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.     

MD-354 potentiates the antinociceptive effect of clonidine in the mouse tail-flick but not hot-plate assay

Albeit conflicting, evidence suggests that 5-HT3 receptor partial agonists as well as alpha2NON-A-adrenoceptor agonists might be involved in antinociception. MD-354 (m-chlorophenylguanidine) can be viewed as the first example of a rather selective 5-HT3/alpha2B-adrenergic ligand. In a tail-flick test in mice, subcutaneous administration of MD-354 doses up to 30 mg/kg did not produce antinociception and failed to antagonize the effect of clonidine (ED50=0.5 mg/kg), but a combination of an inactive de of clonidine (0.25 mg/kg) that produced only 13% maximal possible effect (MPE) with an inactive dose of MD-354 (10 mg/kg, MPE=8%) produced an antinociceptive effect (MPE=83%). In the hot-plate assay, neither subcutaneous administration of MD-354 (3 to 30 mg/kg) alone nor in combination with clonidine (ED50=0.8 mg/kg) produced an antinociceptive effect. MD-354 was demonstrated to potentiate the antinociceptive effect of clonidine in the tail-flick assay, but its underlying mechanism remains to be determined.     

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.     

The antinociceptive effect of intrathecal escin in the rat formalin test

This study investigated the antinociceptive effect of intrathecal escin and examined its effect on the formalin-induced activation of c-Fos and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) in the rat spinal cord. Rats were chronically implanted with lumbar intrathecal catheters, and the ability of intrathecal escin to alter nociceptive behaviours in the rat formalin test was examined. The expression of c-Fos and p-p38 MAPK in the dorsal horn of the spinal cord was detected in the control and escin (40μg) groups using immunohistochemical techniques. Intrathecal escin produced a dose-dependent reduction in formalin-evoked flinching behaviour in rats during the second phase; however, no effect was observed in the first phase. In addition, immunohistochemical experiments showed that the expression of c-Fos and p-p38 MAPK in the spinal cord dorsal horn increased after an injection of formalin into the paw. Interestingly, the 40μg dose of intrathecal escin, which was the larger of the two doses that blocked formalin-induced hyperalgaesia, attenuated the formalin-induced increases in c-Fos and p-p38 MAPK in the dorsal horn of the spinal cord. The decrease in pain-related behaviours and c-Fos expression indicated that escin produced antinociceptive effects in the rat formalin test. Although the specific mechanisms of these effects were not investigated, the reduction in p-p38 MAPK in the dorsal horn of the spinal cord may be involved.     

Peripheral antinociceptive effect of anandamide and drugs that affect the endocannabinoid system on the formalin test in normal and streptozotocin-diabetic rats

Diabetes is often associated with painful neuropathy. The current treatments are symptomatic and ineffective. Cannabinoids have been proposed as promising drugs for chronic pain treatment and its antinociceptive effect has already been related in nerve injury models of neuropathic pain, but little has been investigated in painful diabetic neuropathy models. Thus, the current study aims to investigate the potential antinociceptive effect of drugs that alter endocannabinoid system when injected subcutaneously into the dorsal surface of the ipsilateral hind paw in chemical hyperalgesia induced by formalin in both normoglycemic (Ngl) and streptozotocin-diabetic (Dbt) rats. Diabetic rats exhibited exaggerated flinching behaviors during first and second phases of the formalin test, indicating the presence of hyperalgesia. AM404, an anandamide (AEA) re-uptake inhibitor, AEA (an agonist of CB1/CB2 receptors) or ACEA (a selective CB1 receptor agonist) induced antinociception in both phases of formalin test in Ngl and Dbt rats. In both groups, the antinociceptive effect of ACEA was prevented by AM251, a CB1 inverse agonist while the antinociceptive effect of AEA was prevented by AM251 or AM630, a CB2 receptor antagonist. In Ngl rats, the antinociceptive effect of AM404 was prevented by AM251 or capsazepine only during first phase of the formalin test while in Dbt rats, this effect was blocked by pretreatment with AM251 (both phases) or AM630 (second phase). Taken together, these results demonstrated broad-spectrum antinociceptive properties of cannabinoids in a model of painful diabetic neuropathy. Peripheral activation of both cannabinoid receptors seems to mediate the antinociceptive effect of exogenous or endogenous anandamide.     

