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(Investigative Ophthalmology and Visual Science. 2002;43:632-638.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

A Mouse Model of Keratoconjunctivitis Sicca

Dilek Dursun1, Min Wang1, Dagoberto Monroy1, De-Quan Li1, Balakrishna L. Lokeshwar1,2,3, Michael E. Stern4 and Stephen C. Pflugfelder1

1 From the Ocular Surface and Tear Center, Bascom Palmer Eye Institute, Miami, Florida; the 2 Sylvester Cancer Center and 3 Department of Urology, University of Miami School of Medicine, Miami, Florida; and 4 Allergan, Inc., Irvine, California.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To evaluate the effects of pharmacologic inhibition of aqueous tear production and desiccating environmental stress on aqueous tear production, tear clearance, corneal epithelial permeability, and conjunctival epithelial morphology, proliferation, and conjunctival goblet cell differentiation.

METHODS. Aqueous tear production was inhibited by applying transdermal scopolamine (scop) patches to the depilated midtail of female MC, CBA mice. Desiccating environmental stress was created by placing mice in a hood with a continuous airflow blower. Aqueous tear production and volume, tear clearance, and corneal barrier function were compared in four experimental groups: untreated control mice, mice placed in the blower hood, mice treated with a scop patch, and mice treated with a scop patch and blower hood (scop patch + blower). Cotton threads were used to evaluate aqueous tear production and volume. Tear clearance was assessed by fluorometric measurement of collected tear fluid 15 minutes after instillation of 1% sodium fluorescein. Corneal epithelial barrier function was assessed by fluorometric measurement of carboxyfluorescein uptake. Conjunctival morphology and goblet cell density were evaluated in stained histologic sections. Conjunctival epithelial proliferation was assessed by bromodeoxyuridine (BrdU) labeling.

RESULTS. Significant decreases in cotton thread wetting and tear clearance were observed in mice treated with a scop patch (P < 0.001) or with a scop patch and blower desiccation (P < 0.001), with a greater reduction in tear clearance in the latter group. Significantly increased corneal carboxyfluorescein uptake was noted in the scop patch group compared with untreated mice (P = 0.05) and in the scop patch + blower group compared with all the other groups (P < 0.0001). Changes in conjunctival epithelial morphology and a significant decrease in conjunctival goblet cell density (P < 0.001) were observed in the scop patch + blower group compared with the untreated control group. The number of proliferating conjunctival epithelial cells was significantly greater in the scop patch + blower group.

CONCLUSIONS. Pharmacologic inhibition of tear production in mice is accompanied by ocular surface epithelial changes resembling human keratoconjunctivitis sicca (KCS) that are exacerbated by desiccating environmental stress. Cholinergic stimulated tear production appears to be essential for maintaining a healthy ocular surface.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Dry eye is a common condition that affects 10% of the population between the ages of 30 and 60 years, increasing in prevalence to 15% of the population aged more than 65 years.1 Dry eye results from decreased production, increased evaporation, or decreased clearance of tears.2 3 Dry eye produces ocular irritation and ocular surface disease, termed keratoconjunctivitis sicca (KCS), that causes blurred and fluctuating vision and increases the risk of sight-threatening corneal infection and ulceration.4 The histologic features of KCS are abnormal proliferation and differentiation of the ocular surface epithelium with decreased density of conjunctival goblet cells and decreased and abnormal production of mucus by the ocular surface epithelium.5 These cellular changes are accompanied by altered epithelial barrier function with increased corneal fluorescein uptake.6 7 The most severe KCS has been reported to develop in conditions in which there is loss of the ability to tear in response to neural stimulation.8 9 10

The mechanism by which dry eye causes KCS has not been established. A number of factors have been implicated in its pathogenesis, including increased tear film osmolarity and decreased tear concentrations of lacrimal-gland–secreted factors that support the ocular surface epithelium, such as epidermal growth factor.11 12 13 14 There is also mounting evidence that inflammation plays a role in this process. Increased expression of a number of inflammatory mediators has been identified on the ocular surface of eyes with KCS. These include increased concentrations of proinflammatory cytokines in the conjunctival epithelium and tear fluid13 14 15 ; increased concentration and activity of proteases, such as plasmin and MMP-9, in the tear fluid14 16 ; increased concentration of lipid peroxidases in the tear fluid17 ; and increased expression of immune activation markers, such as HLA-DR, intercellular adhesion molecule (ICAM)-1, and CD-40, by the conjunctival epithelium.18 19 20 The importance of inflammation in the pathogenesis of dry eye is underscored by reports that the signs and symptoms of dry eye markedly improve with anti-inflammatory therapy.21 22

