https://orcid.org/0000-0003-0295-9242
Abstract
It has been shown that transcranial ultrasound stimulation (TUS) is capable of attenuating myelin loss and providing neuroprotection in animal models of brain disorders. In this study, we investigated the ability of TUS to promote remyelination in the lysolecithin (LPC)-induced local demyelination in the hippocampus. Demyelination was induced by the micro-injection of 1.5 μL LPC (1%) into the rat hippocampus and the treated group received daily TUS for 5 or 12 days. Magnetic resonance imaging techniques, including magnetization transfer ratio (MTR) and T2-weighted imaging, were used to longitudinally characterize the demyelination model. Furthermore, the therapeutic effects of TUS on LPC-induced demyelination were assessed by Luxol fast blue (LFB) staining. Our data revealed that reductions in MTR values observed during demyelination recover almost completely upon remyelination. The MTR values in demyelinated lesions were significantly higher in TUS-treated rats than in the LPC-only group after undergoing TUS. Form histological observation, TUS significantly reduced the size of demyelinated lesion 7 days after LPC administration. This study demonstrated that MTR was a sensitive and reproducible quantitative marker to assess remyelination process in vivo during TUS treatment. These findings might open new promising treatment strategies for demyelinating diseases such as multiple sclerosis.
Introduction
Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) and is characterized by myelin loss, inflammation, and axonal loss (Sawcer et al. 2014). The prevalence of MS has been increasing with ~2.8 million people suffering from the disease worldwide (Walton et al. 2020). Current treatments for MS target to the inflammation caused by the disease. Inflammation may indeed be one of the major factors in MS, but the main obstacle in recovery is the remyelination (Trapp and Nave 2008). Until now, many efforts are focused on identifying targets and potential therapies that enhance neuroprotection and the regenerative response leading to remyelination (Emery 2010; Martino et al. 2010). The development of new strategies to promote remyelination is a critical role in the MS treatment, it is essential to monitor this repair process in vivo. However, there is no known imaging marker for the progress of remyelination.
Although traditional magnetic resonance imaging (MRI) methods, such as T1- and T2-weighted imaging, are crucial for the diagnosis of MS, conventional MRI techniques have low sensitivity for the detection of inflammation, demyelination, and reactive glial changes in lesions (Allen et al. 2001). Changes in the magnetization transfer ratio (MTR) have been used as markers of altered myelin levels in models of toxicity-induced demyelination (Merkler et al. 2005; Zaaraoui et al. 2008; Thiessen et al. 2013). MTR measurements are significantly lower in demyelinated areas, and a moderate correlation was identified between MTR values and axonal loss in a myelin lesion model induced by lysolecithin (LPC) administration (Deloire-Grassin et al. 2000). Reductions in MTR values observed during demyelination recover almost completely upon remyelination. LPC-induced damage only affects a limited area for a short period of time, allowing local oligodendrocyte progenitor cells (OPCs) to restore myelin sheaths.
Demyelination is considered to be associated with the development of an inflammatory autoimmune response within the demyelination lesion. Reactive astrocytes and microglia both impact remyelination and produce several pro-inflammatory and anti-inflammatory cytokines (Nair et al. 2008; Lampron et al. 2015). These inflammatory responses not only cause demyelination but also result in neuronal damage (Kutzelnigg et al. 2005; Frischer et al. 2009). Besides, brain-derived neurotrophic factor (BDNF) promotes myelinogenesis by inducing the differentiation of OPCs (McTigue et al. 1998). The astrocyte population may be useful to elaborate BDNF that enhances recovery from demyelination (Fulmer et al. 2014). It has been shown that low-intensity pulsed ultrasound (LIPUS) up-regulates BDNF production in astrocytes through activation of NF-κB via the tropomyosin receptor kinase B (TrkB)–phosphoinositide 3-kinase (PI3K)–protein kinase B (Akt) and calcium–Ca2/calmodulin-dependent protein kinase (CaMK) signaling pathways (Yang et al. 2015; Liu et al. 2017). Moreover, LIPUS treatment significantly enhanced the protein levels of BDNF and attenuated the proinflammatory responses in microglia induced by lipopolysaccharide (LPS; Chang et al. 2020). These findings suggest that LIPUS has potential to serve as a new therapeutic strategy for the treatment of neurodegenerative diseases, such as demyelination disease and MS.
