Diane L. Hatchell

Photocrosslinkable polysaccharides for in situ hydrogel formation.

In situ photopolymerization is an exciting new technique for tissue engineering. Two photocrosslinkable polysaccharides composed of alginate and hyaluronan are described that upon photolysis form soft, flexible, and viscoelastic hydrogels. The degree of methacrylate modification and thus covalent affects mechanical properties such as swelling, compression, and creep compliance. Significant swelling is observed in aqueous solution; these hydrogels can swell up to 14 times their dry weight. Both hydrogels exhibit low phase angles and (G*) values indicative of viscoelastic materials. The hyaluronan based hydrogel is stronger and more resilient than the corresponding alginate gel. SEM and AFM studies on both hydrogels show smooth and uniform surfaces at the macroscopic level with salient features observed only on the nanometer scale. Rapid polymerization by an optical trigger allows for controlled in situ photopolymerization in a minimally invasive manner, indicating that these hydrogels are relevant for biomedical applications such as sealing wounds and reconstructing soft tissues.

Panretinal Photocoagulation and Retinal Oxygenation in Normal and Diabetic Cats

We measured preretinal oxygen tension over laser-treated and intact retina in normal and diabetic cats. The oxygen tension was significantly higher over the area given panretinal photocoagulation than over the intact retina when the cats breathed 100% oxygen and the same trend was seen when the cats breathed 21% oxygen. There was no difference in preretinal oxygen tension between the normal and diabetic retinas. Light and electron microscopy showed laser damage to the mitochondria-rich photoreceptors in the outer retina as well as the retinal pigment epithelium. The inner retina showed no photocoagulation damage. Because panretinal photocoagulation destroys parts of the outer retina, thus reducing its oxygen consumption and allowing oxygen to diffuse from the choroid into the inner retina, it improves the oxygen supply to the inner retina.

The effects of intravitreal triamcinolone acetonide on experimental pre-retinal neovascularization

Corticosteroids, alone or in combination with other drugs, have been shown to inhibit angiogenesis. The purpose of this study was to evaluate the efficacy of triamcinolone acetonide in a new model of preretinal neovascularization. Rabbit eyes were treated with intravitreal triamcinolone acetonide 24 h before partial liquefaction of the posterior vitreous with hyaluronidase and injection of 250000 homologous tissue-cultured dermal fibroblasts. Triamcinolone acetonide effectively inhibited new vessel growth in treated eyes. Only 14% of the treated eyes developed new blood vessels compared to 100% of sham-injected control eyes (P<0.001). These results suggest that intravitreal triamcinolone acetonide might be effective in inhibiting new vessel growth in patients with inflammatory retinal neovascularization, such as that associated with sarcoidosis or other uveitic syndromes.

Daunorubicin treatment in a refined experimental model of proliferative vitreoretinopathy.

A condition similar to proliferative vitreoretinopathy (PVR) in man can be produced by injecting 25000 homologous dermal fibroblasts into rabbit eyes following gas compression of the vitreous. Daunorubicin (15 nmol) was effective in preventing retinal detachment in this model when injected simultaneously with the fibroblasts or in two doses (10 nmol followed by 5 nmol 4 h later) on the 3rd day after fibroblast injection. A single dose of 15 nmol on the 3rd day was not effective in preventing retinal detachment. These results suggest that daunorubicin may be clinically useful in preventing PVR when given by injection both at the time of vitrectomy as well as later, when protein exudation and pigment clumps in the vitreous cavity herald the onset of PVR.

Enhanced superoxide radical production by stimulated polymorphonuclear leukocytes in a cat model of diabetes

This study examines the possibility that polymorphonuclear leukocyte activation, which can cause endothelial injury, may contribute to the capillary closure of diabetic retinopathy. To examine diabetes-related alterations in polymorphonuclear leukocyte activation, we compared the production of superoxide radical by these cells from normal and from diabetic cats that were maintained hyperglycemic. Polymorphonuclear leukocytes isolated from five diabetic and five normal cats were stimulated with 10 ng ml-1 phorbol myristate acetate, and the maximum rate of their superoxide radical production was measured spectrophotometrically. Stimulated polymorphonuclear leukocytes from diabetic cats generated more superoxide radical, at significantly higher rates, than did those from normals (3.32 +/- 0.33 and 2.50 +/- 0.41 nmol O2- min-1 10(-6) cells, respectively; P < 0.02). While addition of insulin or glucagon did not alter stimulated polymorphonuclear leukocyte radical production, glucose in high concentration did mildly impair its production in both groups. The exaggerated respiratory burst of polymorphonuclear leukocytes in diabetes could contribute to microvascular injury in the retina as well as in other tissues.

A photopolymerized sealant for corneal lacerations.

