Barry D. Goldstein

Increase in substance P in the dorsal horn during a chemogenic nociceptive stimulus

Substance P has been implicated as a neuromediator of nociception in the dorsal horn of the spinal cord. This is the first report of quantitative increases in substance P following an acute nociceptive stimulus. Female Wistar rats were injected subcutaneously in the plantar aspect of the right hindpaw with 5% formalin. Substance P levels were determined by quantitative immunohistochemistry 1 h after the injection. The present results show that substance P levels in the upper two laminae of the ipsi-and contralateral dorsal horn were significantly increased 1 h after the formalin injection. These results suggest a direct functional relationship between substance P levels in the upper dorsal horn and an acute chemogenic nociceptive stimulus.

Release of substance P into the superficial dorsal horn following nociceptive activation of the hindpaw of the rat

Substance P (SP) has been widely proposed as being involved in the transmission of nociceptive information in the dorsal horn of the spinal cord. Formalin injected into the hindpaw as a nociceptive stimulus has been shown to increase the amount of immunoreactive SP in the dorsal horn, perhaps by decreasing SP release from primary afferent neurons. Much is known concerning the release of SP from tissue slices or from the entire spinal cord in vivo. However, less is known about the release patterns of SP in the superficial dorsal horn during the activation of peripheral nociceptors. In this study, noxious pinch applied to and formalin injection into the hindpaw were used as nociceptive stimuli while a stereotaxic push-pull cannula was used to perfuse the L5 dorsal horn. Experiments were conducted in unanesthetized decerebrate/spinal rats, and radioimmunoassay was used to determine the SP-like immunoreactivity (SPLI) content of collected perfusates. Results demonstrate that graded intensities of noxious mechanical pinch produced progressively increased release of SPLI into the dorsal horn; SPLI release returned to baseline rates following termination of the stimulus. The injection of 100 microliters of 5% formalin into the hindpaw produced a biphasic inhibition of SPLI release 0-40 min and greater than 60 min after formalin injection. The application of a noxious pinch following formalin injection produced an increase in SPLI release which did not return to baseline rates; this may be indicative of production of a hyperalgesic state caused by formalin injection. The results of this study support the concept that formalin injected into the hindpaw activates segmental antinociceptive systems which block SP release and limit nociceptive transmission.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulatory mechanisms for subtance P in the dorsal horn during a nociceptive stimulus: Axoplasmic transport vs electrical activity

Substance P (SP) is believed to be a neuromediator of nociception in the dorsal horn of the spinal cord. SP precursor is synthesized in the dorsal root ganglia (DRG) and transported via axoplasmic transport to the nerve terminal where it is stored and released as SP. The chemical nociceptive stimulus, formalin, when injected into the hindpaw causes an increase in the level of SP in the dorsal horn. This increase in SP may be the result of increased electrical activity due to activation of free nerve endings or the transport of some chemical or trophic signal to the DRG or to the central terminal. This study investigates the mechanism of the SP increase during the formalin stimulus. Rats were anesthetized and a laminectomy performed. In some experiments the sciatic nerve was exposed. Agar gel pads containing either colchicine or tetrodotoxin (TTX) were applied to the dorsal root or sciatic nerve prior to the injection of 5% formalin or saline into the hindpaw. Electrical activity across the dorsal root distal to the gel pad was monitored to determine the effects of colchicine and TTX on the nerve. Sixty min after the injection into the hindpaw, the animal was perfused and the lumbar spinal cord removed. Ten-micron frozen sections were stained for SP. It was found that the formalin-evoked increase in SP could be partially blocked by either colchicine or TTX applied to the dorsal root and completely blocked by the application of both agents together. TTX or colchicine applied to the sciatic nerve completely blocked the formalin-evoked increase in SP.(ABSTRACT TRUNCATED AT 250 WORDS)

Low but not high rate noxious radiant skin heating evokes a capsaicin-sensitive increase in spinal cord dorsal horn release of substance P

