YIG-381
A potential role for neurosteroid anesthesia in protection from anesthetic neurotoxicity.
Montana M, Swiney B, Li A, Evers A, Noguchi K
Washington University in Saint Louis, Saint Louis, MO, United states
Introduction: General anesthesia in children carries low risk of acute morbidity or mortality. However, data from animals suggest that anesthetics in clinical use may be neurotoxic to the developing brain. Anesthetic neurotoxicity has not been rigorously assessed for older, off-market anesthetics, including neurosteroids. The neurosteroid anesthetic alfaxalalone was the major component of the intravenous anesthetic Althesin, but it was withdrawn from the market due to anaphylactic reactions attributed to its excipient, Cremophor. However, a less toxic excipient, beta-cyclodextrin, has enabled the reformulation of alfaxalone, and it is currently FDA approved as the veterinary anesthetic Alfaxan. Interestingly, neurosteroids inhibit neuronal apoptosis, one of the key steps implicated in anesthetic neurotoxicity. Thus, neurosteroid anesthetics may be non-apoptogenic. The goal of this study is to assess the extent to which the anesthetic neurosteroid alfaxalone causes neuronal apoptosis in mice.
Methods: P7 male and female C57BL/6 mice were injected intraperitoneally with either hydroxypropyl-beta-cyclodextrin, 30 mg/kg alfaxalone (5α-Pregnan-3α-ol-11,20-dione), or 175 mg/kg propofol (2,6-Diisopropylphenol). Both anesthetics were dissolved in cyclodextrin. The dose of alfaxalone and propofol were chosen from preliminary studies performed to identify doses that resulted in loss of righting reflex for approximately 60 minutes. A separate cohort of mice was injected with either normal saline or cyclodextrin to assess any effects of cyclodextrin alone on apoptosis. Stereological counts of the brain were obtained from 75 um midline sagittal sections stained for activated caspase-3 (AC3). Immunostaining was normalized to cyclodextrin treated mice, as background AC3 levels are expected to be non-zero due to the developmental age of the mice.
Results: No significant differences were seen in AC3 immunostaining between animals injected with saline vs. cyclodextrin (6 mice per group). A one-way between subject’s ANOVA was conducted to compare the effect of the anesthetics on normalized AC3 counts. There was a significant main effects difference [F (2,28) = 3.815, p = 0.0343]. Post hoc comparisons using Bonferroni's Multiple Comparison Test indicated that the mean normalized score for propofol injected mice (n=12) (M = 2.2, SD = 1.3) was significantly different (p < 0.05) than cyclodextrin injected mice (n=7). Alfaxalone injected mice (n=12) (M = 1.6, SD = 0.8) did not differ significantly from cyclodextrin mice. However, contrary to results obtained in preliminary studies, the duration of loss of righting reflex was significantly different between the two experimental groups (propofol 150 +/- 74 minutes vs. alfaxalone 89 +/1 41 minutes, p = 0.02).
Discussion: These data suggest that a short duration anesthetic with alfaxalone does not significantly increase the degree of neuronal apoptosis seen in neonatal mice when compared to vehicle injected mice. However, experiments to assess the effects of longer duration anesthetics, and repeated administration of alfaxalone need to be performed. The possibility for alfaxalone administered in the neonatal period to alter biologically relevant behaviors in adulthood should also be assessed.
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