Progenitor Cell

3 days ago - *(I.-C.L., Cell Biology) Mailing address: 259 Wen-Hwa first Road, Kwei-Shan, Tao-Yuan, 33302 Taiwan. E-mail: [email protected]...
0 downloads 0 Views 12MB Size
Download PDF
Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

pubs.acs.org/chemneuro

Dual-Frequency Ultrasound Induces Neural Stem/Progenitor Cell Differentiation and Growth Factor Utilization by Enhancing Stable Cavitation I-Chi Lee,*,†,‡ Hui-Ju Wu,† and Hao-Li Liu*,§,∥ †

Graduate Institute of Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan Neurosurgery Department, Chang Gung Memorial Hospital, Linkou, Taiwan § Department of Electrical Engineering, Chang Gung University, Taoyuan, Taiwan ∥ Department of Neurosurgery, Chang Gung Memorial Hospital, Taoyuan, Taiwan ACS Chem. Neurosci. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/06/19. For personal use only.

‡

ABSTRACT: Neural stem/progenitor cells (NSPCs) have the potential to serve as the basic materials for treating severe neural diseases and injuries. Ultrasound exposure is an effective therapy for nonunion fractures and healing fresh wounds through an easy and noninvasive application. According to the results of our preliminary study, low-intensity ultrasound (LIUS) promotes the attachment and differentiation of NSPCs. However, the parameters of and mechanisms by which LIUS induces NSPC differentiation remain unclear. To the best of our knowledge, no published studies have reported and compared the biological effects of dual-frequency and single-frequency LIUS on NSPCs. The purpose of this study is to systematically compare several LIUS parameters, including single-frequency, single-transducer dual-frequency ultrasound, burst, and continuous cycling stimulation at several intensities. Furthermore, synergistic effects of single-/dual-frequency LIUS combined with neural growth factor addition on NSPCs were also evaluated. Based on the results of the cytotoxicity assay, lowintensity (40 kPa) ultrasound does not damage NSPCs compared with that observed in the control group. The morphology and immunostaining results show that all experimental groups exposed to ultrasound exhibit neurite outgrowth and NSPC differentiation. In particular, dual-frequency ultrasound promotes NSPCs differentiation to a greater extent than singlefrequency ultrasound. In addition, more complicated and denser neural networks are observed in the dual-frequency group. Neural growth factor addition increased the percentage of neurons formed, particularly in the groups stimulated with ultrasound. Among these groups, the dual-frequency group exhibited significant differences in the percentage of differentiated neurons compared with the single-frequency group. This study may the first to prove that dual-frequency LIUS exposure further enhances NSPC differentiation and the utilization of growth factors than single-frequency LIUS. Moreover, the result also revealed that dual-frequency ultrasound generated higher calcium ion influx and extended the channel opening time. A potential explanation is that dual-frequency ultrasound generates more stable cavitation than single-frequency LIUS, which may stimulate cell membrane mechanochannels and enhance calcium ion influx but does not damage them. This in vitro study may serve as a useful alternative for ultrasound therapy. KEYWORDS: Neural stem/progenitor cells, single transducer dual-frequency ultrasound, differentiation, synergistic effect, stable cavitation

■

INTRODUCTION

and cost-effective therapy. Additionally, several studies have described different tissue responses and the effect of low intensity ultrasound (LIUS) on cells. LIUS increases cell differentiation, protein synthesis, and calcium influx.10−12 Moreover, LIUS has already been proven to induce chondrocyte phenotypes in vitro,13 promote chondrogenic progenitor cell migration,14 and improve cartilage and bone repair in animal models.15,16 Consequently, LIUS has been considered a promising physical agent and effective therapy to accelerate bone and tissue regeneration following injury.16−20

Mechanical stimulation is the most widely used biophysical stimulus and has been shown to improve tissue regeneration and cell differentiation in several cell types.1−3 Cells sense changes in their mechanical environment and promote alterations and adaptations to tissue structure and function. Mechanical stimuli regulate fundamental processes such as cell proliferation and differentiation by increasing the production of extracellular matrix (ECM) components or regulating gene expression.3−5 Previous studies have employed diverse mechanical stimuli to induce stem cell differentiation, such as hydrostatic pressure, cyclic compressive loading, mechanical stress and oscillatory fluid flow.6−9 Ultrasound is a clinically effective mechanical stressor that provides simple, noninvasive © XXXX American Chemical Society

