Neuronal tracers for fine caliber spinal primary afferents and their response to peripheral nerve injury
Abstract: The isolectin B4 from Griffonia simplicifolia I (B4) and soybean agglutinin from the plant Glycine max (SBA), have been shown to bind to rat small-diameter dorsal root ganglion (DRG) neurons, and to fibers and presumed terminals in laminae I-II of the spinal cord dorsal horn. in the present thesis, these lectins and/or their horseradish peroxidase (HRP) conjugates, have been investigated in the rat with regard to their usefulness as selective neuronal markers of and axonally transported tracers for somatic and/or visceral spinal primary afferents with fine caliber, including unmyelinated, axons. In addition, binding and transport of B4 and SBA/SBA-HRP have been examined after peripheral nerve injury. In the fifth lumbar DRG, B4 binding and retrogradely transported SBA were found mainly in neurons of small size, and to comprise about half of the total number of DRG neurons, respectively. About half of the SBA cells bound B4, indicating that two different but overlapping sub-populations of small DRG neurons are recognized by these two lectins. Almost all of the B4-, and 85% of the SBA-positive neurons were not immunoreactive for RT97, an antibody which recognizes DRG neurons giving rise to myelinated axons. Calcitonin gene- related peptide (CGRP) immunoreactivity was found in 59% of the B4 binding and in 43% of the SBA transporting DRG cells. Seventeen per cent of the B4 neurons and 33% of the SBA cells contained substance P (SP). A few B4 (9%) and SBA (2.4%) cells were immunoreactive to somatostatin (SOM). Besides, almost all DRG neurons that were positive for fluoride-resistant acid phosphatase (FRAP) were B4-positive. Injections of B4-HRP or SBA-HRP into the sciatic nerve resulted in labeling in the DRG in the same neurons that bound B4 or SBA. In the spinal cord gray matter, such injections gave rise to labeling of axons and presumed terminals in somatotopically appropriate regions of the superficial dorsal horn, most prominently in the inner part of lamina II for both tracers, although intense labeling in lamina I was additionally found for SBA/SBA-HRP. B4-HRP injections into DRGs resulted in labeling in the cutaneous, sural nerve and in the sympathetic, greater splanchnic nerve, but not in the muscle, lateral and medial gastrocnemius nerves. Such injections also resulted in labeling of free and lanceolate-like nerve endings in the hind paw skin. Electron microscopic examination of the labeled nerves showed that B4-HRP labeled exclusively unmyelinated axons. To study spinal afferents from a parasympathetic nerve, tracer injections were made into the urinary bladder wall. These injections resulted in retrograde and transganglionic labeling at the sixth lumbar and the first sacral spinal levels, most effectively when using cholera toxin B subunit (CTB)-HRP. In the DRGs, CTB-positive neurons were mainly RT97- negative (85%) and showed a large overlap with CGRP (71%) and SP (36%). A few CTB neurons showed galanin-, but not SOM-, or vasoactive intestinal polypeptide-immunoreactivity or contained FRAP. In the spinal cord, CTB-HRP labeled fibers were found to form bundles of lateral and medial collateral projections, and to extend into the dorsal gray commissural region and/or into the spinal parasympathetic nucleus. To study neuronal changes of fine caliber primary afferents following peripheral nerve injury, the sciatic nerve was lesioned unilaterally. In the transport studies, B4 or SBA/SBA-HRP was injected into both the previously lesioned and into the contralateral nerve, as control. In the binding studies, no such injections were made. By 2 weeks after nerve transection and ligation, spinal cord binding and transganglionic transport of B4 had disappeared completely, whereas binding and transport of SBA/SBA-HRP were unchanged. At 6-14 weeks, the SBA/SBA- HRP labeling was weaker but not completely depleted, while B4 labeling was still absent. At 8- 16 months, both B4 binding and B4 transport had partially recovered. Results from the crush injury cases, studied by B4, showed a partial decrease of binding and transport by 2 weeks and a complete recovery as early as after 10 weeks. This thesis study suggests that both B4 and SBA are useful markers and transganglionic tracers for two different but overlapping sub-populations of somatic unmyelinated spinal primary afferent neurons. Both of the populations of neurons labeled by B4 and SBA contain FRAP, SOM, CGRP and SP. While B4 is also a useful tracer for primary afferents in the sympathetic, greater splanchnic nerve, CTB is better suited as a tracer for afferents in the parasympathetic, pelvic nerve. Both B4 and SBA are useful for studying effects of peripheral nerve lesions on fine caliber primary afferents, although with somewhat different temporal scales.
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