Brugia Malayi - Clark Science Center - Smith College

Isoflurane Impacts the Actin Cytoskeletal Signalling Pathway by Depleting
Cofilin Reserves in Neonatal Murine Cortical Tissue
Belinda Nhundu and Shimu Liu
General anesthetic toxicity is a major concern in obstetric, pediatric and geriatric medicine. In previous experiments carried in
the Hall laboratory, we tested the neurotoxicity of both inhalant and intravenous general anesthetics on murine cortical neurons
in primary dissociated culture. We discovered that the neonatal neurons were remarkably robust, suffering little degeneration at
clinical concentrations and exposure lengths (Campbell et al, 2011). 1
However, when exposed to clinical concentrations of isoflurane and ketamine, extensive neurite retraction was observed. This
result was supported by Turina et al. (2008) 2 who described how neurites of cultured rat cortical neurons retracted after exposure
to the intravenous anesthetic, propofol, resulting in a retraction bulbs and thin trailing neurite remnants.
In an effort to quantify this retraction, we then developed culturing techniques for cortical neurons from postnatal mice
(P0-P2) based on a culturing protocol described by Jana et al. (2007). This protocol largely removes glial cells resulting in a
sparse plating of isolated neurons We also utilized a largely homogeneous neuroblastoma cell line. Neurons were visualized on a
confocal microscope using DIC , and time-lapse imaging was performed to monitor the changes resulting from volatile anesthetic
exposures. General anesthetic solutions of 1, 2, and 5 MAC of sevo- and isoflurane were introduced (1 MAC, minimum alveolar
concentration ~ the clinical concentration for maintaining a plane of anesthesia). Immediately (~5 min) after exposure we
observed a decrease in overall cell motility and changes in cell morphology including thinning and shortening of neurites.
We hypothesized that a Rho-A signaling pathway may be responsible for the anesthetic-induced retraction process leading to
cytoskeletal rearrangement (whereby ↑ Rho-A activity ↑Rock ↑LIM Kinase-1à Phosphorylation of Cofilin). It has been
reported that it may lead to actin polymerization/depolymerization and hence cytoskeletal reorganization.
Using Western blot analysis we measured the levels of phosphorylated and unphosphorylated cofilin in isoflurane-treated
neuroblastoma. Samples of total protein were collected from differentiated neuroblastoma cell lines exposed to 2 MAC
isoflurane for ten minutes and four hours. Western blot results showed a decrease in both phosphorylated and unphosphorylated
cofilin. These results suggest that isoflurane has an impact on cell morphology and dynamics of immature brain cells through
perturbation of molecular events governing cytoskeltela formation. In October we will be presenting our work at the Society for
Neuroscience conference in New Orleans and continue this work as an honors project in the 2012-13 academic year. (Supported
by the National Institutes of Health and the Blakeslee Fund in the Biological Sciences)
Advisor: Adam Hall
References:
1
L.Campbell, et.al. Assessment of general anaesthetic cytotoxicity in murine cortical neurones in dissociated culture.Toxicology 283(2011), pp.1-7 .
2
D. Turina, V. M. Loitto, K. Björnström, T. Sundqvist, and C. Eintrei.Propofol causes neurite retraction in neurones Br. J. Anaesth. (2008) 101(3): 374-379.
first published online June 27, 2008 doi:10.1093/bja/aen185.
2012
151