Volatile anesthetics, such as isoflurane, acutely ameliorate experimental ischemic brain injury (1–5). Isoflurane provides partial antagonism of the N-methyl-d-aspartate (NMDA) receptor (6,7). In vitro and in vivo data support the hypothesis that isoflurane-mediated neuroprotection can, in part, be explained by dose-dependent NMDA receptor antagonism (8–10). This protection is associated with reduced Ca2+ influx and preservation of mitochondrial membrane potential (10,11).
Ischemic lesions increase in size over time, even after perfusion has been restored (12). Thus, the magnitude of isoflurane neuroprotection may depend on when injury is measured. Several in vivo studies have shown reduction of either selective neuronal necrosis or cerebral infarct size by isoflurane when outcome was assessed at 5–7 days postischemia (3–5). However, one study has shown that isoflurane protection against middle cerebral artery occlusion may be transient (13). Although reduced infarct size was found 24 h postischemia in isoflurane-treated rats, protection could not be detected when infarct size was measured 14 days postischemia. It was postulated that isoflurane inhibits acute necrosis, but because of apoptotic mechanisms initiated as a result of ischemia, cells remain destined to die. This evidence has cast doubt on the ability of isoflurane to meaningfully reduce ischemic brain damage (14).
Sullivan et al. (15) evaluated the effect of 1% isoflurane on delayed neuronal death in vitro by using an organotypic hippocampal slice model subjected to oxygen-glucose deprivation (OGD). Slices treated with isoflurane (1%) or dizocilpine (10 μM) during OGD showed marked and similar protection whether necrosis was measured at 3, 7, or 14 days after OGD. Although the results indicated persistent isoflurane-induced preservation of tissue integrity, apoptotic or apoptotic-like responses were not assessed.
We have reported dose-dependent isoflurane-mediated protection against neuronal necrosis in primary mixed neuronal/glial cell cultures subjected to NMDA excitotoxicity (10). Apoptosis was not assessed. This study was designed to determine whether mixed neuronal/glial cultures protected by isoflurane against NMDA-induced necrosis become apoptotic.
All animal procedures were approved by the Duke University Animal Care and Use Committee. Mixed neuronal/glial cultures were prepared from fetal Sprague-Dawley rat brains (Harlan Sprague Dawley, Inc., Indianapolis, IN) at 18 days of gestation (10,16,17). Brains were harvested and dissected to separate cortex from meninges and subcortical structures by using anatomical landmarks. Cortices were pooled and minced into 2-mm3 pieces in a buffered salt solution (BSS; Hanks’ balanced salt solution; Life Technologies, Gaithersburg, MD) supplemented with 20 mM HEPES buffer (pH 7.4) containing 0.25% trypsin (Life Technologies). The tissue was incubated for 20 min at 37°C in a 5% CO2/95% room air and then washed twice with ice-cold, glutamine-free minimum essential medium (MEM; Life Technologies) containing 15 mM glucose, 5% fetal bovine serum (Gibco Diagnostics, Inc., Madison, WI), 5% horse serum (Gibco), and 1% deoxyribonuclease-I (Sigma Chemical Co., St. Louis, MO). Tissue pieces were dissociated by trituration through a fire-polished 9-inch Pasteur pipette. The resultant suspension was centrifuged at 50g for 10 min, the supernatant was discarded, and the pellet was resuspended in growth medium (MEM supplemented with 15 mM glucose, 5% fetal bovine serum, and 5% horse serum). The dissociated cells were plated to achieve a confluent monolayer (4 × 105 cells per well for neuronal/glial cultures) on poly-d-lysine-coated, 24-well culture plates (Falcon 3047; Becton Dickinson Co., Lincoln Park, NJ). Cultures were maintained undisturbed at 37°C in a humidified 5% CO2/balance room air atmosphere for 10–14 days before use. Previous studies, performed under identical culture conditions (10), demonstrated that cell types are 54% ± 4% neurons and 46% ± 7% glia, as determined by immunohistochemical staining for cell-specific cytoskeletal filaments (neurofilament 160 for neurons and glial fibrillary acidic protein for astrocytes).
