Systemic amiloride inhibits experimentally induced neovascularization.

Amiloride is an inhibitor of urokinase-type plasminogen activator, and might therefore have an inhibitory effect on neovascularization. Neovascularization was induced in rabbit corneas via local implantation of prostaglandin E1 pellets prepared in a slow-release polymer. Animals received daily intraperitoneal injections of 30 mg of amiloride, or an equivalent volume of saline solution for 5 days; both were well tolerated without severe untoward effect. Neovascular response, as documented by corneal photographs, was evaluated after 5 days of injections. The area of induced corneal neovascularization was decreased by 55% in animals receiving amiloride when compared with controls. Thus, amiloride and similar compounds may prove useful in the study and management of neovascularization.

Reversal of conjunctival transdifferentiation by topical retinoic acid.

After resurfacing a total corneal epithelial defect extending 2-3 mm beyond the limbus, conjunctival epithelium gradually loses goblet cells and transforms into a corneal-like epithelium. We examined the effect of topical retinoic acid on the reversal of transdifferentiation on nonvascularized corneas. Four months after total denudation of corneal epithelium using n-heptanol, rabbit corneas without vascularization received topical drops of 0.1% (wt/vol) all-trans retinoic acid in corn oil 3 times a day. Before treatment, the transdifferentiation was complete, as evidenced by the absence of goblet cells on the corneal surface using a topographical assay and routine histology. After treatment for 15 days, goblet cells reappeared 3 mm into the peripheral cornea, and extended in a centripetal density to 4.5 mm after 32 days. To prove that retinoic acid was not angiogenic, retinoid-bearing Elvax-40 pellets were implanted into normal corneal stroma. Taken together, these data indicate that vitamin A or retinoids may be an important factor in the modulation of conjunctival epithelial transdifferentiation.

Conjunctival transdifferentiation induced by systemic vitamin A deficiency in vascularized rabbit corneas.

Conjunctival transdifferentiation, the process in which conjunctival epithelium transforms into a cornea-like epithelium with the loss of goblet cells during the healing of a total corneal epithelial defect, can be retarded or reversed by corneal neovascularization. We have previously shown that this process normally occurring on non-vascularized corneas can be retarded or reversed by topical retinoids, suggesting that vitamin A may be one of the factors from blood circulation which is responsible for modulating transdifferentiation. Herein, we have examined the effect of systemic vitamin A deficiency on vascularized corneas starting 4 months after epithelial denudation, and compared this deficient group with their vascularized and non-vascularized controls. Mean serum retinol level (microgram/dl) (n = 4) measured by HPLC was gradually reduced from 83 of the controls to 20 in a 10 month follow-up. Topographical analysis disclosed a centrifugal loss of goblet cell density with time. Histology showed complete transdifferentiation in vascularized areas at 9 months, initiated by the loss of mucin contents from receding zones first noted at 2 months. Using impression cytology, all corneas were not keratinized and all conjunctivas maintained a normal goblet cell density at 10 months. These results indicate that conjunctival epithelium on corneal surface is more sensitive to the decrease of serum vitamin A levels than that on conjunctiva, and support the hypothesis that the relative vitamin A deficiency on vascularized corneas can also result in the conjunctival transdifferentiation.

Inhibition of conjunctival transdifferentiation by topical retinoids.

During the healing of a total corneal epithelial defect extending beyond the limbus, conjunctival transdifferentiation can be inhibited by corneal vascularization as evidenced by the lack of morphological transformation of the conjunctival epithelium into a cornea-like epithelium and the persistence of goblet cells on the corneal surface. We speculated that corneal vascularization might play a causative role in inhibiting conjunctival transdifferentiation, and examined the hypothesis that vitamin A or retinoids might be one of the blood-borne factors in modulating this process. To test this hypothesis, we created total corneal epithelial defects extending 3 mm beyond the limbus in rabbits using n-heptanol, and segregated the resultant corneas into nonvascularized and vascularized groups. After re-epithelialization, both groups received topical 0.1% Etretinate (Roche-Hoffmann, Nutley, NJ) or 13-cis retinoic acid in corn oil three times a day for 8 weeks. Controls received corn oil only. The extent of transdifferentiation was analyzed by assaying goblet cell density and distribution using flat-mount preparations and Alcian blue and periodic acid-Schiff stains (Fischer Scientific Co., Fair Lawn, NJ) and by conventional histology. Topical retinoid application inhibited conjunctival transdifferentiation in nonvascularized corneas to the same extent as that caused by corneal vascularization, suggesting that vitamin A is an important blood-borne factor for goblet cell maintenance. Its relative deficiency in the normal avascular cornea may explain why conjunctival transdifferentiation occurs.

Immunohistochemical study of the local inflammatory response to chlamydial ocular infection.