A dry-eye animal model that mimics the human dry-eye disease would be a useful tool for investigating the multiple factors that have been implicated in the pathogenesis of KCS. Several different animal models of dry eye have been proposed.23 24 25 26 27 28 29 Androgen deficiency after orchiectomy in rats has been reported to increase the volume of secreted tear fluid, while it reduces the concentration of secretory component and of Ig A in the tear fluid.23 24 Testosterone reverses these changes, whereas hypophysectomy prevents this reversal. Another method for inducing dry eye is surgical excision of tear-producing glands. In rabbits, surgical extirpation of the lacrimal gland, harderian gland, and nictitating membrane resulted in increased tear osmolarity, increased corneal epithelial desquamation, decreased corneal epithelial glycogen, decreased conjunctival goblet cell density and increased ocular surface staining with rose bengal dye.25 Dry eye has also been induced by surgical removal of the exorbital lacrimal glands in monkeys and mechanical inhibition of blinking with a blepharostat for 1 to 3 hours in rabbits.28 29 Progressive lacrimal gland inflammation resembling Sjögren syndrome develops in certain strains of mice; however, it has not been established that KCS develops in these mice.30 31 Pharmacologic blockade of cholinergic muscarinic receptors in the lacrimal glands with topically administered atropine sulfate has been reported to decrease tear production and cause KCS in rabbits.26 The presence of depressed lacrimal gland secretion in response to neural stimulation in many forms of severe dry eye makes cholinergic blockade relevant to human disease. The greater knowledge of the murine inflammatory response, coupled with the ability to experimentally manipulate gene expression in mice, makes them a better choice for an animal model than rabbits.

The purpose of this study was to determine whether an experimentally induced decrease in tear production and clearance would cause phenotypic ocular surface epithelial changes resembling human KCS.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Transdermal scopolamine patches (scop patch) were obtained from Novartis (Summit, NJ); wicks (Transorb) from American Filtrona (Richmond, VA); phenol red impregnated cotton threads (Zone-quick) from Oasis (Glendora, CA); sodium fluorescein from Alcon Laboratories, Inc. (Fort Worth, TX); 0.3% carboxyfluorescein (CF) from Holles Laboratories (Cohasset, MA); and bromodeoxyuridine (BrdU) and anti-BrdU monoclonal antibody from Roche Molecular Biochemicals (Indianapolis, IN). Female MC, CBA mice were obtained from Charles River Laboratories (Wilmington, MA).

Aqueous tear production and volume, tear clearance, and corneal barrier function were compared in four experimental groups: untreated control mice, mice placed in a blower hood, mice treated with the anticholinergic agent scopolamine (scop), and mice treated with scop and placed in a blower hood.

Tear Fluorescein Clearance
Tear fluorescein clearance was determined as previously reported in humans.3 14 32 One microliter 1% fluorescein was applied to the ocular surface. After 15 minutes, fluorescein-stained tear fluid was collected atraumatically with a porous polyester rod (Transorb; American Filtrona) from the tear meniscus in the lateral canthus under a dissecting microscope (SMZ-1; Nikon, Tokyo, Japan), minimizing irritation of the ocular surface or lid margin (Fig. 1 , top left). Immediately after tear collection, the rods were placed in sealed polypropylene tubes and were protected from light until fluorophotometric analysis. The volume of the collected tear fluid was determined by the weight difference between the rod containing the sample and the precollection empty rod (Model GA110 scale; Ohaus, Bern, Switzerland). A volume of phosphate-buffered saline (PBS; 100-µL weight of rod in micrograms) was added to the end of the rod, the tubes were spun at 12,000 cycles per minute for 5 minutes, and the fluid was transferred to wells of a 96-well polycarbonate microtiter plate (Corning 96; Corning Glassworks; Corning, NY). Fluorescence was measured with a fluorescence multiplate reader (Cytofluor II; PerSeptive Biosystems, Framingham, MA) as previously described.3



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  		Figure 1. Top left: collection of tear fluid from the lateral canthus with a polyester wick. Top right: measurement of aqueous tear production and volume with phenol red–impregnated cotton thread. Bottom left: scop patch wrapped around mouse’s depilated midtail. Bottom right: placement of mouse cage into blower hood.

