Neurodevelopmental Impacts of Ketamine and Alfaxalone Anesthesia Evaluated with Mouse MRI
Injectable anesthetics are commonly used in murine experimental procedures. However, these agents may result in neurotoxicity, which should be considered in interpretation of experimental results. We evaluated acute effects of 2 different anesthetic combinations on juvenile mouse brain development using structural MRI to assess impact on the brain. We compared the use of ketamine-xylazine, a commonly used injectable anesthetic combination in mice, to alfaxalone-xylazine in the context of noninvasive procedures requiring immobilization (that is, not a surgical plane of anesthesia). In this longitudinal study, we used MRI to produce three-dimensional scans of mouse brains at 2 time points (postnatal days 14 and 23), analogous to early childhood to prepubescence in humans. At postnatal day 16, mice were either dosed with ketamine-xylazine, alfaxalone-xylazine, or left untreated. From the scans, we quantified whole brain and structure volumes across the brain, comparing growth between time points and modeling the effect of both anesthetics compared with controls. Anesthetic parameters were measured, and general health and welfare were monitored during and after each injectable anesthesia drug condition. Results indicate that systemic and brain toxicity were reduced in mice treated with alfaxalone-xylazine compared with ketamine-xylazine. In addition, both ketamine-xylazine and alfaxalone-xylazine reliably anesthetized all mice, although mice administered ketamine-xylazine showed increased weight loss compared with the alfaxalone-xylazine in the postanesthetic period. These findings highlight alfaxalone-xylazine as a convenient and possibly safer alternative anesthetic for mouse brain development studies when compared with ketamine-xylazine and as a viable option as an injectable anesthetic in juvenile mice.
Introduction
Mice are commonly anesthetized for procedures that require immobilization, such as imaging or radiation treatment.1–3 While inhalant anesthetics such as isoflurane are generally recommended due to their rapid induction, ease of anesthetic depth control, quick recovery, and overall reliability,4,5 certain circumstances require the use of injectable agents. This may be due to equipment constraints—such as the need to place the animal in an enclosed apparatus such as an irradiator—or concerns that inhalants could interfere with specific study parameters. However, some injectable anesthetics have been associated with neurotoxic effects,6,7 which may confound experimental outcomes, necessitating a review and refinement of anesthetic protocols.
Ketamine is one of the most widely used injectable anesthetic agents for mouse anesthesia8,9 due to the high therapeutic index and dosage combination flexibility.4 It functions as an NMDA receptor antagonist, reducing neuronal excitability. While it is both widely available and relatively inexpensive, it is classified as a dissociative anesthetic and its use is subject to restrictions in accordance with regulations. Alfaxalone (with appropriate solubilizing agents10) has gained popularity in veterinary medicine for use in companion animals as a component of intravenous general anesthesia, a sedative, or as an anesthetic induction agent.11,12 It is a neuroactive steroid that acts as a GABAA receptor agonist, resulting in increased inhibitory neurotransmission. Similar to ketamine, alfaxalone has been demonstrated to be effective for mouse anesthesia when combined with α-2 adrenergic agonists such as xylazine or medetomidine.13–19 This suggests that alfaxalone-xylazine (AX), although not widely used in mice, is a potential alternative injectable anesthetic to ketamine-xylazine (KX). However, alfaxalone remains less characterized preclinically.
In this study, we sought to characterize the impacts of ketamine and alfaxalone specifically in the context of the developing mouse brain for applications that require injectable anesthesia for immobilization (such as during cranial radiation20–23). We compared the effects on mouse brain development of KX with AX based on structural analysis with MRI. We administered both types of anesthesia at a juvenile stage (postnatal day [P]16). We performed MRI pretreatment at P14 and then again posttreatment at P23 to compare the effects of anesthesia on neuroanatomy. In addition, anesthetic parameters were measured, and general health and welfare were monitored during and after each injectable anesthesia drug condition. Our results demonstrate that at comparable immobilization responses to the injectable anesthetics, weight loss and induced brain morphology changes appeared milder in mice treated with AX compared with those treated with KX. These findings support the use of AX as a safe and reliable alternative to KX to anesthetize juvenile mice.
Materials and Methods
Ethical review.
All animals were housed in a facility certified by the Canadian Council on Animal Care and registered under the Animals for Research Act of Ontario. All animal procedures were reviewed and approved by the Centre for Phenogenomics Animal Care Committee.
Experimental animals and husbandry.