Immunohistochemical staining of conjunctival biopsies from cynomolgus monkeys (Macaca fascicularis) was performed after they received a single primary ocular infection, a single secondary challenge infection, or repeated ocular inoculations with Chlamydia trachomatis. T cells of the suppressor/cytotoxic (OKT8F) phenotype predominated regardless of the infection protocol, and perifollicular T lymphocytes of both the suppressor/cytotoxic and helper (OKT4A) phenotypes appeared in large numbers during the peak inflammatory reaction. In repeatedly inoculated monkeys, T cells and follicles persisted until cessation of reinfection. IgM-bearing B lymphocytes comprised the majority of cells within follicles, with smaller numbers of IgG- or IgA-positive B cells. The major difference in the response to the various infection protocols was the increased number and persistence of follicles with repeated reinoculation. The finding of large numbers of T-suppressor/cytotoxic and T-helper cells in the infected conjunctiva supports a role for cell-mediated immunity in the local response to C. trachomatis ocular infection.

A role for T lymphocytes in preventing experimental herpes simplex virus type 1-induced retinitis.

We have previously shown that herpes simplex virus Type 1 (HSV-1) inoculated into the anterior chamber (AC) of one eye of BALB/c mice results in retinal destruction in the opposite eye while retinas in virus-injected eyes are preserved. In the present study, immunodeficient mice (athymic BALB/c or normal BALB/c which had received either gamma-irradiation [450 R] or cyclophosphamide [150 mg/kg treatment]), demonstrated bilateral retinal destruction upon unilateral AC inoculation of HSV-1. Reconstitution of these immunodeficient mice with spleen cells obtained from days 10-21 AC-inoculated donor mice, prior to AC inoculation of HSV-1, prevented retinal necrosis in more than 80% of both eyes. In contrast, donor cells from mice inoculated subcutaneously (SC) with HSV-1 preserved only about 30% of recipient retinas, regardless of the cell transfer time after donors received HSV-1. Normal unsensitized syngeneic donor spleen cells failed to prevent bilateral retinal necrosis in either athymic or irradiated BALB/c mice, although they eliminated recipient mortality. T cell depletion of AC- or SC-inoculated donor cells removed their retinal protective effects completely. These studies demonstrate an active role for T lymphocytes in controlling the extent of disease in a murine model of HSV-induced retinitis.

In vitro enhancement of anterior chamber GVH reactions.

The injection of sensitized allogeneic lymphocytes into the anterior chamber of the rabbit eye results in a local graft-verus-host reaction, with focal destruction of the corneal endothelium. This experimental model permits in vitro manipulation of effector cells, and the study of the mechanisms involved in corneal graft rejection. The authors now show that the in vitro activation of sensitized lymphocytes is a one-way mixed lymphocyte reaction yields "supersensitized" effector cells which are quantitatively enriched and qualitatively altered to yield more severe and more rapid endothelial target cell destruction.

Immunology of corneal allograft rejection: in vitro sensitization of effector cells.

Anterior chamber injection of donor rabbit lymphocytes sensitized in vitro to recipient alloantigens leads to the development of small focal areas of endothelial cell destruction (pocks) on the recipient cornea. Damage may be observed through a specular microscope as early as 2 days after injection of sensitized lymphocytes. Recipients of unsensitized allogeneic or sensitized autologous lymphocytes demonstrate little or no endothelial damage and no pock formation. Flat endothelial preparations reveal focal destruction of the endothelium with multiple foci, many infiltrated and surrounded by mononuclear cells. This model provides controlled sensitization to a variety of histocompatibility and corneal antigens that may be responsible for initiation of graft rejection.

Goblet cell density and vascularization during conjunctival transdifferentiation.

After debridement of the entire corneal epithelium with n-heptanol, two groups of rabbit corneas were segregated according to the extent of corneal neovascularization. Using a new topographic goblet-cell counting method and routine histology, the authors have reexamined the process of conjunctival transdifferentiation and compared the changes of goblet-cell density and morphology between nonvascularized and vascularized groups for a follow-up period of 167 days. Analysis of the total goblet-cell density disclosed that no goblet cells appeared on the corneal surface during the entire period of reepithelialization. After that, two phases were identified with respect to goblet-cell density: phase I (day 0-17) and phase II (after day 17). In phase I, both groups had a similar surge of goblet cells, with the peak occurring between days 7 and 11, suggesting little correlation with vascularization. Morphologic studies indicated the presence of a prominent centripetal cellular migration. In phase II, the nonvascularized group showed a rapid decline in goblet-cell density, and as a result the morphologic transdifferentiation into a cornea-like epithelium was completed on day 43. The changes of goblet cells to a smaller size and the presence of a more acidic mucin in the centrifugal receding zone, suggested that transdifferentiation on nonvascularized corneas is a process involving changes of cellular differentiation. In contrast, the vascularized group maintained a high plateau of goblet-cell density and an epithelium with conjunctival characteristics until day 167. This result disclosed that retardation of conjunctival transdifferentiation by corneal vascularization was in phase II. The possible role of vascularization in the modulation of conjunctival transdifferentiation is discussed.