Enucleated Porcine Eyes Research Articles

Microwave thermal keratoplasty for myopia: keratoscopic evaluation in porcine eyes.

Microwave thermal keratoplasty applies microwave energy to elevate the temperature of the paracentral stroma of the cornea to its thermal shrinkage temperature of about 60 degrees C. A suitable pattern of shrinkage in the paracentral cornea can flatten the central cornea. A surface cooling system preserves the epithelium during the procedure. Fourteen enucleated porcine eyes were treated with a prototype microwave thermal keratoplasty applicator that heated in a ring pattern with inner diameter of 3.2 mm and width of 0.7 mm. The change in corneal power was quantified by a videokeratoscope. Slit-lamp microscope examinations and histological assessments were made. The 3-mm simulated keratometry reading showed an average of 6.60+/-6.00 D (standard deviation) of flattening. The region of opacity associated with shrinkage extended to 62% (+/-26%) of the corneal thickness. The epithelium was intact in all eyes. Microwave thermal keratoplasty applied in the paracentral cornea may flatten the central cornea.

Sclerostomy with an Erbium YAG Laser: The Relationship with Pulse Energy

Purpose: To investigate the optimal pulse energy to do sclerostomy with an erbium YAG laser. Materials and Methods: The experiments were performed in enucleated porcine eyes. We changed pulse energy and examined the effects on surrounding tissue. Results: With the increase of pulse energy, the effects of the laser extended to the area surrounding the laser probe. At the threshold energy for doing full-thickness sclerostomy, the total energy was significantly higher than with higher pulse energy. And with pulse energy higher than 2 mJ, the total energy did not show any significant change. Histopathologically, the damaged area around sclerostomy became larger with the increase of pulse energy. Conclusion: The optimal energy to do full-thickness sclerostomy with this system seemed to be 2 mJ.

Determining the accuracy of an eye tracking system for laser refractive surgery.

Patient eye and head movements during laser refractive surgery may result in errors between the surgical beam position and the desired location for optimum correction. This, in turn, may lead to reduced postoperative vision, including increased higher order aberrations of the eye. Active eye tracking systems are often incorporated into laser delivery systems, which aim to reduce the effect of patient eye movement. In this study, the accuracy of an eye tracking system designed for laser refractive surgery was determined. An enucleated porcine eye was attached to a scanning device and the movement measured using the eye tracking system. The recorded position is compared to the preprogrammed position of the scanning device. The system demonstrated an accuracy of 0.06 m for an intact cornea and 0.1 mm for a cornea with a thin flap removed. This compares to an average decentration of ablation of 0.4 mm for patients relying on passive fixation, as measured by previous clinical trials. implementation of this eye tracker would lead to improved alignment between the laser and eye during laser refractive surgery.

First Experimental and Clinical Results With Transscleral Optical Coherence Tomography

To evaluate the potentials of optical coherence tomagraphy (OCT) using long wavelength to penetrate highly scattering tissues of the eye and visualize the anterior chamber angle and the ciliary body. OCT images were generated by an experimental prototype in enucleated porcine eyes using as light source a superluminiscent diode with a wavelength of 1310 nm and a scan frequency of 60 Hz. The number of lateral scans was variable in a range from 100 to 400. Infrared OCT was able to penetrate the sclera. The anterior chamber angle could be visualized completely and the ciliary body could be identified. However, it was not possible to penetrate the highly reflective iris pigment epithelium. The use of infrared OCT allows penetration of the sclera, thus, providing complete visualization of the anterior chamber angle and limited demonstration of the ciliary body. Because of its higher resolution, it may represent an interesting noninvasive alternative to ultrasound biomicroscopy.

A new device for ocular surgical training on enucleated eyes

Objective To develop a reliable inexpensive device for teaching ocular surgical procedures and practicing experimental techniques on enucleated eyes. Design Teaching device trial. Participants Thirty enucleated porcine eyes. Methods A Plexiglas ocular bulb holder was secured with its base support to a polyvinylchloride pillar on a modified polystyrene trial head. Main outcome measure The convenience and reproducibility of both laser and surgical ocular techniques performed with this new device were evaluated. Results This model allows curvilinear capsulorrhexis and phacoemulsification of porcine lenses through a corneal tunnel incision and insertion of a soft foldable acrylic intraocular lens into the capsular bag. Argon and neodymium:YAG laser iridotomy and retinal argon laser photocoagulation can also be performed with this model. Conclusions This inexpensive device is useful for teaching both surgical and laser ocular procedures.

