Top-Down Effect of Direct Current Stimulation on the Nociceptive Response of Rats

Transcranial direct current stimulation (tDCS) is an emerging, noninvasive technique of neurostimulation for treating pain. However, the mechanisms and pathways involved in its analgesic effects are poorly understood. Therefore, we investigated the effects of direct current stimulation (DCS) on thermal and mechanical nociceptive thresholds and on the activation of the midbrain periaqueductal gray (PAG) and the dorsal horn of the spinal cord (DHSC) in rats; these central nervous system areas are associated with pain processing. Male Wistar rats underwent cathodal DCS of the motor cortex and, while still under stimulation, were evaluated using tail-flick and paw pressure nociceptive tests. Sham stimulation and naive rats were used as controls. We used a randomized design; the assays were not blinded to the experimenter. Immunoreactivity of the early growth response gene 1 (Egr-1), which is a marker of neuronal activation, was evaluated in the PAG and DHSC, and enkephalin immunoreactivity was evaluated in the DHSC. DCS did not change the thermal nociceptive threshold; however, it increased the mechanical nociceptive threshold of both hind paws compared with that of controls, characterizing a topographical effect. DCS decreased the Egr-1 labeling in the PAG and DHSC as well as the immunoreactivity of spinal enkephalin. Altogether, the data suggest that DCS disinhibits the midbrain descending analgesic pathway, consequently inhibiting spinal nociceptive neurons and causing an increase in the nociceptive threshold. This study reinforces the idea that the motor cortex participates in the neurocircuitry that is involved in analgesia and further clarifies the mechanisms of action of tDCS in pain treatment.     

Sex differences in nociceptive withdrawal reflex and pain perception

Experimentally induced pain often reveals sex differences, with higher pain sensitivity in females. The degree of differences has been shown to depend on the stimulation and assessment methods. Since sex differences in pain develop anywhere along the physiological and psychological components of the nociceptive system, we intended to compare the nociceptive flexion reflex (NFR) as a more physiological (spinal) aspect of pain procession to the verbal pain report of intensity and unpleasantness as the more psychological (cortical) aspect. Twenty female and twenty male healthy university students were investigated by use of nociceptive flexion reflex threshold (staircase method) after electrical stimulation of the N. suralis. Furthermore, we assessed supra-threshold reflex responses (latency, amplitude and area) by applying 10 stimuli 5 mA above reflex threshold. Following each stimulation, the subjects provided pain ratings of intensity and unpleasantness on a visual analogue scale. Females exhibited marked lower nociceptive flexion reflex thresholds than males, while the supra-threshold reflex response tailored to the individual reflex threshold did not show any significant differences. The verbal pain ratings, corrected for NFR threshold, were not found to differ significantly. The large sex differences in nociception that were present in NFR threshold but not in the pain ratings corroborate the hypothesis that spinal processes contribute substantially to sex differences in pain procession.     

Convergence of nociceptive information on the parabrachio-amygdala neurons in the rat

Neurones were recorded with extracellular micropipettes, in the parabrachial area located in the dorsolateral region of the pons of anaesthetized rats. All the neurones were identified by antidromic stimulation from the nucleus centralis of the amygdala. Numerous parabrachio-amygdala neurones (70%) were exclusively affected by noxious stimuli applied to several areas of the body. The rest of the neurones (30%) were not activated by any of these stimuli. The "nociceptive" neurones were classified in two groups: the neurones in the first group ("specific nociceptive", 55% of the whole population), responded to mechanical nociceptive and thermal nociceptive stimulation (threshold greater than 44 degrees C), with a strong and sustained activation. The neurones in the second group (15% of the whole population) responded by a strong inhibition to the nociceptive stimulation. Transcutaneous electrical stimulation demonstrated that the specific nociceptive parabrachial neurones received messages from A delta and C fibres. These results demonstrate that a spino-(trigemino)-ponto-amygdala nociceptive pathway exists which could be implicated in the emotional responses to noxious events.     

