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BJA Advance Access published online on June 27, 2008
British Journal of Anaesthesia, doi:10.1093/bja/aen185
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© The Board of Management and Trustees of the British Journal of Anaesthesia 2008. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Propofol causes neurite retraction in neurones

D. Turina1,*, V. M. Loitto2, K. Björnström1, T. Sundqvist2 and C. Eintrei1

1 Department of Anaesthesiology
2 Department of Microbiology, Faculty of Health Sciences, Linköping University, 58185 Linköping, Sweden

* Corresponding author. E-mail: dean.turina{at}lio.se

Accepted for publication April 25, 2008.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
Background: The mechanism by which anaesthetic agents produce general anaesthesia is not yet fully understood. Retraction of neurites is an important function of individual neurones and neural plexuses during normal and pathological conditions, and it has been shown that such a retraction pathway exists in developing and mature neurones. We hypothesized that propofol decreases neuronal activity by causing retraction of neuronal neurites.

Methods: Primary cultures of rat cortical neurones were exposed in concentration– and time–response experiments to 0.02, 0.2, 2, and 20 µM propofol or lipid vehicle. Neurones were pretreated with the GABAA receptor (GABAAR) antagonist, bicuculline, the myosin II ATPase activity inhibitor, blebbistatin, and the F-actin stabilizing agent, phalloidin, followed by administration of propofol (20 µM). Changes in neurite retraction were evaluated using time-lapse light microscopy.

Results: Propofol caused a concentration- and time-dependent reversible retraction of cultured cortical neurone neurites. Bicuculline, blebbistatin, and phalloidin completely inhibited propofol-induced neurite retraction. Images of retracted neurites were characterized by a retraction bulb and a thin trailing membrane remnant.

Conclusions: Cultured cortical rat neurones retract their neurites after exposure to propofol in a concentration- and time-dependent manner. This retraction is GABAAR mediated, reversible, and dependent on actin and myosin II. Furthermore, the concentrations and times to full retraction and recovery correspond to those observed during propofol anaesthesia.

Keywords: anaesthetics i.v., propofol; brain, GABA; rat; theories of anaesthetic action, cellular mechanisms


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
The underlying mechanisms by which diverse anaesthetic agents produce anaesthesia are not yet fully understood.1 The normal function of the nervous system depends on precise connections that wire individual neurones together into an orderly network. Selective retraction of newly formed axons occurs extensively in the developing nervous system to eliminate inappropriate axonal projections.2 Hibernating mammals experience a temperature-dependent retraction of the neural microstructure throughout the brain, and recovery of this microstructure to fixed parameters is rapid and complete on return to euthermia.3 Retraction is also an early response of axons to injury.4 Recent investigations indicate that retraction is an active process mediated by alterations in actin filament structure and motor protein-based force generation.5 6 A main function of molecular motor proteins is axonal vesicular transport in neurones. It has been shown that many anaesthetic agents modulate synaptic transmission7 and depress neurosecretion.8

Propofol induces growth cone collapse and neurite retraction in chick explant culture.9 We have previously reported that propofol causes a reorganization of actin to form ring structures in cultured rat neurones and glial cells.10

In the present study, the effects of propofol on neurite morphology were observed, and the response dynamics were analysed using time-lapse light microscopy.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 Acknowledgements
 References
 
Cell culture
The study was approved by the ethics committee for Animal Research at Linköping University. Primary cultures of rat neurones were essentially obtained as described by Hansson and Rönnbäck.11 In short, newborn Sprague–Dawley rats were decapitated and the cortex was dissected free. Cortical tissue was sieved through nylon mesh (80 µm) into Dulbecco's modified Eagle's medium high glucose (Sigma Chemical Co., St Louis, MO, USA), supplemented with glucose (30 mM), insulin (5 µg ml–1), glutamine (2 mM), penicillin (50 U ml–1), streptomycin (50 µg ml–1), and 20% fetal calf serum (FCS). The cells were cultured on poly-L-lysine-coated 25 mm {phi} sterile coverslips at 37°C in a humidified atmosphere of 95% air/5% CO2. After 24 h, the FCS concentration of the medium was changed to 10% and maintained at that level for 7 days. On the sixth day, 10 µM cytosine-1-β-D-arabinofuranoside was added to new 10% media for 24 h, to suppress growth of glial cells. Thereafter, the cultures received new medium containing 5% FCS every second day. The cells were used between days 14 and 35.

