Dual Effects of Isoflurane on Proliferation, Differentiation, and Survival in Human Neuroprogenitor Cells

Abstract

ISOFLURANE has shown neuroprotective properties in response to numerous biological stresses in vitro 1–5and in vivo .6–9However, an increasing number of studies suggest that isoflurane may be neurotoxic in vitro 10–13and in vivo 14–19as well. Isoflurane causes persistent hippocampal-dependent cognitive deficits in rodents,15,16but the mechanisms of such deficits are not clear. Neurogenesis in the hippocampus is involved in memory acquisition,20suggesting that isoflurane may act on neural progenitor cells (NPCs) to impinge on hippocampal-dependent cognitive functions. Accordingly, emerging studies into the mechanisms of anesthetic-induced cognitive deficits have provided some discrepant results on anesthetic-mediated effects on neurogenesis in vivo 17,21and some consistent results in vitro .22,23Because of their importance to cognitive functions and regenerative medicine, it is critical to gain more insight into the mechanisms by which general anesthetics affect neurogenesis.Development of NPCs is regulated by γ-aminobutyric acid and intracellular Ca2+mobilization.24–27Ca2+mobilization through inositol-1,4,5-trisphosphate (InsP3) and ryanodine receptors plays important roles in proliferation and differentiation of nonexcitable cells.28,29Ca2+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 reenter the cell cycle, and promoting the initiation of DNA synthesis at the G1/S transition.30,31The 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.30,32Isoflurane neuroprotective properties in neurons are mediated through an association with smaller isoflurane-evoked Ca2+release via InsP3receptors,33–35whereas the cytotoxic properties of this anesthetic are associated with excessive calcium release via InsP3receptors.12,13,36,37These results raise the possibility that both isoflurane-mediated cytoprotection and cytotoxicity in neurons occur in NPCs through differential InsP3or ryanodine receptor–mediated Ca2+mobilization and control of the neurogenesis process. Thus, we hypothesize that isoflurane affects survival, proliferation, and differentiation of NPCs in a dual manner via activation of InsP3or ryanodine receptors. To that end, we used the immortalized human neural progenitor cell line ReNcell, and show that 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 levels on neurogenesis. Pharmacologic and imaging experiments suggest that these effects are likely attributable to differential activation of InsP3or ryanodine receptors. These results provide some insight into the interaction of anesthetics with neurogenesis and may have implications for studies into cognitive function and transplantation of NPCs under anesthesia.ReNcell CX cells (Millipore, Billerica, MA), an immortalized human neural progenitor cell line, were derived from the cortical region of human fetal (14-weeks’ gestation) 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 described previously.38They 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) as described previously.39,40Cells were plated at a density of 1.5 million cells in T75, 75-cm2, tissue plastic culture flasks precoated with 20 μg/ml laminin (BD Biosciences, San Jose, CA) in Dulbecco’s Modified Eagle Medium/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 48 h. For consistency and practical reasons, all experiments were carried out on cells between passages of 6 and 15. Proliferation was measured by incorporation of 5-bromodeoxyuridine (1:100; Invitrogen, Grand Island, NY) for 2 h after isoflurane exposure. For the differentiation studies, ReNcell CX cells were cultured for 4 days in maintenance medium devoid of growth factors.ReNcell CX NPCs grown on 96-well plates or culture dishes (30,000 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 nitrogen (Boc Gases, Bellmawr, NJ). The flow rate to the tight gas chamber was initially 5 l/min for the first 2 min of the experiment and 0.5 l/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 monitored constantly 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 0.8, 0.4, and 0.2 mM in the culture medium, respectively. Because isoflurane can pass the blood–brain barrier easily, these isoflurane concentrations in the culture medium are approximately 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 NPC 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,3,36making 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.Lactate dehydrogenase (LDH) release into the media following isoflurane exposures was detected using an LDH release assay kit (Promega, Madison, WI) as described previously.3,12,41Briefly, 50 μl of media was mixed with 50 μl of substrate mix in a 96-well plate and incubated for 30 min at room temperature. The reaction was terminated with 50 μl of stop solution and the sample was quantified spectrophotometrically at 490 nm using a plate reader (Opsys MR Absorbance Reader; DYNEX Technologies, Inc., Chantilly, VA). Background signal from the media was measured and subtracted from control signals. Mitochondrial dehydrogenase activity that reduces 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 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 (125 μg/ml; Sigma-Aldrich, St. Louis, MO) in the growth medium for 1 h at 37°C. The medium was then aspirated and the MTT reduction product, formazan, was dissolved in dimethyl sulfoxide and quantified spectrophotometrically at 570 nm. MTT assay detects early and LDH release assay detects 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).ReNcell CX NPCs were seeded onto cover glasses precoated with 20 μg/ml laminin (BD Biosciences) in Dulbecco’s Modified Eagle Medium/F12 (Invitrogen) overnight in proliferation medium (maintenance medium with 20 ng/ml basic fibroblast growth factor and 20 ng/ml epidermal growth factor). 