Received: September 11, 2018 Accepted: November 27, 2018

A

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

(denoted as DB40), single frequency/continuous wave/40 kPa (denoted as SC40), and dual frequency/continuous wave/ 40 kPa (denoted as DC40). For the continuous wave exposure model, the increase in temperature was confirmed to be less than 1 °C, thus avoiding a potential thermal effect. In addition, the parameter of 40 kPa is equal to the intensity of spatial peak temporal average intensity (Ispta) = 533 mW/cm2, which is less than 680 W/cm2, the diagnostic ultrasound level approval by the United States Food and Drug Administration . Furthermore, the output intensities of single- and dualfrequency ultrasound were identical to compare the cavitation effect at the same exposure level. Moreover, compared with continuous mode, burst mode was established with a 20% output duty percentage to assess the biological effect of a fixed acoustic pressure and a decreased total intensity. We postulated that burst mode may reduce thermal and mechanical damage to cells and maintain the differentiation effect on neural stem cells. Figure 1B illustrates the ultrasound waveforms of burst and continuous excitation. NSPCs were cultured on glass coverslips coated with PLL in multiwell plates with/without NGF addition and were subjected to the four experimental parameters of LIUS stimulation. In each experiment, NSPCs were seeded on glass coverslips coated with PLL at an initial density of 200 neurospheroids per well. Figure 1C also displays the frequency spectrum of LIUS, with the receiving signals consisting of single- or dual-frequency components easily identified (blue: single-frequency exposure; red: dual-frequency exposure). The single-frequency exposure presented a single frequency signature at 1138 kHz and dualfrequency exposure presented two frequency signatures at 560 and 1138 kHz. LDH Assay. After isolation, NSPCs were seeded and cultured on glass coverslips coated with PLL and were or were not exposed to LIUS at the different stimulation parameters. The biocompatibility and cytotoxicity of NSPCs exposed to LIUS were assessed by analyzing the morphology and performing LDH assays. Based on the results of the preliminary test, neurite damage and cell detachment were observed when NSPCs were exposed to a higher intensity of LIUS (burst and continuous wave/80 and 100 kPa) (data not shown). Neurite retraction and cell body shrinkage have been observed in neuronal cells over a 10 min exposure period with 1.168 W/cm2 spatial-peak, time-averaged intensity.31 Additionally, LDH release exhibited a slight increase in all of the higher intensity stimulation groups (data not shown). The thermal effect and stronger cavitation may result in acute mechanical stress that affects the viability and adhesion of NSPCs. Hence, only four different LIUS parameters with low intensity exposure (SB40, DB40, SC40, and DC40) were used in subsequent studies to compare the biological effects. Figure 2 reveals the results of the LDH assay using neurospheroids incubated under different conditions after one and two exposures to LIUS. After a 5 day incubation, no significant differences in LDH release were observed between the four LIUS exposure groups and the control group. In addition, the DC40 group released the lowest amount of LDH, a value that was less than that observed in the control group. After a 7 day incubation, higher OD values were recorded in all groups than at 5 days, potentially due to long-term culture without a media change. In addition, after two LIUS exposures, SC40 and DC40 groups showed lower LDH release than the control group. Thus, the application of LIUS with appropriate parameters may exert a protective effect.

However, most of the in vitro and in vivo studies have focused on connective tissue repair and the induction of mesenchymal stem cell differentiation,21−23 whereas only a few studies have explored the biological effects of ultrasound on neural systems and neurons and glial cells. Additionally, most of these studies focused on peripheral nerve regeneration. A previous study has determined the effects of LIUS on the viability and neural differentiation of induced pluripotent stem cell-derived neural crest stem cells and indicated that LIUS may represent an alternative treatment for peripheral nerve regeneration.24 In studies combining LIUS and coculture, the expression of promyelination factors is increased in Schwann cells and the myelination ability of these cells is increased.25 In addition, LIUS stimulation increases levels of the BDNF and VEGF proteins in astrocytes, suggesting that LIUS may exert neuroprotective effects.26 Neural stem/progenitor cells (NSPCs) have the potential to regenerate different types of functional neurons and treat neurodegenerative diseases and neuronal injuries.27,28 After induction, NSPCs differentiate into three lineages: neurons, astrocytes, and oligodendrocytes.27 Therefore, a study of the effects of LIUS exposure on NSPCs should not only evaluate the induction effect but also provide a biomimetic niche to model the brain environment. Based on the findings from our preliminary study, LIUS promotes cell attachment, NSPC differentiation, and neurite outgrowth, and enhances growth factor utilization.29 However, the parameters of and the mechanisms by which LIUS induces NSPC differentiation remain unclear. Liu and Hsieh have proposed a single transducer dual-frequency ultrasound configuration and indicated that dual-frequency ultrasound stimulation enhances acoustic cavitation compared with single-frequency ultrasound stimulation.30 To the best of our knowledge, no published studies have reported and compared the biological effects of dual-frequency and single-frequency LIUS on NSPCs. Therefore, single transducer dual-frequency ultrasound was used to evaluate the biological effects of different exposure parameters and cavitation on the induction of NSPC differentiation in vitro. Consequently, this study systematically compared several LIUS parameters, including single-frequency, single transducer dual-frequency ultrasound, burst, and continuous cycling stimulation with several intensities. Cell morphology, cell viability, phenotype, differentiation, neurite outgrowth and neuron percentages were determined. Moreover, the effects of single and dual-frequency LIUS on differentiation were compared, the synergistic effects of single-/dual-frequency LIUS combined with neural growth factor addition were investigated, and the type of cavitation produced by single-/ dual-frequency LIUS was also quantified.

■

RESULTS AND DISCUSSION Parameter Pretesting and Experimental Design of the Four Groups Exposed to LIUS. Based on the results of our preliminary test, higher intensity (single frequency/burst wave/80−120 kPa and dual frequency/burst wave/80−100 kPa) prevented cell adhesion and resulted in cell damage (data not shown) due to mechanical stress. In particular, single or dual frequency/continuous wave/80−100 kPa resulted in not only sphere nonadhesion but also cell damage due to higher mechanical stress and thermal effects. Hence, four experimental LIUS parameters were established to compare the effects of LIUS, including single frequency/burst wave/40 kPa (denoted as SB40), dual frequency/burst wave/40 kPa B

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

Figure 2. LDH assay of neurospheroids cultured on glass coverslips stimulated with ultrasound using four different simulated parameters (SB40, DB40, SC40, and DC40) after 5 and 7 days of incubation.

