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

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.


Introduction
Astrocytes, the most common glial cell in the central nervous system, play multiple functional roles in brain physiology, including maintaining neuronal function, providing growth factors and metabolic support to neurons, glutamate shuttling, regulating neurotransmitter release, water and electrolyte homeostasis, and maintaining the blood-brain barrier [1][2][3][4].Over time, there has been an increased understanding of how astrocytic dysfunction contributes to overall brain pathology.Studies have indicated the signifcance of astrocytes in normal processes, such as aging, as well as in abnormal development and degeneration of the brain.Tese studies have described the role of astrocytes in the induction, propagation, and resolution of neurodegenerative disease [4].Astrocytes have also been shown to function as both perpetrators and protectors in the pathology of cerebral ischemia, major depressive disorder, stroke, traumatic brain injury, and dementia [5][6][7][8][9].
Anesthesia often involves exposure to volatile anesthetic gases, as well as the administration of intravenous agents such as propofol and ketamine.Propofol [2,6-diisopropylphenol] is frequently used for procedural sedation and general anesthesia in children and adults [10].Ketamine [2-(o-chlorophenyl)-2-(methylamino) cyclohexanone hydrochloride] is commonly used in pediatric patients for procedures [11,12].Tese medications are often chosen because of their rapid efect, short action, and low side efect profle.
Recent advances in anesthesia have led to increased survival of vulnerable populations at the extremes of age, both in the very young and premature as well as in the very old.Tese individuals present with an increased risk of adverse outcomes, including neurocognitive impairment after anesthesia [13][14][15][16].Extensive population-based cohort studies suggest that adult patients who underwent anesthesia and surgery had double the risk of dementia and were diagnosed in a shorter amount of time [17].Clinical studies have also demonstrated the adverse efects of anesthetics on neurodevelopment throughout early life.A study of pediatric patients with repeated anesthetic exposure demonstrated a decreased average processing speed and parental report of executive function, fne motor skills, behavior, and reading ability [18].Another postmortem study of pediatric patients who had undergone anesthesia also showed higher rates of reactive gliosis [19].
However, studies on astrocytes and anesthesia have been limited [20], and there has been a paucity of research investigating the efects of anesthesia on astrocyte viability.Terefore, the present study examines the susceptibility of young and senescent astrocytes in primary cultures to commonly used intravenous anesthetics such as propofol and ketamine.

Cell
Culture and Senescence Induction.Human fetal astrocytes (passage 1) were obtained from ScienCell Research Laboratories (cat.# 1801; Carlsbad, CA, USA) and were grown at 37 °C, 5% CO 2 in an aqueous medium supplemented with 2% fetal bovine serum (FBS), growth supplement, and penicillin/streptomycin as previously described [21].Te cells were seeded at a standard density of 1 × 10 4 cells/cm 2 and cultured until 70%-80% confuence was reached.After each passage, the cells were trypsinized and counted.Te cumulative population doubling (PD) level was then calculated as previously described [8,21] using the formula NH/NI � 2 x , where NH represents the number of cells at harvest, NI represents the initial number of seeded cells or inoculum, and x is the number of population doublings.
Cells were considered to be early passage (young cells) when less than 50% of their replicative life span was completed within PD <10.Cells were used at PD 7.2 for the young astrocyte experiments.Oxidative stress was applied to induce premature senescence, mimicking an aged population of cells as previously described [21,22].Cells were again seeded at 1 × 10 4 cells/cm 2 and then treated with 200 μM hydrogen peroxide (H 2 O 2 ) the following day for two hours.Senescence was reached at least fve days after starting treatment [21].Cells were harvested seven days after treatment and assayed for senescence, characterized by the fattened and enlarged morphology and by the cessation of division.

Administration of Propofol and Ketamine.
Equivalent aliquots of cell suspensions were randomly assigned to the study groups.Propofol (Fresenius Kabi, Lake Zurich, IL 60047) was emulsifed with dimethyl sulfoxide (DMSO) as a vehicle compound to make a stock solution of 15 mM propofol in 74% (v/v) DMSO.Te concentrations of propofol used were 30 μM (low-dose condition), 100 μM (medium-dose condition), and 300 μM (high-dose condition).Tese concentrations were obtained by adding 1.9, 6.4, and 19 μL of the 15 mM propofol to the media for a total volume of 950 μl.DMSO alone was added to culture media in a total volume of 950 µL at the above ratios (1.9, 6.4, and 19 μL), yielding 1% (low-dose condition), 3.34% (medium-dose condition), and 10% v/v (high-dose condition) DMSO as corresponding vehicle controls for propofol.Ketamine (Sigma, St. Louis, MO) was added to culture media at concentrations of either 30 μM (low-dose condition) or 300 μM (high-dose condition).Te concentrations of propofol and ketamine used corresponded to the range of plasma concentrations observed in patients anesthetized with these agents [23,24].In addition, cultures of cells without anesthetic treatment were used as baseline controls.
Te cells were incubated at 37 °C for up to seven hours with DMSO alone, propofol + DMSO, or ketamine at the fnal concentrations indicated in the astrocyte medium described above.After incubation, the culture media containing foating cells were collected, and attached cells were washed once with 1 mL Dulbecco-modifed phosphatebufered saline (PBS, with calcium and magnesium) and then resuspended in 100 μl 0.25% trypsin at 37 °C for three minutes.Te culture media were added back to the cells, and the cellular suspensions were pipetted fve to six times to disperse the cells homogeneously.Cell counts were measured by fow cytometry, whereas cell viability was assessed by cell viability assay.