LPC causes toxicity in myelin and was used to produce focal demyelination in vivo (Hall 1972; Blakemore et al. 1977; Azin et al. 2013). In the present study, we explore the possibility that transcranial ultrasound stimulation (TUS) promotes remyelination in a LPC-established model of local demyelination. MRI was exploited to monitor in vivo during the process of demyelination and remyelination. Specifically, changes in signal intensity and MTR were used as markers of alteration in myelin levels induced by LPC, as confirmed by Luxol fast blue (LFB) staining for postmortem histological assessment.
Material and methods
LPC rat model
Male Sprague Dawley rats weighing from 280 to 320 g were purchased from BioLASCO Taiwan Co., Ltd (Yilan City, Taiwan), housed in a 12-h/12-h light/dark cycle, and provided with ad libitum access to food and water. All procedures involving animals were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals. This study protocol was approved by Animal Care and Use Committee of National Yang Ming Chiao Tung University. An animal model of demyelination was induced through the stereotaxic injection of LPC, as previously described (Pourabdolhossein et al. 2014; Mousavi Majd et al. 2018). Prior to surgery, each animal was anesthetized in the prone position by inhalation of 2% isoflurane mixed with O2 at 2 L/min, and the body temperature was maintained at 37°C using a heating pad. The rat heads were mounted on a stereotaxic apparatus (Stoelting, Wood Dale, IL, United States), and skin on the top of the cranium was shaved. A midline scalp incision was made, and the skull was exposed. A single dose consisting of 1.5 μL of 1% LPC (Sigma-Aldrich, St. Louis, MO) dissolved in 0.9% saline was injected unilaterally into the right hippocampus at 0.5 μL/min at the following coordinates: anteroposterior, −3.6 from bregma; mediolateral, +2.6; dorsoventral, −3.2 mm from the dura surface (Fig. 1A). The sham group received an equal volume of sterile saline injected into the same site. Rats were monitored daily for changes in body weight after LPC administration.
Experimental design. A) Schematic diagram of LPC injections into the hippocampus. B) Schematic showing the time course of the study. Three days after LPC injection, rats were treated with LIPUS for 5 or 12 days. Histological evaluations were performed at 7 days, whereas MRI analyses were performed at baseline, 3 days, 1 week, and 2 weeks after LPC injection. C) Body weights were recorded every day following LPC injection. D) The AUC for body weight measurements. LPC injection and LIPUS treatment did not affect the body weight change. N = 8 for each group. LPC: lysolecithin, LIPUS: low-intensity pulsed ultrasound, MRI: magnetic resonance imaging.
Lesion volume changes and normalized T2-weighted maps over time after LPC injection. A) Example of a T2-weighted MRI showing an LPC-induced lesion (white outline). B) The ratio of the ROI volume on the lesion side to that on the contralateral hemisphere significantly decreased at 1 week and 2 weeks compared with 3 days. * denotes significantly different from 3 days (**P < 0.01 ; ***P < 0.001 ; #P < 0.05, n = 4). No significant differences were found between LPC and LPC + LIPUS at every time points. CL: contralateral, LPC: lysolecithin, LIPUS: low-intensity pulsed ultrasound, MRI: magnetic resonance imaging, ROI: region of interest.
T2-weighted images revealed hyperintensities in response to demyelination. A) Signal inversion was observed in the T2-weighted images of the hippocampus and corpus callosum at baseline, 3 days, 1 week, and 2 weeks after LPC injection (arrow: Hippocampus; arrowhead: Corpus callosum). B) In both the hippocampus and corpus callosum, the normalized T2-weighted signal increased at 3 days and then progressively decreased at 1 week and 2 weeks.
Therapeutic ultrasound system and treatment procedure
The TUS setup was similar to that used in our previous study (Huang et al. 2017). LIPUS was generated using a therapeutic ultrasound generator (ME740, Mettler Electronics, Anaheim, CA) and a 1-MHz plane transducer (ME7413: 4.4 cm2 effective radiating area; Mettler Electronics, Anaheim, CA) with 2-ms burst lengths at a 20% duty cycle and a repetition frequency of 100 Hz. The spatial average intensity (SAI) over the plane transducer head was 500 mW/cm2, and was measured with a radiation force balance (RFB, Precision Acoustics, Dorset, United Kingdom) in degassed water. In order to guide and couple the ultrasound to the brain tissue, the transducer was mounted on a removable aluminum cone with a 10-mm diameter at the cone tip. Sonication was precisely targeted using a stereotaxic apparatus (Stoelting, Wood Dale, IL, United States). The acoustic wave was delivered to the targeted region corresponding to the lesion in the brain. Each rat’s right hemisphere was treated with LIPUS using triple sonication. LIPUS treatment started from maximum level of demyelination at 3 days. Animals in the LPC + LIPUS group underwent MRI analysis and histological evaluations after treatment with LIPUS daily for 5 and 12 days, respectively (Fig. 1B). In order to reduce the thermal effect of ultrasound, the duration of each sonication was 5 min, with a 5-min interval between each sonication treatment. The optimal parameters of the LIPUS exposures were selected based on the results of our previous studies (Lin et al. 2015; Su et al. 2017).
