Why some people wake up under anesthesia and others don’t (Hint: It’s your Hormones)

Dr. Andrzej (Andi) Wasilczuk was the lead author on this study. A recent graduate from Penn Bioengineering, Andi is captivated by the brain’s intricate ability to shift between states of consciousness. His research uses general anesthetics to uncover the neuronal circuits responsible for these shifts, aiming to understand how the brain sustains or disrupts consciousness. By identifying these critical networks, Andi is paving the way for personalized anesthesia and offering new insights into arousal state transitions.

or technically,

Hormonal basis of sex differences in anesthetic sensitivity

[See Original Abstract on Pubmed]

Authors of the study: Andrzej Z Wasilczuk, Cole Rinehart, Adeeti Aggarwal, Martha E Stone, George A Mashour, Michael S Avidan, Max B Kelz, Alex Proekt, ReCCognition Study Group

Waking up during surgery sounds like a nightmare, and did you know that females might be at higher risk for this than males? Through medication, general anesthesia makes a patient unconscious, which allows doctors to perform surgical procedures without the patient’s awareness or discomfort. General anesthesia puts the patient in a sleep-like state, and doing this is an involved process. Anesthesiologists must be highly trained to determine the best course of treatment. When creating a safe treatment plan, anesthesiologists take into account many factors, such as the patient’s body weight or pre-existing conditions. The sex of the patient, however, hasn’t been historically considered as an equally important factor in delivering a safe course of anesthesia.

Previous research about the link between sex and response to anesthesia was ambiguous and conflicting.  Some early clinical trials suggested that females were more likely to wake up under anesthesia, while others found no significant difference between males and females. These clinical trials, however, had diverse patient populations and non-standardized anesthetic protocols, which would make it hard to directly compare anesthetic conditions between patients. Nevertheless, more recently, an analysis done on many of these studies has provided clear cut evidence that females are more resistant to the anesthetic state (Braithwaite et al., 2023). The question of how this sex difference arose, however, remained unanswered.

Dr. Andi Wasilczuk, a former Penn Bioengineering PhD student, and his team wanted to understand why females and males responded differently to anesthesia. To do this, they decided to focus on the hypothalamus, a structure in the brain heavily involved in both sleep-wake and anesthetic induced unconsciousness. The hypothalamus is regulated by hormones, which are the body’s chemical messengers—and the researchers knew that the levels of hormones typically differ between males and females. For example, males typically have a much higher level of the hormone testosterone, whereas females typically have higher levels of the hormone estrogen. 

With these differences in mind, Dr. Wasilczuk wanted to know: Could hormonal differences across sexes alter the effectiveness of general anesthesia?  He framed "effectiveness of general anesthesia" by using the idea of  “anesthetic sensitivity.” Individuals  who are more sensitive to anesthesia need less of the drug to fall and stay unconscious, and wake up smoothly after surgery. On the contrary, individuals with less anesthetic sensitivity, or have anesthetic resistance, require more anesthetic to fall and stay asleep, and wake up sooner once the anesthetic is removed. 

Recognizing this gap in the research, Dr. Wasilczuk’s research group sought to test the influence of sex and sex hormones on anesthetic sensitivity in mice.  First, the researchers compared the dosage of anesthetic required for the mice to be initially anesthetized (induction), and to wake up from anesthesia (emergence). They found that, across all four anesthetics the group tested, female mice required a much higher dose on induction, and were more likely to emerge at higher doses than males. Next, the researchers compared the time, given the same dosage, for female versus male mice to be induced and emerge from anesthesia. Female mice took significantly longer to be induced than males, and also were much quicker to emerge. These experiments indicated that female mice were indeed more resistant to anesthesia, compared to male mice.

Yet, the reason for these results remained unclear: Were these effects due to sex hormone differences? To find out, the researchers changed the mice's hormone levels by surgical removal of the testicles (castration) in male mice or ovaries (oophorectomy) in female mice post puberty. They repeated the experiments, this time using castrated males and oophorectomy females, then compared these mice to the untreated males and females tested before.

