Ketamine/Xylazine Anesthesia-Related Corneal Lesions in Rats With Surgically Implanted Venous Catheters Utilized in Nonclinical Intravenous Studies.

Nonclinical rodent studies with repeat slow intravenous dosing, such as safety assessments of anticancer therapeutics, often require the use of animals with surgically implanted catheters. Catheterization is a relatively short surgical procedure but requires use of anesthesia. Ketamine/xylazine injectable anesthesia is typically used because it has advantages over inhalation anesthesia including ease of administration, safety and predictability of effects, and relatively low cost. However, ketamine/xylazine anesthesia in rodents can also be associated with the development of undesirable corneal lesions of uncertain mechanism such as mineralization of Bowman's membrane or stroma, erosion/ulceration, inflammation, fibroplasia, and neovascularization. Such findings have the potential to confound study interpretation in programs for which the cornea is a potential target tissue. This case report describes the occurrence of ketamine/xylazine-related corneal lesions observed in surgically catheterized rats in a 16-day toxicity study for an oncology compound.

Measuring the middle-ear reflex: A quantitative method to assess effects of industrial solvents on central auditory pathways.

Volatile organic solvents are frequently present in industrial atmospheres. Their lipophilic properties mean they quickly reach the brain following inhalation. Acute exposure to some solvents perturbs the middle ear reflex, which could jeopardize cochlear protection against loud noises. As the physiological mechanisms involved in this protective reflex are highly complex, in vivo rodent models are required to allow rapid and reliable identification of any adverse effects of solvents on the middle ear reflex (MER). In this study, MER amplitude was measured in anesthetized Brown-Norway rats by monitoring the decrease in distortion product otoacoustic emissions (DPOAEs) caused by a contralateral stimulation. Our screening test consisted in measuring the impact of inhalation of solvent vapors at 3000 ppm for 15 min on the MER amplitude. We had previously studied a selection of aromatic solvents with this model; here, we extended the analysis to volatile compounds from other chemical families. The results obtained shed light on the mechanisms involved in the interactions between solvents and their neuronal targets. Thus, benzene and chlorobenzene had the greatest effect on MER (≥ + 1.8 dB), followed by a group composed of toluene, styrene, p-xylene, m-xylene, tetrachloroethylene and cyclohexane, which had a moderate effect on the MER (between + 0.3 and + 0.7 dB). Finally, trichloroethylene, n-hexane, methyl-ethyl-ketone, acetone, o-xylene, and ethylbenzene had no effect on the MER. Thus, the effect of solvents on the MER is not simply linked to their lipophilicity, rather it depends on specific interactions with neuronal targets. These interactions appear to be governed by the compound's chemical structure, e.g. the presence of an aromatic ring and its steric hindrance. In addition, perturbation of the MER by a solvent is independent of its toxic effects on cochlear cells. As the MER plays a protective role against exposure to high-intensity noises, these findings could have a significant impact in terms of prevention for subjects exposed to both noise and solvents.

Preventing Corneal Calcification Associated With Xylazine for Longitudinal Optical Coherence Tomography in Young Rodents.

Purpose: Spectral-domain optical coherence tomography (SD-OCT) is widely used in clinical ophthalmology and recently gained popularity in laboratory research involving small rodents. Its noninvasive nature allows repeated measurements, thereby decreasing the number of animals required. However, when used at a conventional dosage, xylazine (an α2-adrenoceptor) can cause irreversible corneal calcification, especially among young rodents. In the present study, we test whether corneal calcification associated with xylazine is mediated by the α2-adrenoceptor. Methods: Our study tested Sprague-Dawley rats, Long-Evans rats, and CD-1 mice (postnatal day [P]14). Retinal images were captured by SD-OCT. Quantitative PCR (qPCR) was used to study gene expression, whereas receptor localization was examined by immunofluorescent staining followed by confocal microscopy. Calcium deposits were detected via von Kossa staining. Results: When used at dosages appropriate for adult animals, ketamine-xylazine anesthetics led to a high rate of respiratory failure, increased apoptotic activity in the corneal epithelium, and irreversible corneal calcification in P14 rat pups. Meanwhile, OCT image quality decreased drastically as a result of corneal calcification among animals recovering from anesthesia. α2-Adrenoceptor subtypes were highly expressed on P14, in line with rodents' age-specific sensitivity to xylazine. Clonidine, a potent α2-adrenoceptor agonist, dose-dependently induced corneal calcification, which could be prevented by an α2-adrenoceptor antagonist. Conclusions: These data suggest that α2-adrenoceptors contribute to corneal calcification in young rodents. Therefore, we developed a suitable OCT imaging protocol for this cohort, including a carefully tailored ketamine-xylazine dosage (60 mg/kg and 2.5 kg/mg, respectively).

