Pharmacologic consequences of the vascular permeability of chromaffin cell transplants in CNS pain modulatory regions

The transplantation of peripheral neural tissue into the CNS has been shown to alter blood-brain barrier (BBB) permeability to intravascularly injected proteins such as horseradish peroxidase. The pharmacological consequences of such BBB alterations following the transplantation of adrenal medullary tissue, isolated bovine chromaffin cell suspensions, or PC12 cell suspensions into the pain modulatory regions of the periaqueductal gray (PAG) or subarachnoid space of the lumbar spinal cord were studied using agents that normally do or do not readily pass the BBB. The injection of nicotine in animals with adrenal medullary or chromaffin cell transplants produces potent analgesia, most likely due to the stimulated release of opioid peptides and catecholamines from the transplanted cells. This analgesia could be blocked by nicotinic antagonist mecamylamine, which normally passes the BBB, but not by nicotinic antagonist hexamethonium, which normally does not readily pass the BBB. Furthermore, quaternary nicotinic agonists tetramethylammonium and 1,1-dimethyl-phenyl-piperazinium had no effect on pain sensitivity in animals with adrenal medullary implants. The Met-enkephalin peptide analog, D-Ala-Met-enkephalinamide, which normally does not alter pain sensitivity when injected systemically due to limited penetration to the CNS, produced analgesia in animals with adrenal medullary, bovine chromaffin cell, and PC12 cell implants in the PAG, but not in control gelfoam-implanted animals. This analgesia, as well as analgesia induced by nicotine, was completely blocked by naloxone pretreatment, but not by naloxone methobromide, a quaternary derivative of naloxone that does not normally pass the BBB.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relief of intractable cancer pain by human chromaffin cell transplants: Experience at two medical centers

Abstract

In addition to its possible role as a replacement source in CNS degenerative diseases, neural tral)splantation may be used to augment the normal production of neuroactive substances. Our laboratory at the University of Illinois at Chicago has shown, in both acute and chronic pain models, that transplantation of adrenal medullary tissue or isolated chromaffin cells into CNS pain modulatory regions can reduce pain sensitivity in rodents. Chromaffin cells were chosen as the donor source since they produce high levels ofboth opioid peptides and catecholamines, substances which reduce pain sensitivity when injected locally intq the spinal subarachnoid space. The analgesia produced by these transplants probably results from the release of both opioid peptides and catecholamines since it can be blocked or attenuated by both opiate and adrenergic antagonists. Studies indicate that even over long periods there is no apparent development of tolerance. Promising results have been obtained in preliminary clinical studies using allografts of adrenal medulla to relieve cancer pain. This clinical review encompasses results at two Medical Centers-University of Illinois at Chicago and University Paul Sabatier, Toulouse, France- in assessing efficacy of subarachnoid adrenal medullary transplantation for alleviating cancer pain. Our clinical and autopsy data strongly support our previous laboratory studies, i.e., that chromaffin cell transplants into the subarachnoid space represent a promising new approach to the alleviation of chronic pain. It is suggested that further clinical studies are now warranted. [Neural Res 1997; 19: 71-77]     

Attenuation of NMDA-induced spinal hypersensitivity by adrenal medullary transplants