Nitric oxide, but not vasopressin V2 receptor-mediated vasodilation, modulates vasopressin-induced renal vasoconstriction in rats

The renal vascular response to vasopressin and its modulation were evaluated in vivo by infusing the peptide directly into the renal artery of anaesthetized rats. The intra-renal artery (i.r.a) infusion of vasopressin induced a dose-dependent decrease in renal blood flow. Vasoconstriction was obvious at a dose of 3 ng/kg per min and reached a maximum at 100 ng/kg per min. The dose required for a half-maximal response (ED50) was 24+/-4 ng/kg per min (mean+/-SEM, n=8), corresponding to an estimated concentration in renal arterial blood required for a half-maximal response (EC50) of 1.9+/-0.6 nM. Thiobutabarbitone anaesthesia markedly increased plasma vasopressin concentration. This increase was prevented partially by hypotonic hydration of the rats without any change in the renal vascular response to exogenous vasopressin. Vasopressin-induced vasoconstriction dose/response curves were similar in homozygous and heterozygous Brattleboro rats. Infusion of desmopressin (1-1000 ng/kg per min, i.r.a.), a vasopressin V2 receptor-selective agonist, failed to induce renal vasodilation or vasoconstriction. In the presence of SR 49059 (1 mg/kg i.v.), a vasopressin V1A receptor antagonist that completely abolished the vasopressin-induced renal vasoconstriction, desmopressin again failed to induce vasodilation. Inhibition of nitric oxide synthase by N(omega)-nitro-L-arginine (L-NNA, 100 microg/kg for 10 min and 7.5 microg/kg per min, i.r.a.) enhanced vasopressin-induced renal vasoconstriction (EC50 0.6+/-0.1 nM, P<0.05). In contrast, cyclooxygenase blockade by indomethacin (5 mg/kg, i.v.) neither modified the vasopressin-induced decrease in renal blood flow nor altered the potentiation of vasoconstriction by L-NNA. These results show that the constrictor response of the rat renal vascular bed in vivo is observed only with high local concentrations of vasopressin. This hyporeactivity in vivo was not explained by an anaesthesia-elicited increase in endogenous vasopressin, nor by a modulatory effect linked to V2 receptor activation or prostanoid release. In contrast, NO release contributed to the attenuation of vasopressin-induced renal vasoconstriction.     

Sildenafil enhances the peripheral antinociceptive effect of ellagic acid in the rat formalin test

Objective:

Ellagic acid (EA), a major polyphenolic compound of pomegranate juice, produces antinociceptive effects, which are mediated through opioidergic and nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathways. The present study was conducted to elucidate the peripheral antinociceptive effect of EA alone and in combination with sildenafil in the rat formalin test.

Materials and Methods:

Pain was produced by intraplantar injection of formalin (2.5%) in rats and nociceptive behavior was measured as the number of flinches every 5 min in 60 min after injection.

Results:

Local administration of EA and sildenafil dose-dependently increased the nociception threshold in both phases of the test. Moreover, sub-effective doses of sildenafil (25 or 50 mcg/paw, i.p.) significantly and dose-dependently enhanced the antinociception induced by a sub-effective dose of EA (60 mcg/paw, i.pl.) in both phases of the test. The antinociception produced by these drugs alone, or in combination, was due to a peripheral site of action, since the administration in the contralateral paw was ineffective.

Conclusion:

Our results suggest that EA has local peripheral antinociceptive activity, and enhancement of this effect with sildenafil probably occurs through the inhibition of cGMP metabolism.KEY WORDS: Ellagic acid, formalin, peripheral antinociception, sildenafil     

Involvement of serotonergic and opioidergic systems in the antinociceptive effect of ketamine-magnesium sulphate combination in formalin test in rats

