SEX-RELATED DIFFERENCES IN THE ANALGESIC RESPONSE TO THE RAT TAIL IMMERSION TEST

The analgesic response was evaluated by the tail immersion test in adult male (N = 30), female (N = 21) and androgenized female Wistar rats (N = 15). The reaction time for tail withdrawal from the hot water bath was faster for male than for female rats (3.48 +/- 0.12 vs 6.46 +/- 0.42 s). The reaction time of androgenized female rats was similar to that of male rats (3.08 +/- 0.16 s). Blockade of opiate receptors with naloxone (2 mg/kg, ip) decreased the sensitivity to the noxious stimuli in males (4.08 +/- 0.10 s) and in androgenized females (3.69 +/- 0.19 s) but increased it in female rats (5.01 +/- 0.41 s). These data show sex-related differences in the analgesic response evaluated by the tail immersion test and indicate that administration of androgens to newborn female rats affects their pain sensitivity.

Sperm tail flexibility test: a simple test for selecting viable spermatozoa for intracytoplasmic sperm injection from semen samples without motile spermatozoa

PURPOSE: The objective was to describe the results of the injection of immotile spermatozoa with flexible tails when only immotile spermatozoa are present in the semen sample. METHODS: A retrospective study was conducted to analyze the procedure results for 10 couples who participated in our intracytoplasmic sperm injection program. The sperm tail was considered flexible when it moved up and down independently of the head movement, and it was considered inflexible when the movement occurred together (tail plus head). The fertilization and pregnancy rate were analyzed. RESULTS: The normal fertilization rate (presence of 2 pronuclei) was 30.3% (40/132), and the abnormal fertilization rate (presence of less than or more than 2 pronuclei) was 6.81% (9/132). A total of 52 embryos were obtained with 9 transfer procedures performed (pregnancy rate: 11.12%). CONCLUSIONS: The sperm tail flexibility test (STFT) is an easy and cost-effective way for selecting viable immotile spermatozoa and can be used as an alternative method for determining the viability of spermatozoa. This test seems to be a simple and risk-free method when compared to the swelling test.

Determination of the susceptibility of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) to ivermectin and fipronil by Larval Immersion Test (LIT) in Uruguay

Rhipicephalus (Boophilus) microplus is an important cattle pest in Uruguay, and the law regulates its control. It is resistant to organophosphates, synthetic pyrethroids and, as recently discovered, to fipronil. Resistance to macrocyclic lactones (MLs) and amitraz have not been documented; however, veterinarians and farmers have reported treatment failures. The objective of the present work was to study the susceptibility of cattle tick strains from different Uruguayan counties to ivermectin (IVM) and fipronil by using the Larval Immersion Test (LIT). The Mozo strain was used as the susceptible reference strain. From 2007 to 2009, twenty-eight tick populations were collected from different cattle farms with and without history of IVM or fipronil use. A probit analysis estimated dose-mortality regressions, lethal concentrations (LC), and confidence intervals. The resistance ratio (RR) was determined at the LC(50) and LC(90) estimates. To classify a tick population in relation to resistance, three categories based on a statistical analysis of LC and RR between field populations and Mozo strains were defined: susceptible (no differences), incipient resistance (differences and RR(50) < 2) and resistant (differences and RR(50) >= 2). Eighteen field populations were tested with IVM and five of them presented a RR(50) range between 1.35 and 1.98 and the LC(50/90), which is statistically different from the Mozo strain (incipient resistance). However, the RR(90) increases >= 2 in four of the populations, confirming that tick resistance to IVM is emergent. The low RR values obtained could be a result of a low frequency of treatments with IVM. Twenty-seven tick populations were tested with fipronil and six were diagnosed as resistant according to the LIT. Cross-resistance was not observed between fipronil and IVM on these tick populations. The current study presents different R. (B.) microplus populations with an incipient resistance to IVM, and indicates that the fipronil tick resistance is restricted to certain areas in Uruguay. (c) 2011 Elsevier B.V. All rights reserved.