Figure 1. (A) Schematic illustrating the ultrasound stimulation apparatus and device. The customized transducer used in this experiment is a planar type ultrasound transducer (Eleceram Technology, Taoyuan, Taiwan; PZT-4 type, primary resonance frequency = 1138 kHz and secondary resonant frequency = 560 kHz, diameter = 20 mm, thickness = 3 mm). The transducer was powered by a custom-designed multiple-channel driving system. (B) Waveforms of continuous and burst waves from the ultrasound transducer. (C) Comparison of the single- and dual-frequency spectra. Blue and red curves represent the single- and dual-frequency ultrasound, respectively. Figure 3. Phase contrast images showing the phenotypes of cells that migrated from neurospheroids. (A) Images of neurospheroids cultured on glass coverslips with and without LIUS stimulation after 3 and 7 days of incubation. (B) Phase contrast images of neurospheroids cultured on glass coverslips exposed to single- and dual-frequency ultrasound after 3 and 7 days of incubation.

Differentiation Capacity of NSPCs Following Singleand Dual-Frequency LIUS Exposure. Four LIUS parameters were compared in this study to determine the biological effects of different LIUS parameters on NSPCs. Here, SC40 and DC40 were chosen to compare the effects of single- and dual-frequency LIUS on NSPC differentiation and morphology. Figure 3 shows photomicrographs depicting the morphologies of cells that differentiated from embryonic

cerebral cortical neurospheroids cultured on glass coverslips with/without LIUS exposure. As shown in Figure 3A, cells C

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

levels in the DC40 group and cells extended very long processes. Synergistic Effects of Single/Dual LIUS and NGF. Biochemical signals are widely used to regulate stem cell differentiation. Growth factors such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and NGF have been used to enhance the directional NSPCs differentiation efficiency.32 Additionally, LIUS has also been proven to enhance drug delivery and molecule uptake.33 However, no studies have discussed and compared the synergistic effects of single-/dual-frequency LIUS and growth factor treatments. Here, the synergistic effects of SC40 and DC40 with NGF treatments were determined. As shown in Figure 5A, compared

migrated out from their original aggregates. Cells extended short and thin processes in the control group. In contrast, after 7 days of incubation, more cells in the neurospheroids from the DC40 group attached, differentiated, and extended processes. Moreover, as shown in Figure 3B, dual-frequency ultrasound (DC40) promotes NSPC differentiation to a greater extent than single-frequency ultrasound (SC40). In addition, more complicated and denser neural networks were observed in the DC40 group. In summary, the results of the morphological investigation revealed that cells in the LIUS groups displayed longer processes and better neural networks, particularly the DC40 group. NSPCs have the capacity to differentiate into several types of neuronal and glial cells. However, the type of differentiated cell is not identified based on morphology. After 7 days of incubation, immunostaining with specific antibodies was performed to analyze differentiated cell phenotypes. In this study, the expression of the neuron marker MAP-2 (red) and the astrocyte marker GFAP (green) was used to determine the cell types. As shown in Figure 4A, all LIUS stimulation groups

Figure 5. Phase contrast images showing cells cultured on glass coverslips stimulated with LIUS in combination with NGF. (A) Images of neurospheroids cultured on glass coverslips stimulated with and without LIUS and NGF after 7 days of incubation. (B) Images of neurospheroids cultured on glass coverslips stimulated with singleand dual-frequency ultrasound stimulation in combination with NGF after 7 days of incubation.

with the group lacking NGF, cells migrated out from their original aggregates and extended processes in all groups treated with NGF. In addition, process outgrowth was increased in all groups treated with NGF. Moreover, as shown in Figure 5B, extensive process outgrowth, and compact and closely connected networks were observed in the DC40 group treated with NGF. Furthermore, as shown in Figure 6, photomicrographs of immunofluorescence staining revealed the highest expression of MAP-2 in the DC40 group treated with NGF, indicating that more MAP-2-positive neuronal cells migrated away from the neurospheroids after LIUS stimulation. The numbers of MAP-2- and GFAP-positive cells were divided by the total number of cells that migrated out of the neurospheroids to quantify the phenotypes of differentiated cells (Figure 6B). The relative percentages of cells that differentiated into neurons and astrocytes are presented in Figure 6C. As shown in Figure 6B, all groups treated with NGF showed

Figure 4. Photomicrographs of immunofluorescence staining for determining the phenotypes of differentiated cells that migrated from neurospheroids after a 7-day incubation. Anti-MAP-2 (red) and antiGFAP (green) antibodies were used to immunostain neurons and astrocytes, respectively. (A) Images of NSPCs cultured on glass coverslips and exposed to LIUS with four different stimulation parameters (SB40, SC40, DB40, and DC40). (B) Photomicrographs showing the immunofluorescence staining of NSPCs exposed to single- and dual-frequency LIUS.

exhibited increased NSPC differentiation, including MAP-2 and GFAP expression, compared with the control group. Among these four LIUS groups, the dual-frequency LIUS groups (DC40 and DB40) expressed the markers at higher levels than the single-frequency groups (SC40 and SB40). As shown in Figure 4B, GFAP was expressed at particularly high D