Anesthetic Ketamine-Induced DNA Damage in Different Cell Types In Vivo.

The use of a combination of ketamine and xylazine is broadly used either for anesthesia or euthanasia in rodent animal models in research. However, the genotoxicity and mutagenic effects of these drugs are unknown. Therefore, the aim of this study was to evaluate these effects to help the understanding of elevated values in negative controls in genotoxic/mutagenic assays. Sixty CF-1 mice were divided into ten groups of six mice per group: negative control (saline), positive control (doxorubicin, 40 mg/kg), ketamine at 80 mg/kg and xylazine at 10 mg/kg, ketamine at 100 mg/kg and xylazine at 10 mg/kg, ketamine at 140 mg/kg and xylazine at 8 mg/kg, ketamine at 80 mg/kg, ketamine at 100 mg/kg, ketamine at 140 mg/kg, xylazine at 8 mg/kg, and xylazine at 10 mg/kg. After drug induction, the blood cells were analyzed at 1, 12, and 24 h by the comet assay, while the brain cortex, liver, and kidney cells were verified just at 24 h by the comet assay and bone marrow was tested at 24 h by micronucleus test. The positive control was significantly different in relation to the negative control in all times and tissue analyzed. The dose of ketamine at 140 mg/kg plus xylazine at 8 mg/kg and only ketamine at 140 mg/kg exhibited a genotoxic effect in blood and brain cells at all the times analyzed. The doses of ketamine at 80 and 100 mg/kg in association or not with xylazine showed increased DNA damage at 1 and 12 h, but this effect was reversed after 24 h of drug administration. The liver, kidney, and bone marrow cells of animals treated with ketamine or xylazine isolated or combined did not differ when compared with the negative control. Then, our findings emphasize the necessity of more studies that prove safety of the ketamine use, since that anesthetic can be able to induce false-negative results in genotoxic experimental studies.

Cytotoxicity of ketamine, xylazine and Hellabrunn mixture in liver-, heart- and kidney-derived cells from fallow deer.

OBJECTIVES: Chemical restraint of wild animals is practiced to accomplish intended procedures such as capture, clinical examination, collection of diagnostic samples, treatment and/or transport. Extra-label use of animal medicinal drugs is often necessary in wildlife because most approved therapeutics do not list wild species on the labelling. Here, we used cellular in vitro models, a cutting-edge tool of biomedical research, to examine cytotoxicity of anaesthetic agents in fallow deer and extrapolate these data for anaesthetic risks in wildlife. METHODS: We examined the cytotoxic effects of ketamine, xylazine, and ketamine-xylazine, i.e. the Hellabrunn mixture, on liver-, heart- and kidney-derived cell cultures prepared from a fallow deer (Dama dama) specimen. In line with preliminary studies we exposed cells to 10 µM, 50 µM, 100 µM, 1 mM, and 10 mM ketamine or xylazine. The combination of ketamine-xylazine was dosed at 0.025+0.02 mg/ml, 0.05+0.04 mg/ml, 0.75+0.06 mg/ml, 0.1+0.08 mg/ml, and 0.125+0.1 mg/ml per one well containing 10 000 cells. The quantification of cytotoxicity was based on lactate dehydrogenase activity released from damaged cells. RESULTS: Liver-derived cells show higher sensitivity to the cytotoxic effects of both ketamine and xylazine administered as single drugs when compared with cells cultured from the heart and kidney. The Hellabrunn mixture induced significantly higher cytotoxicity for kidney-derived cells ranging from 16.78% to 35.6%. Single and combined exposures to ketamine and xylazine resulted only in high-dose cytotoxicity in the heart-derived cells. CONCLUSIONS: Our results indicate that immobilization drugs significantly differ in their cytotoxic effects on cells derived from various organs of the fallow deer.

