The development and validation of a murine model for studying the role of histamine receptors in acute and chronic itch

Itch (pruritus) is an unpleasant sensation of the skin, which evokes the desire to scratch. The condition commonly presents in clinical practice, as a symptom of systemic disease and various skin disorders, such as atopic dermatitis. Itch has tended to be closely associated with pain, and older evidence suggests that the sensations involve the same sensory nerves, firing at different frequencies. Partly because of this, and the subjective cognitive nature of the condition, itch has been relatively ignored as a research area and there is a general lack of effective animal models for studying itch. This has restricted detailed studies into putative mediators of itch and their mechanism(s) of action.The present studies were undertaken to develop and validate acute and chronic models of itch in mice, based on the combined use of behavioural tests in awake mice and in vivo electrophysiological recordings from itch afferents in anaesthetized animals. The hypothesis was that scratching behaviour can be evoked in mice using intradem1al injections of pruritogenic drugs and that this can be measured automatically and objectively to provide a reliable indicator of itch. A further hypothesis was that electrophysiological recordings made in vivo from murine cutaneous sensory nerves can be used to distinguish between pruritogens and algogens.The model of itch that was developed is based on injection of histamine into the back of the mouse neck to evoke scratching of the area by the hind paws. Histamine is a pruritogen in both humans and mice, although the lack of effectiveness of traditional H 1-receptor antihistamines in treating all clinical itch disorders suggests other mediators are also responsible for pruritus. The studies demonstrated that scratching in mice can be induced using histamine and other pruritogens (e.g. trypsin and 5-HT) in a reproducible dose dependent manner. Scratching was established as a response to itch-provoking agents, but not to painful stimuli. A novel mechanism of histamine evoked scratching involving H4 receptors was discovered. Chronic itching 111 response to topical application of dinitrochlorobenzene was also established. A robust automated method for the detection and measurement of scratching in mice was developed, which considerably enhances accuracy and reduces the time taken, in comparison with manual observation of scratching.In vivo electrophysiological recordings showed that pruritogens evoke a pattern of response in cutaneous nerves distinct in nature from that evoked by algogenic stimuli. However, nerves responded to both stimuli, suggesting that in mice, there are probably no independent 'pruritoceptors', unlike the situation in man. In summary, scratching in mice can be recorded automatically and used as a reproducible quantitative measure of itch. This model can be used for further studies on putative mediators of itch to establish their mechanism of action. Knowledge from such studies should provide understanding of the sensation of itch in man, and should facilitate the development of novel therapies specific for pruritus

Inflammatory responses to platelet-activating factor in human skin

Since its discovery over 20 years ago, platelet- activating factor (PAF) has been the subject of intensive research. In particular, its role in pathophysiology has provoked as much controversy as research interest. The pharmacological profile and ubiquity of PAF have made it a candidate for involvement in a number of inflammatory processes. No clear evidence has been demonstrated however to establish a role in disease in man, although clinical caution about its potential toxic effects has limited the number of available experimental human models. One particular tissue which lends itself to examination of the effects of PAF is the skin. A series of studies has been undertaken to define accurately the characteristics of the acute responses to PAF in human skin and to study the role of histamine in these responses. By way of comparison, the acute responses to other inflammatory mediators (PGE2, substance P, and CGRP) has also been investigated. The effects of histamine antagonism, and depletion on the PAF responses have been examined together with evaluation of local histamine release from the skin after PAF treatment. Additionally, a comparison of the PAF-induced responses in atopic and non-atopic subjects has been undertaken. As a consequence of these studies, quantitative methods have been validated as sensitive and reproducible techniques for evaluating hyperalgesia and histamine-type flare and weal responses in the skin. Using the hyperalgesia model, intradermally injected PAF failed to cause hyperalgesia in the skin. Conversely intradermal PGE2 was shown to provoke dose- related hyperalgesia, which was associated with spreading erythema, lasting for 2-3 hours. Apart from at high doses where the effect was marginal when compared with PGE2, the neuropeptides substance P and CGRP also failed to produce hyperalgesia in the skin. Using the flare and weal model, PAF, substance P and PGE2 were associated with dose-related histamine-like weal and flare responses, although the PGE2 responses were considerably weaker. CGRP did not produce these responses and instead caused a local intense erythema, similar to the later effect of PGE2. Evaluation of the effects of H1-antagonism and histamine depletion, and the quantification of local histamine release have demonstrated that the PAF responses in the skin are largely mediated by histamine. Indomethacin was also found to have a small inhibitory effect on the PAF responses implicating secondary prostaglandin release. The acute inflammatory responses to intradermal PAF have been shown to be similar in atopic subjects compared with non-atopics using the techniques described above. Baseline plasma histamine, however, was found to be higher in atopic subjects