Diode laser thermokeratoplasty: Application strategy and dosimetry

Purpose: To investigate suitable application parameters for efficient hyperopic correction by laser thermokeratoplasty (LTK) using mid-infrared laser diodes.Setting: Medical Laser Center Lubeck, Lubeck, Germany.Method: A tunable continuous-wave laser diode in the spectral range between 1.845 and 1.871 pm was used. Transmitted by waveguides, the laser energy was used to induce coagulations on freshly enucleated porcine eyes to increase corneal curvature. The coagulations were equidistantly applied by a fiber-cornea contact and a noncontact focusing device that were adjusted on a ring concentric to the corneal apex. Different laser parameters and application geometries were evaluated. Refractive changes were measured by computer-assisted corneal topography before and after treatment. Polarization light microscopy and temperature calculations were used to analyze the coagulations.Results: Because of the tunability of the laser diode, the influence of the corneal absorption coefficient (between 0.9 and 1.6 mm-1) on the refractive change could be measured. A laser power between 125 and 200 mW was adequate to achieve refractive changes up to 10.0 diopters. In the preferable focusing device, the refractive change increased almost logarithmically with the irradiation time up to 15 seconds. The number of coagulations on a fixed application ring showed no significant influence on refractive change; however, it showed an almost linear decrease with increasing ring diameter from 5.0 to 10.0 mm. Histological analysis revealed 3 stages of thermal damage.Conclusion: Diode LTK provided defined and uniform coagulations when using a well-adapted focusing device, resulting in sufficient refractive change. The results indicate that diode LTK is superior to pulsed holmium LTK.

Continuous Non-contact Corneal Pachymetry with a High Speed Reflectometer

We developed an instrument that permits non-contact, continuous, high speed and high precision monitoring of corneal thickness and tested the stability and reproducibility of measurements made over extended time periods and under various conditions of low surface reflectivity encountered during protracted exposure of the unmoistened corneal surface to ambient air. The optical pachymeter (basic component of a broad-band, all-fiber Michelson interferometer) was used to monitor changes in the central corneal thickness of enucleated porcine eyes. Measurements were performed on three groups of eight eyes, each with different surface characteristics: intact epithelium, mechanically abraded epithelium, and 90 microm excimer laser keratectomy. Corneal thickness was monitored continuously with values recorded every 2 to 3 minutes for periods up to 1 hour in the absence of surface rinsing. The thicknesses of all unmoistened corneas could be monitored with a precision of 1 microm (ascertained using a calibration glass plate and a living human cornea) over the entire observation period. Under ambient air conditions, deturgescence occurred in each case, and ranged from 1 to 5 microm/min. The rate of corneal thinning was fairly constant during the first 15 minutes of monitoring, but was nonlinear thereafter. Corneas with an intact epithelium had the lowest thinning rate with only 10% of the original thickness lost during the course of 1 hour. Deturgescence increased to 25% in corneas that had mechanical removal of the epithelium and to 28.5% in those that had an anterior excimer laser keratectomy, during a similar time-period. With this new interferometric method, continuous, non-contact measurement of corneal thickness is possible to within a precision of 1 microm for periods up to 1 hour, even under the modified surface conditions after photoablative keratectomy. This device may be useful for on-line monitoring of ablation depths during such procedures.

Acoustic focusing associated with excimer laser ablation of the cornea

Focusing, as well as diffraction effects experienced by the stress wave produced by the argon–fluoride excimer laser ablation of corneal tissue have been studied in artificial and real eye samples. The evolution of the laser-induced acoustic front was imaged by means of fast shadowgraphy in an artificial eye model, while the absolute temporal profile of the pressure transient along the optical axis was directly measured in enucleated porcine eyes by a needle hydrophone inserted into the eyeball. Observations pointed out that the finite size of the acoustic source gave rise to diffraction effects during propagation, as the development of a negative tensile phase in the pulse shape, while the spherical shape of the acoustic source produced focusing of the wave front, causing pressure enhancements up to a factor of 3 in real eyes. The implications to surgical laser procedures as photorefractive keratectomy are discussed.

