Excitatory and inhibitory effects of tricaine (MS-222) on fictive breathing in isolated bullfrog brain stem

This study examined the direct effects of tricaine methanesulfonate (MS-222), a sodium-channel blocking local anesthetic, on respiratory motor output using an in vitro brain stem preparation of adult North American bullfrogs (Rana catesbeiana). Bullfrogs were anesthetized with halothane, and the brain stem was removed and superfused with artificial cerebrospinal fluid containing MS-222 at concentrations ranging from 0.1 to 1,000 micro M. At the lowest concentration of MS-222, respiratory frequency (fR) increased significantly (P < 0.05), but at higher concentrations, fR progressively decreased and was abolished in all preparations at 1,000 micro M (P < 0.01). Respiratory burst amplitude and burst duration were not affected by MS-222. The frequency of nonrespiratory neural activity did not significantly change with the addition of MS-222 below 1,000 micro M. These data indicate that MS-222 has a significant, direct effect on respiratory motor output from the central nervous system, producing both excitation and inhibition of fictive breathing. The results are consistent with other studies demonstrating that low concentrations of anesthetics generally cause excitation followed by depression at higher concentrations. Although the mechanisms underlying the excitatory effects of MS-222 in this study are unclear, they may include increased excitatory neurotransmission and/or disinhibition of inputs to the respiratory central pattern generator.     

Use of Telazol to immobilize female northern sea lions (Eumetopias jubatus) in Alaska

Twenty-nine female northern sea lions (Eumetopias jubatus) were immobilized using Telazol in dosages ranging from 1.8 to 8.1 mg/kg. Best results were achieved with Telazol dosages ranging between 1.8 and 2.5 mg/kg which resulted in smooth induction and recovery. Optimal injection location was in the muscle mass of the lower back and hip. Dosages greater than 3.5 mg/kg resulted in a tendency toward hypothermia. Six mortalities occurred which were partially caused by the location of drug injection and perhaps the high dosage.     

Effects of a high concentration of CO2 on electrocardiograms in the carp, Cyprinus carpio

1. The effects of a high concentration of CO2 (PCO2 = 250 mmHg and PO2 = 360 mmHg in water) and MS222 (tricaine methanesulfonate, 1/8000 or 1/5000) on the electrocardiogram (ECG) in carp were examined using five kinds of bipolar leads from the body surface. 2. In the carp anesthetized with the high concentration of CO2 for 30 min, the QRS duration, PQ interval and and direction of the QRS axis on the frontal plane significantly changed. Even after recovery from anesthesia, delay in the QRS duration was still recognized. 3. The concentration of CO2 used in this study had an anesthetic action to the same degree as 1/8000 of MS222 and had a much more severe effect on the ECG of the carp than 1/5000 of MS222.     

MS-222 (tricaine methane sulfonate) does not kill the amphibian chytrid fungus Batrachochytrium dendrobatidis

MS-222 (tricaine methane sulfonate) is an agent commonly used to anaesthetise or euthanize amphibians used in experiments. It is administered by immersing the animal to allow absorption through the skin. Chytridiomycosis is an important disease of amphibians and research involves experiments with live animals. Batrachochytrium dendrobatidis, the fungus which causes chytridiomycosis, is located in the skin and therefore the organism should come into contact with MS-222 when it is used. B. dendrobatidis is a sensitive organism which could possibly be killed by MS-222. Hence, results of chytridiomycosis studies in which MS-222 is used could be unreliable. A concentration of 2 g l(-1) and an exposure duration of 1 h is at the high end of the range at which MS-222 would be most commonly used. Exposure to 2 g l(-1) MS-222 for 1 h does not kill B. dendrobatidis cultures, suggesting that MS-222 is safe to use in chytridiomycosis studies.     

Evaluation of intramuscular ketoprofen and butorphanol as analgesics in chain dogfish (Scyliorhinus retifer)

The mediation of nociception with analgesic medications has not been well documented in elasmobranchs. The purpose of this study was to determine effective analgesic doses of the opioid agonist‐antagonist, butorphanol, and the non‐steroidal anti‐inflammatory drug, ketoprofen, in an elasmobranch. This was evaluated by repetitively assessing minimum anesthetic concentrations of the immersion anesthetic, tricaine methanesulfonate (MS‐222), required to prevent response to noxious stimuli in chain dogfish (Scyliorhinus retifer) when administered multiple doses of each of the analgesics (0.25, 0.5, 1.0, 2.5, and 5.0 mg/kg butorphanol and 1.0, 1.5, 2.0, and 4.0 mg/kg ketoprofen). Baseline concentrations of MS‐222 required to prevent a response to a noxious stimulus were determined for each animal and served as the controls. Although individual animals displayed a reduction in MS‐222 concentration with various doses of both analgesics, no statistically significant difference was noted between control animals and animals given analgesics. It is plausible that unique elasmobranch anatomy, lack of appropriate medication dose or timing of administration, and other physiological factors not yet identified may have contributed to the lack of apparent efficacy of the analgesics evaluated in this study. Zoo Biol 0:1–10, 2006. © 2006 Wiley‐Liss, Inc.     

