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Purpose: To analyze the retinal radial peripapillary capillary (RPC) network in normal-tension glaucoma (NTG) and nonarteritic anterior ischemic optic neuropathy (NAION) eyes using optical coherence tomography angiography (OCTA)., Material and Methods: Twenty-two patients with NTG, 22 patients with unilateral chronic NAION, and 23 age-matched controls were enrolled. Patients underwent OCTA to obtain en face angiograms of the peripapillary region. The main outcome measures were as follows: (1) the whole en face image perfusion density (WPD) and (2) the circumpapillary perfusion density (CPD)., Results: Mean ± SD age was 66.3 ± 7.0 years in the NTG group, 68.1 ± 4.3 years in the NAION group, and 63.9 ± 7.0 years in the control group (p > 0.05 for all the comparisons). The visual field mean defect (MD) was worse in patients than in controls (p < 0.0001), but did not differ between NTG and NAION (-9.6 ± 2.6 dB and -8.2 ± 2.6 dB, respectively). The WPD was 0.41 ± 0.04 in the NTG group (p < 0.0001 in comparison with healthy subjects and NAION patients), 0.46 ± 0.04 in the NAION group (p < 0.0001 in comparison with the control group), and 0.56 ± 0.03 in the control group. The CPD was significantly reduced in both NTG (0.48 ± 0.04, p < 0.0001) and NAION eyes (0.52 ± 0.05, p < 0.0001), after comparison to control eyes (0.59 ± 0.03). Moreover, the CPD was significantly lower in NTG than in NAION eyes (p = 0.006)., Conclusions: OCTA documented a reduction of the peripapillary perfusion in NTG and unilateral NAION. In presence of similar functional damage, the lower perfusion densities in NTG may indicate greater vascular alterations in chronic compared to acute ischemic optic neuropathies.

Purpose: To investigate, using in vivo confocal microscopy (IVCM), the Meibomian gland (MG) features and conjunctival goblet cell density (GCD) in glaucomatous patients controlled with prostaglandin/timolol fixed combinations (PTFCs)., Materials and Methods: In this cross-sectional study, 60 white patients were treated with PTFCs, 15 with latanoprost+timolol (L+T) unfixed combination, and 15 controls were enrolled. Patients underwent the Ocular Surface Disease Index questionnaire, tear film breakup time, corneal staining, Schirmer test I, and IVCM of MGs and goblet cells. The main outcome measures were: mean Meibomian acinar density (MMAD) and area (MMAA), inhomogeneity of glandular interstice (InI) and acinar wall (InAW), and GCD., Results: PTFCs were: latanoprost/timolol (LTFC, 15 eyes), travoprost/timolol (TTFC, 15), bimatoprost/timolol (BTFC, 15), and preservative-free bimatoprost/timolol (PF-BTFC, 15) fixed combinations. Mean time on therapy did not differ among treatments. IVCM documented lower GCD, MMAD, and MMAA (P<0.001), and greater InI and InAW (P<0.05) in glaucoma patients compared with controls. L+T showed worse values compared with PTFCs and PF-BTFC (P<0.05). Preserved PTFCs showed lower MMAD, MMAA, GCD, and greater InI and InAW compared with PF-BTFC (P<0.05) and controls (P<0.001). Differences were not found among PTFCs. InI and InAW significantly correlated with Ocular Surface Disease Index and breakup time (P<0.001), corneal staining (P<0.05), and GCD (P<0.001); GCD correlated with MMAD (P<0.05)., Conclusions: PTFCs were less toxic towards MGs and goblet cells compared with the L+T unfixed combination, with PF-BTFC presenting the most tolerated profile. These findings should be carefully considered given the role of these structures in the induction of the glaucoma-related ocular surface disease.

Background/aim: In glaucoma, conjunctival epithelial microcysts (CEM) have been extensively investigated by means of laser scanning confocal microscopy. In the present case series, we examined eight glaucomatous patients undergoing trabeculectomy to obtain a 3-dimensional (3-D) characterization of CEM., Materials and Methods: Image acquisition was performed in z-scan automatic volume mode by Heidelberg Retina Tomograph III/Rostock Cornea Module and a series of 40 images of 300×300 μm (384×384 pixels) to a maximum depth of 40 μm were acquired throughout the upper bulbar conjunctiva before (at the site planned for surgery) and eight weeks after trabeculectomy. The 3-D volume tissue reconstruction with maximal size of 300×300×40 μm was obtained., Results: In the enface view, CEM appeared as empty, optically clear, round or oval shaped sub-epithelial structures. The 3-D spatial reconstruction showed microcysts as oval-shaped and optically clear elements, which were close, but clearly separated from the epithelium. CEM were embedded in the extra-cellular spaces and located about 10 μm below the epithelial surface. After trabeculectomy, CEM increased density and area especially along the horizontal axis., Conclusion: The 3-D in vivo confocal reconstruction of CEM permits for better clarification of their microscopic anatomy and patho-physiological significance, confirming their involvement in AH flow through the bleb-wall after filtration surgery for glaucoma., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Ocular surface diseases (OSDs) represent a widely investigated field of research given their growing incidence and the negative impact on quality of life. During OSDs, cytokines generated by damaged epithelia trigger and deregulate the lymphoid cells composing the eye-associated lymphoid tissues, inducing an immune-mediated chronic inflammation that amplifies and propagates the disease during time. The conjunctiva-associated lymphoid tissue (CALT), given its particular position that permits immune cells covering the cornea, might play a crucial role in the development of OSDs. Despite the recognized inflammatory role of mucosa-associated lymphoid tissues in other stations taking contact with the external environment (gut or bronchus), CALT did not gain the deserved consideration. In the last years, the diffusion of the in vivo confocal microscopy (IVCM) stimulated the interest to CALT, especially in dry eye, ocular allergy, and glaucoma. Though the initial stimuli were different, IVCM documented similar changes, represented by increased lymphoid cells within the diffuse layer, follicles and interfollicular spaces. These findings, which need to be validated by immunohistology, support the CALT stimulation during OSDs. However, while an involvement of the CALT in OSDs is hypothesizable, the exact role of this structure in their pathogenesis remains unclear and warrants further investigations.

