The following stepwise approach was used to test the statistical assumptions for ANOVA testing: (1) Distribution: normality was tested using Shapiro-Wilk test. (2) Homogeneity of variances was evaluated by Brown-Forsythe test. All P values for Brown-Forsythe test were >0.05 except for Figures 2A (P = 0.005) and 2B (P = 0.001), Figure 3C (P = 0.01) and 3D (P = 0.02), and Figure 6B (P = 0.005). When P values were <0.05 for Brown-Forsythe test, we relied on the robustness of ANOVA against inequality of population variances in the case of equal samples sizes. (3) Independence of observation was given per study design, since no repeated measurements on the same sample or animal were done and experimental repetitions were true replications.
For the in vivo studies, we sought to detect a 25% increase in RGC death by isoflurane postexposure. Assuming an expected SD of 10% in RGC density counts from our previous use of this technique and based on previously published data and power analysis,34,36 an a priori power analysis (α = 0.05 with two-sided hypothesis, power 80%) indicated that a sample size of 6 animals per group would be sufficient to detect such a difference. One-way ANOVA with post hoc Tukey test was used for between-group comparison of the in vivo data. Data were analyzed with a computerized statistical program (GrapPad Prism 6.0, GraphPad Software). P < 0.05 was considered statistically significant.
Volatile Anesthetic Concentration in the Cell Culture Medium
To first confirm adequate exposure of neuronal cells to the volatile anesthetics in our in vitro gas application system, we used headspace gas chromatography-mass spectrometry to analyze anesthetic concentration in the cell culture medium after 2 hours of exposure to 1.2 MAC (isoflurane 1.4% in gas fraction, sevoflurane 2.4%, and desflurane 7.2%). The preceding calibration showed good linearity (R 2 > 0.99; Fig. 1, A–C) for all gases. The concentration of isoflurane (Fig. 1D) was 0.337 mM [0.231/0.400] (62.2 μg/mL [42.6/73.9]), sevoflurane 0.326 mM [0.295/0.355] (65.1 μg/mL [59.0/70.1]) and desflurane 0.591 mM [0.477/0.640] (99.2 μg/mL [80.2/108]). Measured concentrations correlated well with the expected concentration calculated with the Bunsen partition coefficients (α) for isoflurane, sevoflurane, and desflurane in aqueous solution as derived from the literature37,38:
- Isoflurane: α = 0.54 at 37°C; Caq (mM) = 0.44614 × 0.54 × 1.4 vol-% = 0.337 mM
- Sevoflurane: α = 0.37 at 37°C; Caq (mM) = 0.44614 × 0.37 × 2.4 vol-% = 0.396 mM
- Desflurane: α = 0.225 at 37°C; Caq (mM) = 0.44614 × 0.225 × 7.2 vol-% = 0.723 mM
Isoflurane Does Not Influence Mitochondrial Depolarization
To investigate the effects of volatile anesthetics on neuronal OGD-induced damage, we first measured mitochondrial membrane depolarization as an early indicator of apoptosis. Application of volatile anesthetics before OGD (Fig. 2A, pre-exposure) did not further aggravate the OGD-induced loss of the mitochondrial membrane potential (FL2/FL1 ratio: untreated 4.4 [4.2/4.7] versus OGD 1.3 [1.3/1.4], P < 0.0001; OGD versus isoflurane + OGD 1.4 [1.4/1.5], P = 0.94, versus sevoflurane + OGD 1.1 [1.0/1.3], P = 0.52, and versus desflurane + OGD 1.2 [0.8/1.3], P = 0.39). When volatile anesthetics were applied during the reoxygenation period (Fig. 2B, postexposure), neither isoflurane (P = 0.96, OGD versus OGD + isoflurane) nor sevoflurane (P = 0.34, OGD versus OGD + sevoflurane) influenced mitochondrial membrane potential. Desflurane partly blocked OGD-induced mitochondrial depolarization (FL2/FL1 ratio: OGD 0.8 [0.6/1.1] versus OGD + desflurane 1.6 [1.5/1.7], P = 0.004).
