Characterization of ocular neuropetides

The project aims are defined in three separate parts: Part I: Investigation of the distribution of secretoneurin by immunohistochemistry and biochemical characterization of the peptide by high-pressure liquid chromatography in peripheral tissues of the rat and human eye and in human aqueous humor. Secretoneurin, a 33-amino acid peptide, is a cleavage product of chromogranin C (secretogranin II), which belongs to the chromogranin family. This peptide is widely distributed in the central nervous system, in the periphery, it constitutes a sensory transmitter. The in vivo generation of this peptide has been investigated by the Department of Pharmacology in Innsbruck, and this peptide has already been characterized in the human retina by the applicant. Preliminary detections revealed high concentrations of secretoneurin in the aqueous humor of human uninflamed (cataract) eyes, thus indicating the presence of immunoreactive nerves in the anterior segment of the eye. The aim is therefore to investigate the distribution of secretoneurin-like immunoreactivity in the anterior segment of the eye by means of immunohistochemistry and subsequently to characterize the immunoreactivity by high-pressure liquid chromatography. Part II: Examination of substance P and calcitonin gene-related peptidergic changes both quantitatively and qualitatively in the rat trigeminal ganglion in the streptozotocin-induced diabetic rat model. Diabetes mellitus is associated with alterations of neuropeptide content in several organs, and the changes do not follow a predictable pattern. Thus, levels of particular peptides have been reported to increase, decrease or remain unchanged depending on the target tissue innervation and the time interval of examination. As ocular sensory nerves derive from the trigeminal ganglion, the aim of this project is to characterize peptidergic changes in the trigeminal ganglion both quantitatively (measurements of peptides at the protein level) and qualitatively in the streptozotocin- induced diabetes mellitus model of the rat in a time-dependent manner, i.e. 1, 3, 5, 8 and 12 weeks after streptozotocin injection. There are namely direct (altered expression of peptides) and indirect indications of time- dependent changes of peptide levels (altered nerve growth factor, which regulates neuropeptide expression in sensory ganglia). In preliminary experiments, reduced peptide levels were found, which would help to explain certain ocular disorders, in particular keratitis neuroparalytica. Thus, this project aims to confirm this result. Part III: Investigation of retinal changes in substance P and vasoactive intestinal polypeptide in a timedependent manner in the streptozotocin-induced diabetic rat model by different methods. In the retina of diabetic animals the only peptide examined so far is somatostatin, i.e. a pronounced increase in this peptide has been detected in rat retinal extracts by radioimmunoassay in a time-dependent manner. This project will investigate substance P as this peptide features vasoactive and proliferative responses and it will also investigate vasoactive intestinal polypeptide because of its influences on the metabolism of retinal pigment epithelial cells. The aims of this project are to determine which quantitative and qualitative changes of substance P and vasoactive intestinal polypeptide occur under pathological diabetic metabolism conditions. One of the most serious complications of diabetes mellitus is proliferative diabetic retinopathy. Many growh factors are thought to be involved in the pathogenesis of proliferative diabetic retinopathy, in particular vascular endothelial growth factor, insulin-like growth factor and basic fibroblast growth factor. Interestingly no neovascularizations occur in this diabetes model although these factors are elevated in the retina or vitreous suggesting that further factors may play a role. One of these factors may be substance P which exerts the same vasoproliferative and endothelial cell migratory effect at similar dosages as compared with basic fibroblast growth factor. In preliminary experiments reduced levels of this peptide were found which may represent a missing of proliferative stimulus, thus possibly explaining absence of vasoproliferations. This part aims to become a basic research on peptides in this diabetes model using different methods.

The project proposal P14022-Med consisted of three separate partial projects, namely the investigation of the effect of streptozotocin-induced diabetes mellitus on the levels of the neuropeptides SP and CGRP in the rat trigeminal ganglion in a time-dependent manner, the effect of streptozotocin-induced diabetes mellitus on the levels of the neuropeptides SP and VIP in the rat retina also in a time-dependent manner and finally, the presence and distribution of a previously discovered neuropeptide, secretoneurin, was explored in the anterior segment of the eye. The results revealed new insights into the extent of pathological changes in the case of diabetes mellitus as a reduction of the amounts of the peptides was observed both in the rat trigeminal ganglion and in the rat retina. This has far-reaching consequences since the pathogenesis of trophic lesions on the cornea can now be explained by a reduced trophic support with neuropeptides from the trigeminal ganglion. Trophic corneal lesions are often observed in diabetic patients. On the other hand, the reduced levels in the diabetic retina give an indication on initial disorders in the pathogenesis of diabetic retinopathy. Particularly the reduction of oscillatory potentials which is the first sign of diabetic retinopathy can now be regarded as the consequence of an imbalance in retinal transmitters. The reduced levels also help explain early deficits in this disease, for example the impaired contrast sensitivity. Both SP and VIP represent namely amacrine cell transmitters and are known to modulate the excitability of inner retinal neurons. And finally, secretoneurin was unequivocally characterized as a novel constituent of sensory neurons innervating the anterior segment of the eye and the investigation of its presence and distribution should provide the basis for performing functional studies.