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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Anesthesiology. Author manuscript; available in PMC 2012 October 1.
Published in final edited form as:
PMCID: PMC3432861
NIHMSID: NIHMS303102

Isoflurane Decreases Self-Renewal Capacity of Rat Cultured Neural Stem Cells

Abstract

Background

Isoflurane produces neural and behavioral deficits in in-vitro and in-vivo models. This study tested the hypothesis that neural stem cells are adversely affected by isoflurane such that it inhibits proliferation and kills these cells.

Methods

Sprague Dawley rat embryonic neural stem cells were plated onto 96 well plates and treated with 0.7%, 1.4% or 2.8% isoflurane in 21% oxygen for 6 hours and fixed either at the end of treatment or 6 or 24 hours later. Control plates received 21% oxygen under identical conditions. Cell proliferation was assessed immunocytochemically using 5-ethynyl-2’-deoxyuridine incorporation and death by propidium iodide staining, lactate dehydrogenase release, and nuclear expression of cleaved caspase 3. Data were analyzed at each concentration using an ANOVA; P < 0.05 was considered significant.

Results

Isoflurane did not kill neural stem cells by any measure at any time. Isoflurane 1.4% and 2.8% reduced cell proliferation based upon 5-ethynyl-2’-deoxyuridine incorporation whereas 0.7 % had no effect. At 24 h after treatment, the net effect was a 20–30% decrease in the number of cells in culture.

Conclusions

Isoflurane does not kill neural stem cells in vitro. However, at concentrations at and above the minimum alveolar concentrations required for general anesthesia (1.4 and 2.8%), isoflurane inhibits proliferation of these cells but has no such effect at a sub-minimum alveolar concentrations (0.7%). These data imply that dosages of isoflurane at and above minimum alveolar concentrations may reduce the pool of neural stem cells in vivo but that lower dosages may be devoid of such adverse effects.

Introduction

A growing body of data suggests that exposure to anesthetics during certain periods of development has long-term deleterious effects. At the cellular level, there is evidence that anesthetic agents induce cell death, cause synaptic remodeling, and alter morphology of the developing brain.15 Moreover, in humans, children exposed to anesthesia in early life have a higher incidence of learning deficits in adolescence.6 It is possible that anesthetic effects on neural stem cells (NSCs) may mediate some of these morphological and behavioral phenotypes. NSCs are pluripotent cells in the central nervous system that maintain the capacity for self-renewal and ultimately differentiate into astrocytes, oligodendrocytes, and neurons. Proliferation, differentiation, and migration of cells derived from embryonic NSCs are critical processes for normal brain development.7 These processes are highly regulated and tightly choreographed, especially by γ amino butyric acid (GABA).8,9 GABA is a major inhibitory neurotransmitter in the adult brain but depolarizes NSCs and immature neurons, essentially acting in the developing brain as a trophic factor that regulates neural stem cell proliferation, differentiation, and migration.8,9 Not surprisingly, therefore, excessive or prolonged GABAergic stimulation during a critical period of neurodevelopment can derail neurogenesis and alter neural connectivity and behavior.8,1012

It is relevant, therefore, that many general anesthetic agents, including isoflurane, are GABAA receptor modulators.13 Few studies, however, have investigated the effect of these agents on the capacity of neural stem cells to self-renew—the two main determinants of which are proliferation and death. There is evidence that the commonly used volatile anesthetic isoflurane affects the former but not the latter. Thus, 3.4% isoflurane impaired proliferation of hippocampal neural stem cells in vitro and administration of an ED50 concentration to postnatal day 7 rats decreased hippocampal neurogenesis for at least 5 days.2,14 Cell death was also studied in the in vitro experiment but there was no evidence that isoflurane caused NSCs to die, even though apoptosis is a prominent histological feature of the rodent brain exposed to isoflurane during the early postnatal period.14 Interpretation of these results is difficult because the in-vitro study used an isoflurane concentration (3.4%) significantly greater than the ED50 concentration required to maintain anesthesia and the in-vivo study varied the concentration as required to maintain a constant ED50 for movement (i.e. minimum alveolar concentration), conditions that were associated with abnormal systemic physiology and a 25% death rate.2,14,15 As such, it is unclear if the effects span the clinically relevant range. The present experiment was designed to test the hypothesis that isoflurane impairs proliferation and increases death of NSCs at high but not low concentrations of isoflurane.

