Abstract
purpose. To determine whether endothelial cell loss of human corneas stored in
organ culture before transplantation is due to apoptosis.
methods. The corneal endothelium of human corneas, stored in organ culture at
34°C for varying periods of time, were analyzed for the presence of
apoptotic cells using the TdT-mediated dUTP nick-end labeling (TUNEL)
technique. Corneal endothelial cell apoptosis was confirmed by Hoechst
staining and immunolabeling with anti–caspase 3 active antibody.
results. 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 overall quality of the
corneal endothelium. TUNEL-positive labeling of cells was confirmed as
apoptotic by the presence of morphologic nuclear alterations identified
by Hoechst staining and the presence of immunostaining for caspase-3
activity. Corneal endothelial cell apoptosis was independent of cause
of donor death, death to enucleation time, and death to culture times.
conclusions. Corneal endothelial cell apoptosis appears to determine the suitability
of a cornea for transplantation.
The UK Corneal Transplant Service (CTS) Eye Banks provide
organ-cultured donor corneas for over 2500 corneal transplants per
annum. The success of corneal transplantation is dependent on a healthy
endothelium in the donor cornea,
1 which is essential to
maintain corneal transparency.
2
The nonregenerative capacity of human corneal endothelial cells (CECs)
necessitates the requirement of a storage technique that will maintain
the metabolic function and integrity of donor cadaver corneal
endothelium until transplantation. The CTS Eye Banks in the UK and
Europe have used organ culture storage of donor corneas at 34°C for
this purpose.
3 The use of organ culture for corneal
storage as opposed to other short-term storage media (e.g.,
Optisol in the UK CTS Eye Banks) is generally due to the short
supply of corneas available for transplantation in the United Kingdom.
The extended storage time allows corneas to be stored for up to 28
days, ensuring reduced wastage due to outdating and that a supply of
corneas is always available for emergency procedures. Other benefits of
an extended storage time include routine surgical procedures,
virological testing, and tissue-typing where necessary. Contaminated
corneas are discarded before transplantation as a result of
microbiologic screening, and the evaluation of corneal endothelium
ensures a high quality of donor tissue for transplantation.
The standard prerequisite of an endothelial cell count greater than
2200 cells/mm
2 in all transplantable donor
corneas in the CTS Eye Banks ensures a sufficient cell reserve to
withstand the stresses of transfer from donor to recipient and will
maintain quality of vision for many years after surgery.
4 However, this results in rejection of over 30% of organ-cultured donor
corneas at the time of endothelial assessment before transplantation
due to endothelial deficiencies.
5 Previous studies have
shown that deterioration of the corneal endothelium occurs with
increasing length of organ culture storage time.
5 6 7
Such deterioration of corneal endothelium during organ culture may be a
result of cell loss due to apoptosis or necrosis. Apoptosis is a
physiological and active mode of cell death that is tightly
controlled in multicellular organisms to maintain tissue
homeostasis.
8 Morphologically apoptotic cells can be
identified by condensation and fragmentation of the nucleus. In
contrast to necrotic cell death, the plasma membrane of apoptotic
cells remains intact, and apoptotic cells are rapidly recognized and
phagocytosed without detriment to neighboring viable cells. Biochemical
hallmarks of apoptosis are the nonrandom cleavage of DNA into 50-kbp
fragments that are (in most cell types) subsequently cleaved to 180-bp
integers, which can be readily identified using the TUNEL technique and
agarose gel electrophoresis.
9 Various studies have also
demonstrated the pivotal role of caspases in mammalian cell
apoptosis.
10
The aim of this investigation was to determine whether deterioration of
the human corneal endothelium during organ culture storage resulted
from cell loss due to CEC apoptosis.
Twenty-eight human donor corneas from 20 donors, between 1.5 and
87 years of age, were obtained from the Manchester Eye Bank. All
corneas were received from donors with specific consent for research
purposes and those contraindicated for transplantation due to cause of
death but without endothelial insufficiency. All eyes were retrieved
within 36 hours of cadaver time and were received as whole eyes in
moist chamber at 4°C.
Corneoscleral discs were excised and stored using the organ
culture technique according to standard CTS Eye Bank operating
procedures.
5 After thorough cleansing of whole eyes (four
rinses in sterile 0.9% [wt/vol] NaCl, a 3-minute immersion in 5%
polyvinylpyrrolidone iodine, neutralization in 2% sodium thiosulphate,
and a final wash in 0.9% [wt/vol] NaCl), corneoscleral discs were
excised. Corneoscleral discs were suspended in 80 ml of organ culture
medium in a glass DIN bottle, using a 5/0 Mersilk suture
(Ethicon, AAH, Bristol, UK) through the scleral rim. Organ
culture medium consisted of Eagle’s minimum essential medium (MEM)
with Earle’s salts and 25 mM Hepes buffer (GIBCO, Glasgow, UK),
containing 2% fetal calf serum, 2 mM glutamine, 100 U/ml penicillin,
0.1 mg/ml streptomycin, and 0.25 μg/ml amphotericin B. Corneas were
stored in a standard incubator at 34°C for varying lengths of time,
up to 30 days, with medium remaining unchanged throughout storage.
