Temporal changes in the expression of the translocator protein TSPO and the steroidogenic enzyme 5α-reductase in the dorsal spinal cord of animals with neuropathic pain: Effects of progesterone administration.

Neuropathic pain is a frequent complication of spinal cord injury (SCI), still refractory to conventional treatment. The presence and biological activity of steroidogenic regulatory proteins and enzymes in the spinal cord suggests that neurosteroids locally generated could modulate pain messages. In this study we explored temporal changes in the spinal expression of the 18kDa translocator protein TSPO, the steroidogenic acute regulatory protein (StAr) and the steroidogenic enzyme 5α-reductase (5α-RI/II) in an experimental model of central chronic pain. Male Sprague-Dawley rats were subjected to a SCI and sacrificed at different time points (1, 14 or 28days). The development of mechanical and cold allodynia was assessed. Injured animals showed an early increase in the mRNA levels of TSPO and 5α-RII, whereas in the chronic phase a significant decrease in the expression of 5α-RI and 5α-RII was observed, coinciding with the presence of allodynic behaviors. Furthermore, since we have shown that progesterone (PG) administration may offer a promising perspective in pain modulation, we also evaluated the expression of steroidogenic proteins and enzymes in injured animals receiving daily injections of the steroid. PG-treated did not develop allodynia and showed a marked increase in the mRNA levels of TSPO, StAR, 5α-RI and 5α-RII 28days after injury. Our results suggest that in the acute phase after SCI, the increased expression of TSPO and 5α-RII may represent a protective endogenous response against tissue injury, which is not maintained in the chronic allodynic phase. PG may favor local steroidogenesis and the production of its reduced metabolites, which could contribute to the antiallodynic effects observed after PG treatment.

Aldehyde dehydrogenase-2 regulates nociception in rodent models of acute inflammatory pain.

Exogenous aldehydes can cause pain in animal models, suggesting that aldehyde dehydrogenase-2 (ALDH2), which metabolizes many aldehydes, may regulate nociception. To test this hypothesis, we generated a knock-in mouse with an inactivating point mutation in ALDH2 (ALDH2*2), which is also present in human ALDH2 of ~540 million East Asians. The ALDH2*1/*2 heterozygotic mice exhibited a larger response to painful stimuli than their wild-type littermates, and this heightened nociception was inhibited by an ALDH2-selective activator (Alda-1). No effect on inflammation per se was observed. Using a rat model, we then showed that nociception tightly correlated with ALDH activity (R(2) = 0.90) and that reduced nociception was associated with less early growth response protein 1 (EGR1) in the spinal cord and less reactive aldehyde accumulation at the insult site (including acetaldehyde and 4-hydroxynonenal). Further, acetaldehyde- and formalin-induced nociceptive behavior was greater in the ALDH2*1/*2 mice than in the wild-type mice. Finally, Alda-1 treatment was even beneficial when given after the inflammatory agent was administered. Our data in rodent models suggest that the mitochondrial enzyme ALDH2 regulates nociception and could serve as a molecular target for pain control, with ALDH2 activators, such as Alda-1, as potential non-narcotic, cardiac-safe analgesics. Furthermore, our results suggest a possible genetic basis for East Asians' apparent lower pain tolerance.

Progesterone reduces the expression of spinal cyclooxygenase-2 and inducible nitric oxide synthase and prevents allodynia in a rat model of central neuropathic pain.

BACKGROUND: Spinal cord injury (SCI) results in the development of chronic pain that is refractory to conventional treatment. Progesterone, a neuroprotective steroid, may offer a promising perspective in pain modulation after central injury. Here, we explore the impact of progesterone administration on the post-injury inflammatory cascade involving the enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) at the spinal cord level. We also analyse pain behaviours, the profile of glial cell activation, and IκB-α mRNA levels, as an index of NF-κB transactivation. METHODS: We used biochemical, immunohistochemical and molecular techniques, as well as behavioural studies, to investigate the effects of progesterone in a well-characterized model of central neuropathic pain. RESULTS: Injured animals receiving progesterone presented reduced mRNA levels of the proinflammatory enzymes, as well as decreased COX-2 activity and nitrite levels, as compared to vehicle-treated injured rats. Further, animals receiving the steroid exhibited lower levels of IκB-α mRNA, suggesting decreased NF-κB transactivation. Progesterone administration also attenuated the injury-induced increase in the number of glial fibrillary acidic protein and OX-42 positive cells both at early and late time points after injury, and prevented the development of mechanical and thermal allodynia. Further, when injured rats received early progesterone administration for a critical period of time after injury, they did not display allodynic behaviours even after the treatment had stopped. CONCLUSIONS: Our results suggest that progesterone, by modulating early neuroinflammatory events triggered after SCI, may represent a useful strategy to prevent the development of central chronic pain.

