Comparison of the antinociceptive effect of celecoxib, diclofenac and resveratrol in the formalin test

The peripheral antinociceptive effect of the selective COX-2 inhibitor celecoxib in the formalin-induced inflammatory pain was compared with that of resveratrol (COX-1 inhibitor) and diclofenac (non-selective COX inhibitor). Rats received local pretreatment with saline, celecoxib, diclofenac or resveratrol followed by 50 microl of either 1% or 5% formalin. Peripheral administration of celecoxib did not produce antinociception at either formalin concentration. In contrast, diclofenac and resveratrol produced a dose-dependent antinociceptive effect in the second phase of both 1% and 5% formalin test. The peripheral antinociception produced by diclofenac or resveratrol was due to a local action, as drug administration in the contralateral paw was ineffective. Results indicate that the selective COX-2 inhibitor celecoxib does not produce peripheral antinociception in formalin-induced inflammatory pain. In contrast, selective COX-1 and non-selective COX inhibitors (resveratrol and diclofenac, respectively) are effective drugs in this model of pain.     

Long-lasting antinociceptive effect of DAMGO chloromethyl ketone in rats

Previously, the opioid peptide Tyr-D-Ala-Gly-(NMe)Phe-CH2Cl (DAMCK) has been shown to bind irreversibly to mu opioid receptors in vitro. In the present work, the antinociceptive effect of DAMCK has been evaluated. Rats treated systemically with DAMCK (1-100 pg/kg) displayed a dose-dependent increase in tail-flick analgesia that peaked by 15 min, then stayed about the same until 60 min, followed by some decrease over time. Higher doses of DAMCK (10 ng/kg-100 microg/kg) produced a near-maximal antinociceptive effect that remained stable for 4 h. Significant antinociception was still detected 8 h, but not 24 h postinjection. In comparison, the parent peptide DAMGO (Tyr-D-Ala-Gly-(NMe)Phe-Gly-ol) reached maximal effect by about 30 min, followed by a rapid cessation of its antinociceptive response. Naloxone administered before DAMCK antagonized the antinociceptive response of DAMCK, indicating that it was mediated via opioid receptors. Naloxone administered 45 min after DAMCK attenuated the tail-flick response to some extent, but a substantial part (40-60% depending on the peptide concentration and evaluation time) remained unaffected. Central administration of DAMCK also elicited time- and concentration-dependent, profound, opioid receptor mediated, apparently irreversible antinociception.     

The effect of glucocorticoids on plasma fibronectin levels in normal and arthritic rats

In vivo studies with normal and adjuvant-induced arthritic rats were undertaken in order to measure the effects of glucocorticoids on paw inflammation and plasma fibronectin (Fn) levels. Dexamethasone, methylprednisolone, and corticosterone all enhanced plasma Fn levels in normal animals. All drugs also significantly decreased inflammation in arthritic rats as measured by paw swelling. Of the three glucocorticoids, only corticosterone did not significantly enhance Fn levels in arthritic rats, possibly due to its lesser potency and narrow therapeutic window.     

The antinociceptive effect of tramadol on a model of neuropathic pain in rats

The antinociceptive activity of tramadol was investigated on the vocalization threshold to paw pressure in a rat model of unilateral mononeuropathy produced by loose ligatures around the common sciatic nerve. Despite the analgesic activity of tramadol was clearly established in motor and sensory responses of the nociceptive system in rats, the effect of this atypical opioid on experimental neuropathic pain models is not investigated. The intraperitoneally injected tramadol (2.5, 5 and 10 mg/kg) produced a potent and dose-dependent antinociceptive effect on both lesioned and non-lesioned hind paws. However, the analgesic effect on the lesioned paw was significantly more potent than the non-lesioned paw. This effect was partially antagonized by intraperitoneally administered naloxone (0.1 mg/kg) suggesting an additional non-opioid mechanism. Our results suggest that tramadol may be useful for the alleviation of some symptoms in peripheral neuropathic conditions     

Chemopreventive effect of diclofenac on mammary carcinogenesis in Sprague-Dawley rats

