Enhanced transport of brain interstitial solutes mediated by stimulation of sensorimotor area in rats.

OBJECTIVE: Solute transport in the brain is essential for maintaining cerebral homeostasis. Recent studies have shown that neuronal activity enhances the transport of cerebrospinal fluid solutes, but its impact on interstitial solute transport has not been established. In this study, we investigated whether neuronal activity affects the transport of interstitial solutes. METHODS: Fluorescent Texas Red ovalbumin was injected intracortically into the unilateral sensorimotor area of the Sprague-Dawley rats. Regional neuronal activity around the injection site was elicited by transdermal electrical stimulation of a corresponding forelimb for 90 min ( n  = 6). The control group of rats ( n  = 6) did not receive any electrical stimulation. Subsequently, the spatial distributions of the tracer over the cortical surface and from the brain sections were imaged and compared between two groups. The ovalbumin fluorescence from the cervical lymph nodes was also compared between the groups to evaluate the effect of neuronal activity on solute clearance from the brain. RESULTS: Tracer distribution over the brain surface/sections revealed a significantly higher uptake of ovalbumin in the hemisphere ipsilateral to the injection among the stimulated animals compared to the unstimulated group. This difference, however, was not seen in the hemisphere contralateral to injection. A trace amount of ovalbumin in the lymph nodes was equivalent between the groups, which indicated a considerable time needed for interstitial solutes to be drained from the brain. CONCLUSION: The results suggest that neuronal activity enhances interstitial solute transport, calling for further examination of ultimate routes and mechanisms for brain solute clearance.

Evaluation of advective solute infiltration into porous media by pulsed focused ultrasound-induced acoustic streaming effects.

PURPOSE: Acoustic streaming induced by applying transcranial focused ultrasound (FUS) promotes localized advective solute transport in the brain and has recently garnered research interest for drug delivery and enhancement of brain waste clearance. The acoustic streaming behavior in brain tissue is difficult to model numerically and thus warrants an in vitro examination of the effects of using different sonication parameters, in terms of frequency, intensity, and pulse duration (PD). METHODS: Melamine and polyvinyl alcohol (PVA) foams were used to mimic the porous brain tissue, which contains leptomeningeal fenestrations and perivascular space, while agar hydrogel was used to emulate denser neuropil. FUS was delivered to these media, which were immersed in a phosphate-buffered saline containing toluidine blue O dye, across various frequencies (400, 500, and 600 kHz; applicable to transcranial delivery) in a pulsed mode at two different spatialpeak pulse-average intensities (3 and 4 W/cm2). RESULTS: Image analysis showed that the use of 400 kHz yielded the greatest dye infiltration in melamine foam, while sonication had no impact on infiltration in the agar hydrogel due to the dominance of diffusional transport. Using a fixed spatial-peak temporal-average intensity of 0.4 W/cm2 at 400 kHz, a PD of 75 ms resulted in the greatest infiltration depth in both melamine and PVA foams among the tested range (50-150 ms). CONCLUSION: These findings suggest the existence of a specific frequency and PD that induce greater enhancement of solute/fluid movement, which may contribute to eventual in vivo applications in promoting waste clearance from the brain.

Non-thermal Acoustic Enhancement of Chemotherapeutic Effects of Cisplatin on Xenografted Cervical Cancer in Mice.

BACKGROUND/AIM: We examined the effect of low-intensity focused ultrasound (FUS) on unbinding cisplatin from plasma proteins and enhancing its chemotherapeutic efficacy using a mouse model of xenograft human cervical cancer. MATERIALS AND METHODS: FUS, operating in a pulsed mode, was applied to a dialysis cassette immersed in a normal saline bath containing both bovine serum albumin (BSA) and cisplatin, and the unbound level of cisplatin diffused into the cassette was measured. To assess the in vivo efficacy of the technique, athymic nu/nu mice were inoculated with human cervical cancer cells under four different combinatory conditions, with and without the administration of cisplatin and FUS. FUS was delivered to the tumor mass for 1 h across four separate sessions spanning a period of 10 days, following the intraperitoneal injection of cisplatin. RESULTS: In vitro equilibrium dialysis revealed that non-thermal application of FUS increased the concentration of unbound cisplatin compared to cassettes that were not exposed to sonication, suggesting successful unbinding. Assessment of tumor growth in vivo showed that FUS following cisplatin administration resulted in a significant reduction in tumor growth, whereas the administration of cisplatin alone exhibited plateau growth. Without administration of cisplatin, equivalent rates of aggressive tumor growth were observed regardless of the application of FUS. CONCLUSION: Pulsed application of FUS can unbind cisplatin from albumin and enhance its tumoricidal effects in cervical cancer. Further assessment of intratumoral/systemic cisplatin concentration is required to quantify its selective delivery to the tumor.