Mature cultures (10–14 days in vitro) underwent cell culture media exchange with exposure media: Mg2+-free BSS containing 20 mM HEPES buffer and 1.8 mM CaCl2 (pH 7.4). The cells were exposed to 100 μM NMDA (Sigma), a concentration known to cause necrotic cell death (10). This was reconfirmed in this study with a dose-response curve and lactate dehydrogenase (LDH) release analysis (data not shown). The cultures were returned to the incubator and maintained at 37°C for 30 min. The NMDA-containing medium was then replaced with MEM supplemented with 20 mM glucose.
A stock solution of isoflurane dissolved in culture medium was prepared by using a modification of the method of Blanck and Thompson (18). A 10 mM isoflurane solution was made by injecting 130 μL of liquid isoflurane (1-chloro-2,2,2-trifluoroethyl difluoromethyl ether; Abbott Laboratories Inc., Chicago IL) into 103 mL of BSS in a 100-mL volumetric flask. The flask was sealed with a glass stopper to exclude all air from the neck of the flask. The flask was wrapped in aluminum foil, and the solution was stirred for 24 h to dissolve the isoflurane. Immediately before use, 30 mL of the concentrated stock solution was poured into a 50-mL polypropylene centrifuge tube and vortexed for 5–10 s to produce the working stock. The working stock was then diluted with BSS to produce 0.4 and 1.6 mM isoflurane for treating cell cultures. In previous work, the stability of isoflurane in solution was determined by incubating samples of the working stock under identical conditions used in treating cell cultures for 30 min. Gas chromatography was used to confirm the isoflurane concentration (10).
Mature cultures were exposed to 100 μM NMDA with or without dissolved isoflurane (0, 0.4, and 1.6 mM). The cultures were returned to the incubator and maintained at 37°C for 30 min. The medium was then removed and replaced with MEM supplemented with 20 mM glucose (no isoflurane or NMDA). In different experiments studying the above conditions, Hoechst/propidium iodide and terminal-deoxynucleotidyl transferase end-nick labeling (TUNEL) staining, DNA fragmentation enzyme-linked immunosorbent assay (ELISA), and caspase-3 activity were measured 24 and/or 48 h after NMDA exposure (see below). For each experiment, control conditions constituted no exposure to NMDA, isoflurane, or dizocilpine. The noncompetitive NMDA receptor antagonist dizocilpine maleate (MK-801; Tocris Cookson Inc., St. Louis, MO) (10 μM) was used as a positive control (10,19).
Hoechst/propidium iodide double staining was performed 24 h after NMDA exposure. Cells were exposed to propidium iodide (40 μg/mL) dissolved in BSS for 10 min to identify cells with disrupted membranes. After washing with BSS, the cells were fixed with 4% paraformaldehyde for 10 min, followed by washing with BSS. The cells were then treated with 10 μg/mL Hoechst 33258 (Sigma) in BSS for 4 min in the dark and washed with BSS. The cells were stored in 300 μL of BSS for later assessment of nuclear morphology. All staining procedures were performed at room temperature.
The cells were viewed under an inverted fluorescent microscope with the observer blinded to the treatment condition. Healthy, necrotic, and apoptotic cells were counted. Three fields were chosen for each experimental condition and used for cell counting. The number of cells counted for each field was averaged to produce a single numerical value for each experimental condition. Each experimental condition was performed 12 times. Cellular outcomes were morphologically defined on the basis of the Hoechst and propidium iodide stains (20). Healthy cells were defined as those with no propidium iodide staining and without evidence of nuclear condensation. Necrotic cells were defined as those visibly stained by propidium iodide, which signals cell membrane lysis (20). Cells without propidium iodide staining, but with condensed nuclei, were defined as apoptotic or apoptotic-like (21,22).