Chemopreventive effect of non-steroidal antiinflammatory drugs (NSAIDs) in mammary carcinogenesis was reported in several studies. In this study, the effect of a nonselective cyclooxygenase inhibitor diclofenac (DICLO) in the prevention of N-methyl-N-nitrosourea (NMU)-induced mammary carcinogenesis in Sprague-Dawley female rats was evaluated. NMU was administered to animals intraperitoneally in two doses of 50 mg kg^?1 b.w. within postnatal days 42-48. In experiment A (short-term administration), DICLO was administrated intramuscularly (5 mg kg^?1 b.w.) every other day, starting 3 days before and for subsequent 25 days after first NMU injection. In experiment B (long-term administration), DICLO was administered in tap water (0.01 mg ml^?1) continually, starting 7 days before and for subsequent 22 weeks after first NMU dose. The study was terminated 22 weeks after the first dose of NMU in both experiments. After DICLO treatment, tumor frequency per group was reduced in both variants of drug administration: in experiment A by 38% and in experiment B by 39.5%. Moreover, DICLO decreased tumor incidence by 11.5% and delayed tumor latency by 14 days in experiment B. In our preventive-curative experiments DICLO decreased some parameters of NMU-induced rat mammary carcinogenesis, mainly the tumor frequency.     

The effect of pentobarbital on the antinociceptive action of morphine in morphine-tolerant and non-tolerant rats

The interaction of sodium pentobarbital with morphine sulfate in both morphine-tolerant and non-tolerant rats was investigated using the tail-compression test for analgesia. Male Sprague-Dawley rats (300–350 g) were given pentobarbital (4, 8, or 16 mg/kg) 5 min before morphine (2, 4, 6, or 8 mg/kg). Control animals received two saline injections, or pentobarbital plus saline, or saline plus morphine. All injections were subcutaneous. Prior to the first injection, a baseline nociceptive threshold was determined for each rat by applying a modified micrometer to its tail and increasing the pressure until a squeak was elicited. Test readings were taken every half-hour for 2 hr beginning 30 min after the second injection. For the chronic studies, animals were first made tolerant to morphine by the administration of the narcotic twice a day for 3 days, increasing the dose from 10 to 50 mg/kg/injection. Identical testing procedures were then followed with these rats except that the test dose of morphine given on day 4 was in the range 8–128 mg/kg. It was found that Na pentobarbital, in the subanesthetic doses used, had neither antinociceptive nor hyperalgesic properties. Furthermore, the barbiturate had no effect on the antinociceptive action of morphine in either morphine-tolerant or non-tolerant rats.     

The urea cycle in the liver of arthritic rats

The urea cycle in the liver of adjuvant-induced arthritic rats was investigated using the isolated perfused liver. Urea production in livers from arthritic rats was decreased during substrate-free perfusion and also in the presence of the following substrates: alanine, alanine + ornithine, ammonia, ammonia + lactate, ammonia + pyruvate and glutamine but increased when arginine and citrulline + aspartate were the substrates. No differences were found with ammonia + aspartate, ammonia + aspartate + glutamate, aspartate, aspartate + glutamate and citrulline. Ammonia consumption was smaller in the arthritic condition when the substance was infused together with lactate or pyruvate but higher when the substance was simultaneously infused with aspartate or aspartate + glutamate. Glucose production tended to correlate with the smaller or higher rates of urea synthesis. Blood urea was higher in arthritic rats (+25.6%), but blood ammonia was lower (–32.2%). Critical for the synthesis of urea from various substrates in arthritic rats seems to be the availability of aspartate, whose production in the liver is probably limited by both the reduced gluconeogenesis and aminotransferase activities. This is indicated by urea synthesis which was never inferior in the arthritic condition when aspartate was exogenously supplied, being even higher when both aspartate and citrulline were simultaneously present. Possibly, the liver of arthritic rats has a different substrate supply of nitrogenous compounds. This could be in the form of different concentrations of aspartate or other aminoacids such as citrulline or arginine (from the kidneys) which allow higher rates of hepatic ureogenesis.     