Materials & Methods

The experimental protocol was approved by the Harvard Medical Area Standing Committee on Animals and consisted of treating neural stem cells cultured from embryonic day 18 Sprague Dawley rats to 21% oxygen, with or without isoflurane (0.7, 1.4, or 2.8%), for 6 h. At the conclusion of exposure or 6 h or 24 h later, cell viability and proliferation were evaluated by colorimetric assay or immunocytochemistry and high throughput, unbiased fluorescence microscopy, as appropriate.

Media

PBS+: 500 ml sterile Dulbecco’s phospho-buffered saline (Invitrogen, Carlsbad, CA), 5 ml of Penicillin-Streptomycin (Invitrogen), 5 ml of Fungizone® Antimycotic (Invitrogen). B27 Media: 500 ml Dulbecco's Modified Eagle Medium:F12 high glucose (Invitrogen), 2.5 ml of 200 mM glutamine (Invitrogen), 5 ml of Fungizone® Antimycotic (Invitrogen), 5 ml of Penicillin-Streptomycin (Invitrogen), and 10 ml of B27 supplement without vitamin A (Invitrogen).

Neural Stem Cell Harvest and Culture

Neural stem cells were harvested from timed pregnancy embryonic day 14 Sprague Dawley rats (Harlan Sprague Dawley, Indianapolis, IN). Pregnant rats were sacrificed by carbon dioxide intoxication and embryo cortices harvested and placed in ice cold PBS+ in a 50 ml centrifuge tube. Cortices were washed three times by centrifuging at 3000 RPM, aspirating the supernatant and adding fresh PBS+. The cortices were incubated for 5 minutes in 5 ml of high glucose Dulbecco's Modified Eagle Medium (Invitrogen) to which 10 ul of 4 % papain (Worthington Biochemical Corporation, Lakewood, NJ), 50 ul of Dispase II (neutral protease, Roche Diagnostics, Indianapolis, IN) and 50 ul of Dnase 1 (recominbant Rnase-free Dnase 1, Roche Diagnostics, Indianpolis, IN) were added. Cortices were triturated 10 times with 1000 ul pipet, filtered through a 40 µm filter, washed three times in PBS+, suspended in 5ml of B27 media and the number of cells counted with a hemocytometer. Five-million cells were plated in 75 ml tissue culture flasks (BD Biosciences, Bedford, MA) containing 25 ml of B27 media and placed in a humidified cell culture incubator at 37°C with 5 % CO2. This tissue harvesting always occurred on a Tuesday and the remaining steps were completed over 8 d. The flasks were fed with 5 ml of fresh media on Thursday; cells were passaged the 1st time on Saturday and returned to 75 ml tissue culture flasks; cells were fed with 5 ml of fresh media on Monday; finally, the cells were passaged onto 96 well plates on Tuesday. Cells were treated with isoflurane or oxygen alone the day after plating onto the 96-well plates. For passaging, both media and cells were removed and placed into a 50 ml centrifuge tube and centrifuged at 3000 RPM for 3 minutes the media was removed and Accutase (5 ml, Millipore Corporation, Temecula, CA) was added and incubated at 37°C for 5 minutes at which time the cells were triturated 10 times using a 1000 ul plastic pipet tip. PBS+ was added to the cells and the cells were washed 3 times and plated at a density 5 × 106 cells in 25 ml of B27 media in 75 ml tissue culture flasks. All experiments were performed at the second passage. On the second passage 104 cells in 100 ul of media was added to each well of matched (control and isoflurane) 96 well poly-L-ornithine / laminin coated microplates (BD BioCoat, BD Biosciences, San Jose, CA) using a multichannel pipette (Eppendorf, Westbury, NY). While in flasks, cells were primarily in the form of spheres (Fig. 1 A & B) whereas they grew as individual undifferentiated cells when on plates as reflected by the fact that more than 98% of the cells stained positive for both nestin and sox-2 (Fig. 1C), including in the 24 h control plates.