Repeated exposure to ketamine-xylazine during early development impairs motor learning-dependent dendritic spine plasticity in adulthood.

BACKGROUND: Recent studies in rodents suggest that repeated and prolonged anesthetic exposure at early stages of development leads to cognitive and behavioral impairments later in life. However, the underlying mechanism remains unknown. In this study, we tested whether exposure to general anesthesia during early development will disrupt the maturation of synaptic circuits and compromise learning-related synaptic plasticity later in life. METHODS: Mice received ketamine-xylazine (20/3 mg/kg) anesthesia for one or three times, starting at either early (postnatal day 14 [P14]) or late (P21) stages of development (n = 105). Control mice received saline injections (n = 34). At P30, mice were subjected to rotarod motor training and fear conditioning. Motor learning-induced synaptic remodeling was examined in vivo by repeatedly imaging fluorescently labeled postsynaptic dendritic spines in the primary motor cortex before and after training using two-photon microscopy. RESULTS: Three exposures to ketamine-xylazine anesthesia between P14 and P18 impair the animals' motor learning and learning-dependent dendritic spine plasticity (new spine formation, 8.4 ± 1.3% [mean ± SD] vs. 13.4 ± 1.8%, P = 0.002) without affecting fear memory and cell apoptosis. One exposure at P14 or three exposures between P21 and P25 has no effects on the animals' motor learning or spine plasticity. Finally, enriched motor experience ameliorates anesthesia-induced motor learning impairment and synaptic deficits. CONCLUSIONS: Our study demonstrates that repeated exposures to ketamine-xylazine during early development impair motor learning and learning-dependent dendritic spine plasticity later in life. The reduction in synaptic structural plasticity may underlie anesthesia-induced behavioral impairment.

Toxic effects of xylazine on endothelial cells in combination with cocaine and 6-monoacetylmorphine.

The use of xylazine as a drug of abuse has emerged worldwide in the last 7 years, including Puerto Rico. Clinical findings reported that xylazine users present greater physiological deterioration, than heroin users. The aim of this study was to assess the xylazine toxicity on endothelial cells, as this is one of the first tissues impact upon administration. Human umbilical vein endothelial cells in culture were treated with xylazine, cocaine, 6-monoacetylmorphine (heroin metabolite) and its combinations, at concentrations of 0.10-400 µM, for periods of 24, 48 and 72 h. IC50 were calculated and the Annexin V assay implemented to determine the cell death mechanism. Results indicated IC50 values at 24h as follow: xylazine 62 µM, cocaine 210 µM, 6-monoacetylmorphine 300 µM. When these drugs were combined the IC50 value was 57 µM. Annexin V results indicated cell death by an apoptosis mechanism in cells treated with xylazine or in combination. Results demonstrated that xylazine use inhibits the endothelial cell proliferation, at lower concentrations than cocaine and 6-monoacetylmorphine. These findings contribute to the understanding of the toxicity mechanisms induced by xylazine on endothelial cells.

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC-MS, LC-MSn, and LC-HR-MSn.

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)-mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS(n)). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors' standard urine screening approaches by GC-MS and LC-MS(n). Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Determination of xylazine and 2,6-xylidine in animal tissues by liquid chromatography-tandem mass spectrometry.

