The Castroviejo Lecture. Prolonged eyelid closure is a risk to the cornea.

PURPOSE: The author introduces new concepts and summarizes published evidence suggesting that prolonged eyelid closure puts the cornea at risk. METHODS: Significant clinical and experimental publications are reviewed, and the author's experience is applied relating to the pathogenesis and treatment of a variety of clinical entities thought to be induced, to some degree, by prolonged eyelid closure. RESULTS: Evidence from the scientific literature suggests that the hypoxia or reduced tear volume or both that result after prolonged eyelid closure, especially during sleep and when a soft contact lens is in place, serve as risk factors in the "sucked-on" contact lens syndrome, recurrent corneal erosion, chronic corneal deepithelialization, Pseudomonas keratitis, filamentary keratitis, superior limbic keratoconjunctivitis, sterile midperipheral corneal infiltrates, and corneal vascularization. The limbal stem cells under the upper eyelid are subjected to continuous hypoxic stress and are at special risk to other insults. CONCLUSIONS: Prolonged eyelid closure, such as in patching and in sleep, is a risk factor in the pathogenesis of a variety of corneal conditions.

Pharmacokinetics of newer cephalosporins after subconjunctival and intravitreal injection in rabbits.

Pharmacologic considerations suggest that third-generation cephalosporins might penetrate the vitreous humor better after periocular injection and might be eliminated less readily after intravitreous injection than older agents. We studied the sodium salts of ceftizoxime, ceftriaxone, and ceftazidime, and of an investigational cephalosporin, cefepime, in rabbits. After a single subconjunctival injection in animals with normal eyes, vitreous levels ranged from 3 to 13 mg/L. After five subconjunctival injections in rabbits with infected eyes, vitreous concentrations ranged from 12 to 34 mg/L. These concentrations are not appreciably greater than those found with older beta-lactams. The vitreous half-life of the four drugs after intravitreous injection varied from 5.7 to 20 hours in rabbits with uninflamed eyes and from 9.4 to 21.5 hours in rabbits with infected eyes. Except for ceftizoxime, the half-lives were substantially longer than those for older beta-lactams and suggest predominantly anterior route elimination. Vitreous penetration of these new agents after subconjunctival injection does not appear to be sufficient to overcome the need for intravitreous injections in the treatment of endophthalmitis. However, the longer vitreous half-lives of some of the newer agents may be useful if the drugs are to be given intravitreally.

Effects of corneal extracts on rabbit corneal stromal cells in culture.

Rabbit corneal tissues were treated sequentially with phosphate buffered saline (PBS) and 4 M guanidine hydrochloride (GuHCl), dialyzed, and lyophilized. The interaction of the individual PBS and the GuHCl extracts with cultured rabbit corneal stromal cells was assessed. The PBS extract stimulated stromal cell growth. These cells had a thinner spindle-shaped appearance, a greater tendency toward multilayer formation, and a approximately 40-60% higher final density than the controls. The cells subjected to the GuHCl extract exhibited no such changes. When the PBS extract was heated to 80 degrees C, the stimulatory activity was replaced by an inhibitory activity, indicating that the PBS extract contained both the stimulatory and the inhibitory factors. Using a high performance liquid chromatograph system, such factors could be separated. The effects of corneal extracts on connective tissue synthesis were examined after labeling confluent stromal cultures with either (35S)sulfate or (3H)proline for 20 hr. The PBS and the GuHCl extracts significantly promoted the incorporation of (35S)sulfate into glycosaminoglycans. Neither extract altered the types of glycosaminoglycans synthesized or the collagen synthesis of stromal keratocytes in culture.

Identification of collagens synthesized by cultures of normal human corneal and keratoconus stromal cells.

