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Tear function and ocular surface changes in noninsulin-dependent diabetes mellitus
Tear Function and Ocular Surface Changes in Noninsulin-dependent Diabetes Mellitus Murat Dogru, MD, PhD, Chikako Katakami, MD, PhD, Masanori Inoue, MD, PhD Purpose: To describe the ocular surface disorder in patients with diabetes. Design: A prospective, case-controlled study. Participants: Eighty-eight eyes of 50 noninsulin-dependent diabetes mellitus patients seen at the Department of Ophthalmology, Kobe University School of Medicine, from September 1998 through February 1999, and 40 eyes of 20 healthy control individuals were studied. Intervention: All subjects underwent routine ophthalmic examinations, corneal sensitivity measurements, Schirmer test, tear film break-up time (BUT) analysis, and conjunctival impression cytologic analysis. Main Outcome Measures: Patients and control subjects were compared for corneal sensitivity, tear function parameters, goblet cell density, and squamous metaplasia grade. The relation of diabetic peripheral neuropathy, metabolic control, duration of disease, and status of retinopathy to the ocular surface disorder was also noted. Results: The mean corneal sensitivity was significantly lower in diabetic patients, diabetic patients with peripheral neuropathy, and poorly controlled diabetes compared with control subjects (P ⬍ 0.001). The BUT and Schirmer test values were also significantly lower in the diabetic group, in patients with peripheral neuropathy and poor metabolic control. Impression cytologic analysis showed goblet cell loss and conjunctival squamous metaplasia, both of which again related to peripheral neuropathy, poor diabetic control, and decreased corneal sensitivity. The examined parameters did not relate to duration of disease or status of diabetic retinopathy. Conclusions: The ocular surface disease in diabetes is characterized by a disorder of tear quantity and quality, squamous metaplasia, and goblet cell loss, all of which seem to evolve in close proximity to the status of metabolic control and peripheral neuropathy. Ophthalmology 2001;108:586 –592 © 2001 by the American Academy of Ophthalmology. A number of ocular complications have been reported to be associated with diabetes mellitus, many of which can lead to blindness. Although ocular manifestations such as chronic inflammation of the lids, acute orbital infections, cataract, and diabetic papillopathy and retinopathy have been recognized and studied extensively, the awareness of corneal complications has occurred in recent years.1 Schultz et al2 indicated that 47% to 64% of diabetic patients have primary corneal lesions during their life time. Epithelial fragility, microcystic edema and bleb formation, superficial punctate keratopathy, persistent epithelial defects, recurrent corneal erosions, delayed epithelial healing,3,4 decreased corneal sensitivity [unpublished data; MacRae S, et al. Invest Oph-
thalmol Vis Sci 1982;22(Suppl):200], neurotrophic corneal ulceration, dry eye, filamentary keratitis, and Descemet folds constitute a gamut of diabetic corneal complications.5–7 The cause of these alterations, however, is not clear. In addition, studies relating the changes of tear film and ocular surface in diabetic patients to clinical parameters of diabetes are still rare. Therefore, we aimed to describe the ocular surface disorder in diabetes by performing corneal sensitivity measurements, tear film break-up time analysis, Schirmer test, and conjunctival impression cytologic analysis and to compare the results with those of healthy control subjects.
Materials and Methods Originally received: March 6, 2000. Accepted: November 10, 2000. Manuscript no. 200124. From the Department of Ophthalmology, Kobe University School of Medicine, Kobe, Japan. The preliminary work was presented at the 22nd Japan Congress of Cornea, February 1999, Ube, Yamaguchi, Japan, and at the Fifth Japanese Ophthalmic Diabetology Congress, March 1999, Nagoya, Japan. The finalized work was presented at the annual meeting of the Association for Research in Vision and Ophthalmology, May 2000, Fort Lauderdale, FL. The authors have no proprietary interest in any of the products mentioned in this paper. Address correspondence to Chikako Katakami, MD, PhD, Department of Ophthalmology, Kobe University School of Medicine, Kusunokicho, 7-5-2, Chuoku, Kobe 650-0017, Japan. E-mail: [email protected].
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© 2001 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
Participants and Examinations Eighty-eight eyes of 50 patients with noninsulin-dependent diabetes mellitus (24 males, 26 females) aged between 44 and 75 years (mean, 54.2 years) as well as 40 eyes of 20 healthy subjects aged from 39 to 70 years (mean, 56.4 years; 7 males, 13 females) were recruited from the Department of Ophthalmology of Kobe University Hospital from September 1998 through February 1999. Both groups were similar regarding age and gender characteristics. The presence of diabetes in all patients had been confirmed by the corresponding internal medicine department. The diagnosis of peripheral neuropathy was based on abnormal nerve conduction velocity test results and on the presence of symptoms and signs of ISSN 0161-6420/00/$–see front matter PII S0161-6420(00)00599-6
Dogru et al 䡠 Impression Cytologic Analysis in Diabetes diabetic polyneuropathy such as dysesthesias, paresthesias, intolerable spontaneous pains, abnormal deep tendon reflexes, and neuropathic ulcers. Data on duration of diabetes, glycosylated hemoglobin, and fasting blood glucose levels were also available. Patients with a fasting blood glucose level of less than 140 mg/dl and a glycosylated hemoglobin level of less than 7.8% were accepted as having good control of their diabetes. A duration of more than 10 years was regarded as long-term diabetes in this study. Landolt visual acuities were obtained and binocular fundus examinations were carried out in each patient. The status of retinopathy was also assessed by fundus photography and fluorescein angiography. Early Treatment of Diabetic Retinopathy Study criteria were used to define various stages of diabetic retinopathy. Any patient with a secondary systemic disease associated with proliferative retinopathy was excluded from this study. At ocular examination, particular attention was paid to lid margins, tarsal and bulbar conjunctiva, and cornea. None of the patients with diabetes had a history of Stevens-Johnson syndrome, chemical, thermal or radiation injury, nor any other systemic disorder, nor had any undergone any ocular surgery that would create an ocular