A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 °C to 5 °C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.     

Mechanical and cold allodynia in a rat spinal cord contusion model

This study examined the time course of mechanical and cold allodynia in rat hindpaw after spinal cord contusion. Hindpaw withdrawal threshold to graded von Frey hair stimulation and withdrawal frequency to acetone application were measured in rats subjected to contusions of varying severity, produced by a MASCIS impactor device with a 10 g weight dropped from 6.25, 12.5, or 25 mm. Mechanical and cold allodynia developed following the injury, and differences in the incidence of allodynia and in withdrawal threshold were significant among these groups. The least severe injury (6.25 mm) most consistently caused a decreased hindpaw threshold to mechanical stimulation and an increased withdrawal frequency to cold.     

Mechanical and cold allodynia in a rat spinal cord contusion model

This study examined the time course of mechanical and cold allodynia in rat hindpaw after spinal cord contusion. Hindpaw withdrawal threshold to graded von Frey hair stimulation and withdrawal frequency to acetone application were measured in rats subjected to contusions of varying severity, produced by a MASCIS impactor device with a 10?g weight dropped from 6.25, 12.5, or 25?mm. Mechanical and cold allodynia developed following the injury, and differences in the incidence of allodynia and in withdrawal threshold were significant among these groups. The least severe injury (6.25?mm) most consistently caused a decreased hindpaw threshold to mechanical stimulation and an increased withdrawal frequency to cold.     

早期干预ERK在脊髓的激活可阻断外周神经损伤引起的神经病理性疼痛的发生

本文采用大鼠坐骨神经慢性压迫损伤引起的神经病理痛模型,研究脊髓背角细胞外信号调节激酶(extracellular signal-regulatedkinase,ERK)在外周神经损伤引起的神经病理疼痛发生中的作用.结果显示,单侧坐骨神经压迫性损伤后1天,大鼠损伤侧脊髓背角ERK的磷酸化(激活)水平显著上调,其下游转录因...     

Chronic Constriction of the Sciatic Nerve and Pain Hypersensitivity Testing in Rats

Chronic neuropathic pain, resulting from damage to the central or peripheral nervous system, is a prevalent and debilitating condition, affecting 7-18% of the population^1,2. Symptoms include spontaneous (tingling, burning, electric-shock like) pain, dysaesthesia, paraesthesia, allodynia (pain resulting from normally non-painful stimuli) and hyperalgesia (an increased response to painful stimuli). The sensory symptoms are co-morbid with behavioural disabilities, such as insomnia and depression. To study chronic neuropathic pain several animal models mimicking peripheral nerve injury have been developed, one of the most widely used is Bennett and Xie''s (1988) unilateral sciatic nerve chronic constriction injury (CCI)³ (Figure 1). Here we present a method for performing CCI and testing pain hypersensitivity.CCI is performed under anaesthesia, with the sciatic nerve on one side exposed by making a skin incision, and cutting through the connective tissue between the gluteus superficialis and biceps femoris muscles. Four chromic gut ligatures are tied loosely around the sciatic nerve at 1 mm intervals, to just occlude but not arrest epineural blood flow. The wound is closed with sutures in the muscle and staples in the skin. The animal is then allowed to recover from surgery for 24 hrs before pain hypersensitivity testing begins.For behavioural testing, rats are placed into the testing apparatus and are allowed to habituate to the testing procedure. The area tested is the mid-plantar surface of the hindpaw (Figure 2), which falls within the sciatic nerve distribution. Mechanical withdrawal threshold is assessed by mechanically stimulating both injured and uninjured hindpaws using an electronic dynamic plantar von Frey aesthesiometer or manual von Frey hairs⁴. The mechanical withdrawal threshold is the maximum pressure exerted (in grams) that triggers paw withdrawal. For measurement of thermal withdrawal latency, first described by Hargreaves et al (1988), the hindpaw is exposed to a beam of radiant heat through a transparent glass surface using a plantar analgesia meter^5,6. The withdrawal latency to the heat stimulus is recorded as the time for paw withdrawal in both injured and uninjured hindpaws. Following CCI, mechanical withdrawal threshold, as well as thermal withdrawal latency in the injured paw are both significantly reduced, compared to baseline measurements and the uninjured paw (Figure 3). The CCI model of peripheral nerve injury combined with pain hypersensitivity testing provides a model system to investigate the effectiveness of potential therapeutic agents to modify chronic neuropathic pain. In our laboratory, we utilise CCI alongside thermal and mechanical sensitivity of the hindpaws to investigate the role of neuro-immune interactions in the pathogenesis and treatment of neuropathic pain.     

