Spinal actions of lipoxin A4 and 17(R)-resolvin D1 attenuate inflammation-induced mechanical hypersensitivity and spinal TNF release.

Lipoxins and resolvins have anti-inflammatory and pro-resolving actions and accumulating evidence indicates that these lipid mediators also attenuate pain-like behavior in a number of experimental models of inflammation and tissue injury-induced pain. The present study was undertaken to assess if spinal administration of lipoxin A4 (LXA4) or 17 (R)-resolvin D1 (17(R)-RvD1) attenuates mechanical hypersensitivity in the carrageenan model of peripheral inflammation in the rat. Given the emerging role of spinal cytokines in the generation and maintenance of inflammatory pain we measured cytokine levels in the cerebrospinal fluid (CSF) after LXA4 or 17(R)-RvD1 administration, and the ability of these lipid metabolites to prevent stimuli-induced release of cytokines from cultured primary spinal astrocytes. We found that intrathecal bolus injection of LXA4 and17(R)-RvD1 attenuated inflammation-induced mechanical hypersensitivity without reducing the local inflammation. Furthermore, both LXA4 and 17(R)-RvD1 reduced carrageenan-induced tumor necrosis factor (TNF) release in the CSF, while only 17(R)-RvD1attenuated LPS and IFN-γ-induced TNF release in astrocyte cell culture. In conclusion, this study demonstrates that lipoxins and resolvins potently suppress inflammation-induced mechanical hypersensitivity, possibly by attenuating cytokine release from spinal astrocytes. The inhibitory effect of lipoxins and resolvins on spinal nociceptive processing puts them in an intriguing position in the search for novel pain therapeutics.

Spinal astrocytes in pain processing: non-neuronal cells as therapeutic targets.

The treatment of chronic inflammatory and neuropathic pain is a major concern, and the need for new more effective analgesics with less adverse effects is immense. Traditionally, mechanisms proposed for pain modulation have centered almost exclusively on peripheral or central neurons in the pain pathways. Recent research, however, indicates that activation of microglia and astrocytes in the dorsal horn of the spinal cord is of central importance for the development of chronic pain states. Understanding the intercellular communication among astrocytes, microglia, and neurons in the dorsal horn during acute and chronic pain may be instrumental for the development of new analgesic drugs. The purpose of this review is to highlight the role of astrocytes in regulating pain processing.

Prenatal exposure to nicotine affects substance p and preprotachykinin-A mRNA levels in newborn rat.

Prenatal nicotine exposure influences neuronal development including effects on several neurotransmitter systems. It also attenuates the ventilatory response to hypoxia, known to require a functional substance P-ergic system. Previous studies have shown that nicotine increases the risk for sudden infant death syndrome (SIDS) by 4-fold, and that SIDS-victims have elevated brainstem levels of substance P. We, therefore, studied the effect of prenatal nicotine exposure on the levels of substance P-like immunoreactivity by RIA in the brain in newborn rat pups. The expression of the substance P precursor preprotachykinin A mRNA was also determined by real-time reverse transcriptase-polymerase chain reaction in carotid body, in petrosal/jugular and trigeminal ganglia, in cervical and lumbar dorsal root ganglia, and in the brainstem. We found that prenatal nicotine exposure increased levels of substance P-like immunoreactivity in the brainstem without changing levels in other parts of the brain or in the adrenals. Furthermore, mRNA levels were increased in the carotid bodies and in the petrosal ganglia, in contrast to the decreased levels in the cervical dorsal root ganglia. We conclude that nicotine causes alterations in the substance P-ergic system in the brainstem, possibly linked to the increased risk for SIDS after prenatal nicotine exposure.

Spontaneous nociceptive behaviour in female mice with Freund's complete adjuvant- and carrageenan-induced monoarthritis.

The development of transgenic techniques has accentuated the need for valid disease models in mice. In the present study, we have in female mice characterized adjuvant-induced monoarthritis, an inflammatory model which is commonly used in rats. Freund's complete adjuvant (FCA), 20 microl, was injected into the left knee joint. Pain was inferred from impairment of gait and stance. Two commonly used mouse strains were compared. Outbred NMRI mice showed significant levels of pain behaviour lasting for at least 7 days after injection of FCA while inbred BALB/c mice showed altered behaviour at 3, but not at 5 days. The effects of three commercially available preparations of FCA were compared in NMRI mice. All adjuvants caused highly reproducible spontaneous pain behaviour at 1-3 days with a reduction at 5 days and no significant pain-related behaviour at 10 days. Intraarticular injection of 2% carrageenan performed for comparison, caused pain-related behaviour for less than 24 h. The adjuvant-induced pain behaviour was dose-dependently reduced by 3-30 micromol/kg morphine and 3-30 micromol/kg diclofenac. In conclusion, adjuvant-induced monoarthritis in mice provides a robust model for pharmacological studies of inflammatory pain, but the choice of mouse strain is important for the outcome.

Arthritis-induced increase in cholecystokinin release in the rat anterior cingulate cortex is reversed by diclofenac.