At 48 h after NMDA exposure, caspase-3 activity was measured in cell lysates. Equal volumes of cell lysate and reaction buffer containing 40 mM HEPES, 200 mM NaCl, 2 mM EDTA, 0.2% CHAPS, 20% sucrose, and 20 mM dithiothreitol were combined in a microtiter plate and incubated at 37°C for 1 h with 100 μM Ac-DEVD-AFC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin; Pharmingen International, San Diego, CA). Ac-DEVD-AFC is a synthetic tetrapeptide substrate that is cleaved by active caspase-3. This substrate is cleaved between D and AFC, releasing the fluorogenic AFC (Pharmingen Technical Data Sheet, July 5, 2001). AFC fluorescence was measured at an excitation wavelength of 350 nm and an emission wavelength of 460 nm. Background fluorescence values, determined by measuring samples of cell lysates and reaction buffer, were subtracted from fluorescence units obtained from each sample. Given the results of the 48-h analysis, the study was repeated with caspase-3 activity measured at 1, 6, 12, and 24 h after NMDA/isoflurane or NMDA/dizocilpine exposure.
Cultures were analyzed for apoptosis at 24 and 48 h after NMDA exposure by using the In Situ Cell Death Detection Kit, AP (Boehringer Mannheim, Eugene, OR). TUNEL labels the 3′-OH terminal of DNA strand breaks. Antifluorescein antibody from sheep conjugated with alkaline phosphatase detects the incorporated fluorescein. After substrate reaction with Fast Red (Boehringer Mannheim), the cells were examined under inverted light microscopy with the observer blinded to treatment group. All stained cells with cellular shrinkage and nuclear condensation were considered to be apoptotic and were designated as TUNEL positive (23). Positive-staining cells with evidence of cellular swelling or lysis were considered necrotic and were designated as TUNEL negative. Within each well (six wells per experiment), TUNEL-positive and TUNEL-negative cells were counted, and the percentage of TUNEL-positive cells was calculated from the total number of counted cells.
A sandwich ELISA method was used to measure the enrichment of histone-associated DNA fragments (mononucleosomes and oligonucleosomes). The ELISA uses a monoclonal antihistone antibody as a capture antibody. Nonspecific sites are then saturated by treatment with a commercial incubation buffer. The nucleosomes in the sample bind their histone component to the immobilized antihistone antibody with a peroxidase label. The bound anti-DNA-peroxidase was quantified by using the substrate 2,2′-azino-di-(3-ethyl) benzisothiazoline sulfonate measured at an absorbance of 405 nm. Background absorbance values, determined by measuring samples of cell lysates and reaction buffer, were subtracted from the sample absorbance values (24).
Data were compared by one-way analysis of variance. When indicated by a significant F ratio, the Scheffé test was used to define between condition differences. Values are reported as mean ± sd. A P value <0.05 was considered significant. Statistical analysis was performed with StatView 5.0 (SAS Institute, Cary, NC).
With the Hoechst/propidium iodide double stain, when examined at 24 h, 100 μM NMDA decreased the fraction of healthy cells (i.e., no propidium iodide staining and no evidence of nuclear condensation) (P < 0.001). Isoflurane 1.6 mM caused an increase in the fraction of healthy cells compared with 0 mM isoflurane (P < 0.001). Dizocilpine also increased the fraction of healthy cells and had a greater effect than 1.6 mM isoflurane (P = 0.006). NMDA 100 μM had no effect on the fraction of apoptotic or apoptotic-like cells, and this was not influenced by the addition of either isoflurane (0.4 or 1.6 mM) or 10 μM dizocilpine (Fig. 1).
