Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction

The formalin test: an evaluation of the method.

The formalin test for nociception, which is predominantly used with rats and mice, involves moderate, continuous pain generated by injured tissue. In this way it differs from most traditional tests of nociception which rely upon brief stimuli of threshold intensity. In this article we describe the main features of the formalin test, including the characteristics of the stimulus and how changes in nociceptive behaviour may be measured and interpreted. The response to formalin shows an early and a late phase. The early phase seems to be caused predominantly by C-fibre activation due to the peripheral stimulus, while the late phase appears to be dependent on the combination of an inflammatory reaction in the peripheral tissue and functional changes in the dorsal horn of the spinal cord. These functional changes seem to be initiated by the C-fibre barrage during the early phase. In mice, the behavioural response in the late phase depends on the ambient temperature. We argue that the peripheral tissue temperature as well as other factors influencing the peripheral inflammation may affect the response, possibly confounding the results obtained with the test. Furthermore, we discuss the methods of recording the response and the value of observing more than one aspect of behaviour. Scoring of several behavioural variables provides a means of assessing motor or sensorimotor function as possible causes for changes in behaviour. In conclusion, the formalin test is a valuable addition to the battery of methods available to study nociception.

Peripheral tissue damage or nerve injury often leads to pathological pain processes, such as spontaneous pain, hyperalgesia and allodynia, that persist for years or decades after all possible tissue healing has occurred. Although peripheral neural mechanisms, such as nociceptor sensitization and neuroma formation, contribute to these pathological pain processes, recent evidence indicates that changes in central neural function may also play a significant role. In this review, we examine the clinical and experimental evidence which points to a contribution of central neural plasticity to the development of pathological pain. We also assess the physiological, biochemical, cellular and molecular mechanisms that underlie plasticity induced in the central nervous system (CNS) in response to noxious peripheral stimulation. Finally, we examine theories which have been proposed to explain how injury or noxious stimulation lead to alterations in CNS function which influence subsequent pain experience.

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Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence

http://yael-nissan.weebly.com/uploads/5/2/9/2/5292348/the_introduction_to_pain_an_integrative_review_-_millan.pdf

The induction of pain: an integrative review

The antinociception induced by the intraperitoneal coadministration of combinations of paracetamol with the nonsteroidal anti-inflammatory drugs (NSAIDs) diclofenac, ibuprofen, ketoprofen, meloxicam, metamizol, naproxen, nimesulide, parecoxib and piroxicam was studied by isobolographic analysis in the acetic acid abdominal constriction test of mice (writhing test). The effective dose that produced 50% antinociception (ED50) was calculated from the log dose-response curves of fixed ratio combinations of paracetamol with each NSAID. By isobolographic analysis, this ED50 was compared to the theoretical additive ED50 calculated from the ED(50) of paracetamol and of each NSAID alone obtained from ED50 dose-response curves. As shown by isobolographic analysis, all the combinations were synergistic, the experimental ED50s being significantly smaller than the theoretically calculated ED50s. The results of this study demonstrate potent interactions between paracetamol and NSAIDs and validate the clinical use of combinations of these drugs in the treatment of pain conditions.

/pdf/synergism-between-paracetamol-and-nonsteroidal-anti-5fjmf17tbv.pdf

Synergism between paracetamol and nonsteroidal anti-inflammatory drugs in experimental acute pain.

Antinociceptive effects of Ca2+ channel blockers.

The mechanism of action of tramadol includes the activation of opioid receptors, and the potential ability of the drug to induce tolerance and physical dependence has been evaluated in different animal species and humans. This work was designed to study the involvement of opioid receptors in the antinociceptive activity and the potential ability to develop tolerance, crosstolerance, and/or physical dependence of tramadol. The writhes induced by acetic acid administration was used as algesiometric test. After chronic administration of tramadol, tolerance was evaluated by measuring the antinociceptive activity, and physical dependence was measured by naloxone administration. Morphine was used as drug of comparison. The i.p. administration of tramadol produced a dose-dependent antinociception with an ED50 value of 7.82 +/- 1.16 mg/kg, which was unchanged after chronic administration of either tramadol (39.1 or 100 mg/kg) or morphine (1.05 or 100 mg/kg). By contrast, the ED50 for morphine (0.21 +/- 0.08 mg/kg) was significantly reduced only by chronic pretreatment with both doses of morphine (tolerance). Physical dependence was developed only in mice pretreated with morphine, as evidenced by the presence of jumps, wet-dog shakes, tachypnea, piloerection, seizures, diarrhea, and urination after the administration of naloxone (1 mg/kg). These findings suggest that the antinociceptive activity of tramadol in mice is due to activation of opioid and nonopioid mechanisms, and as opposed to morphine, is not likely to induce tolerance and physical dependence.

Antinociception, tolerance, and physical dependence comparison between morphine and tramadol

The common mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs) is the inhibition of the enzyme cyclo-oxygenase (COX), however, this inhibition is not enough to completely account for the efficacy of these agents in several models of acute pain.


It has been demonstrated that cholinergic agents can induce antinociception, but the nature of the interaction between these agents and NSAIDs drugs has not been studied. The present work evaluates, by isobolographic analysis, the interactions between the cholinergic indirect agonist neostigmine (NEO) and NSAIDs drugs, using a chemical algesiometric test.