Purpose: To examine the preoperative conjunctival dendritic cell density (DCD), goblet cell density (GCD), and stromal meshwork reflectivity (SMR) in glaucomatous patients undergoing filtration surgery, using in vivo confocal microscopy (IVCM)., Methods: Sixty-six patients were enrolled. At baseline, IVCM was performed at the site planned for surgery, and was repeated after 12 months at bleb site. Surgery was successful when a one-third reduction of baseline IOP was obtained at the last follow-up. The main outcomes were baseline DCD, GCD, and SMR, and 12 months IOP. The relations between baseline confocal parameters and 12 months IOP were analyzed., Results: Filtration surgery was successful in 43 patients (group 1: complete success, 25; group 2: qualified success, 18), and unsuccessful in 23 patients (group 3). Baseline IOP (mm Hg) was 27.6 ± 2.8, 28.8 ± 4.1, and 27.7 ± 3.2 in groups 1 to 3, respectively. Preoperative DCD and SMR were lower in group 1 compared with groups 2 (P < 0.001, P < 0.05), and 3 (P < 0.001); preoperative GCD was higher in group 1 compared with groups 2 and 3 (P < 0.001). DCD and GCD were also different between groups 2 and 3 (P < 0.05, P < 0.001). At 12 months, IOP reduced by 43.3%, 38.4%, and 15.8% in groups 1 to 3. Twelve-month IOP reduction negatively correlated with baseline DCD and SMR (P < 0.001, r = -0.786; P < 0.05, r = -0.618), and positively with GCD (P < 0.001, r = 0.752)., Conclusions: Preoperative DCD, GCD, and SMR are parameters correlated with the filtration surgery outcome, with DCD presenting the strongest correlation. IVCM of the conjunctiva may represent an imaging tool to predict the surgical success in glaucoma.

The ciliary body ablation is still considered as a last resort treatment to reduce the intraocular pressure (IOP) in uncontrolled glaucoma. Several ablation techniques have been proposed over the years, all presenting a high rate of complications, nonselectivity for the target organ, and unpredictable dose-effect relationship. These drawbacks limited the application of cyclodestructive procedures almost exclusively to refractory glaucoma. High-intensity focused ultrasound (HIFU), proposed in the early 1980s and later abandoned because of the complexity and side effects of the procedure, was recently reconsidered in a new approach to destroy the ciliary body. Ultrasound circular cyclocoagulation (UC 3 ), by using miniaturized transducers embedded in a dedicated circular-shaped device, permits to selectively treat the ciliary body in a one-step, computer-assisted, and non-operator-dependent procedure. UC 3 shows a high level of safety along with a predictable and sustained IOP reduction in patients with refractory glaucoma. Because of this, the indication of UC 3 was recently extended also to naïve-to-surgery patients, thus reconsidering the role and timing of ciliary body ablation in the surgical management of glaucoma. This article provides a review of the most used cycloablative techniques with particular attention to UC 3 , summarizing the current knowledge about this procedure and future possible developments.

Aims: To evaluate, using anterior segment optical coherence tomography (AS-OCT) and in vivo confocal microscopy (IVCM), the uveo-scleral aqueous humour (AH) outflow pathways after ultrasonic circular cyclocoagulation (UCCC)., Methods: Forty-four patients with refractory glaucoma underwent 4 or 6 s UCCC (group 1, 24 eyes; group 2, 20 eyes). UCCC was successful when the preoperative intraocular pressure (IOP) reduced by one-third. AS-OCT and IVCM were performed at baseline and at month 1 to evaluate the sclera and conjunctiva. The main outcomes were mean intra-scleral hyporeflective spaces area (MIHSA: mm 2 ) at AS-OCT, mean density and area of conjunctival microcysts (MMD: cysts/mm 2 ; MMA: µm 2 ) at IVCM. The relations between MIHSA, MMA and MMD with IOP were analysed., Results: Mean baseline IOP was 26.9±2.8 mm Hg in group 1 and 27.5±4.0 in group 2. Intra-scleral hyporeflective spaces and microcysts were observed in both groups, without significant differences in MIHSA, MMA and MMD. At month 1, UCCC was successful in 63.6% of patients (41.6% in group 1, 80% in group 2), and IOP reduced to 18.8±3.2 (30.1%) and 17.1±2.7 mm Hg (38.7%), respectively (p<0.001). MIHSA showed a twofold and threefold increase in group 1 and 2 (p<0.05), with a significant difference between groups (p<0.05). MMA and MMD increased in both groups (p<0.05), with values higher in group 2 (p<0.05). Significant relations were found between MIHSA and IOP in both groups (p<0.01)., Conclusions: UCCC induced anatomical modifications of sclera and conjunctiva, which suggested that the trans-scleral AH outflow enhancement is one of the possible mechanisms exploited by ultrasounds to reduce IOP., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Purpose: The purpose of this study was to evaluate dendritic cell (DC) distribution, morphology, and DC density in the entire cornea of medically controlled glaucoma patients (MCGP), using in vivo confocal microscopy (IVCM)., Methods: Fifty MCGP were enrolled, 15 patients with dry eye, and 15 healthy subjects served as controls. Patients were asked to complete the Ocular Surface Disease Index (OSDI) questionnaire and then underwent tear film break-up time (BUT), corneal staining, and Schirmer test (ST) I and then IVCM. In vivo confocal microscopy evaluated the limbal and central DC density, the DCs morphology and distribution. Relationships among DC density, OSDI score, and corneal staining were analyzed., Results: Medically controlled glaucoma patients were divided into 2 groups; group 1 (29 eyes) was tested with one drug; group 2 (21 eyes) was tested with ≥2 drugs. Dendritic cells were significantly higher at limbus than at central cornea in both groups. Limbal DCs were found in the 86.7%, 89.7%, 90.4%, and 93.3% of eyes in controls, groups 1 and 2, and DED; central corneal DCs were found in the 26.6%, 75.9%, 80.9%, and 86.6% of eyes in controls, groups 1 and 2, and DED. Dendritic cell density was higher in glaucoma groups and DED than in controls (P < 0.001). Group 2 and DED presented DC density significantly higher compared with group 1 (P < 0.05). In group 1 DC density was higher in patients taking preserved drugs than in those taking preservative-free drugs (P < 0.05). Dendritic cell density was higher in DED than in group 2 (P < 0.05). Dendritic cell density significantly correlated with corneal staining and OSDI (P < 0.001)., Conclusions: Dendritic cells increase in the entire cornea of MCGP, with a higher density at limbus. These modifications may take part in the induction of the glaucoma-related ocular surface disease.

