Using gait analysis to assess weight bearing in rats with Freund׳s complete adjuvant-induced monoarthritis to improve predictivity: Interfering with the cyclooxygenase and nerve growth factor pathways.

Lack of predictive power for drug effects has been a major criticism against animal pain models. It is therefore important to define the utility and limitations of different models. The aim of this study was to extend previous work on gait analysis as a tool to investigate pharmacological effects in monoarthritic rats, specifically to test the hypothesis that monoarthritis induced by Freund׳s complete adjuvant (FCA) provides a better estimate of overall analgesic efficacy of established, and novel, clinically effective and ineffective therapeutic approaches. Male rats injected intra-articularly into one ankle joint with FCA (1.0mg/ml) were treated with the monoclonal antibody to nerve growth factor (NGF), MEDI-578, the inhibitors of tropomyosin receptor kinases A, B and C (pan-Trk) AZ6623 or AZ7092, the transient receptor potential vanilloid 1 (TRPV1) inhibitor AZD1386, or the cyclooxygenase (COX) inhibitors naproxen, ibuprofen, valdecoxib or rofecoxib. Effects on weight bearing during locomotion were tested using video capture of print images. The apparent efficacy in this model was Trk inhibitors≥anti-NGF antibody>COX inhibitors. The TRPV1 inhibitor was ineffective. Together with previous data, the results support using gait-related parameters in the monoarthritis model. FCA as induction agent seems to provide a good overall prediction of analgesic efficacy in disorders with inflammatory joint pain.

Behavioral pharmacology of pain.

Behavioral methods are extensively used in pain research. Rodent modeling tends to rely on evoked responses but there is a growing interest in behavioral readouts that may capture elements of ongoing pain and disability, reflecting the major clinical signs and symptoms. Clinically, analgesics show greater efficacy in acute pain after standard surgery than in chronic conditions but are never completely effective on a population basis. In contrast, experimental pharmacological studies in rodents often demonstrate full efficacy, but there is variability in sensitivity between models and readouts. Full efficacy is rarely seen when more complex or multiple readouts are used to quantify behavior, especially after acute surgery or in studies of clinical pain in animals. Models with excellent sensitivity for a particular drug class exist and are suitable for screening mechanistically similar drugs. However, if used to compare drugs with different modes of action or to predict magnitude of clinical efficacy, these models will be misleading. Effective use of behavioral pharmacology in pain research is thus dependent on selection and validation of the best models for the purpose.

Gait analysis in rats with single joint inflammation: influence of experimental factors.

Disability and movement-related pain are major symptoms of joint disease, motivating the development of methods to quantify motor behaviour in rodent joint pain models. We used observational scoring and automated methods to compare weight bearing during locomotion and during standing after single joint inflammation induced by Freund's complete adjuvant (0.12-8.0 mg/mL) or carrageenan (0.47-30 mg/mL). Automated gait analysis was based on video capture of prints generated by light projected into the long edge of the floor of a walkway, producing an illuminated image of the contact area of each paw with light intensity reflecting the contact pressure. Weight bearing was calculated as an area-integrated paw pressure, that is, the light intensity of all pixels activated during the contact phase of a paw placement. Automated static weight bearing was measured with the Incapacitance tester. Pharmacological sensitivity of weight-bearing during locomotion was tested in carrageenan-induced monoarthritis by administration of the commonly used analgesics diclofenac, ibuprofen, and naproxen, as well as oxycodone and paracetamol. Observational scoring and automated quantification yielded similar results. We found that the window between control rats and monoarthritic rats was greater during locomotion. The response was more pronounced for inflammation in the ankle as compared to the knee, suggesting a methodological advantage of using this injection site. The effects of both Freund's complete adjuvant and carrageenan were concentration related, but Freund's incomplete adjuvant was found to be as effective as lower, commonly used concentrations of the complete adjuvant. The results show that gait analysis can be an effective method to quantify behavioural effects of single joint inflammation in the rat, sensitive to analgesic treatment.

Predictive validity of behavioural animal models for chronic pain.

