http://informahealthcare.com/mor ISSN 1439-7595 (print), 1439-7609 (online) Mod Rheumatol, 2014; Early Online: 1–5 © 2014 Japan College of Rheumatology DOI: 10.3109/14397595.2014.979523
ORIGINAL ARTICLE
In vivo confocal scanning laser microscopy in patients with primary Sjögren’s syndrome: A monocentric experience Giovanna Gabbriellini1, Chiara Baldini2, Valentina Varanini1, Chiara Notarstefano2, Pasquale Pepe2, Francesca Fanucci1, Francesco Ferro2, Nicoletta Luciano2, Marta Mosca2, Marco Nardi1, and Stefano Bombardieri2
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1Ophthalmology Unit, University of Pisa, Pisa, Italy and 2Rheumatology Unit, University of Pisa, Pisa, Italy
Abstract
Keywords
Aims. (i) To analyze the in vivo corneal structure and sub-basal plexus nerves in patients with primary Sjögren’s syndrome (pSS) and no-SS dry eye by confocal scanning laser microscopy (CSLM) and (ii) to correlate CSLM findings with tear function tests and with patients’ subjective dryness. Methods. Seventeen patients with pSS, 16 no-SS dry eye, and 20 healthy volunteers were included. CSLM parameters taken into consideration included: basal epithelial integrity, corneal thickness, epithelial cellular density, keratocyte activation, and sub-basal plexus morphology. Statistical analysis was carried out using SPSS-13 (Chicago IL, USA). Results. CSLM pachymetric data and the superficial epithelium cell density were significantly lower in pSS versus no-SS dry eye (p 0.0001); keratocyte activation and sub-basal nerve abnormalities were also more frequent in pSS patients (p 0.0001). CSLM findings well correlated with both the ocular test results and the patients’ perception of ocular dryness at the baseline and over the follow-up. Conclusion. CSLM might be a useful novel tool in the assessment of the involvement of the lachrymal functional unit in pSS.
Confocal scanning laser microscopy, Dry eye, Keratoconjunctivitis sicca, Lachrymal functional unit, Sjögren’s syndrome
Introduction Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disorder characterized by chronic inflammation and functional impairment of the exocrine glands, usually presenting as a persistent dryness of the mouth and of the eyes. Although the disease might be frequently associated with a variety of scattered extraglandular manifestations, including malignant lymphoproliferative disorders, the hallmark of pSS is represented by chronic and disabling xerostomia and xerophthalmia [1–3]. The assessment of dry eye is currently based upon the combination of tests assessing aqueous tear flow and ocular surface damage, namely Schirmer’s test I, lissamine green staining (LGS), and tear break-up time test (TBUT) [4,5]. Nowadays, although extensively used, there is a general dissatisfaction regarding the clinical value of these traditional ocular tests in differentiating pSS from other dry-eye conditions [6–9]. Thus, a growing interest has arisen in developing new diagnostic tools that allow clinicians to improve pSS dry-eye assessment through an integrated analysis of the different components in the lachrymal unit [10]. This emerging trend reflects the general efforts of the last ten years in searching for novel specific and non-invasive diagnostic biomarkers for pSS and other rheumatic diseases [11,12]. Confocal scanning laser microscopy (CSLM) is a new non-invasive supplementary diagnostic tool for the in vivo assessment of the lachrymal functional unit disorder in many Correspondence to: Chiara Baldini, MD, Rheumatology Unit, University of Pisa, Via Roma 67, 56126 Pisa, Italy. E-mail: chiara.baldini74@gmail. com
History Received 24 July 2014 Accepted 17 October 2014 Published online 10 December 2014
anterior-segment diseases, including dry-eye disorders [13–16]. Preliminary in vivo CSLM studies revealed that, when compared with healthy volunteers, patients with pSS more frequently showed an irregular corneal epithelium with decreased density in the superficial corneal epithelium cells and decreased corneal thickness, an alteration of nerve fiber density, an increased subbasal nerve tortuosity and reflectivity, and an increased density of activated anterior keratocytes and conjunctival inflammatory cells [17,18]. To date, the usefulness of CSLM in differentiating pSS from the no-SS dry-eye diseases has been scarcely investigated and even the relationship between CSLM parameters and the traditional objective ocular tests for dry-eye syndrome are still unknown. In this study, we therefore analyze the in vivo corneal structure and sub-basal plexus nerves in patients with pSS and no-SS dry eye by CSLM, in order to evaluate the usefulness of CSLM in the diagnostic algorithm of the disease. We also correlated CSLM findings with routinely performed tear function tests (Schirmer’s test I, TBUT, and LGS).
Methods Study design This was a single-center case–control study aimed at investigating the ocular anterior-segment microstructure changes in pSS dry eyes and in other dry-eye conditions by CSLM. Subjects provided informed consent, and the study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The Institutional Review Board at the University of Pisa, Italy, approved this study.
