Mouse Formalin Test of Hyperalgesia

Brief Description

Assessment of the in vivo effect of an investigational compound on the reduction of acute and chronic inflammatory pain in mouse.

Introduction

Many of the broad-spectrum ASDs are effective in modulating abnormal neuronal activity in a number of CNS disorders in addition to epilepsy, including bipolar disorder, migraine, and neuropathic pain [1]. Often, these comorbidities of epilepsy can be more detrimental to a patient’s quality of life than the seizures themselves [2]. The 2012 Institute of Medicine’s report acknowledges several painful somatic disorders, such as fibromyalgia and osteoarthritis, as well as painful neurological conditions, such as migraine, chronic pain syndromes, and neuropathic pain, as comorbid conditions associated with epilepsy [3, 4]. Thus, one approach towards finding therapeutics with efficacy for these comorbidities associated with epilepsy is to expand the preclinical screening of the effects of an investigational compound to include models of nociception and neuropathic pain. Like epilepsy itself, neuropathic pain represent pathological states of abnormal neuronal discharges [5]. At the molecular level, most, if not all, of the available ASDs target receptor- and voltage-gated ion channels that contribute to abnormal neuronal discharges. Thus, it is not necessarily unexpected that those ASDs with broad neuromodulatory activities would be clinically useful for the treatment of neurological and somatic disorders associated with acute, inflammatory, or neuropathic pain. Hyperalgesia, which includes both acute and inflammatory processes, is characterized as an increased sensitivity to pain [5]. In an attempt to broaden the therapeutic application of a novel neuromodulatory compound identified in the traditional seizure models, the ETSP has examined the efficacy of the most promising and/or novel investigational ASDs in several models of nociception, including the partial sciatic nerve ligation model of chronic allodynia in rats (Test 23) and the formalin test model of hyperalgesia in mice (Test 22).

Methods

The formalin test is performed according to the method of Tjolsen et al. [6]. A sub-dermal injection of 0.5% formalin is made into the plantar region of the right hindpaw of a mouse. This elicits a distinct behavioral response to the formalin injection characterized by the mouse licking the affected paw. The licking behavior is characteristically biphasic in nature. For example, immediately following the injection the mouse intensely licks the paw for approximately 5-10 min. This initial behavior is considered the acute phase and is thought to be mediated primarily by direct chemical activation of local C-fibers [5, 6]. The acute phase is followed by a brief latent (usually < 5 min) period wherein there is little or no behavioral activity. A more prolonged inflammatory phase (about 20 to 30 min) period of licking then ensues, which are primarily mediated by inflammatory mechanisms and sensitization within the dorsal horn of the spinal cord [5, 6]. Thus, animals are monitored for any effects of the investigational compound on both acute and inflammatory phases of persistent pain. Prior to the administration of the test drug or vehicle, each mouse undergoes a 15-min conditioning period in one of several 6" tall plexiglass cages (4" diameter). The mice (n = 8 / treatment group) are then observed for licking activity for the duration of the experiment. Following the conditioning period, the investigational compound (treatment group) or vehicle (control group) is administered i.p. at a dose approximating the MES ED50 (as determined in Test 4) and the mouse is returned to its home cage. At the time of peak effect (TPE) of the investigational compound, formalin is then injected sub-dermally into the plantar surface of the right hindpaw. Following the formalin injection, each animal is then observed for the first 2 minutes of each 5-min epoch until a total of 40 min has elapsed since the administration of the test compound or vehicle. The cumulative duration of licking (in seconds) during each 2-min recording period is measured for analysis across drug- and vehicle-pretreated groups. Animals are immediately euthanized following the conclusion of the experiment.

Results

Total area under the curve (AUC) and subsequent percent of control licking time for the drug-treated animal groups are determined AUC during both the acute (Phase I) and inflammatory (Phase II) phases. The average and S.E.M. for both the drug- and vehicle-pretreated animals are calculated and tested for significant differences by Student’s t-test, with p < 0.05 considered statistically significant.

Discussion

Recent work has demonstrated that ASDs can frequently attenuate pain in several animal models of inflammatory and neuropathic pain [7]. For example, gabapentin is often clinically prescribed for neuropathic pain, as well as for epilepsy. One explanation for these observations is that epilepsy and neuropathic pain are both arise from pathological states of neuronal hyperexcitability. In epilepsy, such central neuron hyperexcitability manifests in seizures, whereas in neuropathic pain, peripheral neuron hyperexcitability results in chronic repetitive firing that is perceived as pain [1] and, if not addressed adequately, can result in central sensitization via a process very similar to kindling for chronic seizure presentation [8]. Therefore, the ability of investigational compounds to attenuate the acute and/or inflammatory phases of pain in the mouse formalin test has been evaluated at the ETSP. Compounds that, when tested at a dose near the mouse MES ED50, show significant attenuation of paw licking behavior during the acute and/or inflammatory phases following formalin injection into the paw may be effective at this dose as an analgesic or anti-inflammatory agent in this model. Compounds that are found to be effective in a battery of animal models of epilepsy, as well as this mouse pain model, may also ultimately be clinically useful for chronic pain, as modeled in the partial sciatic nerve ligation model in rats (Test 23).