William M. Bourne

Specular Microscopy of Human Corneal Endothelium in VIVO

A clinical specular microscope used in the routine examination fo the corneal endothelium of 40 patients at high magnification, without inconvenience or discomfort to the patients, detected endothelial damage or disease that was not seen by slit-lamp examination. A statistically significant decrease (P less than .001) in the number of central endothelial cells with age was documented.

Cellular changes in transplanted human corneas.

Purpose. To measure endothelial cell and keratocyte densities in transplanted corneas and the changes in these densities with time. Methods. The endothelia of 500 consecutive penetrating corneal transplants were studied longitudinally by specular microscopy for 10 to 20 years. The keratocytes of 36 corneal transplants that varied in postoperative times from 1 month to 20 years were studied cross-sectionally by clinical confocal microscopy. The keratocytes of five transplanted corneas were studied longitudinally by confocal microscopy at 1 day, 1 week, and 1 month postkeratoplasty. Results. Endothelial cell density decreased progressively at an accelerated rate for 20 years after transplantation, with concurrent increases in the coefficient of variation of cell area and corneal thickness and decreases in the percentage of hexagonal cells. Grafts with insufficient endothelial cells developed late endothelial failure, which was the primary cause of graft failure after the first 5 postoperative years. The grafts with late endothelial failure did not lose endothelial cells faster than grafts that did not fail, but instead had fewer cells immediately after transplantation, diminishing to a critically low cell density earlier. The keratocyte density was also decreased in transplanted corneas. Keratocytes became “activated” during the first week after keratoplasty and in grafts with late endothelial failure. Conclusion. It should be possible to prevent or delay late endothelial failure, the primary cause of graft failure, by increasing the number of endothelial cells on transplanted corneas. The status of the keratocytes appears to affect corneal transparency and, thus, visual quality in the grafted eye.

Endothelial Damage Associated with Intraocular Lenses

We examined the corneal endothelium of five patients with the clinical specular microscope before and at intervals after combined cataract extraction and intraocular lens implantation. There was a severe loss of endothelial cells postoperatively in all five patients despite clear, thin corneas. We observed no significant endothelial regeneration (increased number of cells) or continued cell loss during 15 weeks of postoperative observation. The clinical specular microscope was useful in assessing the endothelial effects of ocular surgical procedures.

Cataract Extraction and the Corneal Endothelium

We examined and photographed the central corneal endothelium of 16 patients with the clinical specular microscope before and at intervals after cataract extraction. No detectable loss of the endothelial cells occurred in 75% of the patients, including 12 routine intracapsular cryoextractions and four phacoemulsifications of soft cataracts in young adults. Only one of the four cases of significant endothelial cell loss occurred in a normal cornea without demonstrable operative or postoperative complications. Two of the four corneas that lost central endothelial cells at cataract extraction continued to lose more cells during the ensuing weeks. A significant increase in central endothelial cell density was demonstrated in one patient. The four corneas with endothelial cell loss also had a significantly higher mean increase in corneal thickness postoperatively, although this was transient. All 16 corneas remained clear during the period of observation (maximum, 14 weeks), and long-term studies are needed to measure the chronic effects of the endothelial damage.

The Effect of Age on the Corneal Subbasal Nerve Plexus

Purpose: To measure subbasal nerve density and orientation in normal human corneas across a broad age range. Methods: Sixty-five normal corneas of 65 subjects were examined by using tandem scanning confocal microscopy. Ages of subjects ranged from 15 to 79 years (mean 46 ± 19 years), with 5 subjects from each hemidecade. Subbasal nerve fiber bundles appeared as bright, well-defined linear structures in confocal images of the central cornea. Images from 3 to 8 scans per eye (mean 4.6 ± 1.8 scans) were randomly presented to a masked observer for analysis. The mean subbasal nerve density (total nerve length [μm] within a confocal image [area = 0.166 mm2]), the mean nerve number per confocal scan, and the mean nerve orientation were determined by using a custom software program. Correlations between age and nerve density and age and nerve orientation were assessed by using Pearson correlation coefficients. Results: The subbasal nerve plexus was visible in the central cornea of all subjects. The mean subbasal nerve density was 8404 ± 2012 μm/mm2 (range 4735 to 14,018 μm/mm2). The mean subbasal nerve number was 4.6 ± 1.6 nerves (range 1 to 8 nerves). The mean subbasal nerve orientation was 94 ± 16 degrees (range 58 to 146 degrees). There was no correlation between age and subbasal nerve density (r = 0.21, P = 0.09) or between age and subbasal nerve orientation (r = −0.19, P = 0.12). Conclusion: The density and orientation of the subbasal nerve plexus in the central human cornea does not change with age.

