Biochemical changes induced by intravitreally-injected doxorubicin in the iris-ciliary body and lens of the rabbit eye.

The aim of this study was to examine the chronic effects and mode of action of doxorubicin in ocular tissues. A dose of 10 microg (17.24 nanomoles) of doxorubicin hydrochloride in 20 microl sterile saline were intravitreally injected, under local anaesthesia, in one eye of 13 rabbits and 50 microg (86.20 nanomoles) were similarly injected in one eye of 3 rabbits. The contralateral eye received 20 microl of saline only. The dose of 50 microg induced initially mild uveal inflammation which became chronic and turned into circular iritis. Both doses of the drug induced cataract of the lens and clouding of the cornea within 2-3 months. The activity of superoxide dismutase, in iris-ciliary bodies and lenses treated with either 10 or 50 microg of the compound, was significantly lower relative to that in respective control tissues. In contrast to superoxide dismutase, catalase showed an increased activity in experimental tissues relative to control. The lysosomal hydrolases acid phosphatase, N-acetyl-B-D-glucosaminidase, aryl sulphatase and acid cathepsin, all showed significantly elevated activities in iris-ciliary body tissues one year after injection with the 50 microg doxorubicin. The reduction in superoxide dismutase activity may render ocular tissues susceptible to peroxidative attack and the increased activities of lysosomal hydrolases may contribute to chronic cell injury and inflammation.

Macrophages during fibrosis following scleral fistulising surgery in a rat model.

PURPOSE: Glaucoma filtration surgery can fail in a minority of patients as a result of fibrosis in the subconjunctival bleb space and closure of the scleral fistula. In this study, the rat eye has been used as an experimental model for fistulising surgery in order to evaluate the clinical manifestation of bleb failure with the morphological events of the wound healing process. METHODS: A conjunctival bleb was successfully formed in 25 rats and was examined daily using slit lamp microscopy to evaluate postoperative inflammation and the presence of a bleb. At defined post-operative time points, serial frozen sections of eyes were stained immunohistochemically using a panel of monoclonal antibodies directed against known surface markers on rat immune/inflammatory cells. Positively stained cells were counted (a) in the bleb site, (b) at the sclerostomy and (c) at the suture site. RESULTS: Following an initial post-operative inflammation, a surgically formed sclerostomy and conjunctival bleb underwent a granulation and scarring response so that by 7-19 days the bleb had disappeared. Using the monoclonal antibodies applied in this study, it was possible to show that macrophages most likely play a major and pivotal role throughout the sequence of events that lead to repair of the fistula and closure of the bleb. It was also noted that the presence of an otherwise inert nylon suture used to close the incised conjunctiva can serve as a focus for macrophages. CONCLUSION: The rat has been successfully used as an experimental model of fistulising surgery and its subsequent failure. The use of a panel of monoclonal antibodies directed against specific surface markers on immune-inflammatory cells, highlighted macrophages to be prominent in all stages of this wound healing process.

The effect of topical dexamethasone and preoperative beta irradiation on a model of glaucoma fistulizing surgery in the rabbit.

We studied the effect of topical dexamethasone (1%) and preoperative beta irradiation on a model of glaucoma fistulizing surgery in the rabbit. Intraocular pressure and gross facility of aqueous outflow following surgery were not influenced by either treatment, although blebs persisted longer in the irradiated eyes. Steroids reduced clinically observable inflammation as well as the number of inflammatory cells identifiable by microscopy. Fibroblast production temporarily slowed, and ultra-structural examination demonstrated lipid-filled vacuoles and dilated mitochondria in these eyes. Also, the scar was thinner at 24 days. Beta irradiation delayed wound healing and the scar was thinner in the early postoperative stages, but the light microscopic appearance of the scar was unaltered at 59 days. Inflammation was more pronounced initially, with abundant fibrin in the wound. Recovery of the conjunctival epithelium was delayed. The delay in fibroblast recruitment and wound contraction, the thinner scar tissue, and the increased survival of the bleb are all factors that suggest that beta irradiation may be a useful adjunct to glaucoma surgery.

Review: mediation of the ocular response to injury.

The structure of the anterior segment of the eye provide aqueous humour for metabolic traffic, regulation of intraocular pressure and the maintenance of a functional permeability barrier to separate internal compartments from general systemic influences. Irritative and injurous insults to the eye elicit an acute defensive miotic and vascular response which upsets the aqueous dynamics and provokes the influx of blood plasma proteins into the aqueous chambers. These events are initiated by antidromic activation of sensory elements within the anterior segment, releasing substance P and calcitonin gene-related peptide (CGRP) which, in lower mammals at least, stimulate respectively the miotic and vascular reactions. Considerable species differences can be found in the responsiveness of the eye to injury and in the effects of exogenous CGRP and substance P.

