Jan M. Vetter

Intraocular pressure during corneal flap preparation: comparison among four femtosecond lasers in porcine eyes.

PURPOSE To compare the course of intraocular pressure (IOP) during corneal flap preparation using four different femtosecond lasers in porcine globes. METHODS Forty-eight (12 in each group) enucleated globes were successfully cannulated through the optic nerve. Intraocular pressure was measured continuously through the cannula during a normal lamellar flap creation (regular procedure) using four femtosecond lasers (IntraLase, Abbott Medical Optics; VisuMax, Carl Zeiss Meditec AG; Femtec, Technolas Perfect Vision; and Femto LDV, Ziemer Ophthalmic Systems AG). In an additional measurement (worst-case procedure), the patient interface was pressed against the globe with increasing force until the applanation maneuver was automatically aborted by those devices capable of doing so. RESULTS During the regular procedure, the maximum IOP reached was 135±16 mmHg when using the Intra-Lase, 65±20 mmHg with the VisuMax, 205±32 mmHg with the Femtec, and 184±28 mmHg with the Femto LDV. During the worst-case procedure, a maximum IOP of 260±53 mmHg was reached with the IntraLase, 105±13 mmHg with the VisuMax, and 248±51 mmHg with the Femtec. CONCLUSIONS There is considerable variation in IOP among the tested femtosecond lasers during a regular lamellar flap creation and during the worst-case procedure. The VisuMax femtosecond laser seems to cause the lowest IOP rise in both settings.

Enhanced Insight into the Autoimmune Component of Glaucoma: IgG Autoantibody Accumulation and Pro-Inflammatory Conditions in Human Glaucomatous Retina

Background There is accumulating evidence that autoimmune components, such as autoantibodies and autoantibody depositions, play a role in the pathogenesis of neurodegenerative diseases like Alzheimeŕs disease or Multiple Sclerosis. Due to alterations of autoantibody patterns in sera and aqueous humor, an autoimmune component is also assumed in the pathogenesis of glaucoma, a common reason for irreversible blindness worldwide. So far there has been no convincing evidence that autoantibodies are accumulated in the retina of glaucoma patients and that the local immune homeostasis might be affected. Methods and Results Six human glaucomatous donor eyes and nine samples from donors with no recorded ocular disease were included. Antibody microarrays were used to examine the patterns of pro-inflammatory proteins and complement proteins. Analysis of TNF-α and interleukin levels revealed a slight up-regulation exclusively in the glaucomatous group, while complement protein levels were not altered. IgG autoantibody accumulations and/or cellular components were determined by immunohistology (n = 4 per group). A significantly reduced number of retinal ganglion cells was found in the glaucomatous group (healthy: 104±7 nuclei/mm, glaucoma: 67±9 nuclei/mm; p = 0.0007). Cell loss was accompanied by strong retinal IgG autoantibody accumulations, which were at least twice as high as in healthy subjects (healthy: 5.0±0.5 IgG deposits/100 cells, glaucoma: 9.4±1.9 IgG deposits/100 cells; p = 0.004). CD27+ cells and CD27+/IgG+ plasma cells were observed in all glaucomatous subjects, but not in controls. Conclusion This work provides serious evidence for the occurrence of IgG antibody deposition and plasma cells in human glaucomatous retina. Moreover, the results suggest that these IgG deposits occurred in a pro-inflammatory environment which seems to be maintained locally by immune-competent cells like microglia. Thereby, glaucoma features an immunological involvement comparable to other neurodegenerative diseases, but also shows a multifactorial pathomechanism, which diverges and might be linked to the specific nature of both eye and retina.

The Effect of Long-Term Storage on the Biological and Histological Properties of Cryopreserved Amniotic Membrane