Figure 1
Photomicrographs of neural stem cells in culture. Cells were isolated from embryonic rat brain at day 14 and cultured in proliferation media containing growth factors (see Methods). A. Phase contrast photograph taken prior to fixation of a neurosphere ...

Isoflurane administration

Identical airtight chambers (Billups-Rothenberg, Del Mar, CA) and gas content certified canisters containing 21 % oxygen, 5 % CO2 and nitrogen (Airgas, Hingham, MA) were equilibrated to 37 °C overnight in a heated room. Plates were randomly placed in either control or isoflurane humidified chambers flushed with gas alone (control) or gas containing isoflurane at a rate of 2 liters per minute for 15 minutes followed by 100 ml per minute for a total of 6 hours at 37°C. Isoflurane, oxygen, and carbon dioxide concentrations were measured every 30 minutes with an agent analyzer (Ohmeda 5250 RGM, Louisville, CO). Removing the plates from the chambers terminated treatment. Experiments with 0.7 %, 1.4 % and 2.8 % isoflurane, along with corresponding controls, were performed as distinct experiments on different days with cells from multiple harvests due to the availability of a single vaporizer.

Lactate Dehydrogenase Assay

Cell cytotoxicity was estimated by measuring the release of lactate dehydrogenase (LDH) into the culture medium. LDH was quantified using a commercially available colorimetric cytotoxicity detection kit (Roche Applied Science, Mannheim, Germany). Briefly, 100 ul of supernatant was removed from 12 wells per plate (number based on previous experience), at the end of treatment, or at 6 or 24 hours after treatment and placed into a 96 well plate. 100 ul of reaction mixture was added to each well and incubated (protected from light) for 30–60 minutes at room temperature. The absorbance of the samples was measured using a plate reader (SpectraMax M2, Molecular Devices, Sunnyvale, CA) and the absorbance of the reference wavelength (690 nm) subtracted from the absorbance at 492 nm. All values are expressed as a percent of the corresponding control for each dose at each time point.

EdU incorporation Assay

Cell proliferation was determined based on EdU incorporation using a commercially available kit according to the manufacturers instructions (Click-iT™ EdU Alexa Fluor® High-Throughput Imaging (HCS) Assay, Invitrogen). EdU, like BrdU is a thymidine analogue that is incorporated into cells only during S-phase of cell division and is used to assess cell proliferation.16 Briefly, 100 µL of proliferation media containing 20 µM EdU (final concentration 10µM) was added to 12 wells of a 96 well plate (number based on previous experience with the assay), containing cells in proliferation media either immediately prior to treatment with isoflurane, at end of treatment, or 18 h after treatment for a total of 6 hours. Cells were then fixed with 4% paraformaldehyde in PBS for 15 minutes. The fixative was removed and the cells were washed twice with 100 µL 3% bovine serum albumin (BSA) in PBS, and then incubated in 100 µL of 0.5% Triton® X-100 in PBS for 20 minutes at room temperature. The cells were then washed twice and incubated with 100 µL Click-iT™ reaction cocktail for 30 minutes at room temperature. The reaction cocktail was removed and the cells were washed twice and then blocked with 100 µL / well 3% BSA in PBS and incubated with an anti-nestin (Millipore, Billerica, MA, 1:500 dilution) primary antibody overnight at 4°C. The wells were then washed 3 times with 100 µL / well 3% BSA in PBS and the secondary antibody applied and incubated for 1 hour at room temperature. The wells were then washed 3 times with 100 µL / well 3% BSA in PBS and then incubated with 100 µL 5 µg / mL Hoechst 33342 in PBS for 30 minutes at room temperature and then washed three times and stored in the dark at 4°C until image acquisition and analysis. Hoechst 33342 is a nuclear stain used to determine total cell counts.