Impact of sex on hyperalgesia induced by sleep loss

This study evaluated the impact of sex on the short term consequences of different periods of sleep deprivation and the effect of the respective sleep recovery periods on nociceptive responses. Male and female C57BL/6J mice were assigned to the following groups: paradoxical sleep deprived (PSD) for 72 h, sleep restricted (SR) for 15 days, exposed to respective recovery periods for 24 h, or untreated home-cage controls (CTRL). Mice were submitted to a noxious thermal stimulus to evaluate their nociceptive response after PSD, SR, or recovery periods. Blood was collected for hormonal analysis. The nociceptive response was significantly lower in PSD and SR mice compared to CTRL animals, regardless of the sex. However, SR females had a lower paw withdrawal threshold than males. Sleep recovery was able to restore normal nociceptive sensitivity after PSD in both sexes. The hyperalgesia induced by SR was not reversed by sleep rebound. In females, low concentrations of estradiol were found after SR, and these concentrations continued to decrease after 24 hours of sleep recovery. The PSD male mice exhibited higher concentrations of corticosterone than the CTRL and SR male mice. Corticosterone levels were not affected by SR. Our study revealed that PSD and SR induce hyperalgesia in mice. The SR groups showed marked changes in the nociceptive response, and the females were more sensitive to these alterations. This finding indicates that, although different periods of sleep deprivation change the nociceptive sensitivity in male and female mice, sex could influence hyperalgesia induced by chronic sleep loss.     

Electrophysiological and psychophysical quantification of temporal summation in the human nociceptive system

Animal experiments have shown that the nociceptive reflex can be used as an indicator of central temporal integration in the nociceptive system. The aim of the present study on humans was to investigate whether the nociceptive reflex, evoked by repetitive strong electrical sural nerve stimuli, increased when summation was reported by the volunteers. The reflexes were recorded from the biceps femoris and rectus femoris muscles in eight volunteers following a series of stimulations at 0.1, 1, 2, and 3 Hz. Each series consisted of five consecutive stimuli. Using 0.1- and 1-Hz stimulation, the reflex was not facilitated in the course of the five consecutive stimuli. Following 2- and 3-Hz stimulation, the reflex size (root mean square amplitude) increased significantly during the course of the fifth stimulus. This reflex facilitation was followed by a significant increase (summation) in the pain magnitude when compared with 1- and 0.1-Hz stimulation. Furthermore, the threshold for psychophysical summation could be determined. This threshold (stimulus intensity) decreased when the stimulus frequency (1–5 Hz) of the five consecutive stimuli was increased. The nociceptive reflex and the psychophysical summation threshold might be used to clarify and quantify aspects of temporal summation within the human nociceptive system.     

Effect of centrally administered nitric oxide modulators in Brewer's yeast-induced nociception in rats

Possible modulation of Brewer's yeast-induced nociception by centrally (icv) administered nitric oxide (NO) modulators, viz., NO synthase (NOS) inhibitors, NO precursor, donors, scavengers and co-administration of NO donor (SIN-1) with NOS inhibitor (L-NAME) and NO scavenger (Hb) was investigated in rats. Administration of NOS inhibitors and NO scavenger Hb increased the pain threshold capacity significantly, whereas NO donors SIN-1, SNP and NO precursor L-arginine were found to be hyperalgesic. D-arginine, the inactive isomer of L-arginine and methylene blue, inhibitor of soluble guanylate cyclase failed to alter the nociceptive behaviour in rats. Co-administration of SIN-1 with L-NAME and Hb found to increase the nociceptive threshold. The results indicate, that centrally administered NO modulators alter the nociceptive transmission induced by Brewer's yeast in rats.     

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.     

Thermal Nociceptive Threshold Testing Detects Altered Sensory Processing in Broiler Chickens with Spontaneous Lameness

Lameness is common in commercially reared broiler chickens but relationships between lameness and pain (and thus bird welfare) have proved complex, partly because lameness is often partially confounded with factors such as bodyweight, sex and pathology. Thermal nociceptive threshold (TNT) testing explores the neural processing of noxious stimuli, and so can contribute to our understanding of pain. Using an acute model of experimentally induced articular pain, we recently demonstrated that TNT was reduced in lame broiler chickens, and was subsequently attenuated by administration of Non-Steroidal Anti-Inflammatory Drugs (NSAIDs). This study extended these findings to a large sample of commercial broilers. It examined factors affecting thermal threshold (Part 1) and the effect of an NSAID drug (meloxicam, 5 mg/kg) and of an opioid (butorphanol; 4 mg/kg) (Part 2). Spontaneously lame and matched non-lame birds (n = 167) from commercial farms were exposed to ramped thermal stimulations via a probe attached to the lateral aspect of the tarsometatarsus. Baseline skin temperature and temperature at which a behavioural avoidance response occurred (threshold) were recorded. In Part 1 bird characteristics influencing threshold were modelled; In Part 2 the effect of subcutaneous administration of meloxicam or butorphanol was investigated. Unexpectedly, after accounting for other influences, lameness increased threshold significantly (Part 1). In Part 2, meloxicam affected threshold differentially: it increased further in lame birds and decreased in non-lame birds. No effect of butorphanol was detected. Baseline skin temperature was also consistently a significant predictor of threshold. Overall, lameness significantly influenced threshold after other bird characteristics were taken into account. This, and a differential effect of meloxicam on lame birds, suggests that nociceptive processing may be altered in lame birds, though mechanisms for this require further investigation.     