Presence and actions of vasopressin-like peptides in the rabbit anterior uvea.

The presence of vasopressin-like immunoreactivity (VP-IR) in the rabbit eye was demonstrated by radioimmunoassay. Trigeminal nerve denervation resulted in a significant and selective decrease in the levels of VP-IR in the iris sphincter muscle and the cornea. The isolated iris sphincter muscle contracted in response to low concentrations of [Arg8]vasopressin (AVP) and related peptides. The V1 vasopressin receptor antagonist, d(CH2)5Tyr(Me)AVP, potently inhibited the contractile responses to AVP. AVP was found to induce an increase in the accumulation of inositol phosphates in the iris sphincter muscle but not in the dilator/ciliary body preparation in vitro. The present investigation demonstrates the presence of VP-IR in the rabbit eye and that this substance may be another sensory nerve-derived mediator acting on specific target sites in the anterior uvea.

Mediation of the ocular response to injury and irritation: peptides versus prostaglandins.

The immediate response of the rabbit eye to noxious stimulation is mediated neurogenically through antidromic sensory activation and subsequent action of neuropeptides, CGRP, and substance P or a related tachykinin. The rise in ocular tension and breakdown in the BAB in response to injury are initiated by dilation and increased permeability of the ciliary vessels, which bring about disruptive alterations in the squamous epithelium covering the anterior part of the iridial processes and ciliary ridge. PGs formed during the response to certain types of irritation can enhance the ocular changes to trauma by facilitating the neurogenic pathway. In the rabbit eye substance P acts as a strong miotic in response to injury but has no effect on the IOP rise or disruption of the BAB. Conversely, CGRP is a potent vasodilator that displays no spasmogenic action. Species differences in the ocular response to injury have been observed, with lower mammals such as rabbit and rat generally being more responsive than higher mammals. For the most part, these differences are related to the organization of the iris-ciliary complex and to the sensitivity of the effector elements to the neuromediators. Reciprocal modulation exists between the autonomic and sensory nervous elements and the tissue they innervate, so that altered sensitivity of the effector cells develops in selectively denervated eyes. Chronic sympathetic denervation causes hypersensitivity of the eye to irritation. This increase is owing largely to hypertrophied sensory elements with increased sensory neuropeptide levels and consequently augmented sensory activity. Conversely, chronic sensory denervation is accompanied by an increase in catecholamine-forming enzymes and in the neuropeptide NPY. Future research will no doubt reveal neuromodulation of homeostatic, inflammatory, and vegetative processes and the ability of ocular tissues to recover from wounding. Both the discovery of biogenic neuropeptides in peripheral nervous elements of the eye alongside the classical transmitters and the elucidation of their profound effects on the eye afford scope for the development of potential new therapeutic agents.

The role of inflammation in the development of epiretinal membranes.

Single or multiple (3) injections of "Shigella" endotoxin were administered into the rabbit vitreous body to investigate the development of epiretinal membranes following intraocular inflammation. The evaluation included clinical assessment of the resulting traction retinal detachments, together with histological, autoradiographic, immunohistochemical and ultrastructural studies. Traction retinal detachments were found beneath fibroglial epiretinal membranes (being more extensive in eyes which had received 3 endotoxin injections) in the vicinity of the medullary rays, while purely glial membranes occurred over attached peripheral retina. The primary change at the vitreoretinal interface was an elevation of the inner limiting lamina of the retina followed by the extension of glial cells onto the retinal surface. It is postulated that glial cells breach the inner limiting lamina as a sequel to inflammation involving the vitreoretinal interface and form a scaffold upon which fibroblast-like cells migrate.

Alteration in the histochemical presence of tyrosine hydroxylase and CGRP-immunoreactivities in the eye following chronic sympathetic or sensory denervation.

Changes in the content of calcitonin gene-related polypeptide (CGRP) and tyrosine hydroxylase (TH) immunoreactivities in the anterior segment of the rat eye were assessed histochemically in animals subjected to sympathetic ganglionectomy or to chronic sensory denervation induced by neonatal capsaicin treatment. In the sympathectomised eyes there was a marked depletion of TH and increase in CGRP immunoreactivity while in tissue subjected to sensory denervation the reverse was found, raised presence of TH and absence of CGRP-immunostaining. The results suggest important neurotrophic influences by the host tissue or a trophic interaction of one nerve set on another.

Recent experimental studies on the blood-aqueous barrier: the anatomical basis of the response to injury.