After a defined storage time, endothelial cell density and
viability were evaluated in an area of 1.54 mm
2 using phase-contrast light microscopy according to standard CTS Eye
Banking protocols.
5 The corneal endothelium was stained
for 1 minute in 0.45% trypan blue (Sigma, UK) to identify the number
and distribution of dead cells. After a rinse in sterile 0.9% (wt/vol)
NaCl, hypotonic 1.8% sucrose was applied to the corneal endothelium
for visualization of cell borders. A cell count in the central corneal
endothelium was calculated (expressed as the number of endothelial
cells per square millimeter) using an eyepiece graticule calibrated to
the microscope stage, and the degree of folding was noted. The quality
of the endothelium was assessed and graded 1 to 5, according to the
evaluation criteria demonstrated in
Table 1 . Corneoscleral discs were fixed immediately post assessment in 10%
neutral-buffered formalin and stored at 4°C.
TUNEL Technique.
After fixation, in 10% neutral-buffered formalin as described above,
each cornea was thoroughly washed in three 10-minute changes of
phosphate-buffered saline (PBS). Whole corneas were mounted endothelial
side up on glass slides. Endogenous peroxidase was quenched using 2%
H
2O
2 in PBS for 15 minutes.
Cell membranes were permeabilized by a 20-minute incubation in 20μ
g/ml proteinase K, followed by a 20-minute incubation in 0.1%
Triton X-100. Apoptotic cells in the corneal endothelium were then
identified using the TdT-dUTP terminal nick-end labeling (TUNEL)
technique (Oncor cell death in situ detection kit; Appligene Oncor UK,
Watford, Herts, UK), a histochemical technique that labels the free
ends of the nuclear DNA fragments characteristic of
apoptosis.
9
The corneal endothelium was incubated in TdT in a humidified atmosphere
at 37°C for 2 hours, rinsed in three washes of PBS, and incubated in
peroxidase-tagged anti-digoxigenin antibody for 1 hour at room
temperature. After thorough washing in PBS, diaminobenzidine was
applied to the corneal endothelium for color development of
TUNEL-positive cells. After immersion in distilled water, four radial
cuts were made in the cornea to allow flatmounting of corneas. TdT was
excluded from the DNA nick-end labeling reaction, replaced by distilled
water, for negative controls.
TUNEL-positive cell nuclei were visualized and analyzed using light
microscopy. Apoptosis in each cornea was expressed as a percentage of
cells and as the number of labeled cells per unit area, and quantitated
in 10 fields of view of 0.0625 mm2 in areas of
folds and 5 fields of view of 0.25 mm2 in the
central and the peripheral corneal endothelia. Apoptosis in endothelium
of organ-cultured human corneas was assessed in corneas at different
periods of storage and from different-aged donors.
Hoechst 33345 Staining.
Human corneal endothelium was immersed in 0.2 μg/ml Hoechst 33345
(bis-benzamide; Sigma) in the dark for 10 minutes. After thorough
washing in PBS, four radial cuts were made into the cornea to allow
flatmounting in antifade mountant Gelvatol (Fisons, UK). Nuclear
Hoechst-stained DNA was then observed via UV light using an
Olympus IX-70 microscope (Olympus, London, UK).
Immunohistochemical Detection of Caspase-3 Activity.
Organ culture stored human corneas were fixed in 4% paraformaldehyde
overnight. Each cornea was then rinsed in three washes of PBS, before a
1-hour incubation in 0.3% Triton X-100 in PBS to permeabilize cell
membranes. After three washes in PBS, the cornea was incubated
overnight in anti-human caspase-3 active antibody (R&D Systems,
Abingdon, UK; antibody detects the p20 subunit of caspase-3 but not the
inactive precursor), diluted 1 in 2500 in PBS. In control samples the
primary antibody was replaced with 5% goat serum. The tissue was then
washed in PBS, blocked in 5% normal goat serum for 1 hour and washed
in PBS, before a 2-hour incubation in cy3-conjugated affinipure goat
anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories,
Luton, Beds, UK) diluted 1:300 in PBS. Four cuts were then made in each
cornea to allow flatmounting in antifade mountant Gelvatol (Fisons,
UK). Caspase-3–related immunofluorescence was then observed using an
Olympus IX-70 microscope.
Spearman’s rank correlation was used to test the tendency of
values of y to increase (or decrease) as values of x increase. Spearman’s rank correlation coefficient is
defined as r s. Student’s t-test was used to determine the difference between the
number of TUNEL-labeled cells in areas with and those without folds.
Values of significance were taken as P < 0.05.
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.
The integrity of donor corneal endothelium after organ culture
storage is critical to recipient graft survival. This investigation has
clearly demonstrated that CEC loss during organ culture storage occurs
via apoptosis. Although apoptosis was apparent throughout the corneal
endothelium, the greatest density of apoptotic cells was localized to
corneal folds. The degree of apoptosis showed a positive relationship
with donor age but only a weak correlation with storage time. The
strong correlation between apoptosis, endothelial cell count, and
overall assessment quality of the endothelium indicates that apoptosis
has a major impact on the 33% discard rate of human corneas stored in
organ culture.