Xylazine is a potent α2-adrenergic agonist used in veterinary medicine for sedation, analgesia, muscle relaxation, and so on. Its residue in animal-derived food may cause the food safety problem. Moreover, the metabolite 2,6-xylidine was reported to be a genotoxic and carcinogenic compound. Therefore, it is necessary to develop a high sensitive method for analyzing xylazine and metabolite residue in animal products. Here, we described a LC-MS/MS method for simultaneous determination of xylazine and 2,6-xylidine in 4 animal tissues: liver, meat, kidney, and fat. The samples were extracted by acetonitrile, and further clean up by hexane. The analysis was performed on a C18 reversed-phase column and API 5000 Triple Quadrupole mass spectrometry with positive electrospray ionization interface operating in the multiple-reaction monitoring mode. For all of the investigated sample matrix, the limit of detection (limit of quantitation) for xylazine and 2,6-xylidine were 0.06 (0.2) and 1.5 (5) µg/kg, respectively, the recoveries were between 63.5% and 90.8%. The precision was within the range of required criteria for method development. The presented method is sensitive and reproducible, and thus suitable for accurate quantification of xylazine and metabolite residue in animal-derived food products.

The "other" respiratory effect of opioids: suppression of spontaneous augmented ("sigh") breaths.

The purpose of this study was to examine the effects of a clinically relevant opioid on the production of augmented breaths (ABs) in unanesthetized animals breathing normal room air, using a dosage which does not depress breathing. To do this we monitored breathing noninvasively, in unrestrained animals before and after subcutaneous injection of either morphine, or a saline control. The effect of ketamine/xylazine was also studied to determine the potential effect of an alternative sedative agent. Last, the effect of naloxone was studied to determine the potential influence of endogenous opioids in regulating the normal incidence of ABs. Morphine (5 mg/kg) had no depressive effect on breathing, but completely eliminated ABs in all animals in room air (P = 0.027). However, when animals breathed hypoxic air (10% O(2)), animals did express ABs, although their incidence was still reduced by morphine (P < 0.001). This was not a result of sedation per se, as ABs continued at their normal rate in room air during sedation with ketamine. Naloxone had no effect on breathing or AB production, and so endogenous opioids are not likely involved in regulating their rate of production under normal conditions. Our results show that in unanesthetized animals breathing normal room air, a clinically relevant opioid eliminates ABs, even at a dose that does not cause respiratory depression. Despite this, hypoxia-induced stimulation of breathing can facilitate the production of ABs even with the systemic opioid present, indicating that peripheral chemoreceptor stimulation provides a potential means of overcoming the opioid-induced suppression of these respiratory events.

The effects of anesthesia on measures of nerve conduction velocity in male C57Bl6/J mice.

Animal models, particularly mice, are used extensively to investigate neurological diseases. Basic research regarding animal models of human neurological disease requires that the animals exhibit hall mark characteristics of the disease. These include disease specific anatomical, metabolic and behavioral changes. Nerve conduction velocity (NCV) is the predominant method used to assess peripheral nerve health. Normative data adjusted for age, gender and height is available for human patients; however, these data are not available for most rodents including mice. NCV may be affected by animal age and size, body temperature, stimulus strength and anesthesia. While the effects of temperature, age and size are documented, the direct and indirect effects of anesthesia on NCV are not well reported. Our laboratory is primarily concerned with animal models of diabetic neuropathy (DN) and uses NCV to confirm the presence of neuropathy. To ensure that subtle changes in NCV are reliably assayed and not directly or indirectly affected by anesthesia, we compared the effects of 4 commonly used anesthetics, isoflurane, ketamine/xylazine, sodium pentobarbital and 2-2-2 tribromoethanol on NCV in a commonly used rodent model, the C57Bl6/J mouse. Our results indicate that of the anesthetics tested, isoflurane has minimal impact on NCV and is the safest, most effective method of anesthesia. Our data strongly suggest that isoflurane should become the anesthetic of choice when performing NCV on murine models of neurological disease.

Solid-phase extraction and gas chromatographic- mass spectrometric determination of the veterinary drug xylazine in human blood.

This paper presents a method for the determination of xylazine in whole blood using solid-phase extraction and gas chromatography-mass spectrometry. This technique required only 0.5 mL of sample, and protriptyline was used as internal standard (IS). Limits of detection and quantitation (LOQ) were 2 and 10 ng/mL, respectively. The method was found to be linear between the LOQ and 3.50 microg/mL, with correlation coefficients higher than 0.9922. Precision (intra- and interday) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The analyte was stable in the matrix for at least 18 h at room temperature and for at least three freeze/thaw cycles. Mean recovery, calculated at three concentration levels, was 87%. To the best of our knowledge, this is the first time that solid-phase extraction is used as sample preparation technique for the determination of this compound in biological media. Because of its simplicity and speed when compared to other extraction techniques, the herein described method can be successfully applied in the diagnosis of intoxications by xylazine.

