The genetics of isoflurane-induced developmental neurotoxicity

Abstract

Introduction: Neurotoxicity induced by early developmental exposure to volatile anesthetics is a characteristic of organisms across a wide range of species, extending from the nematode C. elegans to mammals. Prevention of anesthetic-induced neurotoxicity (AIN) will rely upon an understanding of its underlying mechanisms. However, no forward genetic screens have been undertaken to identify the critical pathways affected in AIN. By characterizing such pathways, we may identify mechanisms to eliminate isoflurane induced AIN in mammals.

Methods: Chemotaxis in adult C. elegans after larval exposure to isoflurane was used to measure AIN. We initially compared changes in chemotaxis indices between classical mutants known to affect nervous system development adding mutants in response to data. Activation of specific genes was visualized using fluorescent markers. Animals were then treated with rapamycin or preconditioned with isoflurane to test effects on AIN.

Results: Forty-four mutations, as well as pharmacologic manipulations, identified two pathways, highly conserved from invertebrates to humans, that regulate AIN in C. elegans. Activation of one stress-protective pathway (DAF-2 dependent) eliminates AIN, while activation of a second stress-responsive pathway (endoplasmic reticulum (ER) associated stress) causes AIN. Pharmacologic inhibition of the mechanistic Target of Rapamycin (mTOR) blocks ER-stress and AIN. Preconditioning with isoflurane prior to larval exposure also inhibited AIN.

Discussion: Our data are best explained by a model in which isoflurane acutely inhibits mitochondrial function causing activation of responses that ultimately lead to ER-stress. The neurotoxic effect of isoflurane can be completely prevented by manipulations at multiple points in the pathways that control this response. Endogenous signaling pathways can be recruited to protect organisms from the neurotoxic effects of isoflurane.

Keywords: Anesthetic; C. elegans; Genetics; Isoflurane.

Figure 1

A,C,E,G,I. Chemotaxis indices (CIs) in adults after exposure to isoflurane as L1 larvae. Unexposed animals (solid fill), exposed animals (angled hatching). For all graphs, error bars denote SEM values, N>300 animals for each value. B,D,F,H,J Differences in CIs (ΔCI) between exposed and unexposed animals. Difference between ΔCI of N2 (19.5 +/− 3.7) and each mutant was compared to determine if the mutant affected AIN. ** = ΔCI different from N2, p<0.01, ***= ΔCI different from N2, p<0.005. A,B. Mitochondrial mutants. Chemotaxis in unexposed mitochondrial mutants (gas-1, nuo-6, mev-1, isp-1) was not worsened by isoflurane exposure. The exception was clk-1 which had a ΔCI similar to that of N2. C,D. ROS scavengers/ ER Stress. The effects of defects in ROS scavenging on AIN in C. elegans. CIs of five superoxide disumutatse mutants and hsp-4 (loss of the ER-specific heat shock protein HSP-4). E,F. DAF-2 dependent pathway. The effects of the daf-2 stress pathway on AIN. Loss of DAF-2 removed the AIN effect. The daf-16 mutation removed the effect of daf-2 on AIN. G,H. Kinases. Effects of 5 kinases on neurotoxicity. Loss of cmk-1 and gcn-2, both involved in innate immunity and ER-related stress, eliminated AIN. Loss of ire-1 also eliminated AIN and is discussed later. I,J. Transcription factors. The transcription factors skn-1, hif-1 and xpb-1 all eliminated AIN.

Figure 2. Expression of an DAF-16::GFP fusion protein following exposure to isoflurane

A. A GFP fusion protein with DAF-16 is distributed throughout the cytoplasm when not exposed to isoflurane. B. One hour after exposure to isoflurane, the DAF-16::GFP fusion protein is translocated to the nucleus. These results show that isoflurane exposure leads to nuclear localization of DAF-16 similar to mutations in either DAF-2 or GAS-1 [28, 29].

Figure 3. Expression of an HSP-4 reporter following exposure to isoflurane

The ER specific heat shock protein HSP-4 reporter is upregulated by isoflurane exposure. A. HSP-4::GFP expression at t=0 hours (after removal from anesthetic chamber) without isoflurane exposure. B. HSP-4::GFP expression at t=2 hours (after removal from anesthetic chamber) without isoflurane exposure. C. HSP-4::GFP expression at t=0 (after removal from anesthetic chamber) after isoflurane exposure. D. HSP-4::GFP expression at t=2 hours (after removal from anesthetic chamber) with isoflurane exposure. Expression peaked at 2–3 hours and then slowly decayed (not shown). The reporters for mitochondrial UPR heat shock proteins (HSP-6 and HSP-60) were not upregulated by isoflurane exposure.

Figure 4. Phsp-4::gfp expression in L1 larvae with or without concurrent treatment with rapamycin and isoflurane

A. Phsp-4::gfp expression at t=0 hours (after removal from anesthetic chamber) without isoflurane exposure. B. Phsp-4::gfp expression at t=2 hours (after removal from anesthetic chamber) with isoflurane exposure. C. Phsp-4::gfp expression at t=0 hours (after removal from anesthetic chamber) without isoflurane exposure with rapamycin treatment. D. Phsp-4::gfp expression at t=2 hours (after removal from anesthetic chamber) after 6.5% isoflurane exposure with rapamycin exposure. All plates had 0.2% DMSO on plates to facilitate uptake of rapamycin by the animals. The presence of DMSO mildly increased Phsp-4::gfp expression in the absence of isoflurane. E. Chemotaxis indices (CIs) in adults after exposure to isoflurane as L1 larvae during concurrent treatment with rapamycin. Unexposed animals (dark fill), exposed animals (light fill) and delta values (Δ) for control (left) and rapamycin (right) groups are shown. For all graphs, error bars denote SEM values, N>800 animals for each value. These results show that concurrent treatment with rapamycin completely inhibits AIN in C. elegans.

Figure 5

A. Protocol timeline used to test for neurotoxicity and preconditioning in C. elegans. First stage larva (L1) are first exposed to isoflurane at their EC₉₅ at age zero hours (arrow) and then again at four to eight hours (blue bar). They are then allowed to grow to adulthood (3 days for wildtype, D3). Chemotaxis is then tested as described in the methods and in Figure 1. B. Chemotaxis Index showing the neurotoxic effect in wildtype (N2) C. elegans following isoflurane exposure (black and gray bars). Preconditioning eliminated the neurotoxic effect caused by isoflurane (cross hatched bar). p value compares the difference between preconditioned animals and non-preconditioned animals exposed to isoflurane (cross hatched and gray bars). Error bars are SEM.

Figure 6

Two intersecting pathways that affect AIN in C. elegans. The grouping on the left of the figure (blue ellipses) represents the DAF-2 pathway which, when induced, is capable of inhibiting AIN. The genes on the right (red ellipses) are activated by ROS, leading to ER-stress, apoptosis and causing AIN. The mitochondrial/ROS isoflurane target is at the bottom in green. The pathways are expanded in the upper inserts.