The uncoupling effect of the nonsteroidal anti-inflammatory drug nimesulide in liver mitochondria from adjuvant-induced arthritic rats

The aim of the present study was to evaluate the changes caused by adjuvant-induced arthritis in liver mitochondria and to investigate the effects of the nonsteroidal anti-inflammatory drug nimesulide. The main alterations observed in liver mitochondria from arthritic rats were: higher rates of state IV and state III respiration with beta-hydroxybutyrate as substrate; reduced respiratory control ratio and impaired capacity for swelling dependent on beta-hydroxybutyrate oxidation. No alterations were found in the activities of NADH oxidase and ATPase. Nimesulide produced: (1) stimulation of state IV respiration; (2) decrease in the ADP/O ratio and in the respiratory control ratio; (3) stimulation of ATPase activity of intact mitochondria; (4) inhibition of swelling driven by the oxidation of beta-hydroxybutyrate; (5) induction of passive swelling due to NH(3)/NH(4)+ redistribution. The activity of NADH oxidase was insensitive to nimesulide. Mitochondria from arthritic rats showed higher sensitivity to nimesulide regarding respiratory activity. The results of this work allow us to conclude that adjuvant-induced arthritis leads to quantitative changes in some mitochondrial functions and in the sensitivity to nimesulide. Direct evidence that nimesulide acts as an uncoupler was also presented. Since nimesulide was active in liver mitochondria at therapeutic levels, the impairment of energy metabolism could lead to disturbances in the liver responses to inflammation, a fact that should be considered in therapeutic intervention.     

Gluconeogenesis in the liver of arthritic rats

The gluconeogenic response in the liver from rats with chronic arthritis to various substrates and the effects of glucagon were investigated. The experimental technique used was the isolated liver perfusion. Hepatic gluconeogenesis in arthritic rats was generally lower than in normal rats. The difference between normal and arthritic rats depended on the gluconeogenic substrate. In the absence of glucagon the following sequence of decreasing differences was found: alanine (-71.8 per cent) reverse similarglutamine (-71.7 per cent)>pyruvate (-60 per cent)>lactate+pyruvate (-44.9 per cent)>xylitol (n.s.=non-significant) reverse similarglycerol (n.s.). For most substrates glucagon increased hepatic gluconeogenesis in both normal and arthritic rats. The difference between normal and arthritic rats, however, tended to diminish, as revealed by the data of the following sequence: alanine (-48.9 per cent) reverse similarpyruvate (-47.6 per cent)>glutamine (-33.8 per cent)>glycerol (n.s.) reverse similarlactate+pyruvate (n.s.) reverse similarxylitol (n.s.). The causes for the reduced hepatic gluconeogenesis in arthritic rats are probably related to: (a) lower activities of key enzymes catalyzing most probably steps preceding phosphoenolpyruvate (e.g. phosphoenolpyruvate carboxykinase, pyruvate carboxylase, etc. ); (b) a reduced availability of reducing equivalents in the cytosol; (c) specific differences in the situations induced by hormones or by the individual substrates. Since glycaemia is almost normal in chronically arthritic rats, it seems that lower gluconeogenesis is actually adapted to the specific needs of these animals.     

Analysis of the mechanisms underlying the antinociceptive effect of epicatechin in diabetic rats

Aims

The purpose of this study was to investigate the antinociceptive effect of epicatechin as well as the possible mechanisms of action in diabetic rats.

Main methods

Rats were injected with streptozotocin to produce hyperglycemia. The formalin test was used to assess the nociceptive activity.

Key findings

Acute pre-treatment with epicatechin (0.03–30 mg/kg, i.p.) prevented formalin-induced nociception in diabetic rats. Furthermore, daily or every other day treatment for 2 weeks with epicatechin (0.03–30 mg/kg, i.p.) also prevented formalin-induced nociception in diabetic rats. Acute epicatechin-induced antinociception was prevented by l-NAME (N^ω-nitro-l-arginine methyl ester hydrochloride, 1–10 mg/kg, non-selective nitric oxide synthesis inhibitor), 7-nitroindazole (0.1–1 mg/kg, selective neuronal nitric oxide synthesis inhibitor), ODQ (1H-(1,2,4)-oxadiazolo(4,2-a)quinoxalin-1-one, 0.2–2 mg/kg, guanylyl cyclase inhibitor) or glibenclamide (1–10 mg/kg, ATP-sensitive K⁺ channel blocker). Moreover, epicatechin (3 mg/kg)-induced antinociception was fully prevented by methiothepin (0.1–1 mg/kg, serotonergic receptor antagonist), WAY-100635 (0.03–0.3 mg/kg, selective 5-HT_1A receptor antagonist) or SB-224289 (0.03–0.3 mg/kg, selective 5-HT_1B receptor antagonist). In contrast, BRL-15572 (0.03–0.3 mg/kg, selective 5-HT_1D receptor antagonist) only slightly prevented the antinociceptive effect of epicatechin. Naloxone (0.1–1 mg/kg, opioid antagonist) did not modify epicatechin's effect.

