The intravenous anaesthetics propofol, midazolam and ketamine are routinely used in anaesthesia and emergency medicine practice for premedication or induction and maintenance of general anaesthesia. All three substances have been described as both neuroprotective and neurotoxic. Propofol can be neuroprotective in vivo after ischaemia-reperfusion injury (IRI) by dampening the inflammatory response and activating neuroprotective pathways.1–3 Several studies have demonstrated protection of neuronal cells in in vitro models using hypoxia/glucose deprivation models.4–6 In contrast, other groups report propofol-induced neuroinflammation and cell death in vitro and in vivo.7–9 The same holds true for the benzodiazepine derivate midazolam; reports about its neuroprotective properties10–12 are contrasted by its apoptosis-promoting and neurotoxic effects.13,14 The neurotoxic or neuroprotective effects of pre-clinical ketamine application depend on the combination with other substances15,16 or the exact injury model used.17,18 Although there is no evidence for either neuroprotection or neurotoxicity in the few clinical evaluations in adults,19 the effect of ketamine on the developing infant brain remains controversial.20,21
These differential effects could possibly be explained by different mechanisms of action, but the molecular mechanisms of anaesthetic-mediated neuroprotection remain poorly understood. Both the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which is crucial in the regulation of inflammation, and its upstream cell surface regulator Toll-like receptor 4 (TLR-4) are influenced by propofol,22–24 midazolam25 and ketamine26 in neuronal and non-neuronal cells, but it remains unclear whether this pathway is important in anaesthetic-mediated neuroprotection.
A few studies have directly compared different substances in the same neuronal injury model11,27 and such direct comparative studies are needed to try to elucidate the discordant effects reported in the literature. To clarify the discrepancy regarding their effects on neuronal cells and directly compare their neuroprotective potential, we administered propofol, midazolam and ketamine at clinically relevant doses to injured neuronal cells and hypothesised that all three substances exert neuroprotective effects in vitro and in vivo by inhibition of the TLR-4-NF-κB signalling axis. For this purpose, we utilised our previously described in vitro oxygen-glucose deprivation (OGD) model in neuronal SH-SY5Y cells and the highly reproducible in vivo retinal IRI model to mimic ischaemic insult to neurones.28
Summary materials and methods
SH-SY5Y human neuroblastoma cells (ATCC No. CRL-2266) were exposed to combined glucose deprivation and hypoxia in a sub-culture chamber (Oxycycler C42, Biospherix, Lacona, USA), followed by re-oxygenation. For different treatment modalities, anaesthetics were either administered prior to or after hypoxia. In vitro experiments were repeated six times. Surface expression of TLR-4 (anti-TLR-4 antibody, #ab45126, Abcam, Cambridge, MA, USA) and changes in mitochondrial membrane potential (ΔΨm, Mito-Probe JC-1 Assay, Molecular Probes, Darmstadt, Germany) were determined by flow cytometry. Lactate dehydrogenase (LDH) released from SH-SY5Y cells was analysed in cell culture supernatants using a LDH release detection assay (Cytotoxicity Detection Kit, Roche Diagnostics, Mannheim, Germany). DNA-binding activity of NF-κB p65 and hypoxia-inducible factor 1 α (HIF-1α) were quantified using a DNA-binding ELISA following the manufacturer's instruction (NF-κB p65, HIF-1α TransAM Kit, ActiveMotif, Rixensart, Belgium). For gene expression analysis, cells were transiently transfected with a dual luciferase reporter gene construct of inducible firefly luciferase under control of the NF-κB transcriptional response element and a construct with constitutive renilla luciferase expression (Cignal NFκB Reporter Kit, Hilden, Germany). Luciferase activity was measured using a dual-luciferase assay system (Dual-Glo Luciferase Assay System, Promega) on a microplate luminometer (EG & G-Berthold, Bad Wilbach, Germany).
