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1 From the Department of Biomedical Engineering, Tulane University; and 2 Louisiana State University Eye Center, New Orleans.
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Abstract |
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METHODS. Series I: Adult New Zealand White rabbit corneas were mounted in perfusion chambers. The endothelium was bathed with Ringer’s fluid, and the outer surface was covered with silicone oil. The epithelium of one eye was débrided with a scalpel before mounting, and the cornea of the fellow eye was débrided with a rotating brush after stabilization in the perfusion chamber. Using specular microscope tracking software, it was possible to measure total swelling and local swelling within the cornea. Series II: Diclofenac sodium ophthalmic solution 0.1% or a placebo was applied topically, 1 drop per 45 minutes for 3 hours before animals were euthanatized.
RESULTS. Series I: Corneas with their epithelium scraped with a scalpel before mounting were 37.5 ± 17.5 µm (n = 6; P < 0.001) thicker in vitro than the stromas of perfused, intact fellow corneas. Epithelial débridement with a rotating brush after mounting resulted in an immediate (within 8 minutes) stromal swelling that plateaued in 1 hour at 31.0 ± 5.3 µm (n = 6; P < 0.001). Curiously, in six of six corneas, the anterior stroma swelled more than the posterior stroma. In four of six corneas, the posterior stroma thinned. Analysis showed this pattern to be consistent with a sudden increase in anterior swelling pressure or osmotic pressure and to be inconsistent with a change in endothelial transport properties. Series II: Placebo-treated corneas swelled 30.6 ± 7.7 µm (n = 5) 1 hour after débridement, whereas corneas pretreated with diclofenac sodium swelled only 19.2 ± 3.1 µm (n = 6; P < 0.008).
CONCLUSIONS. The anterior stromal swelling occurs rapidly and near the site of epithelial injury suggesting messenger and/or enzymatic involvement with an effect parallel to apoptosis. Reduction of the swelling response with nonsteroidal anti-inflammatory drugs (NSAIDs) implicates the cyclooxygenase pathway. The swelling is similar to the unexplained acute edema that occurs during inflammation in the rat paw edema model, and may represent a general mechanism for mobilization of inflammatory cells.
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Introduction |
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During attempts to isolate the corneal endothelium and stromal tissue for membrane transport experiments, we observed that the stroma appeared to swell after débridement of the epithelium with a scalpel blade.20 This was unexpected because silicone oil was used to block fluid movements across the débrided surface of the tissue. For this reason, it was surmised that fluid from the anterior chamber perfusate must have been drawn into the stroma through the endothelium to produce such swelling. Because corneal epithelial débridement is used routinely not only in animal investigations, but as a commonplace method to prepare the human cornea for excimer laser photorefractive keratectomy (PRK), we explored this anomalous swelling in the isolated cornea of the rabbit.
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Methods |
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Experimental Series
There were two experimental series conducted to investigate
anomalous stromal swelling after epithelial débridement. In
series I, the magnitude, location within the stroma, and time course of
the swelling response to débridement was investigated. Both the
total and local stromal thicknesses were tracked continuously in
corneas after débridement with a bristle brush. For comparison,
the total thickness of corneas that had been scraped with a scalpel
before mounting was compared at relevant time points. In series II, the
effect of a general nonsteroidal anti-inflammatory drug (NSAID) on the
total stromal swelling response was investigated. In these eyes,
diclofenac sodium ophthalmic solution 0.1% (sterile, unpreserved
formulation, 0.3 ml single dose unit; CIBA Vision Ophthalmics, Atlanta,
GA) or the vehicle alone as a placebo control (CIBA) was applied
topically 1 drop per 45 minutes for 3 hours before the animals were
euthanatized.
Animals
Our procedures conformed to the Louisiana State University Medical
Center Institute Animal Care and Use Committee (IACUC) standards, which
are in accordance with the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research. New Zealand White rabbits of either sex
and of 1.5 to 4.0 kg in body weight were deeply anesthetized with an
intramuscular injection of a ketamine-xylazine mixture (40.0 mg/kg and
5.0 mg/kg, respectively). Subsequently they were euthanatized with a
lethal dose of pentobarbital sodium (100 mg/kg) administered
intravenously through the marginal ear vein.
