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Ketamine/xylazine and barbiturates modulate microglial morphology and motility differently in a mouse model.

ABSTRACT: Microglia, the resident immune cells of the brain, are highly ramified and motile and their morphology is strongly linked to their function. Microglia constantly monitor the brain parenchyma and are crucial for maintaining brain homeostasis and fine-tuning neuronal networks. Besides affecting neurons, anesthetics may have wide-ranging effects mediated by non-neuronal cells and in particular microglia. We thus examined the effect of two commonly used anesthetic agents, ketamine/xylazine and barbiturates, on microglial motility and morphology. A combination of two-photon in vivo imaging and electroencephalography (EEG) recordings in unanesthetized and anesthetized mice as well as automated analysis of ex vivo sections were used to assess morphology and dynamics of microglia. We found that administration of ketamine/xylazine and pentobarbital anesthesia resulted in quite distinct EEG profiles. Both anesthetics reduced microglial motility, but only ketamine/xylazine administration led to reduction of microglial complexity in vivo. The change of cellular dynamics in vivo was associated with a region-dependent reduction of several features of microglial cells ex vivo, such as the complexity index and the ramification length, whereas thiopental altered the size of the cytoplasm. Our results show that anesthetics have considerable effects on neuronal activity and microglial morphodynamics and that barbiturates may be a preferred anesthetic agent for the study of microglial morphology. These findings will undoubtedly raise compelling questions about the functional relevance of anesthetics on microglial cells in neuronal physiology and anesthesia-induced neurotoxicity.

SUBMITTER: Hristovska I

PROVIDER: S-EPMC7410236 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Ketamine/xylazine and barbiturates modulate microglial morphology and motility differently in a mouse model.

Hristovska Ines I Verdonk Franck F Comte Jean-Christophe JC Tsai Eileen S ES Desestret Virginie V Honnorat Jérôme J Chrétien Fabrice F Pascual Olivier O

PloS one 20200806 8

Microglia, the resident immune cells of the brain, are highly ramified and motile and their morphology is strongly linked to their function. Microglia constantly monitor the brain parenchyma and are crucial for maintaining brain homeostasis and fine-tuning neuronal networks. Besides affecting neurons, anesthetics may have wide-ranging effects mediated by non-neuronal cells and in particular microglia. We thus examined the effect of two commonly used anesthetic agents, ketamine/xylazine and barbi ...[more]

PMID: 32760073

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Project description:Recent interest in reversal of the hypnotic effects of anesthesia has mainly focused on overcoming a surge in GABA-mediated inhibitory signaling through activation of subcortical arousal circuits or antagonizing GABA receptors. Here we examine the reversal of anesthesia produced from non-GABA agents ketamine/xylazine and the effects of antagonists of adrenoreceptors. These antagonists vary in selectivity and produce temporally unique waking behavior post-anesthesia. We compared two antagonists with differential selectivity for ?1- vs. ?2-receptors, yohimbine (YOH, 1:40 selectivity) and atipamezole (ATI, 1:8500). Adult mice received intraperitoneal injections of either YOH (4.3 mg/kg), ATI (0.4 mg/kg), or saline after achieving sustained loss of righting following injection of ketamine/xylazine (ketamine: 65.0 mg/kg; xylazine: 9.9 mg/kg). Behaviors indicative of the post-anesthesia, re-animation sequence were carefully monitored and the timing of each behavior relative to anesthesia induction was compared. Both YOH and ATI hastened behaviors indicative of emergence, but ATI was faster than YOH to produce certain behaviors, including whisker movement (YOH: 21.9±1.5 min, ATI: 17.5±0.5 min, p = 0.004) and return of righting reflex (RORR) (YOH: 40.6±8.8 min, ATI: 26.0±1.2 min, p<0.001). Interestingly, although YOH administration hastened early behavioral markers of emergence relative to saline (whisking), the completion of the emergence sequence (time from first marker to appearance of RORR) was delayed with YOH. We attribute this effect to antagonism of ?1 receptors by yohimbine. Also notable was the failure of either antagonist to hasten the re-establishment of coordinated motor behavior (e.g., attempts to remove adhesive tape on the forepaw placed during anesthesia) relative to the end of emergence (RORR). In total, our work suggests that in addition to pharmacokinetic effects, re-establishment of normal waking behaviors after anesthesia involves neuronal circuits dependent on time and/or activity.

Project description:The morphology of astrocytic processes determines their close structural association with synapses referred to as the 'tripartite synapse'. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes. Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as isoform-specific functions.

