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Frogs possess pain receptors and pathways that support processing and perception of noxious stimuli however the level of organization is less well structured compared to mammals. It was long believed that the experience of pain was limited to 'higher' phylums of the animal kingdom. However, it is now commonly accepted that amphibians possess neuro-anatomical pathways conductive of a complete nociceptive experience. Xenopus laevis frogs have been one of the most popular aquatic research models for developmental studies and genetic research. These frogs have been extensively use in research for their eggs, that can be collected following hormonal stimulation either naturally or by surgical intervention. Many anaesthetics have been used in amphibians such as bath solutions of MS-222, benzocaine and eugenol as well as systemic injections of ketamine or tiletamine, barbiturates, propofol and gas administrations of methoxyflurane, halothane and isoflurane. Most of these anaesthetic drugs produce variability in depth and duration of anaesthesia. MS-222 appears to be one of the most reliable anaesthetics. This review will focus on the evidence of pain perception in frogs and will compare the effectiveness and limitations of different anaesthetics used in Xenopus leavis frogs.

The objective of this study was to determine the level of anesthesia attained in Xenopus laevis frogs using a propofol bath administration. Thirty-three nonbreeding female Xenopus laevis frogs were used. At 175 mg/l, all frogs died after bath administration. An appropriate anesthetic dose was determined to be 88 mg/l for 15 min. After administration of this dose, the acetic acid test, withdrawal reflex, righting reflex, heart rate, and respiratory frequency were used to evaluate central nervous system depression. Pharmacokinetics of propofol were calculated after blood concentration determination by tandem liquid chromatography-mass spectrometry analyses. Short-duration anesthesia (less than 30 min) was obtained, and in many frogs, muscular fasciculation was seen during the acetic acid test. The area under the time-concentration curve (AUC0-t) was 24.07 microg.min/ml, and AUCinf was 24.71 microg.min/ml. The elimination half-life was 1.18 h. When administered as a single-bath immersion for 15 min, propofol does not appear to be a safe and effective anesthetic for Xenopus laevis frogs, due to a narrow dose-effect window, short duration, and shallow level of anesthesia obtained.

Neuropathic pain is a type of chronic pain following central or peripheral nervous system lesions that cause allodynia (pain initiated by a non-painful stimulus) and hyperalgesia (increased pain sensation following a painful stimulus). The first objective of the study was to evaluate the pharmacokinetics of eugenol, the principle chemical constituent of clove oil, following a gavage administration (40 mg/kg) in male Sprague-Dawley rats. The second objective was to evaluate the effect of repeated oral administrations of eugenol on hyperalgesia and allodynia using an experimental model of neuropathic pain in rats. Thermal and mechanical sensitivity (Hargreave's test and von Frey filaments) were determined in sciatic nerve cuff-implanted rats. Sensitivities were assessed following repeated oral administrations of 40 mg/kg of eugenol or saline for 5 days (n=6 per group). Pharmacokinetic parameters were calculated using noncompartmental methods. Serial blood samples were collected over 24 h. Concentrations of eugenol in blood and plasma peaked rapidly following oral administration. Mean T(1/2) values of eugenol in plasma and blood were long (14.0 and 18.3 h, respectively), suggesting a potential accumulation of the drug following repeated administrations. Reaction time to thermal stimuli appeared to increase constantly following repeated administrations of eugenol. On the last day of treatment, eugenol treatments resulted in a statistically significant prolongation of the reaction time to thermal stimuli in rats compared to the saline group (Mean+/-S.E.M.: 11.4+/-1.23 vs. 6.1+/-0.53 s, P<0.01). These results support the hypothesis that eugenol may alleviate neuropathic pain and that the cumulative effect of the drug may be in part responsible for this effect following repeated daily administrations.

