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Propofol changes the cytoskeletal function in neurons: An experimental study in cortical cultures
Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Sinnescentrum, Department of Anaesthesiology and Surgery UHL. (Anestesiologi)
2012 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Every day, general anaesthetics are given to a large number of patients around the world but the cellular mechanisms of how anaesthetics act are still not clear. General anaesthetics cause the intended unconsciousness, amnesia and immobility in patients, but also side effects such as a decrease in mean arterial pressure and arrhythmia, both of which contribute to complications such as heart damage and stroke. With more knowledge of the mechanism of anaesthetic drugs, these complications could be reduced.

It has been shown that anaesthetics cause a disruption of the thalamocortical connectivity and brain network connectivity. How the network communication is disrupted however is not known. Propofol and thiopental are both intravenous anaesthetic drugs used widely in clinical anaesthesia. They bind to the GABAA receptor and enhance its function.

The cytoskeleton helps the cell to maintain its shape and participate in cellular movement and transport. Cellular transport to and from a neuron’s cell body and periphery is performed by motor proteins that move vesicles, organelles and proteins along cytoskeletal tracks. We have previously shown that propofol causes a reorganisation of the cytoskeleton protein actin in neurons, but we were further interested to study the effects of propofol and thiopental on the cytoskeletal function of cultured cortical rat neurons.

Our results show that propofol and thiopental cause neurite (axon and dendrite) retraction. Propofol’s effects were time- and dose-dependent, and can be reversed when propofol is removed. We were able to inhibit propofolinduced neurite retraction if we stabilised actin by blocking either the motor protein myosin II or the GABAA receptor. We have previously shown that a small GTP-binding protein, RhoA, inhibits propofol-caused actin reorganisation. Propofol-induced neurite retraction was mediated via a downstream effector of RhoA, ROK, which induces phosphorylation of the myosin light chain and increases contractility. Furthermore, we have shown that propofol causes a switch from anterograde to retrograde transport and increases the average velocity of the moving vesicles in neurites. The propofol induced retrograde vesicle transport was GABAA receptor-mediated.

Orexin A is a neuropeptide which regulates the sleep/awake cycle and has also been shown to reduce anaesthesia in animals when given intracerebroventricularly. We found that orexin A reverses propofol and thiopental-induced neurite retraction and actin reorganisation. Moreover, we have shown that the orexin A inhibition of propofol-induced neurite retraction is mediated via the PLD/PKC intracellular signalling pathway. Propofol and thiopental decreased the tyrosine phosphorilation of the intermediate cytoskeletal protein vimentin which is reversed by orexin A.

Taken together, these results suggest that propofol causes a time- and dose-dependent, reversible and GABAAreceptor-mediated neurite retraction in cultured cortical rat neurons. Propofol also causes a switch from anterograde to retrograde vesicle transport in neurites. Orexin A reverses propofol and thiopental-induced neurite retraction and cytoskeletal reorganisation. Orexin A inhibits propofol-induced neurite retraction via the PLD/PKC intracellular signalling pathway.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2012. , 64 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1305
Keyword [en]
Propofol, cytoskeleton
National Category
Medical and Health Sciences Basic Medicine Neurosciences
Identifiers
URN: urn:nbn:se:liu:diva-77219ISBN: 978-91-7519-910-8 (print)OAI: oai:DiVA.org:liu-77219DiVA: diva2:525640
Public defence
2012-06-08, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (Swedish)
Opponent
Supervisors
Available from: 2012-06-04 Created: 2012-05-08 Last updated: 2012-06-04Bibliographically approved
List of papers
1. Propofol causes neurite retraction in neurons
Open this publication in new window or tab >>Propofol causes neurite retraction in neurons
Show others...
2008 (English)In: British Journal of Anaesthesia, ISSN 0007-0912, E-ISSN 1471-6771, Vol. 101, no 3, 374-379 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
Oxford, UK: Oxford University Press, 2008
Keyword
Anaesthetics i.v., propofol; brain, GABA rat; theories of anaesthetic action, cellular mechanisms
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-78010 (URN)10.1093/bja/aen185 (DOI)000259093300014 ()
Available from: 2012-06-04 Created: 2012-06-04 Last updated: 2017-12-07Bibliographically approved
2. Propofol alters vesicular transport in rat cortical neuronalcultures
Open this publication in new window or tab >>Propofol alters vesicular transport in rat cortical neuronalcultures
2011 (English)In: Journal of Physiology and Pharmacology, ISSN 0867-5910, E-ISSN 1899-1505, Vol. 62, no 1, 119-124 p.Article in journal (Refereed) Published
Abstract [en]