In the mammalian eye the tissue layers which effectively separate the neural retina and the transparent refractive media from the circulating blood are generally known as the blood-retinal barrier (BRB) and blood aqueous barrier (BAB) respectively. These have been delineated by morphologically identifying the ultrastructural features which impede the passage of molecules from the blood to the working parts of the eye; physiologically, there is a certain amount of overlap in their function, as there is free diffusion of solutes between the aqueous humour, the vitreous humour and the interstitial tissue of the neural retina. Notwithstanding, it is convenient and appropriate to maintain the distinction as the more recent work has focused on the fine structural aspects of these barrier systems. The abundant literature which describes the work leading to the identification and characterisation of these barriers has been reviewed in depth with great authority by several eminent researchers, notably the late Giuseppina Raviola, and Jose Cunha-Vaz. Certain pathological conditions, trauma--including surgical manipulation, and chemical irritation of the eye can cause significant disruption of these barriers with important clinical consequences. In our laboratory interest has centred around vascular and epithelial changes which occur when the eye is injured and which can lead to a breakdown of the BAB. More recently attention has been drawn to functional and behavioural differences between species, particularly with regard to the relative stability of the barrier.

Evidence for multiple tachykinin receptor subtypes on the rabbit iris sphincter muscle.

The actions of mammalian tachykinins (substance P, substance K/neurokinin a, neuromedin K/neurokinin b) and non-mammalian tachykinins (eledoisin, kassinin, physalaemin) were compared on the rabbit pupillary sphincter. All acted as direct spasmogens with potencies in the order: eledoisin greater than physalaemin = neurokinin b = substance P greater than kassinin greater than neurokinin a. However, their actions could be divided into at least two categories on the basis of similar kinetics of contractions, differential sensitivity to the tachykinin antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P and specific cross-protection against phenoxybenzamine inactivation by structurally related tachykinins. The relationship between these observations and the suggested "P" and "E" subtypes of tachykinin receptors is discussed.

Enzymatic activities in the iris-ciliary body of the rabbit eye during experimentally induced acute ocular inflammation.

Intravitreal injection of 5 micrograms of Shigella endotoxin, in the rabbit eye, induced an acute inflammatory response which was characterised by conjunctival hyperaemia, limbal and ciliary vascular injection, iritis, aqueous flare, miosis and reduction in intraocular pressure. Iris-ciliary body tissues, from normal and inflamed eyes, were fractionated into subcellular enriched fractions and the activities and distribution of selected enzymes were estimated. Alkaline phosphatase, a plasma membrane-bound enzyme, showed an increase in activity, whereas succinate dehydrogenase and Mn-Superoxide dismutase, both mitochondrial-bound enzymes, exhibited decreased activities. Lysosomal acid phosphatase displayed an increase in free activity and retention of latent activity inside the organelle. No alteration in free activity was shown by acid cathepsin. The cholinesterases did not exhibit any changes in activities nor did the cytosolic enzymes Cu/Zn-superoxide dismutase and lactate dehydrogenase. The decrease activity of the respiratory mitochondrial enzyme succinate dehydrogenase may contribute to the reduction in intraocular pressure, and the ability of the lysosomal organelles to retain their hydrolytic enzymes, ensures recovery of the cell from acute inflammatory attack.

Some effects of phagocytosis on bovine meshwork cells in culture.

In this tissue-culture study, some effects of phagocytosis on the cell were investigated. Third-passage bovine trabecular-meshwork cells were incubated with latex microspheres for periods of up to 60 hr. Cells which had been involved in phagocytosis were found to settle at a faster rate than control cells, but if microspheres were added to the medium of recently settled cells, there was a reduction in their proliferative activity. Incorporation of tritiated proline remained unchanged, whereas uptake of tritiated glucosamine increased with the time period of phagocytosis. The fibronectin scaffold was more extensive and elaborate when cells were pre-incubated with microspheres before settlement. Exposure to a low concentration of microspheres promoted a higher uptake of a second presentation of 125I-labelled microspheres--an effect which was not seen if the original dose was too high.

Morphological changes of sensory CGRP-immunoreactive and sympathetic nerves in peripheral tissues following chronic denervation.

