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(Investigative Ophthalmology and Visual Science. 2003;44:2545-2549.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-1260
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Corneal Innervation and Morphology in Primary Sjögren’s Syndrome

Ilpo S. J. Tuominen,1 Yrjö T. Konttinen,2,3 Minna H. Vesaluoma,1 Jukka A. O. Moilanen,1 Maaret Helintö,1 and Timo M. T. Tervo1

1From the Department of Ophthalmology, University of Helsinki, Finland; the 2Department of Medicine, Invärtes Medicin, Helsinki University Hospital, Finland; and the 3Orton Orthopedic Hospital of the Invalid Foundation, Helsinki, Finland.


   Abstract
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Methods
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 Discussion
 Conclusions
 References
 
PURPOSE. To analyze the in vivo morphology of the different corneal sublayers and corneal nerves in primary Sjögren’s syndrome (SS).

METHODS. Ten eyes of 10 patients with primary SS and 10 eyes of 10 sex- and age-matched control subjects were investigated. Diagnosis was based on American–European consensus criteria. In vivo confocal microscopy with through-focusing was used to investigate corneal morphology and to measure corneal sublayer thickness.

RESULTS. Epithelial punctate staining with fluorescein was observed in 6 of 10 SS and none of 10 control corneas. In addition, Schirmer I test results were significantly lower in SS. Epithelial thickness did not differ between the SS and control groups. Confocal microscopy revealed patchy alterations or irregularities in surface epithelial cells in 6 of 10 SS corneas, whereas the basal epithelium appeared normal in all corneas. Average corneal thickness was lower in the SS group (515.9 ± 22.0 µm) than in the control (547.4 ± 42.0 µm; P = 0.050, t-test). Accordingly, the mean intraocular pressure was lower in the SS group (13.9 ± 2.1 mm Hg) than in the control (16.7 ± 2.9 mm Hg; P = 0.022). The subbasal nerve plexus and stromal nerve fiber bundles were present in all corneas. No difference was noted in nerve density. However, in 4 of 10 SS eyes, the subbasal nerve plexus showed structures resembling nerve sprouting, suggesting ongoing active neural growth. None of the control corneas exhibited such features. Signs of anterior keratocyte activation were observed in 5 of 10 SS corneas.

CONCLUSIONS. In SS, the corneal surface epithelium was irregular and patchy. Anterior keratocytes frequently showed morphologic features of activation. The subbasal nerve fiber bundles revealed abnormal morphology, and the central corneal thickness was reduced by stromal thinning. The findings confirm epithelial, stromal, and neural abnormalities in the corneas of patients with SS.

Dry eye is a common disorder with many causes. It manifests symptoms of varied severity. Sjögren’s syndrome (SS) is a chronic, generalized autoimmune disease, with dry eye as its predominant clinical manifestation. In primary SS, typical symptoms occur in a pure form without an association with any other underlying autoimmune disease such as rheumatoid arthritis or systemic lupus erythematosus.1 2

The prevalence of peripheral neuropathy, most commonly of the distal sensory symmetrical type, ranges from 10% to 30% in primary SS.3 4 5 Often, the neuropathy is subclinical. Trigeminal sensory neuropathy is the best-known cranial neuropathy in SS.6 7 In a Japanese study of patients with primary SS, trigeminal neuropathy was observed in 8 (38%) of 21, whereas in a Finnish study, the condition was noted in only 2 (4%) of 48 patients.8 9 Electrophysiological studies of the trigemino-facial and trigemino-trigeminal reflexes in patients with trigeminal nerve involvement suggest lesions in the neurons of the Gasserian ganglia rather than in the trigeminal axons.10 The pathophysiological mechanisms are unknown, but may involve vasculitis11 or lymphocytic inflammation of nerve cell ganglia.12

Corneal innervation has a trophic effect on corneal epithelial cells.13 Substance P (SP), calcitonin gene-related peptide (CGRP), and nerve growth factor (NGF) regulate corneal epithelial proliferation, integrity, and wound healing.14 15 16 Epithelial wound healing is delayed in denervated corneas after keratectomy, and denervation also predisposes to recurrent corneal erosions.17 In addition, Xu et al.18 showed that corneal sensitivity is decreased in SS-related dry eye. Intact corneal innervation is mandatory for normal blinking and tearing reflexes, because the ocular surface, lacrimal glands, and interconnecting nerves form a functional unit. A compromise in one portion of this reflex arch may result in ocular surface disease.19

Based on current knowledge of peripheral and cranial nerve involvement associated with primary SS and the importance of corneal innervation in lacrimation, we assessed corneal nerve density and morphology, by in vivo confocal microscopy.


