http://informahealthcare.com/bij ISSN: 0269-9052 (print), 1362-301X (electronic) Brain Inj, 2014; 28(13–14): 1758–1765 ! 2014 Informa UK Ltd. DOI: 10.3109/02699052.2014.947624
ORIGINAL ARTICLE
An in vitro study of the neuroprotective effect of propofol on hypoxic hippocampal slice Deng-xing Zhang1, Hao-zhong Ding1, Shan Jiang2, Ying-ming Zeng2, & Qi-feng Tang3 Department of Anesthesiology, The Affiliated Hospital of Jiangnan University, Wuxi, PR China, 2The Key Laboratory of Anesthesiology, Jiangsu Province, Xuzhou Medical College, Xuzhou, PR China, and 3Department of Anesthesiology, Suzhou BenQ Medical Center, Nanjing Medical University, Suzhou, PR China
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Abstract
Keywords
Primary objective: To determine whether propofol has a neuroprotective effect on hypoxic brain injury. Research design: A hippocampal slice, in artificial cerebrospinal fluid (ASCF) with glucose and oxygen deprivation (OGD), was used as an in vitro model for brain hypoxia. Methods and procedures: The orthodromic population spike (OPS) and hypoxic injury potentia1 (HIP) were recorded in the CA1 region when Schaffer collateral was stimulated in the CA3 region of the hippocampal slices during hypoxia. The concentrations of amino acid neurotransmitters in perfusion solution of hippocampal slices were directly measured using high performance liquid chromatography (HPLC). Morphological changes of neurons, astrocytes and mitochondria in CA1 region were observed using histology and electron microscopy. Neuronal apoptosis was evaluated with TUNEL assay. Main outcome and results: Propofol treatment delayed the elimination of OPS and improved the recovery of OPS; decreased frequency of HIP, postponed the onset of HIP and increased the duration of HIP. Propofol treatment also decreased the release of amino acid neurotransmitters such as aspartate, glutamate and glycine induced by hypoxia, but elevated the release of
-aminobutyric acid (GABA). Morphological studies showed that propofol treatment attenuated oedema of pyramid neurons in the CA1 region and reduced apoptosis. Conclusions: Propofol has a neuro-protective effect on hippocampal neuron injury induced by hypoxia.
Hippocampal slice, hypoxic injury, neuroprotection, propofol
Introduction Many pathologic conditions can cause hypoxic brain injury, which may also occur during neurosurgery, cardiovascular surgery and anaesthesia. The mechanism underlying such hypoxic brain injury is still unclear. How to protect the brain from hypoxic injury and how to treat hypoxic brain injury remains clinically challenging. Although hypothermia and pre-ischaemia treatment have been shown to have a protective effect on the brain [1, 2], it is difficult to implement clinically. In contrast, pharmacological treatment seems more clinically practical. During the recent three decades, the neuroprotective effect of anaesthetic drugs has drawn high attention from clinicians. Propofol, an IV anaesthetic drug, has been widely used clinically for anaesthesia induction, maintenance and sedation since the first clinical trial in 1977. Previous studies have
Correspondence: Dr. Qi-feng Tang, Department of Anesthesiology, Suzhou BenQ Medical Center, Nanjing Medical University, Suzhou 215009, PR China. E-mail: [email protected]
History Received 26 November 2013 Revised 23 May 2014 Accepted 20 July 2014 Published online 27 August 2014
shown that propofol could reduce brain artery flow volume, intracranial pressure and metabolism rate that maintains the match of blood supply and oxygen metabolism, as well as improving the oxygen supply during hypoxia, suggesting that propofol protects the brain from hypoxic damage [3–7]. More recent studies suggest that propofol plays a role in central nervous system (CNS) protection through the modulation of Ca2+, oxygen free radicals, -aminobutyric acid (GABA) receptor and N-methyl-D-aspartate (NMDA) receptor [8–12]. In contrast, some data have shown that propofol had no brain protective effect after cardiac surgery and even worsened brain hypoxia [13]. Yet other studies suggest that hypothermia, rather than propofol, is neuroprotective [2]. The aim of this study is to find out if propofol has a neuroprotective effect against hypoxia at 37.5 C to circumvent the hypothermia issue. A hippocampal slice has been used as an in vitro model for neuroprotective study for decades [14–17]. Glucose and oxygen deprivation (OGD) in artificial cerebrospinal fluid (ASCF) can induce hypoxic injury of the hippocampal slice, mimicking brain hypoxia. Compared with cultured cell models, the hippocampal slice more closely resembles the
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whole brain tissue. The current study used an OGD model and extra-cellular electric recording, in which evoked potential was recorded in the CA1 region when Schaffer collateral was stimulated in the CA3 region. It was observed how propofol treatment affects orthodromic population spike (OPS) and hypoxic injury potentia1 (HIP) in the hippocampal slice during hypoxia. Amino acid neurotransmitters, such as aspartate (Asp), glutamate (Glu), glutamine (Gln), glycine (Gly) and
-aminobutyric acid (GABA), are widely distributed and have important functions in CNS. The high concentration of excitatory amino acids in synaptic cleft and extracellular fluid during hypoxia is a common pathway causing excitatory toxicity and neuronal death [10, 18–21]. Most amino acid neurotransmitter assays measure the total amount of amino acids in specific brain areas or nuclei, which may also contain amino acids from protein metabolism. Thus, the total amino acid amount of the hippocampus may not reflect the amount of amino acids as a neurotransmitter. This study used HPLC to directly measure the concentration of amino acid neurotransmitters in perfusion solution of hippocampal slices during hypoxia. In the hippocampus, the CA1 region is most sensitive to hypoxia. Mitochondrion is the organelle playing a key role in the process in which the reversible cellular change becomes irreversible during hypoxia. This study examined the morphological change of neurons, astrocytes and mitochondria in the CA1 region using histology and electron microscopy. The overall goal is to find direct evidences demonstrating that propofol treatment has a brain protective effect in hypoxia.
