Exposure to anesthesia causes lipid clusters to move from an ordered state to a disordered one then back again

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Surgery would be inconceivable without general anesthesia, so it may come as a surprise that despite its 175-year history of medical use, doctors and scientists have been unable to explain how anesthetics temporarily render patients unconscious.

A new study from Scripps Research published Thursday evening in the Proceedings of the National Academies of Sciences (PNAS) solves this longstanding medical mystery. Using modern nanoscale microscopic techniques, plus clever experiments in living cells and fruit flies, the scientists show how clusters of lipids in the cell membrane serve as a missing go-between in a two-part mechanism.

Temporary exposure to anesthesia causes the lipid clusters to move from an ordered state, to a disordered one, and then back again, leading to a multitude of subsequent effects that ultimately cause changes in consciousness.

The discovery by chemist Richard Lerner, MD, and molecular biologist Scott Hansen, PhD, settles a century-old scientific debate, one that still simmers today: Do anesthetics act directly on cell-membrane gates called ion channels, or do they somehow act on the membrane to signal cell changes in a new and unexpected way?

It has taken nearly five years of experiments, calls, debates and challenges to arrive at the conclusion that it’s a two-step process that begins in the membrane, the duo say.

The anesthetics perturb ordered lipid clusters within the cell membrane known as “lipid rafts” to initiate the signal.

“We think there is little doubt that this novel pathway is being used for other brain functions beyond consciousness, enabling us to now chip away at additional mysteries of the brain,” Lerner says.

Lerner, a member of the National Academy of Sciences, is a former president of Scripps Research, and the founder of Scripps Research’s Jupiter, Florida campus. Hansen is an associate professor, in his first posting, at that same campus.

The Ether Dome

Ether’s ability to induce loss of consciousness was first demonstrated on a tumor patient at Massachusetts General Hospital in Boston in 1846, within a surgical theater that later became known as “the Ether Dome.”

So consequential was the procedure that it was captured in a famous painting, “First Operation Under Ether,” by Robert C. Hinckley. By 1899, German pharmacologist Hans Horst Meyer, and then in 1901 British biologist Charles Ernest Overton, sagely concluded that lipid solubility dictated the potency of such anesthetics.

Hansen recalls turning to a Google search while drafting a grant submission to investigate further that historic question, thinking he couldn’t be the only one convinced of membrane lipid rafts’ role.

To Hansen’s delight, he found a figure from Lerner’s 1997 PNAS paper, “A hypothesis about the endogenous analogue of general anesthesia,” that proposed just such a mechanism.

Hansen had long looked up to Lerner–literally. As a predoctoral student in San Diego, Hansen says he worked in a basement lab with a window that looked directly out at Lerner’s parking space at Scripps Research.

“I contacted him, and I said, ‘You are never going to believe this. Your 1997 figure was intuitively describing what I am seeing in our data right now,’” Hansen recalls. “It was brilliant.”

For Lerner, it was an exciting moment as well.

“This is the granddaddy of medical mysteries,” Lerner says. “When I was in medical school at Stanford, this was the one problem I wanted to solve.

Anesthesia was of such practical importance I couldn’t believe we didn’t know how all of these anesthetics could cause people to lose consciousness.”

Many other scientists, through a century of experimentation, had sought the same answers, but they lacked several key elements, Hansen says: First, microscopes able to visualize biological complexes smaller than the diffraction limits of light, and second, recent insights about the nature of cell membranes, and the complex organization and function of the rich variety of lipid complexes that comprise them.

“They had been looking in a whole sea of lipids, and the signal got washed out, they just didn’t see it, in large part for a lack of technology,” Hansen says.

From order to disorder

Using Nobel Prize-winning microscopic technology, specifically a microscope called dSTORM, short for “direct stochastical optical reconstruction microscopy,” a post-doctoral researcher in the Hansen lab bathed cells in chloroform and watched something like the opening break shot of a game of billiards.

Exposing the cells to chloroform strongly increased the diameter and area of cell membrane lipid clusters called GM1, Hansen explains.

What he was looking at was a shift in the GM1 cluster’s organization, a shift from a tightly packed ball to a disrupted mess, Hansen says. As it grew disordered, GM1 spilled its contents, among them, an enzyme called phospholipase D2 (PLD2).

Tagging PLD2 with a fluorescent chemical, Hansen was able to watch via the dSTORM microscope as PLD2 moved like a billiard ball away from its GM1 home and over to a different, less-preferred lipid cluster called PIP2.