Caspase-3 activity was assessed at 48 h after 100 μM NMDA exposure (Fig. 2). There was no increase in caspase-3 activity compared with control (P = 0.97). The addition of isoflurane to 100 μM NMDA also did not alter caspase-3 activity. The addition of 10 μM dizocilpine to 100 μM NMDA increased caspase-3 activity (P = 0.002).
A timed analysis of caspase-3 activity was performed at 1, 6, 12, and 24 h after a 30-min exposure to 100 μM NMDA and 0.4 or 1.6 mM isoflurane or 10 μM dizocilpine to exclude the possibility that caspase-3 activity was increased earlier than the 48-h observation interval described above. An early increase of caspase-3 activity was not seen in the NMDA/isoflurane-treated cultures or in the NMDA/dizocilpine-treated cultures (data not shown).
At 24 h, the TUNEL data were consistent with the Hoechst/propidium iodide data, i.e., an abundance of necrotic neurons with no evidence of apoptosis (Fig. 3). The absence of any increase in TUNEL-positive cells with apoptotic morphology at 24 h in the NMDA-alone condition indicates that the TUNEL analysis with morphologic considerations was effective in excluding necrotic neurons. At 48 h postexposure, 100 μM NMDA caused a 5-fold increase in TUNEL-positive cells relative to control (P = 0.006). Both 0.4 mM (P = 0.006) and 1.6 mM (P = 0.002) isoflurane prevented the emergence of TUNEL-positive cells at 48 h. Dizocilpine was less effective than isoflurane in preventing the emergence of TUNEL-positive cells. This is of note in a population with an increased number of healthy-appearing cells in the NMDA plus dizocilpine condition at 24 h relative to isoflurane, as determined by Hoechst/propidium iodide staining (Fig. 1).
Similar to the Hoechst/propidium iodide and TUNEL experiments, none of the treatment conditions assessed at 24 h was found to exhibit apoptosis that was statistically different from that in the control condition (Fig. 4). At 48 h, all conditions except for 100 μM NMDA plus 1.6 mM isoflurane (P = 0.074) had increased absorbance, i.e., DNA fragmentation, compared with control (P < 0.001).
The key findings are as follows: NMDA (100 μM) induced necrotic cell death in primary mixed neuronal/glial cultures when measured at 24 hours. Isoflurane reduced cellular necrosis (defined on the basis of propidium iodide exclusion) but did not increase the number of apoptotic or apoptotic-like cells, as defined by microscopic morphologic analysis with Hoechst and TUNEL staining. Caspase-3 activity was altered by neither NMDA alone nor coexposure to NMDA and isoflurane, but it was increased by coexposure to NMDA and dizocilpine. Isoflurane (0.4 and 1.6 mM) did not increase the fraction of TUNEL-positive cells or promote DNA fragmentation resulting from 100 μM NMDA exposure at either 24 or 48 hours. Thus, we found no evidence that cells surviving because of isoflurane treatment had become apoptotic.
Volatile anesthetics interact with the NMDA receptor (6,9–11). This interaction has been suggested to account, at least in part, for the neuroprotection provided by volatile anesthetics in both in vivo and in vitro models. Isoflurane anesthesia reduced lesion size in rats subjected to intracortical NMDA injection (9). Isoflurane also reduced cell death and Ca2+ uptake in hippocampal slices exposed to glutamate or NMDA excitotoxicity (11,25). We previously reported that isoflurane reduced NMDA-stimulated LDH release and Ca2+ uptake in primary mixed neuronal/glial cultures within the concentration range of 0.1–1.6 mM (10). The current experimental data support these findings.