Actions of Non-steroidal Anti-Inflammatory Drugs in Sheep

Mechanical and thermal stimuli were used to determine threshold responses in normal experimental sheep which were familiar with both test procedures. The average control response threshold to noxious thermal and mechanical stimulation was 54.

Pharmacodynamics of Non-Steroidal Anti-Inflammatory Agents in Acute Inflammation and Chronic Pain in the Horse.

The analgesic and anti-inflammatory effects of the nonsteroidal anti-inflammatory drugs (NSAIDs) ketoprofen (2.2 and 3.63 mg/kg) and phenylbutazone (4.4 mg/kg) were compared in equine models of acute synovitis and chronic hoof pain. The eicosanoids, prostaglandin E\sb2 (PGE\sb2) and leukotriene B\sb4 (LTB\sb4), increased dramatically in synovial fluid after carrageenan-induced synovitis of the intercarpal joint. PGE\sb2 concentrations in untreated horses peaked at 9 hours while LTB\sb4 concentrations peaked in all horses at 3 hours. Synovial fluid concentrations of both eicosanoids returned to near baseline by 48 hours. Lameness, joint temperature, and synovial fluid volume, protein and nucleated cells increased at 3 to 12 hours with reduction to near baseline levels by 48 hours. NSAIDs when given intravenously decreased joint concentrations of PGE\sb2, but LTB\sb4 levels were unaffected by drug administration. Both drugs decreased the signs of inflammation and lameness, but phenylbutazone was more effective. These data suggest that leukotrienes are involved in equine synovitis and the development of specific leukotriene inhibitors may be of therapeutic value. The plasma half-life of ketoprofen (2.2 mg/kg) in normal horses (0.88 hours) was higher than horses with synovitis (0.55 hours). Synovial fluid levels of ketoprofen in horses with synovitis were 6.5 times higher than normal horses at one hour. The area under the synovial fluid concentration curve for horses with synovitis was greater than in normal horses. These data suggest that the inflamed joint may serve as a site of sequestration for ketoprofen. Digital vein eicosanoid levels from horses with hoof pain from chronic laminitis were not different than those in normal horses. Although hoof pain and lameness could not be attributed to eicosanoids, both effects were reduced by the systemic administration of NSAIDs. Ketoprofen at a dose of 3.63 mg/kg (phenylbutazone equimolar dose) reduced hoof pain and lameness to a greater extent than the 2.2 mg/kg dose and phenylbutazone. These effects were still present at 24 hours for 3 of the 4 measures of hoof pain. These data suggest that phenylbutazone was more potent in alleviating acute joint inflammation whereas ketoprofen at a dosage rate of 1.65 times the therapeutic dose was more potent in alleviating chronic pain and lameness in horses

Effect of peptidoglycan-polysaccharide complex on reproductive efficiency and mastitis in sheep