Project description:ObjectivesTo explore the effect of ketamine-xylazine anesthesia on light-induced retinal degeneration in rats.MethodsRats were anesthetized with ketamine and xylazine (100 and 5 mg, respectively) for 1 h, followed by a recovery phase of 2 h before exposure to 16,000 lux of environmental illumination for 2 h. Functional assessment by electroretinography (ERG) and morphological assessment by in vivo imaging (optical coherence tomography), histology (hematoxylin/eosin staining, TUNEL assay) and immunohistochemistry (GFAP and rhodopsin staining) were performed at baseline (ERG), 36 h, 7 d and 14 d post-treatment. Non-anesthetized animals treated with light damage served as controls.ResultsKetamine-xylazine pre-treatment preserved retinal function and protected against light-induced retinal degeneration. In vivo retinal imaging demonstrated a significant increase of outer nuclear layer (ONL) thickness in the non-anesthetized group at 36 h (p<0.01) and significant reduction one week (p<0.01) after light damage. In contrast, ketamine-xylazine pre-treated animals showed no significant alteration of total retinal or ONL thickness at either time point (p>0.05), indicating a stabilizing and/or protective effect with regard to phototoxicity. Histology confirmed light-induced photoreceptor cell death and Müller cells gliosis in non-anesthetized rats, especially in the superior hemiretina, while ketamine-xylazine treated rats showed reduced photoreceptor cell death (TUNEL staining: p<0.001 after 7 d), thicker ONL and longer IS/OS. Fourteen days after light damage, a reduction of standard flash induced a-wave amplitudes and a-wave slopes (p?=?0.01) and significant alterations in parameters of the scotopic sensitivity function (e.g. Vmax of the Naka Rushton fit p?=?0.03) were observed in non-treated vs. ketamine-xylazine treated animals.ConclusionsOur results suggest that pre-treatment with ketamine-xylazine anesthesia protects retinas against light damage, reducing photoreceptor cell death. These data support the notion that anesthesia with ketamine-xylazine provides neuroprotective effects in light-induced cell damage.

Project description:OBJECTIVE:A simple but reliable and safe anaesthetic procedure is required for surgical interventions in small rodents. Combined ketamine and xylazine injections are often used in rats for less invasive surgery, possibly with spontaneous breathing and without airway management. However, there are important pitfalls to be avoided by special precautions and monitoring, as shown subsequently. STUDY DESIGN:Observational study. ANIMALS:Twenty-four anaesthetic procedures for bile duct ligation, sham operation or carotid artery dilatation in 20 male Sprague-Dawley rats, preoperatively weighing between 440 and 550 g. METHODS:Intolerable high mortality rates occurred in the first 7 postoperative days while establishing a new experimental model in rats using ketamine-xylazine anaesthesia. Rats were spontaneously breathing ambient air during the first 12 surgeries without airway management. An observed high mortality rate in these animals led to a change in the trial protocol: the insufflation of 2 litres of oxygen per minute via nose cone during the following 12 rat surgeries. Retrospective comparison of the outcome (without oxygen vs. with oxygen insufflation) was conducted. RESULTS:The perioperative mortality rate could be significantly reduced from 58% (7/12) to 17% (2/12) (p = 0.036) by oxygen insufflation via nose cone. Significantly different levels of intraoperative oxygen saturation (SpO2; 89 ± 4% [without oxygen] vs. 97 ± 0.5% [with oxygen], p < 0.0001), but no significant differences in heart rate (HR; 267 ± 7 beats minute-1 [bpm] [without oxygen] vs. 266 ± 6 bpm [with oxygen], p = 0.955) were observed. CONCLUSIONS AND CLINICAL RELEVANCE:In summary, rats under ketamine-xylazine anaesthesia are susceptible to hypoxia. This may lead to increased delayed mortality related to hypoxia induced lung failure. Apparently, this is an underestimated problem. We highly recommend using additional oxygen insufflation in spontaneously breathing rats under ketamine-xylazine anaesthesia with basic monitoring such as measurement of oxygen saturation.