Materials and methods Experimental animals and acute hippocampal slice preparation Eight-to-ten weeks old Sprague-Dawley (SD) rats (Experimental Animal Center, Xuzhou Medical College, China), weighing 200–250 grams, were anaesthetized by ether. Hippocampi were dissected and sectioned using vibrating-blade microtome in 300 mm thickness. Hippocampal slices were incubated in ASCF (NaCl 124 mM, KCl 3.3 mM, NaH2PO4 1.24 mM, MgSO4 2.4 mM, NaHCO3 25.7 mM, CaCl2 2.4 mM) with and without glucose (10.0 mM) at 37.5 C for 2–3 hours. The flowing volume was 200 ml min1. All the animal protocols are approved by the Experimental Animal Committee of Xuzhou Medical College. Extra cellular potential recording Electric probes were inserted into Schaffer lateral fiber at CA3. Stimuli (0.6 mA, 0.1 Hz, 100 ms) were given with a 10-second interval. Evoked potentials including orthodromic population spike (OPS) and hypoxia injury potential (HIP) were recorded at CA1. Only brain slices that had stable OPS (43 mv) for at least 20 minutes were selected for experiments. Hypoxic groups were incubated in ASCF without glucose and oxygen (ASCFOGD) for 14 minutes followed by normal ASCF perfusion. Treatment groups were incubated in ASCFOGD with 1, 5, 15 mM propofol (10 mg ml1 injection emulsion,
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AstraZeneca, Caponago, Italy), respectively. The emulsion carrier (soybean oil (100 mg mL1), glycerol (22.5 mg mL1), egg lecithin (12 mg mL1); and disodium edetate (0.005%), pH8.0) without propofol was used in non-propofol-treated hypoxic groups, at a dilution of 1:3500. In hypoxic and treatment groups, every OPS was recorded before its disappearance. Each group contained eight samples. OPS decay is defined as the time period when OPS become undetectable after oxygen deprivation. The amplitude of OPS recovery is defined as the ratio of OPS amplitude after 1 hour of normal ASCF perfusion over OPS amplitude before oxygen deprivation. The ratio of OPS recovery is defined as the percentage of hippocampal slices in which OPS amplitude recovery reach 60% of OPS amplitude before oxygen deprivation. HIP onset is defined as the time period when HIP is recorded after oxygen deprivation. HIP duration is defined as how long HIP lasts. HIP incidence is defined as the percentage of hippocampal slices in which HIP were recorded after oxygen deprivation. High performance liquid chromatographic (HPLC) analysis ASCF from 14 minutes of perfusion from each group was collected and centrifuged. Supernatant was kept for HPLC analysis. The mobile phase A was NaAC (0.5 M, PH 6.0) with 0.05% THF. The mobile phase B was methanol. The following washing gradients (minutes, Phase B %) were used: 0, 30%; 7, 60%; and 9, 30%, with velocity of 0.9 ml min1, temperature at 30 C, lem at 330 nm and lex at 456 nm. Derivation agent was made from OPA, 2-MCE, Boric acid-sodium hydroxide and methanol (PH 9.6). A mixture of 20 ml derivation agent and 30 ml sample was incubated for 2 minutes at RT. Injection volume was 20 ml. The remaining time, regression equations and correlation coefficient were obtained on samples of aspartate (Asp), glutamate (Glu), glutamine (Gln), glycine (Gly) and -aminobutyric acid (GABA) (Table I). Standard curves were prepared using 10, 5, 2.5, 1.25 and 0.625 mM of Asp, Glu, Gln, Gly or GABA. The sensitivity of amino acids is less than 1012 mol/20 mL sample. The concentrations (mM) of Asp, Glu, Gln, Gly and GABA in perfusion ASCF were calculated based on standard curves using Prostar. HE staining Hippocampal slices from each group were fixed in 0.1% methanol and embedded in wax after dehydration. The section thickness was 5 mm. HE staining followed the standard protocol.
Table I. The remaining time, regression equation andcorrelation coefficient of amino acids (n ¼ 8).
Amino acids Asp Glu Gln Gly GABA
Remaining time (min)
Regression equation
Correlation coefficient
3.56 3.92 5.68 6.89 8.37
Y ¼ 1012.54X + 542.62 Y ¼ 2053.23X + 134.77 Y ¼ 9021.48X 378.65 Y ¼ 5993.69X 897.32 Y ¼ 9755.61X 989.24
0.9902 0.9913 0.9899 0.9874 0.9916
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Electron microscopy Hippocampal slices were post-fixed in 4% glutaraldehyde for 24 hours followed by phosphate buffer wash. After post-fix in 1% H2OsO4 for 1 hour followed by ethanol dehydration, samples were incubated in uranium acetate overnight and embedded in Epox-812. Thin sections were stained with 2% uranyl acetate and lead citrate. TUNEL assay Apoptosis in hippocampal slices were detected with terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, performed according to the instructions within the TUNEL assay kit purchased from Beijing Zhongshan Biotechnology (Beijing, China).
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Data analysis Hippocampal slices were randomly divided into five groups: normoxia control group, hypoxia group, 1 mM propofol (P1)
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treated group, 5 mM propofol (P5) treated group and 15 mM propofol (P15) treated group. Results were analysed using ANOVA and Chi square test. Data were presented as mean ± SD. The results were considered statistically significant when the p value was less than 0.05 (p50.05).
Results Effect of propofol treatment on OPS and HIP after hypoxia OPS and HIP were recorded at the CA1 region as described (Figure 1). In the hypoxia group, the average decay time of OPS was 186 ± 26 seconds; the ratio of OPS recovery was 25% and the amplitude of recovery was 30.6 ± 23.8%. In 5 mM and 15 mM propofol treatment groups, the decay time of OPS was significantly prolonged and the ratio of OPS recovery and the amplitude of recovery were increased, compared with the hypoxia group. No significant changes of the decay time, recovery ratio and amplitude of OPS were found in 1 mM propofol treatment group (Table II).
Figure 1. Representative OPS and HIP recordings of the CA1 region of hippocampal slices during hypoxia or hypoxia treated with 15 mM propofol (P15).
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In the hypoxic group, the average onset time of HIP was 598 ± 55 seconds; the average duration of HIP was 113 ± 16 seconds and the incidence of HIP is 100%. The incidence of HIP was reduced; the onset of HIP was postponed and the duration of HIP was extended in 5 mM and 15 mM propofol treatment groups compared with the hypoxic group. The above parameters showed no significant changes in the 1 mM propofol treatment group (Table III).