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Patients eligibility Consecutive unselected patients, who attended the Rheumatology and Ophthalmology Units in the period between 2009 and 2012 due to persistent ( 3 months) subjective dry eye, were enrolled in the study. The diagnosis of pSS was made according to the American and European classification criteria (AECG) [4]. The condition of no-SS sicca syndrome was defined as the presence of xerostomia and xerophthalmia in patients with negative non-organ-specific autoantibodies and negative minor salivary gland biopsy (according to focus scoring system). Exclusion criteria were represented by the following conditions: corneal dystrophy/inflammation; systemic therapies with known corneal toxicity; topical anti-glaucoma drugs, steroids, or non-steroidal anti-inflammatory drugs; use of contact lenses; and previous interventions of ophthalmic surgery. The following baseline data were collected in all the cases: gender, age, disease duration, glandular and extra-glandular disease manifestations, number and type of previous/current disease-modifying anti-rheumatic drugs and prednisolone, and previous/current use of artificial tears. Moreover, at the time of the study entry, every patient had a rheumatologic and ophthalmological examination, complete laboratory tests, an evaluation of the auto-antibodies profile (i.e., anti-nuclear antibodies, extractable nuclear antigen (ENA) antibodies, and rheumatoid factor) and HCV/HBV serological markers, and thyroid function tests. The order of the ocular tests was based on the existing literature following a pre-defined sequence apparently able to minimize the influence of each test to the next test [5,7]. According to this protocol, the Schirmer’s test I was performed first, although potential injuries to the conjunctiva may occur when the test was performed (without anesthesia) [5,7,19]. The Schirmer’s test I (without anesthesia) was followed by instillation of fluorescein dye, determining the TBUT, and grading the corneal fluorescein staining pattern. After fluorescein grading of the cornea, lissamine green dye was applied and the conjunctiva was examined quickly and graded before the dye diffused or the intensity of staining diminished. The total time for these tests was approximately 20 minutes. The Schirmer’s test I was carried out as described elsewhere, using sterile Schirmer’s strips without anesthesia, in a room controlled for lighting (dimlit room), temperature (20–22 C), and humidity (40–60%) [20]. Abnormal value was regarded as 5mm/wetting after 5 min. In addition, GLS was performed as already reported and scored considering a Lissamine green score of 4 as pathological [21]. TBUT values less than 10 seconds were considered consistent with dry eyes [20]. Finally, we also assessed visual acuity (VA; using Snellen acuity charts) and performed intraocular pressure (IOP) measurement by Goldmann applanation tonometry and slit-lamp biomicroscopic examination without pupil dilation in all the cases. The latter was carried out in order to obtain a magnified image of (i) tear film, (ii) ocular surface, (iii) eyelids, and (iv) meibomian glands; furthermore, this test allowed us to carefully examine the anterior portions of the eye, including the anterior chamber and iris. Confocal scanning laser microscopy technique CSLM examination was performed by Confoscan 4.0 Nidek Technologies, equipped with a standard 40x objective. To avoid the diurnal variation of the corneal thickness, the imaging was performed between 09:00 and 11:00 hours. CSLM was performed at least five days after the other ocular tests. A local anesthetic solution (0.4% oxybuprocaine) was used before the CSLM examination in order to avoid blinking. After a drop of viscotears, gel was applied to the tip of the lens and the lens was advanced toward the central cornea until the gel contacted the cornea. The lens was
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aligned with the corneal apex until the operator saw a clear image of corneal endothelium. The alignment of the lens to the corneal apex was performed sometimes with a full automatic method (auto-adjust mode included in Nidek software), while other examinations required the use of a manual alignment; however, in most cases we used a semi-automatic alignment. Each scan recorded 350 z-axis images separated by a distance of 1.5 μm, making two to four complete passages from the lachrymal film to the anterior chamber. Each frame covered an area of approximately 460 345 μm. Z-scan curves (graphs showing the depth coordinate on the z-axis and the level of reflectivity on the y-axis) were used to select the images relating to the superficial and basal epithelium, the anterior and posterior stroma, and Bowman’s membrane or the sub-basal epithelium. Confocal scanning laser microscopy analysis The same ophthalmologist analyzed all the CSLM images and quantified the following variables: basal epithelial integrity, pachymetric data, keratocyte activation, superficial and basal epithelium cell densities, sub-basal corneal nerve density, tortuosity, and fiber reflectivity. Basal epithelial integrity was graded from Grade 0 (normal) to Grade 1 (slight loss) and 2 (severe loss) by the observation of three different frames of this layer [22]. Corneal thickness was defined as the distance in micrometers (μm) between the first surface image of the endothelial layer and the last frame of superficial epithelium on the z-scan axis. The pachymetric data of each eye were the result of the mean between all the pachymetric values obtained from each z-scan curve. Epithelial cell density (cells/mm2) was evaluated at the level of the superficial epithelium and at the basal epithelial layer by counting the cells present in one image with the manual counting procedure program of the instrument. We considered the entire area of the frame. The cells that were partially contained in the frame were considered only on the right and inferior side. The results were derived from the mean of three different counts for each layer [22]. As far as sub-basal plexus nerve was concerned, the number of nerves was defined as the sum of the nerve branches present in one image (nerves/frame) [23]. Reduced sub-basal corneal nerve density was graded according to a five-point severity scale: Grade 1 (very severe decrease), Grade 2 (severe decrease), Grade 3 (moderate decrease), Grade 4 (slight decrease), and Grade 5 (normal). The tortuosity and the reflectivity of the nerve fibers were graded according to the method proposed by Oliveira-Soto and Efron [18]: tortuosity was graded from Grade 0 (the nerve fibers were almost straight) to Grade 4 (the nerve fibers were very tortuous), and reflectivity (brightness) was graded from Grade 0 (the nerve fibers are indistinguishable from the background) to Grade 4 (the reflectivity was much higher than the background) as previously described. In both cases, the severity grading scale assigned a Grade 0 for a normal CSLM observation; a Grade 1 for a slight, not clinically significant abnormality; a Grade 2 for a moderate abnormal CSLM observation; and a Grade 3 and 4 when severe clinical findings and very severe clinical findings, respectively, were observed at the CSLM. Also in this case, the grade of each eye was evaluated by the mean of the values calculated in three different frames of the same layer. Finally, estimates of human keratocyte density was performed using manual analysis by marking each clearly defined cell or nucleus in a pre-defined rectangular frame; the activation of the keratocytes in the anterior stroma was evaluated according to a score varying from Grade 0 (no keratocytes activated) to Grade 1 (until 4 keratocytes activated for frame) and Grade 2 (more than 4 activated keratocytes for frame). We considered the mean between the values obtained from the three frames of each eye with more keratocyte activity [22].
Confocal scanning laser microscopy in SS approved 3
DOI 10.3109/14397595.2014.979523
Table 1. Patients’ clinical features.
