Peripheral nerve injury has recently been shown to be associated with a novel form of sprouting of postganglionic sympathetic afferent fibres. Following either lumbar spinal nerve ligation (Chung, Kim, Na, Park & Chung, 1993), complete sciatic nerve axotomy (McLachlan, Janig, Devor & Michaelis, 1993) or chronic constriction injury of the sciatic nerve (Ramer & Bisby, 1997a), sympathetic fibres sprout into affected ganglia where some sensory neurone cell bodies become wrapped by elaborate pericellular arborizations. These arborizations display synapse-like structures and form functional connections with sensory neurones (McLachlan et al. 1993). In several current animal models of neuropathic pain, as well as in certain clinical circumstances, a complete repertoire of pain behaviour following nerve injury is dependent upon an intact sympathetic nervous system (Kim & Chung, 1991; Blumberg & Jänig, 1994; Desmules, Kayser, Weilfuggaza, Bertrand & Guilbauld, 1995). Under such circumstances an interaction between the sympathetic and sensory nervous system is likely. The sympathetic sprouting within the dorsal root ganglion (DRG) is an important observation, since it may represent an anatomical correlate of the close functional coupling between sympathetic and sensory activity which many clinical and experimental observations imply.
To date the mechanism by which this sprouting occurs is uncertain. In transgenic mice, overexpression of nerve growth factor (NGF) in the skin leads to the formation of tyrosine hydroxylase-immunoreactive (TH-IR) basket structures within the trigeminal ganglia (Davis, Albers, Seroogy & Katz, 1994). Recently, Zhou, Rush & McLachlan (1996) reported that the sympathetic sprouts observed within the DRG following sciatic nerve axotomy were associated with p75-immunoreactive glial cells. The authors suggested that the low-affinity neurotrophin receptor may function as a presenting molecule for neurotrophin-3 (NT3) or NGF to trigger sympathetic sprouting within the DRG. However, a recent study demonstrated significant levels of sympathetic sprouting within the DRG, after axotomy, in mice lacking the p75 low-affinity receptor (Ramer & Bisby, 1997b). This suggests that additional factors may be involved in the sympathetic sprouting observed following axotomy.
A strong candidate molecule is leukemia inhibitory factor (LIF). LIF is a multifunctional cytokine and a member of the haemopoietin cytokine family defined by their interaction with the common receptor motif, gp130. Many of the gp130 cytokines, including LIF, play a role in phenotypic maturation during development of the peripheral nervous system (PNS) and promote neurite extension and morphologic maturation in cultured embryonic neuroblasts (Mehler & Kessler, 1995). In the adult nervous system LIF is normally undetectable (Yamamori, 1991) but is induced at the site of peripheral nerve axotomy (Banner & Patterson, 1994) and is retrogradely transported and accumulates within the DRG in a specific population of nociceptive-specific neurones (Thompson, Vernallis, Heath & Priestley, 1997). Following injury to the mature nervous system LIF also acts as an important phenotypic specifying factor (see Zigmond et al. 1996). Evidence exists therefore that LIF regulates phenotypic responses and may have actions in the mature nervous system in addition to its developmental role. The results from the present study extend the known roles of LIF in the adult PNS demonstrating that LIF is associated with sprouting of sympathetic fibres within the DRG following peripheral nerve axotomy.
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This study demonstrates that intrathecal infusion of LIF induces noradrenergic fibre sprouting and basket formation in intact dorsal root ganglia in the adult rat. Intrathecal delivery does not damage sensory or sympathetic neurones. An important observation therefore is that nerve injury itself is not a priori for sympathetic sprouting into the DRG. There is a large body of evidence on the other hand which indicates that LIF will directly interact with sympathetic neurones. LIF plays an important role in the modulation of neurotransmitter phenotype in both the intact adult sympathetic nervous system (Bamber, Masters, Hoyle, Brinster & Palmiter, 1994) and following sympathetic axotomy (Rao et al. 1993). Although there is evidence that LIF promotes neuronal growth in vitro this is the first observation that LIF is associated with sprouting of the intact adult sympathetic nervous system in vivo.
Although the mechanism by which LIF induces sympathetic sprouting is unclear, it is worthwhile considering likely locations of action of LIF in the present study. Regarding exogenous intrathecal administration, it is possible that LIF was distributed systemically at a sufficient concentration for a direct effect upon sympathetic terminals within peripheral effector organs or within the postganglionic sympathetic chain itself. The most probable site of interaction, however, is likely to be either within the spinal nerve or DRG. Intrathecally delivered substances, such as neurotrophins, penetrate into the spinal cord and dorsal roots and have biological effects within the DRG (Bennett, French, Priestley & McMahon, 1996; Verge, Gratto, Karchewski & Richardson, 1996). The conditions under which endogenous LIF is expressed have been well documented. Axotomy is a potent stimulus for LIF induction. Sciatic nerve axotomy leads to LIF expression within Schwann cells in both proximal and distal nerve stumps (Banner & Patterson, 1994; Sun & Zigmond, 1996) but not within the DRG (Curtis et al. 1994; Sun & Zigmond, 1996). LIF is, however, retrogradely transported to the DRG by sensory neurones (Curtis et al. 1994), in particular by small diameter nociceptive-specific neurones (Thompson et al. 1997). It is unlikely that the presence of LIF within these sensory neurone cell bodies may act directly as a trophic factor for sympathetic sprouting since many TH-IR baskets in this and other studies formed around large diameter cell profiles. Retrogradely transported LIF, or a secondary mediator, however, may be re-released into the DRG and act in a paracrine fashion, but this is yet to be demonstrated. Alternatively, LIF may act directly upon sympathetic neurones to induce sprouting. Transection of postganglionic sympathetic nerve trunks induces a substantial induction of LIF within relevant ganglia (Sun, Rao, Zigmond & Landis, 1994). 125I-labelled LIF may also be retrogradely transported by sympathetic neurones (Ure & Campenot, 1994). Accumulation or expression of LIF within postganglionic sympathetic neurones may therefore be an important step in sympathetic sprouting following nerve injury. The process by which such expression or accumulation triggers sprouting within the DRG is unknown.