Purpose: Cryopreserved amniotic membrane (AM) is widely used in ophthalmology because of its anti-angiogenic, anti-inflammatory, and wound healing promoting capabilities. A common method to conserve the tissue is the storage in cryo-medium containing 50% glycerol at −80°C. The aim of this study was to examine the influence of storage time on the sterility as well as the histological and biological properties of cryopreserved AM. Methods: Amniotic membrane from different donors was stored in cell culture media containing 50% glycerol for different time periods, on average 4 months (group 1), 15 months (group 2), and 24 months (group 3), at −80°C. Samples of the tissue and cryo-medium were examined for bacterial and fungal contamination. Tissue samples were incubated in 0.5 ml/cm2 serum-free medium at 37°C. The medium was changed after 1, 2, and 3 days. The proteins released by AM were TCA-precipitated and the presence of the proteins TIMP-1 and IL-1ra was analyzed using Western blotting and semi quantified by means of image analysis. Integrity of the amniotic epithelium and the basement membrane components collagen IV, collagen VII, laminin, laminin 5, and fibronectin were examined by haematoxylin eosin stain and immunohistochemistry in cryosections of AM. Results: None of the examined samples showed bacterial or fungal contamination. The soluble proteins TIMP-1 and IL-1ra were found in all samples of medium incubated for all time periods. The examined proteins were detectable after one-day incubation but the staining signal diminished significantly in the second and third wash after 48 hr and 72 hr. Differences in the intensity of the Western blot signal between the three particular groups were statistically not significant. The epithelia of all samples were intact. The basement membranes of all samples showed a similar distribution of collagen IV, collagen VII, laminin, laminin 5, and fibronectin. Conclusions: Long-term storage of amniotic membrane in cell culture media with 50% glycerol does not significantly impair sterility, histology, or biological properties of AM.

Tolerability of 24-hour intraocular pressure monitoring of a pressure-sensitive contact lens.

Purpose:To investigate tolerability and safety of a new diagnostic device for 24-hour intraocular pressure monitoring in healthy subjects and age-matched glaucoma patients. Patients and Methods:Twenty healthy subjects (group 1) and 20 age-matched glaucoma patients (group 2) were included in this prospective, single-center, open, observational parallel group study. The SENSIMED Triggerfish Sensor is a soft disposable contact lens embedding a telemetry chip and strain gauge sensor for continuous intraocular pressure monitoring. The Sensor was placed in 1 eye for 24 hours. Tolerability was evaluated using a visual analog scale (range, 0 to 100; 0=no discomfort; 100=very severe discomfort). Safety parameters included best corrected visual acuity, pachymetry, epithelial defects, conjunctival erythema, and corneal topography. Results:Mean age was 61.7 years in group 1 and 65.0 years in group 2. Nineteen healthy subjects and 19 glaucoma patients (95%) completed the 24-hour wearing period. Early discontinuation resulted from pain (n=1) or inappropriate fitting of the sensor due to steep corneal radii (n=1). Mean tolerability was 21.8 in group 1 (range, 7 to 67) and 26.8 in group 2 (range, 0 to 71). Corneal epithelial staining (Modified Oxford scale, grade 0 to 4) changed from 0.4 (group 1) and 1.0 (group 2) at baseline to 1.8 (group 1) and 2.8 (group 2) after monitoring. No statistically significant differences could be detected between both groups. Conclusions:This new pressure-sensitive contact lens is tolerable and safe over a 24-hour wearing period in healthy subjects and glaucoma patients. Both normals and glaucoma patients had a similar safety and tolerability profile.

Irregularity of the posterior corneal surface after curved interface femtosecond laser-assisted versus microkeratome-assisted descemet stripping automated endothelial keratoplasty.

Purpose: During donor tissue preparation for Descemet stripping automated endothelial keratoplasty (DSAEK), either microkeratome or femtosecond laser can be used for intrastromal cutting. We compared morphological and functional outcomes after DSAEK using both cutting techniques. Methods: In this retrospective study, 22 uneventful DSAEK surgeries were reviewed. Eight donor corneas were prepared for DSAEK using the VisuMax femtosecond laser (Carl Zeiss Meditec AG, Jena, Germany). Fourteen corneas were processed using an Amadeus II microkeratome (Ziemer Ophthalmic Systems AG, Port, Switzerland). The postoperative best spectacle-corrected visual acuity was measured. Furthermore, corneal optical coherence tomography scans (RTVue; Optovue, Fremont, CA) were conducted and analyzed for graft cornea thickness and posterior surface irregularities using regression analysis (SPSS; IBM, Chicago, IL) on a second-order polynomial curve as a model for the posterior surface. Results: The graft thickness was 166.3 ± 58.2 &mgr;m (mean ± SD) in the femtosecond laser group and 172.7 ± 48.2 &mgr;m in the microkeratome group. The best-corrected visual acuity of 0.48 ± 0.20 (logarithm of the minimum angle of resolution) in the femtosecond laser group was significantly poorer when compared with 0.33 ± 0.11 in the microkeratome group (P = 0.038). Moreover, the root mean square error between the posterior corneal surface and an ideal parabola surface was significantly higher in the femtosecond laser group (9.9 ± 2.2 &mgr;m) than in the microkeratome group (5.7 ± 2.2 &mgr;m; P < 0.001). Conclusions: Our study underlines the current superiority of a microkeratome-assisted preparation of the stromal–endothelial lamella before DSAEK surgery compared with the curved interface femtosecond laser-assisted processing.