   Methods
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Results
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Patients
Twenty eyes of 20 volunteers were examined. Ten patients with primary SS (nine women and one man) aged 50.1 ± 13.5 years (range, 29–67) were recruited from the Department of Rheumatology, Helsinki University Central Hospital, where they had been thoroughly examined by a rheumatologist. Ten age- and sex-matched healthy control subjects (nine women and one man) aged 48.3 ± 14.5 years (range, 29–65) served as the control group. The mean duration of dry eye symptoms was 16.3 ± 8.2 years, but the average time elapsed from diagnosis was only 8.0 ± 4.6 years.

The SS diagnosis was made according to American–European consensus criteria.20 21 The diagnosis was made if any four of the six following conditions were fulfilled, as long as either item 4 (histopathology) or 6 (autoantibodies) existed. Diagnosis was also made if any three of the four objective criteria were present (that is items 3–6): (1) ocular symptoms (daily, persistent, troublesome dry eyes for more than 3 months and/or recurrent sensation of sand or gravel in the eyes, and/or use of tear substitutes more than three times a day); (2) oral symptoms (daily feeling of dry mouth for more than 3 months and/or recurrently or persistently swollen salivary glands as an adult or need to drink liquids frequently to aid in swallowing of dry food); (3) ocular signs (Schirmer’s I test, performed without anesthesia [≤5 mm in 5 minutes] and/or Rose bengal score or other ocular dye score [≥4 according to van Bijsterveld’s scoring system]; (4) histopathology (focal lymphocytic sialadenitis in minor salivary glands); (5) salivary gland involvement (unstimulated whole salivary flow (<1.5 mL in 15 minutes) and/or parotid sialography showing the presence of diffuse sialectasia and/or salivary scintigraphy showing delayed uptake, reduced concentration, and/or delayed excretion of tracer); and (6) autoantibodies (antibodies to Ro [SS type A] and/or La [SS type A]).

Patient were excluded if they had had head and neck radiation treatment; hepatitis C infection; acquired immunodeficiency disease syndrome (AIDS); preexisting lymphoma, sarcoidosis, or graft-versus-host disease; or if they used anticholinergic drugs. For more detailed information, see American–European consensus criteria.21

All patients used topical ocular lubricants, and five of them received oral anti-rheumatic drugs (methylprednisolone or hydroxychloroquine sulfate). Four patients and two control subjects had hypertension, two had autoimmune thyroiditis and were treated with thyroxin substitution, two had interstitial cystitis, one had autoimmune hepatitis treated with corticosteroids, and one had peripheral symmetrical stocking-like sensory neuropathy in the lower extremities. The latter condition responded to oral corticosteroid therapy. One patient had a history of mammary carcinoma, and another had a history of ocular meningioma. In the latter patient, the contralateral eye was examined in the study. One control subject had bronchial asthma treated with inhaled corticosteroids and ß2-agonists.

The study was approved by the Ethics Review Committee of Helsinki University Eye Hospital and was performed according to the tenets of the Declaration of Helsinki. Informed consent was obtained from all patients and control subjects.

Clinical Evaluation
A careful biomicroscopic examination of the anterior segment of the eye was performed, along with fluorescein staining of the cornea and evaluation of anterior chamber flare and cell reactions. Intraocular pressure (IOP) was measured with Goldmann applanation tonometry. The Schirmer I test was performed with the patient’s eyes closed and under topical anesthesia.

