Intense focused ultrasound as a potential research tool for the quantification of diurnal inflammatory pain

Abstract

Quantifying pain through assay of a human's or animal's response to a known stimulus as a function of time of day is a critical means of advancing chronotherapeutic pain management. Current methods for quantifying pain, even in the context of etiologies involving deep tissue, generally involve stimulation by quantifiable means of either cutaneous (heat-lamp tests, electrical stimuli) or both cutaneous and subcutaneous tissue (von Frey hairs, tourniquets, etc.) or study of proxies for pain (such as stress, via assay of cortisol levels). In this study, we evaluate the usefulness of intense focused ultrasound (iFU), already shown to generate sensations and other biological effects deep to the skin, as a means of quantifying deep diurnal pain using a standard animal model of inflammation. Beginning 5 days after injection of Complete Freund's Adjuvant into the plantar surface of the rat's right hind paw to induce inflammation, the rats were divided into two groups, the light-phase test group (09:00-18:00h) and the dark-phase test group (23:00-06:00h), both of which underwent iFU application deep to the skin. We used two classes of iFU protocol, motivated by the extant literature. One consisted of a single pulse (SP) lasting 0.375s. The other, a multiple pulse (MP) protocol, consisted of multiple iFU pulses each of length 0.075s spaced 0.075s apart. We found the night group's threshold for reliable paw withdrawal to be significantly higher than that of the day group as assayed by each iFU protocol. These results are consistent with the observation that the response to mechanical stimuli by humans and rodents display diurnal variations, as well as the ability of iFU to generate sensations via mechanical stimulation. Since iFU can provide a consistent method to quantify pain from deep, inflamed tissue, it may represent a useful adjunct to those studying diurnal pain associated with deep tissue as well as chronotherapeutics targeting that pain.

Figure 1. Characterization of the stimulating ultrasound iFU field

(A) Two-dimensional mathematical simulation of the intensity field of our iFU transducer as a function of the transverse distance across the transducer face and the axial distance from the transducer, through the proximal surface of the cone (at 55 mm) to the focus of the transducer (at 62 mm) and beyond. (B) Plot of intensity versus axial distance from the proximal face of the ultrasound delivery system, calculated along the center of the ultrasound field. (C) Plot of calculated intensity distribution at the focus as a function of the transverse distance.

Figure 1. Characterization of the stimulating ultrasound iFU field

(A) Two-dimensional mathematical simulation of the intensity field of our iFU transducer as a function of the transverse distance across the transducer face and the axial distance from the transducer, through the proximal surface of the cone (at 55 mm) to the focus of the transducer (at 62 mm) and beyond. (B) Plot of intensity versus axial distance from the proximal face of the ultrasound delivery system, calculated along the center of the ultrasound field. (C) Plot of calculated intensity distribution at the focus as a function of the transverse distance.

Figure 1. Characterization of the stimulating ultrasound iFU field

(A) Two-dimensional mathematical simulation of the intensity field of our iFU transducer as a function of the transverse distance across the transducer face and the axial distance from the transducer, through the proximal surface of the cone (at 55 mm) to the focus of the transducer (at 62 mm) and beyond. (B) Plot of intensity versus axial distance from the proximal face of the ultrasound delivery system, calculated along the center of the ultrasound field. (C) Plot of calculated intensity distribution at the focus as a function of the transverse distance.

Figure 2. Photograph of our experimental design

Here a rat resides within in a Plexiglas box on top of a perforated aluminum plate. The holes in the plate allowed us to deliver ultrasound to the plantar aspect of the rat’s paws in a serial fashion, using our iFU device. That device consists of a transducer – characterized in Figure 1, covered by a water-filled cone to facilitate propagation of the ultrasound from the transducer into the rat’s paw, as well as to aid in the aiming of the iFU into the paw.

Figure 3

Comparison of the intensity and acoustic dose of iFU necessary to produce a reliable withdrawal response in animals using the single pulse (SP) protocol, for the dark-phase group of rats versus the light-phase group of rats.

Figure 4

Comparison of the intensity and acoustic dose of iFU necessary to produce a reliable withdrawal response in animals using the multiple pulse (MP) protocol, for the dark-phase group of rats versus the light-phase group of rats.