Differential effects of anesthetics on electrical properties of the rainbow trout (Oncorhynchus mykiss) heart

We compared electrocardiographic signals in hatchery-reared, non-spinally-transected, immature rainbow trout (Oncorhynchus mykiss Walbaum) under clove oil (25 ppm), tricaine methanesulfonate (tricaine, 60 ppm), and benzocaine (108 ppm) general anesthesia (35 min, 14 degrees C). For all 3 anesthetics, the mean heart rate (HR) and QRS amplitude did not differ, and QRS duration and QT interval were independent of HR. Heart rate variability (HRV) was significantly (4-fold, P=0.032) higher under benzocaine than under clove oil and tricaine, but did not differ between clove oil and tricaine. QRS duration differed between groups (P<0.001, F=121); benzocaine anesthesia resulted in longer QRS complexes compared to clove oil (P<0.001) and tricaine (P<0.001) anesthesia, and QRS complexes under clove oil were longer than those under tricaine (P<0.001). High HRV and QRS amplitude variation with benzocaine were associated with HR oscillations as anesthetic exposure time increased, and suggest benzocaine toxicity which may influence cardiac function studies. Similar clove oil and tricaine ECG patterns suggest comparable autonomic effects, and maintenance of myocardial excitability. Given its low cost, ease of use, and similar ECG profiles to tricaine, clove oil is a viable alternative for studies of cardiac function in anesthetized rainbow trout.     

Behavioral and clinical pathology changes in koi carp (Cyprinus carpio) subjected to anesthesia and surgery with and without intra-operative analgesics

Fish surgery is becoming increasingly common in laboratory and clinical settings. Behavioral and physiologic consequences of surgical procedures may affect experimental results, so these effects should be defined and, if possible, ameliorated. We document behavioral and clinical pathology changes in koi carp (Cyprinus carpio) undergoing surgery with tricaine methanesulphonate (MS-222) anesthesia, with and without intraoperative administration of the opiate butorphanol (0.4 mg/kg intramuscularly) or the nonsteroidal antiinflammatory analgesic ketoprofen (2 mg/kg intramuscularly). For all fish combined, surgery resulted in reduced activity, lower position in the water column, and decreased feeding intensity at multiple time points after surgery. The butorphanol-treated group was the only one not to experience significant (P < 0.05) alterations from presurgical behaviors. Clinical pathology changes at 48 h after anesthesia and surgery included decreased hematocrit, total solids, phosphorus, total protein, albumin, globulin, potassium, and chloride and increased plasma glucose, aspartate aminotransferase, creatine kinase, and bicarbonate. The only clinical pathology difference between treatment groups was a lower increase in creatine kinase in the ketoprofen-treated group. No adverse effects of butorphanol or ketoprofen at these doses were identified. These results suggest a mild behavioral sparing effect of butorphanol and reduced muscle damage from the antiinflammatory activity of ketoprofen.     

A new anesthetic agent for use in the gerbil

Gerbils have been neglected in published reports on anesthesia. This study compared several dosages of Telazol used for anesthesia in the gerbil. Each group of animals injected with Telazol was evaluated for onset and duration of anesthesia and analgesia. Results showed Telazol to be a safe anesthetic and when dosed at 60 mg/kg to be suitable for major surgical procedures. Lower dosages of Telazol, in contrast, provided immobility and analgesia suitable for less nocioceptive and noninvasive experimental manipulations. Dosages of Telazol required for surgical depth of analgesia and anesthesia were accompanied by a prolonged recovery time. Gerbils should be monitored closely to insure a safe recovery when using the higher dosages.     

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.     