surface problem. The patients and control subjects underwent ocular surface examinations including corneal sensitivity measurements, tear film break-up time (BUT), Schirmer test, and conjunctival impression cytologic analysis. Informed consent for the procedures was obtained. No patient was being treated with topical eye medications at the time of impression cytologic analysis. Control subjects did not have any history of ocular or systemic disease or a history of drug or contact lens use that would alter the ocular surface. Measurement of corneal sensitivity was performed by using a Cochet-Bonnet aesthesiometer. The measurements were begun with the nylon filament fully extended. The tip of the nylon filament was applied perpendicular to the surface of the central cornea, making certain not to touch the eyelashes, and was pushed until the fiber’s first visible bending. The length of the fiber was gradually decreased until a blink reflex was observed. The length was recorded in millimeters. A corneal sensitivity measurement of less than 45 mm was regarded as low corneal sensitivity in this study. The standard tear film BUT measurement was performed. Moistened fluorescein strips were introduced to the conjunctival sac with minimal stimulation and were undetected by the patients. The subjects were then instructed to blink several times for a few seconds to ensure adequate mixing of fluorescein. The interval between the last complete blink and the appearance of the first corneal black spot in the stained tear film was measured three times, and the mean value of the measurements was calculated. A BUT value of less than 10 seconds was considered abnormal. For further evaluation of tears, the standard Schirmer test with topical anesthesia (0.4% oxybuprocaine chloride) was performed. The standardized strips of filter paper (Alcon, Fort Worth, Texas) were placed in the lateral canthus away from the cornea and left in place for 5 minutes with the eyes closed. Readings were reported in millimeters of wetting for 5 minutes. A reading of less than 5 mm was referred to as dry eye. The impression cytologic specimens were obtained after administration of topical anesthesia with 0.4% oxybuprocaine. Strips of cellulose acetate filter paper (Millipore HAWP 304, Bedford, MA) that were soaked in distilled water for a few hours and dried at room temperature were applied on the lower nasal bulbar conjunctiva adjacent to the corneal limbus, pressed gently by a glass rod, and then removed. The specimens were then fixed with formaldehyde, stained with periodic acid–Schiff, dehydrated in ascending grades of ethanol and then with xylol, and finally coverslipped. The quantitative studies of conjunctival goblet cells
and squamous metaplasia of conjunctival epithelial cells were conducted by taking photographs using a calibrated grid under a light microscope at a magnification of ⫻400. We photographed five overlapping areas of each sample selected at random and averaged the outcomes for a single sample score. The goblet cell densities were reported as cells per square millimeter with standard deviations. The specimens were also assigned a grade of conjunctival epithelial squamous metaplasia according to an adaptation of Nelson’s grading scheme.8,9
Statistical Analysis Data were processed using Stat View software (Abacus Concepts, Inc, San Diego, CA). The Mann–Whitney U test was used for the analyses of nonparametric values. A probability level less than 1% was considered statistically significant. The analysis of categorized data was performed by the Fisher’s exact probability test with the probability level set at 1% for statistical significance.
Results Clinical Features There were no age- or gender-related statistical differences between patients with diabetes and control subjects. The duration of diabetes varied between 1 and 24 years (mean, 13.6 ⫾ 9.3 years) and was more than 10 years in 30 patients (60%). Twenty-three patients (46%) had good control of their diabetes. Eighteen patients (36%) with diabetes were diagnosed to have peripheral neuropathy after abnormal nerve conduction velocity testing. All patients with peripheral neuropathy had paresthesias, dysesthesias, and spontaneous intolerable pain attacks of the toes and fingers. Ten patients had abnormal deep tendon reflexes and two had neurotrophic foot ulcers. Thirty-one eyes (35.3%) had nonproliferative diabetic retinopathy, and 27 eyes (30.6%) had proliferative diabetic retinopathy. Thirty eyes (34.1%) were not shown to have diabetic retinopathy. Thirty-nine patients (78%) reported symptoms of eye fatigue, irritation, and foreign body sensation. Slitlamp biomicroscopy of the eyelid margins and conjunctiva did not reveal any coexistent blepharitis, meibomian gland disorder, or conjunctivitis. However, 12 eyes (13.6%) had superficial punctate keratopathy, and two eyes (2.3%) with a history of recurrent corneal erosions had corneal leucoma and pannus. None of the control subjects had punctate keratopathy.
Corneal Sensitivity Forty-eight eyes (54.5%) of the patients with diabetes had low corneal sensitivity. The mean corneal sensitivity in the diabetic patients was 44 ⫾ 1.1 mm, compared with 59.4 ⫾ 0.6 mm in the control subjects (Table 1). This difference was statistically significant (P ⬍ 0.001). The corneal sensitivity was significantly lower in diabetics with peripheral neuropathy, as shown in Table 2 (P ⬍ 0.001). Corneal sensitivity measurements were also significantly lower in patients with poorly controlled diabetes compared with control subjects and patients with good control of diabetes, as shown in Table 3 (P ⬍ 0.001). The corneal sensitivity did not relate to either duration of diabetes or status of retinopathy (Tables 4 and 5).
Tear Function Parameters The mean BUT in the diabetic patients was 8.83 ⫾ 0.31 seconds, compared with 12.96 ⫾ 1.39 seconds in the control subjects (Table
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Ophthalmology Volume 108, Number 3, March 2001 Table 1. Comparison of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters between Diabetes Patients and Control Participants
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2)
Diabetes
Control
44.0 ⫾ 1.1* 8.83 ⫾ 0.31* 7.40 ⫾ 0.38* 1.12 ⫾ 0.53*
59.4 ⫾ 0.6 12.96 ⫾ 1.39 13.53 ⫾ 0.50 0.77 ⫾ 0.18
870 ⫾ 134*
1957 ⫾ 156
Table 3. Relation of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters to Control of Diabetes
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2)
Poor Control
Good Control
37.68 ⫾ 1.70* 8.36 ⫾ 0.36* 6.60 ⫾ 0.46* 1.38 ⫾ 0.14*
53.27 ⫾ 1.28 9.72 ⫾ 0.24 9.40 ⫾ 0.53 0.63 ⫾ 0.08
736 ⫾ 244*
1170 ⫾ 180
* P ⬍ 0.001, Fisher’s exact test.
* P ⬍ 0.001, Fisher’s exact test.