In Vivo Confocal Microscopy
A tandem scanning confocal microscope (model 165A, Tandem Scanning Corp., Reston, VA) was used to examine the central cornea of patients at Helsinki University Eye Hospital. The setup and operation of the confocal microscope has been described previously.22 Briefly, a 24x, 0.6 numerical aperture, variable-working-distance objective lens was used. The field-of-view with this lens is 450 x 360 µm, and the z-axis resolution is 9 µm. Images were detected by a low-light-level camera (model VE1000; Dage-MTI, Michigan City, IN) and recorded on SVHS tape. Video images of interest were digitized, with a computer-based imaging system with custom software (University of Texas, Southwestern Medical Center at Dallas, Dallas, TX).

In addition, confocal microscopy through-focusing (CMTF) scans were obtained as previously described.23 Intensity profile curves for digitized CMTF data were calculated. Eight CMTF scans were performed in each individual. Because of eye movement during the examination or inadequate quality, some of the scans were rejected. On average, 5.0 ± 2.1 (range, 2–8) acceptable CMTF scans were obtained from each eye. From each CMTF scan, the epithelial layer, Bowman’s layer, and total corneal thicknesses were measured. The mean of each individual’s measurements was used for statistical analysis.

After the layers of central cornea had been scanned with a confocal microscope, special attention was paid to the morphology of the surface and basal epithelia. Subsequently, the microscope was focused beneath the basal epithelium to evaluate the subbasal nerve plexus in detail. Afterward, images were digitized from the videotape, and the number of nerve fibers in the image was counted by using point-counting principles. The number of nerve fibers in the image with most subbasal nerve fibers was used as a parameter for nerve density. The nerve morphology was assessed from the SVHS-videotape and digitized images. The morphology of the nerve fibers was evaluated, and attention was paid to the following aspects: thickness (fine and faint, normal, thick), beading, side-branching, and sprouting. It must be remembered, however, that the resolution of the confocal microscope enables counting of nerve fiber bundles but not single fibers or terminals.

Statistical Analyses
Statistical analyses were performed with (SPSS for Windows, ver. 9.0; SPSS Sciences, Chicago, IL). Normality was determined with the Kolmogorov-Smirnov test, and Student’s t-test was performed for comparison of the groups. All data are expressed as mean ± SD. Differences were considered to be statistically significant at P < 0.05.


   Results
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Discussion
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Clinical Data
Schirmer I test results in patients under local anesthesia were lower in patients with SS than in the control: 3.0 ± 2.8 and 7.8 ± 4.8 mm (P = 0.015, t-test), respectively. IOP in the SS group was 13.9 ± 2.1 mm Hg and in the control group, 16.7 ± 2.9 mm Hg (P = 0.022, t-test). Fluorescein staining revealed epithelial punctate changes in 6 of 10 corneas of the SS group and in none of the 10 in the control group.

CMTF Data
Epithelial thickness was 49.8 ± 4.9 µm in SS and 51.3 ± 3.6 µm in the control (P = 0.445, t-test). Thickness of the Bowman’s layer was also similar in both groups, 17.5 ± 3.2 and 17.2 ± 2.2 µm, respectively (P = 0.809, t-test). Total corneal thickness was reduced in the SS group compared with the control, with respective averages of 515.9 ± 22.0 and 547.4 ± 42.0 µm, respectively (P = 0.050, t-test).

In Vivo Confocal Microscopy
The surface epithelium was irregular or patchy in 6 of 10 SS corneas (Fig. 1A) , whereas control corneas showed normal morphology (Fig. 1B) . Basal epithelial cells appeared normal in all corneas. The subbasal nerve plexus and stromal nerve fiber bundles were identified in all corneas of both groups. The subbasal nerve density did not differ between SS and control groups: there were 5.4 ± 1.8 or 5.0 ± 1.4 long nerve fiber bundles per microscopic field, respectively (P = 0.584, t-test). A nerve-sprouting pattern (Figs. 2A 2B) indicating neural regeneration was found in 4 of 10 SS corneas at the level of the subbasal nerve plexus. One patient with SS who had severe peripheral neuropathy had very thin subbasal nerve fiber bundles without beading (Fig. 2C) . Control subjects, by contrast, had relatively thick nerve fibers. Only a single highly reflective nerve fiber bundle was observed in one of the control subjects. In addition, subbasal nerve fiber bundles showed abnormal tortuosity in 2 of 10 eyes of the SS group (Fig. 3B) . No such abnormality was seen in the control group (Fig. 3D) . Interindividual variation in the density of the subbasal nerve plexus was wide, ranging between four and nine long nerve fiber bundles per microscopic field. Round particles, measuring approximately 11 to 12 µm in diameter, possibly cells of inflammatory origin, were found in the level of subbasal nerve plexus of one SS cornea (Fig. 3A) .