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Effect of propofol treatment on release of amino acid neurotransmitters in hypoxic hippocampal slice The concentrations of neurotransmitters aspartate (Asp), glutamate (Glu), glutamine (Gln), glycine (Gly) and -aminobutyric acid (GABA) were increased in the hypoxic group compared with the normoxia control group. The concentrations of Asp, Glu and GABA were increased by 51.9%, 58.8% and 29.3%, respectively. In 5 mM and 15 mM propofol treatment groups, the release of Asp, Glu and Gly induced by hypoxia was significantly reduced, while GABA release was significantly increased. Gln release was not affected by propofol. In the 1 mM propofol treatment group, there was no significant change in the release of Asp, Glu, Gln or Gly, except for increased GABA release (Table IV). Morphological improvement and reduced apoptosis in hypoxic hippocampal slice after propofol treatment Using HE staining, it was found that pyramid neurons in the CA1 region of the hippocampus became swollen. The cell boundaries were blurring and the nuclei were dark and Table II. The effect of Propofol on OPS (n ¼ 8).
Groups Hypoxic Propofol 1 mM Propofol 5 mM Propofol 15 mM
Decay time (s)
Recovery rate (%)
Amplitude recovery (%)
186 ± 26 191 ± 37 222 ± 32* 243 ± 29*
25.0 25.0 75.0* 87.5*
31 ± 24 33 ± 18 69 ± 27* 72 ± 31*
*p50.05, compared with the hypoxic group.
Table III. The effect of Propofol on HIP (n ¼ 8). Groups
Onset (s)
Duration (s)
Incidence (%)
Hypoxic Propofol 1 mM Propofol 5 mM Propofol 15 mM
598 ± 55 602 ± 42 644 ± 36* 664 ± 45*
113 ± 16 130 ± 27 181 ± 32* 194 ± 23*
100.0 87.5 50.0* 37.5*
*p50.05, compared with the hypoxic group.
shrunken, suggesting cell damage in the hypoxic group. Those morphologic changes of pyramid neurons induced by hypoxia were ameliorated in the 5 mM and 15 mM propofol treatment groups but not in the 1 mM propofol treatment group (Figure 2). Using EM, the ultra-structure and mitochondria changes of pyramid neurons were observed in the CA1 region. In the hypoxic group the cell membrane of neurons and asytrocytes was broken. The nuclei had fragmented and shrunk the membrane and aggregated chromatin. Organelles such as endoplasmic reticulum were condensed. The mitochondria were swollen, with broken, decreased and even disappeared mitochondria ridges. In 5 mM and 15 mM propofol treatment groups, the membranes of neurons and astrocytes were still intact and the rough endoplasmic reticulum was slightly extended. The mitochondria were only slightly swollen, with partially broken and decreased mitochondria ridges. The ultra-structure damages of neurons and astrocytes were not changed in 1 mM propofol group (Figure 3). TUNEL assay reveals that hypoxia causes pronounced apoptosis of the pyramid neurons in the CA1 region of the hippocampus. The neuronal apoptosis was reduced with propofol treatment at 5 mM and 15 mM, but not 1 mM concentration (Figure 4).
Discussion The hippocampal slice has been widely used in research related to hypoxia-induced brain injury and pharmacologic protection against the injury [14–17]. In the hippocampus, the pyramid neurons are the most sensitive cells to hypoxia. The granule cells and the pyramid neurons in CA1 and CA3 form the hippocampal circuit. The pyramid neurons in CA1 are more vulnerable to hypoxia. The functional recovery of pyramid neurons in CA1 is also slower after hypoxia [14, 22]. The orthodromic population spike (OPS) can be recorded in CA1 when Schaffer collateral in CA3 is stimulated, since there are synapses formed between CA1 and CA3. The OPS presents obvious change of phases during hypoxia, which indicates the effect of hypoxia on the excitability of pyramidal cells. Previous studies have shown that during hypoxia there is reduction of OPS amplitude, increase of OPS width and disappearance of OPS, which indicates synaptic dysfunction and loss of excitatory ability. Those OPS changes are reversible if oxygen supply is restored in time. The delay of OPS disappearance suggests that the neurons are less vulnerable to hypoxia [17]. The decay time of OPS after oxygen deprivation can be used as a parameter to evaluate hypoxia tolerance of neurons. The presence of hypoxic injury potential (HIP) indicates the irreversible hypoxic damage of
Table IV. The effect of Propofol on release of amino acids from hippocampal slices (pmol L1, n ¼ 8). Groups Control Hypoxic Propofol 1 mM Propofol 5 mM Propofol 15 mM
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Asp
Glu
Gln
Gly
GABA
1.89 ± 0.14 2.87 ± 0.12* 2.87 ± 0.11* 2.64 ± 0.20*# 2.65 ± 0.11*#
1.51 ± 0.17 2.40 ± 0.13* 2.35 ± 0.12* 2.07 ± 0.05*# 2.02 ± 0.14*#
0.63 ± 0.03 0.68 ± 0.02* 0.66 ± 0.05 0.67 ± 0.04 0.66 ± 0.03
0.95 ± 0.03 1.20 ± 0.05* 1.19 ± 0.11* 1.07 ± 0.06*# 1.03 ± 0.10*#
1.63 ± 0.06 2.11 ± 0.05* 2.20 ± 0.08*# 2.33 ± 0.09*# 2.35 ± 0.12*#
*p50.05, compared with the normoxia control group; #p50.05, compared with the hypoxic group.
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Figure 2. HE staining (10 10, left; 40 10, right) of hippocampal CA1 pyramidal neurons, indicating the changes in cell membrane (arrow) and nuclei (open arrow head) in normoxia control, hypoxia, hypoxia treated with 1 mM propofol (P1), 5 mM propofol (P5) and 15 mM propofol (P15) brain slices.
hippocampal slices [16, 23]. The onset and duration of HIP is correlated with hypoxic damage. The longer it takes the HIP to appear, the easier the recovery is for hippocampal slice after restoration of oxygen supply. Therefore, neuron damage
will be delayed if onset of HIP is postponed or duration of HIP is extended. This study investigated how propofol at clinic concentrations affects the evoked potentials of hypoxic hippocampal
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Figure 3. Electron micrograph (10 000) of the hippocampal CA1 pyramidal neurons indicating the changes in cell membrane (arrow), nuclei (open arrow head) and mitochondria (filled arrow head) in normoxia control, hypoxia and hypoxia treated with 1 mM propofol (P1), 5 mM propofol (P5) and 15 mM propofol (P15) brain slices.
Figure 4. Micrograph (40 10) of hippocampal CA1 pyramidal neurons showing apoptotic cells identified by TUNEL assay.