Cerebrospinal fluid solute transport associated with sensorimotor brain activity in rodents.

Cerebrospinal fluid (CSF) is crucial for maintaining neuronal homeostasis, providing nutrition, and removing metabolic waste from the brain. However, the relationship between neuronal activity and CSF solute transport remains poorly understood. To investigate the effect of regional neuronal activity on CSF solute transport, Sprague-Dawley rats (all male, n = 30) under anesthesia received an intracisternal injection of a fluorescent tracer (Texas Red ovalbumin) and were subjected to unilateral electrical stimulation of a forelimb. Two groups (n = 10 each) underwent two different types of stimulation protocols for 90 min, one including intermittent 7.5-s resting periods and the other without rest. The control group was not stimulated. Compared to the control, the stimulation without resting periods led to increased transport across most of the cortical areas, including the ventricles. The group that received intermittent stimulation showed an elevated level of solute uptake in limited areas, i.e., near/within the ventricles and on the ventral brain surface. Interhemispheric differences in CSF solute transport were also found in the cortical regions that overlap with the forelimb sensorimotor area. These findings suggest that neuronal activity may trigger local and brain-wide increases in CSF solute transport, contributing to waste clearance.

Non-invasive enhancement of intracortical solute clearance using transcranial focused ultrasound.

Transport of interstitial fluid and solutes plays a critical role in clearing metabolic waste from the brain. Transcranial application of focused ultrasound (FUS) has been shown to promote localized cerebrospinal fluid solute uptake into the brain parenchyma; however, its effects on the transport and clearance of interstitial solutes remain unknown. We demonstrate that pulsed application of low-intensity FUS to the rat brain enhances the transport of intracortically injected fluorescent tracers (ovalbumin and high molecular-weight dextran), yielding greater parenchymal tracer volume distribution compared to the unsonicated control group (ovalbumin by 40.1% and dextran by 34.6%). Furthermore, FUS promoted the drainage of injected interstitial ovalbumin to both superficial and deep cervical lymph nodes (cLNs) ipsilateral to sonication, with 78.3% higher drainage observed in the superficial cLNs compared to the non-sonicated hemisphere. The application of FUS increased the level of solute transport visible from the dorsal brain surface, with ~ 43% greater area and ~ 19% higher fluorescence intensity than the unsonicated group, especially in the pial surface ipsilateral to sonication. The sonication did not elicit tissue-level neuronal excitation, measured by an electroencephalogram, nor did it alter the molecular weight of the tracers. These findings suggest that nonthermal transcranial FUS can enhance advective transport of interstitial solutes and their subsequent removal in a completely non-invasive fashion, offering its potential non-pharmacological utility in facilitating clearance of waste from the brain.

Transcranial focused ultrasound-mediated unbinding of phenytoin from plasma proteins for suppression of chronic temporal lobe epilepsy in a rodent model.

The efficacy of many anti-epileptic drugs, including phenytoin (PHT), is reduced by plasma protein binding (PPB) that sequesters therapeutically active drug molecules within the bloodstream. An increase in systemic dose elevates the risk of drug side effects, which demands an alternative technique to increase the unbound concentration of PHT in a region-specific manner. We present a low-intensity focused ultrasound (FUS) technique that locally enhances the efficacy of PHT by transiently disrupting its binding to albumin. We first identified the acoustic parameters that yielded the highest PHT unbinding from albumin among evaluated parameter sets using equilibrium dialysis. Then, rats with chronic mesial temporal lobe epilepsy (mTLE) received four sessions of PHT injection, each followed by 30 min of FUS delivered to the ictal region, across 2 weeks. Two additional groups of mTLE rats underwent the same procedure, but without receiving PHT or FUS. Assessment of electrographic seizure activities revealed that FUS accompanying administration of PHT effectively reduced the number and mean duration of ictal events compared to other conditions, without damaging brain tissue or the blood-brain barrier. Our results demonstrated that the FUS technique enhanced the anti-epileptic efficacy of PHT in a chronic mTLE rodent model by region-specific PPB disruption.

