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.

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).

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.

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.

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.

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.

The regulatory status of xylazine for use in food-producing animals in the United States.

Xylazine is commonly used in veterinary medicine as a tranquillizer or adjunct to surgical anaesthesia. Although its use is approved in companion animals and certain species of deer, xylazine remains unapproved for use in food-producing animals in the United States. This paper reviews existing toxicological and residue chemistry information on xylazine in food animals, particularly cattle, and discusses the regulatory status of the drug in the US, as well as the conclusions reached by the Joint FAO/WHO Expert Committee on Food Additives in its recent evaluation of xylazine.

Emesis induced in domestic pigs: a new experimental tool for detection of antiemetic drugs and for evaluation of emetogenic potential of new anticancer agents.

The domestic pig was used to develop a new model for evaluating the emetogenic potential of anticancer drugs and determining the antiemetic activity of drugs. Emesis was characterized by expulsion of solid or liquid material. In each animal, the number of vomits after infusion of the emetogenic drug (infusion in ketamine and xylazine anesthesia) was recorded in 1-hr periods during the first 4 hr and then in a 4- and a 16-hr period. Intravenous infusion of cisplatin caused a concentration-dependent emetic response. Anti-cancer drugs other than cisplatin such as carboplatin, dactinomycin, cyclophosphamide, and ifosfamide, also induced emesis, indicating that the domestic pig is suitable to detect the emetogenic potential of chemotherapeutic agents. A cisplatin dose of 2 mg/kg i.v. proved to be most suitable for studying the effect of potential antiemetic drugs (applied as i.v. injection), because this cisplatin dose caused consistent emetic responses without other toxic signs in the 24 hr following its infusion. Emesis induced by cisplatin was reduced by high doses of metoclopramide (25 mg/pig; approximately 0.8 mg/kg). The more selective dopamine D2 receptor antagonists, alizapride and domperidone, even at high doses (25-50 mg/pig; approximately 0.8-1.6 mg/kg), did not inhibit cisplatin-induced emesis, nor did haloperidol up to 20 mg/pig (approximately 0.6 mg/kg). Sulpride (50 mg/pig; approximately 1.6 mg/kg) halved the occurrence of vomits in the first 4 hr after cisplatin, but this effect was followed by an increase in the frequency of vomits; thus, no change in the total number of vomits was observed in the 24-hr observation period.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and morphological alterations in xylazine-induced pulmonary edema.

Sprague-Dawley rats were given 42 mg/kg xylazine intramuscularly, and lungs were lavaged with phosphate-buffered saline 3, 6, and 12 hr later. Total protein, lactate dehydrogenase (LDH), xanthine oxidase (XO), tumor necrosis factor (TNF), and interleukin 1 (IL-1) were measured in bronchoalveolar lavage fluid (BALF). Protein concentration, LDH, XO, and TNF levels were increased (p < 0.05) in the BALF from xylazine-treated rats as compared to controls. IL-1 level was unchanged at 3 and 6 hr and was reduced (p < 0.05) at 12 hr. Another group of rats was given 42 mg/kg xylazine intramuscularly, and lungs were fixed 0.5 and 12 hr later. Histologically, severe pulmonary edema (PE) involving the alveoli and perivascular stroma was observed. Fibrin, increased numbers of eosinophils, and macrophages with foamy cytoplasm were present in the alveoli of all treated animals. Ultrastructurally, endothelial damage, characterized by thinning, detachment from basement membranes, or bleb formation, was observed. The lesions were similar in both xylazine groups, differing mainly in severity with the 12-hr group having more severe lesions than the 0.5-hr group. To determine whether endothelial injury is caused by direct toxicity of xylazine, bovine pulmonary artery endothelial cells (BPAECs) were incubated with xylazine (0.3, 3, and 30 micrograms) for 0.5 or 3 hr. Xylazine did not have any effects on BPAECs, as indicated by phase-contrast microscopy and dye-exclusion viability assay. These results indicate that xylazine-induced PE is due to increased permeability resulting from endothelial injury, which is not caused by direct effect of xylazine on pulmonary endothelium. While oxygen radicals and TNF are possibly involved, IL-1 does not appear to play a role in xylazine-induced PE.

