Iris reconstruction using autologous iris preserved in cold balanced salt solution for 8 hours in iatrogenic total iridodialysis during cataract surgery: a case report.

BACKGROUND: A large iris defect or extensive iridodialysis can be an intractable cause of visual disturbance, photophobia, glare, monocular diplopia, or cosmetic deformity. The implantation of an artificial iris substitute could be an effective option, but this can cause a reduction in endothelial cell density. We succeeded in the anatomical restoration of iris tissue that was totally dialyzed out of the eye, and was preserved in cold balanced salt solution for 8 h. Engrafted iris tissue was maintained within the aqueous humor. CASE PRESENTATION: A 71-year-old man was referred to our clinic for management of an iatrogenic total iridodialysis. The totally dialyzed iris tissue was immediately preserved in sterile cold balanced salt solution and packed in a sterile biopsy bottle that was surrounded with ice cubes. Under general anesthesia, a pars plana vitrectomy was performed to remove the remaining lens cortex and vitreous fiber anterior to the equator. A sulcus-positioned intraocular lens (IOL) was repositioned and fixed by ab externo scleral sutures. Preserved iris tissue was inserted and ironed using both iris spatula and ocular viscoelastic devices. Five-point ab interno scleral sutures were made 1.0 mm posterior to the limbus. CONCLUSIONS: The engrafted iris was successfully maintained for 6 months and did not undergo any atrophic change or depigmentation, which may be caused by primary implantation failure due to a blocked blood supply.

Tumor dormancy in vivo by prevention of neovascularization.

Dormant solid tumors were produced in vivo by prevention of neovascularization. When small fragments of anaplastic Brown-Pearce carcinoma were implanted directly on the iris in susceptible rabbits, they always vascularized. A characteristic growth pattern, consisting of prevascular, vascular, and late phases, was observed, which terminated with destruction of the eye within 2 wk. The beginning of exponential volume increase was shown to coincide with vascularization of the implant, as demonstrated by perfusion with intravenous fluorescein and by histologic sections. In contrast, implants placed in the anterior chamber, at a distance from the iris, did not become vascularized. After initial growth into spheroids, they remained arrested at a small size comparable to prevascular iris implants, for periods as long as 6 wk. Although dormant in terms of expansion, these avascular tumors contained a population of viable and mitotically active tumor cells. When reimplanted on the iris, vascularization was followed by rapid, invasive growth. These observations suggest that neovascularization is a necessary condition for malignant growth of a solid tumor. When a small mass of tumor cells is prevented from eliciting new vessel ingrowth from surrounding host tissues, population dormancy results. These data suggest that the specific blockade of tumor-induced angiogenesis may be an effective means of controlling neoplastic growth.

Nerve growth factor: stimulation of regenerative growth of central noradrenergic neurons.

The growth of new axonal sprouts was studied from transected, ascending noradrenergic axons into transplants of iris tissue in the caudal hypothalamus of the rat. A single intraventricular injection of nerve growth factor, given at the time of axonal damage, resulted in an increased formation and growth of new noradrenaline sprouts 7 days later. The effect seemed to be proportional to the administered dose of nerve growth factor.

[Cosmetic iris implantation can cause irreversible eye complications].

Artificial iris implants were originally developed for therapeutic purposes but have recently been used for cosmetic alteration of the eye colour. A 21-year-old woman presented with bilateral eye redness, visual loss, raised intraocular pressure, corneal oedema and hyphaema following implantation of artificial irises in Tunisia. Combined medical and surgical management led to improvements, but reduced vision and photophobia persisted. Cosmetic iris implantation can lead to persistently sight-threatening eye complications, and we strongly advise against its use.

Intraocular in vivo imaging of pancreatic islet cell physiology/pathology.