Induction of Cross-links in Corneal Tissue

The aim of this study was to investigate the possibility of induction of cross-links in corneal tissue in order to increase the stiffness as a basis for a future conservative treatment of keratectasia. Collagenous biomaterials can be stabilized by chemical and physical agents. The epithelium of enucleated porcine eyes was removed. Eight test groups, 10 eyes each, were treated with UV-light (λ=254 nm), 0.5% riboflavin, 0.5% riboflavin and UV-light (365 nm) blue light (436 nm) and sunlight, and the chemical agents–glutaraldehyde (1% and 0.1%, 10 min) and Karnovsky's solution (0.1%, 10 min). Strips of 5 mm in width and 9 mm in length were cut from each cornea and the stress-strain behaviour of the strips was measured to assess the cross-linking process. For comparison, ten untreated corneas were measured by the same method. Compared to untreated corneas treatment with riboflavin and UV-irradiation as well as weak glutaraldehyde or Karnovsky's solutions resulted in an increased stiffness of the cornea. The biomechanical behaviour of the cornea can be altered by glutaraldehyde, Karnovsky's solution, and with riboflavin and UV-irradiation which offers the potential of a conservative treatment of keratoconus. To optimize this effect further investigation is necessary regarding the dose-response and in-vivo application.

Increased rigidity of the cornea caused by intrastromal cross-linking

To increase the stability of the cornea by artificial cross-linking (radiation or chemical agents) and to investigate a future therapy for keratoconus. The epithelium of enucleated porcine eyes was removed. Ten eyes in each of eight test groups were treated with UV light (lambda = 254 nm), 0.5% riboflavin and UV light (365 nm), blue light (436 nm) and sunlight, and the chemical agents glutaraldehyde (1% and 0.1%, 10 min) and Karnovsky's solution (0.1%, 10 min). Strips of 5 mm in width and 9 mm in length were cut from each cornea and the stress-strain behaviour of the strips was measured. For comparison, eight groups of ten untreated corneas each were measured by the same method. Compared to untreated corneas riboflavin and UV irradiation as well as glutaraldehyde and Karnovsky's solution treatment resulted in significantly increased stiffness of the cornea (p < 0.05). The biomechanical behaviour of the cornea can be altered by low-concentration glutaraldehyde, Karnovsky's solution, and by riboflavin and UV irradiation, which offers potential conservative treatment of keratoconus. To optimize this effect further investigation is necessary regarding the dose-effect relation and the in-vivo conditions.

Real-time control for transscleral cyclophotocoagulation.

In transscleral cyclophotocoagulation, the surgeon cannot directly observe the applied laser effects. Overdosage, possibly resulting in unwanted pop effects, or underdosage with no therapeutic effect therefore often occur. Laser radiation passing through the sclera and ciliary body is partly reflected from the fundus and can be monitored from outside the eye by a detector system. Since all other parameters influencing the intensity of the recorded radiation are constant in time during one laser exposure, the time dependence of this radiation directly reflects the change of transmission of the treated tissue. The laser exposure therefore can be stopped by a computer when certain criteria of the recorded curves are fulfilled. In addition, the transmission curves are displayed on a monitor in real time, permitting the surgeon to interrupt the exposure. A Nd:YAG laser and a diode laser are connected to the device. After successful tests in enucleated porcine eyes which were evaluated histologically and in human cadaver eyes this method is applied to patients suffering from refractory glaucoma. The transmission curves from enucleated porcine eyes show that the 810-nm diode laser is more appropriate for this method than the 1064-nm. Nd:YAG laser, because the radiation output of the diode is more stable in time and the tissue absorption is higher, both resulting in a larger dynamical range of useful signal. The curves from the porcine eyes, the human cadaver eyes and the curves from patients show a typical shape which allows interruption of exposure either by the surgeon or by the computer program before a pop effect occurs. This new method increases the precision and safety of transscleral cyclophotocoagulation.

