Role of P2X7 receptor during low-intensity focused ultrasound- induced blood-brain barrier modulation

Role of P2X7 receptor during low-intensity focused ultrasound- induced blood-brain barrier modulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Role of P2X7 receptor during low-intensity focused ultrasound- induced blood-brain barrier modulation Junwon Park, Young Cheol Na, Jihyeon Lee, Hojin Kim, Sangheon Han, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4140777/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 3 You are reading this latest preprint version Abstract Background Although low-intensity focused ultrasound (LiFUS) with microbubbles is used to temporally open the blood-brain barrier (BBB), the underlying mechanism is not fully understood. This study aimed to analyze BBB-related alterations in the brain microenvironment after LiFUS, with a focus on the involvement of the purinergic P 2 X 7 receptor. Methods Sprague-Dawley rats were sonicated with LiFUS at 0.3 MPa energy. The impact of LiFUS on the P 2 X 7 receptor and inflammatory-related proteins, including NLRP3 and interleukin-1β, was analyzed through western blotting. The BBB-associated tight junction proteins, zonula occludens-1 (ZO-1) and occludin, were also analyzed. BBB permeability was assessed by quantifying the amount of Evans blue dye penetration using spectrophotometry. Furthermore, the safety of the sonication procedure was verified via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and hematoxylin and eosin staining. Results Substantial increases in the P 2 X 7 receptor and its downstream signaling were confirmed after sonicating the BBB with LiFUS for 1 h (p < 0.05). Conversely, for tight junction proteins, the lowest expression was observed at 1 h (p < 0.001). Both responses were normalized back to the original state over time. No evidence of brain damage was observed during the procedure. Furthermore, the P 2 X 7 receptor antagonist-injected group showed reduced Evans blue dye penetration compared to that 1 h after FUS, indicating a mitigated impact of LiFUS on the BBB. Conclusions Herein, we elucidate the underlying mechanism by which LiFUS affects the BBB, with a focus on the involvement of the P 2 X 7 receptor. Our findings demonstrate that the extent of BBB opening varies upon the regulation of the P 2 X 7 receptor. This study provides valuable insights into the mechanisms underlying BBB modulation through LiFUS, thereby laying the foundation for expanding its applications. Low-intensity focused ultrasound Blood-brain barrier P2X7 receptor Tight junction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background The blood brain barrier (BBB) is a complex structure that plays a crucial role in maintaining homeostasis within the central nervous system (CNS) [ 1 ]. The BBB consists of endothelial cells with tight junctions, basal lamina, end-feet of astrocytes, and pericytes embedded in the basement membrane [ 2 , 3 ]. This distinctive arrangement gives the brain immune privilege, while simultaneously presenting a tremendous challenge for the treatment of brain diseases [ 4 , 5 ]. Numerous attempts have been made to facilitate efficient drug penetration through the BBB; however, an optimized approach is yet to be established [ 6 , 7 ]. Low-intensity focused ultrasound (LiFUS) with microbubbles has emerged as a promising technique [ 8 , 9 ]. Moreover, high-intensity focused ultrasound for the thermal coagulation of tissues has been applied, particularly for the treatment of patients with essential tremors [ 10 , 11 ]. These pioneering techniques serve as a foundation for expanding the concept of using ultrasound in the field of neuroscience, specifically in relation to the brain [ 12 , 13 ]. Unlike conventional clinical applications of focused ultrasound, LiFUS induces the oscillation of microbubbles with a relatively small amount of energy [ 14 ]. Many studies have reported that the cavitation effect of bubbles causes loosening of the tight junctions between endothelial cells, leading to a noninvasive, transient, and localized opening of the BBB [ 15 ]. Extensive studies have been conducted to investigate the potential of LiFUS for modulating the BBB. Several studies have analyzed the stability of LiFUS using various parameters [ 16 – 18 ]. Other, drug delivery studies have targeted neurodegenerative diseases such as Parkinson’s disease and Alzheimer’s disease [ 19 – 22 ]. Furthermore, considerable efforts have been made to investigate alterations in the brain microenvironment following the sonication procedure [ 23 – 26 ]. In particular, many studies have evaluated the safety aspects and inflammatory responses after LiFUS [ 27 – 29 ]. However, the molecular mechanisms underlying LiFUS-induced BBB modulation are not fully understood, particularly the primary factors driving this process [ 30 , 31 ]. Once external stress occurs in the brain, the primary response is an increase in extracellular adenosine triphosphate (ATP) [ 32 ]. This ATP increase leads to the activation of purinergic receptors, of which the P 2 X 7 receptor is considered one of the main members [33)]. P 2 X 7 receptors are ATP-gated ion channels that are widely expressed in various components of neurovascular units, including neurons, oligodendrocytes, microglia, and endothelial cells [ 33 , 34 ]. These receptors have been implicated in a broad range of physiological and pathological processes, including inflammation, apoptosis, and cell death [ 35 – 37 ]. Owing to their distinctive characteristics, P 2 X 7 receptors have been mainly investigated in neurodegenerative diseases and tumors, focusing on their involvement in inflammatory responses [ 38 , 39 ]. Recently, a few studies have demonstrated that the regulation of P 2 X 7 receptors can protect against BBB breakdown induced by lipopolysaccharide and mitigate the damage caused by alcohol and nicotine exposure [ 40 – 42 ]. In addition, the expression levels of tight junctions were preserved in animal stroke models by regulating the P 2 X 7 receptor [ 43 ]. These findings highlight the significant effect of P 2 X 7 receptors on BBB permeability [ 44 ]. Gradual upregulation of the mRNA expression of P 2 X 7 receptors was observed in an osteoblast model following LiFUS [ 45 ]. Although this study did not include an in vivo experiment, it showed the potential of LiFUS in modulating the expression of the P 2 X 7 receptor [ 45 ]. This study aimed to analyze BBB-related alterations in the brain microenvironment after LiFUS, with a focus on the involvement of the purinergic P 2 X 7 receptor. Based on the previous results, we hypothesized that BBB modulation induced by LiFUS might be related to the P 2 X 7 receptor and analyzed the factors associated with P 2 X 7 receptors and their potential roles in BBB modulation. Methods All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Health System (IACUC #2019 − 0208). A total of 79 Sprague-Dawley rats (male, 7 weeks old, 270 ± 20 g) were used in this study. Three animals were housed per cage under a light/dark cycle (12 h) in a room with controlled temperature (22 ± 2 ºC) and were allowed free access to food and water. The process and targeted areas for analysis are shown in Fig. 1a and b. The number of animals used in each group is presented in Table 1 . Figure 1. Experimental procedure and MRI images after LiFUS (a) Schematic of the experimental timeline for functional and pathological assessment. (b) Schematic region of the study area used for western blotting, IHC, RT-PCR, and Evans blue quantification. (c) Representative MR images to confirm the opening of the BBB. T2-weighted images (left) and T1-weighted images (right) were used. The opening of the BBB (white box, with LiFUS) can be seen compared to the opposite. Scale bar: 2.5 mm. (d) The enhanced signal in the ROI after LiFUS. *p < 0.05 compared to the control. All data are represented as the mean ± SD (n = 3). Unpaired t-test was performed. LiFUS, low-intensity focused ultrasound; IHC, immunohistochemistry; RT-PCR, reverse transcription-polymerase chain reaction; BBB, blood brain barrier; MRI, magnetic resonance imaging; ROI, region of interest; SD, standard deviation Table 1 Number of animals per group Groups N 1 h 4 h 24 h Total Sham 8 0 0 0 8 FUS only 0 8 8 8 24 FUS + antagonist 0 4 4 4 12 FUS + Evans blue 5 5 5 5 20 FUS + Evans blue + antagonist 0 5 5 5 15 FUS, focused ultrasound Focused ultrasound (FUS) treatment with microbubbles in rats A waveform generator (33220A, Agilent, Palo Alto, CA, USA) was connected to a power amplifier (240 L, ENI Inc., Rochester, NY, USA) to facilitate the experimental setup. Subsequently, a coupler (Pulsar C30-102-481/2N) was used to connect the power amplifier, transducer, and oscilloscope. The 515-kHz single-element transducer (H-107MR, Sonic Concept Inc., Bothell, WA, USA) with a full width at half maximum of 4 mm and a focal depth of 51.74 mm was affixed to a custom-designed cone. The tip of the cone was wrapped in a polyurethane membrane and prefilled with degassed water prior to sonication. The RF signal was monitored continuously using an oscilloscope (DSOX2002A; Keysight Technologies, Santa Rosa, CA, USA). Rats were deeply anesthetized via intraperitoneal injection of ketamine cocktail and positioned in a stereotaxic frame. The scalps of the animals were shaved and subsequently incised, and hemostasis was ensured. To prevent the loss of energy during sonication, an ultrasound transmission gel (ProGel; Dayo Medical Co., Seoul, Korea) was applied to fill the gap between the animal's skull and the tip of the cone. The target region for sonication was the right hippocampal area, located at coordinates − 3.5 mm posterior and 2 mm lateral from the bregma. Intravenous injection of microbubbles (20 µL/kg, Definity, Lantheus Medical Imaging, North Billerica, MA, USA) was performed, with sonication initiated 10 s after injection. A sonication protocol of approximately 0.3 MPa intensity was employed for each session, with a duration of 2 min, burst duration of 10 ms, and pulse repetition frequency of 1 Hz. Animals were divided into four groups (1, 4, and 24 h after FUS and control). The control group did not undergo any sonication procedure. A specific P 2 X 7 receptor antagonist (A430879, HY-15488; MCE, NJ, USA) was administered intraperitoneally at a dose of 100 mg/kg per head 20 min before sonication. Thereafter, identical procedures were performed in all groups. Magnetic resonance imaging (MRI) MRI was performed 1 h after sonication using a Bruker 9.4 T 20-cm bore MRI system (Biospec 94/20 USR; Bruker, Ettlingen, Germany) and a rat head coil. The animals were anesthetized via inhalation of 2% isoflurane during MRI. A gadolinium-based MRI contrast agent, gadobutrol (Gadovist; 0.2 mL/kg), was injected intravenously, and contrast-enhanced T1-weighted images were used to confirm the opening of the BBB (echo time, 2 ms; repetition time, 8.06 ms; echo train length, 2 ms; slice thickness, 1 mm). For statistical analysis, three rats were included in each study group. Immunostaining and histological analyses For this experiment, 5-µm tissue sections were de-paraffinized using xylene and then rehydrated. The antigen was retrieved using 1X citrate buffer 85°C for 15 min. Immunostaining was performed after cooling and rinsing the slides for 20 min. The tissues were blocked for 90 min at room temperature (20°C) using a blocking solution containing phosphate-buffered saline (PBS), 0.2% normal goat serum, and 0.2% Triton X-100. Subsequently, the brains were incubated overnight at 4°C in a blocking solution containing anti-P 2 X 7 (1:300; ab109054; Abcam, UK), anti-Reca-1 (1:300; ab9774; Abcam), anti-MMP9 (1:300; ab76003; Abcam), anti-MMP2 (1:300; ab92536; Abcam), and anti-Iba-1 (1:500; 019-19741; Wako) antibodies. After the primary immunoreaction, the samples were briefly washed with 1× PBS and incubated for 2 h at room temperature with goat anti-rabbit secondary antibodies (1:600; A11008; Thermo Fisher Scientific, USA) conjugated with Alexa Fluor 488 and goat anti-mouse secondary antibodies (1:600; A11005; Thermo Fisher Scientific) conjugated with Alexa Fluor 594. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; 1:1000; D1306; Thermo Fisher Scientific) during conjugation with the secondary antibodies. Hematoxylin and eosin (H&E) (hematoxylin [Vector Laboratories, USA] and eosin [Sigma-Aldrich, USA]) staining was performed on paraffin sections, and images were acquired using an optical microscope (BX51; Olympus, Tokyo, Japan). Histological analysis was performed to examine tissue damage, especially to monitor red blood cell extravasation. Western blotting Rat brains were collected at the indicated time points after FUS (1, 4, and 24 h). Samples were homogenized using lysis buffer (PRO-PREP; Intron Biotechnology, Pyeongtaek, Korea) and placed on ice for 30 min, followed by centrifugation at 13000 rpm for 30 min. The protein concentration in the lysate was measured using the bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific, Fremont, CA, USA). Subsequently, 10–20 µg of each protein sample were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane using a Bio-Rad Trans-Blot apparatus. Membranes were incubated with blocking buffer (5% nonfat dry milk in PBS containing 0.05% Tween 20 [TBST]) for 1 h at room temperature. The membranes were then incubated with the primary rabbit anti-P 2 X 7 (1:1000, Abcam), rabbit anti-occludin (1:1000, Abcam), rabbit anti-ZO-1 (1:1000, Thermo Fisher Scientific), rabbit anti-IL-1β (1:500; Abcam), rabbit anti-NF-κB (1:1000, Cell Signaling Technology, Beverly, MA, USA), and mouse anti-β actin (1:10000, Sigma-Aldrich, MO, USA) antibodies overnight at 4 ºC. Next, the membrane was washed three times for 5 min each and incubated with the corresponding secondary antibodies, either goat anti-rabbit IgG (H + L)-HRP (GenDEPOT, Katy, TX, USA) or goat anti-mouse IgG (H + L)-HRP (GenDEPOT), for 90 min at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescent solution (WEST-Queen; iNtRON Biotechnology, Seongnam, Korea) and developed using LAS 4000 Mini (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The intensity of each band was determined using an analytical system (MultiGauge version 3.0; Fujifilm, Tokyo, Japan). Quantification of Evans blue dye Intravenous injection of Evans blue dye (100 mg/kg, Sigma-Aldrich) diluted in 2% saline solution was administered at specific time points (1, 4, and 24 h) after sonication. The dye was allowed to circulate for 1 h prior to further experiments. Subsequently, the rats were perfused with 500 mL saline to remove the dye localized within the intravascular space. The brains were then harvested and divided along the midline of the brain, and the ipsilateral hemisphere was homogenized. After weighing, samples were homogenized with saline (mg/mL), and centrifuged at 15000× g, 4 ºC for 30 min. Equal volumes of trichloroacetic acid were added to each supernatant. Samples were incubated at 4 ºC overnight and centrifuged. To increase their optic length, 90 µL of 95% ethanol was added to each well containing 30 µL supernatant and mixed by repetitive pipetting. The extravasated Evans blue dye was spectrophotometrically quantified at 620 nm. Real-time reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from the cells using an easy-spin total RNA extraction kit (iNtRON Biotechnology, Seongnam, Republic of Korea), and oligo (dT)-primed cDNA was synthesized from the total RNA using the Maxime RT PreMix kit (iNtRON Biotechnology), according to the manufacturer’s instructions. Quantitative RT-PCR was performed using the StepOnePlus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) with the SYBR Green Dye system (SYBR Premix Ex Taq [Tli RNase Plus] and ROX reference dye [TAKARA Bio Inc., Foster City, CA, USA]). Gene expression level was normalized to glyceraldehyde 3-phosphate dehydrogenase transcript levels. