-assisted groups had injuries of 2.60±1.35 (4 to 0) and 1.80±1.62 (4 to 0), respectively. For the last ten surgeries, the injury degrees were 1.90±1.20 (3 to 0) and 0.80±0.42 (1 to 0). Considering the manual and robot-assisted groups together, 95%, 75% and 60% of the students considered surgical manipulation with the 3D visualization system to be more comfortable, easier and clearer, respectively. The robot-assisted surgical system and 3D visualization system may have value in teaching minimally invasive vitreoretinal surgery.

Impact of freeze-thaw cytoablation on aqueous outflow patterns in ex vivo anterior chamber perfusion cultures and whole eyes

Background: Porcine eyes have been widely used as ex vivo models in glaucoma research, as they share similar features with human eyes. Freeze-thawing is a non-invasive technique that has been used to obliterate living cells in anterior segment ex vivo cultures, to prepare them for further research such as cellular repopulation. This technique has previously been shown to reduce the intraocular pressure (IOP) in porcine eyes. The aim of this study was to investigate whether freeze-thaw cytoablation causes corresponding canalogram outflow changes in perfused anterior segment cultures (AFT) and whole porcine eyes (WFT). We hypothesized that the known IOP drop in AFT after trabecular meshwork ablation by freeze-thaw would be accompanied by a similarly large change in the distal outflow pattern. Methods: Two-dye (fluorescein and Texas red) reperfusion canalograms were used to compare the outflow time before and after two -80°C cycles of freeze-thaw. We assigned 28 freshly enucleated porcine eyes to four groups: perfused anterior segment dye controls (ACO, n = 6), perfused whole eye dye controls (WCO, n = 6), freeze-thaw treated anterior segment cultures (AFT, n = 10), and freeze-thaw treated whole eyes (WFT, n = 6). Results: In control groups ACO and WCO, the two different dyes had similar filling times. In AFT, the outflow pattern and filling times were unchanged. In WFT, the temporal superior quadrant filled more slowly (p = 0.042) while all others remained unchanged. The qualitative appearance of distal outflow spaces was altered only in some eyes. Conclusions: Freeze-thaw cytoablation caused neither loss nor leakage of distal outflow structures. Surprisingly, the loss of an intact trabecular meshwork over the entire circumference did not result in a general acceleration of quadrant outflow times. The results validate freeze-thawing as a method to generate an extracellular matrix without major structural changes.

Estimation of corneal thickness ex vivo and in vivo at instillation of photosensitizer solutions in different modifications of UV corneal crosslinking

Objectives: to evaluate the effectiveness of photosensitizer solutions in the experiment in different modifications of standard UV corneal crosslinking. Materials and Methods. Experiments were performed on 32 enucleated porcine eyes ex vivo and 12 rabbits (24 eyes) in vivo, divided into 4 groups depending on instillations of the photosensitizer solutions: Dextralink, Ribolink, Hypolink and Riboflavin. Evaluation of corneal saturation was performed using two modifications of standard UV CXL: in the first case, instillation of the photosensitizer solution was performed during the entire crosslinking procedure (30 min - saturation and 30 min - ultraviolet irradiation); in the second case, instillations lasted only for the first 30 minutes, the precorneal riboflavin film was removed, and instillation of solution was not performed. Results and Discussion. Instillation of photosensitizer solutions within 60 minutes showed that Dextralink significantly reduced the thickness of the cornea by about 24 % ex vivo and 21 % in vivo, while Hypolink, on the contrary, caused its increase by 9 % ex vivo and 23 % in vivo, respectively. The use of Ribolink and Riboflavin did not change the linear parameters of the cornea during the entire follow-up period. The results of the 2nd series of studies showed that effects of solutions on the cornea during their instillation for 30 minutes were generally preserved during the entire observation period up to 60 minutes. In the group where Dextralink and Hypolink solutions were used, there was a slight tendency to normalization of the initial parameters of the cornea. Conclusion. Safe and effective implementation of UV crosslinking of the cornea is possible on the basis of a rational approach to the performing the stages of stroma saturation with photosensitizer solutions, depending on the initial state of the cornea: Dextralink is recommended for a thickness of more than 450 m, Ribolink - at 400-450 m, Hypolink - from 350 to 400 m. Halting of instillations on the saturated stroma during UV irradiation is advisable to be accompanied by intraoperative control of its thickness. When performing a standard crosslinking technique, where instillations of photosensitizer accompany the stage of UV irradiation of the cornea, it is necessary to take into account the presence of a precorneal film that can absorb some of the radiation energy. The peculiarity of this crosslinking technique can be considered as a potential way to protect intraocular tissues from excessive exposure to UV radiation.