Intraocular pressure during lamellar cuts with two different microkeratomes

The rapid integration of new technology into ophthalmologic practice, e. g. LASIK (laser in situ keratomileusis), is associated with new complications. The limitations of the new technology cannot be precisely determined. During lamellar cuts with microkeratomes intraocular pressure (IOP) elevations occur. The IOP elevation during the lamellar cut cannot be determined by conventional tonometry. We examined the IOP rise during the complete microkeratome treatment. Fresh enucleated porcine eyes were cannulated and direct IOP measurement with a Statham manometer was carried out. Thus we evaluated the IOP during the complete treatment for two differently constructed microkeratome systems. IOP elevations (mean values) between 77 mm Hg for the Corneal Shaper and 229 mm Hg for the Keratek during the vacuum phase and 140 mm Hg for the Corneal Shaper and 360 mm Hg for the Keratek during the lamellar cut were monitored. A sudden IOP decrease to -5 mm Hg was registered for the post-vacuum phase. In our opinion unphysiologic high IOP values could be dangerous for risk eyes.

Corneal ablation profilometry and steep central islands.

Photorefractive keratectomy with large diameter ablations using a uniform laser beam has produced central undercorrections, or "steep central islands" in patients, as seen with videokeratography. Using a custom optical profilometer to measure corneal ablation profiles and a VISX excimer laser system, we measured the effect of ablation algorithms, diameter, depth, and dioptric correction on enucleated porcine eyes and living rabbit eyes. Our profilometer was verified using a 43.00 diopter (D) spherical surface and a 35.00 and 43.00 D bicurve test surface as a model for the ablated cornea. The profilometer measured the test surfaces to within 3 microns of predicted values. Photorefractive keratectomies showed over-ablation peripherally and under-ablation centrally which increased with ablation diameter and dioptric correction. Fixed diameter ablations 2 to 6 mm in diameter and 10 to 80 microns deep showed stromal ablation rates vary spatially but not with ablation depth. These spatially variant ablation profiles were used to re-engineer the ablation algorithm and to produce photorefractive keratectomies with improved sphericity. Steep central islands are caused by the spatial variance of tissue ablated with a uniform laser beam irradiance. This aberration can be corrected by modifying the laser ablation algorithm to correct for the spatial variance of stromal ablation.

Histologic analysis of thermal effects of laser thermokeratoplasty and corneal ablation using Sirius-red polarization microscopy

Purpose: To evaluate how well several histologic techniques differentiate degrees of thermally induced changes in corneal tissue after laser thermokeratoplasty (LTK) or corneal ablation.Setting: Medical Laser Center Lübeck, Germany.Methods: Corneas of freshly enucleated porcine eyes were treated with a continuous wave laser diode (1.86 μm) and a pulsed chromium-thulium-holmium:YAG laser (2.1 μm) to produce LTK lesions or ablated with a Q-switched and a free-running chromium-erbium:YSGG laser (2.70 μm), a free-running erbium:YAG laser (2.94 μm), and an argon-fluoride excimer laser (193 nm). The lesions were evaluated by light microscopy (LM) (hematoxylin and eosin, Azan, van Gieson's, and Masson-Goldner's trichrome stains), transmission electron microscopy (TEM), and polarization microscopy after Sirius-red staining. Sirius-red, a strongly elongated, birefringent molecule binding parallel to collagen molecules, was used to enhance corneal birefringence.Results: With routine LM, it was difficult to discriminate the degrees of thermal alterations in LTK lesions. Combined Sirius-red staining and polarization microscopy distinguished between a strongly coagulated central zone and the transition zone to normal tissue. Sirius-red uptake was increased in both zones, reflecting the availability of new binding sites. The central zone appeared darker under polarization than normal collagen because of a loss of birefringence. Intrinsic birefringence was greatly reduced; however, form birefringence partly remained as long as some collagen fibrils were intact. In the center of very strong lesions, where the collagen was hyalinized, birefringence was completely lost because of the complete disintegration of the fibrillar structure, which was visible under TEM. The transition zone toward normal cornea showed increased birefringence because the natural birefringence was largely preserved and enhanced by the increased Sirius-red uptake. Mechanical stretching between neighboring LTK lesions was manifested by increased birefringence.Conclusion: Sirius red offered an improved and simple histologic method for analyzing thermal collagen changes. It may contribute to a better understanding of the working mechanisms of LTK and improve analysis of thermal effects in corneal ablation.