Microscopy
Cells used for microscopy (on 25 mm round coverslips) were placed in Petri dishes. Each cell culture was rinsed twice in Ca2+-containing medium (CCM)12 before use. The dynamics of neurite morphology were observed before and after the addition of propofol. Cells were exposed to 0.02, 0.2, 2, and 20 µM propofol as the commercially available solution Diprivan® (10 mg ml–1; Astrazeneca Ltd, Cheshire, UK) or lipid vehicle as the commercially available solution Vasolipid (20 mg ml–1; Braun, Melsungen, Germany). Time-lapse images were acquired as described below. Bicuculline (30 µM) (Sigma Chemical Co., St Louis, MO, USA) was added 10 min before exposure to propofol. Phalloidin (5 µM) (Sigma Chemical Co.) was added 10 min and blebbistatin (1 µM) (Calbiochem, EMD Biosciences, Inc., Darmstadt, Germany) 3 min before exposure to propofol. To observe reversibility, propofol was washed out from the culture media after 2 min exposure and the cells were observed for 20 min.

Coverslips with cell cultures were mounted in an RC-21B closed bath imaging chamber and observed by light microscopy using a Zeiss Axiovert 200 (Göttingen, Germany) inverted microscope equipped with an alpha Plan-Fluar 100x/1.45 numerical aperture (NA) oil immersion objective (Carl Zeiss, Microimaging GmbH, Göttingen, Germany). Images were captured using a cooled charge-coupled device (CCD) AxioCam MRm (Carl Zeiss). The temperature in the cell culture was maintained at 37°C using an objective heater (8Pecon GmbH, Erbach, Germany). A time-lapse series was initiated wherein cell images were obtained at 1 min intervals between 0 and 10 min. To visualize a thin trailing remnant and retraction bulb, differential interference contrast images of cells on a 37°C heated stage were captured using a Zeiss Axiovert 135M equipped with a 100x 1.3 NA objective and a ProgRes C10plus CCD camera (Jenoptik, Jena, Germany).

Data quantification and statistical analysis
Neurite retraction was calculated by subtracting the final length from the initial length. Neurite length was measured manually using Adobe PhotoShop 6.0 (Adobe Systems, San Jose, CA, USA) by an observer blinded to the image analysing. The retraction change of the neurite is presented as a proportion of the initial length. Overall significant differences between conditions were determined by two-way analysis of variance for repeated measurements (ANOVA). Post hoc comparisons were performed by the Bonferroni test for multiple comparisons. For propofol, experimental values are expressed as the mean (SD) of 14 independent measurements. In each measurement, one neurone was analysed. The cell cultures were obtained from a total of 5 litters. Concentration–response curves for neurite retraction were analysed (Prism 4.0 software, GraphPad Software, San Diego, CA, USA) by non-linear curve fitting [using the following equation: Y=Bottom+(Top–Bottom)/(1+10(log EC50–X)xHillslope)] to obtain the negative logarithm of the propofol concentration that produced half (pEC50) of the maximal neurite retraction. All statistical analyses and graphing were done using Prism 4.0 software, and P-value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 Acknowledgements
 References
 
Propofol-induced neurite retraction is time-dependent
To study the effects of clinically relevant concentrations of propofol on rat cortical neurites in vitro, we used time-lapse light microscopy (Figs 1A, 2A, and 3A). The response dynamics were evaluated by measuring neurite retraction distance. This was compared with the initial length of the extended neurite which varied between 7 and 40 µm. Images of retracted neurites show that most of them are characterized by a retraction bulb and a thin trailing remnant (Fig. 2A).