Evolution of the MTR values within the hippocampus and corpus callosum at baseline, 3 days, 1 week, and 2 weeks after LPC injection. A) Coronal image of the rat brain showing the selected ROIs within the hippocampus and the corpus callosum where MTR were measured (red outline). B) Representation of 4 MTR images of the same rats from the LPC and LPC + LIPUS groups. MTR maps from which mean MTR values were calculated. Arrow indicates the site of the lesion. Normalized MTR analyzed in the hippocampus C) and corpus callosum D) of LPC and LPC + LIPUS rats. * denotes significantly different from baseline. # Denotes significantly different between LPC and LPC + LIPUS at each time point (*P < 0.05; **P < 0.01; ***P < 0.001; ##P < 0.01; ###P < 0.001, n = 4). LPC: lysolecithin, LIPUS: low-intensity pulsed ultrasound, MRI: magnetic resonance imaging, MTR: magnetization transfer ratio, ROI: region of interest.
Evolution of the MTR values within the hippocampus and corpus callosum from 4 rats at baseline, 3 days, 1 week, and 2 weeks after LPC injection. Intraindividual time courses of 4 different animals in the hippocampus for LPC group A) and LPC + LIPUS group B). Intraindividual time courses of 4 different animals in the corpus callosum for LPC group C) and LPC + LIPUS group D). Note that the MTR values decreased between baseline and 3 days for rats that under LPC injection. All rats of MTR values returned to baseline levels at 2 weeks.
Evaluation of lesion size 7 days after LPC-induced demyelination. A) Representative LFB-stained LPC-injected region in the hippocampus. Scale bar = 2 mm. B) Representative images of LFB staining in the hippocampus for 3 groups. Scale bar = 200 μm. C) The bar graph shows the demyelination lesion size in the 3 groups. * and # denote significantly different from sham and the LPC groups, respectively (***P < 0.001; ###P < 0.001, n = 5). LFB: Luxol fast blue, LPC: lysolecithin, LIPUS: low-intensity pulsed ultrasound.
MRI measurements
MRI of 8 rats was performed using a 7T PET/MR (Biospec AVNEO 70/18 PETMR INLINE, Bruker). Rats were examined by MRI at baseline, 3 days, 1 week, and 2 weeks after LPC injection. The rats were anesthetized with 1.5% isoflurane mixed with O2 and maintained at 1% isoflurane during the imaging procedure. Body temperature was monitored and maintained at 37°C. Transmit-receive volume coils (rat-head volume coil and inner diameter 40 mm) were used. The geometry was identical for all scans: slice thickness = 0.5 mm, field of view (FOV) = 8 × 35 mm, image size = 59 × 258, resolution = 0.136 × 0.136 mm. For the MTR acquisition, a fast low-angle shot (FLASH) sequence was used, with (Ms) and without (M0) an offset magnetization transfer saturation pulse (−2,000 Hz off-resonance, Gaussian-shaped, 10.4-μT strength, 6.65-ms duration, number of pulses: 1, 457-Hz bandwidth, and 400° flip angle), 4 averages, repetition time (TR)/echo time (TE) = 28.5/2.35 ms and flip angle = 10°. A T2-weighted (T2w) rapid acquisition relaxation enhancement (RARE) image was acquired with 3 averages, TR/TE = 3,000/33 ms, and a RARE factor of 8. Brain volumes and lesion volumes were calculated from T2w images processed using ImageJ software (National Institutes of Health). The slice thickness is 0.5 mm. The lesion volume was calculated from T2 hyperintensities. A T1-weighted (T1w) image was acquired with 4 averages and TR/TE = 600/3.88 ms.
