Validation of a simple automated movement detection system for formalin test in rats

Aim: To investigate the validity and sensitivity of an automatic movement detection system developed by our laboratory for the formalin test in rats. Methods:The effects of systemic morphine and local anesthetic lidocaine on the nociceptive behaviors induced by formalin subcutaneously injected into the hindpaw were examined by using an automated movement detection system and manual measuring methods. Results: Formalin subcutaneously injected into the hindpaw produced typical biphasic nociceptive behaviors (agitation). The mean agitation event rate during a 60-min observation period increased linearly following increases in the formalin concentration (0.0%, 0.5%, 1.5%, 2.5%, and 5%, 50 μL). Systemic application of morphine of different doses (1, 2, and 5 mg/kg) 10-min prior to formalin injection depressed the agitation responses induced by formalin injection in a dose-dependent manner, and the antinociceptive effect induced by the largest dose (5 mg/kg) of morphine was significantly antagonized by systemic application of the opioid receptor antagonist naloxone (1.25 mg/kg). Local anesthetic lidocaine (20 mg/kg) injected into the ipsilateral ankle subskin 5-min prior to formalin completely blocked the agitation response to formalin injection. These results were comparable to those obtained from manual measure of the incidence of flinching or the duration time of licking/biting of the injected paw. Conclusion: These data suggest that this automated movement detection system for formalin test is a simple, validated measure with good pharmacological sensitivity suitable for discovering novel analgesics or investigating central pain mechanisms.     

Pharmacological correlation between the formalin test and the neuropathic pain behavior in different species with chronic constriction injury

Research on mechanisms of drug action, and preclinical screening of molecules with a potential activity on neuropathic pain requires extensive animal work. The chronic constriction injury model is one of the best-characterized models of neuropathic pain behavior in rats, but requires extensive time consuming operations and animal handling. The formalin test is easier to perform, and a well validated model. The latter may serve as an effective prescreening test of molecules and may facilitate drug targeting. In the present study the activity of different pharmacological reference compounds was tested in rats and gerbils on the cold plate for animals that had undergone chronic constriction injury and in the second phase of the formalin test. In rats, a comparable outcome in both test conditions was observed for morphine, fentanyl, MK-801 and flunarizine. Clonidine had more activity in the second phase of the formalin test, whereas baclofen, tramadol, amitryptiline, ketamine and topiramate showed more activity in the cold plate. In gerbils, both test conditions yielded comparable results for fentanyl and ketoprofen. Tramadol and CP-96345 tended to have more activity in the second phase of the formalin test, whereas morphine, SR-48968, SR-142801 and R116301 demonstrated more activity in the cold plate test. This study demonstrates a good correlation between the second phase of the formalin test and the cold allodynia in the CCI model for, both for rats and gerbils. Drugs with a proven activity in humans, used as reference compounds, also showed good pharmacological activity in this animal study.     

Role of the endogenous cannabinoid system in the formalin test of persistent pain in the rat

It has been suggested that administration of a cannabinoid CB(1) (SR141716A ?N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1-H-pyrazole-3-carboxamide) and CB(2) (SR144528 ?N-[(1S)-endo-1, 3, 3-trimethyl bicyclo ?2.2.1 heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyr azo le- 3-carboxamide?) receptor antagonists to mice potentiates inflammatory hyperalgesia by removing an endogenous cannabinoid tone. We examined whether the behavioural response to s.c. formalin injection in rats is similarly enhanced. A total of 30 animals received SR141716A (0.5 or 5 mg/kg) or SR144528 (0.3 or 3 mg/kg) 30 min before 1% formalin. Pain behaviour was quantified using the composite weighted pain score technique (CPS-WST(0,1,2)). An overall CPS-WST(0,1,2) was calculated for each phase and groups were compared (analysis of variance). The results obtained in the control group confirmed the characteristic biphasic behavioural response to formalin injection. None of antagonist groups had a significant increase in overall CPS-WST(0,1,2) compared to the control. Indeed, a significant decrease in CPS-WST(0,1,2) scores for both phases was detected in most of all of the groups, except SR141716A at 5 mg/kg. Levels of endogenous cannabinoids (anandamide, palmitoylethanolamide, 2-arachidonylglycerol) were measured from rats hind-paw skin 1 h after s.c. injection of 0.9% saline (100 microl), 1% (50 microl), 2. 5% (50 microl) and 5% (100 microl) formalin. The concentration of endocannabinoids did not differ between control and formalin-induced inflammation groups. The activity of anandamide amidohydrolase in hind-paw skin also did not change after treatment with formalin. In conclusion, cannabinoid antagonists do not enhance formalin-evoked pain behaviour. These results suggest that, in this model, endogenous cannabinoids do not tonically attenuate inflammatory hyperalgesia.     

