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1 From the Ocular Surface and Tear Center, Bascom Palmer Eye Institute, Department of Ophthalmology, Miami, Florida; and the 2 Department of Cell Biology and Anatomy, University of Miami School of Medicine, Florida.
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Abstract |
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METHODS. Transepithelial PRK was performed on both eyes of 30 rabbits. Twenty-six rabbits were divided into 4 groups receiving autologous blood, suturing alone, suturing with amniotic membrane graft, or no treatment as the control. Twenty-four hours later, the ablated zone was analyzed for keratocyte death by TdT-dUTP terminal nick-end label (TUNEL) staining and transmission electron microscopy, for polymorphonuclear cell (PMN) infiltration by hematoxylin–eosin staining, and for oxygen radical–induced lipid peroxidation by malondialdehyde immunohistochemistry. The remaining four rabbits were subjected to PRK or mechanical scraping and analyzed immediately or after culturing for 24 hours.
RESULTS. Compared with the control group where TUNEL-positive keratocytes were found only in the superficial ablated stroma, blood application or suturing caused more and deeper keratocyte death and PMN infiltration (P < 0.05). The amniotic membrane graft group had less keratocyte death and PMN than the control or the suture group (P < 0.05 and P < 0.01, respectively). There was a strong correlation between keratocyte death and PMN infiltration (P < 0.01, correlation factor = 0.786). Transmission electron microscopy revealed that the majority of keratocyte death was due to necrosis. Amniotic membrane stroma trapped and prevented PMN infiltration into the stroma. Malondialdehyde-modified antigen was found on the ablated surface and around infiltrated PMN.
CONCLUSIONS. Transepithelial PRK causes oxygen radical–mediated lipid peroxidation on the superficial stroma and may contribute to superficial keratocyte death even in the absence of inflammation. Mechanical scraping leads to apoptosis without the participation of inflammation. Keratocyte death by necrosis spreads to the deeper part of the stroma and correlates with additional acute inflammation. Amniotic membrane precludes PMN infiltration and decreases lipid peroxidation and keratocyte death. Future studies are needed to discern whether prevention of inflammation-mediated keratocyte necrosis can reduce unwanted scarring caused by PRK.
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Introduction |
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Two types of cell death have been recognized. Apoptosis, or programmed cell death, which exhibits characteristic morphologic changes,1 features a lack of inflammation, and plays an important role in a range of biological processes, including differentiation, development, response to infection, and wound healing.2 3 In contrast, necrosis, which is characterized by a rapid release of cellular contents, is invariably associated with, and frequently incites, inflammation. Wilson et al.4 first provided evidence that removal of the corneal epithelium by mechanical scraping can induce keratocyte apoptosis. This finding explains why there is an early loss of keratocytes in the anterior corneal stroma after epithelial scraping, an intriguing phenomenon first recognized by Dohlman et al.5 and subsequently substantiated by others.6 7 8 It should be noted that such an epithelial injury, which does not damage the basement membrane and Bowman’s layer, frequently results in reepithelialization without stromal haze.
In PRK, epithelial removal can be achieved by either mechanical scraping or transepithelial excimer ablation. Apoptosis of keratocytes caused by the mechanical scraping form of PRK has been recognized,9 and its extent is greater than that caused by transepithelial PRK.10 It is well established that a major component of post-PRK corneal haze is the enhanced backscattering of light from highly reflective intrastromal, migratory fibroblasts, which repopulate the zone of surgically induced keratocyte loss.11 Moller–Pedersen and coworkers,12 13 using in vivo confocal microscopy in a rabbit model, have recently reported that the initial keratocyte loss does not correlate with the subsequent increase in corneal haze. These new findings raise the issue of whether corneal haze is causatively linked with apoptosis and whether apoptosis alone accounts for the total keratocyte loss.
One unsettling variable may be acute inflammation, which varies among different individuals during the early stage of wound healing. Hayashi et al.14 noted that the level of conjugated diene, a product of lipid peroxidation, is higher in the photoablated superficial stroma and postulated that this change is caused by oxygen radicals generated by infiltrating PMNs. However, it remains unclear whether such lipid peroxidation can also be caused by laser irradiation alone, whether PMNs indeed contribute to keratocyte death, or whether keratocyte death is mediated by necrosis or apoptosis when there is acute inflammation. The issue of acute inflammation is worth noting because its reduction by amniotic membrane transplantation has been correlated with reduced haze in a rabbit PRK model.15 In the present study we sought to investigate these questions in a rabbit model of transepithelial PRK. Acute inflammation was experimentally amplified by the application of autologous blood or 10-0 nylon suturing but reduced by amniotic membrane graft. Lipid peroxidation was monitored by immunohistochemistry with an antibody against malondialdehyde (MDA)-modified antigens. The nature of cell death was evaluated by both TdT-dUTP terminal nick-end label (TUNEL) staining and transmission electron microscopy.
