Dual effects of isoflurane on proliferation, differentiation and survival in human neuroprogenitor cells

Abstract

Background

Previous studies have demonstrated that isoflurane can provide both neuroprotection and neurotoxicity in various tissue culture models and in rodent developing brains. The cellular and molecular mechanisms mediating these dual effects are not clear, but the exposure level and duration of isoflurane appear to be determinant factors.

Methods

Using the ReNcell CX human neural progenitor cell line, we investigated the impact of prolonged exposure to varying isoflurane concentrations on cell survival and neurogenesis. In addition, we assessed the impact of short isoflurane preconditioning on elevation of cytosolic Ca²⁺ concentration and cytotoxic effects mediated by prolonged isoflurane exposures and the contribution of InsP₃ or ryanodine receptors activation to these processes.

Results

Short exposures to low isoflurane concentrations promote proliferation and differentiation of ReNcell CX cells, with no cell damage. However, prolonged exposures to high isoflurane concentrations induced significant ReNcell CX cell damage and inhibited cell proliferation. These prolonged exposures suppressed neuronal cell fate, while promoting glial cell fate. Preconditioning of ReNcell CX cultures with short exposures to low concentrations of isoflurane ameliorated the effects of prolonged exposures to isoflurane. Pretreatment of ReNcell cultures with InsP₃ or ryanodine receptor antagonists mostly prevented isoflurane-mediated effects on survival, proliferation, and differentiation. Finally, isoflurane preconditioned cultures showed significantly less isoflurane-evoked changes in calcium concentration.

Conclusion

The commonly used general anesthetic isoflurane exerts dual effects on neuronal stem cell survival, proliferation and differentiation, which may be attributed to differential regulation of calcium release through activation of endoplasmic reticulum localized InsP₃ and/or ryanodine receptors.

Introduction

Isoflurane has shown neuroprotective properties in response to numerous biological stresses in vitro ^1–5 and in vivo ^6–9. However, increasing studies suggest that isoflurane may be neurotoxic in vitro ^10–13 and in vivo ^14–19 as well. Isoflurane causes persistent hippocampal-dependent cognitive deficits in rodents ^15;16, but the mechanisms of such deficits are not clear. Neurogenesis in the hippocampus is involved in memory acquisition ²⁰, suggesting that isoflurane may act on neural progenitor cells (NPCs) to impinge on hippocampal-dependent cognitive functions. Accordingly, emerging studies into the mechanisms of anesthesia-induced cognitive deficits have provided some discrepant results on anesthetic-mediated effects on neurogenesis in vivo ^17;21 and some consistent results in vitro ^22;23. Because of their importance to cognitive functions and regenerative medicine, it is critical to gain more insights into the mechanisms by which general anesthetics affect neurogenesis.

Development of NPCs is regulated by gamma-aminobutyric acid and intracellular Ca²⁺ mobilization ^24–27. Ca²⁺ mobilization through inositol-1,4,5-trisphosphate receptor (InsP₃) and ryanodine receptors plays important roles in proliferation and differentiation of nonexcitable cells ^28;29. Ca²⁺ is one of the key regulators of cell proliferation, via maintaining an oscillatory Ca²⁺ signal, activating the immediate early genes responsible for inducing resting cells (G₀) to re-enter the cell cycle and promoting the initiation of DNA synthesis at the G₁/S transition ^30;31. The Ca²⁺ spiking induced neural cell differentiation by controlling expression of specific neurotransmitters and channels, the behavior of growth cones and the establishment of the specific connections within neuronal circuits ^30;32. Isoflurane neuroprotective properties in neurons are mediated through an association with smaller isoflurane-evoked Ca²⁺ release via InsP₃ receptors ^33–35, whereas the cytotoxic properties of this anesthetic are associated with excessive calcium release via InsP₃ receptors ^12;13;36;37. These results raise the possibility that both isoflurane-mediated cytoprotection and cytotoxicity in neurons occur in NPCs through differential InsP₃ or ryanodine receptor-mediated Ca²⁺ mobilization and control of neurogenesis process. Thus, we hypothesize that isoflurane affects survival, proliferation, and differentiation of NPCs in a dual manner via activation of InsP₃ or ryanodine receptors. To that end, we used the immortalized human neural progenitor cell line, ReNcell, and show that short isoflurane exposures promote or inhibit survival, proliferation, and differentiation in a time or concentration-dependent manner. Preconditioning of these cells with short isoflurane exposures mostly prevented the effects of prolonged exposures to high [isoflurane] on neurogenesis. Pharmacological and imaging experiments suggest that these effects are likely due to differential activation of InsP₃ or ryanodine receptors. These results provide some insights into the interaction of anesthetics with neurogenesis and may have implications for studies into cognitive function and transplantation of NPCs under anesthesia.

Materials and Methods

Cell cultures

ReNcell CX cells, an immortalized human neural progenitor cell line, were derived from cortical region of 14 weeks gestation human fetal brain tissue obtained from Advanced Bioscience Resources (Alameda CA,) following normal terminations and in accordance with nationally (United Kingdom and/or United States) approved ethical and legal guidelines as previously described ³⁸. They were cultured according to the manufacturer’s instructions in ReNcell neural stem cell maintenance medium, supplemented with 20 ng/mL fibroblast growth factor (Millipore) and 20 ng/mL epidermal growth factor (Millipore, Billerica, MA) as previously described ^39;40. Cells were plated at a density of 1.5 million cells in T75 cm² tissue plastic culture flasks precoated with 20 μg/mL laminin (BD Biosciences, San Jose, CA) in DMEM/F12 (Gibco Invitrogen Corp, Grand Island, NY) and maintained as monolayer cultures at 37°C in a humidified incubator with 95% air and 5% CO₂. The culture medium was replaced every 48h. For consistency and practical reasons, all experiments were carried out on cells between passages of 6 and 15. Proliferation was measured by incorporation of Brdu (1:100, Invitrogen, Grand Island, NY) for 2 hours following isoflurane exposure. For the differentiation studies, ReNcell CX cells were cultured for 4 days in maintenance media devoid of growth factors.