Corneal Perfusion
Excised rabbit corneas for both series of experiments were mounted
in thermally controlled perfusion chambers using the method of
Dickstein and Maurice.19
The two chambers were attached to
the base of an automatic specular tracking microscope and could be
alternately rotated under the objective. The endothelial perfusion
solution was the glutathione-bicarbonate Ringer’s solution used by
Klyce and Russell16
with the antibiotic gentamicin
omitted. Successful preparations maintain a steady thickness under
these conditions for 7 to 8 hours. As is customary to prevent anterior
surface (epithelial) fluid movements, a layer of silicone oil (Dow
Corning, Midland, MI) was applied to the epithelium or to the bare
anterior stromal surface if the epithelium had been removed before
mounting.
Epithelial Débridement Protocol
For series I, before enucleation, the cornea of one eye was
débrided with a scalpel and swabbed with silicone oil, and the
fellow eye was left intact under the eyelid. The débrided eye was
then enucleated, and its cornea was excised and mounted in one
perfusion chamber. Without undue delay, the intact, undébrided
companion cornea was enucleated, excised, and mounted in the second
chamber. After completion of both mounting procedures (approximately 35
minutes) and after a brief period for acclimation of the newly mounted
intact cornea (approximately 10 minutes), the total stromal thicknesses
of both corneas were recorded. The total time from cardiac cessation to
these initial thickness measurements was approximately 55 minutes. The
undébrided cornea remained under the microscope, and its total
thickness was tracked briefly to establish the stability of the
preparation. Then, to facilitate observation of the time course of any
rapid swelling response to epithelial débridement, the intact
perfused cornea was débrided in the chamber. To accomplish this,
the perfusion chamber was rotated from under the microscope, most of
the silicone oil was aspirated from the anterior surface, and the
epithelium was removed. Because the cornea was in a recess,
débridement was performed with a rotating bristle brush (405;
Dremel, Racine, WI) with the motor speed reduced by hand-braking
instead of with a scalpel. The epithelial debris and remaining silicone
oil were aspirated. The exposed anterior stroma was then covered with a
fresh layer of silicone oil, and the chamber was returned to its
previous location. After determining with the microscope that the
central epithelium and basement membrane were removed completely, the
total and local stromal thicknesses were tracked continuously for 1
hour. At the end of the hour, the chamber containing the perfused
scalpel-débrided cornea was rotated back under the microscope
objective, and its total stromal thickness was recorded for comparison.
In series II, both the control and experimental corneas were mounted
sequentially in the perfusion chambers; the order of mounting was
randomized. The stabilized, intact cornea of one of the eyes
(experimental or placebo control; also randomized) was débrided
with a bristle brush, and its total stromal thickness was tracked for 1
hour. The procedure was then repeated in the second chamber on the
waiting, perfused fellow cornea. The Pallikaris débridement brush
(designed by Ioannis Pallikaris, Heraklion, Crete21
),
specifically for use in corneal refractive surgery, was used.
Total and Local Stromal Thickness Measurements
For series I, the total stromal thickness of a
scalpel-débrided cornea was compared with its perfused, intact
fellow cornea before and at 1 hour after débridement with a
bristle brush. In addition, the time course of both the total and local
stromal swelling of the bristle brush-débrided cornea was
continuously tracked with the automatic specular microscope for 1 hour.
For series II, the total stromal thickness changes of topical NSAID-
and placebo-treated corneas, after débridement with a bristle
brush, were tracked continuously for an hour. The time resolution,
accuracy, and sensitivity of the scanning specular
microscope22
23
were enhanced by the addition of an
analog-to-digital (A/D) data acquisition board (DT2831; Data
Translation, Marlboro, MA) and custom digital signal processing
software. The signal processor treated the output (optical scans made
bidirectionally across the perfused central cornea) from the specular
microscope’s photomultiplier tube in the following manner: A 7-point
gaussian filter smoothed the original signal, first derivatives were
calculated, a second gaussian filter was applied to the first
derivative output curve, and the second derivatives were calculated.
