Microsc. Microanal. 20, 879–894, 2014 doi:10.1017/S1431927614000324

© MICROSCOPY SOCIETY OF AMERICA 2014

REVIEW ARTICLE In Vivo Laser Scanning Confocal Microscopy of the Ocular Surface in Glaucoma Leonardo Mastropasqua,1,*,a Luca Agnifili,1,a Rodolfo Mastropasqua,2 Vincenzo Fasanella,1 Mario Nubile,1 Lisa Toto,1 Paolo Carpineto,1 and Marco Ciancaglini3 1

Ophthalmic Clinic, Department of Medicine and Aging Science, University G. d’Annunzio of Chieti-Pescara, Chieti, 66100, Italy Ophthalmology Unit, Department of Neurological, Neuropsychological, Morphological and Movement Sciences, University of Verona, Verona, 53593, Italy 3 Ophthalmic Clinic, Department of Surgical Science, University of L’Aquila, L’Aquila, 67100, Italy 2

Abstract: 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. Key words: in vivo confocal microscopy, glaucoma, ocular surface, conjunctiva, cornea, sclero-corneal limbus, meibomian glands, CALT, filtering bleb

I NTRODUCTION Until recently, understanding of the fine anatomy of the ocular surface was derived from light and electron microscopy studies of excised tissue samples. Additional information was gathered using impression cytology (IC), in which superficial cells were removed from the ocular surface (Nelson, 1988). Over the past two decades, knowledge about the ocular surface has greatly increased through the use of in vivo confocal microscopy (IVCM) (Mathers et al., 1995; Mastropasqua & Nubile, 2002; Labbè et al., 2005; Zhivov et al., 2006; Messmer et al., 2006a; Erie et al., 2009; Wang et al., 2011). At present, IVCM can simultaneously provide information on all tissue of the ocular surface, allowing an integrated high-resolution evaluation, and an analysis of the overall behavior of the morpho-functional unit in normal and pathological conditions (Villani et al., 2013a, 2013b, 2013c, 2013d). Thus, confocal microscopy describes physiological tissue modifications during aging, peculiar anatomopathological features in ocular surface

Received October 25, 2013; accepted January 22, 2014 *Corresponding author. [email protected] a L.M. and L.A. equally contributed to this work

diseases and their natural history, and permits an evaluation of the response to therapy. By performing a coronal optical section through the examined tissue, IVCM can provide detailed micro-structural information of the ocular surface and adnexa at the cellular level, in many diseases of the anterior segment of the eye. Confocal microscopy utilizes several principles: tandem scanning, slit scanning, and laser scanning confocal microscopy (LSCM). White light tandem- and slit-scanning confocal microscopes analyze only transparent structures, such as the cornea (Erie et al., 2009). LSCM, which couples the Rostock Cornea Module (RCM) with the Heidelberg Retina Tomograph (HRT II and III; Heidelberg Engineering GmbH, Heidelberg, Germany), represents a major advance compared to the other confocal microscopes since it is capable of also imaging translucent and semi-opaque structures. For these reasons LSCM is currently the most frequently used confocal microscope in ophthalmology for both research purposes and in daily clinical practice (Zhivov et al., 2006). In past years, the application of IVCM has progressively extended to also include glaucoma. In this pathology it was used to evaluate the anatomy and functionality of conjunctival blebs after filtration surgery (Labbè et al., 2004, 2005; Messmer et al., 2006b), and to describe the effects of

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therapy on ocular surface tissues and the patho-physiology of the uveo-scleral aqueous humor (AH) outflow (Ciancaglini et al., 2008a; Mastropasqua et al., 2013a, 2013b). In this review, we summarize current knowledge about in vivo LSCM of the ocular surface in glaucoma and possible applications of this method in routine clinical practice.

M ETHODOLOGY AND RESULTS PubMed searches were performed on September 20, 2013 using the following phrases: “in vivo confocal microscopy”, “ocular surface,” and glaucoma, which identified nine unique publications and ‘‘in vivo confocal microscopy” and glaucoma, which identified 66 unique publications, including those found in the initial search. Thirty-one were excluded, since they did not concern the use of IVCM of the ocular surface in glaucoma. An additional search was conducted using the phrase: “in vivo confocal microscopy” and “ocular surface,” which identified two additional glaucoma articles not found with the previous two searches. Thus, a total of 37 articles analyzing the application of IVCM in glaucoma were included in this review. Three of the considered publications were not published in English (in French, Chinese, and Polish); however, they provided enough information in the English abstract to warrant inclusion.

TECHNICAL CHARACTERISTICS

OF

LSCM

LSCM incorporates the laser scanning optics of the HRT (I–III) with the RCM, a detachable objective system, for imaging the ocular surface and adnexa (Stave et al., 2002). Using laser light at a wavelength of 670 nm, high-contrast and high-quality images are generated. The 384 × 384 pixel images cover an area of 400 × 400 µm, with transversal optical resolution of 2 µm and longitudinal optical resolution of 4 µm (Heidelberg Engineering). Numerous types of information (both quantitative and qualitative) can be obtained when the system is coupled with image analysis software such as RCM (RCM cell count plugin for Image-J software; http://rsb.info.nih.gov/ij/download.html). The optical section of the microscope can be manually advanced through the full thickness of the tissue, but automated control of the optical section is only possible through a depth of 80 µm.

