Fei-Yue Zhao

GW406381, a novel COX-2 inhibitor, attenuates spontaneous ectopic discharge in sural nerves of rats following chronic constriction injury

Abstract There are several lines of evidence to suggest that cyclooxygenase‐2 (COX‐2) plays an important role in the generation and maintenance of neuropathic pain states following peripheral nerve injury. However, COX‐2 inhibitors are generally ineffective in reversing mechanical allodynia and hyperalgesia in models of neuropathic hypersensitivity. Here, we have investigated the effects of GW406381, a novel COX‐2 inhibitor, on mechanical allodynia, hyperalgesia and generation of spontaneous ectopic discharge in rats following chronic constriction injury (CCI) of the sciatic nerve and compared it with rofecoxib. GW406381 (5 mg/kg, 5 days of treatment) significantly reversed the CCI‐induced decrease in paw withdrawal thresholds (PWTs), assessed using both von Frey hair and paw pressure tests, whereas an equi‐effective dose of rofecoxib (5 mg/kg, 5 days of treatment) in inflammatory pain models was ineffective. In rats treated with GW406381, the proportion of fibres showing spontaneous activity was significantly lower (15.58%) than that in the vehicle (32.67%)‐ and rofecoxib (39.66%)‐treated rats. Ibuprofen, a non‐selective COX inhibitor, at 5 mg/kg, orally dosed three times a day for 5 days did not significantly affect the PWTs in CCI rats. In naïve rats, GW406381 did not significantly change the PWTs. These results illustrate that COX‐2 may indeed play an important role in the maintenance of neuropathic pain following nerve injury, but that only certain COX‐2 inhibitors, such as GW406381, are effective in this paradigm. Whilst the mechanisms underlying this differential effect of GW406381 are not clear, differences in drug/enzyme kinetic interactions may be a key contributing factor.

Pharmacodynamic Effects of a d-Amino Acid Oxidase Inhibitor Indicate a Spinal Site of Action in Rat Models of Neuropathic Pain

Inhibition of d-amino acid oxidase (DAAO) activity is a potential target for the treatment of chronic pain. Here we characterized the effects of systemic administration of the DAAO inhibitor 4H-furo[3,2-b]pyrrole-5-carboxylic acid (SUN) in rat models of neuropathic and inflammatory pain. Oral administration of SUN dose dependently attenuated tactile allodynia induced by ligation of the L5 spinal nerve (SNL) and similarly reversed thermal hyperalgesia produced by chronic constriction injury. In addition, SUN was efficacious against complete Freund’s adjuvant-induced thermal hyperalgesia. In these models, maximal reversal of pain-related behaviors corresponded with maximum rates of increase in brain and plasma d-serine concentrations, indicative of full inhibition of DAAO activity. To investigate the possible site(s) of action, we recorded spontaneous nerve activity and mechanically evoked responses of central spinal cord dorsal horn neurons and compared these with spontaneous activity of peripheral dorsal root filaments in anesthetized SNL model animals. Oral SUN reduced spontaneous activity in both central and peripheral recordings at doses and pretreatment times that corresponded to reduced mechanical allodynia in behavioral experiments. After intravenous administration of SUN, the onset of action for this central effect was rapid (maximal effects within 30 minutes), but was abolished by severing afferent inputs to the dorsal horn. Overall, these results indicate that inhibition of DAAO in peripheral afferent spinal circuits reduced spontaneous neuronal activity to attenuate pain-related behaviors in rat models of neuropathic and inflammatory pain.

Norepinephrine can Act via α2-Adrenoceptors to Reduce the Hyper-excitability of Spinal Dorsal Horn Neurons Following Chronic Nerve Injury

BACKGROUND/PURPOSE Rats display behavioral signs of neuropathic pain lasting for months in the chronic constriction injury (CCI) model. During intrathecal anesthesia, the administered drugs mainly diffuse directly into the superficial neurons in the spinal dorsal horn. This study aimed to investigate the effect of bath application of norepinephrine on whole cell patch clamp recordings from spinal cord slices of CCI rats with allodynia. METHODS An assessment of paw withdrawal threshold in response to mechanical stimulation was performed on the operated side on the day before surgery and was repeated after recovery from anesthesia and on the 7(th) and 14(th) days after surgery. Spinal cord slice preparations containing dorsal horn neurons were obtained from both sham-operated rats and CCI rats (after the 14(th) postoperative day behavior test). RESULTS Compared with normal controls, CCI rats had significantly lower levels of both hyperpolarization and spike threshold in single action potentials recorded from lamina I and II neurons of the spinal dorsal horn. In contrast, a series of action potential recordings showed that the percentage of spiking neurons of the spinal dorsal horn of CCI rats were significantly higher than those of normal controls. The CCI-induced reduction in hyperpolarization, as well as the increased numbers of spinal dorsal horn spiking neurons could be significantly reduced by norepinephrine application. The norepinephrine-induced increased hyperpolarization and input resistance could be abolished by the application of an alpha(2)-adrenoceptor antagonist (idazoxan; 200 nM). CONCLUSION The results suggest that chronic nerve injury may induce neuropathic pain by increasing the excitability of spinal dorsal horn neurons. This excitability can be reduced by norepinephrine.