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay A TUNEL assay kit (G3250, Promega, Madison, WI, USA) was used to assess the level of apoptosis in paraffin-embedded rat brain sections according to manufacturer’s instructions. Briefly, the paraffin-embedded tissue sections were de-paraffinized, rehydrated, and treated with proteinase K. Permeabilized tissue sections were incubated with the rTdT reaction mixture for 1 h in a dark and humidified chamber at 37 ℃. The sections were co-stained with DAPI and cover-slipped with mounting solution. The labeled DNA was observed using an LSM700 confocal microscope (Carl Zeiss, Jena, Germany). For the brain sections, the detection wavelengths were those of fluorescein isothiocyanate and DAPI. ATP assay An ATP assay kit (#ab83355, Abcam) was used to analyze the effects of FUS on P 2 X 7 -related ATP levels following the manufacturer’s instructions. ATP content was measured using a colorimetric assay, which generated a quantifiable product detected at 570 nm. Statistical analysis All results are expressed as the mean ± standard deviation and analyzed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). MATLAB software was used to calculate the intensity of the region of interest (ROI) using the T1 images. Data were analyzed using an unpaired t-test (n = 3) and one-way analysis of variance, followed by Tukey’s post-hoc test (n = 3–5). P-values lower than 0.05 were considered to indicate statistical significance. Results Confirmation of BBB opening after LiFUS First, the sonication area of LiFUS and the degree of BBB opening were confirmed. Prior to the analysis of the P 2 X 7 receptor, LiFUS with microbubbles was used to identify the area of the lesion and analyze the changes in MRI signals (Fig. 1c and d). The results confirmed that the energy reached part of the upper layer of the thalamus (DV: 4 mm from the bregma), including the hippocampus, through the T1-contrast-enhanced image. In addition, by quantifying the intensity of the ipsilateral and contralateral signals using the same ROI, it was found that the signals of the sonicated area (FUS: 2.16 ± 0.37) were significantly enhanced (con: 1.16 ± 0.39, p < 0.05, n = 3 for each group). Alterations in P 2 X 7 receptor expression and signaling cascade following LiFUS To investigate whether ultrasound energy affects the P 2 X 7 receptor, we initially employed immunohistochemistry (IHC) to assess the variations in receptor expression levels over time (Fig. 2a). A notable upregulation of receptor expression in the hippocampus within the group 1 h after LiFUS. For a more accurate quantitative analysis, we conducted western blotting and real-time RT-PCR to evaluate the protein and RNA expression levels of the P 2 X 7 receptor. A significant increase in protein expression was observed in the group 1 hour (1 h: 0.89 ± 0.2) after sonication compared to that in the other groups (Fig. 2b, con: 0.57 ± 0.07, p < 0.05; 4 h: 0.56 ± 0.18, p < 0.05; 24 h: 0.56 ± 0.24, p < 0.05, n = 5 for each group). Similarly, the RNA levels showed a significant increase in the group corresponding to 1 h after LiFUS (1 h: 2.7 ± 0.23) compared to the other groups (Fig. 2c, con: 1.16 ± 0.07, p < 0.001; 4 h: 1.91 ± 0.12, p < 0.001; 24 h: 1.61 ± 0.09, p < 0.001, n = 5 for each group). Notably, the RNA expression levels remained elevated even after 24 h (4 h: p < 0.001; 24 h: p < 0.05, compared to the control). Figure 2. Regulation of P 2 X 7 receptors and downstream signaling pathways after sonication (a) Representative IHC images of DAPI (blue), P 2 X 7 receptor (white arrow, green) and RECA-1 (red) in the hippocampal region of the brain after FUS. Scale bar: 100 µm. (b, c) Western blotting and RT-PCR results for P 2 X 7 receptors. The bar graph shows the normalized level of the receptors relative to the housekeeping genes. Band intensity images for each group are also included. *p < 0.05, **p < 0.01, ***p < 0.001; compared to 1 h, #p < 0.05, ##p < 0.01; compared to control. (d, e) Western blot and RT-PCR results for NLRP-3 inflammasome. The analysis method employed for NLRP-3 was the same as that for the P 2 X 7 receptors. **p < 0.01, ***p < 0.001; compared to 1 h, &p < 0.05, &&p < 0.01; compared to 4 h. (f, g) Western blot and RT-PCR results for IL-1ß. The analysis method employed for NLRP-3 was the same as that for the P 2 X 7 receptors. *p < 0.05, **p < 0.01, ***p < 0.001; compared to 1 h, #p < 0.05; compared control. All data are represented as mean ± SD (n = 5). One-way ANOVA with Tukey's multiple comparison test was performed. FUS, focused ultrasound; IHC, immunohistochemistry; RT-PCR, reverse transcription-polymerase chain reaction; SD, standard deviation; DAPI, 4′,6-diamidino-2-phenylindole; ANOVA, analysis of variance We also analyzed NLRP-3 and IL-1β levels, which are known to exhibit a substantial correlation with P 2 X 7 receptor expression (32). The protein expression pattern of NLRP-3 was similar to that of the P 2 X 7 receptor, peaking at 1 h after LiFUS (1 h: 2.22 ± 0.34), but with a slight difference in the significance among groups (Fig. 2d, con: 1.18 ± 0.16, p < 0.001; 4 h: 1.44 ± 0.09, p < 0.001; 24 h: 1.19 ± 0.07, p < 0.001, n = 5 for each group). Similar results were obtained with the RNA levels (Fig. 2e, con: 1.22 ± 0.14, p < 0.001; 4 h: 1.86 ± 0.35, p < 0.001; 24 h: 1.4 ± 0.19, p < 0.001 compared to 1 h: 2.92 ± 0.26, n = 5 for each group). However, NLRP-3 RNA levels remained elevated until 4 h after LiFUS : p < 0.01; 24 h: p < 0.05). IL-1β protein expression level showed similar trends (Fig. 2f, con: 1.18 ± 0.14, p < 0.01; 4 h: 1.69 ± 0.27; 24 h: 1.4 ± 0.18, p < 0.05 compared to the 1 h: 1.89 ± 0.31, n = 5 for each group). The protein levels were found to be upregulated for up to 4 h compared to those in the control group (p < 0.05). In terms of RNA expression, the highest peak was observed at 1 h after sonication (Fig. 2g, con: 1.23 ± 0.1, p < 0.001; 4 h: 1.76 ± 0.47, p < 0.05; 24 h: 1.43 ± 0.28, p < 0.01 compared to 1 h: 2.55 ± 0.55, n = 5 for each group). Taken together, these results showed the upregulation of the receptor and its signaling after LiFUS treatment, with the expression levels gradually returning to baseline within a day. The findings suggest that LiFUS temporally affects the P 2 X 7 receptor and related proinflammatory cytokines. Alterations of BBB-related protein expression after FUS We assessed alterations in BBB-related proteins, such as ZO-1 and occludin, to confirm the impact of LiFUS on BBB integrity using western blotting and RT-PCR. Initially, ZO-1 exhibited the lowest protein expression at 1 h after sonication (1 h: 1.83 ± 0.4, p < 0.01 compared to con), in contrast to the P 2 X 7 receptor, after which ZO-1 protein expression gradually normalized over time (Fig. 3a, con: 3.18 ± 0.36; 4 h: 2.27 ± 0.53, p < 0.05 compared to con; 24 h: 3.79 ± 0.55). The reduced expression level remained significant in the group a day after sonication (1 h and 4 h: p < 0.001 compared to 24 h). This trend was consistent at the ZO-1 RNA level (Fig. 3b. con: 1.18 ± 0.13; 1 h: 0.49 ± 0.1, p < 0.001 compared to con and 24 h; 4 h: 0.82 ± 0.12, p < 0.001 compared to 24 h; 24 h: 1.49 ± 0.38). Occludin, a representative tight junction protein, exhibited similar patterns in both protein and RNA levels (Fig. 3c and d, occludin protein expression : con: 3.13 ± 0.3; 1 h: 1.39 ± 0.3, p < 0.001 compared to con and 24 h; 4 h: 1.87 ± 0.57, p < 0.001 compared to con and 24 h; 24 h: 3.5 ± 0.38 and occludin RNA expression: con: 1.17 ± 0.15; 1 h: 0.46 ± 0.12, p < 0.01 compared to con and p < 0.001 compared to 24 h; 4 h: 0.65 ± 0.17, p < 0.05 compared to con and p < 0.001 compared to con and 24 h; 24 h: 1.37 ± 0.4). In addition, MMP9, which is reportedly correlated to the BBB [ 46 ], was observed through IHC, and its expression was partially elevated at 1–4 h after LiFUS (Fig. 3e). In addition, the expression of MMP2 data for each time point is shown in Additional file. 1. These findings indicate that LiFUS influences BBB integrity and enhances BBB permeability. However, this effect may be transient, as BBB permeability returns to normal within a day. Figure 3. Regulation of BBB-related proteins after LiFUS stimulation (a, b) Western blot and RT-PCR results for ZO-1. The bar graph shows the normalized level of the receptors relative to that of the housekeeping genes. Band intensity images for each group are included. #p < 0.05, ##p < 0.01, ###p < 0.001; compared to control, †††<0.001; compared to 24 h. (c, d) Western blot and RT-PCR results for occludin. The analysis method employed for occludin was the same as that for ZO-1. #p < 0.05, ##p < 0.01, ###p < 0.001; compared to control, †††<0.001; compared to 24 h. All data are presented as mean ± SD (n = 5). One-way ANOVA with Tukey's multiple comparison test was performed. (e) Representative IHC images of DAPI (blue) and MMO-9 (white arrow, green) in the hippocampal region of the brain after LiFUS. Scale bar: 100 µm. LiFUS, low-intensity focused ultrasound; IHC, immunohistochemistry; RT-PCR, reverse transcription-polymerase chain reaction; BBB, blood brain barrier; SD, standard deviation; DAPI, 4′,6-diamidino-2-phenylindole; ANOVA, analysis of variance; ZO-1, zonula occludens-1 Safety assessment of LiFUS-induced BBB modulation After confirming that ultrasound energy was sufficient for BBB modulation, we conducted a comprehensive safety assessment of the process. Initially, we monitored the ATP levels over time following LiFUS treatment (Fig. 4a). Consistent with previous findings, the highest concentration of ATP was found at 1 h after LiFUS (1 h: 1.41 ± 0.37, p < 0.001 compared to con), with continued elevation even after 24 h (con: 1.19 ± 0.39; 4 h: 1.41 ± 0.37, p < 0.01; 24 h: 1.4 ± 0.58, p < 0.05 compared to con, n = 5 for each group). Subsequently, we conducted an analysis of alterations in the NF-κB expression levels in response to the observed temporal increase in IL-1ß. Thus, NF-κB expression levels may represent a signal transduction outcome of P 2 X 7 receptor activation after LiFUS. These findings are consistent with our prior data. Specifically, there was a noticeable elevation in the expression of phosphorylated NF-κB (p-NF-κB) within 1 h (Fig. 4b, 1h: 3.72 ± 0.89). Additionally, their results gradually returned to baseline over time (p-NF-κB, con: 1.19 ± 0.39; 4 h: 1.41 ± 0.37; 24 h: 1.4 ± 0.58, p < 0.001 compared to 1 h, n = 5 for each group). Figure 4. No evidence of death signals after LiFUS stimulation (a) Results of ATP quantification in the lesion. The bar graph shows the normalized level of ATP relative to that of control. #p < 0.05, ##p < 0.01, ###p < 0.001; compared to control, *p < 0.05; compared to 1 h. (b) Western blot results for NF-κB. The bar graph shows the normalized level of the receptor relative to that of the housekeeping genes. Band intensity images for each group are included. ***p < 0.001; compared to 1 h. (c, d) RT-PCR results for caspase 3/9. No significant differences were observed among the groups. All data are represented as mean ± SD (n = 5). One-way ANOVA with Tukey's multiple comparison test was performed. (e) Representative images of the TUNEL assay (white arrow) and H&E images of each group. Scale bar: 20 µm for TUNEL and 100 µm for H&E images. LiFUS, low-intensity focused ultrasound; RT-PCR, reverse transcription-polymerase chain reaction; SD, standard deviation; ANOVA, analysis of variance; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; H&E, hematoxylin and eosin Subsequently, a comprehensive analysis of the cellular damage and apoptosis resulting from sonication was performed. Quantification of the RNA expression levels of caspase 3/9, used as markers of apoptosis, revealed an increased expression level 1 h post-sonication (Fig. 4c and d). However, this increase did not show statistical significance, and caspase 3/9 expression subsequently returned to baseline levels (caspase 3; con: 0.98 ± 0.19; 1 h: 1.34 ± 0.39; 4 h: 1.42 ± 0.49; 24 h: 1.34 ± 0.38, caspase 9; con: 1.26 ± 0.12; 1 h: 1.56 ± 0.36; 4 h: 1.6 ± 0.34; 24 h: 1.43 ± 0.28, n = 5 for each group). Moreover, the TUNEL assay results indicated the absence of activated cell death signals throughout the brain (Fig. 4e). Finally, histopathological examination (H&E staining) confirmed the absence of tissue damage following LiFUS, which was consistent with the findings of the control group. The complete set of whole-brain tissue data for each time point is shown in Additional file 2. Collectively, these results highlight the ability of LiFUS to modulate BBB permeability without inducing brain damage, even in the context of P 2 X 7 receptor activation and transient upregulation of pro-inflammatory cytokines. Decreased BBB permeability after P 2 X 7 receptor antagonist injection To substantiate the involvement of P 2 X 7 receptors in LiFUS-induced BBB opening, quantitative analysis of Evans blue was performed using spectrometry (Fig. 5a, [ 47 ]). The groups used in this study were as follows: N, 1 h, 1 h + A438079, 4 h, 4 h + A438079, 24 h, and 24 h + A438079 (n = 5 for each group). The group that received Evans blue administration at 1 h after sonication (1 h: 0.051 ± 0.01) exhibited