LSCM

OF THE

CONJUNCTIVA

Untreated Ocular Hypertension (OH) In a pioneer study analyzing the bulbar conjunctiva in patients with untreated OH, Ciancaglini et al. (2008a) observed structures within the epithelial layers (20–30 µm depth), named epithelial microcysts (EM) (Fig. 1a). These microcysts were first described within the bleb epithelium after successful filtration surgery and were interpreted as expression of the AH passage through the bleb wall (Labbè et al., 2004, 2005; Messmer et al., 2006b). In this case, microcysts appeared as round- or oval-shaped extra-cellular

structures, ranging from 10 to 200 µm in size, sometimes confluent or clustered, and with an optically clear aspect due to the low internal reflectivity. Occasionally, the inner cavity was hyperreflective due to the accumulation of amorphous material or inflammatory cells (Messmer et al., 2006b). In OH, microcysts were smaller than those observed after filtering surgery (10–90 µm), quite regularly distributed within the epithelium, and without a tendency of clustering. The presence of EM in patients with OH was hypothesized to be an adaptive mechanism in eyes with reduced trabecular outflow, aimed at increasing the AH movement through the sclera. Interestingly, intra-ocular pressure (IOP) did not show a significant correlation with microcyst density and area, which suggested that microcyst formation could be promoted by IOP-related inner mechanical forces only in the very early stages of the disease (Toris et al., 2002). In the chronic phase, the process remains active even if medical therapy is effective in reducing IOP. However, conjunctival microcysts were also occasionally reported in nonglaucomatous ocular conditions such as pterygium, Sjogren’s syndrome, and contact lens wearing (Guthoff et al., 2006; Efron et al., 2010; Wakamatsu et al., 2010). In these conditions, EM were probably features of degenerative processes occurring within the epithelium, when disturbances in cellular maturation created debris field cystic spaces (Wakamatsu et al., 2010). Notably, microcysts were also reported in healthy subjects (Messmer et al., 2006a; Efron et al., 2009a), suggesting that they are not exclusive hallmarks of ocular diseases. Zhu et al. (2010) analyzed different age groups of healthy subjects (from 14 to 73 years), observing EM in all cases with an increase in number with age. These results were in accordance with a further study (Agnifili et al., 2012a) in which EM were observed in both glaucomatous and healthy subjects (Figs. 1b, 1c, respectively). Interestingly, in this study, mean microcyst density (MMD) and area (MMA) were four and five times higher in patients with primary open angle glaucoma (POAG) and low-tension glaucoma (LTG), respectively, compared with healthy subjects. The presence of these structures in healthy eyes could correspond to degenerated goblet cells (GC), occluded GC with retention of their contents, or normal intermediate products that result from cellular development and maturation (Messmer et al., 2006a; Efron et al., 2009a; Zhu et al., 2010). Nevertheless, none of these studies provided information about the IOP of the enrolled subjects. Differently, Agnifili et al. (2012a) proposed that EM in normal eyes could be a sign of physiological trans-scleral (TS) aqueous percolation, which normally occurs as the final phase of the uveo-scleral pathway. Therefore, EM may have more than one implication: it may be a sign of the physiologic TS-AH outflow or an aspect of the GC maturation in healthy conditions, a hallmark of the TS outflow enhancement in the presence of OH or glaucoma, and a sign of epithelial disruption in patients with ocular surface diseases.

In Vivo Confocal Microscopy in Glaucoma

Figure 1. a: Planar reconstruction of the superior bulbar conjunctiva in a patient with OH, showing the presence and distribution of microcysts within the intermediate layers of the epithelium. The final map was created by manually juxtaposing images obtained by consecutively moving the RCM from the nasal to the temporal region (right and left side of the image, respectively) and from the limbus to the upper fornix (bottom and top of the image; from Ciancaglini et al., 2008a with permission from the publisher). b: Planar reconstruction of the superior bulbar conjunctiva in a glaucomatous eye under maximal tolerated medical therapy (three drugs) showing several EM, similar to those described in OH. Arrows indicate a conjunctival fold (from Ciancaglini et al., 2009, with permission from the publisher). c: Distribution and features of EM in a healthy eye showing scattered microcysts within the epithelium, with an evident lower density and area compared to patients with impaired ocular hydrodynamics. (from Agnifili et al., 2012a, with permission from the publisher). Scale bar is expressed in micrometers. OH, ocular hypertension; RCM, Rostock Cornea Module; EM, epithelial microcysts.

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Untreated Open Angle Glaucoma In unpublished data, currently under peer review, we also observed EM in patients with therapy-naïve POAG, with MMD and MMA values three times higher than healthy subjects. Also in this case, the presence of EM may be intended as an adaptive mechanism aimed at increasing alternatively the AH outflow, when the outflow resistance within the physiological pathway is increased. This is probably why the morphological characteristics of microcysts were not different from those observed in OH and in healthy subjects.

Medically Treated Open Angle Glaucoma In medically treated glaucomatous patients, the conjunctiva may present modifications induced by changes in AH hydrodynamics (limited to the epithelium), and alterations directly induced by medications (extending from the epithelium to the stroma). Changes in AH Hydrodynamics Conjunctival epithelium generally shows EM, which also in this case may be considered an in vivo hallmark of the TSAH outflow enhancement. Ciancaglini et al. (2008a) reported that MMD and MMA did not significantly differ between untreated OH and medically controlled glaucoma. They found that the stage of disease and the medical therapy did not affect the microcyst formation process. However, the analysis of the relationship between therapy and conjunctival microcyst parameters produced conflicting results. Eyes treated with unfixed combination therapy [ß-blocker/ prostaglandin analog (PGA)] showed a significantly greater MMD and larger MMA compared to eyes on single drug therapy (preserved PGA or preserved ß-blocker). Thus, unfixed combination therapy seemed to enhance the TS-AH outflow to a greater extent than single drug therapy. Even though the presence of EM in untreated OH does not support the postulation that the preservative [benzalkonium chloride (BAK)]- induced microcyst formation, greater MMD and MMA were observed in patients treated with a combination therapy. Besides, a BAK-induced epithelial disruption, a direct effect of BAK on the scleral architecture and, therefore, on AH outflow (Okabe et al., 2005), or a greater drug penetration and a higher efficacy may be hypothesized. This latter hypothesis was supported by the results of a recent study (unpublished, currently under peer review) in which the effects of preserved and preservative-free (PF)-latanoprost on AH outflow in therapy-naïve glaucomatous patients were evaluated. Both BAK preserved- and PF-latanoprost significantly increased microcyst area after 3 months of therapy, whereas ß-blockers did not. These findings suggested that the higher MMA found in patients treated with preserved latanoprost may account for a higher drug efficacy on AH outflow rather than a direct effect of BAK on the sclera or conjunctiva. Agnifili et al. (2012a) reported that EM were present also in patients with medically controlled LTG. A significant