Statistical analysis Data are presented as percentages along with the number of patients or as the mean (standard deviation). Contingency table analysis, Fisher’s exact test, and analysis of variance (ANOVA) test were used for statistical analysis. Spearman’s rank correlation was applied for detecting correlations between ocular tests results and CSLM findings. Statistical analysis was carried out using SPSS 13 (SPSS Inc., Chicago, IL, USA).
Results
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Study population and clinical data Thirty-four eyes from 17 consecutive females with pSS (AECG criteria) and 32 eyes from 16 consecutive females with a diagnosis of no-SS dry eye were investigated in this study. Twenty healthy women were included as controls. According to previous studies [17,24,25], data from the eye with the highest fluorescein staining score was selected. In the case of equal scores for the two eyes, discriminant criteria considered were, in order of relevance, conjunctival staining and TBUT. The clinical characteristics of the subjects enrolled are provided in Table 1. There was no significant difference in age among the pSS [mean age (SD) 56.5 (13.9) yrs], no-SS dry eye (mean age SD 49.8 15.7 yrs), and healthy volunteer groups [mean age (SD) 53.4 (14.2) yrs]. Moreover, no difference was detected regarding the duration of ocular dryness symptoms between pSS and no-SS dry-eye patients [mean duration (SD) 2.5 (2.7) vs 2 (1.5) yrs, p n.s]. As expected, pSS patients presented a higher frequency of xerostomia, arthralgias, hypergammaglobulinemia, and serologic abnormalities including positivity for anti-nuclear antibodies, anti-Ro/SSA, and rheumatoid factor. All the patients and pathological controls underwent a minor salivary gland biopsy: 14/17 of the pSS patients presented a focal sialoadenitis (FS 1), whereas none of the no-SS dry-eye subjects showed a focal sialoadenitis at the histologic examination. Finally, none of the subjects enrolled had positive serological markers for HCV/ HBV infection. All the patients were using artificial tears and 11 out of 17 pSS patients were also receiving low-dose oral prednisolone (∼ 4mg per day) and hydroxychloroquine (400 mg per day). The ocular test results including Schirmer’s test I, TBUT, and GLS are reported in Table 1. A positive correlation was found between Schirmer’s test I results and TBUT (r 0.504, p 0.003), and a negative correlation was observed between Schirmer’s test I results and GLS (r 453, p 0.009). No statistically significant difference was found between pSS patients and patients with no-SS dry-eye syndrome as far as Schirmer’s test I, TBUT results, and GSL scores were concerned. None of the patients presented any abnormalities in visual acuity and IOP measurement.
Number of patients Age (M SD) Disease duration (M SD, yrs) Xerophthalmia Xerostomia Minor salivary gland biopsy (FS 1) Salivary gland enlargement Arthralgia Low C3/Low C4 Leukopenia Hypergammaglobulinemia Anti-nuclear antibodies Anti-Ro/SSA Anti-La/SSB Rheumatoid factor Thyroiditis Schirmer’s test I (M SD, mm) TBUT (M SD, s) Lissamine green test (score 4)
pSS 17 (15 F:2 M) 56.5 13.9 2.5 2.7
no-SS dry eye 16 (13 F:3 M) 49.8 15.7 2 1.5
p value n.s. n.s. n.s.
17 (100%) 15 (88.2%) 14 (82.3%)
16 (100%) 8 (50%) None
n.s. 0.02 0.0001
4 (23.5%) 8 (47%) 4 (23.5%) 4 (23.5%) 7 (41.2%) 17 (100%) 9 (53%) 4 (23.5%) 10 (59%) 5 (29.4%) 4.7 1.9
None None None 1 (6%) None 2 (12%) None None 1 (6%) 2 (12.5%) 5.4 1.8
n.s. 0.003 n.s. n.s. 0.007 0.0001 0.0009 n.s. 0.002 n.s. n.s.
5.7 1.5 8/17 (47%)
6.7 1.5 7/16 (44%)
n.s. n.s.
F female, M male, M SD mean standard deviation, yrs years, FS focus score
CSLM corneal morphology in pSS, no-SS dry eye, and healthy volunteers Table 2 reports the CSLM parameters evaluating corneal morphology and corneal sub-basal plexus nerves in pSS, no-SS dry eye, and healthy volunteers. From the statistical analysis we retrieved the following data. First, we found that the epithelial corneal thickness was significantly reduced in pSS patients with respect to both healthy volunteers and no-SS dry eye. By contrast, no difference was detected between healthy volunteers and no-SS dry-eye patients. We also found that the cell density of the superficial epithelium was significantly reduced in the pSS group when compared with that in the other two groups. Moreover, the superficial epithelial cell density was significantly lower in pSS patients presenting with severe alterations of the sub-basal nerve plexus (1049 186 vs 1192 129, p 0.019). There was no significant difference in the density of the basal epithelial cell between pSS and no-SS sicca patients. Nonetheless, in both cases the basal epithelial cell density was higher than that in the healthy volunteers group. The basal epithelial integrity was significantly more compromised in pSS and no-SS dry eyes rather than that in healthy volunteers (Figure 1a). Second, regarding the sub-basal nerve plexus, we observed that the number of the nerve fibers visualized in the single photo
Table 2. Corneal morphology and corneal sub-basal plexus nerves in pSS, no-SS dry eye, and healthy volunteers (CTRL).