Figure 1
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  		Fig 1 Propofol induces neurite retraction. (A) Time-lapse imaging reveals dynamics of neurite retraction and morphological changes after the addition of propofol. Cortical cell cultures in CCM were exposed to 2 µM propofol and observed for 10 min. Images shown are 0, 1, 2, 5, 7, and 10 min after addition of propofol. The scale bar is 10 µm. (B) Propofol-induced neurite retraction is time-dependent. Cell cultures were exposed to 0.2, 2, and 20 µM propofol and assessed over 10 min. Values are expressed as percentage neurite retraction. **P<0.01; ***P<0.001 vs vehicle (control) (ANOVA, followed by Bonferroni test). Values represent mean (SD), n=14 for each concentration (n=6 for vehicle).

 


Figure 2
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  		Fig 2 Propofol induces a concentration-dependent neurite retraction. (A) Magnified time-lapse differential interference contrast images of a rat cortical neurite. Treatment with 20 µM propofol-induced neurite retraction characterized by a retraction bulb (thick arrows) and a trailing remnant (thin arrows). The trailing remnant was not included in the length measurements. Images shown are 0 and 10 min after addition of propofol 20 µM. The trailing remnant has become too narrow to permit vesicle transport to the synapse. The scale bar is 10 µm. The percentage of neurite retraction was assessed at (B) 2 and 10 min after exposure to propofol at concentrations of 0.02, 0.2, 2, and 20 µM. Values represent mean (SD), n=14 for each concentration (n=6 for vehicle).

 


Figure 3
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  		Fig 3 Propofol-induced neurite retraction is reversible. (A) Time-lapse images of a rat cortical neurite after propofol washout. Cell cultures in CCM were exposed to 2 µM propofol for 2 min and then washed. Images shown are 0, 10, and 20 min. The scale bar is 10 µm. (B) Reversibility of neurite retraction after washout of propofol. Washout of the chamber and replenishment with fresh bathing solution is denoted by the symbol (w). Values represent mean (SD), n=6.

 
To determine the time course of neurite retraction, we measured neurite length over 10 min after propofol application. Between 1 and 2 min after exposure to 20 µM propofol, neurites had retracted 16.1 (11.5)% and 21.1 (14.6)%, respectively [mean (SD)], P<0.01 compared with vehicle-exposed neurites that showed no change in length [–2.6 (3.9)% and 2.0 (5.9)%, respectively] (Fig. 1B). At 5 min post-exposure to 2 and 20 µM propofol, neurites had retracted 22.8 (10.5) and 34.2 (18), respectively, P<0.001 compared with vehicle-exposed neurites [–5.8 (7.1) and 0.1 (11.4)]. Neurites continued to retract over time such that at 10 min post-propofol application neurite retraction was 34.9% (15.2) and 47.1% (16.8) for 2 and 20 µM, respectively. Vehicle-exposed neurites showed no change in length [–5.9 (7.1) and 1.3 (13.4), respectively]. Propofol 0.02 and 0.2 µM concentrations [1.7 (8.2)% and 1.4 (12.8)% change, respectively] were ineffective. (At 0.2 µM, one of the neurones was excluded due to contractility of the whole cell which made measurement impossible.)

Propofol-induced neurite retraction was concentration-dependent
Propofol-induced neurite retraction was concentration-dependent. No response was seen at 0.02 and 0.2 µM. The EC50 values at 2 and 10 min propofol exposure were 1.1 and 1.5 µM, respectively [pEC50 was –5.97 (0.28) and –5.8 (1.5)] (Fig. 2B). These values agree well with the clinical EC50 concentration for propofol (1.57 µM).13

Propofol-induced neurite retraction was reversible
When 2 µM propofol was washed out 2 min after exposure, neurites continued to retract reaching a minimum length at 10 min. By 20 min, the neurites had returned to pretreatment length (Fig. 3A and B). Propofol-induced retraction was not reversible after prolonged exposure (30 min) or exposure to 20 µM propofol.

