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. 2022 Sep 2;23(17):10034.
doi: 10.3390/ijms231710034.

Ultrasound Neuromodulation Reduces Demyelination in a Rat Model of Multiple Sclerosis

Feng-Yi Yang  1 , Li-Hsin Huang  1 , Meng-Ting Wu  2 , Zih-Yun Pan  1
Affiliations

Ultrasound Neuromodulation Reduces Demyelination in a Rat Model of Multiple Sclerosis

Feng-Yi Yang et al. Int J Mol Sci. .

Abstract

Microglia, astrocytes, and oligodendrocyte progenitor cells (OPCs) may serve as targets for remyelination-enhancing therapy. Low-intensity pulsed ultrasound (LIPUS) has been demonstrated to ameliorate myelin loss and inhibit neuroinflammation in animal models of brain disorders; however, the underlying mechanisms through which LIPUS stimulates remyelination and glial activation are not well-understood. This study explored the impacts of LIPUS on remyelination and resident cells following lysolecithin (LPC)-induced local demyelination in the hippocampus. Demyelination was induced by the micro-injection of 1.5 μL of 1% LPC into the rat hippocampus, and the treatment groups received daily LIPUS stimulation for 5 days. The therapeutic effects of LIPUS on LPC-induced demyelination were assessed through immunohistochemistry staining. The staining was performed to evaluate remyelination and Iba-1 staining as a microglia marker. Our data revealed that LIPUS significantly increased myelin basic protein (MBP) expression. Moreover, the IHC results showed that LIPUS significantly inhibited glial cell activation, enhanced mature oligodendrocyte density, and promoted brain-derived neurotrophic factor (BDNF) expression at the lesion site. In addition, a heterologous population of microglia with various morphologies can be found in the demyelination lesion after LIPUS treatment. These data show that LIPUS stimulation may serve as a potential treatment for accelerating remyelination through the attenuation of glial activation and the enhancement of mature oligodendrocyte density and BDNF production.

Keywords: demyelination; microglia; multiple sclerosis; oligodendrocyte; remyelination; ultrasound.

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Conflict of interest statement

The authors declare that they have no competing interest.

Figures

Figure 1
Figure 1
Evaluation of lesion size and axonal integrity 7 days after LPC-induced demyelination. (A) Immunofluorescent images of NF (green) and MBP (red) at the demyelination lesion in the hippocampus. (B) The percentage area covered by NF+ fibers. (C) The percentage area covered by MBP+ fibers. (D) The percentage of juxtaposed NF+ and MBP+ fibers. (E) The LIPUS-treated rats had a significantly higher remyelination index compared with the sham and LPC-treated rats. * and # denote significant differences from the sham and the LPC groups, respectively (**, p < 0.01; ***, p < 0.001; #, p < 0.05; ###, p < 0.001, n = 5). Scale bar = 100 μm. LPC: lysolecithin, LIPUS: low-intensity pulsed ultrasound, NF: neurofilament, and MBP: myelin basic protein.
Figure 2
Figure 2
LIPUS treatment reduced astrocytic activation and microglial cell density in the vicinity of the demyelination lesion. Immunofluorescent staining image of the astrocytic marker GFAP (A) and the microglial marker Iba1 (B). (C) LIPUS decreased the density of the astrocytic marker GFAP. LIPUS significantly decreased the percentage area covered by GFAP (D) and reduced the GFAP optical density (E) in the vicinity of the demyelinated area of the hippocampus in LPC-treated rats. (F) The LIPUS group showed a significantly reduced density of Iba1+ cells in the vicinity of the demyelination lesion compared to the LPC group. * and # denote significant differences from the sham and LPC groups, respectively (*, p <0.05; **, p < 0.01; ***, p < 0.001; #, p < 0.05; ##, p < 0.01; ###, p < 0.001, n = 5). Scale bar = 100 μm. LPC: lysolecithin, LIPUS: low-intensity pulsed ultrasound, GFAP, glial fibrillary acidic protein, and Iba1: ionized calcium-binding adaptor molecule 1.
Figure 3
Figure 3
Group-specific patterns in Iba1+ microglial morphologies. (A) Five microglial morphologies were analyzed: ramified microglia, hypertrophic microglia, dystrophic microglia, rod-shaped microglia, and amoeboid microglia. Microglial counts are provided for the demyelination lesion in the hippocampus for (B) ramified, (C) hypertrophic, (D) dystrophic, (E) rod-shaped, and (F) amoeboid morphologies. (G) The number of microglia in each of the five distinct classes was plotted as a percentage of the total microglial number for the three groups. The magnification is ×200. # denotes significant differences from the sham and LPC groups, respectively (**, p < 0.01; ***, p < 0.001; #, p < 0.05; ###, p < 0.001, n = 5). LPC: lysolecithin, LIPUS: low-intensity pulsed ultrasound, and Iba1: ionized calcium-binding adaptor molecule 1.
Figure 4
Figure 4
A reduction in Olig2+ cells and an increase in CC-1+ cells and the BDNF level were observed in LPC-induced lesions following LIPUS treatment. (A) Representative immunofluorescent staining of Olig2+ (green) and CC-1+ (red) in the vicinity of the demyelination lesion. Scale bar = 100 μm. Quantification of (B) Olig2+ and (C) CC-1+ cell numbers in the LPC and LPC+LIPUS groups compared to the sham group. (D) Representative immunostaining for BDNF in LPC-induced lesions. Left column: scale bar = 500 μm and, in the right column, 250 μm. (E) The fluorescence intensities of BDNF were significantly decreased in the LPC group compared with the sham group. Compared with the levels observed in the LPC group, the BDNF expression significantly increased in the LPC+LIPUS group. * and # denote significant differences from the sham and LPC groups, respectively (**, p < 0.01; ***, p < 0.001; ###, p < 0.001, n = 5). Scale bar = 100 μm. LPC: lysolecithin, LIPUS: low-intensity pulsed ultrasound, and BDNF: brain-derived neurotrophic factor.
Figure 5
Figure 5
The schematic diagram indicates the potential mechanism through which LIPUS treatment enhances remyelination in a LPC-induced model of demyelination in the hippocampus. Several resident cells impact remyelination after LIPUS treatment, including microglia, astrocytes, oligodendrocyte progenitor cells (OPCs), and oligodendrocytes. Our results demonstrate that LIPUS treatment promotes remyelination in the demyelination lesion. These effects are related to increased expression levels of BDNF and MBP and the inhibition of glial cell activation in LPC-induced demyelination. ↑ and ↓ denote increased and decreased expression, respectively. LPC: lysolecithin, LIPUS: low-intensity pulsed ultrasound, BDNF: brain-derived neurotrophic factor, and MBP: myelin basic protein.
Figure 6
Figure 6
Experimental protocols. (A) Schematic diagram of LPC injections into the hippocampus. (B) The time course of the study. Rats were treated daily with LIPUS for 5 days and then sacrificed at seven days following LPC administration.
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