5-Bromodeoxyuridine was added to the medium at a dilution of 1:100 for 2 h after isoflurane exposure. The cells were then fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. After incubation in blocking solution (10% goat serum, 1% bovine serum albumin/phosphate-buffered saline), cells were stained with anti–5-bromodeoxyuridine antibody (1:100; Invitrogen) overnight at 4°C. After washing with Tris-buffered saline, cells were incubated with fluorescein isothiocyanate–goat anti-mouse immunoglobulin G antibody (1:1,000; Jackson ImmunoResearch Laboratories, Inc., Fairfax, VA) for 1 h. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (1:3,000; Invitrogen) for 2–5 min at room temperature. Cover glasses with immunostained cells were mounted on an IX-70 inverted fluorescence microscope (400×; Olympus USA, Center Valley, PA) and images acquired using IpLab 3.6.5 software (Scanalytics, Inc., Fairfax, VA). 5-Bromodeoxyuridine–positive cells were counted from seven random locations from each slide by two persons blinded to experimental treatments. The percentage of 5-bromodeoxyuridine–positive cells over the total cells was calculated and compared across treatment groups from at least three different cultures.ReNcell CX NPCs were cultured as described above for the proliferation experiments. Before 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 h. Prolonged isoflurane (2.4%) exposures were for 24 h in either preconditioning or nonpreconditioning 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 after 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. Incubation of primary antibodies was accomplished with Tuj1 (1:200; Covance, Princeton, NJ) or glial fibrillary acidic protein (GFAP) (1:1,500; Millipore) for 2 h at 37°C for detection of cells with neuronal or glial phenotypes, respectively. Tuj1 has been used successfully as a neuronal marker in the pluripotent human embryonic carcinoma immortalized cell line NTERA2,42whereas GFAP has been used as a glial marker in immortalized cell lines.43,44For visualization of primary antibody signal, we used the Alexa-488 goat anti-rabbit and Alexa-594 goat anti-mouse immunoglobulin G antibodies (both at 1:1,000; Invitrogen) for 1 h at room temperature. Cell nuclei were counterstained with 4′,6-diamidino-2-phenylindole (1:3,000; Invitrogen) for 2–5 min at room temperature. The cover glasses with immunostained cells were mounted on an IX-70 inverted fluorescence microscope (200× or 600×; Olympus USA) and images acquired with IpLab 3.6.5 software (Scanalytics). Tuj1- or GFAP-positive cells overlapping with 4′,6-diamidino-2-phenylindole signal were counted from seven 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 three different cultures.Changes in cytosolic Ca2+concentration ([Ca2+]c) were measured using Fura-2/AM fluorescence (Molecular Probes, Eugene, OR) with a photometer coupled to an Olympus 1X70 inverted microscope (Olympus America) and the IPLab v3.7 imaging processing and analysis software (Biovision Technologies, Exton, PA). The procedure for [Ca2+]cmeasurement was as described previously.12,13,18,36Briefly, coverslips with ReNcell CX human NPCs were washed three times in Ca2+-free Krebs-Ringer buffer and then loaded with 2.5 μM Fura-2/AM 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 to multiple inflow tubes and one outflow tube, 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 [Ca2+]cin the buffer. Baseline [Ca2+]cwas 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; Beckman Coulter, Fullerton, CA) was used for measurement of isoflurane concentration in the bath solution as described previously.12,36Fluorescence were measured with at and nm and at for to 15 min for each The results are given as a of fluorescence at nm and as an of at least three separate experiments. The assay was used after each imaging experiment to that were from and used 4 software Inc., CA) for all and of for were using by multiple and for were using by the multiple The factor for was for were or concentration and The for all was at the and of isoflurane exposure on ReNcell CX NPC survival. results show that isoflurane induced cell in a and manner as we have for cortical neurons and of ReNcell CX NPCs to 0.6% isoflurane for 24 h no on survival, but exposure to a clinically relevant in cell as measured by the LDH assay clinical not show effects on cytotoxicity as measured by the MTT reduction a more cytotoxicity was noted to 2.4% isoflurane for 24 h induced cytotoxicity as by both LDH and MTT assays exposure with the same concentration (2.4%) of isoflurane for 6 or h not in cytotoxicity These results suggest that of ReNcell CX NPCs on both the concentration and duration of isoflurane gain