Anesthetic exposure

ReNcell CX NPCs grown on 96-well plates or culture dishes (30000 cells/cm²) were exposed to isoflurane in a tight gas chamber (Bellco Glass, Vineland, NJ) placed in a culture incubator (Fisher Scientific, Pittsburgh, PA). Isoflurane was vaporized via an agent-specific vaporizer carried by humidified gas consisting of 5% CO₂, 21%O₂, and balanced N₂ (Boc Gases, Bellmawr, NJ). The flow rate to the tight gas chamber was initially 5L/min for the first two min of the experiment and 0.5L/min thereafter for the remainder of the experimental period. Pilot studies confirmed that gas flow devoid of isoflurane to the chamber does not affect cell survival. Gas phase concentration in the chamber was checked by infrared absorbance of effluent gas and constantly monitored and maintained at the desired concentration throughout experiments using an infrared Ohmeda 5330 agent monitor (Coast to Coast Medical, Fall River, MA). High performance liquid chromatography measurement confirmed that isoflurane concentrations of 2.4%, 1.2% and 0.6% in the chamber yielded isoflurane concentrations of 0.8mM, 0.4mM and 0.2mM, in the culture medium, respectively. Because isoflurane can pass blood brain barrier easily, these isoflurane concentrations in the culture medium are about 0.5 to 2 minimal alveolar concentrations (MAC) used in patients and should be considered clinically relevant concentrations. For the experiments on the impact of exposure duration on ReNcell CX NPCs survival, we exposed these cells to 2.4% isoflurane for 6, 12, or 24 h. Although 24 h isoflurane exposure is rarely seen in clinical settings, it has been consistently used to induce cytotoxicity in different in vitro systems ^3;36, making it a good model for isoflurane-induced cytotoxicity studies. Control ReNcell CX cultures were placed outside the tight gas chamber but inside the same incubator. Following anesthetic exposures, cells were immediately processed for cytotoxicity assays or immunocytochemistry unless noted otherwise.

Cytotoxicity assays

Lactate dehydrogenase (LDH) release into the media following isoflurane exposures was detected using an LDH release assay kit (Promega, Madison, WI) as previously described ^3;12;41. Briefly, 50μl of media was mixed with 50μl substrate mix in a 96-wells plate and incubated for 30 min at room temperature. The reaction was terminated with 50μl stop solution and the sample was quantified spectrophotometrically at 490nm using a plate reader (OPSYS MRTM Absorbance Reader, Dynex Technologies, Chantilly, VA). Background signal from the media was measured and subtracted from control signals. Mitochondrial dehydrogenase activity that reduces 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetra-zolium bromide (MTT) was used to determine cellular redox activity, an initial indicator of cell death, in a quantitative colorimetric assay. Cells were incubated with MTT (125μg/ml, Sigma-Aldrich, St. Louis, MO) in the growth medium for 1h at 37°C. The medium was then aspirated and the MTT reduction product, formazan, was dissolved in dimethyl sulfoxide (DMSO) and quantified spectrophotometrically at 570 nm. MTT assay detect early and LDH release assay detect late cell damage. The results of both LDH and MTT reduction assays were from at least three separate experiments and are expressed as percentage of control first and then compared statistically (n≥5) across three separate isoflurane concentrations (0.6, 1.2, or 2.4%) or durations (6, 12, or 24 h).

Cell proliferation determined by 5-bromodeoxyuridine (BrdU) incorporation and immunostaining

ReNcell CX NPCs were seeded on cover glasses pre-coated with 20 μg/mL laminin (BD Biosciences) in DMEM/F12 (MILLIPORE) overnight in proliferation medium (maintenance medium with 20ng/ml bFGF and 20ng/ml EGF). Brdu was added to the medium at a dilution of 1:100 for 2 hours following isoflurane exposure. The cells were then fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Following incubation in blocking solution (10% goat serum, 1% bovine serum albumin/phosphate buffered saline), cells were stained with anti-Brdu antibody (1:100, Invitrogen) over night at 4°C. After washing with Tris-buffered saline, cells were incubated with fluorescein isothiocyanate-goat anti-mouse IgG antibody (1:1000, Jackson Immunoresearch Laboratories, Inc. Fairfax, VA) for 1h. Cell nuclei were counterstained with DAPI (1:3000, Invitrogen, Carlsbad, CA) for 2–5 min at room temperature. Cover glasses with immunostained cells were mounted on an IX-70 inverted fluorescence microscope (400x, Olympus USA, Center Valley, PA) and images acquired using IpLab 3.6.5 software (Scanalytics, Inc. Fairfax, VA). Brdu positive cells were counted from 7 random locations from each slide by two persons blinded to experimental treatments. The percentage of Brdu positive cells over the total cells was calculated and compared across treatment groups from at least 3 different cultures.