Abnormal sensory hyperexcitability consequent to peripheral injury most likely involves activation of N-methyl-d-aspartate (NMDA) receptors in the spinal cord. This activation may lead to a cascade of neuroplastic events resulting in the exaggeration of sensory responses and the persistence of pathological pain states. Recent studies in our laboratory have demonstrated that the transplantation of adrenal medullary cells into the spinal subarachnoid space can alleviate pathological pain symptoms, possibly by reducing spinal hyperexcitability. The purpose of this study was to assess spinal NMDA activation-induced hypersensitivity to noxious and innocuous stimuli, and determine whether adrenal medullary transplants can intervene favorably to reduce these responses. Animals with either adrenal medullary or control transplants were injected intrathecally with several doses of NMDA, and responses to sensory stimuli were determined over time. NMDA at all doses tested (1–50 nmol) produced significant thermal and mechanical hyperalgesia and tactile allodynia in control transplanted animals, with peak severity at 30 min post-injection. In contrast, both the severity and duration of these exaggerated sensory responses were markedly reduced in animals with adrenal medullary transplants. To assess a possible contribution of released opioid peptides and catecholamines from the transplanted chromaffin cells, animals were pretreated with opiate antagonist naloxone or -adrenergic antagonist phentolamine. While naloxone was ineffective, the phentolamine partially attenuated, but did not completely abolish, the antinociceptive effects of the transplants. The results of these studies demonstrate that adrenal medullary grafts can reduce hypersensitivity responses to NMDA-mediated activation via -adrenergic modulation in addition to other neuroprotective mechanisms.     

Transplants of adrenal medullary chromaffin cells reduce forelimb and hindlimb allodynia in a rodent model of chronic central pain after spinal cord hemisection injury

In the majority of patients, spinal cord injury (SCI) results in abnormal pain syndromes in which non-noxious stimuli become noxious (allodynia). To reduce allodynia, it would be desirable to implant a permanent biological pump such as adrenal medullary chromaffin cells (AM), which secrete catecholamines and opioid peptides, both antinociceptive substances, near the spinal cord. We tested this approach using a recently developed a mammalian SCI model of chronic central pain, which results in development of mechanical and thermal allodynia. Thirty day-old male Sprague-Dawley rats were spinally hemisected at T13 and allowed 4 weeks for recovery of locomotor function and development of allodynia. Nonimmunosuppressed injured animals received either control-striated muscle (n = 7) or AM (n = 10) transplants. Nociceptive behavior was tested for 4 weeks posttransplant as measured by paw withdrawals to von Frey filaments, radiant heat, and pin prick stimuli. Hemisected animals receiving AM demonstrated statistically significant reductions in both fore- and hindlimb mechanical and thermal allodynia, but not analgesia, when compared to hemisected animals receiving striated muscle transplants (P < 0.05). Tyrosine hydroxylase immunoreactivity indicated prolonged transplant survival and production of catecholamines. HPLC analysis of cerebrospinal fluid samples from animals receiving AM transplants demonstrated statistically significant increases in levels of dopamine (sevenfold), norepinephrine (twofold), and epinephrine (threefold), compared to control values several weeks following transplant (P < 0.05). By 28 days posttransplant, however, antinociceptive effects were diminished. These results support the therapeutic potential of transplanted AM in reducing chronic central pain following spinal cord injury.     

Alleviation of neuropathic pain symptoms by xenogeneic chromaffin cell grafts in the spinal subarachnoid space

Persistent sensory abnormalities consequent to injury may involve prolonged neuroplastic changes in the spinal cord similar to those in long-term potentiation. Molecular markers, like the putative nitric oxide synthase, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), can be useful indicators of increased neuronal activity. Peripheral nerve injury markedly increased NADPH-d-labeling in sensory regions of the spinal cord, paralleling induction of abnormal pain (hyperalgesia). Both NADPH-d activation and hyperalgesia were reversed by transplantation of opioid/catecholamine-producing adrenal medullary tissue into the spinal subarachnoid space. These results suggest that adrenal medullary transplants can attenuate abnormal neuronal activity consequent to injury.     