some insight into the mechanisms of isoflurane-mediated cytotoxicity in ReNcell CX we the of calcium release from the of ReNcell CX NPCs with the or the ryanodine isoflurane-induced early cell as by the MTT reduction assay. To the of in isoflurane-mediated cytotoxicity in these exposure to isoflurane (2.4%) was carried out in the of the treatment isoflurane-induced cytotoxicity as measured by the MTT reduction assay with mostly prevented this that plays a in isoflurane-mediated effects on of calcium by the calcium isoflurane-mediated cytotoxicity in ReNcell CX NPCs these results suggest that isoflurane induced cytotoxicity in ReNcell CX NPCs through of intracellular calcium in to be mediated through excessive release of InsP3or ryanodine have that short isoflurane exposure cytotoxicity in cortical neurons and cells induced by prolonged exposure to the same we such a preconditioning in ReNcell CX Preconditioning with 2.4% isoflurane for short exposure for 1 h ReNcell CX NPC cytotoxicity induced by prolonged exposure for h to 2.4% isoflurane at 4 h after completion of preconditioning short exposure of cultures with or prevented the ReNcell CX NPCs from prolonged isoflurane exposures that through InsP3or ryanodine receptors plays important roles in isoflurane-mediated preconditioning and of calcium by not the cytotoxicity induced by prolonged isoflurane exposure but the by preconditioning or short isoflurane exposure To this dual of and cytotoxicity by we measured isoflurane-evoked changes in intracellular [Ca2+]cin and control cells gas Although studies that isoflurane may induce cell by of the and of cytosolic and Ca2+concentration and of is not preconditioning NPCs with minimal exposures the of cytosolic induced by isoflurane exposure. ReNcell CX human NPCs changes in intracellular control cells in response to isoflurane application cells isoflurane preconditioning These results suggest that the preconditioning for isoflurane-mediated of ReNcell CX human NPCs excessive changes in intracellular [Ca2+]cin response to isoflurane exposure, via calcium release from through InsP3or ryanodine receptors as or short for 4 exposure of NPCs to isoflurane proliferation in vitro in vivo with some with to we the impact of concentrations of isoflurane exposure for different durations on proliferation of ReNcell human with control cells exposure of these cells to isoflurane at different concentrations with different in the or of isoflurane preconditioning, to not the of cells that the number of NPCs to of through interaction with the impact of prolonged of ryanodine receptors on ReNcell human NPC treatment of these cells with concentrations of or the number of ReNcell human with of and 20 μM for and These results suggest that normal through InsP3or ryanodine receptors plays a in the of neurogenesis. concentration of 0.6% isoflurane for 1 h proliferation, but the clinically relevant concentration of isoflurane for 1 h no effects and exposure to a high concentration of 2.4% isoflurane for 1 h proliferation of ReNcell human NPCs To the impact of isoflurane exposure on this of proliferation via activation of InsP3or ryanodine we the effects of prolonged isoflurane (2.4%) exposure in the of or in concentrations that not induce of ReNcell human NPC proliferation as in and the of ReNcell human NPC proliferation induced by prolonged exposure of 2.4% isoflurane for 24 h that prolonged of isoflurane ReNcell human NPC proliferation by of InsP3or ryanodine receptors. Because isoflurane preconditioning ReNcell human NPCs from cytotoxicity induced by prolonged isoflurane (2.4%) exposure, we this of cytoprotection has on their proliferation and activation of InsP3or ryanodine receptors plays a preconditioning with 0.6% isoflurane for 1 h of ReNcell CX NPC proliferation induced by 24-h exposure to 2.4% isoflurane of cultures with or prevented the ReNcell CX human NPCs from prolonged isoflurane exposures and that through InsP3or ryanodine receptors plays important roles in the isoflurane-mediated preconditioning on proliferation of these these results suggest that isoflurane-mediated effects on proliferation of ReNcell CX human NPCs activation of InsP3or ryanodine isoflurane exposure has been shown to differentiation of NPCs in vivo in vitro the cellular and mechanisms are not clear. Thus, we exposure of ReNcell CX NPCs to isoflurane can affect differentiation in a manner to effects on proliferation as in this we isoflurane affects differentiation in a manner with preconditioning to control differentiation of ReNcell CX human NPCs into neurons or glial on anesthetic exposure duration 6, to 2.4% isoflurane for 1 h no as measured by Tuj1- 6, and or GFAP-positive cells 6, and prolonged exposure to the same concentration of isoflurane neuronal and glial 6, and with dual effects on cell and proliferation ReNcell CX NPCs were from the of neuronal and of glial cell induced by prolonged isoflurane (2.4%) exposure 6, have that isoflurane induced cytotoxicity and proliferation of ReNcell CX NPCs in a and manner. Prolonged isoflurane exposure neuronal cell and glial cell Isoflurane preconditioning cytotoxicity and the effects on neurogenesis induced by prolonged isoflurane exposure. The dual effects on cytotoxicity and proliferation activation of InsP3or ryanodine