Cell Differentiation determined by immunostaining of Tuj1 and GFAP

ReNcell CX NPCs were cultured as described above for the proliferation experiments. Prior to differentiation experiments, proliferation medium was replaced with differentiation-conditioned or media devoid of growth factors. For short isoflurane exposure or preconditioning, cells were exposed to 2.4% isoflurane for 1 hour. Prolonged isoflurane (2.4%) exposures were for 24 h in either preconditioning or non-preconditioning experiments. For the preconditioning experiments, prolonged isoflurane (2.4%) exposures began 4 h after initial short isoflurane (2.4%) exposure. After isoflurane exposure, ReNcell CX NPCs were allowed to differentiate for an additional 3 days following completion of isoflurane exposures. At the end of the differentiation period, the cells were fixed with 4% formaldehyde and processed for immunocytochemistry as described above for the proliferation experiments. Primary antibodies incubation was with Tuj1 (1:200, Covance, Princeton, NJ) or GFAP (1:1500, Millipore) for 2 h at 37°C for detection of cells with neuronal or glial phenotypes, respectively. Tuj 1 has been used successfully as a neuronal marker in the pluripotent human embryonic carcinoma immortalized cell line NTERA2 ⁴², while GFAP has been used as a glial marker in immortalized cell lines ^43;44. For visualization of 1° antibody signal, we used the Alexa-488 goat anti-rabbit and Alexa-594 goat anti-mouse IgG antibodies (Both at 1:1000, Invitrogen) for 1h at room temperature. Cell nuclei were counterstained with DAPI (1:3000, Invitrogen) for 2–5 min at RT. The cover glasses with immunostained cells were mounted on an IX-70 inverted fluorescence microscope (200x or 600x, Olympus USA, Center Valley, PA) and images acquired with IpLab 3.6.5 software (Scanalytics, Inc. Fairfax, VA). Tuj1 or GFAP positive cells overlapping with DAPI signal were counted from 7 random locations from each slide by two persons blinded to the experimental treatments. The percentage of Tuj1 or GFAP positive cells is given over the total cells and compared across treatment groups from at least 3 different cultures.

Measurement of isoflurane-evoked changes in cytosolic calcium concentration ([Ca²⁺]_c)

Changes in [Ca²⁺]_c was measured using fura-2/AM fluorescence (Molecular probe, Eugene, OR) with a photometer coupled to an Olympus 1×70 inverted microscope (Olympus America Inc.) and the IPLab v3.7 imaging processing and analysis software (Biovision Technologies, Exton, PA). The procedure for [Ca²⁺]c measurement was as previously described ^12;13;18;36. Briefly, coverslips with ReNcell CX human NPCs were washed 3 times in Ca²⁺-free Krebs-Ringer buffer and then loaded with 2.5 μM fura-2/AM (Molecular Probes) in the same buffer for 30 min at room temperature. Cover glasses were then placed in a sealed chamber (Warner Instrument Inc., Hamden, CT) connected with multiple inflow and one outflow tubes, which allowed for constant flow to the chamber. All bubbles in the chamber were flushed out at the beginning so that there was no gas phase in the sealed chamber during measurement of calcium concentration in the buffer. Baseline [Ca²⁺]_c was first recorded for at least 2 min and isoflurane-evoked changes were recorded in response to application of isoflurane (0.64 mM or 2 MAC) for at least 15 min in normal Krebs-Ringer buffer. Isoflurane application was through a separate inflow tube driven by a syringe pump (Braintree Scientific Inc., Braintree, MA). High performance liquid chromatography (System Gold, Beckmam Coulter, Fullerton, CA) was used for measurement of isoflurane concentration in the bath solution as previously described ^12;36. Fluorescence intensities were measured with alternate excitation at 340 and 380nm and emission at 510nM for a period up to 15 min for each treatment. The final results are given as a ratio of fluorescence intensities at 340/380nm (F340/F380) and as average of at least three separate experiments. The trypan blue exclusion assay was used after each imaging experiment to make sure that [Ca²⁺]_c measurements were from healthy and living cells.

Statistical Analysis

We used GraphPad Prism 4 software (GraphPad Software, Inc., San Diego, CA) for all statistical analyses and production of graphs. Data for one-group variable were analyzed using one-way ANOVA followed by Tukey multiple comparisons testing and those for two-group variable were analyzed using two-way ANOVA followed by Bonferroni multiple comparison test. The variance factor for one-way ANOVA was group comparisons, whereas those for two-way ANOVA were time or concentration and group comparisons. The significance level for all statistical comparisons was set at P <0.05.

Results

Isoflurane induced ReNcell CX cytotoxicity in a dose- and time-dependent manner via activation of InsP₃ and/or ryanodine receptors

We determined the dose- and time-dependence of isoflurane exposure on ReNcell CX NPCs survival. Our results show that isoflurane induced cell damages in a dose-(Fig. 1A and B) and time-dependent manner (Fig. 1C and D) as we have previously demonstrated for cortical neurons and PC12 cells ⁴¹. Exposure of ReNcell CX NPCs to 0.6% isoflurane for 24 h had no effect on survival, but exposure to 1.2% isoflurane, a clinically relevant concentration, resulted in significant cell damages as measured by the LDH assay (Fig. 1A). This clinical concentration, however, did not show any significant effects on cytotoxicity as measured by the MTT reduction assay, although a strong trend toward more cytotoxicity was noted (Fig. 1B). Exposure to 2.4% isoflurane for 24 h induced significant cytotoxicity as determined by both LDH and MTT assays (Fig. 1A, B, and C). On the other hand, exposure with the same concentration (2.4%) of isoflurane for 6 or 12 hrs did not result in significant cytotoxicity (Fig. 1C and D). These results suggest that survival of ReNcell CX NPCs depends on both the concentration and duration of isoflurane exposure.

