James L. Henry

Effects of substance P on functionally identified units in cat spinal cord

A correlation was made between the effects of synthetic substance P (ssP) on spinal dorsal horn neurones and the responses of these neurones to natural peripheral stimulation. It was found that ssP caused excitation only of those units which were excited by noxious radiant heat applied to the skin. ssP also caused a small facilitation of the excitatory response to this noxious stimulus and, in two cases, led to a response to noxious heat of units which had previously been unaffected by this stimulus. The ratio of ssP sensitive to insensitive units was highest in the deeper parts of the dorsal horn. Excitation by ssP showed a positive correlation to excitation by bradykinin. These results suggest that substance P may be involved in excitatory spinal processes and that its actions may be associated specifically with nociception.

Substance P reduces tail-flick latency: Implications for chronic pain syndromes

Abstract In the awake restrained rat the intrathecal administration of substance P or the partial substance P homologue eledoisin‐related peptide (ERP) reduced reaction time to a noxious radiant heat stimulus and, at high doses, produced additional behavioural responses suggesting that the animals had reacted to what they perceived as a painful stimulus. The reduction in tail‐flick latency was observed as early as 30 sec following peptide administration peaked at 1 min and persisted for 5–10 min, after which an overshoot of the response (i.e., an increase in reaction time) was observed. The responses varied in their magnitude with the amount of peptide given, substance P being approximately 4 times more potent on a molar basis than ERP. Intrathecal administration of an equal volume of vehicle (artificial cerebrospinal fluid) had no effect on tail‐flick latency and failed to produce any of the other behavioural changes. The following interpretations are made. The decrease in tail‐flick latency suggests that pain threshold was decreased, and the dramatic behavioural effects seen at high doses suggest that an excess of substance P in the spinal cord is capable of producing a painful sensation. The rapid onset of the response suggests rapid penetration of substance P and ERP to the appropriate receptors, and the rapid decay of the response suggests rapid removal. Taken together, these results are consistent with the earlier suggestion that substance P plays a role as an excitatory agent in sensory pathways subserving pain. It is proposed that some conditions of chronic pain in man may therefore be due to an overabundant amount of substance P. This is complementary to a second proposal that other cases of chronic pain may be due to a supersensitivity of substance P receptors. The former is more likely to be associated with organic disorders, the latter with nerve damage, e.g. with causalgia, the neuralgias and perhaps some cases of phantom limb pain.

Novel substance P antagonist, CP-96,345, blocks responses of cat spinal dorsal horn neurons to noxious cutaneous stimulation and to substance P

Responses of dorsal horn neurons to iontophoretic application of substance P (80-120 nA), and to noxious thermal and noxious mechanical stimulations of the receptive field in the hind limb were tested in adult cats before and after the administration of the specific, non-peptide, NK-1 receptor antagonist CP-96,345 (0.5 mg/kg, i.v.). CP-96,345 inhibited the response of the neurones to substance P and also the response of these substance P-sensitive neurones to noxious thermal stimulation. The response of the substance P-insensitive neurones to noxious heat stimulations were, however, unaffected by CP-96,345. The effect of CP-96,345 on the response of neurones to noxious mechanical stimulation was variable. The results confirm the role of substance P in thermal nociception.

Knockdown of spinal metabotropic glutamate receptor 1 (mGluR1) alleviates pain and restores opioid efficacy after nerve injury in rats