Antinociceptive activity of Tilia americana var. mexicana inflorescences and quercetin in the formalin test and in an arthritic pain model in rats

Tilia species are well known around the world for their properties in traditional medicine. Antinociceptive activity of hexane, methanol and aqueous extracts from Tilia americana var. mexicana inflorescences was evaluated in the pain-induced functional impairment model in rats (PIFIR). A preliminar 300 mg/kg dosage of aqueous extracts i.p., but not the same dose of methanol or hexane extract, produced an antinociceptive response in rats similar to that of tramadol (17.8 mg/kg i.p.). A dose-response curve from aqueous extract allowed the determination of ED₅₀ = 364.97 mg/kg in comparison to ED₅₀ = 10.35 mg/kg for tramadol in this model. A previous HPLC-DAD analysis corroborated by an HPLC-MS technique in this study demonstrated the flavonoid composition in this Tilia aqueous extract revealing the presence of glycosides mainly derived from quercetin. Thus, Tilia aqueous extract and quercetin were tested at 30 and/or 100 mg/kg dosages i.p. in the PIFIR and formalin models producing a significant and dose-dependent antinociceptive response resembling that produced by a total and a partial agonist of 5-HT_1A receptors like 8-OH-DPAT (0.1 mg/kg, s.c.) and buspirone (5 mg/kg, i.p.), respectively. In all the treatments, antinociceptive response was inhibited in the presence of WAY 100635 (0.12 mg/kg, i.p.). Our results support the analgesic activity of T. americana var. mexicana inflorescences attributed by folk medicine; they also indicate that quercetin is partly responsible for this pharmacological activity that is likely mediated by serotonin 5-HT_1A receptors.     

The validation of an existing method of scoring the severity of medication administration errors for use in Germany

Objective: The aim of this study was to assess the validity and reliability of an existing method of scoring the severity of medication administration errors, developed in the United Kingdom (UK), for use in Germany.Method: 10 doctors, 10 nurses, and 10 pharmacists from German hospitals were asked to score the potential clinical significance of 49 cases of medication administration errors on a visual analogue scale. Main outcome measure: Generalisability theory was used to determine the minimum number of judges required to obtain a reliable mean score. Validity was assessed by comparing the mean scores given by the judges to the known outcome of the errors for a subset of the cases. German results were compared to original UK data.Results: The scores of 27 judges could be used (nine from each profession). At least three health professionals, one from each profession, were required to achieve a generalisability coefficient of 0.86, indicating acceptable reliability. The mean scores were found to be valid indicators of the potential severity of the errors. German scores were significantly below UK scores for the same cases. Conclusion: The mean score calculated from scores given by one doctor, one nurse and one pharmacist from the population of German health professionals was a valid and reliable measure of the potential clinical significance of medication administration errors. That German health professionals see cases as less dangerous than their UK counterparts is worthy of further investigation.     

Microinjection of histamine into the dentate gyrus produces antinociception in the formalin test in rats

The present study was aimed to investigate the effects of microinjection of histamine, chlorpheniramine (a histamine H₁ receptor antagonist), ranitidine (a histamine H₂ receptor antagonist) and thioperamide (a histamine H₃ receptor antagonist) into the dentate gyrus on the formalin-induced pain. A biphasic pattern (first phase: 0-5 min and second phase: 15-60 min) in nociceptive responses was induced after subcutaneous injection of formalin (50 μl, 2.5%) into the ventral surface of the right hind paw. Microinjection of histamine (1 and 2 μg) into the dentate gyrus decreased the intensity of nociceptive responses. Intra-dentate gyrus microinjection of chlorpheniramine and ranitidine at the same doses of 1 and 4 μg had no effects, whereas thioperamide at a dose of 4 μg suppressed both phases of formalin-induced pain. Pretreatments with chlorpheniramine and ranitidine at the same dose of 4 μg prevented histamine (2 μg)-induced antinociception, while thioperamide (4 μg) increased histamine (2 μg)-induced antinociception. These results indicated that activation of brain neuronal histamine at the levels of dentate gyrus produced antinociception. The post-synaptic H₁, H₂ receptors and pre-synaptic H₃ receptors of histamine may be involved in the histamine-induced antinociception at the level of the dentate gyrus.     