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

derived neurotrophic factor in astrocytes.35 In this biomimetic system, we predicted that LIUS stimulated astrocytes release growth factors that may also increase neuron viability and function. In summary, LIUS stimulation, particularly dualfrequency LIUS, enhanced the utilization of NGF and substantially increased differentiation of neurons. Thus, dualfrequency LIUS stimulation enhanced NSPC differentiation to produce larger quantities of functional neurons that may represent a new opportunity in applications of neural disease therapy. Analysis of Functionally Active Synapses. In addition to the differentiation analysis, Figure 7 shows the results of the functional assessment of active synapses formed by differentiated neurons cultured on glass coverslips substrates with/ without DC40 LIUS exposure. Synaptic vesicles in the differentiated neurons were stained with the FM1-43 membrane dye. Cells were stimulated with a high potassium solution, and the fluorescence intensity of the membrane dye decreased after the second stimulation with the high potassium solution lacking the membrane dye. Fluorescence intensities of the synapses before and after dye removal were analyzed. It is revealed that the synaptic activity of the DC40 LIUS group was higher than that on glass control. Based on this result, the synaptic vesicles were functional and recyclable, supporting hypothesis that LIUS exposure not only induces NSPC differentiation into neurons but also triggers and enhances synaptic function. Live Cell Calcium Imaging and Cavitation Analyses. It is demonstrated that ultrasound stimulation induced Ca2+ signaling in mesenchymal stem cells and affected differentiation in a previous study.36 In addition, neurons stimulated with low intensity ultrasound exhibit increased concentrations of calcium ions and neural excitation.37 Here, we first compared the live NSPC calcium images after 5 min of singleand dual-frequency ultrasound stimulation, and the results are shown in Figure 8. Low intensity ultrasound promoted calcium channel opening and increased calcium ion influx compared with the control group. Furthermore, compared with singlefrequency ultrasound, Figure 8B also reveals that dualfrequency ultrasound generated higher calcium ion influx and extended the channel opening time. Dual-frequency LIUS stimulation enhanced NSPC differentiation and promoted growth factor usage compared with single-frequency LIUS. Stable cavitation and inertial cavitation were quantified to determine an explanation for these findings. Acoustic cavitation is the formation and collapse of preexisting microbubbles in a liquid under the influence of an acoustic pressure field. Cavitation is generally classified into two types: stable cavitation, which results in emissions at subharmonics of the main excitation frequency, and inertial cavitation, which is characterized by broadband noise emissions.38 As shown in Figure 9, stable cavitation occurred more frequently in the dual-frequency ultrasound group than in the single-frequency group, consistent with the study by Liu and co-workers.30 Moreover, stable cavitation increases cytoplasmic calcium concentrations in fibroblasts.39 Dual-frequency ultrasound stimulation at an appropriate intensity may provide more stable cavitation than single-frequency ultrasound, which may stimulate cell membrane mechanochannels but does not damage them. Calcium signaling induced by LIUS through stable cavitation would more efficiently induce cell differentiation. Further research is required to understand the response of the increase in Ca2+ influx and to determine the

Figure 6. Photomicrographs showing immunofluorescence staining of differentiated cells treated with and without NGF after 7 days of incubation. Anti-MAP-2 (red) and anti-GFAP (green) were used to immunostain neurons and astrocytes, respectively. (A) Images of NSPCs cultured on glass coverslips and stimulated with the two different parameters of LIUS combined with NGF. (B) Quantification of the relative percentages of cells that differentiated into neurons and astrocytes (C) Ratio of the area of differentiated neurons and astrocytes divided by the total area of cells that migrated out of neurospheroids for normalization.

significant differences in the percentage of neurons compared with those groups that were not treated with NGF. Moreover, DC40 groups showed significant differences in the percentage of neurons compared with the other groups, as shown in Figure 6C. In contrast, significant differences in the percentage of astrocytes were not observed among all groups exposed to LIUS. We postulated that NGF is a directional differentiation factor, and it exerted a greater synergistic effect on neuron differentiation than on astrocyte differentiation. LIUS enhances inductive molecule uptake and promotes differentiation.34 According to Lin et al., LIUS increases the expression of brainE

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

Figure 7. Photomicrographs showing FM1-43 labeling of recycling synaptic vesicles in cells cultured on glass coverslips and exposed to DC40 LIUS. (A) NSPCs were cultured on glass coverslips and stimulated with/without LIUS before and 100 s after the stimulation with a high concentration of KCl. (B) Quantification of the decrease in the relative fluorescence intensity before and after stimulation in the two groups from three independent experiments.

■

pathway induced by different types of cavitation generated from single and dual-frequency ultrasound. The physical mechanism by which dual-frequency LIUS induced stable cavitation and increased differentiation remains unclear and is the subject of further ongoing studies.

METHODS

Apparatus and Experimental Design of LIUS. As shown in Figure 1A, a customized LIUS apparatus and transducers were used in this study. The customized transducer used in this experiment is a planar type ultrasound transducer (Eleceram Technology, Taoyuan, Taiwan; PZT-4 type, primary resonance frequency = 1138 kHz and secondary resonant frequency = 560 kHz, diameter = 20 mm, thickness = 3 mm). The transducer was powered by a customdesigned multiple-channel driving system. The device generates LIUS with bursts or continuous waves in single (1138 kHz) or dualfrequency (560 and 1138 kHz) excitation mode at a peak pressure of 40 kPa. In burst excitation mode, an exposure of 1 kHz of pulse repetition frequency (PRF) and 20% of duty cycle was employed. The LIUS treatments began after the initiation of cell culture for 3 days for a period of 5 min/2 days and repeated for 2−3 cycles. Glass coverslips used for culture were cleaned with distilled water and then dried under a stream of nitrogen. Before seeding, glass coverslips with diameter of 12 mm were coated with poly-L-lysine (PLL) (Sigma, USA), placed in a 24-well plate, and exposed to ultraviolet light overnight. In the control group, glass coverslips were placed on the same transducers for the same duration, but LIUS was not administered. As shown in Figure 1, the active transducer was