C57BL/6J mice were bred and housed on-site. The in-house colony is periodically refreshed every 10 generations (no. 000664; The Jackson Laboratory, Bar Harbor, ME). Environmental conditions were maintained at 21 to 23 °C, 40% to 60% humidity, with 15 air changes hourly for the room, 70 to 75 air changes hourly for the individually ventilated cages, and a 12-hours light/12-hours dark cycle. Mice were housed in individually ventilated cages (Greenline GM500; Tecniplast USA, West Chester, PA) on disposable bedding (¼-in. corncob; The Andersons, Maumee, OH). Each cage contained a red plastic tunnel (mouse tunnel; Bio-Serv, Flemington, NJ) and nesting material (Enviropak; W.F. Fisher and Son, Somerville, NJ). Mice were fed an extruded pelleted laboratory rodent chow (TD.2918X Inotiv, West Lafayette, IN) and provided reverse osmosis, UV-filtered, acidified water through an automated recirculating water system (Avidity Science, Waterford, WI). Routine health monitoring was performed by testing sentinel-free soiled bedding cages (one cage per rack) via PCR, quarterly. Excluded agents were mouse parvovirus, mouse hepatitis virus, murine rotavirus, Theiler murine encephalomyelitis virus, murine adenovirus 1 and 2, reovirus, pneumonia virus of mice, Sendai virus, ectromelia virus, lymphocytic choriomeningitis virus, Citrobacter rodentium, Mycoplasma pulmonis, Clostridium piliforme, cilia-associated respiratory bacillus, Pseudomonas aeruginosa, Salmonella spp., Bordetella bronchiseptica, Bordetella pseudohinzii, Campylobacter spp., Corynebacterium kutscheri, Corynebacterium bovis, Staphylococcus aureus, Streptococcus pneumonia, Streptococcus moniliformis, Klebsiella pneumoniae, Klebsiella oxytoca, beta-hemolytic Streptococcus groups A, B, C, and G, Proteus mirabilis, Pneumocystis murina, and endoparasites and ectoparasites, including Giardia, Myobia musculi, Myocoptes musculinus, Radfordia affinis, Syphacia spp., Aspiculuris tetraptera, Spironucleus muris, Cryptosporidium, and Demodex. Murine norovirus, Rodentibacter spp., Helicobacter spp., Tritrichomonas, and Entamoeba are endemic in this housing room. Mice were allowed a minimum of 1 week to acclimate to the housing room and cage environment prior to the start of the study.
Study design.
Comparison of KX and AX on brain morphology.
Mice were scanned at P14 using in vivo MRI. At P16, each pup was randomly assigned to a treatment group with approximately equal numbers from each litter. Treatment groups included (1) 75 mg/kg ketamine (100 mg/mL, Narketan; Vetoquinol, Lavaltrie, QC, Canada) combined with 5 mg/kg xylazine (20 mg/mL, Rompun; Elcano Canada, Mississauga, ON, Canada) (KX group) administered intraperitoneally; (2) 20 mg/kg alfaxalone (10 mg/mL, Alfaxan; Jurox, Rutherford, NSW, Australia) combined with 5 mg/kg xylazine (AX group) administered subcutaneously; or (3) no treatment as a control group. Young mice are sensitive to injectable anesthetics leading to higher mortality,4 therefore the doses were reduced from those recommended for adult mice. The dose of ketamine was based on current protocols used in our laboratory to anesthetize mice <21 days old. The alfaxalone dose used was selected based on published doses in adult mice.13–15 We initially administered the alfaxalone dose at 30 mg/kg, which was later reduced to approximately match the duration of anesthesia produced by the ketamine dose to 20 mg/kg. All 30 mg/kg treated animals were excluded from the reported analyses. Ketamine was administered intraperitoneally, which is a common route of administration in mice, whereas alfaxalone was administered subcutaneously. The subcutaneous route for alfaxalone administration was selected based on successful outcomes observed in other studies when used in mice13,15–18 and improved outcomes compared with intraperitoneal administration in some studies.15,17 The mice were weaned at P21 and group-housed with 4 to 5 animals per cage. At P23, mice were scanned with MRI. A summary of the experimental timeline is shown in Figure 1.
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-25-103
Sample size.
A total of 91 C57BL/6J mice (44 females and 47 males) were used for this study with 14 to 16 mice per anesthetic group per sex equally distributed among 14 litters. A power analysis was conducted based on SDs and effect sizes from previous imaging studies.3,24
Inclusion and exclusion criteria.