difference between POAG and LTG was not found, implying the existence of a similar mechanism leading to the TS outflow enhancement, albeit the two types of glaucoma differ in that IOP falls always within the normal range in LTG. Table 1 presents an overview of EM MMD and MMA in healthy subjects, and in patients with OH or open angle glaucoma. Even though EM presented a higher interindividual variability, and a direct comparison is not possible, it was evident that these parameters were markedly greater in the presence of impaired ocular hydrodynamics. Effects of Anti-Glaucoma Medications Anti-glaucoma medications induce several conjunctival changes, which may affect all tissue layers. Both preservatives and active compounds were responsible, even though preservatives played the main role (Mastropasqua et al., 2013a). While toxicity or Th-2-mediated allergic reactions account for alterations produced by BAK (Baudouin et al., 2008), inflammation was responsible for changes induced by active compounds (Baudouin et al., 2004; Liang et al., 2008). Epithelial Modifications The most common epithelial modifications are represented by squamous metaplasia, desquamation, keratinization, thickening, dendritic cell (DC) activation, and GC loss (Mastropasqua et al., 2013a). Figure 2 shows the most common epithelial modifications seen with LSCM. These modifications were more pronounced in patients treated with a combination of three or more drugs, in patients receiving preserved formulations, and in long-term therapy (from 8 months to more than 10 years of treatment; Hong et al., 2006; Baudouin et al., 2010; Pauly et al., 2012; Mastropasqua et al., 2013a). Ciancaglini et al. (2008b) used LSCM and IC to evaluate effects induced by initiating preserved and PF levobunolol hydrochloride 0.5% on bulbar conjunctival epithelium in therapy-naïve glaucomatous patients. Large epithelial cells with irregular nuclei, reduction of intercellular cohesiveness, and phenomena of disruption after 6 months of therapy were mainly observed in patients receiving preserved levobunolol. These results highlighted the primary role of BAK in inducing alterations of the superficial epithelium. Frezzotti et al. (2013) recently confirmed these findings in a 12-month study comparing the effects of preserved timolol with a PF timolol 0.1% gel formulation. In long-term therapy (>2 years) the superficial epithelium presented large and polygonal cells with hyperreflective borders, features of polymorphism and polymegathism, and evident signs of metaplasia (Ciancaglini et al., 2008a, 2009). In addition, activated DC within the epithelium and the basement membrane were frequently observed (Mastropasqua et al., 2013a). These findings support the concept that immune-related inflammation plays an important role in drug-induced conjunctival alterations. GC were the main source of ocular surface mucoproteins and contribute to tear film stability (Doughty & Bergmanson, 2003). These cells were extremely sensitive to several irritating

a = Ciancaglini et al. (2008a). b = Agnifili et al. (2012a). c = unpublished data. *Preserved prostaglandin analogs. †ß-blockers users. §Unfixed combination therapy (ß-blockers/prostaglandin analog). ‡Untreated. MMD, mean microcysts density; MMA, mean microcysts area; OH, ocular hypertension; POAG, primary open angle glaucoma; LTG, low-tension glaucoma; NA, not applicable; NP, not provided.

0 10.9 ± 11.1 19.7 ± 3.5 20.81 ± 3.92 36.1 ± 10.0 20.3 ± 2.5 40.2 ± 5.0 36.8 ± 28.6 45.6 ± 29.0 MMD (cysts/mm ± SD) MMA (µm2 ± SD) 0 1,501.9 ± 1,191.1 4,063.6 ± 921.2 2,158.8 ± 524 4,654.2 ± 1,063.8 4,591.3 ± 609.3 11,319.6 ± 1,610.2 7,904.8 ± 7,050.5 7,946.9 ± 5,227.5 Age (years ± SD) 51.2 ± 6.3 64.07 ± 5.8 53.4 ± 7.4 57.8 ± 4.52 NP NP NP 64.2 ± 8.05 62.5 ± 11.9 IOP (mmHg ± SD) 14.3 ± 1.6 15.1 ± 1.67 24.1 ± 1.83 25.96 ± 2.16 15.4 ± 2.6 16.8 ± 1.9 16.1 ± 2.8 16.3 ± 3.05 12.6 ± 1.75 Mean time on therapy (mo ± SD) NA NA NA NA 25.2 ± 9.2 32.8 ± 9.8 34.5 ± 8.7 29.3 ± 5.8 32.8 ± 7.4 MD (dB ± SD) NP +0.8 ± 0.6 NP − 6.5 ± 2.34 −5.4 ± 2.3 −6.0 ± 2.5 −6.2 ± 3.6 −5.7 ± 1.2 −6.1 ± 1.3 Source a b a‡ c‡ a† a* b* b§ b*

POAG OH Healthy

Epithelial Microcysts Density and Area in Healthy Subjects, in Ocular Hypertension and Glaucoma Table 1.

