Sympathetic nerves sprout aggressively in the neonatal animal, but show restricted outgrowth in the adult. Following excision of a superior cervical ganglion in the mature rat, smooth muscle of the orbit remains denervated indefinitely, while neonatally denervated targets are reinnervated by fibers deriving from the contralateral superior cervical ganglion. Studies in the previous funding period provide evidence that orbital connective tissue structures through which sympathetic nerves travel to their targets undergo a maturational change in their ability to support sympathetic ingrowth, the diminution in sympathetic ingrowth correlates temporally with the loss of the ability to establish contralateral innervation. Preliminary studies indicate that the connective tissue pathway is composed of a modified fibroblast, the myofibroblast, that has phenotypic characteristics intermediate between fibroblasts and smooth muscle, and can under some circumstances transdiffereritlate into smooth muscle cells. We hypothesize that certain types of myofibroblasts with phenotypic features closely aligned to those of smooth muscle may serve as intermediate targets for ingrowing sympathetic fibers and that during maturation, these myofibroblasts assume an altered phenotype which Is lese conducive to sympathetic sprouting. The objectives of the present study are to define the myofibroblast phenotype associated with sympathetic ingrowth, to determine the relationships between sympathetic nerves and myofibroblasts, and to determine whether a phenotypic shift represents a primary factor in diminished target reinnervation in the mature rat. In the first specific aim, we will characterize the structural and molecular phenotypes of orbital myofibroblasts during development using immunocytochemistry, in situ hybridization and electron microscopy.
In aim 2 we will determine if the sympathetic ingrowth that occurs during visceral organ development and wound healing is associated with myofibroblasts of similar molecular and structural phenotype. In the third aim we will determine if the sympathetic fibers form neuroeffector contacts with the myofibroblasts using electron microscopy, retrograde tracer uptake, measurement of tissue contraction, and mympathetic denervation. In the fourth aim we will determine if the diminished ingrowth in the mature rat can be overcome by increasing the number of available neurons in close proximity to the orbit using intracranial sympathetic neuron transplant techniques. This study will provide basic information on the mechanisms of peripheral nerve pathway formation and the role of sympathetic innervation in wound healing, and may lead to therapeutic strategies aimed at facilitating reinnervation following degenerative disease, traumatic injury, and ban transplantation.
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