Propidium iodide (PI) staining

For PI staining, propidium iodide (Invitrogen) was added to each well and allowed to incubate for 5 minutes prior to fixation. PI is a fluorescent molecule that binds DNA; since it is membrane impermeant, PI labels only non-viable cells and immunocytochemical detection of nestin as described above.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde in PBS for 15 minutes either at the end of treatment or 6 or 24 hours after treatment. The fixative was removed and the cells were washed three times with 100 µL 3% BSA in PBS and then blocked with 100 µL / well 3% BSA in PBS for 20 minutes at room temperature and then incubated with the primary antibodies nestin (Millipore, Billerica, MA, 1:500 dilution), sox-2 (Invitrogen,1:200 dilution), or activated caspase 3 (Abcam Inc., Cambridge, MA,1:500 dilution) overnight at 4°C. The wells were then washed 3 times and the secondary antibody (Alexa Fluor® 488 or Alexa Fluor® 555, Invitrogen) applied and incubated for 1 hour at room temperature. The wells were then washed 3 times and then incubated with 100 µL 5 µg / mL Hoechst 33342 in PBS for 30 minutes at room temperature and then washed three times with PBS and stored in the dark at 4°C until image acquisition and analysis. Cells treated with staurosporine (3 µM), a caspase 3 activator, served as a positive control for PI staining and cleaved caspase immunocytochemistry.

Image acquisition and analysis

Eight – twelve images (number based on previous experience with this system) were acquired per well using an IN Cell Analyzer 1000 (GE Healthcare, Piscataway, NJ) in an automated unbiased fashion. This compact bench-top instrument includes an automated Nikon™ (Nikon Inc., Melville, NY) microscope, high-resolution CCD camera, xenon lamp-based illumination, filter wheel based wavelength control, and laser based auto focus and associated image acquisition and analysis software (GE Healthcare). Because image acquisition is automated and large numbers of images can be acquired, cell selection bias is eliminated and the impact of experimental and biological variation is reduced.17 Using a multi-target analysis, cells were first identified based upon having a nuclear area greater than 25µm2 using top-hat segmentation. This threshold setting excludes non-cellular debris from the analysis. Cells were then outlined based upon nestin staining overlapping a nucleus, as defined by staining with Hoechst, using multiscale top-hat segmentation with the characteristic area set at 100µm2 and a sensitivity of 50. For identification and analysis of EdU, PI and CC3 positive cells, we used a two-step filtering process. In the first step, nestin negative cells were excluded; in the second, threshold setting was used to determine the number of EdU, PI, and CC3 positive cells. Thus, only nestin-positive cells were analyzed and included in the final analysis. Imaging parameters were set based on a control plate stained with the fluorophor or antibody of interest and the parameters used to image an entire set of matched control and isoflurane treated plates. Once the parameters were set, images were acquired automatically, meaning there was no cell selection bias. In these experiments, 8 images were acquired per well at a 20× objective from 8 to 12 wells per treatment condition per assay per time (N = 8 – 12) in matched control and isoflurane treated plates. Based on control experiments with staurosporine, a PI or CC3 positive cell was defined as one with a nuclear/cellular intensity ratio greater than 2 or 3, respectively. The nuclear intensity threshold for an EdU positive cell was defined by the intensity of EdU positive staining in matched control cells. A similar two-step filtering process was used to determine the percentage of cells staining positive for nestin and sox-2; cellular intensity of nestin was used as the first screen and nuclear intensity of sox-2 as the second.