Nerve injury often produces long‐lasting spontaneous pain, hyperalgesia and allodynia that are refractory to treatment, being only partially relieved by clinical analgesics, and often insensitive to morphine. With the aim of assessing its therapeutic potential, we examined the effect of antisense oligonucleotide knockdown of spinal metabotropic glutamate receptor 1 (mGluR1) in neuropathic rats. We chronically infused rats intrathecally with either vehicle, or 50 μg day−1 antisense or missense oligonucleotides beginning either 3 days prior to or 5 days after nerve injury. Cold, heat and mechanical sensitivity was assessed prior to any treatment and again every few days after nerve injury. Here we show that knockdown of mGluR1 significantly reduces cold hyperalgesia, heat hyperalgesia and mechanical allodynia in the ipsilateral (injured) hindpaw of neuropathic rats. Moreover, we show that morphine analgesia is reduced in neuropathic rats, but not in sham‐operated rats, and that knockdown of mGluR1 restores the analgesic efficacy of morphine. We also show that neuropathic rats are more sensitive to the excitatory effects of intrathecally injected N‐methyl‐D‐aspartate (NMDA), and have elevated protein kinase C (PKC) activity in the spinal cord dorsal horn, two effects that are reversed by knockdown of mGluR1. These results suggest that activity at mGluR1 contributes to neuropathic pain through interactions with spinal NMDA receptors and PKC, and that knockdown of mGluR1 may be a useful therapy for neuropathic pain in humans, both to alleviate pain directly, and as an adjunct to opioid analgesic treatment.

Effects of substance P on nociceptive and non-nociceptive trigeminal brain stem neurons

&NA; As little information is available on the chemistry of synaptic transmission in trigeminal brain stem nuclei, an iontophoretic study was done on the effects of glutamate and substance P on single neurons in trigeminal nuclei oralis and caudalis in cats anesthetized with chloralose and paralyzed. The neurons were additionally studied for their responses to natural noxious and innocuous cutaneous and intraoral stimuli as well as to bipolar stimulation of the ipsilateral and contralateral canine tooth pulps, the exposed infraorbital and superior laryngeal nerves and forepaw. Glutamate excited all units tested. Substance P also had an excitatory effect, but only on some units. The slow time course of this effect was similar to that reported in other CNS regions. Units excited by substance P were located only in nucleus caudalis, and all responded to noxious cutaneous stimuli and/or to stimulation of tooth pulp; units responding only to innocuous orafacial stimulation were not excited by substance P. Levorphanol and opioid peptides were also applied iontophoretically to some of the neurons and were found to have depressant effects on nociceptive units. The data support the possibility that substance P and endogenous opioids play a role in chemical transmission in nociceptive pathways in trigeminal nucleus caudalis. The regional specificity of substance P excitation adds support to the earlier evidence of a differential distribution of sensory inputs to nuclei oralis vs. caudalis, with facial nociceptive afferents projecting only to caudalis. The functional specificity of substance P excitation also adds to the parallels found between the dorsal horn and nucleus caudalis. In addition, the similarity between the dorsal horn and nucleus caudalis with respect to the effects of substance P and of the opioids suggests a parallel in the neurochemistry of synaptic transmission at the two levels.

Paw withdrawal threshold in the von Frey hair test is influenced by the surface on which the rat stands.

The effect of testing surface on the rat hind paw withdrawal threshold in the von Frey hair test is investigated in this study. The data indicate that wire mesh, which is typically used to apply von Frey hairs, may have an effect on the paw withdrawal threshold. For example, in control rats tested on the wire mesh, variability in the withdrawal threshold was observed between the left and the right hind paws (51.04+/-12.29 and 64.31+/-9.37 g, respectively) and on different days of testing (35.24+/-9.54 and 45.83+/-12.97 g for the left and right hind paws, respectively, 7 days later). In an attempt to reduce this variability, a customized platform was used to measure the von Frey hair-induced paw withdrawal in the rat. It consists of an opaque, flat-surfaced plastic platform with holes through which von Frey hairs are inserted and applied to the plantar surface of the paw. In control rats tested with von Frey hairs using this customized platform, variability in the paw withdrawal thresholds between the left and right hind paws in single rats over time as well as between different rats was reduced (49.86+/-6.97 and 49.29+/-6.56 g for the left and right hind paws, respectively, on day 0; 48.29+/-5.82 and 53.00+/-4.59 g for the left and right hind paws, respectively, 7 days later). Furthermore, in rats in which a 2 mm polyethylene cuff was used to constrict the left common sciatic nerve, the ipsilateral as well as the contralateral hind paw withdrawal thresholds were decreased (2.45+/-0.65 and 26.09+/-5.86 g, respectively, 7 days later). In similar rats tested on the wire mesh, the ipsilateral but not the contralateral paw withdrawal threshold decreased (12.80+/-2.21 and 65.00+/-10.28 g, respectively, at 7 days). The data suggest that the flat surface and opaque properties of the customized platform enable accurate, reliable and repeatable measurements of ipsilateral and contralateral paw withdrawal threshold using von Frey hairs in normal and nerve-injured rats.