CSLM findings
CTRL (M SD)
pSS (M SD)
no-SS dry eye (M SD)
Corneal thickness (μm) Basal epithelium cell density (cells/mm2) Superficial epithelium cell density (cells/mm2) Sub-basal corneal nerve density (grade #2) Tortuosity (grade 3) Reflectivity (grade 3) Keratocyte activation (grade 2) Epithelial integrity disruption (grade 2)
556 37 5793 290 1548 12.6 None 2/20 (10%) 2/20 (10%) 3/20 (15%) 2/20 (10%)
510.5 24.6 5993 337.9 974.7 58.5 13/17 (76.5%) 11/17 (64.7%) 12/17 (70.6%) 15/17 (88.2%) 15/17 (88.2%)
566.6 22.5 6078.5 114 1280.7 90.5 5/16 (31.3%) 4/16 (25%) 4/16 (25%) 8/16 (50%) 8/16 (50%)
p value CTRL vs pSS 0.000 n.s 0.000 0.000 0.001 0.000 0.000 0.000
pSS vs no-SS 0.000 n.s 0.000 0.03 0.037 0.015 0.026 0.026
CTRL vs no-SS n.s. 0.001 0.000 0.000 n.s n.s 0.034 0.011
Note: Statistical analysis was performed by ANOVA test and contingency table (p 0.05). CSLM confocal scanning laser microscopy, CTRL healthy volunteers, pSS primary Sjögren’s syndrome, no-SS non-autoimmune dry-eye condition, M SD mean standard deviation.
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Figure 1. CSLM findings in healthy volunteers (CTRL), patients with no-SS and pSS: (a) epithelial integrity; (b) sub-basal nerve tortuosity; (c) sub-basal nerve reflectivity; (d) keratocyte activation. ***p 0.0001, **p 0.001, *p 0.05.
frame was significantly reduced in both pSS and no-SS dry-eye in comparison with healthy controls (p 0.0001). Moreover, the sub-basal nerve plexus tortuosity and reflectivity were significantly increased in pSS (Figure 1b and c). A statistically significant difference in the density of the nerve fibers (p value 0.0001) was also detected in no-SS dry-eye patients when compared with healthy volunteers, and a significant correlation was found between the density of the nerve fibers and the Schirmer’s test I results (r 0.496, p 0.003). Finally, the keratocyte activation was significantly higher in pSS and in no-SS dry eye than in the healthy volunteers. Keratocyte activation was still significantly higher in pSS patients in comparison to those with no-SS dry eyes (Figure 1d). A negative correlation was found between keratocyte activation and the density of the sub-basal nerves (r 0.712, p 0.000), whereas a positive correlation was found between keratocyte activation and both the sub-basal nerve plexus tortuosity (r 0.470, p 0.006) and reflectivity (r 0.508, p 0.003).
Discussion In this study, we reported a single-center experience on the application of CSLM for the assessment of dry eyes in pSS patients. The results of our study confirmed the value of CSLM for detecting pSS damage of the corneal epithelium layers and a scattered combination of different sub-basal nerve plexus abnormalities. We particularly observed a significant reduction in the epithelial corneal thickness with a decrease in the cell density of the corneal superficial epithelium and a relative increase in the basal epithelial cells. We also described the presence of many irregularities of the basal epithelial integrity, a remarkable increase in the activation of the keratocytes, a reduction in the number of the fibers
of the sub-basal plexus nerves, and an increase in the tortuosity and of the reflectivity of the nervous fibers, thus supporting the hypothesis of chronic inflammation of all the corneal anatomical structures in pSS. These results reflected previous CSLM studies which documented several morphological abnormalities in lachrymal functional unit of patients with pSS. In 2003, Tuominen et al. [26] firstly showed epithelial, stromal, and neural abnormalities in the corneas of 10 patients with pSS. Specifically, they demonstrated that pSS dry eye was characterized by keratocyte activation, abnormalities in the sub-basal nerve fiber morphology, and a reduced thickness of the central cornea. Similarly, in 2004, Benitez del Castillo et al. [27] documented a decrease in the cell density of the superficial epithelium and significant alterations in the corneal innervations in 10 patients with pSS. Subsequently, Villani et al. [17] revealed a significant decrease in the cell density of the superficial epithelium, and a significant increase in the cell density of the basal epithelium in pSS patients with respect to controls. Moreover, the authors showed a statistically significant difference in the number of nerve fibers visualized in the single CSLM photo frame and in the tortuosity of the fibers. In addition to all these findings, with the present work, we also demonstrated that the vast majority of the CSLM pathological findings were significantly interrelated to each other. Indeed, we found a close correlation between superficial epithelial cellular density and the number of nerve fibers per photo frame, and we also observed that the loss of the epithelial integrity and the disruption of the intercellular junctions significantly correlated with the sub-basal plexus nerve alterations and with the activation of the keratocytes. In turn, keratocyte activation was correlated with the sub-basal nerve plexus abnormalities. Significant correlations were also found between the results of the traditional ocular tests, particularly Schirmer’s test I, and CSLM findings.
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DOI 10.3109/14397595.2014.979523
Taken together, our observation and previous observations further support the hypothesis that in pSS, dry eye might represent not only a disorder of the tear film, but rather the consequence of the defective homeostasis of the ocular surface. In other words, the inflammation of the ocular surface and the alteration of corneal innervations might act together resulting in the impairment of the normal lachrymal reflex and in the gradual destruction of the lachrymal functional unit. At the moment, the use of CSLM might not be generalizable to routine differential diagnosis of xerophthalmia, since, reliable cutoff values able to distinguish between pSS and no-SS dryeye conditions have not been validated yet. However, our results encourage the use of CSLM as a non-invasive novel technique for a better understanding of the physiopathology of keratoconjunctivitis sicca associated with pSS. In addition, although CSLM procedures can be time consuming and may exceed practical limits in routine clinical work flow, being able to demonstrate alterations in the superficial epithelial cells and corneal nerves, and immune cell changes at a cellular level that correlate with clinical signs and symptoms of pSS, CMLS may serve as a useful assessment tool supplementary to more traditional clinical diagnostic modalities. More specifically, at least in the setting of clinical trials, this technique may provide a better diagnostic differentiation of pSS from degenerative non-autoimmune forms of dry eyes, and a more accurate treatment stratification and measurement of therapeutic efficacy when taken together with clinical tests.
