Central Consequences of Peripheral Nerve Damage

Introduction

In the first Textbook of Pain , edited by Patrick Wall and Ronald Melzack and published in 1984, basic aspects of the relationship between damage to a peripheral nerve and pain were discussed by Marshall , as well as in several clinically oriented chapters, including one by John on peripheral neuropathies. Devor’s chapter dealt with nerve injury and focused on the pathophysiology and anatomy of the damaged nerve, in particular, on processes leading to the production of abnormal impulse discharges and pain. In the mid-1970s, reported massive spontaneous discharges in the L4 and L5 dorsal rootlets after producing an experimental neuroma by creating a peripheral lesion of the sciatic nerve. and described the time course: a lack of discharges during the first few days followed by spontaneous high activity for several weeks. It was proposed that the nerve damage and neuroma lead to the establishment of ectopically generated abnormal impulses and amplification of impulse discharges and that this effect contributes to chronic neuropathic pain.

At that time, little was known about the dramatic and robust chemical changes occurring in the dorsal root ganglia (DRGs) after peripheral nerve damage, which presumably contributed to the changes in electrical phenomena monitored by Wall, Devor, and their collaborators, as well as others. In this chapter we focus on such chemical changes induced in DRG neurons by nerve injury, including changes in intracellular messengers and their receptors, enzymes, ion channels, and other molecules. We also discuss the issue of nerve injury–induced sprouting of primary afferents in the dorsal horn.

Much of the earlier work was carried out on rats after complete transection of the sciatic nerve, the model introduced by Wall and collaborators, which resulted in robust and reproducible effects. Subsequently, several other “functional” pain models were introduced (see Chapter 11, Chapter 65 ). were the first to show changes in peptide expression in DRGs in the chronic nerve constriction model (four loose ligatures) developed by Bennett and Xie; these changes are similar to those seen after complete nerve transection (see ). The purpose and consequences of these changes are still not well understood, but they may contribute to survival and regeneration of the damaged neuron and also to generation and/or attenuation of pain. It is, however, clear that these changes are not confined to neurons but affect satellite and other non-neuronal cells in the DRG as well. They also extend to the spinal cord as trans-ganglionic effects, which we will discuss with regard to functional, anatomical, and chemical consequences. However, we will not deal with events occurring at higher centers (see Chapter 7 ). It may be anticipated that more complete comprehension of these processes may lead to improved understanding of and ultimately novel treatment strategies for chronic pain. This topic has been dealt with in many reviews, and a few published after the fifth edition of Textbook of Pain may be mentioned ( ) (see also Chapter 5, Chapter 6, Chapter 61 ).

Gene Expression in Dorsal Root Ganglia After Nerve Injury

Sixty years ago, Fred Lembeck in Graz suggested that substance P, an 11–amino acid peptide, could be a transmitter in sensory neurons. The presence of this and several other peptides—for example, calcitonin gene-related peptide (CGRP), somatostatin, and pituitary adenylate cyclase–activating peptide—in subpopulations of normal DRGs in rats and other species could subsequently be demonstrated.

Early evidence that nerve injury could influence the expression of messenger molecules in DRGs was presented by Jessell, Otsuka, and associates, who showed that peripheral transection of the sciatic nerve causes a decrease in substance P levels in the dorsal horn. It could later be demonstrated that this decrease in fact represented down-regulation of substance P synthesis in DRG neurons ( ). Evidence of nerve injury–induced messenger up-regulation in DRG neurons was first obtained for vasoactive intestinal polypeptide (VIP) by . Subsequently, it has turned out that a very large number of molecules—including several neuropeptides such as substance P, CGRP, neuropeptide Y (NPY), galanin, and pituitary adenylate cyclase–activating peptide ( Fig. 63-1 A and B); enzymes such as nitric oxide synthase; and receptors for peptide and classic transmitters synthesized in DRG neurons—are regulated by axotomy (see ). This is also seen in several neuropathic pain models without complete transection.

Importantly, in inflammatory models substance P and CGRP are up-regulated in DRGs, but no distinct effects on galanin, VIP, or NPY have been recorded. Instead, inflammation activates opioid and other peptides in the dorsal horn (see ).

The DRG neurons discussed thus far are often termed the peptide (-positive) population because of their expression of several peptides. They represent 35–40% of all DRG neurons, have unmyelinated or thinly myelinated axons, and are assumed to be involved in nociception.

