Cross-centre replication of suppressed burrowing behaviour as an ethologically relevant pain outcome measure in the rat: a prospective multicentre study.

Burrowing, an ethologically relevant rodent behaviour, has been proposed as a novel outcome measure to assess the global impact of pain in rats. In a prospective multicentre study using male rats (Wistar, Sprague-Dawley), replication of suppressed burrowing behaviour in the complete Freund adjuvant (CFA)-induced model of inflammatory pain (unilateral, 1 mg/mL in 100 µL) was evaluated in 11 studies across 8 centres. Following a standard protocol, data from participating centres were collected centrally and analysed with a restricted maximum likelihood-based mixed model for repeated measures. The total population (TP-all animals allocated to treatment; n = 249) and a selected population (SP-TP animals burrowing over 500 g at baseline; n = 200) were analysed separately, assessing the effect of excluding "poor" burrowers. Mean baseline burrowing across studies was 1113 g (95% confidence interval: 1041-1185 g) for TP and 1329 g (1271-1387 g) for SP. Burrowing was significantly suppressed in the majority of studies 24 hours (7 studies/population) and 48 hours (7 TP, 6 SP) after CFA injections. Across all centres, significantly suppressed burrowing peaked 24 hours after CFA injections, with a burrowing deficit of -374 g (-479 to -269 g) for TP and -498 g (-609 to -386 g) for SP. This unique multicentre approach first provided high-quality evidence evaluating suppressed burrowing as robust and reproducible, supporting its use as tool to infer the global effect of pain on rodents. Second, our approach provided important informative value for the use of multicentre studies in the future.

Cross-centre replication of suppressed burrowing behaviour as an ethologically relevant pain outcome measure in the rat: a prospective multicentre study.

Burrowing, an ethologically relevant rodent behaviour, has been proposed as a novel outcome measure to assess the global impact of pain in rats. In a prospective multicentre study using male rats (Wistar, Sprague-Dawley), replication of suppressed burrowing behaviour in the complete Freund adjuvant (CFA)-induced model of inflammatory pain (unilateral, 1 mg/mL in 100 µL) was evaluated in 11 studies across 8 centres. Following a standard protocol, data from participating centres were collected centrally and analysed with a restricted maximum likelihood-based mixed model for repeated measures. The total population (TP-all animals allocated to treatment; n = 249) and a selected population (SP-TP animals burrowing over 500 g at baseline; n = 200) were analysed separately, assessing the effect of excluding "poor" burrowers. Mean baseline burrowing across studies was 1113 g (95% confidence interval: 1041-1185 g) for TP and 1329 g (1271-1387 g) for SP. Burrowing was significantly suppressed in the majority of studies 24 hours (7 studies/population) and 48 hours (7 TP, 6 SP) after CFA injections. Across all centres, significantly suppressed burrowing peaked 24 hours after CFA injections, with a burrowing deficit of -374 g (-479 to -269 g) for TP and -498 g (-609 to -386 g) for SP. This unique multicentre approach first provided high-quality evidence evaluating suppressed burrowing as robust and reproducible, supporting its use as tool to infer the global effect of pain on rodents. Second, our approach provided important informative value for the use of multicentre studies in the future.

Spinal GABAergic mechanisms in the effects of spinal cord stimulation in a rodent model of neuropathic pain: is GABA synthesis involved?

OBJECTIVES: The effects of spinal cord stimulation (SCS) on the spinal γ-amino butyric acid (GABA) system have previously been studied in animal models of neuropathic pain. These studies, confirming the pivotal role of segmental GABA actions for the efficacy of SCS, have led to the question if the disturbance of the GABA inhibitory system as demonstrated both in basal and clinical studies also encompasses malfunction of the GABA synthesis. METHODS: Rat models of neuropathic pain were submitted to SCS applied with "clinical SCS parameters." The levels of the GABA-synthesizing enzymes, glutamic acid decarboxylase (GAD) 65 and GAD 67, in the spinal dorsal horns (DHs) were analyzed using Western blot and immunohistochemistry comparing responders and nonresponders to SCS, with and without SCS, as well as controls. RESULTS: There were no significant differences in general DH GAD levels between hypersensitive, nonhypersensitive, and intact control animals. Although SCS did not significantly influence these levels, there was a significant local augmentation of GAD 65 expression in lamina II in SCS responders subjected to SCS immediately prior to tissue collection as compared with SCS nonresponders. CONCLUSIONS: Although GABAergic mechanisms are closely related to the effects of SCS, the presence of neuropathic signs and their suppression by SCS are not associated with changes of the general levels of the spinal DH GABA-synthesizing enzymes. However, in SCS responding animals, there was a significant increased expression of GAD 65 in lamina II, presumably reflecting an augmented GABA synthesis following SCS.