Electrophysiological Techniques for Studying Synaptic Activity In Vivo

Understanding the physiology, pharmacology, and plasticity associated with synaptic function is a key goal of neuroscience research and is fundamental to identifying the processes involved in the development and manifestation of neurological disease. A diverse range of electrophysiological methodologies are used to study synaptic function. Described in this unit is a technique for recording electrical activity from a single component of the central nervous system that is used to investigate pre‐ and post‐synaptic elements of synaptic function. A strength of this technique is that it can be used on live animals, although the effect of anesthesia must be taken into consideration when interpreting the results. This methodology can be employed not only in naïve animals for studying normal physiological synaptic function, but also in a variety of disease models, including transgenic animals, to examine dysfunctional synaptic plasticity associated with neurological pathologies. Curr. Protoc. Pharmacol. 64:11.11.1‐11.11.17.

In Vivo Electrophysiological Recording Techniques for the Study of Neuropathic Pain in Rodent Models

Neuropathic pain develops following nerve injury, and is a chronic pain syndrome that can persist long after repair of a wound or removal of the neurological insult. This condition remains poorly treated, not least because of a lack of mechanism‐based therapeutics. Clinically, neuropathic pain is characterized by three major symptoms: thermal or mechanical allodynia (pain sensation in response to previously non‐noxious stimuli); hyperalgesia (enhanced pain sensation to noxious stimulation); and spontaneous, ongoing pain. These clinical symptoms can be modeled in rodent neuropathic pain models using behavioral and electrophysiological readouts. This unit describes techniques designed to record pathophysiological electrical activity associated with neuropathic pain at the level of the periphery, in single fibers of primary sensory neurons, and from wide dynamic range (WDR) neurons of the dorsal horn of the spinal cord. These techniques can be employed in both naïve animals and in animal models of neuropathy to investigate fundamental mechanisms contributing to the neuropathic pain state and the site, mode, and mechanism of action of putative analgesics. Curr. Protoc. Pharmacol. 66:11.15.1‐11.15.26.

146 MURINE MODELS OF NEUROPATHIC PAIN

Spontaneous pain is the primary complaint of neuropathic pain patients. It commonly occurs even without hyperalgesia (Basbaum, 2006). It remains hard to treat because its underlying mechanisms are poorly understood, possibly because most animal studies have focused on evoked pain behaviours. We used two spinal nerve (SN) models of neuropathic pain to test the hypothesis that spontaneous activity (SA) in uninjured dorsal root ganglion (DRG) neurons causes spontaneous pain behaviour. These were axotomy of the L5 SN (SNA) and modified SNA (mSNA) that involved SNA plus loose ligation of the L4 SN with the inflammation-inducing chromic gut. Of these, only mSNA rats showed significant SFL, indicative of spontaneous pain (Koutsikou and Lawson, 2002; Djouhri et al., 2006). Intracellular recordings of evoked and spontaneously occurring action potentials in deeply anesthetised rats were made from: (a) normal L4/L5 DRG neurons in control rats, (b) axotomised L5 neurones in mSNA rats, (c) intact L4 neurons in both mSNA and SNA rats. Neurons were classified as C, Ad, or Aa/b units and as nonnociceptive or nociceptive. Seven days post-operatively, similar increases in percentages of intact nociceptive C-fibre L4 neurons with SA (from 7% to about 35%) in both SNA and mSNA rats occurred. These intact C-nociceptors showed faster spontaneous firing in mSNA (1.8 Hz) than SNA (0.02 Hz) rats, implicating intact C-neurons in SFL. Faster firing rates in intact C-nociceptive neurones, probably resulting from cumulative inflammation/ damage (due to co-mingling degenerating fibres and chromic-gut) are required for spontaneous pain. Acknowledgement

UNIT 11.11 Electrophysiological Techniques for Studying Synaptic Activity In Vivo

Understanding the physiology, pharmacology, and plasticity associated with synaptic function is a key goal of neuroscience research and is fundamental to identifying the processes involved in the development and manifestation of neurological disease. A diverse range of electrophysiological methodologies are used to study synaptic function. Described in this unit is a technique for recording electrical activity from a single component of the central nervous system that is used to investigate pre‐ and post‐synaptic elements of synaptic function. A strength of this technique is that it can be used on live animals, although the effect of anesthesia must be taken into consideration when interpreting the results. This methodology can be employed not only in naïve animals for studying normal physiological synaptic function, but also in a variety of disease models, including transgenic animals, to examine dysfunctional synaptic plasticity associated with neurological pathologies. Curr. Protoc. Pharmacol. 64:11.11.1‐11.11.17.

Electrophysiological Techniques for Studying Synaptic Activity In Vivo: Studying Synaptic Activity In Vivo

Understanding the physiology, pharmacology, and plasticity associated with synaptic function is a key goal of neuroscience research and is fundamental to identifying the processes involved in the development and manifestation of neurological disease. A diverse range of electrophysiological methodologies are used to study synaptic function. Described in this unit is a technique for recording electrical activity from a single component of the central nervous system that is used to investigate pre‐ and post‐synaptic elements of synaptic function. A strength of this technique is that it can be used on live animals, although the effect of anesthesia must be taken into consideration when interpreting the results. This methodology can be employed not only in naïve animals for studying normal physiological synaptic function, but also in a variety of disease models, including transgenic animals, to examine dysfunctional synaptic plasticity associated with neurological pathologies. Curr. Protoc. Pharmacol. 64:11.11.1‐11.11.17.