Project description:Anesthesia for mice is commonly performed through the injection of parenteral agents via the intraperitoneal (IP) route. Variability in anesthetic sensitivities has been noted in mice resulting in inconsistencies in anesthetic depth and/or mortality. Anesthetic protocols that improve consistency and safety are needed. The objectives of this study were to assess the effects of intraperitoneal (IP) ketamine (95 mg/kg) and xylazine (7 mg/kg) alone or combined with lidocaine at 4, 8, or 16 mg/kg on time to loss (LRR) and return (RRR) of righting reflex, duration of immobilization and loss of pedal withdrawal response (PWR), body weight and histopathology in CD-1 mice. In a prospective, randomized trial, 36 male CD-1 mice, 4-6 weeks of age were randomly assigned to 5 groups: saline (SA, n = 4); ketamine-xylazine (KX, n = 8); ketamine-xylazine-lidocaine 4 mg/kg (KXL4, n = 8); ketamine-xylazine-lidocaine 8 mg/kg (KXL8, n = 8); ketamine-xylazine-lidocaine 16 mg/kg (KXL16, n = 8). Two mice in each group were euthanized at day 2 post-injection and the remaining mice were euthanized at day 11 post-injection. After IP injection, LRR and RRR, duration of immobilization and loss of PWR, body weight and histopathology were evaluated. LRR occurred sooner in mice receiving KXL16 compared with KX, with median (range) times of 78 (62-104) and 107 (91-298) seconds, respectively. Loss of PWR occurred in 1, 5, 4, 6 mice for groups KX, KXL4, KXL8, and KXL16 respectively. Median (range) duration of absent PWR was longer in mice receiving KXL16 at 13 (0-30) minutes, compared to KX at 0 (0-9) minutes. Duration of immobilization and RRR were not different between groups. Weight loss occurred 2 days following anesthesia but was not different between groups. Weight gain was significantly greater in all lidocaine groups 11 days post-injection compared to KX. No mortality or histopathologic abnormalities were observed in any group. Lidocaine administered with ketamine and xylazine shortens the onset of anesthesia in mice and improves anesthetic depth without prolonging recovery time.

Project description:Chemical immobilisation is an integral component for the conservation of wild animals and can be stressful if proper protocols are not administered. References on the immobilisation of Arabian striped hyaena (Hyaena hyaena sultana) are scarce. The current study was designed to evaluate the physiological and clinical responses of Arabian striped hyaena, immobilised with ketamine-medetomidine (KM) and ketamine-xylazine (KX); and to compare immobilisation effectiveness of the two combinations in a cross-sectional clinical study. A total of 15 (six males, nine females) (semi-) captive and adult Arabian striped hyaena with an average weight of 31.39 ± 0.36 kg were immobilised 50 times for annual vaccination and translocation purposes from January 2014 till March 2018 on Sir Bani Yas Island, United Arab Emirates. A total of 34 immobilisations were executed with (Mean ± SE) 2.27 ± 0.044 mg/kg ketamine and 0.04 ± 0.001 mg/kg medetomidine; while 16 with 4.95 ± 0.115 mg/kg ketamine and 0.99 ± 0.023 mg/kg xylazine. The drugs were remotely delivered intramuscular. The evaluation of physiological and clinical parameters included monitoring of vital signs through pulse oximetry, blood gas analysis of arterial blood through Istat blood gas analyser, and blood biochemistry and haematology. The quality of induction, anaesthesia and recovery was also assessed. Atipamezole (0.21 ± 0.003 mg/kg) was used to antagonise the effects of KM and 0.09 ± 0.003 mg/kg atipamezole or by 0.23 ± 0.006 mg/kg yohimbine for KX. Data were analysed using the general linear model and inferential statistics. KM was more effective in induction (scores; KM = 1.41 ± 0.10; KX = 1.31 ± 0.12), anaesthesia (KM = 1.00 ± 0.00; KX = 2.0 ± 0.0) and recovery (KM = 1.76 ± 0.15; KX = 2.69 ± 0.12) phases as compared to KX. There was a significant difference (P < 0.05) amongst the two combinations for anaesthesia time (KM = 59.5 ± 2.41; KX = 49.25 ± 1.31 min.), time to stand after reversal (KM = 4.91 ± 0.60; KX = 10.38 ± 1.48 min.) and full loss of the signs of anaesthetics (KM = 12.32 ± 1.37; KX = 21.25 ± 2.16 min.) along with rectal temperature (KM = 37.58 ± 0.29; KX = 36.00 ± 0.68 °C), pulse rate (KM = 50.46 ± 1.90; KX = 61.14 ± 2.79 beats/min), respiration rate (KM = 29.44 ± 0.99; KX = 23.80 ± 1.57 breaths/min.) and partial pressure of oxygen (KM = 89.59 ± 1.34; KX = 82.06 ± 3.92%). The blood oxygen saturation by oximeter indicated hypoxaemia in KX (82.06 ± 3.92), supported by the data from blood gas analyser. KM combination was more suitable for the immobilisation of Arabian striped hyaena, providing a better quality of induction, anaesthesia and recovery compared to KX. However, we strongly suggest further investigation to see the effects of oxygen supplementation for the compensation of hypoxaemia.

Dataset Information

Ketamine/xylazine and barbiturates modulate microglial morphology and motility differently in a mouse model.

Publications

Ketamine/xylazine and barbiturates modulate microglial morphology and motility differently in a mouse model.

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