Reflective structures that produced peaks of light intensity in the
optical scans could then be identified automatically by detecting where
the first derivative was zero, whereas the second derivative was
negative and larger than an adjustable threshold. Wherever the tracking
system recorded a peak in light intensity, it was assumed that a
reflective structure was found. Surface peaks correlate with the
epithelial surface (when present) or the anterior stromal surface and
the endothelial surface. Light peaks within the stroma were identified
as keratocyte nuclei on visual inspection; many of these appeared
consistent enough in the measuring system to be used as reference
sites. During the observation period, changes in the relative positions
of keratocytes must be caused by fluid shifts within and between the
lamellae. Thus, local and total stromal thickness changes can be
tracked with this instrument.
Anterior versus Posterior Stromal Swelling Estimates
To characterize local swelling, thickness measurements were taken
from the anterior and posterior stroma. The thickness of each half
(defined by bounding keratocyte tracks) was calculated immediately
after the débridement procedure and again after 15 minutes.
Computational Modeling
During corneal swelling, merely tracking the total stromal
thickness provides no information about where in the central stroma
(whether anterior, posterior, or uniform) dimensional changes occur.
With both corneal cell layers intact and exposed to fluid, it is
impossible to deduce whether fluid enters from the anterior or the
posterior boundaries. Further, because of the ability of the stroma to
sustain significant hydration gradients, it is also possible that the
tissue is simultaneously swelling and thinning at different locations
across its thickness. Such phenomena have been predicted by a corneal
numerical model16
and verified by
experiment.24
The ability to track keratocyte motions
within the stroma provides information about the magnitude and location
of local swelling (and thinning). These data were combined with the
Klyce and Russell computational model16
in an attempt to
characterize the nature of the forces that drive the observed swelling
response.
To simulate the total and local swelling patterns observed in this
study, the baseline endothelial transport parameters for hydraulic
conductivity [Lp = 42.0 x
10-12 cm3/(dyne x
sec)], reflection coefficient (
NaCl = 0.45),
and permeability (
NaC l = 8.0 x
10-5 cm/sec) were set to those determined by
Klyce and Russell.16
The corresponding epithelial
transport parameters were set to values [0.0
cm3/(dyne x sec), 1.0, and 0.0 cm/sec,
respectively], that generate zero flux to simulate the effect of
silicone oil blocking fluid movement across the bare stromal surface.
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Results |
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After the 3 hours of treatment with drug or placebo in vivo, followed by perfusion of the intact corneas in the perfusion chambers, stromal thicknesses stabilized at 405.0 ± 19.3 µm for the diclofenac sodium–treated corneas compared with 404.6 ± 11.9 µm for those treated with placebo. The difference between these values was not statistically significant (P = 0.969) indicating that NSAID treatment by itself did not alter the swelling properties of the stroma.
The pattern observed in series I, consisting of anterior swelling and posterior thinning (the swelling pattern) after epithelial débridement, also occurred in series II. For the diclofenac sodium–treated corneas, three corneas exhibited the swelling pattern, whereas in three corneas keratocytes could not be tracked and were therefore not analyzable. For the placebo group, four corneas exhibited the swelling pattern, and one was not analyzable. Therefore, pretreatment of the corneas in vivo with diclofenac sodium did not alter the characteristic swelling pattern seen after epithelial débridement.
However, pretreatment of the corneas in vivo with diclofenac sodium was found to reduce the total stromal edema after epithelial débridement. Corneas pretreated with the drug swelled only 19.2 ± 3.1 µm (n = 6) at 1 hour from débridement compared with 30.6 ± 7.7 µm (n = 5) in the corneas of fellow eyes treated with the placebo (vehicle) alone (P < 0.008; Fig. 6 ). This finding strengthened the hypothesis that the unexpected stromal edema was an inflammatory response.
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Discussion |
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Efforts to understand the cause of the observed swelling response led us to re-examine a series of experiments in which the inflammatory response to irritant injections were studied. In 1969, Vinegar et al.25 demonstrated a biphasic swelling response to the injection of an irritant (carrageenan) in the rat paw. The cause of the initial, edematous phase of the inflammatory response, which is virtually identical with the time course of the swelling we observed in the cornea, could not be explained in this or in their later work.25 26 27
In a series of subsequent investigations of burn injuries, Lund et
al.28
measured interstitial pressure in rat skin. It was
found that immediately after tissue damage, the local interstitial
pressure increased in negativity by more than an order of magnitude.
Lund hypothesized that there were three possible mechanisms for the
pressure increase: 1) water loss from the space due to cell swelling or
evaporation, 2) generation and immobilization of new colloids (i.e.,
denatured collagen or proteoglycans), and 3) physical changes in the
interstitial matrix-gel that generated mechanical expansive forces.