a significantly higher fluorescence intensity compared to that in all the other groups, except the group corresponding to 4 h (Fig. 5b, con: 0.038 ± 0.001, p < 0.001; con + A438079: 0.038 ± 0.001, p < 0.001; 1 h + A438079: 0.043 ± 0.002; 4 h: 0.039 ± 0.002, p < 0.01; 4h + A438079: 0.038 ± 0.002, p < 0.001; 24 h: 0.039 ± 0.002, p < 0.001; 24 h + A438079: 0.038 ± 0.001, p < 0.001 compared to 1 h). Similarly, upon antagonist administration, the extent of leakage was most pronounced in the 1-hour post-LiFUS group; however, this result was not statistically significant when compared to the control. Notably, the amount of leakage was comparable to that observed in the group that received Evans blue at 4 h after LiFUS. Figure 5. Alterations in BBB permeability following antagonist administration (a) Representative images of Evans blue dye leakage in each group. (b) Optical density of Evans blue dye in the sonicated area after LiFUS in each group. **p < 0.01, ***p < 0.001; compared to 1 h (c) Western blot results for the P 2 X 7 receptor. The bar graph shows the normalized level of the receptor relative to that of ß-actin. Band intensity images for each group are included. *p < 0.05, **p < 0.01; compared to 1 h. (d) Western blot result for ZO-1. The analysis method employed for the ZO-1 was the same as that for the P 2 X 7 receptor. No statistical differences were observed among the groups. All data are represented as mean ± SD (n = 3–5). *p < 0.05; compared to 1 h. One-way ANOVA with Tukey's multiple comparison test was performed. LiFUS, low-intensity focused ultrasound; BBB, blood brain barrier; SD, standard deviation; ANOVA, analysis of variance; ZO-1, zonula occludens-1 For a detailed understanding of the leakage phenomenon, we conducted western blot quantification of P 2 X 7 and ZO-1 proteins. For the P 2 X 7 receptor, the group corresponding to 1 h after LiFUS (1 h: 293.6 ± 89.56) exhibited a significant expression level (Fig. 5c, con: 114.2 ± 41.15 p < 0.001; 1 h + A438079: 166.7 ± 12.79; 4 h: 170 ± 7.28; 4 h + A438079: 161.5 ± 43.53 p < 0.05; 24 h: 179.9 ± 33.13; 24 h + A438079: 139.4 ± 44.25 p < 0.05 compared to 1 h, n = 3–4 for each group). In contrast, ZO-1 expression was the lowest at 1 h (Fig. 5d, con: 104.8 ± 14.24 p < 0.05 compared to 1 h; 1 h: 60.37 ± 7.55; 1 h + A438079: 80.96 ± 12.06; 4 h: 70.24 ± 15.15; 4 h + A438079: 84.74 ± 19.71; 24 h: 108 ± 28.08; 24 h + A438079: 106.7 ± 29.72, n = 3–4 for each group). The results for the remaining downstream signaling pathways in the antagonist-treated groups are shown in Additional file 3. These findings strongly support a possible role of the P 2 X 7 receptor in LiFUS-mediated BBB modulation. Discussion LiFUS has emerged as a promising technique offering the advantage of temporal modulation of the BBB to enhance drug delivery for the treatment of brain diseases [ 3 , 17 ]. Despite its clinical significance, the precise molecular mechanism underlying LiFUS-mediated BBB modulation remains unclear [ 48 – 50 ]. To address this gap and enable the utilization of the LiFUS technique, we sought to demonstrate the involvement of the P 2 X 7 receptor in BBB modulation [ 51 – 53 ]. The activation of P 2 X 7 receptor and associated factors reached a peak intensity at 1 h after sonication, coinciding with a decrease in tight junction proteins. Notably, there was a substantial disparity in the extent of dye leakage depending on the presence of P 2 X 7 receptors. These findings contribute to our understanding of the role of the P 2 X 7 receptor in LiFUS-mediated BBB modulation (Fig. 6, [ 54 ]). Figure 6. Graphical summary of the study (a) A 0.5-MHz transducer was placed on the skull of rats, and sonication was performed. Microbubbles inside the vessels affected by LiFUS energy loosened the BBB to make it permeable. The hippocampal area was the main region analyzed. (b) Compared to the control group, in the LiFUS treated group, the upregulated expression of P 2 X 7 receptor, NLRP3, and IL-1ß was observed, accompanied by a decrease in the expression level of the tight junction proteins and RNA. Consequently, the leakage of the Evans blue dye in the parenchyma was increased compared to that in the control conditions. In the antagonist-treated group, changes in protein and RNA expression levels were observed, but they did not reach statistical significance. The amount of Evans blue dye leakage was also not significant. (c) Although LiFUS induces the temporal upregulation of markers related to the inflammatory responses, including NLRP-3, IL-1ß, and NF-κB, this technique is safe as it does not reach a level causing cell damage or hemorrhage to the sonicated area. LiFUS, low-intensity focused ultrasound; BBB, blood brain barrier Numerous studies have investigated the effects of LiFUS on the BBB [ 55 , 56 ]. Considering the influence of microbubble cavitation, initial observations predominantly focused on sonication-induced alterations in cerebrovascular vessels [ 57 ]. Notably, several studies have demonstrated ultrastructural changes in endothelial cell morphology and brain microvessels, suggesting enhanced permeability to macromolecules after LiFUS-induced BBB opening [ 58 ]. In addition, analyses of BBB-related proteins have led to the proposal of various possible mechanisms for the passage of substances through the BBB following LiFUS, including paracellular, transcellular, and adsorptive-mediated pathways [ 59 , 60 ]. However, the precise mechanisms underlying BBB modulation are yet to be established. The present study substantially advances our understanding of the effects of LiFUS. Herein, we demonstrated that LiFUS with microbubbles triggers complex alterations within various neurovascular units, with the P 2 X 7 receptor acting as a central mediator in this process. The presence of cavitation in both groups 1 h after sonication offers a possible clue. In particular, the differential leakage of Evans blue dye, contingent on the presence of P 2 X 7 receptors, highlights the multifaceted nature of LiFUS-induced BBB modulation and the significant involvement of the P 2 X 7 receptors. These data underscore the complexity of LiFUS-induced BBB modulation and the integral role of the P 2 X 7 receptors in mediating these effects (44). However, complete restriction of BBB permeability was not achieved even in the antagonist-treated group. Such results are likely due to the involvement of various pathways in LiFUS-induced BBB modulation, as previously suggested [ 59 , 60 ]. Hence, it is necessary to consider the various pathways to gain a comprehensive understanding of BBB modulation. Moreover, our study provides insight into safety considerations associated with the LiFUS technique. Vigilance is crucial in the post-sonication period to monitor potential edema or hemorrhage resulting from BBB modulation and subsequent inflammatory response at the lesion site. Previous studies have emphasized the sterile inflammatory response, with a focus on alterations in the NF-κB pathway following LiFUS [ 61 , 62 ]. In particular, when quantifying protein levels in the brain following sonication, a transient upregulation of cytokines related to inflammation was observed, which eventually normalized over time [ 63 ]. Although the present study revealed similar trends, the outcomes differed significantly. Notably, in our study, the expression of pro-inflammatory cytokines, including NLRP3, IL-1ß, and NF-κB, showed an initial increase up to approximately 4 h after sonication. These elevated levels returned to baseline within 24 h. Moreover, TUNEL staining and H&E analysis consistently verified the absence of damage to the brain tissue. Thus, our study demonstrates that LiFUS induces transient alterations in the BBB and its physiology; however, these changes do not reach a critical or injurious threshold. Comparable or milder inflammatory responses have been observed in studies utilizing energy of similar intensity [ 29 , 63 ]. Our findings suggest that LiFUS can be safely applied under well-defined parameters. We found that the P 2 X 7 receptors are involved in BBB modulation induced by LiFUS under safe conditions. When applying this technique to patients, the transfer of energy is affected by various parameters, such as individual differences in patient status, even in cases where the inflammatory response needs to be well-controlled, such as in Alzheimer’s disease [ 64 , 65 ]. LiFUS energy is applied for a short duration of approximately 2 min. However, the BBB has been shown to be normalized within approximately 1 day [ 3 ]. Notably, the BBB opens up approximately 4 h after sonication, allowing continuous drug accumulation in the brain [ 3 , 30 ]. Under such conditions, the extent of BBB tight junction loosening may be regulated by administering P 2 X 7 receptor antagonists without causing significant damage to the sonicated area, as demonstrated in this study. For the antagonist-treated group (Additional file 3), a slight increase in P 2 X 7 receptor expression was observed, but it did not show statistical significance, and P 2 X 7 receptor expression subsequently returned to baseline levels within 24 h. Given the pivotal role of the P 2 X 7 receptors in brain pathology-related inflammatory pathways, the regulation of receptor activity may serve as a viable approach to attenuate downstream inflammatory processes. Thus, further investigations utilizing P 2 X 7 antagonists and the optimization of treatment parameters are warranted. Nevertheless, this study serves as a foundational investigation with the potential to expand the scope of existing LiFUS applications. Although our findings identify potential modulators of LiFUS-mediated BBB opening that can act without causing brain damage, there are some limitations to this study. First, while our study demonstrated P 2 X 7 receptor activation, which is implicated in BBB modulation, the precise origin of this receptor activation remains unclear [ 34 ]. According to previous studies, no significant change was observed in the expression of Iba-1 (Additional file 4) [ 66 ]). Thus, we speculate that the increased expression of P 2 X 7 receptors may originate from endothelial cells. However, for a more comprehensive understanding, further analyses using techniques such as RNA sequencing should be conducted, particularly considering the clinical applicability of this technology [ 66 , 67 ]. Second, our study focused on analyzing the intracerebral response at an energy level near 0.3 MPa, which translates to approximately 1.6 W/cm 2 in terms of energy per unit area. However, it is important to note that ultrasound energy is employed with a range of powers depending on the specific experimental conditions. Therefore, to assess the effects of LiFUS comprehensively, it is necessary to investigate changes using a broader range of parameters [ 66 , 68 ]. Finally, this study was conducted on rats in a normal physiological state. LiFUS is currently used to treat various brain diseases [ 69 ]. It is important to recognize that in diseases that differ from the normal physiological state, the levels of cytokines related to inflammation and biological responses to ultrasound energy may vary [ 70 ]. Therefore, the findings of this study should be interpreted with caution. To facilitate a more comprehensive understanding and ensure safer application of this technique, future research should encompass animal models representing diverse brain disease conditions. Conclusions Our study confirmed changes in the activity of P 2 X 7 receptors and BBB-related proteins in response to LiFUS. Furthermore, we demonstrated that the presence or absence of P 2 X 7 receptors influences the degree of BBB opening. These findings provide valuable insights into the complex mechanisms underlying LiFUS-mediated BBB modulation. Moreover, they support the feasibility of potentially regulating the degree of BBB opening using LiFUS as a noninvasive strategy for modulating the BBB. Abbreviations Blood brain barrier (BBB) Focused ultrasound (FUS) Low-intensity focused ultrasound (LiFUS) Zonula occludens-1 (ZO-1) Magnetic resonance imaging (MRI) Standard deviation (SD) Region of Interest (ROI) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Adenosine triphosphate (ATP) Phosphate-buffered saline (PBS) Hematoxylin and eosin (H&E) Reverse transcription-polymerase chain reaction (RT-PCR) Declarations Ethics approval All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Health System (IACUC #2019-0208) Consent for publication Not applicable Availability of data and materials The data presented in this study are available on request from the corresponding authors Competing interests The authors declare that they have no competing interests Funding This study was funded by the Basic Science Research Program of the National Research Foundation of Korea supported by the Ministry of Education [RS-2023-00266075] and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) [NRF-2020R1C1C1013535]. Authors’ contributions Y.C.N., J.L., and B.W.S. conceptualized and designed the study. J.L., J.P., H.K., and S.H. assisted with and performed experiments. J.P. Y.C.N.B.W.S. and W.S.C. analyzed the data. W.S.C., Y.C.N., B.W.S., and J.P. edited the manuscript. J.P. and Y.C.N. wrote the original draft. All the authors have read and approved the final version of the manuscript. Acknowledgements We thank the Medical Illustration & Design (MID) team in the Medical Research Support Services of Yonsei University College of Medicine for their excellent support with the medical illustrations and figures made on Biorender.com. References P. Ballabh, A. Braun, M. Nedergaard, The blood-brain barrier: an overview: structure, regulation, and clinical implications, Neurobiol Dis, 16 (2004) 1-13. N.J. Abbott, A.A. Patabendige, D.E. Dolman, S.R. Yusof, D.J. Begley, Structure and function of the blood-brain barrier, Neurobiol Dis, 37 (2010) 13-25. C. Greene, M. 