Behavioral Testing of the Effects of Thermosensitive TRP Channel Agonists on Touch,Temperature, and Pain Sensations

It was recently found that transient receptor potential (TRP) channels play an important role in the transduction of thermal, mechanical, and chemical stimuli underlying the somatic sensation. Several types of TRP channels exhibit sensitivity to increases or decreases in temperature, as well as to the action of chemical ligands that elicit similar thermal or painful sensations. These agents include menthol, mustard oil, cinnamaldehyde (CA), gingerol, capsaicin, camphor, eugenol, and others. Cinnamaldehyde is a pungent chemical obtained from cinnamon, which acts as an agonist of the TRPA1 channels; these channels were originally reported to be activated by cold temperatures (below 18°C). TRPA1 is also implicated in cold nociception. However, its role in the formation of cold pain is more controversial, with discrepant reports that TRPA1s do or do not respond to intense cooling. Menthol derived from plants of the mint family enhances the feeling of coldness by interacting with the cold-sensitive TRPM8 channels, but its effect on pain is less well understood. Using behavioral methods, we showed that unilateral intraplantar injection of CA (5 to 20%) induced a significant concentration-dependent decrease in the latency for ipsilateral paw withdrawal from a noxious heat stimulus, i.e., heat hyperalgesia. Cinnamaldehyde also significantly reduced mechanical withdrawal thresholds for the injected paw, i.e., evoked mechanical allodynia. Bilateral intraplantar injections of CA resulted in a significant cold hyperalgesia (cold plate test) and a weak enhancement of innocuous cold avoidance (thermal preference test). In contrast to CA, menthol in a dose-dependent manner increased the latency for noxious heat-evoked withdrawal, i.e., exerted an antinociceptive effect. Menthol did not affect mechanosensation except for a weak allodynic effect when applied in the highest concentration used (40 %), indicating that it did not exert a local anesthetic effect. Menthol had a biphasic effect on cold avoidance. High concentrations of menthol reduced cold avoidance, i.e., induced cold hypoalgesia, while low menthol concentrations significantly intensified cold avoidance. The highest menthol concentration provided cold hypoalgesia (cold plate test), while lower concentrations had no effect. Taken together, our data support the idea that TRPA1 and TRPM8 channels represent promising peripheral targets for pain modulation.     

Early painful diabetic neuropathy is associated with differential changes in tetrodotoxin-sensitive and -resistant sodium channels in dorsal root ganglion neurons in the rat

Diabetic neuropathy is a common form of peripheral neuropathy, yet the mechanisms responsible for pain in this disease are poorly understood. Alterations in the expression and function of voltage-gated tetrodotoxin-resistant (TTX-R) sodium channels have been implicated in animal models of neuropathic pain, including models of diabetic neuropathy. We investigated the expression and function of TTX-sensitive (TTX-S) and TTX-R sodium channels in dorsal root ganglion (DRG) neurons and the responses to thermal hyperalgesia and mechanical allodynia in streptozotocin-treated rats between 4-8 weeks after onset of diabetes. Diabetic rats demonstrated a significant reduction in the threshold for escape from innocuous mechanical pressure (allodynia) and a reduction in the latency to withdrawal from a noxious thermal stimulus (hyperalgesia). Both TTX-S and TTX-R sodium currents increased significantly in small DRG neurons isolated from diabetic rats. The voltage-dependent activation and steady-state inactivation curves for these currents were shifted negatively. TTX-S currents induced by fast or slow voltage ramps increased markedly in neurons from diabetic rats. Immunoblots and immunofluorescence staining demonstrated significant increases in the expression of Na(v)1.3 (TTX-S) and Na(v) 1.7 (TTX-S) and decreases in the expression of Na(v) 1.6 (TTX-S) and Na(v)1.8 (TTX-R) in diabetic rats. The level of serine/threonine phosphorylation of Na(v) 1.6 and In Na(v)1.8 increased in response to diabetes. addition, increased tyrosine phosphorylation of Na(v)1.6 and Na(v)1.7 was observed in DRGs from diabetic rats. These results suggest that both TTX-S and TTX-R sodium channels play important roles and that differential phosphorylation of sodium channels involving both serine/threonine and tyrosine sites contributes to painful diabetic neuropathy.     