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slice. In hypoxic groups, the average decay time of OPS was 186 seconds and HIP was recorded in all hippocampal slices. The recovery ratio of OPS and amplitude of OPS were both small after restoration of oxygen and glucose supply, indicating the hypoxic damage of synaptic transmission between hippocampal neurons. In the 5 mM and 15 mM propofol treatment groups, both the recovery ratio of OPS and amplitude of OPS were significantly improved after restoration of oxygen and glucose supply, suggesting that propofol treatment increased the hypoxic tolerance of the hippocampal slice. The delay of HIP onset, the extension of HIP duration and decreased incidence of HIP in both 5 mM and 15 mM propofol treatment groups also demonstrated that propofol treatment postpones the occurrence of irreversible hypoxic damage and increases duration of reversible damage. The data, therefore, suggest that propofol treatment reduces the hypoxic damage of neurons and protects the synaptic functions from hypoxic damage. The protective effect of propofol may result from inhibition of glutamate (Glu), a major neurotransmitter in the CA1 region of the hippocampus. Accumulating evidence has shown that excitatory amino acids (EAA), including Glu and Asp, play a key role in the mechanism underlying hypoxic brain damage [24–28]. NMDA receptor is a major receptor of EAA and its activation results in intracellular Ca2+ overload in hypoxia [29, 30]. In this study, Asp and Glu concentrations in perfusion solution had increases of 51.9% and 58.8%, respectively, in the hypoxia group. Propofol treatment reduced the hypoxia-induced release of Asp and Glu, thus reducing the excitatory toxicity. The mechanisms by which propofol decreases Glu concentration may include inhibition of Glu release, enhancement of Glu re-uptake and acceleration of Glu clearance [21, 31, 32]. GABA is an inhibitory neurotransmitter in CNS that can protect neurons from hypoxic damage [33, 34]. This study showed that propofol treatment can increase the concentration of GABA in perfusion fluid, suggesting that propofol-induced GABA increase plays a role in brain protection in hypoxia. The potential mechanisms by which GABA may be elevated in hypoxia include: (1) overload of intracellular Ca2+ induced by Glu excitatory toxicity; (2) inhibition of re-uptake of GABA; and (3) release of GABA induced by activation of Glu receptor to prevent excitatory toxicity [35]. Previous studies have also shown that propofol increases affinity between GABA and its receptor, thus activating the GABA receptor directly to result in enhanced membrane current flow and GABA-induced hypopolarized inhibition [4]. Propofol also may protect CNS from hypoxia through GABA inhibition by extending Cl conductance controlled by GABAA receptor [10] as well as increasing GABA re-uptake at the synaptic cleft [36]. Glycine (Gly) is an EAA modulator that increases the sensitivity of NMDA receptor to EAA, amplifying the excitatory toxicity induced by EAA [18–20]. In the hypoxia group, it was observed that the concentration of Gly was increased in perfusion solution, which may facilitate the excitatory toxicity. Propofol treatment (5 mM and 15 mM) reversed the elevation of hypoxia-induced Gly concentration, suggesting that propofol has a CNS protection effect. The data showed that propofol treatment had no effect on glutamine
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(Gln) release, suggesting that Gln may not be involved in CNS protection by propofol. Brain hypoxia can cause ATP exhaustion, influx of Na+ and Cl, efflux of K+ and H2O that result in cell oedema. On the other hand, excessive release of Glu over-excites the neurons and causes an influx of Ca2+. Furthermore, NMDA dependent Ca2+ influx is also increased. The resulting overload of intracellular Ca2+ initiates enzyme activation, protein hydrolysis, formation of oxygen free radicals, DNA damage and neuron death. The data show that 5 mM and 15 mM propofol treatment reduces cell oedema and mitochondria damage induced by hypoxia. Other potential mechanisms by which propofol protects CNS from hypoxic damage include modulation of Ca2+, clearance of oxygen free radicals, up-regulation of GABA receptor and inhibition of NMDA receptor. Recent studies also have shown that propofol can induce formation of the P13K-AMAR GluR2 sub-unit complex, affect the NF-kB/p53 signalling pathway, inhibit neuron-specific CREB dephosphorylation and reduce the internalization of the AMPAR GluR2 sub-unit [37–40]. In addition, propofol may reduce the metabolism of neurons, delay the energy exhaustion and maintain the activity of Ca2+/Mg2+ ATPase and Na+/K+ ATPase, which is necessary for homeostasis of electrolytes in the cytoplasm. Propofol can also reduce glucose utilization by inhibiting glycolysis and lactic acid production, resulting in attenuated acidosis inside the cell, thus protecting the neurons from oedema and associated hypoxia-induced damage.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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26. Kollegger H, McBean HG, Tipton KF. Reduction of Striatal N-methyl-D-aspartate toxicity by inhibition of nitric oxide synthase. Biochemical Pharmacology 1993;45:260–264. 27. Katsura K, Ekholm A, Siesijo BK. Coupling among changes in energy metabolism, acidbase homestasis, and ion fluxes in ischemia. Canadian Journal of Physiology & Pharmacology 1992; 70(Suppl):S170–175. 28. Martins E, Inamura K, Themner K, Malmqvist KG, Siesjo¨ BK. Accumulation of calcium and loss of potassium in the hippocampus following transient cerebral ischemia: A proton microprobe study. Journal of Cerebral Blood Flow & Metabolism 1988;8: 531–538. 29. Salinska E, Pluta R, Puka M, Lazarewicz JW. Blockade of N-methyl-D-aspartate-sensitive excitatory amino acid receptors with 2-amino-5-phosphovalerate reduces ischemia-evoked calcium redistribution in rabbit hippocampus. Experimental Neurology 1991;112:89–94. 30. Xia YX, Zacharias E, Hoff P, Tegtmeier F. Ion channel involvement in anoxic depolarization induced by cardiac arrest in rat brain. Journal of Cerebral Blood Flow & Metabolism 1995;15:587–594. 31. Rehberg B, Duch DS. Suppression of central nervous system sodium channels by propofol. Anesthesiology 1999;91:512–520. 32. Chen L, Gong Q, Gao H, Xiao C. The effect of propofol, midazolam and sodium thiopental on accumulation of amino acids after reperfusion of ischemic brain in rats. Journal of Chinese Anesthesiology 2001;21:666–668. 33. Lauder JM, Han VKM, Henderson P, Verdoorn T, Towle AC. Prenatal ontogeny of the GABAergic system in the rat brain: an immunocytochemical study. Neuroscience 1986;19:465–493. 34. Fairen CA, Aivarez-Bolado G, Sanchez MP. Prenatal development of intrinsic neurons of rat neocortex: A comparative study of the distribution of GABA- immunoreactive cells and the GABA (A) receptor. Neuroscience 1991;40:375–397. 35. Grondahl TO, Berg-Johnsen J, Langmoen IA. Chloride influx during cerebral energy deprivation. Neurological Research 1998; 20:131–136. 36. Mantz J, Lecharny JB, Laudenbach V, Henzel D, Peytavin G, Desmonts JM. Anesthetics affect the uptake but not the depolarization-evoked release of GABA in rat striatal synaptosomes. Anesthesiology 1995;82:502–511. 37. Wang H, Wang G, Wang C, Wei Y, Wen Z, Wang C, Zhu A. The early stage formation of PI3K-AMPAR GluR2 subunit complex facilitates the long term neuroprotection induced by propofol postconditioning in rats. PLoS One 2013;8:e65187. 38. Cui DR, Wang L, Jiang W, Qi AH, Zhou QH, Zhang XL. Propofol prevents cerebral ischemia-triggered autophagy activation and cell death in the rat hippocampus through the NF-kB/p53 signaling pathway. Neuroscience 2013;246:117–132. 39. Shu L, Li T, Han S, Ji F, Pan C, Zhang B, Li J. Inhibition of neuronspecific CREB dephosphorylation is involved in propofol and ketamine-induced neuroprotection against cerebral ischemic injuries of mice. Neurochemical Research 2012;37:49–58. 40. Wang H, Luo M, Li C, Wang G. Propofol post-conditioning induced long-term neuroprotection and reduced internalization of AMPAR GluR2 subunit in a rat model of focal cerebral ischemia/ reperfusion. Journal of Neurochemistry 2011;119:210–219.