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Konofagou, Natural aging and Alzheimer's disease pathology increase susceptibility to focused ultrasound-induced blood-brain barrier opening, Sci Rep, 13 (2023) 6757. Additional Declarations No competing interests reported. Supplementary Files SuppleFig1.tif SuppleFig2.tif SuppleFig3.tif SuppleFig4.tif Cite Share Download PDF Status: Under Review Version 1 posted Editor assigned by journal 21 Mar, 2024 Submission checks completed at journal 21 Mar, 2024 First submitted to journal 21 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4140777","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282781023,"identity":"d270a4b6-c697-49e0-a691-2b241e0866e5","order_by":0,"name":"Junwon Park","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Junwon","middleName":"","lastName":"Park","suffix":""},{"id":282781024,"identity":"ffed4876-154a-411d-b2d6-0dc216e92d28","order_by":1,"name":"Young Cheol Na","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Young","middleName":"Cheol","lastName":"Na","suffix":""},{"id":282781025,"identity":"6c177054-3500-4eb6-bb6f-a17bd60031b6","order_by":2,"name":"Jihyeon Lee","email":"","orcid":"","institution":"Cleros Biotech","correspondingAuthor":false,"prefix":"","firstName":"Jihyeon","middleName":"","lastName":"Lee","suffix":""},{"id":282781027,"identity":"8faa1efd-f3b8-4c15-97ab-516f2f80cda8","order_by":3,"name":"Hojin Kim","email":"","orcid":"","institution":"Catholic Kwandong University","correspondingAuthor":false,"prefix":"","firstName":"Hojin","middleName":"","lastName":"Kim","suffix":""},{"id":282781028,"identity":"2e9017b5-f5e5-46d6-9cb5-6c39147303b5","order_by":4,"name":"Sangheon Han","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Sangheon","middleName":"","lastName":"Han","suffix":""},{"id":282781030,"identity":"97d5ed39-f683-4258-8244-5c3aa7889855","order_by":5,"name":"Byeong-Wook Song","email":"","orcid":"","institution":"Catholic Kwandong University","correspondingAuthor":false,"prefix":"","firstName":"Byeong-Wook","middleName":"","lastName":"Song","suffix":""},{"id":282781031,"identity":"2bed2a5d-1628-47f9-a188-27293e9f48dd","order_by":6,"name":"Won Seok Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYDACCQjFY3+Y+eADBoYDxGuRYzjOlmxAkhZjhvM8ahJEaeGf3fzs4de2usTGZh62at62O/YM7Icf4LfkzjFzY9m2w4nNzLzHbvO2PUts4EkzwKvFQCLBTFpy24HENma+NKCWwwkMDDn4HWYgkf4NqKUusYeZx6wYqMWegf8NIS05ZpIftzEbSwC1MAO1MDZIELBF4kZOmTTjv8NyBsxsyZJzzh1ObJN4ht8v/DPSt0n+OFPHY8B/+OCHN2WH7fn5kx/gtwYImHmgDCYQg42geiBg/IHOGAWjYBSMglGADACdd0STcyFYwQAAAABJRU5ErkJggg==","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Won","middleName":"Seok","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2024-03-21 04:59:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4140777/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4140777/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53396641,"identity":"099afbd6-2722-4295-92c1-ccc3605aa023","added_by":"auto","created_at":"2024-03-25 13:38:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":777235,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental procedure and MRI images after LiFUS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Schematic of the experimental timeline for functional and pathological assessment. \u003cstrong\u003e(b)\u003c/strong\u003e Schematic region of the study area used for western blotting, IHC, RT-PCR, and Evans blue quantification. \u003cstrong\u003e(c)\u003c/strong\u003e Representative MR images to confirm the opening of the BBB. T2-weighted images (left) and T1-weighted images (right) were used. The opening of the BBB (white box, with LiFUS) can be seen compared to the opposite. Scale bar: 2.5 mm. \u003cstrong\u003e(d)\u003c/strong\u003eThe enhanced signal in the ROI after LiFUS.\u003c/p\u003e\n\u003cp\u003e*p\u0026lt;0.05 compared to the control. All data are represented as the mean ± SD (n=3). Unpaired t-test was performed.\u003c/p\u003e\n\u003cp\u003eLiFUS, low-intensity focused ultrasound; IHC, immunohistochemistry; RT-PCR, reverse transcription-polymerase chain reaction; BBB, blood brain barrier; MRI, magnetic resonance imaging; ROI, region of interest; SD, standard deviation\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/8a466795b0c38a7234e7b7fa.png"},{"id":53396640,"identity":"735456b8-e037-4749-8624-29d26a19aed1","added_by":"auto","created_at":"2024-03-25 13:38:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5724596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation of P\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eX\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e7\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e receptors and downstream signaling pathways after sonication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a)\u003c/strong\u003e Representative IHC images of DAPI (blue), P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor (white arrow, green) and RECA-1 (red) in the hippocampal region of the brain after FUS. Scale bar: 100 µm. \u003cstrong\u003e(b, c)\u003c/strong\u003e Western blotting and RT-PCR results for P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors. The bar graph shows the normalized level of the receptors relative to the housekeeping genes. Band intensity images for each group are also included. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001; compared to 1 h, #p\u0026lt;0.05, ##p\u0026lt;0.01; compared to control. \u003cstrong\u003e(d, e)\u003c/strong\u003e Western blot and RT-PCR results for NLRP-3 inflammasome. The analysis method employed for NLRP-3 was the same as that for the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors. **p\u0026lt;0.01, ***p\u0026lt;0.001; compared to 1 h, \u0026amp;p\u0026lt;0.05, \u0026amp;\u0026amp;p\u0026lt;0.01; compared to 4 h. \u003cstrong\u003e(f, g)\u003c/strong\u003e Western blot and RT-PCR results for IL-1ß. The analysis method employed for NLRP-3 was the same as that for the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors. *p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001; compared to 1 h, #p\u0026lt;0.05; compared control. All data are represented as mean ± SD (n=5). One-way ANOVA with Tukey's multiple comparison test was performed.\u003c/p\u003e\n\u003cp\u003eFUS, focused ultrasound; IHC, immunohistochemistry; RT-PCR, reverse transcription-polymerase chain reaction; SD, standard deviation; DAPI, 4′,6-diamidino-2-phenylindole; ANOVA, analysis of variance\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/911917d99e6d1fa65971535d.png"},{"id":53396643,"identity":"c3cb934a-1b54-41e9-a9e4-3ce00ddf039b","added_by":"auto","created_at":"2024-03-25 13:38:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4851793,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRegulation of BBB-related proteins after LiFUS stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a, b) \u003c/strong\u003eWestern blot and RT-PCR results for ZO-1. The bar graph shows the normalized level of the receptors relative to that of the housekeeping genes. Band intensity images for each group are included. #p\u0026lt;0.05, ##p\u0026lt;0.01, ###p\u0026lt;0.001; compared to control, †††\u0026lt;0.001; compared to 24 h. \u003cstrong\u003e(c, d)\u003c/strong\u003e Western blot and RT-PCR results for occludin. The analysis method employed for occludin was the same as that for ZO-1. #p\u0026lt;0.05, ##p\u0026lt;0.01, ###p\u0026lt;0.001; compared to control, †††\u0026lt;0.001; compared to 24 h. All data are presented as mean ± SD (n=5). One-way ANOVA with Tukey's multiple comparison test was performed. \u003cstrong\u003e(e) \u003c/strong\u003eRepresentative IHC images of DAPI (blue) and MMO-9 (white arrow, green) in the hippocampal region of the brain after LiFUS. Scale bar: 100 µm.\u003c/p\u003e\n\u003cp\u003eLiFUS, low-intensity focused ultrasound; IHC, immunohistochemistry; RT-PCR, reverse transcription-polymerase chain reaction; BBB, blood brain barrier; SD, standard deviation; DAPI, 4′,6-diamidino-2-phenylindole; ANOVA, analysis of variance; ZO-1, zonula occludens-1\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/d28ca3d4ecf6d162e09c56a1.png"},{"id":53396642,"identity":"42879d01-2bbf-435b-9f42-081b0a2d0617","added_by":"auto","created_at":"2024-03-25 13:38:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4809002,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNo evidence of death signals after LiFUS stimulation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eResults of\u003cstrong\u003e \u003c/strong\u003eATP\u003cstrong\u003e \u003c/strong\u003equantification in the lesion.\u003cstrong\u003e \u003c/strong\u003eThe bar graph shows the normalized level of ATP relative to that of control. #p\u0026lt;0.05, ##p\u0026lt;0.01, ###p\u0026lt;0.001; compared to control, *p\u0026lt;0.05; compared to 1 h. \u003cstrong\u003e(b)\u003c/strong\u003e Western blot results for NF-κB. The bar graph shows the normalized level of the receptor relative to that of the housekeeping genes. Band intensity images for each group are included. ***p\u0026lt;0.001; compared to 1 h. \u003cstrong\u003e(c, d)\u003c/strong\u003eRT-PCR results for caspase 3/9. No significant differences were observed among the groups. All data are represented as mean ± SD (n=5). One-way ANOVA with Tukey's multiple comparison test was performed. \u003cstrong\u003e(e)\u003c/strong\u003e Representative images of the TUNEL assay (white arrow) and H\u0026amp;E images of each group. Scale bar: 20 µm for TUNEL and 100 µm for H\u0026amp;E images.\u003c/p\u003e\n\u003cp\u003eLiFUS, low-intensity focused ultrasound; RT-PCR, reverse transcription-polymerase chain reaction; SD, standard deviation; ANOVA, analysis of variance; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; H\u0026amp;E, hematoxylin and eosin\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/97642552f1d273e72804a2ca.png"},{"id":53396645,"identity":"4eb585b2-689c-4528-b76a-9418a7873cf8","added_by":"auto","created_at":"2024-03-25 13:38:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1002254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlterations in BBB permeability following antagonist administration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eRepresentative images of Evans blue dye leakage in each group. \u003cstrong\u003e(b)\u003c/strong\u003e Optical density of Evans blue dye in the sonicated area after LiFUS in each group. **p\u0026lt;0.01, ***p\u0026lt;0.001; compared to 1 h \u003cstrong\u003e(c)\u003c/strong\u003e Western blot results for the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor. The bar graph shows the normalized level of the receptor relative to that of ß-actin. Band intensity images for each group are included. *p\u0026lt;0.05, **p\u0026lt;0.01; compared to 1 h. \u003cstrong\u003e(d)\u003c/strong\u003e Western blot result for ZO-1. The analysis method employed for the ZO-1 was the same as that for the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor. No statistical differences were observed among the groups. All data are represented as mean ± SD (n=3–5). *p\u0026lt;0.05; compared to 1 h. One-way ANOVA with Tukey's multiple comparison test was performed.\u003c/p\u003e\n\u003cp\u003eLiFUS, low-intensity focused ultrasound; BBB, blood brain barrier; SD, standard deviation; ANOVA, analysis of variance; ZO-1, zonula occludens-1\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/8ec7e5de539be7a17a6af62f.png"},{"id":53396647,"identity":"a56e3041-f1db-468f-b177-96c8d65356df","added_by":"auto","created_at":"2024-03-25 13:38:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1527321,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical summary of the study\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003eA\u003cstrong\u003e \u003c/strong\u003e0.5-MHz transducer was placed on the skull of rats, and sonication was performed. Microbubbles inside the vessels affected by LiFUS energy loosened the BBB to make it permeable. The hippocampal area was the main region analyzed. \u003cstrong\u003e(b) \u003c/strong\u003eCompared to the control group, in the LiFUS treated group, the upregulated expression of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor, NLRP3, and IL-1ß was observed, accompanied by a decrease in the expression level of the tight junction proteins and RNA. Consequently, the leakage of the Evans blue dye in the parenchyma was increased compared to that in the control conditions. In the antagonist-treated group, changes in protein and RNA expression levels were observed, but they did not reach statistical significance. The amount of Evans blue dye leakage was also not significant.\u003cstrong\u003e (c) \u003c/strong\u003eAlthough\u003cstrong\u003e \u003c/strong\u003eLiFUS induces the temporal upregulation of markers related to the inflammatory responses, including NLRP-3, IL-1ß, and NF-κB, this technique is safe as it does not reach a level causing cell damage or hemorrhage to the sonicated area.\u003c/p\u003e\n\u003cp\u003eLiFUS, low-intensity focused ultrasound; BBB, blood brain barrier\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/a07e6fd866844ed5c4ab30ac.png"},{"id":53397198,"identity":"72dac8e8-c5d6-4281-a993-b40cb390ebc4","added_by":"auto","created_at":"2024-03-25 13:46:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3773522,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/6bb634b1-b20b-47fc-8f16-bbb7295643ab.pdf"},{"id":53396646,"identity":"be1d2b8d-8853-458b-9023-4c5674ae5e85","added_by":"auto","created_at":"2024-03-25 13:38:32","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12679008,"visible":true,"origin":"","legend":"","description":"","filename":"SuppleFig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/fec26cbea2138c7787de29d7.tif"},{"id":53396650,"identity":"3ccb9744-dafa-4edf-b989-11ecb5bd30bc","added_by":"auto","created_at":"2024-03-25 13:38:33","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":24120436,"visible":true,"origin":"","legend":"","description":"","filename":"SuppleFig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/41ccac88fa44923204c3b156.tif"},{"id":53396644,"identity":"c4a32b86-678a-445d-bec2-5a103bc12071","added_by":"auto","created_at":"2024-03-25 13:38:32","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3727900,"visible":true,"origin":"","legend":"","description":"","filename":"SuppleFig3.tif","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/f7ce3a8d2a05fa6c0b92250a.tif"},{"id":53396649,"identity":"7906bbff-19ba-4c4d-b49c-464b39f67d33","added_by":"auto","created_at":"2024-03-25 13:38:32","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12887668,"visible":true,"origin":"","legend":"","description":"","filename":"SuppleFig4.tif","url":"https://assets-eu.researchsquare.com/files/rs-4140777/v1/720b1479ffe39d69dbd78fa9.