Neuronal Cell Death Is Aggravated by Isoflurane After OGD
To further characterize the influence of volatile anesthetics on neuronal cell death, we next determined apoptotic and necrotic cell death after OGD by AV/Pi staining followed by flow cytometry. OGD induced a strong increase in the percentages of early apoptotic (AV positive) and late apoptotic/necrotic (AV/Pi positive) cells (Fig. 3, A–D, Boxes 1 and 2). In pre-exposure experiments, neuronal cell death was not influenced by application of either gas (Fig. 3A: AV positive cells OGD 16.8% [15.6/19.3] versus isoflurane + OGD 15.9%, P = 0.99; OGD versus sevoflurane + OGD 16.1% [14.7/17.1], P = 0.86; OGD versus desflurane + OGD 13.5% [10.8/16.0]. P = 0.06, 99% CI of differences –0.97 to 8.11; Fig. 3B: AV/Pi positive cells OGD 46.8% [46.2/47.6] versus isoflurane + OGD 45.7 [44.6/47.4], P = 0.41; OGD versus sevoflurane + OGD 48.2% [46.8/48.9], P = 0.79, OGD versus desflurane + OGD 45.3 [42.8/46.9], P = 0.15, 99% CI of differences –0.85 to 4.50). Application of desflurane after OGD inhibited early apoptosis (Fig. 3C; AV positive cells: OGD 18.4% [15.8/22.9] versus OGD + desflurane 11.6% [10.6/13.6], P = 0.001). In contrast, isoflurane postexposure significantly increased the percentage of late-apoptotic/necrotic cells (Fig. 3D; AV/Pi positive cells: OGD 41.1% [39.0/43.3] versus OGD + isoflurane 48.5 [46.4/63.4], P = 0.001).
Isoflurane Increases LDH Release After Hypoxic Insult
To further corroborate the detrimental effect of isoflurane in injured neuronal cells, we next measured LDH release after OGD. While pre-exposure with volatile anesthetics did not increase LDH release after OGD (Fig. 4A), application of isoflurane after OGD significantly increased LDH release compared to sevoflurane and desflurane (Fig. 4B; fold change versus OGD: OGD + isoflurane 1.8 [1.7/2.0] versus OGD + sevoflurane 1.1 [1.1/1.2], P = 0.0004, and OGD + desflurane 1.1 [1.1/1.2], P = 0.0003), indicative of increased cellular damage after isoflurane application.
Isoflurane Increases NF-κB Binding Activity and Expression of NF-κB-Dependent Genes
Since activation of the transcription factor NF-κB can contribute to cell death in neuronal cells, we evaluated whether NF-κB activation was induced by OGD ± volatile anesthetics application. Western blot analyses (Fig. 5A) showed that phosphorylation of p65 at Ser536 (as an indicator of NF-κB activation) was suppressed by sevoflurane and desflurane (Fig. 5A, Lanes 7 and 8) during reoxygenation but not by isoflurane (Fig. 5A, Lane 6). DNA-binding activity of NF-κB was not influenced by isoflurane or desflurane pre-exposure, but sevoflurane inhibited DNA binding activity of NF-κB (Fig. 5B; optical density at 450 nm: OGD 0.26 [0.22/0.30] versus sevoflurane + OGD 0.19 [0.19/0.22], P = 0.008). When volatile anesthetics were applied during the reoxygenation period, isoflurane significantly increased DNA binding of NF-κB (Fig. 5C; OGD 0.24 [0.22/0.26] versus OGD + isoflurane 0.29 [0.26/0.29], P = 0.04), while desflurane reduced DNA-binding activity (OGD 0.24 [0.22/0.26] versus OGD + desflurane 0.16 [0.16/0.17], P = 0.0002).
In a next set of experiments, we wanted to answer the question of whether the induction of NF-κB DNA-binding activity by postexposure with isoflurane resulted in an increased expression of NF-κB-dependent genes. Luciferase assay experiments demonstrated that OGD alone and incubation of cells with PMA/ionomycin as a positive control increased NF-κB-dependent gene expression even further (Fig. 5D; RLU: untreated 56 [54/64] versus OGD 500 [499/637], P = 0.001, and versus PMA/ionomycin 953 [904/978], P = 0.007). When volatile anesthetics were applied during the reoxygenation period (postexposure), a specific effect of isoflurane was detected: isoflurane increased NF-κB-dependent gene expression strongly compared to OGD and PMA/ionomycin (OGD 500 [499/637] versus OGD + isoflurane 1478 [1363/1643], P = 0.001, and PMA/ionomycin 953 [904/978] versus OGD + isoflurane, P = 0.009). This strong activation of NF-κB-dependent gene expression was not detected with sevoflurane or desflurane (OGD versus OGD + sevoflurane, P = 0.35, and versus OGD + desflurane, P = 0.30).