Neuronal intracellular transport is performed by motor proteins, which deliver vesicles, organelles and proteins along cytoskeletal tracks inside the neuron. We have previously shown that the anesthetic propofol causes dose- and time-dependent, reversible retraction of neuronal neurites. We hypothesize that propofol alters the vesicular transport of cortical neurons due to this neurite retraction. Primary cultures of co-cultivated rat cortical neurons and glial cells were exposed to either 2 mu M propofol, control medium or the lipid vehicle, in time-response experiments. Reversibility was tested by washing propofol off the cells. The role of the GABA(A) receptor (GABA(A)R) was assessed with the GABA(A)R antagonist gabazine. Vesicles were tracked using differential interference contrast video microscopy. Propofol caused a retrograde movement in 83.4 +/- 5.2% (mean +/- S.E.M.) of vesicles, which accelerated over the observed time course (0.025 +/- 0.012 mu m.s(-1)). In control medium, vesicles moved predominantly anterograde (84.6 +/- 11.1%) with lower velocity (0.011 +/- 0.004 mu m.s(-1)). Cells exposed to the lipid vehicle showed the same dynamic characteristics as cells in control medium. The propofol-induced effect on vesicle transport was reversible and blocked by the GABA(A)R antagonist gabazine in low concentration. Our results show that propofol causes a reversible, accelerating vesicle movement toward the neuronal cell body that is mediated via synaptic GABA(A)R. We have previously reported that propofol initiates neurite retraction, and we propose that propofol causes vesicle movement by retrograde flow of cytoplasm from the narrowed neurite.

Place, publisher, year, edition, pages
Polish Physiological Society, 2011
Keyword
anesthetics intravenously, brain, cellular mechanism, cerebral cortex, neurotransmission effects, pharmacology, propofol, theories of anesthetic action, vesicular transport
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-67967 (URN)000289542300014 ()
Available from: 2011-05-04 Created: 2011-05-04 Last updated: 2017-12-11Bibliographically approved
3. Orexin A reverses propofol and thiopental induced cytoskeletal rearrangement in primary cortical neuronal culture
Open this publication in new window or tab >>Orexin A reverses propofol and thiopental induced cytoskeletal rearrangement in primary cortical neuronal culture
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Orexin A (OA) is an endogenous peptide regulating awakeness. It is a potential reversing agent of anaesthetics, shown to reduce anaesthesia in animals, but on cellular level its mechanisms are unknown.

Methods: Primary cortical cell cultures from newborn rat brains are used, and live cell light microscopy is performed to measure 1) neurite retraction after propofol, thiopental, barbituric acid and ketamine exposure and 2) the effect of OA application either before or after anaesthetics. Cytoskeletal reorganization of vimentin and actin is evaluated with fluorescence microscopy, protein changes detected with Western blot and proteins identified with mass spectrometry after treatment with anaesthetics and/or OA.

Results: Orexin A reverses and inhibits neurite retraction and the actin ring formation induced by propofol and thiopental. No effect on retraction or actin rings was seen for ketamine (not active on GABAA receptors), the non-anaesthetic barbituric acid, OA or solvents used. OA increases tyrosine phosphorylation of a 50 kDa protein, identified as vimentin. Propofol treatment induces a granular appearance of vimentin, which OA reverses to a smooth distribution throughout the cell.

Conclusions: OA reverses cellular effects known to be mediated via the GABAA receptor of both propofol and thiopental in cultured rat brain cells. The morphologic changes of actin and vimentin caused by propofol and thiopental, and the subsequent reversal by OA, deepens our understanding of the mechanisms of anaesthesia. In the future, an OA agonist could be used to reverse the effects of GABAA receptor dependent anaesthetic drugs.

Keyword
orexin A, propofol, thiopental
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-77167 (URN)
Available from: 2012-05-07 Created: 2012-05-07 Last updated: 2012-06-04Bibliographically approved
4. Orexin A inhibits propofol-induced neurite retraction by a PLD-dependent mechanism in neurons
Open this publication in new window or tab >>Orexin A inhibits propofol-induced neurite retraction by a PLD-dependent mechanism in neurons
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: Propofol retracts neurites and reverses the transport of vesicles in rat cortical neurons in a γ-aminobutyric acid type A (GABAA) receptor dependent manner. Orexin A (OA) is an endogenous peptide regulating wakefulness, and is known to interact with anaesthetics. We aim to investigate whether OA inhibits propofol-induced neurite retraction and elucidate the intracellular signalling involved.

Methods: In primary cortical cell cultures from newborn rat brains, live cell light microscopy was used to measure neurite retraction after propofol (2 μM) with or without OA (10 nM) application after preincubation with the Rhokinase inhibitor (HA-1077), phospholipase D (PLD) inhibitor [5-fluoro-2- indolyl des-chlorohalopemide (FIPI)], protein kinase C (PKC) inhibitor (staurosporine) or PKC activator phorbol 12-myristate 13-acetate (PMA).

Results: The neurite retraction induced by propofol is blocked by HA-1077 and PMA. OA blocks neurite retraction induced by propofol, and this inhibitory effect could be prevented by FIPI, as well as staurosporine.

Conclusions: Rho-kinase is essential for propofol-induced neurite retraction in cortical neuronal cells. Activation of PKC plays an inhibitive role during neurite retraction caused by propofol. OA blocks propofol-induced neurite retraction by a PLD/PKC-mediated pathway.

Keyword
Orexin A, propofol, PLD
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-77168 (URN)
Available from: 2012-05-07 Created: 2012-05-07 Last updated: 2012-06-04Bibliographically approved

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