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of P2X7 receptor during low-intensity focused ultrasound- induced blood-brain barrier modulation","fulltext":[{"header":"Background","content":"\u003cp\u003eThe blood brain barrier (BBB) is a complex structure that plays a crucial role in maintaining homeostasis within the central nervous system (CNS) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The BBB consists of endothelial cells with tight junctions, basal lamina, end-feet of astrocytes, and pericytes embedded in the basement membrane [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This distinctive arrangement gives the brain immune privilege, while simultaneously presenting a tremendous challenge for the treatment of brain diseases [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Numerous attempts have been made to facilitate efficient drug penetration through the BBB; however, an optimized approach is yet to be established [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLow-intensity focused ultrasound (LiFUS) with microbubbles has emerged as a promising technique [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Moreover, high-intensity focused ultrasound for the thermal coagulation of tissues has been applied, particularly for the treatment of patients with essential tremors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These pioneering techniques serve as a foundation for expanding the concept of using ultrasound in the field of neuroscience, specifically in relation to the brain [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Unlike conventional clinical applications of focused ultrasound, LiFUS induces the oscillation of microbubbles with a relatively small amount of energy [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Many studies have reported that the cavitation effect of bubbles causes loosening of the tight junctions between endothelial cells, leading to a noninvasive, transient, and localized opening of the BBB [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Extensive studies have been conducted to investigate the potential of LiFUS for modulating the BBB. Several studies have analyzed the stability of LiFUS using various parameters [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Other, drug delivery studies have targeted neurodegenerative diseases such as Parkinson\u0026rsquo;s disease and Alzheimer\u0026rsquo;s disease [\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, considerable efforts have been made to investigate alterations in the brain microenvironment following the sonication procedure [\u003cspan additionalcitationids=\"CR24 CR25\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In particular, many studies have evaluated the safety aspects and inflammatory responses after LiFUS [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. However, the molecular mechanisms underlying LiFUS-induced BBB modulation are not fully understood, particularly the primary factors driving this process [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOnce external stress occurs in the brain, the primary response is an increase in extracellular adenosine triphosphate (ATP) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This ATP increase leads to the activation of purinergic receptors, of which the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor is considered one of the main members [33)]. P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors are ATP-gated ion channels that are widely expressed in various components of neurovascular units, including neurons, oligodendrocytes, microglia, and endothelial cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These receptors have been implicated in a broad range of physiological and pathological processes, including inflammation, apoptosis, and cell death [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Owing to their distinctive characteristics, P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors have been mainly investigated in neurodegenerative diseases and tumors, focusing on their involvement in inflammatory responses [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Recently, a few studies have demonstrated that the regulation of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors can protect against BBB breakdown induced by lipopolysaccharide and mitigate the damage caused by alcohol and nicotine exposure [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In addition, the expression levels of tight junctions were preserved in animal stroke models by regulating the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. These findings highlight the significant effect of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors on BBB permeability [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Gradual upregulation of the mRNA expression of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors was observed in an osteoblast model following LiFUS [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Although this study did not include an \u003cem\u003ein vivo\u003c/em\u003e experiment, it showed the potential of LiFUS in modulating the expression of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aimed to analyze BBB-related alterations in the brain microenvironment after LiFUS, with a focus on the involvement of the purinergic P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor. Based on the previous results, we hypothesized that BBB modulation induced by LiFUS might be related to the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor and analyzed the factors associated with P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors and their potential roles in BBB modulation.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Health System (IACUC #2019\u0026thinsp;\u0026minus;\u0026thinsp;0208). A total of 79 Sprague-Dawley rats (male, 7 weeks old, 270\u0026thinsp;\u0026plusmn;\u0026thinsp;20 g) were used in this study. Three animals were housed per cage under a light/dark cycle (12 h) in a room with controlled temperature (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2 \u0026ordm;C) and were allowed free access to food and water. The process and targeted areas for analysis are shown in Fig.\u0026nbsp;1a and b. The number of animals used in each group is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;1. Experimental procedure and MRI images after LiFUS\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(a)\u003c/b\u003e Schematic of the experimental timeline for functional and pathological assessment. \u003cb\u003e(b)\u003c/b\u003e Schematic region of the study area used for western blotting, IHC, RT-PCR, and Evans blue quantification. \u003cb\u003e(c)\u003c/b\u003e Representative MR images to confirm the opening of the BBB. T2-weighted images (left) and T1-weighted images (right) were used. The opening of the BBB (white box, with LiFUS) can be seen compared to the opposite. Scale bar: 2.5 mm. \u003cb\u003e(d)\u003c/b\u003e The enhanced signal in the ROI after LiFUS.\u003c/p\u003e \u003cp\u003e*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to the control. All data are represented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3). Unpaired t-test was performed.\u003c/p\u003e \u003cp\u003eLiFUS, low-intensity focused ultrasound; IHC, immunohistochemistry; RT-PCR, reverse transcription-polymerase chain reaction; BBB, blood brain barrier; MRI, magnetic resonance imaging; ROI, region of interest; SD, standard deviation\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNumber of animals per group\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24 h\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSham\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUS only\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUS\u0026thinsp;+\u0026thinsp;antagonist\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUS\u0026thinsp;+\u0026thinsp;Evans blue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFUS\u0026thinsp;+\u0026thinsp;Evans blue\u0026thinsp;+\u0026thinsp;antagonist\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003eFUS, focused ultrasound\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eFocused ultrasound (FUS) treatment with microbubbles in rats\u003c/h2\u003e \u003cp\u003eA waveform generator (33220A, Agilent, Palo Alto, CA, USA) was connected to a power amplifier (240 L, ENI Inc., Rochester, NY, USA) to facilitate the experimental setup. Subsequently, a coupler (Pulsar C30-102-481/2N) was used to connect the power amplifier, transducer, and oscilloscope. The 515-kHz single-element transducer (H-107MR, Sonic Concept Inc., Bothell, WA, USA) with a full width at half maximum of 4 mm and a focal depth of 51.74 mm was affixed to a custom-designed cone. The tip of the cone was wrapped in a polyurethane membrane and prefilled with degassed water prior to sonication. The RF signal was monitored continuously using an oscilloscope (DSOX2002A; Keysight Technologies, Santa Rosa, CA, USA).\u003c/p\u003e \u003cp\u003eRats were deeply anesthetized via intraperitoneal injection of ketamine cocktail and positioned in a stereotaxic frame. The scalps of the animals were shaved and subsequently incised, and hemostasis was ensured. To prevent the loss of energy during sonication, an ultrasound transmission gel (ProGel; Dayo Medical Co., Seoul, Korea) was applied to fill the gap between the animal's skull and the tip of the cone. The target region for sonication was the right hippocampal area, located at coordinates \u0026minus;\u0026thinsp;3.5 mm posterior and 2 mm lateral from the bregma. Intravenous injection of microbubbles (20 \u0026micro;L/kg, Definity, Lantheus Medical Imaging, North Billerica, MA, USA) was performed, with sonication initiated 10 s after injection. A sonication protocol of approximately 0.3 MPa intensity was employed for each session, with a duration of 2 min, burst duration of 10 ms, and pulse repetition frequency of 1 Hz. Animals were divided into four groups (1, 4, and 24 h after FUS and control). The control group did not undergo any sonication procedure.\u003c/p\u003e \u003cp\u003eA specific P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor antagonist (A430879, HY-15488; MCE, NJ, USA) was administered intraperitoneally at a dose of 100 mg/kg per head 20 min before sonication. Thereafter, identical procedures were performed in all groups.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eMagnetic resonance imaging (MRI)\u003c/h2\u003e \u003cp\u003eMRI was performed 1 h after sonication using a Bruker 9.4 T 20-cm bore MRI system (Biospec 94/20 USR; Bruker, Ettlingen, Germany) and a rat head coil. The animals were anesthetized via inhalation of 2% isoflurane during MRI. A gadolinium-based MRI contrast agent, gadobutrol (Gadovist; 0.2 mL/kg), was injected intravenously, and contrast-enhanced T1-weighted images were used to confirm the opening of the BBB (echo time, 2 ms; repetition time, 8.06 ms; echo train length, 2 ms; slice thickness, 1 mm). For statistical analysis, three rats were included in each study group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunostaining and histological analyses\u003c/h2\u003e \u003cp\u003eFor this experiment, 5-\u0026micro;m tissue sections were de-paraffinized using xylene and then rehydrated. The antigen was retrieved using 1X citrate buffer 85\u0026deg;C for 15 min. Immunostaining was performed after cooling and rinsing the slides for 20 min. The tissues were blocked for 90 min at room temperature (20\u0026deg;C) using a blocking solution containing phosphate-buffered saline (PBS), 0.2% normal goat serum, and 0.2% Triton X-100. Subsequently, the brains were incubated overnight at 4\u0026deg;C in a blocking solution containing anti-P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e (1:300; ab109054; Abcam, UK), anti-Reca-1 (1:300; ab9774; Abcam), anti-MMP9 (1:300; ab76003; Abcam), anti-MMP2 (1:300; ab92536; Abcam), and anti-Iba-1 (1:500; 019-19741; Wako) antibodies. After the primary immunoreaction, the samples were briefly washed with 1\u0026times; PBS and incubated for 2 h at room temperature with goat anti-rabbit secondary antibodies (1:600; A11008; Thermo Fisher Scientific, USA) conjugated with Alexa Fluor 488 and goat anti-mouse secondary antibodies (1:600; A11005; Thermo Fisher Scientific) conjugated with Alexa Fluor 594. Nuclei were stained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI; 1:1000; D1306; Thermo Fisher Scientific) during conjugation with the secondary antibodies.\u003c/p\u003e \u003cp\u003eHematoxylin and eosin (H\u0026amp;E) (hematoxylin [Vector Laboratories, USA] and eosin [Sigma-Aldrich, USA]) staining was performed on paraffin sections, and images were acquired using an optical microscope (BX51; Olympus, Tokyo, Japan). Histological analysis was performed to examine tissue damage, especially to monitor red blood cell extravasation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eRat brains were collected at the indicated time points after FUS (1, 4, and 24 h). Samples were homogenized using lysis buffer (PRO-PREP; Intron Biotechnology, Pyeongtaek, Korea) and placed on ice for 30 min, followed by centrifugation at 13000 rpm for 30 min. The protein concentration in the lysate was measured using the bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific, Fremont, CA, USA). Subsequently, 10\u0026ndash;20 \u0026micro;g of each protein sample were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane using a Bio-Rad Trans-Blot apparatus. Membranes were incubated with blocking buffer (5% nonfat dry milk in PBS containing 0.05% Tween 20 [TBST]) for 1 h at room temperature. The membranes were then incubated with the primary rabbit anti-P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e (1:1000, Abcam), rabbit anti-occludin (1:1000, Abcam), rabbit anti-ZO-1 (1:1000, Thermo Fisher Scientific), rabbit anti-IL-1β (1:500; Abcam), rabbit anti-NF-κB (1:1000, Cell Signaling Technology, Beverly, MA, USA), and mouse anti-β actin (1:10000, Sigma-Aldrich, MO, USA) antibodies overnight at 4 \u0026ordm;C. Next, the membrane was washed three times for 5 min each and incubated with the corresponding secondary antibodies, either goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L)-HRP (GenDEPOT, Katy, TX, USA) or goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L)-HRP (GenDEPOT), for 90 min at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescent solution (WEST-Queen; iNtRON Biotechnology, Seongnam, Korea) and developed using LAS 4000 Mini (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The intensity of each band was determined using an analytical system (MultiGauge version 3.0; Fujifilm, Tokyo, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of Evans blue dye\u003c/h2\u003e \u003cp\u003eIntravenous injection of Evans blue dye (100 mg/kg, Sigma-Aldrich) diluted in 2% saline solution was administered