Apoptotic cells were identified in the corneal endothelium of
human organ cultured corneas: Their number and distribution
demonstrated a close correlation with corneal folding and the overall
quality of the corneal endothelium. CEC apoptosis appeared to be
independent of cause of donor death, death to enucleation time, and
death to culture times
(Table 2) .
Apoptosis was confirmed by the presence of morphologic alterations
characteristic of apoptotic cell nuclei
(Figs. 1A 1B 1C) . Chromatin condensation and nuclear blebbing in human CECs
was demonstrated by Hoechst staining
(Fig. 1A) and the TUNEL technique
(Figs. 1B 1C) . Caspase-3 activity was also demonstrated by
immunolabeling in areas between folds and along folds
(Fig. 1D) ,
providing further evidence for CEC apoptosis.
TUNEL labeling of cells was seen throughout the endothelium and along
corneal folds
(Fig. 1E) . The presence of TUNEL-positive apoptotic cells
appeared to be age-dependent. TUNEL-positive cells were not observed in
corneas from a 1.5-year-old donor
(Fig. 1F) , and very few were
identified in corneas from an 18-year-old donor,
Figures 1G(i) and
1G(ii) . However, TUNEL-positive cells were scattered across the corneal
endothelium, often at high density, in all donors over 40 years of age
as is demonstrated in
Figures 1G(i) and
1G(ii) . The total number of
apoptotic cells increased from 1.5% in donors less than 20 years of
age to greater than 15% in donors aged 61 to 80 years. Despite this, a
significant correlation between donor age and percentage apoptosis was
not found (
P = 0.652,
r s = 0.089). In all corneas analyzed
where TUNEL labeling was identified, the spatial distribution of
TUNEL-positive cells demonstrated a higher concentration of apoptotic
endothelial cells along areas with corneal folds,
Figures 1G(ii) and
1H(ii) , significant in both central and peripheral corneas
(
t = −4.643, and
t = −4.681,
P < 0.001, respectively). This association was
demonstrated in
Figure 2 , where a positive significant correlation between the number of cells
in unfolded areas and areas with folds was confirmed using Spearman’s
rank correlation, where
r s = 0.709 and
0.629,
P < 0.001, in both central and peripheral areas
of endothelium, respectively. The overall number of apoptotic cells
appeared to be greater in the peripheral cornea. This is not surprising
because there is an overall increase radially in the number of
endothelial cells from the center of the cornea outward.
The temporal distribution of TUNEL-labeled endothelial cells
demonstrated an insignificant correlation with length of storage time
in organ culture (
Fig. 3 , not significant at
P = 0.882, Spearman’s rank
correlation coefficient,
r s = 0.029).
Except in one 37-year-old cornea, significant numbers of TUNEL-positive
cells (greater than 5% apoptosis) were only apparent in donor corneas
stored for more than 14 days.
As expected, the percentage of TUNEL-positive cells demonstrated a
significant correlation with endothelial cell count (
Fig. 4 , Spearman’s rank correlation coefficient,
r s = −0.543,
P <
0.005) and the overall quality of the endothelium (
Fig. 5 , Spearman’s rank correlation coefficient,
r s = 0.707,
P <
0.001), as determined at the time of endothelial assessment
(see
Table 1 ). Percentage apoptosis was least in corneas that had an excellent
endothelium and greatest in those assessed as having a poor or very
poor endothelium.