Inhibitory effect of certain neuropeptides on the proliferation of human retinal pigment epithelial cells.

AIMS: To define the effect of the neuropeptides substance P, calcitonin gene related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and secretoneurin on the proliferation of human retinal pigment epithelial (RPE) cells. METHODS: ARPE-19 cells were used. The cells were cultured in Dulbecco's modified Eagle's medium. 1000 and 2000 cells were incubated with the peptides for 3 and 5 days, and the effect of the peptides was evaluated by an ATP lite assay dose dependently. Furthermore, specific antagonists at 10(-6) M were used to find out whether the effect would be reversed. RESULTS: In brief, each of the peptides tested had an inhibiting effect. This inhibiting effect was weak but highly significant, averaging 10% to 15%, and was most pronouncedly seen at concentrations between 10(-10) M and 10(-14) M. Each antagonist reversed the inhibiting effect fully. CONCLUSIONS: These results clearly indicate that RPE cells are under neural control and the low effective concentration of the peptides may be the one physiologically acting on these cells. The results are of important relevance both physiologically and pathophysiologically: physiologically, the inhibitory effect may mean that these peptides cause the cells to remain in a differentiated condition. Pathophysiologically, the findings are relevant in proliferative vitreoretinopathy where RPE cells proliferate in excess. The authors hypothesise that the inhibiting effect diminishes when these cells are swept out and actively migrate from their physiological location and thus, dedifferentiate and begin to proliferate. This hypothesis improves the knowledge of the initial processes in the pathogenesis of the disease as there seems to be a discrepancy between facilitatory and inhibitory influences favouring the former in proliferative vitreoretinopathy. Furthermore, these neuropeptides constitute the first endogenous inhibitors of RPE cell proliferation.

Slow release of acetylsalicylic acid by intravitreal silicone oil.

PURPOSE: To assess in vitro the potential of silicone oil as a delivery system for acetylsalicylic acid (ASA) and to evaluate in vivo the pharmacokinetic distribution of salicylic acid (SA) in the eye. METHODS: In an experimental model ASA/silicone oil suspension mixed to a concentration of 1.67 mg/mL was investigated for release rate of ASA and SA. In vivo vitrectomy and intravitreal injection of two different ASA/silicone oil suspensions, both mixed to a concentration of 1.67 mg/mL, was performed on two groups, A and B, of New Zealand white rabbits. Salicylic acid concentrations in ocular tissues, aqueous, vitreous, and blood plasma were evaluated at 6 hours, 24 hours, and 5 days using high performance liquid chromatography. RESULTS: Salicylic acid was detected in all tissues. The highest levels were obtained in the vitreous: 745.4 microg/mL (A) and 640.0 microg/mL (B) at 6 hours. The retina followed with 332.9 ng/mg (A) and 281.3 ng/mg (B) at 6 hours and 31.6 ng/mg (A) and 48.1 ng/mg (B) at day 5. The maximum blood plasma levels were 5.2 microg/mL. CONCLUSION: Silicone oil is an efficacious delivery system of ASA in vitro and in vivo. Higher concentrations of SA were found in all ocular tissues and fluids when compared to intravenous administration of maximum doses.

Intravitreal acetylsalicylic acid in silicone oil: pharmacokinetics and evaluation of its safety by ERG and histology.

BACKGROUND: A new method of intravitreal drug delivery of acetylsalicyclic acid (AS) in silicone oil was investigated for safety and for its pharmacokinetics in the posterior pole of the eye. METHODS: The AS was mixed in silicone oil to a concentration of 1.67 mg/ml. After vitrectomy, 15 NZW rabbits received an intravitreal injection of AS/silicone oil suspension. Clinical examination, pre- and postoperative electroretinography (ERG) and histology were performed. The pharmacokinetics of the distribution of salicylic acid was determined by HPLC analysis at 6 h, 24 h and 5 days in optic nerve, retina, choroid, vitreous, and blood. RESULTS: Clinical examination and histology revealed no adverse effects or signs of toxicity. The ERGs showed no significant difference between the pre- and postoperative results. The salicylic acid concentrations demonstrated peak values in the residual vitreous (640.0 micrograms/ml), choroid (446.0 ng/mg) and retina (281.3 ng/mg) at 6 h. At 24 h, the salicylic acid concentration decreased to 20.9 micrograms/ml in the residual vitreous and to 38.5 ng/mg in the retina. At 5 days the retinal level was still 48.1 ng/mg. CONCLUSIONS: AS delivery by intravitreal administration of loaded silicone oil is a safe method and results in high concentrations of salicylic acid in the posterior segment of the eye while maintaining low blood levels.

Substance P and vasoactive intestinal polypeptide in the streptozotocin-induced diabetic rat retina.

PURPOSE: Little knowledge exists about how neurotransmitters behave in the diabetic retina. In this study, the authors measured the concentration of two neuropeptides, substance P and vasoactive intestinal polypeptide, in the streptozotocin-induced diabetic rat retina in a time-dependent manner. METHODS: The retinas of 1-, 3-, 5-, 8-, and 12-week diabetic rats were processed using a highly sensitive radioimmunoassay for both substance P and vasoactive intestinal polypeptide. Furthermore, the peptide-immunoreactivities were characterized by high-pressure liquid chromatography. RESULTS: Substance P and vasoactive intestinal polypeptide were found to be significantly reduced with a maximum decrease of 28.6% (+/-6.7) and 64.5% (+/-10.7) after 5 weeks, respectively. The peptide-immunoreactivities were found in a major peak coeluting with the synthetic peptides indicating that the quantitative values measured by radioimmunoassay represent the authentic peptides. CONCLUSIONS: The reduction of substance P and vasoactive intestinal polypeptide is in clear contrast to the amino acid transmitters GABA and glycine, which have been shown to be elevated in this early stage of diabetic retinopathy. This finding is important for three reasons: First, the decrease may result in reduced excitability of inner retinal neurons, as both peptides are known to modulate the excitability of these neurons; second, the decrease may be the consequence of a depressing and/or damaging effect by excitotoxins; and third, it may help explain why neovascularizations do not occur in this animal model, although VEGF is massively upregulated, as substance P is a very potent vascular growth factor.