All procedures involving animals complied with the statement of the Association for Research in Vision and Ophthalmology for the use of animals in research and were approved by the Committee of Animal Care of the University of Freiburg (Permit No. 35-9185.81/G-14/123, Chairperson Dr. Schwarzmeier) on 28 January 2015. For in vivo experiments, adult male and female Sprague-Dawley rats (1 : 1, 280 to 350 g body weight, Charles River, Sulzfeld, Germany) were used. Retrograde retinal ganglion cell (RGC)-labelling was done as described previously 7 days prior to retinal ischaemia.29
To evaluate the effect of propofol on neuronal damage, animals were randomised to receive treatment with propofol or phosphate-buffered saline (PBS) immediately following retinal reperfusion. Retinal IRI was performed as described previously.30 Briefly, the anterior chamber of the left eye was cannulated with a 30-gauge needle connected to a reservoir containing 0.9% NaCl. Intraocular pressure was increased to 120 mmHg for 60 min and ocular ischaemia was confirmed microscopically by interruption of the retinal circulation. Reperfusion was initiated by removing the needle tip promptly. Rats without immediate recovery of retinal perfusion at the end of the ischaemic period or those with lens injuries were excluded from the investigation, since the latter prevents RGC death and promotes axonal regeneration.31 Propofol (20 mg kg−1 body weight per hour) was administered intravenously via a tail vein catheter (gauge 26) for 4 h. For RGC quantification, animals were euthanised by CO2 inhalation 7 days after ischaemia. RGC quantification was performed as previously described.32 For Western blot (cleaved Caspase-3 #9665, Caspase-3 #9664, Cell Signaling Technology, Danvers, MA, USA) and RT-PCR [TLR-4 and NF-κB; housekeeping gene: GAPDH (glyceraldehyde 3-phosphate dehydrogenase)] analysis, retinal tissue was harvested 24 h after ischaemia. Data are expressed as the fold change in the IRI ± propofol retina vs the individual, non-ischaemic control retina. The number of animals used for retinal ganglion cell (RGC) quantification and molecular analysis was n = 6 per group.
All data are graphed as boxplots with median, first and third quartiles and max/min for whiskers. In the text, mean ± SD is used. Data with normal distribution were compared using one-way ANOVA (α = 0.05) for between-group comparisons with post-hoc Bonferroni multiple comparison. Non-parametric Kruskal–Wallis one-way ANOVA on ranks with post-hoc Newman–Keuls test was used for data with lack of normal distribution. For the in vivo studies, we wished to detect a 25% decrease in RGC death by propofol intervention. Assuming an expected SD of 10% in RGC density counts from our previous usage of this technique and based on previously published data and power analysis,33,34 an a-priori power analysis (α = 0.05 with two-sided hypothesis, power 80%) indicated that a sample size of six animals per group would be sufficient to detect such a difference. One-way ANOVA with post-hoc Bonferroni test was used for between-group comparison of the RGC quantification data. Two groups (RT-PCR data) were compared using an unpaired, two-tailed t test. Data were analysed with a computerised statistical program (GrapPad Prism 6.0, GraphPad Software). P less than 0.05 was considered statistically significant (see Supplementary file on Materials, Methods and Statistics, http://links.lww.com/EJA/A92).
Propofol stabilises the mitochondrial membrane potential
We first determined the influence of ketamine, midazolam and propofol as pre-treatment and post-treatment agents on neuronal cell death during OGD injury. To this end, we analysed the mitochondrial membrane potential by flow cytometry. Pre-treatment with ketamine (Fig. 1a) showed a mitochondria-stabilising effect only at a concentration of 0.1 μg ml−1. Pre-treatment with midazolam or propofol did not influence mitochondrial depolarisation (Fig. 1b and c).
Post-treatment with ketamine (Fig. 2a) and midazolam (Fig. 2b) stabilised the mitochondrial membrane potential, although the effect was not consistently detectable across the whole dose range. In contrast, propofol post-treatment (Fig. 2c) attenuated OGD-induced mitochondrial depolarisation across a broad concentration range by up to 54% with the most pronounced effect between 1 and 6 μg ml−1, but also at the highest dose used (10 μg ml−1).