Objective: To determine the level of anesthesia attained in Xenopus laevis frogs with eugenol at different doses and by different routes of administration., Study Design: Prospective experimental trial., Animals: Sixty X. laevis nonbreeding female frogs weighing between 90 and 140 g., Methods: Three different routes of administration were tested - subcutaneous injections into the dorsal lymph sacs, topical administration using a gauze patch, and immersion in a bath containing eugenol. Following the determination of the best route of administration, the acetic acid test, the withdrawal reflex, righting reflex, heart rate, and respiratory frequency were used to evaluate central nervous system depression following eugenol bath administration. In an additional group, the response to a surgical incision of the abdominal wall was evaluated. The pharmacokinetics of eugenol were determined following bath immersion administration, and pharmacokinetic parameters were calculated following blood concentration determination by tandem liquid chromatography/mass spectrometry analyses., Results: It was not possible to induce anethesia with subcutaneous and patch administration, independent of the eugenol dose administered. The immersion bath was the only efficacious route for anesthesia inducing surgical anesthesia for at least 30 minutes with postoperative analgesia. Histopathology of selected tissues (heart, lung, liver, kidneys, eyes) showed no evidence of lesions 24 hours following bath immersion. The elimination half-life (T(1/2)) was 4 hours., Conclusions: When administered as a single-bath immersion (dose 350 mg L(-1)) for 15 minutes, eugenol may serve as an effective anesthetic in X. laevis frogs for short surgical procedures.

A rapid, selective and sensitive method was developed for the determination of eugenol concentration using an off-line dansyl chloride derivatization step to enhance signal intensity. The method consisted of a protein precipitation extraction followed by derivatization with dansyl chloride and analysis by full scan liquid chromatography electrospray quadrupole ion trap mass spectrometry (LC-ESI-QIT). The separation was achieved using a 100 x 2 mm C(8) analytical column combined with an isocratic mobile phase composed of 75:25 acetonitrile: 0.1% formic acid in water set at a flow rate of 0.25 mL/min. Signal intensity of the eugenol-dansyl chloride derivative was increased up to 100-fold as compared with the underivatized eugenol in positive electrospray mode. An analytical range of 100-20,000 ng/mL was used in the calibration curve of plasma and blood samples. The LOD observed was 0.5 pg injected on column. The novel method met all requirements of specificity, sensitivity, linearity, precision, accuracy and stability. In conclusion, a rapid and sensitive LC-ESI/MS/MS method using a derivatization agent was developed to enhance signal intensity of eugenol., (Copyright (c) 2006 John Wiley & Sons, Ltd.)

Case of cutaneous necrosis in African Clawed frogs Xenopus laevis after the topical application of eugenol. African Clawed frogs showed necrotic cutaneous lesions after a topical application of high concentrations of eugenol, an analgesic and anesthetic agent. Microscopically, ulceration of the epidermis, a loss of mucous and serous glands as well as an infiltration of inflammatory cells were observed.

A rapid, selective and sensitive method was developed for the determination of propofol concentration using an off-line dansyl chloride derivatization step to enhance signal intensity. The method consisted of a protein precipitation extraction followed by derivatization with dansyl chloride and analysis by liquid chromatography ionspray tandem mass spectrometry (LC-ESI/MS/MS). The separation was achieved using a 100 mm x 2 mm C8 analytical column combined with an isocratic mobile phase composed of 80:20 acetonitrile: 0.5% formic acid in water. Signal intensity of the propofol-dansyl chloride derivative was increased up to 200-fold as compared to the underivatized propofol in positive electrospray mode. An analytical range of 20-20,000 ng/mL was used in the calibration curve of plasma and blood samples. The novel method met all requirements of specificity, sensitivity, linearity, precision, accuracy and stability. A pharmacokinetic study was performed in rats and the novel analytical method was used as a routine analysis to provide enhanced measurements of plasma and blood concentrations of propofol. Blood and plasma pharmacokinetic results show that a very important fraction of propofol distributes into red blood cells. In conclusion, a rapid and sensitive LC-ESI/MS/MS method using a derivatization agent was developed to enhance signal intensity of propofol. Routine analysis with the novel method provided accurate results and enhanced the detection levels of plasma and blood concentrations of propofol to better characterize the in vivo biodisposition of propofol.