Significance

Data suggest the involvement of the nitric oxide–cyclic GMP–K⁺ channel pathway as well as activation of 5-HT_1A and 5HT_1B, and at a lesser extent, 5-HT_1D, but not opioid, receptors in the antinociceptive effect of epicatechin in diabetic rats. Our data suggest that acute or chronic treatment with epicatechin may prove to be effective to treat nociceptive hypersensitivity in diabetic patients.     

The application of direct immunofluorescence to intraoperative neurosurgical diagnosis

A diagnostic problem can occur at the time of intraoperative consultation of neurosurgical tumors as to whether the tumor is of neuroectodermal origin or whether it represents an epithelial metastasis from another site. Intraoperative diagnoses based on hematoxylin and eosin stained frozen sections are often later confirmed by immunocytochemical analysis of formalin-fixed, paraffin-embedded tissue sections that are not available at the time of surgery. The objective of the current study was to demonstrate that the application of direct immunofluorescence to the intraoperative diagnosis of neurosurgical tumors would provide unequivocal, and nearly immediate results. This report describes a new application of an existing technique for an optimized, rapid procedure utilizing direct immunocytochemistry with fluorescence-labeled primary antibodies to analyze surgical biopsies intraoperatively. The examination of five neurosurgical biopsies established a neuroectodermal origin of three tumors via immunolabeling for glial fibrillary acidic protein (GFAP) and lack of labeling with keratin markers, whereas several metastatic lung carcinomas were identified by immunostaining for keratin, but not GFAP, markers. The results of the direct immunolabeling method were unequivocal and required only minutes. The same diagnoses were confirmed by standard immunocytochemical labeling of formalin-fixed, paraffin-embedded sections, though it required several days to obtain the results. Direct immunofluorescence using fluorescently conjugated primary antibodies is a practical and rapid method for deciding whether a neurosurgical tumor is a primary glial or an epithelial metastatic tumor in origin. It is the first reported application of the technique for this aspect of rapid neurosurgical diagnosis.     

The antinociceptive efficacy of buprenorphine administered through the drinking water of rats

Postoperative pain management in laboratory animals is important for animal welfare and required under law in many countries. Frequent injection of analgesics to rodents after surgery is stressful for the animals and labour-intensive for animal care personnel. An alternative dosing scheme such as administration of analgesics in the drinking water would be desirable. However, the efficacy of a chronic oral analgesic treatment via this route has not yet been documented. This study investigated the antinociceptive efficacy of buprenorphine administered ad libitum via the drinking water of laboratory rats. The antinociceptive efficacy of buprenorphine in drinking water was compared with repeated subcutaneous injections. A comparison was also made between buprenorphine in drinking water and the combination of one single subcutaneous injection of buprenorphine followed by buprenorphine in drinking water. Antinociception was assessed by use of an analgesiometric model measuring the rats' latency time to withdrawal from a noxious heat stimulus applied to the plantar surface of the paw. Results revealed that buprenorphine in drinking water (0.056 mg/mL) induced significant increases in paw withdrawal latency times during a three-day period of administration with a maximal effect at 39 h after the start of buprenorphine administration. One single injection of buprenorphine (0.1 mg/kg s.c.) followed by buprenorphine in the drinking water (0.056 mg/mL) induced an earlier onset of antinociception than buprenorphine in drinking water alone. In contrast, buprenorphine (0.1 mg/kg s.c.) injected every 8 h over a period of three days did not result in significant increases in paw withdrawal latency times. In conclusion, our results suggest that one single subcutaneous injection of buprenorphine followed by buprenorphine in drinking water may be a viable treatment option for the relief of pain in laboratory rats, but at the doses used in this study in pain-free rats it was associated with a decrease in water intake and some behavioural changes.     