Pain threshold variations in female rats as a function of the estrus Cycle

In this study, the response of female rats in different phases of the estrus cycle to nociceptive stimulation was evaluated using thermal (hot plate and tail immersion) and chemical (formalin) tests. In the hot plate test, the paw licking latency fell significantly (p < 0.05) in the metestrus and diestrus phases compared with the proestrus and estrus phases. The observations in the tail immersion test also followed the same pattern. The significant reductions in the paw licking and tail withdrawal latencies due to a lowered threshold denote an increase in pain sensitivity in the metestrus and diestrus phases. In the formalin test, the licking time fell significantly from the metestrus to the diestrus phase compared with the proestrus and estrus phases, the reduction in this test which was due to an increased threshold connotes a decrease in pain sensitivity. The results therefore seem test dependent. In conclusion, pain threshold in female rats depends on the estrus state. Keywords: Pain threshold, Variation, Estrus cycle.     

Dissociation of μ- and δ-opioid inhibition of glutamatergic synaptic transmission in superficial dorsal horn

Background

Although it has been widely accepted that the primary somatosensory (SI) cortex plays an important role in pain perception, it still remains unclear how the nociceptive mechanisms of synaptic transmission occur at the single neuron level. The aim of the present study was to examine whether noxious stimulation applied to the orofacial area evokes the synaptic response of SI neurons in urethane-anesthetized rats using an in vivo patch-clamp technique.

Results

In vivo whole-cell current-clamp recordings were performed in rat SI neurons (layers III-IV). Twenty-seven out of 63 neurons were identified in the mechanical receptive field of the orofacial area (36 neurons showed no receptive field) and they were classified as non-nociceptive (low-threshold mechanoreceptive; 6/27, 22%) and nociceptive neurons. Nociceptive neurons were further divided into wide-dynamic range neurons (3/27, 11%) and nociceptive-specific neurons (18/27, 67%). In the majority of these neurons, a proportion of the excitatory postsynaptic potentials (EPSPs) reached the threshold, and then generated random discharges of action potentials. Noxious mechanical stimuli applied to the receptive field elicited a discharge of action potentials on the barrage of EPSPs. In the case of noxious chemical stimulation applied as mustard oil to the orofacial area, the membrane potential shifted depolarization and the rate of spontaneous discharges gradually increased as did the noxious pinch-evoked discharge rates, which were usually associated with potentiated EPSP amplitudes.

Conclusions

The present study provides evidence that SI neurons in deep layers III-V respond to the temporal summation of EPSPs due to noxious mechanical and chemical stimulation applied to the orofacial area and that these neurons may contribute to the processing of nociceptive information, including hyperalgesia.     

Assessment of the relationship between hyperalgesia and peripheral inflammation in magnesium-deficient rats

Magnesium-deficient rats develop simultaneously a significant lowering of nociceptive threshold and a generalized inflammation. We investigated the relationship between these two phenomena by testing drugs that are able to suppress the inflammation in this model. In weaning rats fed a magnesium-depleted diet for ten days, the nociceptive threshold was assessed by the paw pressure test and the inflammation by a clinical score. A non-steroidal anti-inflammatory drug (piroxicam); antagonists of H1 and H2 receptors (astemizole and cimetidine. respectively); a glucocorticoid (dexamethasone); an inhibitor of mastocyte degranulation (cromoglycate); and estradiol benzoate were used to block the inflammatory response. Dexamethasone and estradiol significantly suppressed the inflammation (p < 0.001 vs control group). Cromoglycate showed a delayed anti-inflammatory effect (p < 0.01 vs control group on D10). The combination of astemizole and cimetidine partially blocked the inflammation process, whereas astemizole and piroxicam were without effect. Regardless of the effect of the test drugs on inflammation, no change in the time course of hyperalgesia was observed. These data support the view that hyperalgesia induced by the magnesium-depleted diet is not a consequence of the inflammatory process.     