There were no inclusion/exclusion criteria. All experimental animals were included.
Randomization.
Each pup within its litter was randomly assigned to a treatment group. Mice were block-randomized so that each litter contributed to all treatment groups, with sexes represented as equally as possible when litter sizes allowed. All mice from each litter were included in the study.
Blinding.
All injections and loss of righting reflex (LORR)/return of righting reflex (RORR) monitoring were performed by the same individual, who was not blinded to treatment groups. General health and welfare assessments were conducted by a different individual, who was also not blinded to treatment groups. For manganese chloride injections and MRI, treatment groups were blinded to the experimenter.
Anesthetic parameters.
On the day of injectable anesthesia at P16, the anesthetic drug group (KX, AX, or control), drug dosage, injection time, and injection volumes were recorded. Immediately after the injection, mice were maintained in ventilated cages off the housing rack, with thermal support using electric heating pads. Mice were monitored for LORR, RORR, and time to recovery. LORR was defined as an inability for the mouse to return to standing or sternal recumbency after being placed in dorsal recumbency (checked every 30 seconds once mice stopped voluntarily ambulating until LORR was confirmed). RORR was defined as the ability of the mouse to right itself into sternal recumbency 3 successive times. Recovery was defined as the time the mouse regained purposeful movement and voluntarily propelled itself forward 2 in. or more. Duration of LORR was defined as the time from LORR to RORR and is the time where the mice were immobile. Time to recovery was defined as the time from anesthetic drug injection to recovery. A Welch 2-sample t test was used to compare anesthetic effects between KX and AX groups faceted by sex. Times are presented as the mean ± 95% CI.
Animal monitoring.
Body weight (grams) for each mouse was recorded at P16, P17, P18, P19, and P22. Data were modeled using a linear mixed effects model with age, sex, and anesthetic group as fixed effects and individual mice as random effects. Weights are presented as the mean ± 95% CI. Mice were monitored for general health and welfare by daily visual examination, from P16 to P19, assessing the following parameters: body weight change (%), mobility and ambulation, activity level, appearance and behavior, respiration, social behavior, stereotypies, and abdominal distention.
In vivo magnetic resonance imaging.
In vivo manganese-enhanced MRI scans were obtained using a 7-T MRI scanner (Bruker BioSpin; Ettlingen, Germany) equipped with 4 cryocoils for simultaneous imaging of 4 mice.25 The scans were performed with the following settings: T1-weighted, three-dimensional gradient echo sequence, 75 μm isotropic resolution, repetition time of 26 ms, echo time of 8.25 ms, flip angle of 26°, field of view of 25 × 22 × 22 mm, and matrix size of 334 × 294 × 294. Mice received an intraperitoneal injection of manganese chloride contrast at a dosage of 0.4 mmol/kg body weight ∼24 hours before the imaging procedure.26 During imaging, anesthesia was maintained at 1% to 1.5% isoflurane and respiration rate was monitored. Scan time and duration of anesthesia were ∼1 hour.
Image registration and statistical methods.
Using an automated image registration pipeline from the Pydpiper toolkit, all MR brain images were registered together into a common average reference space.27 To measure structure volumes, the MAGeT brain segmentation algorithm was used to segment images using a published atlas containing 183 bilateral brain regions.28–32 To summarize global changes in brain volumes, summary volumes for gray matter, white matter, and cerebrospinal fluid were reported as a difference (in mm3) between the 2 imaging timepoints. A linear model was used to test for statistical significance for each of the anesthetic groups compared with the controls (sex was excluded from the model after determining it was not significantly impactful). To determine the effect of anesthetic across all 183 structures individually, a linear mixed-effects model33 was employed with age and type of anesthesia as fixed effects. Random effects as intercepts accounted for individual mouse variability. To correct for multiple comparisons across 183 structures, P values were corrected using the false discovery rate method.34 All data are presented as the mean ± 95% CI. The effect of KX or AX was determined for each structure by computing the volume difference from the control mice and normalizing by the estimated control volume, producing a percent change. Neuroanatomical maps were created by overlaying this percent change on the common average image.
As discussed in the Results, volume loss was induced by both anesthetics. To estimate the probability that the AX treatment resulted in larger structure volumes relative to KX (that is, improved outcomes), a Bayesian regression model35 was employed with age and anesthesia type as fixed effects with mouse treated as a random effect. Posterior distributions of the KX and AX effect were drawn 4,000 times. The percentage of instances that the AX estimate was more positive than the KX estimate per structure was deemed to be the probability that the region was bigger in the AX group compared with KX. All statistical tests were performed in the R statistical software environment (version 3.6.3). The RMINC package was used to interface with the MR images.36
Results
Similar anesthetic response to ketamine and alfaxalone resulted in greater acute weight loss for ketamine.