ORIGINAL ARTICLE
An in vitro study of the neuroprotective effect of propofol on hypoxic hippocampal slice Deng-xing Zhang1, Hao-zhong Ding1, Shan Jiang2, Ying-ming Zeng2, & Qi-feng Tang3 Department of Anesthesiology, The Affiliated Hospital of Jiangnan University, Wuxi, PR China, 2The Key Laboratory of Anesthesiology, Jiangsu Province, Xuzhou Medical College, Xuzhou, PR China, and 3Department of Anesthesiology, Suzhou BenQ Medical Center, Nanjing Medical University, Suzhou, PR China
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Abstract
Keywords
Primary objective: To determine whether propofol has a neuroprotective effect on hypoxic brain injury. Research design: A hippocampal slice, in artificial cerebrospinal fluid (ASCF) with glucose and oxygen deprivation (OGD), was used as an in vitro model for brain hypoxia. Methods and procedures: The orthodromic population spike (OPS) and hypoxic injury potentia1 (HIP) were recorded in the CA1 region when Schaffer collateral was stimulated in the CA3 region of the hippocampal slices during hypoxia. The concentrations of amino acid neurotransmitters in perfusion solution of hippocampal slices were directly measured using high performance liquid chromatography (HPLC). Morphological changes of neurons, astrocytes and mitochondria in CA1 region were observed using histology and electron microscopy. Neuronal apoptosis was evaluated with TUNEL assay. Main outcome and results: Propofol treatment delayed the elimination of OPS and improved the recovery of OPS; decreased frequency of HIP, postponed the onset of HIP and increased the duration of HIP. Propofol treatment also decreased the release of amino acid neurotransmitters such as aspartate, glutamate and glycine induced by hypoxia, but elevated the release of
-aminobutyric acid (GABA). Morphological studies showed that propofol treatment attenuated oedema of pyramid neurons in the CA1 region and reduced apoptosis. Conclusions: Propofol has a neuro-protective effect on hippocampal neuron injury induced by hypoxia.
Hippocampal slice, hypoxic injury, neuroprotection, propofol
Introduction Many pathologic conditions can cause hypoxic brain injury, which may also occur during neurosurgery, cardiovascular surgery and anaesthesia. The mechanism underlying such hypoxic brain injury is still unclear. How to protect the brain from hypoxic injury and how to treat hypoxic brain injury remains clinically challenging. Although hypothermia and pre-ischaemia treatment have been shown to have a protective effect on the brain [1, 2], it is difficult to implement clinically. In contrast, pharmacological treatment seems more clinically practical. During the recent three decades, the neuroprotective effect of anaesthetic drugs has drawn high attention from clinicians. Propofol, an IV anaesthetic drug, has been widely used clinically for anaesthesia induction, maintenance and sedation since the first clinical trial in 1977. Previous studies have
Correspondence: Dr. Qi-feng Tang, Department of Anesthesiology, Suzhou BenQ Medical Center, Nanjing Medical University, Suzhou 215009, PR China. E-mail: [email protected]
History Received 26 November 2013 Revised 23 May 2014 Accepted 20 July 2014 Published online 27 August 2014
shown that propofol could reduce brain artery flow volume, intracranial pressure and metabolism rate that maintains the match of blood supply and oxygen metabolism, as well as improving the oxygen supply during hypoxia, suggesting that propofol protects the brain from hypoxic damage [3–7]. More recent studies suggest that propofol plays a role in central nervous system (CNS) protection through the modulation of Ca2+, oxygen free radicals, -aminobutyric acid (GABA) receptor and N-methyl-D-aspartate (NMDA) receptor [8–12]. In contrast, some data have shown that propofol had no brain protective effect after cardiac surgery and even worsened brain hypoxia [13]. Yet other studies suggest that hypothermia, rather than propofol, is neuroprotective [2]. The aim of this study is to find out if propofol has a neuroprotective effect against hypoxia at 37.5 C to circumvent the hypothermia issue. A hippocampal slice has been used as an in vitro model for neuroprotective study for decades [14–17]. Glucose and oxygen deprivation (OGD) in artificial cerebrospinal fluid (ASCF) can induce hypoxic injury of the hippocampal slice, mimicking brain hypoxia. Compared with cultured cell models, the hippocampal slice more closely resembles the
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whole brain tissue. The current study used an OGD model and extra-cellular electric recording, in which evoked potential was recorded in the CA1 region when Schaffer collateral was stimulated in the CA3 region. It was observed how propofol treatment affects orthodromic population spike (OPS) and hypoxic injury potentia1 (HIP) in the hippocampal slice during hypoxia. Amino acid neurotransmitters, such as aspartate (Asp), glutamate (Glu), glutamine (Gln), glycine (Gly) and
-aminobutyric acid (GABA), are widely distributed and have important functions in CNS. The high concentration of excitatory amino acids in synaptic cleft and extracellular fluid during hypoxia is a common pathway causing excitatory toxicity and neuronal death [10, 18–21]. Most amino acid neurotransmitter assays measure the total amount of amino acids in specific brain areas or nuclei, which may also contain amino acids from protein metabolism. Thus, the total amino acid amount of the hippocampus may not reflect the amount of amino acids as a neurotransmitter. This study used HPLC to directly measure the concentration of amino acid neurotransmitters in perfusion solution of hippocampal slices during hypoxia. In the hippocampus, the CA1 region is most sensitive to hypoxia. Mitochondrion is the organelle playing a key role in the process in which the reversible cellular change becomes irreversible during hypoxia. This study examined the morphological change of neurons, astrocytes and mitochondria in the CA1 region using histology and electron microscopy. The overall goal is to find direct evidences demonstrating that propofol treatment has a brain protective effect in hypoxia.