Cell Viability Assay.
Te ViaCount Assay (EMD Millipore, Burlington, MA) was used to distinguish the viability of cells based on diferential permeabilities of two DNA-binding dyes in the Guava ViaCount ® reagent.
A nuclear dye was used to stain only cells with a nucleus, whereas a viability dye stains dying cells.Altogether, this combination of dyes diferentiates between viable and dead cells.Debris was excluded from results based on negative staining with the nuclear dye and by predetermined size cutof in fow cytometry.A volume of 50 μL of cell suspension was mixed with 250 μL of Guava ViaCount reagent (fvefold dilution) in a 1.5-mL microcentrifuge tube per the manufacturer's recommendations.Aliquots (71 μL) were collected from each sample at a concentration of two to ten cells/μL, corresponding to 150 to 800 cells per measurement volume.Samples were mixed well, incubated for about fve minutes at room temperature, and kept protected from light.All treatments were made in triplicate wells.Via-Count measurements were made on a Guava EasyCyte system (Millipore).Cells were kept on ice while waiting for measurement.

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Anesthesiology Research and Practice

Statistical Analysis. Te baseline control astrocytes
grown in media without additives were evaluated for cell count.Tese cells were obtained from the same batch and cultured on the same multi-well culture plate of each set of experiments, simultaneously with the other experimental groups.For relative quantifcation of cell growth, the well showing the best growth of astrocytes in control media was defned to be 100% and referred to as baseline control.Te data from the experimental groups were presented as the means of % best growth, 95% confdence intervals of the baseline control in the wells run on the same day of the experiment.One-way ANOVA with Bonferroni correction for post hoc comparisons was used.All analyses utilized SigmaPlot 13.0 (Systat Software, Inc.San Jose, CA).Statistical signifcance was determined at p < 0.05.

Efects of Senescence on Astrocytes' Growth in Culture.
Overall, 99.1 ± 1.7% of young astrocytes (N � 375 ± 56 cells) in 71 μL measurement volume remained viable after incubation in culture media with no vehicle or drug for seven hours, compared to 87.3 ± 20.5% of senescent cells (N � 349 ± 41 cells; p � N.S. vs. young).Senescent human astrocytes showed morphological changes indicative of cellular senescence, including fattened cell bodies and the presence of cytoplasmic vacuoles compared to young cells (Figure 1).

Efects of Diferent Media on Young Astrocyte Growth in Primary
Culture.Young astrocytes were incubated with control media, DMSO, propofol + DMSO, and ketamine for seven hours and were then analyzed based on resultant total cell count, dose-response cell count, and total viability assays.

Total Cell Count.
Cell counts of young astrocytes were combined between all doses of each assigned medium.Te efects of DMSO, propofol + DMSO, and ketamine on young primary culture human astrocytes are shown in Figure 2.
Baseline control media demonstrated a total cell count of 375 ± 56 cells per aliquot.Astrocytes incubated for seven hours in propofol dissolved with DMSO saw a 39.2% reduction of total cell count as opposed to the baseline control, which is the greatest reduction in total cell count as compared to either DMSO vehicle alone or ketamine (p < 0.01).
Propofol dissolved in DMSO also had a signifcant 26% cell count reduction versus DMSO as well as a 29% reduction versus ketamine (both at p < 0.01).Tere was no signifcant diference in total cell count between the baseline control group and either DMSO or ketamine.

