Andrew Allchorne

1. Peripheral inflammation is characterized by heightened pain sensitivity. This hyperalgesia is the consequence of the release of inflammatory mediators, cytokines and growth factors. A key participant is the induction of the neurotrophin nerve growth factor (NGF) by interleukin-1 beta (IL-1 beta). 2. Tumour necrosis factor alpha (TNF alpha) has been shown both to produce hyperalgesia and to upregulate IL-1 beta. We have now examined whether the induction of TNF alpha in inflammatory lesions contributes to inflammatory sensory hypersensitivity by inducing IL-1 beta and NGF. 3. The intraplantar injection of complete Freund's adjuvant (CFA) in adult rats produced a localized inflammation of the hindpaw with a rapid (3 h) reduction in withdrawal time in the hot plate test and in the mechanical threshold for eliciting the flexion withdrawal reflex. 4. The CFA-induced inflammation resulted in significant elevation in the levels of TNF alpha, IL-1 beta and NGF in the inflamed paw. In the case of TNF alpha, an elevation was detected at 3 h, rose substantially at 6 h, peaked at 24 h and remained elevated at 5 days, with similar but smaller changes in the contralateral non-inflamed hindpaw. No increase in serum TNF alpha was detected at 24 h post CFA injection. 5. Intraplantar recombinant murine TNF alpha injections produce a short-lived (3-6 h) dose-dependent (50-500 ng) increase in thermal and mechanical sensitivity which was significantly attenuated by prior administration of anti-NGF antiserum. 6. Intraplantar TNF alpha (100-500 ng) also elevated at 6 but not 48 h the levels of IL-1 beta and NGF in the hindpaw. 7. A single injection of anti-TNF alpha antiserum, 1 h before the CFA, at a dose sufficient to reduce the effects of a 100 ng intraplantar injection of TNF alpha, significantly delayed the onset of the resultant inflammatory hyperalgesia and reduced IL-1 beta but not NGF levels measured at 24 h. 8. The elevation of TNF alpha in inflammation, by virtue of its capacity to induce IL-1 beta and NGF, may contribute to the initiation of inflammatory hyperalgesia.

1. Peripheral inflammation is associated with the local production of neuroactive inflammatory cytokines and growth factors. These may contribute to inflammatory pain and hyperalgesia by directly or indirectly altering the function or chemical phenotype of responsive primary sensory neurones. 2. To investigate this, inflammation was produced by the intraplantar injection of complete Freund's adjuvant (CFA) in adult rats. This resulted in a significant elevation in interleukin-1 beta (IL-1 beta) and nerve growth factor (NGF) levels in the inflamed tissue and of the peptides, substance P and calcitonin gene-related peptide (CGRP) in the L4 dorsal root ganglion 48 h post CFA injection. 3. The effects of a steroidal (dexamethasone) and a non-steroidal (indomethacin) anti-inflammatory drug on the levels of NGF and IL-1 beta in inflamed tissue were investigated and compared with alterations in behavioural hyperalgesia and neuropeptide expression in sensory neurones. 4. Systemic dexamethasone (120 micrograms kg-1 per day starting the day before the CFA injection) had no effect on the inflammatory hyperalgesia. When the dose was administered 3 times daily, a reduction in mechanical and to a lesser extent thermal sensitivity occurred. Indomethacin at 2 mg kg-1 daily (i.p.) had no effect on the hyperalgesia and a dose of 4 mg kg-1 daily was required to reduce significantly mechanical and thermal hypersensitivity. 5. The increase in NGF produced by the CFA inflammation was prevented by both dexamethasone and indomethacin, but only at the higher dose levels. Dexamethasone at the lower and higher dose regimes diminished the upregulation of IL-1 beta whereas indomethacin had an effect only at the higher dose. 6. The increase in SP and CGRP levels produced by the CFA inflammation was prevented by dexamethasone and indomethacin at the lower and higher dose regimes. 7. Intraplantar injections of IL-1 beta (0.01, 0.1 and 1 ng) produced a brief (6 h) thermal hyperalgesia and an elevation in cutaneous NGF levels which was prevented by pretreatment with human recombinant IL-1 receptor antagonist (IL-1 ra) (0.625 microgram, i.v.). The thermal hyperalgesia but not the NGF elevation produced by intraplantar IL-1 beta (1 ng) was prevented by administration of a polyclonal neutralizing anti-NGF serum. 8. IL-1 ra significantly reduced the mechanical hyperalgesia produced by CFA for 6 h after administration as well as the CFA-induced elevation in NGF levels. Anti-NGF pretreatment substantially reduced CFA-induced mechanical and thermal hyperalgesia without reducing the elevation in IL-1 beta. 9. Intraplantar NGF (0.02, 0.2 and 2 microg) injections produced a short lasting thermal and mechanical hyperalgesia but did not change IL-1beta levels in the hindpaw skin.10. Our results demonstrate that IL-1beta contributes to the upregulation of NGF during inflammation and that NGF has a major role in the production of inflammatory pain hypersensitivity.

BACKGROUND: Rat oligonucleotide microarrays were used to detect changes in gene expression in the dorsal root ganglion (DRG) 3 days following sciatic nerve transection (axotomy). Two comparisons were made using two sets of triplicate microarrays, naïve versus naïve and naïve versus axotomy. RESULTS: Microarray variability was assessed using the naïve versus naïve comparison. These results support use of a P < 0.05 significance threshold for detecting regulated genes, despite the large number of hypothesis tests required. For the naïve versus axotomy comparison, a 2-fold cut off alone led to an estimated error rate of 16%; combining a >1.5-fold expression change and P < 0.05 significance reduced the estimated error to 5%. The 2-fold cut off identified 178 genes while the combined >1.5-fold and P < 0.05 criteria generated 240 putatively regulated genes, which we have listed. Many of these have not been described as regulated in the DRG by axotomy. Northern blot, quantitative slot blots and in situ hybridization verified the expression of 24 transcripts. These data showed an 83% concordance rate with the arrays; most mismatches represent genes with low expression levels reflecting limits of array sensitivity. A significant correlation was found between actual mRNA differences and relative changes between microarrays (r2 = 0.8567). Temporal patterns of individual genes regulation varied. CONCLUSIONS: We identify parameters for microarray analysis which reduce error while identifying many putatively regulated genes. Functional classification of these genes suggest reorganization of cell structural components, activation of genes expressed by immune and inflammatory cells and down-regulation of genes involved in neurotransmission.