Janet A. Anderson

Air/liquid corneal organ culture: a light microscopic study

Air/liquid organ culture of tissues with stratified epithelial layers has been shown to encourage tight packing of cells and promote cellular differentiation. In this study human corneas cultured in a air/liquid environment were compared to paired, conventionally-cultured corneas to determine if the long-term morphology could be improved. Fourteen paired human corneas were cultured at 37 degrees C in covered culture dishes for 1 to 3 weeks. Air/liquid cultured corneas were placed epithelial-side up in a fixed position and culture medium was added to a level so that during rocking the corneal epithelia were intermittently exposed to air/liquid environments. Mate corneas were cultured using the conventional method. In this method corneas are fully submerged, epithelial-side down, in culture medium. After 3 weeks of culture significantly less epithelial intercellular edema was noted for the air/liquid cultures (p = 0.033), compared to conventional cultures. Significant improvements in cellular structure of the endothelial layers, after 1 and 3 weeks incubation (p = 0.029 and 0.000) and stromal layers, after 3 weeks in culture (p = 0.024), were also noted. We have shown that slight modifications of the organ culture environment lead to improvements in corneal morphology. Air/liquid corneal organ culture has promise for use in corneal wound healing studies and long-term culture.

Human excimer laser keratectomy. Clinical and histopathologic correlations.

PURPOSE To understand the healing capabilities of the diseased human cornea after excimer laser photoablation by morphologic analysis of laser-treated corneas. METHODS Twelve corneal specimens were obtained 5 to 16 months after lamellar or full-thickness keratoplasty following phototherapeutic keratectomy for undercorrected myopic epikeratoplasty (2 eyes), corneal leukomas (2 eyes), herpes zoster corneal scarring (1 eye), band keratopathy (2 eyes), adenoviral subepithelial opacity (1 eye), keratoconus (1 eye), herpes simplex corneal scarring (2 eyes), granular corneal dystrophy (1 eye), and recurrent lattice dystrophy (1 eye). The morphology of the corneas was examined by light and electron microscopy. RESULTS Epithelial hyperplasia, abnormal epithelial attachment, and disorganized stromal matrices were observed. Evidence of residual disease frequently observed in these specimens indicated that the pathology either was not excised at the time of laser keratectomy or was recurrent. CONCLUSIONS The response of the diseased cornea to excimer laser treatment has similar characteristics to the responses previously observed in animal studies. Incomplete ablation of diseased tissue and/or recurrence of the initial disease was the major reason for failure of the treatment. Possible causes for the inability to remove diseased tissues and superficial scars with the excimer laser include (1) insufficiently achieved ablation depth and/or diameter and (2) decreased laser ablation rates of scarred cornea.

In vitro model for corneal wound healing; organ-cultured human corneas

Healing of linear, non-perforating thermal burns was studied in 56 human corneas in an air/liquid organ culture system in serum free medium or in media supplemented with 10% fetal bovine serum, 10% human serum or 10% human plasma. The extent of epithelial wound healing was determined by measuring epithelial growth into the wound using digitized computer scanning of light micrographs of 1 micron sections. The cross-sectional area of this epithelial growth entering the wound was significantly greater for corneas incubated with either human serum (16,350 +/- 12,088 microns 2/day; p < 0.0001) or human plasma (20,571 +/- 12,276 microns 2/day; p = 0.0004) than for those incubated in serum free (1,784 +/- 1,957 microns 2/day) medium. There was no significant difference between epithelial growth in the serum free and fetal bovine serum supplemented (3,779 +/- 2,580 microns 2/day) media or between that in human serum and human plasma supplemented media. The thickness of the epithelium adjacent to the wound was greater in corneas cultured in fetal bovine serum supplemented media than in corneas cultured in the presence of human serum. Similarly, the build-up of epithelium at the wound edge for corneas in either serum free or fetal bovine serum supplemented media was significantly greater than for either human serum or human plasma supplemented media. The percentage of basal epithelial nuclei which incorporated bromodeoxyuridine (BrdU) increased during the first three days of culture when it reached a plateau. Comparison of paired wounded and unwounded corneas showed that wounding stimulated an increase in DNA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

A modified trichrome stain for light microscopic examination of plastic-embedded corneal tissue.

Corneal histopathology is essential to understanding the mechanisms of corneal disease and wound healing. We have developed a light microscopic trichrome stain for 1-µm, aldehyde-fixed, osmicated, epon/resin-embedded corneal sections. The stain consists of azure II, methylene blue, and basic fuchsin. This combination of stains provides a fast and effective method to clearly differentiate corneal cells from their surrounding extracellular matrix, and chromatically separates various ECM materials within the cornea. Correlative examination by light and transmission electron microscopy (TEM) provides multilevel understanding of corneal morphology and ultrastructure. Tissues examined using this staining procedure may be subsequently examined by TEM for ultrastructural morphology. This stain has proved beneficial in our research of both cellular and extracellular constituents of the cornea.

Antiproteolytic Activities Found in Human Tears

Human tears were found to inhibit the thiol-dependent protease, papain. Inhibitory activity in normal tears was compared with that in patients with blepharitis, infectious and allergic conjunctivitis, and herpes simplex. Activities lower than normal were found in some patients with infectious conjunctivitis and blepharitis. Higher than normal activities were found in patients with herpes simplex and allergic conjunctivitis. Differences from normal values were found to be statistically significant in allergic conjunctivitis and blepharitis by analyses of sample medians, means, and geometric means. A function of this inhibitor in external ocular inflammations is suggested.

Keratocyte Cell Culture

Keratocytes, or cornea1 fibroblasts, are the primary cell type of the cornea1 stroma. They lie between and are oriented parallel to the orthogonally arranged collagen lamellae, forming a continuous interconnecting cellular network that has been hypothesized to transmit information throughout the cornea concerning the status of the tissue (1). Under normal conditions, the keratocyte in the adult cornea is a relatively quiescent cell. However, in the event of a cornea1 injury or trauma, the keratocytes near the injured area differentiate into active, synthesizing cells and rapidly replace damaged stromal matrix (3, 3).

Recombinant enkephalinase effectively inhibits substance P-induced miosis in the rabbit eye cup model

Enkephalinase (EC 3.4.24.11) is a naturally occurring, membrane-bound peptidase that degrades substance P in vivo and in vitro. Addition of this neutral endopeptidase to a rabbit eye cup model partially inhibits substance P-induced contraction of the iris sphincter muscle. Inactivation of substance P is reversed by thiorphan, a specific inhibitor of enkephalinase. These results show that enkephalinase degradation of substance P produces metabolites that are physiologically inactive in iris contraction. We also observed that atropine acts synergistically with enkephalinase to completely abolish substance P-induced iris contraction suggesting that the action of substance P on the iris contains an acetylcholine-stimulatory effect which is not lost by enkephalinase treatment.

Cornea1 Organ Culture

Long-term organ culture of human corneas offers a tool for conducting a variety of biochemical, histological, and wound-healing studies on human tissue in vitro. A requirement of any organ culture system is that cell differentiation and structural integrity of the tissue be maintained or even improved during culture The latter is particularly relevant in cornea1 organ culture, since available donor corneas are sometimes of poor quality These corneas are usually unsuitable for transplantation and are from older donors (>65 yr), from donors with known bacterial or viral infections at the time of death (e g., septicemia, hepatitis), or are rejected because of a low endothelial cell count, such as might occur following ocular surgery.