Kiran Yashpal

Intracellular Messengers Contributing to Persistent Nociception and Hyperalgesia Induced by L‐Glutamate and Substance P in the Rat Formalin Pain Model

The contribution of the intracellular messengers nitric oxide, arachidonic acid and protein kinase C to persistent nociception in response to tissue injury in rats was examined following the subcutaneous injection of formalin into the hindpaw. Formalin injury‐induced nociceptive behaviours were reduced by intrathecal pretreatment with inhibitors of nitric oxide synthase (NG‐nitro‐L‐arginine methyl ester, L‐NAME), arachidonic acid (dexamethasone) or protein kinase C [protein kinase C (19–26) and 1–95‐(isoquinolinesulphonyl)‐2‐methylpiperazine dihydrochloride, H‐7]. Each of these agents affected the tonic, but not the acute, phase of the formalin response. Furthermore, none of these agents affected mechanical or thermal flexion reflex thresholds in rats not injected with formalin. Conversely, formalin‐induced nociceptive responses were enhanced by stimulators of nitric oxide (sodium nitroprusside), arachidonic acid metabolism (arachidonic acid) or protein kinase C [(±)‐1‐oleoyl‐2‐acetyl‐glycerol], and were slightly reduced by inositol trisphosphate. Mechanical flexion reflexes were also reduced by arachidonic acid, while thermal flexion reflexes were reduced after treatment with sodium nitroprusside, arachidonic acid or [(±)‐1 ‐oleoyl‐2‐acetyl‐glycerol]. The enhancement of formalin nociceptive behaviours (hyperalgesia) in rats treated with L‐glutamate or substance P was reversed by pretreatment with inhibitors of nitric oxide (L‐NAME), arachidonic acid (dexamethasone) or protein kinase C (H‐7). The results suggest that central sensitization and persistent nociception following formalin‐induced tissue injury, and the hyperalgesia in the formalin test induced by L‐glutamate and substance P, are dependent on the intracellular messengers nitric oxide, arachidonic acid and protein kinase C.

Quantitative autoradiographic distribution of multiple neurokinin binding sites in rat spinal cord

As a means of evaluating the role of neurokinins (NKs) in spinal function, the present study examines the quantitative autoradiographic distribution in the rat spinal cord of [125I]Bolton-Hunter-substance P, (2-[125I]iodohistidyl1)-neurokinin A and [125I]Bolton-Hunter-eledoisin as respective radioligands for NK-1, NK-2 and NK-3 receptors. These putative NK receptor sub-types are clearly differentially distributed at the various levels of the spinal cord. NK-1 sites represent the most abundant population of spinal NK receptors. They are most concentrated in the dorsal and ventromedial borders of the dorsal horn, the intermediolateral nucleus of the thoracic cord and the phrenic motor nucleus in the cervical ventral horn. NK-2 and NK-3 sites are also present in the spinal cord, although in much lower quantities than NK-1 sites. NK-2 sites are mostly found along the dorsal and ventromedial borders of the dorsal horn, in a narrow band connecting the two lateral horns of the thoracic cord, around the central canal of the lumbar and sacral segments and lamina IX of the cervical ventral horn. NK-3 sites are most dense in the dorsal border of the dorsal horn, with moderate amounts in the lateral horn of the thoracic cord and around the central canal of lumbar and sacral segments. The differential distribution of these 3 classes of NK sites in the spinal cord suggests that each NK receptor sub-type could mediate specific sensory, autonomic and/or motor functions at the spinal level.

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.

Differential effects of NMDA and group I mGluR antagonists on both nociception and spinal cord protein kinase C translocation in the formalin test and a model of neuropathic pain in rats.