Pain relief by spinal cord stimulation involves serotonergic mechanisms: an experimental study in a rat model of mononeuropathy.

The aim of the present study was to examine the role of the spinal serotonergic system in the pain relieving effect of spinal cord stimulation (SCS) using a rat model of mononeuropathy. Tactile withdrawal thresholds, cold responses and heat withdrawal latencies were assessed before and after SCS. In some rats, SCS produced an attenuation of the hypersensitivity following nerve injury (SCS responding rats). When SCS was applied immediately prior to sacrifice, the 5-HT content in the dorsal quadrant of the spinal cord ipsilateral to the nerve injury was increased in SCS responding rats. But there was no change in responding rats without stimulation, or in SCS non-responding rats with or without stimulation or in controls. Immunohistochemical examination showed a high density of 5-HT stained terminals in the dorsal horn superficial laminae (I-II) in SCS responding rats following stimulation. It was also found that i.t. administration of a sub-effective dose of serotonin in SCS non-responding rats markedly enhanced the pain relieving effect of SCS on tactile and cold hypersensitivity, while there was no effect on heat hyperalgesia. This enhanced effect on tactile hypersensitivity could be partially blocked by a GABA(B) receptor antagonist (CGP 35348) but not by a muscarinic M(4) receptor antagonist (Muscarinic toxin 3) administered i.t. shortly before the 5-HT injection. In conclusion, there is evidence that the spinal 5-HT system plays an important role in the mode of action of SCS involving the activation of descending serotonergic pathways that may inhibit spinal nociceptive processing partially via a GABAergic link.

Cholinergic mechanisms involved in the pain relieving effect of spinal cord stimulation in a model of neuropathy.

The mechanisms underlying the pain relieving effect of spinal cord stimulation (SCS) on neuropathic pain remain unclear. We have previously demonstrated that suppression of tactile hypersensitivity produced by SCS may be potentiated by i.t. clonidine in a rat model of mononeuropathy. Since the analgesic effect of this drug is mediated mainly via cholinergic mechanisms, a study exploring the possible involvement of the spinal cholinergic system in SCS was undertaken. The effect of SCS was assessed with von Frey filaments in rats displaying tactile hypersensitivity after partial ligation of the sciatic nerve and both SCS-responding and non-responding as well as normal rats were subjected to microdialysis in the dorsal horn. Acetylcholine (ACh) was analyzed with HPLC before, during and after SCS. SCS produced significantly increased release of ACh in the dorsal horn in rats responding to SCS whereas the release was unaffected in the non-responding animals. Furthermore, the basal release of ACh was significantly lower in nerve lesioned than in normal rats. In another group of rats it was found that the response to SCS was completely eliminated by i.t. atropine and a muscarinic M(4) receptor antagonist while a partial attenuation was produced by M(1) and M(2) antagonists. Blocking of nicotinic receptors did not influence the SCS effect. In conclusion, the attenuating effect of SCS on pain related behavior is associated with the activation of the cholinergic system in the dorsal horn and mediated via muscarinic receptors, particularly M(4,) while nicotinic receptors appear not to be involved.

Spinal NMDA receptor phosphorylation correlates with the presence of neuropathic signs following peripheral nerve injury in the rat.

Substantial evidence has established that activation of the NMDA receptor in the spinal dorsal horn is essential for central sensitization-a phenomenon which comprises various pathophysiological mechanisms underlying neuropathic pain-like signs in animal models. In the present study, a partial sciatic nerve ligation in the rat was used to produce a model of nerve injury-induced pain represented by hypersensitivity to innocuous stimuli ("allodynia"). The aim was to assess whether alteration of NMDA receptor expression correlates with the presence of neuropathic signs. Our approach was to compare spinal NMDA receptor subunit expression and especially subunit 1 phosphorylation, assessed with immunohistochemistry and Western blot at late postoperative times, between nerve-injured rats with marked signs of neuropathy in terms of mechanical and cold hypersensitivity and nerve-injured rats that lacked robust behavioral signs of neuropathy. Quantification of receptor expression was based on comparisons between the dorsal horns ispi- and contralateral to the nerve lesion. The phosphorylated NR1 subunit of the NMDA receptor was found to be significantly increased in the ipsilateral dorsal horn in hypersensitive, but not in non-hypersensitive nerve-injured rats. We did not detect any differences in immunoreactivity in any of the non-phosphorylated NR1, NR2A, NR2B, NR2C or the NR2D subunits. These data suggest that phosphorylation of the NMDA receptor 1 subunit is correlated to the presence of signs of neuropathy (stimulus evoked pain-like behavior) and possibly also to persistent pain following nerve injury. This may represent a mechanism involved in spinal sensitization.