Roswell R. Pfister

The Combined Effect of Citrate/ascorbate Treatment in Alkali-injured Rabbit Eyes

The incidence of ulceration and perforation in the cornea of alkali-injured eyes is significantly reduced by treatment with trisodium citrate or sodium ascorbate. Topical citrate reduces the inflammatory response in the cornea by inhibiting polymorphonuclear leukocytes. Topical ascorbate elevates the depressed level of this vitamin in the alkali-injured cornea, reversing a scorbutic condition. The purpose of the current study was to determine whether combined treatment with topical citrate and ascorbate has an additional therapeutic value when compared with citrate alone. Adsorbotear without EDTA was used as the vehicle for both medications. Rabbit eyes were injured with IN NaOH for 35 s using a 12-mm well and were rinsed. Group I (47 eyes) received two drops of 10% citrate every hour on the hour and Adsorbotear on the half-hour for 14 h/day. Group II (48 eyes) received two drops of 10% citrate every hour on the hour and 10% ascorbate every hour on the half-hour for 14 h/day. The citrate/ascorbate group had significantly fewer ulcerations during the experiment than did the group receiving citrate alone (2 of 48 versus 10 of 47, 0.01 < p < 0.02). Both anterior ulcers in the citrate/ascorbate group and five ulcers in the citrate group healed to no ulcer by the end of the experiment (0 of 48 versus 5 of 47, 0.02 < p < 0.05). The average depth of ulceration was significantly less for the citrate/ascorbate group (p < 0.001). No descemetoceles or perforations were observed in either group. No differences were observed between the two groups with respect to the onset of ulceration or the number of total vascularizations. We conclude that the combined therapy of citrate and ascorbate was a significant improvement over citrate treatment alone and almost eradicated corneal ulceration.

Fibrin sealant in corneal stem cell transplantation.

PURPOSE To determine if transplanted corneal epithelial stem cells are safely and efficiently attached to the deficient limbal niche with use of fibrin sealant. The primary outcome is measured with respect to the stability of the transplant, with secondary qualitative evaluations of inflammation, patient comfort, speed of operation, and incidence of complications. METHODS This retrospective case study examined a total of 114 corneal stem cell reconstructions performed in 95 patients from 1996 to 2004 using corneal stem cells primarily, with a minority of amnion alone, or both. Fibrin sealant was used as the only technique of stem cell adhesion for limbal reconstruction for primary or recurrent pterygia and various stem cell-deficient diseases from 2000 to 2004. RESULTS The fibrin sealant group showed 1 small recurrence of pterygium but no complications. With sutures, there were 3 recurrences in the pterygia group. After completion of all surgical procedures, all patients were free of pterygia. Miscellaneous stem cell deficiencies were included to demonstrate that corneal stem cell transplants can be used in other corneal procedures in addition to pterygia. CONCLUSIONS Fibrin sealant alone effectively and safely attached corneal stem cell transplants to the limbal niche. The additional qualitative observations of a reduction in operation time, postoperative pain, and inflammation augurs for more extensive use of fibrin sealants in ophthalmology.

The Efficacy of Sodium Citrate in the Treatment of Severe Alkali Burns of the Eye Is Influenced by the Route of Administration

Rabbit corneas subjected to 12 mm, 1 N NaOH alkali burns for 35 seconds were treated with 10% sodium citrate topically or by subcutaneous injections. In the topically treated group, 18.8% of the citrate-treated corneas ulcerated compared to 78.6% of the controls (0.01 > p > 0.001). The ulcers developing in the citrate-treated group were not only statistically fewer but were significantly less severe in degree. Band keratopathy developed in 64.3% of the control eyes but in none of the citrate-treated eyes. Subcutaneous citrate reduced the incidence of ulceration from 75% in the control to 43% in the treated animals. Although this may represent a trend, it is not statistically significant (p = 0.073). Combining the descemetoceles and perforations in the parenterally treated groups yields an incidence of 56% in the controls and 14% in the citrate-treated animals. This significant difference (p = 0.017) clearly shows the greater severity of ulceration in the control group of the subcutaneously treated animals. These results show that sodium citrate has a most favorable effect in the prevention of corneal ulceration and perforation after alkali burns when applied topically.

Chemical burns to the eye: paradigm shifts in treatment.