CEC apoptosis has been unequivocally characterized by a variety of
techniques: the TUNEL technique, histochemical staining to demonstrate
morphologic nuclear changes typical of apoptotic cells, and positive
immunostaining for active caspase-3. The lack of evidence of cell
membrane permeability, as determined by trypan blue exclusion indicates
that apoptosis rather than necrosis is the principal form of cell death
in the corneal endothelium under organ culture conditions.
Our observation of CEC apoptosis during Eye Bank storage is supported
by a number of studies. The most recent by Komuro et al. reported
apoptosis in human corneas stored in optisol at 4°C for up to 21
days.
11 Apoptosis occurred at low levels (up to 3%) but
then increased significantly after 16 days of storage.
11 Cell culture studies have demonstrated that endothelial cell apoptosis
can be mediated by staurosporine, oxidative stress, and Fas/Fas ligand
interaction.
12 13 14 15 The latter system is particularly
important because the Fas system is known to be present in the cornea
and has been shown to be implicated in keratocyte cell death in the
anterior stroma during corneal wound healing.
16
Apoptosis is a tightly controlled physiological mechanism that can be
triggered by a variety of stimuli such as the Fas/APO system,
growth factors or their absence, oxidative damage, transformation,
viral infection, chemotherapeutic agents, and mechanical
stress.
17 The trigger for CEC apoptosis in organ culture
is unknown, although a variety of factors may contribute and are likely
to be dependent on the local environment of the cell. Such factors,
including nutrient deprivation, mechanical stress, endotoxins, and/or
loss of survival factors, are discussed below in more detail. In vivo
essential metabolites are supplied to the cornea via tears (oxygen) and
the aqueous humor (glucose). The latter contains proteins that
upregulate bcl-2 gene transcription and protect cultured CECs
from apoptosis.
15 Submersion of the cornea in a closed
organ culture storage system provides a totally artificial environment
that limits oxygen supply and results in glucose depletion and lactate
accumulation.
18 19 These factors have all been implicated
in apoptosis in other tissues.
20 Komura et al. have
reported that 4°C storage of human corneas can result in cell death
in all layers of the cornea, being greatest in
keratocytes.
11 They also demonstrated that this was
predominantly due to apoptosis with some necrosis that may have
resulted secondary to apoptosis. This is suggestive of considerable
cross talk between the three cell types in the cornea that may promote
apoptosis under adverse conditions. Our study did not investigate cell
death in the other layers of the cornea because our main concern was
the endothelium.
Mechanical induction is likely to play an important role in CEC
apoptosis because there was a strong association with corneal folds,
which occur as a consequence of stromal swelling.
6 21 Stromal swelling is known to deform the cornea such that posterior
strain is increased,
22 thereby providing a stimulus for
mechanical disruption of cell–cell and/or cell–matrix interactions
leading to anoikis. Consistent with this is the overall lower
level of endothelial cell apoptosis identified in optisol-stored human
corneas in which stromal swelling is greatly reduced compared with that
in organ culture storage.
11 Mechanical stress has also
been shown to modulate myocyte apoptosis as a result of
stretch-mediated activation of p53 in vitro, an abnormal myocardial
load in heart disease in vivo,
23 whereas vascular
endothelial cell death occurs after removal of hemodynamic
forces.
24
Bacterial lipopolysaccharides or endotoxins, identified in“
sterile” organ culture medium,
25 have been shown to
instigate caspase-mediated cleavage of cell–cell and cell–matrix
adherens junctions
26 and are thus potential activators of
CEC apoptosis. However, inhibition of endotoxin-mediated endothelial
cell loss in porcine corneas can be achieved by increasing the
concentration of fetal calf serum in organ culture.
27
Our findings suggest that prevention of endothelial cell apoptosis
either by inhibition of stromal swelling and/or cell death would reduce
the discard rate of human corneas, which in turn will help relieve the
Europe-wide shortage of donor tissue. Although the cause of corneal
swelling in organ culture is unknown, a mechanism by which to regulate
stromal water intake and/or output would be pertinent to reduce corneal
distension, corneal folds, and, thus, CEC apoptosis. Further studies
are necessary in this area.
Factors known to inhibit apoptosis in other cells include the growth
factors insulin-like growth factor-1, fibroblast growth
factor, and platelet-derived growth factor.
20 28 29 30 CEC
apoptosis itself has been suppressed by an uncharacterized aqueous
humor borne factor.
14 15 Thus, an understanding and
identification of anti-apoptotic factors are required to limit
deterioration of the endothelium during organ culture storage and,
therefore, reduce the discard rate of donor corneas. It is critical
that the donor corneal endothelium has sufficient viable cells to
guarantee a clear corneal transplant and to survive a postoperative
endothelial cell loss of up to 48%.
31 Successful
identification of such survival factors may also be relevant to the
potential prevention of postoperative CEC death in vivo.