Bicuculline, blebbistatin, and phalloidin inhibit propofol-induced retraction
To examine propofol cell signalling pathway(s), we applied pharmacological inhibitors to cultured neurones. The GABAA-receptor blocker bicuculline (30 µM) inhibited the propofol-mediated neurite retraction (Fig. 4). It has been shown that axon retraction is dependent on an interaction between myosin and actin.6 To confirm this, we applied the myosin II blocker, blebbistatin (1 µM), and the F-actin stabilizing agent, phalloidin (5 µM), both of which completely inhibited propofol-induced neurite retraction (Fig. 4).


Figure 4
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  		Fig 4 Bicuculline, phalloidin, and blebbistatin inhibit propofol-induced neurite retraction. Time course for changes in percentage neurite retraction. Cell cultures were pretreated with 30 µM bicuculline for 10 min, 5 µM phalloidin for 10 min, or 1 µM blebbistatin for 3 min before the addition of 20 µM propofol. Cells were subsequently assessed for 10 min. Phalloidin and blebbistatin significantly prevents propofol-induced retraction after 1 and 2 min. **P<0.01; ***P<0.001 (ANOVA, followed by Bonferroni test). All inhibitors prevented neurite retraction after 5 and 10 min. ***P<0.001. Values represent mean (SD), n=6 for each inhibitor.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 Acknowledgements
 References
 
We have examined the effect of the anaesthetic agent propofol on neurite retraction in vitro using rat cortical neurone cultures. Compared with vehicle-treated neurites, propofol caused a GABAA receptor (GABAAR)-mediated, concentration-dependent neurite retraction. The concentrations of propofol used in the cell cultures were clinically relevant and we found an EC50 of 1.1 and 1.5 µM, after 2 and 10 min propofol exposure, which is very close to the anaesthetic EC50 concentration of propofol (1.57 µM).13 The retraction is time-dependent and reversible. We could inhibit propofol-mediated neurite retraction by using the myosin II ATPase activity inhibitor blebbistatin,14 which shows that retraction is a myosin II-dependent and active process. These findings support our hypothesis that propofol activates the GABAAR leading to an actin–myosin contraction. Treatment with propofol initiates the movement of the neurite cytoplasm towards the cell body, causing the neurite to form retraction bulbs, leaving observable thin trailing remnants along its retracted path. In our study, stabilization of F-actin with phalloidin completely blocked neurite retraction. There is increasing evidence that axon retraction is dependent on an interaction between myosin and actin.5 6 The coordinated activation of myosin II and the formation of intra-axonal F-actin bundles for myosin II-driven force generation cause axon retraction.5 The axonal microfilament system is subject to strong contractile forces generated by myosin, and those forces are counterbalanced by forces between the microfilament and microtubule arrays generated by cytoplasmic dynein.6 Furthermore, retraction is regulated by a GTPase RhoA-dependent mechanism and negative regulation of RhoA and its downstream Rho-associated coiled coil-forming protein kinase (ROCK) prevents the retraction of established axons in vivo.15 In the cell, ROCK activated by the GTPase RhoA increases phosphorylation of myosin regulatory light chains by phosphorylating and inhibiting the myosin light chain (MLC) phosphatase. The motor protein myosin II is regulated by MLC kinase that directly phosphorylates myosin regulatory light chains.16 A similar actin–myosin-dependent retraction has been observed for lysophosphatidic acid (LPA) in neurofilament rearrangements.17 LPA is an extracellular signalling phospholipid that produces actin rearrangement to influence cell morphology and motility in various cell types, including neural and non-neural cells.18 Retraction is an important function of individual neurones and neural plexuses during normal and pathological conditions and it has been shown that such an axon retraction pathway exists in developing2 and mature neurones.15

