Poster 28 - Astrocytic Responses to Injury by Laser-Induced Shockwave

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Yiqing Chen

High school volunteer
UCSD IEM
Walnut, California, United States

Co-Author(s)

WB

Wendao Bao

High School Volunteer
UCSD IEM, United States

Co-Author(s)

Sonia Kang

Volunteer research intern
University of California, San Diego: Institute of Engineering in Medicine
San Diego, California, United States

Primary Investigator(s)

Linda Shi, PhD (she/her/hers)

OPALS Program Director, Project Scientist, Co-director IEM
University of California, San Diego: Institute of Engineering in Medicine
La Jolla, California, United States

Co-Author(s)

CW

Chengbiao Wu

Professor
UCSD School of Medicine Dept of Neuroscience, Outreach Program for Advancing Learning in STEM (OPALS), United States
VG

Veronica Gomez-Godinez

Scientist
University of California, San Diego
La Jolla, California, United States

Introduction: Astrocytes play a crucial role in supporting neuron recovery after damage by taking up damaging molecules and debris and recruiting repair factors to injury sites. Traumatic brain injury (TBI), caused by large forces striking the brain, often results in significant neuron cell death. To investigate the astrocyte responses in such circumstances, this study uses laser-induced shockwave (LIS) to simulate blast-induced TBI (bTBI) for a 30-minute imaging window. Calcium is the universal signal transmitter in a wide range of cellular processes, therefore, by analyzing the calcium level changes in the astrocytes using Fluo-4 dye, we were able to detect the excitement level within the cells responding to the LIS. Through this study, we aim to advance the understanding of cellular processes involved in brain repair and potential therapeutic targets for TBI.

Materials and

Methods: In this study, a laser-induced shockwave was generated in a container with calcium-stained astrocyte cells. Time-lapse imaging was conducted at 3-second intervals over a 30-minute duration, encompassing the period before and after shockwave induction. Two filters were utilized to differentiate cells: the Fluo4-AM filter to identify living cells and the HCred filter to mark dead cells. Additionally, images without any filters were captured for baseline comparison. To process the images, 600 sequential images and four phase images (pre- and post-shockwave for each filter type) were loaded into ImageJ to determine the exact frame immediately preceding the shockwave event. A MATLAB program was utilized to identify and circle cells, referencing the phase images to exclude dead cells or those that moved during imaging. MATLAB was further used to record calcium brightness intensities over time, measure distances from the shockwave origin (592, 678) to each circled cell, and calculate cell half-lives. Another MATLAB script generated calcium spike peaks for each cell, with peak data exported to Excel for analysis. Finally, data compilation and graphical analysis were conducted in Graphpad Prism to assess the significance of observed effects.

Results, Conclusions, and Discussions: The experiment showed that upon shockwave, there is a larger calcium increase in AD cells than that of WT cells, as shown in Figure (a). There is also a slight difference in the amount of calcium increase between AD cytoplasm and AD nuclei, where the nuclei exhibited a greater increase than that of the cytoplasm. Furthermore, the peak rise rate of both AD nuclei and cytoplasm is greater than that of WT nuclei and cytoplasm, as shown in Figure (b). The peak rise rate of AD nuclei and cytoplasm is shown to be approximately 3 s-1 greater than the rate of WT nuclei and cytoplasm. The different responses exhibited by AD/WT nuclei and cytoplasm indicate an impairment in calcium regulation upon cell injury. Calcium dysregulation contributes to secondary neuronal death post-injury, which could potentially lead to Alzheimer’s disease, thus a more complete understanding of the dysregulation pathway is necessary. Repeating the experiment with a larger sample size will provide more complete information on the differences between the two cell types.

Acknowledgements (Optional): This material was based upon work supported by a gift from Beckman Laser Institute Inc. to LS. The high school students (*) participated in the UCSD OPALS internship. Special thanks to Dr. Shu Chien for supporting the OPALS program.

References (Optional):