Purpose: We analyzed the preoperative conjunctival goblet cell density (GCD), MUC5AC, and HLA-DR in glaucomatous patients undergoing trabeculectomy, using laser scanning confocal microscopy (LSCM) and impression cytology (IC)., Methods: We enrolled 57 patients undergoing trabeculectomy. At baseline LSCM and IC were performed at the site planned for surgery; LSCM was repeated after 12 months at the bleb site. The main outcomes were: GCD, mean microcyst density (MMD) and area (MMA) at LSCM, MUC5AC, and HLA-DR positivity at IC, and IOP. The relationships between baseline GCD, and 12-month IOP, MMD, and MMA were analyzed., Results: Trabeculectomy was successful in 39 patients (complete success in 27, Group 1; qualified in 12, Group 2), and unsuccessful in 18 (Group 3). At baseline IOP (mm Hg) was 27.2 ± 3.12, 27.5 ± 2.23, and 27.7 ± 1.90 in Groups 1 to 3, respectively; GCD and MUC5AC positivity were higher in Group 1 compared to Groups 2 and 3 (P < 0.05); HLA-DR, MMD, and MMA were not significantly different among the groups. At 12 months, IOP reduced by 45.3%, 35.4%, and 12.8% in Groups 1 to 3, respectively. Goblet cell density did not change in Group 1, whereas it was reduced in Groups 2 and 3 (P < 0.05), with values lower in Group 3. Mean microcyst density and MMA increased in Groups 1 and 2 (P < 0.05), with values higher in Group 1 (P < 0.05). Baseline GCD positively correlated with 12-month IOP reduction (P < 0.001, r = 0.641), MMD (P < 0.05, r = 0.454), and MMA (P < 0.001, r = 0.541)., Conclusions: Goblet cells positively affect the filtration ability after trabeculectomy; therefore, preoperative GCD could be considered as a potential in vivo biomarker of surgical success.

Meibomian glands (MGs) play a crucial role in the ocular surface homeostasis by providing lipids to the superficial tear film. Their dysfunction destabilizes the tear film leading to a progressive loss of the ocular surface equilibrium and increasing the risk for dry eye. In fact, nowadays, the meibomian gland dysfunction is one of the leading causes of dry eye. Over the past decades, MGs have been mainly studied by using meibography, which, however, cannot image the glandular structure at a cellular level. The diffusion of the in vivo laser scanning confocal microscopy (LSCM) provided a new approach for the structural assessment of MGs permitting a major step in the noninvasive evaluation of these structures. LSCM is capable of showing MGs modifications during aging and in the most diffuse ocular surface diseases such as dry eye, allergy, and autoimmune conditions and in the drug-induced ocular surface disease. On the other hand, LSCM may help clinicians in monitoring the tissue response to therapy. In this review, we summarized the current knowledge about the role of in vivo LSCM in the assessment of MGs during aging and in the most diffuse ocular surface diseases.