Comparison of cardiovascular effects of tiletamine-zolazepam, pentobarbital, and ketamine-xylazine in male rats.

We allocated 35 male Sprague-Dawley rats into 7 groups and anesthetized each by using one of the following regimens: ketamine 50 mg+xylaxine 5 mg; ketamine 75 mg+xylazine 5 mg; pentobarbital 45 mg; and Telazol 30, 40, 50, and 60 mg/kg; supplemental doses were used as required. Respiratory rate, heart rate, mean arterial pressure, cardiac index, and stroke index were measured every 30 min for 4 h. The Telazol groups showed a dose-dependent increase in duration of anesthesia. Duration of anesthesia was significantly shorter for the ketamine and pentobarbital groups than for any of the Telazol doses. Heart rate showed a dose-dependent decrease among the Telazol groups, but overall heart rate in these groups was higher than in the ketamine and pentobarbital groups. Mean arterial pressure in the Telazol 40 and 50 groups was significantly higher than the pentobarbital and higher ketamine groups yet lower than that of the Telazol 60 group. Overall animals anesthetized with Telazol showed the highest cardiac index, ketamine intermediate, and pentobarbital the lowest; cardiac index was higher in the Telazol 50 group than in either the Telazol 30 or pentobarbital groups. The pentobarbital group exhibited the lowest stroke index, whereas ketamine-treated animals had an intermediate stroke index. These differing effects of anesthetics on cardiovascular parameters must be considered when choosing an anesthesia regimen or comparing data from different studies. In our model, the Telazol 40 and 50 groups appeared to exhibit the fewest adverse cardiovascular effects.

Efficacy and safety of stored and newly prepared tribromoethanol in ICR mice.

This study, performed in conjunction with an in vitro evaluation of tribromoethanol (TBE), consisted of three trials with three objectives. The first objective was to compare anesthetic efficacy and short-term pathologic findings of TBE, ketamine-xylazine (K-X), and sodium pentobarbital (NaP). The second objective was to evaluate how changes of TBE that occur during the perceived most favorable and least favorable storage conditions (8 weeks at 5 degrees C in the dark [5D] and 25 degrees C with exposure to light [25L], respectively) affect anesthetic efficacy and short-term pathology when compared to newly prepared TBE. The third objective was to perform a 6-week clinical assessment of animals that received newly prepared TBE. All animals that received TBE (400 mg/kg) and 14 of 15 that received K-X (K, 120 mg/kg; X, 16 mg/kg) were anesthetized, as defined by loss of pedal reflex. In comparison, only 8 of 15 animals administered NaP (60 mg/kg) were anesthetized. Anesthetic duration for animals that received K-X was 31.7 min, which was significantly (P = 0.0085) longer than animals that received TBE (18.5 min). Recovery times for TBE and K-X were not significantly different (26.5 and 27.5 min, respectively). Pathologic lesions associated with TBE administration were significantly (P = 0.001) greater than those associated with K-X. NaP was not associated with any pathologic lesions. The pH of newly prepared and 5D TBE was 6.5 to 7.0, whereas that for 25L TBE was 3.0. Anesthetic induction, duration, recovery times, and pathologic lesions were not significantly different, regardless of the pH or storage condition of the solution. It was noted, however, that the average anesthetic duration for animals administered newly prepared TBE in the second trial was longer (37.7 min) than the first trial that used newly prepared TBE. For the third trial (long-term clinical assessment), the average anesthetic duration for TBE was 46.5 min, significantly (P < 0.025) longer when compared to the first trial that used newly prepared TBE. During the third trial, 10 animals were found dead or moribund. All animals that were found moribund were necropsied and found to exhibit a marked ileus. Because of the variability in anesthetic effectiveness, pathology, and morbidity and mortality associated with the use of TBE, we do not recommend the use of this anesthetic agent in ICR mice.

