Christopher A. Paterson

Quantification of ocular inflammation: Evaluation of polymorphonuclear leucocyte infiltration by measuring myeloperoxidase activity

Myeloperoxidase (MPO) activity was measured in rabbit cornea and iris-ciliary body to quantitate the infiltration and accumulation of polymorphonuclear leucocytes (PMNs) following an inflammatory stimulus. Following injection of clove oil into the cornea, MPO activity could be detected in the cornea at 6 hr, reaching a maximum at 12 hr, and falling to non-detectable levels at 72 hr. MPO activity was only detected in the iris-ciliary body 24 hr after intracorneal clove oil injection. MPO activity in the iris-ciliary body increased in a dose-dependent manner following intravitreal injection of endotoxin. No MPO activity could be detected in cornea. Topical administration of dexamethasone inhibited MPO activity in cornea and iris-ciliary body 24 hr after intracorneal clove oil and intravitreal endotoxin injection, respectively. Measurement of MPO activity in ocular tissues could provide a useful tool to quantitatively evaluate the severity and time course of inflammation.

Detection of EP2, EP4, and FP receptors in human ciliary epithelial and ciliary muscle cells

We have examined the expression of three prostaglandin (PG) receptors, EP2, EP4, and FP, in a nonpigmented ciliary epithelial cell line (ODMCl-2) and in human ciliary muscle (HCM) cells. Total RNA preparations from either ODMCl-2 or HCM cells were subjected to reverse transcription-polymerase chain reaction (RT-PCR) with sense and antisense primers for each of the three PG receptors. The RT-PCR generated DNA products of predicted sizes corresponding to the EP2, EP4, and FP receptors in both ODMCl-2 and HCM cells. PCR products corresponding to each receptor were hybridized with specific 32P-labeled probes and, for further confirmation, digested with appropriate restriction enzymes. Pharmacological studies with the EP2 receptor-selective agonist butaprost resulted in a significant increase in the cyclic AMP level in ODMCl-2 cells. The stimulation of cyclic AMP in ODMCl-2 cells by PGE2 and 11-deoxy PGE1, the respective EP1/EP2/EP3/EP4 and EP2/EP3/EP4 receptor agonists, was concentration-dependently inhibited by the EP4 receptor-selective antagonist AH23848. These results conclusively demonstrate the presence of both mRNA and protein for EP2, EP4, and FP receptors in ODMCl-2 and HCM cells.

Nuclear Magnetic Resonance Studies and Water "Ordering" in the Crystalline Lens

Nuclear magnetic resonance studies of the relaxation times of the water in the crystalline lens show that, as in all interfacial systems, these parameters are markedly reduced from their values in pure water, that T2 is less than T1, and that both depend on water content. Determination of diffusion coefficients and studies on physiologically inert lenses indicate that reduced relaxation times do not provide direct evidence for ordering of the bulk of the cell water.

Aqueous Humor pH Changes After Experimental Alkali Burns

Following application of 2N sodium hydroxide, or 8.1N ammonium hydroxide to rabbit cornea, the aqueous humor pH reached a maximum of 10.2, 11.9, and 12 within six minutes following 20-, 50-, and 100-mu1 sodium hydroxide burns, respectively; after two hours the pH had fallen to 8.5, 10, and 10.5. The maximum rise following application of 100-mu1 of ammonium hydroxide was 10.8, declining to about 9 at two hours. The fall in pH following a 100-mu1 sodium hydroxide burn was not greatly influence by external lavage. However, the pH was significantly lowered (12 to 10) by paracentesis alone and further reduced by immediate or delayed intracameral administration of phosphate buffer. On the basis of these results moderately severe and severe alkali burns of the eye should be treated by paracentesis and if possible with anterior chamber reformation with a sterile solution.

Characteristics of polymorphonuclear leukocyte infiltration into the alkali burned eye and the influence of sodium citrate

Polymorphonuclear leukocytes (PMNs) are considered to play a central role in the corneal ulceration process subsequent to an alkali burn. We have described the time course of PMN infiltration into the ocular tissues following an alkali burn. In addition, we examined the effect of sodium citrate upon the accumulation of PMNs in the alkali burned cornea. The accumulation of PMNs into the cornea and iris-ciliary body was quantified by measuring myeloperoxidase (MPO), an enzyme marker for these inflammatory cells. Leukocytes in aqueous humor aspirates and corneal washes were counted directly under a microscope. In the alkali burned cornea, we found an initial transient, yet substantial, infiltration of PMNs, peaking at about 12-24 hr and limited to the peripheral cornea; this subsided by about 72 hr. By 14 days, the MPO activity, and hence the number of leukocytes, had risen again, and by 21 days the level had increased by several fold. Qualitatively similar biphasic patterns of leukocyte infiltration were observed in the iris-ciliary body and aqueous humor. Leukocyte numbers in corneal washes only increased between 4-24 hr following the alkali burn. The exceptionally high degree of leukocyte infiltration into the cornea at 21 days corresponded with the presence of ulceration. Topical administration of sodium citrate (10%) inhibited the early and late phase of PMN accumulation in the alkali burned cornea, i.e. at 24 hr (-63%) and 21 days (-92%). The inhibition of PMN infiltration by sodium citrate correlates with the reduced corneal ulceration observed following treatment with this compound.

Effect of oxidation on Ca2+ -ATPase activity and membrane lipids in lens epithelial microsomes.