Development of a wireless ultrasonic brain stimulation system for concurrent bilateral neuromodulation in freely moving rodents.

Bilateral brain stimulation is an important modality used to investigate brain circuits and treat neurological conditions. Recently, low-intensity pulsed ultrasound (LIPUS) received significant attention as a novel non-invasive neurostimulation technique with high spatial specificity. Despite the growing interest, the typical ultrasound brain stimulation study, especially for small animals, is limited to a single target of sonication. The constraint is associated with the complexity and the cost of the hardware system required to achieve multi-regional sonication. This work presented the development of a low-cost LIPUS system with a pair of single-element ultrasound transducers to address the above problem. The system was built with a multicore processor with an RF amplifier circuit. In addition, LIPUS device was incorporated with a wireless module (bluetooth low energy) and powered by a single 3.7 V battery. As a result, we achieved an ultrasound transmission with a central frequency of 380 kHz and a peak-to-peak pressure of 480 kPa from each ultrasound transducer. The developed system was further applied to anesthetized rats to investigate the difference between uni- and bilateral stimulation. A significant difference in cortical power density extracted from electroencephalogram signals was observed between uni- and bilateral LIPUS stimulation. The developed device provides an affordable solution to investigate the effects of LIPUS on functional interhemispheric connection.

High Incidence of Intracerebral Hemorrhaging Associated with the Application of Low-Intensity Focused Ultrasound Following Acute Cerebrovascular Injury by Intracortical Injection.

Low-intensity transcranial focused ultrasound (FUS) has gained momentum as a non-/minimally-invasive modality that facilitates the delivery of various pharmaceutical agents to the brain. With the additional ability to modulate regional brain tissue excitability, FUS is anticipated to confer potential neurotherapeutic applications whereby a deeper insight of its safety is warranted. We investigated the effects of FUS applied to the rat brain (Sprague-Dawley) shortly after an intracortical injection of fluorescent interstitial solutes, a widely used convection-enhanced delivery technique that directly (i.e., bypassing the blood-brain-barrier (BBB)) introduces drugs or interstitial tracers to the brain parenchyma. Texas Red ovalbumin (OA) and fluorescein isothiocyanate-dextran (FITC-d) were used as the interstitial tracers. Rats that did not receive sonication showed an expected interstitial distribution of OA and FITC-d around the injection site, with a wider volume distribution of OA (21.8 ± 4.0 µL) compared to that of FITC-d (7.8 ± 2.7 µL). Remarkably, nearly half of the rats exposed to the FUS developed intracerebral hemorrhaging (ICH), with a significantly higher volume of bleeding compared to a minor red blood cell extravasation from the animals that were not exposed to sonication. This finding suggests that the local cerebrovascular injury inflicted by the micro-injection was further exacerbated by the application of sonication, particularly during the acute stage of injury. Smaller tracer volume distributions and weaker fluorescent intensities, compared to the unsonicated animals, were observed for the sonicated rats that did not manifest hemorrhaging, which may indicate an enhanced degree of clearance of the injected tracers. Our results call for careful safety precautions when ultrasound sonication is desired among groups under elevated risks associated with a weakened or damaged vascular integrity.

Enhancement of cerebrospinal fluid tracer movement by the application of pulsed transcranial focused ultrasound.