This activated key molecules within PIP2 clusters, among them, TREK1 potassium ion channels and their lipid activator, phosphatidic acid (PA). The activation of TREK1 basically freezes neurons’ ability to fire, and thus leads to loss of consciousness, Hansen says.

This is a slide from the study
An ordered cholesterol cluster in a cell membrane briefly becomes disordered on exposure to chloroform. Image is credited to Hansen lab, Scripps Research.

“The TREK1 potassium channels release potassium, and that hyper-polarizes the nerve–it makes it more difficult to fire–and just shuts it down,” Hansen says.

Lerner insisted they validate the findings in a living animal model. The common fruit fly, drosophila melanogaster, provided that data. Deleting PLD expression in the flies rendered them resistant to the effects of sedation. In fact, they required double the exposure to the anesthetic to demonstrate the same response.

“All flies eventually lost consciousness, suggesting PLD helps set a threshold, but is not the only pathway controlling anesthetic sensitivity,” they write.

Hansen and Lerner say the discoveries raise a host of tantalizing new possibilities that may explain other mysteries of the brain, including the molecular events that lead us to fall asleep.

Lerner’s original 1997 hypothesis of the role of “lipid matrices” in signaling arose from his inquiries into the biochemistry of sleep, and his discovery of a soporific lipid he called oleamide. Hansen and Lerner’s collaboration in this arena continues.

“We think this is fundamental and foundational, but there is a lot more work that needs to be done, and it needs to be done by a lot of people,” Hansen says. Lerner agrees.

“People will begin to study this for everything you can imagine: Sleep, consciousness, all those related disorders,” he says. “Ether was a gift that helps us understand the problem of consciousness. It has shined a light on a heretofore unrecognized pathway that the brain has clearly evolved to control higher-order functions.”


Clinical Significance

Intravenous Anesthetics

The first step in inducing general anesthesia in most surgical procedures is the administration of a hypnotic drug which is then followed by maintenance with inhalational anesthetic.

Patients better tolerate intravenous (IV) induction, but inhalational induction is often used in children or where IV access is problematic. All IV anesthetics produce rapid unconsciousness, and redistribution from the brain to muscle and adipose tissue leads to awakening. Barbiturates are lipid soluble, alkalotic compounds which produce deep unconsciousness. Providers should exercise caution using these agents if the airway is difficult to maintain as this can cause apnea.

Barbiturates also cause pronounced myocardial depression and vasodilation. Phenobarbital is a long-acting barbiturate which begins working within five minutes and its effects last 4 to 48 hours.

Etomidate is an IV anesthetic related to antifungal drug ketoconazole. Use of etomidate is usually limited to induction, and repeated doses or infusions should not be used. Pain and phlebitis are common side effects which can be reduced with prior IV injection of lidocaine.

Risk of nausea or vomiting makes etomidate a less ideal drug for use in an ambulatory setting. Propofol is a phenol agent with rapid onset and short duration of action and can be used for induction and maintenance of anesthesia.

Profound respiratory depression can be caused by an induction dose, similar to barbiturates. Propofol offers the advantage of effortless awakening with minimal residual sedation even with prolonged infusion.

Additionally, it has antiemetic properties making it popular for outpatient procedures. Ketamine is a dissociative anesthetic meaning it distorts perception of sight and sounds as well as producing feelings of detachment from environment and self.

Unique among IV anesthetics, ketamine produces intense analgesia. Important side effects of ketamine include increased secretions, the risk of laryngospasm and hallucinations. Dexmedetomidine is a selective alpha-2 receptor agonist with sedative, sympatholytic and analgesic properties. A

dvantages of dexmedetomidine include better patient tolerance, hemodynamic stability, and preservation of patent airway. These qualities make it a preferred agent for conscious fiberoptic intubation.

Inhalational Anesthetics

Inhalational anesthetics are liquids at ambient temperature and pressure. These liquids are transformed by vaporization into gas for rapid absorption in and elimination by the pulmonary circulation.

These medications are absorbed in alveoli, and the anesthetic concentration in the brain is directly related to alveolar concentration. Inhalational agents are commonly used for maintenance of anesthesia.

A key measure of these medications is the minimal alveolar concentration (MAC), which is the concentration that will prevent movement in 50% of patients in response to a painful stimulus like a surgical incision.