Purpose. To determine whether a novel photocrosslinkable polymer synthesized from hyaluronic acid would seal experimental full-thickness corneal lacerations in a rabbit model. Methods. A solution of hyaluronic acid was modified with methacrylate groups (HA-MA), precipitated, dried, reconstituted in an aqueous solution, and sterilized before use. The viscous polymer solution was applied to 38 of 43 experimental corneal lacerations in rabbits and subsequently irradiated with a low-intensity argon laser beam to produce a clear flexible polysaccharide hydrogel patch. The ability of this sealant to repair corneal lacerations was evaluated in four types of full-thickness, 3-mm corneal wounds (linear, linear + epithelium removed, stellate, and stellate + epithelium removed). Slit-lamp examinations, measurements of intraocular pressure, Seidel tests, and histologic studies were performed at selected intervals to evaluate the wound and determine the rate of healing. Results. Corneal perforations were completely sealed and the anterior chambers had reformed by 6 hours in HA-MA–treated eyes. There was no evidence of leakage at this or later times in 37 of the 38 eyes. Intraocular pressure had risen to near-normal levels by day 7 in all four groups, and the sealant was still present in most eyes at day 7. In contrast, the anterior chambers did not re-form in control eyes (five) with untreated perforations because of aqueous leakage through the wounds. Minimal inflammation was observed clinically or in histologic sections of treated corneas. There was extensive proliferation of stromal cells and formation of new extracellular matrix at the wound edges, which became tightly adherent between days 4 and 7. Conclusion. Our novel photocrosslinkable methacrylated hyaluronan polymer sealed 97% (37/38) of the experimental corneal lacerations. HA-MA may prove useful for sealing corneal lacerations in patients and for other sutureless ophthalmic surgical procedures.

Histopathology of Silicone Oil Keratopathy in Humans

&NA; Corneal endothelial decompensation is a frequent complication when silicone oil is used as a tamponade following vitrectomy for treatment of proliferative vitreoretinopathy in an aphakic eye. We evaluated the clinical, histopathological, and ultrastructural features of silicone oil‐induced keratopathy in 10 patients who developed corneal complications requiring penetrating keratoplasty. Clinically, some cases showed corneal edema, corneal hypesthesia, endothelial opacification, band keratopathy, and peripheral corneal vascularization. Histopathologically, retrocorneal membranes were present, and different degrees of stromal hypercellularity, superficial stromal calcification, and vascularization were noted. The ultrastructural features of endothelial cell loss and retrocorneal membrane formation are consistent with changes previously reported in rabbits and cats receiving intracameral silicone oil injections.

The effect of photocoagulation on the oxygenation and ultrastructure of avascular retina

Panretinal photocoagulation has been shown to raise preretinal oxygen tension in cats and monkeys breathing 100% oxygen. However, in normoxia, there is no significant difference between normal and photocoagulated areas. This is thought to be due to autoregulatory vasoconstriction of the retinal circulation. In avascular rabbit retina, photocoagulation effects on retinal oxygenation can be studied without the influence of retinal vascular autoregulation. We measured normoxic preretinal oxygen tension of normal and photocoagulated rabbit retina at 1, 7, 14 and 28 days after photocoagulation using polarographic oxygen electrodes. For all time points, preretinal oxygen tension of photocoagulated retina (42 +/- 14 torr; mean +/- S.D.) was higher than untreated retina (14 +/- 10 torr; mean +/- S.D., P less than 0.001). Light and electron microscopy of lasered retina showed that the improved oxygenation corresponded to loss of mitochondria-rich inner segments of the photoreceptors.

Electron immunocytochemical analysis of posterior hyaloid associated with diabetic macular edema.

Background: Tangential traction in the macula from a thickened posterior hyaloid of the vitreous has been implicated as a cause of diffuse diabetic macular edema. Vitrectomy with peeling of the posterior hyaloid has been shown to reduce retinovascular leakage and improve vision in select patients. We report a clinicopathologic correlation using electron microscopy and electron immunocytochemistry to characterize the membrane infiltrating the posterior hyaloid in two such patients. Methods: Two patients presented with vision loss associated with diffuse diabetic macular edema and an attached, thickened, and taut posterior hyaloid. The patients underwent vitrectomy with peeling of the posterior hyaloid. The premacular posterior hyaloid specimens then were analyzed by electron microscopy with immunocytochemical staining for cytokeratin and glial fibrillary acidic protein. Results: Both posterior hyaloid specimens contained collagen and a large cellular component. Immunogold labeling showed cells positive for glial fibrillary acidic protein or cytokeratin. With double labeling, no cells expressed both proteins simultaneously. Clini‐cally, both patients had vision improvement and macular edema resolution after surgery. Conclusions: The thickened, taut posterior hyaloid observed in our patients with dia‐betic macular edema contained cells of glial and epithelial origin. This cellular infiltration may contribute to abnormal vitreomacular adherence and could play a role in the patho‐genesis of macular edema in some patients with diabetes.

Effect of pentoxifylline on the flow of polymorphonuclear leukocytes through a model capillary.

Pentoxifylline is a methylxanthine derivative used to increase blood flow in peripheral atherosclerosis. Pentoxifylline is known to increase whole blood filtration rate, and recent evidence suggests that pentoxifylline increases the filtration rate of polymorphonuclear leukocytes (PMNs). The purpose of this study was to directly observe and quantitate the effect of pentoxifylline on the flow of individual PMNs into a model capillary. Short-term incubation of human PMNs with 10 mM pentoxifylline inhibited cell activation, as judged by a significant reduction in the number of neutrophils forming pseudopods. Furthermore, incubation of PMNs from 6 healthy men with 0.1, 1.0 and 10 mM pentoxifylline significantly decreased the time required for individual cells to be aspirated into a 4 μm pipet under constant pressure by 16 ± 5%, 21 ± 7%, and 41 ± 8%, respectively (mean ± SEM, p≤0.05), compared with control. These experiments are the first direct demonstration of increased deformability in neutrophils treated with pentoxifylline. The results are consistent with the hypothesis that the beneficial effect of pentoxifylline on microvascular perfusion is partly due to an inhibition of PMN stiffness and activation.