Adrenal medullary transplants reduce formalin-evoked c-fos expression in the rat spinal cord

Previous studies have indicated that adrenal medullary chromaffin cells transplanted into the spinal subarachnoid space can alleviate pain behaviors in several animal models. The goal of this study was to assess whether decreased activation of spinal dorsal horn neurons responsive to nociceptive stimuli may contribute to these antinociceptive effects. In order to address this, expression of neural activity marker c-fos in response to intraplantar formalin was evaluated in animals with intrathecal adrenal medullary or control striated muscle transplants. Adrenal medullary transplants significantly attenuated formalin-induced flinching behaviors in both acute and tonic phases of the formalin response, in comparison with control transplanted animals. Fos-like-immunoreactive (Fos-LI) cell numbers were markedly reduced in the dorsal horns of animals with adrenal medullary transplants in comparison to robust Fos-LI expression in control transplanted animals. This reduction was observed in both superficial and deep laminae of the dorsal horn, but the magnitude of the decrease was greatest in lamina V. Similar to reports using other antinociceptive treatments, some residual c-fos expression was observed, particularly in laminae I-II, in animals with adrenal medullary transplants. The results of these studies suggest that adrenal medullary transplants produce antinociception in part by inhibiting spinal dorsal horn neuronal activation in response to noxious stimuli.     

Neuropeptide FF and modulation of pain

Neuropeptide FF (NPFF) and the related longer peptide neuropeptide AF (NPAF) derive from a single gene in several mammalian species. The gene product is expressed mainly in the CNS, where the posterior pituitary and dorsal spinal cord contain the highest concentrations. Evidence from biochemical and immunohistochemical studies combined with in situ hybridization using NPFF gene-specific probes suggest that all NPFF-like peptides may not derive from the characterized NPFF gene, but that other genes can exist which give rise to related peptides. Intraventricular NPFF exerts antiopioid effects, but intrathecal NPFF potentiates the analgesic effects of morphine. NPFF mRNA expression is upregulated in the dorsal horn of the spinal cord after carrageenan-induced inflammation in the hind paw of the rat, but not in the neuropathic pain model induced by ligation of the spinal roots. NPFF produces a submodality-selective potentiation of the antinociceptive effect induced by brain stem stimulation in the spinal cord during inflammation, and this effect is independent of naloxone-sensitive opioid receptors. In neuropathic animals, NPFF injected into the periaqueductal grey produces a significant attenuation of tactile allodynia, which is not modulated by naloxone. NPFF thus modulates pain sensation and morphine analgesia under normal and pathological conditions through both spinal and brain mechanisms.     

A new electrochemical HPLC method for analysis of enkephalins and endomorphins

Endogenous opioid peptides, enkephalins and endomorphins, are located in key regions involved in pain transmission and analgesia, including the spinal cord. These endogenous peptides activate opioid receptors to produce analgesia and reduce pain. We describe a new method to measure enkephalin and endomorphins by high performance liquid chromatography with electrochemical detection. This method allows use of a small sample volume to measure met-enkephalin, leu-enkephalin, endomorphin-1 and endomorphin-2 simultaneously. Using push-pull perfusion of the spinal cord, there were detectable concentrations of met-enkephalin, leu-enkephalin, and endomorphin-2. Further infusion of 100mM potassium chloride evoked release of met-enkephalin and endomorphin-2 but not leu-enkephalin. Thus, we have developed a method to simultaneously measure enkephalins and endomorphins in small sample volume that allows measurement of these opioid peptides in vivo.     

Comparison of tyrosine hydroxylase and preproenkephalin expression in rat adrenal medullary explants in vitro and transplanted into subarachnoid space

When adrenal medullary cells are cultured in vitro, tyrosine hydroxylase (TH) mRNA, preproenkephalin (PPEnk) mRNA, and methionine enkephalin (Mek) immunoreactivity was markedly increased compared with intact adrenal medullary cells in situ, suggesting an increased biosynthesis of catecholamines and enkephalin-containing peptides. In transplanted adrenal medullary cells in vivo, TH mRNA and TH immunoreactivity are still apparent for at least 1 year after transplantation, indicating continued capacity for catecholamine biosynthesis. PPEnk mRNA levels in surviving adrenal medullary grafted cells increased, particularly in the first week after transplantation, and remained above levels found in the intact adrenal gland for at least 1 year after transplantation. These results support other studies in our laboratory, suggesting that adrenal medullary transplants reduce pain by synthesis and secretion of both catecholamines and enkephalin-containing peptides. The differences in expression of TH mRNA and PPEnk mRNA in the adrenal medulla in situ, in explants in culture and in transplants in the spinal subarachnoid space, indicate that the mechanisms regulating the expression of neurohumoral factors depend upon environmental factors extrinsic to the medullary cells themselves.     