 
Measurement of Aqueous Tear Production
Tear production was measured with cotton threads (Zone-quick; Oasis). The threads were held with jeweler forceps and applied to the ocular surface in the lateral canthus for 60 seconds. Wetting of the thread was measured in millimeters, using the scale on the cotton thread (Fig. 1 , top right).

Induction of Dry Eye with Cholinergic Receptor Blockade and Blower Hood Desiccation
Two methods were used to deliver anti-cholinergic agents to block lacrimal gland muscarinic cholinergic receptors: topically applied atropine sulfate and transdermally delivered scopolamine. Topically treated mice received 1 µL of 1% atropine sulfate in each eye. A separate group of mice served as a negative control and received 1 µL sterile saline. Transdermal scopolamine (scop) patches were applied by cutting the patches into four pieces, wrapping them around the depilated midtail, and securing them with cellophane tape (Fig. 1 , bottom left). Patches were reapplied on days 0 and 2. To induce environmental stress, some mice were placed in a blower hood (LabSafety Hood; Labconco, Kansas City, MO), with a flow rate of 300 ft/min at 7 seconds, for 1 hour, 3 times per day for 4 days (scop patch + blower; Fig. 1 , bottom right). Tear production and clearance were evaluated before treatment, then 1, 4, 12, 24, and 48 hours after treatment.

Corneal Fluorescein Staining
Corneal fluorescein staining was evaluated and photographed with a slit lamp biomicroscope (Humphrey-Zeiss, Dublin, CA) using a cobalt blue light 10 minutes after application of 1 µL 1% sodium fluorescein.

Permeability to CF
Uptake of CF by corneas in normal and dry-eye mice was determined by modifying a previously reported technique.33 34 CF (1 µL of a 0.3% solution) was applied to the ocular surface. After a 10-minute delay, the mice were killed with high-dose carbon dioxide. Corneas without scleral rims were then excised, rinsed twice with 200 µL balanced salt solution (BSS; Alcon Laboratories, Inc.), cut into four pieces, and placed in 200 µL BSS. The solution containing the corneal tissue was protected from light and placed on an orbital shaker for 90 minutes. The CF concentration (in units per microliter) was measured with a fluorometer (excitation wavelength-bandwidth, 485 nm; emission wavelength-half bandwidth, 530 nm; gain, 40; Cytofluor II; PerSeptive Biosystems).

Conjunctival Histopathology and Epithelial Proliferation
Conjunctival biopsy specimens, measuring approximately 1 x 2 mm, were obtained from the same regions in the superior and the inferior bulbar conjunctiva, in control eyes and eyes of mice treated with the scop patch + blower. Tissues were fixed immediately in 2% formaldehyde and embedded in paraffin. Six- to eight-micrometer-thick sections were cut and stained with hematoxylin and eosin, or with the periodic acid-Schiff (PAS) reagent.

Conjunctival epithelial morphology was evaluated, and the number of conjunctival epithelial goblet cells was counted along the length of three separate tissue sections that spanned the entire length of a microscope field (Axiophot 2; Nikon), by two independent masked observers, using a x20 objective. Three mice were evaluated in each treatment group.

As a control to study the direct effects of scopolamine on the ocular surface, 1 µL 0.25% scopolamine solution was applied to the ocular surface, 4 hours before the biopsy was performed. Conjunctival biopsies were then performed to evaluate conjunctival goblet cell density and epithelial morphology.

Conjunctival epithelial proliferation was assessed by injecting BrdU (0.5 mg in 0.2 mL) subcutaneous in the flank 24 and 2 hours before death. BrdU was immunodetected in 5-µm frozen tissue sections using an anti-BrdU monoclonal antibody after treating sections with 2 N HCl for 60 minutes at 37°C and then 0.1 M borate buffer. The number of labeled cells was counted in three separate sections in specimens from three mice in each treatment group.