Ocular Penetrance and Safety of the Dopaminergic Prodrug Etilevodopa.

PurposeAnimal models have demonstrated the role of dopamine in regulating axial elongation, the critical feature of myopia. Because frequent delivery of dopaminergic agents via peribulbar, intravitreal, or intraperitoneal injections is not clinically viable, we sought to evaluate ocular penetration and safety of the topically applied dopaminergic prodrug etilevodopa.MethodsThe ocular penetration of dopamine and dopaminergic prodrugs (levodopa and etilevodopa) were quantified using an enzyme-linked immunosorbent assay in enucleated porcine eyes after a single topical administration. The pharmacokinetic profile of the etilevodopa was then assessed in rats. A four-week once-daily application of etilevodopa as a topical eye drop was conducted to establish its safety profile.ResultsAt 24 hours, the studied prodrugs showed increased dopaminergic derivatives in the vitreous of porcine eyes. Dopamine 0.5% (P = 0.0123) and etilevodopa 10% (p = 0.370) achieved significant vitreous concentrations. Etilevodopa 10% was able to enter the posterior segment of the eye after topical administration in rats with an intravitreal half-life of eight hours after single topical administration. Monthly application of topical etilevodopa showed no alterations in retinal ocular coherence tomography, electroretinography, caspase staining, or TUNEL staining.ConclusionsAt similar concentrations, no difference in ocular penetration of levodopa and etilevodopa was observed. However, etilevodopa was highly soluble and able to be applied at higher topical concentrations. Dopamine exhibited both high solubility and enhanced penetration into the vitreous as compared to other dopaminergic prodrugs.Translational RelevanceThese findings indicate the potential of topical etilevodopa and dopamine for further study as a therapeutic treatment for myopia.

Impact of freeze-thaw cytoablation on aqueous outflow patterns in ex vivo anterior chamber perfusion cultures and whole eyes

Background: Porcine eyes have been widely used as ex vivo models in glaucoma research, as they share similar features with human eyes. Freeze-thawing is a non-invasive technique that has been used to obliterate living cells in anterior segment ex vivo cultures, to prepare them for further research such as cellular repopulation. This technique has previously been shown to reduce the intraocular pressure (IOP) in porcine eyes. The aim of this study was to investigate whether freeze-thaw cytoablation causes corresponding canalogram outflow changes in perfused anterior segment cultures (AFT) and whole porcine eyes (WFT). We hypothesized that the known IOP drop in AFT after trabecular meshwork ablation by freeze-thaw would be accompanied by a similarly large change in the distal outflow pattern. Methods: Two-dye (fluorescein and Texas red) reperfusion canalograms were used to compare the outflow time before and after two -80°C cycles of freeze-thaw. We assigned 28 freshly enucleated porcine eyes to four groups: perfused anterior segment dye controls (ACO, n = 6), perfused whole eye dye controls (WCO, n = 6), freeze-thaw treated anterior segment cultures (AFT, n = 10), and freeze-thaw treated whole eyes (WFT, n = 6). Results: In control groups ACO and WCO, the two different dyes had similar filling times. In AFT, the outflow pattern and filling times were unchanged. In WFT, the temporal superior quadrant filled more slowly (p = 0.042) while all others remained unchanged. The qualitative appearance of distal outflow spaces was altered only in some eyes. Conclusions: Freeze-thaw cytoablation caused neither loss nor leakage of distal outflow structures. Surprisingly, the loss of an intact trabecular meshwork over the entire circumference did not result in a general acceleration of quadrant outflow times. The results validate freeze-thawing as a method to generate an extracellular matrix without major structural changes.

Intraocular pressure and injection forces during intravitreal injection into enucleated porcine eyes

Injection of biological molecules into the intravitreous humor is of increasing interest for the treatment of posterior segment eye diseases such as age-related degenerative macular degeneration. The injection volume is limited by an increase in intraocular pressure (IOP) and 50–100 µL are typically used for most intravitreally (IVT) applied commercial products. Direct measurement of IOP is difficult and has not been studied dependent on solution properties and injection rates. We used an instrumental set-up to study IOP ex vivo using healthy enucleated porcine eyes. IOP was determined as a function of injection volume for viscosities between 1 and 100 mPas, injection rates of 0.1, 1, and 1.5 mL/min, and needle length and diameter (27/30G and 0.5/0.75″) using Dextran solutions. IOP increased exponentially for injection volumes larger than 100 µL. We did not observe differences in IOP dependent on viscosity, injection rate, and needle diameter. However, variability increased significantly for injection volumes larger than 100 µL and, unexpectedly, declined with higher viscosities. We demonstrate that the exponential increase in IOP is not reflected by injection force measurements for typical configurations that are used for IVT application. The present findings may guide injection volumes for intravitreal injection and inform injection force considerations during technical drug product development.