Chromaffin allografts into arachnoid of spinal cord reduce basal pain responses in rats.

In this present study, behavioral responses to a subcutaneous formalin test for pain are evaluated in rats that previously received an allograft of adrenal chromaffin tissue into arachnoid of the dorsal spinal cord and in control animals. In the group of rats with grafts, a significant basal analgesia, reversed by the opioid antagonist naloxone, is found. These findings suggest that the grafts secrete some substance that reduces the response to painful stimulation and whose action is blocked by naloxone.     

Intrathecal xenogeneic chromaffin cell grafts reduce nociceptive behavior in a rodent tonic pain model

Adrenal medullary chromaffin cells synthetize and secrete a combination of pain-reducing neuroactive compounds including catecholamines and opioid peptides. Previous reports have shown that implantation of chromaffin cells into the spinal subarachnoid space can reduce both acute and chronic pain in several animal models. We recently demonstrated that human chromaffin cell grafts in the cerebrospinal fluid (CSF) could alleviate intractable cancer pain after failure of systemic opiates. However, wider application of this approach was limited by the limited availability of allogeneic donor material. Alternatively, chromaffin cells from xenogeneic sources such as bovine adrenal medulla were successful in the experimental treatment of pain, but recent concern over risk of prion transmission precluded use of bovine grafts in human clinical trials. The objective of the present study was to investigate the possibility of developing a new xenogeneic porcine source of therapeutic chromaffin cells because this strategy is currently considered the safest for transplantation in man. In the present study, we report the isolation and the characterization of primary porcine chromaffin cells (PCC) compared to bovine cells. We show, for the first time, that these cells grafted in the rat subarachnoid space can attenuate pain-related behaviors as assessed by the formalin test, a model of tonic pain. Moreover, in addition to behavioral studies, immunohistochemical analysis revealed robust survival of chromaffin cells 35 days after transplantation. Taken together, these results support the concept that porcine chromaffin cells may offer an alternative xenogeneic cell source for transplants delivering pain-reducing neuroactive substances.     

Effects of adrenal medullary transplants on pain-related behaviors following excitotoxic spinal cord injury

Previous studies have shown that intraspinal injection of quisqualic acid (QUIS) produces excitotoxic injury with pathological characteristics similar to those associated with ischemic and traumatic spinal cord injury (SCI). Significant changes in the functional properties of sensory neurons adjacent to the site of injury have also been observed in this model. Additionally, following QUIS injections, mechanical and cold allodynia, combined with excessive grooming behavior have been shown to be the behavioral correlates of these pathological and physiological changes. These behaviors are believed to be related to the clinical conditions of spontaneous and evoked pain following SCI. Given the therapeutic properties of adrenal chromaffin cell transplantation in conditions of neuropathic and cancer pain, it is proposed that the neuroactive substances released from chromaffin cells can alter or prevent the onset and progression of QUIS-induced behavioral changes. The effects of adrenal transplants were evaluated in 14 male Long–Evans rats that received intraspinal injections of QUIS. Pain behaviors, including the progression of excessive grooming behavior (n=8) and hypersensitivity to mechanical and thermal stimuli (n=6) were evaluated following transplantation. A 53% increase in mechanical thresholds was observed following adrenal transplants along with a 70% reduction in the area of skin targeted for excessive grooming. These behaviors were not affected in 11 animals receiving transplants of skeletal muscle. The effects of adrenal transplants on cold allodynia consisted of a stabilization of response latencies in contrast to the continued decrease in latencies, i.e., increased sensitivity, following transplants of skeletal muscle. The results are consistent with previous studies showing the therapeutic efficacy of adrenal chromaffin cell transplants in neuropathic pain, and support the use of this treatment strategy for the alleviation of chronic pain following spinal cord injury.     

