A thin glycoprotein coating of a synthetic lenticule does not cause nutritional deficiency of the anterior cornea.

PURPOSE: This study investigated whether a glycoprotein coating that will be used to enhance corneal epithelialization affects in situ nutritional passage through a permeable membrane. METHODS: Sixteen adult cats were equally divided into two groups. Polycarbonate membranes with pore size of 0.1 microm and total pore area (porosity) of 3.1% were used as implant materials. The membranes for Group 1 were coated with a thin layer of Collagen I, while the membranes for Group 2 were uncoated. Each membrane with 8-mm diameter was implanted into an interlamellar pocket of the cornea. The eyes were observed for approximately 35 days to monitor clinical signs of nutritional deficiency of the cornea, and then 7 membranes were removed from the eyes. The permeability of the explanted membranes to glucose, inulin and albumin was used to predict the in situ difference between the coated and uncoated groups in regard to nutritional passage through the membranes. To investigate the long-term effect of the surface coating on corneal health, two animals from Group 1 were followed for up to two years and then both eyes of each animal underwent histological examination. RESULTS: Clinically, no post-surgical complications associated with nutritional deficiency were observed in any of the eyes. Nutritional permeability tests showed no significant differences between the coated and uncoated membranes. Histologically, the long-term animals showed no abnormal morphology associated with nutritional deficiency in the cornea anterior or posterior to the membranes. CONCLUSIONS: A thin glycoprotein coating on a permeable membrane does not appear to affect the nutritional supply of the anterior cornea and therefore can be used to enhance epithelialization of synthetic corneal onlays in vivo.

An ocular strain of Pseudomonas aeruginosa is inflammatory but not virulent in the scarified mouse model.

Pseudomonas aeruginosa is the most common pathogen among contact lens-associated infections. This study investigated the response of the murine cornea to infection with an ocular strain of P. aeruginosa isolated from a subject with an inflammatory adverse response to contact lens wear termed CLARE. Although this bacterium was isolated in confluency (greater than 2000 cfu lens-1) from the lens at the time of the inflammatory episode, no infection of the cornea subsequently developed. Male C57BL/6J mice (20 per strain) had their corneas scratched with a 26 gauge needle (3 parallel 1.0 mm wounds in the left eye only). The incisions were centered over the pupillary axis and penetrated the epithelial cell basal lamina and into the superficial stroma. The CLARE strain was found to persist (viable bacteria could be cultured from corneal homogenates) up to 8 hr, as did the virulent control strain ATCC 19660. At 24 hr, only ATCC 19660 could be cultured, indicating an inability of the strain isolated from CLARE, Paer1, to persist in the eye consistent with the human inflammatory episode. Histological examination of the mouse tissue showed further differences between infection by the two strains. Infection with ATCC 19660 resulted in tissue necrosis and a large population of polymorphonuclear leukocytes (PMNs) recruited to the wound site. In contrast, during infection with the CLARE strain, PMN recruitment was reduced and temporally delayed. The CLARE strain grew as well as ATCC 19660 in vitro but produced less protease activity, in particular less elastase. The decreased PMN response and decreased protease production by the CLARE strain may have been responsible for the lack of ocular damage and apparent healing of the wound. P. aeruginosa strains are considered to be invasive or cytotoxic to corneal tissue, however this strain may represent a third inflammatory type consistent with its differing pathology.

Persistent adhesion of corneal epithelial tissue on synthetic lenticules in vivo.

PURPOSE: In intact cornea, firm anchorage of the epithelium to its underlying stroma is provided by a system of adhesive structures, which include the epithelial basement membrane, adhesion complexes and associated alpha6beta4 integrin receptors. In the present study we sought to determine whether the sustained epithelialization of synthetic lenticules observed in vivo involved the formation of adhesive structures at the epithelial-lenticule interface similar to those present in intact tissue. METHODS: Collagen I-coated microporous polycarbonate membranes were implanted in the anterior stroma of adult cats. Successfully epithelialized lenticules were maintained on eye for 9-12 weeks, after which time the epithelial-lenticule interface was examined for evidence of adhesive structures using immunohistochemistry and electron microscopy. RESULTS: Immunohistochemistry identified laminin, hemidesmosomal plaque, collagen VII and alpha6 integrin at the tissue lenticule interface. Ultrastructural examination showed evidence of assembly of these components into a basement membrane and adhesion complexes. CONCLUSIONS: The formation of these adhesive structures is likely to have contributed to the sustained epithelialization observed clinically on the collagen-coated synthetic lenticules.