Further explorations of these potential mechanisms began to clarify
the origin of the initial phase of the acute edema response to
local burn injury and application of irritants, including
carrageenan.28
29
30
31
32
Using Lund’s third hypothesis,
coupled with the supposition that the dermal cells provide the
mechanism to alter the local extracellular matrix in the short
term,33
34
Reed et al.35
re-examined the
onset of local edema and found that there was a cell–matrix
interaction mediated by ß1 integrins. This result led to the proposal
that the hydration of the dermal interstitium is maintained, in part,
by the local fibroblasts, and that these cells can modulate matrix
properties in the relatively short term.35
In subsequent
investigations, the same laboratory found that 
-trinositol mitigates
the extent of interstitial pressure increase after disruption of the
ß1-integrin system and that prostaglandins may also mediate cellular
control of local hydration.36
37
Thus, a plausible theory
for cellular control and a potential pathway to modulation of acute
edema and local hydration has been advanced for connective tissue.
Similar to the dermis, the normal corneal stromal imbibition pressure is negative relative to the atmosphere. At normal hydration, the in vivo imbibition pressure is -40 mm Hg and the in vitro value is -60 mm Hg, the difference being the in vivo presence of the 20-mm Hg intraocular pressure.4 38 The classic concept of the maintenance of corneal hydration and transparency implies that this stromal swelling tendency provided by the imbibition pressure is balanced by the combination of active transport of HCO3- into the aqueous humor by the endothelium9 12 and the barrier properties of the bounding membranes. These factors act in concert to limit the passive stromal absorption of fluid.10 However, stromal swelling properties generally had been thought to remain constant (for a given hydration) in response to environmental stress (osmotic challenges, hypoxia, and inflammation). The current findings challenge this assumption.
The well-known ability of the corneal stroma to imbibe fluid during impairment of ion transport or after membrane barrier compromise has been attributed to the osmotic properties and the repulsion of the fixed negative charges of the stromal GAGs.2 Our results indicate that after epithelial débridement, the stromal swelling pressure has locally increased negativity. Lund’s third hypothesis, which would require keratocyte-mediated structural control of corneal hydration, is not likely to apply to the cornea, because corneal swelling in the anterior to posterior direction is observed to proceed virtually unrestricted under a variety of stimuli. This suggests that, at least in the cornea, local edema of the stromal tissue after injury does not appear to be immediately controlled by the connective tissue cells.
It is clear from our results that the behavior of the stromal tissue after injury is not consistent with the traditional paradigms that define corneal hydration control, nor is it consistent with the new cellular-based theories of connective tissue edema modulation. Thus, the traditional models of corneal hydration maintenance must be modified to include a mechanism that rapidly alters local stromal swelling pressure. We postulate that a local increase in the osmotic pressure is responsible for the observed anterior stromal edema (cf. Figs. 3 4 5 ). However, the constituent molecules that are responsible for the increase in osmotic pressure have not yet been identified. It is possible that some of the increased osmolarity is due to the proteolytic action of enzymes on the macromolecular constituents of the tissue’s extracellular matrix. This suggested cause is similar to Lund’s second hypothesis, which holds that local matrix constituents are modified or damaged to produce higher oncotic pressures.28 Indeed, proteases specific for stromal core protein-GAG linkage molecules (serine) have been identified in the anterior keratocytes and in the tears after injury.39 40 However, lysing of GAG molecules from their protein cores is not expected to significantly increase their osmotic pressure contribution. Urban et al.41 demonstrated that most of the osmotic pressure associated with GAG molecules depends primarily on the concentration of their fixed negative charges (Donnan effects) and not on their molecular weight or aggregation state. Because lysing of the GAG from its core protein changes only the latter two quantities, we cannot attribute the observed large change in swelling pressure in such a short time to lysis of these particular molecules. Another possible mechanism explaining the swelling is that local stromal keratocytes release hyperosmotic products of proteolysis as they undergo apoptosis.42 Unfortunately, the paucity of available biochemical data with regard to the process of keratocyte apoptosis requires acknowledgment that the cause of anterior stromal swelling is not fully understood at this time.
