Effects of Thermal Preconditioning on Tissue Susceptibility to Histotripsy

Abstract

Histotripsy is a non-invasive ablation method that mechanically fractionates tissue by controlling acoustic cavitation. Previous work has revealed that tissue mechanical properties play a significant role in the histotripsy process, with stiffer tissues being more resistant to histotripsy-induced tissue damage. In this study, we propose a thermal pretreatment strategy to precondition tissues before histotripsy. We hypothesize that a thermal pretreatment can be used to alter tissue stiffness by modulating collagen composition, thus changing tissue susceptibility to histotripsy. More specifically, we hypothesize that tissues will soften and become more susceptible to histotripsy when preheated at ∼60°C because of collagen denaturation, but that tissues will rapidly stiffen and become less susceptible to histotripsy when preheated at ∼90°C because of collagen contraction. To test this hypothesis, a controlled temperature water bath was used to heat various ex vivo bovine tissues (tongue, artery, liver, kidney medulla, tendon and urethra). After heating, the Young's modulus of each tissue sample was measured using a tissue elastometer, and changes in tissue composition (i.e., collagen structure/density) were analyzed histologically. The susceptibility of tissues to histotripsy was investigated by treating the samples using a 750-kHz histotripsy transducer. Results revealed a decrease in stiffness and an increase in susceptibility to histotripsy for tissues (except urethra) preheated to 58°C. In contrast, preheating to 90°C increased tissue stiffness and reduced susceptibility to histotripsy for all tissues except tendon, which was significantly softened due to collagen hydrolysis into gelatin. On the basis of these results, a final set of experiments were conducted to determine the feasibility of using high-intensity focused ultrasound to provide the thermal pretreatment. Overall, the results of this study indicate the initial feasibility of a thermal pretreatment strategy to precondition tissue mechanical properties and alter tissue susceptibility to histotripsy. Future work will aim to optimize this thermal pretreatment strategy to determine if this approach is practical for specific clinical applications in vivo without causing unwanted damage to surrounding or overlying tissue.

Keywords: High-intensity focused ultrasound; Histotripsy; Thermal therapy; Tissue stiffness.

Figure 1. Experimental setup

(A) After heating, tissue Young’s modulus (compression) was measured using a tissue elastometer.(B) Susceptibility to histotripsy was tested using a 750 kHz therapy transducer with the focused aligned to the tissue surface. Histotripsy was applied for five minutes using 5 cycle pulses, a pulse repetition frequency of 500 Hz, and pressure of 19/60 MPa (p−/P+).

Figure 2. Young’s modulus plots

Elastometer measurements for tissues heated at 58ºC demonstrated a significant decrease in the Young’s modulus of tongue, artery, kidney medulla, and tendon (A:C, E), no significantly change for liver (D), and a significant increased for urethra (F). Heating at 90ºC significantly increased the Young’s modulus of tongue, artery, kidney medulla, liver, and urethra (A:D, F) and significantly decreased the Young’s modulus of tendon (E).

Figure 3. Representative Force vs. Strain Curves

Plots show representative Force vs. Strain curves measured by the elastometer for tissues samples comparing unheated control samples with samples that were heated for 4 hours at 58°C and 90°C, respectively.

Figure 4. Histology: 58°C Samples

Images show histology slides stained with a trichrome blue staining for samples heated at 58°C for 4, 8, and 12 hours. Results for tongue, artery, and tendon demonstrated a decrease in collagen (blue) density with heating. Furthermore, some hydrolysis into gelatin (red) was observed in tendon after 8 and 12 hours. Urethra results demonstrated an increase in collagen density after heating compared to control.

Figure 5. Histotripsy Treatment Pictures: 58°C Samples

Morphological analysis of bovine tissues after histotripsy demonstrated that tongue, artery, liver, kidney, and tendon were more susceptible to histotripsy-induced tissue damage after heating at 58°C. Urethra was observed to be less susceptible to histotripsy after heating at 58°C.

Figure 6. Histology: 90°C Samples

Images show histology slides stained with a trichrome blue staining for samples heated at 90°C for 4, 8, and 12 hours. Results for tongue, artery, and urethra demonstrated an increase in collagen (blue) density with heating. In tendon, collagen was observed to hydrolyze into gelatin (red) with heating. Some hydrolysis was also observed in the urethra after 12 hour heating.

Figure 7. Histotripsy Treatment Pictures: 90°C Samples

Morphological analysis of bovine tissues after histotripsy demonstrated tongue, artery, liver, kidney, and urethra were less susceptible to histotripsy-induced tissue damage after heating at 90°C for 4 hours while tendon was more susceptible to histotripsy after heating. All tissues became more susceptible to histotripsy after heating was continued for 8 and 12 hours (compared to 4 hour samples).

Figure 8. HIFU Thermal Treatment

Plots show example temperature vs. time curves for HIFU heating of tendon measured using hypodermic needle thermocouples. For all experiments, 3 thermocouples were placed near the focal region, and HIFU was applied to rapidly increase the temperature to (A) ~60°C or (B) ~90°C. Once the desired temperature was reached, the PRF was lowered to stabilize the temperature at a near constant level for the duration of the heating. (B) For the higher temperature HIFU treatments, the temperature measured by the thermal couples did not remain above 90°C due to thermocouple movement caused by tissue contraction. Similar curves were obtained for HIFU treatments in all tissue types.

Figure 9. Histotripsy Treatment Pictures: Ultrasound Heated Samples

Morphological analysis of bovine tissues treated with thermal HIFU followed by histotripsy demonstrated similar results to controlled heating experiments. Results showed an increase in susceptibility to histotripsy for tongue, liver, and tendon heated with ultrasound at a peak temperature of ~60°C. Tongue and liver samples heated with ultrasound at a peak temperature of ~90°C were less susceptible to histotripsy while tendon became more susceptible to histotripsy after heating at ~90°C.