Tear production after unilateral removal of the main lacrimal gland in squirrel monkeys.

OBJECTIVE: To study the effects of lacrimal gland removal on basal and reflex tear production and on the ocular surface in the squirrel monkey. METHODS: Unilateral main lacrimal gland removal in 6 squirrel monkeys was followed by Schirmer testing, slit-lamp examination with fluorescein, and collection of basal and reflex (stimulated) tears for analysis of tear protein spectra between 0 and 20 kd, as well as histological evaluation. RESULTS: Schirmer test results showed an 80% decrease in basal tears and a 90% decrease in reflex tears during week 1, and a 32.2% and 33.3% decrease, respectively, at week 20 after surgery, compared with the contralateral control side. However, no gross abnormalities or fluorescein staining were seen in 5 of the 6 monkeys, and the conjunctival surfaces remained normal. The main and accessory lacrimal glands appeared to secrete similar types of proteins. No histological changes were seen in corneal, conjunctival, or eyelid tissues 20 weeks after surgery. CONCLUSIONS: Tears from accessory lacrimal glands were sufficient to maintain a stable tear layer on the cornea, suggesting that so-called basal tear flow is made up of fluid from both main and accessory lacrimal glands and that decreased tear production by the main lacrimal gland is not a causative factor in keratoconjunctivitis sicca. CLINICAL RELEVANCE: This study shows that total removal of the main lacrimal gland does not in itself lead to keratoconjunctivitis sicca. However, the nature of neural control of the accessory glands is not yet clear.

Confocal microscopy in posterior polymorphous corneal dystrophy.

PURPOSE: To report the distinguishing characteristics of posterior polymorphous corneal dystrophy (PPMD) using confocal microscopy. MATERIAL AND METHODS: Two consecutive patients with PPMD were prospectively examined using a white-light tandem scanning confocal microscope with a 24x/0.60 contact objective. RESULTS: At the level of Descement's membrane, roundish hyporeflective images were found in 1 patient. In the other patient, hyporeflective bands were detected. In both patients, patchy hyperreflective areas were identified. CONCLUSION: Confocal microscopy may allow diagnosis of PPMD by demonstrating the alterations in Descement's membrane. This technique is especially valuable in cases of endothelial decompensation, where slit-lamp and specular microscopy may fail to demonstrate changes in Descement's membrane.

Interface inflammation after laser in situ keratomileusis. Sands of the Sahara syndrome.

PURPOSE: To determine the source of the interface debris that causes the interface inflammation known as "sands of the Sahara" after laser in situ keratomileusis (LASIK). SETTING: Department of Ophthalmology, LSU Eye Center, Louisiana State University Medical Center School of Medicine, New Orleans, USA. METHODS: A microkeratome (Automated Corneal Shaper) was used to make a LASIK flap in 8 eyes of 4 rabbits. In 4 eyes, the blade was used directly from the sterile pack; in the contralateral 4 eyes, the blade was cleaned prior to use. In vivo confocal microscopy of the corneas was performed 1 day after surgery. An unused, cleaned blade and an unused, uncleaned blade, as well as blades used in the rabbit eyes, were examined by scanning electron microscopy. RESULTS: Confocal microscopy revealed numerous fragments of debris surrounded by inflammatory cells in the LASIK flap interfaces created by blades taken directly from the sterile package. Interfaces created by the cleaned blades showed only rare, scattered bits of debris. Scanning electron microscopy of the unused blades showed debris on the uncleaned blade removed directly from the sterile package. CONCLUSION: Post-LASIK interface inflammation may be caused by debris on the microkeratome blade, although other sources are possible. The interface debris and inflammation can be reduced or eliminated by cleaning the microkeratome blade before use.

In vivo confocal imaging of corneal neovascularization.

PURPOSE: To investigate the cellular dynamics of vessel formation during corneal neovascularization in the living eye by confocal microscopy. METHODS: Corneal neovascularization was initiated by placing a 7-0 silk suture through the corneal stroma 3 mm from the limbus at the 12 o'clock position in both eyes of 10 New Zealand white rabbits. The corneas were examined for vessel ingrowth at intervals from 1 to 15 days after suture placement using a tandem scanning confocal microscope with a 20X water immersion objective, as well as a slit-lamp biomicroscope. Changes in the limbal vessels were recorded on videotape for later analysis. As early vessel growth appeared to be associated with corneal nerves, the total number of sprouts and the number of sprouts along nerves were counted in confocal images, and the results analyzed for statistical significance. Vessel growth and the structural relationship between vascular buds and the deep stromal nerves were examined by light and transmission electron microscopy. RESULTS: The early events of cell migration from the limbal microvessels were found to be associated with the deep stromal nerves; although this association was easily visualized by confocal microscopy, it could not be documented by slit-lamp biomicroscopy. By 18 h after suture placement, the limbal vessels were dilated and the first vascular buds appeared as short, pointed, or flat-topped protrusions from the deep limbal capillaries. By 96 h, the capillary buds had increased in density and had begun to form lumens. Movement of red blood cells was established between 72 and 80 h after the first signs of bud formation, at the same time that cells of immune origin were seen. Confocal microscopy revealed and transmission electron microscopy verified that new bud formation began with the formation of vascular tubes by endothelial migration along the deep stromal nerves. The total number of sprouts and the number of sprouts associated with stromal nerves were similar on days 1 and 2 but differed on days 3-7, suggesting an association between sprouts and nerves in the early stages of neovascularization. CONCLUSION: Using real-time white light confocal microscopy, we were able, for the first time, to observe the process of corneal neovascularization in the living eye, from the earliest stages within hours after initiation to 2 weeks. The deep stromal nerves appear to serve as a focus for the growth of new vessels, by attracting and supporting vessel growth and/or by providing a potential space for movement of the endothelial cells. Confocal microscopy may provide a new approach to achieving a better understanding of the mechanisms involved in corneal neovascularization.