Possible mechanisms of morphine analgesia.

The body has an endogenous analgesic system that prevents excess pain from interfering with the normal body functions. Depression of pain sensations occurs within the dorsal horn of the spinal cord where the primary pain fibers, which transmit pain sensations from the periphery, synapse with neurons that transmit pain to the higher centers. There appear to be two mechanisms by which the transmission of pain sensations are depressed; these include hyperpolarization of interneurons within the dorsal cord and depressing the release of the neurotransmitters associated with pain transmission. Activation of the analgesic mechanisms results from an interaction between specific neurotransmitters, such as enkephalin, serotonin, or norepinephrine, and specific receptors located on the neurons that transmit pain. The spinal analgesic mechanisms can be activated by either pain or nonpainful sensations arriving from the periphery or by supraspinal mechanisms. The supraspinal mechanisms originate in specific structures within the brainstem that include the periaqueductal gray matter, locus ceruleus, and nuclei in the medulla. These systems are activated either by ascending pain impulses or by higher centers such as the cortex or hypothalamus that, in turn, activate the spinal analgesic systems. There are three systems associated with activation of the supraspinal mechanisms. These include the opioid system associated with the release of the endorphins, the adrenergic system associated with the release of norepinephrine, and the serotonergic system associated with the release of serotonin. The interaction between these systems activates the spinal analgesic system. When the endogenous analgesic systems fail to control pain, analgesic drugs can be used to enhance the endogenous systems. Opiate drugs, such as morphine, interact with opioid receptors and produce analgesia by the same mechanisms as enkephalin, i.e., hyperpolarization of interneurons and depressing the release of transmitters associated with transmission of pain. In addition, morphine can interact with opioid receptors located in the supraspinal structures and activate the supraspinal system. Adrenergic drugs that interact with specific receptors also produce analgesia and it has been suggested that morphine interacts with the adrenergic system to produce analgesia.     

Proinflammatory cytokines oppose opioid-induced acute and chronic analgesia

Spinal proinflammatory cytokines are powerful pain-enhancing signals that contribute to pain following peripheral nerve injury (neuropathic pain). Recently, one proinflammatory cytokine, interleukin-1, was also implicated in the loss of analgesia upon repeated morphine exposure (tolerance). In contrast to prior literature, we demonstrate that the action of several spinal proinflammatory cytokines oppose systemic and intrathecal opioid analgesia, causing reduced pain suppression. In vitro morphine exposure of lumbar dorsal spinal cord caused significant increases in proinflammatory cytokine and chemokine release. Opposition of analgesia by proinflammatory cytokines is rapid, occurring 5 min after intrathecal (perispinal) opioid administration. We document that opposition of analgesia by proinflammatory cytokines cannot be accounted for by an alteration in spinal morphine concentrations. The acute anti-analgesic effects of proinflammatory cytokines occur in a p38 mitogen-activated protein kinase and nitric oxide dependent fashion. Chronic intrathecal morphine or methadone significantly increased spinal glial activation (toll-like receptor 4 mRNA and protein) and the expression of multiple chemokines and cytokines, combined with development of analgesic tolerance and pain enhancement (hyperalgesia, allodynia). Statistical analysis demonstrated that a cluster of cytokines and chemokines was linked with pain-related behavioral changes. Moreover, blockade of spinal proinflammatory cytokines during a stringent morphine regimen previously associated with altered neuronal function also attenuated enhanced pain, supportive that proinflammatory cytokines are importantly involved in tolerance induced by such regimens. These data implicate multiple opioid-induced spinal proinflammatory cytokines in opposing both acute and chronic opioid analgesia, and provide a novel mechanism for the opposition of acute opioid analgesia.     

