Diabetes and corneal cell densities in humans by in vivo confocal microscopy.

PURPOSE: Diabetes is accompanied by an increased autofluorescence of the cornea, probably because of accumulation of advanced glycation end products (AGEs). The pathogenic mechanism is still unknown. This study aimed to quantify differences in corneal cell densities between diabetic patients and healthy controls. METHODS: The left cornea of 15 patients with non-insulin-dependent diabetes mellitus (NIDDM) with level of retinopathy 20 according to the Early Treatment of Diabetic Retinopathy Study (ETDRS) and of 15 healthy controls were examined by noninvasive in vivo confocal microscopy in an observational prospective study. The cell densities in 6 corneal layers were determined along the optical axis of the cornea by using stereologic methods. RESULTS: The average cell density per unit area in the superficial and basal epithelium and the endothelial layer was 725 +/- 171, 5950 +/- 653, and 2690 +/- 302 cells/mm in controls and 815 +/- 260, 5060 +/- 301, and 2660 +/- 364 cells/mm in diabetic patients. The cell density per unit volume in the anterior, mid-, and posterior stroma was 26,300 +/- 4090, 19,390 +/- 3120, and 25,700 +/- 3260 cells/mm in controls and 27,560 +/- 3880, 21,930 +/- 2110, and 25,790 +/- 3090 cells/mm in patients with diabetes. In both groups, the density in the midstroma was significantly lower than in both the anterior stroma and the posterior stroma (P < 0.02). The cell density in the basal layer of diabetic patients was significantly lower than in healthy controls (-15.0%, P < 0.0004). In the other layers, no significant differences between both groups (P > 0.07) were observed. CONCLUSIONS: The lower basal cell density found in patients with diabetes may result from a combination of different mechanisms including decreased innervation at the subbasal nerve plexus, basement membrane alterations, and higher turnover rate in basal epithelial cells. The lower cell density in the midstroma of diabetic patients and healthy controls may be attributed in part to differences in oxygen concentration in the stromal layers (depths). Changes in cellular density did not seem to be responsible for the increased autofluorescence in diabetes.

Corneal cell density measurement in vivo by scanning slit confocal microscopy: method and validation.

PURPOSE: Method and validation of a technique to quantify cell density in vivo in 6 corneal layers with a scanning slit confocal microscope (SSCM). METHOD: A confocal image of a small volume in a corneal layer is registered on videotape. Cells or nuclei according to a layer classification are counted manually using an unbiased frame. Surface cell density is calculated from an image on the screen, and volumetric density is obtained using stereological methods. RESULTS: Image distortion on the screen is less than 3%. The classification of a cell layer is verified by determining the position of the measurement volume in the cornea. Validation of density measurements is performed by comparing confocal results with those obtained by histology. The difference between the two methods varies from -24.1% (posterior stroma) to +16.4% (basal layer). Intersession and intrasession repeatability are 8.3 and 5.8%, respectively. The cell density (mean +/- SD) in 20 healthy controls in the superficial, basal and endothelial layers was 759 +/- 162, 5,817 +/- 632 and 2,743 +/- 285 cells.mm(-2) (surface), and in the anterior, mid and posterior stroma 28,616 +/- 3,081, 19,578 +/- 4,421 and 26,073 +/- 5,962 cells.mm(-3) (volumetric). These results are in accordance with those of other investigators. CONCLUSIONS: The SSCM can produce repeatable quantitative measurements of corneal cell density in conscious humans.

Corneal autofluorescence in relation to permeability of the blood-aqueous barrier in diabetic patients with clinically significant macular edema and in an age-matched control group.

PURPOSE: Corneal autofluorescence is related to advanced glycation end products formed by glucose that reaches the cornea via the aqueous humour. The aim of the study was to examine the influence on autofluorescence of changes in permeability of the blood aqueous barrier. METHODS: Corneal autofluorescence was measured in 50 diabetic patients with clinically significant macular edema and in 28 age-matched control subjects. Permeability of the blood aqueous barrier was assessed using the diffusion coefficient of fluorescein. RESULTS: Corneal autofluorescence was higher in diabetic subjects than in the control group, mean (SD) at an excitation wavelength of 458 nm was 41.2 ng f-eq/ml (11.7) in diabetic patients and 26.5 ng f-eq/ml (7.3) in the control group, p < 0.001. The mean permeability of the blood aqueous barrier, Kd(F), was 492.0.10(-6) min(-1 )in the diabetic patients and 484.2. 10(-6) min(-1)in the control group. There was no association between permeability of the blood aqueous barrier and corneal autofluorescence, p = 0.99 for the diabetic patients and p = 0.15 for the control group (458 nm). CONCLUSIONS: Corneal autofluorescence was unaffected by permeability of the blood aqueous barrier suggesting that formation of advanced glycation products is limited by other factors than the concentration of glucose in the aqueous humour, or that other factors unrelated to nonenzymatic glycation of stromal proteins are involved.