1). The difference was statistically significant (P ⬍ 0.001). Sixtyone eyes (69.3%) of the patients with diabetes had a BUT value of less than 10 seconds, whereas none of the controls had poor BUT results. Tear film BUT values were significantly lower in patients with peripheral neuropathy and decreased corneal sensitivity, as shown in Tables 2 and 6 (P ⬍ 0.001). Patients with poor control of diabetes had lower BUT scores compared with patients with good diabetic control (P ⬍ 0.001). Tear film BUT scores did not relate to duration of diabetes or status of retinopathy (Tables 5 and 6). Schirmer test values averaged 7.40 ⫾ 0.38 mm in patients with diabetes versus 13.53 ⫾ 0.50 mm in the control subjects (P ⬍ 0.001). Twenty eyes (22.7%) of the diabetic patients had Schirmer test values measuring less than 5 mm. Schirmer test results were significantly lower in patients with peripheral neuropathy and low corneal sensitivity (Tables 2 and 3). Schirmer test results were also lower in patients with poorly controlled diabetes compared with diabetics with good control (P ⬍ 0.001). Schirmer test scores did not relate to duration of diabetes or status of retinopathy.
Diabetics with peripheral neuropathy had a significantly higher grade of squamous metaplasia (Table 2). The impression cytologic specimen from a patient with severe peripheral neuropathy is shown in Figure 3. The average squamous metaplasia grade was also significantly higher in patients with decreased corneal sensitivity (Table 3). Patients with poor control of diabetes had significantly higher grades of squamous metaplasia compared with diabetics with good control (Table 4). The impression cytologic specimen from a patient hospitalized for poor diabetes control and ketoacidotic coma showed marked loss of cellular cohesion with isolated epithelial cells in every field and no goblet cells (Fig 4). Duration of diabetes and status of retinopathy did not seem to affect the grade of squamous metaplasia (P ⬎ 0.001).
Impression Cytologic Analysis Conjunctival imprints from healthy control subjects and diabetics contained conjunctival epithelial cells, variable amounts of goblet cells, and mucin (Figs 1 and 2). Specimens from patients with peripheral diabetic neuropathy and poor control of diabetes did not show any mucin pickup. Squamous metaplasia and goblet cell density of each specimen was graded and calculated as described in “Materials and Methods.”
Squamous Metaplasia The average grade of squamous metaplasia in the diabetic patients was 1.12 ⫾ 0.53 and in the controls was 0.77 ⫾ 0.18 (P ⬍ 0.001). Table 2. Comparison of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters to Peripheral Neuropathy in Diabetes Patients
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2) * P ⬍ 0.001, Fisher’s exact test.
588
Peripheral Neuropathy
No Peripheral Neuropathy
32.50 ⫾ 1.73* 8.14 ⫾ 0.35* 5.50 ⫾ 0.29* 1.48 ⫾ 0.15*
53.66 ⫾ 1.17 9.50 ⫾ 0.25 9.30 ⫾ 0.48 0.74 ⫾ 0.10
510 ⫾ 231*
1230 ⫾ 290
Goblet Cell Density The average goblet cell densities were significantly lower in patients with diabetes when compared with those of the control group. Patients with peripheral neuropathy also displayed significantly decreased goblet cell numbers (Table 2). Likewise, patients with a low corneal sensitivity had significantly lower goblet cell densities (P ⬍ 0.001). Patients with poor diabetic control had lower goblet cell counts with statistical significance. Goblet cell density did not seem to relate to duration of diabetes and status of retinopathy in this study.
Discussion Several clinical and experimental studies have reported structural, metabolic, and functional abnormalities in the conjunctiva and cornea of diabetic patients and have suggested that these abnormalities may be responsible for the Table 4. Relation of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters to Duration of Diabetes
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2) * P ⬍ 0.001.
More than 11 yrs
10 yrs or Fewer
49.95 ⫾ 8.75 9.28 ⫾ 1.83 7.70 ⫾ 3.11 0.88 ⫾ 0.76
51.62 ⫾ 11.73 8.94 ⫾ 2.33 8.55 ⫾ 4.26 1.05 ⫾ 1.02
898 ⫾ 249
1146 ⫾ 112
Dogru et al 䡠 Impression Cytologic Analysis in Diabetes Table 5. Relation of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters to Diabetic Retinopathy Status of Retinopathy in Diabetes Patients
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2)
Proliferative
Simple
None
43.51 ⫾ 8.85 8.48 ⫾ 2.04 6.21 ⫾ 2.09 1.46 ⫾ 0.93 771 ⫾ 270
50.19 ⫾ 11.22 8.58 ⫾ 2.46 8.50 ⫾ 2.89 0.91 ⫾ 0.87 845 ⫾ 217
57.56 ⫾ 3.62 10.03 ⫾ 1.27 9.96 ⫾ 4.27 0.58 ⫾ 0.61 1336 ⫾ 233
* P ⬍ 0.001.
clinical corneal manifestations of diabetes.10 –18 The tear film has also been reported to be unstable19 and to have a higher osmolarity in diabetes (unpublished data; Aragona P, et al. Invest Ophthalmol Vis Sci 1999;40(Suppl):S542). A recent study demonstrated decreased Schirmer test readings and pronounced signs of conjunctival squamous metaplasia in patients with insulin-dependent diabetes.20 How these ocular surface disorders and tear film changes relate to clinical parameters of diabetes are still controversial. In this study, we performed corneal sensitivity measurements, tear film BUT analysis, Schirmer test, and conjunctival impression cytologic analysis to analyze the relation between the clinical features and these examinations and also compared the results with those of healthy control subjects. Corneal sensitivity was observed to be significantly lower in our diabetic patients compared with the control subjects. We found that all patients with corneal lesions and peripheral neuropathy had decreased corneal sensitivity, suggesting that keratopathy and corneal neuropathy may be manifestations of the distal peripheral neuropathy of diabetes. Indeed, experimental evidence in diabetic animal models points out Schwann cell basal lamina thickening and thinning, irregular distribution in the nerve beading pattern, and occasional axonal degeneration of the unmyelinated corneal nerves.21,22 The reduction in corneal sensitivity did not relate to duration of diabetes and the status of retinopathy. Although reports stating association of patient age, duration of disease, and proliferative diabetic retinopathy with decreased corneal sensitivity exist, normal corneal sensitivity with long-standing diabetes has also been documented.23–26 Interestingly, we observed that corneal sensitivity was significantly decreased in patients with poor control of diabetes. Changes of intraneural concentration of myoinositols and increased sorbitol levels within the Table 6. Relation of Tear Function and Impression Cytologic Analysis Parameters to Corneal Sensitivity in Diabetes Patients
Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2) * P ⬍ 0.001, Fisher’s exact test.