The non-peptide DRG neurons represent a second class ( ). They account for 20–30% of the DRG neurons and are characterized by several markers, such as the non-lysosomal fluoride-resistant acid phosphatase, the P2X ₃ purinoceptor, the vanilloid receptor 1 (VR1, now called transient receptor potential vanilloid 1 [TRPV1]), and receptor components of glial cell line–derived neurotrophic factor (GDNF), especially RET mRNA. They bind the lectin Griffonia simplicifolia isolectin B4 (IB4). Some of these markers, such as TRPV1 and P2X ₃ , are up-regulated after peripheral nerve injury, whereas others are down-regulated. These neurons are also small with non-myelinated axons and are assumed to represent nociceptors.

The remaining 30–40% of DRG neurons are large and medium sized. These neurons have fast-conducting, myelinated Aα/β-range axons and receive input from peripheral mechanoreceptors.

It has long been assumed that glutamate is the principal transmitter in DRG neurons. However, only after the three vesicular glutamate transporters (VGLUT1–3) had been discovered was it possible to identify, with certainty, glutamatergic neurons with the microscope (see ). In fact, with this approach it now seems that in rodents all DRG neurons express one or more VGLUTs ( ), thus confirming the general view that they use glutamate as a transmitter. Axotomy causes a reduction in VGLUT1 and -2 in DRG neurons, although a population of small neurons show increased staining ( ).

For a comprehensive account of neuron types, their receptors, and ion channels expressed in DRGs (and examples of their regulation), we refer readers to a review by .

Some Methodological Aspects

Many early nerve injury studies are based on complete transection of the sciatic nerve of the rat at the mid-thigh level, and in general, percentages of DRG neuron profiles expressing a particular molecule (immunohistochemistry) or a transcript (in situ hybridization) are calculated. It is important to note that this lesion will affect only approximately 70–80% of the L5 DRG neurons projecting into the sciatic nerve because the remaining ones branch off central to the transection ( ). Moreover, an important issue is to what extent the nerve injury causes cell loss, which has not been monitored in most studies. More recently, stereological techniques have shown that in the rat no significant loss of neurons occurs up until 4 weeks after a lesion at the mid-thigh level ( , but see ) whereas a spinal nerve lesion (close to the cell bodies) causes a progressive loss of neuronal cells ( ). In the mouse, axotomy at the mid-thigh level causes a 24% loss after 7 days and a 50% loss after 28 days ( ), possibly because of the short distance between the lesion and cell bodies in this small animal. To what extent neurogenesis occurs in DRGs, particularly after nerve injury, has not been established. For animal pain models, see Chapter 11 .

Global Gene Expression Studies

Global gene expression has been analyzed with array techniques to monitor genes expressed in DRGs after peripheral nerve injury versus those expressed in non-lesioned ganglia ( ). In these analyses many different types of genes show marked changes ( Table 63-1 ).

found changes in 178 or 240 genes (depending on the cutoff) after axotomy. showed greater than two-fold up-regulation of 102 genes and greater than two-fold down-regulation of 46 genes in a spinal nerve ligation neuropathic pain model. As shown in Figure 63-2 , approximately 2.3 and 2.8% of the genes have been down- and up-regulated, respectively, if genes with a more than 1.5-fold regulation and P < 0.05 are considered. The genes have been categorized as indicated in Figure 63-2 , and bidirectional changes are found in all classes except for translational regulation genes, where no down-regulation was observed. In many cases, approximately the same number of genes go in either direction, although up-regulation dominates in the categories apoptosis, cytoskeleton, and immunologically related genes. Down-regulation prevails in the categories ion channels, neurotransmission, vesicle trafficking, and unknown genes.

examined 7523 genes and sequence tags expressed after axotomy, of which 122 and 51, respectively, were strongly changed (see Table 63-1 ). In a similar manner as the Costigan study (2002), several families were identified, although under somewhat different headings and confined to 10 classes ( Table 63-1 ).

Xiao and colleagues used a two-fold change in signal intensity as a cutoff line. They identified 122 genes and 51 expressed sequence tags, which represented 2.3% of the total genes examined. Among these the vast majority (86%) had not previously been identified in DRGs after nerve injury ( ). Thus, these array studies have not only provided evidence of regulatory changes but also showed the presence of a number of genes hitherto not identified in DRGs. Interestingly, the changes are long lasting, often throughout the entire period studied (28 days) (see Table 63-1 ). It is known from other studies that changes in expression can last for long periods if regeneration is efficiently prevented. If not, regeneration will take approximately a month in the rat after complete mid-thigh transection, and most changes then revert to normal. The significance of some of these results has been discussed by Xiao and co-authors (2002, and references therein) and is summarized here.