Dose-Response Cell
Count.Dose-response cell counts of young astrocytes were examined between the low and high doses of each medium type.Te efects of DMSO, propofol + DMSO, and ketamine on young primary culture human astrocytes are shown in Figure 3. Dose-response studies demonstrated variable efects of medication on cell count reductions.Low doses of incubation media were set at 30 μM.Propofol dissolved in DMSO demonstrated signifcant cell count reductions versus the baseline control (p < 0.01) and versus DMSO alone (p < 0.05).High doses corresponded with the administration of 300 μM of media.Once more, propofol dissolved in DMSO had a signifcant cell count reduction versus the baseline control (p < 0.01) and versus DMSO alone (p < 0.05).In the high-dose condition, propofol dissolved in DMSO also showed a signifcant cell count reduction compared to ketamine (p < 0.05).
Tere were no signifcant diferences between the baseline control and DMSO alone or ketamine, nor between DMSO alone and ketamine at either concentration.

Efects of Diferent Media on Senescent Astrocyte
Growth in Primary Culture.Senesced astrocytes were incubated with control media, DMSO, propofol dissolved in DMSO, and ketamine.After seven hours, they were then analyzed based on the resultant total cell count, doseresponse cell count, and total viability assays.

Total Cell Count.
Cell counts of senesced astrocytes were combined between doses of each assigned media.Te efects of DMSO, propofol + DMSO, and ketamine on senesced primary culture human astrocytes are shown in Figure 5.When compared to the baseline control, there were signifcant reductions in total cell count in DMSO alone (p < 0.05) and in propofol dissolved in DMSO (p < 0.05).In addition, when compared to ketamine, there were also signifcant cell count reductions in DMSO alone (p < 0.05) and in propofol dissolved in DMSO (p < 0.05).Tese results indicate that the cell count reduction seen in the senesced group exposed to propofol emulsifed in DMSO may not have been due to exposure to the anesthetic but rather to DMSO itself.

Dose-Response Cell
Count.Dose-response cell counts of senesced astrocytes were examined between the low and high doses of each medium type.Te efects of DMSO, propofol + DMSO, and ketamine on senesced primary culture human astrocytes are shown in Figure 6.Tere were no signifcant diferences in cell counts between high-, Anesthesiology Research and Practice medium-, and low-dose conditions of diferent medium incubations.

Viability.
Te dose-response viability of senescent astrocytes was examined between the low and high doses of each medium type.Te efects of DMSO, propofol + DMSO, and ketamine on senesced primary culture human astrocytes are shown in Figure 7. Tere were no signifcant diferences in percent viability between the diferent medium incubation groups.

