Optimal timing for drug delivery into the hippocampus by focused ultrasound: A comparison of hydrophilic and lipophilic compounds.

Aims: Previous studies have reported that focused ultrasound (FUS) helps modulate the blood-brain barrier (BBB). These studies have generally used the paracellular pathway owing to tight junction proteins (TJPs) regulation. However, BBB transport pathways also include diffusion and transcytosis. Few studies have examined transcellular transport across endothelial cells. We supposed that increased BBB permeability caused by FUS may affect transcytosis. We investigated drug delivery through transcytosis and paracellular transport to the brain after BBB modulation using FUS. Main methods: FUS and microbubbles were applied to the hippocampus of rats, and were euthanized at 1, 4, 24, and 48 h after sonication. To investigate paracellular transport, we analyzed TJPs, including zona occludens-1 (ZO-1) and occludin. We also investigated caveola-mediated transcytosis by analyzing caveola formation and major facilitator superfamily domain-containing 2a (Mfsd2a) levels, which inhibit caveola vesicle formation. Key findings: One hour after FUS, ZO-1 and occludin expression was the lowest and gradually increased over time, returning to baseline 24 h after FUS treatment. Compared with that of TJPs, caveola formation started to increase 1 h after FUS treatment and peaked at 4 h after FUS treatment before returning to baseline by 48 h after FUS treatment. Decreased Mfsd2a levels were observed at 1 h and 4 h after FUS treatment, indicating increased caveola formation. Significance: FUS induces BBB permeability changes and regulates both paracellular transport and caveola-mediated transcytosis. However, a time difference was observed between these two mechanisms. Hence, when delivering drugs into the brain after FUS, the optimal drug administration timing should be determined by the mechanism by which each drug passes through the BBB.

Amygdala electrical stimulation for operant conditioning in rat navigation.

There have been several attempts to navigate the locomotion of animals by neuromodulation. The most common method is animal training with electrical brain stimulation for directional cues and rewards; the basic principle is to activate dopamine-mediated neural reward pathways such as the medial forebrain bundle (MFB) when the animal correctly follows the external commands. In this study, the amygdala, which is the brain region responsible for fear modulation, was targeted for punishment training. The brain regions of MFB, amygdala, and barrel cortex were electrically stimulated for reward, punishment, and directional cues, respectively. Electrical stimulation was applied to the amygdala of rats when they failed to follow directional commands. First, two different amygdala regions, i.e., basolateral amygdala (BLA) and central amygdala (CeA), were stimulated and compared in terms of behavior responses, success and correction rates for training, and gene expression for learning and memory. Then, the training was performed in three groups: group R (MFB stimulation for reward), group P (BLA stimulation for punishment), and group RP (both MFB and BLA stimulation for reward and punishment). In group P, after the training, RNA sequencing was conducted to detect gene expression and demonstrate the effect of punishment learning. Group P showed higher success rates than group R, and group RP exhibited the most effective locomotion control among the three groups. Gene expression results imply that BLA stimulation can be more effective as a punishment in the learning process than CeA stimulation. We developed a new method to navigate rat locomotion behaviors by applying amygdala stimulation.

Long-lasting restoration of memory function and hippocampal synaptic plasticity by focused ultrasound in Alzheimer's disease.

BACKGROUND: Focused ultrasound (FUS) is a medical technology that non-invasively stimulates the brain and has been applied in thermal ablation, blood-brain barrier (BBB) opening, and neuromodulation. In recent years, numerous experiences and indications for the use of FUS in clinical and preclinical studies have rapidly expanded. Focused ultrasound-mediated BBB opening induces cognitive enhancement and neurogenesis; however, the underlying mechanisms have not been elucidated. METHODS: Here, we investigate the effects of FUS-mediated BBB opening on hippocampal long-term potentiation (LTP) and cognitive function in a 5xFAD mouse model of Alzheimer's disease (AD). We applied FUS with microbubble to the hippocampus and LTP was measured 6 weeks after BBB opening using FUS. Field recordings were made with a concentric bipolar electrode positioned in the CA1 region using an extracellular glass pipette filled with artificial cerebrospinal fluid. Morris water maze and Y-maze was performed to test cognitive function. RESULTS: Our results demonstrated that FUS-mediated BBB opening has a significant impact on increasing LTP at Schaffer collateral - CA1 synapses and rescues cognitive dysfunction and working memory. These effects persisted for up to 7 weeks post-treatment. Also, FUS-mediated BBB opening in the hippocampus increased PKA phosphorylation. CONCLUSION: Therefore, it could be a promising treatment for neurodegenerative diseases as it remarkably increases LTP, thereby improving working memory.

