Immunofluorescence study of corneal wound healing after excimer laser anterior keratectomy in the monkey eye.

We performed anterior keratectomies on six monkey eyes, four by excimer laser large-area ablation at 193 nm and two by mechanical keratectomy. Immunofluorescence was used to study the wound healing response histopathologically. The distribution of fibrinogen, fibronectin, laminin, collagen types III, IV, and VI, and keratan sulfate was determined at postoperative intervals of 24 hours, 6 days, and 1 month. At 24 hours, fibrinogen and fibronectin coated the ablated surface, but corneal epithelial cells had not yet migrated over the wound. By 6 days and persisting at 1 month, an epithelial ingrowth of seven to 10 layers, mild stromal hypercellularity, and new collagen formation were present in the repair region. At 1 month, fibrinogen, fibronectin, laminin, and type III collagen were strongly detected in the repair region. Type VI collagen was present in both normal and healed corneal stroma at all intervals, and type IV collagen was present in Descemet's membrane only. Sulfated keratan sulfate was absent from the newly synthesized collagen stroma at all intervals. Slit-lamp photographs demonstrated corneal haze in the ablation zone in all cases at 24 hours, persisting for 1 month. The fluorescence patterns produced by excimer laser ablation and mechanical keratectomy were qualitatively identical.

Corneal ablation by nanosecond, picosecond, and femtosecond lasers at 532 and 625 nm.

We produced corneal excisions with nanosecond (ns)-, picosecond-, and femtosecond (fs)-pulsed lasers at visible wavelengths. The threshold energy for ablation was proportional to the square root of the pulse duration and varied from 2.5 microjoules (microJ) at 100 fs to 500 microJ at 8 ns. Excisions made with picosecond and femtosecond lasers was ultrastructurally superior to those made with nanosecond lasers and, at pulse energies near threshold, showed almost as little tissue damage as excisions made with excimer lasers at 193 nm. We conclude that ultrashort-pulsed lasers at visible and near-infrared wavelengths are a possible alternative to excimer lasers for corneal surgery and might have advantages over conventional ophthalmic neodymium-YAG lasers for some intraocular applications.

A new model of experimental choroidal neovascularization in the rat.

Choroidal neovascularization in rat eyes was induced by krypton laser photocoagulation. Lesions were studied weekly by ophthalmoscopy, fundus photography, and fluorescein angiography. Morphologic correlation was provided by serial sectioning of lesions for light and transmission electron microscopy. In addition, vascular casts were prepared for scanning electron microscopy. Choroidal neovascularization occurred in 25 (60%) of 42 lesions, as evidenced by growth of capillaries through breaks in Bruch's membrane. In addition, 24 (28%) of 86 lesions studied by fluorescein angiography demonstrated leakage. This study provides the most complete angiographic, histologic, and ultrastructural documentation of experimental choroidal neovascularization in the rat. This model may be useful for in vivo studies of choroidal angiogenesis and its modulation via drug therapy.

Infrared laser surgery of the cornea. Studies with a Raman-shifted neodymium:YAG laser at 2.80 and 2.92 micron.

Tissue absorption lengths for infrared radiation at 2.8 to 3.1 micron are very short due to strong absorption by water. Corneal ablation using pulsed lasers at these wavelengths can potentially produce incisions similar in quality to cuts produced by excimer lasers at 193 nm. The authors have used 8-ns pulses at 2.80 and 2.92 micron, generated by a Raman-shifted neodymium:YAG (Nd:YAG) laser, to make slit-like incisions in bovine and human corneas. At 2.8 micron, etch depth per pulse increases sigmoidally from 0.15 micron at 390 mJ/cm2 to 3.8 micron at 2200 mJ/cm2. No ablation occurs at fluences below 250 mJ/cm2. Light and transmission electron microscopy show smooth-walled incisions bordered by a thermally damaged region that varies in width from 1.5 micron at 600 mJ/cm2 to 10 micron at 2200 mJ/cm2. The small amount of tissue damage produced at low fluences suggests that infrared ablation may be useful in keratorefractive surgery.

Distribution of R-cognin and choline acetyltransferase in the ganglion cell layer of developing chick neural retina.