Materials and methods Experimental animals and acute hippocampal slice preparation Eight-to-ten weeks old Sprague-Dawley (SD) rats (Experimental Animal Center, Xuzhou Medical College, China), weighing 200–250 grams, were anaesthetized by ether. Hippocampi were dissected and sectioned using vibrating-blade microtome in 300 mm thickness. Hippocampal slices were incubated in ASCF (NaCl 124 mM, KCl 3.3 mM, NaH2PO4 1.24 mM, MgSO4 2.4 mM, NaHCO3 25.7 mM, CaCl2 2.4 mM) with and without glucose (10.0 mM) at 37.5 C for 2–3 hours. The flowing volume was 200 ml min1. All the animal protocols are approved by the Experimental Animal Committee of Xuzhou Medical College. Extra cellular potential recording Electric probes were inserted into Schaffer lateral fiber at CA3. Stimuli (0.6 mA, 0.1 Hz, 100 ms) were given with a 10-second interval. Evoked potentials including orthodromic population spike (OPS) and hypoxia injury potential (HIP) were recorded at CA1. Only brain slices that had stable OPS (43 mv) for at least 20 minutes were selected for experiments. Hypoxic groups were incubated in ASCF without glucose and oxygen (ASCFOGD) for 14 minutes followed by normal ASCF perfusion. Treatment groups were incubated in ASCFOGD with 1, 5, 15 mM propofol (10 mg ml1 injection emulsion,
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AstraZeneca, Caponago, Italy), respectively. The emulsion carrier (soybean oil (100 mg mL1), glycerol (22.5 mg mL1), egg lecithin (12 mg mL1); and disodium edetate (0.005%), pH8.0) without propofol was used in non-propofol-treated hypoxic groups, at a dilution of 1:3500. In hypoxic and treatment groups, every OPS was recorded before its disappearance. Each group contained eight samples. OPS decay is defined as the time period when OPS become undetectable after oxygen deprivation. The amplitude of OPS recovery is defined as the ratio of OPS amplitude after 1 hour of normal ASCF perfusion over OPS amplitude before oxygen deprivation. The ratio of OPS recovery is defined as the percentage of hippocampal slices in which OPS amplitude recovery reach 60% of OPS amplitude before oxygen deprivation. HIP onset is defined as the time period when HIP is recorded after oxygen deprivation. HIP duration is defined as how long HIP lasts. HIP incidence is defined as the percentage of hippocampal slices in which HIP were recorded after oxygen deprivation. High performance liquid chromatographic (HPLC) analysis ASCF from 14 minutes of perfusion from each group was collected and centrifuged. Supernatant was kept for HPLC analysis. The mobile phase A was NaAC (0.5 M, PH 6.0) with 0.05% THF. The mobile phase B was methanol. The following washing gradients (minutes, Phase B %) were used: 0, 30%; 7, 60%; and 9, 30%, with velocity of 0.9 ml min1, temperature at 30 C, lem at 330 nm and lex at 456 nm. Derivation agent was made from OPA, 2-MCE, Boric acid-sodium hydroxide and methanol (PH 9.6). A mixture of 20 ml derivation agent and 30 ml sample was incubated for 2 minutes at RT. Injection volume was 20 ml. The remaining time, regression equations and correlation coefficient were obtained on samples of aspartate (Asp), glutamate (Glu), glutamine (Gln), glycine (Gly) and -aminobutyric acid (GABA) (Table I). Standard curves were prepared using 10, 5, 2.5, 1.25 and 0.625 mM of Asp, Glu, Gln, Gly or GABA. The sensitivity of amino acids is less than 1012 mol/20 mL sample. The concentrations (mM) of Asp, Glu, Gln, Gly and GABA in perfusion ASCF were calculated based on standard curves using Prostar. HE staining Hippocampal slices from each group were fixed in 0.1% methanol and embedded in wax after dehydration. The section thickness was 5 mm. HE staining followed the standard protocol.
Table I. The remaining time, regression equation andcorrelation coefficient of amino acids (n ¼ 8).
Amino acids Asp Glu Gln Gly GABA
Remaining time (min)
Regression equation
Correlation coefficient
3.56 3.92 5.68 6.89 8.37
Y ¼ 1012.54X + 542.62 Y ¼ 2053.23X + 134.77 Y ¼ 9021.48X 378.65 Y ¼ 5993.69X 897.32 Y ¼ 9755.61X 989.24
0.9902 0.9913 0.9899 0.9874 0.9916
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Electron microscopy Hippocampal slices were post-fixed in 4% glutaraldehyde for 24 hours followed by phosphate buffer wash. After post-fix in 1% H2OsO4 for 1 hour followed by ethanol dehydration, samples were incubated in uranium acetate overnight and embedded in Epox-812. Thin sections were stained with 2% uranyl acetate and lead citrate. TUNEL assay Apoptosis in hippocampal slices were detected with terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, performed according to the instructions within the TUNEL assay kit purchased from Beijing Zhongshan Biotechnology (Beijing, China).