The anesthetic effects of KX and AX were compared at P16. Time to recovery for a 75 mg/kg ketamine dose and 20 mg/kg alfaxalone dose showed no statistical significance for females (KX: 85.3 ± 3.0 minutes compared with AX: 80.9 ± 4.5 minutes, P = 0.45) or males (KX: 85.2 ± 4.1 minutes compared with AX: 81.6 ± 4.3 minutes, P = 0.59) as shown in Figure 2A. Similarly, the duration of LORR was not significantly different between KX and AX groups for females (KX: 87.6 ± 2.4 minutes compared with AX: 88.5 ± 5.6 minutes, P = 0.89) or males (KX: 88.1 ± 3.7 minutes compared with AX: 86.4 ± 3.9 minutes, P = 0.65) as shown in Figure 2B.
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-25-103
Mouse weights were measured at P16 to P19 and P22. A linear mixed effects model was used to evaluate weight change between groups. Due to statistically significant sex–treatment interactions, mice were visualized separately by sex (Figure 2C). Overall, systemic effects as assessed by mouse weight appeared greater in the KX group compared with the AX group. Females in the KX group exhibited statistically significant weight loss compared with control mice starting from P18 (−3.1% ± 1.7%, P < 0.01) to P22 (−4.6% ± 1.6%), whereas males exhibited significant weight loss at P17 (−3.7% ± 1.6%, P < 0.05) and P18 (−4.0% ± 1.6%, P < 0.05). Equivalent measurements in the AX group were not considered statistically significant (P > 0.05).
Each mouse was assessed at P16 to P19 for changes to mobility and ambulation, activity level, appearance and behavior, respiration, social behavior, stereotypies, and abdominal distention. Other than the changes in weight described above, mice in the KX, AX, and control groups showed no changes in health and welfare except for 2 male mice in the KX group who developed mild ambulation impairment after anesthetic recovery that resolved later that same day.
Ketamine and alfaxalone both induced volume change in the brain, although ketamine exhibited a greater impact.
To summarize brain volume changes induced by KX or AX treatment, whole-brain volume, gray matter, white matter, and CSF volumes were compared between control and anesthetic groups. Volume changes between MRI timepoints were calculated by subtracting the P23 volume from the P14 baseline volume. The P14 to P23 change in brain volume (Figure 3A) in the KX group was significantly smaller than in the control group (−21% ± 5.2%, P < 0.001) while reductions in the AX group were more subtle (−7.9% ± 5.2%, P = 0.14). The KX group also exhibited significant differences in the P14 to P23 volume change of total gray (Figure 3B, −23.1% ± 6.6%, P < 0.001) and white matter (Figure 3C, −17.6% ± 5.2%, P < 0.01), whereas the AX group only exhibited significant reductions in white matter (−10.5% ± 5.1%, P < 0.05). The KX group also exhibited a significant decrease in the CSF volume change (Figure 3D, −24.4% ± 11.6%, P < 0.05). The AX group exhibited a larger CSF volume change relative to controls (−35.1% ± 11.5%, P < 0.01) than did the KX group. No sex–treatment interaction terms in volume change were considered significant (P > 0.05).
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-25-103
Ketamine and alfaxalone impact similar brain structures, with alfaxalone-treated mice likely to show smaller volume differences.
Brain volumes for 183 regions were evaluated using a linear mixed effects model. After correction for multiple comparisons, no regions were considered statistically significant relative to control for either the KX or AX group (q > 0.1). By comparing uncorrected P values, 37 structures in the KX group had P values <5% compared with 12 in the AX group. Unthresholded effects on neuroanatomy in the KX and AX groups are shown in Figure 4A with more structures highlighted in the KX group compared with the AX group. Many structures appeared in both groups (38 out of 48 possible structures that had at least a 2% change). Using a Bayesian approach, we present a neuroanatomical map (Figure 4B) that depicts the probability that a given region is larger in the AX group than in the KX group (for example, 50% indicates equal likelihood, 100% indicates AX is always larger, and 0% indicates KX is always larger). There were 133 structures likely to be larger in the AX group (>50% probability) compared with 50 in the KX group (<50% probability). This suggests that structures on average are likely to be larger in the AX group compared with the KX group.
