Poster 73 - Evaluating the Impact of Laser-Induced Shock Waves on Drosophila Brain Tissue: A Study of Morphology and Volume Influence on Tissue Displacement

Saturday, October 26, 2024

10:00 AM - 12:00 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Rehaan Hassan

IEM OPALS Intern
UCSD Institute of Engineering in Medicine
Carlsbad, California, United States

Co-Author(s)

HH

Huiying Huang, n/a

Undergraduate student
University of California, San Diego, United States

Co-Author(s)

JH

Jiayu Hu

IEM OPALS Intern
UCSD Institute of Engineering in Medicine, United States
XY

Xuning Ye

IEM OPALS Intern
UCSD Institute of Engineering in Medicine, United States

Co-Author(s)

Sophia Liu (she/her/hers)

IEM OPALS Intern
UCSD Institute of Engineering in Medicine, California, United States
Sirasit Prayotamornkul (he/him/his)

Undergraduate Student Researcher
UCSD Shu Chien-Gene Lay Department of Bioengineering
San Diego, California, United States
Matthew Callahan (he/him/his)

Undergraduate Student Researcher
UCSD Shu Chien-Gene Lay Department of Bioengineering
Indio, California, United States
Avriza D. Bestafa (he/him/his)

Undergraduate Research Assistant
University of California, San Diego
La Jolla, California, United States
Jenna Cecil (she/her/hers)

Undergraduate Research Assistant
University of California, San Diego, United States
YL

Yajuan Li

Postdoctoral Researcher
University of California, San Diego, United States
LS

Lingyan Shi

Advisor, Associate Professor, Postdoctoral Research Scientist
UCSD Shu Chien-Gene Lay Department of Bioengineering, 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

Last Author(s)

VG

Veronica Gomez-Godinez

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

Introduction: This study explores the impact of laser-induced shockwaves on brain tissues in Drosophila melanogaster. Renowned for its genetic tractability and neurobiological parallels to higher organisms, Drosophila serves as an optimal model for investigating tissue responses to mechanical damage. Utilizing advanced imaging techniques, this research quantifies tissue changes following controlled shockwave exposure.
The primary objective is to assess the effects of targeted shockwaves, generated via precise laser applications, on the tissue architecture of the Drosophila brain. Laser-induced shockwaves effectively simulate mechanical damage, akin to traumatic brain injuries (TBI) commonly observed in clinical scenarios[1]. The ability to calibrate the extent and localization of damage with precision allows for an in-depth analysis of tissue responses. The findings from this study are poised to enhance our understanding of neurobiological repair mechanisms by modeling brain tissue responses to injurious stimuli such as TBI.

Materials and

Methods: To isolate the Drosophila brain tissue, the eyes and mandibles were carefully excised. The brain tissue was subsequently stained with Dead-Red (EthD-1) and NucBlue (Hoechst 33342), enabling the visualization of both total cellular populations and apoptotic cells. Imaging was performed using a Zeiss Axiovert 200M microscope, with environmental conditions maintained at 37°C, 5% CO2, and 70% humidity. The laser-induced shock wave system employed a 1032 nm laser, calibrated to 700 mW output prior to the 20X NA 0.6 Zeiss objective lens. A Coherent Flare 532 nm 100 Hz repetition rate system with a 2 ns pulse width and 450 µJ pulse energy was used to induce a shockwave. A rotating optical polarizer mounted on a stepper-motor controlled rotating mount was used to attenuate the power. One to multiple pulses were allowed to enter the microscope by the use of a mechanical shutter with a 10–15 ms duty cycle [2]. The Z-plane focal adjustment was executed via Micromanager software to align with the microscope's focal plane. Quantification of the Drosophila brain tissue area was conducted using ImageJ software.
This study successfully dissected and stained brain tissues from fruit flies to visualize total cellular populations and apoptotic cells using Dead-Red (EthD-1) and NucBlue (Hoechst 33342) dyes. The staining enabled the identification of an initial focal plane that offered optimal visualization, with clear differentiation of dead cells in the Dead-Red channel and sharp delineation of the brain tissue edges under Phase 3 illumination.

Results, Conclusions, and Discussions: Results/

Discussion: The experiment was conducted at this initial focal plane for all brain tissues, where laser-induced shock waves were applied at 5-micron intervals, progressing downward through the tissue until the majority was displaced. Two distinct brain tissue morphologies were examined: one with a shorter height and wider base area, and the other with a taller height and tighter base area. The flatter structure measured a base area of 194,132.11 µm² (Figure 1a), multiple laser-induced shock waves were applied without significant cell damage, as observed in the Dead-Red channel (Figure 1b). The laser focus was then shifted vertically by a total of 15 µm over three consecutive intervals, with shock waves applied at each shift until the tissue was fully displaced (Figure 1c-1d). The total volume exposed to the laser-induced shock waves for this wider base brain tissue was 2,911,981.69 µm³.
The second tissue had a base area of 116,924.39 µm² measured from the phase image (Figure 1e) and the Dead-Red channel (Figure 1f) before being subjected to laser-induced shock waves. The laser focus was shifted vertically by a total of 25 µm over five consecutive intervals, with shock waves applied at each shift until the tissue was completely displaced, as shown in the phase image (Figure 1g) and Dead-Red channel (Figure 1h). The total volume exposed to the laser-induced shock waves for this tissue was 2,923,109.70 µm³.
Despite the morphological differences, both exhibited similar displacement behavior when subjected to the same laser-induced shockwave energy. This consistency in response suggests tissue volume, rather than morphology, is a key factor in determining resistance to laser-induced shock wave damage.

Conclusions: This study demonstrates that laser-induced shock waves can effectively displace brain tissues from fruit flies, regardless of their morphological differences. The key finding that tissue volume, rather than shape or structure, predominantly influences the tissue's resistance to laser-induced short wave damage provides valuable insight into the mechanical properties of biological tissues. These results highlight the potential of using laser-induced shock waves as a tool for studying tissue biomechanics and suggest further exploration into its applications in tissue engineering and regenerative medicine.

Acknowledgements (Optional): This material was based upon work supported by a gift from Beckman Laser Institute Inc. to Dr. Linda Shi. The high school students (*) participated in UCSD OPALS internship. Special thanks to Dr. Shu Chien from UCSD Bioengineering and Dr. Lizhu Chen from CorDx Inc. for their donations.

References (Optional): [1] Gomez, Veronica G., et. al. Laser-Induced Shockwave (LIS) to Study Neuronal Ca2+ Responses, Front. Bioeng. Biotechnol., 2021, 15(9), 598896.
[2] Gomez, Veronica G., et. al. Effects of laser induced shockwave on neurons from an Alzheimer’s mouse model, OTOM XX, 2023, 12649, 200-204.