2

LTG

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stimuli and their number progressively decreased with aging (Zhu et al., 2010; Wei et al., 2011). They were shown to dramatically decrease after short-term exposure to BAK and after long-term therapy with BAK-containing anti-glaucoma drugs (Pisella et al., 2004; Baudouin, 2008; Ciancaglini et al., 2008b). GC loss has a noteworthy clinical impact since it induces dry eye, reduces clearance on the ocular surface, and conveys cytotoxic inflammatory mediators, thus inducing conjunctival inflammation (Baudouin, 2008; Baudouin et al., 2010). The previously mentioned study of Ciancaglini et al. (2008b) found that levobunolol hydrochloride significantly decreased GC density after 6 months of therapy either in patients receiving preserved or PF formulations, but a greater decrease was observed in the BAK preserved group (61 and 17%, respectively). Inversely, PGAs showed a different effect on GC. Mastropasqua et al. (2013b) reported a short-term favorable effect of PF-tafluprost on GC, observing with both LSCM and IC a significant increase of the GC density 1 and 6 months after initiating therapy. Differently, GC density increased after 1 month in patients receiving BAK-preserved latanoprost, whereas density was reduced to baseline values after 6 months. These results were in accordance with previous IC studies, which documented a transient increase of GC density after a short duration of treatment with preserved latanoprost, before density decreased after longer periods (Moreno et al., 2003; Russ et al., 2007). Figure 3 presents an overview of the GC density in healthy subjects, and changes induced by different anti-glaucoma medications. Rossi et al. (2013) used LSCM to evaluate ocular surface changes in patients treated with preserved anti-glaucoma medications shifted to PF-tafluprost. The authors observed an increased number of epithelial cells, reduced keratocyte activation, increased number of corneal nerves, a decreased number of bead-like formations, and reduced nerve tortuosity 12 months after the modification of therapy. All these findings highlighted the toxicity of BAK on several ocular surface structures, and supported the need to develop PF-formulations or alternative preservatives (Liang et al., 2012a). Even though all preservatives presented toxic effects on the conjunctiva, polyquaternium-1 (polyquad, an alternative preservative) was reported to be less toxic than BAK (Labbè et al., 2006). Of note, all these epithelial modifications are responsible for the signs and symptoms of ocular surface disease reported by the majority of medically treated glaucomatous patients [as documented by impaired break-up-time (BUT), Schirmer test, corneal staining tests, and ocular surface disease index (OSDI) questionnaire score]. Stromal Modifications Even though the epithelium was more frequently altered by anti-glaucoma drugs, the stroma may also be involved (Fig. 2). Inflammatory cell (lymphocytes and mast cells) infiltration, fibroblast proliferation and activation, and an increase in connective tissue represented the most common stromal modifications (Sherwood et al., 1989; Baudouin, 2008). By using LSCM, Mastropasqua et al. (2013a) reported

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Figure 2. a–d: LSCM of the conjunctiva in a healthy subject. Superficial epithelium shows a regular cellular mosaic (a) with numerous goblet cells (arrowhead) (b); sub-epithelium appears regular, without cellular elements (c) (arrowhead represents a conjunctival fold), while stroma appears loosely arranged (d). e–h: LSCM of the conjunctiva in a glaucomatous patient on medical therapy (two drugs: ß-blocker and PGA, unfixed combination; 10 years of treatment). The epithelium shows hyperreflective and desquamating cells (asterisk), with dendritic cell activation (e) and goblet cell loss (f); the basement membrane shows several dendritic cells (arrow) (g). The stroma is markedly hyperreflective with a reticular pattern, as expression of connective tissue production and fibrosis (h). (from Mastropasqua et al., 2013a, with permission of the publisher). Scale bar is 50 µm. LSCM, laser scanning confocal microscopy.

features of stromal hyperreflectivity, indicative of connective tissue deposition, in patients treated with associative unfixed combination therapy for at least 10 years. The increase in collagen fibers was critical since fibrosis was reported to be associated with a higher risk of bleb failure after filtration surgery (Broadway et al., 1994).

Surgically Treated Open Angle Glaucoma To date, slit lamp examination using well-defined classification systems such as the Indiana Bleb Appearance and the Moorfields Bleb Grading System, is still the gold standard approach for assessing filtering bleb status (Wells et al., 2006). Nevertheless, slit lamp appearance may not completely reflect the drainage ability of a bleb, since clinical analysis is a qualitative assessment, markedly influenced by inter-observer variability. LSCM proved valuable in defining the AH pathways after trabeculectomy, and in characterizing bleb wall features associated with success or failure. Thus, confocal microscopy may quantitatively indicate the capability of AH drainage within the bleb wall after filtering surgery. Epithelial Features of the Filtering Bleb As initially reported by Labbè et al. (2004, 2005), and subsequently confirmed by other studies (Guthoff et al., 2006; Messmer et al., 2006b; Jurowska-Liput et al., 2008; Ciancaglini et al., 2008c; Sbeity et al., 2009; Zeng et al., 2011; Morita et al., 2012), the bleb wall epithelium presented well

defined and opposing features in functioning compared to nonfunctioning cases. Numerous inhomogeneous microcysts were the epithelial hallmarks of functioning filtering blebs (Fig. 4). These microcysts were very similar to those described in untreated and medically treated glaucomatous patients and, thus, may be intended as vesicles transporting AH through the bleb wall layers. Microcysts were usually detected within the bleb epithelium, at a depth of 6–45 μm, with a greater concentration in the conjunctiva adjacent to the limbus and with a size that ranged from 10 and 150 µm. The intra-operative use of mitomycin-C-induced large confluent microcysts, sized from 10 to 300 µm, occasionally showing hyperreflective microdots that probably corresponded to necrotic epithelial cells or inflammatory cells. Oppositely, nonfunctioning blebs presented very few or no EM that could be an inadequate expression or absent AH percolation through the conjunctiva (Fig. 5). Messmer et al. (2006b) clarified the features of EM as encapsulated, filled with amorphous material or with an open lumen, and occasionally containing round and hyperreflective cells (most probably lymphocytes). Guthoff et al. (2006) observed that these round hyperreflective cells were early (2–4 days postoperatively) features of both functioning and malfunctioning blebs. Conversely, late functioning blebs (5–42 months after trabeculectomy) presented few inflammatory cells and microcysts within the epithelium. The presence of EM was in accordance with ex vivo histological studies, which reported intra-epithelial cystic spaces in functioning

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Figure 3. LSCM of goblet cells (arrowheads) in a healthy subject (a) and in therapy-naïve glaucomatous patients receiving PF-PGA (b), preserved PGA (c), PF-ß-blocker (d), preserved ß-blocker (e), and unfixed associative combined therapy (f: PGA + ß-blocker). The patient treated with PF-PGA presented numerous and frequently clustered (arrow) GC, with an overall density quite similar to healthy subjects (asterisk indicates a microcyst). In all other patients GC density was evidently lower compared to healthy subjects and patients receiving PGA, especially in eyes treated with preserved drugs. Scale bar is 50 µm. LSCM, laser scanning confocal microscopy; PF, preservative-free; PGA, prostaglandin analog; GC, goblet cells.