Proglumide enhances the antinociceptive effect of cyclooxygenase inhibitors in diabetic rats in the formalin test

The purpose of this study was to assess the effect of the non-selective cholecystokinin receptor antagonist proglumide on the antinociceptive activity of ketorolac and meloxicam in non-diabetic and diabetic rats. Streptozotocin (60 mg/kg) injection caused hyperglycemia which was maintained for 2 weeks. Formalin-evoked flinching was increased in diabetic rats as compared to non-diabetic rats. Local peripheral ipsilateral, but not contralateral, administration of ketorolac and meloxicam produced antinociception in non-diabetic and diabetic rats. However, the antinociceptive effect of both drugs was significantly reduced in diabetic animals. Proglumide was ineffective by itself and it did not affect the antinociception induced by the cyclooxygenase inhibitors in non-diabetic rats. Contrariwise, proglumide reduced formalin-induced nociception and it increased ketorolac- or meloxicam-induced antinociception in diabetic rats. These results suggest that peripheral cholecystokinin plays an important role in diabetes-induced sensitization as well as in the reduction of the antinociceptive effects of ketorolac and meloxicam in diabetic rats. The combination of cholecystokinin receptor antagonists and ketorolac or meloxicam may be a useful strategy to reduce nociception in diabetic patients.     

Pharmacological validation of ritanserin and risperidone in the drug discrimination test procedure in the rat

The results presented here indicate that 0.16 mg/kg LSD, 2.50 mg/kg 8-OHDPAT, 1.25 mg/kg d-amphetamine, 10.00 mg/kg cocaine, 40.00 mg/kg chlordiazepoxide, 2.50 mg/kg xylazine, and 0.04 mg/kg fentanyl can be used as discriminative stimuli in a two-lever drug discrimination test procedure in the rat. The central 5-HT₂ antagonist ritanserin and the 5-HT₂ and catecholamine (CA)-antagonist risperidone were tested for stimulus generalization with, and possible antagonism of, the discriminative stimulus properties of the various training drugs. With both drugs at all doses tested, no stimulus generalization was observed with any of the training drugs. Ritanserin completely blocked the discriminative stimulus properties of LSD at 40.00 mg/kg but was, at doses up to 40.00 mg/kg, unable to block the discriminative stimulus properties of any of the other training drugs. Risperidone completely antagonized the stimulus properties of LSD and d-amphetamine, partially blocked cocaine, and possessed minor effects on 8-OHDPAT and fentanyl. Whereas ritanserin was almost without any effects or response rate, risperidone mostly reduced response rate at doses starting between 0.16 and 0.63 mg/kg. However, the complete antagonism of the LSD and d-amphetamine was observed without effects on response rate. Globally, these results confirm ritanserin to be a selective 5-HT₂ antagonist without any effects on conditioned behaviour. Risperidone was found to be a potent 5-HT₂ and DA antagonist, affecting conditioned behaviour by interfering with response rate and with the response-reinforcement contingency.     

Effects of formalin pain on hippocampal c-Fos expression in male and female rats

Immediate early genes are crucial intermediates in a cascade linking membrane stimulation to long-term alterations of neuronal activity. In the present experiment, we performed immunohistochemistry for c-Fos to determine the effects of persistent pain on cells of the hippocampus of male and female rats. Animals were subcutaneously injected with formalin (50 microl, 10%) and perfused: 2 h later, time 2; 24 h later, time 24; 24 h later after 20 min of the open-field test, time 24/OF. Controls were left undisturbed. In control, c-Fos was higher in females than in males in all hippocampal fields. In males at time 2, formalin increased c-Fos in the dentate gyrus (DG) and CA3 fields; at time 24, c-Fos returned to the control level; at time 24/OF, c-Fos was higher than in control in the DG, but not in the other fields. In the formalin-treated females at time 2 and at time 24, c-Fos levels were lower, or tended to be lower, than in control in all hippocampal fields; at time 24/OF, c-Fos levels in the DG were higher than in control and in males. In conclusion, persistent pain had different effects on c-Fos in the hippocampal subfields, depending on the time after treatment and the sex of the subject.     