&NA; Coincident with nociception, both noxious chemical stimulation of the hind paw and chronic constriction injury (CCI) of the sciatic nerve produce an increase in protein kinase C (PKC) translocation in the spinal cord of rats. Noxious stimulus‐induced PKC translocation likely depends on glutamate activity at either N‐methyl‐D‐aspartate (NMDA) receptors or group I metabotropic glutamate receptors (mGluR1/5) in the spinal cord dorsal horn. This study compares nociceptive responses to, and the alterations in membrane‐associated PKC, induced by noxious chemical stimulation of the hindpaw and CCI of the sciatic nerve, as well as their modulation by both NMDA and mGluR1/5 receptor antagonists. Three groups of rats were given a single intrathecal (i.t.) injection of either vehicle, dizocilpine maleate (MK‐801, 60 nmol), an NMDA receptor antagonist, or (S)‐4‐carboxyphenylglycine (S)‐4CPG, (150 nmol), an mGluR1/5 antagonist, 10 min prior to a 50 &mgr;l of 2.5% formalin injection into the ventral surface of one hind paw. Another three groups of rats were given twice daily injections of either vehicle, MK‐801 (30 nmol) or (S)‐4CPG (90 nmol) i.t. for 5 days starting 30 min before CCI or sham injury of the sciatic nerve. Nociceptive responses were assessed for a 60 min period after the formalin injection in the first three groups, and tests of mechanical and cold allodynia were performed on days 4, 8, 12 and 16 after CCI for the latter three groups. Furthermore, changes in the levels of membrane‐associated PKC, as assayed by quantitative autoradiography of the specific binding of [3H]‐phorbol 12,13‐dibutyrate ([3H]‐PDBu) in the dorsal horn of the lumbar spinal cord sections, were assessed in formalin‐injected rats (at 5, 25 and 60 min) and in neuropathic rats 5 days after CCI, treated (as above) with vehicle, MK‐801 or (S)‐4CPG. The results indicate that i.t. treatment with MK‐801 significantly reduced nociceptive scores in the formalin test and also produced a significant suppression of formalin‐induced increases in [3H]‐PDBu binding in laminae I–II, III–VI and X of the lumbar spinal cord. In contrast, i.t. treatment with (S)‐4CPG failed to significantly affect either nociceptive behaviours in the formalin test or formalin‐induced increases in [3H]‐PDBu binding in laminae I–II and III–VI of the lumbar spinal cord. On the other hand, i.t. treatment with either MK‐801 or (S)‐4CPG produced a significant reduction in mechanical and cold hypersensitivity, as well as [3H]‐PDBu binding in laminae I–II and III–VI of the lumbar spinal cord, after CCI. These results suggest that while NMDA, but not mGluR1/5, receptors are involved in translocation of PKC and nociception in a model of persistent acute pain, both types of receptors influence the translocation of PKC in dorsal horn and mechanical and cold allodynia in a model of chronic neuropathic pain.

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.

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)

Effects of Preemptive or Postinjury Intrathecal Local Anesthesia on Persistent Nociceptive Responses in Rats: Confounding Influences of Peripheral Inflammation and the General Anesthetic Regimen

Background Although experimental evidence indicates that preemptive intrathecal treatment with local anesthetics reduces postinjury neuronal hyperexcitability, clinical evidence indicates that preemptive treatments do not consistently reduce postoperative pain. The current study used experimental models of postinjury nociception, in which rats received subcutaneous or intraarticular injections of the irritant formalin, to evaluate the effects of peripheral inflammation, or the use of agents supplemental to anesthesia, as possible confounding influences on the effectiveness of preinjury and postinjury intrathecal local anesthetic treatments. Methods In experiment 1, lumbar intrathecal lidocaine (30 micro liter, 2%), given either 5 min before or 5 min after hind paw injection of 50 micro liter of varying concentrations of formalin, was compared with intrathecal cerebrospinal fluid, for their effects on nociceptive responses in the late phase of the formalin test. Furthermore, the effect of hind paw injection of 50 micro liter of 2.5, 3.75, or 5.0% formalin on peripheral inflammation was assessed by measuring plasma extravasation in the hind paws of rats given Evans Blue dye (50 mg/kg, intravenous). In experiment 2, rats received a deep tissue injury (100 micro liter of 5.0% formalin into the knee joint) while under halothane anesthesia. In addition to halothane (3-4%), rats received either saline, pentobarbital (13 mg/kg, intraperitoneal), or pentobarbital + morphine (0.5 mg/kg, intravenous), with or without preinjury or postinjury spinal anesthesia using intrathecal bupivacaine (30 micro liter, 0.75%), to assess the effects of supplemental treatments on the preemptive effects of intrathecal bupivacaine. Results Lumbar intrathecal lidocaine pretreatment, but not posttreatment, significantly reduced late phase nociceptive responses to hind paw injections of 2.5% formalin. The preemptive effects of lidocaine were overridden in rats that received hind paw injections of 3.75 and 5.0% formalin. Hind paw injection of 50 micro liter of 3.75 or 5.0%, but not 2.5% formalin produced an increase in plasma extravasation. Either pentobarbital or pentobarbital + morphine treatment, or a pentobarbital + morphine treatment and postinjury treatment with intrathecal bupivacaine failed to produce a significant reduction in the nociceptive response to the deep tissue injury. However, rats that received pentobarbital + morphine treatments and intrathecal bupivacaine before the injury had significantly reduced nociceptive responses to deep tissue injury when compared to the saline control group, but not to the group that received pentobarbital + morphine treatment and postinjury treatment with bupivacaine. Conclusions The current results attest to the important effects of ongoing inputs from inflamed tissue, and the use of supplemental treatments, as important confounding factors that may influence the effectiveness of preemptive spinal anesthesia for postoperative pain.