Membrane oxidation may contribute to cataractogenesis. In our pursuit to understand the etiology of cataracts, we assessed the effect of membrane oxidation products on the activity of the lens epithelium calcium pump. Microsome preparations from bovine lens epithelium were oxidized to varying degrees with a ferrous and ferric ascorbate system to generate hydrogen peroxide and superoxide. Ca2+ -ATPase activity was measured using a colorometric assay. Lipid oxidation was quantified by infrared spectroscopy. Ca2+ -ATPase activity decreased as a function of ascorbate concentration between 0 and 200 microM. The level of Ca2+ -ATPase inhibition was correlated to both the level of lipid oxidation and the degree of lipid hydrocarbon chain order. At 25 degrees C when lipids are more ordered, the Ca2+ -ATPase activity was similar to that observed in the oxidized system measured at 37 degrees C. Glutathione, mercaptoethanol, and iodoacetate were able to reverse the oxidative inhibition of the calcium pump, suggesting that the ascorbate/iron oxidant directly oxidized the protein sulfhydryl moieties. To further probe the mechanism of Ca2+ ATPase inhibition, hydrogen peroxide was used to oxidize muscle sarcoplasmic reticulum Ca2+ -ATPase reconstituted in its native lipid vesicles, egg phosphatidylcholine, and dihydrosphingomyelin, with saturated hydrocarbon chains. In these systems, oxidation inhibited the Ca2+ -ATPase pump by 60-80%. There was no statistical difference between the level of oxidative inhibition and the percentage of dihydrosphingomyelin. Because dihydrosphingomyelin cannot be oxidized, whereas egg phosphatidylcholine (PC) can, and because the percentage of inhibition was the same for reconstituted systems using either lipid, the mechanism of inhibition is likely not via a secondary process involving oxidation-induced lipid structural changes or products of lipid oxidation.

Ascorbic acid inhibits the activity of polymorphonuclear leukocytes in inflamed ocular tissues

Myeloperoxidase (MPO) is present at high levels in polymorphonuclear leukocytes (PMNs) and has been used as a marker to quantify the accumulation of PMNs in inflamed tissues. MPO activity in inflamed ocular tissues was inhibited by aspirates of aqueous humor. This inhibition could be duplicated by the addition of ascorbic acid at concentrations equivalent to those present in the aliquots of aqueous humor. Similarly, aqueous humor and ascorbic acid inhibited MPO from isolated rabbit leukocytes. Therefore, ascorbic acid appears to inhibit the functional activity of the peroxidase in PMNs, thus preventing potential tissue damage by this enzyme when released during leukocyte degranulation in inflammation. Ascorbic acid might fulfill a role as an endogenous anti-inflammatory agent in the eye.

Evidence for two sodium pumps in the crystalline lens of the rabbit eye

Abstract The effect of ouabain on the ATPase activity, the electrical potential, 86Rb uptake, and sodium content in the crystalline lens of the rabbit were studied. The ouabain-sensitive ATPase activity of the lens epithelium was compared to that of segments of lens cortex. The lens fibres were found to possess one-third of the total ouabain-sensitive ATPase activity of the lens. Dose-response studies of ATPase inhibition by ouabain gave a KI of about 1·4 × 10−6 m for the epithelium and about 3·3 × 10−5 m for the anterior cortex. Although ouabain sensitive ATPase was present in the posterior cortex, ouabain had no effect on 86Rb uptake across the posterior lens surface. Application of ouabain to the anterior surface of the lens resulted in an 8 mV decrease in the absolute value of the lens potential, a reduction of 86Rb uptake to about one-third of its value in unpoisoned lenses, and a significant increase in sodium content after 2 hr of incubation. Dose-response studies of these effects suggested that the Na K ATPase of the anterior cortex, but not that of the epithelium, makes an electrogenic contribution to the measured lens potential. On the other hand, inhibition of the epithelial ATPase effects a greater reduction in 86Rb uptake than inhibition of the cortical ATPase. The maintenance of lens sodium content cannot be ascribed solely to the activity of the sodium pump in the lens epithelium. The results of these studies suggest that two types of Na K ATPase with different anatomical localizations contribute to the maintenance of cation balance in the rabbit lens.

Effect of hybrid peptides of cecropin A and melittin in an experimental model of bacterial keratitis.

Synthetic peptides, ranging from 12 to 18 residues, containing partial sequences from natural cecropin A and melittin were tested for activity in an experimental pseudomonas keratitis model in rabbits. In separate experiments, two Pseudomonas aeruginosa strains: (a) a clinical isolated strain, and (b) an American Type Culture Collection (ATCC) strain, were inoculated into the stroma of one cornea of each rabbit. Peptides were topically applied at 0.1% in phosphate-buffered saline (PBS) and compared with PBS alone and 0.3% gentamicin eye drops. Clinical evaluation, based on the McDonald-Shadduck scale, was performed during a > 48-h period after the bacterial inoculation. The peptide-treated animals showed significantly lower (p < 0.05) inflammatory signs and lower anterior-segment bacterial damage compared with PBS-treated animals, after the first 6 h. The antiinflammatory/antimicrobial activity was non significantly differnt (p > 0.05) from that in animals treated with gentamicin. We conclude that peptides keeping the sequence KWKLFKK from cecropin A and at least the sequence VLKVL from melittin show promise as novel agents in topical ocular therapy of bacterial keratitis.

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.