Combined Effects of Focused Ultrasound and Photodynamic Treatment for Malignant Brain Tumors Using C6 Glioma Rat Model.

PURPOSE: Glioblastoma (GBM) is an intractable disease for which various treatments have been attempted, but with little effect. This study aimed to measure the effect of photodynamic therapy (PDT) and sonodynamic therapy (SDT), which are currently being used to treat brain tumors, as well as sono-photodynamic therapy (SPDT), which is the combination of these two. MATERIALS AND METHODS: Four groups of Sprague-Dawley rats were injected with C6 glioma cells in a cortical region and treated with PDT, SDT, and SPDT. Gd-MRI was monitored weekly and 18F-FDG-PET the day before and 1 week after the treatment. The acoustic power used during sonication was 5.5 W/cm² using a 0.5-MHz single-element transducer. The 633-nm laser was illuminated at 100 J/cm². Oxidative stress and apoptosis markers were evaluated 3 days after treatment using immunohistochemistry (IHC): 4-HNE, 8-OhdG, and Caspase-3. RESULTS: A decrease in tumor volume was observed in MRI imaging 12 days after the treatment in the PDT group (p<0.05), but the SDT group showed a slight increase compared to the 5-Ala group. The high expression rates of reactive oxygen species-related factors, such as 8-OhdG (p<0.001) and Caspase-3 (p<0.001), were observed in the SPDT group compared to other groups in IHC. CONCLUSION: Our findings show that light with sensitizers can inhibit GBM growth, but not ultrasound. Although SPDT did not show the combined effect in MRI, high oxidative stress was observed in IHC. Further studies are needed to investigate the safety parameters to apply ultrasound in GBM.

Preclinical Research on Focused Ultrasound-Mediated Blood-Brain Barrier Opening for Neurological Disorders: A Review.

Several therapeutic agents for neurological disorders are usually not delivered to the brain owing to the presence of the blood-brain barrier (BBB), a special structure present in the central nervous system (CNS). Focused ultrasound (FUS) combined with microbubbles can reversibly and temporarily open the BBB, enabling the application of various therapeutic agents in patients with neurological disorders. In the past 20 years, many preclinical studies on drug delivery through FUS-mediated BBB opening have been conducted, and the use of this method in clinical applications has recently gained popularity. As the clinical application of FUS-mediated BBB opening expands, it is crucial to understand the molecular and cellular effects of FUS-induced microenvironmental changes in the brain so that the efficacy of treatment can be ensured, and new treatment strategies established. This review describes the latest research trends in FUS-mediated BBB opening, including the biological effects and applications in representative neurological disorders, and suggests future directions.

Enhanced delivery of a low dose of aducanumab via FUS in 5×FAD mice, an AD model.

BACKGROUND: Aducanumab (Adu), which is a human IgG1 monoclonal antibody that targets oligomer and fibril forms of beta-amyloid, has been reported to reduce amyloid pathology and improve impaired cognition after administration of a high dose (10 mg/kg) of the drug in Alzheimer's disease (AD) clinical trials. The purpose of this study was to investigate the effects of a lower dose of Adu (3 mg/kg) with enhanced delivery via focused ultrasound (FUS) in an AD mouse model. METHODS: The FUS with microbubbles opened the blood-brain barrier (BBB) of the hippocampus for the delivery of Adu. The combined therapy of FUS and Adu was performed three times in total and each treatment was performed biweekly. Y-maze test, Brdu labeling, and immunohistochemical experimental methods were employed in this study. In addition, RNA sequencing and ingenuity pathway analysis were employed to investigate gene expression profiles in the hippocampi of experimental animals. RESULTS: The FUS-mediated BBB opening markedly increased the delivery of Adu into the brain by approximately 8.1 times in the brains. The combined treatment induced significantly less cognitive decline and decreased the level of amyloid plaques in the hippocampi of the 5×FAD mice compared with Adu or FUS alone. Combined treatment with FUS and Adu activated phagocytic microglia and increased the number of astrocytes associated with amyloid plaques in the hippocampi of 5×FAD mice. Furthermore, RNA sequencing identified that 4 enriched canonical pathways including phagosome formation, neuroinflammation signaling, CREB signaling and reelin signaling were altered in the hippocami of 5×FAD mice receiving the combined treatment. CONCLUSION: In conclusion, the enhanced delivery of a low dose of Adu (3 mg/kg) via FUS decreases amyloid deposits and attenuates cognitive function deficits. FUS-mediated BBB opening increases adult hippocampal neurogenesis as well as drug delivery. We present an AD treatment strategy through the synergistic effect of the combined therapy of FUS and Adu.