blebs (Picht & Green, 1998a, 1998b). Amar et al. (2008) provided additional clarifications on the nature and function of EM. In an immune-cytological and LSCM study the authors found that the surface of functioning blebs presented numerous atypical GC that did not or only weakly colored in their periphery with MUC5AC immune staining. These GC were closely organized in some areas, whereas they appeared confluent in others, showing empty spaces that contained several nuclei. The empty spaces appeared to match at least in part the microcysts observed with LSCM. In contrast, very few GC were observed at the surface of nonfunctioning filtering blebs. Given the topographic correspondence between MUC5AC-postive GC and epithelial empty spaces, the authors suggested that microcysts corresponded to GC-containing AH. Thus, the trans-cellular pathway of AH could be hypothesized to occur at the level of GC. These findings open important concerns in medical strategies: anti-glaucoma medications must preserve the integrity of this critical cell population, because the final filtering ability of a bleb could depend on GC density available before, and surviving surgery. Stromal Features of Filtering Blebs Functioning and nonfunctioning blebs presented specific and opposing stromal features by LSCM. A condensed and hyperreflective stroma (expression of fibrosis) with rare and

encapsulated cystic spaces were features associated with bleb failure, whereas a loosely arranged collagen-like network with numerous, large, and unencapsulated cysts was an expression of a good functioning bleb (Labbè et al., 2004, 2005; Guthoff et al., 2006; Messmer et al., 2006b; Jurowska-Liput et al., 2008; Ciancaglini et al., 2008c; Sbeity et al., 2009; Zeng et al., 2011; Morita et al., 2012). As for EM, stromal cystic spaces were interpreted as a sign of AH passage through the bleb wall. Guthoff et al. (2006) observed that early and late functioning blebs were characterized by a trabecular or reticular stromal pattern, respectively, whereas early and late malfunctioning blebs presented a compacted or corrugated pattern. Moreover, good functioning blebs were associated with a less stromal vascularization and vessel tortuosity (Messmer et al., 2006b). To summarize, high density and area of EM, a loosely arranged sub-epithelial connective tissue, large and unencapsulated cystic spaces within the stroma, less stromal vascularization, and less tortuosity of stromal vessels, was an expression of good bleb function. Conversely, failing blebs presented contrasting features. The use of confocal microscopy after filtering surgery may help clinicians in identifying the early signs of failure (before they appear at slit lamp), thus permitting a appropriate use of recovery procedures (such as laser suture lysis, anti-metabolite injection, and needling) before IOP rises and blebs irremediably fail.

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Figure 4. Planar reconstruction of the entire superior bulbar conjunctiva 5 weeks after a successful MMC-augmented trabeculectomy. The bottom of the image represents the bleb site. Numerous EM are evident, showing an inhomogeneous distribution and larger area than those found in medically treated eyes, surrounded by a low reflective and thinner wall, frequently with a clustered distribution (arrows) that was more evident at the bleb site. The inner spaces of EM occasionally appeared filled with amorphous material and/or round cells (arrowhead). (from Ciancaglini et al., 2009, with permission from the publisher). Scale bar is expressed in micrometers. MMC, mitomycin-C; EM, epithelial microcysts.

Figure 5. Planar reconstruction of the entire superior bulbar conjunctiva 4 months after a failed MMC-augmented trabeculectomy. EM are scattered, mainly located in the middle and posterior regions of the bleb, sometimes organized in clusters (arrows), and smaller than those observed after successful filtration surgery. The limbal side of the bleb shows rare microcysts. Numerous hyperreflective elements (arrowheads), corresponding to mono-nucleate inflammatory cells infiltrating the epithelium, are widespread throughout the bleb. Scale bar is expressed in micrometers. MMC, mitomycin-C.

Carpineto et al. (2011) reported another potential application of LSCM after filtering surgery. In bleb-associated endophthalmitis, confocal microscopy proved valuable in defining conjunctival and corneal alterations induced by the infective process, in evaluating the effects of topical drugs and modulating the dose regimen during therapy. In the acute phase, a diffuse mononuclear inflammatory cell infiltration (presumably granulocytes and lymphocytes) within the conjunctival epithelium, and in and around microcysts,

was found. Two weeks after diagnosis, the sub-epithelium presented activated DC and 6 weeks later the blebs presented characteristics similar to noninfected functioning blebs. The cornea showed an evident stromal edema, keratocyte activation, and inflammatory precipitates on the endothelium. LSCM was able to document both the positive response to therapy, showing progressive improvement of the conjunctival and corneal status, and the side effects of topical fortified antibiotics on corneal epithelium. An irregular epithelial

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Figure 6. LSCM of the CALT at the superior tarsum. In a representative healthy eye (a), the CALT presents typical oval-shaped lymphatic follicles (asterisk), with a loosely arranged reticular pattern. In medically controlled glaucomatous patients (b,c), the CALT presents hyperreflective elements, corresponding to lymphocytes infiltrating the follicles (arrowheads), with a tendency to clustering (arrow). These features were more evident in multi-treated patients (c: PGA/ß-blocker unfixed combination) than in patients controlled with a single medication (b: preserved PGA). The follicular reticular pattern of glaucomatous eyes appeared more prominent and hyperreflective compared to healthy subjects, probably due to the deposition of connective fibers. Scale bar is 50 µm. LSCM, laser scanning confocal microscopy; CALT, conjunctiva-associated lymphoid tissue.