Inhibition or Silencing of p75NTR Signaling Counteracts Isoflurane Neurotoxicity
These results prompted us to search for possible isoflurane targets that could serve as upstream regulators of NF-κB activation and are responsible for isoflurane neurotoxicity. Therefore, we next analyzed whether the detrimental effect of isoflurane is mediated via activation of p75NTR. Western blot analyses (Fig. 6A) confirmed the expression of p75NTR in neuronal SH-SY5Y cells. Flow cytometry after AV/Pi staining demonstrated that blocking p75NTR signaling with the blocking peptide TAT-pep5 dose-dependently abolished the harmful effect on neuronal cells exerted by isoflurane application after OGD (Fig. 6B; AV/Pi positive cells: OGD + isoflurane 56.9% [56.3/57.4] versus p75-Inh. 500 nM OGD + isoflurane 51.1% [49.2/51.3], P = 0.02, and versus p75-Inh. 1 μM OGD + isoflurane 50.2% [50.0/50.9], P = 0.02).
p75NTR silencing by RNA interference was confirmed by Western blot (Fig. 6C). Mock transfection or transfection with negative control siRNA did not affect the detrimental influence of isoflurane on OGD-injured neuronal cells (data not shown). However, silencing of p75NTR with specific siRNA reversed the detrimental effects of isoflurane (Fig. 6D; OGD 42.2% [40.0/45.3] versus OGD + isoflurane 44.9% [44.2/46.6], P = 0.07, 99% CI of differences –5.87 to 0.97).
Isoflurane Does Not Influence Retinal p75NTR Expression
In a last set of experiments, we translated the in vitro results into our previously well-described34 in vivo model of neuronal cell damage, namely retinal IRI. RGCs are particularly susceptible to cell death via apoptosis when subjected to IRI. We first examined the distribution and degree of retinal p75NTR expression by immunohistochemistry in retinal cross-sections (Fig. 7A). p75NTR staining was observed in the ganglion cell layer and in the inner and the outer plexiform layers. No difference in p75NTR expression was observed with or without the p75NTR inhibitor or isoflurane because TAT-pep5 is a downstream inhibitor of this receptor and does not cause a change in receptor expression itself. Injury of the retina is always accompanied by glial activation. We therefore analyzed Mueller cell activation by GFAP staining. There was a moderate increase in GFAP expression in all IRI-treated groups, which further confirmed retinal damage.
Isoflurane Enhances RGC Death After IRI In Vivo via p75NTR
The quantification of fluorogold-labeled RGC showed that retinal tissue from animals subjected to IRI followed by exposure to isoflurane (1.4 vol-%) had significantly lower ganglion cell densities compared to animals subjected to IRI and room air (Fig. 7, B and C; IRI 1479 RGC/mm2 [1311/1697] versus IRI + isoflurane 1170 [1093/1211], P = 0.03). In vivo inhibition of p75NTR with TAT-pep5 before isoflurane exposure reversed the detrimental effect mediated by isoflurane (Fig. 7, B and C; IRI + isoflurane + TAT-pep5 1577 RGC/mm2 [1436/1624] versus IRI + isoflurane 1170 [1093/1211], P = 0.02), while the inhibitor alone had no harmful effects (Fig. 7, B and C; room air 2828 RGC/mm2 [2708/3021] versus isoflurane + TAT-pep5 2699 [2508/2878], P = 0.80).
Of note, isoflurane treatment without any IRI significantly reduced the number of vital RGC in vivo compared to eyes in animals receiving room air without IRI (Fig. 7, B and C; room air 2828 RGC/mm2 [2708/3021] versus isoflurane 2491 [2244/2820], P = 0.04).
The main findings of this study can be summarized as follows: Isoflurane, but not desflurane or sevoflurane, aggravates neuronal cell death in SH-SY5Y cells when applied after hypoxic injury. Isoflurane strongly induces NF-κB activation, whereas inhibition or knockdown of the p75NTR receptor, which is an upstream regulator of NF-κB, counteracts the effects exerted by isoflurane in SH-SY5Y cells. Furthermore, isoflurane inhalation after retinal IRI increases neuronal cell death of RGC neurons in rats. Inhibition of p75NTR before isoflurane inhalation rescues RGC from increased death. We acknowledge that the link between p75NTR and NF-κB demands further investigation, but we can conclude that both molecules are involved in the damaging effects of isoflurane to pre-injured mature neuronal cells in vitro and in vivo. Of note, the 2 other volatile anesthetics studied in vitro did not exert the same detrimental effects as isoflurane, indicating a difference between the volatile anesthetics currently used in clinical practice concerning their influence on neuronal damage.