Discussion
Incubation of young astrocytes for seven hours in diferent medium types demonstrated a diferential susceptibility of these cultures to certain commonly used anesthetics.Young astrocytes incubated in baseline control media grew to 375 ± 56 cells/aliquot.When the total cell counts among the diferent treatment groups were compared, astrocytes incubated with propofol dissolved in DMSO demonstrated a 39% reduction in cell count compared to the baseline control media.In contrast, young cells in propofol + DMSO had a 26% cell count reduction compared to DMSO alone and a 29% cell count reduction compared to ketamine.Interestingly, the efect of DMSO alone on primary cultures of young astrocytes was not as signifcant as that of propofol + DMSO, indicating a signifcant alteration of astrocytic survivability with exposure to this common anesthetic.Tese results contrast the efect of the anesthetics on senesced cultures of astrocytes, in which only total cell counts indicated a change in astrocytic survivability.Cultures exposed to DMSO alone and DMSO + propofol demonstrated a signifcant loss of cell count as opposed to the baseline control and ketamine.Tere was no dose-response efect of these anesthetics nor an alteration in our measures of viability.In no experimental group or condition did ketamine have any signifcant efect as compared to the baseline controls.
Te growing understanding of the function of astrocytes intensifes the need to characterize the anesthesia efects on Figure 2: Cell counts of young astrocytes in DMSO, propofol, and ketamine.Te average number of young astrocytes per exposure at all doses after seven hours of incubation in diferent media was expressed as a percentage of the best growth in control media for each set of experiments and presented as means, 95% confdence 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.young and aged astrocytes.Tis goal is of particular signifcance as it impacts all populations, even beyond the extremes of age, as only about 30-50% of astrocytes are senescent in an adult [8].Previous studies on the efect of aging on rat astrocytes suggest that these cells demonstrate declining function in culture that corresponds and is similar to observed changes in life, including characteristics of cellular senescence [25][26][27].Tis observation may be why the results of the present study demonstrated a diferential susceptibility of young, but not senesced, astrocytes to commonly used intravenous anesthetics when added to culture media, with ketamine showing a more favorable profle than propofol.Propofol is frequently used for procedural sedation as well as for general anesthesia in children and adults.Propofol can act as a gamma-aminobutyric acid (GABA A )  6 Anesthesiology Research and Practice agonist [10].Propofol has been shown to induce apoptosis in neurons, but its efect on astrocytes is less clear [28].Ketamine is often used in infants and toddlers for elective procedures.Tis anesthetic is often utilized due to its short duration of action and use in procedures requiring dissociative amnesia with swift recovery time [29].Ketamine may act as a noncompetitive blocker of N-methyl-d-aspartate (NMDA) receptor ion channels [29,30].Ketamine has been shown to induce apoptosis in cultured rat cortical neurons [31].However, little is known about whether ketamine induces cell death in astrocytes.Despite the widespread use of these agents, research on the efects of anesthetics on the brain has been scant and sometimes contradictory.Tis notion is complicated by the fact that several brain regions may be more vulnerable to anesthetics than others and thus may have diferential impacts on cell populations within the nervous system [28,32].Multiple studies have found altered cognition and cellular development following anesthetic administration, particularly in vulnerable or developing populations [15,16,20,23,33].
Propofol has been found to have both neuroprotective and neurotoxic properties, depending on the conditions of the study, in a cell type, age, and time-dependent manner.Some studies have reported that propofol protects the brain [3,24,34] and have proposed propofol as an antioxidant agent that may protect neurons through regulation of oxidative stress, brain-derived neurotrophic factor (BDNF) pro-survival signaling, Na + /H + exchange, and blood-brain barrier maintenance [24,34,35], as well as by reducing the efects of neuroinfammation on the nervous system [36].Other studies have concluded that propofol may not signifcantly afect astrocytic viability, cell cycle, apoptosis, morphology, or the presence of reactive oxygen species [3,28,37,38].Conversely, propofol has been suspected to cause astrocytes to become activated and neurotoxic when exposed for more extended periods.It may also induce astrocyte apoptosis, as indicated by glial fbrillary acidic protein (GFAP) expression, cleaved caspase-3, cytokines, or other protein levels [20,23,39].Recent research examining microRNAs has implicated propofol in increased expression of rno-miR-665, which preferentially binds and inhibits Bcl2l1, a signifcant anti-apoptotic protein [20,23,39].Propofol has also been shown to change cerebral blood fow, alter astrocyte communication, decrease brainstem and cortical lactate release, increase neuronal calcium levels, impair memory and learning functions, and alter the electron transport chain in the mitochondria [16,32,[40][41][42][43][44][45].Our results indicate that propofol's efect on astrocytes may be more toxic.Te data also indicated a strong dose-response of cell count in young primary cultures with exposure to propofol, over and above that of the DMSO vehicle alone.
Te present study has focused on astrocytic cell count and viability as measures of astrocyte well-being.It is possible that other forms of viability may be impacted by these drugs that are not encapsulated in the Guava ViaCount assay that was conducted.However, the congruence between cell count and viability results signifes a detrimental efect of propofol on young astrocytes in culture.Te diferential efect of these anesthetics on young and senesced cultures of astrocytes may be partly due to their mechanisms of action.Te observed cell count reduction may be related to the adverse efects of propofol on mitochondrial function, as described in previous studies [45].We also observed the detrimental efects of DMSO on young astrocytes in culture, compared to baseline controls.Te fndings on cell counts and viability by DMSO support the observed additional adverse efect of propofol on young astrocytes and its absence in cultures of senesced astrocytes as these cells have already been subjected to oxidative stress as part of their senescence process.
Te reports on ketamine's efects are also controversial.Ketamine acts as a noncompetitive blocker of Nmethyl-d-aspartate (NMDA) receptor ion channels [29].Some studies have indicated ketamine's ability to induce reactive gliosis or astrocytic apoptosis [9,46,47] and neuronal apoptosis [33,48].Other studies have found that ketamine alone does not alter astrocyte survival or cognitive impairment following anesthetic administration [49].Even further, some research has indicated that ketamine may also exhibit neuroprotective efects by increasing synaptogenesis, AMPA receptor expression, and arborization of dendrites [7].Our study found no detrimental efects of ketamine on neither young nor senesced astrocytes in primary cultures.
It is important to note that while our results indicate that propofol may detrimentally impact young astrocytes, there are limitations to the applications of these data.For one, astrocytes in vivo are surrounded by a diverse network of other cells and environments.Our cells were not in any such framework.Additionally, we cannot conclude any clinical efect at this time due to this limitation.
We speculate that ketamine may have a more favorable safety profle than propofol in infants and young children.Further investigations should be conducted to translate this research into clinical settings and to elucidate the mechanism of anesthetic-astrocyte involvement.

Figure 1 :Figure 3 :Figure 4 :
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 fattened cell bodies and the presence of cytoplasmic vacuoles.

Figure 5 :
Figure5: Cell counts of senesced astrocytes in DMSO, propofol, and ketamine.Te average number of senescent astrocytes per exposure at all doses after seven hours of incubation in diferent media, expressed as a percentage of the best growth in control media for each set of experiments and presented as means, 95% confdence 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.