Statistical Analysis

With the exception of PI staining, which failed normality testing, the remainder of the data from each assay at each time point from matched control and isoflurane treated cells (plates) were analyzed using SigmaStat (Systat Software, Inc, Chicago, IL) software using 2-way ANOVA’s with treatment condition and time as the between group factors and Bonferroni corrections for multiple comparisons. Data for PI staining were analyzed using Kruskal-Wallis 1-way ANOVA’s followed by Dunn’s testing for multiple comparisons. Staurosporine experiments used as a positive control for apoptosis (cleaved caspase 3) and cell death (PI) were analyzed using an unpaired two-tailed Student’s t-test. Cell number and sox-2 intensity 24 hours after treatment were analyzed using unpaired two-tailed Student’s t-test with a Bonferroni correction for multiple comparisons. Data are expressed as mean ± SD; P < 0.05 was considered statistically significant.

Results

In our culture system, > 98% of control cells plated on poly-d-lysine laminin coated plates stained positive for both nestin and sox-2 at the end of treatment and 6 h and 24 h later, confirming the cells studied were NSC’s (Fig. 1). As mentioned previously, cells that were not nestin-positive were excluded from subsequent immunocytochemical analysis.

Cell Proliferation

Isoflurane 0.7% had no effect on proliferation of NSC’s (P = 0.60; Fig. 2) but there was an effect of time post-treatment, with increased proliferation 24 h after treatment in both the control and isoflurane treated groups (P < 0.001) but no interaction between treatment condition and time post-treatment (P = 0.42; Fig. 2C). With 1.4 % isoflurane, there was an effect of treatment condition, with impaired proliferation in isoflurane treated cells relative to controls (P = 0.004) but no effect of time post-treatment (P = 0.82) and no interactions between treatment condition and time post-treatment (P = 0.63; Fig. 2D). With 2.8% isoflurane, there was an effect of treatment condition (P < 0.001) and time post-treatment (P < 0.001) and an interaction between isoflurane treatment and time post-treatment (end of treatment, control 39 ± 3%, isoflurane 27 ± 3 %; 6 hours after treatment, control 40 ± 3 %, isoflurane 26 ± 3 %; 24 hours after treatment, control 44 ± 2 %, isoflurane 40 ± 4 %; P < 0.001, Fig. 2E). This resulted in there being more EdU positive cells 24 h after the experiment began under both control and isoflurane conditions but the 2.8% isoflurane treated plates had fewer EdU positive cells compared to time-matched control plates. These data suggest that isoflurane has no effect on NSC proliferation at low concentrations but sustained effects at higher concentrations.

Figure 2
Isoflurane and stem cell proliferation. Using matched plates, neural stem cells were exposed to control conditions or 0.7, 1.4, or 2.8 % isoflurane for 6 h. Dividing cells were labeled at the end of treatment or 6 h or 24 h later by incorporation of EdU. ...

Staurosporine and Cell Death

Staurosporine (6 h), as expected, increased the percentage of cleaved caspase 3 positive cells from 0.6 ± 0.1 under control conditions to 11.7 ± 1.6 and the percentage of PI positive cells from 0.90 ± 0.40 under control conditions to 6.0 ± 0.40 (P < 0.001). There was clear differentiation between positive and negative cells when the nuclear / cellular intensity of cleaved caspase 3 and PI were > 3 and 2, respectively. Accordingly, we used these parameters to set thresholds for defining caspase 3 and PI positive cells in subsequent experiments.

Effects of isoflurane on NSC Cytotoxicity and Cell Death

The concentration of LDH in the culture media was analyzed with a colorometric assay (Fig. 3). At isoflurane 0.7%, there was no effect of treatment condition (P = 0.55) or time post-treatment (P = 0.31) and no interaction between the two (P = 0.31; Fig. 3A). Likewise, there was no effect of treatment condition (P = 0.72) or time post-treatment (P = 0.39) and no interaction between the two (P = 0.42) with 1.4% isoflurane (Fig. 3B). In contrast, with isoflurane 2.8%, there was an effect of treatment condition (P = 0.002) and time post-treatment (P = 0.006) and an interaction between the two (P = 0.006), with a decrease in LDH to 88 ± 3 percent of control 24 hours after isoflurane was withdrawn (Fig. 3C), perhaps because there are fewer cells at that time.