Purpose: To investigate morphologic changes of the corneoscleral limbus in glaucoma patients using laser scanning confocal microscopy (LSCM) and impression cytology (IC)., Methods: Eighty patients with glaucoma and 20 with dry eye were enrolled; 20 healthy subjects served as controls. Patients underwent the Ocular Surface Disease Index (OSDI) questionnaire, tear film break-up time, corneal staining, Schirmer test I, and LSCM of the limbus. Laser scanning confocal microscopy evaluated the limbal transition epithelium (LTE) regularity, dendritic cell (DC) density, and palisades of Vogt (POV). Impression cytology was performed and samples stained with HLA-DR and IL6., Results: Glaucomatous patients were divided into three groups: Group 1 (40 eyes): one drug; Group 2 (20): two drugs; and Group 3 (20): three or more drugs. Limbal transition epithelium regularity was worse, and DC density higher in Groups 2, 3, and dry eyes compared with Group 1 and controls (P < 0.01). Preserved drugs worsened LTE regularity and induced higher DC density compared with preservative-free (PF) drugs (P = 0.041; P = 0.004). Despite typical POV architecture was preserved, signs of inflammation were found in glaucoma groups. HLA-DR and IL-6 positivity were higher in Groups 2, 3, and dry eye compared with controls (P < 0.001), and in preserved versus PF drugs (P < 0.05; P < 0.001). Dendritic cell density and LTE regularity correlated with HLA-DR, IL-6, and OSDI score in glaucoma groups and dry eyes (P < 0.001)., Conclusions: Laser scanning confocal microscopy and IC documented antiglaucoma therapy induced morphologic alterations of limbus, which may play a role in the glaucoma-related ocular surface disease. Further studies are required to determine if limbal changes affect stem cell viability., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Aim: To investigate the features of optic nerve head (ONH) microvasculature in primary open angle glaucoma using fractal geometry analysis., Patients and Method: ONH blood flow was analyzed at the level of the lamina cribrosa by means of confocal scanning laser Heidelberg Doppler flowmetry (HRF) in medically-controlled early and advanced glaucoma. Fractal dimension D of vasculature map was calculated using the Box Counting., Results: Our data demonstrated that, in patients with advanced glaucoma, fractal dimension D was significantly lower than in controls, whereas, in the early stage of disease, its value was similar. Fractal dimension D of microcirculation was significantly and negatively correlated with the cup-disk area ratio in both early and advanced glaucoma groups, whereas linear cup-disk ratio of the disk, cup shape measure and nerve fiber layer thickness, where correlated only in advanced stage of the disease., Conclusion: These findings demonstrate that fractal dimension D of ONH appeared significantly reduced in advanced glaucoma and correlated with the optic disc damage., (Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Purpose: To examine the circadian intraocular pressure (IOP) patterns in healthy subjects, in primary open angle and normal tension glaucoma (POAG; NTG) using a contact lens sensor (CLS; Sensimed Triggerfish, Lausanne, Switzerland)., Methods: This was an observational, nonrandomized study. Ten healthy subjects (Group 1, 10 eyes) and 20 glaucomatous patients [20 eyes, 10 with POAG (Group 2) and 10 with NTG (Group 3)] were enrolled. All patients were controlled with prostaglandin analogues. The 24-hr IOP pattern was the main outcome. The morning (6AM-11AM), afternoon/evening (noon-11PM) and night (midnight-5AM) subperiod patterns, peaks and prolonged peaks (>1 hr) were secondary outcomes., Results: Mean 24-hr IOP pattern showed a nocturnal acrophase in all groups. Patterns were significantly different among groups (p = 0.02), with highest nocturnal IOP values in POAG. Prolonged peaks were more common in patients with glaucoma (70%) than in healthy subjects (33.3%) (p < 0.001). Significant differences were found for Groups 2 and 3 in the morning versus afternoon/evening (p = 0.019 and p = 0.035, Bonferroni correction), morning versus night (p = 0.005 and p < 0.0001) and afternoon/evening versus night periods comparisons (p < 0.0001 for both groups). In Group 1, patterns significantly differed in the morning versus night and afternoon/evening versus night period comparisons (p < 0.0001)., Conclusions: Continuous 24-hr IOP monitoring with the CLS revealed a nocturnal acrophase in healthy subjects and, more markedly, in glaucoma. Because the diurnal IOP profile seems not to predict the nocturnal rhythm, the circadian IOP pattern should be evaluated in clinical practice. These findings may be worthwhile for the management of glaucoma., (© 2014 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.)

Glaucoma is a multifactorial disease that is the leading cause of irreversible blindness. Recent data documented that glaucoma is not limited to the retinal ganglion cells but that it also extends to the posterior visual pathway. The diagnosis is based on the presence of signs of glaucomatous optic neuropathy and consistent functional visual field alterations. Unfortunately these functional alterations often become evident when a significant amount of the nerve fibers that compose the optic nerve has been irreversibly lost. Advanced morphological and functional magnetic resonance (MR) techniques (morphometry, diffusion tensor imaging, arterial spin labeling, and functional connectivity) may provide a means for observing modifications induced by this fiber loss, within the optic nerve and the visual cortex, in an earlier stage. The aim of this systematic review was to determine if the use of these advanced MR techniques could offer the possibility of diagnosing glaucoma at an earlier stage than that currently possible.

Glaucoma, the first cause of irreversible blindness worldwide, is a neurodegenerative disease characterized by the progressive loss of retinal ganglion cells. There are different subtypes of glaucoma, all expression of a common optic neuropathy; primary open-angle glaucoma (POAG) is the most diffuse subtype in western countries. To date, unfortunately, several questions still remain unsolved in the glaucoma management, such as the availability of powerful methods for screening high-risk populations, early diagnosis, timely detection of damage progression, and prediction of response to therapy. Over the last years, biomarkers have gained immense scientific and clinical interest to solve these issues, with countless molecules that have been candidate as potential biomarkers. In the present review, we summarize the current knowledge about the most robust molecular biomarkers proposed in POAG, distinguishing noninvasive from minimally invasive, and invasive biomarkers, according to the procedure adopted to collect fluid samples., (© 2015 Elsevier B.V. All rights reserved.)

Normal-tension glaucoma (NTG) is a multifactorial disease where mechanical stresses and vascular alterations to the optic nerve head probably represent the key pathogenic moments. Although intraocular pressure (IOP) plays a crucial role in the retinal ganglion cell loss, the IOP reduction does not necessarily reduces the disease progression. Therefore, several IOP-independent factors such as glutamate toxicity, oxidative stress, autoimmunity, and vascular dysregulation have been considered in the pathogenesis of NTG. Numerous evidences documented an impairment of the ocular blood flow, involved both in the onset and progression of the disease. The IOP reduction remains the main strategy to reduce the damage progression in NTG. Recently, new treatment strategies have been proposed to improve the control of the disease. Neuroprotection is a rapidly expanding area of research, which represents a promising tool. In the present review, we summarize the recent scientific advancements in the pathogenesis and treatment of NTG., (© 2015 Elsevier B.V. All rights reserved.)

Purpose: To evaluate the effects of intravitreal ranibizumab monotherapy on predominantly hemorrhagic choroidal neovascularization with foveal involvement associated with age-related macular degeneration., Materials and Methods: Twenty-two consecutive eyes with hemorrhagic neovascularization were treated with 3 monthly intravitreal ranibizumab injections. Additional injections were administered according to retreatment criteria during 12 months of follow-up., Results: A mean of 6.64 ± 1.36 injections was administered. Overall, the mean visual acuity increased from 10.90 ± 6.02 to 12.81 ± 8.34 ETDRS letters (p > 0.05) at 12 months. The 'early treatment group' gained a mean of 2.83 ± 2.24 ETDRS letters (p < 0.05), while the 'late treatment group' gained a mean of 0.30 ± 1.25 ETDRS letters (p > 0.05) with significant differences between the groups (p < 0.05). A progressive resolution of macular bleeding was registered in 20 patients (mean time: 5.3 ± 1.6 months)., Conclusions: Ranibizumab injections can be considered a beneficial approach for the management of predominantly hemorrhagic choroidal neovascularization with foveal involvement associated with age-related macular degeneration. Furthermore, the time interval between hemorrhage and the first injection seems to be an important predicting factor of final visual acuity., (© 2014 S. Karger AG, Basel.)