morphology characterized by polygonal-shaped and hyperreflective cells with poorly distinguishable borders were observed in 75% of patients after only a few days of therapy. Finally, LSCM was also used to describe the characteristics of the trabeculo-Descemet membrane (TDM) after nonpenetrating deep sclerectomy (Mansouri et al., 2011), in order to predict the IOP-lowering efficacy of Nd:YAG goniopuncture. When a carpet of epithelial cells covered the TDM, goniopuncture was able to create an opening and lower IOP. Conversely, the presence of hyperreflective and dense fibrotic tissue covering the TDM hampered perforation and IOP reduction. Conjunctiva After Bleb-Less Glaucoma Surgery To overcome the risk of bleb dysfunction and reduce the need for postsurgical management, different bleb-less procedures have been proposed over the last years. The gold micro shunt (GMS) implantation is the insertion of a biocompatible gold shunt in the supra-choroidal space aimed at directing the AH flow from the anterior chamber toward the choroid, without percolating through the conjunctiva. Currently, GMS has been abandoned due to the occurrence of fibrosis in and around the device, and the high rate of early surgical failure (Agnifili et al., 2012b). Canaloplasty, one of the most promising new surgical approaches for glaucoma, applies tension to and dilates the circumferential lumen of Schlemm’s canal and the trabecular meshwork by using a 10-0 Prolene suture. Thus, it attempts to restore the physiological trabeculo-canalicular outflow system. The contribution of LSCM to clarify the AH outflow pathways involved in the final IOP lowering in both procedures was that it showed a concurrent increase of the TS-AH outflow at the site of surgery (besides the main mechanism) (Mastropasqua et al., 2010, 2012). In fact, due to the reduction of scleral thickness produced by surgery, the mean area of EM

significantly increased after successful canaloplasty or GMS implantation (by fourfold and sixfold compared to baseline, respectively).

LSCM OF THE CONJUNCTIVA-A SSOCIATED LYMPHOID TISSUE (CALT) CALT is located in the lamina propria of the conjunctiva and variably consists of a diffuse layer of a specialized secretory lympho-epithelium, lymphoid follicles, and crypt-associated lymphoid structures (Knop & Knop, 2000). Follicles are mainly observed in the tarsal and orbital conjunctiva of the upper and lower lids, with fewer in the fornix and rarely in the bulbar conjunctiva. In humans, its presence has been extremely difficult to observe, for both anatomical and technical reasons. Thus, to date, LSCM studies were conducted especially in animals. In an acute toxic model, Liang et al. (2012b) found that preserved PGA (latanoprost, travoprost, and bimatoprost) stimulated inflammatory cell infiltration in the dome and intra-follicular layers of the rabbit CALT. Moreover, a marked decrease of MUC-5AC + mucocytes around the CALT structures was found. Conversely, PF-tafluprost and polyquad-preserved travoprost did not induce changes, supporting the primary role of BAK in inducing CALT alterations. In an unpublished in vivo LSCM study conducted by our group on human eyes (currently under peer review) we found results consistent with those reported by Liang et al. (2012b) in rabbits. At the 3-month follow-up, therapy-naïve glaucomatous patients receiving preserved latanoprost showed inflammatory cell infiltration within the follicles and intra-follicular crypts, while these findings were not present in patients treated with PF tafluprost. In addition, the cellular infiltration was greater in patients who started therapy with an unfixed PGA–ß-blocker associative regimen (Fig. 6).

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These findings suggested that anti-glaucoma medications induced inflammatory changes in the lymphoid structures (for the most part BAK related), with the possibility of developing an immune-related ocular surface disorder. Further immune-cytological and clinical studies are mandatory to better clarify these modifications at the molecular level, and to evaluate whether mucosal immunological changes play a role in inducing symptoms referred by patients and in increasing the risk for surgical failure. These findings also support the conclusion that glaucoma should be treated with PF medications, when available.

LSCM

OF THE

CORNEA

Modifications Induced by Medical Therapy Anti-glaucoma medications may induce several modifications of all corneal layers. Epithelial Modifications In a confocal microscopy study that evaluated the long-term effects of PF and preserved anti-glaucoma drugs on the ocular surface, Martone et al. (2009) documented a reduction of cellular density in the superficial epithelium in patients treated with preserved formulations. These changes were interpreted as a direct toxic effect of BAK on superficial epithelium. But, basal epithelium density was greater in patients receiving preserved drugs, most probably due to a proliferative stimulus from the superficial layer. Stromal Alterations In the same study, stromal keratocyte activation was observed in patients receiving single preserved therapy, or treated with ß-blockers/PGA unfixed combination therapy. Epithelial cell modifications were hypothesized to be promoters of stromal changes, where inflammation may induce stromal apoptotic phenomena and increase the stromal proteolytic activity. Bergonzi et al. (2010) reported similar results, showing that PGA increased the keratocyte density in the entire corneal stroma, especially in the anterior layer. The authors speculated that this increase could result from collagen degradation in the extracellular matrix due to activation of metalloproteinases and inhibition of tissue inhibitors of metalloproteinases. A possible long-lasting effect of these modifications on central corneal thickness was hypothesized. Different studies observed reduction in the number and density of sub-basal plexus nerves (Baratz et al., 2006; Martone et al., 2009; Labbè et al., 2012). These alterations were evident in all therapy regimens, but were more pronounced in patients receiving preserved drugs. Bead-like formations and tortuosity of nerve fibers were also described as significantly higher in glaucoma patients than controls, without statistically significant differences between different groups of therapy (Martone et al., 2009). Labbè et al. (2012) found similar changes in patients with dry eye, suggesting that anti-glaucoma medications may lead to an iatrogenic neurotrophic keratopathy with dry eye. A significant

correlation between nerve modifications and ocular surface tests indicative of dry eye (Schirmer test, BUT test, and corneal sensitivity) were found (Martone et al., 2009; Labbè et al., 2012). Endothelial Alterations In a scanning confocal study in patients with pseudo-exfoliative and keratopathy, the endothelium presented a normal cell count, although with evident signs of polymegathism and pleomorphism (Wali et al., 2009). Differently, in an LSCM study, Ranno et al. (2011) reported a reduction of the cell density in patients treated for at least 2 years either with ß-blockers or PGA. All these findings highlighted the role of anti-glaucoma medications and preservatives in inducing epithelial and stromal corneal changes. Along with conjunctival modifications, such corneal alterations are responsible for signs and symptoms of ocular surface disease during long-term therapy. Thus, PF anti-glaucoma medications and fixed combination therapy should also be used for corneal safety when available. Figure 7 displays the most common corneal changes described in medically treated glaucomatous patients.