Role of endogenous opiates and extracellular K+ accumulation in the inhibition of frog spinal reflexes by electrical skin stimulation

Electrical skin stimulation of the hind limb (10-100 Hz, 30 s-5 min) at the intensity which leads only to the excitation of low threshold afferents depressed (for 1-30 min) the flexor reflex evoked in spinal frogs by nociceptive stimuli. The inhibition, which lasted for longer than 5 min was blocked by naloxone. Short-term poststimulation effects were associated with an increase of extracellular K+ concentration (delta [K]e) and were not blocked by naloxone. Enkephalins or morphine applied to the spinal cord surface increased the threshold for flexor reflexes while naloxone decrease their threshold. The stimulation was followed by short-term hyperpolarization of primary afferents (PAH; 1-5 min) and by depression of dorsal root potentials (DPRs) which had a similar time course to the delta [K]e, and were not blocked by naloxone. This period was frequently followed by longlasting PAH and enhancement of DRPs (5-30 min), which were abolished by naloxone. Superfusion of the isolated spinal cord with opioids produced PAH and enhanced DRPs evoked by nociceptive stimuli, while naloxone or increase of [K] in Ringer solution depolarized primary afferents and depressed DRPs. It is suggested that the antinociceptive effects of electrical stimulation of low threshold cutaneous afferents in spinal frogs involves at least two mechanisms. The short-term effect may result from delta [K]e, especially at high stimulus strength and is equally effective against noxious and non-noxious stimuli. The longlasting effects selectively affecting nociceptive transmission appear to be produced by endogenous opioids.     

Interaction of Tonic and Phasic Pain in Rabbit Ontogenesis

In the experiments on the 20–25-day-old and adult rabbits, effects of tonic pain focus (a subcutaneous injection of formalin into leg dorsal surface) on behavioral and electrophysiological characteristics of acute pain were studied. The effect of the 40–60-min-long tonic pain was seen as a decrease of defensive reaction threshold and an increase of inhibitory effect of brain rewarding zones on evoked potential recorded in thalamus parafascicular complex in response to a nociceptive electrocutaneous stimulation in narcotized rabbits. The changes observed were biphasic and coincided in time with an enhancement of the earlier described [26] specific behavioral responses to formalin injection. It is established that the effect of tonic pain is more expressed by its intensity and duration in the 20–25-day-old than in adult rabbits.     

Investigating the role of the hypothalamic supraoptic nucleus in nociception in the rat

We investigated the role of the hypothalamic supraoptic nucleus (SON) in nociception in the rat. Electrical stimulation of the SON or microinjection of a small dose of L-glutamate sodium into the SON elevated the nociceptive threshold in a dose-dependent manner, while cauterization of the SON decreased the nociceptive threshold. Pituitary removal did not influence the antinociceptive effect of L-glutamate sodium in the SON. The data suggested that the neurons and not the nervous fibers in the SON played an important role in antinociception.     

Lesions of the hypothalamic arcuate nucleus produce a temporary hyperalgesia and attenuate stress-evoked analgesia

In an attempt to elucidate the role of β-endorphin in the modulation of ‘basal’ nociceptive threshold and in the mediation of the antinociception (analgesia) evoked by stress, a series of lesions of the arcuate nucleus, the origin of the central system of β-endorphinergic neurones, were performed. These lesions produced an ～80% depression in the level of β-endorphin immunoreactivity in both the hypothalamus and periventricular β-endorphinergic fibre-containing tissue. A 50% decrease in the neurointermediate lobe content of immunoreactivity, but no change in the levels of this in the anterior lobe was also observed. Arcuate lesioned rats were significantly hyperalgesic in comparison to sham animals on day 4 post-operation, but on days 10 and 12, the basal nociceptive threshold of lesioned and sham groups did not differ significantly. On day 12 post-surgery upon exposure to 5 min foot-shock stress, lesioned rats developed a significantly smaller increase in tail-flick latency than did sham animals. These data are evidential of the importance of the arcuate nucleus in the determination of basal nociceptive threshold and in the generation of the analgesia which accompanies stress and are, further, suggestive of a role of central β-endorphin in the mediation of these processes.     