Not unexpectedly, neuropeptides belonged to the group of compounds showing the most dramatic changes, particularly with regard to up-regulation (see Table 63-1 ). One reason is that neuropeptides, in contrast to enzymes and other proteins, are released from the neuron and have to be replaced via ribosomal synthesis in the cell soma. This is reflected by distinct changes in peptide and, especially, mRNA levels and can be conveniently recorded in single cells by in situ hybridization or immunohistochemistry. In contrast, most other messenger molecules, such as the classic transmitters (e.g., acetylcholine and noradrenaline), are synthesized by enzymes in all parts of neurons, and efficient transporter molecules allow reuptake and recycling of the transmitter. Down-regulation of excitatory peptides (substance P and CGRP) will attenuate transmission, and some up-regulated peptides (VIP, galanin, and NPY) affect transmission but in addition improve survival and regeneration (see, for example, ).

Compounds of importance for survival—such as heat shock protein 27, endoplasmic reticular stress protein, and the LIM domain protein CLP36—are transiently up-regulated. In addition, the transcription factor Jun-D and eukaryotic initiation factor 4E, a translation initiation factor, were up-regulated. Interestingly, two important growth factors, brain-derived neurotrophic factor (BDNF) and glial cell line–derived neurotrophic factor (GDNF), were increased less than twofold. However, basic fibroblast growth factor was strongly up-regulated.

With regard to synaptic transmission, most molecules were down-regulated, and only synaptotagmin IV showed an increase. However, synaptotagmin IV can form hetero-oligomers with synaptotagmin I, and this results in less efficient Ca ²⁺ coupling. Thus, in general, the changes in synaptic vesicle proteins tend to work toward attenuation of transmission in the dorsal horn.

The expression of receptors for messenger molecules is often changed. For example, several of the neuropeptide receptors—such as the cholecystokinin B receptor (CCK _B , CCK2) and the NPY Y ₂ and Y ₅ receptors, as well as a nicotinic acetylcholine receptor subtype (α ₇ ), a purinoceptor (P2Y ₁ ), the α _2A -adrenergic receptor, and the benzodiazepine receptor of the peripheral type—were up-regulated, whereas others were down-regulated. Clearly, it is a difficult puzzle to understand the functional significance of these complex regulations.

Peripheral nerve injury also causes molecular modification in the dorsal horn of the spinal cord. Using a cDNA array, found that expression of the genes encoding 14 channels, 25 receptors, and 42 signal transduction–related molecules is strongly regulated in the spinal dorsal horn following peripheral axotomy. Although knowledge about these regulated genes and the related functional consequences is very limited, current findings suggest that novel signal pathways may be generated in the dorsal spinal cord after peripheral nerve injury. For example, there is a significant increase in the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors ( ), which may be accessible to the elevated levels of glutamate resulting from the decrease in glutamate uptake ( ). These changes may be involved in mechanisms underlying the increased neuronal sensitivity after nerve injury ( ).

Classic Growth Factors

Classic growth factors play a major role in the development, physiology, and pathophysiology of DRGs and sensory mechanisms. They are often target derived and therefore localized in non-neuronal cells, whereas their tyrosine kinase (Trk) receptors (TrkA, TrkB, and TrkC) are often present in neurons. Many studies have been devoted to analysis of these growth factors in non-neuronal cells (including satellite and Schwann cells) and their receptors under normal circumstances and after injury, but this topic is not discussed here (but see ). However, we will address BDNF and its reaction to nerve injury and inflammation because it is neuronally localized. (GDNF and nerve growth factor [NGF] have, however, been suggested to have a neuronal localization in some studies.)

Under normal circumstances, BDNF is constitutively expressed in 11–38% of DRG neurons, mainly in small TrkA-positive neurons ( ; ; ; ; ; ). Axotomy of a peripheral nerve remote from the cell body induces up-regulation of BDNF mRNA, mainly in medium-sized and large-diameter neurons expressing TrkB and TrkC, that is, NGF-insensitive neurons ( ). NPY is up-regulated in the same neurons ( ). BDNF expression in most TrkA cells was, however, unchanged ( ).

In contrast, spinal nerve ligation increases BDNF expression mainly in small-sized neurons ( ), thus showing clear differential regulation depending on the type of nerve injury. Peripheral tissue inflammation increases BDNF mRNA in DRG neurons ( ), whereas capsaicin causes a marked down-regulation ( ). Interestingly, BDNF is packaged in large dense-core vesicles (the same vesicles that store neuropeptides) and then transported into the dorsal horn of the spinal cord ( ), which suggests that it can be released and control spinal cord excitability in a transmitter-like fashion (see ). In Figure 63-1 C, the relationship of several growth factor receptors and other molecules to some major DRG neuron markers (CGRP, IB4, and neurofilament 200 [NF200]) has been summarized.

