Patrick Raboisson

Application of the formalin test to the study of orofacial pain in the rat

A modification of the formalin test for assessing pain and analgesia in the orofacial region of the rat is described. A formalin solution (5%) was subcutaneously injected into the upper lip, then the length of time the animal spent rubbing the injected zone was recorded. Two distinct periods of intensive rubbing activity were identified: an early phase between 0 and 3 min after the injection and a late phase between 18 and 42 min after the injection. Acetylsalicylic acid, paracetamol and morphine all had an antinociceptive effect during the two phases although incomplete during the early phase. Our results indicate that this orofacial formalin test is a valid technique for the study of orofacial pain.

The orofacial formalin test

The subcutaneous injection of formalin into the rat upper lip generates behavioral responses that last several minutes. The time course of the response is similar to what is observed following formalin injection into the paw, i.e. biphasic, with an early and short-lasting first phase followed, after a quiescent period by a second, prolonged (tonic) phase. The applied chemical stimulus (formalin) can be qualified as noxious since it produces tissue injury, activates Adelta and C nociceptors as well as trigeminal and spinal nociceptive neurons and is felt as painful in man. In addition, increasing the concentration of formalin causes a parallel aggravation of histological signs of tissue inflammation and injury. The measured behavioral response (face rubbing) is a relevant end-point: prolonged face rubbing is evoked by formalin but not saline injection and a positive relationship between the amplitude of the response and the formalin concentration is observed, at least up to 2.5%. At higher formalin concentrations, the use of other or additional end-points should be considered. Finally, the behavioral response in the orofacial formalin test is sensitive to various opioid and non-opioid analgesics. The orofacial formalin test can then be considered as a reliable way of producing and quantifying nociception in the trigeminal region of the rat.

Evidence for a peripheral origin of the tonic nociceptive response to subcutaneous formalin

&NA; The orofacial formalin test in the rat is a valid and reliable model of nociception and is sensitive to various classes of analgesic drugs. The noxious stimulus consists in an injection of diluted formalin (2.5% in saline) into the upper lip. The behavioural nociceptive response is measured in terms of the amount of time the animal spends rubbing the injected area. Two distinct periods of intense rubbing activity can be identified, a first phase occurring in the first 3 min and a second phase lasting from 12 to 39 min after formalin injection. The present study verified the peripheral origin of the first phase of the formalin response and examined whether the second phase is produced by peripheral activation of afferent fibres and/or by a phenomenon of central facilitation induced by the neural activity that occurs during the first phase. This was determined by assessing the effect of a local anaesthetic agent (lidocaine) administered into the formalin injection site, before or after the first phase of the formalin response. Local injection of 50 &mgr;l of lidocaine prior to formalin completely abolished the first phase of the formalin response but this blockade did not significantly influence the appearance and development of the second phase. Thus, the primary afferent activity that normally occurs during the first phase of the formalin response is not a prerequisite for the expression of the second phase. A higher dose of lidocaine (150 &mgr;l) induced, in addition, inhibition of the first part of the second phase. Fifty or 150 &mgr;l of lidocaine injected after the first phase produced a blockade of the second phase, with a dose‐dependent duration (6 min and 18 min, respectively). Our results demonstrated that both the first and second phases of the nociceptive response to formalin depend on primary afferents activation and, consequently, that the second phase cannot be mediated by central sensitisation alone.

The orofacial formalin test in rats : effects of different formalin concentrations

&NA; In this study of the orofacial formalin test in rats, the effects of different formalin concentrations (0.2%, 0.5%, 1.5%, 2.5%, 5% and 10%) on the behavioural nociceptive response (face rubbing) was investigated. The histological responses of the skin were also evaluated. Increasing the concentration of formalin caused a parallel aggravation of histological signs of tissue inflammation and injury. All concentrations provoked an early phase of nociceptive response, but its intensity was not concentration‐dependent. The 2nd phase of response to formalin only occurred for concentrations of 1.5% and higher. A positive relationship between the formalin concentration and the amplitude of the rubbing activity measured between 12 and 45 min after injection could be observed until 2.5% but with the highest concentrations (5 and 10%), the amplitude of the response decreased. Our findings indicate that the orofacial formalin test should be carried out using concentration between 0.5 and 2.5%. This is essential to assess increase as well as decrease in pain intensity. Moreover, this will have the effect of minimizing the suffering of the experimental animal.