Low Corneal Sensitivity
Normal Corneal Sensitivity
7.45 ⫾ 0.37* 5.50 ⫾ 0.47* 1.59 ⫾ 0.17*
10.10 ⫾ 0.22 8.91 ⫾ 0.46 0.67 ⫾ 0.09
424 ⫾ 39*
1071 ⫾ 183
Schwann cell basal lamina leading to mechanical compression or toxic axonal damage have been shown in the corneas of diabetic animal models.27–29 A recent clinical study also showed that corneal sensitivity was reduced during phases of hyperglycemia as a result of changes in corneal hydration control.30 Not only corneal sensitivity, but also tear function and impression cytologic parameters showed marked changes in this study. Among our subjects, 22.7% of the patients had Schirmer test values measuring less than 5 mm and 69.3% had poor BUT scores. Seventy-eight percent of patients reported eye fatigue, irritation, and foreign body sensation. All these findings strongly suggest that dry eye is a significant feature of the diabetic ocular surface disease. Schirmer test values did not relate to duration of diabetes or retinopathy status, but were significantly lower in diabetics with poor metabolic control and peripheral neuropathy, suggesting a neuropathy involving the innervation of the lacrimal gland. Likewise, fluctuations in the glycemic control may affect the lacrimal gland secretory function. Yet, we believe in the necessity of further work to support this hypothesis. The results of BUT testing showed marked instability of the tear film in diabetics with poor control and peripheral neuropathy, but no significant relation with disease duration and retinopathy status could be found in this study. To provide more clues in relation to these tear function changes, we carried on with impression cytologic analysis, which provided evidence that prominent squamous metaplasia and goblet cell loss existed in diabetics compared with control subjects and was much more prominent in those diabetics with peripheral neuropathy and poor metabolic control. Impression cytologic analysis parameters again did not relate to duration of disease and status of retinopathy. Reduction in goblet cell numbers may account for the shortening of the BUT and instability of the tear film as a result of decreased mucin production, because goblet cells are the major source of mucin in the tear film.31 Although we had a noteworthy observation that mucin pick up by the filter papers in patients with peripheral neuropathy and poor metabolic control was almost nonexistent, we can not answer whether dry eye in diabetes results from mucin deficiency, because sources of mucin other than the goblet cells are present, and we did not measure the mucin content of tears in our patients this time. We believe that studies on the mucin gene expression of the diabetic ocular surface would provide very useful information. Goblet cell counts are known to reflect the health status of the ocular surface.
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Figure 1. Impression cytologic specimen from a normal subject shows abundant periodic acid–Schiff-positive oval-plum goblet cells and sheets of small, round nonsecretory epithelial cells with nucleocytoplasmic ratio 1:1 or 1:2.
Figure 3. Impression cytologic specimen from a patient with advanced peripheral neuropathy and diabetic foot. Note the total loss of goblet cells, decrease of cellular cohesion, pyknosis, and absence of mucin pick up. The squamous metaplasia grade was 2.4 ⫾ 0.2.
Goblet cell loss is a well-known sign of squamous metaplasia and goes along with an increase of cellular stratification and keratinization.32–34 Concurrent involvement of the conjunctival and corneal epithelial surfaces in our patients, as evidenced by prominent squamous metaplasia, markedly decreased goblet cell population, and superficial punctate keratopathy, may be viewed as a ‘primary ocular surface disease.’ The mechanisms of these ocular surface changes during the course of diabetic disease are still not clear, but loss or decrease of trophic effects of trigeminal sensory nerves on the conjunctiva and cornea may be responsible.35,36 We believe that fluctuations in and insufficiency of metabolic control may be more important than the duration of diabetes and may affect the diabetic ocular surface much more adversely. We would also expect loss of neurotrophic effects evidenced by corneal hypesthesia and alternating hyperglycemia and euglycemia to induce conjunctival squamous metaplasia. We believe that squamous metaplasia of the conjunctival epithelium may account for most, if not all,
the clinical morbidity of corneal complications in diabetes. With such a hypothesis, one would conclude that the ocular surface epithelium be diseased under certain circumstances and not only be in jeopardy because of tear film inadequacies. Treatment in such conditions would thus be directed toward the epithelium, rather than the tear film. Apart from the conventional dry eye therapies including artificial tear drops, lubricants, and therapeutic soft contacts, promising approaches using aldose reductase inhibitors like sorbinil, CT-112, and ONO-2235 have recently been tested and are being reported. Such treatment methods have also been shown to improve nerve conduction, corneal sensitivity, corneal epithelial disease, and even tear function parameters.37– 42 Topical retinoids, which have been shown to promote the production of new basement membrane components and cellular differentiation and to maintain cellular growth, may be promising for the treatment of diabetic conjunctival and corneal epithelial disease as well.43– 47
Figure 2. Impression cytologic specimen from another control subject shows periodic acid–Schiff-positive staining with small, round goblet cells and sheets of conjunctival epithelial cells with strong cohesion.
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Figure 4. Impression cytologic specimen from a patient hospitalized as a result of poorly controlled diabetes. Note the grade 3 squamous metaplasia changes with large and isolated epithelial cells that have pale staining cytoplasm and pyknotic nuclei.
Dogru et al 䡠 Impression Cytologic Analysis in Diabetes Impression cytologic analysis will help in the decision making to treat such a disease process. In conclusion, our data suggest that peripheral neuropathy and poor metabolic control are important determinants of diabetic ocular surface disease. It is our belief that further clinical and experimental work about the changes of tear function and impression cytologic parameters after introduction of aldose reductase inhibitors or topical retinoids will lead to better understanding and management of diabetic ocular surface disease.
19. 20. 21. 22.
23.