Xylazine-induced pulmonary edema in rats.

Inhibitors of cytochrome P450, such as SK&F 525-A, prolong the duration of xylazine-ketamine anesthesia and cause pulmonary edema (PE) and death in rats. To determine the cause of PE, Sprague-Dawley rats were given a single dose of xylazine (21 mg/kg, im) alone or in combination with ketamine (45 mg/kg, im) and/or SK&F 525-A (50 mg/kg, ip) and percentage lung to body weight (%LW/BW) ratios (as an indicator of PE) were compared. The results indicated that xylazine caused PE which was independent of ketamine and was enhanced by SK&F 525-A. Subsequently, it was determined that 42 mg/kg xylazine, im, is an optimal edemagenic dose. Xylazine (42 mg/kg, im) increased the %LW/BW ratio as compared to control. Pleural effusion (PLE) of various amounts was observed in 75% of the animals. The pleural fluid to serum protein ratio for xylazine was similar to that obtained for alpha-naphthylthiourea (5 mg/kg, ip). Extensive serous PLE and alveolar edema with hemorrhage were found at necropsy in xylazine-treated rats. Pretreatment with yohimbine (4.2 mg/kg), prazosin (20 mg/kg), tolazoline (20 mg/kg), yohimbine (4.2 mg/kg) plus prazosin (20 mg/kg), atropine (20 mg/kg), dimethyl sulfoxide (DMSO) (7.8 g/kg), allopurinol (50 mg/kg), superoxide dismutase (20,000 U/kg), catalase (20,000 U/kg), BW755C (50 mg/kg), ibuprofen (50 mg/kg), cystathionine (100 mg/kg) plus taurine (100 mg/kg) did not affect the %LW/BW ratio. PLE was increased by yohimbine, yohimbine plus prazosin, and allopurinol, reduced by DMSO, and not changed in other groups. The results indicate that xylazine caused increased-permeability PE characterized by rapid onset, cellular damage and protein-rich pleural fluid. PE may not be mediated by adverse cardiovascular effects of xylazine and oxygen radicals are possibly involved in its etiology.

Effects of xylazine and ketamine on epinephrine-induced arrhythmia in the dog.

Ten dogs were studied to determine the effects of xylazine, ketamine, and xylazine combined with ketamine on the dosage of epinephrine required to produce ventricular arrhythmia. Untreated dogs required an arrhythmogenic dose (AD) of 5.88 +/- 2.85 micrograms/kg/min. The AD was 4.28 +/- 3.25 micrograms/kg/min in xylazine-treated dogs, 3.05 +/- 2.3 micrograms/kg/min in ketamine-treated dogs, and 2.96 +/- 1.95 micrograms/kg/min in xylazine/ketamine-treated dogs. The latter two dosages were significantly less than that of the controls (p less than 0.025). The duration of increased arrhythmogenicity was also examined. Four hours after drug administration, the AD for xylazine-treated dogs was decreased further to 3.87 +/- 2.52 micrograms/kg/min (p less than 0.05). Ketamine-treated dogs had returned partially to normal with an AD of 4.09 +/- 3.09 micrograms/kg/min, as had xylazine/ketamine-treated dogs, at 4.22 +/- 2.71 micrograms/kg/min.

Effects of xylazine administration on serum amylase, pancreas-specific isoamylase and pancreatic polypeptide in normal dogs.

Changes in serum total amylase, pancreas-specific isoamylase and pancreatic polypeptide activities were measured in six dogs following multiple intramuscular injections of xylazine at 0.5 mg/kg of body weight. The activities of those analytes did not change over 24 hours of study.