All procedures in the study protocol adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Aqueous Tear Production and Clearance
The initial phase of this study compared the effects of topically applied atropine to the eye and transdermal scop patches applied to the tail of mice, to determine which method produces the greatest pharmacologic inhibition of tear production. Topical atropine produced a significant reduction in aqueous tear production 1 hour after instillation, compared with mice that were treated with a saline drop (P = 0.001). However, the effect was short-lived with tear production returning to baseline levels within 4 hours. In contrast, the transdermal scop patches produced a statistically significant decrease in aqueous production (mean, 0.5 ± 0.7 mm) and tear clearance (mean, 985.625 ± 312 fluorescence units [FU]) at 24 hours, compared with control animal (mean, 2.98 ± 0.6 mm and 283 ± 96 FU, P = 0.001 for each). Placement of mice treated with the scop patch into a blower hood further worsened tear clearance (P < 0.001, Table 1 ), but it did not cause a greater reduction in the cotton thread test results than that in mice treated with the scop patch alone.


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  		Table 1. Effects of Experimentally Induced Dry Eye

 
Ocular Surface Fluorescein Staining and CF Uptake
Visual evaluation of corneal fluorescein staining and corneal CF uptake were used as measures of the effect of dry eye on corneal epithelial barrier function. Minimal scattered punctate staining or no staining was observed on the corneas of control mice, after instillation of fluorescein dye (Fig. 2 , top left). Mild central punctate fluorescein staining was observed in most mice treated with a scop patch (Fig. 2 , top right). Patches of punctate and diffuse corneal fluorescein staining were observed in all scop patch + blower–treated mice (Fig. 2 , bottom). Consistent with the visually observed staining was an increase in CF uptake by the corneas of mice treated with a scop patch compared with untreated mice (P = 0.05) and in the scop patch + blower–treated mice compared with the other groups (P < 0.0001; Table 1 ). These results indicate that the experimental manipulations decreased aqueous tear production and tear clearance and altered corneal epithelial barrier function. Based on findings that the worst KCS developed in mice treated with a scop patch and blower, this combination was used to evaluate the effects of dry eye on the conjunctival epithelium.



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  		Figure 2. Mouse corneas viewed and photographed under cobalt blue light 10 minutes after application of 1 µL 1% sodium fluorescein to the ocular surface. Top left: normal control mouse. Top right: mouse 4 days after application of scop patch. Bottom left: mouse 4 days after application of scop patch and placement in a blower hood 1 hour per day. Bottom right: mouse treated with scop patch + blower. This mouse blinked infrequently and developed a central corneal epithelial defect.

 
Conjunctival Epithelial Morphology, Goblet Cell Density, and Proliferation
The conjunctiva had a cuboidal basal epithelial layer with three to four overlying squamous layers with interspersed goblet cells in control untreated mice (Figs. 3A 3B) . The conjunctival epithelial morphology in dry-eye mice showed two patterns: a thinner-than-normal layer with loss of the cuboidal basal layer or a hyperplastic appearance with six or more epithelial cell layers (Figs. 3C 3D) . Goblet cell density was significantly reduced in the scop patch + blower–treated mice compared with normal mice (Table 2 , compare Fig. 3A 3B with Fig. 3C 3D ). Mice treated with topical scopolamine showed normal epithelial cell morphology and conjunctival goblet cell density. The number of proliferating conjunctival epithelial cells labeled with BrdU (Fig. 4) was significantly greater in the scop patch + blower–treated eyes (41.00 ± 17.69 cells per x20 microscope field) than in untreated control mice (11.00 ± 2.71 cells per x20 microscope field, P = 0.02).



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  		Figure 3. Histologic sections of paraffin-embedded mouse conjunctiva stained with periodic acid Schiff (PAS) reagent. (A) Superior bulbar conjunctiva of normal mouse. Goblet cells are stained pink and are located in the superficial epithelium. (B) Inferior bulbar conjunctiva of normal mouse. (C) Superior bulbar conjunctiva of mouse after 4 days of treatment with scop patch + blower. Epithelium was thinned with loss of goblet cells. (D) Inferior bulbar conjunctiva of mouse after 4 days of treatment with scop patch + blower. Epithelium had an increased number of cell layers and showed loss of goblet cells.