Purpose: To analyze, using in vivo laser scanning confocal microscopy (LSCM), the conjunctival features in glaucomatous patients receiving prostaglandin analogues (PGA)., Methods: Eighty eyes of 80 consecutive glaucomatous patients naive for therapy were enrolled; 30 eyes of 30 healthy subjects served as a control. Patients were randomized to: preservative-free (PF) and preserved latanoprost (groups 1 and 2, respectively), PF and preserved timolol (groups 3 and 4), and controls to vehicle of latanoprost or physiological buffered saline solution (groups 5 and 6). All subjects underwent LSCM of bulbar conjunctiva at baseline and 3 months after initiating therapy. The main outcomes were: mean density (MMD: cysts/mm(2)) and mean area (MMA: cysts/mm(2)) of epithelial microcysts. The relations between MMA and MMD with intraocular pressure (IOP), age, and mean defect (MD), were analyzed., Results: At baseline, microcysts were found in all subjects. At month three, MMD did not change in all groups (p > 0.05). MMA significantly increased only in group 1 from 2,158.81 ± 524.09 to 3,877.77 ± 867.31, and in group 2 from 2,019.71 ± 541.03 to 5,560.39 ± 1,176.14, with values significantly higher in group 2 (p < 0.001). Significant relations were not found between MMD and MMA with IOP, MD, and age (p > 0.05)., Conclusions: PGA increased MMA in therapy-naive glaucomatous patients, indicating a possible enhancement of the trans-conjunctival aqueous humor outflow. Therefore, conjunctiva seems an additional target tissue to evaluate the hydrodynamic pathways in glaucoma and modifications induced by medical therapy.

Purpose: To investigate modifications with aging of the presence, distribution and morphologic features of conjunctiva-associated lymphoid tissue (CALT) in healthy human subjects using laser scanning in vivo confocal microscopy (IVCM)., Methods: A total of 108 (age range, 17-75 years) subjects were enrolled. In vivo confocal microscopy of the tarsal and bulbar conjunctiva, and impression cytology (IC) with CD3 (intra-epithelial T-lymphocytes) and CD20 (intra-epithelial B-lymphocytes) antibody immunofluorescence staining were performed. The main outcomes were subepithelial lymphocyte density (LyD), follicular density (FD), and follicular area (FA). The secondary outcomes were follicular reflectivity (FR), and lymphocyte density (FLyD), and CD3 and CD20 positivity., Results: Conjunctiva-associated lymphoid tissue was observed in all subjects (97% only superior and 3% in both superior and inferior tarsum). Lymphocyte density ranged from 7.8 to 165.8 cells/mm(2) (46.42 [18.37]; mean [SD]), FD from 0.5 to 19.4 follicles/mm(2) (5.3 [3.6]), and FA from 1110 to 96,280 mm(2) (26,440 [26,280]). All three parameters showed a highly significant inverse cubic relationship with age (P < 0.001); that is, in the first and last parameters a steep decline up to 35 years and above 65 years of age, with a plateau phase between these ages, whereas FA had a gradually decreasing rate of loss over the studied age range. CD3 and CD20 IC were consistent with these results., Conclusions: In vivo confocal microscopy was effective in revealing CALT and modifications these structures undergo with aging. Aging correlated with an involution of all parameters defining lymphoid structures. These modifications may account for the decrease of mucosal immune response and increase of ocular surface diseases in the elderly., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Over the past decade, knowledge about the ocular surface in glaucoma has significantly increased through the use of in vivo laser scanning confocal microscopy (LSCM). This in vivo imaging method can show modifications at the cellular level induced by anti-glaucoma drugs on ocular surface structures and adnexa in the eye. High-quality images of the conjunctiva, cornea, limbus, meibomian glands, and lymphoid structures during therapy can be obtained. In addition, LSCM opened new fields of research on the patho-physiology of aqueous humor (AH) hydrodynamics in untreated, and in medically or surgically treated glaucomatous patients. In these conditions, an enhancement of the trans-scleral AH outflow contributed to clarification of the mechanism of action of different anti-glaucoma medications and surgical approaches. Finally, the use of LSCM represented a huge advance in evaluation of bleb functionality after filtration surgery, defining the hallmarks of AH filtration through the bleb-wall and distinguishing functional from nonfunctional blebs. Thus, signs seen with LSCM may anticipate clinical failure, guiding the clinician in planning the appropriate timing of the various steps in bleb management. In this review we summarize the current knowledge about in vivo LSCM of the ocular surface in glaucoma.

Time domain (TD) and spectral domain (SD) optical coherence tomography (OCT) are cross-sectional, noncontact, high-resolution diagnostic modalities for posterior and anterior segment (AS) imaging. The AS-OCT provides tomographic imaging of the cornea, iris, lens, and anterior chamber (AC) angle in several ophthalmic diseases. In glaucoma, AS-OCT is utilized to evaluate the morphology of AS structures involved in the pathogenesis of the disease, to obtain morphometric measures of the AC, to evaluate the suitability for laser or surgical approaches, and to assess modifications after treatment. In patients undergoing surgery, AS-OCT is crucial in the evaluation of the filtering bleb functionality, permitting a combined qualitative and quantitative analysis. In this field, AS-OCT may help clinicians in distinguishing between functioning and nonfunctioning blebs by classifying their macroscopic morphology, describing bleb-wall features, bleb cavity, and scleral opening. This information is critical in recognizing signs of filtration failure earlier than the clinical approach and in planning the appropriate timing for management procedures in failing blebs. In this review, we summarize the applications of AS-OCT in the conjunctival bleb assessment.