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  		FIGURE 1. (A) Surface epithelium in the cornea of a 41-year-old woman with primary SS. The epithelium showed patchy and irregular changes. Highly reflecting epithelial cells were also visible. (B) Surface epithelium in the cornea of a 30-year-old woman in the control group. The ocular surface was normal. Epithelial cells had halos around the nuclei and sharp borders. (C) Activated keratocytes in the anterior stroma of a 29-year-old woman with SS. The optical density of keratocytes was higher than in the control (D), and they formed vacuoles. (D) Normal anterior keratocytes in a 48-year-old woman in the control group. The size of each image is 300 x 240 µm.

 


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  		FIGURE 2. (A) Nerve sprouting (arrow) in the subbasal nerve plexus in a 35-year-old woman with a 15-year history of SS. The subbasal nerve fiber bundles in this field were fine and faint. (B) Nerve sprouting (arrow) in a subbasal nerve fiber bundle in a 41-year-old woman with a 20-year history of SS symptoms. (C) Extremely thin and faintly visible subbasal nerve fiber bundles in the cornea of a 67-year-old woman with SS. The patient also had a history of peripheral neuropathy in the lower limbs and a 25-year history of SS symptoms. (D) Normal subbasal nerve plexus in a 58-year-old male in the SS group. The size of each image is 300 x 240 µm.

 


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  		FIGURE 3. (A) Abnormal particles, possibly cells of inflammatory origin, at the level of the subbasal nerve plexus of a 59-year-old woman with a five-year history of SS. The diameter of the particles is approximately 11 to 12 µm. (B) Abnormally tortuous nerve fiber bundles from same patient as in (A). (C) Beading in the subbasal nerve fiber bundles of a 37-year-old woman with a 19-year history of SS. (D) Normal subbasal nerve plexus in a 41-year-old woman in the control group. The size of each image is 300 x 240 µm.

 
The anterior keratocytes showed signs of activation, hyperreflectivity, or visible processes24 in 5 of 10 SS corneas (Fig. 1C) . Three of these corneas also showed nerve sprouting (Figs. 1A 1B) . Normal-appearing stromal nerves were observed in all subjects.


   Discussion
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Corneal Thickness
The thickness of the normal cornea is relatively constant, varying only a few percentage points throughout the day because of changes in evaporation from the surface. Maintenance of corneal thickness depends on the pump and barrier functions of the epithelium and endothelium, the latter being mainly responsible for corneal dehydration. Our results are in line with those of Liu and Pflugfelder,25 who studied aqueous tear-deficiency dry eye, and with the findings of Guzey et al.,26 who studied trachomatous dry eye. They both found central corneal thickness to be decreased. However, the methods used are different. We used CMTF, which has been shown to be a very accurate and precise technique, comparable to pachymetry. In addition, it allows corneal sublayer pachymetry, which is not possible with other in vivo techniques.27 Liu and Pflugfelder25 hypothesized that increased production of inflammatory and catabolic cytokines, such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1 explains the corneal thinning. Moreover, an abnormally rough ocular surface contributing to increased shear force may mechanically affect corneal thickness. Our findings demonstrate, however, that the thicknesses of the epithelium and Bowman’s layer were normal. Consequently, the thinner cornea in SS must result from the thinning of the stroma. If shear forces play a significant role in thinning, then thinning of the epithelial cell layer would be expected. The present findings may support the idea of an ongoing inflammatory process leading to stromal thinning. Phenomena such as apoptosis28 29 and increased stromal proteolysis (Selzer M, Afonso A, Monroy D, Lokeshwar B, Pflugfelder SC, ARVO Abstract 3072, 1998)30 have been attributed to SS.

In the SS group, the apparent IOP was lower than in the control group. The central corneal thickness influences the results of Goldmann applanation tonometry, which may explain our finding. According to a recent meta-analysis by Doughty and Zaman,31 in eyes with chronic diseases, a 10% difference in central corneal thickness results in 2.5 mm Hg difference in IOP. We noted a 6% (31.5 µm) decrease in the central corneal thickness and 2.8 mm Hg lower mean IOP.