Different strains of Pseudomonas aeruginosa isolated from ocular infections or inflammation display distinct corneal pathologies in an animal model.

PURPOSE: The present investigation sought to define the responses of mouse eyes to challenge with three different strains of P. aeruginosa isolated from human corneas or contact lenses: two different strains produced an ulcerative keratitis, and one strain produced contact lens-induced acute red eye (CLARE). METHODS: The corneas of BALB/c mice were inoculated with three different strains of P aeruginosa. The strains were allowed to interact with the corneas for up to 24 h. In addition, strain Paerl, isolated from CLARE, was subjected to in vitro assays to measure its ability to invade corneal epithelial cells, or to produce cytotoxicity in these cells. Both these assays used cultured rabbit corneal epithelial cells. RESULTS: Both MK isolates were able to infect the corneas of mice, but the CLARE isolate was non-infective. The predominant response to infection with the cytotoxic strain was severe corneal edema and infiltration of the corneal stroma with polymorphonuclear leukocytes (PMNs). The predominant response with the invasive MK isolate was corneal ulceration and infiltration with PMNs. The CLARE strain produced only low levels of PMN infiltration. In in vitro assays the CLARE strain was non-invasive and non-cytotoxic. CONCLUSIONS: This study has identified that P. aeruginosa produces at least three different types of corneal pathology and that not all strains are able to infect mouse corneas.

Interactions of living astrocytes in vitro: evidence of the development of contact spacing.

We have studied the behaviour of living, process-bearing astrocytes in vitro, observing groups of cells at daily intervals for up to 7 days. Each cell initially formed two processes, appearing bipolar in shape, and with further time in culture, grew additional processes and appeared stellate. As their processes grew, the interactions between astrocytes underwent characteristic changes. While bipolar, the cells appeared to avoid making contact, lying parallel to each other. As they became stellate, the astrocytes made extensive contact with neighbours, gradually forming extended, contacting networks in which their somas were regularly spaced (as previously described). The interactions which led to the establishing of such arrays were also evident. If two cells were initially close or adjacent, they extended short processes to contact each other; then, as their processes grew, their somas moved apart, until they were separated by 60-120 microns. If two cells were initially well separated, each directed processes towards the other until contact was made, often with striking precision, and their somas then moved together, until they were separated by 60-120 microns. These behaviours of contact, separation, and approach caused astrocytes to form clusters, within which their somas appeared regularly spaced, and may represent the interactions which occur among astrocytes during normal development to produce the regularly spaced arrays of astrocytes described in earlier studies of intact central nervous tissue.

The role of Müller cells in the formation of the blood-retinal barrier.

We have compared the ability of Müller cells and astrocytes to induce the formation of barrier properties in blood vessels. Müller cells cultured from the rabbit retina, and astrocytes and meningeal cells cultured from the rat cerebral cortex, were injected into the anterior chamber of the rat eye, where they formed aggregates on the iris. We have examined the barrier properties of the vessels in those aggregates and, for comparison, the barrier properties of vessels in the retina, ciliary processes and iris. Two tracers were perfused intravascularly to test barrier properties. The movement of Evans Blue was assessed by light microscopy, and the movement of horseradish peroxidase by light and electron microscopy. Our results indicate that Müller cells share the ability of astrocytes to induce the formation of barrier properties by vascular endothelial cells, and we suggest that Müller cells play a major role in the formation of barrier properties in retinal vessels.

Contact-spacing among astrocytes is independent of neighbouring structures: in vivo and in vitro evidence.