Factors Associated with Energy Efficiency of Focused Ultrasound Through the Skull: A Study of 3D-Printed Skull Phantoms and Its Comparison with Clinical Experiences.

While focused ultrasound (FUS) is non-invasive, the ultrasound energy is attenuated by the skull which results in differences in energy efficiency among patients. In this study, we investigated the effect of skull variables on the energy efficiency of FUS. The thickness and density of the skull and proportion of the trabecular bone were selected as factors that could affect ultrasound energy transmittance. Sixteen 3D-printed skull models were designed and fabricated to reflect the three factors. The energy of each phantom was measured using an ultrasonic sound field energy measurement system. The thickness and proportion of trabecular bone affected the attenuation of transmitted energy. There was no difference in the density of the trabecular bone. In clinical data, the trabecular bone ratio showed a significantly greater correlation with dose/delivered energy than that of thickness and the skull density ratio. Currently, for clinical non-thermal FUS, the data are not sufficient, but we believe that the results of this study will be helpful in selecting patients and appropriate parameters for FUS treatment.

The synergistic effect of focused ultrasound and biophotonics to overcome the barrier of light transmittance in biological tissue.

Optical technology is a tool to diagnose and treat human diseases. Shallow penetration depth caused by the high optical scattering nature of biological tissues is a significant obstacle to utilizing light in the biomedical field. In this paper, light transmission enhancement in the rat brain induced by focused ultrasound (FUS) was observed and the cause of observed enhancement was analyzed. Both air bubbles and mechanical deformation generated by FUS were cited as the cause. The Monte Carlo simulation was performed to investigate effects on transmission by air bubbles and finite element method was also used to describe mechanical deformation induced by motions of acoustic particles. As a result, it was found that the mechanical deformation was more suitable to describe the transmission change according to the FUS pulse observed in the experiment.

A novel rat robot controlled by electrical stimulation of the nigrostriatal pathway.

OBJECTIVE: Artificial manipulation of animal movement could offer interesting advantages and potential applications using the animal's inherited superior sensation and mobility. Although several behavior control models have been introduced, they generally epitomize virtual reward-based training models. In this model, rats are trained multiple times so they can recall the relationship between cues and rewards. It is well known that activation of one side of the nigrostriatal pathway (NSP) in the rat induces immediate turning toward the contralateral side. However, this NSP stimulation-induced directional movement has not been used for the purpose of animal-robot navigation. In this study, the authors aimed to electrically stimulate the NSP of conscious rats to build a command-prompt rat robot. METHODS: Repetitive NSP stimulation at 1-second intervals was applied via implanted electrodes to induce immediate contraversive turning movements in 7 rats in open field tests in the absence of any sensory cues or rewards. The rats were manipulated to navigate from the start arm to a target zone in either the left or right arm of a T-maze. A leftward trial was followed by a rightward trial, and each rat completed a total of 10 trials. In the control group, 7 rats were tested in the same way without NSP stimulation. The time taken to navigate the maze was compared between experimental and control groups. RESULTS: All rats in the experimental group successfully reached the target area for all 70 trials in a short period of time with a short interstimulus interval (< 0.7 seconds), but only 41% of rats in the control group reached the target area and required a longer period of time to do so. The experimental group made correct directional turning movements at the intersection zone of the T-maze, taking significantly less time than the control group. No significant difference in navigation duration for the forward movements on the start and goal arms was observed between the two groups. However, the experimental group showed quick and accurate movement at the intersection zone, which made the difference in the success rate and elapsed time of tasks. CONCLUSIONS: The results of this study clearly indicate that a rat-robot model based on NSP stimulation can be a practical alternative to previously reported models controlled by virtual sensory cues and rewards.

Non-invasively enhanced intracranial transplantation of mesenchymal stem cells using focused ultrasound mediated by overexpression of cell-adhesion molecules.

