Robert S. Baird

Number of Inflammatory Cells in the Normal Conjunctiva

We counted inflammatory cells per cubic millimeter in both the epithelium and the substantia propria of samples from upper tarsal conjunctiva of 15 normal subjects and from lower forniceal conjunctiva of ten normal subjects. The upper limit of normal for number of cells was nearly 500,000/mm3. Lymphocytes accounted for about 70% of the inflammatory cells. Neutrophils and lymphocytes were almost always present in both the epithelium and the substantia propria. Plasma cells and mast cells were present in the substantia propria of all subjects, but never in the epithelium. Neither eosinophils nor basophils were found in any specimen. We concluded that conjunctiva of the white and quiet eye usually contains heavy infiltrates of inflammatory cells.

Vernal Conjunctivitis and Contact Lens-Associated Giant Papillary Conjunctivitis Compared and Contrasted

We compared quantitative histologic counts of ten subjects with vernal conjunctivitis to counts of 15 subjects with contact lens-associated giant papillary conjunctivitis and to counts of 15 normal subjects. Both vernal conjunctivitis and contact lens-associated giant papillary conjunctivitis subjects had abnormalities of mast cells in the epithelium, and eosinophils and basophils in epithelium and substantia propria. No normal individuals had these abnormalities. An additional 28 subjects with ocular inflammatory conditions had tissue evaluation for abnormalities of mast cells, eosinophils, and basophils. A few eosinophils were found in four subjects. The histologic abnormalities of vernal conjunctivitis are shared by contact lens-associated giant papillary conjunctivitis but not by conjunctival inflammation in general. Vernal conjunctivitis and contact lens-associated giant papillary conjunctivitis may represent different subtypes of a general category of conjunctival abnormality characterized by giant papillae.

Tear Histamine Levels in Vernal Conjunctivitis and Other Ocular Inflammations

Histamine levels were measured in tears from normal volunteers (geometric mean, 5 ng/ml), patients with vernal conjunctivitis (16 ng/ml), and patients with a wide variety of external ocular inflammatory disease (3 ng/ml). Histamine levels were significantly higher in patients with vernal conjunctivitis than in normal subjects (P < 0.01) or patients with other types of ocular inflammation (P < 0.001). We postulate that histamine is an important mediator in the expression of the inflammatory response in vernal conjunctivitis, and that its elevation may be caused by abnormalities in the number of mast cells, the stability of the mast cells or in the system that regulates histamine.

Ocular anaphylaxis induced in the rat by topical application of compound 48/80. Dose response and time course study.

Abstract In the present study we sought to develop a model of ocular anaphylaxis based on the topical application of compound 48/80 to the surface of the rat eye. Doses ranging from 50 to 1000 μg were found to produce graded edema of the conjunctiva and swelling of the lid. On histologic examination, 50 μm compound 48/80 produced no changes distinguishable from those in PBS‐treated controls, 150 μm produced mild alterations, and 250, 500, and 1000 μg compound 48/80 produced a marked increase in degranulated mast cells and a mild influx of neutrophils. The time course of the response to 250 μg and 1000 μg of compound 48/80 was evaluated over a 72‐h period. Both doses elicited epithelial damage. A mild reduction in the number of mast cell was seen at 6 h in rats receiving 250 or 1000 μg. The reduction persisted to 72 h in rats receiving 1000 μg. The number of neutrophils was increased at 1 and 6 h in eyes treated with 250 (ig and at 1,6, and 24 h in eyes treated with 1000 [ig compound 48/80. The clinical and histologic changes induced by application of 250 jig compound 48/80 resemble those seen in patients with allergic conjunctivitis suggesting that a model of ocular anaphylaxis based on the topical application compound 48/80 will be clinically relevant and experimentally practical.

Effects of Eye Rubbing on the Conjunctiva as a Model of Ocular Inflammation

We assessed the effects of eye rubbing on the histologic characteristics and inflammatory cell infiltrate of the conjunctiva. The upper eyelids of 20 adult rats were rubbed during a five-minute period, and then the animals were killed immediately, or at four, eight, or 24 hours after trauma. One eye of each animal was rubbed; the unrubbed contralateral eye served as a control. Counts of mast cells, degranulated mast cells, and inflammatory cells (neutrophils, eosinophils, lymphocytes, plasma cells, and macrophages) were recorded from conjunctival samples from the upper eyelid. Immediately after eye rubbing the conjunctival epithelium was histologically disrupted and 50% of the mast cells showed evidence of degranulation. At four hours after trauma the increase in the number of neutrophils was more than 2,300%. Neutrophils were in the margins in the conjunctival vessels, had migrated into the substantia propria, and were aligned subjacent to the epithelial basement membrane; large numbers of neutrophils populated the epithelium. The four-hour stage was the most dramatic phase of inflammation that occurred from the eye rubbing. At 24 hours there was a significant increase in the number of macrophages. The numbers of lymphocytes, plasma cells, and eosinophils were not significantly changed throughout the study. Our findings demonstrate that eye rubbing histologically disrupts the epithelium and induces significant alteration in the inflammatory cell infiltrate of the conjunctiva. These changes may influence the course of ocular disease.

