Central Plasticity of Cutaneous Afferents Is Associated with Nociceptive Hyperreflexia after Spinal Cord Injury in Rats.

Electrical stimulations of dorsal cutaneous nerves (DCNs) at each lumbothoracic spinal level produce the bilateral cutaneus trunci muscle (CTM) reflex responses which consist of two temporal components: an early and late responses purportedly mediated by Aδ and C fibers, respectively. We have previously reported central projections of DCN A and C fibers and demonstrated that different projection patterns of those afferent types contributed to the somatotopic organization of CTM reflex responses. Unilateral hemisection spinal cord injury (SCI) was made at T10 spinal segments to investigate the plasticity of early and late CTM responses 6 weeks after injury. Both early and late responses were drastically increased in response to both ipsi- and contralateral DCN stimulations both above (T6 and T8) and below (T12 and L1) the levels of injury demonstrating that nociceptive hyperreflexia developed at 6 weeks following hemisection SCI. We also found that DCN A and C fibers centrally sprouted, expanded their projection areas, and increased synaptic terminations in both T7 and T13, which correlated with the size of hemisection injury. These data demonstrate that central sprouting of cutaneous afferents away from the site of injury is closely associated with enhanced responses of intraspinal signal processing potentially contributing to nociceptive hyperreflexia following SCI.

SOX2 expression is upregulated in adult spinal cord after contusion injury in both oligodendrocyte lineage and ependymal cells.

The upregulation of genes normally associated with development may occur in the adult after spinal cord injury (SCI). To test this, we performed real-time RT-PCR array analysis of mouse spinal cord mRNAs comparing embryonic day (E)14.5 spinal cord with intact adult and adult cord 1 week after a clinically relevant standardized contusion SCI. We found significantly increased expression of a large number of neural development- and stem cell-associated genes after SCI. These included Sox2 (sex determining region Y-box 2), a transcription factor that regulates self-renewal and potency of embryonic neural stem cells and is one of only a few key factors needed to induce pluripotency. In adult spinal cord of Sox2-EGFP mice, Sox2-EGFP was found mainly in the ependymal cells of the central canal. After SCI, both mRNA and protein levels of Sox2 were significantly increased at and near the injury site. By 1 day, Sox2 was upregulated in NG2(+) oligodendrocyte progenitor cells (OPC) in the spared white matter. By 3 days, Sox2-EGFP ependymal cells had increased proliferation and begun to form multiple layers and clusters of cells in the central lesion zone of the cord. Expression of Sox2 by NG2(+) cells had declined by 1 week, but increased numbers of other Sox2-expressing cells persisted for at least 4 weeks after SCI in both mouse and rat models. Thus, SCI upregulates many genes associated with development and neural stem cells, including the key transcription factor Sox2, which is expressed in a pool of cells that persists for weeks after SCI.

Central expression of synaptophysin and synaptoporin in nociceptive afferent subtypes in the dorsal horn.

Central sprouting of nociceptive afferents in response to neural injury enhances excitability of nociceptive pathways in the central nervous system, often causing pain. A reliable quantification of central projections of afferent subtypes and their synaptic terminations is essential for understanding neural plasticity in any pathological condition. We previously characterized central projections of cutaneous nociceptive A and C fibers, selectively labeled with cholera toxin subunit B (CTB) and Isolectin B4 (IB4) respectively, and found that they expressed a general synaptic molecule, synaptophysin, largely depending on afferent subtypes (A vs. C fibers) across thoracic dorsal horns. The current studies extended the central termination profiles of nociceptive afferents with synaptoporin, an isoform of synaptophysin, known to be preferentially expressed in C fibers in lumbar dorsal root ganglions. Our findings demonstrated that synaptophysin was predominantly expressed in both peptidergic and IB4-binding C fiber populations in superficial laminae of the thoracic dorsal horn. Cutaneous IB4-labeled C fibers showed comparable expression levels of both isoforms, while cutaneous CTB-labeled A fibers exclusively expressed synaptophysin. These data suggest that central expression of synaptophysin consistently represents synaptic terminations of projecting afferents, at least in part, including nociceptive A-delta and C fibers in the dorsal horn.

Differential cardiovascular responses to cutaneous afferent subtypes in a nociceptive intersegmental spinal reflex.

Electrical stimulation to segmental dorsal cutaneous nerves (DCNs) activates a nociceptive sensorimotor reflex and the same afferent stimulation also evokes blood pressure (BP) and heart rate (HR) responses in rats. To investigate the relationship between those cardiovascular responses and the activation of nociceptive afferents, we analyzed BP and HR responses to electrical stimulations at each DCN from T6 to L1 at 0.5 mA to activate A-fiber alone or 5 mA to activate both A- and C-fibers at different frequencies. Evoked cardiovascular responses showed a decrease and then an increase in BP and an increase and then a plateau in HR. Segmentally, both cardiovascular responses tended to be larger when evoked from the more rostral DCNs. Stimulation frequency had a larger effect on cardiovascular responses than the rostrocaudal level of the DCN input. Stimulation strength showed a large effect on BP changes dependent on C-fibers whereas HR changes were dependent on A-fibers. Additional A-fiber activation by stimulating up to 4 adjacent DCNs concurrently, but only at 0.5 mA, affected HR but not BP. These data support that cutaneous nociceptive afferent subtypes preferentially contribute to different cardiovascular responses, A-fibers to HR and C-fibers to BP, with temporal (stimulation frequency) and spatial (rostrocaudal level) dynamics.

Peripheral and central anatomical organization of cutaneous afferent subtypes in a rat nociceptive intersegmental spinal reflex.

Stimulation of rat segmental dorsal cutaneous nerves (DCNs) evokes the nociceptive intersegmental cutaneus trunci muscle (CTM) reflex. The reflex consists of early and late responses, mediated by Aδ and C fibers, respectively, based on required stimulation strengths, and shows segmental differences in terms of amplitude and duration. We have now investigated whether the peripheral or central anatomy of nociceptive afferent subtypes in different DCNs also vary in a segmental manner. The numbers of different axon subtypes, determined by axon diameter, were analyzed across peripheral DCNs from T6 to L1. The central projections of T7 and T13 DCN afferents were traced using DCN injections of cholera toxin subunit B (CTB) for myelinated A fibers and isolectin B4 (IB4) for unmyelinated C fibers and both labels were quantified in the dorsal horns. Peripheral axon subtype numbers did not differ significantly across DCNs. Centrally, IB4+ , but not CTB+ , projection areas were different between T7 and T13, consistent with different segmental CTM neurogram responses. At both levels, A fibers projected to deeper layers of the dorsal horn than did C fibers. These termination sites are consistent with both mono- and polysynaptic connections between these afferents and the ascending propriospinal interneurons of the reflex. Also analyzed were the spatial distribution, the synaptic termination, and the glutamatergic transporter profiles of DCN A and C fibers and their relationship to calcitonin gene-related peptide (CGRP) staining in the dorsal horn.