Involvement of alpha(1B)-adrenoceptors in the anti-immobility effect of imipramine in the tail suspension test

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Absence of LPA1 receptor results in altered pattern of limbic activation after tail suspension test

Stress serves as an adaptive mechanism and helps organisms to cope with life-threatening situations. However, individual vulnerability to stress and dysregulation of this system may precipitate stress-related disorders such as depression. The neurobiological circuitry in charge of dealing with stressors has been widely studied in animal models. Recently our group has demonstrated a role for lysophosphatidic acid (LPA) through the LPA1 receptor in vulnerability to stress, in particular the lack of this receptor relates to robust decrease of adult hippocampal neurogenesis and induction of anxious and depressive states. Nevertheless, the specific abnormalities in the limbic circuit in reaction to stress remains unclear. The aim of this study is to examine the differences in the brain activation pattern in the presence or absence of LPA1 receptor after acute stress. For this purpose, we have studied the response of maLPA1-null male mice and normal wild type mice to an intense stressor: Tail Suspension Test. Activation induced by behaviour of brain regions involved in mood regulation was analysed by stereological quantification of c-Fos immunoreactive positive cells. We also conducted multidimensional scaling analysis in order to unravel coativation between structures. Our results revealed hyperactivity of stress-related structures such as amygdala and paraventricular nucleus of the hypothalamus in the knockout model and different patterns of coactivation in both genotypes using a multidimensional map. This data provides further evidence to the engagement of the LPA1 receptors in stress regulation and sheds light on different neural pathways under normal and vulnerability conditions that can lead to mood disorders.

Cannabinoid withdrawal syndrome is reduced in pre-proenkephalin knock-out mice

The functional interactions between the endogenous cannabinoid and opioid systems were evaluated in pre-proenkephalin-deficient mice. Antinociception induced in the tail-immersion test by acute Delta9-tetrahydrocannabinol was reduced in mutant mice, whereas no difference between genotypes was observed in the effects induced on body temperature, locomotion, or ring catalepsy. During a chronic treatment with Delta9-tetrahydrocannabinol, the development of tolerance to the analgesic responses induced by this compound was slower in mice lacking enkephalin. In addition, cannabinoid withdrawal syndrome, precipitated in Delta9-tetrahydrocannabinol-dependent mice by the injection of SR141716A, was significantly attenuated in mutant mice. These results indicate that the endogenous enkephalinergic system is involved in the antinociceptive responses of Delta9-tetrahydrocannabinol and participates in the expression of cannabinoid abstinence.

Antinociceptive effects of Cremophor EL orally administered to mice

Surfactants are frequently used to improve solubilization of lipophilic drugs. Cremophor EL (CrEL) is a polyoxyethylated castor oil surfactant used to solubilize water-insoluble drugs such as anesthetic, antineoplastic, immunosuppressive and analgesic drugs, vitamins and new synthetic compounds, including potential analgesics. The antinociceptive effect of CrEL (3.2, 6.4 and 10.6 g/kg, in 10 ml/kg body weight, by gavage) on the abdominal writhing response induced by intraperitoneal administration of acetic acid (0.8%, 10 ml/kg body weight) and on the tail immersion test was investigated in mice. Control animals received castor oil (10 ml/kg body weight) or saline (0.9% NaCl, 10 ml/kg body weight). CrEL reduced nociception in a dose-dependent manner in both tests. At 10.6 g/kg, CrEL caused antinociception similar to that induced by dipyrone (300 mg/kg, by gavage) in the abdominal writhing test, and antinociception similar to that induced by morphine (20 mg/kg, by gavage) in the tail immersion test. The effect of castor oil was similar to that of saline in both assays. These data indicate that the appropriate controls should be used when evaluating the effects of potential antinociceptive agents dissolved in CrEL.