Peripheral inflammatory hyperalgesia depends on the COX increase in the dorsal root ganglion.

It is well established that dorsal root ganglion (DRG) cells synthesize prostaglandin. However, the role that prostaglandin plays in the inflammatory hyperalgesia of peripheral tissue has not been established. Recently, we have successfully established a technique to inject drugs (3 µL) directly into the L5-DRG of rats, allowing in vivo identification of the role that DRG cell-derived COX-1 and COX-2 play in the development of inflammatory hyperalgesia of peripheral tissue. IL-1ß (0.5 pg) or carrageenan (100 ng) was administered in the L5-peripheral field of rat hindpaw and mechanical hyperalgesia was evaluated after 3 h. Administration of a nonselective COX inhibitor (indomethacin), selective COX-1 (valeryl salicylate), or selective COX-2 (SC-236) inhibitors into the L5-DRG prevented the hyperalgesia induced by IL-1ß. Similarly, oligodeoxynucleotide-antisense against COX-1 or COX-2, but not oligodeoxynucleotide-mismatch, decreased their respective expressions in the L5-DRG and prevented the hyperalgesia induced by IL-1ß in the hindpaw. Immunofluorescence analysis demonstrated that the amount of COX-1 and COX-2, constitutively expressed in TRPV-1(+) cells of the DRG, significantly increased after carrageenan or IL-1ß administration. In addition, indomethacin administered into the L5-DRG prevented the increase of PKCε expression in DRG membrane cells induced by carrageenan. Finally, the administration of EP1/EP2 (7.5 ng) or EP4 (10 µg) receptor antagonists into L5-DRG prevented the hyperalgesia induced by IL-1ß in the hindpaw. In conclusion, the results of this study suggest that the inflammatory hyperalgesia in peripheral tissue depends on activation of COX-1 and COX-2 in C-fibers, which contribute to the induction and maintenance of sensitization of primary sensory neurons.

Blockade of peripheral and spinal Na+/H+ exchanger increases formalin-induced long-lasting mechanical allodynia and hyperalgesia in rats.

The Na(+)/H(+) exchanger (NHE) is involved in the regulation of intracellular pH and volume by mediating the electroneutral transport of H(+) against an influx of Na(+) ions. Since NHE1 regulates pH in neurons and astrocytes and it is expressed in nociceptive nerve fibers, it is likely that NHE may modulate neuronal excitability and pain transmission. The purpose of this study was to assess the participation of peripheral and spinal NHE in the secondary allodynia/hyperalgesia induced by formalin. In addition, we determined whether formalin injection modifies the expression of NHE1 in lumbar dorsal root ganglia (DRG) and dorsal spinal cord. Subcutaneous injection of 0.5% formalin into the dorsal surface of the hind paw produced acute nociceptive behaviors (flinching and licking/lifting) followed by long-lasting bilateral secondary mechanical allodynia/hyperalgesia. Peripheral and intrathecal pre-treatment (-10min) with selective NHE inhibitors 5-(N,N-dimethyl)amiloride hydrochloride (DMA, 0.3-30µM), 5-(N-ethyl-N-isopropyl)amiloride (EIPA, 0.3-30µM) and [1-(quinolin-5-yl)-5-cyclopropyl-1H-pyrazole-4-carbonyl] guanidine dihydrochloride (zoniporide, 0.03-3µM) significantly increased 0.5% formalin-induced bilateral long-lasting secondary allodynia/hyperalgesia. Contrariwise, local peripheral or intrathecal post-treatment (day 6 postinjection) with these NHE inhibitors did not affect formalin-induced nociceptive behaviors. Formalin injection reduced NHE1 expression in ipsilateral and contralateral spinal dorsal horns from day 1 to 12. In addition, formalin diminished NHE1 protein expression in DRG at day 12. These results suggest that NHE1 plays a role in pain processing at peripheral and spinal levels in formalin-induced long-lasting nociceptive behaviors. Additionally, these results suggest that proteins involved in pH regulation could be targets for the development of new analgesic drugs.

Stimulation of peripheral kappa opioid receptors inhibits inflammatory hyperalgesia via activation of the PI3Kγ/AKT/nNOS/NO signaling pathway.