at specific time points (1, 4, and 24 h) after sonication. The dye was allowed to circulate for 1 h prior to further experiments. Subsequently, the rats were perfused with 500 mL saline to remove the dye localized within the intravascular space. The brains were then harvested and divided along the midline of the brain, and the ipsilateral hemisphere was homogenized. After weighing, samples were homogenized with saline (mg/mL), and centrifuged at 15000\u0026times; g, 4 \u0026ordm;C for 30 min. Equal volumes of trichloroacetic acid were added to each supernatant. Samples were incubated at 4 \u0026ordm;C overnight and centrifuged. To increase their optic length, 90 \u0026micro;L of 95% ethanol was added to each well containing 30 \u0026micro;L supernatant and mixed by repetitive pipetting. The extravasated Evans blue dye was spectrophotometrically quantified at 620 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eReal-time reverse transcription-polymerase chain reaction (RT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from the cells using an easy-spin total RNA extraction kit (iNtRON Biotechnology, Seongnam, Republic of Korea), and oligo (dT)-primed cDNA was synthesized from the total RNA using the Maxime RT PreMix kit (iNtRON Biotechnology), according to the manufacturer\u0026rsquo;s instructions. Quantitative RT-PCR was performed using the StepOnePlus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA) with the SYBR Green Dye system (SYBR Premix Ex Taq [Tli RNase Plus] and ROX reference dye [TAKARA Bio Inc., Foster City, CA, USA]). Gene expression level was normalized to glyceraldehyde 3-phosphate dehydrogenase transcript levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay\u003c/h2\u003e \u003cp\u003eA TUNEL assay kit (G3250, Promega, Madison, WI, USA) was used to assess the level of apoptosis in paraffin-embedded rat brain sections according to manufacturer\u0026rsquo;s instructions. Briefly, the paraffin-embedded tissue sections were de-paraffinized, rehydrated, and treated with proteinase K. Permeabilized tissue sections were incubated with the rTdT reaction mixture for 1 h in a dark and humidified chamber at 37 ℃. The sections were co-stained with DAPI and cover-slipped with mounting solution. The labeled DNA was observed using an LSM700 confocal microscope (Carl Zeiss, Jena, Germany). For the brain sections, the detection wavelengths were those of fluorescein isothiocyanate and DAPI.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eATP assay\u003c/h2\u003e \u003cp\u003eAn ATP assay kit (#ab83355, Abcam) was used to analyze the effects of FUS on P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e-related ATP levels following the manufacturer\u0026rsquo;s instructions. ATP content was measured using a colorimetric assay, which generated a quantifiable product detected at 570 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll results are expressed as the mean \u0026plusmn; standard deviation and analyzed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA, USA). MATLAB software was used to calculate the intensity of the region of interest (ROI) using the T1 images. Data were analyzed using an unpaired t-test (n\u0026thinsp;=\u0026thinsp;3) and one-way analysis of variance, followed by Tukey\u0026rsquo;s post-hoc test (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;5). P-values lower than 0.05 were considered to indicate statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eConfirmation of BBB opening after LiFUS\u003c/h2\u003e \u003cp\u003eFirst, the sonication area of LiFUS and the degree of BBB opening were confirmed. Prior to the analysis of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor, LiFUS with microbubbles was used to identify the area of the lesion and analyze the changes in MRI signals (Fig.\u0026nbsp;1c and d). The results confirmed that the energy reached part of the upper layer of the thalamus (DV: 4 mm from the bregma), including the hippocampus, through the T1-contrast-enhanced image. In addition, by quantifying the intensity of the ipsilateral and contralateral signals using the same ROI, it was found that the signals of the sonicated area (FUS: 2.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37) were significantly enhanced (con: 1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;3 for each group).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAlterations in P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor expression and signaling cascade following LiFUS\u003c/h2\u003e \u003cp\u003eTo investigate whether ultrasound energy affects the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor, we initially employed immunohistochemistry (IHC) to assess the variations in receptor expression levels over time (Fig.\u0026nbsp;2a). A notable upregulation of receptor expression in the hippocampus within the group 1 h after LiFUS. For a more accurate quantitative analysis, we conducted western blotting and real-time RT-PCR to evaluate the protein and RNA expression levels of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor. A significant increase in protein expression was observed in the group 1 hour (1 h: 0.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2) after sonication compared to that in the other groups (Fig.\u0026nbsp;2b, con: 0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; 4 h: 0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; 24 h: 0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, n\u0026thinsp;=\u0026thinsp;5 for each group). Similarly, the RNA levels showed a significant increase in the group corresponding to 1 h after LiFUS (1 h: 2.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23) compared to the other groups (Fig.\u0026nbsp;2c, con: 1.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 4 h: 1.91\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 24 h: 1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;5 for each group). Notably, the RNA expression levels remained elevated even after 24 h (4 h: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 24 h: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, compared to the control).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;2. Regulation of P\u003c/b\u003e \u003csub\u003e \u003cb\u003e2\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eX\u003c/b\u003e \u003csub\u003e \u003cb\u003e7\u003c/b\u003e \u003c/sub\u003e \u003cb\u003ereceptors and downstream signaling pathways after sonication\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003e(a)\u003c/b\u003e Representative IHC images of DAPI (blue), P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor (white arrow, green) and RECA-1 (red) in the hippocampal region of the brain after FUS. Scale bar: 100 \u0026micro;m. \u003cb\u003e(b, c)\u003c/b\u003e Western blotting and RT-PCR results for P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors. The bar graph shows the normalized level of the receptors relative to the housekeeping genes. Band intensity images for each group are also included. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; compared to 1 h, #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ##p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; compared to control. \u003cb\u003e(d, e)\u003c/b\u003e Western blot and RT-PCR results for NLRP-3 inflammasome. The analysis method employed for NLRP-3 was the same as that for the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors. **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; compared to 1 h, \u0026amp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u0026amp;\u0026amp;p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; compared to 4 h. \u003cb\u003e(f, g)\u003c/b\u003e Western blot and RT-PCR results for IL-1\u0026szlig;. The analysis method employed for NLRP-3 was the same as that for the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; compared to 1 h, #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; compared control. All data are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;5). One-way ANOVA with Tukey's multiple comparison test was performed.\u003c/p\u003e \u003cp\u003eFUS, focused ultrasound; IHC, immunohistochemistry; RT-PCR, reverse transcription-polymerase chain reaction; SD, standard deviation; DAPI, 4\u0026prime;,6-diamidino-2-phenylindole; ANOVA, analysis of variance\u003c/p\u003e \u003cp\u003eWe also analyzed NLRP-3 and IL-1β levels, which are known to exhibit a substantial correlation with P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor expression (32). The protein expression pattern of NLRP-3 was similar to that of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor, peaking at 1 h after LiFUS (1 h: 2.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34), but with a slight difference in the significance among groups (Fig.\u0026nbsp;2d, con: 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 4 h: 1.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 24 h: 1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, n\u0026thinsp;=\u0026thinsp;5 for each group). Similar results were obtained with the RNA levels (Fig.\u0026nbsp;2e, con: 1.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 4 h: 1.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 24 h: 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to 1 h: 2.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26, n\u0026thinsp;=\u0026thinsp;5 for each group). However, NLRP-3 RNA levels remained elevated until 4 h after LiFUS : p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; 24 h: p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). IL-1β protein expression level showed similar trends (Fig.\u0026nbsp;2f, con: 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; 4 h: 1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27; 24 h: 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to the 1 h: 1.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31, n\u0026thinsp;=\u0026thinsp;5 for each group). The protein levels were found to be upregulated for up to 4 h compared to those in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In terms of RNA expression, the highest peak was observed at 1 h after sonication (Fig.\u0026nbsp;2g, con: 1.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 4 h: 1.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; 24 h: 1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 compared to 1 h: 2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55, n\u0026thinsp;=\u0026thinsp;5 for each group). Taken together, these results showed the upregulation of the receptor and its signaling after LiFUS treatment, with the expression levels gradually returning to baseline within a day. The findings suggest that LiFUS temporally affects the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor and related proinflammatory cytokines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAlterations of BBB-related protein expression after FUS\u003c/h2\u003e \u003cp\u003eWe assessed alterations in BBB-related proteins, such as ZO-1 and occludin, to confirm the impact of LiFUS on BBB integrity using western blotting and RT-PCR. Initially, ZO-1 exhibited the lowest protein expression at 1 h after sonication (1 h: 1.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 compared to con), in contrast to the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor, after which ZO-1 protein expression gradually normalized over time (Fig.\u0026nbsp;3a, con: 3.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36; 4 h: 2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to con; 24 h: 3.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55). The reduced expression level remained significant in the group a day after sonication (1 h and 4 h: p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to 24 h). This trend was consistent at the ZO-1 RNA level (Fig.\u0026nbsp;3b. con: 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13; 1 h: 0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to con and 24 h; 4 h: 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to 24 h; 24 h: 1.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38). Occludin, a representative tight junction protein, exhibited similar patterns in both protein and RNA levels (Fig.\u0026nbsp;3c and d, occludin protein expression : con: 3.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3; 1 h: 1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to con and 24 h; 4 h: 1.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to con and 24 h; 24 h: 3.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 and occludin RNA expression: con: 1.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15; 1 h: 0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 compared to con and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to 24 h; 4 h: 0.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to con and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to con and 24 h; 24 h: 1.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4). In addition, MMP9, which is reportedly correlated to the BBB [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], was observed through IHC, and its expression was partially elevated at 1\u0026ndash;4 h after LiFUS (Fig.\u0026nbsp;3e). In addition, the expression of MMP2 data for each time point is shown in Additional file. 1. These findings indicate that LiFUS influences BBB integrity and enhances BBB permeability. However, this effect may be transient, as BBB permeability returns to normal within a day.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;3. Regulation of BBB-related proteins after LiFUS stimulation\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(a, b)\u003c/b\u003e Western blot and RT-PCR results for ZO-1. The bar graph shows the normalized level of the receptors relative to that of the housekeeping genes. Band intensity images for each group are included. #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ##p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ###p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; compared to control, \u0026dagger;\u0026dagger;\u0026dagger;\u0026lt;0.001; compared to 24 h. \u003cb\u003e(c, d)\u003c/b\u003e Western blot and RT-PCR results for occludin. The analysis method employed for occludin was the same as that for ZO-1. #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ##p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ###p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; compared to control, \u0026dagger;\u0026dagger;\u0026dagger;\u0026lt;0.001; compared to 24 h. All data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;5). One-way ANOVA with Tukey's multiple comparison test was performed. \u003cb\u003e(e)\u003c/b\u003e Representative IHC images of DAPI (blue) and MMO-9 (white arrow, green) in the hippocampal region of the brain after LiFUS. Scale bar: 100 \u0026micro;m.