Rodent models of chronic pain may elucidate pathophysiological mechanisms and identify potential drug targets, but whether they predict clinical efficacy of novel compounds is controversial. Several potential analgesics have failed in clinical trials, in spite of strong animal modelling support for efficacy, but there are also examples of successful modelling. Significant differences in how methods are implemented and results are reported means that a literature-based comparison between preclinical data and clinical trials will not reveal whether a particular model is generally predictive. Limited reports on negative outcomes prevents reliable estimate of specificity of any model. Animal models tend to be validated with standard analgesics and may be biased towards tractable pain mechanisms. But preclinical publications rarely contain drug exposure data, and drugs are usually given in high doses and as a single administration, which may lead to drug distribution and exposure deviating significantly from clinical conditions. The greatest challenge for predictive modelling is, however, the heterogeneity of the target patient populations, in terms of both symptoms and pharmacology, probably reflecting differences in pathophysiology. In well-controlled clinical trials, a majority of patients shows less than 50% reduction in pain. A model that responds well to current analgesics should therefore predict efficacy only in a subset of patients within a diagnostic group. It follows that successful translation requires several models for each indication, reflecting critical pathophysiological processes, combined with data linking exposure levels with effect on target.

Pharmacokinetic-pharmacodynamic modeling of the inhibitory effects of naproxen on the time-courses of inflammatory pain, fever, and the ex vivo synthesis of TXB2 and PGE2 in rats.

PURPOSE: To quantify and compare the time-course and potency of the analgesic and antipyretic effects of naproxen in conjunction with the inhibition of PGE(2) and TXB(2). METHODS: Analgesia was investigated in a rat model with carrageenan-induced arthritis using a gait analysis method. Antipyretics were studied in a yeast-induced fever model using telemetrically recorded body temperature. Inhibition of TXB(2) and PGE(2) synthesis was determined ex vivo. Pharmacokinetic profiles were obtained in satellite animals. Population PKPD modeling was used to analyze the data. RESULTS: The IC(50) values (95% CI) of naproxen for analgesia (27 (0-130) µM), antipyretics (40 (30-65) µM) and inhibition of PGE(2) (13 (6-45) µM) were in similar range, whereas inhibition of TXB(2) (5 (4-8) µM) was observed at lower concentrations. Variability in the behavioral measurement of analgesia was larger than for the other endpoints. The inhibition of fever by naproxen was followed by an increased rebound body temperature. CONCLUSION: Due to better sensitivity and similar drug-induced inhibition, the biomarker PGE(2) and the antipyretic effect would be suitable alternative endpoints to the analgesic effects for characterization and comparisons of potency and time-courses of drug candidates affecting the COX-2 pathway and to support human dose projections.

Using the CatWalk method to assess weight-bearing and pain behaviour in walking rats with ankle joint monoarthritis induced by carrageenan: effects of morphine and rofecoxib.

The CatWalk automated quantitative gait analysis technique has been validated as a method to quantify behaviour in rodent models of neuropathic and arthritic pain. Its suitability for pharmacological testing of pain relief has been questioned, however, based on findings using paw soft tissue plantar inflammation as stimulus. In this study, we investigated the effectiveness of morphine and rofecoxib in reducing pain behaviour in monoarthritic rats. The CatWalk was used to assess print area, weight load and duration of stance for each paw, as well as interlimb coordination, before and 3, 5 and 24h after injection of lambda-carrageenan into one ankle joint. The monoarthritic rat showed a reduced print area, weight load and duration of stance for the injected paw at all times tested, and a significant loss of interlimb coordination at 3 and 5h after injection. Both morphine (3.75 and 15 micromol/kg s.c.) and rofecoxib (7.5 and 30 micromol/kg p.o.) reduced the effects of carrageenan. In conclusion, behavioural effects interpreted as reflecting movement-related pain in monoarthritic rats and pharmacological treatment of the monoarthritis can objectively and efficiently be quantified in detail by the CatWalk method.

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.

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.

COX-inhibiting nitric oxide donators (CINODs) - a new paradigm in the treatment of pain and inflammation.

