Cell Death during Corneal Storage at 4°C

Abstract

purpose. To evaluate cell death in human donor corneas stored at 4°C, to determine whether terminal deoxynucleotidyl transferase–mediated dUTP-fluorescein nick-end labeling (TUNEL) discriminates between apoptosis and necrosis in corneas stored at 4°C.

methods. Ten human corneas were stored in Optisol (Chiron Ophthalmics, Irvine, CA) at 4°C for periods ranging from 0 to 21 days and then fixed for histologic examination. Central corneal sections from each cornea were examined by transmission electron microscopy (TEM) and by the TUNEL assay. Electron micrographs of at least 15 keratocytes each from the anterior, middle, and posterior stroma were examined by three masked observers who graded each cell as normal, apoptotic, or necrotic. Central sections from the same corneas were processed by the TUNEL assay and evaluated with a laser scanning confocal microscope to determine the percentage of apoptotic cells.

results. By TEM, apoptosis occurred in 23% of the keratocytes and necrosis in 12%. By TUNEL assay, apoptosis occurred in 11% of the keratocytes, with the results in individual corneas being similar to the findings by TEM for apoptosis, rather than for necrosis. By TUNEL assay, apoptosis occurred in 13% of the epithelial cells and in 8% of the endothelial cells. The percentage of apoptotic cells and storage time correlated significantly for the epithelium, but not for the keratocytes or endothelium in this small sample.

conclusions. Both apoptosis and necrosis occur in cells during corneal storage at 4°C, with apoptosis appearing to predominate. The TUNEL assay identifies cells undergoing apoptosis, but not necrosis, in corneal tissue. Inhibition of apoptosis in corneas stored at 4°C may prolong acceptable storage times.

Various methods have been devised to prolong the viability of excised corneas destined for transplantation. The number of viable corneal cells decreases with time during storage at both 4°C ^{1 2 3 4} and 34°C. ^{5 6 7 8} The gradual death of corneal cells during preservation may occur by two pathways: necrosis and apoptosis. ⁹ Because it is possible to inhibit the apoptotic pathway under certain circumstances, ^{10 11 12 13 14} it becomes important to learn by what mechanisms the death of corneal cells occurs during preservation. If the cells undergo apoptosis, the addition of appropriate molecules to the storage media may increase viability and prolong storage times. 

Apoptosis is an active process of self destruction requiring the synthesis of macromolecules and occurring throughout normal development. It differs from necrosis, the other form of cell death, both morphologically and biochemically. Necrosis is characterized by swelling of mitochondria and other organelles, often with cytoplasmic vacuolization, followed by dissolution of nuclear, organelle, and plasma membranes. ⁹ Conversely, apoptosis has been characterized ultrastructurally by cell shrinkage and loss of normal cell contact, maintenance of plasma and nuclear membranes, dense chromatin condensation and fragmentation, cellular blebbing, and formation of membrane-bound protuberances from the cell surface called apoptotic bodies. ^{15 16} It has been characterized biochemically by increased endogenous endonuclease activity that cleaves internucleosomal DNA to form a ladder of oligonucleosome fragments. ¹⁷ Based on these characteristics, apoptotic cells have been identified mainly by gel electrophoresis of extracted DNA or by the typical electron microscopic changes in cell nuclei. 

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), ¹⁸ has been widely used to detect cells with DNA fragmentation, assumed to be apoptotic cells. It has been reported, however, that the TUNEL assay also detects necrotic cells, as demonstrated in rat liver, ¹⁹ rat brain, ^{20 21} and human endometrium and placenta. ²² Without ultrastructural evidence, the TUNEL assay alone may not be sufficient to differentiate whether corneal cells are dying by apoptotic or necrotic mechanisms. An additional complicating factor is that during refrigerated storage, cell death takes place at 4°C, and metabolic processes are retarded. In this investigation, therefore, we examined each cornea by both the TUNEL assay and by transmission electron microscopy (TEM). Examination of stromal keratocytes by TEM, with its well-defined criteria for apoptosis and necrosis, ⁹ was used to validate the results of the TUNEL assay. We examined a series of human corneas thathad been preserved at 4°C, which is the most commonly used temperature for corneal preservation in the United States today. 