Transplantation of embryonic spinal cord-derived neurospheres support growth of supraspinal projections and functional recovery after spinal cord injury in the neonatal rat

Great interest exists in using cell replacement strategies to repair the damaged central nervous system. Previous studies have shown that grafting rat fetal spinal cord into neonate or adult animals after spinal cord injury leads to improved anatomic growth/plasticity and functional recovery. It is clear that fetal tissue transplants serve as a scaffold for host axon growth. In addition, embryonic Day 14 (E14) spinal cord tissue transplants are also a rich source of neural-restricted and glial-restricted progenitors. To evaluate the potential of E14 spinal cord progenitor cells, we used in vitro-expanded neurospheres derived from embryonic rat spinal cord and showed that these cells grafted into lesioned neonatal rat spinal cord can survive, migrate, and differentiate into neurons and oligodendrocytes, but rarely into astrocytes. Synapses and partially myelinated axons were detected within the transplant lesion area. Transplanted progenitor cells resulted in increased plasticity or regeneration of corticospinal and brainstem-spinal fibers as determined by anterograde and retrograde labeling. Furthermore, transplantation of these cells promoted functional recovery of locomotion and reflex responses. These data demonstrate that progenitor cells when transplanted into neonates can function in a similar capacity as transplants of solid fetal spinal cord tissue.     

Nicotinic modulation of GABAergic synaptic transmission in the spinal cord dorsal horn

While the mechanisms underlying nicotinic acetylcholine receptor (nAChR)-mediated analgesia remain unresolved, one process that is almost certainly involved is the recently-described nicotinic enhancement of inhibitory synaptic transmission in the spinal cord dorsal horn. Despite these observations, the prototypical nicotinic analgesic (epibatidine) has not yet been shown to modulate inhibitory transmission in the spinal cord. Furthermore, while nAChRs have been implicated in short-term modulation, no studies have investigated the role of nAChRs in the modulation of long-term synaptic plasticity of inhibitory transmission in dorsal horn. Whole-cell patch clamp recordings from dorsal horn neurons of neonatal rat spinal cord slices were therefore conducted to investigate the short- and long-term effects of nicotinic agonists on GABAergic transmission. GABAergic synaptic transmission was enhanced in 86% of neurons during applications of 1 microM nicotine (mean increased spontaneous GABAergic inhibitory postsynaptic current (sIPSC) frequency was approximately 500% of baseline). Epibatidine (100 nM) induced an increase to an average of approximately 3000% of baseline, and this effect was concentration dependent (EC50=43 nM). Nicotinic enhancement was inhibited by mecamylamine and DHbetaE, suggesting an important role for non-alpha7 nAChRs. Tetrodotoxin (TTX) did not alter the prevalence or magnitude of the effect of nicotine, but the responses had a shorter duration. Nicotine did not alter evoked GABAergic IPSC amplitude, yet the long-term depression (LTD) induced by strong stimulation of inhibitory inputs was reduced when paired with nicotine. These results provide support for a mechanism of nicotinic analgesia dependent on both short and long-term modulation of GABAergic synaptic transmission in the spinal cord dorsal horn.     

Analgesia produced by electrical stimulation of the brain

1. Electrical stimulation of the brain can produce a selective and potent modulation of responding to noxious stimuli in animals and man. The influence of various stimulation parameters is discussed.

2. Brain stimulation at numerous loci results in analgesia. The most well characterized regions are the mesencephalic periaqueductal gray matter and the medullary raphe nuclei.

3. One pain inhibitory system activated by brain stimulation involves a neural circuit from the PAG to the medullary raphe nuclei. Output from there descends via the DLF to modulate pain transmission in the dorsal horn of the spinal cord. Other analgesia systems are also activated by brain stimulation.