Although there have been reports of promising results regarding the transplantation of mesenchymal stem cells (MSCs) for neurodegenerative diseases through the use of neuronal differentiation or control of the microenvironment, traditional surgical transplantation methods like parenchymal or intravenous injection have limitations such as secondary injuries in the brain, infection, and low survival rate of stem cells in the target site. Focused ultrasound (FUS) treatment is an emerging modality for the treatment of brain diseases, including neurodegenerative disorders. The various biological effects of FUS treatment have been investigated; therefore, the goal is now to improve the delivery efficiency and function of MSCs by capitalizing on the advantages of FUS. In this study, we demonstrated that FUS increases MSC transplantation into brain tissue by >2-fold, and that this finding might be related to the activation of intercellular adhesion molecule-1 in endothelial and subendothelial cells and vascular adhesion molecule-1 in endothelial cells.

Focused ultrasound-induced blood-brain barrier opening improves adult hippocampal neurogenesis and cognitive function in a cholinergic degeneration dementia rat model.

BACKGROUND: The persistence of adult hippocampal neurogenesis (AHN) is sharply decreased in Alzheimer's disease (AD). The neuropathologies of AD include the presence of amyloid-ß deposition in plaques, tau hyperphosphorylation in neurofibrillary tangles, and cholinergic system degeneration. The focused ultrasound (FUS)-mediated blood-brain barrier opening modulates tau hyperphosphorylation, the accumulation of amyloid-ß proteins, and increases in AHN. However, it remains unclear whether FUS can modulate AHN in cholinergic-deficient conditions. In this study, we investigated the effect of FUS on AHN in a cholinergic degeneration rat model of dementia. METHODS: Adult male Sprague-Dawley rats (n = 48; 200-250 g) were divided into control (phosphate-buffered saline injection), 192 IgG-saporin (SAP), and SAP+FUS groups; in the two latter groups, SAP was injected bilaterally into the lateral ventricle. We applied FUS to the bilateral hippocampus with microbubbles. Immunohistochemistry, enzyme-linked immunosorbent assay, immunoblotting, 5-bromo-2'-deoxyuridine labeling, an acetylcholinesterase assay, and the Morris water maze test were performed to assess choline acetyltransferase, acetylcholinesterase activity, brain-derived neurotrophic factor expression, neural proliferation, and spatial memory, respectively. Statistical significance of differences in between groups was calculated using one-way and two-way analyses of variance followed by Tukey's multiple comparison test to determine the individual and interactive effects of FUS on immunochemistry and behavioral analysis. P < 0.05 was considered significant. RESULTS: Cholinergic degeneration in rats significantly decreased the number of choline acetyltransferase neurons (P < 0.05) in the basal forebrain, as well as AHN and spatial memory function. Rats that underwent FUS-mediated brain-blood barrier opening exhibited significant increases in brain-derived neurotrophic factor (BDNF; P < 0.05), early growth response protein 1 (EGR1) (P < 0.01), AHN (P < 0.01), and acetylcholinesterase activity in the frontal cortex (P < 0.05) and hippocampus (P < 0.01) and crossing over (P < 0.01) the platform in the Morris water maze relative to the SAP group after sonication. CONCLUSIONS: FUS treatment increased AHN and improved spatial memory. This improvement was mediated by increased hippocampal BDNF and EGR1. FUS treatment may also restore AHN and protect against neurodegeneration, providing a potentially powerful therapeutic strategy for AD.

Optimization of Medial Forebrain Bundle Stimulation Parameters for Operant Conditioning of Rats.

BACKGROUND: The medial forebrain bundle (MFB) is involved in the integration of pleasure and reward. Previous studies have used various stimulation parameters for operant conditioning, though the effectiveness of these parameters has not been systematically studied. OBJECTIVES: The purpose of the present study was to investigate the optimal MFB stimulation parameters for controlling the conditioned behavior of rats. METHODS: We evaluated four factors, including intensity, frequency, pulse duration, and train duration, to determine the effect of each on lever pressure applied by animals. We further compared burst and tonic stimulation in terms of learning and performance abilities. RESULTS: The number of lever presses increased with each factor. Animals in the burst stimulation group exhibited more lever presses. Furthermore, the average speed in the maze among burst stimulation group subjects was higher. CONCLUSION: We determined the optimal parameters for movement control of animals in operant conditioning and locomotor tasks by adjusting various electrical stimulation parameters. Our results reveal that a burst stimulation is more effective than a tonic stimulation for increasing the moving speed and number of lever presses. The use of this stimulation technique also allowed us to minimize the training required to control animal behavior.