Bacterial infections associated with mastitis reduce pregnancies in cattle. Effects on pregnancy and incidence of mastitis in sheep were investigated after immunization with peptidoglycan-polysaccharide (PG-PS) and killed cells from Strep. pyogenes. Rambouillet (n = 100) and mixed breed (n = 18) ewe lambs were immunized (∼d42 and 22 before breeding) with PG-PS (30mugPG/kg/bw) or killed cells or were not immunized (Control, n = 117). IgG antibodies were detected by ELISA. Ewes were bred at synchronized estrus. All immunized and half of non-immunized ewes were challenged with PG-PS (60mugPG/kg/bw) d5 after breeding. Although proportion of ewes pregnant at d42 did not differ, probability of pregnancy decreased with total dose of PG-PS (p \u3c .05). Incidence of mastitis in mixed breed ewes in the middle and at the end of lactation did not differ. In conclusion, immunization of ewe lambs with PG-PS or killed cells of Strep. pyogenes did not improve pregnancy rate or incidence of mastitis after PG-PS challenge

Mechanisms of pain processing: spinal protein translation in the rat

The word ‘pain’ is described by the International Association for the Study of Pain (IASP) as an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage. Shifts in pain thresholds and responsiveness are an expression of neural plasticity, a process whereby changes in the nervous system modulate the response to a given stimulus. It is widely believed that this process may contribute to chronic pain. Forms of long-term plasticity specifically require protein synthesis and such mechanisms are widely believed to be cellular counterparts of long-term memory. Relevant to mammalian pain nociceptors is the realisation that messenger ribonucleic acid (mRNA) in dendrites, axons, axon terminals as well as cell bodies, is essential for long-term synaptic plasticity and may therefore be important in pain processing. The protein kinase mammalian target of rapamycin (mTOR) is a key regulator of protein translation and can be specifically inhibited by the drug rapamycin and the rapamycin analogue cell cycle inhibitor (CCI)-779 (CCI-779). This was investigated in rats under physiological conditions and also pathophysiological conditions relevant to clinical pain syndromes i.e. persistent pain-like states. A variety of techniques were utilised: in vivo electrophysiology was used to obtain extracellular single unit recordings of spinal cord lamina V wide dynamic range (WDR) neurones that respond to innocuous and noxious stimuli to peripheral sites i.e. the rat hind paw; behavioural studies were used to assess the progression of pain-like states and assess the effects of rapamycin/CCI-779 on this behaviour and immunohistochemistry was used to visualise active components of rapamycin-sensitive protein translation pathways at the spinal level. In addition, the dependence of these spinal mechanisms on descending serotonergic pathways from higher brain centres was investigated pharmacologically by selectively activating or blocking serotonergic spinal 5-HT3 receptors. These pathways have already been proven to be pivotal in the maintenance of persistent pain-like states. The results suggest that mTOR has a continuous role in maintaining persistent pain-like states via rapid local protein translation, which can be influenced by descending facilitatory controls from higher centres in the brain

Central nervous system mast cells in peripheral inflammatory nociception

<p>Abstract</p> <p>Background</p> <p>Functional aspects of mast cell-neuronal interactions remain poorly understood. Mast cell activation and degranulation can result in the release of powerful pro-inflammatory mediators such as histamine and cytokines. Cerebral dural mast cells have been proposed to modulate meningeal nociceptor activity and be involved in migraine pathophysiology. Little is known about the functional role of spinal cord dural mast cells. In this study, we examine their potential involvement in nociception and synaptic plasticity in superficial spinal dorsal horn. Changes of lower spinal cord dura mast cells and their contribution to hyperalgesia are examined in animal models of peripheral neurogenic and non-neurogenic inflammation.</p> <p>Results</p> <p>Spinal application of supernatant from activated cultured mast cells induces significant mechanical hyperalgesia and long-term potentiation (LTP) at spinal synapses of C-fibers. Lumbar, thoracic and thalamic preparations are then examined for mast cell number and degranulation status after intraplantar capsaicin and carrageenan. Intradermal capsaicin induces a significant percent increase of lumbar dural mast cells at 3 hours post-administration. Peripheral carrageenan in female rats significantly increases mast cell density in the lumbar dura, but not in thoracic dura or thalamus. Intrathecal administration of the mast cell stabilizer sodium cromoglycate or the spleen tyrosine kinase (Syk) inhibitor BAY-613606 reduce the increased percent degranulation and degranulated cell density of lumbar dural mast cells after capsaicin and carrageenan respectively, without affecting hyperalgesia.</p> <p>Conclusion</p> <p>The results suggest that lumbar dural mast cells may be sufficient but are not necessary for capsaicin or carrageenan-induced hyperalgesia.</p