Some kinds of nociception appear to be partially mediated by the release of substance P (SP) in the spinal cord dorsal horn from terminals of primary afferent nociceptors. Only some nociceptors contain and release SP however. Specifically, SP appears to be released by unmyelinated (C) nociceptive afferents when activated by noxious stimulation to the skin, but does not appear to be contained in cutaneous myelinated (A delta) nociceptive afferents. We have proposed a model of nociception in rats that uses different rates of noxious skin heating to allow for differential assessment on behavioral responses mediated by the activation of A delta or C fiber nociceptors. As one means of testing the validity of this model we have examined the effects of using high and low rate noxious skin heating on the dorsal horn release of substance P-like immunoreactivity (SPLI) in decerebrate/spinal transected animals. Consistent with the model, low rate skin heating evokes a significant increase in dorsal horn SPLI release indicating C fiber mediation, whereas high rate skin heating did not evoke SP release, indicating mediation by afferents other than C afferents, i.e. A delta nociceptive afferents. Also consistent with behavioral effects, topical application of capsaicin, which sensitizes C nociceptors, increased the SPLI release evoked by low but not high rate skin heating. These data provide additional evidence that foot withdrawals evoked by low rate skin heating are mediated by C fiber activation, whereas foot withdrawals evoked by high rate skin heating are evoked by A delta fiber activation.

Time course of the alteration in dorsal horn substance p levels following formalin: blockade by naloxone

Substance P (SP) found in the dorsal horn of the spinal cord has been proposed as a mediator of nociception. Formalin injected into the hind paw of a rat as a nociceptive stimulus has been shown to increase the amount of immunoreactive SP in the dorsal horn, perhaps by decreasing SP release from primary afferent neurons. These SP changes may be due to the actions of endogenous opiates which can block SP release from primary afferent neurons. In order to determine the time course of SP changes in the dorsal horn and their modulation by naloxone, anesthetized rats pretreated subcutaneously with naloxone or saline were injected in the right hind paw with 0.4 ml of either saline or 5% formalin. After various time intervals, the animals were perfused and the lumbar enlargement of the spinal cord removed. Immunohistochemical staining and manual photometry were used to quantitate SP-like immunoreactivity (SPLI) in the dorsal horn. The results show that saline injection produced an increase in SPLI lasting 20 min, while formalin produced a biphasic effect with early (0-20 min) and late (20-60 min) increases in SPLI. Naloxone pretreatment 30 min prior to hind paw injection partially blocked the initial SPLI increase due to saline or formalin. However, this was not the case if naloxone was injected 2 min following hind paw injection. The formalin-induced late SPLI increase was blocked by naloxone only if it was administered prior to the formalin. This blockade of SPLI increases in the dorsal horn by naloxone implies that endogenous opioid systems play a role in the control of SP levels in the dorsal horn during nociception.

δ-Opioid receptor modulation of the release of substance P-like immunoreactivity in the dorsal horn of the rat following mechanical or thermal noxious stimulation

The present study was undertaken to investigate the effects of the opioid peptide Met-enkephalin (met-enk) on the release of substance P-like immunoreactivity (SPLI) in the lumbar dorsal horn during the application of a noxious mechanical or thermal stimulus to the ipsilateral hind paw and lower limb of the rat. A push-pull cannula was introduced to the lumbar dorsal horn in non-anesthetized decerebrate/spinal transected rats. The dorsal horn was perfused with artificial CSF and the collected perfusates were assayed for SPLI using radioimmunoassay. A noxious mechanical or thermal stimulus was applied to different areas of the ipsilateral hind paw and lower limb. Met-enk (500 nM) applied to the dorsal horn through the perfusate reduced the basal release of SPLI by 29 +/- 9% and prevented the increase in the release of SPLI evoked by the noxious mechanical or thermal stimulus. The effect of met-enk was blocked by the selective delta-opioid receptor antagonist naltrindole (500 nM). Naltrindole (NTD) alone elicited a 75 +/- 30% increase in the basal release of SPLI. These data show that met-enk inhibits the thermally or mechanically evoked release of SPLI in the dorsal horn by activating the delta opioid receptors. These receptors are also involved in the tonic spinal regulation of the release of SPLI.

Naloxone blocks the formalin-induced increase of substance P in the dorsal horn

&NA; Substance P (SP) has been proposed as a mediator of nociception in the dorsal horn of the spinal cord. Activation of nociceptive pathways by stimuli such as formalin injected into the hind paw has been shown to produce an increase in the amount of immunoreactive SP in primary afferent neurons. Opiate agonist and antagonist binding in the dorsal horn has been shown to affect the SP levels and release. In order to determine the effects of opiates on SP mediated nociception in the spinal cord, anesthetized rats pretreated subcutaneously with morphine, naloxone, or saline were injected in the right hind paw with 0.4 ml of either saline or 5% formalin. After 1 h, the animals were perfused and the lumbar enlargement of the spinal cord removed. SP‐like immunoreactivity (SPLI) in the dorsal horns was quantitated using immunohistochemical staining and manual photometry. The results show that formalin injection increases the SPLI in the dorsal horn after 1 h, as does pretreatment with morphine. Morphine pretreatment combined with formalin injection further increases SPLI, but not significantly higher than either treatment alone. The morphine‐induced increases could be blocked by naloxone, which had no effects on saline‐treated controls. Most importantly, naloxone was able to block the formalin‐induced increase in SPLI, implying that endogenous opioid systems play a role in the SP increases seen during formalin‐induced nociception.

