Ana M. Valentim

Lower Isoflurane Concentration Affects Spatial Learning and Neurodegeneration in Adult Mice Compared with Higher Concentrations

Background:Volatile anesthetics such as isoflurane are widely used in clinical and research contexts. Concerns have been raised that the effects of these drugs on the central nervous system may result in long-term impairment after surgery or general anesthesia. Hence, this study aimed to detect how different isoflurane concentrations influence spatial learning and cell death in adult mice. Methods:Fifty-two C57BL/6 mice were randomly divided in four groups. Mice in three groups were exposed to different concentrations of isoflurane (1, 1.5, and 2%) for 1 h; the control group was not exposed to anesthesia. Five mice per group were killed 3 h after anesthesia to perform histopathologic and immunohistochemical analyses (hematoxylin-eosin staining; caspase-3 activation). Eight mice per group were used for behavioral tests (open field, T-maze spontaneous alternation, and water maze) on subsequent days. Results:There were no differences between groups in the T-maze spontaneous alternation test or in the open field (no confounding effects of stress or locomotion). The group anesthetized with 1% isoflurane performed worse in the water maze task on day 1 (550.4 ±162.78 cm) compared with the control group (400.1 ± 112.88 cm), 1.5% isoflurane (351.9 ± 150.67 cm), and 2% isoflurane (364.5 ± 113.70 cm; P ≤ 0.05) and on day 3 (305.0 ± 81.75 cm) compared with control group (175.13 ± 77.00 cm) and 2% isoflurane (204.11 ± 85.75 cm; P ≤ 0.038). In the pyramidal cell layer of the region cornu ammonis 1 of the hippocampus, 1% isoflurane showed a tendency to cause more neurodegeneration (apoptosis) (61.4 ± 26.40, profiles/mm2) than the group with 2% of isoflurane (20.6 ± 17.77, profiles/mm2; P = 0.051). Conclusion:Low isoflurane concentration (1%) caused spatial learning impairment and more neurodegeneration compared with higher isoflurane concentrations. Results for mice receiving the latter concentrations were similar to those of control 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.

Embryonic Stage-Dependent Teratogenicity of Ketamine in Zebrafish (Danio rerio)

Ketamine, a widely used anesthetic, has been shown to have NMDA receptor dependent and independent actions during zebrafish (Danio rerio) embryogenesis. Notwithstanding, the effects of developmental toxicity and the mechanisms of ketamine action on fish embryos are still not well understood, and its implications for early vertebrate development remains to be clarified. In this work, zebrafish embryos were exposed to ketamine (0.2, 0.4, and 0.8 mg mL(-1)) in order to study the stage-developmental toxicity of this pharmaceutical. During 256-cell (2.5 h post-fertilization, hpf), 50% epiboly (5.5 hpf) and 1-4 somites (10.5 hpf), embryos were exposed to the referred ketamine concentrations for a period of 20 min and were allowed to grow until 144 hpf. Both lethal and nonlethal parameters were evaluated. Skeletal development was assessed by alcian blue and calcein staining. Additionally, the expression of the developmental genes sonic hedgehog a (shh a) and noggin 3 (nog3) was evaluated. Similar to our previous work, bone and cartilage malformations were observed after 256-cell exposure. During 50% epiboly, ketamine exposure induced concentration-dependent mortality and malformations, such as lordosis and/or kyphosis and microcephaly, namely, at higher concentrations. Conversely, exposure during 1-4 somites showed the induction of nonspecific effects with no rise in mortality. The quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed differences in shh a and nog3 expressions comparatively to the control group. Overall, this study shows that the ketamine toxic profile is developmental phase-dependent with 256-cell being the most susceptible phase. The effects observed may result from ketamine interaction with cellular signaling pathways that merits further investigation.

