Oxygen-loaded microbubble-mediated sonoperfusion and oxygenation for neuroprotection after ischemic stroke reperfusion

doi:10.21203/rs.3.rs-2761060/v1

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Research Article

Oxygen-loaded microbubble-mediated sonoperfusion and oxygenation for neuroprotection after ischemic stroke reperfusion

https://doi.org/10.21203/rs.3.rs-2761060/v1

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published 06 Jul, 2023

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Background: Ischemic stroke-reperfusion (S/R) injury is a crucial issue in the protection of brain function after thrombolysis. The vasodilation induced by ultrasound (US)-stimulated microbubble cavitation has been applied to reduce S/R injury through sonoperfusion. The present study uses oxygen-loaded microbubbles (OMBs) with US stimulation to provide sonoperfusion and local oxygen therapy for the reduction of brain infarct size and neuroprotection after S/R.

Methods: The murine S/R model was established by photodynamic thrombosis and thrombolysis at the remote branch of the anterior cerebral artery. In vivo blood flow, partial oxygen pressure (pO₂), and brain infarct staining were examined to analyze the validity of the animal model and OMB treatment results. The animal behaviors and measurement of the brain infarct area were used to evaluate long-term recovery of brain function.

Results: The percentage of blood flow was 45±3%, 70±3%, and 86±2% after 60 min stroke, 20 min reperfusion, and 10 min OMB treatment, respectively, demonstrating sonoperfusion, and the corresponding pO₂ level was 60±1%, 76±2%, and 79±4%, showing reoxygenation. After 14 days of treatment, a 87±3% reduction in brain infarction and recovery of limb coordination were observed in S/R mice. The expression of NF-κB, HIF-1α, IL-1β, and MMP-9 was inhibited and that of eNOS, BDNF, Bcl2, and IL-10 was enhanced, indicating activation of anti-inflammatory and anti-apoptosis responses and neuroprotection. Our study demonstrated that OMB treatment combines the beneficial effects of sonoperfusion and local oxygen therapy to reduce brain infarction and activate neuroprotection to prevent S/R injury.

Ultrasound

thrombolysis

perfusion

ischemia-reperfusion injury

inflammatory responses

mechanical effect

Ischemic stroke is one of the leading causes of disability and mortality worldwide. Obstruction of blood flow delivery due to thrombus after ischemic stroke induces neuronal damage and dysfunction that result in limb incoordination, cognitive deterioration, and central nervous system diseases. Intravenous injection of tissue plasminogen activator (tPA) for thrombolysis has been approved by the FDA for the treatment of acute ischemic stroke within 3 hours of onset [1]. However, the reperfusion of blood flow after thrombolysis can further damage the ischemic vessels and penumbra, causing ischemic stroke-reperfusion (S/R) injury [2]. The induction process of S/R injury includes reactive hyperemia, hypoperfusion, and increased paracellular permeability, resulting in blood–brain barrier (BBB) disruption, nutritional deficiency, and neutrophil adhesion that increase inflammatory and oxidative stress [3]. Finally, the irreversible ischemic penumbra will enlarge the infarct size and aggravate neuron death, reducing the prognosis of ischemic stroke after thrombolysis.

Current thrombolytic therapy for ischemic stroke is usually combined with neuroprotective agents to protect ischemic neurons and prevent S/R injury by clearing free radicals and inhibiting inflammatory and apoptotic factors [4]. However, these neuroprotective agents showed potential in animal models but have failed in clinical trials [5]. The main reasons for failure of this strategy might be an insufficient treatment dose of neuroprotective agents within the ischemic brain, unfavorable long-term vessel recanalization, and inability to restore the microcirculation [5, 6]. Furthermore, establishing the balance of neuroprotective agents between an effective treatment dose and induction of systemic side effects (such as disruption of the central nervous system or gastrointestinal system or reversible altered mentation) should be considered [4]. Since the nutrient supply for neurons relies on the adjacent microcirculation, effective reperfusion within the microcirculation has been reported as a key factor to maintain neuronal function [7].

Ultrasound (US) has been widely applied to provide real-time medical imaging for clinical diagnosis. One commercial contrast agent for US imaging is microbubbles (MBs), which are designed on the basis of the high difference in acoustic impedance between soft tissue and gas [8]. Improved contrast imaging due to the presence of intravascular MBs provides further information about microvascular distribution and blood perfusion to evaluate vascular health, tumor progression, or cardiovascular diseases [9]. Upon US stimulation of intravascular MBs, the cavitation effects produced by MB oscillations generate additional mechanical forces on the vessel wall that can induce enhanced permeability for drug penetration, vasoconstriction for blood supply blockage, or vasodilation for enhanced blood perfusion [10–12]. US-stimulated MB cavitation has been proposed as a potential way to prevent ischemia-reperfusion injury in myocardial infarction, ischemic stroke, and peripheral arterial diseases via acousto-mechanical vasodilation, termed sonoperfusion [13–15]. Furthermore, MBs can also be used to carry drugs, genes, or specific gases for local delivery in the reperfusion regions to reduce injury and systemic side effects [16–18].