Retina cognin, a cell membrane glycoprotein which mediates cell-cell recognition and adhesion in vitro, is initially present throughout the retina and becomes confined to the ganglion cell layer at 14-15 days of embryogenesis. Within this layer it is found on membranes of virtually all ganglion and displaced amacrine cells, but not on membranes of retinal glial cells (Müller fibers) which traverse this layer. The distribution of cognin as determined by immunocytochemistry is described and compared with that of choline acetyltransferase. The significance of cognin as a possible address marker during development of neural retina is discussed.

Distribution of R-cognin in late embryonic and post-hatching chicken retina.

The histological distribution of R-cognin in chick retinas was determined from embryonic day 8 through 13 wk post-hatching by indirect immunofluorescence using polyclonal anticognin. On embryonic day 8, at a developmental stage without distinct retina layers, most of the cells within the tissue exhibited fluorescence. By embryonic day 12, when the strata of the retina are delineating and initial synapses are beginning to form, R-cognin fluorescence became concentrated in the nascent ganglion cell, and to a lesser extent, inner nuclear layers. By embryonic day 16 (extending through post-hatching day 26), the staining of R-cognin was specific for the ganglion cell and nerve fiber layers. Fluorescence was prominent on the cell somal membranes but also was present on the processes of these cells at the ganglion cell layer-inner plexiform layer interface. Pre-immune serum and anticognin after preabsorption with R-cognin exhibited no fluorescence. The results demonstrated that the known decrease in R-cognin found in the retina during the latter half of embryonic development in the chick is not uniform across the retina, but that R-cognin is preferentially retained on cells within the ganglion cell layer. While cells within the ganglion cell layer also exhibit alpha-bungarotoxin binding, the majority of the latter is found in the inner plexiform layer. Thus, the observations are consistent with a role for R-cognin in the formation or maintenance of functional cell-cell connections within the entire retina prior to developmental day 11, or in retinal ganglion cell layer formation or stability subsequent to embryonic day 11.

Prostaglandin binding activity and myoblast fusion in aggregates of avian myoblasts.

Myoblast aggregates provide a system for studying cell interactions which have several advantages over standard, stationary cultures. In gyrotory rotation, aggregate size can be controlled and is independent of cell migration. In muscle aggregates, fibroblasts are excluded, yet myoblast differentiation and fusion occur in a highly synchronous fashion. Specific PG binding occurs in chick or quail myoblast aggregates: in chick the peak of binding is at 35-36 hr. Aggregation is complete 16 hr before PG binding activity appears. This suggests either that gyrotory aggregation is not identical to myoblast recognition, or that PG binding activity occurs subsequent to myoblast recognition. Myoblast aggregates begin to release PG before 18 hr. The amount detected remains constant until binding begins at 34 hr when PG binding to the aggregates begins. Thus, both the release of PG and PG receptor activity are characteristics of the myoblasts and release of prostaglandin precedes appearance of the binding activity. As a first step in identifying the PG receptor and determining its appearance on the myoblast cell surface, we have prepared antisera against myoblast surfaces which blocks receptor-ligand interaction and have absorbed it against both peripheral and intrinsic membrane fractions. The results indicate that the PG receptor is a myoblast peripheral membrane macromolecule.

Cognin distribution during differentiation of embryonic chick retinal cells in vitro.

Differentiation of individual retina neurons is closely linked to development of retina function. This differentiation may be intrinsic to the cell or determined by the position of the cell within the developing tissue. Retina cognin, a cell-cell recognition protein, which may itself mediate position-dependent cell interactions in vivo exhibits a characteristic change in distribution during embryonic chick development. Cognin is progressively lost from the outer retina in a manner which appears position-dependent. We asked if this change in cognin distribution was actually position-dependent or intrinsic to the retina cells. Neural retina cells from 8-day-old chick embryos were cultured in vitro. Continued differentiation of the cultured cells was demonstrated by neurite outgrowth and characteristic increases in choline acetyltransferase and glutamic acid decarboxylase activity. In such cultures, the characteristic developmentally related disappearance of retina cognin occurred as in vivo. This indicated that this aspect of retina neuronal differentiation was independent of position within the tissue and likely intrinsic to individual cells after 8 days of embryonic development.