Physiological evidence that the `interphase' in the formalin test is due to active inhibition

Injection of a dilute solution of formalin into a rat hindpaw produces a biphasic nociceptive response consisting of an early phase during the first 5 min after formalin injection and a later phase starting after 15 min and lasting for 40-50 min. The period between the two phases of nociceptive responding is generally considered to be a phase of inactivity. We compared the nociceptive behaviors produced by a single hindpaw injection of 50 microl of formalin with those produced by two formalin injections given 20 min apart. A single formalin injection at concentrations of either 1 or 2.5%, produced the typical biphasic nociceptive responses. In rats given a second injection of either 1 or 2.5% formalin 20 min after the first, a triphasic response occurred, with a second diminution of nociceptive behavior observed between 10 and 15 min after the second injection. When a second injection of 2.5% formalin was given 5 min after the first, there was no difference from the scores in the group given only one injection. In electrophysiological experiments on single dorsal horn nociceptive neurons, a second injection of 2.5% formalin into the peripheral cutaneous receptive field, 40 min after the first and at the same site of injection as the first formalin injection, depressed neuronal activity for approximately 15-20 min. From the data it can be concluded that the interphase period in the formalin test is due to active inhibition. Furthermore, the inhibition which we are reporting here is independent of the concentration of formalin used, and thus of any so-called inflammatory component. The lack of additive nociceptive effects when the inter-injection interval was only 5 min, suggests that a maximum inhibition was provoked by 2.5% formalin; it can also be concluded that the active inhibition is of overriding importance physiologically, compared with the nociceptive activity. Data from parallel electrophysiological experiments on spinal dorsal horn neurons demonstrated a diminution in excitability after a second formalin injection into the cutaneous receptive field. As these data were obtained from pentobarbital-anesthetized, spinalized rats, the data suggest further that the two excitatory phases and the active inhibition are mediated by spinal mechanisms and that the inhibition is not under regulation of a GABAergic mechanism. The implication of the results is not only further evidence of physiological control mechanisms interacting to regulate pain, but they also indicate the overriding priority of intrinsic inhibitory mechanisms. This, in turn, suggests that the clinical management of pain may be enhanced by efforts to potentiate mechanisms of inhibition.

Parametric Studies on Electroacupuncture-Like Stimulation in a Rat Model: Effects of Intensity, Frequency, and Duration of Stimulation on Evoked Antinociception

We have found that electroacupuncture-like stimulation of defined sites in the hindlimb of the rat inhibits a nociceptive withdrawal reflex. The lightly anaesthetized rat was used and tall withdrawal from a noxious radiant heat stimulus was the nociceptive reflex. Standard stimulation of hindlimb meridian points femur-futu (ST-32), fengshi (GB-31), and zusanli (ST-36) consisted of a 2-ms square voltage pulse at 4 Hz for a duration of 20 min, applied at 20 times the threshold to evoke muscle twitch. This produced two types of inhibition of the reflex; one was an increase in the latency of up to 80% during the stimulation, termed the brief antinociception, and the other was a post stimulation increase of up to 60% lasting greater than 1 h, termed the persistent antinociception. When the stimulus intensity was reduced to 10 times threshold, the latency during stimulation increased up to 50%, but the persistent response did not occur. Stimulation at threshold produced neither effect. When the train duration was altered, 10 min of stimulation produced only the brief effect, whereas 40 min of stimulation produced both effects, although the persistent effect lasted only 20 min. Stimulation at 6 Hz produced responses similar to those at 4 Hz, whereas stimulation at 2 Hz produced smaller effects. At 8 Hz, only the brief antinociception was elicited. With a pulse duration of 0.2 ms, the brief response was observed but the persistent response was markedly attenuated, whereas 5 ms produced responses similar to those with 2 ms. These data suggest that high-intensity, low-frequency electrical stimulation of meridian points in the rat hindlimb produces both brief and persistent antinociceptive effects on the tail withdrawal reflex, and both effects are dependent upon the parameters of stimulation. The persistence of the latter effect beyond the period of stimulation suggests events occurring after direct synaptic activity, possibly mediated via plastic changes at spinal and/or supraspinal levels.

