Longitudinal effects of ketamine on cell proliferation and death in the CNS of zebrafish.

Zebrafish is known for its widespread neurogenesis and regenerative capacity, as well as several biological advantages, which turned it into a relevant animal model in several areas of research, namely in toxicological studies. Ketamine is a well-known anesthetic used both in human as well as veterinary medicine, due to its safety, short duration and unique mode of action. However, ketamine administration is associated with neurotoxic effects and neuronal death, which renders its use on pediatric medicine problematic. Thus, the evaluation of ketamine effects administration at early stages of neurogenesis is of pivotal importance. The 1-41-4 somites stage of zebrafish embryo development corresponds to the beginning of segmentation and formation of neural tube. In this species, as well as in other vertebrates, longitudinal studies are scarce, and the evaluation of ketamine long-term effects in adults is poorly understood. This study aimed to assess the effects of ketamine administration at the 1-4 somites stage, both in subanesthetic and anesthetic concentrations, in brain cellular proliferation, pluripotency and death mechanisms in place during early and adult neurogenesis. For that purpose, embryos at the 1-4 somites stage (10.5 h post fertilization - hpf) were distributed into study groups and exposed for 20 min to ketamine concentrations at 0.2/0.8 mg/mL. Animals were grown until defined check points, namely 50 hpf, 144 hpf and 7 months adults. The assessment of the expression and distribution patterns of proliferating cell nuclear antigen (PCNA), of sex-determining region Y-box 2 (Sox 2), apoptosis-inducing factor (AIF) and microtubule-associated protein 1 light chain 3 (LC3) was performed by Western-blot and immunohistochemistry. The results evidenced the main alterations in 144 hpf larvae, namely in autophagy and in cellular proliferation at the highest concentration of ketamine (0.8 mg/mL). Nonetheless, in adults no significant alterations were seen, pointing to a return to a homeostatic stage. This study allowed clarifying some of the aspects pertaining the longitudinal effects of ketamine administration regarding the CNS capacity to proliferate and activate the appropriate cell death and repair mechanisms leading to homeostasis in zebrafish. Moreover, the results indicate that ketamine administration at 1-4 somites stage in the subanesthetic and anesthetic concentrations despite some transitory detrimental effects at 144 hpf, is long-term safe for CNS, which are newly and promising results in this research field.

Euthanasia using gaseous agents in laboratory rodents.

Several questions have been raised in recent years about the euthanasia of laboratory rodents. Euthanasia using inhaled agents is considered to be a suitable aesthetic method for use with a large number of animals simultaneously. Nevertheless, its aversive potential has been criticized in terms of animal welfare. The data available regarding the use of carbon dioxide (CO2), inhaled anaesthetics (such as isoflurane, sevoflurane, halothane and enflurane), as well as carbon monoxide and inert gases are discussed throughout this review. Euthanasia of fetuses and neonates is also addressed. A table listing currently available information to ease access to data regarding euthanasia techniques using gaseous agents in laboratory rodents was compiled. Regarding better animal welfare, there is currently insufficient evidence to advocate banning or replacing CO2 in the euthanasia of rodents; however, there are hints that alternative gases are more humane. The exposure to a volatile anaesthetic gas before loss of consciousness has been proposed by some scientific studies to minimize distress; however, the impact of such a measure is not clear. Areas of inconsistency within the euthanasia literature have been highlighted recently and stem from insufficient knowledge, especially regarding the advantages of the administration of isoflurane or sevoflurane over CO2, or other methods, before loss of consciousness. Alternative methods to minimize distress may include the development of techniques aimed at inducing death in the home cage of animals. Scientific outcomes have to be considered before choosing the most suitable euthanasia method to obtain the best results and accomplish the 3Rs (replacement, reduction and refinement).

The anaesthetic combination of ketamine/midazolam does not alter the acquisition of spatial and motor tasks in adult mice.

The ketamine/midazolam association of a dissociative with a sedative agent is used for the induction and maintenance of anaesthesia in laboratory animals. Anaesthesia may interfere with research results through side-effects on the nervous system, such as memory impairment. It is known that ketamine and midazolam affect cognition; however, their effects have not been clarified when used in a context of balanced anaesthesia. Thus, this study evaluated the effects of ketamine/midazolam on the acquisition of motor and of a spatial memory task in adult mice. Twenty-eight C57BL/6 adult male mice were divided into three groups: untreated control, treated with ketamine/midazolam (75 mg/kg / 10 mg/kg) and treated with midazolam (10 mg/kg) groups. Respiratory rate, heart rate and systolic pressure were measured every 5 min in the animals treated with ketamine/midazolam, as this was the only group that exhibited loss of the righting reflex. One day after treatment, animals were tested in the open field, rotarod and radial arm maze. There were no differences between treatments regarding open-field activity, rotarod performance or number of working and reference memory errors in the radial arm maze task. In conclusion, the learning process of spatial and motor tasks was not disrupted by the anaesthetic combination of ketamine/midazolam. These results suggest its safe use in adult mice in projects where acquisition of a spatial and motor task is necessary.