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Ophthalmology Volume 108, Number 3, March 2001 of corneal sensation and tear dynamics in diabetic patients by oral aldose reductase inhibitor, ONO-2235: preliminary study. Cornea 1996;15:368 –72. 42. Tseng SCG, Farazdaghi M. Reversal of conjunctival transdifferentiation by topical retinoic acid. Cornea 1988;7:273–9. 43. Tseng SCG, Maumenee AE, Stark WJ, et al. Topical retinoid treatment for various dry eye disorders [published erratum appears in Ophthalmology 1989;96:730]. Ophthalmology 1985;92:717–27. 44. Ubels JL, Edelhauser HF, Foley KM, et al. The efficacy of
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retinoic acid ointment for treatment of xerophthalmia and corneal epithelial wounds. Curr Eye Res 1985;4:1049 –57. 45. Ubels JL, Edelhauser HF, Austin KH. Healing of experimental corneal wounds treated with topically applied retinoids. Am J Ophthalmol 1983;95:353– 8. 46. Smolin G, Okumoto M. Vitamin A acid and corneal epithelial wound healing. Ann Ophthalmol 1981;13:563– 6. 47. Hatchell DL, Ubels JL, Stekiel T, Hatchell MC. Corneal epithelial wound healing in normal and diabetic rabbits treated with tretinoin. Arch Ophthalmol 1985;103:98 –100.
thalmol Vis Sci 1982;22(Suppl):200], neurotrophic corneal ulceration, dry eye, filamentary keratitis, and Descemet folds constitute a gamut of diabetic corneal complications.5–7 The cause of these alterations, however, is not clear. In addition, studies relating the changes of tear film and ocular surface in diabetic patients to clinical parameters of diabetes are still rare. Therefore, we aimed to describe the ocular surface disorder in diabetes by performing corneal sensitivity measurements, tear film break-up time analysis, Schirmer test, and conjunctival impression cytologic analysis and to compare the results with those of healthy control subjects.
Materials and Methods Originally received: March 6, 2000. Accepted: November 10, 2000. Manuscript no. 200124. From the Department of Ophthalmology, Kobe University School of Medicine, Kobe, Japan. The preliminary work was presented at the 22nd Japan Congress of Cornea, February 1999, Ube, Yamaguchi, Japan, and at the Fifth Japanese Ophthalmic Diabetology Congress, March 1999, Nagoya, Japan. The finalized work was presented at the annual meeting of the Association for Research in Vision and Ophthalmology, May 2000, Fort Lauderdale, FL. The authors have no proprietary interest in any of the products mentioned in this paper. Address correspondence to Chikako Katakami, MD, PhD, Department of Ophthalmology, Kobe University School of Medicine, Kusunokicho, 7-5-2, Chuoku, Kobe 650-0017, Japan. E-mail: [email protected].
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© 2001 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
Participants and Examinations Eighty-eight eyes of 50 patients with noninsulin-dependent diabetes mellitus (24 males, 26 females) aged between 44 and 75 years (mean, 54.2 years) as well as 40 eyes of 20 healthy subjects aged from 39 to 70 years (mean, 56.4 years; 7 males, 13 females) were recruited from the Department of Ophthalmology of Kobe University Hospital from September 1998 through February 1999. Both groups were similar regarding age and gender characteristics. The presence of diabetes in all patients had been confirmed by the corresponding internal medicine department. The diagnosis of peripheral neuropathy was based on abnormal nerve conduction velocity test results and on the presence of symptoms and signs of ISSN 0161-6420/00/$–see front matter PII S0161-6420(00)00599-6
Dogru et al 䡠 Impression Cytologic Analysis in Diabetes diabetic polyneuropathy such as dysesthesias, paresthesias, intolerable spontaneous pains, abnormal deep tendon reflexes, and neuropathic ulcers. Data on duration of diabetes, glycosylated hemoglobin, and fasting blood glucose levels were also available. Patients with a fasting blood glucose level of less than 140 mg/dl and a glycosylated hemoglobin level of less than 7.8% were accepted as having good control of their diabetes. A duration of more than 10 years was regarded as long-term diabetes in this study. Landolt visual acuities were obtained and binocular fundus examinations were carried out in each patient. The status of retinopathy was also assessed by fundus photography and fluorescein angiography. Early Treatment of Diabetic Retinopathy Study criteria were used to define various stages of diabetic retinopathy. Any patient with a secondary systemic disease associated with proliferative retinopathy was excluded from this study. At ocular examination, particular attention was paid to lid margins, tarsal and bulbar conjunctiva, and cornea. None of the patients with diabetes had a history of Stevens-Johnson syndrome, chemical, thermal or radiation injury, nor any other systemic disorder, nor had any undergone any ocular surgery that would create an ocular surface problem. The patients and control subjects underwent ocular surface examinations including corneal sensitivity measurements, tear film break-up time (BUT), Schirmer test, and conjunctival impression cytologic analysis. Informed consent for the procedures was obtained. No patient was being treated with topical eye medications at the time of impression cytologic analysis. Control subjects did not have any history of ocular or systemic disease or a history of drug or contact lens use that would alter the ocular surface. Measurement of corneal sensitivity was performed by using a Cochet-Bonnet aesthesiometer. The measurements were begun with the nylon filament fully extended. The tip of the nylon filament was applied perpendicular to the surface of the central cornea, making certain not to touch the eyelashes, and was pushed until the fiber’s first visible bending. The length of the fiber was gradually decreased until a blink reflex was observed. The length was recorded in millimeters. A corneal sensitivity measurement of less than 45 mm was regarded as low corneal sensitivity in this study. The standard tear film BUT measurement was performed. Moistened fluorescein strips were introduced to the conjunctival sac with minimal stimulation and were undetected by the patients. The subjects were then instructed to blink several times for a few seconds to ensure adequate mixing of fluorescein. The interval between the last complete blink and the appearance of the first corneal black spot in the stained tear film was measured three times, and the mean value of the measurements was calculated. A BUT value of less than 10 seconds was considered abnormal. For further evaluation of tears, the standard Schirmer test with topical anesthesia (0.4% oxybuprocaine chloride) was performed. The standardized strips of filter paper (Alcon, Fort Worth, Texas) were placed in the lateral canthus away from the cornea and left in place for 5 minutes with the eyes closed. Readings were reported in millimeters of wetting for 5 minutes. A reading of less than 5 mm was referred to as dry eye. The impression cytologic specimens were obtained after administration of topical anesthesia with 0.4% oxybuprocaine. Strips of cellulose acetate filter paper (Millipore HAWP 304, Bedford, MA) that were soaked in distilled water for a few hours and dried at room temperature were applied on the lower nasal bulbar conjunctiva adjacent to the corneal limbus, pressed gently by a glass rod, and then removed. The specimens were then fixed with formaldehyde, stained with periodic acid–Schiff, dehydrated in ascending grades of ethanol and then with xylol, and finally coverslipped. The quantitative studies of conjunctival goblet cells
and squamous metaplasia of conjunctival epithelial cells were conducted by taking photographs using a calibrated grid under a light microscope at a magnification of ⫻400. We photographed five overlapping areas of each sample selected at random and averaged the outcomes for a single sample score. The goblet cell densities were reported as cells per square millimeter with standard deviations. The specimens were also assigned a grade of conjunctival epithelial squamous metaplasia according to an adaptation of Nelson’s grading scheme.8,9
Statistical Analysis Data were processed using Stat View software (Abacus Concepts, Inc, San Diego, CA). The Mann–Whitney U test was used for the analyses of nonparametric values. A probability level less than 1% was considered statistically significant. The analysis of categorized data was performed by the Fisher’s exact probability test with the probability level set at 1% for statistical significance.