Figure 3
Effect of isoflurane on lactate dehydrogenase (LDH) release. Using matched plates, neural stem cells were exposed to control conditions or 0.7, 1.4, or 2.8 % isoflurane for 6 h. LDH was assayed in the media by colorimetric assay at the end of treatment ...

There were few dead or dying cells, as determined by PI staining, at any time under control conditions (< 2% on average; Fig. 4), indicating the cultures were healthy. There was no effect of treatment condition or time post-treatment with 0.7% isoflurane on the percentage of cells that stained positive for PI (P = 0.49, Fig. 4C). At isoflurane 1.4%, there was no effect of treatment condition but there was an effect of time post-treatment, with a larger percentage of cells staining positive for PI 6 and 24 hours after isoflurane treatment compared to those fixed at the end of treatment (P < 0.001; Fig. 4D). The effect of 2.8% isoflurane was similar; there was no effect of treatment condition but a larger percentage of cells stained positive for PI 6 and 24 hours after isoflurane compared to those that were fixed at the end of treatment (P < 0.01, Fig. 4E).

Figure 4
Effect of isoflurane on cell death. Using matched plates, neural stem cells were exposed to control conditions or 0.7, 1.4, or 2.8 % isoflurane for 6 h. The percentage of dead cells was determined by propidium iodide (PI) staining at the end of treatment ...

Similiarly, few cells stained positive for cleaved caspase 3 under control conditions (< 2% on average; Fig. 5). With 0.7% and 1.4% isoflurane, there was no effect of treatment condition (P = 0.73 and 0.87, respectively) or time post-treatment (P = 0.82 and 0.68, respectively) and nor was there an interaction between the two (P = 0.99 and 0.06, respectively) (Figs. 5C and 5D). For isoflurane 2.8%, there also was no effect of treatment condition (P = 0.97) but there was an effect of time post-treatment (P < 0.001) and an interaction between the two (P = 0.03), indicating high dose isoflurane reduces natural cell death in NSCs during the time of exposure but not subsequently (Fig. 5E).

Figure 5
Effect of Isoflurane on cleaved caspase-3 (CC3). Using matched plates, neural stem cells were exposed to control conditions or 0.7, 1.4, or 2.8 % isoflurane for 6 h. The percentage of CC3 positive cells was determined at the end of treatment or 6 h or ...

Effect of isoflurane on Sox-2 expression

The percentage of cells expressing sox-2 did not change with isoflurane regardless of concentration but the intensity of expression did (Fig. 6). Thus, at 24 h after treatment with 0.7% isoflurane, the nuclear intensity of sox-2 was no different than in control cells (107 ± 9 percent of control; P = 0.13) but it was 7% (93 ± 5 percent of control; P = 0.024) and 14% (86 ± 7 percent of control; P = 0.012) lower in cells treated with 1.4% and 2.8% isoflurane, respectively.

Figure 6
Isoflurane decreases sox-2 expression. Using matched plates, neural stem cells were exposed to control conditions or 0.7, 1.4, or 2.8 % isoflurane for 6 h. Cells were fixed and stained 24 h later with sox-2 (Alexa Fluor 555; red) and Hoechst 33342 (blue). ...

Ultimately, as determined by Hoechst staining and comparison to matched controls, these effects on self-renewal translated into there being 20–30% fewer cells in culture 24 h after treatment with 1.4% and 2.8% isoflurane (Fig. 7).

Figure 7
Effect of isoflurane on number of neural stem cells (NSCs) in culture. Using matched plates, neural stem cells were exposed to control conditions or 0.7, 1.4, or 2.8 % isoflurane for 6 h. The absolute number of cells was determined 24 h after anesthesia ...