Modulation of central corneal thickness by various riboflavin eyedrop compositions in porcine corneas

PURPOSE: To evaluate the modulatory effect of various riboflavin 0.1% and 0.2% compositions on the central corneal thickness (CCT) in fresh porcine corneas. SETTING: Department of Ophthalmology, Johannes Gutenberg University of Mainz, Mainz, Germany. DESIGN: Experimental study. METHODS: The CCT in freshly enucleated porcine globes was measured by ultrasound pachymetry before and after (if applicable) deepithelialization and every 10 minutes thereafter during 120 minutes of eyedrop application. In Groups 1 and 2 (controls), no eyedrops were applied. In Groups 3 and 4, isotonic riboflavin eyedrops were used. In Groups 5 to 9, hypotonic riboflavin eyedrops were given. In Groups 10 and 11, preparations for transepithelial crosslinking were applied. In Groups 2 to 9, deepithelialization was performed. The final CCT in the groups was compared by analysis of variance. RESULTS: One hundred ten freshly enucleated porcine globes were used. The mean final CCT compared with preoperative values was 97% ± 4% (SD) in Group 1, 91% ± 4% in Group 2, 66% ± 5% in Group 3, 151% ± 13% in Group 4, 65% ± 2% in Group 5, 105% ± 3% in Group 6, 120% ± 4% in Group 7, 130% ± 4% in Group 8, 132% ± 4% in Group 9, 114% ± 2% in Group 10, and 114% ± 4% in Group 11. The differences between Group 1 and each of Groups 3, 4, 5, 7, 8, and 9 were statistically significant (P<.05). CONCLUSION: There was considerable variation in the final CCT as a result of varying riboflavin eyedrop compositions. Financial Disclosure: No author has a financial or proprietary interest in any material or method mentioned.

Identification of the Muscarinic Acetylcholine Receptor Subtype Mediating Cholinergic Vasodilation in Murine Retinal Arterioles

PURPOSE To identify the muscarinic acetylcholine receptor subtype that mediates cholinergic vasodilation in murine retinal arterioles. METHODS Muscarinic receptor gene expression was determined in murine retinal arterioles using real-time PCR. To assess the functional relevance of muscarinic receptors for mediating vascular responses, retinal vascular preparations from muscarinic receptor-deficient mice were studied in vitro. Changes in luminal arteriole diameter in response to muscarinic and nonmuscarinic vasoactive substances were measured by video microscopy. RESULTS Only mRNA for the M(3) receptor was detected in retinal arterioles. Thus, M(3) receptor-deficient mice (M3R(-/-)) and respective wild-type controls were used for functional studies. Acetylcholine concentration-dependently dilated retinal arterioles from wild-type mice. In contrast, vasodilation to acetylcholine was almost completely abolished in retinal arterioles from M3R(-/-) mice, whereas responses to the nitric oxide (NO) donor nitroprusside were retained. Carbachol, an acetylcholinesterase-resistant analog of acetylcholine, also evoked dilation in retinal arterioles from wild-type, but not from M3R(-/-), mice. Vasodilation responses from wild-type mice to acetylcholine were negligible after incubation with the non-subtype-selective muscarinic receptor blocker atropine or the NO synthase inhibitor N(ω)-nitro-L-arginine methyl ester, and were even reversed to contraction after endothelial damage with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. CONCLUSIONS These findings provide evidence that endothelial M(3) receptors mediate cholinergic vasodilation in murine retinal arterioles via activation of NO synthase.

Comparison of intraocular pressure during corneal flap preparation between a femtosecond laser and a mechanical microkeratome in porcine eyes.