Medicine and surgery of amphibians

Amphibians are most notably characterized by their glandular skin, which they shed regularly and ingest routinely. It is advisable to handle amphibians only with protective gloves to avoid damaging their skin. These animals absorb water readily across the skin as a means of maintaining hydration. They also easily absorb drugs and anesthetics that are applied directly to the skin. Investigators commonly utilize cutaneous respiration in amphibians and evaluate skin abnormalities via wet mount preparations, skin scrapes, and biopsy. The examination of blood samples can be useful in evaluating the status of ill amphibians, although the similarity in function of amphibian blood cell types and those of other species is largely unknown. If surgery is required, it is necessary to fast the animals before surgery, and to monitor their hydration. The wet environment required for amphibian surgery makes sterile technique challenging, and it is advisable to institute prophylactic antibiotic therapy before the procedure. The anesthetic of choice for amphibian surgery is tricaine methanesulfonate (MS-222). Postoperative recommendations include fluids, nutritional support if necessary, and analgesia. If euthanasia is required, MS-222 overdose or pentobarbital injection are the preferred methods.     

Susceptibility of the Antarctic fish Pagothenia borchgrevinki to MS-222 anaesthesia

Summary Induction of anaesthesia and distribution of the routinely used fish anaesthetic, MS-222 (ethyl m-amino-benzoate methanesulfonate), were monitored in the Antarctic fish Pagothenia borchgrevinki. As expected from the extreme low metabolic rate of Antarctic fish, induction times were significantly longer than those from rainbow trout. Onset of anaesthesia in the Antarctic fish also failed to correlate with either brain or blood MS-222 concentrations, in contrast to the trends observed in freshwater salmonids. High levels of free MS-222 in liver and in the aglomerular kidney, and the presence of acetylated derivatives in the blood, suggest these organs as primary sites of drug metabolism. MS-222 anaesthesia in P. borchgrevinki is also accompanied by dose-dependent changes in haematological parameters.     

Failure of tetracycline as a biomarker in batch-marking juvenile frogs

Recent widespread amphibian declines call for better techniques to assess population dynamics. Tetracycline as a biomarker in capture-recapture studies is one technique used successfully in fish, reptiles, and mammals. A two-phase experimental study was conducted to evaluate tetracycline as a biomarker in green frogs (Rana clamitans) and pickerel frogs (Rana palustris). In the first experimental phase tadpoles were exposed to water containing either 250 mg/l or 500 mg/l tetracycline for a period of 24 hr. During the second phase, juvenile frogs were exposed to tetracycline in water at 500 mg/l or given injections of tetracycline at the dose rate of 100 mg/kg body weight. At selected times several weeks later, under tricaine methanesulfonate anesthesia, a toe was surgically excised from each animal, sectioned and viewed under an ultraviolet microscope. No significant differences were found between the various treatments and control animals (untreated). Therefore, the use of tetracycline as a biomarker in anurans using these techniques is not recommended.     

Naltrexone modulates body and brain development in rats: a role for endogenous opioid systems in growth

Preweaning rats receiving daily injections of 20, 50, or 100 mg/kg naltrexone, a potent opiate antagonist, had body and brain weights that were increased 16-22% and 6-13%, respectively, from control levels on day 21 (weaning). All of these dosages of naltrexone blocked the opiate receptor for 24 hr/day as measured in opiate challenge experiments. Dosages of 0.1, 1, and 10 mg/kg naltrexone, which blocked the opiate receptor for less than 12 hr/day, inhibited growth. Repetitive administration of low dosages (3 mg/kg naltrexone, 3 times daily), which blocked the receptor 24 hr/day, increased body and brain development by 31% and 10%, respectively, whereas a cumulative dosage of 9 mg/kg naltrexone given once daily retarded growth. These results show that developmental events are dictated by the duration of opiate receptor blockade and provide compelling evidence that endogenous opioid systems play a crucial role in growth.     

Efficacy of Tricaine Methanesulfonate (MS-222) as an Anesthetic Agent for Blocking Sensory-Motor Responses in Xenopus laevis Tadpoles

Anesthetics are drugs that reversibly relieve pain, decrease body movements and suppress neuronal activity. Most drugs only cover one of these effects; for instance, analgesics relieve pain but fail to block primary fiber responses to noxious stimuli. Alternately, paralytic drugs block synaptic transmission at neuromuscular junctions, thereby effectively paralyzing skeletal muscles. Thus, both analgesics and paralytics each accomplish one effect, but fail to singularly account for all three. Tricaine methanesulfonate (MS-222) is structurally similar to benzocaine, a typical anesthetic for anamniote vertebrates, but contains a sulfate moiety rendering this drug more hydrophilic. MS-222 is used as anesthetic in poikilothermic animals such as fish and amphibians. However, it is often argued that MS-222 is only a hypnotic drug and its ability to block neural activity has been questioned. This prompted us to evaluate the potency and dynamics of MS-222-induced effects on neuronal firing of sensory and motor nerves alongside a defined motor behavior in semi-intact in vitro preparations of Xenopus laevis tadpoles. Electrophysiological recordings of extraocular motor discharge and both spontaneous and evoked mechanosensory nerve activity were measured before, during and after administration of MS-222, then compared to benzocaine and a known paralytic, pancuronium. Both MS-222 and benzocaine, but not pancuronium caused a dose-dependent, reversible blockade of extraocular motor and sensory nerve activity. These results indicate that MS-222 as benzocaine blocks the activity of both sensory and motor nerves compatible with the mechanistic action of effective anesthetics, indicating that both caine-derivates are effective as single-drug anesthetics for surgical interventions in anamniotes.     