Given a hypothesised role for CCK in the anterior cingulate cortex (ACC) for the sensation of pain, the aim of the present study was to investigate whether the increased CCK release could be affected by two different analgesic drugs, morphine and the non-selective cyclooxygenase inhibitor diclofenac. Since opioids stimulate CCK release in other CNS regions we have also studied the effect of morphine by itself on the CCK-LI release in the ACC of non-arthritic rats. Three to seven hours after intraarticular carrageenan injection, at the time when the animals are known to show pain-related behaviour, in vivo microdialysis in awake rats revealed increased CCK-LI release in the ACC. The CCK-LI release was significantly attenuated by diclofenac (25 mg/kg i.m.), but not by morphine (10 mg/kg s.c.). Neither diclofenac (25 mg/kg i.m.) nor morphine (5 or 10 mg/kg s.c.) affected the CCK-LI release in the ACC in non-arthritic rats. The results obtained with diclofenac indicate that prostaglandins contribute to the increased CCK-LI release in the ACC during monoarthritis. However, the lack of effect of morphine suggests that the CCK release in the ACC is not directly related to the sensation of pain. Further on, the failure of morphine to affect the extracellular level of CCK-LI in the ACC in control animals as well as in animals with carrageenan-induced monoarthritis is in contrast to previous studies on the frontal cortex or the dorsal horn of the spinal cord, in which similar doses of morphine stimulate CCK release. Thus, compared to these regions, CCK release may be differently regulated in the ACC.

Neurokinin 1 receptor signaling affects the local innate immune defense against genital herpes virus infection.

We show that genital infection with neurotropic HSV type 2 (HSV-2) induced a significant increase of the neuropeptide substance P (SP) within the genital tract of mice. SP was shown to weakly interfere with the HSV-2 replication. Furthermore, lack of SP signaling through the use of mice deficient in the SP receptor, neurokinin 1 receptor (NK1R), revealed an important role for SP in the innate defense against HSV-2. NK1R-deficient mice had significantly enhanced levels of HSV-2 in the genital tract and in the CNS following infection and a significantly accelerated disease progression, which was associated with an impaired NK cell activity locally in the vagina. Lack of NK1R signaling did, however, not impair the animals' ability to mount a protective immune response to HSV-2 following vaccination with an attenuated virus. Both NK1R+/+ and NK1R-/- mice developed strong HSV-2-specific Th1 T cell responses following vaccination. No genital viral replication was observed in either vaccinated NK1R-deficient or NK1R+/+ control animals following a genital HSV-2 challenge, and all of these animals survived without any symptoms of disease. In conclusion, the present results indicate that SP and NK1R signaling contributes to the innate resistance against HSV-2 infection in mice.

Detection of beta-endorphin in the cerebrospinal fluid after intrastriatal microinjection into the rat brain.

We have investigated to what extent microinjected beta-endorphin could migrate from the rat brain parenchyma into the CSF compartment. Exogenous rat beta-endorphin (0.1 nmol) was microinjected into the left striatum 1 mm from the lateral ventricle in anesthetized male rats. CSF samples were collected at different time points up to 2 h post-injection from a catheter affixed to the atlanto-occipital membrane of the cisterna magna. Radioimmunoassay and mass spectrometry were performed on the CSF samples, and brain sections were immunostained for beta-endorphin and mu-opioid receptors. The beta-endorphin injected rats showed a marked increase in beta-endorphin immunoreactive (IR) material in the CSF, with a peak at 30-45 min post-injection, and this beta-endorphin-IR material existed mainly as the intact beta-endorphin peptide. The immunohistochemistry results revealed the appearance of distinct beta-endorphin-IR cell bodies in the globus pallidus and the bed nucleus of stria terminalis supracapsular part, regions distant from the injection site, at 2 h post-injection of exogenous beta-endorphin. The beta-endorphin-IR in several of the globus pallidus cell bodies colocalized with the mu-opioid receptor-IR at the cell surface. These findings show that upon delivery of synthetic beta-endorphin, there is a significant intracerebral spread of the injected peptide, reaching regions far from the site of injection via diffusion in the extracellular space and flow in the cerebrospinal fluid. This may be of relevance when interpreting studies based on intracerebral injections of peptides, and advances our knowledge regarding the migration of compounds within the brain.

Capsaicin-evoked substance P release in rat dorsal horn increases after peripheral inflammation: a microdialysis study.

Numerous in vitro studies suggest that inflammation is associated with enhanced release of substance P (SP) in the dorsal horn. To test the hypothesis that inflammation increases the evoked concentration of SP in the intact animal, we used in vivo microdialysis with a highly sensitive radioimmunoassay to monitor SP-like immunoreactivity (SP-LI) in the dorsal horn. Seven days after the induction of persistent unilateral inflammation with hindpaw injection of complete Freund's adjuvant, perfusion of the microdialysis probe with 10 microM capsaicin (a concentration which failed to induce SP-LI release in rats without inflammation) induced a significant increase of microdialysate SP-LI. Inclusion of an NMDA antagonist in the perfusion fluid completely blocked this capsaicin-evoked SP release. Administration of a five-fold higher dose of capsaicin did not further increase SP release. These results in a rat model of chronic arthritis suggest that persistent inflammatory signaling facilitates capsaicin-evoked SP release in the dorsal horn in vivo.