In the present study, oxygen-loaded MBs (OMBs) were used to examine the concept of local oxygen (O₂) therapy triggered by US to prevent S/R injury. Since O₂ plays an important role in cell metabolism through the generation of energy, additional compensation for O₂ loss in the ischemic brain might be a promising way to maintain neuronal function. US-stimulated OMB treatment could provide sonoperfusion and local O₂ therapy to enhance effective reperfusion in the microcirculation and promote repair of injured cells through aerobic respiration. We established a murine S/R model after tPA-induced thrombolysis to investigate the mechanisms and results of OMB treatment. In vivo tracking of blood flow and tissue O₂ levels revealed the real-time OMB treatment conditions of a targeted vessel. Long-term follow-up of brain infarct size and animal behavior was performed to assess recovery of brain function. Finally, protein and mRNA expression was evaluated to determine the mechanisms of OMB treatment.

OMB preparation

The details and optimal fabrication of OMB were followed our previous study [19, 20]. The lipid shell of OMB contained 1,2 Distearoyl-sn-glycero-3-phosphorylcholine (DSPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[10-(trimethoxysilyl)undecanamide(polyethylene glycol)-2000] (DSPE-PEG2000; Avanti Lipids Polar, Alabaster, USA) with a weighted ratio of 10:4. The gas core contained perfluoropropane (C₃F₈) and O₂ with a volume ratio of 7:5, which was regulated by syringes to exchange some of the C₃F₈ with O₂ in a sealed vial. The OMBs provided both local O₂ therapy and sonoperfusion. MBs loaded with pure C₃F₈ (CMBs) were used as control MBs for evaluation of sonoperfusion. The size distribution and concentration of OMBs and CMBs were detected using a particle size analyzer (Multisizer 3, Beckman Coulter, Fullerton, USA).

Stability

The in vitro stability of OMBs was evaluated by contrast enhancement under US B-mode imaging since MBs function as a US contrast agent. A cylindrical hollow chamber (Ø=5 mm) within a 2% cuboid agarose phantom was made to simulate the vascular lumen within tissues. The phantom was placed in a water tank maintained at a temperature of 37°C. Diluted OMB emulsion (2×10⁷ MBs/mL, the same MB dose as for in vivo experiments) was placed in the cylindrical hollow chamber and a commercial US imaging system (Terason, Model 3000, Burlington, USA) with a 7-MHz linear array transducer was applied to record transverse sectional images of the chamber. The time-sequence US images were collected with 5 images at each time point (0 to 60 min) and then analyzed using MATLAB (MathWorks, Natick, USA) software. A region of interest (ROI; 2.5 x 2.5 mm²) was placed at the center of the cylindrical hollow on US images to quantify the increase in contrast intensity in the presence of OMBs.

Destruction threshold

For local release of O₂ from OMB disruption triggered by US, the destruction threshold of OMBs under various acoustic pressures was evaluated. A hollow pipe (Ø=0.58 mm) within a 2% cuboid agarose phantom was connected to a PE50 tube (BD Corp., Franklin Lakes, USA) and a syringe pump (flow rate 1.6 µL/s) to generate a flowing condition that simulated OMB perfusion in the blood circulation. The concentration of OMB emulsion was 2×10⁷ MBs/mL. A 1-MHz focused US transducer (Spherical focus PFT, V302, Olympus, USA) was used to transmit 5000-cycle US pulses with different acoustic pressures (peak negative pressures of 0-600 kPa). The pulse repetition frequency (PRF) of 1 Hz was set according to the flow rate to confirm that OMBs were stimulated once within the US focal zone (diameter of 3 mm and length of 26 mm). A commercial US imaging system (Prodigy 128, L18.0VM, S-Sharp) with an 18-MHz linear array transducer was used to record transverse sectional images of the hollow pipe downstream of the US focal area. The intensity of contrast enhancement on US B-mode images was quantified using MATLAB software. A ROI (0.25 x 0.25 mm²) was placed at the center of the hollow pipe on US images to quantify the contrast intensity induced by the remaining OMBs after US stimulation.

O2 release

The in vitro O₂ release from OMB disruption was demonstrated by detecting the partial pressure of O₂ (pO₂) of the OMB emulsion. The experimental set up was the same as for the stability study. The concentration of CMB and OMB emulsion was 2×10⁷ MBs/mL. For real-time detection of the pO₂ level, a fiberoptic probe was inserted into the cylindrical hollow chamber of the phantom. The pO₂ levels were received by an OxyLite 2000 system (Oxford Optronics) at pre, during, and post 10-min US sonication (1 MHz, 5000-cycle, PRF 1 Hz, 300 kPa). In addition, OMB emulsions with different concentrations of MBs (1x10⁷, 2x10⁷, 4x10⁷ MBs/mL) were used to evaluate the amount of O₂ release during US sonication and demonstrate the encapsulation of O₂ within OMBs.