Materials and Methods

Ten human corneas, preserved in Optisol ²³ or Optisol-GS (Chiron Ophthalmics, Irvine, CA), at 4°C for varying periods, were obtained from the Mayo Clinic Eye Bank (Table 1) . The mean donor age was 52.5 ± 18.3 (SD) years, mean time from death to enucleation was 4.2 ± 4.9 hours, and mean time from enucleation to preservation was 3.9 ± 4.8 hours. One of the corneas (cornea 1) was obtained as a normal control from an eye removed during orbital exenteration for a lacrimal gland tumor. The cornea was excised and placed directly in fixative approximately one-half hour after enucleation. 

TEM

Corneas stored at 4°C were placed without warming into cold Trump’s fixative (1% glutaraldehyde and 4% formaldehyde in 0.1 M phosphate buffer, pH 7.2) and bisected. The central cornea from one half was cut into 1-mm cubes. Specimens were postfixed with osmium tetroxide and embedded in Spurr’s resin. Thin sections (70 nm) were cut and placed on copper grids and double stained with uranyl acetate and lead citrate. The tissue was examined and photographed with a transmission electron microscope (1200 EXII; JEOL, Peabody, MA) operating at 60 kV. 

The corneal stroma was divided into three anteroposterior regions of equal thickness by using a low-magnification photograph. Photographs were taken at high magnification (more than ×5000) of at least 15 randomly selected keratocytes (mean, 23 ± 6 [SD]; range, 15–36 keratocytes) in each region of each cornea. Keratocytes were classified in a masked fashion by three examiners into one of three groups: normal, apoptosis, or necrosis and vacuolization, according to the following definitions (Fig. 1) . A normal keratocyte was defined as having a nucleus with peripheral heterochromatin and a thin rim of cytoplasm. Apoptotic keratocytes were defined as having chromatin condensation and fragmentation, nuclear membrane blebbing, cell shrinkage, and loss of cytoplasm with preservation of the integrity of organelles. Necrotic keratocytes were defined as those with irregular clumping of nuclear chromatin, dissolution of organelles, and rupture of the cell membrane or vacuolization of the cytoplasm. Each cell was given a final classification based on results from the three masked examiners. There was agreement between two of the three masked examiners about the final classification of all cells and agreement among all three in 68% of the 685 micrographs examined. 

TUNEL Assay

The other half of each bisected cornea was embedded in paraffin, and 5-μm sections from the central cornea were mounted onto glass slides. Sections were then deparaffinized by heating for 20 minutes at 60°C followed by washing twice for 5 minutes each in xylene. The tissue sections were hydrated by transferring the slides through the following solutions: 100% ethanol twice for 5 minutes each, 95% ethanol for 3 minutes, 70% ethanol for 3 minutes, distilled water for 5 minutes, and phosphate-buffered saline (PBS) for 5 minutes. Protein present in the sections was digested with 20 μg/ml proteinase K (Boehringer–Mannheim, Indianapolis, IN) for 20 minutes at room temperature. After the sections were rinsed twice with PBS, DNA strand breaks were fluorescein labeled according to the instructions of a commercial kit (In Situ Cell Death Detection Kit, Fluorescein; Boehringer Mannheim). Appropriate positive and negative controls were used. The slides were counterstained with 4′6-diamidino-2-phenylindole (DAPI), which binds to double-stranded DNA and thereby stained both normal and apoptotic nuclei. 

The sections were examined using a confocal laser scanning microscope (LSM 510, Carl Zeiss, Oberkochen, Germany). For excitation, the 488-nm wavelength of an argon-krypton laser and the UV (351–364 nm) wavelength of an argon ion laser were used. The samples were viewed through a ×10 (0.45 numeric aperture) water immersion objective lens(C-Apochromat; Carl Zeiss). Images were digitized using a 385- to 475-nm emission filter for DAPI and a 505- to 550-nm emission filter for fluorescein. The digitized images were analyzed using a commercial system (KS-400; Carl Zeiss). Three sections from each cornea were analyzed and the results averaged. The epithelium and endothelium in each image were outlined manually so that they could be separated from the stroma during analysis. Then the stroma was divided into three equal-thickness anteroposterior regions. Nuclei stained with DAPI (blue) and TUNEL (green) were discriminated and counted in the epithelium, endothelium, and each stromal region (Fig. 2) . Keratocyte density was estimated from the number of DAPI-positive stromal cells and expressed as cells per square millimeter for each region. The stromal thickness was also calculated from the digitized images. 

