Use of pulsed high intensity focused ultrasound to study the response of brain endothelial cells to mechanical injury

This work investigated the significance of a sublethal primary mechanical injury induced by pulsed high intensity focused ultrasound (pHIFU) and mechanical stretching techniques to mouse brain endothelial cell (bEND.3) nuclei in vitro followed by secondary hypoxia. The lateral size of the pHIFU beam’s focal spot (~ 1.7 mm) was obtained by characterizing the pHIFU system, and the transparency of the membrane on which the cells were to be cultured was determined theoretically and experimentally. Preliminary assessment of injury using a MTT assay (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) at high acoustic pressures (3.15 MPa and 1.04 MPa for 50 ms) demonstrated cell damage. In the subsequent experiments, the cells were sublethally exposed to either pHIFU (0.57 MPa for 50ms) or stretch injuries (0.012 – 0.014 MPa for 50 ms). After each primary injury, the cells were either fully normoxic for 24 h or were subjected to a 6 h hypoxic environment followed by an 18 h recovery under normoxic conditions. The morphological parameters (area and circularity) of bEND.3 nuclei post-treatment were measured using an image analysis tool (ImageJ) for three primary treatments (control, pHIFU and stretch) and evaluated by plotting the scatterplots. Compared to the control group, a statistically significant decrease in circularity of bEND.3 nuclei was observed when the cells were subjected to 6 h hypoxia following a primary injury whereas nuclear area remained unchanged. The percentage of deformed nuclei also increased to ~ 19 % for pHIFU and stretch injury groups after 18 h normoxia following primary and secondary insult (6 h hypoxia), demonstrating cells’ inability to recover due to injury. However, when this hypoxic-normoxic regimen was not preceded by a pHIFU (or mechanical stretch) primary injury, nuclei recovered their original shape successfully. In conclusion, as shown in this study, cells became more susceptible to secondary hypoxic injury after being exposed to an initial mechanical insult.