BACKGROUND: In addition to their central effects, opioids cause peripheral analgesia. There is evidence showing that peripheral activation of kappa opioid receptors (KORs) inhibits inflammatory pain. Moreover, peripheral µ-opioid receptor (MOR) activation are able to direct block PGE(2)-induced ongoing hyperalgesia However, this effect was not tested for KOR selective activation. In the present study, the effect of the peripheral activation of KORs on PGE(2)-induced ongoing hyperalgesia was investigated. The mechanisms involved were also evaluated. RESULTS: Local (paw) administration of U50488 (a selective KOR agonist) directly blocked, PGE(2)-induced mechanical hyperalgesia in both rats and mice. This effect was reversed by treating animals with L-NMMA or N-propyl-L-arginine (a selective inhibitor of neuronal nitric oxide synthase, nNOS), suggesting involvement of the nNOS/NO pathway. U50488 peripheral effect was also dependent on stimulation of PI3Kγ/AKT because inhibitors of these kinases also reduced peripheral antinociception induced by U50488. Furthermore, U50488 lost its peripheral analgesic effect in PI3Kγ null mice. Observations made in vivo were confirmed after incubation of dorsal root ganglion cultured neurons with U50488 produced an increase in the activation of AKT as evaluated by western blot analyses of its phosphorylated form. Finally, immunofluorescence of DRG neurons revealed that KOR-expressing neurons also express PI3Kγ (≅ 43%). CONCLUSIONS: The present study indicates that activation of peripheral KORs directly blocks inflammatory hyperalgesia through stimulation of the nNOS/NO signaling pathway which is probably stimulated by PI3Kγ/AKT signaling. This study extends a previously study of our group suggesting that PI3Kγ/AKT/nNOS/NO is an important analgesic pathway in primary nociceptive neurons.

Inflammatory muscle pain is dependent on the activation of kinin B1 and B2 receptors and intracellular kinase pathways.

BACKGROUND AND PURPOSE: B(1) and B(2) kinin receptors are involved in pain transmission but they may have different roles in the muscle pain induced by intense exercise or inflammation. We investigated the contribution of each of these receptors, and the intracellular pathways involved, in the initial development and maintenance of the muscle pain associated with inflammation-induced tissue damage. EXPERIMENTAL APPROACH: Mechanical hyperalgesia was measured using the Randall-Selitto apparatus after injecting 5% formalin solution into the gastrocnemius muscle in mice treated with selective antagonists for B(1) or B(2) receptors. The expression of kinin receptors and cytokines and the activation of intracellular kinases were monitored by real-time PCR and immunohistochemistry. KEY RESULTS: The i.m. injection of formalin induced an overexpression of B(1) and B(2) receptors. This overexpression was associated with the mechanical hyperalgesia induced by formalin because treatment with B(1) receptor antagonists (des-Arg(9) [Leu(8)]-BK, DALBK, and SSR240612) or B(2) receptor antagonists (HOE 140 and FR173657) prevented the hyperalgesia. Formalin increased myeloperoxidase activity, and up-regulated TNF-α, IL-1ß and IL-6 in gastrocnemius. Myeloperoxidase activity and TNF-α mRNA expression were inhibited by either DALBK or HOE 140, whereas IL-6 was inhibited only by HOE 140. The hyperalgesia induced by i.m. formalin was dependent on the activation of intracellular MAPKs p38, JNK and PKC. CONCLUSIONS AND IMPLICATIONS: Inflammatory muscle pain involves a cascade of events that is dependent on the activation of PKC, p38 and JNK, and the synthesis of IL-1ß, TNF-α and IL-6 associated with the up-regulation of both B(1) and B(2) kinin receptors.

The neuronal NO synthase participation in the peripheral antinociception mechanism induced by several analgesic drugs.