BACKGROUND: Diabetes mellitus has reached epidemic proportions and requires new strategies for treatment. Unfortunately, the efficacy of treatment regimens on maintaining/re-gaining functional beta cell mass can, at the present, only be determined indirectly. Direct monitoring of beta cell mass is complicated by the anatomy of the endocrine pancreas, which consists of thousands to a million of discrete micro-organs, i.e. islets of Langerhans, which are scattered throughout the pancreas. SCOPE OF REVIEW: Here, we review the progress made over the last years using the anterior chamber of the eye as a transplantation site for functional imaging of pancreatic islet cells in the living organism. Islets engrafted on the iris are vascularized and innervated and the cornea, serving as a natural body-window, allows for microscopic, non-invasive, longitudinal evaluation of islet/beta cell function and survival with single-cell resolution in health and disease. MAJOR CONCLUSIONS: Data provided by us and others demonstrate the high versatility of this imaging platform. The use of 'reporter islets' engrafted in the eye, reporting on the status of in situ endogenous islets in the pancreas of the same animal, allows the identification of key-events in the development and progression of diabetes. This will not only serve as a versatile research tool but will also lay the foundation for a personalized medicine approach and will serve as a screening platform for new drugs and/or treatment protocols. 'Metabolic' islet transplantation, in which islets engrafted in the eye replace the endogenous beta cells, will allow for the establishment of islet-specific transgenic models and 'humanized' mouse models as well as serving as the basis for a new clinical transplantation site for the cure of diabetes.

Characteristics of iris and retinal pigment epithelial cells cultured on collagen type I membranes.

PURPOSE: Transplantation of pigment epithelial cells is a promising treatment modality to repair retinal damage in age-related macular degeneration. For this purpose, it is necessary to establish cell culture techniques that allow acquisition of proper functional and morphological characteristics by the cells to be transplanted. METHODS: Primary retinal pigment epithelial (RPE) and iris pigment epithelial (IPE) cells grown to confluence on collagen membranes were examined for morphology, adhesion, proliferation, apoptosis, as well as viability after ex vivo transplantation. RESULTS: Pigment epithelial cells adhere, proliferate, form monolayers, acquire differentiated properties, and remain viable during transplantation to the subretinal space. CONCLUSIONS: Pigment epithelial cells cultured on collagen membranes acquire differentiated characteristics and are amenable to be transplanted as cell monolayers.

Glial fibrillary acidic protein (GFAP)-like immunoreactivity in the rodent eye. Comparison between peripheral glia of the anterior uvea and central glia of the retina.

Immunohistochemistry with antiserum raised against glial fibrillary acidic protein (GFAP) revealed a dense plexus of GFAP-positive fibers in normal rodent iris. These fibers were not stained with 2 monoclonal GFAP antibodies which readily stain astrocytes, suggesting that they contain a polypeptide closely related, but not identical, to CNS GFAP. The GFAP-positive iris fibers did not disappear after short-term intraocular grafting or culturing of irides; instead a conspicuous system of fluorescent, star-shaped cells appeared. In the retina Müller glia were intensely fluorescent using GFAP antiserum whereas positive staining was observed with GFAP monoclonals only after injury to the retina. These antibodies, however, readily stained astrocytes in the inner layers of the normal retina. Taken together, these findings support the idea of GFA proteins as a group of closely related but not identical polypeptides.

Parasympathetic neurotrophic activity in the rat iris: determination after different denervations.

The iris of the adult rat contains one or several neurotrophic factors that enhance the survival of dissociated parasympathetic neurons (from the embryonic chick ciliary ganglion) in culture. To assay survival activity, iris homogenates were serially diluted with culture medium and the percentage of neurons surviving for 2 days in a collagen matrix in culture determined. The extract induced survival curves that were similar for denervated and normal irides. Similarly no differences in fibre outgrowth from cultured whole ciliary ganglia were found. The results suggest that the apparent level of parasympathetic growth factor(s) is not under strict control of the innervation of the iris.

Substance P-containing sensory nerves in the rat iris. Normal distribution, ontogeny and innervation of intraocular iris grafts.