Figure 1. Isoflurane induces ReNcell CX human neural progenitor cell damage in a dose- and time- dependent manner.

Cells were exposed to different concentrations (0.6%, 1.2% and 2.4%, respectively) of isoflurane (A, B) for 24 h or different durations (6hr, 12hr and 24 h respectively) at 2.4% isoflurane (C, D). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction and lactate dehydrogenase (LDH) release assays were used to determine early or late cell damage respectively. Data were obtained from at least 3 separate cultures, given as mean±SEM, and analyzed by two-way ANOVA followed by Bonferroni multiple comparison tests (n≥6 for each condition). *, ** or *** indicates P<0.05, P<0.01 or P<0.001, respectively, compared to untreated controls. ^{#, ##} or ^### indicates P<0.05, P≪0.01 or P<0.001, respectively, compared to 0.6% ispoflurane (A, B) or 6 h treatment groups (D). ^^ and ^^^ indicate P<0.01 or P<0.001 compared to 1.2% (A) or 12 h treatment groups (D). Sample size (n) represents the number of replicates assayed for LDH and MTT experiments.

To gain some insights into the mechanisms of isoflurane-mediated cytotoxicity in ReNcell CX NPCs, we investigated the role of calcium release from the ER. Pretreatment of ReNcell CX NPCs with the InsP₃R antagonist, xestospongin C (Xc, Fig. 2A) or the ryanodine receptor antagonist, dantrolene (Fig. 2B) significantly inhibited isoflurane-induced early cell damage as determined by the MTT reduction assay. To assess the role of InsP₃ release in isoflurane-mediated cytotoxicity in these cells, exposure to isoflurane (2.4%) was carried out in presence of the cholinergic agonist, carbachol. This treatment condition potentiated isoflurane-induced cytotoxicity as measured by the MTT reduction assay (Fig. 2C). Pretreatment with Xc mostly prevented this effect (Fig. 2C), suggesting that that InsP₃ release plays a role in mediating isoflurane-mediated effects on cytotoxicity. Similarly, depletion of ER calcium by the serca Ca²⁺ pump inhibitor, thapsigargin (TG), potentiated isoflurane-mediated cytotoxicity in ReNcell CX NPCs (Fig. 2D). Overall, these results suggest that isoflurane induced cytotoxicity in ReNcell CX NPCs through disruption of intracellular calcium homeostasis. This disruption in Ca²⁺ homeostasis appears to be mediated through excessive release of Ca²⁺ via InsP₃ or ryanodine receptor activation.

Figure 2. Isoflurane induces ReNcell CX human neural progenitor cells damage through activation of InsP₃ or ryanodine receptors.

A: InsP₃ receptor antagonist Xestospongin (Xc, 200 nM) inhibited ReNcell CX cell damage induced by 2.4% isoflurane (Iso) for 24 h (n=6 and 10, respectively). B: Ryanodine receptor antagonist dantrolene (Dan, 20 μM) inhibited ReNcell CX cell damage induced by 2.4% Iso for 24 h (n=12 and 10, respectively). C: Carbachol (10 μM) enhanced ReNcell CX cell damage induced by 2.4% isoflurane (Iso) for 24 h through activation of InsP₃ receptors (n=6 and 18, respectively). D: The sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (TG, 100 nM) potentiated ReNcell CX cell damage induced by 2.4% Iso for 24 h (n=6 and 12, respectively). All data are given as mean±SEM from at least three separate experiments, and analyzed by one-way ANOVA followed by Tukey multiple comparison tests. *, ** or *** indicates P<0.05, P<0.01 or P<0.001 respectively. Sample size (n) represents the number of replicates assayed for MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction experiments.

Isoflurane preconditioning ameliorated ReNcell CX cell damage induced by prolonged isoflurane exposure through activation of InsP₃ or ryanodine receptors

We have previously demonstrated that short isoflurane exposure inhibits cytotoxicity in cortical neurons and PC12 cells induced by prolonged exposure to the same anesthetic ³. Thus, we wondered whether such preconditioning mechanism operates in ReNcell CX NPCs. Preconditioning with 2.4% isoflurane for short exposure for 1 h nearly abolished ReNcell CX NPC cytotoxicity induced by prolonged exposure for 12 h to 2.4% isoflurane initiated at 4 h after completion of 1 h preconditioning short exposure (Figure 3A and B). Pretreatment of cultures with Xc or dantrolene prevented the protection afforded isoflurane-preconditioned ReNcell CX NPCs against toxic insults from prolonged isoflurane exposures (Fig. 3A), suggesting that Ca²⁺ flux through InsP₃ or ryanodine receptor plays important roles in isoflurane-mediated preconditioning and cytoprotection. In addition, depletion of endoplasmic reticulum (ER) calcium by thapsigargin not only potentiated the cytotoxicity induced by prolonged isoflurane exposure it eliminated the protection afforded by preconditioning or short isoflurane isoflurane exposure (Fig. 3B). To further understand, this dual effect of protection and cytotoxicity by isoflurane, we measured isoflurane-evoked changes in intracellular [Ca²⁺]_c in preconditioned and control cells (carrier gas exposed). Although our previous studies clearly demonstrated that isoflurane may induce cell apoptosis by over activation of the InsP₃ receptor and subsequent abnormal elevation of cytosolic and mitochondria Ca²⁺ concentration and decrease of ER Ca²⁺ concentration ^36;37, it is not clear if preconditioning neuronal with minimal exposures will ameliorate the abnormal elevation of cytosolic Ca²⁺ concentrations induced by subsequent detrimental isoflurane exposure. Isoflurane preconditioned ReNcell CX human NPCs showed significantly greater changes in intracellular [Ca²⁺]_c than control cells in response to isoflurane application then those cells without previous isoflurane preconditioning (Fig. 4). These results suggest that the preconditioning mechanism for isoflurane-mediated protection of ReNcell CX human NPCs prevents excessive changes in intracellular [Ca²⁺]_c in response to isoflurane exposure, possibly via calcium release from ER through InsP₃ or ryanodine receptor as demonstrated previously ^36;37;41