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Methods |
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Blood Application, Suturing, and Amniotic Membrane Graft
Both corneas of 26 rabbits received transepithelial PRK,
consisting of 47 µm, 6-mm-diameter phototherapeutic keratectomy for
epithelial removal and -9 D, 5-mm diameter PRK for refractive cut
using a Kera Technology dual-beam 193 nm scanning laser (Model
IsoBeam D200; Kera Technology, Orlando, FL). They were subdivided into
the following four groups. Seventeen eyes were used as the control
group without any additional treatment. Twelve eyes received
application of autologous whole blood for 5 minutes immediately after
PRK, and the blood clot was removed thereafter (the blood group). Nine
eyes received 5 interrupted 10-0 nylon sutures placed outside of the
ablation zone and also served as a control for the amniotic membrane
graft group (the suture group). Nine eyes were covered with an 8-mm
disc of preserved human amniotic membrane (kindly provided by
Bio-Tissue, South Miami, FL), with the stromal side facing the corneal
surface, on the ablated surface with five interrupted 10-0 nylon
sutures placed outside of the ablation zone (the AM graft group). To
prevent the amniotic membrane from being dislodged by the movement
generated by the nasal nictitating membrane, we placed two sutures
instead of one in that quadrant. All rabbits were killed 24 hours
later.
Immediate Suture
Four additional rabbits received transepithelial PRK in one eye
and the fellow eye received either removal of a 6-mm-diameter area of
the central corneal epithelium by mechanical scraping in two rabbits or
no treatment as a control in the other two rabbits. These rabbits were
killed immediately, and their corneoscleral buttons were excised,
rinsed in Hanks’ balanced salt solution with gentamicin (50 µg/ml),
and cultured with the epithelial side up in a 30-mm petri dish
containing supplemental hormonal epithelial media (SHEM; Dulbecco’s
modified Eagle’s medium/Ham’s F-12 medium with 5% fetal bovine
serum, 5 µg/ml insulin, 100 ng/ml cholera toxin, 10 ng/ml epidermal
growth factor, 0.5% dimethyl sulfoxide, and 50 µg/ml gentamicin).
The dishes were incubated at 37°C in humidified air with 5%
CO2 for 24 hours before analysis.
Histology and TUNEL Staining
After euthanatization, corneoscleral rims were excised, flattened
by four radial relaxing cuts, and bisected into two halves through the
center of the ablated center at the 12 and 6 o’clock axis. Bisected
corneas were embedded in OCT compound (Sakura Finetek, Torrance, CA)
for frozen sections. Serial 6-µm-thick sections were cut, and
sections were stained with hematoxylin and eosin (H & E) alone until
both epithelial edges of the ablation zone were identified together
with the ablation zone in the center. From there on, the 5th section
was selected for the H & E staining alone, and the 6th section was
stained for the TUNEL assay. TUNEL staining, which detects cell death
by apoptosis predominantly and necrosis to a lesser extent, used an in
situ fluorescein-based Apoptosis Detection Kit and followed procedures
recommended by the manufacturer (Oncor, Gaithersburg, MD). An
epifluorescence microscope (Carl Zeiss, Thornwood, NY) with
appropriate excitation and emission filters was used to photograph all
propidium iodide–stained cell nuclei (as red fluorescence) and the
fluorescein-stained apoptotic nuclei (as green fluorescence). The
counting of cells was performed (by WCP) and verified (by SCGT) in a
masked fashion. The H & E slide was used for counting total number of
PMNs; the TUNEL slide was used for counting apoptotic cells. The
counting was conducted under 400x magnification of a high power field
(HPF), which covers the stromal area bordered by the epithelium
superiorly and the endothelium inferiorly. A total of four HPFs, with
each tangential to the other and together spanning the majority of the
ablated stroma, were counted for each slide.