Increased corneal thickness in patients with ocular hypertension

BACKGROUNDnCentral corneal thickness greater than 0.520 mm causes true intraocular pressure to be overestimated when the technique of applanation tonometry is used to measure intraocular pressure.nnnOBJECTIVEnTo compare the corneal thickness measurements of patients enrolled in a study of ocular hypertension with those of age-matched control subjects with normal intraocular pressure.nnnMETHODSnCentral corneal pachymetry using an optical pachymeter was performed on each study subject (n = 55) at baseline and in an independent sample of control subjects. A 2 sample, 2-tailed T test was used to compare the 2 populations.nnnRESULTSnThe patients with ocular hypertension had significantly higher mean corneal thickness measurements (mean +/- SD, 0.594 +/- 0.037 mm) than the control group (0.563 +/- 0.027 mm) (P<.001).nnnCONCLUSIONnCorneal thickness may be a confounding factor in the measurement of intraocular pressure, and this may modify the risk for progression to glaucoma in patients with ocular hypertension.

The Effect of Contact Lens Wear on the Central and Peripheral Corneal Endothelium

PURPOSEnTo compare central and peripheral corneal endothelial cell morphometry in normal subjects and long-term contact lens wearers.nnnMETHODSnEndothelial cell density (ECD), coefficient of variation of cell area (CV), and percentage of six-sided cells were measured by contact specular microscopy in the corneal center and temporal periphery of both eyes of 43 long-term contact lens wearers and in 84 normal subjects who had never worn contact lenses. The latter group included 43 age- and sex-matched controls for the contact lens wearers. ECDs were corrected for magnification changes due to corneal thickness.nnnRESULTSnCentral ECD (2,723+/-366 cells/mm2, mean +/- SD) was significantly higher than peripheral ECD (2,646+/-394 cells/mm2) for the normal group (p = 0.01) but not for the contact lens wear group (2,855+/-428 cells/mm2 central, 2,844+/-494 cells/mm2 peripheral, p = 0.84). Peripheral CV was significantly higher than central for normal subjects and contact lens wearers and was significantly higher in both center and periphery in contact lens wearers than in controls. Central percentage of six-sided cells was significantly higher than peripheral for normal subjects and contact lens wearers and was lower in both center and periphery in contact lens wearers than in controls.nnnCONCLUSIONSnCentral ECD was significantly higher by 3% than peripheral ECD in normal subjects, but not in contact lens wearers. The results suggest that contact lens wear causes a mild redistribution of endothelial cells from the central to the peripheral cornea. A reversal of this redistribution after contact lens wear is discontinued for refractive surgery could mask mild central endothelial damage from the refractive procedure.

Comparison of corneal endothelial cell images from a noncontact specular microscope and a scanning confocal microscope

Purpose: We compared endothelial cell density (ECD) from images recorded by the ConfoScan 3 confocal microscope and a noncontact specular microscope. Methods: Endothelial micrographs of 50 normal corneas of 25 subjects were acquired by a Konan Noncon Robo noncontact specular microscope (Konan Medical, Inc., Hyogo, Japan) and a ConfoScan 3 confocal microscope (Nidek Technologies, Inc, Greensboro, NC). ECD was determined in images from both instruments by using the HAI CAS System Corners Method (HAI Labs, Inc., Lexington, MA). Distances in the images from both machines were calibrated from images of an external scale. Images from the ConfoScan 3 were also assessed using the automated endothelial analysis software provided by the manufacturer, with and without manual correction. Results: The ECD was 2634 ± 186 cells/mm2 (mean ± SD) and 2664 ± 173 cells/mm2 by the Robo and ConfoScan 3 Corners methods, respectively. Differences between these 2 methods were not significant. When the automated analysis software was used, however, significant differences were found (P = 0.001). The uncorrected analysis program provided with the ConfoScan 3 indicated a higher ECD (2742 ± 284 cells/mm3) than the Corners method did with images from the Robo and ConfoScan 3. The ECD from the manually corrected ConfoScan 3 method was 2716 ± 229 cells/mm3, not significantly different from the ConfoScan 3 Corners method but significantly different from the Robo Corners method. Conclusions: The ConfoScan 3 can be used interchangeably with the Robo when the Corners method is used to assess ECD and the magnification of both microscopes is calibrated with an external scale. If the proprietary software provided with the ConfoScan 3 is used, it should be manually corrected.

