Expression and subcellular localization of USH1C/harmonin in human retina provides insights into pathomechanisms and therapy.

Usher syndrome (USH) is the most common form of hereditary deaf-blindness in humans. USH is a complex genetic disorder, assigned to three clinical subtypes differing in onset, course and severity, with USH1 being the most severe. Rodent USH1 models do not reflect the ocular phenotype observed in human patients to date; hence, little is known about the pathophysiology of USH1 in the human eye. One of the USH1 genes, USH1C, exhibits extensive alternative splicing and encodes numerous harmonin protein isoforms that function as scaffolds for organizing the USH interactome. RNA-seq analysis of human retinae uncovered harmonin_a1 as the most abundant transcript of USH1C. Bulk RNA-seq analysis and immunoblotting showed abundant expression of harmonin in Müller glia cells (MGCs) and retinal neurons. Furthermore, harmonin was localized in the terminal endfeet and apical microvilli of MGCs, presynaptic region (pedicle) of cones and outer segments (OS) of rods as well as at adhesive junctions between MGCs and photoreceptor cells (PRCs) in the outer limiting membrane (OLM). Our data provide evidence for the interaction of harmonin with OLM molecules in PRCs and MGCs and rhodopsin in PRCs. Subcellular expression and colocalization of harmonin correlate with the clinical phenotype observed in USH1C patients. We also demonstrate that primary cilia defects in USH1C patient-derived fibroblasts could be reverted by the delivery of harmonin_a1 transcript isoform. Our studies thus provide novel insights into PRC cell biology, USH1C pathophysiology and development of gene therapy treatment(s).

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 Alzheimers 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.

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 µm (mean ± SD) in the femtosecond laser group and 172.7 ± 48.2 µm 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 µm) than in the microkeratome group (5.7 ± 2.2 µm; 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.

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.

Irregularity of the posterior corneal surface during applanation using a curved femtosecond laser interface and microkeratome cutting head.

PURPOSE: To evaluate the irregularity of the posterior corneal surface and intrastromal dissection during the preparation of donor tissue for Descemet stripping automated endothelial keratoplasty (DSAEK) using a curved interface femtosecond laser and microkeratome. METHODS: Sixteen human donor corneas unsuitable for transplantation were divided into two groups: a femtosecond (FS) laser group (n=7) using the VisuMax femtosecond laser (Carl Zeiss Meditec) and a microkeratome group (n=9) using the Amadeus II microkeratome (Ziemer Ophthalmic Group). The corneas were fixed on artificial anterior chambers. Horizontal cross-sections were obtained using spectral-domain optical coherence tomography prior to applanation, during applanation, as well as during and after intrastromal dissection at 450-µm corneal depth. The posterior surface and the dissection line were evaluated for irregularity by fitting a second-order polynomial curve using regression analysis and obtaining the root-mean-square error (RMSE). Groups were compared using analysis of variance. RESULTS: The RMSE of the posterior surface prior to applanation was 9.7 ± 3.1 µm in the FS laser group and 10.2 ± 2.3 µm in the microkeratome group. The RMSE increased to 50.7 ± 9.4 µm and 20.9 ± 6.1 µm during applanation and decreased again to 10.6 ± 1.4 µm and 8.1 ± 1.8 µm after applanation in the FS laser and microkeratome groups, respectively. The RMSE of the intrastromal cut was 19.5 ± 5.7 µm in the FS laser group and 7.7 ± 3.0 µm in the microkeratome group (P<.001). CONCLUSIONS: Our results show significantly greater irregularity with the curved interface femtosecond laser-assisted cleavage compared to microkeratome-assisted corneal dissection, possibly due to applanation-derived deformation of the posterior cornea.

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.

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.

Survival after transplantation of corneas from a rabies-infected donor.

PURPOSE: To examine the tissue samples of 2 corneal recipients from a rabies-infected donor for the presence of rabies to explain their survival. METHODS: Interventional case series with a review of the literature. The explanted corneal donor buttons were examined via nested reverse transcriptase polymerase chain reaction. The patients were followed up ophthalmologically and neurologically. Antirabies antibodies were measured in blood samples, and skin biopsies were examined by direct fluorescent antibody staining. RESULTS: Two patients received corneas from the same multiorgan donor. Six weeks after transplantation, 3 of the donor's organ recipients became symptomatic and rabies virus was confirmed in tissue from the donor's central nervous system. Immediately, both the corneal recipients underwent active and passive postexposure treatment. The corneal buttons were replaced. Examination of the explanted donor corneas, skin biopsies, and serum and saliva samples showed no signs of rabies infection. The 2 corneal recipients were followed up at our hospital and, to date, are without symptoms of infection. CONCLUSIONS: Transmission of the potentially deadly rabies virus by corneal transplantation has been described previously. To our knowledge, this is the first report in which no rabies virus transmission occurred without immediate postexposure treatment.

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.

Surgical management of acute angle-closure glaucoma after toric implantable contact lens implantation.

A case of pupillary block after implantation of an implantable contact lens (ICL) is reported, and surgical management and prevention are discussed. In a myopic patient, the best corrected visual acuity in the right eye was 20/50 with -15.50 -3.00 x 175. After uneventful implantation of an ICL, painful acute glaucoma developed with an intraocular pressure beyond measurable values. Apparent anterior vaulting of the ICL suggested a sizing problem. In a situation of a mid-wide dilated pupil, immediate explantation of the ICL was performed. Then, using a preoperative iris photography as guidance, an anterior chamber iris-claw toric phakic intraocular lens was implanted. On postoperative examination, the anterior chamber was deep, the angle open, the natural lens clear, and uncorrected visual acuity was 20/40.