■

CONCLUSIONS LIUS promotes NSPC differentiation, and the effect may be enhanced when LIUS is combined with growth factors. In particular, significant differences in the percentage of differentiated neurons and network formation were observed between the single transducer dual-frequency ultrasound and single-frequency ultrasound group. We postulate that dualfrequency ultrasound stimulation may induce more stable cavitation than single-frequency ultrasound and subsequently stimulates cell membrane mechanochannels and enhances calcium ion influx without damaging them. This study is the first to show the effect of dual-frequency ultrasound on NSPC differentiation and suggest that this treatment represents an alternative method for inducing stem cell differentiation. F

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

Figure 8. Increase in the intracellular Ca2+ concentration triggered by ultrasound. Single- and dual-frequency ultrasound was applied to NSPCs for 5 min. (A) Fluo-4 AM labeling in NSPCs triggered by dual-frequency ultrasound after 1, 6, 8, and 20 min. (B) Normalized changes in the fluorescence intensity of the calcium indicator, Fluo-4 AM. Delayed Ca2+ responses were monitored in NSPCs exposed to single- and dualfrequency ultrasound after 25 min.

Figure 9. Analysis of cavitation induced by dual-frequency and single-frequency ultrasound. (A) Stable cavitation and inertial cavitation. (B) Analysis of the magnitudes of the two types of cavitation. pelleted by centrifugation and resuspended in serum-free medium. Cells were counted using a hemocytometer. Then, cells were incubated in T25 culture flasks (Corning, USA) in serum-free culture medium supplemented with basic fibroblast growth factor (Invitrogen, USA) at 37 °C in a humidified atmosphere of 95% air/5% CO2. After 3 days, adherent cells were discarded and only suspended neurospheroids were collected. Neurospheroids were dissociated into single cells and subcultured at a density of 50,000 cells/cm2. The subculture procedure was repeated 2−3 times to purify NSPC spheroids and allow the cells to proliferate.

arranged in a chess distribution and red wells were seeded with NSPCs and exposure to LIUS to minimize mutual coupling interference when cells were exposed to LIUS with different parameters. Isolation and Culture of Neural Stem/Progenitor Cells (NSPCs). NSPCs were isolated from the cerebral cortices of ED 14−15 Wistar rat embryos using our previously reported protocol, with some modifications.40 Briefly, cerebral cortices were isolated from rat embryos, dissected, cut into small pieces, and mechanically crushed in Hank’s balanced salt solution. Dissociated cells were G

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience Cytotoxicity Assay. The cytotoxicity of NSPCs exposed to four LIUS parameters of SB40, DB40, SC40, and DC40 was evaluated using a lactate dehydrogenase (LDH) kit (Roche, Mannheim, Germany). The principle is to measure extracellular LDH levels in the culture media released from cells with plasma membrane damage using an enzymatic reaction. The culture medium was collected directly from four experimental groups and control group, incubated with the reaction mixture according to the manufacturer’s protocol and then measured spectrophotometrically. Absorbance was recorded at 492 nm using a microplate reader with a reference wavelength of 630 nm. Immunocytochemistry. Immunocytochemistry detects specific antigens in preserved cell populations using an antibody labeling strategy. After 5 days of culture, cells were collected and fixed with ice-cold 4% paraformaldehyde in PBS for 20 min and rinsed three times with PBS. Then, the primary monoclonal antibodies were diluted to the appropriate concentration using blocking solution. Cells were incubated with the following primary antibodies at the indicated dilutions in a solution containing 0.3% Triton X-100 and 10% bovine serum albumin (BSA) for 2 h at 37 °C: antimicrotubule associated protein 2 polyclonal antibody (anti-MAP-2; 1:1000; Millipore) for detecting neurons and rabbit antiglial fibrillary acidic protein polyclonal antibody (anti-GFAP; 1:1000; Millipore) for detecting astrocytes. Cells were then incubated with FITC- and rhodamineconjugated secondary antibodies (AP187F; AP181 R; 1:250; Millipore) for 30 min at room temperature to visualize the signal. DAPI (1:2000) was diluted in 1% blocking solution and incubated with the cells for 10 min, and then a drop of mounting medium was added to each slide and sealed with a coverslip. Immunostained cells were visualized using a fluorescence microscope (Axiovert 100TV, Germany). The antibodies used in this study were pretested in preliminary studies. Analysis of the Percentages of Differentiated Neurons and Astrocytes. The immunostaining results were quantified with a microscope equipped with standard fluorescence illumination and a digital camera. For each experimental condition, images of at least three randomly selected microscopic fields were captured. The number of cells expressing a specific phenotypic marker was divided by the total number of cells that migrated out of neurospheroids to calculate and the ability of NSPCs to differentiate into neurons and astrocytes under different experimental conditions. Finally, relative percentages of cells that differentiated into neurons and astrocytes were presented. Data were recorded from three independent experiments and analyzed using ImageJ software. Labeling of Active Synapses Following Exposure to LIUS or the Control Condition. Synapses were loaded with FM1-43 and destained to determine the activity of synaptic vesicles. Standard procedures for culturing NSPCs with/without LIUS exposure and FM1-43 dye were performed as described in previous study.41 After culture with/without two exposures to the LIUS stimulus, a 90 mM KCl solution containing 2 μM FM1-43 membrane probe (N-(3triethylammoniumpropyl)-4-(4-(dibutylamino) styryl) pyridinium dibromide) (Invitrogen) was added and incubated with the cells for 60 s. Then, the cells were rinsed with normal saline three times for 5 min each to remove surface-bound FM1-43. Then, the synapses were destained with the 90 mM KCl solution lacking FM1-43 for 150 s. Images of the FM1-43-loaded synaptic vesicles in cells were captured with a confocal microscope (LSM 510 META, Zeiss, Germany). Live Cell Calcium Imaging and Quantification. The intracellular calcium concentration was monitored and measured using cell-permeable Fluo-4 AM (Thermo Fisher, MA, F-14201). After stimulation with/without LIUS, cultured NSPCs were washed with buffer and incubated with Fluo-4 AM (2 μM) at room temperature for 1 h. Afterward, the dish was washed twice. A confocal microscope (LSM 510 META, Zeiss, Germany) was used for live cell imaging of intracellular calcium signaling. Changes in the fluorescence intensity of the calcium indicator in ROIs were also recorded. Cavitation Analysis. Passive cavitation emissions were received via the piezoelectric receiving-ring transducer and commercial water immersion transducer to characterize stable cavitation (characterized