Altered neuropathic pain behaviour in a rat model of depression is associated with changes in inflammatory gene expression in the amygdala

The association between chronic pain and depression is widely recognized, the comorbidity of which leads to a heavier disease burden, increased disability and poor treatment response. This study examined nociceptive responding to mechanical and thermal stimuli prior to and following L5‐L6 spinal nerve ligation (SNL), a model of neuropathic pain, in the olfactory bulbectomized (OB) rat model of depression. Associated changes in the expression of genes encoding for markers of glial activation and cytokines were subsequently examined in the amygdala, a key brain region for the modulation of emotion and pain. The OB rats exhibited mechanical and cold allodynia, but not heat hyperalgesia, when compared with sham‐operated counterparts. Spinal nerve ligation induced characteristic mechanical and cold allodynia in the ipsilateral hindpaw of both sham and OB rats. The OB rats exhibited a reduced latency and number of responses to an innocuous cold stimulus following SNL, an effect positively correlated with interleukin (IL)‐6 and IL‐10 mRNA expression in the amygdala, respectively. Spinal nerve ligation reduced IL‐6 and increased IL‐10 expression in the amygdala of sham rats. The expression of CD11b (cluster of differentiation molecule 11b) and GFAP (glial fibrillary acidic protein), indicative of microglial and astrocyte activation, and IL‐1β in the amygdala was enhanced in OB animals when compared with sham counterparts, an effect not observed following SNL. This study shows that neuropathic pain‐related responding to an innocuous cold stimulus is altered in an animal model of depression, effects accompanied by changes in the expression of neuroinflammatory genes in the amygdala .     

Evidence for a supraspinal analgesic effect with cyclazocine and pentazocine

The antinociceptive effect of the benzomorphan class of opioid analgesics have been difficult to measure utilizing some of the standard animal pain models. This may be due, in part, to the sedative and/or motor effects associated with these drugs. In addition, it has been proposed that the major site of action for drugs with agonist activity at the kappa opiate receptor is exclusively at the spinal level opposed to both spinal and supraspinal as with the mu receptor agonists such as morphine. The present study examines the antinociceptive effect of the mixed agonist-antagonists cyclazocine and pentazocine utilizing electrical stimulation of the midbrain reticular formation (MRF) as the aversive stimulus in the rat. Animals were trained to escape MRF stimulation by turning a cylindrical manipulandum. An escape threshold was determined by varying the current intensity according to a modification of the psychophysical method of limits. In addition to the determination of the escape threshold the response latency and strength of response was also measured. Both cyclazocine (0.25-1.0 mg/kg) and pentazocine (2.5-12.5 mg/kg) raised the escape threshold in a dose-dependent manner without any concomitant change in the response latency or strength of response. These data suggest that the observed threshold elevation is due to a specific antinociceptive effect. Since the aversive stimulation was delivered supraspinally, the data also suggest that there are supraspinal mechanisms mediated by kappa receptors responsible for this analgesic effect.     