Trigeminal, substance P-containing nerves have been studied in the stretch-prepared rat iris with immunohistochemical techniques. The normal iris exhibited a slightly irregular plexus of individual fibres in the dilator, intermingled with thin, meandering axon bundles. The sphincter contained more circumferentially oriented fibres. Occasional free nerve endings were present in all parts of the iris; no obvious association with blood vessels was detected. All substance P-positive nerves in the iris disappeared after lesioning the trigeminal nerve. Irides of neonates showed scattered, smooth fibres in a sparse plexus, without visible axon bundles. Over the first two postnatal weeks, the density of innervation developed rapidly, reaching a transiently supranormal level and fluorescence intensity, compared to adulthood. From 3 weeks on, the pattern and density of substance P-containing fibres approached the normal adult appearance. In irides grafted to the anterior eye chamber, the intrinsic substance P nerves degenerated, disappearing completely after 5 days. Reinnervation from the host irides transpired over the next few weeks, approximating normal density after 3 weeks, and organotypic density and distribution from 4 weeks on. No obvious hyperinnervation was encountered after longer postoperative times (3 months). In the host iris, many substance P fibres disappear or exhibit low fluorescence intensity during the first postoperative week, recovering fully during the next 2 weeks. Over longer postoperative periods irregular, moderate hyperinnervation developed with increased numbers of axons in bundles. In conclusion, we show normal distribution and plasticity during ontogeny and maturity of substance P-containing iris nerves in the rat, with a sensitive immunohistochemical technique in iris whole mounts.

Effects of age and GDNF on noradrenergic innervation of the hippocampal formation: studies from intraocular grafts.

Recent studies have suggested that factors in the target tissue influence the degree of plasticity and regeneration following aging and/or specific insults. We have investigated whether young or aged targets differ in their noradrenergic innervation from fetal locus coeruleus (LC) neurons, and also if a specific growth factor, glial cell line-derived neurotrophic factor (GDNF) can affect this innervation pattern. Tissue pieces of fetal brainstem and young (3 months) or old (18 months) iris tissue were transplanted simultaneously into the anterior chamber of the eye of adult hosts. We found that aged iris transplants became innervated to a significantly lesser degree by the cografted LC neurons than young iris transplants. Fetal hippocampal tissue was then grafted to adult hosts, and a fetal brainstem graft containing LC neurons was placed adjacent to the first graft, either at 3 or 21 months post-grafting. Thus, old/young chimeras of the noradrenergic coeruleo-hippocampal pathway were created. Aged hippocampal grafts received a much less dense innervation from co-grafted LC neurons than young hippocampal grafts. Tyrosine hydroxylase-positive-immunoreactive innervation was only found in the outskirts of aged grafts, while the young hippocampal grafts contained an even innervation pattern. The innervation density of hippocampal grafts was significantly enhanced by GDNF treatment. These findings demonstrate that target-derived factors may regulate neuronal plasticity, and that the age of the target is more important for innervation properties than the age of the neuron innervating a particular target.

Heart conditioned medium elicits post-lesion muscarinic receptor recovery in vivo.

Intraseptal administration of heart conditioned medium (HCM) stimulates the growth of injured cholinergic fibers into iris implants placed in the anterodorsal hippocampus. The aims of the present report were to monitor muscarinic cholinergic receptor concentrations during denervation and central innervation of the peripheral tissue targets, and to evaluate the effect of HCM on these changes. For 0, 4, 8, 16 and 28 days after mechanical injury to septohippocampal axons, animals received either intraseptal injections of HCM or control vehicle. Binding of [3H]quinuclidinylbenzilate ([3H]QNB) within iris implants was used as an index of cholinergic muscarinic receptor concentration. The results indicate that within the iris implants: (1) a dramatic drop in the number of muscarinic receptors is observed 4 days after denervation; (2) under control conditions, central cholinergic innervation is not associated with muscarinic receptor recovery; and (3) after administration of HCM, muscarinic receptor levels begin to increase within two weeks and approach the pre-lesion endogenous concentration following 28 days of treatment. These results support the hypothesis that trophic factors may facilitate the restoration of effective, appropriate connections between nerve fibers and their targets.

Reinnervation, myelination and organization of iris tissue implanted into the rat midbrain--an ultrastructural study.

The ultrastructure of autologous irides implanted into the midbrain of mature Sprague-Dawley rats was studied over a time period of 4-14 days. Most features of the normal iris still persisted throughout this time, including typical iris blood vessels and amelanotic melanocytes. At 5-4 days after implantation, the normal innervation of the implanted iris had degenerated, except for the presence of intact Schwann cells and some myelin debris. Reinnervation, beginning about the seventh day, proceeded rapidly. By the fourteenth day, extensive reinnervation of the implant was evidenced by the presence of numerous small (0.1-0.6 micrometer) and large (2-4 micrometer) non-myelinated axons ensheathed in Schwann cell cytoplasm. Axonal varicosities, filled either with dense core or clear core vesicles, formed junctions with axons. These junctions were characterized by an accentuation of areas of the axonal membrane and pre- or post-junctional thickenings; however, we did not observe typical synaptic complexes. Some large axons within the myelinated iris developed thick myelin sheaths of the peripheral nervous system (PNS) type; we believe this is the first reported instance in which myelination of central axons by Schwann cells within the brain parenchyma has been produced by the implantation of PNS elements.