Citation: Journal of the American Association for Laboratory Animal Science 2025; 10.30802/AALAS-JAALAS-25-103
Anesthesia affected several of the same structures, although to varying extents. The hippocampus (Figure 4C), calculated by summing its subregions, showed reduced volume relative to controls in both KX (−0.85% ± 0.94%, P < 0.05) and AX (−0.82% ± 0.58%, P < 0.05) groups. Similarly, the olfactory bulbs (Figure 4D) exhibited decreased volume in the AX group (−0.92% ± 0.42%, P < 0.05) and an even greater reduction in the KX group (−1.6% ± 0.4%, P < 0.001). To compare the magnitude of these effects across brain structures, Figure 4E shows the percent change in 183 structures due to anesthesia, with structures sorted by the KX group. Many structures in the AX group exhibited a more positive effect size than in the KX group (134 out of 183 structures). Moreover, Figure 4F demonstrates that there is a high correlation in structures affected by KX compared with AX (R2= 0.3, P = 6.4 × 10−16); the slope of the line suggests that the AX effect is on average 51% that of KX, although with considerable variability by structure.
Discussion
Alfaxalone has been investigated as an alternative to ketamine for mouse anesthesia.13–19 However, its effects in juvenile mice—of particular interest in study of neurodevelopment—remain poorly characterized. After the selection of an appropriate alfaxalone dose, we found AX was able to produce comparable anesthetic effects to KX, supporting alfaxalone as a reliable alternative for juvenile mouse anesthesia. Mice treated with alfaxalone showed no significant weight loss, whereas those treated with ketamine experienced weight loss shortly after treatment. Although both anesthetic combinations resulted in modest reductions in brain growth over the observation period, AX administration resulted in approximately half the change observed compared with KX. These findings suggest that alfaxalone may be a viable, potentially less impactful alternative to ketamine when anesthesia is required for neurodevelopment studies in mice. We also note that we focused on neuroimaging outcomes over only a week following anesthesia; it may also be of interest to evaluate the evolution of brain changes into adulthood.
Both time to recovery and duration of LORR indicate that AX can be as effective as KX for achieving anesthesia in juvenile mice. Due to the young age of the animals and our focus on immobilization rather than a surgical plane of anesthesia, the doses used were lower than those reported in previous studies.13–17 Importantly, note that a surgical plane of anesthesia was not evaluated as part of this study, and no noxious stimuli (for example, toe pinch) were applied to confirm loss of reflexes. Higher doses would likely be necessary to attain a surgical plane in this age group, and further investigation is needed to establish appropriate dosing for that purpose. In addition, more detailed analysis of physiologic parameters such as respiratory rate, heart rate, and stress biomarkers would enhance our understanding of alfaxalone use in mice. The existing literature on the use of alfaxalone in mice is limited, warranting further study.
Previous studies have reported that strain- and sex-dependent differences in alfaxalone dosage are required in mice.15 For example, one study found that male mice required higher doses than females to reach a surgical plane, and that the duration of anesthesia was longer in female C57BL/6J mice compared with males.15 Other studies reported strain-dependent differences but did not find sex-based differences in dose or duration.13 In our study, we did not evaluate other mouse strains, but we observed no sex differences in the anesthetic parameters evaluated.
Alfaxalone, as an alternative to ketamine, was associated with overall reduced acute toxicity as evaluated by weight and brain morphology changes. Ketamine significantly reduced body weights in both female and male mice, with females exhibiting more weight loss. This aligns with a previous report indicating a greater vulnerability to ketamine in female mice.37 In contrast, alfaxalone did not significantly reduce body weight in either sex at the measured posttreatment timepoints, suggesting less physiologic impact compared with ketamine and the potential for improved welfare. Ketamine also induced significant global brain volume reductions across gray matter, white matter, and CSF, whereas alfaxalone treatment led to significant volume decreases only in white matter and CSF. Interestingly, the spatial patterns of volume change were highly correlated between the 2 anesthetics, indicating significant overlap in neurotoxicity profile. While alfaxalone had a milder overall impact on the brain, it still resulted in comparable levels of neurotoxicity in the developing brain (as measured by MRI), despite reports of neuroprotective properties.38,39 Notably, hippocampal volume, critical for working memory,40 was similarly reduced following both treatments, which could adversely affect studies examining neurocognitive outcomes. Several studies have associated such brain morphology alterations with abnormal behavioral phenotypes in mice.41,42 Nevertheless, it appears that alfaxalone produced a milder impact than ketamine, supporting its use as an anesthetic in experiments where minimizing neurotoxicity during neurodevelopment is a priority.