Ketamine/xylazine anesthesia for radiologic imaging of neurologically impaired rats: dose response, respiratory depression, and management of complications.

In vivo imaging of rats represents an important tool for outcome evaluation in research on stroke, brain trauma, and other neurologic diseases. Since sedation of animals is necessary to avoid artifacts, a mixture of ketamine and xylazine is frequently used for anesthesia. We assessed the suitable dosage of narcotics and its correlation to severe respiratory adverse events in 269 cases of ketamine/xylazine anesthesia in male Wistar rats for performance of magnetic resonance imaging after middle cerebral artery occlusion (MCAO) or sham surgery. Anesthesia depth was not measured. Anesthesia was efficacious in avoiding movement artifacts during imaging. Necessary dosage was lower if rodents were subjected to MCAO instead of sham surgery, if body weight was below baseline, and if time since surgery was short. If anesthesia was induced during the first 2 days after surgery in animals with body weight loss, necessary dose rates were 27% below doses required for rats more than 10 days post-surgery with body weight above baseline (91.4/8.3 versus 125.1/11.3 mg of ketamine/xylazine/kg). A dose adaptation scale for the prediction of necessary dose rates was developed. Apnea developed in 3.3% of all animals. Use of ketamine/xylazine anesthesia for imaging procedures is feasible and safe, though it is associated with a small risk of respiratory arrest. In case of apnea, inspiration can be provoked by a puff of air into the rat's pelt. If unsuccessful, endotracheal intubation and mechanical ventilation are needed until spontaneous breathing is restored or xylazine effects are antagonized.

Dose-threshold for thyroid tumor-promoting effects of xylazine in rats.

To clarify the threshold dose of thyroid tumor-promoting effects of xylazine hydrochloride (XZ), male F344 rats received pulverized basal diet containing 0, 250, 500, or 1000 ppm XZ for 26 weeks with or without initiation of 2400 mg/kg N-bis(2-hydroxypropyl)nitrosamine (DHPN). Thyroid weights significantly increased in the groups with or without DHPN initiation that were given 500 ppm XZ or more. The serum thyroxine (T4) and triiodothyronine (T3) levels decreased significantly in the XZ 250 and XZ 1000 ppm groups, respectively, although there were no remarkable changes in the serum thyroid-stimulating hormone (TSH) levels. Histopathologically, follicular cell hyperplasias and adenomas were induced in the DHPN-alone and DHPN+XZ groups, and the incidences and multiplicities of these lesions in the DHPN groups treated with 500 ppm XZ or more were significantly higher than those in the DHPN alone group. These results suggest that the threshold dose of rat thyroid tumor-promoting effects of XZ is between 250 and 500 ppm under the present experimental condition.

Corneal toxicity of xylazine and clonidine, in combination with ketamine, in the rat.

PURPOSE: To compare the corneal toxicity of xylazine (XYL)/ketamine (KET) with that of clonidine (CLO)/KET in the rat, in the presence or not of the alpha(2)-adrenergic antagonist yohimbine (YOH). METHODS: XYL (10 mg/kg) and CLO (0.15 mg/kg) were administered subcutaneously in the rat in combination with KET (50 mg/kg), in the presence or not of YOH (2 mg/kg). RESULTS: The corneas immediately lost transparency and luster, but recovered within 120 min. By both light and electron microscopy, a marked stromal edema and alterations of all layers were observed. In addition, XYL/KET altered the permeability of the cornea as indicated by the augmented levels of (14)C-indomethacin, topically administered 30 min after the anesthetic combination. CONCLUSIONS: The mechanism of the corneal toxicity of XYL and CLO in the rat is unclear but we speculate that: (a) proptosis and inhibition of normal blinking did not play a major role because topical application of hyaluronic acid did not protect against it; corneal decompensation, edema and opacification could be due to (b) osmotic or (c) mechanical endothelial stress: the first resulting from the sudden increase of the glucose concentration in the aqueous humor due to the well-known inhibition of insulin release by alpha(2)-adrenergic agonists, and the second from the acute elevation of intraocular pressure caused by these alpha(2)-adrenergic mydriatics in the rat; (d) addition, XYL and CLO could act by directly interacting with local alpha(2)- or, possibly, alpha(1)-adrenergic receptors, whose function is still not clear but probably essential for corneal homeostasis.