TUNEL-positive cells were distinguished as keratocytes by their slender and scanty cytoplasm and round nuclei, but as PMNs by their multi-lobed nuclei. When in doubt, we performed on the 4th section fluorescein-based TUNEL staining without adding propidium iodide and double-labeled it with a rhodamine-conjugated anti-vimentin antibody, which stains mesenchymal cells including fibroblasts and keratocytes.16 If TUNEL-positive cells (green nuclear fluorescence) also showed vimentin-positive staining (red cytoplasmic fluorescence), these cells were counted as apoptotic keratocytes (an example is given in Figs. 3G and 3H ). For identifying PMNs, we relied primarily on the nuclear characteristics as described above (see Figs. 4C and 4D for an example). When in doubt, on the 4th section we also performed fluorescein-based TUNEL staining without adding propidium iodide and double-labeled it with a rhodamine-conjugated anti-myeloperoxidase antibody (Sigma, St. Louis, MO), which stains the granules of PMNs.17
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MDA Immunohistochemistry
To evaluate the involvement of oxygen radical–mediated lipid
peroxidation, immunohistochemical staining was performed with a rat
polyclonal antibody raised against MDA-modified albumin using a
Vectastain Elite ABC kit in combination with and an affinity-purified
biotinylated goat anti-rat IgG secondary antibody (Vector, Burlingame,
CA) according to the manufacturer’s recommendation, followed by color
reaction with diaminobenzidine tetrahydrochloride as the peroxidase
substrate. We developed such an antibody using a previously reported
protocol by Hall et al.18
In brief, MDA was prepared by
acid-catalyzed hydrolysis of MDA bis dimethyl acetal (Sigma). This
solution was then mixed with rat serum albumin (RaSA; Sigma) at 37°C
for 72 hours. The reaction mixture was then dialyzed against a total 4
liters of phosphate-buffered saline with 6 changes over a 72-hour
period. Six rats were immunized by an intramuscular injection of 0.1 mg
of MDA–RaSA antigen mixed with complete Freund’s adjuvant (Sigma).
Ten days later, 0.05 mg of MDA–RaSA antigen was similarly administered
in incomplete Freund’s adjuvant (Sigma) and repeated twice at 10-day
intervals. The specificity of these antisera obtained at 7 days after
the last injection was tested by demonstrating their immunoreactivity
with MDA–RaSA but not with unmodified RaSA, using an enzyme-linked
immunosorbent assay identical to that previously
described.18
Furthermore, its specificity was identical to
that of a similar antibody from a commercial source and confirmed by a
well-described rat model of mid–cerebral artery occlusion–induced
infarction, which is known to produce MDA-modified antigens via lipid
peroxidation (unpublished observation by B. Watson, PhD, Department of
Neurology, University of Miami, Florida, 1998).
Statistical Analysis
Statistical comparisons between groups were performed using the
Mann–Whitney and Wilcoxon signed rank tests with the help of the
Department of Biostatistics, Bascom Palmer Eye Institute, University of
Miami. P < 0.05 was considered statistically
significant. All results are expressed as mean ± SD.
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Results |
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Compared with the suture group (Figs. 3E and 3F) , the AM graft group showed a reduced number of PMNs and TUNEL-positive cells in the stroma (Figs. 4A and 4B ). In this particular example, only two keratocytes were TUNEL-positive (indicated by arrowheads, Fig. 4B ), whereas the remainder of TUNEL-positive cells was found in the amniotic membrane itself (also see the higher magnification shown in Figs. 4C and 4D ). This notion was confirmed by partially detaching the amniotic membrane from the underlying corneal stroma before embedding and staining (Fig. 4E) . In the latter situation, the majority of the TUNEL-positive cells were noted in the amniotic membrane predetached from the ablated surface before embedding and corresponded to PMNs. As a control, cells normally present in the amniotic membrane did not reveal any TUNEL positivity before transplantation (Fig. 4F) . Under high power magnification (Fig. 4C) , we confirmed that PMNs in contact with the amniotic membrane stromal matrix underwent cell death (i.e., mostly exhibiting green fluorescence) were more frequent than those in contact with the basement membrane side (i.e., mostly exhibiting red fluorescence; Fig. 4D ).
Investigation of Cell Death by Transmission Electron Microscopy
Compared with the morphology of the normal keratocyte that was
present in the unablated region of the control cornea (Fig. 5A
), numerous keratocytes found in the superficial stroma of the blood
group showed disruption of the cell membrane and intracellular
organelles and dissolution of the chromatin matrix (Fig. 5B)
.