Species Differences

Most studies on the effect of nerve injury on the expression of various molecules in DRGs have been done on rats, but mice are increasingly also being analyzed and have demonstrated no obvious qualitative differences. However, in other species, differences have been encountered (see ). Thus, in the guinea pig, up-regulation of galanin is much more restricted than in the rat and mouse, whereas NPY regulation is similar. In addition, up-regulation of galanin is very modest in the cat. Up-regulation of galanin is strong in the monkey ( Macaca mulatta ), whereas NPY expression is hardly detectable. In human ganglia, galanin is expressed in some 10–15% of the DRG neuron profiles versus around 40–50% of CGRP-positive neurons ( ), a proportion that is similar to that found in the monkey. Thus, galaninergic mechanisms may exist at the spinal level in humans as in certain rodents and M. mulatta .

Role of Voltage-Gated Sodium Channels

Voltage-gated sodium channels in DRG neurons play a crucial role in chronic pain. Many subtypes of sodium channels are localized in DRG neurons (see ). Based on their sensitivity to tetrodotoxin (TTX), these sodium channels are classified as TTX-sensitive or TTX-resistant subtypes. Both electrophysiological experiments and pain behavior tests show that the TTX-sensitive subtypes of sodium channels play important roles in generating ectopic discharges in injured sensory neurons and in maintaining allodynic behavior in animal models of neuropathic pain ( ). Moreover, following peripheral nerve injury, expression of the TTX-sensitive sodium channels Na _v 1.3 and Na _v 2 is up-regulated in primary sensory neurons, whereas Na _v 1.1, Na _v 1.2, Na _v 1.6, Na _v 1.7, Na _v 1.8, and Na _v 1.9 are down-regulated ( ). These findings suggest that TTX-sensitive sodium channels, especially Na _v 1.3 and some other sodium channels, are potentially important in generating and maintaining neuropathic pain.

Mechanisms Underlying Injury-Induced Phenotypic Changes

The mechanisms underlying up- and down-regulation of the various molecules in sensory ganglia are only incompletely understood. In several cases, classic neurotrophic factors play a role (see ). A common denominator does not seem to exist, and every molecule may have its own regulatory pathway. These problems have been analyzed in vivo in DRGs for substance P, CGRP, and galanin, whereas knowledge of VIP and NPY mainly stems from in vitro work and studies on cell lines. In the case of the distinct down-regulation of the two excitatory peptides substance P and CGRP, a clear mechanism has, however, been identified. Thus it was early shown that NGF is essential for the expression of these two peptides and that axotomy-induced down-regulation can be reversed by the administration of exogenous NGF (see ). The nerve transection interrupts the centripetal transport of peripheral, target-derived NGF to the cell body; even if the injury increases NGF production in Schwann cells and fibroblasts around the injury site, this cannot compensate for the loss of NGF produced in the skin (see ).

There is some evidence of the mechanisms underlying axotomy-induced regulation of galanin. Galanin up-regulation is markedly impaired in mice lacking the gene for leukemia inhibitory factor (LIF) ( ). LIF is produced in DRG neurons during development and is up-regulated in Schwann cells after nerve injury ( ). Also, NGF may play a role by having antagonistic action on LIF induction. The nerve injury–induced inhibition of NGF transport to the cell bodies may stimulate the synthesis of LIF and thus enhance galanin production. In addition, the cytokine interleukin-6 (IL-6) may participate, because IL-6 transcript levels increase in DRG neurons and Schwann cells after nerve injury and galanin up-regulation is impaired in IL-6 knockout mice ( ). It was therefore suggested that the effect of LIF on galanin synthesis is mediated through IL-6 ( ).

In the case of VIP, focus has been on ciliary neurotrophic factor, a member of the neuropoietic cytokine family just like LIF (see and references therein). Ciliary neurotrophic factor and fibroblast-transforming growth factor-β (FGF-β) induce transcription of VIP through a 180–base pair cytokine response element in the VIP promoter. These molecules act synergistically, with ciliary neurotrophic factor inducing signal transducer and activator of transcription (STAT)- and small (body size) mothers against decapentaplegic (Smad)-containing complexes.

With regard to the NPY gene, both NGF and BDNF stimulate the NPY promoter ( and references therein). In vivo studies suggest that members of the FGF family can attenuate NPY up-regulation in large DRG neurons, in agreement with the presence of these growth factors and their receptors on DRG neurons and associated cells ( ).

Subsequently, focus turned to GDNF and related molecules, and it was demonstrated that they not only partially or completely reverse many of the nerve injury–induced morphological and neurochemical changes described earlier but also block the associated neuropathic pain state ( ). These findings open up new venues for understanding the mechanisms underlying neuropathic pain and its treatment.