Efficient transport of solutes in the cerebrospinal fluid (CSF) plays a critical role in their clearance from the brain. Convective bulk flow of solutes in the CSF in the perivascular space (PVS) is considered one of the important mechanisms behind solute movement in the brain, before their ultimate drainage to the systemic lymphatic system. Acoustic pressure waves can impose radiation force on a medium in its path, inducing localized and directional fluidic flow, known as acoustic streaming. We transcranially applied low-intensity focused ultrasound (FUS) to rats that received an intracisternal injection of fluorescent CSF tracers (dextran and ovalbumin, having two different molecular weights-Mw). The sonication pulsing parameter was determined on the set that propelled the aqueous solution of toluidine blue O dye into a porous media (melamine foam) at the highest level of infiltration. Fluorescence imaging of the brain showed that application of FUS increased the uptake of ovalbumin at the sonicated plane, particularly around the ventricles, whereas the uptake of high-Mw dextran was unaffected. Numerical simulation showed that the effects of sonication were non-thermal. Sonication did not alter the animals' behavior or disrupt the blood-brain barrier (BBB) while yielding normal brain histology. The results suggest that FUS may serve as a new non-invasive means to promote interstitial CSF solute transport in a region-specific manner without disrupting the BBB, providing potential for enhanced clearance of waste products from the brain.

Trans-Spinal Focused Ultrasound Stimulation Selectively Modulates Descending Motor Pathway.

Compared to current non-invasive methods utilizing magnetic and electrical means, focused ultrasound provides greater spatial resolution and penetration depth. Despite the broad application of ultrasound stimulation, there is a lack of studies dedicated to the investigation of acoustic neuromodulation on the spinal cord. This study aims to apply focused ultrasound on the spinal cord to modulate the descending pathways in a non-invasive fashion. The application of trans-spinal focused ultrasound (tsFUS) was examined on the motor deficit mouse model. tsFUS was achieved using a single-element focused ultrasound transducer operating at 3 MHz. The sonication was performed on anesthetized 6 week-old mice targeting T12 and L3 vertebrae. The effect was analyzed by comparing electromyography responses from the hindlimb induced by electrical stimulation of the motor cortex. Further, the mouse model with the Harmaline-induced essential tremor (ET) was selected to investigate the potential clinical application of tsFUS. The safety was verified by histological assessment. Sonication at the T12 area inhibited motor response, while sonication over the L3 region provided signal enhancement. Sonication of T12 of the ET mouse also showed the ability of ultrasound to suppress tremors. Meanwhile, the histological examination did not show any abnormalities with the highest applied acoustic pressure. In this work, a non-invasive motor signal modulation was achieved using tsFUS. Moreover, the results showed the ability of focused ultrasound to manage tremors in a safe manner. This study provides a stepping stone for the trans-spinal application of focused ultrasound to motor-related disorders.

Wearable Transcranial Ultrasound System for Remote Stimulation of Freely Moving Animal.

OBJECTIVE: Transcranial focused ultrasound (tFUS) has drawn considerable attention in the neuroscience field as a noninvasive approach to modulate brain circuits. However, the conventional approach requires the use of anesthetized or immobilized animal models, which places considerable restrictions on behavior and affects treatment. Thus, this work presents a wireless, wearable system to achieve ultrasound brain stimulation in freely behaving animals. METHODS: The wearable tFUS system was developed based on a microcontroller and amplifier circuit. Brain activity induced by tFUS was monitored through cerebral hemodynamic changes using near-infrared spectroscopy. The system was also applied to stroke rehabilitation after temporal middle cerebral artery occlusion (tMCAO) in rats. Temperature calculations and histological results showed the safety of the application even with prolonged 40 min sonication. RESULTS: The output ultrasonic wave produced from a custom PZT transducer had a central frequency of 457 kHz and peak to peak pressure of 426 kPa. The device weight was 20 g, allowing a full range of motion. The stimulation was found to induce hemodynamic changes in the sonicated area, while open-field tests showed that ultrasound applied to the ipsilateral hemisphere for 5 consecutive days after the stroke facilitated recovery. CONCLUSION: The wearable tFUS system has been designed and implemented on moving rats. The results showed the ability of device to cause both short- and long lasting effects. SIGNIFICANCE: The proposed device provides a more natural environment to investigate the effects of tFUS for behavioral and long-term studies.

Non-invasive measurement of hemodynamic change during 8 MHz transcranial focused ultrasound stimulation using near-infrared spectroscopy.