Acknowledgements We wish to thank Dr. Francesca Sernissi and Miss Wend Doherty for their valuable contribution in reviewing the text.
Conflict of interest None.
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ORIGINAL ARTICLE
In vivo confocal scanning laser microscopy in patients with primary Sjögren’s syndrome: A monocentric experience Giovanna Gabbriellini1, Chiara Baldini2, Valentina Varanini1, Chiara Notarstefano2, Pasquale Pepe2, Francesca Fanucci1, Francesco Ferro2, Nicoletta Luciano2, Marta Mosca2, Marco Nardi1, and Stefano Bombardieri2
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1Ophthalmology Unit, University of Pisa, Pisa, Italy and 2Rheumatology Unit, University of Pisa, Pisa, Italy
Abstract
Keywords
Aims. (i) To analyze the in vivo corneal structure and sub-basal plexus nerves in patients with primary Sjögren’s syndrome (pSS) and no-SS dry eye by confocal scanning laser microscopy (CSLM) and (ii) to correlate CSLM findings with tear function tests and with patients’ subjective dryness. Methods. Seventeen patients with pSS, 16 no-SS dry eye, and 20 healthy volunteers were included. CSLM parameters taken into consideration included: basal epithelial integrity, corneal thickness, epithelial cellular density, keratocyte activation, and sub-basal plexus morphology. Statistical analysis was carried out using SPSS-13 (Chicago IL, USA). Results. CSLM pachymetric data and the superficial epithelium cell density were significantly lower in pSS versus no-SS dry eye (p 0.0001); keratocyte activation and sub-basal nerve abnormalities were also more frequent in pSS patients (p 0.0001). CSLM findings well correlated with both the ocular test results and the patients’ perception of ocular dryness at the baseline and over the follow-up. Conclusion. CSLM might be a useful novel tool in the assessment of the involvement of the lachrymal functional unit in pSS.
Confocal scanning laser microscopy, Dry eye, Keratoconjunctivitis sicca, Lachrymal functional unit, Sjögren’s syndrome
Introduction Primary Sjögren’s syndrome (pSS) is a systemic autoimmune disorder characterized by chronic inflammation and functional impairment of the exocrine glands, usually presenting as a persistent dryness of the mouth and of the eyes. Although the disease might be frequently associated with a variety of scattered extraglandular manifestations, including malignant lymphoproliferative disorders, the hallmark of pSS is represented by chronic and disabling xerostomia and xerophthalmia [1–3]. The assessment of dry eye is currently based upon the combination of tests assessing aqueous tear flow and ocular surface damage, namely Schirmer’s test I, lissamine green staining (LGS), and tear break-up time test (TBUT) [4,5]. Nowadays, although extensively used, there is a general dissatisfaction regarding the clinical value of these traditional ocular tests in differentiating pSS from other dry-eye conditions [6–9]. Thus, a growing interest has arisen in developing new diagnostic tools that allow clinicians to improve pSS dry-eye assessment through an integrated analysis of the different components in the lachrymal unit [10]. This emerging trend reflects the general efforts of the last ten years in searching for novel specific and non-invasive diagnostic biomarkers for pSS and other rheumatic diseases [11,12]. Confocal scanning laser microscopy (CSLM) is a new non-invasive supplementary diagnostic tool for the in vivo assessment of the lachrymal functional unit disorder in many Correspondence to: Chiara Baldini, MD, Rheumatology Unit, University of Pisa, Via Roma 67, 56126 Pisa, Italy. E-mail: chiara.baldini74@gmail. com
History Received 24 July 2014 Accepted 17 October 2014 Published online 10 December 2014
anterior-segment diseases, including dry-eye disorders [13–16]. Preliminary in vivo CSLM studies revealed that, when compared with healthy volunteers, patients with pSS more frequently showed an irregular corneal epithelium with decreased density in the superficial corneal epithelium cells and decreased corneal thickness, an alteration of nerve fiber density, an increased subbasal nerve tortuosity and reflectivity, and an increased density of activated anterior keratocytes and conjunctival inflammatory cells [17,18]. To date, the usefulness of CSLM in differentiating pSS from the no-SS dry-eye diseases has been scarcely investigated and even the relationship between CSLM parameters and the traditional objective ocular tests for dry-eye syndrome are still unknown. In this study, we therefore analyze the in vivo corneal structure and sub-basal plexus nerves in patients with pSS and no-SS dry eye by CSLM, in order to evaluate the usefulness of CSLM in the diagnostic algorithm of the disease. We also correlated CSLM findings with routinely performed tear function tests (Schirmer’s test I, TBUT, and LGS).
Methods Study design This was a single-center case–control study aimed at investigating the ocular anterior-segment microstructure changes in pSS dry eyes and in other dry-eye conditions by CSLM. Subjects provided informed consent, and the study was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. The Institutional Review Board at the University of Pisa, Italy, approved this study.