Suppression of nociceptive reactions in mice under low-intensity microwave irradiation of acupuncture points

We studied nociceptive responses to subcutaneous injections of formalin and electrical stimulation of the limbs in control mice and in mice whose acupuncture points (AP) were subjected to low-intensity microwave irradiation. In the latter animals, nociceptive reactions were significantly weaker than those in the control mice. The analgesic effect depended on what AP was selected and irradiated and on the duration and timing of microwave irradiation. In different experimental series, the duration of a formalin injection-induced nociceptive behavioral reaction decreased by 23.3–59.6%. The threshold of vocalization responses to stimulation on an electrified floor increased by 25.8±28.0%. The results demonstrate that a technique of analgesia by influencing the AP with microwave irradiation of a nonthermal intensity is rather effective.     

Autophagy impairment in a mouse model of neuropathic pain

Background

High frequency electrical stimulation (HFS) of primary nociceptive afferents in humans induce a heightened sensitivity in the surrounding non-stimulated skin area. Several studies suggest that this heterotopic effect is the result of central (spinal) plasticity. The aim of this study is to investigate HFS-induced central plasticity of sensory processing at the level of the brain using the electroencephalogram (EEG). To this end we measured evoked potentials in response to noxious electrical pinprick-like stimuli applied in the heterotopic skin area before, directly after and 30 minutes after HFS.

Results

We observed potential cortical electrophysiological correlates of heterotopic facilitation. Two different cortical correlates were found; the first one was a lateralized effect, i.e. a larger N100 amplitude on the conditioned arm than the control arm 30 minutes after end of HFS. This was comparable with the observed lateralized effect of visual analogue scale (VAS) scores as response to the mechanical punctate stimuli. The second correlate seems to be a more general (non-lateralized) effect, because the result affects both arms. On average for both arms the P200 amplitude increased significantly 30 minutes after end of HFS with respect to baseline.

Conclusions

We suggest that for studying heterotopic nociceptive facilitation the evoked brain response is suitable and relevant for investigating plasticity at the level of the brain and is perhaps a more sensitive and reliable marker than the perceived pain intensity (e.g. VAS).     

Systemic and anti-nociceptive effects of prolonged lidocaine,ketamine, and butorphanol infusions alone and in combination in healthy horses

Background

Prolonged drug infusions are used to treat horses with severe signs of pain, but can be associated with altered gastrointestinal transit. The purpose of this study was to determine the effects of prolonged constant rate infusions (CRI) of lidocaine (L), butorphanol (B), and ketamine (K) alone and in combination on gastrointestinal transit, behavior, and thermal nociceptive threshold in healthy horses.

Methods

Eight healthy adult horses were used in a randomized, cross-over, blinded, prospective experimental trial. Interventions were saline, L, K, B, LK, LB, BK, and LBK as an intravenous CRI for 96 hours. Drugs were mixed or diluted in saline; following a bolus, CRI rate was 0.15mL/kg/hr with drug doses as follows: L – 1.3 mg/kg then 3 mg/kg/hr; B – 0.018 mg/kg then 0.013 mg/kg/hr; K – 0.55 mg/kg then 0.5 mg/kg/hr. Two-hundred plastic beads were administered intragastrically by nasogastric tube immediately prior to the bolus. Feces were collected every 2 hours, weighed, and beads manually retrieved. Behavior was scored every 2 hours, vital parameters every 6 hours, and thermal nociceptive threshold every 12 hours for 96 hours. Drug concentrations in the LBK solution were tested every 6 hours for 72 hours.

Results

Four of 64 trials (3 LBK, 1 BK) were discontinued early due to signs of abdominal discomfort. There were no apparent differences between groups in vital parameters or thermal threshold. Transit time was delayed for LB and LBK with a corresponding decrease in fecal weight that was most severe in the final 24 hours of infusion. Significant changes in behavior scores, vital parameters, or thermal threshold were not observed. The concentration of each drug in the combined solution declined by less than 31% over the sampling period.

Conclusions

Drug combinations containing butorphanol cause an apparent delay in gastrointestinal transit in healthy horses without substantially affecting somatic nociception at the doses studied. Combinations of lidocaine and ketamine may have less impact on gastrointestinal transit than infusions combined with butorphanol. Further work is needed to determine the effects of these drugs in painful or critically ill patients.