Importantly, nitrous oxide MAC is very high (104%) meaning it is unlikely to produce general anesthesia as a single agent. Nitrous oxide (NO) is an odorless nonhalogenated agent that can be combined with a halogenated anesthetic to hasten induction and emergence.

NO can support combustion especially if delivered with a high oxygen concentration, thus should be avoided in laser endoscopy. Halothane was a commonly used agent historically but has been replaced by other halogenated agents like sevoflurane, which offers smoother mask induction, quicker emergence, and less myocardial depression and arrhythmogenic potential than halothane.

Halothane also carries a risk of allergic hepatitis and malignant hyperthermia. Sevoflurane and desflurane are non-flammable, volatile halogenated agents which are completely fluorinated analogues of isoflurane. The fluorinated agents produce rapid awakening compared to isoflurane especially in obese patients following prolonged surgery.

Isoflurane which contains fluoride is not completely fluorinated. Desflurane notably can cause coughing or laryngospasm. Inhalational agents are especially advantageous for use in head and neck surgery as they decrease bronchoconstriction by relaxing the bronchial smooth muscle.

This provides controlled muscle relaxation without the use of neuromuscular blocking drugs, allowing monitoring of nerve function. Small concentrations of inhalational agents may severely depress ventilatory response to acute hypoxia so patients should be closely monitored during transport to the post-anesthetic care unit.

Intravenous Sedatives

Benzodiazepines are often used as premedication for general anesthesia or for anxiolysis in patients who are undergoing regional anesthesia. Midazolam (Versed) is the most commonly used preoperative sedative and can provide anxiolysis, sedation, and amnesia.

Diazepam (Valium) causes veno-irritation on injection in contrast to midazolam which is painless. Midazolam also offers quicker onset and shorter duration of action than lorazepam.

Lorazepam is a long-acting sedative hypnotic not commonly used for anesthesia. All benzodiazepines suppress the ventilatory response to hypercarbia. Therefore, providers must be careful in patients with COPD or respiratory insufficiency.

Synthetic Opioids

Synthetic opioids are particularly potent opioids, which restricts their routine use to the operating room where ventilatory support is readily available. As with other opioids, these drugs can cause meiosis, respiratory depression, bradycardia, constipation and urinary retention. Synthetic opioids include alfentanil, sufentanil, remifentanil, and fentanyl.

These medications produce rapid and intense analgesia. Fentanyl is one hundred times, and sufentanil one thousand times more potent than morphine.

Remifentanil is an expensive, ultrashort-acting opioid resulting in minimal “drug hangover” and no residual analgesia. These qualities can be beneficial in endoscopic procedures and neurosurgery; however, rapid tolerance can occur resulting in increased opioid requirements postoperatively. These agents can cause profound respiratory depression and chest wall rigidity.

Neuromuscular Blocking Drugs

Neuromuscular blocking drugs (NMBDs) act on the postsynaptic membrane of nicotinic cholinergic receptors. These can be subclassified into competitive (non-depolarizing) and noncompetitive (depolarizing).

Succinylcholine is a noncompetitive NMBD which binds strongly to the receptor site and mimics the effects of acetylcholine leading to fasciculations. It can cause prolonged paralysis or bradycardia if used as intermittent bolus or infusion. It carries the highest risk of malignant hyperthermia of all NMBDs.

It should only be used in pediatrics with a clear indication as it can cause rhabdomyolysis, hyperkalemia and cardiac arrest in patients with undiagnosed myopathy. Succinylcholine reaches a maximum block in less than one minute and has a short duration of action (less than 10 minutes).

This makes succinylcholine a commonly used agent in rapid sequence intubation. Competitive NMBDs bind loosely with nicotinic cholinergic receptors and compete with acetylcholine. These drugs include the following: atracurium, cisatracurium, pancuronium, vecuronium, and rocuronium.

The maximum block is reached in 1 to 3 minutes with, and duration of action is greater than 40 minutes with each of these medications. [Oh SK, Kwon WK, Park S, Ji SG, Kim JH, Park YK, Lee SY, Lim BG. Comparison of Operating Conditions, Postoperative Pain and Recovery, and Overall Satisfaction of Surgeons with Deep vs. No Neuromuscular Blockade for Spinal Surgery under General Anesthesia: A Prospective Randomized Controlled Trial. J Clin Med. 2019 Apr 12;8(4) ]


Source:
Scripps Research Institute
Media Contacts:
Stacey Singer DeLoye – Scripps Research Institute

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