The influence of different volatile anesthetics on neuronal cells has been extensively studied before. However, concerning the neurotoxic effects of isoflurane, recent research has mainly focused on the neonatal and developing brain,5,7–10,18,19,39–42 whereas only few studies have explored the neurotoxic effects of isoflurane on mature neuronal cells.4,6,43,44 Results showing isoflurane-related neurotoxicity are contrasted by numerous in vitro and in vivo injury studies where isoflurane has been shown to be neuroprotective.1–3,17,45–52 Importantly, these dual effects of isoflurane are highly dependent on several factors, including length of exposure,9,10 concentration used,8,44 and time point of exposure in relation to injury.50 This notion is supported by our in vitro data showing that pre-exposure with isoflurane did not affect neuronal cell death, whereas exposure after injury exerted significant neuronal injury.
These same dual effects, far less extensively studied, can be attributed to sevoflurane11–13,53–56 and desflurane.14–16 Only few studies have directly compared different volatile anesthetics in the same injury model and experimental setting. Of note, in these studies, no differences in respect to protection14 or neurotoxicity15 have been reported for the different anesthetics. Our results are a contrast to this uniform effect of the different volatile anesthetics because we found a specific neurotoxic effect of isoflurane exposure after injury, which was not seen with sevoflurane or desflurane.
Specific solubility coefficients for isoflurane, sevoflurane, and desflurane in aqueous solutions have been published.37,38 Our gas chromatographic measurements of volatile anesthetic concentration in the cell media matched the expected concentrations derived from these coefficients and were reproducible. The measurements also showed that volatile anesthetics were used in presumably clinically relevant concentrations, excluding that the neurotoxic effects seen with isoflurane were due to nonphysiologic doses. In addition, we used exposure times of 2 hours, which is also a realistic clinical scenario. This is important to note, since many studies showing isoflurane-induced neurotoxicity have used high isoflurane concentrations and/or long exposure times up to 24 hours.4,6,10,39,44,57
We acknowledge the limitations of our in vitro model of neuronal cell death in SH-SY5Y cells as a transformed neuroblastoma cell line. However, this cell line has been used in numerous neuronal cell death models58–60 and has been used to evaluate isoflurane pre-exposure in simulated IRI.61 In addition, SH-SY5Y cells have been shown to highly express p75NTR 21 and respond to p75NTR activation with cell death,62 supporting the usefulness of this cell line in studying neuronal cell death related to the p75NTR pathway.
The differential effect of isoflurane on AV/Pi positivity with lack of effect on the mitochondrial membrane potential could be explained by a predominant aggravation of necrosis, in which case no change in mitochondrial membrane potential will be observed. However, it could also be due to the time point of analysis and the kinetics of cell death with mitochondrial depolarization occurring during early apoptosis only. The AV/Pi data do not allow us to differentiate between late apoptosis and necrosis.
Besides its important role in neurogenesis and neuronal development by regulation of cell differentiation,21 neurite outgrowth,22 and axonal degeneration,23 p75NTR signaling also determines the fate of mature neuronal cells after BDNF stimulation24,25 and most importantly after neuronal ischemic injury.26–28 Recently, the neurotoxic effects of isoflurane and propofol have been linked to the activation of p75NTR after exposure to these anesthetics.18–20 However, downstream signaling of the p75NTR-RhoA kinase pathway in mediation of neuronal apoptosis has not been studied. Some evidence suggests that activation of transcription factor NF-κB by p75NTR signaling induces apoptosis in neuronal cells,29–31 which is supported by our results showing a specific effect of isoflurane on NF-κB activity and dependence of neuronal cell death on p75NTR signaling.