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Patients eligibility Consecutive unselected patients, who attended the Rheumatology and Ophthalmology Units in the period between 2009 and 2012 due to persistent ( 3 months) subjective dry eye, were enrolled in the study. The diagnosis of pSS was made according to the American and European classification criteria (AECG) [4]. The condition of no-SS sicca syndrome was defined as the presence of xerostomia and xerophthalmia in patients with negative non-organ-specific autoantibodies and negative minor salivary gland biopsy (according to focus scoring system). Exclusion criteria were represented by the following conditions: corneal dystrophy/inflammation; systemic therapies with known corneal toxicity; topical anti-glaucoma drugs, steroids, or non-steroidal anti-inflammatory drugs; use of contact lenses; and previous interventions of ophthalmic surgery. The following baseline data were collected in all the cases: gender, age, disease duration, glandular and extra-glandular disease manifestations, number and type of previous/current disease-modifying anti-rheumatic drugs and prednisolone, and previous/current use of artificial tears. Moreover, at the time of the study entry, every patient had a rheumatologic and ophthalmological examination, complete laboratory tests, an evaluation of the auto-antibodies profile (i.e., anti-nuclear antibodies, extractable nuclear antigen (ENA) antibodies, and rheumatoid factor) and HCV/HBV serological markers, and thyroid function tests. The order of the ocular tests was based on the existing literature following a pre-defined sequence apparently able to minimize the influence of each test to the next test [5,7]. According to this protocol, the Schirmer’s test I was performed first, although potential injuries to the conjunctiva may occur when the test was performed (without anesthesia) [5,7,19]. The Schirmer’s test I (without anesthesia) was followed by instillation of fluorescein dye, determining the TBUT, and grading the corneal fluorescein staining pattern. After fluorescein grading of the cornea, lissamine green dye was applied and the conjunctiva was examined quickly and graded before the dye diffused or the intensity of staining diminished. The total time for these tests was approximately 20 minutes. The Schirmer’s test I was carried out as described elsewhere, using sterile Schirmer’s strips without anesthesia, in a room controlled for lighting (dimlit room), temperature (20–22 C), and humidity (40–60%) [20]. Abnormal value was regarded as 5mm/wetting after 5 min. In addition, GLS was performed as already reported and scored considering a Lissamine green score of 4 as pathological [21]. TBUT values less than 10 seconds were considered consistent with dry eyes [20]. Finally, we also assessed visual acuity (VA; using Snellen acuity charts) and performed intraocular pressure (IOP) measurement by Goldmann applanation tonometry and slit-lamp biomicroscopic examination without pupil dilation in all the cases. The latter was carried out in order to obtain a magnified image of (i) tear film, (ii) ocular surface, (iii) eyelids, and (iv) meibomian glands; furthermore, this test allowed us to carefully examine the anterior portions of the eye, including the anterior chamber and iris. Confocal scanning laser microscopy technique CSLM examination was performed by Confoscan 4.0 Nidek Technologies, equipped with a standard 40x objective. To avoid the diurnal variation of the corneal thickness, the imaging was performed between 09:00 and 11:00 hours. CSLM was performed at least five days after the other ocular tests. A local anesthetic solution (0.4% oxybuprocaine) was used before the CSLM examination in order to avoid blinking. After a drop of viscotears, gel was applied to the tip of the lens and the lens was advanced toward the central cornea until the gel contacted the cornea. The lens was
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aligned with the corneal apex until the operator saw a clear image of corneal endothelium. The alignment of the lens to the corneal apex was performed sometimes with a full automatic method (auto-adjust mode included in Nidek software), while other examinations required the use of a manual alignment; however, in most cases we used a semi-automatic alignment. Each scan recorded 350 z-axis images separated by a distance of 1.5 μm, making two to four complete passages from the lachrymal film to the anterior chamber. Each frame covered an area of approximately 460 345 μm. Z-scan curves (graphs showing the depth coordinate on the z-axis and the level of reflectivity on the y-axis) were used to select the images relating to the superficial and basal epithelium, the anterior and posterior stroma, and Bowman’s membrane or the sub-basal epithelium. Confocal scanning laser microscopy analysis The same ophthalmologist analyzed all the CSLM images and quantified the following variables: basal epithelial integrity, pachymetric data, keratocyte activation, superficial and basal epithelium cell densities, sub-basal corneal nerve density, tortuosity, and fiber reflectivity. Basal epithelial integrity was graded from Grade 0 (normal) to Grade 1 (slight loss) and 2 (severe loss) by the observation of three different frames of this layer [22]. Corneal thickness was defined as the distance in micrometers (μm) between the first surface image of the endothelial layer and the last frame of superficial epithelium on the z-scan axis. The pachymetric data of each eye were the result of the mean between all the pachymetric values obtained from each z-scan curve. Epithelial cell density (cells/mm2) was evaluated at the level of the superficial epithelium and at the basal epithelial layer by counting the cells present in one image with the manual counting procedure program of the instrument. We considered the entire area of the frame. The cells that were partially contained in the frame were considered only on the right and inferior side. The results were derived from the mean of three different counts for each layer [22]. As far as sub-basal plexus nerve was concerned, the number of nerves was defined as the sum of the nerve branches present in one image (nerves/frame) [23]. Reduced sub-basal corneal nerve density was graded according to a five-point severity scale: Grade 1 (very severe decrease), Grade 2 (severe decrease), Grade 3 (moderate decrease), Grade 4 (slight decrease), and Grade 5 (normal). The tortuosity and the reflectivity of the nerve fibers were graded according to the method proposed by Oliveira-Soto and Efron [18]: tortuosity was graded from Grade 0 (the nerve fibers were almost straight) to Grade 4 (the nerve fibers were very tortuous), and reflectivity (brightness) was graded from Grade 0 (the nerve fibers are indistinguishable from the background) to Grade 4 (the reflectivity was much higher than the background) as previously described. In both cases, the severity grading scale assigned a Grade 0 for a normal CSLM observation; a Grade 1 for a slight, not clinically significant abnormality; a Grade 2 for a moderate abnormal CSLM observation; and a Grade 3 and 4 when severe clinical findings and very severe clinical findings, respectively, were observed at the CSLM. Also in this case, the grade of each eye was evaluated by the mean of the values calculated in three different frames of the same layer. Finally, estimates of human keratocyte density was performed using manual analysis by marking each clearly defined cell or nucleus in a pre-defined rectangular frame; the activation of the keratocytes in the anterior stroma was evaluated according to a score varying from Grade 0 (no keratocytes activated) to Grade 1 (until 4 keratocytes activated for frame) and Grade 2 (more than 4 activated keratocytes for frame). We considered the mean between the values obtained from the three frames of each eye with more keratocyte activity [22].
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Table 1. Patients’ clinical features.