Results Clinical Features There were no age- or gender-related statistical differences between patients with diabetes and control subjects. The duration of diabetes varied between 1 and 24 years (mean, 13.6 ⫾ 9.3 years) and was more than 10 years in 30 patients (60%). Twenty-three patients (46%) had good control of their diabetes. Eighteen patients (36%) with diabetes were diagnosed to have peripheral neuropathy after abnormal nerve conduction velocity testing. All patients with peripheral neuropathy had paresthesias, dysesthesias, and spontaneous intolerable pain attacks of the toes and fingers. Ten patients had abnormal deep tendon reflexes and two had neurotrophic foot ulcers. Thirty-one eyes (35.3%) had nonproliferative diabetic retinopathy, and 27 eyes (30.6%) had proliferative diabetic retinopathy. Thirty eyes (34.1%) were not shown to have diabetic retinopathy. Thirty-nine patients (78%) reported symptoms of eye fatigue, irritation, and foreign body sensation. Slitlamp biomicroscopy of the eyelid margins and conjunctiva did not reveal any coexistent blepharitis, meibomian gland disorder, or conjunctivitis. However, 12 eyes (13.6%) had superficial punctate keratopathy, and two eyes (2.3%) with a history of recurrent corneal erosions had corneal leucoma and pannus. None of the control subjects had punctate keratopathy.
Corneal Sensitivity Forty-eight eyes (54.5%) of the patients with diabetes had low corneal sensitivity. The mean corneal sensitivity in the diabetic patients was 44 ⫾ 1.1 mm, compared with 59.4 ⫾ 0.6 mm in the control subjects (Table 1). This difference was statistically significant (P ⬍ 0.001). The corneal sensitivity was significantly lower in diabetics with peripheral neuropathy, as shown in Table 2 (P ⬍ 0.001). Corneal sensitivity measurements were also significantly lower in patients with poorly controlled diabetes compared with control subjects and patients with good control of diabetes, as shown in Table 3 (P ⬍ 0.001). The corneal sensitivity did not relate to either duration of diabetes or status of retinopathy (Tables 4 and 5).
Tear Function Parameters The mean BUT in the diabetic patients was 8.83 ⫾ 0.31 seconds, compared with 12.96 ⫾ 1.39 seconds in the control subjects (Table
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Ophthalmology Volume 108, Number 3, March 2001 Table 1. Comparison of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters between Diabetes Patients and Control Participants
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2)
Diabetes
Control
44.0 ⫾ 1.1* 8.83 ⫾ 0.31* 7.40 ⫾ 0.38* 1.12 ⫾ 0.53*
59.4 ⫾ 0.6 12.96 ⫾ 1.39 13.53 ⫾ 0.50 0.77 ⫾ 0.18
870 ⫾ 134*
1957 ⫾ 156
Table 3. Relation of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters to Control of Diabetes
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2)
Poor Control
Good Control
37.68 ⫾ 1.70* 8.36 ⫾ 0.36* 6.60 ⫾ 0.46* 1.38 ⫾ 0.14*
53.27 ⫾ 1.28 9.72 ⫾ 0.24 9.40 ⫾ 0.53 0.63 ⫾ 0.08
736 ⫾ 244*
1170 ⫾ 180
* P ⬍ 0.001, Fisher’s exact test.
* P ⬍ 0.001, Fisher’s exact test.
1). The difference was statistically significant (P ⬍ 0.001). Sixtyone eyes (69.3%) of the patients with diabetes had a BUT value of less than 10 seconds, whereas none of the controls had poor BUT results. Tear film BUT values were significantly lower in patients with peripheral neuropathy and decreased corneal sensitivity, as shown in Tables 2 and 6 (P ⬍ 0.001). Patients with poor control of diabetes had lower BUT scores compared with patients with good diabetic control (P ⬍ 0.001). Tear film BUT scores did not relate to duration of diabetes or status of retinopathy (Tables 5 and 6). Schirmer test values averaged 7.40 ⫾ 0.38 mm in patients with diabetes versus 13.53 ⫾ 0.50 mm in the control subjects (P ⬍ 0.001). Twenty eyes (22.7%) of the diabetic patients had Schirmer test values measuring less than 5 mm. Schirmer test results were significantly lower in patients with peripheral neuropathy and low corneal sensitivity (Tables 2 and 3). Schirmer test results were also lower in patients with poorly controlled diabetes compared with diabetics with good control (P ⬍ 0.001). Schirmer test scores did not relate to duration of diabetes or status of retinopathy.
Diabetics with peripheral neuropathy had a significantly higher grade of squamous metaplasia (Table 2). The impression cytologic specimen from a patient with severe peripheral neuropathy is shown in Figure 3. The average squamous metaplasia grade was also significantly higher in patients with decreased corneal sensitivity (Table 3). Patients with poor control of diabetes had significantly higher grades of squamous metaplasia compared with diabetics with good control (Table 4). The impression cytologic specimen from a patient hospitalized for poor diabetes control and ketoacidotic coma showed marked loss of cellular cohesion with isolated epithelial cells in every field and no goblet cells (Fig 4). Duration of diabetes and status of retinopathy did not seem to affect the grade of squamous metaplasia (P ⬎ 0.001).