Pharmacological evidence for the activation of Ca2+-activated K+ channels by meloxicam in the formalin test

The possible participation of K+ channels in the antinociceptive action of meloxicam was assessed in the 1% formalin test. Local peripheral administration of meloxicam produced a dose-dependent antinociception only during the second phase of the formalin test. K+ channel blockers alone did not modify formalin-induced nociceptive behavior. However, local peripheral pretreatment of the paw with charybdotoxin and apamin (large- and small-conductance Ca2+-activated K+ channel inhibitors, respectively), 4-aminopyridine and tetraethylammonium (non-selective voltage-dependent K+ channel inhibitors), but not glibenclamide or tolbutamide (ATP-sensitive K+ channel inhibitors), dose-dependently prevented meloxicam-induced antinociception. It is concluded that meloxicam could open large- and small-conductance Ca2+-activated K+ channels, but not ATP-sensitive K+ channels, in order to produce its peripheral antinociceptive effect in the formalin test. The participation of voltage-dependent K+ channels was also suggested, but since non-selective inhibitors were used the data await further confirmation.     

Noradrenergic and opioidergic influences on the antinociceptive effect of clomipramine in the formalin test in rats

Although tricyclic antidepressant are especially useful in the treatment of chronic pain conditions, most of the work about its mechanism of action has been made on acute pain tests. The present study was aimed at studying the role played by noradrenergic and opioidergic influences on the antinociceptive activity of subchronically administered clomipramine in the formalin test (a tonic pain model) in rats. Clomipramine produced antinociception after 7 days, administration (2.5 mg/kg/day), an effect equivalent to that obtained by acute morphine (5 mg/kg). The antinociceptive effect of clomipramine was inhibited by the following: nonspecific blocking of alpha₁-and alpha₂-adrenoceptors by phentolamine, specific blocking of alpha₁-adrenoceptors by prazosin; stimulation of alpha₂ receptors by clonidine; and blocking of the opioid receptors by naloxone. Blocking the alpha₂-receptors with yohimbine did not antagonize the effect of clomipramine. These results suggest that clomipramine produces antinociception in this test, partly via the participation of the endogenous opioid system and partly by further activating or potentiating previously activated noradrenergic pathways which are involved in the control of pain information.     

Yohimbine produces antinociception in the formalin test in rats: involvement of serotonin1A receptors

Rationale: Previous studies have suggested that the α₂-adrenergic receptor antagonist yohimbine produced antinociceptive effects in the formalin test in rats. However, yohimbine is also an agonist at serotonin (5-HT)_1A receptors, suggesting the possibility that the antinociceptive effects of yohimbine might be mediated via these receptors. Objective: The purpose of the present studies was to evaluate the potential role of 5-HT_1A receptors in mediating the antinociceptive effects of yohimbine. Methods: The antinociceptive effects of yohimbine were evaluated using the formalin test in rats. Results: Yohimbine (2.5–10 mg/kg s.c.) produced dose-related antinociception during both phase I and phase II of the formalin test, and was approximately equipotent and equiefficacious to morphine. The selective 5-HT_1A receptor antagonist WAY 100,635 (0.03–3.0 mg/kg s.c.) produced a partial reversal of yohimbine. In comparison, the selective 5-HT_1A receptor agonist (±)8-hydroxy- dipropylaminotetralin HBr (8OH-DPAT; 1.0 mg/kg s.c.) also produced a dose-related antinociception in the formalin test, although 8OH-DPAT was completely reversed by WAY 100,635 (3.0 mg/kg s.c.). The antinociceptive effects of yohimbine were not antagonized by the 5-HT_1B/1D antagonist GR 127935 (1.0 mg/kg and 3.0 mg/kg s.c.), the 5-HT₂ antagonist LY53857 (1.0 mg/kg s.c.), or the 5-HT₃ antagonist zatosetron (3.0 mg/kg s.c.). Conclusions: The present results demonstrate that yohimbine produces a dose-related antinociception in the formalin test in rats which is mediated in part by the agonistic actions at 5-HT_1A receptors. Received: 10 September 1999 / Final version: 5 November 1999     

Chronic administration of desipramine and zimelidine changes the behavioural response in the formalin test in rats.