Propofol inhibits LDH-release following neuronal injury
We next analysed whether this stabilising effect on the mitochondrial membrane potential would result in improved cell survival after OGD. We chose medium concentrations of anaesthetic substances from the initial dose response and determined neuronal cell death by measuring LDH-release. Both pre-treatment (Fig. 3a) and post-treatment (Fig. 3b) with 5 μg ml−1 propofol reduced the release of LDH after OGD by 41 and 21%, respectively, whereas pre-treatment and post-treatment with midazolam 100 ng ml−1 or ketamine 5 μg ml−1 had no influence on LDH release, indicating a propofol-specific effect on neuronal cell survival after OGD.
Propofol diminishes the oxygen-glucose deprivation-induced induction of Toll-like receptor 4 surface expression on neurones
We next aimed to explore the TLR-4-NFκB pathway as a putative mechanism for propofol-specific neuroprotection. Surface expression of TLR-4 on neurones and downstream TLR-4 signalling plays a role in neuronal cell death.35 As we found a selective effect of propofol on neuronal cell survival, we next asked the question whether this concurred with changes in neuronal TLR-4 expression and subsequent intracellular HIF-1 α and NFκB signalling. Analysing TLR-4 surface expression after OGD ± propofol by flow cytometry, we found that propofol incubation after OGD diminished the OGD-induced increase in TLR-4 surface expression on neuronal cells (Fig. 4a).
DNA-binding activity of hypoxia-inducible factor 1 α is preserved by propofol in oxygen-glucose deprivation-injured neurones
We next asked the question whether potential downstream targets of TLR-4 signalling were affected by propofol. Stabilisation of HIF-1α in neurones has been implicated as a neuroprotective factor by regulating downstream target genes important for cellular survival.36,37 Pre-treatment or post-treatment with midazolam or ketamine did not preserve DNA-binding activity of HIF-1α (data not shown). Pre-treatment with propofol did not change HIF-1α DNA-binding activity (data not shown). In contrast, OGD-induced decrease in HIF-1α DNA-binding activity was partially antagonised by propofol post-treatment (Fig. 4b).
Propofol reduces the DNA-binding activity of nuclear factor kappa-light-chain-enhancer of activated B cells p65
Since HIF-1α can negatively regulate transcriptional NF-κB activity38,39 and TLR-4 represents the established upstream regulator of NF-κB signalling, we next determined the influence of OGD ± anaesthetics on DNA-binding activity of NF-κB p65 by ELISA. Reduction of NF-κB p65 DNA-binding activity was not observed after treatment with ketamine or midazolam (data not shown). In pre-treatment experiments (data not shown), no influence on NF-κB p65 DNA-binding by propofol was detectable. Post-treatment with propofol reduced the DNA-binding of NF-κB p65 after OGD (Fig. 4c).
Propofol treatment results in decreased transcriptional activity of nuclear factor kappa-light-chain-enhancer of activated B cells
To determine whether the differential effect on NF-κB p65 DNA-binding activity seen by the different anaesthetic substances would concur with changes in transcriptional NF-κB activity and expression changes of NF-κB-dependent genes, we transiently transfected a luciferase reporter gene construct with inducible luciferase expression under control of the NF-κB transcriptional response element into neuronal cells. In pre-treatment (Fig. 5a) and post-treatment (Fig. 5b) experiments, propofol exerted an inhibitory effect on the transcriptional activity of NF-κB. For midazolam and ketamine, this differential effect on transcriptional activity of NF-κB was not detected (data not shown).
Propofol application after retinal ischaemia improves survival of retinal ganglion cells
To translate these in vitro findings into a relevant in vivo situation, we utilised the well established and previously published model of retinal IRI in rats. To this end, rats were randomised to receive either PBS or propofol injection during reperfusion after 1 h of retinal ischaemia. When analysing the density of fluorogold-labelled RGCs 7 days after ischaemia and reperfusion, fluorescence microscopy (Fig. 6a) revealed that the loss of RGCs because of ischaemia-reperfusion was attenuated in animals treated with propofol at the initiation of reperfusion. The respective non-ischaemic control retinae did not differ in RGC density. Quantification of RGC density was performed (RGC mm−2, Fig. 6b) and provided quantifiable confirmation for the effects seen in representative images from Fig. 5a. Propofol significantly increased the RGC-density after ischaemia-reperfusion in comparison to treatment with PBS and reduced the IRI-induced RGC loss by 55%.