Purpose: To evaluate the density of conjunctival goblet cells (GCs) in glaucomatous patients treated with preservative-free (PF) tafluprost, using laser scanning confocal microscopy (LSCM) and impression cytology (IC)., Methods: Thirty glaucomatous patients (30 eyes) naive for therapy and thirty healthy subjects (30 eyes) were enrolled. Conjunctiva was examined by means of Heidelberg Retina Tomography/Rostock cornea module. Afterwards, the specimens for IC were obtained. Patients were randomized to PF-tafluprost (Group 1) or preserved latanoprost (Group 2) and controls to the vehicle of latanoprost (Group 3) or physiological buffered saline solution (Group 4). Both LSCM and IC were performed at baseline, and after the 1st and 6th months of therapy, GC density (GCD) (cells/mm(2) ) was the main outcome measurement., Results:   Baseline. Mean GCD was 240.69 ± 25.43 and 232.65 ± 23.52, for LSCM, and 162.10 ± 23.44 and 164.71 ± 21.03 for IC in Group 1 and 2, respectively. GC density values were not significantly different in Group 3 and 4 (p > 0.05%). Month one. Mean GCD increased to 284.16 ± 43.88 and 230.62 ± 48.32 in Group 1 (p < 0.001) and to 297.86 ± 26.87 and 221.78 ± 43.02 in Group 2 (p < 0.05), measured with LSCM and IC, respectively. In Group 3, GCD decreased to 205.88 ± 25.04 and 139.54 ± 17.37 measured with LSCM and IC, respectively (p < 0.05). Month six. Mean GCD did not change in Group 1 (p > 0.05) whereas it decreased in Group 2 (p < 0.05), compared to month 1. In Group 3, GCD further decreased to 166.32 ± 22.31 and 120.76 ± 11.66, measured with LSCM and IC, respectively (p < 0.05); in Group 4, mean GCD did not change during the study period (p > 0.05)., Conclusions: Treatment with PF tafluprost was associated with an increase in conjunctival GCD in glaucomatous eyes naïve for therapy. Further studies are mandatory to verify this finding because its validation may have important consequences in the medical management of glaucoma., (© 2013 Acta Ophthalmologica Scandinavica Foundation. Published by Blackwell Publishing Ltd.)

Aim: To investigate, using laser scanning confocal microscopy (LSCM), the morphological changes of meibomian glands (MGs) in patients with glaucoma., Methods: A total of 80 patients who were glaucomatous were enrolled, and 20 healthy subjects were used as controls. After completing an Ocular Surface Disease Index (OSDI) questionnaire, all subjects underwent tear film break-up time (BUT), fluorescein staining, Schirmer test I (STI) and LSCM examination of the MGs. The main outcome measures were: eyelid margin epithelial cell density, mean acinar density (MAD) and area (MAA), glandular orifice area, secretion reflectivity and inhomogeneous appearance of interstice and acinar wall., Results: According to the number of anti-glaucoma medications they were taking, patients were divided into three groups: group 1 (30 eyes), one drug; group 2 (23 eyes), two drugs; group 3 (27 eyes), three or more drugs. LSCM showed lower MAD and MAA, greater secretion reflectivity and glandular orifice area in groups 2 and 3 than in controls (p<0.05). The inhomogeneity of the interstice and acinar wall was significantly greater in all groups compared to controls (p<0.05). Preserved prostaglandin analogues (PGAs) induced more pronounced modifications of all parameters than preservative free (PF)-PGAs (p<0.05). No significant differences were found between preserved and PF-β-blockers. Significant relations were found among MAD, MAA, secretion reflectivity and OSDI score, BUT and ST (p<0.05) and between secretion reflectivity and orifice area (p<0.001)., Conclusions: In vivo LSCM is an effective tool in revealing morphological changes of MGs induced by anti-glaucoma medications. Given the key role in the ocular surface health, the evaluation of MG status in patients who are glaucomatous is worthwhile.

Lowering intra-ocular pressure, either medically or surgically, is the proven strategy to control glaucoma, though profound changes to the ocular surface and conjunctiva are caused. Toxicity and allergy initiated by medical therapy induce modifications, which progressively worsen with the length of treatment and number of drugs. Conjunctival changes lead to symptoms of ocular surface disease, reduced quality of life, reduced therapeutic compliance and increased risk of surgical failure. Surgery modifies conjunctiva by inducing bleb formation in fistulizing techniques, and by activating secondary aqueous humour outflow pathways, such as trans-scleral routes, in both filtration and bleb-less approaches. The use of unpreserved medications, limitation of intra-operative conjunctival damage and development of bleb-less surgery are advisable., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: The consistent association between choroid neovascularization (CNV) and increased VEGF-A expression provides a strong reason for exploring the therapeutic potential of anti-VEGF agents in the treatment of neovascular age-related macular degeneration (AMD). The authors report the systemic side effects secondary to intravitreal administration of these compounds, that is, the main cardiovascular effects, as well as the less frequent cerebrovascular accidents, myocardial infarction, transient ischemic attacks, deep vein thrombosis, pulmonary embolism and thromboflebitis., Areas Covered: The authors reviewed major Clinical Trials and publications concerning systemic adverse events of anti-VEGF drugs in order to identify the main thromboembolic events related to the use of these agents and their occurrence. Anti-VEGF efficacy, safety and tolerability are also discussed., Expert Opinion: Three compounds (pegaptanib, ranibizumab and aflibercept) have been approved for the treatment of AMD; a fourth agent, bevacizumab, is used off-label. Anti-VEGF therapy has not shown the ability to fully eradicate the CNV, so that recurrences are common when the intravitreal injections are suspended. Although no evident rise in anti-VEGF-induced thromboembolic side effects was reported, more data are required to evaluate hemodynamic and pharmacokinetics of these compounds. Since only few studies have focused on these aspects, further researches are mandatory to determine distribution, effects and duration of these substances.