SCD/ICD

The stable cavitation dose (SCD) and inertial cavitation dose (ICD) of OMBs were determined to evaluate the type of MB oscillation during US stimulation. The demarcation of acoustic pressures between SCD and ICD would allow release O₂ from OMB disruption while avoiding damage to cells and vessels. The diluted OMB emulsion (2×10⁷ MBs/mL) was infused into a cellulose tube (Ø=200 µm; Spectrum Labs, USA) that mimicked vessels with a flow rate of 0.375 mL/h. A 1-MHz focused US transducer was used to transmit therapeutic US pulses with different acoustic pressures (5000-cycle, PRF 1 Hz, 0-600 kPa). A 0.5-MHz or 5-MHz US receiving transducer (Spherical focus PFT, V301 and V307, Olympus, USA) was placed perpendicular to the 1-MHz focused US transducer. The US receiving transducers were used to detect the scattered signals from the oscillating OMBs during US stimulation. The detailed methods of signal analysis were described in our previous study [21].

Murine ischemia-stroke reperfusion model

All animal experimental procedures were performed with approval from the Animal Experiment Committee at National Yang Ming Chiao Tung University (approval number: 111025A). 182 Mice (C57BL/6JNarl, male, 8–14 weeks old, 25–30 g) were purchased from the National Laboratory Animal Center (Taipei, Taiwan). Before the experiment, mice were intraperitoneally anesthetized with a mixture of Zoletil 50 (Virbac, Carros, France) and Rompun 2% (Bayer HealthCare, Leverkusen, Germany). The photodynamic dyes, drugs, and MBs were retro-orbitally administrated using a manual insulin needle. The body temperature of mice was maintained at 35–37°C using a heating pad (THM100, Indus Instruments, Houston, USA).

In this study, photodynamic thrombosis was used to generate a clot at the murine anterior cerebral artery remote branch (ACArb) to establish the ischemic stroke model. The experimental set up is illustrated in Fig. 1. Under anesthesia, a 2.5 mm×2.5 mm piece of the skull located posterior and lateral to bregma (right brain) was removed to expose the ACArb (Fig. 1A). Mice were retro-orbitally injected with 50 µL of the photosensitizer rose bengal (RB, 10 or 20 mg/kg; Alfa Aesar, Bellingham, USA) for 30 s circulation and then exposed to a laser (532 nm, 0.5 mW) for 10, 15, or 20 min at the ACArb (~ 1.0 mm lateral to bregma; each N = 3–5) for the generation of clots and establishment of the ischemic stroke model. After 60 min of ischemia, mice were retro-orbitally injected with tPA (10 mg/kg; Actilyse, Boehringer Ingelheim, Ingelheim, Germany) for thrombolysis and reperfusion. Methods of tPA administration were (1) two 50 µL bolus injections with an interval of 10 min, designated 50 + 50 or (2) 10 µL bolus injection and 90 µL introduced by a syringe pump (KDS120, KD Scientific, New Hope, PA, USA) with a constant flow rate of 4.5 µL/min (total injection time: 20 min), designated 10 + 90 (each N = 3–5).

During establishment of the murine S/R model, a fiberoptic probe (NX-BF/OFT/E, Oxford Optronics, Oxford, UK) linked to OxyLite and OxyFlo 2000 systems (Oxford Optronix) was non-invasively placed downstream of ACArb (~ 0.5 mm lateral to bregma) for real-time detection of in vivo blood flow and pO₂. A stereo microscope (SZ61, Olympus, Tokyo, Japan) was used to observe the process of thrombosis and thrombolysis at ACArb. After 24 h of reperfusion, the mice were sacrificed and perfused with 0.9% normal saline. The brains were removed, sliced into coronal sections with thickness of 2 mm, and stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC; Sigma-Aldrich, St. Louis, USA) for 20 min at 37°C. Finally, the brain sections were observed and images were recorded using a stereo microscope. Tissue images were analyzed using MATLAB software to quantify the infarct areas labeled white by TTC staining within the brain section. The percentage of infarct area for each total section was calculated. The blood flow, pO₂, and infarct area were used to evaluate the optimal RB doses, laser exposure times, and tPA administration method for establishment of the murine S/R model.

Biosafety and BBB opening

Since MB cavitation could enhance vascular permeability, open the BBB, and even disrupt vessel walls, the biosafety of OMB cavitation should be considered. Our murine S/R model was used to evaluate the bioeffects after OMB treatment. A 1-MHz US transducer with acoustic pressure of 300 or 400 kPa was focused on the S/R site of ACArb. At 20 min after tPA injection, mice were retro-orbitally injected with different concentrations of OMB emulsion (0.5x10⁷, 1x10⁷, and 2x10⁷ OMBs in 50 µL; each N = 3) for 30 s circulation and then subjected to US sonication for 5 min to disrupt the OMB for local O₂ therapy and sonoperfusion. The OMB treatment procedure included two rounds of OMB administration and US sonication; the second dose of OMBs was injected immediately after the first sonication. The total OMB treatment therefore consisted of 1x10⁷, 2x10⁷, or 4x10⁷ OMBs and 10 min US sonication. After OMB treatment, 50 µL of Evans blue dye (2.5 mL/kg) was retro-orbitally injected into the mice to visualize the BBB opening induced by OMB cavitation. After 3 h, mice were sacrificed and perfused with 0.9% normal saline. The brains were removed and sliced into coronal sections with a thickness of 2 mm. The brain sections were observed and images were recorded using a stereo microscope to evaluate BBB opening and brain hemorrhage. The bioeffects induced by different OMB doses and acoustic pressures were used to define the optimal OMB treatment protocol for subsequent experiments.