Antinociceptive effect of novel pyrazolines in mice

The antinociceptive effect of six novel synthetic pyrazolines (3-ethoxymethyl-5-ethoxycarbonyl-1H-pyrazole (Pz 1) and its corresponding 1-substituted methyl (Pz 2) and phenyl (Pz 3) analogues, and 3-(1-ethoxyethyl)-5-ethoxycarbonyl-1H-pyrazole (Pz 4) and its corresponding 1-substituted methyl (Pz 5) and phenyl (Pz 6) analogues) was evaluated by the tail immersion test in adult male albino mice. The animals (N = 11-12 in each group) received vehicle (5% Tween 80, 10 ml/kg, sc) or 1.5 mmol/kg of each of the pyrazolines (Pz 1-Pz 6), sc. Fifteen, thirty and sixty minutes after drug administration, the mice were subjected to the tail immersion test. Thirty minutes after drug administration Pz 2 and Pz 3 increased tail withdrawal latency (vehicle = 3.4 ± 0.2; Pz 2 = 5.2 ± 0.4; Pz 3 = 5.9 ± 0.4 s; mean ± SEM), whereas the other pyrazolines did not present antinociceptive activity. Dose-effect curves (0.15 to 1.5 mmol/kg) were constructed for the bioactive pyrazolines. Pz 2 (1.5 mmol/kg, sc) impaired motor coordination in the rotarod and increased immobility in the open-field test. Pz 3 did not alter rotarod performance and spontaneous locomotion, but increased immobility in the open field at the dose of 1.5 mmol/kg. The involvement of opioid mechanisms in the pyrazoline-induced antinociception was investigated by pretreating the animals with naloxone (2.75 µmol/kg, sc). Naloxone prevented Pz 3- but not Pz 2-induced antinociception. Moreover, naloxone pretreatment did not alter Pz 3-induced immobility. We conclude that Pz 3-induced antinociception involves opioid mechanisms but this is not the case for Pz 2.

Antinociceptive effects of the essential oil of Mentha x villosa leaf and its major constituent piperitenone oxide in mice

Mentha x villosa Huds (Labiatae) is an aromatic herb widely used in folk medicine. Since the essential oil of the herb has many pharmacological activities, including antispasmodic effects, we determined whether the oil and its major constituent, piperitenone oxide (PO), have antinociceptive activity. The essential oil of M. x villosa (EOMV) and PO administered orally at 200 mg/kg (vehicle: 0.1% Tween 80 in water) significantly reduced the writhings induced by acetic acid from control values of 59.5 ± 3.1 s (N = 10) to 31.9 ± 2.8 s (N = 10) and 23.8 ± 3.4 s (N = 10), respectively. When administered at 100 and 200 mg/kg, EOMV reduced the paw licking time for the second phase of the formalin test from the control value of 20.6 ± 2.1 s (N = 13) to 5.3 ± 2.2 s (N = 12) and 2.7 ± 1.2 s (N = 18), respectively. At 100 and 200 mg/kg, PO reduced this second phase to 8.3 ± 2.7 s (N = 12) and 3.0 ± 1.2 s (N = 10), respectively. This effect of EOMV and PO was not reversed by naloxone. EOMV and PO had no significant effect on the first phase of the formalin test. As evaluated by the hot-plate and tail immersion test, EOMV and PO, at doses up to 200 mg/kg, showed no analgesic activity. These results show that EOMV and PO have antinociceptive activity and suggest that this effect is probably an indirect anti-inflammatory effect, which does not involve the central nervous system.

