Panagiotis Laspas

Retinal Ganglion Cell Loss Is Accompanied by Antibody Depositions and Increased Levels of Microglia after Immunization with Retinal Antigens

Background Antibodies against retinal and optic nerve antigens are detectable in glaucoma patients. Recent studies using a model of experimental autoimmune glaucoma demonstrated that immunization with certain ocular antigens causes an immun-mediated retinal ganglion cell loss in rats. Methodology/Principal Findings Rats immunized with a retinal ganglion cell layer homogenate (RGA) had a reduced retinal ganglion cell density on retinal flatmounts (p = 0.007) and a lower number of Brn3+retinal ganglion cells (p = 0.0001) after six weeks. The autoreactive antibody development against retina and optic nerve was examined throughout the study. The levels of autoreactive antibodies continuously increased up to 6 weeks (retina: p = 0.004; optic nerve: p = 0.000003). Additionally, antibody deposits were detected in the retina (p = 0.02). After 6 weeks a reactive gliosis (GFAP density: RGA: 174.7±41.9; CO: 137.6±36.8, p = 0.0006; %GFAP+ area: RGA: 8.5±3.4; CO: 5.9±3.6, p = 0.006) as well as elevated level of Iba1+ microglia cells (p = 0.003) was observed in retinas of RGA animals. Conclusions/Significance Our findings suggest that these antibodies play a substantial role in mechanisms leading to retinal ganglion cell death. This seems to lead to glia cell activation as well as the invasion of microglia, which might be associated with debris clearance.

Autoreactive antibodies and loss of retinal ganglion cells in rats induced by immunization with ocular antigens.

PURPOSE In an experimental autoimmune animal model, retinal ganglion cell (RGC) loss was induced through immunization with glaucoma-related antigens. The target of this study was to investigate the pathomechanism behind this decline and the serum antibody reactivity against ocular and neuronal tissues after immunization with glaucoma- and non-glaucoma-associated antigens. METHODS Rats immunized with optic nerve antigen homogenate (ONA) or keratin (KER) were compared to control rats (CO). Intraocular pressure (IOP) was measured, and the fundi were examined regularly. Four weeks afterward, cells were counted in retinal flat mounts. Retina, optic nerve, and brain sections from healthy animals and optic nerve sections from immunized animals were incubated with serum collected at different time points. The occurrence of autoreactive antibodies was examined. Signs of antibody deposits, microglia activation, and demyelination were sought in optic nerves of immunized animals. Brain sections were examined for abnormalities. RESULTS No IOP or fundus changes were observed. Animals immunized with ONA showed a significant cell loss compared with the CO group. Elevated autoreactive antibodies against retina, optic nerve, and brain were observed. Animals immunized with KER, despite their immunologic response against KER, demonstrated neither RGC loss, nor increased development of autoreactive antibodies. Optic nerve from animals immunized with ONA demonstrated antibody accumulation, glia activation, and demyelination. No such observations were made in the KER or CO groups. Brain sections were without pathologic findings. CONCLUSIONS Systemic autoimmunity against ocular and neuronal epitopes, mediated by accordant autoreactive antibodies, is involved in the inflammatory processes that cause RGC degeneration in this experimental animal model.

A New Method for Revision of Encapsulated Blebs after Trabeculectomy: Combination of Standard Bleb Needling with Transconjunctival Scleral Flap Sutures Prevents Early Postoperative Hypotony

Purpose A simple needling procedure is the standard method for restoring the function of an encapsulated bleb after trabeculectomy. However, postoperative hypotony represents a possible hazard. This study describes a new surgical approach for treating encapsulated blebs with reduced risk of early postoperative hypotony: bleb needling combined with transconjunctival sutures tightening the scleral flap directly. Methods The study included two groups of 23 patients with failing bleb following trabeculectomy: “Group 1” underwent simple needling revision of the filtering bleb and served as a control group, while “Group 2” received needling revision with additional transconjunctival scleral flap sutures, if intraoperatively the intraocular pressure was estimated to be very low. Intraocular pressure (IOP), postoperative management and complications were analyzed over a follow-up period of 4 weeks postoperatively. Results were compared using t-test or Mann-Whitney U-tests. Results Adverse effects occurred with a higher frequency after sole needling of the bleb (5 cases of choroidal effusion and 1 case of choroidal hemorrhage) than after the combined method with additional scleral sutures (1 case of choroidal effusion). The IOP on the first postoperative day was significantly lower in group 1, with 9.43 ± 9.01 mm Hg vs. 16.43 ± 8.35 mm Hg in group 2 (P = 0.01). Ten patients with ocular hypotony (IOD of 5 mmHg or lower) were found in group 1 and only two in group 2. One week and one month after surgery the intraocular pressure was similar in both groups (P>0.05). Conclusions This new needling technique with additional transconjunctival scleral flap sutures appears to reduce postoperative hypotony, and may thus protect from further complications, such as subchoroidal hemorrhage.