Ischemia and excitotoxic insults induce a spectrum of mechanisms that lead to cellular demise, defined in part by insult severity (26). Our experiments examined a moderately severe insult (100 μM NMDA). This severity caused rapid necrotic cell death that was attenuated by isoflurane. Although isoflurane treatment partially suppressed necrotic mechanisms, it may have allowed delayed apoptosis to proceed. Thus, it can be argued that a post-NMDA exposure observation interval longer than the 48-hour interval used in this experiment could have produced a different result. Although this remains possible, several lines of evidence argue against this. First, apoptosis was already present at 48 hours in sister cultures coexposed to dizocilpine and NMDA, as defined by morphologic analysis of TUNEL-stained samples, caspase-3 activity, and histone-bound DNA fragmentation. Thus, the induction of apoptosis at 48 hours in isoflurane-treated cultures should have been detected if it was present. Second, Kawaguchi et al. (27) studied isoflurane-anesthetized rats subjected to focal cerebral ischemia. The number of apoptotic neurons was similar at both one day and seven days postischemia. Finally, organotypic hippocampal slices, protected by isoflurane against OGD, did not deteriorate over a 14-day post-OGD observation interval (15).
Leist and Jaattela (28) proposed four patterns of neuronal cell death (necrosis, necrosis-like, apoptosis-like, and apoptosis). Propidium iodide staining is specific for necrotic death (20). To assess apoptotic responses to NMDA excitotoxicity, we used four independent assays. Nuclear condensation, as observed with Hoechst staining, is consistent with apoptosis but may also detect cells undergoing apoptotic-like cell death (26,29). TUNEL staining is often used as a marker for apoptosis but, again, is not sufficient to exclude necrotic mechanisms (30). Although these assays allowed morphological evaluation at the intact cellular level, the lack of specificity could allow necrotic-like or apoptotic-like cells to be to be falsely counted as being apoptotic, thus leading to an overestimation of the number of apoptotic cells. Measurement of DNA fragmentation and caspase-3 activity provides a more specific analysis for apoptosis (31); thus, these assays were included in our experimental design.
Both isoflurane and dizocilpine reduced the number of TUNEL-positive cells caused by NMDA exposure. The reduction was larger with isoflurane than with dizocilpine. We then used DNA fragmentation and caspase-3 activation assays but still found no evidence of apoptosis in the isoflurane-treated cultures. Thus, these techniques agree with and support the hypothesis that cells surviving NMDA excitotoxicity, as a result of coexposure to isoflurane, would not become apoptotic.
NMDA alone induced apoptotic changes, as defined by nuclear condensation in TUNEL-stained samples and DNA fragmentation ELISA, but caspase-3 activity was not altered at 48 hours. It is possible that the 48-hour observation interval missed caspase-3 activation that had occurred at earlier intervals in any of the treatment conditions. However, a time-course study from 1 to 24 hours after OGD demonstrated no increase in capsase-3 activity after NMDA exposure, with or without isoflurane or dizocilpine coexposure. In contrast, NMDA in the presence of dizocilpine did increase caspase-3 activity at 48 hours. This positive control is consistent with other investigations (32,33). Therefore, although the data appear strong enough to exclude caspase-3-mediated apoptosis in isoflurane-protected cells, we cannot exclude that delayed cell death may have occurred by mechanisms independent of caspase-3 activation (34).
In an in vitro model similar to ours, evidence of apoptosis was observed at 48 hours after OGD in cultures treated with 10 μM dizocilpine (35). This supports our findings that a specific glutamate antagonist may contribute to apoptosis in the presence of an excitotoxic stress such as NMDA. NMDA antagonist-induced apoptosis has also been demonstrated in models of immature brain (36,37). These studies may have the greatest relevance to our investigation, because our cell cultures were derived from fetal brain tissue. Thus, our finding of a proapoptotic effect of dizocilpine is not surprising.
In conclusion, this study replicates earlier in vitro work that demonstrated mild protection against NMDA-induced excitotoxic necrotic injury by isoflurane in primary mixed neuronal/glial cell cultures (10,11,15). No evidence was found in this study to indicate that those cells protected by isoflurane had become apoptotic or apoptotic-like. These in vitro data do not support the hypothesis that isoflurane protection against acute excitotoxic necrosis allows residual apoptotic cell death to proceed.
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