Janet Winter

Nerve growth factor contributes to the generation of inflammatory sensory hypersensitivity

Experimental inflammation produced by an intraplantar injection of complete Freunds adjuvant results in local sensory hypersensitivity and up-regulates the neuropeptides substance P and calcitonin gene related peptide in the primary sensory neurons innervating the inflamed tissue. The inflammation also elevates nerve growth factor levels in the skin. Systemic administration of anti-NGF neutralizing antibodies prevent the behavioral sensitivity, the up-regulation of neuropeptides and the inflammation-induced expression of the immediate early gene c-fos in dorsal horn neurons, without modifying swelling and erythema. Elevation of the neurotrophin NGF in the periphery is a major contributor, therefore, of inflammatory pain.

Contribution of interleukin‐1β to the inflammation‐induced increase in nerve growth factor levels and 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 Freunds adjuvant (CFA) in adult rats. This resulted in a significant elevation in interleukin‐ip (IL‐1β) 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‐1β in inflamed tissue were investigated and compared with alterations in behavioural hyperalgesia and neuropeptide expression in sensory neurones. 4 Systemic dexamethasone (120 μg 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‐1β 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‐1β (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 μg, i.v.). The thermal hyperalgesia but not the NGF elevation produced by intraplantar IL‐1β (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‐1β. 9 Intraplantar NGF (0.O2, 0.2 and 2 μg) injections produced a short lasting thermal and mechanical hyperalgesia but did not change IL‐1β levels in the hindpaw skin. 10 Our results demonstrate that IL‐1β contributes to the upregulation of NGF during inflammation and that NGF has a major role in the production of inflammatory pain hypersensitivity.

VR1 protein expression increases in undamaged DRG neurons after partial nerve injury

Changes in phenotype or connectivity of primary afferent neurons following peripheral nerve injury may contribute to the hyperalgesia and allodynia associated with neuropathic pain conditions. Although earlier studies using partial nerve injury models have focused on the role of damaged fibres in the generation of ectopic discharges and pain, it is now thought that remaining undamaged fibres may be equally important. We have examined the expression of the sensory neuron‐specific cation channel Vanilloid Receptor 1 (VR1), an important transducer of noxious stimuli, in three models of nerve injury in the rat, using anatomical separation or fluorescent retrograde tracers to identify damaged or undamaged sensory neurons. After total or partial sciatic nerve transection, or spinal nerve ligation, VR1‐immunoreactivity (IR) was significantly reduced in the somata of all damaged dorsal root ganglion (DRG) neuronal profiles, compared to controls. However, after partial transection or spinal nerve ligation, VR1 expression was greater in the undamaged DRG somata than in controls. Unexpectedly, after L5 spinal nerve ligation, VR1‐IR of the A‐fibre somata increased ≈ 3‐fold in the uninjured L4 DRG compared to controls; a much greater increase than seen in the somata with C‐fibres. Furthermore, we found that VR1‐IR persisted in the transected sciatic nerve proximal to the lesion, despite its down‐regulation in the damaged neuronal somata. This persistence in the nerve proximal to the lesion after nerve section, together with increased VR1 in DRG neurons left undamaged after partial nerve injury, may be crucial to the development or maintenance of neuropathic pain.

Inhibition of spinal microglial cathepsin S for the reversal of neuropathic pain

A recent major conceptual advance has been the recognition of the importance of immune system–neuronal interactions in the modulation of brain function, one example of which is spinal pain processing in neuropathic states. Here, we report that in peripheral nerve-injured rats, the lysosomal cysteine protease cathepsin S (CatS) is critical for the maintenance of neuropathic pain and spinal microglia activation. After injury, CatS was exclusively expressed by activated microglia in the ipsilateral dorsal horn, where expression peaked at day 7, remaining high on day 14. Intrathecal delivery of an irreversible CatS inhibitor, morpholinurea-leucine-homophenylalanine-vinyl phenyl sulfone (LHVS), was antihyperalgesic and antiallodynic in neuropathic rats and attenuated spinal microglia activation. Consistent with a pronociceptive role of endogenous CatS, spinal intrathecal delivery of rat recombinant CatS (rrCatS) induced hyperalgesia and allodynia in naïve rats and activated p38 mitogen-activated protein kinase (MAPK) in spinal cord microglia. A bioinformatics approach revealed that the transmembrane chemokine fractalkine (FKN) is a potential substrate for CatS cleavage. We show that rrCatS incubation reduced the levels of cell-associated FKN in cultured sensory neurons and that a neutralizing antibody against FKN prevented both FKN- and CatS-induced allodynia, hyperalgesia, and p38 MAPK activation. Furthermore, rrCatS induced allodynia in wild-type but not CX3CR1-knockout mice. We suggest that under conditions of increased nociception, microglial CatS is responsible for the liberation of neuronal FKN, which stimulates p38 MAPK phosphorylation in microglia, thereby activating neurons via the release of pronociceptive mediators.