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Data analysis Hippocampal slices were randomly divided into five groups: normoxia control group, hypoxia group, 1 mM propofol (P1)
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treated group, 5 mM propofol (P5) treated group and 15 mM propofol (P15) treated group. Results were analysed using ANOVA and Chi square test. Data were presented as mean ± SD. The results were considered statistically significant when the p value was less than 0.05 (p50.05).
Results Effect of propofol treatment on OPS and HIP after hypoxia OPS and HIP were recorded at the CA1 region as described (Figure 1). In the hypoxia group, the average decay time of OPS was 186 ± 26 seconds; the ratio of OPS recovery was 25% and the amplitude of recovery was 30.6 ± 23.8%. In 5 mM and 15 mM propofol treatment groups, the decay time of OPS was significantly prolonged and the ratio of OPS recovery and the amplitude of recovery were increased, compared with the hypoxia group. No significant changes of the decay time, recovery ratio and amplitude of OPS were found in 1 mM propofol treatment group (Table II).
Figure 1. Representative OPS and HIP recordings of the CA1 region of hippocampal slices during hypoxia or hypoxia treated with 15 mM propofol (P15).
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In the hypoxic group, the average onset time of HIP was 598 ± 55 seconds; the average duration of HIP was 113 ± 16 seconds and the incidence of HIP is 100%. The incidence of HIP was reduced; the onset of HIP was postponed and the duration of HIP was extended in 5 mM and 15 mM propofol treatment groups compared with the hypoxic group. The above parameters showed no significant changes in the 1 mM propofol treatment group (Table III).
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Effect of propofol treatment on release of amino acid neurotransmitters in hypoxic hippocampal slice The concentrations of neurotransmitters aspartate (Asp), glutamate (Glu), glutamine (Gln), glycine (Gly) and -aminobutyric acid (GABA) were increased in the hypoxic group compared with the normoxia control group. The concentrations of Asp, Glu and GABA were increased by 51.9%, 58.8% and 29.3%, respectively. In 5 mM and 15 mM propofol treatment groups, the release of Asp, Glu and Gly induced by hypoxia was significantly reduced, while GABA release was significantly increased. Gln release was not affected by propofol. In the 1 mM propofol treatment group, there was no significant change in the release of Asp, Glu, Gln or Gly, except for increased GABA release (Table IV). Morphological improvement and reduced apoptosis in hypoxic hippocampal slice after propofol treatment Using HE staining, it was found that pyramid neurons in the CA1 region of the hippocampus became swollen. The cell boundaries were blurring and the nuclei were dark and Table II. The effect of Propofol on OPS (n ¼ 8).
Groups Hypoxic Propofol 1 mM Propofol 5 mM Propofol 15 mM
Decay time (s)
Recovery rate (%)
Amplitude recovery (%)
186 ± 26 191 ± 37 222 ± 32* 243 ± 29*
25.0 25.0 75.0* 87.5*
31 ± 24 33 ± 18 69 ± 27* 72 ± 31*
*p50.05, compared with the hypoxic group.
Table III. The effect of Propofol on HIP (n ¼ 8). Groups
Onset (s)
Duration (s)
Incidence (%)
Hypoxic Propofol 1 mM Propofol 5 mM Propofol 15 mM
598 ± 55 602 ± 42 644 ± 36* 664 ± 45*
113 ± 16 130 ± 27 181 ± 32* 194 ± 23*
100.0 87.5 50.0* 37.5*
*p50.05, compared with the hypoxic group.
shrunken, suggesting cell damage in the hypoxic group. Those morphologic changes of pyramid neurons induced by hypoxia were ameliorated in the 5 mM and 15 mM propofol treatment groups but not in the 1 mM propofol treatment group (Figure 2). Using EM, the ultra-structure and mitochondria changes of pyramid neurons were observed in the CA1 region. In the hypoxic group the cell membrane of neurons and asytrocytes was broken. The nuclei had fragmented and shrunk the membrane and aggregated chromatin. Organelles such as endoplasmic reticulum were condensed. The mitochondria were swollen, with broken, decreased and even disappeared mitochondria ridges. In 5 mM and 15 mM propofol treatment groups, the membranes of neurons and astrocytes were still intact and the rough endoplasmic reticulum was slightly extended. The mitochondria were only slightly swollen, with partially broken and decreased mitochondria ridges. The ultra-structure damages of neurons and astrocytes were not changed in 1 mM propofol group (Figure 3). TUNEL assay reveals that hypoxia causes pronounced apoptosis of the pyramid neurons in the CA1 region of the hippocampus. The neuronal apoptosis was reduced with propofol treatment at 5 mM and 15 mM, but not 1 mM concentration (Figure 4).
Discussion The hippocampal slice has been widely used in research related to hypoxia-induced brain injury and pharmacologic protection against the injury [14–17]. In the hippocampus, the pyramid neurons are the most sensitive cells to hypoxia. The granule cells and the pyramid neurons in CA1 and CA3 form the hippocampal circuit. The pyramid neurons in CA1 are more vulnerable to hypoxia. The functional recovery of pyramid neurons in CA1 is also slower after hypoxia [14, 22]. The orthodromic population spike (OPS) can be recorded in CA1 when Schaffer collateral in CA3 is stimulated, since there are synapses formed between CA1 and CA3. The OPS presents obvious change of phases during hypoxia, which indicates the effect of hypoxia on the excitability of pyramidal cells. Previous studies have shown that during hypoxia there is reduction of OPS amplitude, increase of OPS width and disappearance of OPS, which indicates synaptic dysfunction and loss of excitatory ability. Those OPS changes are reversible if oxygen supply is restored in time. The delay of OPS disappearance suggests that the neurons are less vulnerable to hypoxia [17]. The decay time of OPS after oxygen deprivation can be used as a parameter to evaluate hypoxia tolerance of neurons. The presence of hypoxic injury potential (HIP) indicates the irreversible hypoxic damage of
Table IV. The effect of Propofol on release of amino acids from hippocampal slices (pmol L1, n ¼ 8). Groups Control Hypoxic Propofol 1 mM Propofol 5 mM Propofol 15 mM
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Asp
Glu
Gln
Gly
GABA
1.89 ± 0.14 2.87 ± 0.12* 2.87 ± 0.11* 2.64 ± 0.20*# 2.65 ± 0.11*#
1.51 ± 0.17 2.40 ± 0.13* 2.35 ± 0.12* 2.07 ± 0.05*# 2.02 ± 0.14*#
0.63 ± 0.03 0.68 ± 0.02* 0.66 ± 0.05 0.67 ± 0.04 0.66 ± 0.03
0.95 ± 0.03 1.20 ± 0.05* 1.19 ± 0.11* 1.07 ± 0.06*# 1.03 ± 0.10*#
1.63 ± 0.06 2.11 ± 0.05* 2.20 ± 0.08*# 2.33 ± 0.09*# 2.35 ± 0.12*#
*p50.05, compared with the normoxia control group; #p50.05, compared with the hypoxic group.