Figure 3. Preconditioning with short isoflurane exposure inhibits ReNcell CX human neural progenitor cells damage induced by prolonged isoflurane exposure through activation of InsP₃ or ryanodine receptors.

2.4% isoflurane (Iso) for short exposure (1 h) protected ReNcell CX cell from damages induced by prolonged exposure of 2.4% Iso for 12 h and this cytoprotective effect was prevented by, xestospongin C (Xc, 200 nM) or dantrolene (Dan, 20 μM) (A). Thapsigargin (TG, 100 nM) potentiated ReNcell CX cells damage induced by prolong exposure of 2.4% Iso for 12 h and prevented the neuroprotective effects of preconditioning by short exposure to 2.4% Iso for 1 h (B). All data are given as mean±SEM from at least three separate experiments, and analyzed by one-way ANOVA followed by Tukey multiple comparison tests (n≥ 16 for each condition). *, ** or *** indicates P<0.05, P<0.01 or P<0.001, respectively. Sample size (n) represents the number of replicates assayed for Lactate dehydrogenase (LDH) release experiments.

Figure 4. Isoflurane preconditioning ameliorated isoflurane-evoked elevation in cytosolic calcium concentration in ReNcell CX neuronal progenitor cells.

Changes in Fura2-AM intensities were measured in isoflurane (Iso) preconditioned ReNcell CX neuronal progenitor cells with either 2.4% isoflurane (Precon Iso+Iso, 2.4% for 1 h) or carrier gas (Control+Iso) in response to application of isoflurane (0.4 mM). F/340/F380 ratio values were normalized to baseline. A. Averaged typical response of cytosolic calcium concentrations to isoflurane. B. Peak elevations of cytosolic calcium level as percentage of control were compared (n≥38). C. Averaged elevation of cytosolic calcium level was measured using the area under the curve (AUC) as percentage of control (n≥38). Data are given as mean±SEM from at least three separate experiments. ^*** indicates P<0.001 by two tailed student t-test and the sample number (n) represents the number of cells or regions of interest analyzed (B and C).

Dual effects of isoflurane on ReNcell CX cell proliferation through activation of InsP₃ or ryanodine receptors

Single (4–6 h) or repeated short (45 min/day for 4 days) exposure of rodent NPCs to isoflurane decrease proliferation in vitro ^22;23 and in vivo, albeit with some discrepancies with regard to adult NPCs ^17;21. Thus, we assessed the impact of varying concentrations of isoflurane exposure for different durations on proliferation of ReNcell human NPCs. Compared to control (Figure 5A), exposure of these cells to isoflurane at diffconcentrations with different durations, in the presence or absence of isoflurane preconditioning, seemed not change the shape of cells (Figure 5B–F). Given that the number of NPCs appears to require modulation of Ca²⁺ influx through interaction with InsP₃ receptors ⁴⁵, we assessed the impact of prolonged inactivation of InsP₃ and ryanodine receptors on ReNcell human NPC proliferation. Indeed, treatment of these cells with varying concentrations of Xc or dantrolene decreased the number of proliferating ReNcell human NPCs, with effective doses of 100nM and 20μM for Xc and dantrolene, respectively (Fig 5G and H). These results suggest that normal Ca²⁺ flux through InsP₃ or ryanodine receptors plays a role in the regulation of neurogenesis. Low concentration of 0.6% isoflurane for 1 h enhanced proliferation, but the clinically relevant concentration of 1.2% isoflurane for 1 h had no effects (Fig 5A, B and I). However, exposure to high concentration of 2.4% isoflurane for 1 h decreased proliferation of ReNcell human NPCs (Fig 5A and I). To understand the impact of isoflurane exposure on this basal regulation of proliferation via modulating activation of InsP₃ or ryanodine receptors, we investigated the effects of prolonged isoflurane (2.4%) exposure in presence of Xc (50nM) or dantrolene (1μM) in concentrations that would not induce significant inhibition of ReNcell human NPC proliferation alone as demonstrated in Fig 5G and H. Both Xc and dantrolene significantly inhibited the suppression of ReNcell human NPC proliferation induced by prolonged exposure of 2.4% isoflurane for 24 hr (Fig 5J), suggesting prolonged use of isoflurane inhibit ReNcell human NPCs proliferation by over activation of InsP₃ or ryanodine receptors. Since isoflurane preconditioning protect ReNcell human NPCs from cytotoxicity induced by prolonged isoflurane (2.4%) exposure, we wondered whether this mechanism of cytoprotection has any implication on their proliferation and whether activation of InsP₃ or ryanodine receptors plays a role. Indeed, preconditioning with 0.6% isoflurane for 1 h inhibited suppression of ReNcell CX NPC proliferation induced by 24 h exposure to 2.4% isoflurane (Fig. 5A, B, C, D, and K). Pretreatment of cultures with Xc (50nM) or dantrolene (1μM) prevented the protection afforded isoflurane-preconditioned ReNcell CX human NPCs against toxic insults from prolonged isoflurane exposures (Fig. 5E, F and K), suggesting that Ca²⁺ flux through InsP₃ or ryanodine receptors also plays important roles in isoflurane-mediated preconditioning effect on proliferation of these cells. Altogether, these results suggest that isoflurane-mediated effects on proliferation of ReNcell CX human NPCs require activation of InsP₃ or ryanodine receptors.