The rostral part of the trigeminal sensory complex is involved in orofacial nociception.

Abstract Single units responsive to noxious mechanical stimulation of orofacial receptive fields were recorded within the ventrobasal complex of the rat thalamus. The induced activities were compared before and after deafferentation of the subnucleus caudalis by a trigeminal tractotomy performed at the obex level. The receptive fields activated by noxious stimulation were classified as ‘oral’ when included in the oral, perioral or paranasal areas, and as ‘facial’ when included in facial regions distant from the oral cavity. After tractotomy, the unit responses to noxious stimulation of an oral field remained unchanged in 8 cases, decreased in 3 cases, and were suppressed in 4 cases. For units responding to noxious stimulation of a facial field, the responses were suppressed in 8 cases, decreased in two cases and remained unchanged in two other cases. So it appears that the rostral part of the trigeminal sensory complex (1) receives nociceptive afferents mainly from the oral and perioral areas and (2) is a relay in ascending pathways which convey painful sensations.

Recent Advances in Non-Competitive mGlu5 Receptor Antagonists and their Potential Therapeutic Applications

Extensive research into the functions of glutamate and glutamate receptors in the central nervous system (CNS) has shown an essential role of metabotropic glutamate (mGlu) receptors in normal brain functions, but also in neurological and psychiatric disorders. The precise functions of these receptors remain undefined, and progress toward understanding their functions has been hampered by the lack of selective ligands with appropriate pharmacokinetic properties. The Group I mGlu receptor, mGlu5, is well positioned to regulate and fine-tune neuronal excitability and synaptic transmission through its modulation of various signal transduction pathways and interactions with other transmitter systems. Therefore, the mGlu5 receptor may be an important therapeutic target for the treatment of disorders of the central nervous system. The discovery of MPEP 3, a non-competitive mGlu5 receptor antagonist, provided a potent, selective, systemically active tool compound for proof of concept studies in animal models of various disease states. These studies have led to greater understanding of possible therapeutic applications of mGlu5 receptor antagonists in recent years, suggesting their use in a number of disease states, including chronic pain, various psychiatric and neurological disorders, substance abuse and withdrawal, obesity and gastroesophageal reflux disease (GERD). Together, these findings have intensified efforts to find other non-competitive mGlu5 receptor antagonists and have led to the discovery of several second-generation compounds, a few of which are in preclinical evaluations. There have been several recent reviews on mGlu receptor. This article highlights recent efforts on the design, synthesis and development of novel, non-competitive mGlu5 receptor antagonists and studies to understand their in vitro mechanisms of action and in vivo pharmacological profiles. Emphasis is also given to recent advances in the potential therapeutic applications of non-competitive mGlu5 receptor antagonists.

Organization of efferent projections from the spinal cervical enlargement to the medullary subnucleus reticularis dorsalis and the adjacent cuneate nucleus: A PHA-L study in the rat