The production of nitric oxide (NO) from l-arginine is catalyzed by NO synthase (NOS), which exists as the following three isoforms: endothelial (eNOS), neuronal (nNOS), and inducible (iNOS). The participation of this pathway in peripheral antinociception has been extensively established by our group with the use of several types of drugs, including opioids, cannabinoids, cholinergic, and α(2C) adrenoceptor agonists and nonsteroidal anti-inflammatory drugs (NSAIDS), and even non-pharmacological procedures such as electroacupuncture. In this study, we aimed to refine the previous data to investigate which type of NOS isoform is involved in the peripheral antinociception mechanism induced by anandamide, morphine, SNC80, bremazocine, acetylcholine, xylazine, baclofen, dipyrone, and diclofenac. After hyperalgesia was induced by intraplantar injection of prostaglandin E(2) in male Wistar rats, we measured peripheral nociception with the paw pressure test. All drugs that were used induced a peripheral antinociception effect that was completely blocked by injection of the selective neuronal NO synthase inhibitor, L-NPA (24µg/paw). The exception was the GABA(B) agonist baclofen, which induced an effect that was not antagonized. We used the inhibitors L-NIO and -NIL (24µg/paw) to exclude the involvement of endothelial and inducible NO synthase, respectively. These drugs were ineffective against the antinociception effect induced by all analgesic drugs that we utilized. Based on the experimental evidence, we conclude that the local injection of analgesic drugs activates nNOS to release NO and induce peripheral antinociception.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection.

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 microg in 50 microL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48% by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed's lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469%). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20%), reaching the greatest increase (60%) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Participation of neuronal nitric oxide synthase in experimental neuropathic pain induced by sciatic nerve transection

Nerve injury leads to a neuropathic pain state that results from central sensitization. This phenomenom is mediated by NMDA receptors and may involve the production of nitric oxide (NO). In this study, we investigated the expression of the neuronal isoform of NO synthase (nNOS) in the spinal cord of 3-month-old male, Wistar rats after sciatic nerve transection (SNT). Our attention was focused on the dorsal part of L3-L5 segments receiving sensory inputs from the sciatic nerve. SNT resulted in the development of neuropathic pain symptoms confirmed by evaluating mechanical hyperalgesia (Randall and Selitto test) and allodynia (von Frey hair test). Control animals did not present any alteration (sham-animals). The selective inhibitor of nNOS, 7-nitroindazole (0.2 and 2 µg in 50 µL), blocked hyperalgesia and allodynia induced by SNT. Immunohistochemical analysis showed that nNOS was increased (48 percent by day 30) in the lumbar spinal cord after SNT. This increase was observed near the central canal (Rexed’s lamina X) and also in lamina I-IV of the dorsal horn. Real-time PCR results indicated an increase of nNOS mRNA detected from 1 to 30 days after SNT, with the highest increase observed 1 day after injury (1469 percent). Immunoblotting confirmed the increase of nNOS in the spinal cord between 1 and 15 days post-lesion (20 percent), reaching the greatest increase (60 percent) 30 days after surgery. The present findings demonstrate an increase of nNOS after peripheral nerve injury that may contribute to the increase of NO production observed after peripheral neuropathy.

Protease-activated receptors and inflammatory hyperalgesia.

Recent advances in basic science pointed to a role for proteinases, through the activation of proteinase-activated receptors (PARs) in nociceptive mechanisms. Activation of PAR1, PAR2 and PAR4 either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Sub-inflammatory doses of PAR2 agonist still induced hyperalgesia and allodynia while PAR2 has been shown to be implicated in the generation of hyperalgesia in different inflammatory models. In contrast, sub-inflammatory doses of PAR1 increases nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as an analgesic agent. PARs are present and functional on sensory neurons, where they participate either directly or indirectly to the transmission and/or inhibition of nociceptive messages. Taken together, the results discussed in this review highlight proteinases as signaling molecules to sensory nerves. We need to consider proteinases and the receptors that are activated by proteinases as important potential targets for the development of analgesic drugs in the treatment of inflammatory pain.

Protease-activated receptors and inflammatory hyperalgesia

Recent advances in basic science pointed to a role for proteinases, through the activation of proteinase-activated receptors (PARs) in nociceptive mechanisms. Activation of PAR1, PAR2 and PAR4 either by proteinases or by selective agonists causes inflammation inducing most of the cardinal signs of inflammation: swelling, redness, and pain. Sub-inflammatory doses of PAR2 agonist still induced hyperalgesia and allodynia while PAR2 has been shown to be implicated in the generation of hyperalgesia in different inflammatory models. In contrast, sub-inflammatory doses of PAR1 increases nociceptive threshold, inhibiting inflammatory hyperalgesia, thereby acting as an analgesic agent. PARs are present and functional on sensory neurons, where they participate either directly or indirectly to the transmission and/or inhibition of nociceptive messages. Taken together, the results discussed in this review highlight proteinases as signaling molecules to sensory nerves. We need to consider proteinases and the receptors that are activated by proteinases as important potential targets for the development of analgesic drugs in the treatment of inflammatory pain.