Propofol reduces caspase-3 cleavage and Toll-like receptor 4/nuclear factor kappa-light-chain-enhancer of activated B cells expression in the retina
In a last set of experiments, we wanted to answer the question whether the differential effects by propofol on TLR-4 and NF-κB signalling and on neuronal apoptosis seen in vitro would also apply to the in vivo retinal ischaemia-reperfusion model. Analyses of retinal caspase-3 expression and cleavage and expression of TLR-4 and NF-κB showed that propofol reduced the IRI-induced increase in retinal caspase-3 expression (Fig. 7a) and cleavage (Fig. 7b and c) and reduced the expression of NF-κB (Fig. 7e) to baseline control values. TLR-4 expression was significantly mitigated in the ischaemic retinae by propofol intervention (Fig. 7d).
Results from this study indicate that propofol given after neuronal injury at clinically relevant concentrations in vitro can protect neuronal cells, indicated by preservation of the mitochondrial membrane potential and reduced LDH-release. In direct comparative studies, we found that the beneficial effect in neuronal injury and the concurrent changes in cellular signalling are specific to propofol and not observed with other anaesthetic substances such as midazolam or ketamine. Moreover, while some of the effects seen by post-treatment with propofol were also evident in pre-treatment experiments, preconditioning effects were not consistently seen across all parameters studied, indicating that the timing of propofol application in relation to neuronal injury seems to determine the beneficial effects on injured neuronal cells. Pivotal to the beneficial effects of propofol was the modulation of neuronal TLR-4 expression with corresponding changes in downstream HIF-1α and NF-κB signalling. Furthermore, these in vitro findings were corroborated in an in vivo neuronal ischaemia-reperfusion model in the retina. Propofol treatment prevented RGC loss and reduced the expression of caspase-3 with concomitant inhibition of TLR-4-NF-κB signalling.
The neuroprotective effects of propofol have been extensively studied in previous investigations.1–6,40 However, several reports have questioned the neuroprotective properties of this drug and have reported propofol-induced neurotoxicity, especially in the developing infant brain.7–9,41 For the other drugs used in this study, a similar contradictory role in neuronal injury has been reported.10,12–14,17,19 Few studies have directly compared the effect of different intravenous anaesthetics in the same injury model,11 and until now no differential effect has been reported. We found a specific propofol-mediated neuroprotective effect that was not evident after treatment with midazolam or ketamine (Fig. 8a).
The doses used for all three intravenous agents in vitro span a dose range that corresponds well to the dose range seen during general anaesthesia in humans.42–45 In addition, incubation times of 2 h were used, which also corresponds to a realistic clinical scenario. Regarding the in vivo experiments, propofol doses were used in accordance with the current literature.46,47 Propofol doses in rodents are in general higher than in humans but the doses used in our experiments were within the usual dose range for rodent procedures. We decided to utilise the clinically used formulations of all three substances for better comparison and clinical relevance. However, one has to take into consideration the potential antioxidant and protective effects of the additives in the formulations, especially α-tocopherol in soybean oil, which is used as a solvent for propofol.48
TLR-4 represents an important upstream regulator of NF-κB,49 which in turn can promote neuronal apoptosis when activated.50,51 Even though earlier studies have suggested that TLR-4 expression on neurones is negligible,52 it has more recently been shown that neurones are capable of expressing TLR-453–55 with subsequent intracellular NF-κB signalling. It is known now that neurones express several Toll-like receptors such as TLR-4.56 Cerebral ischaemia reperfusion injury is associated with a strong inflammatory response with increased levels of cytokines.57 Some studies indicate that cerebral inflammation results in increased TLR-4 expression. Conversely, TLR-4 deficient animals show minor inflammation after ischaemic stroke.58 Our results demonstrate an increased RGC count as a sign of propofol's protective effect. This might be because of a direct inhibition of TLR-4 or a general suppression of the inflammatory response. Nevertheless, our findings support the notion that both TLR-4 and NF-κB are involved in pathways that determine the fate of injured neuronal cells and that this signalling pathway is differentially influenced by propofol but not by other intravenous anaesthetics.