Aim: To assess the epithelial features of the bulbar conjunctiva using in vivo confocal microscopy in patients with glaucoma undergoing canaloplasty., Methods: Thirty consecutive patients with glaucoma were enrolled. Canaloplasty was considered successful if the preoperative intraocular pressure (IOP) was reduced by one-third. The conjunctiva was examined using confocal laser-scanning microscopy 1 week before (baseline) and 12 weeks after surgery. The mean density (MMD, cysts/mm(2)) and mean area (MMA, μm(2)) of conjunctival microcysts and IOP were measured. Anterior segment optical coherence tomography was performed to evaluate post-operative trabecular distension and scleral modifications at the surgery site., Results: Twelve weeks after surgery, canaloplasty was successful in 23 patients (group 1) but unsuccessful in 7 (group 2). At baseline, IOP was 28.1±2.98 and 28.3±2.81 mm Hg, MMD was 10.61±4.31 and 11.35±5.6 and MMA was 2845.02±411.85 and 2700.56±518.85 in groups 1 and 2, respectively (p>0.05). Twelve weeks after canaloplasty, mean IOP was 13.2±4.48 (p<0.05) and 24.6±3.48 mm Hg in groups 1 and 2, respectively. In group 1, MMD and MMA were 37.86±21.4 and 11997.84±8630.35, respectively, a fourfold increase compared to baseline (p<0.001); no significant differences were found in group 2. Conjunctival bleb was not documented in any case., Conclusions: Conjunctival microcysts were evident in all glaucomatous eyes prior to surgery, and tended to increase in density and surface area after successful canaloplasty. These findings indicated enhanced aqueous humour filtration across the sclera and conjunctiva after canaloplasty.

Primary open angle (POAG) and pseudoexfoliative glaucoma (PXG) are the most common primary and secondary forms of glaucoma, respectively. Even though the patho-physiology, aqueous humor composition, risk factors, clinical features, therapy and drug induced ocular surface changes in POAG and PXG have been widely studied, to date information concerning tear protein characterization is lacking. Tears are a source of nourishment for ocular surface tissues and a vehicle to remove local waste products, metabolized drugs and inflammatory mediators produced in several ophthalmic diseases. In glaucoma, the proteomic definition of tears may provide insights concerning patho-physiology of the disease and ocular surface modifications induced by topical therapy. Our study aimed at characterizing protein patterns in tears of patients with medically controlled POAG and PXG. A comparative tears proteomic analysis by label-free LC-MS(E) highlighted differences in the expression of several proteins in the two glaucoma sub-types and control subjects, highlighting inflammation pathways expressed in both diseases. Results were independently reconfirmed by SDS-PAGE and linear MALDI-TOF MS, validating altered levels of Lysozyme C, Lipocalin-1, Protein S100, Immunoglobulins and Prolactin Inducible Protein. Moreover, we found a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies. The most relevant results suggest that in both pathologies there may be active inflammation pathways related to the disease and/or induced by therapy. We show, for the first time, tear protein patterns expressed under controlled intraocular pressure conditions in POAG and PXG subjects. These findings could help in the understanding of molecular machinery underlying these ophthalmologic diseases, resulting in early diagnosis and more specific therapy.

Purpose: To analyse the epithelial features of the bulbar conjunctiva in hyperbaric and low-tension glaucoma (LTG) using in vivo confocal microscopy (IVCM)., Methods: Thirty-six eyes of 36 patients [18 affected by primary open-angle glaucoma (POAG) and 18 with LTG] were studied; control group was constituted by 28 eyes of 28 healthy subjects. All eyes were examined using digital confocal laser-scanning microscopy (HRT II Rostock Cornea Module). The main IVCM outcome measurements were mean density (MMD: cysts/mm(2)) and mean total area (MMA: μm(2)) of the epithelial microcysts., Results: The mean intraocular pressure level (mmHg ± SD) was 15.1 ± 1.7, 16.3 ± 3.1 and 12.6 ± 1.8 in healthy, POAG and LTG eyes, respectively. Conjunctival microcysts were found in all patients and subjects: for healthy subjects, MMD = 10.9 ± 11.1 cysts/mm(2) and MMA = 1501.9 ± 1191.1 μm(2); for patients infected with POAG, MMD = 36.8 ± 28.6 cysts/mm(2) and MMA = 7904.8 ± 7050.5 μm(2); and for patients infected with LTG MMD = 45.6 ± 29.0 cysts/mm(2) and MMA =7946.9 ± 5227.5 μm(2). MMD and MMA were not significantly different between patients infected with POAG and those with LTG, whereas they were significantly greater in patients (fourfold and fivefold, respectively) than healthy subjects., Conclusions: The present study demonstrated that conjunctival microcysts represent an in vivo feature in all eyes with medically controlled POAG and LTG. Therefore, conjunctiva deserves careful analysis, because its accurate microscopic definition could help clarify the pathophysiology of aqueous outflow in glaucoma., (© 2011 The Authors. Acta Ophthalmologica © 2011 Acta Ophthalmologica Scandinavica Foundation.)