The exact molecular mechanisms explaining why the p75NTR-NF-κB signal transduction pathway is specifically activated by isoflurane but not by sevoflurane or desflurane remain unclear and require further investigation. RGCs are the retinal cell population most susceptible to cell death after ischemic injury.63,64 The role of p75NTR as a cell death inducing signaling pathway in retinal ischemia is suggested by the fact that p75NTR expression is induced in RGC after retinal ischemia.27 Furthermore, p75NTR activation in the retina has directly been linked to RGC death, even though p75NTR signaling in non-neuronal cells seems to be crucially important in this matter.24 Our immunohistochemistry results did not confirm an ischemia-induced upregulation. We rather found constitutive p75NTR expression that was not changed after isoflurane exposure, indicating that isoflurane does not exert its effect on the transcriptional level but rather by directly interacting with the p75 receptor. As expected, no difference in p75NTR expression was observed with or without the inhibitor TAT-pep5, because it is a downstream inhibitor of this receptor, which does not cause an acute and direct change of expression. TAT-pep5 inhibits the interaction of p75NTR with Rho guanosine diphosphate (GDP) dissociation inhibitor, whose interaction with p75 would normally initiate RhoA activation.65
The neuronal cell type and injury model kinetics we used in vitro and in vivo differed from one another, due to the characteristics of the SH-SY5Y cell line in respect to their response to hypoxia as discussed above. However, since SH-SY5Y cells undergo cell death due to the lack of oxygen after OGD similar to RGC in response to IRI, both models are comparable with respect to their usefulness to study neuronal cell death after oxygen deprivation and suitable to study the modulating effects of volatile anesthetics. We chose the retinal IRI model over other neuronal ischemic injury models because it has been proven to be a model with a highly reproducible degree of injury, little interindividual variability, and direct visual control over ischemia and reperfusion33,34,36 compared to other neuronal injury models. It is therefore most suitable to study the effect of volatile anesthetics on injured primary neurons.
Apart from its still widespread use for surgical procedures, isoflurane has been tested for the treatment of conditions with subsequent neuronal injury, such as status epilepticus66 or subarachnoid hemorrhage (SAH)-induced vasospasm.67 The results of our study question the notion that isoflurane might be beneficial in these conditions. Interestingly, neurotoxicity in status epilepticus patients treated with isoflurane has been suggested earlier.68 We must stress that our model and the use of isoflurane in status epilepticus are not directly comparable due to very different cumulative dosages being used because epilepsy patients are usually treated with isoflurane for much longer. This is different for the treatment of SAHe-induced vasospasm with isoflurane because dosage and duration might be very comparable to our model.67 Our data suggest that isoflurane might not be the ideal candidate for the alternative treatment of SAH-induced vasospasm due to the direct damaging effects on preinjured neurons.
Taken together, this study demonstrates that isoflurane, but not sevoflurane or desflurane exposure, aggravates neuronal cell injury in pre-injured SY5Y and RGCs via activation of p75NTR and NF-κB. We acknowledge the fact that additional neuronal cell death due to isoflurane exposure is small and clinical relevance of our findings has to be further evaluated in the operating room. In contrast, long-term sedation in intensive care units may be achieved using volatile nebulizer systems (like the AnaConDa), in which the isoflurane exposure period is considerably extended, making isoflurane-induced neurotoxicity even more likely in this clinical setting. Therefore, these findings may have future implications for the choice of volatile anesthetic being used in patients with or at risk for neuronal injury. Specifically, it questions the use of isoflurane for surgical procedures in patients with a stroke or history of stroke and for surgical procedures in which neuronal injury could occur with a high probability, such as cardiac surgery or neurovascular interventions. These findings also question the use of isoflurane in other settings such as sedation of the critically ill, rescue treatment of status epilepticus, or treatment of vasospasm in SAH.
Name: Nils Schallner, MD.
Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.
Attestation: Nils Schallner has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Felix Ulbrich, MD.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Felix Ulbrich has seen the original study data and approved the final manuscript.
Name: Helen Engelstaedter, MD.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Helen Engelstaedter has seen the original study data and approved the final manuscript.
Name: Julia Biermann, MD.
Contribution: This author helped conduct the study and analyze the data.
Attestation: Julia Biermann approved the final manuscript.
Name: Volker Auwaerter, PhD.
Contribution: This author helped conduct the study and analyze the data
Attestation: Volker Auwaerter approved the final manuscript.
Name: Torsten Loop, MD.
Contribution: This author helped design the study and write the manuscript.
Attestation: Torsten Loop approved the final manuscript.
Name: Ulrich Goebel, MD.
Contribution: This author helped design the study, analyze the data, and write the manuscript.
Attestation: Ulrich Goebel has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
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