Discussion

The salient results of our study are that isoflurane decreases NSC proliferation but does not kill cultured embryonic neural stem cells. Importantly, we demonstrate these effects occur at concentrations of isoflurane that span the clinical range but not at a sub-minimum alveolar concentration. In fact, at the highest concentration, isoflurane reduced natural cell death as measured by PI staining and caspase activation, which confirms the previously identified ability of this agent to reduce or delay apoptosis in both in vitro and in vivo models.18,19 Isoflurane’s anti proliferative effect occurred only at 1.4% and 2.8% and persisted for at least 24 h only at the high concentration but, compared to matched controls, the net effect was a 20–30% decrease in the number of NSCs in culture 24 h after treatment. Furthermore, NSCs treated with isoflurane express less sox-2, a transcription factor important for maintaining an undifferentiated phenotype, which supports previous observations that this agent promotes early differentiation and neuronal fate selection.14 Therefore, our results demonstrate that isoflurane impairs embryonic neural stem cell proliferation at dosages at and above minimum alveolar concentrations, which implies it may impair neurogenesis and deplete the neural stem cell pool in vivo at clinically relevant but not sub-minimum alveolar concentrations.

This is the first study to examine effects of isoflurane at concentrations that span the clinical range on neural stem / progenitor cells but it is not the first to investigate the impact of isoflurane on the death or proliferation of NSC’s. A recent study using 3.4% isoflurane and NSC’s harvested from postnatal day 2 rats reported a decrease in activated caspase 9 but not caspase 3/7 during and 2 h after exposure to isoflurane but no change in LDH release 18 h following withdrawal of the agent.14 Our results generally agree with those of that study14 inasmuch as we found no evidence for cytotoxicity in this model using either PI staining or cleaved caspase 3. This is interesting given that the concentrations of isoflurane (3.4% vs. 0.7, 1.4, and 2.8%), timing of assessment (during and 2 h after vs. during, 6 h, and 24 h), and age of the animals used for the stem cell harvest (postnatal rats vs. E14 animals) are different. This latter point is noteworthy because the expression of receptors and signaling systems relevant for the action of isoflurane (e.g. GABA and N-Methyl-D-aspartic acid receptors) are developmentally regulated8,20,21 such that sensitivity to isoflurane might be expected to vary with developmental age. Thus, the fact that neither study found a cytotoxic effect is persuasive evidence that NSCs in vitro, as compared to neurons, are resistant to the cytotoxic effects of isoflurane.

Isoflurane’s effect on proliferation is a different story. Whereas isoflurane 0.7% had no effect at any time, both 1.4% and 2.8% reduced EdU incorporation significantly, with the latter change lasting at least 24 h. This decrease in NSC proliferation is broadly consistent with other in vitro and in vivo work with isoflurane. Studies report a 20–40% decrease in proliferation of NSCs in culture or in the hippocampus of postnatal day 7 animals exposed to isoflurane concentrations of 1.4 – 3.4%, and brief repeated exposure to 1.7% produces a 20% decrease in NSC proliferation acutely and a 42% decrease in the radial glia-like stem cell pool in P14 rats.1,2,14,15 Although we found changes of a similar magnitude in our culture model, there are two important differences between the in vitro and in vivo data. First, whereas the anti-proliferative effect of isoflurane is temporary at low dosages in vitro (recovering within 24 h at all but the two highest concentrations; Fig. 2), it persists for days or even weeks in vivo.1,2 This implies that while isoflurane initiates cell cycle arrest by a direct effect on NSCs, the sustained anti proliferative effect in vivo may be maintained by secondary effects of isoflurane on surrounding neural tissue. Importantly, our results indicate isoflurane 0.7% is without effect on NSC proliferation, implying low concentrations isoflurane may have minimal or no adverse effects on developmental processes that depend on NSC self-renewal.