Purpose: To assess the reproducibility and repeatability of peripapillary retinal nerve fibre layer (RNFL) thickness measurements using a spectral-domain optical coherence tomography (SD-OCT) device in healthy subjects., Methods: In this observational study, 68 young Caucasian healthy volunteers (68 eyes) were subjected to Cirrus™ high-definition (HD) OCT (Zeiss) peripapillary RNFL thickness measurements by two experienced examiners in two different sessions. Average, 4-quadrant and 12-clock-hour sector RNFL thicknesses were analysed. For each option, intra-observer, intrasession repeatability and interobserver, intersession reproducibility were tested. To assess the repeatability of measurements, the Bland and Altman plots were used and the coefficient of repeatability was calculated. Interobserver and intersession reproducibilities were analysed by means of concordance correlation coefficients (CCCs)., Results: The sample age ranged from 21 to 39 years (mean 29.09, standard deviation ±5.21). The average RNFL thickness ranged from 90.97 to 91.46 and from 91.34 to 91.78 μm, for the first and the second operator, respectively. The highest repeatability and reproducibility were obtained for average RNFL thickness with coefficients of repeatability of 5.30 and 6.05 μm for the first and the second operator, interoperator CCCs of 0.95 and 0.96 for the first and the second session, and intersession CCCs of 0.96 and 0.97 for the first and the second operator, respectively., Conclusions: Cirrus OCT peripapillary average RNFL thickness measurement in young healthy subjects showed high interoperator and intersession reproducibility. Intrasession repeatability as tested by coefficient of repeatability was next to the device resolution, with very similar results between the two operators. When analysing quadrant and clock hour sector RNFL thickness measurements, both repeatability and reproducibility tend to decrease., (Copyright © 2012 S. Karger AG, Basel.)

Purpose: To report the conjunctival and corneal findings in delayed onset glaucoma filtering bleb-associated endophthalmitis (BAE), by using in vivo confocal microscopy (IVCM)., Methods: This was an observational case series. Four eyes of four glaucomatous patients who previously underwent mytomicin C augmented filtering surgery and affected with delayed onset BAE, underwent IVCM of conjunctival bleb and cornea at diagnosis, after 2 and 8 weeks of therapy. The inflammatory status of the conjunctival epithelium and sub-epithelium was microscopically investigated. Corneal epithelial cells, stromal and endothelial morphology were also evaluated. A group of eight patients with functioning conjunctival filtering bleb was used as control., Results: At diagnosis, a diffuse inflammatory cell infiltration within the conjunctival epithelium presenting evident microcysts was found; conversely, there were no such alterations in the sub-epithelium. An evident stromal oedema, keratocytes activation and diffuse endothelial inflammatory precipitates were the major corneal hallmarks. After 2 weeks of therapy, besides a remarkable improvement of epithelial inflammation and an evident reduction in endothelial precipitates, dendritic cells appeared within conjunctival sub-epithelium and corneal epithelium showed aspects of cellular disruption. After 8 weeks, the conjunctival and corneal features consistently improved, except for the endothelium which still presented high-reflective residual precipitates., Conclusions: In vivo confocal microscopy proved valuable in the analysis of conjunctival bleb and cornea in patients affected with delayed onset BAE, permitting an evaluation of the course of the disease, the response to therapy and the modulation of dose regimen., (© 2009 The Authors. Journal compilation © 2009 Acta Ophthalmol.)

Purpose: The study was conducted to analyze, by in vivo confocal microscopy (IVCM), the conjunctival epithelial characteristics in untreated ocular hypertension (OH) and in topically treated primary open-angle glaucoma (POAG)., Methods: The study included 30 eyes affected with untreated OH, 96 eyes with POAG receiving medical therapy, and 15 healthy control eyes. The main outcome measures were the mean density and the mean area of conjunctival epithelium microcysts. The relations among the microscopic parameters intraocular pressure (IOP), and age in both hypertensive and glaucomatous eyes and between mean defect (MD) of visual fields and the time on therapy in patients with glaucoma were analyzed., Results: There was no evidence of conjunctival microcysts in any of the healthy eyes examined; conversely, conjunctival microcysts were found in all ocular hypertensive eyes (mean microcyst density of 19.7 +/- 3.5 cysts/mm(2) and mean total microcyst area of 4063.6 +/- 921.2 mum(2)). All patients with POAG showed conjunctival microcysts (mean density of 28.7 +/- 2.7 cysts/mm(2) and a mean total microcyst area of 6564.2 +/- 671.4 mum(2)). No significant differences were found between OH and POAG subjects for microcyst parameters and no significant relations were found in either OH or POAG eyes for microcyst density, area, IOP, MD, and time on therapy., Conclusions: The results of the study show that conjunctival microcysts are features present in all hypertensive and glaucomatous eyes. Based on these findings, conjunctiva could be an additional potential target tissue available for the investigation by a noninvasive in vivo approach of glaucoma-induced pathologic modifications.

Purpose: To provide a microscopic and macroscopic analysis by a clinical, in vivo confocal microscopy (IVCM) and anterior segment optical coherence tomography (AS-OCT) assessment of filtering blebs after glaucoma surgery., Material and Methods: We retrospectively analyzed 60 eyes of 48 glaucomatous patients by slit-lamp, IVCM, and AS-OCT examinations. Patients were divided into 2 groups: successful blebs (group 1, 16 patients, 27 eyes) were defined as a one-third reduction in preoperative intraocular pressure without antiglaucoma medications and failed blebs (group 2, 32 patients, 33 eyes) as a less than one-third reduction in preoperative intraocular pressure without therapy. The examinations were performed from 1 to 96 months postoperatively., Results: Diffuse or cystic clinical patterns were indicative for good functionality whereas flat or encapsulation was indicative for poor functionality. When comparing successful with failed blebs, the IVCM analysis showed a greater number (P=0.014), density (P=0.009), and total area of epithelial microcysts (P=0.017) and a lower density of connective tissue (P=0.006). The AS-OCT analysis showed a lower degree of bleb wall reflectivity (P<0.001). A significant correlation was found between the clinical and AS-OCT parameters, particularly for the cystic (100%) and diffuse (74%) patterns., Conclusions: All IVCM parameters did correlate well with the bleb functionality whereas, among the AS-OCT parameters, only the bleb wall reflectivity was significantly related to the filtering capability. Clinical and AS-OCT bleb classification showed a significant degree of concordance. As a consequence, simultaneous approach by clinical, microscopic, and tomographic assessment improves the clinician's ability in the postsurgery understanding and management of blebs.