Synergistic effect of the interaction between curcumin and diclofenac on the formalin test in rats

The association of non-steroidal anti-inflammatory drugs with certain plant extracts can increase antinociceptive activity, permitting the use of lower doses and thus limiting side effects. Therefore, the aim objective of the current study was to examine the effects of curcumin on the nociception and pharmacokinetics of diclofenac in rats. Antinociception was assessed using the formalin test. Diluted formalin was injected subcutaneously into the dorsal surface of the right hind paw. Nociceptive behavior was quantified as the number of flinches of the injected paw during 60 min after injection, and a reduction in formalin-induced flinching was interpreted as an antinociceptive response. Rats were treated with oral diclofenac (1–31 mg/kg), curcumin (3.1–100 mg/kg) or the diclofenac–curcumin combination (2.4–38.4 mg/kg). To determine the possibility of a pharmacokinetic interaction, the oral bioavailability of diclofenac (10 mg/kg) was studied in presence and the absence of curcumin (31 mg/kg). Diclofenac, curcumin, or diclofenac–curcumin combination produced an antinociceptive effect on the formalin test. ED₃₀ values were estimated for the individual drugs, and an isobologram was constructed. The derived theoretical ED₃₀ for the antinociceptive effect (19.2 mg/kg) was significantly different from the observed experimental ED₃₀ value (9.8 mg/kg); hence, the interaction between diclofenac and curcumin that mediates the antinociceptive effect was synergistic. Notwithstanding, the interaction does not appear to involve pharmacokinetic mechanisms, as oral curcumin failed to produce any significant alteration in oral diclofenac bioavailability. Data suggest that the diclofenac–curcumin combination can interact at the systemic level and may have therapeutic advantages for the clinical treatment of inflammatory pain.     

An antinociceptive effect of the intraperitoneal injection of nifedipine in rats,measured by tail-flick test

The tail-flick (TF) technique was used to assess the antinociceptive properties of nifedipine (NIF) given intraperitoneally (i.p.). First, the most suitable intensity of the noxious stimulus (temperature of the bulb) has been ascertained and used in the main study. Male Sprague-Dawley rats received NIF, dissolved in dimethyl sulfoxide (DMSO) at the doses of 0.0, 0.5, 2, 5, 10 and 15 mg/kg, or control with no injection. For the main study, the noxious stimulus was limited to 15 sec (cut-off time) and TF latencies were recorded up to 120 min. The antinociceptive response was expressed as the area under the curve for each rat and analyzed by one-way ANOVA. The antinociceptive response to the lower doses of NIF (0.5 and 2 mg/kg) did not differ from control (no injection) and DMSO alone. Significance was found at 5, 10 and 15 mg NIF with no difference among the doses. However, there was an increasing tendency of the mean values from 0.5 to 15mg NIF resulting in a positive correlation. The correlation coefficient was 0.32483 (p=0.015) and regression equation Y=(19.37) × dose + 1320. Our data suggest that spinal mechanisms are involved in NIF-induced antinociception.     

Anti-inflammatory effect of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) in arthritic rats

Chronic arthritis induces hypermetabolism and cachexia. Ghrelin is a gastrointestinal hormone that has been proposed as a treatment to prevent cachexia. The aim of this work was to examine the effect of administration of the ghrelin agonist growth hormone-releasing peptide-2 (GHRP-2) to arthritic rats. Male Wistar rats were injected with Freund's adjuvant, and 15 days later arthritic and control rats were daily injected with GHRP-2 (100 microg/kg) or with saline for 8 days. Arthritis induced an increase in serum ghrelin (P < 0.01) and a decrease in serum concentrations of leptin (P < 0.01), whereas GHRP-2 administration increased serum concentrations of leptin. GHRP-2 increased food intake in control rats but not in arthritic rats. However, in arthritic rats GHRP-2 administration ameliorated the external symptoms of arthritis, as it decreased the arthritis score (10.4 +/- 0.8 vs. 13.42 +/- 0.47, P < 0.01) and the paw volume. In addition, circulating IL-6 and nitrites/nitrates were increased by arthritis, and GHRP-2 treatment decreased the serum IL-6 levels (P < 0.01). To elucidate whether GHRP-2 is able to modulate IL-6 release directly on immune cells, peritoneal macrophage cultures were incubated with GHRP-2 or ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor. Both GHRP-2 (10(-7) M) and ghrelin (10(-7) M) prevented endotoxin-induced IL-6 and decreased nitrite/nitrate release from peritoneal macrophages in vitro. These data suggest that GHRP-2 administration has an anti-inflammatory effect in arthritic rats that seems to be mediated by ghrelin receptors directly on immune cells.     