The MTR was calculated according to the following equation: MTR = (1 − Ms/M0) × 100, where Ms is the magnitude of the signal with MT saturation and M0 is the magnitude of the signal without MT saturation (Dousset et al. 1998). Signal magnitudes (Ms and M0) were measured on the injection side and on the contralateral side. The region of interest (ROI) was of the same size for both sides analyzed in each animal. The MTR on the lesion side was expressed as a percentage of the MTR on the contralateral side for each animal. The data were compared with the histological results.
Tissue processing
Seven days after LPC injection, 5 rats were sacrificed by transcardial perfusion with phosphate-buffered saline (PBS), and tissues were fixed with 4% paraformaldehyde. Brains were collected, postfixed in 4% paraformaldehyde overnight, and transferred to PBS containing 30% sucrose for cryoprotection. Serial coronal sections were obtained using a microtome. Each section was serially cut in 10-μm coronal slices. The images of 3 brain sections from the center of the demyelination lesion were acquired in 5 rats per group for image analysis.
LFB staining
Paraffin-embedded brains were coronally cut at a 10-μm thickness. LFB staining was performed to assess the extent of the demyelination lesion in the hippocampus. Three brain sections from the center of the demyelination lesion were imaged in 4 rats per group at 10× magnification under a bright field microscope (Olympus BX-63, Tokyo, Japan). The demyelinated area was delineated and measured using Image J software.
Statistical analysis
All data are presented as the mean ± standard error of the mean (SEM). Two-way repeated-measures analysis of variance (ANOVA) followed by Tukey’s post-hoc test was used to determine significant differences in MRI data. One-way ANOVA with Tukey’s post-hoc test was used to evaluate histological data. Significance was established at a P value ≤ 0.05.
Results
Body weight changes
Changes in body weight (Fig. 1C) and the area under the curve (AUC) values for body weight measurements in rats following LPC administration with or without LIPUS treatment did not differ from those for the sham group. No significant difference was found in Fig. 1D.
In vivo MRI analysis
The LPC-induced brain lesion spread from the injection site (Fig. 2A). The evolution of demyelinating lesions was monitored using T2w MRI at 3 days, 1 week, and 2 weeks after LPC injection (Fig. 2B). At the corresponding time points, no significant attenuation of the lesions was identified in LPC-administered rats following LIPUS treatment compared with LPC-administered rats without LIPUS treatment. As the LPC-induced lesion progressed, contrast inversion was evident in the hippocampus and corpus callosum (Fig. 3A). Compared with the baseline group, the normalized T2w signal increased at 3 days and then progressively decreased at 1 and 2 weeks in both the hippocampus and corpus callosum (Fig. 3B).
We used MTR to assess demyelination and recovery following LPC administration. ROIs on T2w MRI included the hippocampus and the corpus callosum (Fig. 4A). Figure 4B shows the MTR maps used to assess myelin recovery after LPC injection. To evaluate the longitudinal evolution of the MTR, data from 4 rats in each group were examined at 3 days, 1 week, and 2 weeks after LPC injection (Fig. 4C and D). At 3 days, the MTR measured within the demyelinated lesion of hippocampus significantly decreased compared with baseline (P < 0.001, Fig. 4C). MTR values measured within the corpus callosum also significantly decreased at 3 days compared with baseline (P < 0.001, Fig. 4D). The mean MTR values in both regions were higher in LIPUS-treated rats after LPC induction than in LPC-induced rats without LIPUS treatment at all 3 time points, and these differences were significant at 1 week (27.53 ± 0.26 vs. 25.91 ± 0.34, P < 0.01; n = 4, Fig. 4C) and 2 weeks (30.32 ± 0.19 vs. 28.06 ± 0.32, P < 0.001; n = 4, Fig. 4C) in the hippocampus and at 1 week in the corpus callosum (31.73 ± 0.31 vs. 29.20 ± 0.35, P < 0.01; n = 4, Fig. 4D).
Because the evaluation in Fig. 4C and D may have been caused by interindividual differences, the intraindividual follow-up studies were carried out in an additional cohort of 4 different rats. To assess longitudinal evolution of the MTR, values from 4 rats followed at baseline, 3 days, 1 week, and 2 weeks after LPC injection are showed in Fig. 5. The MTR values decreased significantly between baseline and 3 days. All rats of MTR values showed an increase between 3 days and 2 weeks and returned to the baseline levels.