Quantitative Characterization of High- and Low-Affinity Binding Sites for Basic Fibroblast Growth Factor on Trabecular Cells of the Eye

By radioligand binding followed by Scatchard analysis, we characterized and quantitated the specific binding sites for bFGF on cultured trabecular meshwork cells obtained from freshly enucleated porcine eyes. We detected two binding sites: 1.67×104±5.75×103high-affinity receptors per cell with a Kdof 33.4±7.90 pM, and 1.70×106±7.57×105low-affinity binding sites per cell with a Kdof 3.84±1.41 nM. At low concentrations of125I-bFGF (<1.50 ng ml−1), binding was primarily determined by the high-affinity receptors and, at high concentrations (>2.50 ng ml−1), binding was dependent on the low-affinity binding sites. By phase-contrast time-lapse video micrography and sequential photomicrography, we demonstrated that at a concentration of 1 ng ml−1, bFGF significantly stimulated the rate of mitosis of the trabecular meshwork cells in G0-phase compared with control cultures maintained in serum-free medium alone. Treatment with higher concentrations of bFGF did not reveal more potent effects on these cells. Our findings demonstrate that trabecular meshwork cells do possess low- and high-affinity receptors for bFGF and that bFGF induces these cellsin vitroto re-enter the cell cycle.Because the low-affinity interactions of125I-bFGF were reduced by 75% following pretreatment of the trabecular meshwork cells with heparinase, these sites represent cell-associated heparin-like molecules and heparan sulfate proteoglycans, and may control the bioavailability of bFGF to ocular tissues. Heparinase treatment also resulted in a 30% reduction in high-affinity binding, which may be secondary to the decreased low-affinity binding. This finding agrees with the well-established scheme for bFGF-receptor interaction. We conclude that bFGF at the concentration present in aqueous humor is capable of stimulating the mitotic activity of trabecular meshwork cellsin vitro,suggesting a possible paracrine role of aqueous humour bFGFin vivo.The results obtained in this study, together with our previous findings on bFGF mRNA expression by trabecular meshwork cells and protein deposition in this tissue, also indicates that trabecular cells of the eye may utilize bFGF by an autocrine mechanism.

Intraocular measurements of pressure transients induced by excimer laser ablation of the cornea.

The evolution of pressure waves induced by argon-fluoride laser ablation of the cornea in the typical operative conditions of clinical laser keratectomy has been studied experimentally and analyzed. Freshly enucleated porcine eyes were irradiated at a laser fluence of 180 mJ/cm2 with various spot diameters in the range 1-6.5 mm. Pressure transients were detected by a fast rise time needle hydrophone inserted into the eyeball from the posterior pole and moved along the eye optical axis toward the cornea. Pressure peaks as high as 90 bar and 50 ns pulse duration (FWHM) were measured in the anterior chamber. Observation of the pulse shape evolution during propagation put in evidence the onset of a marked rarefaction phase following the compressional spike, with intense negative peaks (up to -40 bar) located at increasing distances from the corneal surface for increasing spot diameters. This behavior was explained by means of simplified models describing pressure pulse generation and diffraction effects occurring during its propagation. Implications to clinical procedures, as possible damages due to tissue stretching and cavitation formation, are also discussed.

