Investigating the Fundamental Physics of Anesthetic Action

Abstract

Purpose: The fundamental purpose of this thesis is to establish methods for culturing, anesthetizing, recording, and analyzing the behavior of C. elegan nematodes to quantitatively characterize their response to anesthesia in terms of motion inhibition. Anesthesia has been a staple of effective medicine for nearly 200 years, with a variety of agents being characterized and implemented for various procedures around the world. However, despite this successful and long withstanding history, the fundamental mechanisms of anesthetic action remain to be fully elucidated for any anesthetic drugs. While many hypotheses have come and gone, this overarching project intends to investigate a recent proposal involving quantum physics. It was discovered that Xenon’s anesthetic potency is dependent on nuclear spin, and involvement of an entangled radical electron pair may be able to explain this phenomenon. This hypothesis also suggests that influence from external magnetic fields could alter the potency of xenon and other agents with a similar action mechanism. Demonstrating a potency-altering impact of an external field on anesthesia would provide the first experimental evidence in support of this idea, and that demonstration is the goal of this interdisciplinary project. First, however, a foundation establishing the ability to effectively induce anesthesia and quantify results with a model suited for mechanistic investigation is necessary. The scope of this thesis involves identifying behavioral endpoints of our test subjects, C. Elegans, and characterizing their response to traditional anesthesia before deploying these tools on xenon and other agents. Methods: This thesis involves the development of the team, equipment, methods, and ground truth data to allow for future investigations of external magnetic field effects on anesthesia. Adult N2-strain (wild type) C. elegan nematodes were anesthetized in 5% isoflurane for two independent experiments, RP1and RP2, for 10 minutes and 1 hour respectively. In RP2 they were also exposed to 1-Phenoxy-2-Propanol for 3 minutes as a positive control. The C. elegans were perpetually maintained in agar petri dish cultures with an E. coli food source, and a cohort of young adults was age-synchronized before each experiment. An anesthesia delivery setup was built by connecting a SomnoSuite small animal anesthesia delivery device to a sealed acrylic chamber with an optical window centered on the lid. Plates exposed in the chamber were backlighted and imaged with a Fiber-Lite DC-950 backlight and Canon EOS250 digital camera to record movement before, during, and after anesthetic exposure. A 12V vibration motor was also adhered to the acrylic chamber to stimulate worm movement post-exposure. Videos were analyzed manually by counting body bend and motion reversal movements over designated time intervals. They were also analyzed automatically with the Tierpsy Tracker software, which outputs motion datapoints including speed, acceleration, and eigenworm values for individual elegans over time. Differences between treatment arms were analyzed, 95% confidence intervals were calculated with Welch’s t-tests, and contrast to noise ratios were calculated by dividing the inter-arm difference by the sum of each arm’s 95% confidence interval. All these methods were developed in a newly acquired laboratory. Results: The elegans’ motion was not successfully inhibited with the use of isoflurane as demonstrated with 4 endpoints: body bends, direction reversals, total movements, and average speed. A 10-minute exposure in manual analysis did not provide any statistically significant endpoints. For a 1-hour exposure and vibratory stimulus, however, we observed statistically significant reductions of 20.34 ± 6.29 body bends per minute (p < 0.0001 & CNR = 3.23), -1.41 ± 1.04 direction reversals per minute (p = 0.009 & CNR = 1.36), 9.05 ± 7.65 total movements per minute (p = 0.0218 & CNR = 1.18), and 0.26 ± 0.09 pixels per frame (p < 0.0001 & CNR = 2.83) in the isoflurane group compared to the control group. There were no differences between treatment arms after 1 hour of exposure in the absence of a vibration stimulus. After an additional hour post-exposure, however, we observed statistically significant reductions of 9.04 ± 4.09 body bends per minute (p < 0.0001 & CNR = 2.21), 2.79 ± 2.35 direction reversals per minute (p < 0.0001 & CNR = 1.19), 11.82 ± 7.57 total movements per minute (p < 0.0001 & CNR = 1.56), and 0.08 ± 0.06 pixels per frame (p < 0.0087 & CNR = 1.38) between the same groups. This demonstrated motion inhibition that was only apparent upon stimulus after 1 hour of exposure, but observable in natural motion after a delay period. of motion. 3 minutes of 1P2P produced the lowest number of body bends of any treatment arm and time point at 3.79 ± 4.10 and zero direction reversals. All endpoints demonstrated inter-arm consistency for the pre-exposure time point and inter-plate consistency (within arms) for all time points except for the eigenworm values, invalidating the posture analysis for comparison between treatment arms. Conclusion: The significant responses of these identified endpoints demonstrate the use of C. elegans as a viable subject for observing anesthetic effects and have sufficient CNRs to provide sensitivity to fractional changes in potency. While responses in RP2 were significant, CNR could likely be improved by extended exposure times as indicated by the effect delay and effectiveness of 1-Phenoxy-2-Propanol as a positive control. Each endpoint’s response to isoflurane was characterized by multiple time points, so that potency changes in follow-up experiments may be quantified in terms of well understood metrics. The successful identification and characterization of these endpoints, along with the experimental logistics resolved throughout the course of the project, marks the completion of necessary steps to initiate anesthetic studies of external magnetic fields. This will allow for potential answers to questions regarding the fundamental physics of anesthetic action, quantum consciousness, and quantum medicine. The work in this thesis successfully established a new collaboration between the departments of medical physics, anesthesia, and physics, established and optimized the methods and materials in a new lab space, demonstrated ability to quantify the desired observables in the C. elegan model, and established a clear direction forward to pursue investigation into the fundamental physics of anesthesia.