Apoptotic neurodegeneration and spatial memory are not affected by sedative and anaesthetics doses of ketamine/medetomidine combinations in adult mice.

BACKGROUND: Ketamine is increasingly popular in clinical practice and its combination with α(2)-agonists can provide good anaesthetic stability. Little is known about the effects of this combination in the brain. Therefore, we investigated the effects of different concentrations of ketamine combined with medetomidine on cognition and its potential apoptotic neurodegenerative effect in adult mice. METHODS: Seventy-eight C57BL/6 adult mice were divided into six different groups (saline solution, 1 mg kg(-1) medetomidine, 25 mg kg(-1) ketamine+1 mg kg(-1) medetomidine, 75 mg kg(-1) ketamine+1 mg kg(-1) medetomidine, 25 mg kg(-1) ketamine, and 75 mg kg(-1) ketamine). Eight animals per group were tested in the T-maze, vertical pole, and open-field test. Five animals per group were used for histopathological [haematoxylin and eosin (HE) staining] and immunohistochemical analyses [caspase-3 activation and expression of neurotrophin brain-derived neurotrophic factor (BDNF)]. Cells showing clear HE staining and positive immunoreactions for caspase-3 and BDNF in the retrosplenial cortex, visual cortex, pyramidal cell layer of the cornu Ammonis 1 and cornu Ammonis 3 areas of the hippocampus, and in the granular layer of the dentate gyrus were counted. RESULTS: There were no differences between groups regarding the number of dead cells and cells showing positive immunoreactions in the different areas of the brain studied. Similarly, no differences were detected in the number of trials to complete the T-maze task. Nevertheless, α(2)-agonist decreased hyperlocomotion caused by ketamine in the open field. CONCLUSIONS: Neither apoptotic neurodegeneration nor alterations in spatial memory were observed with different concentrations of ketamine combined with medetomidine in adult mice.

Intraperitoneal anaesthesia with propofol, medetomidine and fentanyl in mice.

Fast recoveries are essential when looking for a safe anaesthetic protocol to use on mice. Propofol is a short-acting anaesthetic agent, which provides a smooth, fast recovery. A recent study carried out in our laboratory showed that the intraperitoneal (i.p.) administration of propofol combined with a fast-acting opioid does not provide a sufficiently stable anaesthesia. In this experiment, we hypothesized that the additional application of medetomidine would increase muscle relaxation and analgesia. Fifty-four male CD1 mice, divided into six groups of five and three groups of eight, were used to test nine different combinations of propofol, medetomidine and fentanyl administered i.p. and reversed with atipamezole 30 min after induction. These combinations were composed in the following manner: propofol 75 mg/kg, medetomidine 1 and 2 mg/kg and fentanyl 0.1, 0.15 and 0.2 mg/kg. The depth of anaesthesia, loss of righting reflex, loss of pedal withdrawal reflex, pulse rate and respiratory rate were recorded along with the duration and quality of the recovery. The combination of propofol and medetomidine provided a predictable induction, hypnosis and muscle relaxation, but surgical anaesthesia (loss of pedal withdrawal reflex) was not achieved. The addition of fentanyl increased analgesia leading to surgical anaesthesia. We concluded that a combination of 75/1/0.2 mg/kg of propofol, medetomidine and fentanyl, respectively, is a safe, easy and reversible technique for i.p. anaesthesia in mice, providing a surgical window of 15 min and restraint for 30 min with a fast recovery.

Intraperitoneal propofol and propofol fentanyl, sufentanil and remifentanil combinations for mouse anaesthesia.

The combination of propofol and a rapid-acting opioid, such as fentanyl, sufentanil or remifentanil, is a relatively safe, total intravenous anaesthesia technique, commonly used in humans and which has been investigated in laboratory animals. The objective of this study was to evaluate these combinations for anaesthesia of mice by the intraperitoneal (i.p.) route. Sixty-seven mice, divided into groups of four, were used to test 28 combinations of propofol alone and propofol with fentanyl, sufentanil or remifentanil administered i.p. The dose ranges of drugs studied were propofol 50-200 mg/kg, fentanyl 0.2-0.4 mg/kg, sufentanil 0.05-0.1 mg/kg and remifentanil 0.2-1.0 mg/kg. The loss of righting reflex (RR) and the loss of pedal withdrawal reflex (PWR) were recorded along with the duration and quality of recovery. The results obtained in these studies were unpredictable. The same dose combinations of propofol and opioids were associated with different responses in different individuals. Higher doses did not induce loss of RR and PWR in all animals and were associated with high mortality rates. An adequate hypnotic level was only observed with higher doses of propofol. The synergistic effect of propofol and the opioids was not sufficient to allow surgical procedures. Animals that reached PWR loss showed tail rigidity, shaking limbs and scratched their heads with their forefeet. Higher opioid doses induced respiratory depression and higher death rates. The inconsistency between and within groups may be associated with the i.p. route. The results reported here show that the i.p. route is not appropriate for mouse anaesthesia using propofol alone or in combination with fentanyl, sufentanil or remifentanil.