Figure 5. Dual effects of isoflurane on proliferation and modulation of Ca²⁺ release from the endoplasmic reticulum (ER) via activation of InsP₃ or ryanodine receptors in ReNcell CX neural precursor cells.

(A–F) Representative micrographs of ReNcell neural precursors with incorporated Bromodeoxyuridine (Brdu, arrows) in the presence or absence of isoflurane (Iso) at various concentrations and durations, with or without InsP₃R antagonist xestospongin C (Xc) or ryanodine receptor antagonistdantrolene (Dan). Scale bar=100 μm. Proliferation of ReNcell CX neural progenitors requires calcium release from ER via InsP₃ (G) or ryanodine (H) receptors. Exposure to 2.4% isoflurane for 1 h (I) or 24 h (C, J, and K) significantly suppressed proliferation of ReNcell CX neural progenitors through activation of InsP₃ or ryanodine receptors (J). However, exposure of ReNcell CX neural progenitors to 0.6% for 1 h promoted proliferation (A, B, I, and K) through activation of InsP₃ (E and K) and ryanodine (F and K) receptors, while exposure to 1.2% isoflurane had no effects. Isoflurane (0.6%) preconditioning of ReNcell CX cells for 1 h mostly prevented the suppression of ReNcell CX neural progenitors proliferation induced by prolonged (24 h) isoflurane (2.4%) exposure (D and K). This preconditioning effect requires activation of InsP₃R (D, E, and K) or ryanodine receptors (D, F, and K). All data are given as mean±SEM from at least three separate experiments, and analyzed by one-way (J and K) or two-way (G–I) ANOVA followed by Bonferroni multiple comparison tests (n≥7). *, and ** or *** indicates P<0.05, P<0.01 or P<0.001 respectively compared to controls (G–I) or as indicated (J and K). The sample number (n) represents the number of cover glasses used to assess the % of Brdu positive cells from at least 7 random locations on each cover glass.

Dual effects of isoflurane on ReNcell CX cell differentiation

Acute isoflurane exposure has been shown to increase differentiation of NPCs in vivo ¹⁷ and in vitro ²³, but the cellular and molecular mechanisms are not clear. Thus, we wondered whether exposure of ReNcell CX NPCs to isoflurane can affect differentiation in a manner similar to its effects on proliferation as demonstrated in this study (Fig. 6). More specifically, we wondered whether isoflurane affects differentiation in a time-dependent manner with preconditioning features. Compared to its control (Figure 6A), Differentiation ReNcell CX human NPCs into neurons or glial fate dependent on anesthetic exposure duration (Figure 6B, C and D). Exposure to 2.4% isoflurane for 1 h had no effect as measured by Tuj1 (Fig. 6A, B and E) or GFAP positive cells (Fig. 6A, B and F). However, prolonged exposure to the same concentration of isoflurane significantly suppressed neuronal fate, while promoting glial fate (Fig. 6A, C, E and F). Consistent with its dual effects on cell survival (Fig. 3) and proliferation (Fig. 5), isoflurane preconditioned ReNcell CX NPCs were protected from the suppression of neuronal fate and promotion of glial cell fate selections induced by prolonged (24 h) isoflurane (2.4%) exposure (Fig. 6A, D, E and F).

Figure 6. Isoflurane preconditioning ameliorated the suppression of ReNcell CX neural progenitors differentiation induced by prolonged isoflurane exposure.

(A–D) Representative micrographs of differentiated and non-differentiated ReNcell CX neural progenitors under various pharmacological conditions (Scale bar=100 μm). ReNcell CX progenitors were exposed to 2.4% isoflurane (Iso) for 1 h (B) or for 24 h (C) in the absence or presence of preconditioning with 2.4% isoflurane for 1h (D). Tuj1 (neuronal class III β-tubulin) positive (red) or Glial fibrillary acidic protein (GFAP) positive (green) cells (A–D) were counted (n≥6) and expressed as % of controls (E and F). All data are given as mean±SEM and analyzed by one-Way ANOVA followed by Tukey multiple comparison tests (E, F). * or *** indicates P<0.05, or P<0.001, respectively. The sample number (n) represents the number of cover glasses used to assess the % of Tuj1 or GFAP positive cells from at least 7 random locations on each cover glass.