BackgroundKetamine and magnesium sulphate showed synergic interaction in the tail-immersion test and additive interaction in the rat formalin test. Aim of study was to evaluate the influence of serotonergic and opioidergic system of this combination in the formalin test in rats.MethodsAntinociceptive activity was assessed by the formalin test in male Wistar rats (200–250 g). Antagonists (naloxone and methysergide) were administrated 5 min before and magnesium sulphate 5 min after ketamine injection. Formalin (2.5%, 100 μL) was injected into the right hind paw surface (intraplantar) of rats 5 min after ketamine/magnesium combination. Data were recorded as the total time spent in pain related behavior after the injection of formalin or vehicle (0.9% NaCl).ResultsIn the intermediate phase of the formalin test, methysergide at a dose of 0.2 mg/kg did not have any effect, but at doses of 0.5 and 1 mg/kg it had a pronociceptive effect. Methysergide (0.2, 0.5 and 1 mg/kg) inhibited the antinociceptive effect of ketamine-magnesium sulphate combination. In the intermediate phase, naloxone at a dose of 0.2 mg/kg did not have any effect, but at a dose of 3 mg/kg it produced a pronociceptive effect. Naloxone (0.2 and 3 mg/kg) antagonized the antinociceptive effect of the ketamine (5 mg/kg)-magnesium sulphate (5 mg/kg) combination.ConclusionThe results of the present study suggest that serotonergic and opioidergic systems are involved, at least in part, in the antinociceptive effect of the ketamine-magnesium sulphate combination in the model of inflammatory pain in rats.     

Mechanisms involved in the antinociceptive effect caused by diphenyl diselenide in the formalin test

This study investigated the mechanisms involved in the antinociceptive action induced by diphenyl diselenide ((PhSe)₂) in the formalin test. Mice were pre‐treated with (PhSe)₂ by the oral route (0.1–100 mg kg⁻¹), 30 min before formalin injection. To address some of the mechanisms by which (PhSe)₂ inhibits formalin‐induced nociception mice were treated with different drugs. The antinociceptive effect of (PhSe)₂ was shown in the first and second phases of the formalin test. The antinociceptive effect caused by (PhSe)₂ (10 mg kg⁻¹, p.o.) was prevented by intrathecal injection of K⁺ channel blockers such as apamin and charybdotoxin (small‐ and large‐conductance Ca²⁺‐activated K⁺ channel inhibitors, respectively) and tetraethylammonium (TEA, a non‐selective voltage‐dependent K⁺ channel inhibitor), but not glib‐enclamide (an ATP‐sensitive K⁺ channel inhibitor). The antinociceptive action caused by (PhSe)₂ (10 mg kg⁻¹, p.o.) was also blocked by a nitric oxide (NO) synthase inhibitor (N^ω‐nitro‐l ‐arginine, L‐NOARG) and the soluble guanylate cyclase inhibitors 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) and methylene blue. These results suggest the participation of NO/cyclic GMP/Ca²⁺ and K⁺ channel pathways in the antinociceptive effect caused by (PhSe)₂.     

Pharmacokinetic-pharmacodynamic modeling of the antinociceptive effect of baclofen in mice.

The aim of this study was the development of a pharmacokinetic-pharmacodynamic (PK/PD) model of the antinociceptive effect of baclofen in mice. We studied the dose response curve of the analgesic action of baclofen in mice by hot plate test. Baclofen produced a dose dependent antinociceptive effect with doses between 1-3 mg/kg administered intraperitoneally (i.p.) (ED50: 1.94 mg/kg of racemate) and this effect fits to a linear pharmacodynamic model. Blood and brain concentrations of (-)3H-baclofen were determined by Thin-Layer Chromatography (TLC) and counted in the scintillation-counter. The PK/PD models were analyzed with the PC-TOPFIT V.2.0 and the tests for distinguishing between models were several adjustment parameters as Akaike information criterion (AIC), Imbimbo criterion (Ip), standard deviation (SD) and the correlation coefficient (r2). Accordingly with these adjustment parameters, a 2 compartment open model was selected where plasma is the central compartment and brain is in the peripheral compartment. In this model, the effect is linked to the peripheral compartment. When the antinociceptive effect of baclofen was plotted against blood concentration, the resulting curve exhibited an anticlockwise hysteresis loop, but on the other hand, when the antinociceptive effect was plotted against the brain concentration, the hysteresis was collapsed. These results confirmed the selected model in our study, as the best adjustment was shown when the pharmacological response was linked to the peripheral compartment.     