Purpose: To compare the increase in intraocular pressure (IOP) during corneal flap preparation in porcine eyes when using a femtosecond laser or a mechanical microkeratome. Setting: The present study was conducted at a university hospital and a private clinic. Methods: The vitreous IOP was directly measured with a cannula through the optic nerve in 24 porcine globes (n = 12 for each device). In the first procedure (worst-case procedure), the eye interface was lowered against the globe until abortion of the docking maneuver when using the IntraLase femtosecond laser (Abbott Medical Optics, Santa Ana, CA) or the suction ring was pressed very firmly against the globe when using the Amadeus microkeratome (Ziemer Ophthalmic Systems AG, Port, Switzerland). In the second procedure (regular procedure), a normal lamellar flap creation was performed using each of these devices. Results: During the worst-case procedure, a maximum IOP of 260 ± 53 mm Hg (mean ± SD) was reached with the IntraLase and 318 ± 59 mm Hg was reached with the Amadeus. During the regular procedure, the maximum IOP increased to 135 ± 16 mm Hg when using the IntraLase femtosecond laser and to 152 ± 24 mm Hg with the Amadeus microkeratome. Conclusions: In the worst-case procedure, the maximum IOP levels were lower in the IntraLase group. No significant difference in maximum IOP levels could be obtained during the regular procedure. The duration of the suction phase is longer when using the IntraLase femtosecond laser.

Intraocular pressure measurements during flap preparation using 2 femtosecond lasers and 1 microkeratome in human donor eyes

PURPOSE: To evaluate and compare intraocular pressures (IOPs) during flap preparations performed using 2 femtosecond lasers and a mechanical microkeratome in human donor globes. SETTING: University Medical Center Mainz, Mainz, and Euroeyes Clinic Stuttgart, Stuttgart, Germany. DESIGN: Experimental study. METHODS: A cannula was inserted through the optic nerve in human globes. The IOP was obtained continuously during flap preparation using the 60 kHz Intralase femtosecond laser, the 200 kHz Visumax femtosecond laser, or the Amadeus II microkeratome. For each experiment, a normal lamellar flap preparation (regular procedure) and a worst‐case procedure (femtosecond laser interface was pressed against globe until docking maneuver was aborted) were performed. RESULTS: During the regular procedure, the mean maximum IOP measured was 181.3 mm Hg (range 159.1 to 194.8 mm Hg) with the 60 kHz femtosecond laser, 77.6 mm Hg (range 58.1 to 100.3 mm Hg) with the 200 kHz femtosecond laser, and 198.1 mm Hg (range 162.8 to 299.6 mm Hg) with the microkeratome. During the worst‐case procedure, the maximum measured IOP was 319.7 mm Hg (range 299.1 to 341.2 mm Hg) with the 60 kHz laser and 120.4 mm Hg (range 118.1 to 134.7 mm Hg) with the 200 kHz laser. CONCLUSION: Maximum IOPs during corneal flap preparations in human enucleated eyes were lower during performance of a regular procedure and a worst‐case procedure with the 200 kHz femtosecond laser than with the 60 kHz femtosecond laser and the mechanical microkeratome. Financial Disclosure: Dr. Sekundo is a member of the Scientific Advisory Board of Carl Zeiss Meditec AG, Jena, Germany. No author has a financial or proprietary interest in any material or method mentioned.

Functional role of α1-adrenoceptor subtypes in murine ophthalmic arteries.

PURPOSE To identify the α(1)-adrenoceptor (α(1)-AR) subtypes mediating vascular adrenergic responses in murine ophthalmic arteries. METHODS Expression of mRNA was quantified for individual α(1)-AR subtypes in murine ophthalmic arteries using real-time PCR. To assess the functional relevance of α(1)-ARs for mediating vascular responses, ophthalmic arteries from mice deficient in one of the three α(1)-AR subtypes (α(1A)-AR(-/-), α(1B)-AR(-/-), and α(1D)-AR(-/-), respectively) and wild-type controls were isolated, cannulated with micropipettes, and pressurized. Changes in luminal artery diameter in response to the α(1)-AR agonist phenylephrine, the sympathetic transmitter noradrenaline, and to the nonadrenergic vasoconstrictor arginine vasopressin (AVP) were measured by video microscopy. RESULTS Using real-time PCR, mRNA for all three α(1)-AR subtypes was detected in ophthalmic arteries from wild-type mice. In functional studies, phenylephrine and noradrenaline produced dose-dependent constriction of ophthalmic arteries that was similar in wild-type, α(1B)-AR(-/-), and α(1D)-AR(-/-) mice. Strikingly, responses to phenylephrine and noradrenaline were almost completely abolished in α(1A)-AR(-/-) mice. In contrast, the nonadrenergic agonist AVP produced dose-dependent vasoconstrictor responses that did not differ between any of the mouse genotypes tested. CONCLUSIONS These findings provide evidence that the α(1A)-AR subtype mediates adrenergic vasoconstriction in murine ophthalmic arteries.