Pain related behaviour in two models of osteoarthritis in the rat knee

Abstract Osteoarthritis (OA) is a major healthcare burden, with increasing incidence. Pain is the predominant clinical feature, yet therapy is ineffective for many patients. While there are considerable insights into the mechanisms underlying tissue remodelling, there is poor understanding of the link between disease pathology and pain. This is in part owing to the lack of animal models that combine both osteoarthritic tissue remodelling and pain. Here, we provide an analysis of pain related behaviours in two models of OA in the rat: partial medial meniscectomy and iodoacetate injection. Histological studies demonstrated that in both models, progressive osteoarthritic joint pathology developed over the course of the next 28 days. In the ipsilateral hind limb in both models, changes in the percentage bodyweight borne were small, whereas marked mechanical hyperalgesia and tactile allodynia were seen. The responses in the iodoacetate treated animals were generally more robust, and these animals were tested for pharmacological reversal of pain related behaviour. Morphine was able to attenuate hyperalgesia 3, 14 and 28 days after OA induction, and reversed allodynia at days 14 and 28, providing evidence that this behaviour was pain related. Diclofenac and paracetamol were effective 3 days after arthritic induction only, coinciding with a measurable swelling of the knee. Gabapentin varied in its ability to reverse both hyperalgesia and allodynia. The iodoacetate model provides a basis for studies on the mechanisms of pain in OA, and for development of novel therapeutic analgesics.Abbreviations: OA: osteoarthritis; DRGs: dorsal root ganglia; NSAIDs: non‐steroidal anti‐inflammatory drugs; PWT: paw withdrawal thresholds; p.o.: orally; s.c.: sub cutaneously.

A rat model of bone cancer pain

&NA; This study describes the first known model of bone cancer pain in the rat. Sprague–Dawley rats receiving intra‐tibial injections of syngeneic MRMT‐1 rat mammary gland carcinoma cells developed behavioural signs indicative of pain, including: mechanical allodynia, difference of weight bearing between hind paws and mechanical hyperalgesia. The development of the bone tumour and structural damage to the bone was monitored by radiological analysis, quantitative measurement of mineral content and histology. Intra‐tibial injections of 3×103 or 3×104 syngeneic MRMT‐1 cells produced a rapidly expanding tumour within the boundaries of the tibia, causing severe remodelling of the bone. Radiographs showed extensive damage to the cortical bone and the trabeculae by day 10–14 after inoculation of 3×103 MRMT‐1 cells, and by day 20, the damage was threatening the integrity of the tibial bone. While both mineral content and mineral density decreased significantly in the cancerous bone, osteoclast numbers in the peritumoural compact bone remained unchanged. However, tartarate‐resistant acid phosphatase staining revealed a large number of polykariotic cells, resembling those of osteoclasts within the tumour. No tumour growth was observed after the injection of heat‐killed MRMT‐1 cells. Intra‐tibial injections of 3×103 or 3×104 MRMT‐1 cells, heat‐killed cells or vehicle did not show changes in body weight and core temperature over 19–20 days. The general activity of animals after injection with live or heat‐killed MRMT‐1 cells was higher than that of the control group, however, the activity of the MRMT‐1 treated group declined during the progress of the disease. Rats receiving intra‐tibial injections of MRMT‐1 cells displayed the gradual development of mechanical allodynia and mechanical hyperalgesia/reduced weight bearing on the affected limb, beginning on day 12–14 or 10–12 following injection of 3×103 or 3×104 cells, respectively. These symptoms were not observed in rats receiving heat‐killed cells or vehicle. Behavioural data suggest a reasonable time window for evaluation of anti‐nociceptive agents between day 14 and 20 after cancer cell inoculation in this model. Acute treatment with morphine (1–3 mg/kg, subcutanously (s.c.)) produced a dose‐dependent reduction in the response frequency of hind paw withdrawal to von Frey filament stimulation 17 or 19 days following intra‐tibial injections of 3×103 MRMT‐1 cells. A significant reduction in the difference in hind limb weight bearing was also observed. Acute treatment with celebrex (10–30 mg/kg, s.c.) did not affect mechanical allodynia or difference in weight bearing in rats 20 days following treatment with 3×103 MRMT‐1 cells. Although the pathophysiology of cancer pain is largely unknown, significant enhancement of glial fibrillary acidic protein (GFAP) staining in the corresponding segments of the ipsilateral spinal cord highlights the possible involvement of astrocytes. In summary, the induction of bone cancer in the rat by the syngeneic MRMT‐1 mammary tumour cell line provides a valid pre‐clinical model for pain associated with bone metastases. Significant mechanical hyperalgesia and allodynia develops in association with the progression of the tumour in the bone marrow cavity, while the general condition of the animal remains satisfactory. While acute treatment with morphine has some analgesic effect on hind limb sparing the selective COX‐2 inhibitor, celebrex, has no influence on the pain‐related behavioural changes in this model.

