Anesthetics interfere with the polarization of developing cortical neurons

Abstract

Numerous studies from the clinical and preclinical literature indicate that general anesthetic agents have toxic effects on the developing brain, but the mechanism of this toxicity is still unknown. Previous studies have focused on the effects of anesthetics on cell survival, dendrite elaboration, and synapse formation, but little attention has been paid to possible effects of anesthetics on the developing axon. Using dissociated mouse cortical neurons in culture, we found that isoflurane delays the acquisition of neuronal polarity by interfering with axon specification. The magnitude of this effect is dependent on isoflurane concentration and exposure time over clinically relevant ranges, and it is neither a precursor to nor the result of neuronal cell death. Propofol also seems to interfere with the acquisition of neuronal polarity, but the mechanism does not require activity at GABAA receptors. Rather, the delay in axon specification likely results from a slowing of the extension of prepolarized neurites. The effect is not unique to isoflurane as propofol also seems to interfere with the acquisition of neuronal polarity. These findings demonstrate that anesthetics may interfere with brain development through effects on axon growth and specification, thus introducing a new potential target in the search for mechanisms of pediatric anesthetic neurotoxicity.

Figure 1. Isoflurane inhibits axon specification

A: On DIV1 under control conditions dissociated cortical neurons, visualized here with immunolabeling for βIII tubulin, show a mixed population of polarized (arrowheads) and unpolarized neurons (cell bodies without arrowheads), as assessed by the Banker length criteria. B: Cultures treated for four hours with 2.4% isoflurane immediately after plating and assayed at DIV1 demonstrate a reduced percentage of polarized neurons (arrowhead). C: Quantitative analysis shows a significant reduction in the percentage of polarized neurons relative to control that is concentration-dependent over a range from 1.2% to 3% isoflurane for four hours. D: The magnitude of this effect also varies with exposure time, and there is a significant reduction in neuronal polarity with 2.4% isoflurane treatment in exposures lasting four hours or longer. E–G: At DIV2 neurons immunolabeled for the axon-specific protein L1CAM (E, green in G) and stained for F-actin to define the cell (F, red in G), show a segregation of L1CAM signal to the axon (arrowhead). H–J: By contrast, neurons treated with isoflurane at a one millimolar for six hours and labeled for L1CAM (H, green in J) and F-actin (I, red in J) show diffuse labeling for that is seen in the axon (arrowhead) and also in minor processes (arrows). Scale bar in A is 20 μm for A and B. n = 2151 cells in 210 fields for C and n = 1,523 cells in 150 fieldsfor D. Scale bar in E is 10 μm for E–J. * = p<0.05.

Figure 2. Isoflurane delays the acquisition of neuronal polarity without causing cell death

A: Neuronal polarity was assayed at DIV1, DIV2, and DIV3 after a four hour treatment with 2.4% isoflurane at the time of plating. The mean percent of polarized neurons is markedly reduced on DIV1, somewhat reduced on DIV2, and not significantly different on DIV3, when nearly all neurons have polarized. B: The number of neurons per microscopic field does not differ significantly between control and isoflurane treated cultures on DIV1, DIV2, or DIV3. C: An MTT assay for cell viability shows no significant reduction in absorbance for isoflurane treated cultures over the same time frame, confirming that cell death does not occur with this isoflurane exposure paradigm. n = 1,682 cells in 180 fields for A and B. n = 50 samples for C. * = p<0.05, ns = not significant.

Figure 3. Propofol inhibits the acquisition of neuronal polarity

A: Cortical neuron cultures were treated with 3 μM propofol for four hours immediately after plating and assayed for neuronal polarity on DIV1. Similarly to isoflurane, propofol significantly reduces the mean percentage of polarized neurons. B: The GABA_AR agonist muscimol does not alter mean percentage of polarized neurons in the same exposure paradigm, suggesting that the effects of isoflurane and propofol on axon specification do not occur via activity at GABA_ARs. n = 666 cells in 61 fields for A and n = 527 cells in 60 fields for B. * = p<0.05

Figure 4. Isoflurane reduces outgrowth of pre-polarized neurites

A, B: Neurolucida tracings were made of unpolarized neurons from control (A) and 2.4% isoflurane treated cultures (B) at DIV1. The isoflurane treated group has similar numbers of neurites, but the lengths appear shorter on average. C: Quantitiatve analysis shows that neurite length is reduced, indicating that the delay in acquisition of neuronal polarity seen with isoflurane treatment is due to a reduction in neurite outgrowth speed. Scale bar in A is 25 μm for A and B. n = 128 neurons and 42 neurons. * = p<0.05.