 

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  		Table 2. Average Conjunctival Goblet Cell Density

 


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  		Figure 4. BrdU labeling of proliferating conjunctival epithelial cells in a normal untreated mouse (top) and a mouse treated with scop patch + blower for 4 days (bottom). Dotted line: junction of the conjunctival epithelium and substantia propria; arrows: BrdU-labeled cells.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In this study, experimentally induced dry eye resulted in ocular surface epithelial changes, with features that mimic human KCS, such as corneal fluorescein staining, altered corneal epithelial barrier function, reduced conjunctival goblet cell density, and increased conjunctival epithelial proliferation.2 4 5 6 7 8 Pharmacologic inhibition of tear secretion with the anti-cholinergic agent scopolamine significantly decreased tear production and clearance compared with untreated mice and caused a significant increase in corneal epithelial permeability to CF within 4 days of treatment. Exposing the scopolamine treated mice to desiccating environmental stress significantly worsened the severity of the ocular surface disease. This finding suggests that normal production and clearance of tears is required for maintaining a healthy, well-lubricated ocular surface and for protecting it from environmental insults.

Regulated tear secretion by the lacrimal glands is controlled by the autonomic nervous system. The rat lacrimal gland is densely innervated with parasympathetic nerves that contain acetylcholine and vasoactive intestinal peptide (VIP).35 36 These nerves were observed to surround the basolateral membranes of most lacrimal gland secretory acini.

Immunocytochemical examination of lacrimal gland neural innervation in mice shows that most of their nerve fibers are also parasympathetic, containing both acetylcholine and VIP.37 It has been demonstrated that sympathetic nerves containing norepinephrine are also present in the lacrimal gland, innervating some acini and blood vessels, although the density of sympathetic nerves is less than in the parasympathetics.38 39

Systemic administration of anti-cholinergic drugs, such as atropine, is well recognized to decrease tear secretion.40 41 Decreased tear production and the development of dry spots on the corneal surface, after 2 days of topical atropine sulfate administration in rabbits, were reported by Burgalassi et al.26 Indeed, in our preliminary studies, we observed that topical administration of atropine sulfate also decreased aqueous tear production in mice. However, this effect was short-lived, lasting for only a few hours before returning to normal.

The short effective duration of topical atropine sulfate prompted us to evaluate the effectiveness of a transdermal delivery system that would control absorption and the rate of drug delivery into the blood stream over an extended period. A transdermal patch delivery system is available for the anti-cholinergic medication scopolamine. Scopolamine transdermal patches have become a frequently used and effective means of delivering this medication for treatment of motion sickness and for nausea and vomiting related to chemotherapy.42

Similar to atropine, scopolamine is a competitive inhibitor of acetylcholine for muscarinic cholinergic receptors.43 Quantitatively, scopolamine is more potent than atropine in its action on the iris, ciliary body, and certain secretory (salivary, bronchial, and sweat) glands but is less potent in its actions on the heart, and intestinal and bronchial smooth muscle. Transdermal scop patches were noted to significantly decrease tear production in the CBA mice that were used in our study. Maximum decrease in aqueous tear production was noted by 12 hours, and it persisted for more than 24 hours. By 48 hours, the cotton thread test results tended to return to normal. Based on these results, scop patches were reapplied after 2 days.

The air draft in the blower hood created an environmental stress that increased evaporation and further desiccated the ocular surface. The cotton thread test, tear fluorescein clearance, and corneal CF uptake data in mice placed in the blower hood alone were similar to those in untreated mice. It is possible that the air draft stimulated reflex tearing in these mice that compensated for the increased evaporative tear loss. This hypothesis is supported by the finding that the worst ocular surface disease was observed in the mice that were placed in the blower hood and treated with a scop patch that blocked their ability to produce reflex tears. The aqueous tear production in the scop patch + blower group was not different from that in the group that received the patch alone. However, their tear clearance was significantly decreased, probably because fluorescein tear clearance is a better measure of dynamic movement of tear fluid across the ocular surface, indirectly assessing tear production, tear drainage, tear volume, and evaporative loss.3 44 It is commonly noted that among human patients with dry eye who have the same level of aqueous tear production, some have more severe ocular surface disease, whereas others have minimal or none. We have recently reported that tear clearance shows a stronger correlation with the severity of corneal fluorescein staining in human patients with dry eye than does the level of aqueous tear production measured with the Schirmer test.44 Certainly, our findings in this mouse model of dry eye support the concept that poor tear clearance leads to ocular surface disease.

