Intravitreal injection of fibroblasts: the pathological effects on the ocular tissues of the rabbit following an intravitreal injection of autologous skin fibroblasts.

The intravitreal injection of autologous cultured fibroblasts has been used by many groups to study proliferative vitreoretinopathy. Ninety-five New Zealand white rabbits were used to study the pathological effects on the ocular tissues following such an injection over various time periods up to six months. The ocular tissues were studied by light microscopy, electron microscopy, immunohistochemistry, and autoradiography. The cells which contributed to the inflammatory response (initially neutrophils, then later macrophages and lymphocytes) were found to gain entry into the vitreous via the pars plana, pars plicata, and the vessels associated with the optic nerve head. In the experimental eyes the detached retinae had a reduced ability to incorporate 3H proline. Both epiretinal and subretinal membranes were found on the retinal surfaces. The majority of the glial cells within the membranes were identified as Müller cells. The retinal pigment epithelium beneath the detached retinae incorporated 3H thymidine and detached into the subretinal space. Clear evidence was obtained of both epithelial cell migration through the retina and involvement within epiretinal membranes.

Shape changes produced in detergent extracted bovine retinal pigment epithelium when exposed to ATP. A comparison with fibroblasts and smooth muscle cells.

The response of single detergent treated bovine retinal pigment epithelial cells in culture to ATP was measured with an image analyser. The most pronounced contraction was produced by 1.0 mM ATP with most change taking place in the first 10 min. At 1 h the area had decreased by about 33%, perimeter 22% and maximum length 25%. By way of comparison rabbit skin fibroblasts had a decreased area of approximately 40%, perimeter 25% and maximum length 22%. Bovine aortic smooth muscle cells on the other hand decreased in area by 55%, perimeter 40% and maximum length 36%. It is hoped that this assay may be used to evaluate drugs which could counteract contractile events in proliferative vitreoretinopathy.

Contraction of scar tissue in the rabbit vitreous.

Sheets of vitreous membrane (scar tissue) and associated retinal detachment were produced in the right eye of 86 adult New Zealand white rabbits by intravitreal injection of cultured autologous skin fibroblasts. The membranes were examined by light and electron microscopy and time-lapse cinephotomicrography. Immunohistochemistry was used to demonstrate alterations in the distribution of cytoplasmic contractile proteins. While retinal detachment and membrane contraction were taking place, there was pronounced increase in the numbers of fibroblasts with an elongated spindle shape. These spindle-shaped cells had some similarities to myofibroblasts including the presence of 'stress cables'. However, the myofibroblast-like cells stained much less avidly for cytoplasmic (actin) microfilaments than migratory fibroblasts seen at early stages of membrane development. The significance of migrating fibroblasts and myofibroblasts in scar contraction is discussed.

Experimental scar membranes in the rabbit's vitreous. An autoradiographic and quantitative morphological study.

A morphological and autoradiographic assessment was made of scar tissue development in an experimental rabbit model of proliferative vitreoretinopathy which involves the intravitreal injection of cultured autologous skin fibroblasts. Within a few hours after injection as many as 40% of the cultured cells had autolysed. However, the remaining cells proliferated and formed membranes. The incorporation of [3H] thymidine was maximal at 1 week. Between 2 and 4 weeks spindle-shaped myofibroblasts were prominent, and this form of fibroblast has been considered to have an important role in scar tissue contraction. At the end of 4 weeks it was noted that all eyes had developed retinal detachments. Subsequently the membranes became progressively more fibrous and lipid-like material accumulated in the cytoplasm of many cells. The findings were discussed in relation to the use of this model to test the effectiveness of various drugs which may be of value in the treatment of proliferative vitreoretinopathy.

The effects of injections of cultured fibroblasts into the rabbit vitreous.

A comparison was made between rabbit's eyes which had an intravitreal injection of either 400,000 autologous or homologous skin fibroblasts. The follow-up period was up to 6 months with autologous and 10 weeks with homologous cells. The evaluation involved the clinical assessment of membrane formation, retinal detachment and anterior segment inflammation. In addition, a detailed histological, electron microscopic and autoradiographic study was made of membrane development and changes in the surrounding tissue. Clinical evaluations revealed little difference between the two models. However, on the basis of macroscopic and microscopic examination, homologous cells produced more extensive detachments and more pronounced retinal gliosis. Plasma cells were evident in the choroid 6 weeks after homologous fibroblast injection, but they were not associated to any appreciable extent with the autologous model. Fibrovascular proliferation from around the optic nerve head made a major contribution to the formation of membranes in the experiments with homologous fibroblasts but not those using the autologous cells.

Epiretinal membranes in vitro.

Monolayers of cells were grown from epiretinal membranes using standard tissue culture techniques. The growing cells were filmed by time-lapse cinephotomicrography to record their locomotory characteristics and cell-to-cell interactions. The established layers of cells were examined in the scanning electron microscope. In addition, the cultured cells were studied immunohistochemically to identify specific cell types and the distribution of contractile elements (e.g. actin). The locomotory patterns, the topography and the immunohistochemistry of cells from epiretinal membranes were compared to the movement, appearance and staining characteristics of a wide range of normal cells in culture. From the growth characteristics in tissue culture, early membranes appeared more hypercellular than longer standing membranes. Cell outgrowth was established more quickly and was more widespread in the young epiretinal membranes than in older, more fibrous, specimens. The locomotory and immunohistochemical studies demonstrated that a wide variety of cell types may contribute to epiretinal membranes. A remarkably wide range of cell forms were observed in cultures of individual membranes. Epithelial and glial cells were found in many of the monolayers. However, they did not constitute the majority of cells in the cultures. The predominating cell types had the morphological and locomotory characteristics of fibroblasts or macrophages.