Throughout the entire section from the superficial stroma to the deep
posterior stroma, we did not find chromatin condensation characteristic
of apoptosis in any of the keratocytes surveyed. Scattered in the
superficial stroma were some multinucleated PMNs, some of which were
adjacent to the keratocytes showing the above degenerative changes
(Fig. 5C) . Compared with the PMNs in the stroma of the blood group
(Fig. 5C) or the suturing group (not shown), PMNs trapped in the
amniotic membrane stroma showed marked vacuolation of the cytoplasm,
and swollen mitochondria and chromatin condensation at the nuclear
membrane characteristic of apoptosis (Fig. 5D
, see arrows).
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Discussion |
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One way to induce keratocyte death in the deeper part of the stroma by transepithelial PRK is to create acute inflammation caused by the application of autologous blood or corneal sutures. Once PMNs appear in the deeper part of the stroma, they can release agents to cause more damage to cells and their matrices. Oxygen radicals generated by PMN respiratory burst or degranulation are such agents.22 23 In this study we did find positive MDA staining in the region where there was intense PMN infiltration, supporting the idea that lipid peroxidation by oxygen radicals released by PMNs indeed can occur as speculated by Hayashi et al.14 and reported by others.19 20 Future studies are still needed to resolve whether oxygen radicals, lipid peroxidation, or both are directly responsible for such keratocyte death in the deeper part of the stroma.
Another way to induce more keratocyte death in the deeper part of the stroma is by mechanical scraping to remove the epithelium as demonstrated in Figure 7C . This result is consistent with a recent report.10 It has been reported that mechanical scraping–PRK induces more inflammation than transepithelial PRK.24 This study further showed keratocyte death even after the cornea was removed immediately for 24 hours of culturing. This result clearly indicates that this process of cell death, characterized as apoptosis,9 does not require the participation of PMNs. Because mechanical scraping did not generate MDA staining, we also conclude that lipid peroxidation is not involved.
In contrast to the apoptosis explanation for keratocyte death caused by mechanical scraping, necrosis was identified as the major mechanism for keratocyte death associated with acute inflammation (Fig. 5) . In this study we distinguished keratocytes from PMNs by nuclear morphology and double-labeling; we cannot be absolutely certain that the process of counting TUNEL-positive keratocytes may have included some PMNs. Furthermore, TUNEL assay detects cell death by apoptosis predominantly and necrosis to a lesser extent. Therefore, necrotic keratocyte death induced by PMNs might have been underrepresented in this study. Moreover, our study draws a correlation but not a causative relationship between inflammation and keratocyte loss. Necrosis involves the rapid release of intracellular contents and is invariably associated with inflammation and the incitement of inflammation. It has been reported that PMNs first appear at the ablated margin at 6 hours and increase at 24 hours after PRK.25 The finding of intense MDA staining around the infiltrating PMNs in the stroma is important because oxidized lipids act as chemoattractants26 and may help amplify inflammation.19 20 It remains to be determined whether keratocyte necrosis, but not apoptosis, is responsible for subsequent fibroblast activation and transformation into scar-forming myofibroblasts.
One effective measure to suppress keratocyte death is to use amniotic membrane as a temporary patch. Within 24 hours, the suture-induced PMN influx from the denuded stroma was mitigated, and as a result keratocyte death was significantly reduced and MDA staining was limited to the PMN-infiltrated amniotic membrane. The finding that PMNs are excluded by amniotic membrane has previously been observed.15 27 We further noted that more TUNEL-positive PMNs were adherent to the amniotic membrane stromal matrix than those adherent to the basement membrane. Transmission electron microscopy revealed these PMN undergoing apoptosis (Fig. 5) . Because facilitation of PMN apoptosis is an effective and physiological way of suppressing tissue inflammation,28 future research is needed to determine whether amniotic membrane stromal matrix promotes PMN adherence and subsequent apoptosis. Such an action better explains how amniotic membrane transplantation reduces inflammation as a graft29 30 31 32 and reduces PRK-induced corneal haze in rabbits as a temporary patch used for 2 days15 or 1 week.27 Research in this direction may uncover other new applications.
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Footnotes |
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Submitted for publication June 15, 1999; revised January 11 and April 8, 2000; accepted April 27, 2000.
Commercial relationships policy: P.
Presented, in part, at the annual meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May, 1998.
Corresponding author: Scheffer C. G. Tseng, Bascom Palmer Eye Institute, William L. McKnight Vision Research Center, 1638 NW 10th Avenue, Miami, FL 33136. stseng{at}bpei.med.miami.edu
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, a statistically significant difference
when compared with the suture group (P < 0.01).
, membrane
stroma).