Dynorphin-(1–8) inhibits the release of substance P-like immunoreactivity in the spinal cord of rats following a noxious mechanical stimulus

This study was conducted to determine the effect of the opioid peptide dynorphin-(1-8) on the release of substance P-like immunoreactivity in the dorsal horn during mechanical activation of peripheral nociceptors. A push-pull cannula was used to perfuse the dorsal horn of decerebrate/spinal transected rats before, during and following the application of a noxious mechanical stimulus to the ipsilateral hindpaw and lower limb. The collected perfusates were assayed for substance P-like immunoreactivity using radioimmunoassay. Dynorphin-(1-8) applied to the spinal cord at a concentration of 1 microM reduced the basal release of substance P-like immunoreactivity by 28 +/- 11% and prevented the mechanically evoked release of substance P-like immunoreactivity. This effect of dynorphin-(1-8) was reversed by 2 microM of the selective kappa-opioid receptor antagonist nor-binaltorphimine. Moreover, blockade of the kappa-opioid receptors by nor-binaltorphimine resulted in a 33 +/- 5% increase in the basal release of substance P-like immunoreactivity. These data show that activation of nor-binaltorphimine-sensitive sites by dynorphin-(1-8) results in inhibition of the release of substance P-like immunoreactivity in the dorsal horn of the rat.

Substance P receptor desensitization in the dorsal horn: possible involvement of receptor-G protein complexes

The repeated administration of a high dose of substance P (SP) onto the spinal cord has been shown to attenuate behavioral responses to an intense heat (tail-flick) or noxious mechanical stimulus (paw pressure). Studies performed to investigate the action of spinal SP have suggested that changes in behavioral responses involve endogenous opiate or neurokinin systems. This study was performed to investigate whether the binding characteristics of SP receptors in the dorsal horn are altered following successive administration of SP. Two populations of [3H]-SP binding sites were distinguished on the basis of their binding affinity. Gpp(NH)p, a stable analogue of GTP, decreased the size and affinity of the high affinity binding component selectively labelled with [125I]-Bolton Hunter-SP. Repeated intrathecal administration of SP (15 micrograms) which reduced behaviors also reduced the number and affinity of high affinity binding sites. Thus, attenuated behaviors in response to repeated administration of SP are paralleled by an alteration of SP binding in the dorsal horn. The altered agonist affinity seen under desensitizing conditions raises the possibility that SP receptor desensitization involves an uncoupling of receptor-G protein complexes.

Primary afferent terminal function following acrylamide: Alterations in the dorsal root potential and reflex

Abstract The dorsal root potential (DRP) and the dorsal root reflex (DRR) were studied in acrylamide (ACR)-induced axonopathy to determine the nature and extent of primary afferent terminal (PAT) dysfunction. Cats were administered 30 mg/kg/day ACR for either 5 (ACR 5D) or 10 (ACR 10D) days. The day after the last injection, the spinal cord as isolated in situ and the DRP and DRR were elicited. It was found that only 21% of the ACR 10D animals exhibited a DRP. Futhermore, in that 21%, the DRP appeared to degrade over distance differently from the control group. There was no change in the DRP evoked in the ACR 5D group. ACR affected the DRR when evoked from the cutaneous sural nerve (SU) to a greater extent than when evoked from the medial gastrocnemius (MG) nerve. A SU-evoked DRR could not be elicited in any of the animals in the ACR 10D group and in only 20% of the ACR 5D group. There was no difference in the ability to elicit a MG-evoked DRR in either ACR-treated group when compared to control. However, the maximum-evoked area under the DRR elicited from the MG nerve was significantly smaller in the ACR-treated groups than in control. These data show that ACR does indeed impair PAT function. ACR preferentially affects the PAT processing of the SU when compared to the MG nerve, which may indicate that the selective vulnerability of the largest diameter fibers to ACR is not necessarily true.