mGlu5 receptors and nociceptive function II. mGlu5 receptors functionally expressed on peripheral sensory neurones mediate inflammatory hyperalgesia

Previous studies have demonstrated that the metabotropic glutamate receptor subtype 5 (mGlu5 receptor) is expressed in the cell bodies of rat primary afferent neurones. We have further investigated the function and expression of mGlu5 receptors in primary afferent neurones, and their role in inflammatory nociception. Freunds complete adjuvant-induced inflammatory hyperalgesia of the rat hind paw was significantly reduced by intraplantar, but not by intracerebroventricular or intrathecal microinjection of the selective mGlu5 receptor antagonist, 2-methyl-6-(phenylethynyl)-pyridine (MPEP). Pharmacological comparison in vivo of the nociceptive effects of glutamate, and ionotropic and metabotropic glutamate (mGlu) receptor agonists applied to the rat hind paw, indicated that group I mGlu receptor agonists induce a dose-dependent decrease in paw withdrawal threshold (mechanical hyperalgesia). Group I mGlu agonist-induced hyperalgesia was inhibited by co-microinjection of MPEP, but not by the mGlu1 receptor antagonist (S)-4-carboxy-phenylglycine (4-CPG). Carrageenan-induced inflammatory hyperalgesia was inhibited by pre-treatment of the inflamed hind paw with MPEP, but not following MPEP injection into the contralateral hind paw. Dorsal horn neurones receiving peripheral nociceptive and non-nociceptive afferent input were recorded in anaesthetized rats following microinjection of CHPG into their peripheral receptive fields. CHPG significantly increased the frequency and duration of firing of dorsal horn wide dynamic range (WDR) neurones and this activity was prevented by co-administration of CHPG and MPEP into their receptive fields. Immunohistochemical experiments revealed the co-expression of mGlu5 receptor protein and betaIII tubulin in skin from naive rats, indicating the constitutive expression of mGlu5 receptors on peripheral neurones. Double-labelling of adult rat DRG cells with mGlu5 receptor and vanilloid receptor subtype 1 antisera also supports the expression of mGlu5 receptors on peripheral nociceptive afferents. These results suggest that mGlu5 receptors expressed on the peripheral terminals of sensory neurones are involved in nociceptive processes and contribute to the hyperalgesia associated with inflammation.