A striking finding in the dry-eye mice was a marked decrease in the number of conjunctival goblet cells compared with those in the untreated normal mice. Cytologic studies have indicated that decreased goblet cell density is a consistent finding accompanying aqueous tear deficiency in humans.45 46 Conjunctival goblet cells are a main source of the ocular surface mucoproteins that lubricate and protect the ocular surface.47 48 It is likely that the poorly lubricated ocular surface resulting from reduced mucin production by the goblet cells and nongoblet ocular surface, epithelia in KCS, makes the ocular surface more susceptible to environmental insults. The reason for the disappearance of PAS-stained goblet cells in our mice was not investigated. It is possible that these cells are still present but are not visualized, because their mucin production is inhibited by conditions on the ocular surface. Alternatively, the dry-eye environment may promote conjunctival epithelial proliferation rather than goblet cell differentiation. This is supported by our finding of a significantly greater number of conjunctival epithelial cells in the S phase of the cell cycle in the mice in which KCS developed. This mouse model provides an excellent tool to study these alternative hypotheses.

Taken together, our findings indicate that there are many similarities between this mouse model of dry eye and human aqueous tear deficiency. This model may be a useful tool for investigating the pathophysiological mechanisms for KCS.


   Footnotes
 
Supported in part by National Eye Institute Grant EY11915 (SCP) and National Cancer Institute Grant R01-CA61038 (BLL), National Institutes of Health, Bethesda, Maryland; an unrestricted grant from Research to Prevent Blindness; the Drs. David and Maureen Smith Ocular Surface and Tear Research Fund, and Allergan, Inc.

Submitted for publication October 18, 2000; revised August 6, 2001; accepted November 2, 2001.

Commercial relationships policy: N.

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: Stephen C. Pflugfelder, Cullen Eye Institute, 6565 Fannin Street, NC-205, Houston, TX 77030; stevenp{at}bcm.tmc.edu