The results were striking. In both experiments, castrated males and oophorectomized females showed a similar resistance to anesthesia as untreated females. Oophorectomy did not change a female mouse’s anesthetic sensitivity. Castration, however, produced a female-like anesthetic sensitivity in males. Eliminating male sex hormones, therefore, seemed to remove the sex differences in response to anesthesia!

The researchers also directly measured the effect of testosterone. Under a steady dose of anesthetic, untreated males and castrated males were injected with testosterone, and continually tested for responsiveness using the righting reflex. Testosterone administration increased anesthetic sensitivity for both groups of mice in a dose-dependent manner. This finding could explain why males, who typically have higher testosterone, are more sensitive to general anesthetics, and therefore are at lower risk of waking up under anesthesia than females.

Intrigued, the researchers wondered: Can these sex differences be seen in brain activity? The conventional measure of anesthetic depth (how unconscious someone is in response to anesthesia) during surgery is the Electroencephalogram (EEG). EEG measures electrical brain activity through electrodes attached to the scalp. The researchers found that sex differences were not reflected in the EEG of the mice they tested. Similar conclusions were made when re-analyzing human data from another study. In this study, female volunteers displayed resistance to general anesthesia based on assessments of behavior and cognitive function, but not based on information gathered from the EEG.

Looking at the activity of individual neurons, however, clearly revealed sex differences. They looked for elevated levels of the protein c-Fos, an indicator of neuronal activity, throughout the whole brain. Compared to anesthetized male mice, anesthetized female mice had fewer neurons expressing c-Fos in sleep-promoting hypothalamic cells. In other words, anesthesia activates fewer sleep-promoting circuits in females than males, correlating with females’ greater resistance to anesthetics. 

Compared to untreated male mice, castrated male mice also had reduced c-Fos expression in similar hypothalamic structures. Fewer sleep-promoting circuits were activated in castrated males (which displayed a similar aesthetic sensitivity to females) than untreated males. Thus, sex-dependent activity patterns, seen in hypothalamic structures, reflected anesthetic sensitivity trends!

Dr. Wasilczuk’s groundbreaking paper reveals why researching sex-dependence is incredibly important: females may need different anesthetic management than males due to their higher resistance to anesthesia. After years of standard general anesthesia administration to millions of patients, and using EEGs to measure anesthetic depth, Dr. Wasilczuk’s findings have huge clinical implications supporting personalized anesthetic care. 

About the brief writer: Sydney Liu

Sydney is a guest writer for Brains in Briefs! She is a Penn undergraduate in Dr. Shinjae Chung’s lab researching what makes us sleep and the brain transitions between sleep states. She is a Junior majoring in neuroscience, and is interested in teaching. In her free time, she likes to draw!

Citations:

1. E. Braithwaite et al., Impact of female sex on anaesthetic awareness, depth, and emergence: A systematic review and meta-analysis. Br. J. Anaesth. 131, 510–522 (2023).

Interested in learning more about how anesthetic sensitivity is different in males and females? Check out Andi’s paper here!

Keeping your brain's symphony in sync

Dr. Brenna Shortal was one of the two lead authors of this publication. Her graduate and undergraduate research focused on understanding the neurological mechanisms of consciousness, and she has published a number of papers on the topic. While she was a student at UPenn, Dr. Shortal was the director of Brains in Briefs, and her passion for science communication led her to pursue a career as a medical writer for Red Nucleus following her graduation in 2021. She hopes to continue working to communicate and advocate for scientific research to broad audiences.

or technically,

Weakly correlated local cortical state switches under anesthesia lead to strongly correlated global states

[See Original Abstract on Pubmed]

Authors of the study: Ethan B Blackwood, Brenna P Shortal, Alex Proekt

The most complicated piece of machinery you will ever encounter is sitting right between your ears: your brain. Our ability to move, sense, and think is thanks to billions of individual neurons that interact in varied and complicated ways. With this level of complexity, it’s miraculous that our brains work at all, let alone as well or as long as they do. Even more impressively, when our brain gets knocked off track, like from a seizure or anesthesia, it can quickly go back to typical patterns of activity. How does such a complex thing keep itself in sync?