Lack of fluorophotometric evidence of aqueous-vitreous barrier disruption after posterior capsulorhexis.

PURPOSE: To evaluate the integrity of the aqueous-vitreous barrier by assessing the flow of fluorescein from the anterior chamber to the anterior vitreous using fluorophotometry in eyes with a posterior continuous curvilinear capsulorhexis (PCCC) and in eyes without a PCCC. SETTING: University Hospital Antwerp, Edegem, Belgium. METHODS: Ten patients had bilateral extracapsular cataract extraction with implantation of an intraocular lens. In 1 eye, a PCCC was performed; the other eye served as a negative control. The eyes of 2 other patients who had complicated cataract surgery with posterior capsule and anterior hyaloid membrane rupture served as positive controls. All patients had fluorophotometry of both eyes 12 to 18 months after surgery to measure the flow of fluorescein from the anterior chamber to the anterior vitreous. RESULTS: There were no statistically significant differences in the distribution pattern of fluorescein between eyes with PCCC and eyes without PCCC. In contrast, enhanced flow was detected in both eyes with rupture of the posterior capsule and the anterior hyaloid. CONCLUSIONS: In this fluorophotometry study, a PCCC did not seem to disrupt the aqueous-vitreous barrier. Results indicate that an intact anterior vitreous membrane is crucial to maintain the barrier function between the anterior and the posterior segments of the eye.

Derivation of lenticular transmittance from fluorophotometry.

PURPOSE: To derive transmittance spectra for the human lens using the ratio between posterior and anterior autofluorescence of the lens as measured by fluorophotometry. METHODS: Transmittance spectra of the lens can be described with a one-parameter model to a high degree of accuracy. The parameter m of this model defines the differences between lens transmittance spectra of individuals. In fluorophotometry literature another parameter related to lens transmittance, T, has been defined as the square root of the ratio between posterior and anterior lenticular autofluorescence. T can be predicted from parameter m, given the spectra of the excitation light, of the fluorescence emitted by the lens and of the detecting device are known, and assuming that the anterior and posterior fluorescence efficiencies of the lens are equal. When this relation is inverted, parameter m can be derived from T, giving the complete transmittance spectrum on the basis of T. RESULTS: A transformation curve was calculated to determine T from m and vice versa. The light transmittance spectrum of the lens was calculated as a function of T. The validity of this approach was evaluated using an independent method for assessment of lenticular transmittance. This method consisted of making color slitlamp slides, grading the observed color of these slides with the LOCS III NC grading system, and transforming these grades into the model parameter m using published transformation curves. CONCLUSIONS: The total transmittance spectrum can be calculated reliably from a fluorophotometric scan of the human lens.

Reflex and steady state tears in patients with latent stromal herpetic keratitis.

PURPOSE: To compare tear production in patients with stromal herpetic keratitis with that in healthy control subjects. METHODS: After instillation of 2 microL fluorescein into both eyes, the tear-fluorescein concentration was measured by fluorophotometry. During the first 10 minutes, steady state tear turnover (TTO-1) was determined. After a nasal alcohol stimulus to induce reflex tears, a second steady state tear turnover (TTO-2) was obtained during 15 minutes. The index of reflex lacrimation (IRL) was calculated as the percentage decrease in tear fluorescein concentration directly after the stimulus. TTO-1, TTO-2, and IRL were determined in the patients' affected eyes (n = 12), in the patients' healthy contralateral eyes, if possible (n = 9), and in one eye of healthy control subjects (n = 24). RESULTS: The TTO-1 in the affected and healthy eyes of patients was approximately two times lower than the TTO-1 in eyes of healthy control subjects (P = 0.012 and P = 0.024, respectively) and almost equal to the TTO-2 in eyes of healthy control subjects (P = 0.32 and P = 0.40). There were no significant differences in the values of TTO-1, IRL, and TTO-2 between affected and healthy eyes of patients (P > 0.5). IRL and TTO-2 did not differ significantly among the three groups (P > 0.5). CONCLUSIONS: Both eyes of the patients were dry. The dryness could be due to a defective reflex lacrimation under physiological conditions that can still be induced by nonphysiological nasal excitation. The cause of this may be demyelination of both trigeminal root entry zones as a result of a unilateral eye infection by the herpes virus. Another possibility is that dryness predisposes to herpetic infection or recurrent inflammation.