Taurine replacement attenuates hyperalgesia and abnormal calcium signaling in sensory neurons of STZ-D rats

The etiology of painful diabetic neuropathy is poorly understood, but may result from neuronal hyperexcitability secondary to alterations of Ca2+ signaling in sensory neurons. The naturally occurring amino acid taurine functions as an osmolyte, antioxidant, Ca2+ modulator, inhibitory neurotransmitter, and analgesic such that its depletion in diabetes may predispose one to neuronal hyperexcitability and pain. This study reports the effects of taurine replacement on hyperalgesia and sensory neuron Ca2+ homeostasis in streptozotocin-diabetic (STZ-D) rats. Nondiabetic and STZ-D rats were treated with a 2% taurine-supplemented diet for 6-12 wk. Thermal hyperalgesia and mechanical allodynia were determined by measuring hindpaw withdrawal latency to radiant heat and the withdrawal threshold to the von Frey anesthesiometer. Intracellular Ca2+ signaling was explored in neurons from L4-L6 dorsal root ganglia (DRG), using fura 2 fluorescence. Taurine replacement of diabetic rats attenuated deficits of nerve conduction and prevented reductions of mechanical and thermal withdrawal threshold and latency, respectively. In small DRG sensory neurons from diabetic rats, recovery of intracellular Ca2+ concentration ([Ca2+]i) in response to KCl was slowed and 73% corrected by taurine. The amplitudes of caffeine and ATP-induced [Ca2+]i transients were decreased by 47 and 27% (P < 0.05), respectively, in diabetic rat DRG sensory neurons and corrected by 74 and 93% (P < 0.05), respectively, by taurine replacement. These data indicate that taurine is important in the regulation of neuronal Ca2+ signaling and that taurine deficiency may predispose one to nerve hyperexcitability and pain, complicating diabetes.     

Effect of chronic intermittent exposure to AM radiofrequency field on responses to various types of noxious stimuli in growing rats

There are several reports of altered pain sensation after exposure (from a few minutes to hours in single or repeated doses for 2-3 weeks) to electromagnetic fields (EMF) in adults. The commonly utilized noxious stimulus is radiant heat. The nociceptive responses are known to be influenced by characteristics of stimulus, organism, and environment. We studied the pattern of nociceptive responses to various noxious stimuli in growing rats exposed to radiofrequency field (73.5 MHz amplitude modulated, 16 Hz power density 1.33 mw/cm(2), SAR = 0.4 w/kg) for 45 d (2 h/d). Threshold current for stimulation of nociceptive afferents to mediate motor response of tail (TF), vocalization during stimulus (VD), and vocalization after discharge (VA); the withdrawal latency of tail (TFL) and hind paw (HPL) to thermal noxious stimulus and tonic pain responses were recorded in every rat. The TFL was not affected, HPL was decreased (p < 0.01), and the thresholds of TF and VD were not affected, while, that of VA was significantly decreased. The tonic pain rating was decreased (p < 0.01). A decrease in the threshold of VA (p < 0.01) is indicative of an increase in the emotional component of the response to the phasic pain, whereas a decrease in the pain rating indicates analgesia in response to the tonic pain. The results of our study suggest that chronic (45 d), intermittent (2 h/d) amplitude modulated RF field exposure to the peripubertal rat increases the emotional component of phasic pain over a basal eaualgesic state, while late response to tonic pain is decreased. The data suggest that amplitude modulated RF field differentially affects the mechanisms involved in the processing of various noxious stimuli.     

Acute and chronic tactile sensory testing after spinal cord injury in rats

Spinal cord injury (SCI) impairs sensory systems causing allodynia. To identify cellular and molecular causes of allodynia, sensitive and valid sensory testing in rat SCI models is needed. However, until recently, no single testing approach had been validated for SCI so that standardized methods have not been implemented across labs. Additionally, available testing methods could not be implemented acutely or when severe motor impairments existed, preventing studies of the development of SCI-induced allodynia(3). Here we present two validated sensory testing methods using von Frey Hair (VFH) monofilaments which quantify changes in tactile sensory thresholds after SCI. One test is the well-established Up-Down test which demonstrates high sensitivity and specificity across different SCI severities when tested chronically. The other test is a newly-developed dorsal VFH test that can be applied acutely after SCI when allodynia develops, prior to motor recovery. Each VFH monofilament applies a calibrated force when touched to the skin of the hind paw until it bends. In the up-down method, alternating VFHs of higher or lower forces are used on the plantar L5 dermatome to delineate flexor withdrawal thresholds. Successively higher forces are applied until withdrawal occurs then lower force VFHs are used until withdrawal ceases. The tactile threshold reflects the force required to elicit withdrawal in 50% of the stimuli. For the new test, each VFH is applied to the dorsal L5 dermatome of the paw while the rat is supported by the examiner. The VFH stimulation occurs in ascending order of force until at least 2 of 3 applications at a given force produces paw withdrawal. Tactile sensory threshold is the lowest force to elicit withdrawal 66% of the time. Acclimation, testing and scoring procedures are described. Aberrant trials that require a retest and typical trials are defined. Animal use was approved by Ohio State University Animal Care and Use Committee.     