The clinical utility of non-selective non-steroidal anti-inflammatory drugs (NSAIDs) for pain relief is tempered by their propensity to cause gastrointestinal toxicity. Cyclooxygenase (COX)-inhibiting nitric oxide donators (CINODs) are a new class of drugs designed to provide analgesic efficacy through COX inhibition and gastrointestinal safety through the protective effects of controlled nitric oxide donation. Pre-clinical studies assessing the pharmacology, efficacy and gastrointestinal safety of AZD3582 [4-(nitrooxy)butyl-(2S)-2-(6-methoxy-2-naphthyl)propanoate] support this concept. Based on these studies, AZD3582 was the first CINOD to enter clinical development for the treatment of acute and chronic pain. The potential clinical utility of this new class is illustrated by a study of AZD3582 in healthy volunteers in which it caused significantly less acute gastrointestinal toxicity than an equimolar dose of naproxen. The results of the animal studies and the initial clinical study warrant long-term tolerability studies of AZD3582 along with evaluation of its anti-inflammatory and analgesic effects in humans.

Pathogenesis of sciatic pain: a study of spontaneous behavior in rats exposed to experimental disc herniation.

STUDY DESIGN: A new way to study pain in experimental animals without handling of the animals and based on registration of spontaneous behavior using video recordings. OBJECTIVES: To evaluate if experimental disc herniation in the rat may induce changes in spontaneous behavior. SUMMARY OF BACKGROUND DATA: The knowledge regarding the basic pathophysiologic mechanisms of sciatica has increased dramatically during the last decade. However, studies have mainly assessed nerve dysfunction rather than pain. Existing methods to study pain generally comprise a certain amount of handling and registration of changes in sensory thresholds. In the present study we introduce a new way to assess pain that focuses on changes in behavior rather than on changes in thresholds. METHODS: Forty rats were divided equally into four experimental series: sham exposure of the left L4 dorsal root ganglion, exposure of the left L4 dorsal root ganglion and incision of the L4-L5 disc, exposure and slight displacement of the left L4 dorsal root ganglion, and combination of disc incision and displacement. The rats were videotaped the day before surgery and on day 1, 3, 7, 14, and 21 after surgery. Spontaneous behavior was categorized into 10 behaviors and recorded during 20 minutes of observation. RESULTS: Disc incision and displacement per se did not induce any behaviors different from that observed in the sham-operated group. In the series with the combination of disc incision and displacement there was increased focal pain, seen as increased lifting of the hind paw on the operated side and increased rotation of the head toward the operated side. This pain pattern was most pronounced the day after surgery. Fourteen days after surgery there were no detectable differences in behavior between this group and the sham group. At day 21 after surgery, however, another picture of increased immobility and decreased locomotion was seen in this group, possibly indicating more generalized pain. CONCLUSIONS: The study demonstrates that it is possible to detect changes in spontaneous behavior after experimental disc herniation. However, such changes may only be seen if disc incision and slight mechanical deformation are combined. This is in agreement with previous clinical and experimental observations. The present model allows for convenient assessment of pain in a way that focuses on spontaneous behavior rather than changes in pain thresholds and that reduces the interference of the researcher and environment on the outcome of the assessment.

The NMDA antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) has antinociceptive effect after intrathecal injection in the rat.

This behavioral study was performed in order to delineate the antinociceptive effects of and the influence on motor function of a highly potent, competitive NMDA receptor antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP). After intrathecal (i.t.) administration of CPP to chronically catheterized rats, antinociception was studied in 3 different nociceptive tests: the tail-flick test, the hot-plate test, and the formalin test. The lowest dose producing visible motor dysfunction was 1 nmol, with 2 of 8 animals showing slight ataxia. Dose-related motor dysfunction and apparent sedation was present after 5 and 10 nmol. Dose-related antinociception was evident in the thermal tests following doses that produced little or no motor dysfunction. In the tail-flick test, the antinociceptive effect was attenuated at higher doses, resulting in a bell-shaped dose-response relationship. Dose-related antinociception was found in both the first and second phase of the formalin test following doses from 0.25 up to 1 nmol. The present study shows that the competitive NMDA antagonist CPP has an antinociceptive effect in doses that do not affect motor function. Furthermore, antinociception was evident in both phasic and tonic nociceptive tests. Finally, the dose-response relationship in the tail-flick test was bell-shaped. As discussed this indicates that NMDA receptors may be involved in functionally divergent nociceptive systems.

Intrathecal co-administration of substance P and NMDA augments nociceptive responses in the formalin test.