Corneal Findings in Secondary Glaucoma In a recent study, Sbeity et al. (2011) evaluated a noncontact lens prototype of the RCM to detect exfoliative material (XFM) and exfoliative-related changes in the cornea, iris, and lens capsule in patients with unilateral exfoliation syndrome (XFS). The prototype included a Nikon 50xCF Plan/ NA 0.45 EPI SLWD, with a working distance 13.8 mm, an estimated digital lateral/axial resolution of 1–2/10 µm, and a field of view of 500 × 500 µm. The lateral and axial resolutions were 0.8 and 8 µm, respectively. The authors observed hyperreflective structures (corresponding to XFM) in the granular and central disc areas, and hyporeflective spaces in the intermediate zones of the lens capsule in patients with clinical evidence of XFS. Of note, lenticular hyperreflective structures along with endothelial deposits were also described in the clinically unaffected fellow eye. Thus, LSCM may allow preclinical early detection of XFS, and guide the ophthalmologist in surveillance decisions. LSCM was also used to investigate corneal features in primary congenital glaucoma. Reduced mean keratocyte densities in the anterior and posterior stroma, and reduced mean endothelial cell density with signs of pleomorphism and polymegathism, and multiple white-dot-like lesions were the main features observed (Gatzioufas et al., 2013). These findings were in accordance with a previous study of Mastropasqua et al. (2002), which examined two adult patients with congenital glaucoma and megalocornea using slit scanning confocal microscopy. In addition, this study reported an abnormal appearance of the stromal nerve fibers, which presented a “coil-shaped” configuration. The reduced endothelial density was thought to be a consequence of the corneal distention that occurs as a result of ocular globe enlargement.

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Figure 7. LSCM of the cornea. In healthy eyes superficial epithelium appears quite regular, with good homogeneity in cellular shape and size (a); sub-basal nerve plexus shows several fibers (white arrowhead), regularly distributed (b); and the endothelial mosaic is very regular (c). In a glaucomatous eye (three drugs: PGA + ß-blocker/α-agonist fixed combination; 4 years of therapy), the superficial epithelium presents signs of polymorphism and polymegathism, and cell desquamation (black arrowhead) (d); the sub-basal nerve plexus presents an evident reduction of the number of nerves with higher tortuosity and several activated dendritic cells (arrow) (e); endothelial cell density appears similar to the normal eye, but with signs of polymorphism (asterisks) (f). Scale bar is 50 µm. LSCM, laser scanning confocal microscopy; PGA, prostaglandin analog.

LSCM

OF THE

SCLERO-CORNEAL L IMBUS

LSCM was used to evaluate the human sclero-corneal limbus (SL) in healthy subjects and in patients affected with limbus deficiency (Nubile et al., 2013). LSCM of normal SL showed the presence of palisades of Vogt and a progressive morphologic transition of epithelial cells from the conjunctival to the corneal phenotype in the peripheral cornea adjacent to the limbus (Patel et al., 2006). To date, studies of SL modifications in glaucoma are still lacking. Recently, we performed LSCM and IC with immunofluorescence for HLA-DR and IL-6 to study the immunoinflammatory effects of anti-glaucoma medications on SL structures (unpublished data, currently under peer review). Patients on mono-therapy for 3.5 ± 1.2 years presented mild irregularity of the transition epithelium with some scattered activated DC within the sub-epithelium. These features were more common in patients receiving BAK-preserved drugs (PGA and ß-blockers) compared to patients receiving PF drugs. On the other hand, in multi-treated patients (≥2 drugs; 3.8 ±2.1 years of therapy) LSCM found a marked irregularity of the transition epithelium (with signs of metaplasia), DC activation, and fibrosis of the Vogt’s palisades around the entire limbal circumference (Fig. 8). In all

groups of patients HLA-DR and IL-6 were significantly more expressed than controls. Patients controlled with a single BAK preserved medication and multi-therapy patients presented higher expression of both HLA-DR and IL-6 compared to PF-mono-therapy. These findings are noteworthy since, as documented, inflammation decreased the number of limbal stem cells or altered their function, resulting in varying degrees of stem cell deficiency (Puangsricharern & Tseng, 1995; Dua & Azuara-Blanco, 2000). This finding was in accordance with previous studies conducted by Schwartz & Holland (1998, 2001) who identified the concept of iatrogenic limbal stem cell deficiency in eyes treated with anti-glaucoma eye drops. Further immune-cytological studies evaluating the expression of molecular biomarkers of limbal stem cell deficiency (integrins, p63, vimentin, connexin 43, laminin 5, and desmoglein 3) are mandatory to assess the risk for limbal stem cell damage in patients undergoing long-term therapy.

LSCM

OF

MEIBOMIAN G LANDS (MG)

MG represent the most important structures involved in production of the lipoid portion of the tear film, which normally limits tear film evaporation.

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Figure 8. LSCM of the sclero-corneal limbus. a: Normal features of the transition epithelium (seen from the corneal side), showing small islands (arrowhead) of corneal epithelial cells (regular mosaic with well-defined cell borders) within the sheet of conjunctival epithelium (cells with indistinct boundaries). b: The sub-epithelium is regular and quite homogeneous feature, without inflammatory elements. c: Regular architecture of the limbal palisades of Vogt, characterized by hyperreflective linear radial stromal ridges, alternating with columns of basal epithelial cells. In a medically treated glaucomatous patient (3 years of therapy with PGA/ß-blocker unfixed combination), transition epithelium (d) appears irregular and hyperreflective, with signs of metaplasia at the conjunctival side; sub-epithelium (e) shows several activated DC (arrow) and, palisades of Vogt (f) appear hyperreflective due to connective fiber deposition. Scale bar is 50 µm. LSCM, laser scanning confocal microscopy; DC, dendritic cell.