Carbon-Fiber Based Microelectrode Array Embedded with a Biodegradable Silk Support for In Vivo Neural Recording.

BACKGROUND: Recently, carbon fibers have been utilized to develop a depth-type microelectrode array for chronic neural recording. Since the diameter of carbon fibers is smaller than the conventional electrodes made of metal wires or microfabricated silicon, the carbon fiber electrodes showed an improved capability for chronic neural recording with less tissue damages. However, the carbon fiber based microelectrodes have a limitation of short insertion depth due to a low stiffness. METHODS: We proposed a carbon fiber based microelectrode array embedded with a mechanical support structure to facilitate the penetration into the deeper brain. The support is made of biodegradable silk fibroin to reduce the reactive tissue responses. The 4-channel carbon fiber based microelectrode arrays were fabricated and accessed in terms of electrochemical impedance, recording capability for 1-month implantation in rat hippocampi. The electrodes with tungsten supports were fabricated and tested as a control group. Immunohistochemical analysis was performed to identify the reactive glial responses. RESULTS: The carbon fiber based electrode arrays with silk supports showed about 2-fold impedance increase 2 weeks after implantation while the number of active electrodes decreased simultaneously. However, after 1 month, the electrode impedance decreased back to its initial value and the percentage of active electrodes also increased above 70%. Immunohistochemical staining clearly showed that the electrodes with silk supports induced less reactive glial responses than that with tungsten supports. CONCLUSION: The proposed carbon fiber based microelectrode array is expected to be used for long-term in vivo neural recording from deep brain regions with the minimized reactive tissue response.

Focused ultrasound-mediated noninvasive blood-brain barrier modulation: preclinical examination of efficacy and safety in various sonication parameters.

OBJECTIVE The application of pharmacological therapeutics in neurological disorders is limited by the ability of these agents to penetrate the blood-brain barrier (BBB). Focused ultrasound (FUS) has recently gained attention for its potential application as a method for locally opening the BBB and thereby facilitating drug delivery into the brain parenchyma. However, this method still requires optimization to maximize its safety and efficacy for clinical use. In the present study, the authors examined several sonication parameters of FUS influencing BBB opening in small animals. METHODS Changes in BBB permeability were observed during transcranial sonication using low-intensity FUS in 20 adult male Sprague-Dawley rats. The authors examined the effects of FUS sonication with different sonication parameters, varying acoustic pressure, center frequency, burst duration, microbubble (MB) type, MB dose, pulse repetition frequency (PRF), and total exposure time. The focal region of BBB opening was identified by Evans blue dye. Additionally, H & E staining was used to identify blood vessel damage. RESULTS Acoustic pressure amplitude and burst duration were closely associated with enhancement of BBB opening efficiency, but these parameters were also highly correlated with tissue damage in the sonicated region. In contrast, MB types, MB dose, total exposure time, and PRF had an influence on BBB opening without conspicuous tissue damage after FUS sonication. CONCLUSIONS The study aimed to identify these influential conditions and provide safety and efficacy values for further studies. Future work based on the current results is anticipated to facilitate the implementation of FUS sonication for drug delivery in various CNS disease states in the near future.

Rostral Agranular Insular Cortex Lesion with Motor Cortex Stimulation Enhances Pain Modulation Effect on Neuropathic Pain Model.

It is well known that the insular cortex is involved in the processing of painful input. The aim of this study was to evaluate the pain modulation role of the insular cortex during motor cortex stimulation (MCS). After inducing neuropathic pain (NP) rat models by the spared nerve injury method, we made a lesion on the rostral agranular insular cortex (RAIC) unilaterally and compared behaviorally determined pain threshold and latency in 2 groups: Group A (NP + MCS; n = 7) and Group B (NP + RAIC lesion + MCS; n = 7). Also, we simultaneously recorded neuronal activity (NP; n = 9) in the thalamus of the ventral posterolateral nucleus and RAIC to evaluate electrophysiological changes from MCS. The pain threshold and tolerance latency increased in Group A with "MCS on" and in Group B with or without "MCS on." Moreover, its increase in Group B with "MCS on" was more than that of Group B without MCS or of Group A, suggesting that MCS and RAIC lesioning are involved in pain modulation. Compared with the "MCS off" condition, the "MCS on" induced significant threshold changes in an electrophysiological study. Our data suggest that the RAIC has its own pain modulation effect, which is influenced by MCS.