A New Anaesthetic Protocol for Adult Zebrafish (Danio rerio): Propofol Combined with Lidocaine

Background The increasing use of zebrafish model has not been accompanied by the evolution of proper anaesthesia for this species in research. The most used anaesthetic in fishes, MS222, may induce aversion, reduction of heart rate, and consequently high mortality, especially during long exposures. Therefore, we aim to explore new anaesthetic protocols to be used in zebrafish by studying the quality of anaesthesia and recovery induced by different concentrations of propofol alone and in combination with different concentrations of lidocaine. Material and Methods In experiment A, eighty-three AB zebrafish were randomly assigned to 7 different groups: control, 2.5 (2.5P), 5 (5P) or 7.5 μg/ml (7.5P) of propofol; and 2.5 μg/ml of propofol combined with 50, (P/50L), 100 (P/100L) or 150 μg/ml (P/150L) of lidocaine. Zebrafish were placed in an anaesthetic water bath and time to lose the equilibrium, reflex to touch, reflex to a tail pinch, and respiratory rate were measured. Time to gain equilibrium was also assessed in a clean tank. Five and 24 hours after anaesthesia recovery, zebrafish were evaluated concerning activity and reactivity. Afterwards, in a second phase of experiments (experiment B), the best protocol of the experiment A was compared with a new group of 8 fishes treated with 100 mg/L of MS222 (100M). Results In experiment A, only different concentrations of propofol/lidocaine combination induced full anaesthesia in all animals. Thus only these groups were compared with a standard dose of MS222 in experiment B. Propofol/lidocaine induced a quicker loss of equilibrium, and loss of response to light and painful stimuli compared with MS222. However zebrafish treated with MS222 recovered quickly than the ones treated with propofol/lidocaine. Conclusion In conclusion, propofol/lidocaine combination and MS222 have advantages in different situations. MS222 is ideal for minor procedures when a quick recovery is important, while propofol/lidocaine is best to induce a quick and complete anaesthesia.

Effects of depth of isoflurane anaesthesia on a cognition task in mice

Editor—Several clinical studies have suggested the existence of postoperative cognitive dysfunction (POCD), especially in elderly patients. 2 However, human studies carry many variables. As the sole effects of anaesthesia are difficult to study, we used mice without performing surgery to study the role of anaesthesia in cognitive dysfunction. Thirty 10–12-week-old inbred male mice (approved by DGV, Portugal) were randomly assigned into three groups: control group (animals not anaesthetized); Group I (anaesthetized with 1% isoflurane); and Group II (anaesthetized with 2% isoflurane). The animals were placed individually in an induction chamber, and anaesthesia was induced with 3% isoflurane (Isoflo, Esteve Farma, Carnaxide, Portugal) in 100% oxygen with a delivery rate of 5 litre min until loss of righting reflex. After induction, the animals were moved into a plastic zip bag and placed in dorsal recumbence. Anaesthesia was then maintained with isoflurane in 100% oxygen with a flow of 1.5 litre min. Heart rate and ventilatory frequency were recorded over intervals of 10 min. Body temperature was maintained at 37+28C by a homeothermic blanket (N-HB101-S-402, Panlab, Barcelona, Spain) placed under the zip bag. Animals remained anaesthetized for 1 h. Isoflurane concentration was monitored in the exhausted air with an agent gas monitor (Datex Capnomac Ultima, Helsinki, Finland), and no stimuli were applied. At recovery, all animals received 100% oxygen until the gain of righting reflex. Animals from the control group were not anaesthetized; however, they were manipulated and placed inside the induction chamber for 1 min (average time until loss of the righting reflex in anaesthetized animals) and were thereafter returned to their home cage to avoid isolation stress. One week before the procedure, a food restriction schedule was established to ensure feeding motivation in the spatial learning task. At 28 and 56 h after anaesthesia, mice were tested in an eight-arm radial maze (RAM) where animals had to find a piece of cereal. During the test, only one of the choice arms was baited, and this arm remained the same throughout the experiment. Each trial was over when the mouse entered the correct arm and ate the reward. Each animal made 15 test trials per session. An error was recorded when a mouse entered an unrewarded arm. Number of errors before correct choice in each trial, and time to complete the trial was also recorded. Student’s t-testing for independent samples showed that Group II had a significantly higher heart rate (P,0.0004) and a lower ventilatory frequency (P,0.0002) 20 min after induction of anaesthesia compared with Group I. No differences were detected between the groups concerning the number of errors and time to complete the task using repeated measures ANOVA. Twenty-eight hours post-anaesthesia, the control group had a higher number of trials with zero errors and a lower number of trials with one error compared with Group II, using Kruskal–Wallis and Mann– Whitney test (Fig. 1). At 56 h post-anaesthesia, animals did not show any difference at this level.