Chemical immobilization of free-ranging dhole (Cuon alpinus), binturong (Arctictis binturong), and yellow-throated marten (Martes flavigula) in Thailand

No published information exists on the chemical immobilization of free-ranging dholes (Cuon alpinus), binturongs (Arctictis binturong), or yellow-throated martens (Martes flavigula). We chemically immobilized these species in Thailand using a mixture of ketamine hydrochloride with xylazine hydrochloride (KH–XH) and tiletamine hydrochloride with zolazepam hydrochloride (TH–ZH). Mean (±SD) dose of KH–XH was 18.1±5.3 and 0.9±0.1 mg/kg for dholes (n=2), 19.7±4.1 and 1.3±0.4 mg/kg for binturongs (n=8), and 28.7±3.3 and 1.1±0.1 mg/kg for yellow-throated martens (n=5), respectively. Mean dose of TH–ZH was 4.1±0.1 mg/kg for dhole (n=2). Induction time, duration of anesthesia, and recovery time were satisfactory for standard field research procedures including radio-collaring, although the effects on yellow-throated martens and binturongs were more variable. Respiration and muscle rigidity were monitored during sedation with no observed adverse physiological effects. Individuals were released after full recovery and monitored via radio telemetry for 4–23 months with no observed detrimental effects due to chemical immobilization. We conclude that KH–XH and TH–ZH are safe and effective immobilization agents for these carnivores; however, we suggest testing different KH–XH ratios and dosages, and other immobilizing agents for these species.     

Plasma cortisol response of seawater-adapted mummichogs (Fundulus heteroclitus) during deep MS-222 anesthesia

Anesthetics are used to reduce stress in fishes during handling and transfer. However, deep anesthesia of seawater-adapted mummichogs (Fundulus heteroclitus) results in a time-related increase in plasma cortisol, indicating a primary (neuroendocrine) stress response. Groups of seven fish were bled within 1 to 12 min of exposure to the anesthetic MS-222. Plasma cortisol rose more rapidly in fish removed from the MS-222 solution immediately after 1 min and held between wet paper towels than in fish that remained immersed. The difference between methods was significant (P < 0.001) with variation restricted to the later sampling periods. Differences were not significant in fish sampled immediately after 1 min (P > 0.05).     

MS-222对两种规格的异齿裂腹鱼麻醉效果研究

通过MS-222对2种规格的异齿裂腹鱼进行麻醉行为特征研究, 为高原鱼类的麻醉以及运输提供技术支持。试验表明: MS-222麻醉大规格(25.0±1.5) cm和小规格(14.8±2.3) cm异齿裂腹鱼时, 在麻醉时期3期以内, 呼吸频率增加并之间无显著性差异, 在麻醉4期以后呼吸频率才开始显著下降。MS-222麻醉大规格和小规格异齿裂腹鱼的有效质量浓度分别为150—180 mg/L和150 mg/L。在此浓度范围内, 大规格异齿裂腹鱼在MS-222麻醉液中, 5min之内达到4级麻醉状态, 5min之内苏醒恢复, 且在麻醉液中浸浴20min后成活率为100%时的浓度; 小规格异齿裂腹鱼在MS-222麻醉液中, 5min之内达到4级麻醉状态, 7min之内苏醒恢复, 且在麻醉液中浸浴20min后成活率为100%时的浓度。大规格异齿裂腹鱼在180 mg/L的MS-222溶液中麻醉5min, 在空气中进行暴露0—15min, 复苏时间无显著性差异(P>0.05); 小规格异齿裂腹鱼在150 mg/L的MS-222溶液中麻醉5min, 在空气中进行暴露0—15min, 复苏时间存在显著性差异(P<0.05)。     

