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Multiplexing Focused Ultrasound Stimulation with Fluorescence Microscopy
Published on: January 7, 2019
In vivo effects of focused shock waves on tumor tissue visualized by fluorescence staining techniques
Petr Lukes1, Jan Zeman2, Vratislav Horak3
1Institute of Plasma Physics AS CR, v.v.i., Za Slovankou 3, 182 00 Prague 8, Czech Republic.
Abstract:
Shock waves can cause significant cytotoxic effects in tumor cells and tissues both in vitro and in vivo. However, understanding the mechanisms of shock wave interaction with tissues is limited. We have studied in vivo effects of focused shock waves induced in the syngeneic sarcoma tumor model using the TUNEL assay, immunohistochemical detection of caspase-3 and hematoxylin-eosin staining. Shock waves were produced by a multichannel pulsed-electrohydraulic discharge generator with a cylindrical ceramic-coated electrode. In tumors treated with shock waves, a large area of damaged tissue was detected which was clearly differentiated from intact tissue. Localization and a cone-shaped region of tissue damage visualized by TUNEL reaction apparently correlated with the conical shape and direction of shock wave propagation determined by high-speed shadowgraphy. A strong TUNEL reaction of nuclei and nucleus fragments in tissue exposed to shock waves suggested apoptosis in this destroyed tumor area. However, specificity of the TUNEL technique to apoptotic cells is ambiguous and other apoptotic markers (caspase-3) that we used in our study did not confirmed this observation. Thus, the generated fragments of nuclei gave rise to a false TUNEL reaction not associated with apoptosis. Mechanical stress from high overpressure shock wave was likely the dominant pathway of tumor damage.
Insights
Focused shock waves cause significant tumor tissue damage. While TUNEL assay suggested apoptosis, caspase-3 markers indicated mechanical stress, not programmed cell death, was the primary damage mechanism.
Area of Science:
- Biophysics
- Oncology
- Biomedical Engineering
Background:
- Shock waves exhibit cytotoxic effects on tumor cells and tissues.
- Mechanisms of shock wave-tissue interaction require further elucidation.
Purpose of the Study:
- To investigate the in vivo effects of focused shock waves on a syngeneic sarcoma tumor model.
- To differentiate between apoptosis and mechanical stress as mechanisms of shock wave-induced tumor damage.
Main Methods:
- Utilized a multichannel pulsed-electrohydraulic discharge generator for shock wave production.
- Employed TUNEL assay, caspase-3 immunohistochemistry, and hematoxylin-eosin staining for tissue analysis.
- High-speed shadowgraphy was used to visualize shock wave propagation.
Main Results:
- Observed a distinct cone-shaped region of damaged tumor tissue correlating with shock wave propagation.
- TUNEL assay indicated nuclear fragmentation, initially suggesting apoptosis.
- Caspase-3 staining did not confirm apoptosis, indicating false positives from the TUNEL assay due to mechanical damage.
Conclusions:
- Focused shock waves induce significant mechanical damage in sarcoma tumors.
- Nuclear fragmentation observed via TUNEL assay is not a reliable indicator of apoptosis in this context.
- Mechanical stress, rather than apoptosis, is the dominant mechanism of tumor damage induced by high-pressure shock waves.