by subharmonic and harmonic components), and every pulse was recorded on a personal computer using a PCI-based oscilloscope with a sampling rate of 60 MHz and 32,768 sampling points. The oscilloscope was synchronized based on triggers from the US exposure system. Energy spectral density (ESD, in unit of V2·s· Hz−1) at the selected frequency ωt was used to calculate cavitation dose42 and defined as follows: ESD =

ωt + B /2

∫ω −B/2

|F(jω)|2 dω

(1)

t

where F(jω) is the fast Fourier transform of the passive emission signal in the time domain and B is the designated computation bandwidth. In our experiment designed to specifically examine the spectral information with respective to specific cavitation type, we defined the integral of the stable cavitation dose (SCD) magnitude at the selected frequency including subharmonics (i.e., fo/2), ultraharmonics (i.e., 3fo/2, 5fo/2, ...), where fo is the center frequency (i.e., 560 kHz in this study). In this study, the bandwidth was typically set to 15% (with respect to the center frequency). In contrast, the inertial cavitation dose (ICD) was calculated as the sum of the wideband emission ESD over the entire spectrum while excluding the baseband (i.e., fo = 1138 kHz), subharmonics (i.e., fo/2), harmonics (i.e., 2fo, 3fo, ...), and other ultraharmonics (i.e., 3fo/2, 5fo/2, ...). Statistical Analysis. Data are presented as the means ± standard deviations (SD) of 4−6 independent experiments. Image quantification results are presented as the means ± standard deviations (SD) of at least three microscopic fields/substrate from three independent experiments. The results were analyzed using one-way analysis of variance (ANOVA). Statistical significance is indicated as follows: *, # p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001.

■

AUTHOR INFORMATION

Corresponding Authors

*(I.-C.L., Cell Biology) Mailing address: 259 Wen-Hwa first Road, Kwei-Shan, Tao-Yuan, 33302 Taiwan. E-mail: iclee@ mail.cgu.edu.tw. Tel: +886-3-2118800 ext. 5985. *(H.-L.L., Ultrasound) Mailing address: 259 Wen-Hwa first Road, Kwei-Shan, Tao-Yuan, 33302 Taiwan. E-mail: haoliliu@ mail.cgu.edu.tw. Tel: +886-3-2118800 ext. 5677. ORCID

I-Chi Lee: 0000-0001-6527-0077 Author Contributions

I.-C.L. designed the experiments analyzing neural stem cells and the biological analyses and guided H.-J.W. in analyzing the data. H.-L.L. designed the ultrasound experiments and analyzed the ultrasound data. H.-J.W. performed the experiments and collected the data. I.-C.L. and H.L.L. wrote the manuscript and revised the article. Funding

We thank Chang Gung University for providing the financial support, Summit Project Grants of Chang Gung Memorial Hospital (CMRPD1G0221-2, CIRPD2E0051-3, and CMRPD2D0111-3). Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We acknowledge equipment support provided by the Chang Gung University Microscopy Center. REFERENCES

(1) Ogawa, R (2011) Mechanobiology of scarring. Wound Repair Regener. 19, s2−s9. H