Statistical analysis Data are presented as percentages along with the number of patients or as the mean (standard deviation). Contingency table analysis, Fisher’s exact test, and analysis of variance (ANOVA) test were used for statistical analysis. Spearman’s rank correlation was applied for detecting correlations between ocular tests results and CSLM findings. Statistical analysis was carried out using SPSS 13 (SPSS Inc., Chicago, IL, USA).
Results
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Study population and clinical data Thirty-four eyes from 17 consecutive females with pSS (AECG criteria) and 32 eyes from 16 consecutive females with a diagnosis of no-SS dry eye were investigated in this study. Twenty healthy women were included as controls. According to previous studies [17,24,25], data from the eye with the highest fluorescein staining score was selected. In the case of equal scores for the two eyes, discriminant criteria considered were, in order of relevance, conjunctival staining and TBUT. The clinical characteristics of the subjects enrolled are provided in Table 1. There was no significant difference in age among the pSS [mean age (SD) 56.5 (13.9) yrs], no-SS dry eye (mean age SD 49.8 15.7 yrs), and healthy volunteer groups [mean age (SD) 53.4 (14.2) yrs]. Moreover, no difference was detected regarding the duration of ocular dryness symptoms between pSS and no-SS dry-eye patients [mean duration (SD) 2.5 (2.7) vs 2 (1.5) yrs, p n.s]. As expected, pSS patients presented a higher frequency of xerostomia, arthralgias, hypergammaglobulinemia, and serologic abnormalities including positivity for anti-nuclear antibodies, anti-Ro/SSA, and rheumatoid factor. All the patients and pathological controls underwent a minor salivary gland biopsy: 14/17 of the pSS patients presented a focal sialoadenitis (FS 1), whereas none of the no-SS dry-eye subjects showed a focal sialoadenitis at the histologic examination. Finally, none of the subjects enrolled had positive serological markers for HCV/ HBV infection. All the patients were using artificial tears and 11 out of 17 pSS patients were also receiving low-dose oral prednisolone (∼ 4mg per day) and hydroxychloroquine (400 mg per day). The ocular test results including Schirmer’s test I, TBUT, and GLS are reported in Table 1. A positive correlation was found between Schirmer’s test I results and TBUT (r 0.504, p 0.003), and a negative correlation was observed between Schirmer’s test I results and GLS (r 453, p 0.009). No statistically significant difference was found between pSS patients and patients with no-SS dry-eye syndrome as far as Schirmer’s test I, TBUT results, and GSL scores were concerned. None of the patients presented any abnormalities in visual acuity and IOP measurement.
Number of patients Age (M SD) Disease duration (M SD, yrs) Xerophthalmia Xerostomia Minor salivary gland biopsy (FS 1) Salivary gland enlargement Arthralgia Low C3/Low C4 Leukopenia Hypergammaglobulinemia Anti-nuclear antibodies Anti-Ro/SSA Anti-La/SSB Rheumatoid factor Thyroiditis Schirmer’s test I (M SD, mm) TBUT (M SD, s) Lissamine green test (score 4)
pSS 17 (15 F:2 M) 56.5 13.9 2.5 2.7
no-SS dry eye 16 (13 F:3 M) 49.8 15.7 2 1.5
p value n.s. n.s. n.s.
17 (100%) 15 (88.2%) 14 (82.3%)
16 (100%) 8 (50%) None
n.s. 0.02 0.0001
4 (23.5%) 8 (47%) 4 (23.5%) 4 (23.5%) 7 (41.2%) 17 (100%) 9 (53%) 4 (23.5%) 10 (59%) 5 (29.4%) 4.7 1.9
None None None 1 (6%) None 2 (12%) None None 1 (6%) 2 (12.5%) 5.4 1.8
n.s. 0.003 n.s. n.s. 0.007 0.0001 0.0009 n.s. 0.002 n.s. n.s.
5.7 1.5 8/17 (47%)
6.7 1.5 7/16 (44%)
n.s. n.s.
F female, M male, M SD mean standard deviation, yrs years, FS focus score
CSLM corneal morphology in pSS, no-SS dry eye, and healthy volunteers Table 2 reports the CSLM parameters evaluating corneal morphology and corneal sub-basal plexus nerves in pSS, no-SS dry eye, and healthy volunteers. From the statistical analysis we retrieved the following data. First, we found that the epithelial corneal thickness was significantly reduced in pSS patients with respect to both healthy volunteers and no-SS dry eye. By contrast, no difference was detected between healthy volunteers and no-SS dry-eye patients. We also found that the cell density of the superficial epithelium was significantly reduced in the pSS group when compared with that in the other two groups. Moreover, the superficial epithelial cell density was significantly lower in pSS patients presenting with severe alterations of the sub-basal nerve plexus (1049 186 vs 1192 129, p 0.019). There was no significant difference in the density of the basal epithelial cell between pSS and no-SS sicca patients. Nonetheless, in both cases the basal epithelial cell density was higher than that in the healthy volunteers group. The basal epithelial integrity was significantly more compromised in pSS and no-SS dry eyes rather than that in healthy volunteers (Figure 1a). Second, regarding the sub-basal nerve plexus, we observed that the number of the nerve fibers visualized in the single photo
Table 2. Corneal morphology and corneal sub-basal plexus nerves in pSS, no-SS dry eye, and healthy volunteers (CTRL).