The release of drugs from iontophoretic pipettes

A theoretical account is given of the relationship between applied current and release of drug ions from an iontophoretic pipette. The Nernst-Planck equation predicts a non-linear relationship except in the region of large ejecting currents. No finite value of retaining current can completely suppress spontaneous diffusional leakage. These conclusions apply to both conical and cylindrical tip geometries. An expression is derived giving release as a function of pulsed charge applied to a conical pipette. This relation is also non-linear. Dose-response curves constructed using charge as a measure of drug ejection should be interpreted with some caution.     

The spinal antinociceptive effect of FR140423 is mediated through kyotorphin receptors

We investigated the antinociceptive effect of a novel anti-inflammatory and analgesic drug, 3-(difluoromethyl)-1-(4-methoxyphenyl)-5-[4-(methylsulfinyl)phenyl]pyraz ole (FR140423), in the tail-pinch test in mice, and evaluated the mechanism of action of FR140423 using L-leucyl-L-arginine (Leu-Arg), a kyotorphin (endogenous Met-enkephalin releaser) receptor antagonist, L-NG-nitroarginine methylester (L-NAME), an inhibitor of nitric oxide (NO) synthase, and methylene blue (MB), an inhibitor of activation of guanylate cyclase. Oral administration of FR140423, at doses of 5-80 mg/kg, produced a dose-dependent antinociceptive effect with an ED50 value of 18 mg/kg. This antinociception was reversed by intrathecal (i.t.) (10 microg/mouse), but not by intracerebroventricular (i.c.v.) (100 microg/mouse), injection of Leu-Arg. Moreover, the antinociceptive effect of i.t. injection of FR140423 with an ED50 value of 3.7 microg/mouse was completely antagonized by co-administered Leu-Arg 10 microg/mouse. However, L-NAME (2000 mg/kg s.c.) and MB (200 mg/kg s.c.) did not antagonize the antinociception of FR140423. These findings suggest that FR140423 plays a role in nociceptive modulation in the spinal cord, being antinociceptive via the kyotorphin-Met-enkephalin pathway but not via the peripheral NO-cyclic GMP pathway.     

Respiratory effects of intrathecal capsaicin in arthritic and non-arthritic rats

The study determined the effects of intrathecal injection of 50 μg of capsaicin on respiration in rats with adjuvant arthritis as well as in control animals. Whole body plethysmographic measurements of steady-state frequency, tidal volume, and minute volume of respiration were made 3 hours and for up to 11 days after intrathecal injection. Capsaicin increased minute volume within 3 hours of its intrathecal injection in control animals. Intrathecal capsaicin also reduced the respiratory response to adjuvant arthritis in the experimental animals; the latter effect was apparent 11 days after injection. This biphasic pattern of capsaicin effects is consistent with a possible role of substance P in the chronic pain which is presumably associated with adjuvant arthritis in the rat.     

Action of local iontophoretic application of acetylcholine and serotonin to neurons of the rabbit superior cervical ganglion

The action of ionotophoretic application of acetylcholine and serotonin (5-hydroxytryptamine) on neurons of the isolated rabbit superior cervical ganglion was investigated by intracellular recording. The soma of neurons in the ganglion was shown to have no muscarinic receptors and to have only nicotinic receptors scattered irregularly over the whole surface of the neuron soma membrane. Acetylcholine has an excitatory action on presynaptic endings. In about half of the neurons of the ganglion the soma was shown to possess serotonin receptors.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 5, pp. 519–524, September–October, 1978.