Corneal stromal roughness after VisuMax and Intralase femtosecond laser photodisruption: An atomic force microscopy study.

PurposeTo compare the induced corneal stromal bed roughness measured with atomic force microscopy (AFM) after LASIK flap creation with the IntraLase 60 kHz and the VisuMax femtosecond laser platforms.MethodsThree freshly enucleated porcine eyes were operated with each femtosecond laser in this experimental study. Standard LASIK treatment parameters were used for the experiment. After LASIK flap creation, the corneal stromal roughness was assessed using a JPK NanoWizard II® AFM in contact mode immersed in liquid. Olympus OMCL-RC800PSA commercial silicon nitride cantilever tips were used. Surface measurements were made in 10 regions of the central cornea of each sample measuring 20 x 20 microns, at 512 x 512 point resolution. Roughness was measured using the root-mean-square (RMS) value within the given regions.ResultsMeasurements from 30 regions of the 3 eyes (10 measurements per eye) in the Intralase (FS1) group, and 30 regions of the 3 eyes (10 measurements per eye) in the VisuMax (FS2) group were analyzed. There was a statistically significant difference in mean ± standard deviation RMS values between the FS1 and the FS2 groups (360 ± 120 versus 230 ± 100 nm respectively; P< 0.00001).ConclusionThis AFM study indicates that the surface of the stromal bed after LASIK flap creation is smoother in the FS2 group than the FS1 group.

Measurement of the Intraocular Pressure Elevation During Laser-Assisted In Situ Keratomileusis Flap Creation Using a Femtosecond Laser Platform.

PurposeThe purpose of this study was to measure the intraocular pressure (IOP) elevation during laser assisted in situ keratomileusis (LASIK) flap creation using the WaveLight FS200 femtosecond (FS) laser platform.MethodsWe conducted an ex vivo experimental study in an animal model. The WaveLight FS200 FS laser platform was used to perform the corneal LASIK flap in freshly enucleated porcine eyes. We measured the changes in IOP from the application of the suction ring (suctioning phase) through the creation of the lamellar corneal flap (cutting phase). The IOP was recorded using a manometric technique with direct cannulation to the anterior chamber.ResultsNine freshly enucleated porcine eyes were included in the study. The mean baseline IOP before the procedure was 20.33 ± 5.9 mm Hg. The mean IOP increase over baseline IOP was 32.33 ± 11.3 mm Hg at the suctioning phase, and 38.22 ± 11.3 mm Hg at the cutting phase. The total surgical time needed to complete the procedure was 29.5 ± 4.4 seconds.ConclusionsThe WaveLight FS200 FS laser platform produces a low to moderate increase in IOP during LASIK flap creation.Translational RelevanceThe WaveLight FS200 is a safe FS laser platform because it induces a low to moderate IOP increase during LASIK flap creation.

Effect of variations of corneal physiology on novel non-invasive intraocular pressure monitoring soft contact lens.

Glaucoma is the leading cause of irreversible blindness around the world. With its slow asymptomatic progression, there is an emphasis on early detection and frequent monitoring. A novel microfluidic contact lens has been established as a potential way to track the fluctuations of the intraocular pressure (IOP) which is a key indicator for diagnosing and monitoring glaucoma progression. The purpose of this article is to determine the effect of physiological variations of the eye on the performance of the microfluidic contact lens. Ultrasound biomicroscopy (UBM) was used to measure the central corneal thickness (CCT) and radius of corneal curvature (RCC) for a series of 16 fresh enucleated porcine eyes. The effect of these corneal anatomic features on device performance was then assessed by systematically adjusting intraocular pressure from 10 to 34mmHg and monitoring the device indicator response. The performance of the microfluidic contact lens was determined by finding the amount the indicator fluid shifted in position as a result of 1 mmHg IOP increase. The relationship between IOP and indicator fluid was found to be linear for all eyes. The slope of the indicator fluid movement as a result of the IOP was evaluated against the CCT and RCC of each porcine eye. This yielded low correlation coefficients, 0.057 for CCT and 0.024 for RCC, meaning that these physiological differences showed no systematic impact on the measurements made with the contact lens.