The distribution and organization of projections from the spinal cervical enlargement to subnucleus reticularis dorsalis (SRD) and the neighbouring Cuneate nucleus (Cu) area was studied in the rat by using microinjections of Phaseolus vulgaris leucoagglutinin (PHA‐L) into different laminae around the C7 level. The Cu received very dense projections from the dorsal horn, with the highest density being observed following injections into the medial part of laminae III–IV. The SRD received dense projections from laminae V–VII of the cervical enlargement, particularly from the reticular and medial aspects of lamina V, lamina VI, and the dorsal part of lamina VII. By contrast, the superficial part of the dorsal horn (laminae I to IV) and the dorsal part of lamina X provided only sparse projections to the SRD. Clusters of labelled terminals and boutons were observed mainly in the SRD areas subjacent to the Cu. In the caudorostral axis, labelled terminals were spread along the whole SRD from the cervicomedullary junction up to the caudal‐most part of the area postrema. Contralateral projections to the SRD were scarce and were observed mainly after injections into the medial part of laminae VI–VII. These data give further support to the proposal that there are two parallel systems in neighbouring structures of the caudal medulla, viz. the Cu and the SRD, which, respectively, relay lemniscal and nociceptive information from the spinal cord to the thalamus.

Responses of trigeminal subnucleus oralis nociceptive neurones to subcutaneous formalin in the rat

Extracellular recordings of 33 single nociceptive neurones of the trigeminal subnucleus oralis (SNO) were made in rats under halothane nitrous oxide anaesthesia. These neurones were tested for their responses to a s.c. injection of formalin in their receptive field. Such a chemical noxious stimulation is known to induce a biphasic response of nociceptive dorsal horn neurones, the second period of which would be due to inflammation. Twenty-three neurones were characterized as nociceptive non-specific (NnS) and 10 as nociceptive specific neurones (NS). Formalin activated both SNO NS and NnS neurones, but, when they responded, NS neurones (n = 5) showed only the first phase of activity while NnS neurones showed either one (n = 13) or two phases (n = 6). Biphasic responses were most often observed for NnS neurones with A delta- and C-fibre inputs. These results indicate that the time-course similarity between the behavioural and the neuronal responses to formalin exists only for some SNO convergent neurones and that therefore the SNO does not seem to be very involved in the inflammatory component of the pain caused by formalin.

A positron emission tomography study in healthy volunteers to estimate mGluR5 receptor occupancy of AZD2066 — Estimating occupancy in the absence of a reference region

AZD2066 is a new chemical entity pharmacologically characterized as a selective, negative allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). Antagonism of mGluR5 has been implicated in relation to various diseases such as anxiety, depression, and pain disorders. To support translation from preclinical results and previous experiences with this target in man, a positron emission tomography study was performed to estimate the relationship between AZD2066 plasma concentrations and receptor occupancy in the human brain, using the mGluR5 radioligand [(11)C]-ABP688. The study involved PET scans on 4 occasions in 6 healthy volunteers. The radioligand was given as a tracer dose alone and following oral treatment with different doses of AZD2066. The analysis was based on the total volume of distribution derived from each PET-assessment. A non-linear mixed effects model was developed where ten delineated brain regions of interest from all PET scans were included in one simultaneous fit. For comparison the analysis was also performed according to a method described previously by Lassen et al. (1995). The results of the analysis showed that the total volume of distribution decreased with increasing drug concentrations in all regions with an estimated Kipl of 1170 nM. Variability between individuals and occasions in non-displaceable volume of distribution could explain most of the variability in the total volume of distribution. The Lassen approach provided a similar estimate for Kipl, but the variability was exaggerated and difficult to interpret.

Differential effects of trigeminal tractotomy on Aδ- and C-fiber-mediated nociceptive responses

In this study we have tested in the rat, whether trigeminal tractotomy, which deprives the spinal trigeminal nucleus caudalis (Sp5C) of its trigeminal inputs, affected differentially nociceptive responses mediated by C- vs. Adelta-nociceptors from oral and perioral regions. Tractotomy had no effect on the threshold of the jaw opening reflex, induced by incisive pulp stimulation (Adelta-fiber-mediated), but blocked the formalin response (mainly C-fiber-mediated). These results suggest that nociceptive responses mediated by trigeminal C-fibers completely depend on the integrity of the Sp5C, while intraoral sensations triggered Adelta-fibers (especially of dental origin) are primarily processed in the rostral part of the spinal trigeminal nucleus.