Myelin staining
To confirm the demyelinating size in the hippocampus by LPC, we used LFB staining (Fig. 6A; Mozafari et al. 2011). Saline injections did not result in any obvious demyelination activity in the hippocampus of the sham group 7 days postinjection (Fig. 6B). The LPC-injected hippocampus showed significant demyelination activity compared with the hippocampus of the sham group (0.27 ± 0.02 vs. 0.05 ± 0.02, P < 0.001; n = 5, Fig. 6C). The size of the LPC-induced demyelination lesion significantly decreased in LPC + LIPUS rats compared with LPC-only rats (0.04 ± 0.02 vs. 0.27 ± 0.02, P < 0.001; n = 5, Fig. 6C). No significant difference in the size of the demyelination lesion was observed between the sham and LPC + LIPUS groups.
Discussion
More recent studies showed TUS can ameliorate myelin loss in the animal models of brain disorders (Yang et al. 2015; Huang et al. 2017). LPC demyelinates the axons in the injection site and provides a suitable model for exploring the remyelinating response. This study demonstrates that TUS could enhance remyelination and reduce demyelinating lesion size in the hippocampus of LPC-treated rats. Moreover, MTR was a sensitive, quantitative marker for assessing changes in myelin content in vivo during remyelination following TUS treatment.
Although MS presents with hyperintense lesions on T2w MRI, conventional MRI suffers from a lack of specificity, which may explain the lack of significant difference observed in the lesion sizes measured on T2w MRI between the LPC + LIPUS and LPC-only groups (Fig. 2B). However, significant changes occurred in both the T2w and MTR values of the hippocampus and corpus callosum 3 days after LPC administration. This corresponds to the significant demyelinating lesion size observed in LFB stained histology after LPC administration (Fig. 6C). MTR has been utilized as a semiquantitative method that may be sensitive to myelin content changes in MS (Dousset et al. 1992; Grossman 1994). The marked MTR increase thus indicates that the lesion undergoes remyelination. The MTR could be exploited to monitor not only demyelinatin but also remyelination (Waxman et al. 1979; Ford et al. 1990). Our data confirm that an increase in T2w and a reduction in MTR are related to demyelination following LPC administration. In agreement with previous studies, the observed decrease in MTR during demyelination recovered almost completely upon remyelination (Fig. 4C and D; Deloire-Grassin et al. 2000; Giorgetti et al. 2019). Based on in vivo longitudinal evaluations, our results showed that MTR could serve as a sensitive, quantitative marker for assessing myelin content changes during remyelination after TUS treatment.
LPC is a lipid-disrupting agent known to induce focal demyelination in the CNS (Plemel et al. 2018). Several studies have demonstrated that microinjection of LPC into the corpus callosum induced demyelination at 1 week postinjection which can be detected by MRI (Deloire-Grassin et al. 2000; Magalon et al. 2012). In the present study, the LPC-induced demyelination in the hippocampus and corpus callosum was shown reached their maximum level at 3 days and was reduced at 1 week and 2 weeks postinjection. The anti-inflammatory and reduced demyelinating effects of LIPUS were previously mentioned in animal models of different neurological disorders and in vitro (Yang et al. 2015; Huang et al. 2017; Chen et al. 2019). Our MTR analysis confirmed that treatment with TUS effectively promoted the remyelination at 1 week and 2 weeks compared with related times in LPC groups (Fig. 4C and D). In particular, TUS enhanced the MTR even to the values more than that in the baseline in the hippocampus at 2 weeks after LPC administration. A beneficial therapeutic effect on MS could be caused either by decreasing demyelination or by increasing remyelination. Here, our results raise the possibility that the therapeutic effect of TUS in LPC-induced model may in part be the result of enhanced remyelination because the TUS treatment started from maximum level of demyelination at 3 days. Therefore, the effect of TUS on demyelination should be investigated in our future studies.
This study suggests that LIPUS stimulation can enhance the remyelinating effects in an LPC-induced model of demyelination. Our data show that MTR measurements represent a valuable in vivo technique with a potential application for assessing the TUS-mediated promotion of remyelination. The MTR profile could be used to monitor in vivo the myelin content and possible therapeutic action promoting remyelination.
Funding
This study was supported by grants from the Ministry of Science and Technology of Taiwan (no. MOST 110-2314-B-A49A-502-MY3 and MOST 108–2314-B-010-034-MY3), FEMH-NYMU Joint Research Program (nos. 111DN27 and 110DN33). Conflict of interest statement: None declared.
References
Author notes
Li-Hsin Huang, Zih-Yun Pan, Yi-Ju Pan contributed equally to this work.