Increased cholecystokinin release in the rat anterior cingulate cortex during carrageenan-induced arthritis.

Several human and animal studies indicate that the anterior cingulate cortex (ACC) plays an important role in the affective component of pain. The neuropeptide cholecystokinin (CCK) is especially abundant in the ACC. CCK has been suggested to be involved in the mediation of anxiety and in the modulation of opioid effects in the spinal cord and medulla oblongata. However, its possible role in pain transmission or modulation in the brain is far less clear. In this study, a model of subchronic inflammatory pain in rats, carrageenan-induced monoarthritis, was used to study the effect of pain on the release of CCK-like immunoreactivity (CCK-LI) in the ACC. Pain-related behaviour quantified by weight bearing and stance scoring, as well as inflammation measured by ankle oedema, was increased for at least 24 h after carrageenan injection with a maximum at 5 h. Using microdialysis in freely moving rats, extracellular concentrations of CCK-LI was measured in the ACC during a time period when the animals showed significant pain behaviour. In animals with carrageenan-induced arthritis, both basal and potassium-evoked release of CCK-LI were significantly increased compared to controls. HPLC analysis of dialysates from the ACC during potassium stimulation showed that the main part of the immunoreactive material was sulphated CCK-8. Because CCK has been implicated in anxiety, we suggest that an altered CCK-ergic activity in the ACC may be of importance for the affective component of pain, but an involvement in the modulation of nociception is also possible.

Microdialysis in pain research.

In vivo microdialysis has been used in preclinical pain research for more than a decade. This valuable tool allows correlations between nociceptive behavior and neurotransmitter release in pain-related CNS sites. However, several methodological issues must be considered to adequately interpret microdialysis data. Thus, the aim of this review is to describe key considerations, potential pitfalls, and important control experiments. We focus on animal experiments which evaluate the effects of noxious stimulation on CNS neurotransmitter release, particularly those that address clinically relevant problems in patients with long-lasting painful conditions.

Gabapentin reverses mechanical allodynia induced by sciatic nerve ischemia and formalin-induced nociception in mice.

The anticonvulsant drug gabapentin has been demonstrated to alleviate symptoms of painful diabetic neuropathy as well as other types of neuropathic pain. The aim of the present study was to investigate the effect of gabapentin in a recently developed mouse model of peripheral neuropathy. This model is based on a photochemical ischemic lesion of the sciatic nerve generated by laser-induced activation of the photosensitizing dye erythrosin B. Following laser irradiation of the sciatic nerve for 2, 5, or 10 min, tactile allodynia was observed during at least 3 weeks. The degree of allodynia was most marked following 10 min of irradiation. Subcutaneous administration of gabapentin [175-300 micromol/kg ( approximately 30-51 mg/kg), cumulative doses, at 1-h intervals] significantly reversed tactile allodynia induced by 10-min laser irradiation. The maximal dose of gabapentin increased the withdrawal threshold from approximately 0.55 to approximately 1.85 g (i.e., about 77% of the threshold in normal animals, approximately 2.4 g). Gabapentin did not affect the tactile withdrawal threshold in intact animals. A dose of gabapentin (100 micromol/kg, sc) that had no effect on allodynia was found to significantly reduce the pain behavior during phase 2 of the formalin test. The present study demonstrates that systemic administration of gabapentin suppresses both allodynia induced by an ischemic lesion of the sciatic nerve and pain behavior in the formalin test.

Spinal substance P release in vivo during the induction of long-term potentiation in dorsal horn neurons.

Long-term potentiation (LTP) in wide dynamic range (WDR) neurons in the dorsal horn has been suggested to contribute to central sensitization and the development of chronic pain. Indirect experimental evidence indicates an involvement of substance P (SP), in this respect. The aim of the present study was to monitor the extracellular level of substance P-like immunoreactivity (SP-LI) in the dorsal horn of the rat during and after induction of LTP in WDR neurons in vivo. Electrophysiological recordings of single (WDR) neurons were performed in parallel with microdialysis in the dorsal horn under urethane-anaesthesia. The amount of SP-LI in the microdialysate was determined by radioimmunoassay. As previously shown, high frequency conditioning stimulation of the sciatic nerve induced an increased firing response of WDR neurons. An increased response to C-fibre stimulation, but not A-fibre stimulation, could be determined. A significant increase of the extracellular level of SP-LI in the dorsal horn was detected during, but not after, induction of LTP. These data suggest that SP may be involved in the induction of LTP by high frequency stimulation. However, the maintenance of spinal LTP following high frequency peripheral nerve stimulation does not seem to depend on an increased release of SP.