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience (2) Sarraf, C. E., Otto, W. R., and Eastwood, M. (2011) In vitro mesenchymal stem cell differentiation after mechanical stimulation. Cell Proliferation 44, 99−108. (3) Fahy, N., Alini, M., and Stoddart, M. H. (2018) Mechanical stimulation of mesenchymal stem cells: Implications for cartilage tissue engineering. J. Orthop. Res. 36, 52−63. (4) Engler, A. J., Sweeney, H. L., Discher, D. E., and Schwarzbauer, J. E. (2007) Extracellular matrix elasticity directs stem cell differentiation. J. Musculoskelet. Neuronal Interact. 7, 335. (5) Engler, A. J., Sen, S., Sweeney, H. L., and Discher, D. E. (2006) Matrix elasticity directs stem cell lineage specification. Cell 126, 677− 689. (6) Luu, Y. K., Capilla, E., Rosen, C. J., Gilsanz, V., Pessin, J. E., Judex, S., and Rubin, C. T. (2009) Mechanical stimulation of mesenchymal stem cell proliferation and differentiation promotes osteogenesis while preventing dietary-induced obesity. J. Bone Miner. Res. 24, 50−61. (7) Sim, W. Y., Park, S. W., Park, S. H., Min, B. H., Park, S. R., and Yang, S. S. (2007) A pneumatic micro cell chip for the differentiation of human mesenchymal stem cells under mechanical stimulation. Lab Chip 7, 1775−1782. (8) Juncosa-Melvin, N., Matlin, K. S., Holdcraft, R. W., Nirmalanandhan, V. S., and Butler, D. L. (2007) Mechanical stimulation increases collagen type I and collagen type III gene expression of stem cell-collagen sponge constructs for patellar tendon repair. Tissue Eng. 13, 1219−1226. (9) Juncosa-Melvin, N., Shearn, J. T., Boivin, G. P., Gooch, C., Galloway, M. T., West, J. R., Nirmalanandhan, V. S., Bradica, G., and Butler, D. L. (2006) Effects of mechanical stimulation on the biomechanics and histology of stem cell-collagen sponge constructs for rabbit patellar tendon repair. Tissue Eng. 12, 2291−2300. (10) Raza, H., Saltaji, H., Kaur, H., Flores-Mir, C., and El-Bialy, T. (2016) Effect of Low-Intensity Pulsed Ultrasound on Distraction Osteogenesis Treatment Time. J. Ultrasound Med. 35, 349−358. (11) Hasanova, G. I., Noriega, S. E., Mamedov, T. G., Thakurta, S. G., Turner, J. A., and Subramanian, A. (2011) The effect of ultrasound stimulation on the gene and protein expression of chondrocytes seeded in chitosan scaffolds. J. Tissue Eng. Regener. Med. 5, 815−822. (12) Juffermans, L. J. M., Kamp, O., Dijkmans, P. A., Visser, C. A., and Musters, R. J. P. (2008) Low-Intensity Ultrasound-Exposed Microbubbles Provoke Local Hyperpolarization of the Cell Membrane Via Activation of BKCa Channels. Ultrasound in Medicine & Biology 34, 502−508. (13) Lee, H. J., Choi, B. H., Min, B. H., Son, Y. S., and Park, S. R. (2006) Low-intensity ultrasound stimulation enhances chondrogenic differentiation in alginate culture of mesenchymal stem cells. Artif. Organs 30, 707−715. (14) Jang, K. W., Ding, L., Seol, D., Lim, T.-H., Buckwalter, J. A., and Martin, J. A. (2014) Low-Intensity Pulsed Ultrasound Promotes Chondrogenic Progenitor Cell Migration via Focal Adhesion Kinase Pathway. Ultrasound Med. Biol. 40, 1177−1186. (15) Shimazaki, A., Inui, K., Azuma, Y., Nishimura, N., and Yamano, Y. (2000) Low-intensity pulsed ultrasound accelerates bone maturation in distraction osteogenesis in rabbits. J. Bone Jt. Surg., Br. Vol. 82, 1077−1082. (16) Jung, Y. J., Kim, R., Ham, H.-J., Park, S. I., Lee, M. Y., Kim, J., Hwang, J., Park, M.-S., Yoo, S.-S., Maeng, L.-S., Chang, W., and Chung, Y.-A. (2015) Focused Low-Intensity Pulsed Ultrasound Enhances Bone Regeneration in Rat Calvarial Bone Defect through Enhancement of Cell Proliferation. Ultrasound in Medicine & Biology 41, 999−1007. (17) Machen, M. S., Tis, J. E., Inoue, N., Meffert, R. H., Chao, E. Y., and McHale, K. A. (2002) The effect of low intensity pulsed ultrasound on regenerate bone in a less-than-rigid biomechanical environment. Biomed. Mater. Eng. 12, 239−247. (18) Sun, J. S., Hong, R. C., Chang, W. H., Chen, L. T., Lin, F. H., and Liu, H. C. (2001) In vitro effects of low-intensity ultrasound stimulation on the bone cells. J. Biomed. Mater. Res. 57, 449−456.