CSLM findings
CTRL (M SD)
pSS (M SD)
no-SS dry eye (M SD)
Corneal thickness (μm) Basal epithelium cell density (cells/mm2) Superficial epithelium cell density (cells/mm2) Sub-basal corneal nerve density (grade #2) Tortuosity (grade 3) Reflectivity (grade 3) Keratocyte activation (grade 2) Epithelial integrity disruption (grade 2)
556 37 5793 290 1548 12.6 None 2/20 (10%) 2/20 (10%) 3/20 (15%) 2/20 (10%)
510.5 24.6 5993 337.9 974.7 58.5 13/17 (76.5%) 11/17 (64.7%) 12/17 (70.6%) 15/17 (88.2%) 15/17 (88.2%)
566.6 22.5 6078.5 114 1280.7 90.5 5/16 (31.3%) 4/16 (25%) 4/16 (25%) 8/16 (50%) 8/16 (50%)
p value CTRL vs pSS 0.000 n.s 0.000 0.000 0.001 0.000 0.000 0.000
pSS vs no-SS 0.000 n.s 0.000 0.03 0.037 0.015 0.026 0.026
CTRL vs no-SS n.s. 0.001 0.000 0.000 n.s n.s 0.034 0.011
Note: Statistical analysis was performed by ANOVA test and contingency table (p 0.05). CSLM confocal scanning laser microscopy, CTRL healthy volunteers, pSS primary Sjögren’s syndrome, no-SS non-autoimmune dry-eye condition, M SD mean standard deviation.
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Figure 1. CSLM findings in healthy volunteers (CTRL), patients with no-SS and pSS: (a) epithelial integrity; (b) sub-basal nerve tortuosity; (c) sub-basal nerve reflectivity; (d) keratocyte activation. ***p 0.0001, **p 0.001, *p 0.05.
frame was significantly reduced in both pSS and no-SS dry-eye in comparison with healthy controls (p 0.0001). Moreover, the sub-basal nerve plexus tortuosity and reflectivity were significantly increased in pSS (Figure 1b and c). A statistically significant difference in the density of the nerve fibers (p value 0.0001) was also detected in no-SS dry-eye patients when compared with healthy volunteers, and a significant correlation was found between the density of the nerve fibers and the Schirmer’s test I results (r 0.496, p 0.003). Finally, the keratocyte activation was significantly higher in pSS and in no-SS dry eye than in the healthy volunteers. Keratocyte activation was still significantly higher in pSS patients in comparison to those with no-SS dry eyes (Figure 1d). A negative correlation was found between keratocyte activation and the density of the sub-basal nerves (r 0.712, p 0.000), whereas a positive correlation was found between keratocyte activation and both the sub-basal nerve plexus tortuosity (r 0.470, p 0.006) and reflectivity (r 0.508, p 0.003).
Discussion In this study, we reported a single-center experience on the application of CSLM for the assessment of dry eyes in pSS patients. The results of our study confirmed the value of CSLM for detecting pSS damage of the corneal epithelium layers and a scattered combination of different sub-basal nerve plexus abnormalities. We particularly observed a significant reduction in the epithelial corneal thickness with a decrease in the cell density of the corneal superficial epithelium and a relative increase in the basal epithelial cells. We also described the presence of many irregularities of the basal epithelial integrity, a remarkable increase in the activation of the keratocytes, a reduction in the number of the fibers
of the sub-basal plexus nerves, and an increase in the tortuosity and of the reflectivity of the nervous fibers, thus supporting the hypothesis of chronic inflammation of all the corneal anatomical structures in pSS. These results reflected previous CSLM studies which documented several morphological abnormalities in lachrymal functional unit of patients with pSS. In 2003, Tuominen et al. [26] firstly showed epithelial, stromal, and neural abnormalities in the corneas of 10 patients with pSS. Specifically, they demonstrated that pSS dry eye was characterized by keratocyte activation, abnormalities in the sub-basal nerve fiber morphology, and a reduced thickness of the central cornea. Similarly, in 2004, Benitez del Castillo et al. [27] documented a decrease in the cell density of the superficial epithelium and significant alterations in the corneal innervations in 10 patients with pSS. Subsequently, Villani et al. [17] revealed a significant decrease in the cell density of the superficial epithelium, and a significant increase in the cell density of the basal epithelium in pSS patients with respect to controls. Moreover, the authors showed a statistically significant difference in the number of nerve fibers visualized in the single CSLM photo frame and in the tortuosity of the fibers. In addition to all these findings, with the present work, we also demonstrated that the vast majority of the CSLM pathological findings were significantly interrelated to each other. Indeed, we found a close correlation between superficial epithelial cellular density and the number of nerve fibers per photo frame, and we also observed that the loss of the epithelial integrity and the disruption of the intercellular junctions significantly correlated with the sub-basal plexus nerve alterations and with the activation of the keratocytes. In turn, keratocyte activation was correlated with the sub-basal nerve plexus abnormalities. Significant correlations were also found between the results of the traditional ocular tests, particularly Schirmer’s test I, and CSLM findings.
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DOI 10.3109/14397595.2014.979523
Taken together, our observation and previous observations further support the hypothesis that in pSS, dry eye might represent not only a disorder of the tear film, but rather the consequence of the defective homeostasis of the ocular surface. In other words, the inflammation of the ocular surface and the alteration of corneal innervations might act together resulting in the impairment of the normal lachrymal reflex and in the gradual destruction of the lachrymal functional unit. At the moment, the use of CSLM might not be generalizable to routine differential diagnosis of xerophthalmia, since, reliable cutoff values able to distinguish between pSS and no-SS dryeye conditions have not been validated yet. However, our results encourage the use of CSLM as a non-invasive novel technique for a better understanding of the physiopathology of keratoconjunctivitis sicca associated with pSS. In addition, although CSLM procedures can be time consuming and may exceed practical limits in routine clinical work flow, being able to demonstrate alterations in the superficial epithelial cells and corneal nerves, and immune cell changes at a cellular level that correlate with clinical signs and symptoms of pSS, CMLS may serve as a useful assessment tool supplementary to more traditional clinical diagnostic modalities. More specifically, at least in the setting of clinical trials, this technique may provide a better diagnostic differentiation of pSS from degenerative non-autoimmune forms of dry eyes, and a more accurate treatment stratification and measurement of therapeutic efficacy when taken together with clinical tests.
Acknowledgements We wish to thank Dr. Francesca Sernissi and Miss Wend Doherty for their valuable contribution in reviewing the text.
Conflict of interest None.
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