Elevated levels of calcitonin gene-related peptide in aqueous humor of patients with proliferative vitreoretinopathy.

PURPOSE: To detect the levels of the sensory peptide calcitonin gene-related peptide in aqueous humor of patients with proliferative vitreoretinopathy and to compare them with those of uninflamed eyes (cataract and uncomplicated rhegmatogenous retinal detachment). MATERIALS AND METHODS: Using a highly specific and sensitive radioimmunoassay the concentration of calcitonin gene-related peptide was detected in fresh samples of aqueous humor obtained via paracentesis. Furthermore, calcitonin gene-related peptide-like immunoreactivities were characterized by high-pressure liquid chromatography. RESULTS: The mean level of calcitonin gene-related peptide was 6.11 fmol/ml in cataract controls and 14.77 fmol/ml in uncomplicated rhegmatogenous retinal detachment. In the cataract group, 9 of 18 cases were below the detection limit and in the retinal detachment group, 5 of 16. In proliferative vitreoretinopathy, the peptide averaged 76.92 fmol/ml and none of the samples was below the detection limit. High-pressure liquid chromatography revealed one major peak corresponding to synthetic calcitonin gene-related peptide. CONCLUSION: In recent studies, we found elevated levels of the sensory peptide substance P in aqueous humor of patients with proliferative vitreoretinopathy. This fact and the present result, the elevation of calcitonin gene-related peptide in aqueous humor of eyes with proliferative vitreoretinopathy, clearly point to an involvement of sensory peptides in the pathobiology of the disease. The source of the elevation is not clear, but we hypothesize that it originates from a neurogenic mechanism, i.e. an acceleration of the peptides by their enhanced release from the iris/ciliary body complex subsequent to sensitization of sensory neurons, thus representing a very interesting epiphenomenon of proliferative vitreoretinopathy. Our results constitute novel aspects in the pathophysiological concept of proliferative vitreoretinopathy and extend the knowledge about the pathobiology of the disease process.

Retinal thickness analysis in subjects with different refractive conditions.

PURPOSE: The aim of the present study is to evaluate a measure of retinal thinning at the posterior pole in eyes with increasing axial myopia. METHODS: 129 eyes of 79 healthy persons with different refractive conditions were examined using a commercially available prototype of retinal thickness analyzer. RESULTS: Increasing myopia has been found to be accompanied by bulbus elongation, and calculation of the values obtained with the instrument revealed a significant decrease in retinal thickness at the posterior pole in eyes with increasing axial myopia, i.e. 4.8 microm/spherical equivalent in the foveolar region and 6.6 microm/spherical equivalent in the other areas of the posterior pole. CONCLUSION: Our data show retinal thinning at the posterior pole in myopic eyes based for the very first time on in vivo measurements. Furthermore, our findings might influence measurement data for other purposes, if the degree of myopia is not taken into account.

The effect of streptozotocin-induced diabetes mellitus on substance P and calcitonin gene-related peptide expression in the rat trigeminal ganglion.

Substance P (SP) and calcitonin gene-related peptide (CGRP) constitute the main sensory peptides in the trigeminal ganglion (TG). The objective of this study was to characterize peptidergic changes in the streptozotocin-induced diabetes mellitus rat model both quantitatively and qualitatively. Diabetes mellitus was induced by a single intraperitoneal injection of streptozotocin (65 mg/kg) and the levels of SP and CGRP were measured by means of radioimmunoassay (RIA) in a time-dependent manner. Peptide immunoreactivities were characterized by high pressure liquid chromatography (HPLC). The expression of both neuropeptides was examined 5 weeks after streptozotocin injection using in situ hybridization with 35S-labelled oligonucleotides. Saline-injected rats served as controls. SP was significantly decreased in the diabetic rat TG, i.e. , a 44.6% (+/-10.9) decrease after 1 week, 40.2% (+/-11.8) after 3 weeks and 72.3% (+/-14.6) after 5 weeks. CGRP was decreased only after 5 weeks (19.6% decrease +/-3.9), whereas at later stages, both peptide levels returned to normal values. HPLC revealed one major peak coeluting with the synthetic peptides. By using in situ hybridization, a significantly increased signal of both peptide-encoding mRNAs was found (43.8%), which seems to act to restore a diabetes-associated depletion of neuropeptides in the diabetic rat TG. The decreased SP- and CGRP levels in the diabetic rat TG reflect a diabetes-associated deficit which may be clinically relevant. Diabetes mellitus is associated with a variety of ocular complications, even corneal complications, including decreased corneal sensitivity, which in many ways resemble those after interruption of the normal trophic innervation of the eye. Our results point to reduced availability of neuropeptides for corneal innervation and may thus support the idea of a partial loss of trophic influences from the trigeminal nerve in diabetics.