Early exercise in spinal cord injured rats induces allodynia through TrkB signaling

Rehabilitation is important for the functional recovery of patients with spinal cord injury. However, neurological events associated with rehabilitation remain unclear. Herein, we investigated neuronal regeneration and exercise following spinal cord injury, and found that assisted stepping exercise of spinal cord injured rats in the inflammatory phase causes allodynia. Sprague-Dawley rats with thoracic spinal cord contusion injury were subjected to assisted stepping exercise 7 days following injury. Exercise promoted microscopic recovery of corticospinal tract neurons, but the paw withdrawal threshold decreased and C-fibers had aberrantly sprouted, suggesting a potential cause of the allodynia. Tropomyosin-related kinase B (TrkB) receptor for brain-derived neurotrophic factor (BDNF) was expressed on aberrantly sprouted C-fibers. Blocking of BDNF-TrkB signaling markedly suppressed aberrant sprouting and decreased the paw withdrawal threshold. Thus, early rehabilitation for spinal cord injury may cause allodynia with aberrant sprouting of C-fibers through BDNF-TrkB signaling.     

Effects of thermal acclimation on nervous conduction and muscle contraction in the frog Rana temporaria

The effects of season and acclimation temperature on the latency of the leg withdrawal reflex and three of its components have been studied: conduction velocity in the sciatic nerve, spinal conduction time, and contraction time of gastrocnemius muscle. The latency of the leg withdrawal reflex was markedly shortened by cold acclimation: the reaction times were at 6 degrees C 1.54 s in 4 degrees C acclimated and 3.97 s in 24 degrees C acclimated winter frogs. Also, the temperature dependence of the reflex latency was reduced by cold acclimation. Thus, frogs acclimated to cold responded to external stimuli in cold more rapidly than warm-acclimated ones. This cold adaptation of the reflex could not be explained by changes in its studied components. These made up only one-tenth of the reflex response time, and either did not show significant cold acclimation (muscle contraction and spinal conduction times in summer) or showed inverse acclimation, especially when measured at high temperatures (i.e. conduction velocities were reduced by acclimation to cold). Thus, the cold acclimation of the reflex response probably resides in the sensory component of the response. The inverse temperature adaptation response of conduction velocities may reflect a reduced ion permeability across cellular membranes in cold which decreases metabolic energy expenditure during inactive periods.     

The location of olfactory receptor sites. Inferences from latency measurements.

Excitatory responses recorded from vertebrate olfactory sensory neurons are characterized by long latencies compared with those from other sensory receptors. Explanations which assume free access of the stimuli to receptor molecules presumably located on the olfactory cilia necessarily imply an intrinsic delay in the transduction mechanism. In contrast, the possibility of restricted or delayed access due to diffusion of the stimulus to molecular receptors located on the dendritic know or proximal portions of the cilia suggests transduction processes having time courses similar to those in other sensory systems. We show that the threshold stimulus concentrations and the latency of the excitatory response of the salamander can be predicted primarily on the basis of a diffusional delay and that the receptor molecules are well below the surface of the mucus. Examination of response latencies for other species reported in the literature support the generality of diffusional delay. The predicted location of molecular receptor sites is largely insensitive to assumptions based on the mode of clearance of the stimuli. Additional access restrictions are discussed but are shown to generate qualitatively different latency functions than does diffusion, suggesting that they exert only minor influences on latency and threshold characteristics.     