(19) Cheung, W.-H., Chin, W.-C., Wei, F.-Y., Li, G., and Leung, K.S. (2013) Applications of Exogenous Mesenchymal Stem Cells and Low Intensity Pulsed Ultrasound Enhance Fracture Healing in Rat Model. Ultrasound in Medicine & Biology 39, 117−125. (20) Angle, S. R., Sena, K., Sumner, D. R., Virkus, W. W., and Virdi, A. S. (2014) Combined Use of Low Intensity Pulsed Ultrasound and rhBMP-2 to Enhance Bone Formation in a Rat Model of Critical-Size Defect. Journal of orthopaedic trauma 28, 605−611. (21) Lim, K., Kim, J., Seonwoo, H., Park, S. H., Choung, P.-H., and Chung, J. H. (2013) In Vitro Effects of Low-Intensity Pulsed Ultrasound Stimulation on the Osteogenic Differentiation of Human Alveolar Bone-Derived Mesenchymal Stem Cells for Tooth Tissue Engineering. BioMed Res. Int. 2013, 15. (22) Chiu, C.-Y., Tsai, T.-L., Vanderby, R., Jr, Bradica, G., Lou, S.-L., and Li, W.-J. (2015) Osteoblastogenesis of Mesenchymal Stem Cells in 3-D Culture Enhanced by Low-Intensity Pulsed Ultrasound through Soluble Receptor Activator of Nuclear Factor Kappa B Ligand. Ultrasound in Medicine & Biology 41, 1842−1852. (23) Jang, K. W., Ding, L., Seol, D., Lim, T.-H., Buckwalter, J. A., and Martin, J. A. (2014) Low-Intensity Pulsed Ultrasound Promotes Chondrogenic Progenitor Cell Migration via Focal Adhesion Kinase Pathway. Ultrasound in Medicine & Biology 40, 1177−1186. (24) Lv, Y., Zhao, P., Chen, G., Sha, Y., and Yang, L. (2013) Effects of low-intensity pulsed ultrasound on cell viability, proliferation and neural differentiation of induced pluripotent stem cells-derived neural crest stem cells. Biotechnol. Lett. 35, 2201−2212. (25) Yue, Y., Yang, X., Zhang, L., Xiao, X., Nabar, N. R., Lin, Y., Hao, L., Zhang, D., Huo, J., Li, J., Cai, X., and Wang, M. (2016) Lowintensity pulsed ultrasound upregulates pro-myelination indicators of Schwann cells enhanced by coculture with adipose-derived stem cells. Cell Proliferation 49, 720−728. (26) Yang, F.-Y., Lu, W.-W., Lin, W.-T., Chang, C.-W., and Huang, S.-L. (2015) Enhancement of Neurotrophic Factors in Astrocyte for Neuroprotective Effects in Brain Disorders Using Low-intensity Pulsed Ultrasound Stimulation. Brain Stimulation 8, 465−473. (27) Temple, S. (2001) The development of neural stem cells. Nature 414, 112. (28) Ager, R. R., Davis, J. L., Agazaryan, A., Benavente, F., Poon, W. W., LaFerla, F. M., and Blurton-Jones, M. (2015) Human neural stem cells improve cognition and promote synaptic growth in two complementary transgenic models of Alzheimer’s disease and neuronal loss. Hippocampus 25, 813−826. (29) Lee, I. C., Lo, T.-L., Young, T.-H., Li, Y.-C., Chen, N. G., Chen, C.-H., and Chang, Y.-C. (2014) Differentiation of Neural Stem/ Progenitor Cells Using Low-Intensity Ultrasound. Ultrasound in Medicine & Biology 40, 2195−2206. (30) Liu, H.-L., and Hsieh, C.-M. (2009) Single-transducer dualfrequency ultrasound generation to enhance acoustic cavitation. Ultrason. Sonochem. 16, 431−438. (31) Hu, Y., Zhong, W., Wan, J. M. F., and Yu, A. C. H. (2013) Ultrasound can Modulate Neuronal Development: Impact on Neurite Growth and Cell Body Morphology. Ultrasound in Medicine & Biology 39, 915−925. (32) Kuhn, H. G., Winkler, J., Kempermann, G., Thal, L. J., and Gage, F. H. (1997) Epidermal Growth Factor and Fibroblast Growth Factor-2 Have Different Effects on Neural Progenitors in the Adult Rat Brain. J. Neurosci. 17, 5820−5829. (33) Mitragotri, S. (2005) Healing sound: the use of ultrasound in drug delivery and other therapeutic applications. Nat. Rev. Drug Discovery 4, 255. (34) El-Bialy, T., Alhadlaq, A., Wong, B., and Kucharski, C. (2014) Ultrasound Effect on Neural Differentiation of Gingival Stem/ Progenitor Cells. Ann. Biomed. Eng. 42, 1406−1412. (35) Liu, S.-H., Lai, Y.-L., Chen, B.-L., and Yang, F.-Y. (2017) Ultrasound Enhances the Expression of Brain-Derived Neurotrophic Factor in Astrocyte Through Activation of TrkB-Akt and CalciumCaMK Signaling Pathways. Cerebral Cortex 27, 3152−3160. (36) Yoon, C. W., Jung, H., Goo, K., Moon, S., Koo, K. M., Lee, N. S., Weitz, A. C., and Shung, K. K. (2018) Low-Intensity Ultrasound I

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience Modulates Ca2+ Dynamics in Human Mesenchymal Stem Cells via Connexin 43 Hemichannel. Ann. Biomed. Eng. 46, 48−59. (37) Tyler, W. J., Tufail, Y., Finsterwald, M., Tauchmann, M. L., Olson, E. J., and Majestic, C. (2008) Remote Excitation of Neuronal Circuits Using Low-Intensity, Low-Frequency Ultrasound. PLoS One 3, e3511. (38) Datta, S., Coussios, C.-C., McAdory, L. E., Tan, J., Porter, T., De Courten-Myers, G., and Holland, C. K. (2006) CORRELATION OF CAVITATION WITH ULTRASOUND ENHANCEMENT OF THROMBOLYSIS. Ultrasound in Medicine & Biology 32, 1257−1267. (39) Tsukamoto, A., Higashiyama, S., Yoshida, K., Watanabe, Y., Furukawa, K. S., and Ushida, T. (2011) Stable cavitation induces increased cytoplasmic calcium in L929 fibroblasts exposed to 1-MHz pulsed ultrasound. Ultrasonics 51, 982−990. (40) Lee, I. C., and Wu, Y.-C. (2014) Assembly of Polyelectrolyte Multilayer Films on Supported Lipid Bilayers To Induce Neural Stem/Progenitor Cell Differentiation into Functional Neurons. ACS Appl. Mater. Interfaces 6, 14439−14450. (41) Lei, K. F., Lee, I. C., Liu, Y.-C., and Wu, Y.-C. (2014) Successful Differentiation of Neural Stem/Progenitor Cells Cultured on Electrically Adjustable Indium Tin Oxide (ITO) Surface. Langmuir 30, 14241−14249. (42) Tsai, C. H., Zhang, J. W., Liao, Y. Y., and Liu, H. L. (2016) Real-time monitoring of focused ultrasound blood-brain barrier opening via subharmonic acoustic emission detection: implementation of confocal dual-frequency piezoelectric transducers. Phys. Med. Biol. 61, 2926−2946.

J

DOI: 10.1021/acschemneuro.8b00483 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX