Expression of eight neuropeptides in intraocular spinal cord grafts: Organotypical and disturbed patterns as evidenced by immunohistochemistry

Abstract

The present study examines the distribution of several neuropeptides, as revealed by immuno-histochemistry in the isolated cord. Fetal rat spinal cord was grafted to the anterior chamber of the adult Sprague-Dawley albino rats. After intraocular maturation for 2–3 months, the amount and distribution of somatostatin, neuropeptide Y, substance P, enkephalin, vasoactive intestinal peptide, peptide histidine-isoleucine, calcitonin gene-related peptide and cholecystokinin immunoreactive terminals and cell bodies were analysed using indirect fluorescence immunohistochemistry. The visualization of immunoreactive cell bodies in the grafts was enhanced using a novel intraocular colchicine treatment. In the graft a rich network of somatostatin-positive terminals was found with a high density in well-demarcated areas reminiscent of substantia gelatinosa of the dorsal horn of normal spinal cord. A large number of small- to medium-sized somatostatin neurons was found throughout the grafts without colchicine treatment. This is in contrast to noimal spinal cord, where positive neurons were difficult to visualize without colchicine and were mainly confined to the dorsal horn. Neuropeptide Y had a distribution in the grafts similar to that of somatostatin and neuropeptide Y cells were found throughout the grafts without colchicine treatment. In normal spinal cord, neuropeptide Y-positive fibers were found mainly in substantia gelatinosa with a sparse network in the ventral horn. Enkephalin-positive fibers were found throughout the grafts. The distribution of fibers resembled that of somatostatin and neuropeptide Y with distinct zones of high fiber density in well-demarcated areas, whereas the density of nerve fibers in the rest of the graft neuropil was moderate to low. The distribution of substance P was similar to that of enkephalin. After colchicine treatment, both enkephalin- and substance P-positive cell bodies were visualized. In the intact spinal cord both peptides were seen in the entire gray matter with the highest concentrations in the superficial laminae of the dorsal horn. Antisera against calcitonin gene related-peptide, revealed a sparse terminal network and many large cells, which might represent motoneurons. A sparse network of varicose cholecystokinin-immunoreactive fibers was found evenly distributed in the grafts. In normal spinal cord a dense cholecystokinin-positive network of primary sensory afferent origin was found in the dorsal horn. In the grafts cholecystokinin cell bodies were seen after colchicine treatment. Vasoactive intestinal peptide and peptide histidine-isoluecine were found in a sparse, regular network. After colchicine treatment, both vasoactive intestinal peptide and peptide histidine-isoleucine cell bodies were visualized. Normal spinal cord expressed fine, sparse vasoactive intestinal peptide and peptide histidine-isoleucine networks. The present results demonstrate that several peptides normally found in spinal cord are expressed in isolated spinal cord grafts. Ths distribution of immunoreactive terminals shows similarities to that found in normal spinal cord, supporting earlier findings that the fetal spinal cord possesses a considerable degree of intrinsic determination for its normal development. Development in total isolation from the rest of the central nervous system, however, lead to disturbances in the expression of some peptides, possibly suggesting a retarded maturation of the grafts, perhaps due to lack of normal spinal connections or with peripheral target tissues.