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. 2012 Mar 20;29(5):925-35.
doi: 10.1089/neu.2011.2007. Epub 2011 Nov 4.

Spinal cord injury triggers an intrinsic growth-promoting state in nociceptors

Supinder S Bedi  1 , Michael T Lago, Luke I Masha, Robyn J Crook, Raymond J Grill, Edgar T Walters
Affiliations

Spinal cord injury triggers an intrinsic growth-promoting state in nociceptors

Supinder S Bedi et al. J Neurotrauma. .

Abstract

Although most investigations of the mechanisms underlying chronic pain after spinal cord injury (SCI) have examined the central nervous system (CNS), recent studies have shown that nociceptive primary afferent neurons display persistent hyperexcitability and spontaneous activity in their peripheral branches and somata in dorsal root ganglia (DRG) after SCI. This suggests that SCI-induced alterations of primary nociceptors contribute to central sensitization and chronic pain after SCI. Does SCI also promote growth of these neurons' fibers, as has been suggested in some reports? The present study tests the hypothesis that SCI induces an intrinsic growth-promoting state in DRG neurons. This was tested by dissociating DRG neurons 3 days or 1 month after spinal contusion injury at thoracic level T10 and measuring neuritic growth 1 day later. Neurons cultured 3 days after SCI exhibited longer neurites without increases in branching ("elongating growth"), compared to neurons from sham-treated or untreated (naïve) rats. Robust promotion of elongating growth was found in small and medium-sized neurons (but not large neurons) from lumbar (L3-L5) and thoracic ganglia immediately above (T9) and below (T10-T11) the contusion site, but not from cervical DRG. Elongating growth was also found in neurons immunoreactive to calcitonin gene-related peptide (CGRP), suggesting that some of the neurons exhibiting enhanced neuritic growth were nociceptors. The same measurements made on neurons dissociated 1 month after SCI revealed no evidence of elongating growth, although evidence for accelerated initiation of neurite outgrowth was found. Under certain conditions this transient growth-promoting state in nociceptors might be important for the development of chronic pain and hyperreflexia after SCI.