The effects of intrathecal administration of substance P and N-methyl-D-aspartate (NMDA) were studied in the formalin test in mice. Both substances were administered 5 min before injection of formalin into the hind paw. Co-administration of substance P and NMDA intensified the response in both the 1st (0-10 min) and the 2nd phase (20-30 min) of the formalin test, and increased the duration of the response. The increase in the response to formalin depended on the formalin concentration and was significant with 1% and 5% concentrations of formalin but not with a 0.05% concentration. No increase in the response was observed when NMDA or substance P was given alone. These findings indicate that concurrent activation of spinal NMDA and substance P receptors induces an enhancement of spinal transmission of nociception, and that this enhancement is dependent on the intensity or the quality of the peripheral stimulus.

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.

Apparent hyperalgesia after lesions of the descending serotonergic pathways is due to increased tail skin temperature.

It has been suggested that the descending serotonergic pathways exercise a tonic inhibition on nociception in the spinal cord. In this study 5,6-dihydroxytryptamine (5,6-DHT, 20 micrograms base) injected intrathecally in rats reduced spinal serotonin concentration to 3.5% of control levels without significantly affecting spinal noradrenaline. The lesion reduced the mean tail-flick latency by approximately 35% and increased the mean tail skin temperature by approximately 3.5 degrees C; both parameters gradually returned to normal values within 2-3 weeks. Both in controls and in lesioned animals there was a highly significant negative correlation between tail skin temperature and tail-flick latency. Multiple regression analysis showed that the effect of lesioning on tail-flick latency was non-significant when the effect of skin temperature was taken into account. Thus the reduced tail-flick latency after lesions of descending serotonergic pathways, usually interpreted as increased nociception, may be due to changes in tail skin temperature.

Dissociation between antinociceptive and anti-inflammatory effects of acetylsalicylic acid and indomethacin in the formalin test.

It is assumed that the mild analgesia produced by acetylsalicylic acid (ASA) and indomethacin is due to a common mode of action, namely inhibition of the cyclo-oxygenase reaction in the synthesis of prostaglandins. It has, however, been difficult to separate the influence of the anti-inflammatory activity from pure analgesia in standard animal tests using a fully developed inflammatory state. In the present experiments a modification of the formalin test in mice is used. Licking of the injected paw is recorded after the injection of a small nociceptive amount of formalin (20 microliters, 1%). The results show that the response to formalin is biphasic with an early (0-5 min) and a late (20-30 min) phase of high licking activity. ASA had a dose-dependent antinociceptive effect during both the early and the late phases. In contrast, antinociceptive effect of indomethacin was found only during the late phase. On the basis of these results it may be suggested that inhibition of the cyclo-oxygenase reaction has no major effect on the early phase in the formalin test. This also suggests that ASA and indomethacin are antinociceptive through partially different modes of action. In addition to an anti-inflammatory effect common to both drugs, ASA may have a direct antinociceptive action.

Effects of 5-HT receptor agonists and antagonists on a reflex response to radiant heat in normal and spinally transected rats.

Tail-flick latency (TFL) was tested in intact and spinally transected rats. Spinal transection permanently lowered the TFL by 25-30%. A diurnal rhythm in nociception was found in intact but not in spinal rats with maximum sensitivity during the early light period. Administration of the 5-hydroxytryptamine (5-HT) receptor antagonists metergoline (0.06-2.0 mg/kg), mianserin (1.0 and 5.0 mg/kg) or methiothepin (0.1-2.5 mg/kg) reduced the TFL of normal rats to the same level as that of the transected animals. The diurnal variation in TFL was also abolished by metergoline. Neither drug changed the TFL of the transected rats. The 5-HT agonist 5-methoxy-N,N-dimethyltryptamine (5-MeODMT, 0.5-2.0 mg/kg) induced the 5-HT syndrome in intact rats and elicited spinal reflexes in the transected animals. Concomitantly, the TFLs of both groups were elevated to the same maximum level, approximately 25% above normal TFL of intact rats. Transected rats consistently responded to lower doses than intact rats, indicating 5-HT receptor supersensitivity. The 5-MeODMT effects were reversed by administration of 5-HT antagonists. Administration of the 5-HT precursor dl-5-hydroxytryptophan (200 mg/kg), after inhibition of peripheral decarboxylation by carbidopa (75 mg/kg), also elevated the TFL and induced other behavioral signs of 5-HT stimulation. It is concluded tht there exists a tonic inhibitory influence with diurnal variations on spinal nociceptive reflexes in the rat. The data reported suggest that this inhibition is mediated by descending 5-HT pathways.