Browsing by Subject "Anesthesia"

The overarching goal of this dissertation was to improve our understanding of the neural basis of consciousness by approaching the problem along two separate, complementary facets: examining the levels of consciousness and the contents of consciousness.

Chapter 2 examines how the level of consciousness changes under general anesthesia for surgery, and how neural (EEG) markers of this change relate to postoperative cognitive impairments afflicting many older adults. Older adult patients underwent neurocognitive testing before and after surgery, and their 32-channel EEG was recorded both before and during general anesthesia for surgery. Results showed that one of the most profound changes from the awake to the anesthetized brain—the anteriorization of alpha-band (8-12 Hz) activity—correlated with preoperative cognitive scores, which are themselves predictors for postoperative cognitive impairments. These results have added to our understanding of how manipulations of the level of consciousness under general anesthesia ramify into potentially long-lasting impairments to cognition, and how these impairments might be monitored and avoided.

Chapters 3 and 4 examined how the contents of consciousness relate to the selection mechanism of attention. Chapter 3 investigated the dissociability of these two phenomena by examining the neural mechanisms underlying the orienting of spatial attention without awareness. High-density (64-channel) EEG was recorded while subjects performed a novel task that combined classic spatial cueing with object-substitution masking to manipulate subjects’ awareness of the cues on ~half of the trials, allowing a direct comparison of orienting with and without awareness, controlled for having identical sensory stimulation. Results confirmed that attention could be oriented without awareness, leading to improved behavior (faster reaction times and better accuracy) and enhanced sensory processing (indexed by the P1 event-related potential, ERP) for validly (compared to invalidly) cued targets. Interestingly, the hallmark ERP for the orienting of attention in response to a cue, the N2pc, was only observed for conscious orienting, pointing to an alternate mechanism for unconscious orienting, such as via the subcortical retinotectal pathway.

Chapter 4 investigated the mechanisms and temporal dynamics of the attentional selection of conscious internal representations in working memory. EEG was recorded while subjects performed a modified delayed match-to-sample task where one of two sample objects, a face or a house, was retroactively cued on each trial. A multivariate classifier was trained on the pattern of alpha-band activity to determine if and when information about the selected object could be decoded from the alpha signal following the retrocue. Results showed that alpha could be used to decode the selected object, pointing to its general role as a top-down attentional control signal. This decoding was relatively transient, rather than sustained, which accords with recent proposals of “activity-silent” working memory and argues against accounts of working memory that posit sustained internal attention as the underlying mechanism. Together the results of Chapters 3 and 4 help inform our understanding of how attention operates both externally and internally to select the contents of consciousness.

In vivo imaging is an important scientific tool for studying bio-molecular interactions, but lack of preserved functionality during imaging restricts scientists’ abilities to gain critical knowledge. Structure can be preserved while using high-resolution optical imaging by utilizing window chambers in murine models1, yet the use of anesthesia for immobilization is problematic. Anesthesia affects tissue oxygenation2, blood cell velocities3, immunosuppression4, and allowable duration of imaging5–thus its usage restricts in vivo bio-molecular imaging accuracy and duration.

Developing a portable imaging system that attaches to murine dorsal window chambers enables imaging without anesthesia, avoiding previous drawbacks of window chamber models. A raspberry pi camera (RPI-CAM-V2, Raspberry Pi) was modified for microscopy and used alongside 3D printed panels for attaching the camera, optical filters, and LED light source to murine window chambers. Multiple applications for the portable system were developed, each requiring their own setup of filters and stimulating LEDs. The system is powered by a Raspberry Pi 3 Model B single-board computer (RASPBERRYPI3-MODB-1GB, Raspberry Pi), allowing for stream-lined data acquisition.

Imaging tissue oxygenation was the first application developed for the portable system. Oxygen sensing boron nanoparticles were injected into window chambers, while a UV LED was used to stimulate fluorescent and phosphorescent signals. When stimulated by UV light, the boron nanoparticles emit fluorescence and phosphorescence. Fluorescence is stable regardless of oxygenation, while phosphorescence signal from the nanoparticles is quenched in the presence of higher oxygenations. The ratio of fluorescence to phosphorescence was used to calculate oxygen concentration maps of window chamber tissue. Tissue oxygenations in awake and anesthetized mice inhaling varied oxygen concentrations were analyzed. In 5 awake nude mice inhaling 20% O2, the median partial pressure of oxygen was measured as 49 mmHg within their window chambers. From a one-tailed t-test with a false positive correction, 3 of the mice had significantly higher (p ≤ 0.05) tissue oxygenation while anesthetized compared to the awake measurements.

Developing the portable systems ability to image blood cells was another focus of this project. Blood cells were visible with white LED exposure. A frame rate of 30 frames/second was adequate for tracking cell motion while allowing for the highest resolution possible with the system. Blood cell velocities in a mouse awake and anesthetized were analyzed, while also observing change in blood cell velocities during sepsis that was induced by cecal ligation puncture (CLP). Three days after CLP, the mean awake blood cell velocity was measured as 0.21 ± 0.03 mm/s, while the mean anesthetized blood cell velocity was measured as 0.080 ± 0.002 mm/s. Six days after CLP, the awake measurement had reduced to 0.019 ± 0.005 mm/s, while the anesthetized measurement was reduced to 0.031 ± 0.002 mm/s (91% decrease in awake measurement, 61% decrease in anesthetized measurement). A two-way ANOVA on the factors of anesthesia and time post-CLP performed on multiple vessel regions calculated significance (p ≤ 0.05) for both of these factors on blood cell velocities within the pilot mouse’s window chamber.

Noting the differences between data collected on awake and anesthetized mice, our system has been validated as a tool for real-time imaging of tissue without the observed effects of anesthesia. By avoiding anesthesia, the developed device allows for continual data acquisition to increase from hours to days. The system is generalizable, and while only two applications are presented in this study, the system could be modified for imaging fluorescently labeled cells/proteins for other bio-molecular interactions.