OMB treatment

The treatment procedures of the murine S/R model involved the following steps (Fig. 1B): (1) 60 min stroke induction by photodynamic thrombosis; (2) 20 min of reperfusion induced by tPA injection; (3) 10 min OMB treatment including two rounds of OMB administration (1x10⁷ MBs/mouse/injection) and US sonication (1-MHz, 5000-cycle, PRF 1 Hz, 300 kPa, 10 min). The S/R mice were separated into six groups: S/R, CMB, OMB, S/R+, CMB+, and OMB + groups, where the plus (+) symbol presents US sonication (each group N = 5). Real-time recording of blood flow and pO₂ downstream of the ACArb was performed to evaluate establishment of the S/R model and OMB treatment effects. The mean percentages of blood flow and pO₂ were determined during the periods of pre-treatment (pre), during OMB treatment (T), and post-treatment (post). After 24 h of reperfusion the mice were sacrificed for TTC staining to estimate the treatment efficacy based on changes in brain infarct size. It should be noted that the brain infarct size was calculated as the percentage of infarct areas within the whole brain, which was different from the method used in establishment of the S/R model.

Post-treatment evaluation of animal behaviors and brain infarct size

Animal behaviors and brain infarct size were tracked over 14 days to evaluate the long-term recovery of brain function. Since the brain infarct was located at the primary and secondary motor areas, the beam walking test and hanging wire test were used to evaluate the motor coordination and muscle performance of S/R mice. Mice were separated into four groups (normal, S/R, CMB+, and OMB + groups, each N = 4–5) for evaluation of behaviors at 1, 3, 7, and 14 days after treatment. The beam walking test was performed five times to calculate the average walking times (Fig. 1C). In the hanging wire test, the average recorded time calculated from five repetitions was multiplied by the mouse weight to account for the influence of body size (Fig. 1D). In addition, the long-term effect of inflammatory responses on infarction was evaluated by determining the percentage of infarct areas within the whole brain at 1, 3, 7, and 14 days after treatment (each N = 3). The time point of maximum infarct size in the S/R group was chosen for further histological analysis and measurement of protein and mRNA expression.

Histological qualitative assessment

At 3 days after OMB treatment the mice were sacrificed and perfused with 0.9% normal saline. The experimental groups included normal, S/R, CMB+, and OMB + groups (each N = 3). The brain tissue was removed for frozen sectioning at a thickness of 20 µm using a cryostat microtome (Leica CM1850 Cryostat, Leica Biosystems GmbH, Wetzlar, Germany). Hematoxylin and eosin staining (H&E) was used to observe brain tissue structure. Since brain infarction causes differentiation of microglia, apoptosis of neuron cells, and population with astrocytes, immunohistochemical assessments of staining for CD206 (anti-mannose receptor antibody, ab64693, Abcam, Cambridge, UK), CD11b (purified rat anti-CD11b, 550282, BD Biosciences, Franklin Lakes, USA), NeuN (recombinant anti-NeuN antibody [EPR12763]-Neuronal Marker, ab177487, Abcam), and GFAP (anti-GFAP antibody, ab4674, Abcam) were performed to evaluate the distribution of anti-inflammatory M2 microglia, pro-inflammatory M1 microglia, mature neurons, and astrocytes, respectively, within the brain infarct areas.

Enzyme-linked immunosorbent assay

At 3 days after OMB treatment the mouse brain was separated into the right S/R cerebral hemisphere and left contralateral cerebral hemisphere. The perfused cerebral hemispheres were collected and homogenized in 1 mL phosphate-buffered saline using a gentleMACs dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) with a M-tube. Brain samples were centrifuged at 5000 rcf for 5 min at 4°C and the supernatant was collected for enzyme-linked immunosorbent assay. The experimental groups included normal, S/R, CMB+, and OMB + groups (each N = 5). The expression of endothelial nitric oxide synthase (eNOS; Cusabio, Wuhan, China), brain derived neurotrophic factor (BDNF; Cusabio), and NF-κB (ab176648, Abcam) was measured using commercial kits and protocols. Protein expression was detected by the optical density at 450 nm using a plate reader system (Tecan Infinite M200, Tecan Trading AG, Männedorf, Switzerland). The final protein expression was presented as the ratio relative to the normal cerebral hemisphere.

Quantitative polymerase chain reaction

The procedure of homogeneous brain sample collection for quantitative polymerase chain reaction was as described above. The experimental groups included normal, S/R, CMB+, and OMB + groups (each N = 5–9). Total mRNA was isolated from brain sample using Tri-reagent (TRIzol™ Reagent, Thermo Fisher Scientific, Waltham, USA). Primers for hypoxia-inducible factor 1-alpha (HIF-1α), B-cell lymphoma 2 (BCL2), interleukin 1 beta (IL-1β), interleukin 10 (IL-10), and matrix metallopeptidase 9 (MMP-9) were synthesized by Genomics BioSci and Tech Ltd. (New Taipei City, Taiwan) for SYBR green-based analysis. The final mRNA levels were normalized to β-actin expression and presented as ratios relative to normal brain. Details of the primers are provided in the supplementary data (Table S1).