We have examined the collagens synthesized by cultures of normal human corneal stromal cells. Radioactively labeled products, accumulated in the culture medium during a 24-h labeling period, were treated with pepsin and analyzed by SDS-polyacrylamide gel electrophoresis. The cell layer collagen was characterized by 2.6 M and 4.4 M salt fractionation at neutral pH. CM-cellulose column chromatography, SDS-gel electrophoresis, and cyanogen bromide peptide mapping. Type I alpha 1 and alpha 2 chains were the predominant components in both the cell layer and the medium fractions of normal human stromal cultures; type III collagen was found mostly in the culture medium; and type V collagen was associated with the cell layer. Immunofluorescent techniques used to visualize collagen deposition in the cell layer confirmed the presence of these collagen types. Keratoconus is a disease characterized by thinning and scarring of the central cornea. Stromal cells grown from keratoconus corneas produced similar types of collagen (types I, III, and V) as normal human controls. Cells from keratoconus patients, however, contained more type V collagen in the cell layer than did normal cells. The difference was seen only in the 4.4 M salt precipitates. Since type V collagen is one component of cell surfaces, the primary defect in cultures from keratoconus corneas could involve cell membrane and cell surface components.

Effects of ascorbic acid on cultured rabbit corneal endothelial cells.

Rabbit corneal endothelial primary cultures and subcultures with initial high seeding densities (split ratio 1 : 2) and low seeding densities (split ratio 1 : 4) were maintained routinely in Falcon Petri dishes. Ascorbate, 75 microgram/ml, was added daily to experimental cultures to evaluate its effect on growth of endothelial cells. With supplemental ascorbic acid, the cells grew at a slow rate, had a shorter lifespan, and reached a lower cell density at confluency than did control cells. The inhibitory effect of ascorbic acid on cell growth was more pronounced with cultures plated at low cell densities and with cells in later passages. Cultures grown with ascorbate contained a greater number of elongated cells and large-sized cells with vacuoles. A basal lamina was observed even in control cultures with no supplemental ascorbate.

Ocular findings in Bell's palsy.

Thirty-three patients with Bell's palsy underwent ophthalmic examination, including Schirmer testing, corneal sensitivity measurement, and electromyography. Sixteen patients returned for follow-up after one year. Sixty-one percent of the patients initially showed punctate fluorescein staining of the inferior cornea. Electromyographic studies demonstrate a high correlation between orbicularis function and the presence of corneal epithelial staining. Our results demonstrate a high frequency of minor corneal involvement in the acute phase of Bell's palsy. Healing of the epithelial defects follows improvement in orbicularis function.

Initial therapy of suspected microbial corneal ulcers. I. Broad antibiotic therapy based on prevalence of organisms.

The initial treatment of bacterial ulcers of the cornea should consist of a combination of antibiotics that are effective against the major pathogens in the community. A gram stain may be misleading and therefore may suggest inappropriate therapy. Antibiotic therapy should include subconjunctival injections and concentrated eye drops, but not systemic administration except following perforation. Initially, we use cefazolin and tobramycin or gentamicin. Bacitracin may be substituted as a topical medication. Antibiotic therapy should be changed only if the pathogen is reported to be resistant to initial therapy and if the corneal ulcer continues to worsen.

Mass culture of human corneal endothelial cells.

Human corneal endothelial cells have been grown in mass culture for the first time to our knowledge. Cultures from donors who were under 20 years of age grew well. Cells from older donors were difficult to grow or did not grow at all. Initial outgrowth usually began one week after explantation. After an initial lag phase, mass increased by 15% weekly, and confluency was approached in eight to nine weeks. The lag phase of growth correlated with the age of the donor. A basal lamina was secreted by human corneal endothelial cells in culture. Chromosomal counts demonstrated a modal number of 46. With this technique, sufficient human corneal endothelial cells may be obtained for biochemical investigation.

Origin of the retrocorneal membrane in the rabbit.