Discussion

We have demonstrated that isoflurane induced cytotoxicity and affected proliferation of ReNcell CX NPCs in a dose- and time-dependent manner. Prolonged isoflurane exposure inhibited neuronal cell fate, while promoting glial cell fate. Isoflurane preconditioning abolished cytotoxicity and the effects on neurogenesis induced by prolonged isoflurane exposure. The dual effects on cytotoxicity and proliferation required activation of InsP₃ or ryanodine receptors. To our knowledge, this is the first study to demonstrate dual effects of isoflurane on NPCs survival and a preconditioning effect on neurogenesis.

Dual effects of cytoprotection and cytotoxicity by general anesthetics have been demonstrated in various in vitro ^{3–5;10;22;35} and in vivo model systems ^{6;9;15;46–49}. Here we demonstrated that isoflurane induced cytotoxicity at high doses and cytoprotection at low doses in ReNcell CX NPCs (Fig 1–3). This is remarkably consistent with observations in 7 day old or in utero developing rat brains ^6;14. The mechanisms of neuroprotection by isoflurane in ReNcell CX NPCs are not clear, but our results suggest a role for ER localized InsP₃ or ryanodine receptors.

Isoflurane has been shown to be neurotoxic ^10–19, but rodent NPCs are resistant its toxic insults ^17;21–23. However, we report that isoflurane induced cytotoxicity in ReNcell CX NPCs in a dose- and time-dependent manner. The difference in exposure time or duration may explain the discrepancies between our study and others. Indeed, we only noted significant differences in cytotoxicity after 24 h of exposure at 1.2% or 2.4% of isoflurane, whereas others have reported data for acute or repeated exposures lasting ≤6 h ^17;21–23. Nonetheless, the results herein are consistent with isoflurane-mediated cytotoxicity in cardiac progenitors ^50;51. As demonstrated in cortical neurons and PC12 cells ³, isoflurane preconditioning of ReNcell CX NPCs protected these cells from cytotoxicity induced by prolonged isoflurane exposure. This is consistent with the protective effects by isoflurane or sevoflurane previously noted in cardiac ^50;51 or endothelial progenitors derived from human embryonic stem cells ⁵², respectively. This cytoprotective effect by isoflurane on stem cells has been described in various cell types in response to many biological stresses ^1–5. The InsP₃ receptor has been implicated in the maintenance of adult NPC number in bax knockout mice ⁴⁵, suggesting that isoflurane-mediated effects on ReNcell CX NPCs survival may require activation of these receptors. We found that to be the case for both isoflurane-mediated protection and cytotoxicity and, most surprisingly, the ryanodine receptor appears to be equally involved in these processes. Interestingly, isoflurane preconditioned ReNcell CX NPCs are less sensitive to isoflurane-evoked changes in [Ca²⁺]_c, suggesting that the dual effects of isoflurane on cytotoxicity and cytoprotection are possibly mediated through changes in Ca²⁺ homeostatic balance. In support of this notion, depletion of ER Ca²⁺ with TG exacerbated the cytotoxic effects of isoflurane, while preventing its cytoprotective effects.

Proliferation and differentiation of NPCs provide a great opportunity for studies into neurogenesis and replacement therapies under anesthesia. Thus, it is of particular importance to understand the basic mechanisms of anesthetic effects on neurogenesis. Here, we used the human ReNcell CX progenitor line to investigate the hypothesis that isoflurane affects survival and neurogenesis in a dual manner via activation of InsP₃ or ryanodine receptors. Although immortalized, these cells express the intermediate filament nestin, a marker of NPCs ³⁸. In addition, they maintain the ability to proliferate and differentiate into astrocytes, oligodendrocytes, and neurons ³⁸. Immortalized NPCs derived from cortical (ReNcell CX) and midbrain (ReNcell VM) tissues maintain stable phenotypes across passages compared to normal human NPCs ³⁸, but extrapolation of data from these cells to normal neurons is quite challenging given the paucity of studies into the biochemical and electrophysiological characterization of these cells. Differentiated ReNcell NPCs express GFAP (astrocyte), βIII-tubulin (neurons), or O1/Gal C (oligodendrocyte) and initial electrophysiological characterization of voltage-gated potassium (ReNcell CX) and sodium currents or action potentials (ReNcell VM) confirmed the specificity of these markers in these progenitors ³⁸, making them ideal for mechanistic studies into human neurogenesis.

Recent studies suggest that isoflurane affects proliferation of NPCs in an age-, dose-, and session-dependent manner. Exposure of postnatal rats to isoflurane, above 1 MAC, transiently ¹⁷ or persistently ²¹ decreased proliferation. Single exposure (4 h) of adult rats to 2.4% isoflurane initially decreased (for 1 day) and then increased proliferation of NPCs 5 to 10 days after anesthesia ¹⁷, whereas short exposure to 1.7% isoflurane had no effect ²¹. However, NPCs isolated from embryonic ²² or early postnatal ²³ rats consistently exhibited reductions in proliferation following single exposures to isoflurane (4–6 h) in a dose-dependent manner ²². By contrast, exposure of ReNcell NPCs to 0.6% isoflurane for 1 h enhanced proliferation in this study, but the clinically relevant concentration of 1.2% had no effects. Exposure to 2.4% isoflurane for 1 h, however, decreased proliferation of ReNcell CX NPCs as previously reported for rodent NPCs in vitro ^22;23 and for young rats in vivo ^17;21. Evidently, the duration of isoflurane exposure may influence proliferation in addition to doses, session number, and age. It is, however, clear from this study that activation of InsP₃ or ryanodine receptors may be an important modulator of isoflurane mediated-effects on proliferation of ReNcell CX NPCs, irrespective of the aforementioned factors. This is further supported by the requirement of the modulatory effect of Ca²⁺ influx and InsP₃ receptor activation in regulating NPC numbers in Bax knockout mice ⁴⁵.