Impression Cytologic Analysis Conjunctival imprints from healthy control subjects and diabetics contained conjunctival epithelial cells, variable amounts of goblet cells, and mucin (Figs 1 and 2). Specimens from patients with peripheral diabetic neuropathy and poor control of diabetes did not show any mucin pickup. Squamous metaplasia and goblet cell density of each specimen was graded and calculated as described in “Materials and Methods.”
Squamous Metaplasia The average grade of squamous metaplasia in the diabetic patients was 1.12 ⫾ 0.53 and in the controls was 0.77 ⫾ 0.18 (P ⬍ 0.001). Table 2. Comparison of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters to Peripheral Neuropathy in Diabetes Patients
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2) * P ⬍ 0.001, Fisher’s exact test.
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Peripheral Neuropathy
No Peripheral Neuropathy
32.50 ⫾ 1.73* 8.14 ⫾ 0.35* 5.50 ⫾ 0.29* 1.48 ⫾ 0.15*
53.66 ⫾ 1.17 9.50 ⫾ 0.25 9.30 ⫾ 0.48 0.74 ⫾ 0.10
510 ⫾ 231*
1230 ⫾ 290
Goblet Cell Density The average goblet cell densities were significantly lower in patients with diabetes when compared with those of the control group. Patients with peripheral neuropathy also displayed significantly decreased goblet cell numbers (Table 2). Likewise, patients with a low corneal sensitivity had significantly lower goblet cell densities (P ⬍ 0.001). Patients with poor diabetic control had lower goblet cell counts with statistical significance. Goblet cell density did not seem to relate to duration of diabetes and status of retinopathy in this study.
Discussion Several clinical and experimental studies have reported structural, metabolic, and functional abnormalities in the conjunctiva and cornea of diabetic patients and have suggested that these abnormalities may be responsible for the Table 4. Relation of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters to Duration of Diabetes
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2) * P ⬍ 0.001.
More than 11 yrs
10 yrs or Fewer
49.95 ⫾ 8.75 9.28 ⫾ 1.83 7.70 ⫾ 3.11 0.88 ⫾ 0.76
51.62 ⫾ 11.73 8.94 ⫾ 2.33 8.55 ⫾ 4.26 1.05 ⫾ 1.02
898 ⫾ 249
1146 ⫾ 112
Dogru et al 䡠 Impression Cytologic Analysis in Diabetes Table 5. Relation of Corneal Sensitivity, Tear Function, and Impression Cytologic Analysis Parameters to Diabetic Retinopathy Status of Retinopathy in Diabetes Patients
Corneal sensitivity (mm) Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2)
Proliferative
Simple
None
43.51 ⫾ 8.85 8.48 ⫾ 2.04 6.21 ⫾ 2.09 1.46 ⫾ 0.93 771 ⫾ 270
50.19 ⫾ 11.22 8.58 ⫾ 2.46 8.50 ⫾ 2.89 0.91 ⫾ 0.87 845 ⫾ 217
57.56 ⫾ 3.62 10.03 ⫾ 1.27 9.96 ⫾ 4.27 0.58 ⫾ 0.61 1336 ⫾ 233
* P ⬍ 0.001.
clinical corneal manifestations of diabetes.10 –18 The tear film has also been reported to be unstable19 and to have a higher osmolarity in diabetes (unpublished data; Aragona P, et al. Invest Ophthalmol Vis Sci 1999;40(Suppl):S542). A recent study demonstrated decreased Schirmer test readings and pronounced signs of conjunctival squamous metaplasia in patients with insulin-dependent diabetes.20 How these ocular surface disorders and tear film changes relate to clinical parameters of diabetes are still controversial. In this study, we performed corneal sensitivity measurements, tear film BUT analysis, Schirmer test, and conjunctival impression cytologic analysis to analyze the relation between the clinical features and these examinations and also compared the results with those of healthy control subjects. Corneal sensitivity was observed to be significantly lower in our diabetic patients compared with the control subjects. We found that all patients with corneal lesions and peripheral neuropathy had decreased corneal sensitivity, suggesting that keratopathy and corneal neuropathy may be manifestations of the distal peripheral neuropathy of diabetes. Indeed, experimental evidence in diabetic animal models points out Schwann cell basal lamina thickening and thinning, irregular distribution in the nerve beading pattern, and occasional axonal degeneration of the unmyelinated corneal nerves.21,22 The reduction in corneal sensitivity did not relate to duration of diabetes and the status of retinopathy. Although reports stating association of patient age, duration of disease, and proliferative diabetic retinopathy with decreased corneal sensitivity exist, normal corneal sensitivity with long-standing diabetes has also been documented.23–26 Interestingly, we observed that corneal sensitivity was significantly decreased in patients with poor control of diabetes. Changes of intraneural concentration of myoinositols and increased sorbitol levels within the Table 6. Relation of Tear Function and Impression Cytologic Analysis Parameters to Corneal Sensitivity in Diabetes Patients
Tear film break-up time (sec) Schirmer test (mm) Conjunctival squamous metaplasia (grade) Goblet cell density (cell/mm2) * P ⬍ 0.001, Fisher’s exact test.