Statistics

The data from the nine preserved corneas and the normal control specimens were combined for the overall comparisons and correlations. Comparisons between TEM and TUNEL and between regions of the cornea were made by using a paired t-test when the data were distributed normally and a signed-rank test when they were not. Correlations between the percentage of apoptotic cells and the other variables were investigated by using the Pearson correlation coefficient (r_p) when the data were distributed normally and the Spearman correlation coefficient (r_s) when they were not. P ≤ 0.05 was considered statistically significant. 

Results

By TEM, both apoptosis and necrosis occurred in the stromal keratocytes in the majority of stored corneas (Table 2) , with apoptotic cells (23% ± 32%) exceeding necrotic cells (12% ± 11%), but not significantly so (P = 0.37). Comparing the percentage of apoptotic keratocytes among the anterior, middle, and posterior stromal regions, we found a significant difference only between the anterior (14% ± 25%; median 5%) and middle (24% ± 35%; median 7%; P < 0.05) regions. There were no significant correlations between the percentage of apoptotic keratocytes or of necrotic keratocytes and storage time, donor age, ventilator time, death to enucleation time, enucleation to preservation time, or combined death to preservation time. 

TUNEL-positive stromal cells were observed in the majority of stored corneas (Table 2) . There were no significant differences between the percentage of TUNEL-positive cells in the anterior, middle, and posterior stromal regions. There were no significant correlations between the percentage of TUNEL-positive cells in the stroma and storage time, donor age, ventilator time, death to enucleation time, enucleation to preservation time, or combined death to preservation time. 

The percentage of apoptotic keratocytes defined by TEM in the full-thickness stroma (23% ± 32%; median 5%) was significantly greater than the percentage of TUNEL-positive keratocytes (11% ± 20%; median 2%; P < 0.05). In cases in which the percentages of apoptotic and necrotic cells were markedly different, the percentage of TUNEL-positive cells mirrored that of the apoptotic cells rather than that of the necrotic cells (Table 2) . 

TUNEL-positive epithelial cells were observed in all the stored corneas (Table 2) . In cornea 1, a control cornea that was not preserved at 4°C, apoptotic cells were observed only in the most superficial epithelial layer. In the preserved corneas, TUNEL-positive cells were observed at all levels of the epithelium (Fig. 2) . The percentage of TUNEL-positive cells in the epithelium (13% ± 17%) was significantly correlated with both the percentage of TUNEL-positive cells in the stroma (r_s = 0.83; P = 0.003) and endothelium (r_s = 0.83; P = 0.003) and with storage time (r_s = 0.78; P = 0.008). TUNEL-positive endothelial cells were observed in the majority of stored corneas. The percentage of TUNEL-positive cells in the endothelium (8% ± 20%) was significantly correlated with percentage of TUNEL-positive cells in the stroma (r_s = 0.75; P = 0.01), but not with storage time (r_s = 0.56; P = 0.09). 

The total density of keratocytes ranged from 283 to 1034 cells/mm² and was inversely proportional to stromal thickness (r_s = −0.96; P < 0.0001), which ranged from 338 to 785 μm (Table 2) . Stromal thickness was significantly correlated with the percentage of TUNEL-positive keratocytes (r_s = 0.70; P = 0.03), but not with storage time, death to enucleation time, enucleation to preservation time, or combined death to preservation time. 

Discussion

Our goal in this study was to determine whether apoptosis plays a role in the death of corneal cells during storage at 4°C. Because the TUNEL assay identified both apoptotic and necrotic cells in some systems, ^{19 20 21 22} we used TEM, with its well-defined criteria for apoptosis and necrosis, as a gold standard to validate the results of the TUNEL assay for corneal cells stored in situ at 4°C. We chose stromal keratocytes for examination by TEM because they were isolated cells that could be photographed individually for subsequent analysis by three masked examiners. We then compared the percentage of apoptotic stromal cells by TEM with the percentage found by the TUNEL assay on sections from the same central corneas (Table 2) . The results showed that the TUNEL assay detected apoptotic, but not necrotic, cells (e.g., corneas 2, 3, 5, 8, and 9). This comparison validated the TUNEL assay for the detection of apoptotic cells in corneas stored at 4°C. Thus, we were reasonably confident in accepting the results of the TUNEL assay for the corneal epithelium and endothelium without simultaneous morphologic confirmation by TEM, which is much more time consuming and expensive. 