Cell fate specification is a critical step in wiring of the central nervous system and the events underlying this process are under the combinatorial control of intrinsic and extrinsic factors ⁵³. Indeed, single isoflurane exposure at or above 1 MAC for 4 h promotes neuronal fate selection in primary cultures of early postnatal rat NPCs ²³ and in adult rats ¹⁷. Here we show that isoflurane exposure affected differentiation in a time-dependent manner. Exposure of ReNcell CX NPCs to 2.4% isoflurane for 1 h had no effect on neuronal or glial cell fate selection. However, prolonged exposure (24 h) suppressed neuronal fate, while promoting glial fate. Interestingly, isoflurane preconditioning of ReNcell CX NPCs prevented the suppression of neuronal fate and enhancement of glial fate selections induced by 24 h isoflurane exposure. The suppression of neuronal fate by prolonged isoflurane exposure in this study is inconsistent with previous reports ^17;23. The difference in exposure duration is a possible reason for the discrepancies. Although exposure to 2.4 % isoflurane for 24 h is rarely used in clinical settings, it served as a reliable approach for mechanistic insights into neurogenesis and survival of ReNcell CX NPCs. In addition, the contribution of InsP₃ and ryanodine receptors to isoflurane dual effects on ReNcell CX NPCs is inferred from highly specific pharmacological antagonists for these receptors, but nonspecific effects occasionally associated with pharmacological drugs cannot be ruled out completely. Thus, the findings of our study should not be used as a guide directly in anesthesia practice.

Our data suggest a model in which moderate Ca²⁺ release through InsP₃ receptor receptors promotes neurodegenesis (Fig. 7). By contrast, excessive Ca²⁺ release due to prolonged activation of these receptors may suppress neurogenesis (Fig. 7). The preconditioning effects of isoflurane are likely due to a non-detrimental reduction in ER [Ca²⁺]after short anesthetic exposure, which then mitigates excessive Ca²⁺ release in response to subsequent and prolonged isoflurane exposures (Fig. 4). Indeed, isoflurane preconditioned ReNcell CX NPCs displayed significant less isoflurane evoked changes in [Ca²⁺]_c (Fig 4). Accordingly, neurogenesis and survival of ReNcell CX NPCs are likely to correlate with the duration and level of isoflurane induced cytoplasmic Ca²⁺ elevation, with short and moderate Ca²⁺ inducing cytoprotection, whereas sustained and excessive Ca²⁺ elevation due to prolonged stimulation by isoflurane are expected to induce cytotoxicity (Figs. 4 and 7). Given the versatility of Ca²⁺ as a second messenger, isoflurane induced Ca²⁺ elevation via InsP₃ or ryanodine receptors may not be sufficient for the noted effects on ReNcell CX NPCs. Additional studies on other signaling pathways upstream and downstream of InsP₃ and ryanodine receptor activation should shed more light into other possible contributing factors into isoflurane-mediated dual effects in these cells. It should be noted that isoflurane has been shown to increase cytoplasmic Ca²⁺ level through N-methyl-D-aspartate ^1;54 and gamma-Aminobutyric acid receptors ^55;56, both upstream of ER Ca²⁺ signaling and major players in neurogenesis ^{24; 26}. Isoflurane prolongs gamma-Aminobutyric acid A receptor activation ⁵⁷ during the critical period of brain development and disrupts neurogenesis ²⁴. These results are remarkably consistent with the effects of isoflurane in this study, suggesting that gamma-Aminobutyric acid may act upstream of the ER to activate InsP₃ or ryanodine receptors to impinge on the noted isoflurane dual effects.

Figure 7. Model for Dual effects of isoflurane on neurogenesis.

Left side of the figure, Exposure to low and moderate concentrations of isoflurane for short durations induces moderate calcium release from the endoplasmic reticulum (ER) via activation of InsP₃R and/or ryanodine receptor calcium channels on ER membrane. This low or moderate increase in cytosolic calcium concentrations then promote neurogenesis by stimulating neuronal stem cell (NSC) proliferation, thus providing neuroprotection. Right side of figure, Prolonged exposures to high concentrations of isoflurane induce excessive Ca²⁺ release from the ER via over activation of InsP₃R and/or ryanodine receptors. This results in abnormally elevated cytosolic calcium concentrations and inhibition of neurogenesis by suppressing NSC proliferation, thus contributing to isoflurane mediated neurotoxicity.

In summary, our findings suggest that isoflurane may affect ReNcell CX NPC survival and neurogenesis in dual manner through differential activation of InsP₃ or ryanodine receptors located on the ER membrane. Given the complexity of Ca²⁺ signaling, we cannot attribute these effects solely on levels of Ca²⁺ elevation through InsP₃ and ryanodine receptors. However, our results suggest a strong association between isoflurane induced activity on these receptors and the dual effects on human ReNcell CX NPC survival and neurogenesis.

Final Boxed Summary Statement.

What we already know about this topic

• Isoflurane produces both experimental neuroprotective and neurotoxic effects on the developing brain, depending on the duration and level of exposure

What this article tells us that is new

• Using a human neural progenitor cell line, the authors confirmed and extended the dual effect of isoflurane exposures, and demonstrated the pivotal role of differential regulation of intracellular calcium in the cellular and molecular mechanisms of these effects