Ethan Blackwood is a fifth-year neuroscience graduate student in the lab of Dr. Alex Proekt. Before coming to Penn and as a rotation student with Dr. Proekt, his research focused on how neural oscillations ("brain waves") change over time or with stimulation and what this means for behavior. More recently, he has been zooming in to the individual neuron level and studying how the firing of large groups of neurons changes during learning.

Neuroscience PhD student Ethan Blackwood and Drs. Brenna Shortal and Alex Proekt at the University of Pennsylvania sought to answer this question by studying brain activity in rats under anesthesia. Anesthesia is a useful way to study the coordination of brain activity because it is easy to put animals under anesthesia in the lab and because researchers already know a lot about the patterns of brain activity that occur when people are under anesthesia. The team studied this phenomenon in rats because they were able to directly record the activity of the neurons in the rat’s brain, something that is rarely possible in the human brain.  

The team had two ideas about how the brain keeps itself in sync. Their ideas are easiest to understand if we think of the brain as a symphony with your neurons as the musicians. Just like an orchestral piece comes together because the musicians move in sync from one part of the music to the next, so too do the groups of neurons in your brain. The researchers’ first idea about how the brain might keep its symphony together was that there is a conductor who dictates how all the groups of neurons behave. The second possibility was that there is no conductor, but nearby neurons listen to each other so that the whole orchestra stays together.

To distinguish between these two possibilities, the team recorded a kind of brain signal called a local field potential in two parts of the rat brain. They did this by placing electrodes in the rat’s brain and listening to the activity of nearby neurons. This is like listening to a few microphones placed in the cello and violin sections to understand how the whole orchestra works. Each microphone captures sound produced by several nearby musicians, but it can’t capture the whole orchestra’s sound.

The team started by identifying what musical melodies, which they call brain states, each electrode recorded and noting when the nearby neurons switched from one state to the next. By doing this for all the electrodes, they showed that there were only a small number of brain states that the neurons played, and the same states appeared in different rats. The relatively small number of brain states they found is something other neuroscientists have observed, and it’s key to how the brain keeps itself in sync. If every musician in the orchestra played their own tune, it would be hard to make sense of what was going on. However, by moving through different sections of the same piece of music in sync, the instruments create a beautiful piece of music together. The same is true of your brain’s symphony. Rather than coordinating billions of songs, each sung by different neurons, your brain’s symphony sings just a few, transitioning between a small number of brain states over time.

Now that they had their brain recordings, the team could see which of their two proposals about how the neural symphony stays in sync was true. If their first prediction, that there is a conductor that signals when to transition from one state to another, was true, the team expected to see all the groups of neurons transitioning between states at similar times. On the other hand, if their second prediction was true, that the neural symphony stays in sync by listening to nearby neurons, the researchers would expect to see groups of nearby neurons transitioning between themes mostly together, with nearby neurons more likely to move together than neurons that are further apart. When they measured the neurons’ activity, they found that transitions between states measured on different electrodes corresponded only weakly to each other, but that the closer the electrodes were, the more the state transitions were related. This supported their second prediction, that neurons listen to their neighbors to decide when to transition from one state to another.

This is an exciting step toward understanding how the brain coordinates the movements between states that help keep our complex brains in sync. Understanding this process is important because it can help us develop therapies that mimic it for patients whose brain activity can’t always keep up healthy patterns, such as seizure patients. Beyond medical uses, understanding nature’s elegant solution to managing the complexity of brain signaling can teach us how to build computer systems and models that can handle increasingly more complexity to do things like power robots. And if none of these applications excite you, hopefully you can appreciate the wonder of understanding a little more about what makes us tick and how our neural symphonies stay in sync.

About the brief writer: Catrina Hacker

Catrina Hacker is a PhD candidate working in Dr. Nicole Rust’s lab. She is broadly interested in the neural correlates of cognitive processes and is currently studying how we remember what we see. She also co-directs PennNeuroKnow.

Want to learn more about how our brain activity changes during anesthesia? Read this paper to learn more!