The percentage of apoptotic cells detected by the TUNEL assay was significantly less than that detected by TEM. This could mean that the TUNEL assay is less sensitive than direct morphologic examination, in contrast to the conclusions of Gavrieli et al. ¹⁸ Indeed, DNA fragmentation, which is the basis for the TUNEL assay, is not required for apoptosis. ²⁴ Moreover, chromatin condensation as identified by TEM can also occur in the absence of DNA fragmentation. ²⁵ Taken together, our observations suggest that chromatin condensation precedes DNA fragmentation in the cornea at 4°C, making TEM more sensitive than the TUNEL assay for the detection of this process. 

The percentage of apoptotic stromal cells was twice that of necrotic cells. Although the difference was not statistically significant, it is still reasonable to conclude that apoptosis accounts for most cell death in the cornea during preservation at 4°C. The addition to storage media of molecules that inhibit apoptosis ^{10 11 12 13 14} may thus hold promise for increasing the number of viable cells that remain, thereby prolonging corneal storage times. 

Because cell death during storage necessarily takes place at 4°C, the process could be expected to be prolonged and the number of cells involved to be cumulative, in that dead cells cannot be removed by macrophages or absorbed. Our small sample of nine corneas preserved for 2 to 21 days, with only one cornea at each storage time, lacked sufficient power to detect all but the strongest correlations with donor factors such as storage time. In fact, a statistically significant increase in apoptotic cells with storage time was present in the epithelium, but not in the stroma or endothelium. In fresh, unpreserved rabbit corneas, Ren and Wilson ²⁶ found apoptosis only in superficial epithelial cells. Our findings were similar in cornea 1, which was immersed in fixative 30 minutes after enucleation from a living donor, without preservation at 4°C. In the remaining nine corneas, however, which were preserved for varying periods at 4°C, apoptotic cells were observed at all layers of the epithelium (Fig. 2) . In clinical studies, the loss of endothelial cells measured 2 months after keratoplasty is significantly related to storage time. ²⁷ In addition to apoptosis during storage, however, these cells could also have died by necrosis, which was not measured in endothelial cells in the present study, or as the result of an increased susceptibility to surgical trauma. 

A greater percentage of apoptotic keratocytes were present in corneas that were more swollen during preservation. Stromal thickness increases in stored corneas when the colloidal molecules present in Optisol (chondroitin sulfate and dextran) ²³ enter the stroma across the epithelial and endothelial barriers. These barriers are likely to be decreased in corneas with more apoptotic keratocytes because of the positive correlations between apoptosis in the stroma, epithelium, and endothelium. Stromal thickness was not related to any donor or storage variable except for the anticipated inverse correlation with keratocyte density resulting from the larger cross-sectional area of swollen corneas. 

The morphologic results show that keratocyte cell death occurred in stored corneas by both apoptotic and necrotic mechanisms, although apoptosis was more common. This coexistence of both types of cell death has been reported in other tissues. ^{28 29 30} The high percentage of necrotic cells in some corneas (Table 2) is unexplained, although a relationship to the high incidence of acquired chromosome abnormalities in keratocytes could be postulated. ³¹ Whether cells die by apoptosis or necrosis may depend in part on adenosine triphosphate (ATP) concentration, with apoptosis predominating in cells with sufficient ATP. ^{32 33} In human donor corneas, Redbrake et al. ³⁴ found that ATP concentration was decreased if death resulted from septicemia. Only one of the donors in the present study appeared to be septic (cornea 2). When examined by TEM, 29% of the cells in the cornea from this donor were necrotic, whereas no cells were apoptotic (Table 2) . This finding is at least consistent with the idea that sufficient ATP is necessary for apoptosis to proceed. We were unable to detect a relationship between apoptosis and any donor factor in the 10 corneas in the present study except for a positive correlation between TUNEL-positive cells in the epithelium and storage time. 

In a study of human donor corneas by Moller–Pedersen, keratocyte density ranged from 108 to 315 nuclei/mm² in corneas kept in organ culture for 2 to 28 days. ³⁵ As expected, the keratocyte density was greater than this in the corneas that we measured (283–1034 nuclei/mm²), presumably because they were thinner than the organ-cultured corneas, which were incubated in tissue culture medium without chondroitin sulfate or dextran. Direct comparisons between the present study and that of Moller–Pedersen et al. ³⁵ are not possible, because neither stromal thickness nor the concentration of formalin used for fixation was reported in the latter study. Moller–Pedersen et al. found that organ culture at 30°C for 2 to 28 days had no significant influence on the number of keratocytes. During 4°C storage, our results also failed to find a correlation between storage time and keratocyte density. 