Intraperitoneal (i.p.) or intrathecal (i.t.) administration of NEO and of the different NSAIDs produced dose-dependent antinociception in the acetic acid writhing test of the mouse.


The i.p. or i.t. co-administration of fixed ratios of ED50 fractions of NSAIDs and NEO, resulted to be synergistic or supra-additive for the combinations ketoprofen (KETO) and NEO, paracetamol (PARA) and NEO) and diclofenac (DICLO) and NEO administered i.p. However, the same combinations administered i.t. were only additive. In addition, the combinations meloxicam (MELO) and NEO and piroxicam (PIRO) and NEO, administered either i.p. or i.t., were additive.


The results suggest that the co-administration of NEO with some NSAIDs (e.g. KETO, PARA or DICLO) resulted in a synergistic interaction, which may provide evidence of supraspinal antinociception modulation by the increased acetylcholine concentration in the synaptic cleft of cholinergic interneurons. The interaction obtained between neostigmine and the NSAIDs could carry important clinical implications.





Keywords: Writhing test, NSAIDs, neostigmine, isobolographic analysis, antinociception


Introduction
It is generally accepted that the common mechanism of action of non-steroidal anti-inflammatory drugs (NSAIDs) is the inhibition of the enzyme cyclo-oxygenase (COX). Two isoforms of COX, referred to as COX-1 and COX-2, have been identified (Vane et al., 1998; Marnett & Kalgutkar, 1999). Each enzyme is encoded by a separate gene with a different pattern of expression and function. COX-1 is constitutively expressed and high levels can be found in most tissues, whereas COX-2 is inducible and usually, under physiological conditions, low levels are detectable in some tissues, but levels are rapidly increased during inflammation (Vane et al., 1998; Marnett & Kalgutkar, 1999). Several works have demonstrated that the inhibition of COX mediates the anti-inflammatory, antipyretic and analgesic effects of NSAIDs (Cryer & Feldman, 1998; Warner et al., 1999; Mitchell & Warner, 1999; Smith et al., 2000; Paik et al., 2000). In spite of this emphasis on the COX mediated-antinociceptive activity of NSAIDs, the selective inhibition of COX seems not to be enough to completely account for the efficacy of these agents, since in several models of pain without inflammation, such as models of chemical and thermal acute pain, NSAIDs are powerful analgesics (Miranda et al., 2001; Pinardi et al., 2001).

The role of the cholinergic system in nociception has been recognized. Thus, it has been suggested that acetylcholine is an endogenous antinociceptive compound that may act through monoaminergic pathways (Gillberg et al., 1989; Iwamoto & Marion, 1993; Guimaraes & Prado, 1994). Cholinergic agents such as neostigmine (NEO), produce analgesia after systemic and intrathecal administration (Eisenach & Gebhart, 1995; Naguib & Yaksh, 1997; Chiari et al., 1999; Eisenach, 1999). On the other hand, cholinergic agonists exert a positive modulatory action enhancing analgesia induced by opioid or clonidine (Abram & Winne, 1995; Eisenach & Gebhart, 1995; Xu et al., 1996; Hood et al., 1997).

Although previous studies have demonstrated that cholinergic agents can induce antinociception, a study of the characteristics of the interaction between these agents and NSAIDs has not been performed. The purpose of the current study was to examine, by isobolographic analysis, the interactions between the cholinergic indirect agonist NEO and NSAIDs drugs, using a chemical algesiometric test.

Neostigmine interactions with non steroidal anti-inflammatory drugs

The nonsteroidal anti-inflammatory drugs (NSAIDs) clonixin, diclofenac, piroxicam, ketoprofen, meloxicam, and paracetamol induced antinociception after intraperitoneal or intrathecal administration in mice submitted to an acute thermal algesiometric test without inflammation (tail-flick). Antinociception was evaluated by the increase in reaction time difference (Delta latency), between readings obtained before and after the administration of drugs. The antinociception induced by doses of NSAIDs producing between 20% and 30% of the maximum possible effect (MPE) 30 min after intraperitoneal and 15 min after intrathecal injections was compared with the antinociception obtained after pretreatment with 1 mg/kg atropine ip, 30 min before. Systemic atropine (1 mg/kg) significantly antagonized NSAID-induced antinociception in all cases, both after intraperitoneal and intrathecal administration. Cholinergic depletion by intracerebroventricular hemicholinium-3 (HC-3, 5 microg) 5 h before prevented the antinociceptive effect of all NSAIDs. These observations suggest that intrinsic muscarinic cholinergic facilitatory pathways represent an important modulating system in pain perception in this animal model of acute thermal pain. The results of the present work support the increasingly accepted notion that NSAIDs are effective analgesics even when inflammation is not present, acting by mechanisms that involve actions on spinal and supraspinal nociceptive transmission. It is suggested that, similar to morphine and clonidine, the active mechanism of NSAIDs may involve the release of acetylcholine (ACh) in the spinal cord.

Hugo F. Miranda

Papers

Synergism between paracetamol and nonsteroidal anti-inflammatory drugs in experimental acute pain.

Antinociceptive effects of Ca2+ channel blockers.

Antinociception, tolerance, and physical dependence comparison between morphine and tramadol

Neostigmine interactions with non steroidal anti-inflammatory drugs

Atropine reverses the antinociception of nonsteroidal anti-inflammatory drugs in the tail-flick test of mice.