Steffen Sperling

APPLANATION TONOMETRY AND CENTRAL CORNEAL THICKNESS

Readings with the Goldmann applanation tonometer were made at various intraocular hydrostatic pressures and compared with central corneal thickness and radius in rabbit and in man. Linear correlations were established between hydrostatic pressure and applanation readings, with correlation coefficients close to 1.0. In rabbits the tonometer readings were generally too low. In human eyes with a normal corneal thickness tonometer readings and hydrostatic pressure coincided, with thick corneas the readings were too high, with thin corneas too low. The correlation between corneal thickness and the error of applanation tonometry (ΔP) was statistically highly significant (P < 0.001). No statistical correlation could be established between corneal radius and ΔP. Multiple regression, taking thickness as well as corneal radius into consideration, revealed only slightly higher correlation coefficients. It is concluded that the central corneal thickness is a parameter which should be taken into consideration when evaluating applanation tonometer readings. A Table is presented showing the correction to be added to the applanation reading at differing corneal thickness.

HUMAN CORNEAL ENDOTHELIUM IN ORGAN CULTURE: The Influence of Temperature and Medium of Incubation

Forty‐four human corneas from patients between 21 and 86 years were incubated in Eagles minimum essential medium with Earles salts 10–46 h post mortem. The influence of incubation temperature and composition of the medium on endothelial survival was evaluated. Whole corneas were stained by alizarine red. Recent cell loss was indicated by morphological alterations in the endothelial pattern. After 20–28 h of incubation minimum cell loss was found at 31 °C when 8% Dextrane‐250 and 20% serum or 8% Dextrane‐500 and 10% serum was added to the medium.

THE PRECISION OF UNBIASED ESTIMATES OF NUMERICAL DENSITY OF ENDOTHELIAL CELLS IN DONOR CORNEAS

The precision of estimates of central corneal endothelial density was studied in 16 human corneas stained by alizarine red and trypane blue. Estimates based on central counts and estimates based on peripheral counts were considered separately. The numerical density was estimated employing an unbiased sampling technique. From central counts estimates with an error of less than five per cent could be obtained. From peripheral counts a maximum precision of mean ± 12.2 per cent could be obtained. The theoretical maximum precision was calculated by application of a variance component model. The precision of estimates was calculated for 1, 2 and 4 test areas of different sizes. Economy of sampling was evaluated by comparison of the actual precision to the theoretical maximum precision of estimates.

Early morphological changes in organ cultured human corneal endothelium.

Nineteen human cadaver corneas with few damaged endothelial cells were incubated under tissue culture conditions for time periods ranging from five min to 48 h. Morphological alterations of the endothelial cells were studied in whole wet mounts stained by alizarine red‐alkohol‐trypane blue and by scanning electron microscopy. Joint meetings of three cells are characterisic for normal corneal endothelium. After 15–60 min of incubation, damaged cells were expelled from the coherent cell sheet by expanding neighbouring cells. Joint meetings of 5–8 expanding cells were formed. After 24 h of incubation, joint meetings of four cells were the dominating morphological abnormality. Morphological changes during reduction of the numbers of cells in joint meetings are described.

COMBINED STAINING OF CORNEAL ENDOTHELIUM BY ALIZARINE RED AND TRYPANE BLUE

Combined staining by alizarine red and trypane blue is an easy and reliable method for in vitro visualization of corneal endothelial nuclei, cellular borders and uncovered parts of the membrane of Descemet. A stained full thickness wet mount can be prepared in less than five min. The staining method and preliminary findings concerning endothelial morphology in the centre and in the periphery of normal corneas are presented.

Fresh and cultured corneal grafts compared by post-operative thickness and endothelial cell density.

Thirty‐nine corneas were removed within 6 h post mortem and stored in a moist chamber at 4°C before grafting. The mean donor age was 33 years and the average time between death and grafting was 11 h. Thirty cadaver corneas were selected after trypane blue staining and cultured at 31°C for 24 h before grafting. The mean donor age was 61 years and the mean time between death and culture was 18 h. During the first 10 postoperative days fresh grafts were thinner than cultured grafts. One year after the transplantation the two groups did not differ significantly in regard to the clinical result, corneal thickness, or endothelial cell loss. This indicates that corneas from old donors with extended post mortem time can be used for transplantation after individual evaluation and corneal culture.

Evaluation of the endothelium of human donor corneas by induced dilation of intercellular spaces and trypan blue

The endothelium of 30 pairs of human cadaver corneas was stained by trypan blue and the intercellular spaces were visualized by induced dilation prior to corneal culture. Trypan blue staining and induced dilation of intercellular spaces by 0.9% and 0.45% NaCl were found to be atraumatic. Only a fraction of damaged cells were stained by trypan blue. Endothelial cell losses in culture did not correlate with the number of trypan-blue stained cells, the post-mortem time, or donor age.

CRYOPRESERVATION OF HUMAN CADAVER CORNEAS REGENERATED AT 31°N A MODIFIED TISSUE CULTURE MEDIUM

Human corneas were obtained 2–82 h post mortem, cultured for 20–28 h, in a modified tissue culture medium, frozen at a controlled rate, and thawed rapidly. The thawed corneas were subjected to 20 –28 h of additional culture. Immediately after thawing, a mean endothelial cell damage of 11% was indicated by trypane blue staining. The mean endothelial cell loss during the subsequent culture was 34%. This cell loss was not related to post mortem time, to donor age, to cell loss during the primary culture, or to endothelial cell density.

POST-OPERATIVE THICKNESS AND ENDOTHELIAL CELL DENSITY IN CULTIVATED, CRYOPRESERVED HUMAN CORNEAL GRAFTS

Seventeen corneas obtained 2–72 h post mortem, from donors aged 17–78 years, were cultivated for 24 h, cryopreserved, thawed, cultivated for another 24 h and grafted. One year postoperatively 12 of the 17 grafts were clear. In 10 of these 12 cases the visual acuity was ≥ 0.33. One primary graft failure occurred, while 4 primarily clear grafts became cloudy due to glaucoma (2), phthisis bulbi (1) and herpetic reinfection (1). One year after the transplantations the central thickness of the clear grafts was 0.51 mm, and the endothelial cell density was 1028 cells/mm2, corresponding to 32% of the cell density before cryopreservation. This endothelial cell loss was not correlated to donor age or to the time between the death of the donor and the primary cultivation.

THYMIDINE INCORPORATION BY HUMAN CORNEAL ENDOTHELIUM DURING ORGAN CULTURE

Nine corneas from 5 human adult donors were obtained 11 to 31 h post mortem. In 4 corneas the endothelium was wounded by freezing and in 1 cornea by mechanical means. Care was taken to minimize endothelial damage in the remaining 4 corneas. The corneas were incubated at 31°C for 6 days in a tissue culture medium containing 3H thymidine. Autoradiographs were made of the flat preparations of the endothelium. All corneas contained radioactive endothelial cell nuclei, with the highest concentration of labelled nuclei being in the wound areas. The greatest number of labelled cells was found in the cornea from the youngest donor, 19 years of age, but thymidine uptake also occurred in the oldest cornea, 89‐years‐old, which additionally had signs of endothelial dystrophy.