Cilioretinal Artery Occlusion

BACKGROUND The aim of this study was to evaluate characteristic findings and functional outcome of this rare type of retinal vascular occlusion in a large patient series. PATIENTS AND METHODS A retrospective chart review of all patients with a cilioretinal artery obstruction (CAO) seen at the University Eye Clinic Mainz (Germany) over a ten-year period between 1/1999 and 12/2008 was undertaken. The mean follow-up amounted to 42 months. RESULTS Thirty-three eyes were diagnosed with CAO. Three distinct groups were differentiated: (i) isolated CAO in 20/33 eyes; visual outcome achieving 20/50 in 17/20 eyes; (ii) CAO combined with central retinal vein occlusion in 9/33 eyes; visual outcome achieving 20/50 in 5/9 eyes; (iii) CAO combined with anterior ischaemic optic neuropathy in 4/33 eyes; associated with giant cell arteriitis in 4/4 eyes; visual outcome achieving 20/160 in 1/4 eyes. CONCLUSIONS The visual prognosis of CAO is significantly better than for other types of retinal artery occlusions, with the exception of cases associated with giant cell arteriitis.

Effect of the M1 Muscarinic Acetylcholine Receptor on Retinal Neuron Number Studied with Gene-Targeted Mice

Pharmacological activation of the M1 muscarinic receptor subtype was suggested to promote the survival of retinal neurons. We examined the hypothesis that the M1 receptor is crucial for retinal neuron survival in vivo by using mice devoid of the M1 receptor gene. Muscarinic receptor gene expression was determined in the retina using real-time PCR. The amount of neurons in the retinal ganglion cell layer and of axons in the optic nerve was determined in retinal wholemounts stained with cresyl blue and in optic nerve cross-sections stained with toluidine blue, respectively. mRNA of all five muscarinic receptor subtypes (M1–M5) was detected in the retina from wild-type mice. Remarkably, M2 and M3 receptor mRNA were most abundant. In retinas from M1 receptor-deficient mice, M4 receptor mRNA expression was increased compared to that of wild-type mice, while no marked changes in the mRNA expression levels of the other muscarinic receptor subtypes were observed. The amount of cells in the retinal ganglion cell layer and the amount of axons in the optic nerve did not differ between M1 receptor-deficient and wild-type mice. The present findings suggest that the M1 receptor is not essential for the survival of retinal neurons in vivo.

Role of α1-Adrenoceptor Subtypes in Pupil Dilation Studied With Gene-Targeted Mice

PURPOSE The α₁A-adrenoceptor (α₁A-AR) subtype was suggested to mediate contraction and trophic effects in the iris dilator muscle, and thus its pharmacological blockade may be involved in intraoperative floppy iris syndrome. We tested the hypothesis that the α₁A-AR mediates pupil dilation and trophic effects in the mouse iris. METHODS The α₁-AR subtype mRNA expression was quantified in iris tissue by real-time PCR. To assess the role of individual α₁-ARs for mediating pupil dilation, the α₁-AR agonist phenylephrine was topically applied to the ocular surface of mice deficient in one of the three α₁-AR subtypes (α₁A-AR(-/-), α₁B-AR(-/-), α₁D-AR(-/-), respectively) and wild-type controls. Changes in pupil diameter were measured under a microscope in restrained mice. Moreover, iris and iris muscle thickness were determined in cryosections. RESULTS Messenger RNA for all three α₁-AR subtypes was detected the iris of wild-type mice with a rank order of abundance of α₁A ≥ α₁B > > α₁D. The lack of a single α₁-AR gene did not affect mRNA expression of the remaining two receptor subtypes. Phenylephrine induced pupil dilation in wild-type mice that was reduced in extent and duration in α₁A-AR(-/-) and, less so, in α₁B-AR(-/-) but not in α₁D-AR(-/-) mice. The lack of a single α₁-AR subtype had no effect on iris or iris muscle thickness. CONCLUSIONS The α₁-AR-induced mydriasis in mice is mediated mainly by the α₁A-AR, with a smaller contribution of the α₁B-AR, matching the relative abundance of these subtypes at the mRNA level. The lack of a single α₁-AR subtype does not appear to cause atrophy in the mouse iris.

Revision of encapsulated blebs after trabeculectomy: Long-term comparison of standard bleb needling and modified needling procedure combined with transconjunctival scleral flap sutures.

Purpose To compare two surgical approaches for treating encapsulated blebs after trabeculectomy with mitomycin C, in terms of the development of intraocular pressure and progression of glaucoma in a long-term follow up: 1. bleb needling alone vs. 2. a combined approach of needling with additional transconjunctival scleral flap sutures, to prevent early ocular hypotony. Methods Forty-six patients with failing blebs after trabeculectomy with mitomycin C were enrolled in this study. Patients received either needling revision alone (group 1; n = 23) or a combined needling with additional transconjuctival flap sutures, if intraoperatively the intraocular pressure was estimated to be low (group 2; n = 23). Intraocular pressure (IOP), visual acuity, visual fields, and optic nerve head configuration by means of Heidelberg Retina Tomograph (HRT®) were analysed over time. Results from both groups were compared using Mann-Whitney U-test for single timepoints. Results IOP did not differ significantly between the two groups during follow-up at three months (P = 0.13), six months (P = 0.12), one year (P = 0.92) and two years (P = 0.57) after surgery. Furthermore, there was no significant difference in the course of glaucoma concerning the optic nerve anatomy between the two groups (Rim Area Change in the Moorfields Regression Analysis of HRT®) till two years after surgery (P = 0.289). No functional impairment in visual acuity and visual fields was found in the groups of the study. Conclusions Single needling procedure is a standard successful method for restoring the function of encapsulated blebs. Postoperative hypotony represents a possible hazard, which can be minimized by additional transconjunctival flap sutures. Long-term results suggest that this modification is equally effective in lowering the IOP and preventing the progression of glaucoma as the standard needling procedure. To our knowledge this is the first study to investigate the long-term effect of tranconjunctival sutures for the prevention of hypotony.