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Orexin A reverses propofol and thiopental induced cytoskeletal rearrangement in primary cortical neuronal culture
Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. (Anestesiologi)
Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences. (Anestesiologi)
Linköping University, Department of Medical and Health Sciences, Anesthesiology. Linköping University, Faculty of Health Sciences.
(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 [en]
orexin A, propofol, thiopental
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-77167OAI: oai:DiVA.org:liu-77167DiVA: diva2:525266
Available from: 2012-05-07 Created: 2012-05-07 Last updated: 2012-06-04Bibliographically approved
In thesis
1. Propofol changes the cytoskeletal function in neurons: An experimental study in cortical cultures
Open this publication in new window or tab >>Propofol changes the cytoskeletal function in neurons: An experimental study in cortical cultures
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
Propofol, cytoskeleton
National Category
Medical and Health Sciences Basic Medicine Neurosciences
Identifiers
urn:nbn:se:liu:diva-77219 (URN)978-91-7519-910-8 (ISBN)
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

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Turina, DeanGlavas, AlenkaBjörnström, Karin

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