Radial keratotomy increases outflow facility in the porcine eye in vitro

PURPOSE. To investigate the biomechanical effect induced by radial keratotomy on outflow facility in paired, enucleated whole porcine eyes. METHODS. Freshly enucleated porcine eyes were perfused at a constant pressure of 10 mm Hg. Radial keratotomy (RK), with a 3.5 mm central clear zone and eight radial incisions, was performed using a diamond knife with the blade length set at 100% of the paracentral corneal thickness, as measured by pachymetry. The fellow eye of each pair received sham RK as a control. RESULTS. Seven pairs of eyes were perfused (RK group n = 7, control n = 7). There was no significant difference in the mean baseline of outflow facility between the paired experimental and control eyes preoperatively (p = 0.5). After RK, outflow facility increased by 46% (p < 0.001) in the treatment group, compared to a 7% (p < 0.04) increase in the control group, resulting in a 39% increase in outflow facility attributed to RK (p < 0.001). CONCLUSIONS. Radial keratotomy produced a statistically significant acute increase in outflow facility in freshly enucleated porcine eyes. We believe that this increase results from steepening of the peripheral corneal curvature and the concomitant stretching of the iridocorneal angle. Further studies are needed to evaluate this effect in human eyes and in vivo.

Acoustic Transients following Excimer Laser Ablation of the Cornea

Pressure transients occurring inside the eyeball as a consequence of excimer laser ablation of the cornea were measured at various distances along the optical axis, on enucleated porcine eyes. Positive (compressive) pressure spikes up to 90 bars were observed, lasting 100 ns, and developing, as they propagated in the eyeball, satellite rarefaction pulses with negative pressure as high as -40 bar. Such rarefaction wavefronts can trigger the formation of cavitation bubbles, in both the anterior and posterior chamber, depending on the ablation geometry. The potential risks associated with these photoacoustic phenomena, particularly for retina and corneal endothelium, are outlined.

Steps to optimize transscleral photocoagulation.

In transscleral photocoagulation, the desired effect is coagulation of parts of the ciliary body or of the peripheral retina. However, the application is often limited by the unwanted effect of coagulation of the sclera. To reduce this effect, the ratio of incident radiation flux to radiation flux transported through the sclera (and able to coagulate the target tissue) should be minimized by the incident beam characteristics. Monte Carlo simulations for the radiation transport problem of multiple scattering in the sclera were used to calculate the ratio of transported to incident radiation for different parameter settings of beam diameters, optical thicknesses of the sclera and beam angles. To verify the theoretical calculations, an simple optical device utilizing a bulb instead of a laser source was constructed and applied to enucleated porcine eyes. The theoretical calculations showed that the ratio of incident to transported radiation flux can typically be decreased by a factor of three by increasing the beam radius from 0.35 mm (as used in state-of-the-art laser devices) to 2 mm. This was confirmed by the experiments. Coagulations of the ciliary body or of the peripheral retina were possible with power densities an order of magnitude below the values normally applied with laser sources. To improve transscleral photocoagulation, beam diameters should be increased.

The influence of the cutting procedure on the loss of corneal endothelial cells of the recipient eye in mechanically guided trephination

Excessive damage to the endothelial monolayer during corneal trephination may contribute to transplant failure. For this reason, we performed several trephining experiments to determine the influence of various cutting conditions (sharpness, rotation rate, and cutting power) on the endothelial damage in the recipient bed. A mechanically guided motor trephine (Mikro-Keratron) was used for cutting experiments in fresh, enucleated porcine eyes. Eight different, reproducible combinations of cutting conditions were applied, and ten epithelial, full-thickness trephinations were performed for each combination. The endothelial defect was measured with a light microscope (60 x). In addition, the bend radii of the porcine cornea were measured. The mechanically guided Mikro-Keratron used with a sharp blade, high rotation rate (840 rev/min), and moderate cutting power (49 mN) caused a 31-micron-wide band of damaged endothelium in the recipient bed. Decrease of the rotation rate to 30 rev/min resulted in an increase of the endothelial defect to 168%. A cutting power of 392 mN combined with a rotation rate of 840 rev/min increased the defect to 126%. The system allowed full-thickness 360 degrees trephination. The corneal bend radii in porcine eyes measured 8.28 mm horizontally and 8.20 mm vertically. Our preliminary results indicate that the mechanically guided motor trephine, used under optimal conditions, is a suitable cutting device and may improve the outcome of penetrating keratoplasty; however, further investigation of this new approach is necessary.