\u003c/p\u003e \u003cp\u003eLiFUS, low-intensity focused ultrasound; IHC, immunohistochemistry; RT-PCR, reverse transcription-polymerase chain reaction; BBB, blood brain barrier; SD, standard deviation; DAPI, 4\u0026prime;,6-diamidino-2-phenylindole; ANOVA, analysis of variance; ZO-1, zonula occludens-1\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eSafety assessment of LiFUS-induced BBB modulation\u003c/h2\u003e \u003cp\u003eAfter confirming that ultrasound energy was sufficient for BBB modulation, we conducted a comprehensive safety assessment of the process. Initially, we monitored the ATP levels over time following LiFUS treatment (Fig.\u0026nbsp;4a). Consistent with previous findings, the highest concentration of ATP was found at 1 h after LiFUS (1 h: 1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to con), with continued elevation even after 24 h (con: 1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39; 4 h: 1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; 24 h: 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to con, n\u0026thinsp;=\u0026thinsp;5 for each group). Subsequently, we conducted an analysis of alterations in the NF-κB expression levels in response to the observed temporal increase in IL-1\u0026szlig;. Thus, NF-κB expression levels may represent a signal transduction outcome of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor activation after LiFUS. These findings are consistent with our prior data. Specifically, there was a noticeable elevation in the expression of phosphorylated NF-κB (p-NF-κB) within 1 h (Fig.\u0026nbsp;4b, 1h: 3.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89). Additionally, their results gradually returned to baseline over time (p-NF-κB, con: 1.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39; 4 h: 1.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37; 24 h: 1.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to 1 h, n\u0026thinsp;=\u0026thinsp;5 for each group).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;4. No evidence of death signals after LiFUS stimulation\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(a)\u003c/b\u003e Results of ATP quantification in the lesion. The bar graph shows the normalized level of ATP relative to that of control. #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, ##p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ###p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; compared to control, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; compared to 1 h. \u003cb\u003e(b)\u003c/b\u003e Western blot results for NF-κB. The bar graph shows the normalized level of the receptor relative to that of the housekeeping genes. Band intensity images for each group are included. ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; compared to 1 h. \u003cb\u003e(c, d)\u003c/b\u003e RT-PCR results for caspase 3/9. No significant differences were observed among the groups. All data are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;5). One-way ANOVA with Tukey's multiple comparison test was performed. \u003cb\u003e(e)\u003c/b\u003e Representative images of the TUNEL assay (white arrow) and H\u0026amp;E images of each group. Scale bar: 20 \u0026micro;m for TUNEL and 100 \u0026micro;m for H\u0026amp;E images.\u003c/p\u003e \u003cp\u003eLiFUS, low-intensity focused ultrasound; RT-PCR, reverse transcription-polymerase chain reaction; SD, standard deviation; ANOVA, analysis of variance; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; H\u0026amp;E, hematoxylin and eosin\u003c/p\u003e \u003cp\u003eSubsequently, a comprehensive analysis of the cellular damage and apoptosis resulting from sonication was performed. Quantification of the RNA expression levels of caspase 3/9, used as markers of apoptosis, revealed an increased expression level 1 h post-sonication (Fig.\u0026nbsp;4c and d). However, this increase did not show statistical significance, and caspase 3/9 expression subsequently returned to baseline levels (caspase 3; con: 0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19; 1 h: 1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39; 4 h: 1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49; 24 h: 1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38, caspase 9; con: 1.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12; 1 h: 1.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36; 4 h: 1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34; 24 h: 1.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28, n\u0026thinsp;=\u0026thinsp;5 for each group). Moreover, the TUNEL assay results indicated the absence of activated cell death signals throughout the brain (Fig.\u0026nbsp;4e). Finally, histopathological examination (H\u0026amp;E staining) confirmed the absence of tissue damage following LiFUS, which was consistent with the findings of the control group. The complete set of whole-brain tissue data for each time point is shown in Additional file 2. Collectively, these results highlight the ability of LiFUS to modulate BBB permeability without inducing brain damage, even in the context of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor activation and transient upregulation of pro-inflammatory cytokines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eDecreased BBB permeability after P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor antagonist injection\u003c/h2\u003e \u003cp\u003eTo substantiate the involvement of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors in LiFUS-induced BBB opening, quantitative analysis of Evans blue was performed using spectrometry (Fig.\u0026nbsp;5a, [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]). The groups used in this study were as follows: N, 1 h, 1 h\u0026thinsp;+\u0026thinsp;A438079, 4 h, 4 h\u0026thinsp;+\u0026thinsp;A438079, 24 h, and 24 h\u0026thinsp;+\u0026thinsp;A438079 (n\u0026thinsp;=\u0026thinsp;5 for each group). The group that received Evans blue administration at 1 h after sonication (1 h: 0.051\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01) exhibited a significantly higher fluorescence intensity compared to that in all the other groups, except the group corresponding to 4 h (Fig.\u0026nbsp;5b, con: 0.038\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; con\u0026thinsp;+\u0026thinsp;A438079: 0.038\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 1 h\u0026thinsp;+\u0026thinsp;A438079: 0.043\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002; 4 h: 0.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; 4h\u0026thinsp;+\u0026thinsp;A438079: 0.038\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 24 h: 0.039\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 24 h\u0026thinsp;+\u0026thinsp;A438079: 0.038\u0026thinsp;\u0026plusmn;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 compared to 1 h). Similarly, upon antagonist administration, the extent of leakage was most pronounced in the 1-hour post-LiFUS group; however, this result was not statistically significant when compared to the control. Notably, the amount of leakage was comparable to that observed in the group that received Evans blue at 4 h after LiFUS.\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;5. Alterations in BBB permeability following antagonist administration\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(a)\u003c/b\u003e Representative images of Evans blue dye leakage in each group. \u003cb\u003e(b)\u003c/b\u003e Optical density of Evans blue dye in the sonicated area after LiFUS in each group. **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; compared to 1 h \u003cb\u003e(c)\u003c/b\u003e Western blot results for the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor. The bar graph shows the normalized level of the receptor relative to that of \u0026szlig;-actin. Band intensity images for each group are included. *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; compared to 1 h. \u003cb\u003e(d)\u003c/b\u003e Western blot result for ZO-1. The analysis method employed for the ZO-1 was the same as that for the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor. No statistical differences were observed among the groups. All data are represented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (n\u0026thinsp;=\u0026thinsp;3\u0026ndash;5). *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; compared to 1 h. One-way ANOVA with Tukey's multiple comparison test was performed.\u003c/p\u003e \u003cp\u003eLiFUS, low-intensity focused ultrasound; BBB, blood brain barrier; SD, standard deviation; ANOVA, analysis of variance; ZO-1, zonula occludens-1\u003c/p\u003e \u003cp\u003eFor a detailed understanding of the leakage phenomenon, we conducted western blot quantification of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e and ZO-1 proteins. For the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor, the group corresponding to 1 h after LiFUS (1 h: 293.6\u0026thinsp;\u0026plusmn;\u0026thinsp;89.56) exhibited a significant expression level (Fig.\u0026nbsp;5c, con: 114.2\u0026thinsp;\u0026plusmn;\u0026thinsp;41.15 p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; 1 h\u0026thinsp;+\u0026thinsp;A438079: 166.7\u0026thinsp;\u0026plusmn;\u0026thinsp;12.79; 4 h: 170\u0026thinsp;\u0026plusmn;\u0026thinsp;7.28; 4 h\u0026thinsp;+\u0026thinsp;A438079: 161.5\u0026thinsp;\u0026plusmn;\u0026thinsp;43.53 p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; 24 h: 179.9\u0026thinsp;\u0026plusmn;\u0026thinsp;33.13; 24 h\u0026thinsp;+\u0026thinsp;A438079: 139.4\u0026thinsp;\u0026plusmn;\u0026thinsp;44.25 p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to 1 h, n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4 for each group). In contrast, ZO-1 expression was the lowest at 1 h (Fig.\u0026nbsp;5d, con: 104.8\u0026thinsp;\u0026plusmn;\u0026thinsp;14.24 p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 compared to 1 h; 1 h: 60.37\u0026thinsp;\u0026plusmn;\u0026thinsp;7.55; 1 h\u0026thinsp;+\u0026thinsp;A438079: 80.96\u0026thinsp;\u0026plusmn;\u0026thinsp;12.06; 4 h: 70.24\u0026thinsp;\u0026plusmn;\u0026thinsp;15.15; 4 h\u0026thinsp;+\u0026thinsp;A438079: 84.74\u0026thinsp;\u0026plusmn;\u0026thinsp;19.71; 24 h: 108\u0026thinsp;\u0026plusmn;\u0026thinsp;28.08; 24 h\u0026thinsp;+\u0026thinsp;A438079: 106.7\u0026thinsp;\u0026plusmn;\u0026thinsp;29.72, n\u0026thinsp;=\u0026thinsp;3\u0026ndash;4 for each group). The results for the remaining downstream signaling pathways in the antagonist-treated groups are shown in Additional file 3. These findings strongly support a possible role of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor in LiFUS-mediated BBB modulation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eLiFUS has emerged as a promising technique offering the advantage of temporal modulation of the BBB to enhance drug delivery for the treatment of brain diseases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Despite its clinical significance, the precise molecular mechanism underlying LiFUS-mediated BBB modulation remains unclear [\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. To address this gap and enable the utilization of the LiFUS technique, we sought to demonstrate the involvement of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor in BBB modulation [\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The activation of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor and associated factors reached a peak intensity at 1 h after sonication, coinciding with a decrease in tight junction proteins. Notably, there was a substantial disparity in the extent of dye leakage depending on the presence of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors. These findings contribute to our understanding of the role of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor in LiFUS-mediated BBB modulation (Fig.\u0026nbsp;6, [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003e \u003cb\u003eFigure\u0026nbsp;6. Graphical summary of the study\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e(a)\u003c/b\u003e A 0.5-MHz transducer was placed on the skull of rats, and sonication was performed. Microbubbles inside the vessels affected by LiFUS energy loosened the BBB to make it permeable. The hippocampal area was the main region analyzed. \u003cb\u003e(b)\u003c/b\u003e Compared to the control group, in the LiFUS treated group, the upregulated expression of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor, NLRP3, and IL-1\u0026szlig; was observed, accompanied by a decrease in the expression level of the tight junction proteins and RNA. Consequently, the leakage of the Evans blue dye in the parenchyma was increased compared to that in the control conditions. In the antagonist-treated group, changes in protein and RNA expression levels were observed, but they did not reach statistical significance. The amount of Evans blue dye leakage was also not significant. \u003cb\u003e(c)\u003c/b\u003e Although LiFUS induces the temporal upregulation of markers related to the inflammatory responses, including NLRP-3, IL-1\u0026szlig;, and NF-κB, this technique is safe as it does not reach a level causing cell damage or hemorrhage to the sonicated area.\u003c/p\u003e \u003cp\u003eLiFUS, low-intensity focused ultrasound; BBB, blood brain barrier\u003c/p\u003e \u003cp\u003eNumerous studies have investigated the effects of LiFUS on the BBB [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Considering the influence of microbubble cavitation, initial observations predominantly focused on sonication-induced alterations in cerebrovascular vessels [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Notably, several studies have demonstrated ultrastructural changes in endothelial cell morphology and brain microvessels, suggesting enhanced permeability to macromolecules after LiFUS-induced BBB opening [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. In addition, analyses of BBB-related proteins have led to the proposal of various possible mechanisms for the passage of substances through the BBB following LiFUS, including paracellular, transcellular, and adsorptive-mediated pathways [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. However, the precise mechanisms underlying BBB modulation are yet to be established.