In our study, the concentration-dependent propofol retraction of neurites could be blocked by the GABAAR antagonist bicuculline. This demonstrates involvement of the GABAAR in propofol cell signalling. We have previously shown that propofol induces tyrosine phosphorylation in the GABAAR β2 and β3-subtypes and increases the neuronal content of F-actin.19 Our previous findings have demonstrated that propofol mediates a calcium-dependent, reversible, and concentration-dependent actin reorganization.10 When propofol was washed out 2 min after exposure, neurites continued to retract reaching a minimum length at 10 min. The continued retraction after propofol washout may be explained by the fact that it takes time to reverse the retraction process, a time that is similar to recovery after propofol anaesthesia. By 20 min, the neurites had returned to pretreatment length. We believe that the time scale is not dissimilar from that of clinical anaesthesia. There is a significant retraction of neurites after 1 and 2 min; it takes at least 2 min for patients to fall asleep when given propofol. The recovery period for patients after propofol anaesthesia is also within the timeframe we have seen (minutes, not seconds or hours). It must be taken into account that clearance by blood flow is more rapid than found in our Petri dish.

In line with our findings, Al-Jahdari and colleagues9 recently reported that propofol at 5 and 20 µM concentrations induce mild growth cone collapse in chick-embryonic neurones.

With support from previous studies, we suggest a signalling pathway leading to cytoskeletal rearrangement that involves a Rho pathway activation and MLC phosphorylation which increase the contractility of myosin, followed by reorganization of actin and neurite retraction.15 The phenomenon of neurite retraction may explain the anaesthetic effect of propofol since neurite retraction has also been reported in hibernation3 and neurotrauma.4 Retraction of neurites in the cortex, thalamus, and hippocampus, together with decreased synaptic protein clustering without protein loss, has previously been shown in hibernating ground squirrels.3 20 In the awake state, neurites returned to their pre-hibernation locations.21

Several anaesthetic agents including propofol profoundly decrease ongoing neuronal activity in isolated cortical networks.22 It has been shown that propofol and other anaesthetics modulate synaptic transmission7 and depress neurosecretion.8 At each synaptic contact, the magnitude of the postsynaptic response is related to the number of vesicles released and the amount of transmitter in each vesicle and alterations in receptor properties. In this study, we have shown that retracted neurites left a retraction bulb and a thin trailing remnant, which probably serve as a pathway for the neurite to find its way back when the effects of propofol are gone. It is very likely that the existing neuronal network prevails during anaesthesia, which could explain the rapid recovery.

Remaining electrophysical action potentials support our findings that there is still a contact between the neurones. Our hypothesis is that the trailing neurite remnants become too narrow to allow vesicles through to the synapse which leads to inhibition of neurotransmitter release.

The findings in this study are obtained from in vitro experiments, which may not faithfully replicate in vivo conditions. As always, extrapolation of data obtained from laboratory-based studies to the clinical setting is difficult.

In conclusion, using brain cortical neurone cultures, we have shown that propofol causes a concentration- and time-dependent reversible neurite retraction. This retraction is GABAAR-mediated and dependent on actin and myosin II. Furthermore, the concentrations and times to full retraction and recovery correspond to the anaesthetic effects of propofol.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
Acknowledgements
 References
 
This study was financially supported by the Östergötland County Council and Linköping Society of Medicine, Sweden.


   Acknowledgements
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 Acknowledgements
References
 
The authors wish to thank Sievert Lindström for advice and Peter Cox for linguistic help.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 Acknowledgements
 References
 
1 Franks NP. Molecular targets underlying general anaesthesia. Br J Pharmacol (2006) 147:72–81.[CrossRef]

2 Luo L, O'Leary DDM. Axon retraction and degeneration in development and disease. Annu Rev Neurosci (2005) 28:127–56.[CrossRef][Web of Science][Medline]

3 von der Ohe CG, Darian-Smith C, Garner CC, Heller HC. Ubiquitous and temperature-dependent neural plasticity in hibernators. J Neurosci (2006) 26:10590–8.[Abstract/Free Full Text]

4 Houle JD, Tessler A. Repair of chronic spinal cord injury. Exp Neurol (2003) 182:247–60.[CrossRef][Web of Science][Medline]

5 Gallo G. RhoA-kinase coordinates F-actin organization and myosin II activity during semaphoring-3A-induced axon retraction. J Cell Sci (2006) 119:3413–23.[Abstract/Free Full Text]