The morphological relationship between sensory and sympathetic nerves was studied in tissues of the eye and the oral cavity following chronic sympathetic or sensory denervation. Immunoreactivities for calcitonin gene-related peptide (CGRP) and tyrosine hydroxylase (TH) were used as indexes to assess the changes of the two nerve populations after denervation. Following surgical sympathectomy, a marked increase of CGRP-containing fibres was seen in all tissues studied, while TH-imunoreactive fibres were totally depleated. Conversely, after capsaicin treatment, an increase of TH-immunoreactive nerves was found in the same tissues, concomitant with a sharp decrease of CGRP-immunoreactive nerves. These changes were particularly evident in iridial stroma and around blood vessels in all tissue, where sensory and sympathetic nerves have a closely overlapping distribution pattern. The altered proportion of sensory peptide- and catecholamine-containing nerves following sympathetic and sensory denervation suggest that there is a reciprocal trophic influence between the two nerve subsets, possibly with the intervention of neurotrophic substances such as nerve growth factor. These results indicate a close interaction between sensory peptidergic and sympathetic nervous systems in peripheral organs.

Phagocytosis of latex microspheres by bovine meshwork cells in culture.

The ability of cultured bovine trabecular meshwork cells to phagocytose latex microspheres was studied by electron microscopy. In addition, the incorporation of 125I gamma-globulin-coated microspheres by the meshwork cells was monitored. The cultured cells, on the basis of quantitation by scanning electron microscopy, did not show significant preference between biotic particles (gamma-globulin-coated microspheres) and abiotic particles (uncoated microspheres). Surface microvilli appeared to be involved in the initial stages of phagocytosis; indeed a significant negative correlation was demonstrated between the incidence of microvilli and the commitment to phagocytosis of preconfluent cells. Two weeks postconfluent cells were shown to be significantly less phagocytic than preconfluent cells. The importance of these findings in terms of the behaviour of meshwork cells in vivo is discussed.

Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye.

The occurrence, distribution, and origin of immunoreactive calcitonin gene-related peptide (CGRP) in nerves of rat, guinea pig, cat, and monkey eyes were investigated by immunocytochemistry, radioimmunoassay, and chromatography. A rich network of CGRP-immunoreactive nerve fibres was noted in the anterior uvea, which was widely distributed in both dilator and constrictor pupillae muscles and extended to the ciliary body and uveal blood vessels. Numerous CGRP-immunoreactive neuronal cells were present in the trigeminal ganglion. The extractable CGRP was 8.6 +/- 1.8 pmoles/gm of tissue in the iris and 44.0 +/- 8.1 pmoles/gm in the trigeminal ganglion. Following damage to the Gasserian ganglion a marked decrease of CGRP immunoreactivity was observed in the anterior uvea (control 11.3 +/- 1.6 pmoles/gm; operated 1.4 +/- 0.1 pmoles/gm) confirming the origin of the immunoreactive fibres from trigeminal primary sensory neurons. The sensory nature of the CGRP-immunoreactive fibres was substantiated by the depletion of CGRP immunoreactivity observed after treatment with capsaicin, which is known to cause selective degeneration of sensory neurons. Comparative studies on the distribution and colocalisation of CGRP and the putative sensory neurotransmitter substance P revealed a closely parallel distribution of the two peptides in certain regions of the uvea and their coexistence in a subpopulation of trigeminal primary sensory neurons. This study suggests that the sensory nervous system in the eye is more heterogeneous in terms of its putative neurotransmitters than previously indicated.

The morphological features of bovine meshwork cells in vitro and their synthetic activities.

The morphological characteristics of 3rd-passage cultured bovine meshwork cells were investigated, as were some of their synthetic activities. Growing meshwork cells had the ultrastructural characteristics of metabolically active cells whereas postconfluent cells formed gap junctions at their lateral borders and were closer in their fine structure to meshwork cells in vivo. The incorporation of 3H proline and 3H glucosamine was significantly greater in growing than postconfluent cultures. The normal bovine outflow system was rich in type I collagen but type V collagen and fibronectin was also evident. The 3rd-passage cells were shown to synthesize type I collagen, type V collagen and fibronectin. However, ultrastructural studies of the extracellular matrix which was produced in vitro demonstrated the presence of small fibrils with no distinctive banding pattern, unlike the larger collagen fibrils with a 640 A banding pattern seen in vivo.

Calcitonin gene-related polypeptide as a mediator of the neurogenic ocular injury response.

Calcitonin gene-related polypeptide (CGRP) has been localised immunochemically within the rat and guinea pig anterior uvea to nerve fibres of trigeminal origin. As with substance P (1-3) the level of CGRP in the iris-ciliary body is depleted after thermal damage to the Gasserian ganglion and elevated in chronically sympathectically denervated eyes. Unlike substance P, a potent pupillary constrictor (4,5), CGRP has no notable miotic action, but does, however, cause an elevation of the intraocular pressure (IOP) accompanied by disruption of the blood-aqueous barrier. It is proposed that the diverse actions of these two sensory neuropeptides conjointly mediate the antidromic ocular injury response.