Every ophthalmologist knows that prompt irrigation is critical after a chemical injury of the eye. This should be continued on the way to the emergency room where standard procedure requires topical anesthetics, cleaning the fornices of any particulate material, and copious irrigation with fluid until litmus tests of the tear film approach neutrality. The value of standard intravenous solutions as ocular irrigants is now being called into question. In most countries, physiological saline is the standard used in industries, laboratories, and hospital emergency rooms. Countering this approach, Paterson et al have shown that physiological saline lavage of an alkali-injured rabbit eye results in only a negligible decrease of aqueous pH. Only paracentesis and reformation of the anterior chamber with phosphate buffer rapidly lowered the pH to physiological levels. Perhaps surprisingly, lavage with tap water is more effective than saline. More importantly, however, Reim et al have demonstrated, again in rabbits, that a borate buffer (Cederroth Eye Wash; Cederroth AB, Upplands Väsby, Sweden) is far more effective in reducing aqueous pH after alkali injury. Alternatively, an amphoteric class of substances (Diphoterine or Previn solutions; Prevor, Cologne, Germany) can be effective. These irrigating solutions are now in place at many workplaces and in hospitals, particularly in Europe. The rest of the world, including the United States, should follow. For acid injuries, which are less penetrating, the composition of the solution is far less critical. After lavage of the eye, what should come next? The use of topical steroids is somewhat controversial. No comprehensive human studies are available for guidance. Topical steroids might be used in milder injuries, along with prophylactic antibiotics and a bandage contact lens for a brief period. If topical steroids are given in a severe injury, then the use must be limited to a week or 2, and then rapidly tapered to avoid stromal ulceration and perforation. These latter events are the result of unbridled inflammation and ineffective corneal stromal repair. There is now substantial experimental and human data indicating that concomitant use of topical ascorbate 10% and topical citrate 10% reduces the risk of ulceration by enhancing fibroblastic repair of collagen and glycosaminoglycans and inhibiting the invasion and degranulation of neutrophils in the cornea, respectively. Collagenase inhibitors (tetracyclines, acetylcysteine, and so on) might diminish ulceration. Surgical placement of an amniotic membrane overlay, to protect naked stroma, can suppress the inflammatory process. Despite the fact that no medication is known to successfully clear the cornea after a severe chemical injury, limitation of the inflammation has a salutary effect on subsequent attempts at rehabilitation. What then constitutes the most significant threat to eventual visual outcome? The answer is glaucoma! High-pressure intraocular spikes can occur immediately after an alkali injury and can persist indefinitely. They are often missed when attention is directed at the cornea. Applanation tonometry is often imprecise or unreadable on an injured cornea, and any other device might be deemed undesirable for fear of damage to any existing or regenerating epithelium. Intraocular pressure measurement by digital palpation of the eye is crude but can be helpful. Paracentesis during the initial phases shortly after the injury, repeated if necessary, could relieve pressure over the short term by the exit of necrotic tissue and protein clogging the trabecular meshwork. The secondary glaucoma resulting from chemical insult is less responsive to traditional glaucoma medications, which must be used.

Synthetic complementary peptides inhibit a neutrophil chemoattractant found in the alkali-injured cornea.

Purpose. We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense–antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP. Methods. The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4. Results. The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 &mgr;M). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 &mgr;M) but had no effect on LTB4. Conclusions. A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.

NMR conformational analysis of cis and trans proline isomers in the neutrophil chemoattractant, N-acetyl-proline-glycine-proline.

Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.

The effects of citrate on fMLP-induced polymorphonuclear leukocyte stimulation and locomotion.