receptors. To this is the first to dual effects of isoflurane on NPC and a preconditioning on effects of cytoprotection and cytotoxicity by general anesthetics have been in in vitro and in vivo model this we that isoflurane induced cytotoxicity at high and cytoprotection at in ReNcell CX NPCs is consistent with in or in mechanisms of by isoflurane in ReNcell CX NPCs are not but results suggest a for InsP3or ryanodine has been shown to be NPCs are to we that isoflurane induced cytotoxicity in ReNcell CX NPCs in a and manner. The in exposure or duration may the between and we noted in cytotoxicity after 24 h of exposure at or 2.4% of have for or exposures or to 6 the results in this are consistent with isoflurane-mediated cytotoxicity in in cortical neurons and preconditioning of ReNcell CX NPCs these cells from cytotoxicity induced by prolonged isoflurane exposure. is consistent with the effects of isoflurane or noted in derived from human embryonic stem of isoflurane on stem cells has been described in cell in response to biological has been in the maintenance of NPC number in that isoflurane-mediated effects on ReNcell CX NPC may activation of these receptors. that to be the for both isoflurane-mediated and cytotoxicity the ryanodine to be involved in these ReNcell CX NPCs are to isoflurane-evoked changes in that the dual effects of isoflurane on cytotoxicity and cytoprotection are mediated through changes in of this of the cytotoxic effects of isoflurane and prevented and differentiation of NPCs provide a for studies into neurogenesis and under Thus, it is of importance to the basic mechanisms of anesthetic effects on neurogenesis. we used the human ReNcell CX progenitor line to the that isoflurane affects and neurogenesis in a dual manner via activation of InsP3or ryanodine receptors. Although these cells the a marker of the to and differentiate into and NPCs derived from cortical and across passages compared with normal human of from these cells to normal neurons is given the of studies into the and of these ReNcell NPCs GFAP or and initial of and or confirmed the of these in these for studies into human studies suggest that isoflurane affects proliferation of NPCs in an and manner. of to above 1 exposure of to 2.4% isoflurane initially 1 and then proliferation of NPCs days after short exposure to isoflurane no NPCs from early consistently in proliferation following exposures to isoflurane in a exposure of ReNcell NPCs to 0.6% isoflurane for 1 h proliferation in this but the clinically relevant concentration of no to 2.4% isoflurane for 1 proliferation of ReNcell CX NPCs as for NPCs in vitro for in vivo the duration of isoflurane exposure may proliferation in to and it is from this that activation of InsP3or ryanodine receptors may be an important of isoflurane-mediated effects on proliferation of ReNcell CX of the factors. is by the of the of and activation in NPC in is a critical in the of the and the this are under the control of and isoflurane exposure at or above 1 for 4 h neuronal in primary cultures of early in this we show that isoflurane exposure differentiation in a manner. of ReNcell CX NPCs to 2.4% isoflurane for 1 h no on neuronal or glial cell prolonged exposure neuronal and glial isoflurane preconditioning of ReNcell CX NPCs prevented the of neuronal and of glial induced by 24-h isoflurane exposure. The of neuronal by prolonged isoflurane exposure in this is with in exposure duration is a for the Although exposure to 2.4% isoflurane for 24 h is rarely used in clinical settings, it as a for insight into neurogenesis and of ReNcell CX the of ryanodine receptors to the dual effects of isoflurane on ReNcell CX NPCs is from specific for these but effects associated with be out Thus, the of should not be used as a in suggest a model in which Ca2+release through ryanodine receptors neurogenesis excessive Ca2+release by prolonged activation of these receptors may neurogenesis The preconditioning effects of isoflurane are likely attributable to a reduction in Ca2+concentration short anesthetic exposure, which then excessive Ca2+release in response to and prolonged isoflurane exposures ReNcell CX NPCs isoflurane-evoked changes in Accordingly, neurogenesis and of ReNcell CX NPCs are likely to with the duration and of isoflurane-induced with short and inducing and excessive from prolonged by isoflurane is to induce cytotoxicity the of a isoflurane-induced via InsP3or ryanodine receptors may not be for the noted effects on ReNcell CX studies on and of ryanodine activation should more onto into isoflurane-mediated dual effects in these should be noted that isoflurane has been shown to through γ-aminobutyric acid of and in γ-aminobutyric acid the critical of brain and results are consistent with the effects of isoflurane in this that γ-aminobutyric acid may act of the to InsP3or ryanodine receptors to impinge on the noted isoflurane dual suggest that isoflurane may affect ReNcell CX NPC and neurogenesis in a dual manner through differential activation of InsP3or ryanodine receptors on the the of we these effects to levels of through ryanodine receptors. results suggest a association between isoflurane-induced activity on these receptors and the dual effects on human ReNcell CX NPC and from of and of of and of of