Increased expression of NGF after spinal cord injury induces sprouting of primary nociceptive axons. Exogenous application of NGF also results in extensive sprouting of these axons and causes chronic pain in uninjured animals. During development, semaphorin3A is thought to act as a repulsive guidance cue for NGF-responsive nociceptive afferents, restricting their projections to the superficial dorsal horn. We investigated the ability of semaphorin3A to selectively reduce NGF-induced sprouting and neuropathic pain in adult rats. The chemorepulsive effect of virus-mediated semaphorin3A expression was shown to counteract the sprouting induced by NGF in a dosedependent manner, both in vitro and in adult rat spinal cords. Coexpression of semaphorin3A and NGF at moderate to low concentrations within the adult spinal cord reduced sprouting of calcitonin gene-related peptide and substance P-containing axons compared with GFP and NGF coexpression controls. At high expression levels of NGF, there was no difference in sprouting between the semaphorin3A-treated and control groups. The distribution of endogenous primary nociceptive afferents in the spinal cord appeared to be unaffected by semaphorin3A treatment in these experiments. Behavioral assessment shows that semaphorin3A coexpression with NGF led to decreased mechanical allodynia but no significant reductions in thermal hyperalgesia. These findings demonstrate directly that mature sensory afferents maintain their responsiveness to semaphorin3A, suggesting that this molecule might be used therapeutically to control aberrant sensory sprouting involved in pain or autonomic dysfunction.

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An in vitro preparation of rabbit cornea was used to compare anatomical specialization and electrophysiological responses of A delta and C fiber sensory afferents which terminate as free nerve endings. Living nerve endings were visualized using epifluorescence microscopy and the vital dye 4-di2-ASP, and response properties were determined using microstimulation and recording of fiber discharge activity. Fiber type was determined based on conduction velocity measurement and preferred stimulus energy (modality) of each fiber. Four modality-specific fiber populations were identified: (1) slowly adapting C fiber cold receptors (conduction velocity of 0.25-1.6 m/sec), (2) C fiber chemosensitive units with mixed phasic and tonic activity (1.1-1.8 m/sec), (3) rapidly adapting mechanosensitive A delta fibers (1.5-2.8 m/sec), and (4) high-threshold mechano/heat (> 350 dyne or > 40 degrees C) phasic A delta afferents (3.5-4.4 m/sec). In addition to these physiological differences, anatomical specialization was also noted. A delta fiber nerve endings were distinguished from those of C fibers by thin, elongated sensory endings that ran parallel to the corneal surface; C fiber endings formed short, branching clusters that ran mostly perpendicular to the surface. The elongated structure of A delta nerve endings was associated with directional selectivity for mechanical stimuli. These results substantiate previous suggestions that free nerve endings can exhibit both structural and functional specialization.

During development, semaphorins (collapsin, fasciclin) mediate repulsive and inhibitory guidance of neurons. Semaphorin III, a secretable member of this family, is expressed by the ventral spinal cord at the time corresponding to projection of sensory afferents from the dorsal root ganglion (DRG) into the spinal cord. The inhibitory effect of E14 ventral cord is active only on nerve growth factor (NGF)-responsive sensory afferents (small-diameter A-delta and C fibers subserving sensations of temperature and pain). Similarly, COS cells secreting recombinant semaphorin III are able to selectively repel DRG afferents whose growth is stimulated by NGF and not NT-3. However, it is not known whether these molecules can exert a functional role in the fully developed adult peripheral nervous system. In this study, we demonstrated that gene gun transfection and production of semaphorin III in corneal epithelial cells in adult rabbits in vivo can cause repulsion of established A-delta and C fiber trigeminal sensory afferents. In addition, it is shown that, following epithelial wounding and denervation, semaphorin III is able to inhibit collateral nerve sprouts from innervating the reepithelialized tissue. These findings are significant in that they provide direct evidence that small-diameter adult sensory neurons retain the ability to respond to semaphorin III. In addition, the corneal gene gun technique may be generally used to study the in vivo effects of neural growth modulators by quantifying the amount of sensory nerve growth.

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The thermal sensitivity of the eyelid and cornea was compared using an automated apparatus to produce stimulus pulses of known magnitude and duration over the range 33--45 degrees C. Subjects reported only temperature sensation when the skin of the upper eyelid was tested; however, corneal stimulation in the same subjects was always perceived as nociceptive. The possibility that other ocular tissues may be involved in the pain responses was shown to be unlikely by direct experimentation or by calculation of heat flow in those tissues. Cornea and eyelid thresholds were compared in relationship to the structural and physical properties of these tissues. It was found that the nerve endings of the corneal epithelium are less sensitive to temperature change when compared to the thermal receptors of the eyelid. It is concluded that the cornea is useful for the experimental study of pain.