The significance of an alteration in stromal swelling characteristics in response to débridement is of paramount importance to both experimental corneal transport physiology and corneal surgery. In these disciplines, the epithelium is removed or damaged routinely with the traditional assumption that the underlying tissue remains stable. However, it has been known for some time that epithelial débridement or trauma can induce apoptosis in the anterior stromal keratocytes43 on a time scale similar to the swelling response reported in this article (Steve E. Wilson, personal communication, May 1999). In wound-healing studies it has been shown that subepithelial keratocytes undergo apoptosis as quickly as the tissue can be processed after traumatic débridement.42 44 Of particular concern, epithelial débridement is typically used in PRK to expose the stromal surface (Bowman’s layer) to the excimer laser for reshaping. Surgeons have used manual débridement with a scalpel or blunt spatula as well as a rotating bristle brush. Damaged epithelial cells remain in contact with the stromal surface from 30 seconds to several minutes with these procedures. During this time, cytokines are released to the anterior stroma where they initiate keratocyte apoptosis that can involve the anterior half or more of the stroma. Apoptosis is greatly attenuated when the epithelium is removed cleanly (without crushing) with the excimer laser.45 This has particular relevance to our investigation, in that it suggests a potentially less traumatic approach to the anterior stroma than using a scalpel or a spatula.
PRK ablations after manual débridement of the epithelium are not nearly as accurate as ablations of inert materials and are prone to generating central islands of unablated tissue. We believe that excess fluid in the central anterior cornea arising from the acute inflammatory response could be a contributing factor in the formation of central islands. With this scheme, fluid is drawn anteriorly as shown in the experiments reported here, resulting in extra laser energy’s being spent in its removal rather than tissue ablation. Because the cornea swells preferentially in the center under a variety of uniformly applied stimuli,46 47 energy absorption is likely to be exacerbated in that region. In addition to the increase in anterior hydration, fluid may collect on the bare stromal surface further reducing ablation efficiency through absorption and reflection of the incident laser energy.48
The success of refractive surgery depends on the management of inflammation and wound healing, without which tissue remodeling and stromal scarring can occur. Although topical prednisolone has been used for this purpose, NSAIDs, used before and after surgery, have the additional benefit of ameliorating postoperative pain.49 We have shown that the edema produced by corneal epithelial débridement can be diminished using pretreatment of the cornea with the NSAID diclofenac sodium, a nonspecific cyclooxygenase inhibitor.
We suspect that this acute edema may have a cause similar to that of the inflammatory response seen in the rat paw model, as noted earlier. If this is the case, we may have uncovered a basic early process in the response of tissues to injury that leads to a swelling and loosening of the connective tissue that may provide an avenue through which migrating inflammatory cells may respond. Until we learn more, it may be prudent to minimize contact between the scraped epithelium and the underlying stroma during surgical procedures to reduce hydration-related complications.
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Acknowledgements |
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Footnotes |
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Presented in part to the Graduate Faculty of Tulane University, New Orleans, in partial fulfillment of the requirements for a Doctor of Philosophy in the Department of Biomedical Engineering; at Aegean Cornea IV, June 26, 1998, Santorini Island, Greece; at the XIII International Congress of Eye Research, July 1998, Paris, France; and at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, Florida, May 1999.
Supported in part by US Public Health Service Grants EY03311 and EY02377 from the National Eye Institute, National Institutes of Health.
Submitted for publication June 15, 1999; revised November 22, 1999, January 19, 2000 and March 28, 2000; accepted April 19, 2000.
Commercial relationships policy: N.
Corresponding author: Jeffrey W. Ruberti, 2145 Sheridan Road Tech. E-310, Biomedical Engineering Department, Northwestern University, Evanston, IL 60208. j-ruberti{at}nwu.edu
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-trinositol exerts its edema-preventing effect through modulation of ß1 integrin function Circ Res 75,942-948 analogs on collagen gel compaction in vitro and interstitial pressure in vivo Am J Physiol 274,H663-H671This article has been cited by other articles:
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  M. D. Karon and S. D. Klyce Effect of Inhibition of Inflammatory Mediators on Trauma-Induced Stromal Edema Invest. Ophthalmol. Vis. Sci., June 1, 2003; 44(6): 2507 - 2511. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) and correlates them with the position of the microscope objective
to permit tracking of surfaces and internal reflections for thickness
calculations.