Ketamine-induced oxidative stress at different developmental stages of zebrafish (Danio rerio) embryos

Ketamine, a widely used anesthetic in a variety of species, has been shown to exert a potential teratogenic effect during the early life stages of zebrafish. A number of mechanisms have been suggested for the etiology of teratogens. One of the most studied involves reactive oxygen species (ROS) formation and oxidative damage. In this study, zebrafish embryos were used to analyze oxidative stress as a potential mechanism of ketamine-induced toxicity. The changes in the accumulation and in vivo patterns of ROS, enzymatic activities (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE)), glutathione levels (oxidized (GSSG) and reduced (GSH)), oxidative damage (lipid peroxidation (LPO) and protein carbonyls (CO)) and gene expression (gclc, gstp1, sod1 and cat) were evaluated at 8 and 24 hours post fertilization (hpf) in zebrafish embryos exposed during 20 minutes to 0.2, 0.4 and 0.8 mg mL−1 ketamine in the course of blastula (2.5 hours post fertilization-hpf), gastrula (5.5 hpf) and segmentation (10.5 hpf). Although no changes in ROS patterns were visible after all ketamine exposures, an increase in GSH levels was observed after exposure during blastula, indicating possible alterations in cell oxidative capacity. After exposure in gastrula, an increase in SOD and CAT enzymatic activities along with an increase in GSSG levels were observed at 8 hpf. At 24 hpf, CAT activity remained higher in ketamine exposed groups. The expression of the cat gene was also augmented at this time point. The changes were related with the ability of the embryo to handle oxidative stress and to a turning point during development of the oxidative defense system. At segmentation, the exposure to ketamine induced changes in the accumulation of ROS and sod gene expression which were related to protective mechanisms against ketamine-induced oxidative stress. Changes in acetylcholinesterase were also observed which may be related to changes in ROS. The overall results show that ketamine induces phase-dependent oxidative stress misregulation that could be the key factor for ketamine toxicity and could help to elucidate and provide more information on the mechanism of embryotoxicity of ketamine.

A single intraperitoneal injection of ketamine does not affect spatial working, reference memory or neurodegeneration in adult mice: An animal study.

BACKGROUND Ketamine is an anaesthetic and analgesic drug used in research and clinical practice. Little is known about the effects of different doses of this drug on memory and brain cellular death. OBJECTIVE To study the effects of different doses of ketamine on working and reference memory, and neurodegeneration in adult mice. DESIGN A randomised study. SETTINGS The study was carried out in a basic science laboratory, between March 2011 and August 2012. ANIMALS Forty-eight 7-month-old, male C57BL/6 mice were used. INTERVENTION Animals received a single intraperitoneal injection of physiological saline solution or one of three doses of ketamine (25, 75 or 150 mg kg−1). Each group consisted of 12 animals (seven animals for behavioural tests and five animals for histopathological and immunohistochemical studies). The animals used for histopathology studies were sacrificed 3 h after anaesthesia. MAIN OUTCOME MEASURES Working and reference memories were assessed using the radial-maze test over 12 consecutive days. The equilibrium was tested using the vertical pole (4 and 24 h after injection), whereas locomotion was assessed using the open field (24, 48 and 72 h after injection). Histopathological (haematoxylin-eosin staining) and immunohistochemical analyses (procaspase-3 and activated caspase-3 detections) were performed 3 h after injection to assess neurodegeneration in the retrosplenial and visual cortices, pyramidal cell layer of the cornu Ammonis 1 and cornu Ammonis 3 areas of the hippocampus, in the granular layer of the dentate gyrus, in the laterodorsal thalamic nucleus, striatum and accumbens nucleus. RESULTS No significant differences were observed between the groups regarding the number of dead cells and cells showing positive immune-reactivity in the different regions of the brain studied. The performance in the vertical pole test and the number of reference and working memory errors in the radial-maze were similar in all groups. Nevertheless, the animals treated with ketamine 75 mg kg−1 were transiently more active, walking a greater total distance at a greater speed in the open field than other groups (power of 0.96). CONCLUSION These data indicate that a single intraperitoneal injection of ketamine at subanaesthetic and anaesthetic doses does not impair working memory, reference memory or neurodegeneration in adult mice, but an intermediate dose of ketamine produces transitory hyperlocomotion.

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.