Ultrasound therapy with optimal intensity facilitates peripheral nerve regeneration in rats through suppression of pro-inflammatory and nerve growth inhibitor gene expression

Abstract

Background Therapeutic ultrasound (US) is a promising physical therapy modality for peripheral nerve regeneration. However, it is necessary to identify the most effective US parameters and clarify the underlying mechanisms before its clinical application. The intensity of US is one of the most important parameters. However, the optimum intensity for the promotion of peripheral nerve regeneration has yet to be determined. Objectives To identify the optimum intensity of US necessary for the promotion of peripheral nerve regeneration after crush injuries in rats and to clarify the underlying mechanisms of US by mRNA expression analysis. Methods We inflicted sciatic nerve crush injuries on adult Lewis rats and performed ultrasound irradiation using 4 different US intensities: 0 (sham stimulation), 30, 140, and 250 mW/cm 2 with frequency (5 days/week) and duration (5 min/day). We evaluated peripheral nerve regeneration by quantitative real-time PCR one week after injury. Histomorphometric analyses and motor function analysis were evaluated 3 weeks after injury. Results US stimulation enhanced re-myelination as well as sprouting of axons, especially at an intensity of 140 mW/cm 2. mRNA expression revealed that US suppressed the expression of the inflammatory cytokines TNF and IL-6 and the axonal growth inhibitors SEMA3A and GSK3β.

Therapeutic Low-Intensity Ultrasound for Peripheral Nerve Regeneration – A Schwann Cell Perspective

“…First, LIU intensity needs to be between 200 and 500 mW/cm 2 . At lower intensity (≤100 mW/cm 2 ), the beneficial effect of LIU is not observable (Daeschler et al, 2018a;Ito et al, 2020), while at higher intensity (≥1 W/cm 2 ) the beneficial effect is reduced or absent (Hong et al, 1988;Mourad et al, 2001;Akhlaghi et al, 2012;Jiang et al, 2016). Second, to improve nerve regeneration, LIU should be applied repetitively, either every day or every other day and for a short period of time (between 1 and 10 min per application).…”

“…In summary, multiple advances made from pre-clinical studies lead to the current consensus that LIU application promotes peripheral nerve regeneration after peripheral nerve injuries (Hong et al, 1988;Mourad et al, 2001;Crisci and Ferreira, 2002;Chang and Hsu, 2004;Chang et al, 2005;Raso et al, 2005;Chen et al, 2010;Park et al, 2010;Akhlaghi et al, 2012;Oliveira et al, 2012;Jahromy et al, 2013;Kim et al, 2013;Lv et al, 2015;Jiang et al, 2016;Ni et al, 2017;Ito et al, 2020;Wang et al, 2021). More precisely, it was shown that LIU could: (i) increase the number, diameter, or the myelination of axon distal to the lesion site; (ii) improve nerve conduction velocities (NCV) and compound muscle action potentials (CMAP); and (iii) enhance functional recovery after nerve injury (Table 1) (for review, see Peng et al, 2020).…”

“…More precisely, it was shown that LIU could: (i) increase the number, diameter, or the myelination of axon distal to the lesion site; (ii) improve nerve conduction velocities (NCV) and compound muscle action potentials (CMAP); and (iii) enhance functional recovery after nerve injury (Table 1) (for review, see Peng et al, 2020). In addition, a few studies have shown that application of LIU on injured nerves is sufficient to alter gene regulation of neurotrophic factors, cytokines, or promyelinating genes during peripheral nerve regeneration ( Chen et al, 2010;Jahromy et al, 2013;Ito et al, 2020;Wang et al, 2021). However, despite these encouraging studies, LIU application on peripheral nerve injury is not considered, possibly because the precise cellular and molecular mechanisms supporting the therapeutic effect of LIU during peripheral nerve regeneration are still not understood.…”

“…Neurotrophic factors have been shown to promote neuroprotection, axonal regrowth and even myelinogenesis following peripheral nerve injury (for review, see Li et al, 2020 ). Six studies, using various therapeutic regiment, have looked at the effect of LIU on neurotrophic factor expression in Schwann cells ( Zhang et al, 2009 ; Chen et al, 2010 ; Jahromy et al, 2013 ; Ren et al, 2018 ; Ito et al, 2020 ; Wang et al, 2021 ). However, it remains unclear how parameter changes in LIU show contrasting effects on neurotrophic factor secretion and how neurotrophic factor secretion will be modulated by LIU after more severe injury (nerve cut).…”

Ultrasound stimulation improves inflammatory resolution, neuroprotection, and functional recovery after spinal cord injury

“…Moreover, several recent clinical trials and experimental studies have reported that US can elicit an anti-inflammatory response, promote tissue repair, and reduce pain [27][28][29][30] . US stimulation reduces inflammatory mediators, such as cyclooxygenase (COX)-1/2, inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β, as well as immune cell infiltration, in numerous inflammatory diseases, including peripheral nerve injury [31][32][33] . In particular, Zachs et al demonstrated that daily US stimulation targeting the spleen could induce an anti-inflammatory response via immunomodulation of both T and B cells, conferring protective and therapeutic effects in inflammatory arthritis 34 .…”