Habituation and dishabituation mediated by the peripheral and central neural circuits of the siphon of aplysia

The siphon withdrawal response evoked by a weak tactile (water drop) or light stimulus is mediated primarily by neurons in the siphon. Central neurons (abdominal ganglion) contribute very little since the response amplitude and latency are not changed following removal of the abdominal ganglion. Similarly, habituation and dishabituation of this withdrawal response are not different after removal of the abdominal ganglion, indicating that the peripheral neural circuit in the isolated siphon can mediate habituation itself, and thus has many of the properties attributed to central neurons. Responses evoked by electrical stimulation of the siphon nerve habituate, depending upon the stimulus intensity and interval. These habituated responses may be dishabituated by tactile or light stimulation of the siphon. These results show that each neural system, peripheral and central, has an excitatory modulatory influence on the other. Normally adaptive siphon responses must be shaped by the integrated activity of both of these neural systems.     

Habituation and dishabituation mediated by the peripheral and central neural circuits of the siphon of Aplysia.

The siphon withdrawal response evoked by a weak tactile (water drop) or light stimulus is mediated primarily by neurons in the siphon. Central neurons (abdominal ganglion) contribute very little since the response amplitude and latency are not changed following removal of the abdominal ganglion. Similarly, habituation and dishabituation of this withdrawal response are not different after removal of the abdominal ganglion, indicating that the peripheral neural circuit in the isolated siphon can mediate habituation itself, and thus has many of the properties attributed to central neurons. Response evoked by electrical stimulation of the siphon nerve habituate, depending upon the stimulus intensity and interval. These habituated responses may be dishabituated by tactile or light stimulation of the siphon. These results show that each neural system, peripheral and central, has an excitatory modulatory influence on the other. Normally adaptive siphon responses must be shaped by the integrated activity of both of these neural systems.     

Mathematical models of threshold phenomena in the nerve membrane

The types of mathematical model which have been used to represent all-or-none behavior in the nerve membrane may be classified as follows: (1) thediscontinuous threshold phenomenon, in which differential equations with discontinuous functions provide both a discontinuity of response as a function of stimulus intensity at threshold and a finite maximum latency, (2) thesingular-point threshold phenomenon which exists in a phase space having analytic functions in its differential equations and having a singular point with one characteristic root positive and the rest with negative real parts, the latency being unbounded, and (3) thequasi threshold phenomenon, which has a finite maximum latency and continuous functions, but neither a true discontinuity in response nor an exact threshold. Several models of the nerve membrane in the literature are classified accordingly, and the applicability of the different types of threshold phenomena to the membrane is discussed, including an extension to a stochastic model.     

Dry Ice Fixation of Myofibrils for Scanning Electron Microscopy

A rapid method of fixation of myofibrils using dry ice is reported. A glass slide or coverslip containing a drop of glutaraldehyde-fixed suspension of myofibrils is placed on dry ice causing the myofibrils to adhere to the glass surface. The specimens are then dehydrated through the alcohols, air dried and metal coated. This technique gives the myofibrils a corrugated appearance under the scanning electron microscope corresponding to the sarcomere banding.     

The Spared Nerve Injury (SNI) Model of Induced Mechanical Allodynia in Mice

Peripheral neuropathic pain is a severe chronic pain condition which may result from trauma to sensory nerves in the peripheral nervous system. The spared nerve injury (SNI) model induces symptoms of neuropathic pain such as mechanical allodynia i.e. pain due to tactile stimuli that do not normally provoke a painful response [1]. The SNI mouse model involves ligation of two of the three branches of the sciatic nerve (the tibial nerve and the common peroneal nerve), while the sural nerve is left intact [2]. The lesion results in marked hypersensitivity in the lateral area of the paw, which is innervated by the spared sural nerve. The non-operated side of the mouse can be used as a control. The advantages of the SNI model are the robustness of the response and that it doesn’t require expert microsurgical skills.The threshold for mechanical pain response is determined by testing with von Frey filaments of increasing bending force, which are repetitively pressed against the lateral area of the paw [3], [4]. A positive pain reaction is defined as sudden paw withdrawal, flinching and/or paw licking induced by the filament. A positive response in three out of five repetitive stimuli is defined as the pain threshold. As demonstrated in the video protocol, C57BL/6 mice experience profound allodynia as early as the day following surgery and maintain this for several weeks.