Peripheral nerve injury induces cannabinoid receptor 2 protein expression in rat sensory neurons

We have localized cannabinoid receptor 2 protein in rat and mouse somatic sensory nervous system, using an antibody that recognizes mouse cannabinoid receptor 2. Little or no cannabinoid receptor 2 immunoreactivity was found in sections of naive rat or mouse dorsal root ganglia or spinal cord. This was in accord with the lack of detectable cannabinoid receptor 2 mRNA in (dorsal root ganglion) neurons by in situ hybridization experiments described in the literature. However, we could detect cannabinoid receptor 2 immunoreactivity following unilateral nerve damage-either by sciatic nerve section, or by spinal nerve ligation. It was localized to the superficial laminae of the dorsal horn of the spinal cord, ipsilateral to the nerve damage, coincident with the area of termination of damaged afferents which was marked by loss of isolectin B4 binding. This upregulation was not seen in cannabinoid receptor 2 null mice. The cannabinoid receptor 2 protein in spinal cord appeared to be expressed on sensory neuron afferent terminals as it colocalized with two markers of damaged afferents, namely growth associated protein-43 and the neuropeptide galanin. Moreover, it did not colocalize with markers of activated microglial cells (OX-42) or astroglial cells (glial fibrillary acidic protein) in rat spinal cord. In the peripheral nerve, accumulation of cannabinoid receptor 2 immunoreactivity was seen in nerve sections proximal, but not distal, to the ligation site, suggesting transport down the nerve from the cell bodies. Although convincing cannabinoid receptor 2 immunoreactivity was seen in neither uninjured nor injured dorsal root ganglion neuron cell bodies in tissue sections, expression was detectable in isolated, cultured neurons that had received a prior axotomy in vivo. This clear demonstration of CB(2) receptors on sensory neurons suggests an additional cellular target for CB(2) agonist induced analgesia, at least in neuropathic models.

Nerve growth factor (NGF) regulates adult rat cultured dorsal root ganglion neuron responses to the excitotoxin capsaicin

An overlap between subpopulations of nerve growth factor (NGF)-responsive and capsaicin-sensitive dorsal root ganglion (DRG) sensory neurons has been suggested from a number of in vivo studies. To examine this apparent link in more detail, we compared the effects of capsaicin on adult rat DRG neurons cultured in the presence or absence of NGF. Capsaicin sensitivity was assessed histochemically by a cobalt staining method, by measuring capsaicin-induced 45Ca2+ uptake, and by electrophysiological recording of capsaicin-evoked membrane currents. When cultured with NGF, approximately 50% of these adult DRG neurons were capsaicin-sensitive, whereas adult sympathetic neurons or ganglionic nonneuronal cells were insensitive. DRG cultures grown in the absence of NGF, however, were essentially unresponsive to capsaicin. Capsaicin sensitivity could be regained fully within 4-6 days of replacement of NGF. These results indicate that, at least in vitro, NGF can modify the capsaicin sensitivity of adult DRG neurons.

Pharmacological differences between the human and rat vanilloid receptor 1 (VR1).

Vanilloid receptors (VR1) were cloned from human and rat dorsal root ganglion libraries and expressed in Xenopus oocytes or Chinese Hamster Ovary (CHO) cells. Both rat and human VR1 formed ligand gated channels that were activated by capsaicin with similar EC50 values. Capsaicin had a lower potency on both channels, when measured electrophysiologically in oocytes compared to CHO cells (oocytes: rat=1.90±0.20 μM; human=1.90±0.30 μM: CHO cells: rat=0.20±0.06 μM; human=0.19±0.08 μM). In CHO cell lines co‐expressing either rat or human VR1 and the calcium sensitive, luminescent protein, aequorin, the EC50 values for capsaicin‐induced responses were similar in both cell lines (rat=0.35±0.06 μM, human=0.53±0.03 μM). The threshold for activation by acidic solutions was lower for human VR1 channels than that for rat VR1 (EC50 pH 5.49±0.04 and pH 5.78±0.09, respectively). The threshold for heat activation was identical (42°C) for rat and human VR1. PPAHV was an agonist at rat VR1 (EC50 between 3 and 10 μM) but was virtually inactive at the human VR1 (EC50>10 μM). Capsazepine and ruthenium red were both more potent at blocking the capsaicin response of human VR1 than rat VR1. Capsazepine blocked the human but not the rat VR1 response to low pH. Capsazepine was also more effective at inhibiting the noxious heat response of human than of rat VR1.