In studies of the effect on nociception of chronic administration of antidepressants, the stress of the injections may influence the results. In this experiment, desipramine or zimelidine were administered in the drinking water of rats, in a concentration yielding a dose of approximately 8 mg/kg/24 hr. Desipramine, given both for a short time (24 hr) and chronically (14 days), induced antinociception in the increasing temperature hot-plate test; zimelidine did not significantly influence the results of this test. In the tail-flick test, neither short-term nor chronic administration of these antidepressants had any effect on nociception, when correction was made for the changes in the temperature of the tail skin. In the formalin test, nine behavioural categories were scored for 1 hr and the data were treated statistically, using a multivariate analysis. Chronic administration of desipramine increased nociceptive behaviour during the first 10 min of the test. Desipramine and, to a lesser extent, zimelidine, changed the response in the late phase (10-60 min), showing less focussed pain-related behaviour (jerks and shaking, licking and biting of the injected paw) and more non-focussed pain-related behaviour (activity states with elevation or protection of the injected paw). It was concluded that desipramine is antinociceptive in the increasing temperature hot-plate test. Desipramine and zimelidine, administered chronically, modify the late phase of the formalin test towards less focussed pain-related behaviour, suggesting an antinociceptive effect. Multivariate analysis of the data of the formalin test seemed to be of value for the interpretation of the data.     

Sildenafil increases diclofenac antinociception in the formalin test

The antinociceptive activity of an inhibitor of phosphodiesterase 5, alone or combined with diclofenac, was assessed in the formalin test. Local administration of diclofenac produced a significant antinociception in both phases of the formalin test in female Wistar rats. In contrast, 1-[4-ethoxy-3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [3,4-d]pyrimidin-5-yl)phenylsulfonyl]-4-methyl piperazine (sildenafil, an inhibitor of phosphodiesterase 5) produced significant antinociception, only during the second phase of the formalin test. Non-effective doses of sildenafil (25-100 microg/paw) significantly increased the antinociceptive effect of an inactive dose of diclofenac (25 microg) in both phases of the test. The antinociception produced by the drugs alone or the combination was due to a local action, as its administration in the contralateral paw was ineffective. Since sildenafil is a potent and selective inhibitor of phosphodiesterase 5, our results suggest that this drug produced its antinociceptive activity, and increased that of diclofenac, probably through the inhibition of cyclic GMP degradation.     

Comparison of the effects of anticonvulsant drugs with diverse mechanisms of action in the formalin test in rats

The purpose of the present studies was to compare anticonvulsant drugs with diverse mechanisms of action in a persistent pain model, the formalin test. In addition, the anticonvulsant effects of the compounds were determined in the threshold electroshock tonic seizure test and the 6-Hz limbic seizure test. The effects of the compounds were also determined on locomotor activity. Carbamazepine, oxcarbazepine, lamotrigine, gabapentin and ethosuximide all produced statistically significant analgesic effects in the formalin test whereas phenytoin, topiramate, zonisamide, phenobarbital, tiagabine, valproate and levetiracetam did not. All compounds were anticonvulsant. In addition, morphine and phenobarbital increased locomotor activity while ethosuximide had no effect and all other compounds decreased locomotor activity. For those compounds that were analgesic, the doses required to produce analgesia were larger in magnitude than the anticonvulsant ED50 values in the threshold electroshock and 6-Hz tests, as well as larger than doses that altered locomotor activity. The present results demonstrate that the anticonvulsant and analgesic effects of clinically used antiepileptic drugs do not necessarily correlate and therefore suggest that the anticonvulsant and analgesic efficacy of these drugs may be due to different pharmacologic mechanisms.     