Additive interaction between peripheral and central mechanisms involved in the antinociceptive effect of diclofenac in the formalin test in rats

It has been proposed that the antinociception of systemic diclofenac is the outcome of peripheral and central actions. Hence, our purpose was to examine if systemic diclofenac is able to achieve effective concentrations at local and spinal sites and to characterize the interaction between its local and spinal actions. Pain was produced in the rat using the formalin test. Oral diclofenac (1-10 mg/kg) reduced formalin-induced pain. The antinociceptive effect of oral diclofenac (10 mg/kg) was abolished by local or spinal administration of either L-NAME (1-100 microg and 1-50 microg) or glibenclamide (12.5-100 microg and 25-75 microg). These results suggest that oral diclofenac achieves effective concentrations producing an antinociceptive effect involving participation of the NO-potassium channel pathway at both, the local and spinal levels. In an additional experimental series, diclofenac was administered either locally (25-200 mug) or spinally (12.5-100 mug), yielding an antinociceptive effect by both routes. Then, diclofenac was given simultaneously by these two routes in a fixed-ratio, and antinociception was assayed. Isobolographic analysis revealed an additive interaction between the local and spinal effects of diclofenac. Hence, our results provide evidence that the overall antinociceptive effect induced by systemic diclofenac is the outcome of central and peripheral mechanisms.     

Nitric oxide modulates apomorphine-induced recognition memory deficits in rats

Nitric oxide (NO) is an important intracellular messenger in the brain. The implication of NO in schizophrenia is well documented although it is not yet clear whether net over or underproduction of NO is typical of this disease. In line with this, either NO donors or NO synthase (NOS) inhibitors were found to abolish psychotomimetic effects, including cognition deficits, produced by N-methyl-d-aspartate (NMDA) receptor hypofunction. In addition, there is poor experimental evidence concerning the efficacy of NO to modulate memory deficits produced by dopamine (DA) dysfunction. The present study was designed to investigate the ability of NO modulators (NO donors and NOS inhibitors to reverse recognition memory impairments produced by the DA D₁/D₂ mixed receptor agonist apomorphine in rats. For these studies, the novel object recognition test (NORT) was used as the memory test. Apomorphine (0.05, 0.1, 0.5 and 1.0 mg/kg), dose-dependently, disrupted performance in this recognition memory procedure in rats. The NO donors molsidomine (2.0 and 4.0 mg/kg) and SNP (0.3 and 1.0 mg/kg), reversed the impairing effects of apomorphine (1.0 mg/kg) in the NORT. Administration of the NOS inhibitors L-NAME (1.0 and 3.0 mg/kg) or 7-NI (1.0 and 3.0 mg/kg) produced similar results. The present findings indicate a) that apomorphine dose-dependently impaired recognition memory and b) that a cognitive deficit produced by DA dysfunction is sensitive to NO.     

Evaluation of interaction between valproate and baclofen in the formalin test in mice.

Valproate and baclofen dose-dependently inhibited both phases of the formalin test. Combination of valproate and baclofen exerted the additive antinociceptive effect on both phases of the formalin test.     

Nitric oxide modulates the gastrointestinal plasma extravasation following intraabdominal surgical manipulation in rats.

The actions of nitric oxide (NO) on gastrointestinal plasma loss, assessed by the leakage of [125I]human serum albumin, provoked by intraabdominal surgery and organ manipulation has been investigated in pentobarbitone-anaesthesized rats. Gentle manipulation (3 min) of the stomach or the small intestine following laparotomy leads to an increase in albumin extravasation in the stomach, duodenum, jejunum and colon over 1 h. Administration of the NO synthase inhibitors, N(G)-nitro-L-arginine methyl ester (1-5 mg kg(-1), s.c.) and N(G)-monomethyl-L-arginine (12.5-50 mg kg(-1), s.c.), provoked a further substantial elevation of gastrointestinal albumin extravasation in the surgically manipulated rat, but not in control rats. This effect could be prevented by the pretreatment (15 min) with L-arginine (300 mg kg(-1), s.c.) or by the concurrent infusion of the NO donor, S-nitroso-glutathione (5 microg kg(-1) min(-1), i.v.). Endogenous NO, most likely formed by endothelial NO synthase, thus appears to maintain microvascular integrity during surgery and organ manipulation of the gastrointestinal tract.     