Low Corneal Sensitivity
Normal Corneal Sensitivity
7.45 ⫾ 0.37* 5.50 ⫾ 0.47* 1.59 ⫾ 0.17*
10.10 ⫾ 0.22 8.91 ⫾ 0.46 0.67 ⫾ 0.09
424 ⫾ 39*
1071 ⫾ 183
Schwann cell basal lamina leading to mechanical compression or toxic axonal damage have been shown in the corneas of diabetic animal models.27–29 A recent clinical study also showed that corneal sensitivity was reduced during phases of hyperglycemia as a result of changes in corneal hydration control.30 Not only corneal sensitivity, but also tear function and impression cytologic parameters showed marked changes in this study. Among our subjects, 22.7% of the patients had Schirmer test values measuring less than 5 mm and 69.3% had poor BUT scores. Seventy-eight percent of patients reported eye fatigue, irritation, and foreign body sensation. All these findings strongly suggest that dry eye is a significant feature of the diabetic ocular surface disease. Schirmer test values did not relate to duration of diabetes or retinopathy status, but were significantly lower in diabetics with poor metabolic control and peripheral neuropathy, suggesting a neuropathy involving the innervation of the lacrimal gland. Likewise, fluctuations in the glycemic control may affect the lacrimal gland secretory function. Yet, we believe in the necessity of further work to support this hypothesis. The results of BUT testing showed marked instability of the tear film in diabetics with poor control and peripheral neuropathy, but no significant relation with disease duration and retinopathy status could be found in this study. To provide more clues in relation to these tear function changes, we carried on with impression cytologic analysis, which provided evidence that prominent squamous metaplasia and goblet cell loss existed in diabetics compared with control subjects and was much more prominent in those diabetics with peripheral neuropathy and poor metabolic control. Impression cytologic analysis parameters again did not relate to duration of disease and status of retinopathy. Reduction in goblet cell numbers may account for the shortening of the BUT and instability of the tear film as a result of decreased mucin production, because goblet cells are the major source of mucin in the tear film.31 Although we had a noteworthy observation that mucin pick up by the filter papers in patients with peripheral neuropathy and poor metabolic control was almost nonexistent, we can not answer whether dry eye in diabetes results from mucin deficiency, because sources of mucin other than the goblet cells are present, and we did not measure the mucin content of tears in our patients this time. We believe that studies on the mucin gene expression of the diabetic ocular surface would provide very useful information. Goblet cell counts are known to reflect the health status of the ocular surface.
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Figure 1. Impression cytologic specimen from a normal subject shows abundant periodic acid–Schiff-positive oval-plum goblet cells and sheets of small, round nonsecretory epithelial cells with nucleocytoplasmic ratio 1:1 or 1:2.
Figure 3. Impression cytologic specimen from a patient with advanced peripheral neuropathy and diabetic foot. Note the total loss of goblet cells, decrease of cellular cohesion, pyknosis, and absence of mucin pick up. The squamous metaplasia grade was 2.4 ⫾ 0.2.
Goblet cell loss is a well-known sign of squamous metaplasia and goes along with an increase of cellular stratification and keratinization.32–34 Concurrent involvement of the conjunctival and corneal epithelial surfaces in our patients, as evidenced by prominent squamous metaplasia, markedly decreased goblet cell population, and superficial punctate keratopathy, may be viewed as a ‘primary ocular surface disease.’ The mechanisms of these ocular surface changes during the course of diabetic disease are still not clear, but loss or decrease of trophic effects of trigeminal sensory nerves on the conjunctiva and cornea may be responsible.35,36 We believe that fluctuations in and insufficiency of metabolic control may be more important than the duration of diabetes and may affect the diabetic ocular surface much more adversely. We would also expect loss of neurotrophic effects evidenced by corneal hypesthesia and alternating hyperglycemia and euglycemia to induce conjunctival squamous metaplasia. We believe that squamous metaplasia of the conjunctival epithelium may account for most, if not all,
the clinical morbidity of corneal complications in diabetes. With such a hypothesis, one would conclude that the ocular surface epithelium be diseased under certain circumstances and not only be in jeopardy because of tear film inadequacies. Treatment in such conditions would thus be directed toward the epithelium, rather than the tear film. Apart from the conventional dry eye therapies including artificial tear drops, lubricants, and therapeutic soft contacts, promising approaches using aldose reductase inhibitors like sorbinil, CT-112, and ONO-2235 have recently been tested and are being reported. Such treatment methods have also been shown to improve nerve conduction, corneal sensitivity, corneal epithelial disease, and even tear function parameters.37– 42 Topical retinoids, which have been shown to promote the production of new basement membrane components and cellular differentiation and to maintain cellular growth, may be promising for the treatment of diabetic conjunctival and corneal epithelial disease as well.43– 47
Figure 2. Impression cytologic specimen from another control subject shows periodic acid–Schiff-positive staining with small, round goblet cells and sheets of conjunctival epithelial cells with strong cohesion.
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Figure 4. Impression cytologic specimen from a patient hospitalized as a result of poorly controlled diabetes. Note the grade 3 squamous metaplasia changes with large and isolated epithelial cells that have pale staining cytoplasm and pyknotic nuclei.
Dogru et al 䡠 Impression Cytologic Analysis in Diabetes Impression cytologic analysis will help in the decision making to treat such a disease process. In conclusion, our data suggest that peripheral neuropathy and poor metabolic control are important determinants of diabetic ocular surface disease. It is our belief that further clinical and experimental work about the changes of tear function and impression cytologic parameters after introduction of aldose reductase inhibitors or topical retinoids will lead to better understanding and management of diabetic ocular surface disease.
19. 20. 21. 22.
23.
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Ophthalmology Volume 108, Number 3, March 2001 of corneal sensation and tear dynamics in diabetic patients by oral aldose reductase inhibitor, ONO-2235: preliminary study. Cornea 1996;15:368 –72. 42. Tseng SCG, Farazdaghi M. Reversal of conjunctival transdifferentiation by topical retinoic acid. Cornea 1988;7:273–9. 43. Tseng SCG, Maumenee AE, Stark WJ, et al. Topical retinoid treatment for various dry eye disorders [published erratum appears in Ophthalmology 1989;96:730]. Ophthalmology 1985;92:717–27. 44. Ubels JL, Edelhauser HF, Foley KM, et al. The efficacy of
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retinoic acid ointment for treatment of xerophthalmia and corneal epithelial wounds. Curr Eye Res 1985;4:1049 –57. 45. Ubels JL, Edelhauser HF, Austin KH. Healing of experimental corneal wounds treated with topically applied retinoids. Am J Ophthalmol 1983;95:353– 8. 46. Smolin G, Okumoto M. Vitamin A acid and corneal epithelial wound healing. Ann Ophthalmol 1981;13:563– 6. 47. Hatchell DL, Ubels JL, Stekiel T, Hatchell MC. Corneal epithelial wound healing in normal and diabetic rabbits treated with tretinoin. Arch Ophthalmol 1985;103:98 –100.