Electron microscopic studies have offered presumptive evidence that the origin of both cellular and extracellular components of the retrocorneal membrane (RCM) derive from corneal endothelial cells. In an attempt to demonstrate conclusively the origin of the cell in the RCM, we performed 8-mm exchange, penetrating keratoplasties between male and female rabbits. After allowing for complete healing and using only clear corneas, we produced central RCM by freezing the central cornea after inducing intraocular inflammation. Care was taken to ensure that the membrane was surrounded by normal donor endothelium and Descemet's membrane was intact. Sex chromatin counts of the cells in the RCM of a female host with a male graft showed an average of 2% Barr bodies. Cells in the membrane of a male host with a female graft showed an average of 40% Barr bodies. The experimental and control sex chromatin counts were almost identical. This study provides conclusive evidence that central retrocorneal, fibroblast-like cells and membranes in rabbits are derived from corneal endothelial cells.

Maintenance of mydriasis with topical indomethacin during and after penetrating keratoplasty in the rabbit.

The use of indomethacin eyedrops, 0.5%, in combination with topical atropine and phenylephrine, prior to and immediately following an 8-mm penetrating keratoplasty in rabbits maintains maximal pupillary dilation. With this technique, we achieved clear grafts without synechiae in virtually every instance.

Postlenticular membrane associated with Smith-Lemli-Opitz syndrome.

A 6-day-old boy with Smith-Lemli-Opitz syndrome developed bilateral cataracts, posterior synechiae, and a dense postlenticular membrane. No other signs of inflammation were noted that could account for the development of either the posterior synechiae or membrane. To our knowledge, this is the first published report of such an associated membrane.

The synthesis of glycosaminoglycans by cultures of corneal stromal cells from patients with keratoconus.

Keratoconus is a disease that results in thinning and ectasia of the central cornea. Cultures of corneal stromal cells from patients with keratoconus were established and the synthesis of glycosaminoglycans compared with the synthesis of glycosaminoglycans by normal human corneal stromal cells in culture. Keratoconus and normal control cell cultures were incubated with sodium [(35)S]sulfate and [(3)H]glucosamine for 4 h. After incubation, the labeled glycosaminoglycans were isolated from the medium fractions and cells. Keratoconus and normal control cultures synthesized similar amounts of sulfated glycosaminoglycans independent of the age of donors and(or) the number of subcultures. In contrast to normal control cultures, most of the newly synthesized glycosaminoglycans produced by keratoconus cells were found in the growth medium and much less were in the cell layer. Treatment with glycosaminoglycan-degrading enzymes followed by paper chromatography showed that keratoconus cells, as normal control cells, produced hyaluronic acid and various sulfated glycosaminoglycans. The production of cell layer-related heparan sulfate was markedly reduced in keratoconus cultures. Because heparan sulfate has been shown to be associated with cell surfaces, the decreased heparan sulfate content could reflect changes at this location.

Intraocular levels of cefamandole compared with cefazolin after subconjunctival injection in rabbits.

We compared the intraocular pharmacokinetics of cefazolin with those of cefamandole, a recently marketed cephalosporin with enhanced activity against gram-negative bacilli. Following subconjunctival injection of 12.5 mg into infected eyes (S. aureus endophthalmitis) of pigmented rabbits, both drugs reached peak concentrations greater than 100 microgram/gm in cornea, sclera, and choroid-retina. The half-life was markedly shorter in sclera and choroid-retina than in cornea. Levels in the aqueous humor rose and fell more slowly than those in ocular tissues, reaching a maximum of only 5 to 10 microgram/ml. The pharmacokinetics of the two drugs were virtually identical in most intraocular sites. When cefazolin, which was less irritating than cefamandole by the subconjunctival route, was given in a dosage of 100 mg, levels in ocular tissues were increased by twofold to fourfold and in aqueous humor by 15-fold, compared to the concentrations produced by the 12.5 mg dosage. Levels in the vitreous humor were exceedingly low with both drugs; mean peak concentrations were 0.24 microgram/ml after the 12.5 mg dosage of cefamandole and less than 1.6 microgram/ml after the 100 mg dose of cefazolin.