Optimization of intraperitoneal injection anesthesia in mice: drugs, dosages, adverse effects, and anesthesia depth.

PURPOSE: The goals of the study were to find a safe intraperitoneal injection anesthesia protocol for medium-duration surgery in mice (e.g., embryo transfer/vasectomy) coupled with a simple method to assess anesthesia depth under routine laboratory conditions. METHODS: Eight anesthetic protocols consisting of combinations of dissociative anesthetics (ketamine, tiletamine), alpha2-agonists (xylazine, medetomidine), and/or sedatives (acepromazine, azaperone, zolazepam) were compared for their safety and efficacy (death rate, surgical tolerance), using observations and reflex tests. The four best protocols were further evaluated during vasectomy: physiologic measurements (respiratory rate, electrocardiogram, arterial blood pressure, body temperature, blood gas tensions, and acid-base balance) were used to characterize the quality of anesthesia. The reactions of physiologic parameters to surgical stimuli were used to determine anesthesia depth, and were correlated with reflex test results. RESULTS: The protocol with the highest safety margin and the longest time of surgical tolerance (54 min) was ketamine/ xylazine/acepromazine. Three further anesthetic combinations were associated with surgical tolerance: ketamine/ xylazine, ketamine/xylazinelazaperone, and tiletamine/xylazine/zolazepam (Telazol/xylazine). The protocols consisting of ketamine/medetomidine and ketamine/azaperone were not associated with clearly detectable surgical tolerance. The most reliable parameter of surgical tolerance under routine laboratory conditions was the pedal withdrawal reflex. CONCLUSIONS: The best intraperitoneal injection anesthesia regimen consisted of ketamine/xylazine/acepromazine. The dose must be adapted to the particulars of each experimental design (mouse strain, sex, age, mutation). This is best done by measuring surgical tolerance, using the pedal withdrawal reflex.

Toxicity and blood concentrations of xylazine and its metabolite, 2,6-dimethylaniline, in rats after single or continuous oral administrations.

To cast light on whether the carcinogenic risk of 2,6-dimethylaniline (DMA), a metabolite of xylazine, may increase by ingestion of edible tissues from domestic animals treated with xylazine, the following studies of xylazine and DMA were performed. In Experiment I, male F344 rats received a single oral administration of 150 mg/kg of xylazine hydrochloride. Rats showed symptoms suggesting loss of sensation and pain immediately after the treatment. These signs had disappeared after 3 hr, but the animals died of hydrothorax and pulmonary edema by 9 hr. The plasma concentration of xylazine was 2.88 +/- 0.95 micrograms/ml at 15 min, and then decreased to 0.10 +/- 0.01 microgram/ml at 6 hr. The plasma level of DMA remained at 0.03 to 0.04 microgram/ml during the measurement period. In Experiment II, male F344 rats were fed a diet containing 1000 ppm of xylazine hydrochloride, regarded as the maximum tolerated dose, for 4 weeks. No clear clinical signs were evident and the plasma levels of xylazine and DMA were at the detection limit (0.02 microgram/ml) or less, although follicular cell hypertrophy of the thyroid was observed in all the treated animals. In Experiment III, male F344 rats were fed a diet containing 3000 ppm or 300 ppm of DMA for 4 weeks. Histological changes, such as atrophy of Bowman's gland and irregular arrangement of olfactory epithelial cells, were only observed in the olfactory epithelium of the 3000 ppm group. The plasma levels of DMA were 0.20 to 0.36 microgram/ml in the 3000 ppm group, but under the detection limit in the 300 ppm group. These results suggest that the probability of nasal carcinogenic effects of DNA on consumers via ingestion of edible tissues from food-producing animals treated with xylazine is extremely low, since DMA levels in the blood of rats subjected to continuous administration of high doses of xylazine remained under the detection limit.