We have examined the morphology of astrocytes and the arrays they form in two situations, in retinas from which ganglion cells and blood vessels have been caused to degenerate, and in vitro. These observations were made to test whether the regularity of the spacing of astrocytes within normal central nervous tissue results from interaction among astrocytes, or from interaction between astrocytes and other elements of that tissue. Both in the partially degenerated cat retina, and in cultures of astrocytes from neonatal rat cortex, astrocytes make and maintain contact with neighbouring astrocytes, yet space their somas apart, giving regularity to the arrays. These results support the hypothesis that the regularity observed in arrays of astrocytes in intact tissue results from an interaction among astrocytes, independent of neighbouring structures, and lead us to suggest that the cell-cell interactions involved in contact spacing serve to distribute astrocytes through the central nervous system, and may, in other tissues, underlie the formation of epithelia.

Vascular changes and their mechanisms in the feline model of retinopathy of prematurity.

This study documents changes to retinal vasculature during the feline form of retinopathy of prematurity (ROP). The authors describe the closure and obliteration of retinal vessels during exposure to high oxygen, the pattern and tempo of growth of proliferative vasculature, which, after the return of the animal to room air, extends from the optic disc in a spectacular "rosette" pattern, the formation of preretinal vascular growths, and an initial lack of barrier properties in the new vessels. Finally, the response of the vasculature to the relief of hypoxia is reported, including the gradual establishment of barrier properties in the intraretinal vessels, the partial normalization of the proliferative vessels, and the abnormalities that persist. It is suggested that the vascular changes occur in successive stages: closure and obliteration during hyperoxia, vasoproliferation induced by hypoxia, and normalization after the relief of hypoxia with distinct cellular mechanisms and stimuli. It is argued that the same stages can be seen in the human form of ROP; two possible stimuli for the fibroplasia that damages the retina in human ROP are discussed.

The development of astrocytes in the albino rabbit retina and their relationship to retinal vasculature.

We have examined the development of astrocytes in the albino rabbit retina, using antibodies to glial fibrillary acidic protein (GFAP) and vimentin. Vimentin immunoreactive (vimentin+) astrocyte-like cells first appear at the 24th postconceptional day (24 PCD), in a pattern similar to that of the adult. GFAP immunoreactivity was first detected in astrocytes at the 29 PCD, in a similar pattern. Vessels enter the retina from 29 PCD. The presence of astrocytes in a mature distribution prior to the ingrowth of vessels indicates that astrocytes are not dependent on the vessels for their early positioning and differentiation. In contrast with the rat and cat, we found no evidence of migration of astrocytes into the rabbit retina from the optic nerve.

Motoneurone survival and neuritic outgrowth promoted by different cell types in embryonic muscle.

Different cell types within developing chick skeletal muscle were assayed for their ability to release factors into culture media which could affect the survival and neuritic development of labelled motoneurones and lateral motor column explants. Enriched cultures of myotubes, myoblasts, fibroblasts and mesenchyme were prepared by selective preplating and trypsinisation techniques. Degrees of enrichment were assessed immunofluorescently and morphologically; fibroblasts were the main contaminating cell type. Medium conditioned over each cell type was then tested in dose-response assay against both explants and dissociated motoneurones. In both cases the myotube conditioned medium (MCM) promoted the greatest levels of both survival and neuritic outgrowth, and had the greatest relative potency of all of the cell types. When MCM was preincubated over polycationic substrata, it lost the ability to promote neuritic growth; this could be restored if fresh conditioned medium (CM) was added to the cultures. Thus it was demonstrated that within the MCM there are physically separable agents responsible for neurone survival and neurite expression. The neurite-promoting factor (NPF) within the MCM was stable to collagenase, deoxyribonuclease, neuraminidase and chondroitinase ABC, but was destroyed by trypsin and heparinase. These results imply that a heparan sulfate proteoglycan is essential for the activity of the factor.

Formulation and bioavailability of directly compressed oxytetracycline hydrochloride tablets.

Different tablet formulations containing various vehicles were selected for this study. These tablets were prepared using different proportions of either single or binary vehicles, and were found to possess different disintegration times. A significant correlation was obtained between the dissolution time (T50) and the urinary excretion data as well as their disintegration time. The dissolution rate of the produced tablets therefore can affect their physiological availability which in turn was dependent upon the type of vehicle used and its actual concentration in the formula. Other factors such as the type and concentration of the lubricant(s), hardness values of the tablets prepared, solubility of the drug as well as interactions between the drug and vehicles used may also play a part.