麻醉剂MS-222对斑马鱼行为的影响

实验室条件下观察并测定了斑马鱼(Danio rerio)在不同MS-222浓度处理下的麻醉行为、斑马鱼在高剂量MS-222致死过程中的行为变化、MS-222对斑马鱼摄食条件反射的影响。试验表明:(1)斑马鱼的麻醉行为是一个渐变的过程,可分为不被麻醉(Ⅰ)、轻度麻醉(Ⅱ)、中度麻醉(Ⅲ)深度麻醉(Ⅳ);(2)不同MS-222浓度下斑马鱼进入不同麻醉程度的时间有差异;(3)经MS-222处理900s过程中,80-90mg·L^-1浓度组进入Ⅲ级麻醉程度并有100%的存活率,而麻醉浓度在100mg·L^-1以上时可以迅速在49s内使鱼进入Ⅲ级麻醉程度以及在178s后即可进入Ⅳ级麻醉程度,并在中度麻醉和深度麻醉的过渡中死亡,存活率不超过10%;(4)MS-222对摄食条件反射影响的试验中,光刺激-不麻醉、无光刺激-不麻醉、光刺激-麻醉、无光刺激-麻醉四组班马鱼从投喂到摄食所用时间分别为12.06±1.34s、13.20±1.13s、56.56±56.48s、36.20±25.74s,麻醉组的摄食速度慢于对照组,说明MS-222影响了斑马鱼的摄食条件反射。     

Daily rhythms of toxicity and effectiveness of anesthetics (MS222 and eugenol) in zebrafish (Danio rerio)

Although the chronotoxicity of xenobiotics is relatively well known in mammals, the existence of daily rhythms of drug toxicity and effectiveness in fish has been neglected to date. The aim of this research was to investigate the influence of the time (middle of the light phase [ML] versus middle of the dark phase [MD]) of exposure to two anesthetic substances (MS-222 or clove oil) commonly used with fish on the median lethal concentration (LC(50)) and swimming activity of zebrafish (Danio rerio). To this end, adult zebrafish were kept under a 12 h:12 h light-dark (LD) cycle and exposed to different concentrations of the anesthetics for 15 min at ML or MD. LC(50) calculations were performed using the Spearman-Karber program, whereas swimming activity was video-recorded and analyzed with specialized software. Zebrafish exhibited a mostly diurnal activity pattern (77.9% of activity occurring during daytime). The acute toxicity and mortality caused by MS-222 and eugenol varied with the time of exposure. For MS-222, the LC(50) was 170.6 ± 7.4 mg/L in fish exposed at ML and 215.6 ± 3.9 mg/L at MD, whereas for eugenol the LC(50) was 70.3 ± 3.1 mg/L at ML and 104.9 ± 5.4 mg/L at MD. Exposure to sublethal concentrations of MS-222 and eugenol altered the swimming patterns of zebrafish in a different manner depending on the time of exposure. Thus, the time required for decreasing swimming activity during exposure to anesthetics was shorter at ML than at MD, whereas the recovery period was longer during the day. In conclusion, these results revealed that the toxicity and effectiveness of both anesthetic substances is highest during daytime, the active phase of fish, thus suggesting a link between the daily rhythms of behavior and toxicity.     

β-细辛醚对谷蠹成虫体内四种酶活性的影响

采用石菖蒲Acorus gramineus根茎提取物β-细辛醚对谷蠹Rhizopertha dominica成虫进行拌粮处理,测定β-细辛醚对谷蠹体内乙酰胆碱酯酶、谷胱甘肽S-转移酶、羧酸酯酶和酯酶同工酶的时间效应和剂量效应。结果表明：β-细辛醚对试虫体内4种酶的酶活性均表现出较强的时间效应。LC₅₀（94.49 mg/kg）剂量的β-细辛醚处理后,谷蠹成虫体内乙酰胆碱酯酶和谷胱甘肽S-转移酶活性随处理时间的延长整体表现为抑制作用,对羧酸酯酶和酯酶同工酶则表现诱导增加作用。低剂量（67.5 mg/kg）β-细辛醚对乙酰胆碱酯酶具有显著的诱导作用,但是随着处理剂量的升高,对乙酰胆碱酯酶的活力多数表现为抑制作用。低剂量（≤100.0 mg/kg）β-细辛醚对谷胱甘肽S 转移酶具有诱导作用,而高剂量（≥133.3 mg/kg）β-细辛醚对谷胱甘肽S-转移酶具有抑制作用。β-细辛醚对羧酸酯酶的活性多数表现为诱导作用,提高β-细辛醚的处理剂量可提高羧酸酯酶的活力。不同剂量的β-细辛醚处理对谷蠹酯酶同工酶均具有显著的诱导作用,但诱导效果与处理剂量关系并不显著。