Peripheral and central antinociceptive actions of ethylketocyclazocine in the formalin test

The antinociceptive actions of ethylketocyclazocine and morphine were examined in rats in a thermal nociceptive test (tail-immersion) and a test involving minor tissue injury (formalin). In the formalin test, the antinociceptive effects of high doses of ethylketocyclazocine, but not morphine, were attenuated by the peripherally acting antagonist naloxone methylbromide. Naloxone methylbromide had no effect on antinociception produced by ethylketocyclazocine in the tail-immersion test. When ethylketocyclazocine was injected intraventricularly, only partial antinociception was observed in the formalin test. Conversely, naloxone given intraventricularly only partially attenuated the antinociception produced by ethylketocyclazocine given systemically. The data indicate that the antinociceptive effects of ethylketocyclazocine in the tissue injury-induced nociception are a result of summation of central and peripheral actions. Morphine antinociception reaches ceiling at doses that are devoid of such peripheral actions. The data imply that it may be possible to develop a new class of peripherally acting analgesics that are effective in acute inflammatory pain.     

Nitric oxide but not prostacyclin is an autocrine endothelial mediator.

Using porcine aortic endothelial cells, the present study investigates whether stimulation of prostacyclin (PGI2) and nitric oxide also causes elevation of the respective second messengers cAMP and cGMP in the endothelial generator cells. The calcium ionophore A23187 at 0.3-3 microM increased endothelial cGMP levels up to 27-fold in an L-arginine-dependent manner as assessed through complete inhibition by NG-monomethyl-L-arginine (100 microM). The 36-fold PGI2 stimulation by 3 microM A23187 was not accompanied by an intracellular increase in cAMP or an enhanced cAMP efflux. Correspondingly, the PGI2 mimetic iloprost (10 pM-100 microM) did not change endothelial cAMP levels. However, forskolin (1-100 microM) and prostaglandin E2 (PGE2) (0.1-10 microM) produced concentration-dependent increases in cAMP with a 9-fold and 8-fold stimulation at 100 microM forskolin and 10 microM PGE2, respectively. These results demonstrate that in contrast to NO, PGI2 acts as a strictly paracrine hormone without affecting the respective second messenger cAMP in the endothelial generator cells.     

Role of the NO-cGMP pathway in the systemic antinociceptive effect of clonidine in rats and mice

The mechanism underlying the analgesic effect of clonidine, an alpha(2)-adrenoceptor agonist, remains uncertain. Activation of alpha(2)-adrenoceptor induces the release of nitric oxide (NO) from endothelial cells, which has led us to test the hypothesis that the observed antinociceptive effect induced by the systemic administration of clonidine depends on the NO-cGMP pathway. The possible involvement of an opioid link in the antinociceptive effect of clonidine was also evaluated. The antinociceptive effect induced by systemic administration (intravenous or intraperitoneal) of clonidine was evaluated using the rat paw formalin, mice tail-flick and writhing tests. Clonidine (3-120 microg/kg) induces a dose-dependent antinociceptive effect in the formalin, tail-flick and writhing tests. The antinociceptive effect of clonidine in a dose that had no sedative effect assessed by rota rod test, was significantly reduced by NO-synthase and guanylyl cyclase inhibition. The antinociceptive effect of morphine, but not clonidine, was inhibited by naloxone. Our current results suggest that the antinociceptive effect of systemic clonidine does not involve the opioid receptor and is modulated by the NO-cGMP pathway.     

Roles of adenosine receptor subtypes in the antinociceptive effect of intrathecal adenosine in a rat formalin test

The contributions of adenosine receptor subtypes to antinociception produced by adenosine were determined at the spinal level. There are 4 types of adenosine receptors, namely A1, A(2A), A(2B) and A3. The authors investigated the properties of the subtypes of spinal adenosine receptors in terms of nociceptive modulation. The nociceptive state was induced by subcutaneously injecting formalin solution (5%, 50 microl) into the hind paws of male Sprague-Dawley rats. After observing the effect of intrathecal adenosine during the formalin test, the effects of intrathecal adenosine A1 (CPT), A(2A) (CSC), A(2B) (alloxazine) and A3 (MRS 1220) receptor antagonists on the action of adenosine were examined. Intrathecal adenosine inhibited phase 2 flinching response without affecting phase 1 response. CPT, CSC, alloxazine and MRS 1220 antagonized the antinociceptive action of adenosine during phase 2 of the formalin test. These results suggest that spinal adenosine A1, A(2A), A(2B) and A3 receptors may play an important role in the antinociception of adenosine in the formalin-induced facilitated state.