Enhancement of central cholinergic sprouting by prior injury: correlation with endogenous trophic content of hippocampus.

Injury to neural tissue elicits the release of neurotrophic substances which support the survival of cultured cholinergic neurons. The aim of the present report was to determine the effect of prior hippocampal injury on the sprouting of mature intrinsic cholinergic septal afferents in vivo. Levels of choline acetyltransferase (CAT) in intrahippocampal iris implants were used as an index of cholinergic regeneration. The results show that prelesioning the hippocampus 1 week prior to insertion of the iris increased CAT levels of implants left for 8 days in situ. Levels of iris CAT were correlated with the trophic content of the surrounding hippocampal tissue. These observations suggest that endogenous trophic factors released as a consequence of neural injury facilitate cholinergic sprouting in vivo.

Regeneration of central cholinergic neurones in the adult rat brain.

The regrowth of lesioned central acetylcholinesterase (AChE)-positive axons in the adult rat was studied in irides implanted to two different brain sites: in the caudal diencephalon and hippocampus, and in the hippocampal fimbria. At both implantation sites the cholinergic septo-hippocampal pathways were transected. At 2-4 weeks after lesion, newly formed, probably sprouting fibres could be followed in abundance from the lesioned proximal axon stumps into the iris transplant. Growth of newly formed AChE-positive fibres into the transplant was also observed from lesioned axons in the anterior thalamus, and to a minor extent also from the dorsal and ventral tegmental AChE-positive pathways and the habenulo-interpeduncular tract. The regrowth process of the sprouting AChE-positive, presumed cholinergic fibres into the iris target was studied in further detail in whole-mount preparations of the transplants. For this purpose the irides were removed from the brain, unfolded, spread out on microscope slides, and then stained for AChE. During the first 2-4 weeks after transplantation the sprouting central fibres grew out over large areas of the iris. The new fibres branched profusely into a terminal plexus that covered maximally about half of the iris surface, and in some areas the patterning of the regenerated central fibres mimicked closely that of the normal autonomic cholinergic innervation of the iris. In one series of experiments the AChE-staining was combined with fluorescence histochemical visualization of regenerated adrenergic fibres in the same specimens. In many areas there was a striking congruence in the distributional patterns of the regenerated central cholinergic and adrenergic fibres in the transplant. This indicates that - as in the normal iris - the sprouting cholinergic axons (primarily originating in the lesioned septo-hippocampal pathways) and adrenergic axons (primarily originating in the lesioned axons of the locus neurones) regenerate together along the deneravated Schwann cell sheaths. From a comparison between the central reinnervation process and the process of reinnervation of the iris by peripheral cholinergic axons after transplantation to the anterior eye chamber, it is concluded that the regenerative capacity of central cholinergic neurones (above all the septo-hippocampal system) is not much inferior to that of their peripheral counterparts when given similar growth conditions. Moreover, central cholinergic neurones seem partly able to replace the peripheral ones in the reinnervation of a denervated peripheral target.

Experimental ocular angiogenesis.

In 29 experiments, vascular autologous retina from the contralateral rabbit eye was implanted into corneal pockets in New Zealand white rabbits. The implants induced a neovascular growth of limbal vessels that anastomosed with the vessels of the implant in 15 of the cases. Implantation of vascular autologous iris, choroid, optic nerve extraocular muscle, and conjunctiva also induced neovascularization. Avascular peripheral retina, cornea, and scleral implants did not cause a neovascular response, nor did boiled vascular retina, boiled iris, and boiled choroid. Neovascularization from the retinal and iris intracorneal implants was induced by V2 carcinoma.

Descemet's membrane as membranous support in RPE/IPE transplantation.