Characterization of fibrous retrocorneal membrane by confocal microscopy.

PURPOSE: To study the appearance of a fibrous retrocorneal membrane as seen by confocal microscopy. METHODS: A 67-year-old white woman with a history of multiple ocular surgeries, including repeated penetrating keratoplasties for aphakic bullous keratopathy, developed a retrocorneal membrane in the right eye. The membrane was first noticed 3 years after the last corneal transplant and remained stable subsequently. The patient was examined by in vivo white light tandem-scanning confocal microscopy. RESULTS: At the level of the retrocorneal membrane, confocal microscopy disclosed the presence of a hyperreflective fibrous-appearing layer. Normal endothelial cells could not be found. Anterior to the hyperreflective layer, activated keratocytes were identified. CONCLUSION: Confocal microscopy may allow noninvasive diagnosis of fibrous retrocorneal membrane. Additionally, our data suggest that the posterior keratocytes might play a role in the production and deposition of fibrous tissue.

Detection of substance P in human tears by laser desorption mass spectrometry and immunoassay.

PURPOSE: To determine whether substance P is present in human tears. METHODS: Tear samples (1-2 microliters) were collected from one eye of each of 12 subjects. Two of the eyes had dry eye syndrome, two wore contact lenses and had dry eye syndrome, and eight were normal. Five of the eight normal eyes were scheduled to undergo excimer laser refractive surgery, and tears were collected from these eyes before and after surgery. Tear samples were analyzed by laser desorption mass spectrometry. Pooled samples from one individual were subjected to enzyme-linked immunoabsorbent assay. RESULTS: Laser desorption mass spectra of the 18 tear samples displayed well defined peaks with mass to charge (m/z) ratios ranging from 1343.7 to 1355.9 and/or 1356.9 to 1364.7, corresponding to an average m/z of 1349.8 +/- 1.13 for protonated substance P and 1361.2 +/- 0.54 for oxidized substance P obtained from 14 mass spectra of standards formulated with substance P concentrations ranging from 10(-4) M to 10(-12) M. As confirmation, an enzyme-linked immunoabsorbent assay performed twice on pooled tears from one eye detected substance P in both replicates at a concentration of 125 pg/ml (9.26 x 10(-11) M). CONCLUSIONS: These findings demonstrate that substance P is a component of tears obtained from normal eyes of men and women ranging in age from 26 to 60 years, from eyes fitted with contact lenses, from eyes with dry eye syndrome, and from eyes 1 and 2 days after excimer laser refractive surgery. Whether the concentration of substance P in tears varies with sex, age, or eye condition, the source of substance P in tears, and its role in tears remains to be discovered.

Analysis of rabbit tear fluid using capillary electrophoresis with UV or laser-induced fluorescence detection.

In preliminary studies of the development of tear analysis methodology that may eventually be useful in the clinical setting, the authors evaluated various protocols for analyzing rabbit tears by capillary zone electrophoresis (CZE). Conditions included the use of a 50-mM monosodium phosphate buffer, pH 2.5, or a 400-mM sodium borate buffer, pH 8.9, both with ultraviolet (UV) detection, as well as a 50-mM borate buffer, pH 8.5, with laser-induced fluorescence (LIF) detection of ATTO-TAG CBQ (Molecular Probes, Inc., Eugene, OR, U.S.A.) derivatized tears. All CZE analyses were performed with a P/ACE System 2100 instrument equipped with System Gold software (Beckman Instruments, Fullerton, CA, U.S.A.), using a 50 microns x 57 cm (50 cm to the window) fused-silica capillary, at 25 degrees C, with constant voltage of 20 kV for UV detection and 11 kV for LIF detection. Tear samples were collected from normal rabbit eyes by means of 10-microL glass micropipets. The volume of each sample was approximately 2 microL. Analysis using the phosphate buffer with UV detection produced as many as 35 peaks in each sample, of which 11 peaks were readily discerned. This compared favorably with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, which produced 32 bands with silver staining and 11 quantifiable bands with Coomassie brilliant blue staining. Many of the tear protein components have yet to be identified. CZE analysis with the high-ionic-strength borate buffer with UV detection produced only four peaks, and the low-ionic-strength borate buffer with LIF detection produced only six peaks. CZE analysis was completed in less than 1 hr, compared with 7-8 hr for SDS-PAGE. In summary, CZE analysis of tear fluid is comparable to CZE analysis of other bodily fluids and shows great potential for use in clinical diagnosis as well as for enhancing our understanding of the cellular actions of tears on the front of the eye.