Statistical Analysis

Data were presented as the mean ± standard deviation from at least 3 independent experiments. Results were statistically analyzed by two-tailed, unpaired Student’s t-test for two-group comparisons. One-way ANOVA followed by Bonferroni's multiple comparisons test was used for comparisons of more than two groups. Statistically significant differences were considered for p < 0.05.

In vitro OMB disruption by US sonication increased pO ₂ level.

The lipid-shell CMBs and OMBs had a mean diameter of 1.27 ± 0.61 and 1.33 ± 0.69 µm, respectively (Fig. 2A). The size distribution revealed that the diameter range was smaller than 4 µm in both the CMB and OMB groups. The in vitro US images showed only a slight decrease in contrast enhancement over time, demonstrating good stability of the CMBs and OMBs (Figure S1). The contrast intensity of OMBs decreased from 24 ± 0.7 dB at 0 min to 23 ± 0.3 dB at 60 min. An acoustic pressure of 300 kPa was determined to be the destruction threshold for O₂ therapy, and disrupted 93 ± 6.2% of CMBs and 98 ± 1.9% of OMBs (Fig. 2B). The intensity of contrast enhancement under US imaging decreased with an increase in acoustic pressures (Figure S2). The results revealed no significant differences in size distribution, stability, and destruction threshold between CMBs and OMBs.

After US sonication, the pO₂ levels increased by 0 ± 2, 0 ± 1, and 9 ± 2 mmHg in the PBS+, CMB+, and OMB + group, respectively (Fig. 2C). Final pO₂ levels of the OMB group without and with US sonication were 158 ± 1 and 162 ± 0 mmHg, respectively, demonstrating local O₂ release from OMBs triggered by US. The elevation of pO₂ levels increased with increased OMB dose, further confirming the release of O₂ from OMBs (Fig. 2D).

The murine S/R model revealed non-complete recovery in blood flow, pO ₂, and brain infarct after thrombolysis.

During establishment of the murine S/R model, the dissecting microscopic images revealed clot formation at ACArb at 60 min after stroke induction and thrombolysis after 60 min of reperfusion (Fig. 3A). The optimal procedure for establishment of the murine S/R model was separated into stroke and reperfusion components. For the stroke component, photodynamic thrombosis with 20 mg/kg RB and 20 min laser exposure (20 + 20) significantly decreased the percentage of blood flow to 45 ± 3% and pO₂ to 60 ± 1% at 60 min to establish the ischemic stroke model (Figure S3A). TTC staining identified the brain infarcts as white regions at the right cerebral primary and secondary motor areas (Figure S3B). The percentage of the infarct area was 0.0 ± 0.0, 5.1 ± 1.0, 5.4 ± 2.8, 8.0 ± 0.7, and 11.9 ± 1.1% in the normal, 10 + 20, 20 + 10, 20 + 15, and 20 + 20 groups, respectively.

In the reperfusion component, the 50 + 50 group with two bolus injections of tPA showed non-significant recovery in blood flow and pO₂ (Fig. 3B). In contrast, blood flow and pO₂ increased to 70 ± 3% and 76 ± 2% respectively in the 10 + 90 group, indicating successful thrombolysis for reperfusion after continuous injection of tPA. TTC staining showed a decrease in infarct size after reperfusion (Fig. 3C), with percentage infarct area of 11.9 ± 1.1, 7.5 ± 1.7, and 5.2 ± 0.6% in the stroke, 50 + 50, and 10 + 90 groups, respectively. Thus, in the present study the optimal procedure for generation of the murine S/R model was induction of stroke by photodynamic thrombosis with 20 mg/kg RB and 20 min laser exposure, followed by reperfusion by thrombolysis with 10 µL bolus injection and 90 µL continuous injection of tPA.

OMB treatment promoted BBB opening without hemorrhage by regulating the stable cavitation of OMBs.

In the S/R brain, leakage of Evans blue within the brain tissue indicated damage of the BBB due to the S/R injury (Fig. 4A). The degree of BBB opening was visibly enhanced after OMB cavitation; the blue areas within the S/R brain intensified after a dose of 1x10⁷ OMBs/mouse with acoustic pressures of 300 and 400 kPa. When the OMB dose was increased to 2x10⁷ OMBs/mouse, the areas of BBB opening were enlarged under an acoustic pressure of 300 kPa, but hemorrhage was induced at 400 kPa. Serious brain hemorrhage was observed in the groups treated with a dose of 4x10⁷ OMBs/mouse under acoustic pressures of both 300 and 400 kPa.

To evaluate the correlation between brain hemorrhage and OMB cavitation, the in vitro characteristics of OMB cavitation (2×10⁷ MBs/mL) under various acoustic pressures were examined. The stable cavitation of MBs produced at the acoustic pressures of 200 and 300 kPa due to the significant reduction of SCD, and then decreased at acoustic pressures of 400 kPa or higher (Fig. 4B). With conversion of OMB cavitation from stable to inertial cavitation, the ICD started to increase significantly at an acoustic pressure of 400 kPa (Fig. 4C). Thus, the optimal protocol of OMB treatment in the murine S/R model was defined as a treatment dose of 2x10⁷ OMBs/mouse with acoustic pressure of 300 kPa, which disrupted OMBs to release O₂ and produced stable cavitation to promote BBB opening without brain hemorrhage.

Blood flow and pO ₂ were recovered to reduce the infarct size after OMB treatment in the murine S/R model.