Late-phase reactions in ocular anaphylaxis in the rat

We assessed whether anaphylactic stimulation of rat ocular tissue produces a late-phase reaction at the histologic level. Clinical changes of swelling and redness started within minutes, peaked at about 20 min, and then subsided. Neutrophils increased at 1/2 hr after stimulation, peaked at 6 hr, and subsided to normal at 24 hr. Eosinophils reached a significant increase at 6 hr. Compaction of vessel contents was present at 1/2 hr; vessels were normal thereafter. Extravasated red blood cells and debris in the tissue were prominent at 6 hr. Macrophages had accumulated significantly at 6 hr and maintained that level at 24 hr. Any effect of anaphylaxis on macrophage accumulation was masked because control-injected ocular tissue also showed an accumulation of macrophages at 24 hr. Our results demonstrate that in ocular tissue, as in skin, the early acute phase of immediate hypersensitivity is but one stage of a multiphasic reaction.

Percentage of Degranulated Mast Cells in Vernal Conjunctivitis and Giant Papillary Conjunctivitis Associated with Contact-Lens Wear

Using light microscopy, we determined the percentage of granulated and degranulated mast cells in sections of tissue from ten persons with vernal conjunctivitis, ten with giant papillary conjunctivitis associated with contact-lens wear, and ten normal subjects. Tissues from both groups of patients had a significantly higher percentage of degranulated mast cells (greater than 80%) than did normal tissue (less than 25%). The fully granulated mast cells in the three groups did not appear morphologically different, nor did the degranulated mast cells in the three groups. The percentage of degranulated mast cells in vernal conjunctivitis did not differ significantly from that in giant papillary conjunctivitis associated with contact-lens wear. The histamine level in the tears of patients with vernal conjunctivitis, which is four times higher than that of normal subjects and that of patients with giant papillary conjunctivitis associated with contact-lens wear, cannot be explained by a difference in the percentage of degranulated cells detectable by light microscopy.

Technical Aspects of Histamine Determination in Human Tears

We evaluated various technical aspects of tear collection to assess their influence on the measurement of tear histamine levels. We included several methods of collecting tears, storage of tears with and without freezing, storage for three vs ten days, and removal vs nonremoval of cells before freezing. We assayed tear samples in various groups of people with inflamed eyes. None of the various means of collecting or storing the tear samples influenced the histamine measured. The measuring technique is a stable one that requires minimal treatment of samples before assay. To date, we have found that only patients with vernal conjunctivitis have elevated tear histamine levels. Any increase of tear histamine over 10 ng/ml (geometric mean plus 2 S.D.) or any significant decrease is probably related to the disease itself and not to factors related to the technique used to measure tear histamine.

Worm antigen-induced ocular anaphylaxis in rats infected with Nippostrongylus brasiliensis

A model of topically induced conjunctival anaphylaxis has been developed. Male Sprague-Dawley rats were immunized by infection with 3000 Nippostrongylus brasiliensis larvae and challenged topically on the eye 4 weeks later. Application of worm antigen alone did not induce clinical (conjunctival edema) or histologic (mast-cell degranulation) signs of anaphylaxis. Topical challenge with antigen 15 min after topical application of dithiothreitol (DTT), a mucolytic agent, elicited conjunctival edema and mast-cell degranulation within the first hour after challenge. At 6 and 24 hr, no clinical change was evident and conjunctival mast cells had again become granulated. At none of the three intervals (1, 6 and 24 hr) was there a significant increase in neutrophils, lymphocytes, eosinophils or macrophages in tissues from DTT-pretreated, antigen-eyes. The present model of ocular anaphylaxis resembles the ocular component of human hay fever in that sensitization prior to challenge is essential, the antigen is presented topically to the ocular tissues, conjunctival edema is the clinical manifestation and mast cell degranulation characterizes the histologic changes.

Effect of anaphylaxis on conjunctival goblet cells

Rats undergoing ocular anaphylaxis induced by systemic or local injection of antigen, topical application of antigen, or topical application of compound 48/80 were evaluated conjunctival goblet cell changes that might be related to anaphylaxis. The number of goblet cells in 1 micron, alkaline Giemsa-stained sections averaged 500/mm2 of epithelium in normal rats; this number was not significantly changed in any of the experimental groups. Goblet cells in control rats occasionally demonstrated evacuation of their contents (less than 1%) or upward displacement of the intracellular bolus of mucus (about 1%); these percentages were not increased in anaphylaxis. Topical application of 2.0 micrograms of histamine induced an intracellular displacement of mucus in both control animals and animals undergoing anaphylaxis. These findings suggest that in ocular anaphylaxis the amount of histamine released may be insufficient to produce such intracellular changes. Our results indicate that in ocular anaphylaxis in the rat, there is no light microscopic evidence of increased mucus discharge from conjunctival goblet cells. Increased mucus in secretions of patients with ocular allergic syndromes may not be attributable to anaphylactic mechanisms alone.