Effects of dorsal rhizotomy on neurokinin receptor sub-types in the rat spinal cord: a quantitative autoradiographic study.

Although abundant evidence suggests a major role for substance P (SP) and other neurokinins (NK) in the transmission of nociceptive information, it is not known whether the various NK receptor classes are differentially located in the substantia gelatinosa of the spinal cord where primary afferent fibres mostly terminate. In order to investigate this issue, we studied the effects of unilateral dorsal rhizotomy on binding of 125I-Bolton-Hunter-SP, (2-[125I]iodohistidyl1)-neurokinin A, and 125I-Bolton-Hunter-eledoisin as respective radioligands for the NK-1, NK-2 and NK-3 receptor sub-types. Seven, 14, 21 and 28 days following unilateral lumbosacral dorsal horn deafferentiation, NK receptor binding parameters were evaluated using quantitative receptor autoradiography. Rhizotomy produced an increase in the densities of NK-1, NK-2 and NK-3 binding sites in the superficial laminae of the dorsal horn. Increases were maximal at 14 days, post-operatively, for both NK-1 and NK-2 sites; slight recovery being observed thereafter. For NK-3 sites, unilateral rhizotomy induced a progressive increase in binding without evidence of recovery over time, at least up to 28 days post-lesion. NK-1 receptor binding parameters around the central canal and in the ventral horn were not affected by the dorsal rhizotomy. These data suggest that all 3 NK receptor classes are located post-synaptically to afferent fiber terminals in laminae I, II and X of the dorsal horn of the spinal cord.

NMDA receptor antagonist blocks the facilitation of the tail flick reflex in the rat induced by intrathecal administration of substance P and by noxious cutaneous stimulation.

This study examined effects of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonovaleric acid (APV), on facilitation of the tail flick reflex (1) by intrathecal administration of 6.5 nmol of substance P at the lumbar spinal level in awake rats and (2) by noxious cutaneous stimulation in anesthetized rats (by immersing the tip of the tail in hot water at 55 +/- 1 degrees C for 1.5 min). Reaction time was decreased by about 70% by intrathecal administration of substance P and by about 40% by tail immersion. Intrathecal administration of APV (2 nmol) or cerebrospinal fluid (CSF) failed to alter the baseline responses. However, APV but not CSF blocked the facilitation induced by intrathecal administration of substance P and by tail immersion. These results indicate that while NMDA receptors do not appear to be involved in mediating the tail flick reflex, they may be involved in expression of the facilitation of this reflex by substance P and/or by a related peptide.

Substance p anatogue blocks sp-induced facilitation of a spinal nociceptive reflex

Intrathecal administration of 10 micrograms (6.5 nmoles) of substance P to the lumbar spinal cord of the awake, restrained rat had a biphasic effect on reaction time in the tail-flick test. This effect consisted of an initial decrease at one min after administration, followed five min later by a smaller increase in reaction time. When substance P was given after prior intrathecal administration of 1.55, 3.1 or 6.2 nmoles of [D-Pro2, D-Phe7, D-Trp9]-substance P, the reduction in latency could be blocked in a dose-related manner. The analogue alone failed to alter reaction time but did produce a flaccid paralysis in some cases. Our results support the possibility that substance P is involved in transmission of nociceptive information at the spinal level, but indicate that it does not participate directly in the fast tail withdrawal response. The mechanism of the flaccid paralysis is not understood, but it appears to be a cumulative effect of more than one dose and is likely associated with motor rather than sensory neurones.