The mechanisms by which isoflurane impairs proliferation has not been investigated. Isoflurane is a pleiotropic agent that modulates GABA receptors and antagonizes N-Methyl-D-aspartic acid receptors but these are not the only targets of isoflurane that might be relevant to its effect on NSC proliferation.13 Embryonic NSCs express GABA and N-Methyl-D-aspartic acid receptors but the latter are inactive at this stage of development,8,20,21 leaving GABA receptors as a likely target in these cells. GABAA receptors are excitatory at this stage of neurodevelopment (in contrast to their inhibitory effect in adulthood).8,22 GABA is tonically released by NSCs and acts as a trophic factor regulating key developmental processes including proliferation of neural stem cells, neuronal differentiation, and migration.8,9 Not surprisingly, therefore, excessive or prolonged GABAergic stimulation such as with ethanol or valproic acid, which like isoflurane are GABA agonists / modulators, decreases NSC proliferation, increases differentiation, and alters neural connectivity.11,12 As such, we theorize that the excessive GABA receptor-mediated excitation produced in NSCs during exposure to isoflurane may cause decreased proliferation of these cells. Indeed, although the percentage of cells expressing sox-2 was unchanged by isoflurane treatment, the intensity of sox-2 expression was reduced at 1.4% and 2.8% isoflurane, which are the same concentrations that decreased proliferation. Sox-2 is a key transcription factor for maintaining the self-renewal capacity of undifferentiated embryonic stem cells.23 Therefore, our data suggest that isoflurane may reduce NSC proliferation, and potentially promote early differentiation of NSCs, via a sox-2 dependent mechanism.

Our study is limited in several important ways. First, EdU is a thymidine analogue that is incorporated into DNA only during the S-phase of cell division or during DNA repair. Accordingly, an alternative explanation for our results is that isoflurane damages DNA and the EdU incorporation reflects DNA repair rather than cell division. This seems unlikely, however, given that isoflurane decreased the absolute number of NSCs relative to the control state. Second, we can only speculate about the pharmacological and molecular mechanisms responsible for the effects we have observed. Third, we do not know whether isoflurane has consequences for brain development beyond or in addition to affecting the self-renewal capacity of NSCs. GABA, for example, plays a prominent role in neuronal migration but that aspect of isoflurane’s action has yet to be examined.10 Fourth, the sox-2 results are based on changes in intensity of immunostaining, which is less reliable than cell counting. As such, conclusions about the effect of isoflurane on NSC differentiation must be made cautiously and will require independent confirmation.

The biological significance of these results is unclear. Normal brain development depends upon a readily available pool of neural stem cells to generate, in the right location, the proper numbers of neurons and glia.10 NSCs are also present in the mature brain, albeit in lower numbers, where they are implicated in processes ranging from learning and memory to mood and epilepsy and in brain maintenance and repair.24 Thus, even a transient adverse effect of isoflurane on self-renewal of NSCs might have far reaching consequences for brain development and function across the lifespan. To date, such developmental consequences have not yet been demonstrated. However a 15–20% decrease in neurogenesis in vivo, which resembles the effect observed here, is sufficient to impair hippocampal dependant memory in rodents25 and, even more striking, recall of remote spatial memory in adult animals depends on recruiting as few as 1–4% newly born neurons into hippocampal circuits.26 Therefore, these results may have implications for the use of isoflurane at critical periods of brain development as well as in adulthood.

Summary Statement

This study demonstrates that concentrations of isoflurane at and above minimum alveolar concentrations impair proliferation of rat embryonic neural stem cells in culture but that lower concentrations do not.

Acknowledgements

The authors thank Marcie A. Glicksman, PhD, Director, Leads Discovery and Thomas Gainer, MS, Scientist, Laboratory for Drug Discovery in Neurodegeneration (LDDN), Harvard NeuroDiscovery Center, Harvard Medical School, Brigham & Women’s Hospital, Cambridge, MA for their for helpful comments and advice on high-content screening.

This study was received from the Department of Anesthesia, Harvard Medical School, Brigham & Women’s Hospital, Boston, MA 02115, USA and supported by National Institutes of Health Grants (Bethesda, MD, USA) RO1 AG20253 and RO1 GM088817 (GC); K08NS048140, R21AG029856 and R01 GM088801 (ZX); KO8 GM077057 (DJC) and the Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Boston, MA, (USA).

Footnotes

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References

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