Pharmacological studies of prostanoids and other substances on sensory nerves in arthritic rats

A novel model of unilateral, localized adjuvant-induced arthritis in the rat was assessed for its use in pharmacological studies on the pain associated with chronic joint inflammation. Levels of inflammation, changes in behaviour and joint pathology were examined. Electrophysiological recordings of afferent activity were made in anaesthetized rats in vivo and in vitro from a fine branch of the medial plantar nerve innervating the tarsal joint. The effects of various putative inflammatory mediators on the discharge of articular nociceptors both in normal and arthritic joints were also examined. Where possible, selective agonists and antagonists were used to characterize the pharmacological receptors involved in the actions of these substances.Injection of Freunds Complete Adjuvant around the left ankle joint caused a localized swelling, nociceptive thresholds to mechanical stimuli were lowered and use of the injected joint was significantly reduced. Histological studies revealed the presence of an arthritis which was confined to the injected ankle. Electrophysiological investigations showed that in arthritic joints there were a larger number of identifiable receptive fields for articular mechanonociceptors compared to those found in normal joints. Receptors had lower activation thresholds, and often displayed a resting discharge not seen in normal joints.Intra-arterial (i.a.) injection of 5-hydroxytryptamine (5-HT), excited articular nociceptors from normal and arthritic joints. Responses consisted of two components: (a) a fast transient burst of activity mediated by a 5-HT₃-receptor, followed by (b) a delayed, longer-lasting excitation mediated by a 5-HT₂-receptor. These responses were shown to occur both in vivo and in vitro. 5-HT also increased the mechanical responsiveness of articular mechanonociceptors via a 5-HT₃-receptor. Sensory receptors in arthritic joints were more sensitive to 5-HT than those from normal joints. Administration of the 5-HT₃- or 5-HT₂ receptor antagonists caused short-lasting reductions in background activity in arthritic joints, as well as in normal joints in which activity had increased following administration of 5-HT.In normal joints, i.a. injection of PGE₂, PGI₂ or the selective IPreceptor agonist cicaprost, excited and caused mechanical sensitization of articular mechanonociceptors. Potentiation of the short-lived excitatory and sensitizing effects of i.a. injected bradykinin on these receptors was also shown. Examination of the effects of PGE₂, PGI₂ and cicaprost in vivo or PGE₂, cicaprost, PGD₂, and PGF₂ alpha in vitro, produced a rank order of potency ofPGI₂ - cicaprost » PGE₂ » PGD₂ - PGF₂ alpha In arthritic rats injection of cicaprost, and to a lesser extent PGE₂, was effective in increasing the mechanical responsiveness and resting discharge of articular mechanonociceptors previously depressed by i.v. lysine acetylsalicylate. These results provide evidence for the involvement of IP-receptors, and perhaps EP-receptors, in the excitatory and sensitizing actions of the prostanoids on articular nociceptors, and suggest that PGI₂ is the major endogenous prostanoid responsible for the mechanonociceptor sensitization seen in arthritic rat ankle joints.Overall the results suggest that more than one mediator is required to produce conditions of mechanonociceptor sensitization seen in arthritic rat ankle joints. However, as modulators of the responsiveness of nociceptors to other mediators, and as potent excitants themselves, the prostanoids, and in particular PGI₂, probably play a major role in the alterations in nociceptor sensitivity seen in arthritic joints