Lawrence Kruger - US grants

It appears likely that the transducer mechanism responsible for excitation and prolonged sensitization of nociceptive afferent discharges is a chemically mediated process linked to injury of innervated tissue, called 'neurogenic inflammation.' This nerve-dependent aspect of the inflammatory process is believed to underlie cutaneous hyperalgesia and many of the conditions of chronic pain. A first step in understanding this process is to identify, characterize and determie the distribution and relationship of cutaneous nociceptive fibers and to distinguish those specialized biochemical features that might account for the specificity of inflammatory action on nociceptors. A series of experiments are planned to be performed on skin of laboratory rats with emphasis on the unmyelinated axons forming the majority of the sensory nerve population. Sensory axons will be identified by injecting axon-transported labels into dorsal root ganglion and, in some experiments, by comparing neurotoxins specific for thin sensory fibers (capsaicin) or unmyelinated sympathetic axons (6-hydroxydopamine). Specific markers will be employed to determine the distribution of sensory axons involved in tachykinin (e.g. substance P) mediated antidromic vasodilatation and plasma extravasation, their receptor binding sites (e.g. specific tritiated substance P binding), the binding sites of known endogenous algogenic substances (e.g. bradykinin), and the immunocytochemical detection of highly specific receptors for the Fc domain of IgG2b molecules on the surface of macrophages, the binding of which is believed to trigger release of arachidonic acid and synthesis of prostanoids. Co-localization studies with multiple labels should provide criteria for distinguishing the variety of unmyelinated sensory axon patterns and the correlates of efferent effects mediated by these axons of possible relevance in neurogenic inflammation. A variety of new methods are to be employed for multiple markers of transported proteins, neuropeptides, enzymes and antigenic sites specific to the small ganglion cells known to emit thin, primarily nociceptive, axons and to describe the unexplored fine structure of unmyelinated nociceptors. The findings should reveal the specificity of biochemical correlates with sense organ and efferent terminal patterns at the electron microscopic level and provide clues concerning the distribution of nociceptive axons in relation to other elements invoked in the inflammatory processes underlying prolonged pain.

The aim of this project is to characterize the chemical and morphological properties of the small sensory ganglion cells that emit thin afferent axons implicated in nociceptive discharge. Emphasis is placed upon understanding 1) those properties of peripheral afferent terminals underlying efferent regulatory function and effector response to injury in peripheral tissues, and 2) the neural mechanisms responsible for prolonged pain of pathological origin. These studies could provide new therapeutic approaches to recovery following injury and control of chronic pain syndromes. The objective is to provide a morphological basis for identifying and defining the heterogeneous subpopulations of putative peripheral 'nociceptor' axon terminals in visceral tissues suitable for flat-mount study and dominated by unmyelinated innervation, cf. tunica vasculosa of testis and mesenteries. Electrophysiologically characterized nociceptive spots will be analyzed for patterns of peptide expression and for a variety of undetermined co-localization factors in axons labeled by lectins (markers for axon membrane glycoconjugates) with emphasis on the purinergic and adrenergic influences implicated in inflammatory processes and pain. The project shifts from description of innervation in different tissues to electron microscopic studies relying heavily on post-embedment immunogold techniques. Experiments aimed at hypotheses concerning axonal sprouting and regulation of peptide expression will be carried out using immunocytochemical and in situ hybridization methods to define and quantify, at the transcriptional and translational level, changes in sensory neurons induced by sympathectomy and different types of nerve lesions, including axotomy, dorsal rhizotomy, constriction neuropathy, and neuroma formation. The axonal-growth specific protein GAP-43, and its mRNA, will serve as a tool for studying axon outgrowth. Emphasis is also placed on the morphological components underlying the contribution of norepinephrine and adenosine to hyperpathias in rat models affected by these substances. Establishing a peripheral mechanism of action can have direct implications for guiding pain therapy.