There is considerable variation in the extent and time course of the sympathetic sprouting in the DRG between the various models of neuropathic pain currently employed. Lumbar spinal nerve transection produces a rapid (2 days) sprouting of adrenergic fibres into the DRG and peripheral nerve (Chung, Lee, Yoon & Chung, 1996) whilst the time course of basket formation following sciatic axotomy is much slower (Ramer & Bisby, 1997a). Comparison of the extent of basket formation between experimental groups in the present study would suggest that the time course of LIF-induced TH-IR basket formation most closely follows that observed following sciatic axotomy. Interestingly, the number of TH-IR fibres observed within the spinal nerve in the present study was not significantly different between experimental groups. It is likely that the increase in TH-IR fibre density within the spinal nerve in all three groups progresses more rapidly than the formation of TH-IR baskets within the DRG.
Bathing of the proximal end of the sciatic nerve with an antibody against the LIF receptor component gp130 significantly reduced the number of TH-IR baskets within the lumbar ganglia. This demonstrates that a significant portion of the sympathetic sprouting response to axotomy may be initiated by peripherally released factors which bind the gp130 motif. gp130 is the defining component of a number of closely related cytokines which include interleukins 6 and 11 (IL-6, IL-11), oncostatin-M (OSM), cardiotrophin-1 (CT-1) and ciliary derived neurotrophic factor (CNTF). Whilst the role of some of these factors such as OSM and CT-1 within the nervous system is undetermined, it is possible that others such as IL-6 or CNTF may play a significant part in the response to injury of either sympathetic or sensory nervous systems. Although CNTF may mimic the effect of LIF on peptide induction in sympathetic neurones under certain circumstances in vitro (Sun et al. 1994), there is good evidence that many of the biological actions of the gp130 family of cytokines are not strictly interchangeable (Piquet-Pellorce, Grey, Mereau & Heath, 1994). There is no evidence therefore that CNTF may play a role in sympathetic ganglia following axotomy in vivo (Zigmond et al. 1996). Indeed it is well known that, following peripheral nerve axotomy, expression of CNTF is significantly decreased (Sendtner, Stocki & Thoenen, 1992). A definitive answer to this point, however, awaits the development of gp130 ligand-specific antagonists.
In contrast to its effect upon the DRG, anti-gp130 was without effect upon the number of fibres present within the spinal nerve. Assuming significant inactivation of this receptor component by the antibody, it is likely that further factors may act as a stimulus to sympathetic sprouting. Neurotrophins are obvious candidates. Sympathetic axons in the central nervous system sprout in response to NGF (Isaacson, Saffran & Crutcher, 1992); however, data regarding the role of NGF following peripheral nerve axotomy are equivocal. Although there is a modest rise in NGF mRNA within the DRG following sciatic axotomy (Sebert & Shooter, 1993), and at the site of injury (Heumann, Korsching, Badtlow & Thoenen, 1987), retrograde transport of NGF to the DRG declines (Heumann et al. 1987) together with a reduced trkA synthesis (Verge, Riopelle & Richardson, 1989). Indeed many axotomy-induced responses within the DRG may be reversed by exogenous application of NGF. It is likely that multiple factors are involved in the sympathetic sprouting observed following peripheral axotomy and that the contributions of each factor may differ depending upon the type (transection vs. constriction) and location (proximal vs. distal) of the lesion. It has been demonstrated recently that the extent of sympathetic sprouting within the DRG is inversely related to the distance between the DRG and the injury site (Kim, Na, Nam, Park, Hong & Kang, 1996).
Previous studies have shown a correlation between sympathetic sprouting in the DRG and the development of behavioural signs of neuropathic pain (Chung et al. 1996; Ramer & Bisby, 1997a). Exogenous LIF will also induce mechanical allodynia in nerve-intact rats (Thompson, Dray & Urban, 1996). Although the effect of LIF in these latter experiments was acute, measured in terms of hours, it is tempting to speculate upon a link between LIF expression and the sympathetic nervous system in the development of neuropathic pain behaviours following peripheral nerve injury. It remains to be demonstrated whether a functional connection occurs between LIF-induced sympathetic sprouts and sensory neurones within the DRG. Interventions, however, which reduce the expression of LIF at the site of nerve injury or within sympathetic ganglia, may reduce sympathetic sprouting and may have a concomitant effect upon the degree of neuropathic pain experienced following peripheral nerve trauma.