Subbasal Innervation, Anterior Keratocytes, and NGF
Corneal nerves are mainly derived from the ophthalmic branch of the trigeminal nerve.32 33 The cornea is the most densely innervated peripheral tissue in the human body. Three modalities of sensation, mechanical, thermal, and chemical, are represented in the human cornea. Most of the nerves display polymodal nociceptors.34 35 Nerve fibers terminate as free nerve endings between the epithelial cells. Even single epithelial and stromal cells may be innervated.36 37 The beading (Fig. 3C) is thought to be characteristic of metabolically active, transmitter-containing nerve fibers.38 Peptidergic nerves containing neuropeptides such as GCRP and SP have been demonstrated in the human cornea.14 15 The neurotrophic influence of these neuropeptides on corneal epithelial cells has been demonstrated or suggested in many experimental studies.17 38

A somewhat unexpected result of our study was that subbasal nerve density was not reduced in SS. Instead, eyes of 4 of 10 patients with SS showed nerve sprouting in the subbasal nerve fiber bundles. We hypothesize that this indicates regeneration of the sensory nerves. Corneal nerves show sprouting after neonatal treatment with capsaicin, the pungent component of chili peppers, which induces release of neuropeptides from primary afferent sensory neuron nerve endings.39 40 Overexpression of NGF has been found to induce hypertrophy of the peripheral nervous system.41 Focally applied neutralizing antibodies to neurotrophic factors reduce collateral axonal branching after a peripheral nerve lesion.42 Our patient with severe stocking-like peripheral sensory neuropathy had remarkably fine and faint subbasal nerves without beading. However, nerve sprouting was observed as well. Three corneas with nerve sprouting also showed signs of activation in the anterior stromal keratocytes. Of interest, keratocytes are also activated after corneal wounding24 and after certain inflammations, such as Acanthamoeba keratitis43 or herpes simplex keratitis.44 Because NGF has been shown to be expressed concomitantly with {alpha}-smooth muscle actin in dermal myofibroblasts,45 which can be interpreted to be analogous to activated corneal anterior keratocytes, we postulate that activated corneal keratocytes also express NGF.

We hypothesize that chronic inflammation and diminished volume of tear fluid enriched in proinflammatory cytokines, such as IL-1 and -6,46 47 lead to activation of keratocytes, which is followed by synthesis of NGF or other neuronal growth factors. NGF and its high-affinity receptor TrkA are expressed in human corneal stroma and epithelium.16 48 NGF facilitates transmission of pain (hyperalgesia), possibly due to its effect on ion channels.49 50 51 Overexpression of neuronal growth factors could explain not only the observed changes in corneal nerves but also the tenderness, or even the chronic pain, often experienced by these patients.


   Conclusions
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 Abstract
 Methods
 Results
 Discussion
 Conclusions
References
 
Two distinct features were shown in corneas affected by primary SS dry eye: The central corneal thickness (stroma) was decreased, possibly because of metabolic changes and/or inflammation and corneal subbasal nerves were present but showed abnormal morphology, such as sprouting of nerve fiber bundles, which implies ongoing neural regeneration. The morphologic neural alterations may interfere with sensory function, contributing to decreased tear fluid secretion in patients with SS.


   Footnotes
 
Presented in part at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 2000.

Supported by the Finnish Medical Society Duodecim, Special State Grant for Research, the University of Helsinki; Helsinki University Central Hospital; the Finnish Eye and Tissue Bank Foundation; and the Finnish Eye Foundation.

Submitted for publication December 10, 2002; revised February 5 and 17, 2003; accepted February 21, 2003.

Disclosure: I.S.J. Tuominen, None; Y.T. Konttinen, None; M.H. Vesaluoma, None; J.A.O. Moilanen, None; M. Helintö, None; T.M.T. Tervo, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Ilpo S. J. Tuominen, Helsinki University Eye Hospital, PO Box 220, Fin-00029 HUS, Finland; ilpo.tuominen{at}hus.fi.


   References
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 References
 

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