CP-96,345, but not its stereoisomer, CP-96,344, blocks the nociceptive responses to intrathecally administered substance P and to noxious thermal and chemical stimuli in the rat.

The effects of subcutaneous administration of the non-peptide NK-1 (substance P) receptor antagonist, CP-96,345, and its stereoisomer, CP-96,344, were tested in three nociceptive paradigms in the rat. In the first paradigm, tail flick responses were monitored before and after intrathecal administration of substance P (6.5 nmol) in rats pretreated subcutaneously with saline, CP-96,344 (5 mg/kg) or CP-96,345 (5 mg/kg). In the control groups, pretreated with saline (n = 6) or with CP-96,344 (n = 5), substance P reduced the tail flick reaction time at 1 min after administration to 38.3 +/- 5.1 (mean +/- S.E.M.) and 32.1 +/- 7.7% of the mean baseline value, respectively. In contrast, in the group pretreated with CP-96,345 (n = 6) the reaction time following administration of substance P was 98.8 +/- 3.3% of the baseline reaction time; this value was not significantly different from the baseline value of this group, indicating a block (P < 0.01) of the substance P-induced facilitation of the tail flick response. In the second paradigm, rats were anesthetized with a mixture of chloral hydrate (120 mg/kg, i.p.) and sodium pentobarbital (20 mg/kg, i.p.), and the effects were determined on tail flick reaction time of a sustained noxious cutaneous stimulation, immersing the tip of the tail in hot water at 55 degrees C. In the groups of rats pretreated with saline (n = 4) or with CP-96,344 (n = 7), this noxious stimulus produced a transient decrease in reaction time to 62-74% of the baseline value.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo antinociceptive activity of anti-rat mGluR1 and mGluR5 antibodies in rats.

TO examine the specific roles of group I metabotropic glutamate receptors (mGluRs) in nociceptive processing, we examined the effects of intrathecal (i.t.) treatment with antibodies raised against the C-terminals of mGluR1 and mGluR5 in various rat pain models. The effects of anti-mGluR1 IgG and anti-mGluR5 IgG were assessed in a model of persistent pain induced by intrathecal administration of the mGluR1/5 agonist DHPG, as well as in models of heat pain (plantar test), chemical pain (formalin test) and neuropathic pain. DHPG-induced spontaneous nociceptive behaviours (SNB) were significantly attenuated by i.t. treatment with either anti-mGluR1 IgG (30 μg) or anti-mGluR5 IgG (10 and 3 0μg). Neither anti-mGluR1 IgG (30 μg) nor anti-mGluR5 IgG (30 μg) significantly increased response latencies to noxious heat in the plantar test, compared with anti-rat IgG (control IgG). Moreover, neither antibody (3 0μg) significantly reduced formalin pain scores as compared to control IgG. However, i.t. treatment with anti-mGluR1 IgG (30 μg) or anti-mGluR5 IgG (30 μg) significantly reduced cold hyper-sensitivity exhibited 8 days after constriction injury of the sciatic nerve, supporting the contention that group I mGluRs play a role in the development of neuropathic pain. Because these antibodies were effective against neuropathic pain, and not acute heat or chemical noxious stimuli, these results suggest that mGluRs are involved in nociceptive processing in chronic pain states rather than signaling acute noxious stimuli, and that DHPG-induced pain may be mediated by similar mechanisms as neuropathic pain.