Differential Susceptibility to Propofol and Ketamine in Primary Cultures of Young and Senesced Astrocytes

Abstract

The adverse effects of general anesthesia on the long-term cognition of young children and senior adults have become of concern in recent years. Previously, mechanistic and pathogenic investigations focused on neurons, and little is known about the effect of commonly used intravenous anesthetics such as propofol and ketamine on astrocytes. Recently, astrocyte dysfunction has been implicated in a wide range of age-related brain diseases. In this study, we examined the survival and viability of both young and senescent astrocytes in culture after adding propofol and ketamine to the media at varying strengths. Oxidative stimulus was applied to commercially available fetal cell lines of human astrocytes in vitro to induce morphological changes in cellular senescence. Our results indicate that propofol reduces the survival of young astrocytes as compared to controls, as well as to ketamine. These effects were seen in comparisons of total cell count and at both high and low dose concentrations. High doses of propofol also significantly reduced cell viability compared to those exposed to baseline controls and ketamine. Senescent astrocytes, on the other hand, demonstrated cell count reductions as compared to baseline controls and ketamine when exposed to either DMSO or propofol. The data show differential susceptibility of young astrocytes to propofol than to ketamine. The observed cell count reduction may be related to the adverse effects of propofol on mitochondrial function and free radical production, as described in previous studies. We speculate that ketamine may have a more favorable safety profile in infants and young children.

Figure 1

Representative cell viability assay of primary astrocytes. Representative phase-contrast microscopic images (10X) showing young human fetal astrocytes in primary cultures without any additives (a) and dying and detaching young astrocytes in cultures containing 300 μM propofol in DMSO ((b), arrows). (c) Senescent human astrocytes show morphological changes indicative of cellular senescence, including flattened cell bodies and the presence of cytoplasmic vacuoles.

Figure 2

Cell counts of young astrocytes in DMSO, propofol, and ketamine. The average number of young astrocytes per exposure at all doses after seven hours of incubation in different media was expressed as a percentage of the best growth in control media for each set of experiments and presented as means, 95% confidence intervals. Control (N = 4; white bar), DMSO (N = 12 with low, medium, and high preparations included; light gray bar), propofol + DMSO (N = 12 with low, medium, and high preparations included; dark gray bar), and ketamine (N = 6 with low and high preparations included; closed bar). ^∗∗p < 0.01 vs control; ⁺⁺p < 0.01 vs DMSO; ^##p < 0.01 vs propofol + DMSO. ANOVA with Bonferroni correction for post hoc comparisons.

Figure 3

Cell count dose response of young astrocytes to DMSO, propofol, and ketamine. The average number of young astrocytes per dose after seven hours of incubation in escalating strengths of different media (30 μM, low; 300 μM, high; 100 μM of DMSO and propofol + DMSO not shown), expressed as percent of best growth in control media for each set of experiments and presented as means, 95% confidence intervals. Control (N = 4; white bar), low DMSO corresponding to 74% DMSO used to prepare 30 μM propofol (N = 3; light gray bar, left), high DMSO corresponding to 74% DMSO used to prepare 300 μM propofol (N = 6; light gray bar, right), low propofol + DMSO (N = 3; 30 μM dark gray bar, left), high propofol + DMSO (N = 6; 300 μM dark gray bar, right), low ketamine (N = 3; 30 μM closed bar, left), and high ketamine (N = 3; 300 μM closed bar, right). ^∗∗p < 0.01 vs control; ⁺p < 0.05 vs DMSO; ^#p < 0.05 vs propofol + DMSO. ANOVA with Bonferroni correction for post hoc comparisons.

Figure 4

Viability dose response of young astrocytes to DMSO, propofol, and ketamine. Average percent viability of young astrocytes per dose after seven hours of incubation in escalating strengths of different media (30 μM, low; 300 μM, high; 100 μM of DMSO and propofol + DMSO not shown), presented as means, 95% confidence intervals. Control (N = 4; white bar), low DMSO corresponding to 74% DMSO used to prepare 30 μM propofol (N = 3; light gray bar, left), high DMSO corresponding to 74% DMSO used to prepare 300 μM propofol (N = 6; light gray bar, right), low propofol + DMSO (N = 3; 30 μM dark gray bar, left), high propofol + DMSO (N = 6; 300 μM dark gray bar, right), low ketamine (N = 3; 30 μM closed bar, left), and high ketamine (N = 3; 300 μM closed bar, right). ^∗p < 0.05 vs. control; ^#p < 0.05 vs. propofol + DMSO. ANOVA with Bonferroni correction for post hoc comparisons.

Figure 5

Cell counts of senesced astrocytes in DMSO, propofol, and ketamine. The average number of senescent astrocytes per exposure at all doses after seven hours of incubation in different media, expressed as a percentage of the best growth in control media for each set of experiments and presented as means, 95% confidence intervals. Control (N = 3; white bar), DMSO (N = 9 with low, medium, and high preparations included; light gray bar), propofol + DMSO (N = 9 with low, medium, and high preparations included; dark gray bar), and ketamine (N = 6 with low and high preparations included; closed bar). ^∗p < 0.05 vs control; ⁺p < 0.05 vs DMSO; ^#p < 0.05 vs propofol + DMSO. ANOVA with Bonferroni correction for post hoc comparisons.

Figure 6

Cell count dose response of senesced astrocytes to DMSO, propofol, and ketamine. The average number of senescent astrocytes per dose after seven hours of incubation in escalating strengths of different media (30 μM, low; 300 μM, high; 100 μM of DMSO and propofol + DMSO not shown), expressed as a percent of the best growth in control media for each set of experiments and presented as means, 95% confidence intervals. Control (N = 3; white bar), low DMSO corresponding to 74% DMSO used to prepare 30 μM propofol (N = 3; light gray bar, left), high DMSO corresponding to 74% DMSO used to prepare 300 μM propofol (N = 3; light gray bar, right), low propofol + DMSO (N = 3; 30 μM dark gray bar, left), high propofol + DMSO (N = 3; 300 μM dark gray bar, right), low ketamine (N = 3; 30 μM closed bar, left), and high ketamine (N = 3; 300 μM closed bar, right).

Figure 7

Viability dose response of senesced astrocytes to DMSO, propofol, and ketamine. The average percent viability of senescent astrocytes per dose after seven hours of incubation in escalating strengths of different media (30 μM, low; 300 μM, high; 100 μM of DMSO and propofol + DMSO not shown), presented as means, 95% confidence intervals. Control (N = 3; white bar), low DMSO corresponding to 74% DMSO used to prepare 30 μM propofol (N = 3; light gray bar, left), high DMSO corresponding to 74% DMSO used to prepare 300 μM propofol (N = 3; light gray bar, right), low propofol + DMSO (N = 3; 30 μM dark gray bar, left), high propofol + DMSO (N = 3; 300 μM dark gray bar, right), low ketamine (N = 3; 30 μM closed bar, left), and high ketamine (N = 3; 300 μM closed bar, right).