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Schein, OD, Muñoz, B, Tielsch, JM, Bandeen-Roche, K, West, S. (1997) Prevalence of dry eye among the elderly Am J Ophthalmol 124,723-728[Medline][Order article via Infotrieve]
  2. Lemp, MA, chairman (1995) Report on the National Eye Institute/Industry workshop on clinical trials in dry eyes CLAO J 21,221-232[Medline][Order article via Infotrieve]
  3. Afonso, A, Monroy, D, Tseng, SCG, Stern, M, Pflugfelder, SC (1999) Diagnostic sensitivity and specificity of Schirmer test and fluorescein clearance test for ocular irritation Ophthalmology 106,803-810[Medline][Order article via Infotrieve]
  4. Murillo-Lopez, F, Pflugfelder, SC (1996) Dry eye Krachmer, J Mannis, M Holland, E eds. The Cornea ,663-686 Mosby St. Louis.
  5. Jones, DT, Ji, Z, Monroy, D, Pflugfelder, SC (1998) Alterations of ocular surface gene expression in Sjögren’s syndrome Adv Exp Med Biol 438,533-536[Medline][Order article via Infotrieve]
  6. Gobbels, M, Spitznas, M. (1992) Corneal epithelial permeability of dry eyes before and after treatment with artificial tears Ophthalmology 99,873-878[Medline][Order article via Infotrieve]
  7. Yokoi, N, Kinoshita, S. (1995) Clinical evaluation of corneal epithelial barrier function with a slit-lamp fluorophotometer Cornea 14,485-489[Medline][Order article via Infotrieve]
  8. Pflugfelder, SC, Tseng, SCG, Sanabria, O, et al (1998) Evaluation of subjective assessments and objective diagnostic tests for diagnosing tear-film disorders known to cause ocular irritation Cornea 17,38-56[Medline][Order article via Infotrieve]
  9. Tsubota, K. (1991) The importance of the Schirmer test with nasal stimulation Am J Ophthalmol 111,106-108[Medline][Order article via Infotrieve]
  10. Heigle, TJ, Pflugfelder, SC (1996) Aqueous tear production in patients with neurotrophic keratitis Cornea 15,135-138[Medline][Order article via Infotrieve]
  11. Gilbard, JP, Rossi, SR, Gray, CL, Hanninen, LA (1989) Natural history of disease in a rabbit model for keratoconjunctivitis sicca Acta Ophthalmol Suppl 192,95-101
  12. Pflugfelder, SC (1998) Tear fluid influence on the ocular surface Adv Exp Med. Biol. 438,611-617[Medline][Order article via Infotrieve]
  13. Pflugfelder, SC, Jones, DT, Ji, Z, Afonso, A, Monroy, D. (1999) Altered cytokine balance in the tear fluid and conjunctival epithelium of patients with Sjögren’s syndrome keratoconjunctivitis sicca Curr Eye Res 19,201-211[Medline][Order article via Infotrieve]
  14. Afonso, A, Sobrin, L, Monroy, DC, Selzer, M, Lokeshwar, B, Pflugfelder, SC (1999) Tear fluid gelatinase B activity correlates with IL-1ß concentration and fluorescein tear clearance Invest Ophthalmol Vis Sci 40,2506-2512[Abstract/Free Full Text]
  15. Tishler, M, Yaron, I, Geyer, O, Shirazi, I, Naftaliev, E, Yaron, M. (1998) Elevated tear interleukin-6 levels in patients with Sjögren syndrome Ophthalmology 105,2327-2329[Medline][Order article via Infotrieve]
  16. Virtanen, T, Konttinen, YT, Honkanen, N, Harkonen, M, Tervo, T. (1997) Tear fluid plasmin activity of dry eye patients with Sjögren’s syndrome Acta Ophthalmol Scand 75,137-141[Medline][Order article via Infotrieve]
  17. Augustin, AJ, Spitznas, M, Kaviani, N, et al (1995) Oxidative reaction in the tear fluid of patients suffering from dry eyes Graefes Arch Clin Ophthalmol 233,694-698
  18. Jones, DT, Yen, M, Monroy, D, Ji, X, Atherton, SS, Pflugfelder, SC (1994) Evaluation of cytokine expression in the conjunctival epithelia of Sjögren’s syndrome patients Invest Ophthalmol Vis Sci 35,3493-3504[Abstract/Free Full Text]
  19. Baudouin, C, Brignole, F, Becquet, F, Pisella, PJ, Goguel, A. (1997) Flow cytometry in impression cytology specimens: a new method for evaluation of conjunctival inflammation Invest Ophthalmol Vis Sci 38,1458-1464[Abstract/Free Full Text]
  20. Brignole, F, Pisella, PJ, Goldschild, M, De Saint, Jean M, Goguel, A, Baudouin, C (2000) Flow cytometric analysis of inflammatory markers in conjunctival epithelial cells of patients with dry eyes Invest Ophthalmol Vis Sci 41,1356-1363[Abstract/Free Full Text]
  21. Marsh, P, Pflugfelder, SC (1999) Topical non-preserved methylprednisolone therapy of keratoconjunctivitis sicca Ophthalmology 106,811-816[Medline][Order article via Infotrieve]
  22. Sall, K, Stevenson, OD, Mundorf, TK, Reis, BL, . the CSA phase 3 study group (2000) Two, multicenter, randomized studies of the efficacy and safety of cyclosporin ophthalmic emulsion in moderate to severe dry eye Ophthalmology 107,631-639[Medline][Order article via Infotrieve]
  23. Sullivan, DA, Allansmith, MR (1986) Hormonal modulation of tear volume in the rat Exp Eye Res 42,131-139[Medline][Order article via Infotrieve]