6 Ahmad FJ, Hughey J, Wittmann T, Hyman A, Greaser M, Baas PW. Motor proteins regulate force interactions between microtubules and microfilaments in the axon. Nat Cell Biol (2000) 2:276–80.[CrossRef][Web of Science][Medline]

7 Richards CD. Anaesthetic modulation of synaptic transmission in the mammalian CNS. Br J Anaesth (2002) 89:79–90.[Abstract/Free Full Text]

8 Kitamura A, Marszalec W, Yeh JZ, Narahashi T. Effects of halothane and propofol on excitatory and inhibitory synaptic transmission in rat cortical neurons. J Pharmacol Exp Ther (2003) 304:162–71.[Abstract/Free Full Text]

9 Al-Jahdari WS, Saito S, Nakano T, Goto F. Propofol induces growth cone collapse and neurite retractions in chick explant culture. Can J Anaesth (2006) 53:1078–85.[Web of Science][Medline]

10 Jensen AG, Lindroth M, Sjölander A, Eintrei C. Propofol induces changes in the cytosolic free calcium concentrations and the cytoskeletal organisation of cultured human glial cells and primary embryonic rat brain cells. Anesthesiology (1994) 81:1220–9.[CrossRef][Web of Science][Medline]

11 Hansson E, Rönnbäck L. Primary cultures of astroglia and neurons from different brain regions. In: A Dissection and Tissue Culture Manual of Nervous System—Shahar A, de Vellis J, Vernadakis A, Haber B, eds. (1989) New York: Alan R. Liss. 92–104.

12 Sjölander A, Grönroos E, Hammarström S, Andersson T. Leukotriene D4 and E4 induce transmembrane signalling in human epithelial cells: single cell analysis reveals diverse pathways at the G-protein level for the influx and the intracellular mobilization of Ca2+. J Biol Chem (1990) 265:20976–81.[Abstract/Free Full Text]

13 Sewell JC, Sear JW. Can molecular similarity-activity models for intravenous general anaesthetics help explain their mechanism of action? Br J Anaesth (2002) 88:166–74.[Abstract/Free Full Text]

14 Straight AF, Cheung A, Limouze J, et al. Disecting temporal and spatial control of cytokinesis with a myosin II inhibitor. Science (2003) 299:1743–7.[Abstract/Free Full Text]

15 Billuart P, Winter CG, Maresh A, Zhao X, Luo L. Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway. Cell (2001) 107:195–207.[CrossRef][Web of Science][Medline]

16 Luo L. Rho GTPases in neuronal morphogenesis. Nat Rev Neurosci (2000) 1:173–80.[CrossRef][Web of Science][Medline]

17 Fukushima N. LPA in neural cell development. J Cell Biochem (2004) 92:993–1003.[CrossRef][Web of Science][Medline]

18 Fukushima N, Weiner JA, Chun J. Lysophosphatidic acid (LPA) is a novel extracellular regulator of cortical neuroblast morphology. Dev Biol (2000) 228:6–18.[CrossRef][Web of Science][Medline]

19 Björnström K, Eintrei C. The difference between sleep and anaesthesia is in the intracellular signal. Propofol and GABA use different subtypes of the GABAA receptor β subunit and vary in their interaction with actin. Acta Anaesthesiol Scand (2003) 47:157–64.[CrossRef][Web of Science][Medline]

20 von der Ohe CG, Garner CC, Darian-Smith C, Heller HC. Synaptic protein dynamics in hibernation. J Neurosci (2007) 27:84–92.[Abstract/Free Full Text]

21 Magarinos AM, McEwen BS, Saboureau M, Pevet P. Rapid and reversible changes in interhippocampal connectivity during the course of hibernation in European hamsters. Proc Natl Acad Sci USA (2006) 103:18775–880.[Abstract/Free Full Text]

22 Antkowiak B. Different actions of general anesthetics on the firing patterns of neocortical neurons mediated by the GABAA receptor. Anesthesiology (1999) 91:500–11.[CrossRef][Web of Science][Medline]


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