Effects of intraplantar morphine in the mouse formalin test

We studied the effects of intraplantar morphine in the formalin test in mice. Intraplantarly administered morphine (30 - 300 microg) induced analgesic effects at lower doses than intraperitoneally administered morphine. Following the administration of [3H]morphine, the % of radioactivity present in brain was the same by either route. In contrast, higher radioactivity values appeared in the injected paw in those mice intraplantarly injected. Since local morphine induces analgesia at doses lower than the intraperitoneally administered drug, especially in the second phase of the test, and the access to brain is undistinguishable, we propose that local morphine enhances central opiate analgesia in the formalin test in mice.     

Antinociceptive effect of gabapentin in the formalin test.

Gabapentin is an adjunctive drug for the treatment of resistant partial seizures. The antinociceptive effect of this drug was assessed by using the formalin pain test in rats. Although low dose gabapentin (10 mg/kg s.c.) was unable to alter pain scores, higher doses (30 and 90 mg/kg s.c.) significantly reduced them. Results suggest that gabapentin may modulate the pain perception produced by chemical irritants like formalin in rats.     

Development and validation of an automated solid phase extraction and liquid chromatographic method for the determination of piperaquine in urine

A sensitive and specific bioanalytical method for determination of piperaquine in urine by automated solid-phase extraction (SPE) and liquid chromatography (LC) has been developed and validated. Buffered urine samples (containing internal standard) were loaded onto mixed phase (cation-exchange and octylsilica) SPE columns using an ASPEC XL SPE robot. Chromatographic separation was achieved on a Chromolith Performance RP-18e (100 mm x 4.6 mm I.D.) LC column with phosphate buffer (pH 2.5; 0.1 mol/L)-acetonitrile (92:8, v/v). Piperaquine was analysed at a flow rate of 3 mL/min with UV detection at 347 nm. A linear regression model on log-log transformed data was used for quantification. Within-day precision for piperaquine was 1.3% at 5000 ng/mL and 6.6% at 50 ng/mL. Between-day precision for piperaquine was 3.7% at 5000 ng/mL and 7.2% at 50 ng/mL. Total-assay precision for piperaquine over 4 days using five replicates each day (n = 20) was 4.0%, 5.2% and 9.8% at 5000, 500 and 50 ng/mL, respectively. The lower limit of quantification (LLOQ) was set to 3 ng/mL using 1 mL of urine, which could be lowered to 0.33 ng/mL when using 9 mL of urine and an increased injection volume.     

A national validation study of the acute-toxic-class method—an alternative to the LD50 test

In a national colloborative study an alternative to the classical LD₅₀ test — the acute-toxic-class method — was validated. With this testing procedure mortality ranges are determined between defined dose levels that are used for classification and labelling in the European Community. The results were compared with LD₅₀ data obtained from the literature which were categorized according to the defined dose levels. The results of this collaborative study have shown that the acute-toxic-class method allows allocation to the toxicity classes of very toxic, toxic, harmful and unclassified in the same manner as on the basis of the classical LD₅₀ tests. The acute-toxic-class method uses fewer animals and subjects fewer animals to pain and distress than the LD₅₀ test and yields the same information on toxic signs in the treated animals. Identical classifications were obtained by the six participating laboratories in 86% of the tests. This demonstrates that the acute-toxic-class method results in excellent reproducibility in comparison to the classical LD₅₀ test and that this new method is a reliable alternative to the LD₅₀ test.     

Apparent lack of tolerance in the formalin test suggests different mechanisms for morphine analgesia in different types of pain

Tolerance to morphine analgesia was examined using the Formalin test in which pain lasting about 2 hrs associated with minor tissue injury is produced by subcutaneous injection of dilute Formalin. To distinguish behavioral from pharmacological tolerance, different groups of rats received their daily morphine injection (7 mg/kg) in the test environment or in their home environment for 5 days. Another group of rats was given morphine for 15 days in the home cage followed by 5 days in the test environment. None of the morphine injected groups differed from saline injected control groups in the amount of analgesia. These findings add to previous evidence that the Formalin test measures a type of pain which is different from that assessed in withdrawal reflex tests, and which more closely resembles clinical pain in man. Moreover, the fact that analgesia in the Formalin test shows little tolerance while analgesia in withdrawal tests shows rapid tolerance suggests that the underlying neural mechanisms are different.