Selective reactions of cutaneous and muscle afferent neurons to peripheral nerve transection in rats

Abstract

To determine whether peripheral nerve injury has similar effects on all functional types of afferent neuron, we retrogradely labeled populations of neurons projecting to skin and to muscle with FluoroGold and lesioned various peripheral nerves in the rat. Labeled neurons were counted after different periods and related to immunohistochemically identified ectopic terminals and satellite cells in lumbar dorsal root ganglia. After 10 weeks, 30% of cutaneous afferent somata labeled from transected sural nerves had disappeared but, if all other branches of the sciatic nerve had also been cut, 60% of cutaneous neurons were lost. Small-diameter sural neurons preferentially disappeared. In contrast, the number of muscle afferent somata was not affected by transection of various nerves. p75 was downregulated in axotomized cutaneous neurons but in not axotomized muscle neurons. Conversely, p75 was upregulated in satellite cells around cutaneous but not muscle neurons. Consistent with this, perineuronal rings containing tyrosine hydroxylase, calcitonin gene-related peptide, galanin, or synaptophysin were formed preferentially around cutaneous neurons. Selective lesions of predominantly cutaneous nerves triggered the formation of rings, but none were detected after selective lesions of muscle nerves. We conclude that cutaneous neurons are both more vulnerable and more associated with ectopic nerve terminals than muscle neurons in dorsal root ganglia after transection and ligation of peripheral nerves.

Figure 1.

Loss of small-diameter cutaneous afferent neurons in L5 DRGs after sciatic nerve transection. Somata labeled retrogradely with FG applied to the cut axons of the left sural nerve. A, C, Histograms of soma diameter after nerve transection and ligation. Sural X, 1 wk, One week after cutting and labeling the sural nerve (illustrated in B). Sural X, 10 wks, Ten weeks after cutting and labeling the sural nerve. Sciatic X, 10 wks, Ten weeks after cutting and labeling the sural nerve and also cutting and ligating the tibial and peroneal branches of the sciatic nerve at the same operation (illustrated in D). Scale bar: (in D) A-D, 50 μm.

Figure 2.

Number and size of neurons labeled from the sural nerve after different lesions. Somata labeled retrogradely FG+ from the cut sural nerve. Histograms show the number of labeled somata in each section (A) and their mean diameter (B). The lesions are indicated between the plots together with the time in weeks (wks) since the lesion: SurX, only sural nerve cut; SciX, sciatic nerve cut. The numbers below each column indicate the number of animals (in A) and the number of neuronal profiles (in B). The number of sural neurons declined more if the entire sciatic nerve was cut; as mean diameter increased, small neurons had disappeared.

Figure 4.

Number and size of neurons labeled from muscles after different lesions. Somata labeled retrogradely with FG+ from injections into muscles of the lower hindlimb. Histograms show the number of labeled somata in each section (A) and their mean diameter (B). The lesions are indicated between the plots together with the time in weeks (wks) since the lesion: NC, naive control; C/l, contralateral to the lesions at 1 and 10 weeks (pooled); MusX, muscle nerves cut; SciX, sciatic nerve cut; SpiX, L5 spinal nerve cut. The numbers below each column indicate the number of animals (in A) and the number of neuronal profiles (in B). There was no significant loss of muscle afferents after any lesion. The cells atrophied slightly after spinal nerve transection.

Figure 3.

Changes in diameter of muscle afferent neurons in L5 DRGs after peripheral nerve transection. Somata labeled retrogradely with FG injected into the muscles of the lower hindlimb. A, C, Histograms of soma diameter with and without nerve transection and ligation. Control, One week after muscle injections with no nerve lesion (illustrated in B). Sciatic X, 10 wks, Ten weeks after cutting and ligating the sciatic nerve 1 week after muscle injections (illustrated in D1). Spinal X, 10 wks, Ten weeks after cutting and ligating the spinal nerve 1 week after muscle injections (illustrated in D2). Scale bar: (in B) A-D, 50 μm.

Figure 5.

Expression of p75 in satellite cells and neurons 10 weeks after sciatic nerve transection. Perineuronal rings are associated with p75+ satellite cells around cutaneous somata, whereas p75 is expressed in muscle rather than cutaneous somata after the lesion. A, Cutaneous afferent neurons labeled with FG. B, Muscle afferent neurons labeled with FG. 1, Varicose tyrosine hydroxylase (TH)-positive terminals. 2, FG-labeled neurons. 3, p75 immunoreactivity. Matching letters in the three micrographs indicate the same neuron. TH+ terminals were associated with large-diameter cutaneous neurons surrounded by a multiple layers of p75-positive satellite cells. In contrast, muscle neurons expressed p75 but lacked the ensheathment of p75+ satellite cells. Scale bar: (in B2) A1-B3, 50 μm.

Figure 6.

Density of perineuronal rings after different nerve lesions. Histograms show the number of perineuronal rings of terminals per section present in the L5 DRG 10 weeks after various lesions indicated at the bottom of the figure. Rings containing tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), galanin, and synaptophysin are shown for different lesions: SciX, sciatic nerve cut (black; n = 10 rats); SpiX, spinal nerve cut (left bold hatching; n = 3); SurX, sural nerve cut (right faint hatching; n = 4); SkinX, four skin nerves cut (white; n = 4); MusX, muscle nerves cut (vertical hatching; n = 4). The effects of sural, skin, and muscle nerve transections were all significantly different from sciatic and spinal nerve transections (p < 0.001). Asterisks indicate other differences: ^*p < 0.05; ^**p < 0.01. Note that there were virtually no rings formed after muscle nerve transection.

Figure 7.

Different types of immunoreactive perineuronal ring structures in DRGs 10 weeks after peripheral nerve transection. A, A ring of varicose galanin-positive terminals surrounds a cutaneous afferent neuron (arrows). B, A ring of varicose tyrosine hydroxylase-positive terminals surrounds a muscle afferent neuron (arrows). C, A ring of varicose synaptophysin-positive terminals. D, A small CGRP+ neuron soma surrounded by a coiled smooth CGRP+ axon, probably arising from the same cell. A, C, D, After sciatic nerve transections; B, after spinal nerve transection. Scale bar: (in D) A-D, 50 μm.

Figure 8.

Comparison of the density of perineuronal rings after different nerve lesions. Histograms show the ratios of the density of rings in DRG sections after sciatic, spinal, sural, and skin nerve lesions. Rings containing TH (black), CGRP (white), galanin (GAL, left bold hatching), and synaptophysin (Syn, right hatching). Except for the number of CGRP rings that includes coils (∼50% of total in each case), the ratios are similar. The effect of spinal nerve transection was similar to that of sciatic nerve transection. The effects of cutting four skin nerves was almost the same as that of cutting only the sural nerve except for the number of GAL+ rings. Syn+ rings were more numerous because Syn is present in varicosities of all types.