PARTICIPATION OF OPIOID RECEPTORS IN THE MODULATION OF THE ANALGESIC RESPONSE BY ADRENAL AND GONADAL GLANDS

The involvement of opioid receptors in the analgesic response was evaluated by the tail-immersion test in simultaneously adrenalectomized and ovariectomized female Wistar rats (210-250 g). The reaction time (mean +/- SEM) for tail withdrawal from hot water decreased significantly 2 weeks after surgery (3.52 +/- 0.20 s) when compared to intact animals (6.09 +/- 0.23 s). Hormonal replacement with dexamethasone (50-mu-g/day) did not affect reaction time (3.38 +/- 0.19 s). However, this response was restored by combined adrenal and gonadal steroid substitution (estradiol 5-mu-g/day and progesterone 1.5-mu-g 6 h before the tests) therapy (5.11 +/- 0.45 s in animals treated with dexamethasone plus estradiol and 5.04 +/- 0.43 s in animals treated with dexamethasone plus estradiol plus progesterone). Naloxone (2 mg/kg) decreased the reaction time of animals treated with adrenal and gonadal steroids (5.11 +/- 0.45 vs 4.15 +/- 0.44 s and 5.04 +/- 0.43 vs 3.87 +/- 0.28 s, respectively, before and after naloxone) but failed to decrease it in rats treated with dexamethasone only (3.88 +/- 0.18 vs 4.34 +/- 0.25 s, before and after naloxone). These observations indicate that gonadal steroids are the most important steroid factors involved in the reaction time to tail immersion in hot water and confirm other reports that the opioid pathways modulating the neuronal circuitry require the presence of these hormones.

Antinociceptive effects of the essential oil of Mentha x villosa leaf and its major constituent piperitenone oxide in mice

Mentha x villosa Huds (Labiatae) is an aromatic herb widely used in folk medicine. Since the essential oil of the herb has many pharmacological activities, including antispasmodic effects, we determined whether the oil and its major constituent, piperitenone oxide (PO), have antinociceptive activity. The essential oil of M. x villosa (EOMV) and PO administered orally at 200 mg/kg (vehicle: 0.1% Tween 80 in water) significantly reduced the writhings induced by acetic acid from control values of 59.5 ± 3.1 s (N = 10) to 31.9 ± 2.8 s (N = 10) and 23.8 ± 3.4 s (N = 10), respectively. When administered at 100 and 200 mg/kg, EOMV reduced the paw licking time for the second phase of the formalin test from the control value of 20.6 ± 2.1 s (N = 13) to 5.3 ± 2.2 s (N = 12) and 2.7 ± 1.2 s (N = 18), respectively. At 100 and 200 mg/kg, PO reduced this second phase to 8.3 ± 2.7 s (N = 12) and 3.0 ± 1.2 s (N = 10), respectively. This effect of EOMV and PO was not reversed by naloxone. EOMV and PO had no significant effect on the first phase of the formalin test. As evaluated by the hot-plate and tail immersion test, EOMV and PO, at doses up to 200 mg/kg, showed no analgesic activity. These results show that EOMV and PO have antinociceptive activity and suggest that this effect is probably an indirect anti-inflammatory effect, which does not involve the central nervous system.

In vivo analgesic activity and safety assessment of Vitis vinifera L and Punica granatum L fruits extracts

Purpose: To investigate the analgesic properties of fruit extracts of Vitis vinifera (grape) and Punica granatum (pomegranate) in Albino mal mice. Methods: The analgesic activity of fruit extracts of V. vinifera and P. granatum were examined in vivo using thermal stimulus assays (i.e., tail immersion and hot plate) and acetic acid-induced writhing test using acetylsalicylic acid (0.1 g/kg, per os) as standard. The extracts were administered orally in doses of 1.0, 2.0 and 3.0 g/kg. Results: In acetic acid writhes test, both fruit extract pretreatments (1.0, 2.0 and 3.0 g/kg, per os) significantly decreased the number of writhes (p < 0.0001) in a dose-dependent manner compared to control. The Index of Pain Inhibition (IPI) values following V. vinifera extract treatments were 36.52 % (1.0 g/kg), 66.67 % (2.0 g/kg) and 89.71 % (3.0 g/kg) which were significantly different from those for P. granatum extracts (45.39 %, 1.0 g/kg), 70.93 %, 2.0 g/kg) and 86.88 %, 3.0 g/kg) at equivalent doses of 2.0 and 3.0 g/kg of the extracts The fruit extracts of both species increased the reaction latency time. In tail-immersion assay, only the fruit extract of P. granatum significantly increased the response to heat stimulus at doses of 2.0 g/kg (p < 0.05). Conclusion: The hydroalcohol fruit extracts of P. granatum and V. vinifera have potential analgesic effects. Further studies are needed to determine the active component responsible for this effect.

