Sympathetic sprouting near sensory neurons after nerve injury occurs preferentially on spontaneously active cells and is reduced by early nerve block

Abstract

Some chronic pain conditions are maintained or enhanced by sympathetic activity. In animal models of pathological pain, abnormal sprouting of sympathetic fibers around large- and medium-sized sensory neurons is observed in dorsal root ganglia (DRGs). Large- and medium-sized cells are also more likely to be spontaneously active, suggesting that sprouting may be related to neuron activity. We previously showed that sprouting could be reduced by systemic or locally applied lidocaine. In the complete sciatic nerve transection model in rats, spontaneous activity initially originates in the injury site; later, the DRGs become the major source of spontaneous activity. In this study, spontaneous activity reaching the DRG soma was reduced by early nerve blockade (local perfusion of the transected nerve with TTX for the 1st 7 days after injury). This significantly reduced sympathetic sprouting. Conversely, increasing spontaneous activity by local nerve perfusion with K(+) channel blockers increased sprouting. The hyperexcitability and spontaneous activity of DRG neurons observed in this model were also significantly reduced by early nerve blockade. These effects of early nerve blockade on sprouting, excitability, and spontaneous activity were all observed 4-5 wk after the end of early nerve blockade, indicating that the early period of spontaneous activity in the injured nerve is critical for establishing the more long-lasting pathologies observed in the DRG. Individual spontaneously active neurons, labeled with fluorescent dye, were five to six times more likely than quiescent cells to be co-localized with sympathetic fibers, suggesting a highly localized correlation of activity and sprouting.

Figure 1

Early nerve blockade decreased the incidence of spontaneous activity in DRG neurons following sciatic nerve transection. Sciatic nerve transection caused high incidence of spontaneous activity in both large and medium-sized DRG neurons. Early nerve blockade (perfusion with TTX) inhibited the abnormal change in spontaneous activity and almost normalized it in nerve injured DRG; perfusion with vehicle (ACSF), or perfusion of the uninjured contralateral nerve with TTX, did not inhibit spontaneous activity. *, significantly different from normal group; #, significantly different from axotomy group. Standard error bars are based on comparisons between different animals.

Figure 2

The distribution of spontaneous activity patterns among spontaneous activity in large or medium-sized DRG neurons. Spontaneous activity was classified as bursting, irregular, or tonic, and the overall incidence of each subtype is indicated for each experimental group.

Figure 3

Anti-TH immunostaining of sympathetic fibers in whole mount DRG. Sympathetic nerve fibers could be observed on the dorsal (top) and ventral (bottom) surfaces of both normal (A, B) and axotomized (C, D) DRGs, however, the axotomized DRGs had much higher fiber density on both sides of the ganglion. Scale bar=200μm.

Figure 4

Manipulation of spontaneous activity in injured nerve alters sympathetic fiber density in DRG. TH-positive fiber density was measured in 40 μm DRG sections as described. Sciatic nerve was perfused in all experimental groups. A: Sprouting, measured on POD 35–42 (i.e, 28 – 35 days after the end of the nerve blockade period), is significantly increased in transected vs. uninjured nerve (p< 0.001); perfusion of the transected nerve with TTX during the first 7 days after injury significantly (p<0.001) reduces this sprouting, back to levels statistically indistinguishable from that seen in normal uninjured nerve (one-way ANOVA with Tukey’s post test). B. Perfusing the transected nerve with potassium channel blockers TEA (10 mM) or 4-AP (10 mM) significantly (p<0.001) increased sympathetic fiber density compared to perfusion with vehicle (ACSF). The differences between the two channel blocker groups were not significant (one-way ANOVA with Tukey’s post test). In this experiment sprouting was measured at POD 14, 7 days after the end of channel blocker perfusion, hence the overall sprouting density in axotomized axons was lower than in Figure 4A. Sympathetic nerve sprouting is higher in L4 DRG than in L5 in this model, so data from these ganglia are presented and analyzed separately.

Figure 5

Examples of spontaneously active (left, center panels) or quiescent neurons (right panels) filled with fluorescent dye after recording followed by TH staining in whole mount DRGs. Compared with normal DRG (a & b), sympathetic fiber endings were more often co-localized with spontaneously active neurons after sciatic nerve transection. Basket structures (arrows) are formed around (d & e) or near (h) spontaneously active neurons. The quiescent neurons (c, f, i) were less specifically co-localized with sympathetic fiber endings. Scale=50 μm.

Figure 6

The percentages of labeled spontaneously active and quiescent cells that had sympathetic fiber endings localized near their soma are indicated for each experimental group. In normal animals, the differences between spontaneously active and quiescent cells were not significant (p = 0.5); * - in all groups with sciatic nerve transection the differences were significant (Fishers exact test; p = 0.02 for the TTX group and p<0.001 for the axotomized and axotomized perfused with ACSF groups). Standard error bars are based on differences between experiments in different animals.