Comparison of recording systems and analysis methods in specular microscopy

PURPOSEnTo compare corneal endothelial cell images from contact and automated noncontact specular microscopes and to compare endothelial image analysis by the Konan Robo Center Method and the Bio Optics Bambi Corners Method.nnnMETHODSnTwenty-six normal corneas of 13 subjects and 41 penetrating keratoplasties (PKs) of 38 patients were photographed with a Keeler-Konan contact specular microscope and a Konan Noncon Robo automated noncontact specular microscope. (i) After measuring and calibrating the magnification of each instrument, we digitized the cellular apices and analyzed the images from both instruments by using the Corners Method modified to accept x and y calibrations. (ii) Using the internal calibration marks of the Konan Noncon Robo specular microscope for calibration of magnification (as required for the Center Method), we evaluated identical cells on images from this microscope by both the Center Method and the Corners Method. (iii) We evaluated the reproducibility of both methods by repeating measurements on the same image.nnnRESULTSn(i) When the images were properly calibrated for magnification by using an external scale, endothelial cell density (ECD) of normal corneas was 2,703 +/- 354 (mean +/- SD) cells/mm2 by contact and 2,685 +/- 357 cells/mm2 by noncontact techniques (p = 0.51). ECD of PK corneas was 1,767 +/- 773 cells/mm2 by contact and 1,807 +/- 775 cells/mm2 by noncontact techniques (p = 0.31). (ii) When images from the Konan Noncon Robo specular microscope were calibrated for magnification on the internal marks, the measured ECD from the same noncontact photographs was 6% less (p < 0.001). ECD was then 2,519 +/- 294 cells/mm2 (means +/- SD) by the Center Method and 2,523 +/- 305 cells/mm2 by the Corners Method (p = 0.55) in normal corneas and 1,715 +/- 748 cells/mm2 by the Center Method and 1,731 +/- 763 cells/mm2 by the Corners Method (p = 0.04) in PK corneas. (iii) The coefficient of variation of repeated measurements on the same normal image was 0.0025 for the Centers Method and 0.0099 for the Corners Method.nnnCONCLUSIONSn(i) Images from the automated noncontact specular microscope may be used interchangeably with those from the contact specular microscope to measure ECD, but only if both are properly calibrated by measuring an external scale. (ii) As a method of analysis, the Center Method is equivalent to the Corners Method in normal corneas, but the proprietary internal calibration of the Center Method, which is required for its use, yields ECDs approximately 6% less than when an external scale is used for distance calibration. (iii) Cell density measurements by both the Center Method and the Corners Method were reproducible within 1%.

Effects of glaucoma medications on corneal endothelium, keratocytes, and subbasal nerves among participants in the ocular hypertension treatment study

Purpose: To compare subbasal corneal nerve and keratocyte density and endothelial characteristics of ocular hypertensive patients treated with medications or observation. Methods: Participants in the Ocular Hypertensive Treatment Study (OHTS) randomized at Mayo Clinic to medication or observation were evaluated with specular microscopy annually for 6 years. Confocal microscopy was performed 78 to 108 months after enrollment. Subbasal nerve density was calculated by manual tracing and digital image analysis. Keratocyte density was determined by manual counting methods. Data were compared using a t test and a rank sum test. Results: After 6 years, corneal endothelial cell density, percent hexagonal cells, and coefficient of variation of cell area for the observation (n = 21) and medication groups (n = 26) were similar (2415 ± 300 vs. 2331 ± 239 cells/mm2; 63% ± 11% vs. 65% ± 10%; and 0.32 ± 0.07 vs. 0.30 ± 0.06, respectively). Of 38 participants undergoing confocal examination, the medication group (n = 19) had fewer nerves (3.8 ± 2.1 vs. 5.9 ± 2.0 nerves/frame; P = 0.02) and a lower nerve density (5643 ± 2861 vs. 9314 ± 3743 μm/mm2; P = 0.007) than the observation patients (n = 10). An additional 9 patients in the observation group, who began medication before confocal scanning, had intermediate nerve densities. Full-thickness keratocyte density was similar, with 22,257 ± 2419 and 23,430 ± 3285 cell/mm3 in the observation and medication groups, respectively. Conclusions: Chronic administration of glaucoma medications causes a decrease in the number and density of corneal subbasal nerve fiber bundles but does not affect keratocyte density or corneal endothelial characteristics.