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Nerve injury, particularly of the brachial plexus, may result in lifelong disability and chronic pain, despite technically excellent reconstructive surgery. Studies of molecular changes in injured nerves may identify new treatments to enhance the success of nerve repair, such as with recombinant human neurotrophic factors. Interleukin-6 (IL-6) is a member of the neuropoietic cytokine family that includes ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), and oncostatin M. As there is increasing evidence of a neurotrophic role for IL-6 in animal models of nerve injury and inflammation,1 2 we have studied, for the first time in humans, IL-6 protein in injured and control peripheral nerve and dorsal root ganglia, using specific immunoassay, immunocytochemistry, and western blotting. We report a most remarkable increase of IL-6 concentrations in acutely avulsed dorsal root ganglia and injured nerves.
Proximal and distal injured nerve segments were obtained from six adult patients with traction brachial plexus injury, ranging from 2 weeks to 10 weeks after trauma. Injured dorsal root ganglia were collected from seven adult patients with brachial spinal root avulsion injuries (central axotomy), ranging from 3 days to 15 months after trauma. Tissue removal was a necessary part of the surgical repair procedure; in all cases informed consent was obtained for tissue collection, and the study had ethics committee approval. Control dorsal root ganglia were obtained from four subjects and segments of normal nerve from five subjects at postmortem, with a mean delay of 36 hours after death; all died from myocardial causes. Tissue extracts prepared as previously described3 4 were analysed for IL-6 using an enzyme immunoassay (Pelikine CompactTM, Eurogenetics, UK Ltd, Middlesex, UK) with recombinant human IL-6 standard calibrated against the World Health Organisation (WHO) First International Standard 89/548. For immunohistochemistry, frozen sections (8 μm) were fixed in 4% paraformaldehyde in phosphate buffered saline for 30 minutes. Sections were incubated with monoclonal antibodies to IL-6 (CLB.IL-6/7 20 μg/ml, diluted 1:400; gift from K Nordlind, Sweden, or ref No 1618–01, Genzyme, USA) and immunoreactivity to IL-6 visualised using a standard immunoperoxidase method (ABC; Vector Labs, UK) with nickel enhancement as described elsewhere.3 4 Specific immunoreactivity was extinguished by a concentration of rhIL-6 (ImmunoKontact, Frankfurt, ref 111–40–136) in the range 0.01–0.1 μg/ml. For western blotting, tissue extracts were separated by gel electrophoresis on 15% acrylamide gels and then electrophoretically transferred to nitrocellulose membranes (Hybond Super, Amersham) using a semidry transblotter. Strips were blocked in a solution of 5% non-fat milk in phosphate buffered saline (PBS) containing 0.05% Tween-20 for 1 hour and then probed with CLB.IL-6/7 (with or without IL-6 peptide, 20 μl) at a final titre of 1:1000, for 2 hours. The strips were then incubated with anti-rabbit HRP (Sigma) at 1:10,000 dilution for a further 30 minuttes. Bands were then visualised on x ray film after treatment with ECL reagents (Amersham).
Concentrations of IL-6 were increased in injured nerves (figure). The increase was greater in nerve segments distal to injury, but IL-6 concentrations in both proximal and distal segments were both significantly increased compared with controls (p<0.01, Mann-Whitney test). Concentrations of IL-6 were greatly raised in two avulsed dorsal root ganglia obtained 3 and 4 days after injury (354 pg/mg and 128 pg/mg). At longer operative delays after injury, IL-6 concentrations in avulsed dorsal root ganglia approached the range of values obtained for postmortem controls (4.9 (SD 2.9) pg/mg for operative delays from 1 week to 15 months, and 0.2 (SD 0.06) pg/mg for controls). Western blotting showed the presence of an expected strong 29 kDa band in detergent extracts of injured nerve, which was abolished in the presence of excess synthetic IL-6. Immunohistochemical studies with both antibodies demonstrated IL-6 staining within the soma of avulsed dorsal root ganglion neurons of all sizes, particularly of small size. Immunostaining of postmortem ganglia seemed similar in pattern, but was generally weaker. Interleukin-6 immunoreactivity was also seen in nerve-like structures within the dorsal root ganglia and distal injured nerve segments.
The pattern of changes of IL-6 in injured nerves and dorsal root ganglia differs from that seen for other neurotrophic factors, such as nerve growth factor (NGF) and glial-derived neurotrophic factor (GDNF).3 4 Concentrations of IL-6 were usually higher in distal nerve segments when compared with those proximal to the site of injury: this seems to result from IL-6 synthesis in Schwann cells in Wallerian degeneration, as shown by immunostaining, and previous animal model studies.2 Interleukin-6 and its receptor have been shown to be required for normal nerve regeneration in animal models, which may be enhanced with exogenous IL-6.1 The injured nerves presumably take up IL-6 synthesised in the Schwann cells, and transport it proximally, which accounts for the higher IL-6 concentrations in proximal segments of injured nerves in comparison with controls. Anterogradely transported IL-6, if released in the spinal cord, may play a part in pain processing, for which there is some evidence from animal models. In this study, the most remarkable finding was the very high concentration of IL-6 in extracts of the acutely avulsed dorsal root ganglia, with much smaller increases at later times after injury. The acute increase of IL-6 could originate from sensory cell bodies themselves, as has been shown with IL-6 mRNA in situ hybridisation studies in rat sensory ganglia after peripheral nerve injury,5 or from inflammatory cells. This increase may have autocrine/paracrine effects, which may aid cell survival, or have a role in sensory or sympathetic sprouting.
We conclude that IL-6 is a significant factor in the events after nerve injury in humans, particularly in sensory neurons. The potential therapeutic role in nerve repair for recombinant human IL-6, and agents that modulate its action, deserves further investigation.
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