Figure 9. LSCM of meibomian glands of the lower eyelid. In a representative healthy subject (a) acinar units (lower eyelid) present a normo-reflective glandular wall, a silent interstice, and a normo-reflective acinar secretion with a medium-sized glandular orifice. In a representative multi-treated glaucomatous patient (three drugs), MG presents a lower density and area, an inhomogeneity and hyperreflectivity of the acinar wall (arrowhead) and inter-glandular interstice (arrow), hyperreflectivity of the acinar secretion (asterisk) with dilation of the glandular orifice (b–d) (from Agnifili et al., 2013, with permission from the publisher). Scale bar is 50 µm. LSCM, laser scanning confocal microscopy; MG, meibomian glands.

Different studies used LSCM to characterize the features of MG in healthy subjects and in patients affected with meibomian gland dysfunction (MGD), one of the major causes of dry eye (Matsumoto et al., 2008; Efron et al., 2009b; Ibrahim et al., 2010). Recently, LSCM was also used to evaluate the impact of medical therapy on MG in patients with glaucoma (Agnifili et al., 2013). Patients treated with two or more drugs presented a reduction of glandular density

and area, an increased reflectivity of the acinar secretion, ductal orifice dilation, and signs of inflammation of the glandular wall and interstice (inhomogeneous patterns) (Fig. 9). These modifications were in part different from those described in patients with MGD, since the glandular area appeared reduced in glaucoma. Thus, anti-glaucoma medications could induce a secondary MGD probably determined by different pathogenetic

In Vivo Confocal Microscopy in Glaucoma

mechanisms. As known, MGD is a primitive disease and may be associated with other ocular surface conditions or diseases such as normal aging, use of contact lenses, and dry eye, that have well defined and different inductive pathogenic mechanisms (Villani et al., 2011a, 2011b, 2013b). The induction mechanisms probably involved in the pathogenesis of MGD secondary to anti-glaucoma therapy may be an active compound- and preservative-related toxicity, a primary iatrogenic glandular atrophy, and an inflammatory or immune-mediated response. When considering the effect of BAK and active compounds on glandular modifications, preserved drugs appeared more toxic than PF formulations, with PGA being more toxic than PF-PGA. No differences between ß-blockers and PF-ß-blockers have been reported. Thus, PGA might play a key role in inducing MG alterations, with an important adjunctive effect of the preservative. Inflammatory parameters were the only features that differed between patients treated with a single medication and healthy controls, suggesting that inflammation might be the first step in the cascade of glandular modifications. These findings presented an important clinical impact since MG alterations correlated with indicators of dry eye (OSDI index, break-up time, and Schirmer test). Thus, preservation of the structural and functional integrity of MG and, therefore, modification of the tear film represents an essential challenge during glaucoma therapy. Further studies evaluating the relation between MG changes with adherence to and persistence of treatment could clarify the role of MG damage on worsening patient compliance during long-term therapy.

L IMITATIONS OF THE LSCM Although the possibility of assessing the fine structure of ocular surface tissues represents a true advance with respect to the past, LSCM gives only morphological details, and identification of cell phenotypes still requires ex vivo histology. The different morphological aspects of cells as they appear in confocal images are based on their reflectivity and light-scattering phenomenon: thus requiring final interpretation by clinicians. This is true for every cell type. For most of the cells, confocal morphology is well established, and we can easily discriminate cells belonging to different populations in each structure analyzed. But, how can we be sure that conjunctival GC are really mucin-secreting cells or modified nongoblet epithelial cells exhibiting squamous metaplasia in response to inflammatory cells, as reported in aging normal subjects or in patients affected with Sjogren syndrome (Villani et al., 2011)? Are we sure that so-called EM are vesicles of AH percolating through the conjunctiva? Or are they features of degenerative processes occurring within the epithelium or degenerated GC? How can a dysplastic epithelial cell be characterized? How can we be sure that DC, appearing within the cornea, limbus, or conjunctiva, are really antigenpresenting cells? In addition, some ocular surface structures (i.e., conjunctival lymphatics) have not been identified by LSCM or

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cannot be investigated due to anatomical difficulties (such as lacrimal glands) or to the depth (sclera). A comprehensive clinical examination is still fundamental, and LSCM is often complementary. Only clinical and pathological correlations will help define the real nature of what we observe in confocal images, particularly in diseased eyes. Future technical advancements in the resolution and scan depth of the RCM will probably overcome these limitations.

SUMMARY

AND

CONCLUSIONS

Until recently, definition of the microscopic morphology of the ocular surface tissues in glaucoma was very limited, and was based on IC (which is, however, based on a limited sampling of superficial epithelial layers) with or without immune-fluorescence, or on ex vivo histology (which presents limitations due to tissue degeneration, artifacts, and impossibility of evaluating over time a chronic and progressing disease). The recent rapid diffusion of LSCM included also glaucoma, allowing in vivo, safe, and repeatable examinations of living ocular surface tissues for their entire depth. LSCM permitted several advancements in the knowledge of ocular surface microanatomy in patients with glaucoma, some of them presenting a notable clinical impact such as characterization of the filtering ability of blebs. Moreover, LSCM provided essential information on the effects of anti-glaucoma medications on the ocular surface, highlighting the main role of preservatives and documenting the relation between these changes and symptoms referred by patients. Thus, the need for long-term unpreserved therapies seems to be increasingly supported. Further prospective studies correlating presurgical in vivo conjunctival features with the risk of filtration failure, will probably permit planning surgery at a more appropriate moment during the natural history of glaucoma, before drugs have irreversibly modified the conjunctival status. Besides these clinical applications, LSCM was also used for research purposes; especially for evaluating AH outflow. In this field LSCM has played a role in clarifying the mechanism of action of different surgical procedures for glaucoma, and IOP-lowering medications such as PGA. Finally, LSCM could open new fields of research with a huge potential further clinical impact, linked to the activation of mucosal immunity and the modification of stem cell behavior during glaucoma therapy.

ACKNOWLEDGMENTS The authors have no proprietary or commercial interests in any concepts or products discussed in this article.

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