Citrate reduces the incidence of corneal ulceration and perforation in alkali burned eyes and prevents polymorphonuclear leukocyte (PMN) accumulation in certain inflamed ocular tissues. Chelation of Ca-2+ Mg-2+ by citrate appears to be the mechanism causing strong inhibition of in vitro PMN stimulation by opsonized zymosan. It is important to know if other activating agents of PMNs, with differing sensitivities to divalent cations, are inhibited by citrate. Citrate (12 mM) partially inhibits fMLP stimulation of the respiratory burst (50%) and degranulation (65%) of PMNs in a divalent cation free media, while having no effect or only a small effect in the presence of 1 mm Ca-2+. Citrate also caused significant inhibition of fMLP (12 mM = 50%) induced locomotion of PMN when incubated in media containing 500 μM Ca2+ 600 μM Mg-2+ in a modified Boyden chamber. When used together, Ca-2+ (6 mM) and Mg2+ (6 mM) reduced this inhibition to only 20%. Citrate apparently inhibits fMLP-induced stimulation in cation free media by chelating CA-2+ effluxed from intracellular storage sites. In the chemotactic studies, citrate probably chelates extracellular Ca2+ Mg2+. The divalent cation requirements of the activating agents and/or the PMN function may determine the degree of inhibition by citrate. It is therefore important to identify the mediators in alkali burned corneas as well as other inflammatory conditions.

Degradation of bovine corneal collagen by alkali.

This study was undertaken to demonstrate the effect of alkali on the molecular size of collagen. Type I and V collagen from bovine cornea were incubated with different concentrations of NaOH at room temperature and 37°C for various times. The samples were then neutralized and analyzed by high-pressure liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degradation of collagen molecules to a heterogeneous mixture of peptides with molecular weights <20,000 daltons occurred at concentrations >0.25N, and the effect of alkali was much faster at 37°C than at room temperature. In contrast, the molecular size of collagen was not affected by similar concentrations of HC1 under similar conditions of time and temperature.

An excess of topical calcium and magnesium reverses the therapeutic effect of citrate on the development of corneal ulcers after alkali injury.

Our purpose was to determine whether chelation of Ca2 + and Mg2+ is the mechanism by which sodium citrate inhibits corneal ulceration in the alkali-injured rabbit eye. The right eyes of 60 albino rabbits (2-2.5 kg) were alkaliinjured by filling a 12-mm-diameter plastic well placed on the corneal surface with 0.4 ml of 1 N NaOH. After 35 s the alkali was aspirated, and the well was rinsed with physiological saline. Animals were randomly distributed to three treatment groups of equal size. Two drops of the following topical medications were administered on the hour (14 times per day) for 35 days: physiological saline, 10% citrate in saline, and 346 mM Ca2+, 346 mM Mg2 +, and 10% citrate in saline. During the experiment, significantly fewer ulcerations occurred in the citrate-treated eyes (five of 20,25%) than in the saline-treated eyes (13 of 20, 65%) or in the calcium-magnesium-citrate-treated eyes (15 of 20, 75%). When ulcerations did develop in the citrate group, they occurred significantly later and were less severe than those in the saline and calcium-magnesium- citrate groups. There was a significant increase in the number of eyes with signs of band keratopathy and translucent areas in the calcium-magnesium-citrate group when compared with the other two groups. As in previous studies, sodium citrate significantly inhibited the development of corneal ulcers after alkali injury. The annulment of the favorable effect of citrate on ulceration in the alkali-injured eye by the addition of calcium and magnesium shows that the mechanism of action of citrate is the chelation of these divalent cations.

A visual assay for quantitating neutrophil chemotaxis in a collagen gel matrix

The chemotactic behavior of polymorphonuclear leukocytes (PMNs) suspended in a three-dimensional gel of native collagen fibers was analyzed using a new visual assay aided by computer assisted tracking. Cell behavior was observed in a 7 microliters chamber closed at either end with capillary tubes tipped with dialysis membrane. The chemoattractant, LTB4, was placed in one capillary tube and the control substance in the opposite tube. Under microscopic observation neutrophils were videotaped, their images digitized, and the x and y coordinates of the cell centroids captured at 30 s intervals for 15 min and subsequently analyzed. The data generate a global perspective of neutrophil behavior in a medium simulating a collagenous tissue. The results show that when leukotriene B4 was substituted for HBSS the PMN population underwent chemotactic displacement. PMN chemotaxis was increased greatly when the concentration of LTB4 was increased from 10 nM to 1 microM in separate experiments. This result was partly achieved by movement of an increasing percentage of the PMN population, less frequent stops, and longer durations of motility for individual cells. The most dramatic effect of LTB4 on neutrophil behavior was a large increase in directional movement toward the chemotactic source. The effects of LTB4 fell dramatically when the gradient source concentration was increased to 10 microM. The visual assay described here provides clear evidence that LTB4 induces true neutrophil chemotaxis in a collagenous matrix.