\u003c/p\u003e \u003cp\u003eThe present study substantially advances our understanding of the effects of LiFUS. Herein, we demonstrated that LiFUS with microbubbles triggers complex alterations within various neurovascular units, with the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor acting as a central mediator in this process. The presence of cavitation in both groups 1 h after sonication offers a possible clue. In particular, the differential leakage of Evans blue dye, contingent on the presence of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors, highlights the multifaceted nature of LiFUS-induced BBB modulation and the significant involvement of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors. These data underscore the complexity of LiFUS-induced BBB modulation and the integral role of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors in mediating these effects (44). However, complete restriction of BBB permeability was not achieved even in the antagonist-treated group. Such results are likely due to the involvement of various pathways in LiFUS-induced BBB modulation, as previously suggested [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Hence, it is necessary to consider the various pathways to gain a comprehensive understanding of BBB modulation.\u003c/p\u003e \u003cp\u003eMoreover, our study provides insight into safety considerations associated with the LiFUS technique. Vigilance is crucial in the post-sonication period to monitor potential edema or hemorrhage resulting from BBB modulation and subsequent inflammatory response at the lesion site. Previous studies have emphasized the sterile inflammatory response, with a focus on alterations in the NF-κB pathway following LiFUS [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In particular, when quantifying protein levels in the brain following sonication, a transient upregulation of cytokines related to inflammation was observed, which eventually normalized over time [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Although the present study revealed similar trends, the outcomes differed significantly. Notably, in our study, the expression of pro-inflammatory cytokines, including NLRP3, IL-1\u0026szlig;, and NF-κB, showed an initial increase up to approximately 4 h after sonication. These elevated levels returned to baseline within 24 h. Moreover, TUNEL staining and H\u0026amp;E analysis consistently verified the absence of damage to the brain tissue. Thus, our study demonstrates that LiFUS induces transient alterations in the BBB and its physiology; however, these changes do not reach a critical or injurious threshold. Comparable or milder inflammatory responses have been observed in studies utilizing energy of similar intensity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Our findings suggest that LiFUS can be safely applied under well-defined parameters.\u003c/p\u003e \u003cp\u003eWe found that the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors are involved in BBB modulation induced by LiFUS under safe conditions. When applying this technique to patients, the transfer of energy is affected by various parameters, such as individual differences in patient status, even in cases where the inflammatory response needs to be well-controlled, such as in Alzheimer\u0026rsquo;s disease [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. LiFUS energy is applied for a short duration of approximately 2 min. However, the BBB has been shown to be normalized within approximately 1 day [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Notably, the BBB opens up approximately 4 h after sonication, allowing continuous drug accumulation in the brain [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Under such conditions, the extent of BBB tight junction loosening may be regulated by administering P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor antagonists without causing significant damage to the sonicated area, as demonstrated in this study. For the antagonist-treated group (Additional file 3), a slight increase in P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor expression was observed, but it did not show statistical significance, and P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor expression subsequently returned to baseline levels within 24 h. Given the pivotal role of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors in brain pathology-related inflammatory pathways, the regulation of receptor activity may serve as a viable approach to attenuate downstream inflammatory processes. Thus, further investigations utilizing P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e antagonists and the optimization of treatment parameters are warranted. Nevertheless, this study serves as a foundational investigation with the potential to expand the scope of existing LiFUS applications.\u003c/p\u003e \u003cp\u003eAlthough our findings identify potential modulators of LiFUS-mediated BBB opening that can act without causing brain damage, there are some limitations to this study. First, while our study demonstrated P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor activation, which is implicated in BBB modulation, the precise origin of this receptor activation remains unclear [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. According to previous studies, no significant change was observed in the expression of Iba-1 (Additional file 4) [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]). Thus, we speculate that the increased expression of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors may originate from endothelial cells. However, for a more comprehensive understanding, further analyses using techniques such as RNA sequencing should be conducted, particularly considering the clinical applicability of this technology [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Second, our study focused on analyzing the intracerebral response at an energy level near 0.3 MPa, which translates to approximately 1.6 W/cm\u003csup\u003e2\u003c/sup\u003e in terms of energy per unit area. However, it is important to note that ultrasound energy is employed with a range of powers depending on the specific experimental conditions. Therefore, to assess the effects of LiFUS comprehensively, it is necessary to investigate changes using a broader range of parameters [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Finally, this study was conducted on rats in a normal physiological state. LiFUS is currently used to treat various brain diseases [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. It is important to recognize that in diseases that differ from the normal physiological state, the levels of cytokines related to inflammation and biological responses to ultrasound energy may vary [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. Therefore, the findings of this study should be interpreted with caution. To facilitate a more comprehensive understanding and ensure safer application of this technique, future research should encompass animal models representing diverse brain disease conditions.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur study confirmed changes in the activity of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors and BBB-related proteins in response to LiFUS. Furthermore, we demonstrated that the presence or absence of P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptors influences the degree of BBB opening. These findings provide valuable insights into the complex mechanisms underlying LiFUS-mediated BBB modulation. Moreover, they support the feasibility of potentially regulating the degree of BBB opening using LiFUS as a noninvasive strategy for modulating the BBB.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBlood brain barrier (BBB)\u003c/p\u003e\n\u003cp\u003eFocused ultrasound (FUS)\u003c/p\u003e\n\u003cp\u003eLow-intensity focused ultrasound (LiFUS)\u003c/p\u003e\n\u003cp\u003eZonula occludens-1 (ZO-1)\u003c/p\u003e\n\u003cp\u003eMagnetic resonance imaging (MRI)\u003c/p\u003e\n\u003cp\u003eStandard deviation (SD)\u003c/p\u003e\n\u003cp\u003eRegion of Interest (ROI)\u003c/p\u003e\n\u003cp\u003eTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)\u003c/p\u003e\n\u003cp\u003eAdenosine triphosphate (ATP)\u003c/p\u003e\n\u003cp\u003ePhosphate-buffered saline (PBS)\u003c/p\u003e\n\u003cp\u003eHematoxylin and eosin (H\u0026amp;E)\u003c/p\u003e\n\u003cp\u003eReverse transcription-polymerase chain reaction (RT-PCR)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Health System (IACUC #2019-0208)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data presented in this study are available on request from the corresponding authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Basic Science Research Program of the National Research Foundation of Korea supported by the Ministry of Education [RS-2023-00266075] and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT)\u0026nbsp;[NRF-2020R1C1C1013535].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.C.N., J.L., and B.W.S. conceptualized and designed the study. J.L., J.P., H.K., and S.H. assisted with and performed experiments. J.P. Y.C.N.B.W.S. and W.S.C. analyzed the data. W.S.C., Y.C.N., B.W.S., and J.P. edited the manuscript. J.P. and Y.C.N. wrote the original draft. All the authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Medical Illustration \u0026amp; Design (MID) team in the Medical Research Support Services of Yonsei University College of Medicine for their excellent support with the medical illustrations and figures made on Biorender.com.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eP. Ballabh, A. Braun, M. Nedergaard, The blood-brain barrier: an overview: structure, regulation, and clinical implications, Neurobiol Dis, 16 (2004) 1-13.\u003c/li\u003e\n\u003cli\u003eN.J. Abbott, A.A. Patabendige, D.E. Dolman, S.R. Yusof, D.J. 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Dhiman, P2X7 receptor in multifaceted cellular signalling and its relevance as a potential therapeutic target in different diseases, Eur J Pharmacol, 906 (2021).\u003c/li\u003e\n\u003cli\u003eR.L. Noel, A.J. Batts, R. Ji, A.N. Pouliopoulos, S. Bae, A.R. Kline-Schoder, E.E. Konofagou, Natural aging and Alzheimer\u0026apos;s disease pathology increase susceptibility to focused ultrasound-induced blood-brain barrier opening, Sci Rep, 13 (2023) 6757.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Low-intensity focused ultrasound, Blood-brain barrier, P2X7 receptor, Tight junction","lastPublishedDoi":"10.21203/rs.3.rs-4140777/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4140777/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAlthough low-intensity focused ultrasound (LiFUS) with microbubbles is used to temporally open the blood-brain barrier (BBB), the underlying mechanism is not fully understood. This study aimed to analyze BBB-related alterations in the brain microenvironment after LiFUS, with a focus on the involvement of the purinergic P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eSprague-Dawley rats were sonicated with LiFUS at 0.3 MPa energy. The impact of LiFUS on the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor and inflammatory-related proteins, including NLRP3 and interleukin-1β, was analyzed through western blotting. The BBB-associated tight junction proteins, zonula occludens-1 (ZO-1) and occludin, were also analyzed. BBB permeability was assessed by quantifying the amount of Evans blue dye penetration using spectrophotometry. Furthermore, the safety of the sonication procedure was verified via terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and hematoxylin and eosin staining.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eSubstantial increases in the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor and its downstream signaling were confirmed after sonicating the BBB with LiFUS for 1 h (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, for tight junction proteins, the lowest expression was observed at 1 h (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Both responses were normalized back to the original state over time. No evidence of brain damage was observed during the procedure. Furthermore, the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor antagonist-injected group showed reduced Evans blue dye penetration compared to that 1 h after FUS, indicating a mitigated impact of LiFUS on the BBB.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eHerein, we elucidate the underlying mechanism by which LiFUS affects the BBB, with a focus on the involvement of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor. Our findings demonstrate that the extent of BBB opening varies upon the regulation of the P\u003csub\u003e2\u003c/sub\u003eX\u003csub\u003e7\u003c/sub\u003e receptor. This study provides valuable insights into the mechanisms underlying BBB modulation through LiFUS, thereby laying the foundation for expanding its applications.\u003c/p\u003e","manuscriptTitle":"Role of P2X7 receptor during low-intensity focused ultrasound- induced blood-brain barrier modulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-25 13:38:26","doi":"10.21203/rs.3.rs-4140777/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorAssigned","content":"","date":"2024-03-21T16:50:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-21T10:13:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Fluids and Barriers of the CNS","date":"2024-03-21T04:52:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"fluids-and-barriers-of-the-cns","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"fbcn","sideBox":"Learn more about [Fluids and Barriers of the CNS](http://fluidsbarrierscns.biomedcentral.com/)","snPcode":"12987","submissionUrl":"https://submission.nature.com/new-submission/12987/3","title":"Fluids and Barriers of the CNS","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7e781664-0619-4f86-a185-199908f97f60","owner":[],"postedDate":"March 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-03-25T13:38:26+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-25 13:38:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4140777","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4140777","identity":"rs-4140777","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}