To prevent S/R injury, S/R mice were subjected to OMB treatment at 20 min of reperfusion. The mean percentages of blood flow and pO₂ were not significantly different between the pre, T, and post periods in the S/R, CMB, OMB, and S/R + groups (Fig. 5A). In the CMB + group, the blood flow was 75 ± 4%, 80 ± 2%, and 76 ± 2% at the pre, T, and post periods, respectively. A transient but significant enhancement of blood flow was induced by CMB cavitation but there was no significant difference in the pO₂ levels. After OMB treatment (OMB+), the mean percentages of blood flow and pO₂ were significantly increased by 7 ± 5% and 14 ± 4% at the T period relative to the pre period. In comparison with the T period, the blood flow decreased from 79 ± 4–75%3% (p > 0.05) and the pO₂ decreased from 86 ± 2% to 72 ± 4% (p < 0.05) at the post period.

The white areas indicating infarct in brain sections stained by TTC showed a reduction in infarct size in the CMB + and OMB + groups (Fig. 5B). The percentages of infarct area within the whole brain were 3.2 ± 0.3%, 2.8 ± 0.6%, 2.6 ± 1.1%, 2.3 ± 0.8%, 1.3 ± 0.0%, and 1.3 ± 0.1% in the S/R, CMB, OMB, S/R+, CMB+, and OMB+, respectively. A significant reduction in infarct size in whole brain demonstrated the efficacy of S/R injury prevention by both CMB and OMB treatment.

Long-term tracing of animal behaviors and brain infarct size revealed rapid recovery of brain function after OMB treatment.

The recovery of brain function after OMB treatment was evaluated through long-term tracking of mice behaviors and TTC staining. The results of the beam walking test and hanging wire test showed a significant difference between the normal and S/R groups that demonstrated damage to brain function after S/R (Fig. 6A and 6B). In the OMB + group, the time of the beam walking test decreased after day 1 and showed no significant difference from the normal group at day 7. There was no significant difference among all groups at day 14, with values of 18 ± 4, 25 ± 6, 24 ± 3, 20 ± 2 s in the normal, S/R, CMB+, and OMB + groups, respectively. The results of the hanging wire test at 14 days after treatment showed a significant increase in the CMB + and OMB + groups but the values did not recover to the normal level.

TTC staining showed the maximum brain infarct size at day 3 and a subsequent reduction in size with time in each group (Fig. 6C). The brain infarct areas were 3.6 ± 0.2%, 2.1 ± 0.3%, and 1.6 ± 0.1% at day 3 in the S/R, CMB+, and OMB + group, respectively, compared with 1.6 ± 0.3%, 0.7 ± 0.0%, and 0.2 ± 0.1% at day 14. In comparison with CMB treatment, OMB treatment resulted in a faster recovery of animal behaviors and brain infarct sizes.

Reoxygenation and sonoperfusion protected neurons through inhibition of the activation of inflammatory and apoptotic factors.

After 3 days of OMB treatment, histological staining, protein expression, and mRNA levels in the brain were evaluated. The histological images revealed sparse tissue in the S/R group that became denser in the CMB + and OMB + groups (Fig. 7A). Immunohistochemical staining of CD206 showed numerous anti-inflammatory M2 microglia in the S/R, CMB+, and OMB + groups (Fig. 7B). In comparison to the S/R group, the CMB + and OMB + groups showed a decrease in CD11b labeling demonstrating a reduction in pro-inflammatory M1 microglia distribution. The absence of mature neurons and astrocyte distribution in the S/R group reflected the abnormal animal behaviors caused by the dysfunctional brain.

Expression of proteins and mRNA in the S/R cerebral hemispheres was also detected to evaluate the mechanisms of local O₂ therapy and sonoperfusion induced by OMB treatment. Consistent with the mechanical effect of MB cavitation on the vessel wall, the expression of eNOS for vasodilation was significantly enhanced in the CMB + and OMB + groups relative to the S/R group (Fig. 8A). The expression of BDNF was 1.0 ± 0.0, 0.5 ± 0.2, 1.0 ± 0.2, and 1.1 ± 0.2 in the normal, S/R, CMB+, and OMB + groups, respectively, indicating the recovery of BDNF expression for promotion of neuron growth after treatment (Fig. 8B). The significant decrease in expression of NF-κB in the CMB + and OMB + groups indicated inhibition of the inflammatory response after MB cavitation (Fig. 8C). The mRNA ratios showed significant decreases in HIF-1α, IL-1β, and MMP-9, representing reoxygenation, reduction of pro-inflammatory M1 microglia, and BBB recovery, respectively, after OMB treatment (Fig. 8D, 8E, and 8F). Moreover, the increase in Bcl2 and IL-10 mRNA levels revealed anti-apoptosis activity and an increase in anti-inflammatory M2 microglia differentiation in the OMB + group (Fig. 8G and 8H). Changes in mRNA levels in the CMB group were not significantly different from the S/R group except for the significant reduction of IL-1β. Comparisons between the normal, S/R, and contralateral cerebral hemispheres are shown in the supplementary data (Figure S5).