Apparent insensitivity of the hotplate latency test for detection of antinociception following intraperitoneal, intravenous or intracerebroventricular M6G administration to rats

Although morphine-6-glucuronide (M6G) has been shown to be analgesically active, the relative involvement of spinal and supraspinal structures in mediating M6G's pain-relieving effects following central and systemic administration to rats is unclear. As the tail flick and hotplate latency tests are reported to quantify antinociception mediated primarily by spinal and supraspinal mechanisms respectively, these methods were used to determine the comparative apparent levels of antinociception (expressed as percentage maximum possible effect, % MPE) achieved after M6G or morphine administration. Following i.v. or i.p. M6G (1.9-5.4 mu mol) dosing or i.p. morphine (10 mu mol) dosing, high levels of antinociception (>50% MPE) were achieved using the tail flick test whereas base-line levels of antinociception were observed 30 sec later in the same rats using the hotplate test. By contrast, antinociception evoked by i.v. morphine (10 mu mol) exceeded 50% MPE using both the hotplate and tail flick tests although the apparent potency was approximately 2.5 times greater using the tail flick test. After i.c.v. dosing, M6G (0.22-3.3 nmol) was significantly (P < .05) more potent when assessed using the tail flick compared with the hotplate test. Taken together, these data strongly indicate that following central and systemic administration, M6G's antinociceptive effects are mediated primarily by spinal structures whereas both spinal and supraspinal mechanisms contribute to systemic morphine's antinociceptive effects.

Antinociceptive activity and toxicology of the lectin from Canavalia boliviana seeds in mice

The aim of the present study was to evaluate the potential antinociceptive and toxicity of Canavalia boliviana lectin (CboL) using different methods in mice. The role of carbohydrate-binding sites was also investigated. CboL given to mice daily for 14 days at doses of 5 mg/kg did not cause any observable toxicity. CboL (1, 5, and 10 mg/kg) administered to mice intravenously inhibited abdominal constrictions induced by acetic acid and the two phases of the formalin test. In the hot plate and tail immersion tests, the same treatment of CboL induced significant increase in the latency period. In the hot plate test, the effect of CboL (5 mg/kg) was reversed by naloxone (1 mg/kg), indicating the involvement of the opioid system. In the open-field and rota-rod tests, the CboL treatment did not alter animals` motor function. These results show that CboL presents antinociceptive effects of both central and peripheral origin, involving the participation of the opioid system via lectin domain.

Pharmacological screening of Ageratum conyzoides L. (mentrasto)

The pharmacological activities of a water extract (WE) of Ageratum conyzoides L, a plant populary known for its analgesic and anti-inflamatory properties, were studied in vivo and in vitro preparations. Oral administration (p.o.) of the water extract (WE, 0.1 to 5 g/Kg) to rats and mice induced quietness and reduced the spontaneous motility. the sleeping time induced by sodium pentobarbital (50 mg/Kg, i.p.) in mice was not altered by previous treatment with We (2 g/Kg, p.o.). The same treatment did not influence the paw edema induced by carrageenan or dextran, nor did it reduce the chronic paw edema induced by complete Freund's adjuvant or formaldehyde in rats. The tail flick response in immersion test and writhings induced by 0.8%acetic acid in mice were not altered by WE either. In isolated guinea-pig ilea WE (0.4 to 4 mg/ml) did not alter the EC50 values of histamine or acetylcholine, but reduced the maximal response to the agonists by 20 to 50%. We (0.01 to 10 mg/ml) produced tonic contractions of the ileal smooth muscle proportional to the doses, reaching a maximum of 75% relatively to the maximum obtained with histamine. Those contractions were blocked by diphenhydramine (10 nM) and reduced by 32% in presence of atropine (10 nM). The results indicated that oral treatment of rodents with A. conyzoides L neither reduced the inflammatory edema nor did it decrease the reaction to pain stimuli. In vitro the extract presented an unexpected histamine-like activity characteristic of a partial agonist. The results did not confirm the popular medicinal indications of the plant.