Modulation of Pain-Associated Behaviors in Sickle Mice Using Low-Intensity Transcranial Focused Ultrasound

Pain is a major comorbidity of sickle cell disease (SCD), which is the most genetic diathesis affecting mostly African Americans in the United States. Opioids are used to reduce pain, however the increased risk of drug abuse, overdose, and addiction has led to an urgent need to develop non-pharmacologic approaches to treat pain safely and effectively. Low-intensity transcranial focused ultrasound (tFUS) is a non-invasive brain stimulation method featured with high precision and deep brain penetration, which holds great promise in managing pain. Here, we report how tFUS modulates pain-related behaviors in wild-type mice and humanized sickle mice by targeting specific brain regions involved in the processing and perception of pain.

We used wild-type mice (C57BL/6J) and humanized transgenic BERK mice on a mixed genetic background (FVB/N, 129, DBA/2, C57BL/6, and Black Swiss) murine α and β globin knockouts (α^−/−, β^−/−) with sickle mice expressing >99% human sickle hemoglobin S (HbS), demonstrating severe SCD. Mice were generated in-house by cross-breeding homozygous (SS) males with hemizygous (AS) females. All studies in mice were approved by IACUC of Carnegie Mellon University and University of California at Irvine, and complied with NIH guidelines. The tFUS was applied to hindlimb of the primary somatosensory cortex (S1HL) at the left hemisphere of the brain, and the effect of tFUS stimulation was further investigated by comparing with control groups such as sham treatment (applying tFUS to a vicinity region of S1HL) and negative control (not applying tFUS while keeping the same experimental procedures). A modified hot-plate behavior test was used to assess unilateral effects of tFUS by comparing withdrawal responses of contralateral (right) and ipsilateral (left) hind paws. The nociceptive responses to noxious heat stimuli were blindly assessed and analyzed in terms of ∆hind paw withdrawal latency (∆hPWL) (s) (= contralateral hPWL- ipsilateral hPWL) before and after specific treatment.

We observed a marked reduction in sensitivity to noxious heat stimuli in tFUS-treated male and female wild-type mice. Compared to the baseline value, tFUS stimulation with lower pulse repetition frequency (PRF) (i.e., 40 Hz) at left S1HL led to a significant increase in the averaged ∆hPWL (***p<0.001, N=8 male wild-type mice for 3 min after tFUS; *p<0.05, N=8 for 8 min after tFUS; **p<0.01, N=8 female wild-type mice for 3 min after tFUS; *p<0.05, N=8 for 8 min after tFUS; Friedman test), but not with the sham treatment (p>0.05, N=8 male and female wild-type mice for 3 and 8 min after sham treatment; Friedman test). Next, we assessed whether tFUS could modulate heat hyperalgesia in female sickle mice. Fig. 1 shows that tFUS stimulation with PRF of 40 Hz at left S1HL resulted in a significant change of ∆hPWL in the sickle mice compared to pre-stimulation baseline (**p<0.01, N=10 for 3 and 8 min after tFUS; Friedman test). This effect was specific to tFUS stimulation as a remarkable difference of averaged ∆hPWL between the sham treatment/negative control and pre-sham/negative control baseline was not observed (p>0.05, N=10 for 3 and 8 min after sham treatment and negative control; Friedman test), which suggests that tFUS stimulation with a lower PRF at S1HL can attenuate heat hyperalgesia in the sickle mice. In addition, when tFUS with higher PRF (i.e., 3 kHz) was applied at left S1HL, we found a negative averaged value of ∆hPWL based on the decreased contralateral (right) hind paw latency compared to latency of ipsilateral (left) hind paw, which is significantly different from the behavioral responses to heat stimuli with the PRF of 40 Hz (^###p<0.001, N=10 for 3 min after tFUS with PRF of 40 and 3 kHz; ^####p<0.0001, N=10 for 8 min after tFUS with PRF of 40 and 3 kHz; Kruskal-Wallis test). Since, tFUS stimulation with a higher PRF leads to exacerbation of heat hyperalgesia in the sickle mice, it is critical to optimize the level of PRF to achieve an analgesic effect.

We show that tFUS stimulation can be effective in non-invasively attenuating heat pain sensitivity in wild-type mice and heat hyperalgesia in humanized sickle mice at a lower PRF, but exacerbating at a higher PRF by stimulating pre-identified pain-processing brain circuit. These observations have a translational potential for establishing an effective, non-addictive, non-pharmacological, non-invasive device-based neuromodulation technique for managing sickle cell pain.

Kim:Carnegie Mellon University: Patents & Royalties: pending application. Argueta:Cayenne Foundation: Honoraria; Cyclerion: Honoraria. Gupta:Novartis: Honoraria, Research Funding; CSL Behring: Honoraria; Cyclerion: Research Funding; 1910 Genetics: Research Funding; Grifols: Research Funding; Zilker LLC: Research Funding; UCI Foundation: Research Funding; SCIRE Foundation: Research Funding; Tautona Group: Honoraria.