We evaluated the influence of only ketamine and alfaxalone in this study. It is possible that these results extrapolate to related NMDA receptor antagonists and GABAA receptor agonists. If consistent with the current findings, drugs that increase activation of inhibitory transmitters, as compared with excitatory receptor antagonists, may reduce risk of weight loss or neurodevelopmental change. However, other factors, including drug solvent, can have important implications for toxicity,10 so such extrapolation should be considered cautiously. Moreover, local logistical considerations, such as availability or regulatory requirements (for example, for controlled substances such as ketamine), may influence drug preferences for individual laboratories.
Conclusion
AX provides effective anesthesia in juvenile mice and represents an alternative to KX for preclinical research. When administered at P16, alfaxalone caused less weight loss compared with ketamine. Both anesthetics produced a similar profile of brain morphology changes as evaluated by MRI, although alfaxalone appeared to produce milder changes. These findings support the use of alfaxalone as a convenient alternative to ketamine in mouse studies, particularly when minimizing neurodevelopmental toxicity is a priority. Studies examining long-term neurodevelopmental outcomes and optimizing dosing strategies will further inform best practices. Refining anesthetic protocols remains essential to ensuring both scientific rigor and ethical responsibility in animal research, and alfaxalone presents a promising avenue for continued refinement.
Conflict of Interest
The authors have no conflicts of interest to declare.
Funding
This study was supported by Canadian Institutes of Health Research Grant 192014.
Protocol Registration
No protocol was registered before the study.
Data Availability
Data will be made available on request.
Experimental timeline for studying the effects of injectable anesthesia in wild-type C57BL/6J male and female mice. Mice were scanned at P14 and P23 using in vivo MRI under isoflurane anesthesia. Manganese chloride (MnCl2) was administered 24 h before scans as a contrast agent. At P16, mice in the study were injected with ketamine-xylazine (KX) or alfaxalone-xylazine (AX) or left untreated (control). M, male; F, female.
(A and B) Comparisons of the anesthetic effects between the ketamine-xylazine (KX) group compared with the alfaxalone-xylazine (AX) group. There were no statistically significant differences between groups in both males and females for time to recovery. Similarly, duration of LORR was also not statistically significant between groups in both males and females. Points and whiskers represent mean times and bootstrapped 95% CIs. (C) Weight measurements over time between control (Ctrl), KX, and AX groups. Weights were measured on treatment day (at P16) where treatment is visualized graphically as the vertical green shade. Posttreatment weights show statistically significant weight loss in KX groups relative to the Ctrl for females and males whereas changes in the AX group were minimal. Points and whiskers represent mean structure volume and bootstrapped 95% CIs after removal of fitted random effects. Groups and data points were jittered horizontally for visualization. #, P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Summary volume changes in the ketamine-xylazine (KX) or alfaxalone-xylazine (AX) group relative to controls (Ctrl). Volume changes were calculated by taking the difference between the P23 and P14 volume for each mouse. (A) Whole (total) brain volume, (B) total gray matter, (C) total white matter, and (D) CSF volumes are plotted. KX and AX groups both exhibit reductions in global brain volumes relative to controls. Points and whiskers represent mean structure volume and bootstrapped 95% CIs. Data points were jittered horizontally for visualization. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Effect of ketamine-xylazine (KX) and alfaxalone-xylazine (AX) on brain structure volume. (A) Neuroanatomical maps representing the effects of KX and AX relative to control. Regions that are highlighted represent effects with a 2% volume threshold. (B) Bayesian probability that the AX effect in a particular region is larger than the KX effect. A probability of 50% represents equal chance that AX or KX is larger than one another. (C and D) Volumes compared between control, KX, and AX group for the hippocampus and olfactory bulbs. Points and whiskers represent mean structure volume and bootstrapped 95% CIs. Data points were jittered horizontally for visualization. (E) Induced volume change compared between ketamine compared with alfaxalone on a per structure basis. The effect of AX on most structures is less than the KX effect. (F) Scatter plots for the KX compared with AX effect where each point represents a unique structure. A line of best fit is shown with 95% CIs. In C and D: *, P < 0.05; **, P < 0.01; ***, P < 0.001. HP, hippocampus; OB, olfactory bulb.
Contributor Notes
These authors contributed equally to this study.