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Figures

FIG. 1.
FIG. 1.
Examples of neurite growth in small dorsal root ganglia (DRG) neurons dissociated from rats that were naïve (A), that had received sham surgery (B), or that had received spinal cord injury (SCI) 3 days before dissociation (C). In each case a digital image was taken of a fixed DRG neuron from spinal level T11 to produce a camera-lucida representation of all visible branches. The longest neurite in the neuron sampled from the SCI animal is indicated (white highlight), along with each branch point on the neurite (arrowheads), and the longest unbranched segment on the longest neurite (adjacent dashed black line; scale bar=100 μm).
FIG. 2.
FIG. 2.
Spinal cord injury (SCI) increases the length of neurites growing from dorsal root ganglia (DRG) neurons taken from segments immediately above and below the injury, and from the lumbar region 3 days after T10 contusion injury. (A) Mean length of longest neurite in small neurons (soma diameter < 30 μm) 1 day after dissociation. Sample sizes (number of cells/number of rats) were, respectively, for the naive, sham-treated, and SCI groups at each spinal level: cervical (14/5, 13/6, 14/7), above the injury (11/4, 25/7, 41/8), below the injury (8/3, 40/6, 36/6), and lumbar (9/5, 14/5, 10/5) levels. (B) Mean length of the longest neurite in medium-sized neurons (31–45 μm). Sample sizes were, respectively, for the naive, sham-treated, and SCI groups: cervical (9/5, 31/7, 31/7), above the injury (13/5, 50/7, 71/7), below the injury (20/5, 61/7, 49/7), and lumbar (10/3, 18/6, 32/7) levels. (C) Mean length of the longest neurite in large neurons (>45 μm). Sample sizes were, respectively, for the naive, sham-treated, and SCI groups: cervical (7/2, 18/5, 7/3), above the injury (6/3, 32/5, 19/6), below the injury (13/5, 31/6, 19/4), and lumbar (4/5, 21/6 15/5) levels. Sample sizes are the same for the other morphological parameters measured 3 days after injury (Figs. 3 and 4). In this and subsequent figures error bars denote standard error of the mean, and significant differences between the SCI and naïve groups are indicated by asterisks (*p<0.05, **p<0.01, ***p<0.001), while plus signs indicate the corresponding significant differences between the SCI and sham groups (+, ++, +++), and pound signs indicate the differences between the sham and naïve groups (#, ##, ###). (Open bars, naïve group; gray bars, sham group; black bars, SCI group.)
FIG. 3.
FIG. 3.
Spinal cord injury (SCI) does not increase the number of branches per longest neurite 3 days after T10 contusion injury. (A) Mean number of branches on the longest neurite in small neurons (soma diameter <30 μm) at each level sampled 1 day after dissociation. (B) Mean number of branches in medium-sized neurons (31–45 μm). (C) Mean number of branches on the longest neurite in large neurons (>45 μm). (Open bars, naïve group; gray bars, sham group; black bars, SCI group.)
FIG. 4.
FIG. 4.
Spinal cord injury (SCI) increases the length of the longest unbranched segment in the longest neurite 3 days after T10 contusion injury. (A) Mean length of the longest segment in small neurons (soma diameter <30 μm) 1 day after dissociation. (B) Mean length of the longest segment in medium-sized neurons (31–45 μm). (C) Mean length of the longest segment in large neurons (>45 μm). (Open bars, naïve group; gray bars, sham group; black bars, SCI group.)
FIG. 5.
FIG. 5.
Examples of neurite growth in dorsal root ganglia (DRG) neurons immunostained for calcitonin gene-related peptide (CGRP, green) and neurotubulin (red). Photomicrographs show neurons dissociated from DRG with dorsal root entry zones at spinal level T11 in sham-treated (A) and SCI (B) animals. Notice the longer processes extending from the DRG neuron taken from the SCI rat.
FIG. 6.
FIG. 6.
Spinal cord injury (SCI) promotes elongating growth of neurites from calcitonin gene-related peptide (CGRP)-immunoreactive dorsal root ganglia (DRG) neurons from lumbar and thoracic segments. Neurons were fixed 1 day after dissociation, which occurred 3 days after SCI or sham treatment. (A) Increased length of the longest neurite in the SCI group. (B) Increased length of the longest unbranched segment on the longest neurite in the SCI group. (C) Lack of significant effect of SCI on the number of branches on the longest neurite. (D) Decrease in the number of neurons exhibiting uniform neuritic CGRP staining in neurons sampled from DRGs immediately below the thoracic SCI site. A lack of uniform neuritic staining in neurons with CGRP-immunoreactive somata was defined as either the absence of detectable neuritic staining or staining solely in isolated neuritic puncta. Sample sizes (number of cells/number of rats in parts A–D) were, respectively, for the naive, sham-treated, and SCI groups at each spinal level: cervical (15/2, 13/2, 14/2), above the injury (15/2, 13/2, 14/2), below the injury (15/2, 13/1, 14/1), and lumbar (15/2, 13/2, 14/2) levels. (Open bars, naïve group; gray bars, sham group; black bars, SCI group.)
FIG. 7.
FIG. 7.
Spinal cord injury (SCI) enhances neurite elongation and growth initiation 3 days after injury, but only neurite growth initiation is enhanced 1 month after injury. (A and B) SCI increased the length of the longest neurite on small dorsal root ganglia (DRG) neurons sampled from lumbar and thoracic DRG 3 days after injury, but not 1 month after injury. The 3-day data are pooled from the results shown at each level in Figure 2A (excluding the cervical DRG neurons). Injury also affected the initiation of neurite outgrowth, as monitored by the fraction of neurons having neurite lengths≥the diameter of the cell soma (C and D). Over each bar are shown the number of neurons having neurites over the total number of neurons measured. (C) Neurons dissociated 3 days after injury from both SCI and sham-treated animals were more likely than neurons from naïve animals to have neurites 1 day later. (D) After 1 month, only neurons from SCI animals had a higher incidence of neurites 1 day after dissociation.
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