US-stimulated MB cavitation has been widely applied in the treatment of various diseases because of its combined local drug delivery and acousto-mechanical vascular effects. The present study used OMB treatment to prevent murine S/R injury after thrombolysis. Sonoperfusion induced during OMB treatment might be responsible for the 9.4 ± 5.1% enhancement in blood flow observed relative to the S/R group. The improvement of blood perfusion signals at the peripheral microcirculation of ACArb after OMB treatment was visualized in the laser speckle images (Figure S4). Since MB cavitation can activate release of the vasodilator nitric oxide (NO), sonoperfusion might promote effective reperfusion in the microcirculation to protect neurons [18, 22]. In addition, the local release of O₂ during OMB treatment promoted a 10.3 ± 3.1% increase in pO₂ level relative to the S/R group, allowing reoxygenation of the hypoxic brain. This enhanced reoxygenation could reduce the pathological effects of hypoxia and ischemia, effectively reducing the infarct size of the ischemic brain. In our study, the infarct size was not significantly different between the CMB + and OMB + groups at days 1 to 3, but significantly decreased in the OMB + group relative to the CMB + group at days 7 to 14. This reduction of infarct size demonstrated that the reoxygenation effect of OMB treatment might provide long-term recovery of the ischemic penumbra to protect brain function related to limb coordination [23].

Based on the mechanism of O₂ therapy, hyperbaric O₂ therapy (HBOT) and normobaric O₂ therapy (NBOT) have been applied the prevention of S/R injury in clinical applications or trials [23–25]. The production of ROS at non-lethal levels during HBOT activates the expression of antioxidant enzymes to balance the oxidative stress [26, 27]. The increase of pO₂ levels in the cerebral ischemic penumbra could reduce the infarct formation and active neuroprotective effects [23, 28]. However, the whole-body effect of HBOT and NBOT should be concerned the duration and repetition times to avoid excessive production of ROS and central nervous system O₂ toxicity [29, 30]. Therefore, the local O₂ release of OMB treatment might represent a promising strategy to achieve safer and more precise O₂ therapy to prevent the systemic side effects. To date, OMB treatment has been used in tumor therapy to reoxygenate hypoxic tumors and improve the efficacy of radiotherapy and chemotherapy [19, 20, 31, 32]. The increase in reactive oxygen species (ROS) production via OMB cavitation has also been exploited to improve the efficacy of sonodynamic therapy [33, 34]. However, when applying OMB treatment in normal organs or tissues, the production of cytotoxic ROS during OMB cavitation should be considered to avoid further injury. Importantly, ROS do not only induce cytotoxicity, but also regulate cellular metabolism for cell proliferation, differentiation, migration, and repair [35]. Intracellular ROS levels in normal cells should therefore be maintained at a homeostatic level through effective antioxidant production [36]. In the current study, the prevention and repair of S/R injury by OMB treatment was demonstrated by the recovery of brain morphology and function. Changes in ROS levels in the murine S/R model should be measured to investigate the level of oxidative stress after OMB treatment.

The mechanisms of OMB treatment included the activation of eNOS for vasodilation, BDNF for neuron repair, IL-10 for anti-inflammatory M2 microglia differentiation, and Bcl2 for anti-apoptosis activity. Inhibition of HIF-1α, NF-κB, and IL-1β expression indicated a reduction of brain hypoxia and inflammatory responses by OMB treatment. The decrease in MMP-9 after OMB treatment would reduce disruption of the BBB induced by S/R injury. Moreover, the contralateral cerebral hemispheres in each group showed no significant difference in the expression of eNOS, IL-1β, MMP-9, and BCL2 relative to the normal cerebral hemispheres (Figure S5). The expression of anti-inflammatory IL-10 in the contralateral cerebral hemispheres was enhanced in each group, possibly to balance the pro-inflammatory IL-1β in the S/R cerebral hemispheres. The regulation of inflammatory responses in the contralateral cerebral hemispheres might promote the expression of growth factors and immune modulators for neuron proliferation and survive, which might maintain the brain function and support the S/R cerebral hemispheres [37]. HIF-1α expression in the contralateral cerebral hemispheres was not significantly different from than that in the S/R cerebral hemispheres in the S/R and CMB + groups but was significantly higher for the OMB + group. Stabilization of HIF-1α in the contralateral cerebral hemispheres might help to decrease injury of the S/R cerebral hemispheres. Hypoxia-mimetic agents (e.g., HIF-prolyl hydroxylase inhibitor) have been reported to prevent S/R injury by neuroprotection, angiogenesis, and erythropoiesis through the stabilization of HIF-1α expression [38]. However, the efficacy of hypoxia-mimetic agents for preventing S/R injury is a controversial issue, and the timing and levels of HIF-1α expression during the treatment should be further investigated to avoid aggravation of injury [39]. Even though the expression of MMP-9 was reduced after OMB treatment, the induction of BBB opening by MB cavitation might enhance penetration of inflammatory factors into the injured brain tissue and impede recovery [40]. In normal brain, the expression of inflammatory factors was significantly increased at 6 h and 24 h after BBB opening induced by MB cavitation and returned to the baseline level after 72 h [41]. Thus, the optimal time point of OMB treatment, the duration of BBB opening, and the possibility of BBB recovery should be evaluated to avoid aggravation of S/R injury.