4. Compelling evidence implicates endogenous opiates in SPA. Monoaminergic neurotransmitters are also involved in SPA.

5. Brain stimulation has proven to be useful for the management of some forms of intractable pain in man.     

Glia: novel counter-regulators of opioid analgesia

Development of analgesic tolerance and withdrawal-induced pain enhancement present serious difficulties for the use of opioids for pain control. Although neuronal mechanisms to account for these phenomena have been sought for many decades, their bases remain unresolved. Within the past four years, a novel non-neuronal candidate has been uncovered that opposes acute opioid analgesia and contributes to development of opioid tolerance and tolerance-associated pain enhancement. This novel candidate is spinal cord glia. Glia are important contributors to the creation of enhanced pain states via the release of neuroexcitatory substances. New data suggest that glia also release neuroexcitatory substances in response to morphine, thereby opposing its effects. Controlling glial activation could therefore increase the clinical utility of analgesic drugs.     

Role of spinal muscarinic and nicotinic receptors in clonidine-induced nitric oxide release in a rat model of neuropathic pain

Intrathecal administration of alpha(2) adrenergic agonists, such as clonidine, is capable of alleviating neuropathic pain. Recent studies suggest that spinal nitric oxide (NO) mediates the analgesic effect of intrathecal clonidine. Furthermore, compared to nicotinic receptors, spinal muscarinic receptors play a greater role in the analgesic effect of intrathecal clonidine. In the present study, we tested a hypothesis that clonidine-evoked NO release is dependent primarily on muscarinic receptors in the spinal cord after nerve injury. A rat model of neuropathic pain was induced by ligation of the left L(5)/L(6) spinal nerves. Using an in vitro spinal cord perfusion preparation, the effect of muscarinic and nicotinic receptor antagonists on clonidine-evoked nitrite (a stable product of NO) release was determined. Both muscarinic and nicotinic antagonists dose-dependently attenuated clonidine-elicited nitrite release. In spinal cords from the neuropathic rats, the inhibitory effect of muscarinic receptor antagonists (atropine and scopolamine) on clonidine-elicited nitrite release was more potent than that of nicotinic receptor antagonists (mecamylamine and hexamethonium). However, in spinal cords obtained from sham animals, the inhibitory effect of muscarinic and nicotinic antagonists did not differ significantly. These results indicate that muscarinic, as well as nicotinic, receptors mediate clonidine-induced NO release in the spinal cord. These data also suggest that after nerve injury, the cascade of activation of alpha(2) adrenergic receptors-muscarinic receptors-NO in the spinal cord likely plays a predominant role in the analgesic effect of intrathecal clonidine on neuropathic pain.     

Cannabinoid CB1 receptor facilitation of substance P release in the rat spinal cord,measured as neurokinin 1 receptor internalization

The contribution of CB1 receptors in the spinal cord to cannabinoid analgesia is still unclear. The objective of this study was to investigate the effect of CB1 receptors on substance P release from primary afferent terminals in the spinal cord. Substance P release was measured as neurokinin 1 (NK1) receptor internalization in lamina I neurons. It was induced in spinal cord slices by dorsal root stimulation and in live rats by a noxious stimulus. In spinal cord slices, the CB1 receptor antagonists AM251, AM281 and rimonabant partially but potently inhibited NK1 receptor internalization induced by electrical stimulation of the dorsal root. This was due to an inhibition of substance P release and not of NK1 receptor internalization itself, because AM251 and AM281 did not inhibit NK1 receptor internalization induced by exogenous substance P. The CB1 receptor agonist ACEA increased NK1 receptor internalization evoked by dorsal root stimulation. The effects of AM251 and ACEA cancelled each other. In vivo, AM251 injected intrathecally decreased NK1 receptor internalization in spinal segments L5 and L6 induced by noxious hind paw clamp. Intrathecal AM251 also produced analgesia to radiant heat stimulation of the paw. The inhibition by AM251 of NK1 receptor internalization was reversed by antagonists of μ‐opioid and GABA_B receptors. This indicates that CB1 receptors facilitate substance P release by inhibiting the release of GABA and opioids next to primary afferent terminals, producing disinhibition. This results in a pronociceptive effect of CB1 receptors in the spinal cord.