  24. Sullivan, DA, Bloch, KJ, Allansmith, MR (1984) Hormonal influence on the secretory immune system of the eye: androgen regulation of secretory component levels in rat tears Immunology 52,234-246
  25. Gilbard, JP, Rossi, SR, Gray, KL, Hanninen, LA, Kenyon, KR (1988) Tear film osmolarity and ocular surface disease in two rabbit models for keratoconjunctivitis sicca Invest Ophthalmol Vis Sci 29,374-378[Abstract/Free Full Text]
  26. Burgalassi, S, Panichi, L, Chetoni, P, Seattone, FM, Boldrini, E. (1999) Development of a simple dry eye model in the albino rabbit and evaluation of some tear substitutes Ophthalmic Res 31,229-235[Medline][Order article via Infotrieve]
  27. Kaswan, RL, Salisbury, MA, Ward, DA (1989) Spontaneous canine keratoconjunctivitis sicca: a useful model for human keratoconjunctivitis sicca: treatment with cyclosporine eye drops Arch Ophthalmol 107,1210-1216[Abstract]
  28. Maitchouk, DY, Beuerman, RW, Ohta, T, Stern, ME, Varnell, RJ (2000) Tear production after unilateral removal of the main lacrimal gland in squirrel monkeys Arch Ophthalmol 118,246-252[Abstract/Free Full Text]
  29. Fujihara, T, Nagano, T, Nakamura, M, Shirasawa, E. (1995) Establishment of a rabbit short-term dry eye model J Ocul Pharmacol 11,503-508
  30. Kessler, HS, Cubberly, M, Manski, W. (1971) Eye changes in autoimmune NZB and NZB X NZW mice Arch Ophthalmol 85,211-219[Medline][Order article via Infotrieve]
  31. Jabs, DA, Enger, C, Prendergast, RA (1991) Murine models of Sjögren’s syndrome Invest Ophthalmol Vis Sci 32,371-380[Abstract/Free Full Text]
  32. Jones, DT, Monroy, D, Pflugfelder, SC (1997) A novel method of tear collection: comparison of glass capillary micropipettes with porous polyester rods Cornea 16,450-458[Medline][Order article via Infotrieve]
  33. Bernal, DL, Ubels, JL (1991) Quantitative evaluation of the corneal epithelial barrier: effect of artificial tears and preservatives Curr Eye Res 10,645-656[Medline][Order article via Infotrieve]
  34. Watsky, MA, Mc.Demott, ML, Edelhauser, HF (1989) In vitro corneal endothelial permeability in rabbit and human: the effects of age, cataract surgery and diabetes Exp Eye Res 49,751-767[Medline][Order article via Infotrieve]
  35. Nikkinen, A, Lehtosalo, JI, Uusitalo, HP, Palkama, A, Panula, P. (1984) The lacrimal gland of the rat and guinea pig are innervated by nerve fibers containing immunoreactivities for substance P and vasoactive intestinal peptide Histochemistry 92,23-27
  36. Dartt, DA (1989) Signal transduction and control of lacrimal gland protein secretion: a review Curr Eye Res 8,619-636[Medline][Order article via Infotrieve]
  37. Dartt, DA, Baker, AK, Vaillant, C, Rose, PE (1984) Vasoactive intestinal polypeptide stimulation of protein secretion from rat lacrimal gland acini Am J Physiol 247,G502-G509[Abstract/Free Full Text]
  38. Ehinger, B. (1966) Ocular and orbital vegetative nerves Acta Physiol Scand 67(suppl),17-32
  39. Nikkinen, A, Uusitalo, HP, Lehtosalo, JI, Palkama, A. (1985) Distribution of adrenergic nerves in the lacrimal glands of guinea pig and rat Exp Eye Res 40,751-756[Medline][Order article via Infotrieve]
  40. Mader, TH, Stulting, RD (1991) Keratoconjunctivitis sicca caused by diphenoxylate hydrochloride with atropine sulphate (Lomotil) Am J Ophthalmol 111,377-378[Medline][Order article via Infotrieve]
  41. Crandall, DC, Leopold, IH (1979) The influence of systemic drugs on tear constituents Ophthalmology 86,115-125[Medline][Order article via Infotrieve]
  42. Dahl, E, Offer-Ohlsen, D, Lillevold, PE, Sandvik, L. (1984) Transdermal scopolamine, oral meclizine, and placebo in motion sickness Clin Pharmacol Ther 36,116-120[Medline][Order article via Infotrieve]
  43. Clissold, SP, Heel, RC (1985) Transdermal hyoscine (scopolamine) a preliminary review of its pharmacodynamic properties and therapeutic efficacy Drugs 29,189-207[Medline][Order article via Infotrieve]
  44. Macri, A, Pflugfelder, SC (2000) Correlation of Schirmer 1 test and fluorescein clearance tests with the severity of corneal epithelial and eyelid disease Arch Ophthalmol 118,1632-1638[Abstract/Free Full Text]
  45. Nelson, JD, Wright, JC (1984) Conjunctival goblet cell densities in ocular surface disease Arch Ophthalmol 102,1049-1051[Abstract]
  46. Pflugfelder, SC, Tseng, SCG, Yoshino, K, Monroy, D, Felix, C, Reis, B. (1997) Correlation of goblet cell density and mucosal epithelial mucin expression with rose bengal staining in patients with ocular irritation Ophthalmology 104,223-235[Medline][Order article via Infotrieve]
  47. Lemp, MA (1973) The preocular tear film and dry eye syndrome: the mucin-deficient dry eye Int Ophthalmol Clin 13,185-189[Medline][Order article via Infotrieve]
  48. Gipson, IK, Inatomi, T. (1997) Mucin genes expressed by the ocular surface epithelium Prog Retinal Eye Res 16,61-98



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