PURPOSE: The correct orientation of retinal pigment epithelium (RPE) cells is necessary for the integrity and proper function of the retina. For transplantation of RPE/iris pigment epithelium (IPE) grafts to the subretinal space in age-related macular degeneration, this cellular orientation is most effectively provided by a membranous support. The goal of this study was to establish an autologous or homologous membrane as a substratum for the growth of RPE/IPE. METHODS: Porcine and bovine RPE and IPE were placed in primary culture on a dissected sheet (5 x 5 mm) of autologous porcine and bovine Descemet's membrane in slide chambers and grown to confluence. RESULTS: RPE and IPE cells cultured on Descemet's membrane form an intact monolayer. Light and electron microscopy showed the formation of both an intact monolayer and microvilli in both cell types. CONCLUSION: Since the slow host-graft rejection appears to play an important role in the failure of RPE transplantation in the subretinal space, it is critical to be able to transplant autologous materials. The techniques presented here establish a novel means to culture RPE or IPE cells on autologous Descemet's membrane where they form a "cell monolayer patch," consisting of a fragment of Descemet's membrane with cultured RPE or IPE, which can be easily manipulated and transplanted, using an established glass pipette method.

[Retinal pigment epithelial cell transplantation: perspective].

Age-related macular degeneration is one of the most serious diseases in elderly people because of its disasterous visual outcome and its prevalence. Even if the submacular and choroidal neovascular membranes could be surgically excised, severe damage or evacuation of retinal pigment epithelium is inevitable in the operated area. Pigmentary dystrophy is also a devastating hereditary eye disease with severe visual disturbance. Up to now, there have been no effective treatments for either of them. We conducted basic experiments on retinal pigment epithelium (RPE) culture, transplantation of the cells to the subretinal space of animals, especially, the Royal College of Surgeon's (RCS) rat, a model of hereditary retinal degeneration, and observed their effects in preventing photoreceptor cell death. 1) We reviewed recent reports of RPE function in relation to cytokine production and autocrine/paracrine function of these ligands. Some cytokines with strong mitogenic effects as nerve trophic/growth factors were able to rescue photoreceptor cell death in dystrophic, ischemic, and light-damaged retinas in the rats. We transplanted allograft pigmented RPE from Long Evans rats or xenograft, human and bovine RPE into the subretinal space of RCS rats, and could observe the retardation of the photoreceptor cell death. 2) As a source of human transplantable RPE in clinical practice, we could use patients' own RPE cells as autografts or those from aborted human fetus eyes as allografts. At present, we cannot use RPE cells from different species as xenografts. We tried to obtain enough RPE cells for culture in vitro from patients with large or giant retinal tears, but were unsuccessful. Cells were easily obtained from fetus eyes, and could be cultured and transplanted as fresh, primary, or multiple passage cells. We also tried cryopreservation of these cells for up to 3 months. Enzymatic expression of tyrosinase, tyrosinase related protein I and II and some other enzymes was examined by proliferating chain reaction to detect possible transformation during the procedure. The cell characteristics were well preserved. In the future, if these RPE cells could be safely kept and available in deep-frozen condition, we could use them clinically at the appropriate time and in appropriate numbers for patients as an "RPE bank" just like an "eye bank" for corneal transplantation. 3) Immunological reaction is very important if we consider this technique for clinical application. Up to now, in experimental animals, no immunological reaction has been reported even for xenograft human RPE in rats, in funduscope and histological examination, because the intraocular space is an immunologically privileged site. But transplantation of human RPE cells with a collagen sheet into the anterior chamber in rabbits caused a definite reaction detected by suppression of the electroretinogram and macrophage infiltration into the subretinal space, not only in the operated eye but also in the contralateral non-operated eye. These results suggest that we must be cautious in clinical use of heterogeneous RPE transplantation. The expression of MHC class II cells was observed in the course of photoreceptor cell degeneration in the RCS rats but it was suppressed if they were rescued by the transplantation of human cultured RPE in these animals. 4) For clinical application of this technique, autografts are naturally much better than the xeno grafts or allografts. We tried to use iris pigment epithelium (IPE) for transplantation because it consists of pigmented cells of neural origin and enough could be obtained with ease by peripheral iridectomy. We also tried transfection of a vector (pCNX2) or vector-inserted cDNA of rat bFGF into the rat IPE and transplanted into the subretinal space of RCS rats. These transfected cells expressed strong mRNA of bFGF. The photoreceptors were well preserved and immunological reaction could not be detected by funduscopical or histological examinat