Apoptosis of corneal keratocytes occurs after epithelial injury but is limited in vivo to the anterior stroma. ^{36 37} Wilson et al. ³⁸ reported that the apoptotic changes are mediated by soluble cytokines, such as interleukin-1 or the Fas-Fas ligand system, that are apparently released by injured epithelial cells. In our corneas preserved at 4°C, keratocyte apoptosis was not limited to the anterior stroma, but occurred throughout the stroma and was more common in the midstroma than anteriorly. These findings suggest that keratocyte apoptosis during corneal storage at 4°C may be mediated by factors other than soluble cytokines released from epithelial cells. 

In conclusion, we have demonstrated that the TUNEL assay identifies cells undergoing apoptosis, but not necrosis, in corneas stored at 4°C. Cell death occurs by both apoptosis and necrosis in these stored corneas, with apoptosis appearing to predominate. Therefore, inhibition of apoptosis may increase cell survival and thereby prolong the maximum period corneas awaiting transplantation may be stored at 4°C. 

 Supported in part by National Institutes of Health Grant EY02037; Research to Prevent Blindness and Mayo Foundation.

 Submitted for publication December 10, 1998; revised April 14, 1999; accepted June 21, 1999.

 Commercial relationships policy: N.

 

Table 1.

 

Donor Corneas

Cornea	Storage Time (days)	Donor Age (yr)	Time between Death and Enucleation (h)	Time between Enucleation and Preservation (h)	Cause of Death	Time on Ventilator (days)
1	0	64	0	0.5	Liver donor, orbital tumor	0
2	2	62	3	2	Cardiogenic shock, probable sepsis	9
3	5	57	4	2	Acute pneumonia	0
4	6	4	3	1	Congenital heart disease	18
5	9	63	1	14	Atherosclerotic disease	0
6	12	61	3	1	Perforation of colon	0
7	14	59	3	0	Ischemic heart disease	1
8	16	56	4	6	Intracranial bleed	0
9	19	59	1	1	Closed head injury	3
10	21	40	15	1	Myocardial infarction	0

Figure 1.

 

Transmission electron micrographs of normal, apoptotic, and necrotic or vacuolated keratocytes. (A) Normal keratocyte. Note the normal chromatin pattern in the cell nucleus that occupies almost the entire visible portion of the cell. (B) Apoptotic keratocyte. Note chromatin condensation and fragmentation along with cell shrinkage and almost complete loss of cytoplasm. (C) Necrotic keratocyte. Note the irregular clumping of chromatin and disintegration of organelles. (D) Vacuolated keratocyte. Note the normal chromatin pattern in the cell nucleus and swelling of organelles.

Figure 2.

 

Analysis of section from cornea 7 (stored 14 days) stained for DNA (all cells) with DAPI and for apoptotic cells with the TUNEL assay. (A) Keratocyte nuclei stained with DAPI were discriminated. (B) TUNEL-positive nuclei were discriminated.

Table 2.

 

Analysis of Corneas Stored at 4°C

Cornea	Storage Time (days)	Stromal Region	TEM of Stroma			TUNEL-Positive Cells (%)					Stromal Thickness (μm)	Keratocyte Density (cells/mm2)
						Apoptosis (%)	Necrosis (%)	Stroma	Epithelium	Endothelium
1	0	Anterior	0	0	0	1	1	345	1034
		Mid	0	0	0
		Posterior	0	0	0
2	2	Anterior	0	7	0	2	0	543	496
		Mid	0	46	2
		Posterior	0	35	0
3	5	Anterior	7	0	8	12	1	785	303
		Mid	5	45	4
		Posterior	26	35	25
4	6	Anterior	6	6	0	3	0	423	805
		Mid	7	0	0
		Posterior	7	0	0
5	9	Anterior	4	17	0	5	1	338	1000
		Mid	7	41	0
		Posterior	0	13	0
6	12	Anterior	32	27	3	6	3	571	443
		Mid	67	0	34
		Posterior	70	4	6
7	14	Anterior	7	29	9	22	2	532	522
		Mid	52	19	18
		Posterior	91	5	23
8	16	Anterior	0	0	0	4	0	390	742
		Mid	0	7	0
		Posterior	5	16	0
9	19	Anterior	81	0	51	55	63	766	283
		Mid	100	0	76
		Posterior	95	0	68
10	21	Anterior	4	0	0	23	9	369	850
		Mid	7	0	5
		Posterior	0	7	6

The authors thank James E. Tarara for assistance in software development for the confocal image analysis system.