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Figure 2. HE staining (10 10, left; 40 10, right) of hippocampal CA1 pyramidal neurons, indicating the changes in cell membrane (arrow) and nuclei (open arrow head) in normoxia control, hypoxia, hypoxia treated with 1 mM propofol (P1), 5 mM propofol (P5) and 15 mM propofol (P15) brain slices.
hippocampal slices [16, 23]. The onset and duration of HIP is correlated with hypoxic damage. The longer it takes the HIP to appear, the easier the recovery is for hippocampal slice after restoration of oxygen supply. Therefore, neuron damage
will be delayed if onset of HIP is postponed or duration of HIP is extended. This study investigated how propofol at clinic concentrations affects the evoked potentials of hypoxic hippocampal
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Figure 3. Electron micrograph (10 000) of the hippocampal CA1 pyramidal neurons indicating the changes in cell membrane (arrow), nuclei (open arrow head) and mitochondria (filled arrow head) in normoxia control, hypoxia and hypoxia treated with 1 mM propofol (P1), 5 mM propofol (P5) and 15 mM propofol (P15) brain slices.
Figure 4. Micrograph (40 10) of hippocampal CA1 pyramidal neurons showing apoptotic cells identified by TUNEL assay.
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slice. In hypoxic groups, the average decay time of OPS was 186 seconds and HIP was recorded in all hippocampal slices. The recovery ratio of OPS and amplitude of OPS were both small after restoration of oxygen and glucose supply, indicating the hypoxic damage of synaptic transmission between hippocampal neurons. In the 5 mM and 15 mM propofol treatment groups, both the recovery ratio of OPS and amplitude of OPS were significantly improved after restoration of oxygen and glucose supply, suggesting that propofol treatment increased the hypoxic tolerance of the hippocampal slice. The delay of HIP onset, the extension of HIP duration and decreased incidence of HIP in both 5 mM and 15 mM propofol treatment groups also demonstrated that propofol treatment postpones the occurrence of irreversible hypoxic damage and increases duration of reversible damage. The data, therefore, suggest that propofol treatment reduces the hypoxic damage of neurons and protects the synaptic functions from hypoxic damage. The protective effect of propofol may result from inhibition of glutamate (Glu), a major neurotransmitter in the CA1 region of the hippocampus. Accumulating evidence has shown that excitatory amino acids (EAA), including Glu and Asp, play a key role in the mechanism underlying hypoxic brain damage [24–28]. NMDA receptor is a major receptor of EAA and its activation results in intracellular Ca2+ overload in hypoxia [29, 30]. In this study, Asp and Glu concentrations in perfusion solution had increases of 51.9% and 58.8%, respectively, in the hypoxia group. Propofol treatment reduced the hypoxia-induced release of Asp and Glu, thus reducing the excitatory toxicity. The mechanisms by which propofol decreases Glu concentration may include inhibition of Glu release, enhancement of Glu re-uptake and acceleration of Glu clearance [21, 31, 32]. GABA is an inhibitory neurotransmitter in CNS that can protect neurons from hypoxic damage [33, 34]. This study showed that propofol treatment can increase the concentration of GABA in perfusion fluid, suggesting that propofol-induced GABA increase plays a role in brain protection in hypoxia. The potential mechanisms by which GABA may be elevated in hypoxia include: (1) overload of intracellular Ca2+ induced by Glu excitatory toxicity; (2) inhibition of re-uptake of GABA; and (3) release of GABA induced by activation of Glu receptor to prevent excitatory toxicity [35]. Previous studies have also shown that propofol increases affinity between GABA and its receptor, thus activating the GABA receptor directly to result in enhanced membrane current flow and GABA-induced hypopolarized inhibition [4]. Propofol also may protect CNS from hypoxia through GABA inhibition by extending Cl conductance controlled by GABAA receptor [10] as well as increasing GABA re-uptake at the synaptic cleft [36]. Glycine (Gly) is an EAA modulator that increases the sensitivity of NMDA receptor to EAA, amplifying the excitatory toxicity induced by EAA [18–20]. In the hypoxia group, it was observed that the concentration of Gly was increased in perfusion solution, which may facilitate the excitatory toxicity. Propofol treatment (5 mM and 15 mM) reversed the elevation of hypoxia-induced Gly concentration, suggesting that propofol has a CNS protection effect. The data showed that propofol treatment had no effect on glutamine
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(Gln) release, suggesting that Gln may not be involved in CNS protection by propofol. Brain hypoxia can cause ATP exhaustion, influx of Na+ and Cl, efflux of K+ and H2O that result in cell oedema. On the other hand, excessive release of Glu over-excites the neurons and causes an influx of Ca2+. Furthermore, NMDA dependent Ca2+ influx is also increased. The resulting overload of intracellular Ca2+ initiates enzyme activation, protein hydrolysis, formation of oxygen free radicals, DNA damage and neuron death. The data show that 5 mM and 15 mM propofol treatment reduces cell oedema and mitochondria damage induced by hypoxia. Other potential mechanisms by which propofol protects CNS from hypoxic damage include modulation of Ca2+, clearance of oxygen free radicals, up-regulation of GABA receptor and inhibition of NMDA receptor. Recent studies also have shown that propofol can induce formation of the P13K-AMAR GluR2 sub-unit complex, affect the NF-kB/p53 signalling pathway, inhibit neuron-specific CREB dephosphorylation and reduce the internalization of the AMPAR GluR2 sub-unit [37–40]. In addition, propofol may reduce the metabolism of neurons, delay the energy exhaustion and maintain the activity of Ca2+/Mg2+ ATPase and Na+/K+ ATPase, which is necessary for homeostasis of electrolytes in the cytoplasm. Propofol can also reduce glucose utilization by inhibiting glycolysis and lactic acid production, resulting in attenuated acidosis inside the cell, thus protecting the neurons from oedema and associated hypoxia-induced damage.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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