An interdependence of NF-κB and HIF-1α has been suggested earlier. Although some studies showed that transcriptional activity of both transcription factors are simultaneously und uniformly changed upon upstream signalling,59,60 our results support the notion that transcriptional activity of NF-κB and HIF-1α are regulated diametrically in that HIF-1α negatively regulates NF-κB activity.38,39 This diametrical effect was seen in our in vitro model for propofol, but not for midazolam or ketamine. Furthermore, regulation of HIF-1α by propofol suggests that the protective effects of propofol could be partly explained by its antioxidant effects.61
The neuronal injury models used in vitro and in vivo differed from each another in respect to kinetics and duration of hypoxia. This is mainly because of the nature of SH-SY5Y cells as a transformed neuroblastoma cell line, which reacts very differently to hypoxia and neuronal injury. However, both SH-SY5Y cells and RGCs undergo cell death because of the lack of oxygen in the respective model (OGD in vitro and IRI in vivo). Therefore, both models are comparable in regard to their usefulness to study neuronal cell death and equally suitable to study the modulating effects of anaesthetics. The retinal ischaemia reperfusion injury model was selected in favour of other neuronal ischaemic injury models because retinal ischaemia provides a highly reproducible degree of injury, little inter-individual variability and direct visual control over ischaemia and reperfusion.32,34 It is, therefore, a very suitable injury model to study the effects of anaesthetics on neuronal injury.
Ketamine, xylazine and isoflurane are commonly used for anaesthesia in rodents.62 After administration of ketamine and xylazine, side-effects like hypotension, bradycardia, hypotension and respiratory depression may occur, affecting cerebral circulation and oxygenation.63,64 Similar haemodynamic and respiratory processes were detectable when using isoflurane.65 Furthermore, potential neuroprotective effects of ketamine and isoflurane are still discussed controversially.66,67 In summary, these substances might have an impact on RGCs and should be mentioned as a potential limitation of our study. However, because of the fact that all animals in both groups (PBS vs propofol) received the exact same treatment, effects seen in vivo can reasonably be attributed to propofol.
We acknowledge the limitations of studying neuronal cell death in transformed neuroblastoma cells. However, the SY5Y cell line has previously been used in neuronal cell death models68,69 and has been used to evaluate the effects of propofol on neuronal cell death.4,70,71 Furthermore, we acknowledge that proofing inhibition of TLR-4-HIF-1α-NF-κB signalling by propofol as the mechanism of action will require further studies with loss of function and rescue experiments. However, the concurrent influence of propofol on TLR-4 surface expression and NF-κB activity strongly suggests a role for this signal transduction pathway in the effects seen.
In summary, we found in this experimental study that propofol, in contrast to midazolam and ketamine, reduces neuronal cell injury in SY5Y neuroblastoma cells and RGCs when given at the time point of re-oxygenation and reperfusion, respectively. The proposed mechanism of action for propofol-mediated neuroprotection in this study is the inhibition of TLR-4-NF-κB signalling resulting in reduced neuronal cell death. Even though both midazolam and ketamine provided some degree of protection, their neuroprotective effect was not consistently detectable across the whole dose range used. As we used clinically relevant doses for our experiments, it would be interesting to explore the differential effects of propofol vs midazolam or ketamine in clinical situations. We acknowledge that the anaesthetics used in the study have different areas of application in clinical anaesthesia and therefore a direct clinical comparison would be challenging. Nevertheless, our study provides evidence for the beneficial effect of propofol and outlines a potential signalling pathway. As we have previously shown that isoflurane aggravates neuronal cell death in the same injury model,28 it would be interesting to compare propofol with isoflurane anaesthesia in future studies where general anaesthesia for patients with neuronal injury is necessary.
Acknowledgements relating to this article
Assistance with study: none.
Financial support and sponsorship: this study was supported by departmental funding.
Conflicts of interest: none.
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