Sonothrombolysis has been applied to promote fibrinolytic therapy via MB cavitation [13]. The mechanical force induced by MB cavitation disrupts the thrombus, allowing tPA to penetrate into deep sites for thrombolysis [42]. The recovery of blood flow and vascular recanalization play a crucial role in preventing ischemia-reperfusion injury in thrombolysis. Corro et al. designed a NO donor-loaded MB to induce rat femoral vasodilation and clot degradation by sonothrombolysis for femoral artery recanalization [43]. The local release of NO from NO donor-loaded MBs enhanced the relaxation of rat aortic rings to achieve the NO-related vasodilation effect. In the present study, OMB treatment was performed after blood reperfusion for 20 min to prevent S/R injury. However, different time schedules for ischemia stroke, thrombolytic reperfusion, and OMB treatment should be further investigated to determine the optimal treatment procedure for preventing S/R injury. Figure S6A shows initial results for OMB treatment at the late stage of stroke (S), early stage of reperfusion (S-R), and post reperfusion (R). The percentage of infarct areas within whole brain at 24 h after treatment was 2.4 ± 0.7, 1.0 ± 0.4, and 1.6 ± 0.1 in the S, S-R, and R groups, respectively (Figure S6B). The combination of tPA administration and OMB treatment in the S-R group promoted sonothrombolysis to significantly shrink the subsequent brain infarct area.

This study demonstrated that US-stimulated OMB cavitation could prevent and repair S/R injury. The enhanced blood flow induced by sonoperfusion promoted blood reperfusion at the peripheral microcirculation to supply nutrients to neurons. Reoxygenation of the ischemic brain by local O₂ therapy inhibited hypoxia-inducible inflammation and apoptosis to prevent subsequent injury. Recovery of the reversible ischemic penumbra reduced brain infarct size. Brain morphology and function in S/R mice were protected to maintain the ability of limb coordination due to the survival of neurons and astrocytes. This local and non-invasive OMB treatment might be a promising strategy for the development of a precise therapy to prevent ischemia-reperfusion injury in various organ systems.

Tissue plasminogen activator (tPA)

Ischemic stroke-reperfusion (S/R)

Blood–brain barrier (BBB)

Ultrasound (US)

Microbubbles (MBs)

Oxygen-loaded MBs (MBs)

Pulse repetition frequency (PRF)

Partial pressure of O₂ (pO₂)

Stable cavitation dose (SCD)

Inertial cavitation dose (ICD)

Anterior cerebral artery remote branch (ACArb)

Rose bengal (RB)

2,3,5-triphenyltetrazolium chloride (TTC)

Hematoxylin and eosin staining (H&E)

Endothelial nitric oxide synthase (eNOS)

Brain derived neurotrophic factor (BDNF)

Hypoxia-inducible factor 1-alpha (HIF-1α)

B-cell lymphoma 2 (BCL2)

Interleukin 1 beta (IL-1β)

Interleukin 10 (IL-10)

Matrix metallopeptidase 9 (MMP-9)

Nitric oxide (NO)

Hyperbaric O₂ therapy (HBOT)

Normobaric O2 therapy (NBOT)

Reactive oxygen species (ROS)

Ethics approval and consent to participate

All animal experimental procedures were performed with approval from the Animal Experiment Committee at National Yang Ming Chiao Tung University (approval number: 111025A).

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research, authorship, and publication of this article was funded by National Science and Technology Council of Taiwan (MOST 111-2628-E-A49-020- to Dr Ho; 110-2221-E-400-003-MY3 to Dr Liao; 112-2636-B-007-008 to Dr Lin; 108-2221-E-007-040-MY3, 110-2221-E-007-019-MY3, and 111-2221-E-007-019-MY3 to Dr Yeh). Dr Ho was funded by National Yang Ming Chiao Tung University of Taiwan (Grant number 111W20205). Dr Lin and Dr Yeh were funded by National Tsing Hua University of Taiwan (Grant number 111Q2713E1). Dr Tsai and Dr Yeh was funded by Chang Gung Memorial Hospital - National Tsing Hua University joint research program (CGMH-NTHU-111-No. CORPG3M0091).

Authors' contributions

Dr Ho and Yeh contributed to the study conception and design. Most material preparation, data collection, and analysis were performed by HL Cheng. Dr Liao assisted murine S/R model building and laser speckle imaging system. Dr Lin provided supports on molecular biological experiments and instruments. Dr Tsai and Dr Yeh provided funding and supervise the project. The first draft of the article was written by Dr Ho. All authors read and approved the final article.

Acknowledgements

Not applicable.

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Oxygen-loaded microbubble-mediated sonoperfusion and oxygenation for neuroprotection after ischemic stroke reperfusion

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Abstract

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Background

Methods

OMB preparation

Stability

Destruction threshold

O2 release

SCD/ICD

Murine ischemia-stroke reperfusion model

Biosafety and BBB opening

OMB treatment

Post-treatment evaluation of animal behaviors and brain infarct size

Histological qualitative assessment

Enzyme-linked immunosorbent assay

Quantitative polymerase chain reaction

Statistical Analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

Ethics approval and consent to participate

Consent for publication

Availability of data and materials

Competing interests

Funding

Authors' contributions

Acknowledgements

References

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