Extracellular matrix molecules influence innervation density in rat cerebral blood vessels

We have investigated the contribution of the acellular component (i.e., the extracellular matrix, or ECM) in determining the innervation density of peripheral target tissue, and the possible involvement of ECM molecules in age-related axonal atrophy. We addressed this issue using cerebral blood vessels where both the overall density and the density of sympathetic innervation decrease by 50% in aged rats. In order to study the capacity of the ECM to influence pattern and density of innervation, cerebral blood vessels from young (6 weeks) and old (24 months) rats were repeatedly freeze-thawed to kill the cellular component of the vessel wall before transplantation in oculo into young host rats. After 4 weeks, the nature and degree of reinnervation of transplants by host nerves was studied by quantitative immunohistochemistry using PGP9.5 (a general neuronal marker) or CGRP (calcitonin gene-related peptide, a sensory marker), or catecholamine histochemistry using the glyoxylic acid technique to visualise noradrenergic sympathetic nerves. The density of reinnervation was measured by computerized image analysis. The PGP-immunoreactive reinnervation on old transplanted cerebral blood vessels was significantly less dense compared to young ones. However, nerve density on both young and old transplants was lower than on control, ex vivo blood vessels. CGRP-positive nerves on both young and old transplants were sparse, but similar to control tissues, whereas catecholamine-containing nerves were very sparse on all transplants, and significantly less than on control blood vessels. These results suggest that alterations occur in the aged ECM, that make it less receptive to innervation. However, they also indicate that, along with the ECM, factors produced by the cellular components of the blood vessel wall are essential, at least for the maintenance of the sympathetic innervation.