Effects of Noninvasive Low-Intensity Focus Ultrasound Neuromodulation on Spinal Cord Neurocircuits In Vivo

Abstract

Although neurocircuits can be activated by focused ultrasound stimulation, it is unclear whether this is also true for spinal cord neurocircuits. In this study, we used low-intensity focused ultrasound (LIFU) to stimulate lumbar 4-lumbar 5 (L4-L5) segments of the spinal cord of normal Sprague Dawley rats with a clapper. The activation of the spinal cord neurocircuits enhanced soleus muscle contraction as measured by electromyography (EMG). Neuronal activation and injury were assessed by EMG, western blotting (WB), immunofluorescence, hematoxylin and eosin (H&E) staining, Nissl staining, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry (IHC), somatosensory evoked potentials (SEPs), motor evoked potentials (MEPs), and the Basso-Beattie-Bresnahan locomotor rating scale. When the LIFU intensity was more than 0.5 MPa, LIFU stimulation induced soleus muscle contraction and increased the EMG amplitudes (P < 0.05) and the number of c-fos- and GAD65-positive cells (P < 0.05). When the LIFU intensity was 3.0 MPa, the LIFU stimulation led to spinal cord damage and decreased SEP amplitudes for electrophysiological assessment (P < 0.05); this resulted in coagulation necrosis, structural destruction, neuronal loss in the dorsal horn by H&E and Nissl staining, and increased expression of GFAP, IL-1β, TNF-α, and caspase-3 by IHC, ELISA, and WB (P < 0.05). These results show that LIFU can activate spinal cord neurocircuits and that LIFU stimulation with an irradiation intensity ≤1.5 MPa is a safe neurostimulation method for the spinal cord.

Figure 1

Timeline of study I (a) and II (b) experimental protocols. The rats were killed, and the safety was examined on days 0 and 3 post-low-intensity focused ultrasound (LIFU) stimulation.

Figure 2

(a) Schematic of the LIFU pulsing strategy. (b) Schematic of LIFU stimulation of the spinal cord and electromyography (EMG) examination. The signal was generated by the generator, amplified by the amplifier, and then converted into an acoustic signal by the ultrasound probe. The ultrasound probe was fixed by the clamp on the back of the rats at the segment L4–L5 spinal cord level. The recruitment of the soleus muscle was recorded at the time of LIFU spinal cord stimulation. (c–e) Parameters of focal ultrasound, including the acoustic-intensity distribution map from transverse and sagittal planes.

Figure 3

Electromyography (EMG) shows the recruitment of the soleus (Sol) muscle by different intensities of LIFU stimulation. (a) The red arrow shows the moment at which the LIFU was initiated, and the black arrow shows the moment at which the LIFU was stopped. The duration (LIFU on) from the red arrow to the black arrow was 1 s and that from the black to the red arrow (LIFU turned off) was 4 s. The black dotted rectangle shows the recruitment of Sol muscle and activation of EMG. (b) Amplitude of EMG after different irradiation intensity stimulations. ^∗P < 0.05, ^∗∗∗P < 0.01, and ^∗∗∗∗P < 0.0001. Each symbol represents the mean ± SEM; paired t-test; n = 6 rats per assay.

Figure 4

The effect of LIFU stimulation on neuron and synaptic activation in the lumbar spinal cord (×40 and ×400). Scale bar = 500 μm and 20 μm. (a, c) Representative immunofluorescence pictures showing the c-fos-positive (a) and GAD65-positive (c) cells after different intensities of LIFU stimulation of the lumbar spinal cord. (b, d) C-fos- and GAD65-positive cells after different intensities of stimulation. ^∗P < 0.05, ^∗∗∗P < 0.01, and ^∗∗∗∗P < 0.0001. Each symbol represents the mean ± SEM; one-way ANOVA, followed by LSD test for pairwise comparisons; n = 3 rats per assay.

Figure 5

(a) Somatosensory evoked potentials (SEPs) were used to detect somatosensory conduction from the spinal cord. (b) SEP amplitude analyses for different intensities of stimulation. Each symbol represents the mean ± SEM; paired t-test; n = 6 rats per assay.

Figure 6

Enzyme-linked immunosorbent assay (ELISA) for detection of the inflammatory factors IL-1β (a) and TNF-α (b). ^∗P < 0.05 and ^∗∗P < 0.01. Each symbol represents the mean ± SEM; one-way ANOVA, followed by LSD test for pairwise comparisons; n = 3 rats per assay.

Figure 7

Hematoxylin and eosin (H&E) staining for histological examination of spinal cord injury (×40 and ×400). Scale bars = 1 mm and 100 μm. Comparison of H&E staining among the spinal cord sections after different irradiation intensities of LIFU stimulation. There was no significant difference in histological results among 0 MPa, 0.5 MPa, and 1.5 MPa stimulation groups. In the 3.0 MPa stimulation group, coagulative necrosis was clear at the dorsal horn of the spinal cord (red arrow).

Figure 8

Nissl staining for histological examination of spinal cord injury (×100 and ×400). Scale bars = 200 μm and 100 μm. Comparison of Nissl staining among the spinal cord sections after different irradiation intensities of LIFU stimulation. After 0 MPa, 0.5 MPa, and 1.5 MPa stimulation, the neuron arrangement was regular, the structure was clear, the morphology was normal, and the Nissl body was clear (shown in the black square). After 3.0 MPa irradiation intensity stimulation, the neuron arrangement was irregular, the structure was unclear, the morphology was abnormal, and the Nissl body was unclear (shown in the black square).

Figure 9

Results of immunohistochemistry (IHC) staining of astrocyte activation in the spinal cord sections after different irradiation intensities of LIFU stimulation (×40 and ×400). Scale bars = 1 mm and 100 μm. (a) Black arrows show the GFAP-positive astrocytes, and the red arrow shows coagulative necrosis of the spinal cord. A diagram indicating a 300 µm × 300 µm square area was defined for further analysis of the positive cells. (b) Intensity analysis of the GFAP-positive area using ImageJ showed that 3.0 MPa stimulation increased the intensity of GFAP. ^∗∗∗∗P < 0.0001. Each symbol represents the mean ± SEM; one-way ANOVA, followed by LSD test for pairwise comparisons; n = 3 rats per assay.

Figure 10

LIFU stimulation induces the expression of caspase-3 and Bcl-2 as markers of apoptosis induction and apoptosis inhibition. (a–c) Western blot results showed that LIFU stimulation did not significantly alter the expression levels of caspase-3 and Bcl-2 on day 0 (P > 0.05), while (d–f) 3.0 MPa irradiation intensity stimulation increased the expression levels of caspase-3 and Bcl-2 compared to 0 MPa and 0.5 MPa irradiation intensities on day 3 after LIFU stimulation (P < 0.05). There were no significant differences among the 0 MPa, 0.5 MPa, and 1.5 MPa irradiation intensity stimulation groups (P > 0.05). Values were normalized to β-actin. Each symbol represents the mean ± SEM; ^∗P < 0.05, ^∗∗P < 0.01; one-way ANOVA, followed by LSD test for pairwise comparisons; n = 3 rats per assay.