BACKGROUND: Transcranial focused ultrasound (tFUS) attracts wide attention in neuroscience as an effective noninvasive approach to modulate brain circuits. In spite of this, the effects of tFUS on the brain is still unclear, and further investigation is needed. The present study proposes to use near-infrared spectroscopy (NIRS) to observe cerebral hemodynamic change caused by tFUS in a noninvasive manner. RESULTS: The results show a transient increase of oxyhemoglobin and decrease of deoxyhemoglobin concentration in the mouse model induced by ultrasound stimulation of the somatosensory cortex with a frequency of 8 MHz but not in sham. In addition, the amplitude of hemodynamics change can be related to the peak intensity of the acoustic wave. CONCLUSION: High frequency 8 MHz ultrasound was shown to induce hemodynamic changes measured using NIRS through the intact mouse head. The implementation of NIRS offers the possibility of investigating brain response noninvasively for different tFUS parameters through cerebral hemodynamic change.

The hemodynamic changes during cupping therapy monitored by using an optical sensor embedded cup.

Cupping therapy is one form of alternative medicine that is used widely across the world. Although the applications of cupping therapy including pain relief have a 1000-year history, the therapeutic effect of cupping is still questionable due to a lack of scientific evidence. Therefore, in the present study, we embedded a near-infrared spectroscopic sensor into a suction cup to monitor the hemodynamic changes on the treated site while the hemodynamics at the surrounding tissue of the cup was also simultaneously monitored by another near-infrared spectroscopic sensor. The results from 10 healthy male subjects show a dramatic increase of the oxy-hemoglobin (OHb) and deoxy-hemoglobin (RHb) concentrations at the treatment site while the OHb and RHb levels were decreased at the surrounding tissue. Moreover, after the treatment, we observed that the OHb concentrations were maintained at a higher level than before treatment at both sites, which may demonstrate how cupping therapy works for treatment. In summary, the results showed that cupping therapy increases blood volume and tissue oxygenation at the treatment site while those were slightly decreased at the surrounding tissue. This study showed that the embedding of near-infrared spectroscopy in a cupping system could offer a better understanding of the mechanism of cupping therapy.

Mobile Wireless Low-intensity Transcranial Ultrasound Stimulation System for Freely Behaving Small Animals.

Transcranial ultrasound stimulation (tUS) is a promising noninvasive approach to modulate brain circuits. While low-intensity tUS is putatively safe and has already been used for human participants, pre-clinical studies that aim to determine the effects of tUS on the brain still need to be carried out. Conventional tUS stimulation, however, requires the use of the anesthetized or immobilized animal model, which can place considerable restrictions on behavior. Thus, this work presents a portable, low cost, wireless system to achieve ultrasound brain stimulation in freely behaving animals. The tUS system was developed based on a commercial 16 MHz microcontroller and amplifier circuit. The acoustic wave with a central frequency of 450 kHz was generated from a 5mm PZT with a peak pressure of 426 kPa. The wireless tUS with a total weight of 20 g was placed on the back of the rat allowing the animal a full range of unimpeded motion. The mobile ultrasound system was able to induce a robust ear movement as a response to stimulation of the motor cortex. The outcome demonstrates the ability of wireless tUS to modulate the brain circuit of a freely behaving rat. The portability of the whole system provides a more natural environment for investigating the effect of tUS on behavior and chronic studies.

Simultaneous Monitoring of Hemodynamic Response in the Pre-Frontal Cortex and Genital Organ During Sexual Arousal Using Near-Infrared Spectroscopy.

BACKGROUND: The monitoring of brain activity along with genital organ response to sexual stimulation can play an important role in understanding the under-lying mechanisms of sexual arousal as well as diagnosing erectile dysfunction. Several studies have observed brain activity corresponding to sexual stimuli, but only a few studies have shown a simultaneous measurement of brain activation and penile response. AIM: To introduce near-infrared spectroscopy (NIRS) as a portable, easily implemented, and low-cost technique to simultaneously record brain activity and hemodynamics in the genital organ during sexual arousal. METHODS: Hemodynamic measurements of 15 healthy men were obtained using a home-built NIRS system. In the initial experiment, hemodynamics in the pre-frontal cortex (N = 10) were measured during visual sexual stimulation (VSS) and neutral visual stimulation (NVS) to identify brain activity related to sexual arousal. In the subsequent experiment, cerebral and penile hemodynamics were simultaneously measured (N = 5) using NIRS during VSS and NVS. RESULTS: The pre-frontal cortex showed activity related to VSS but not to NVS. Simultaneous measurements showed a corresponding increase of penile oxygenated and deoxygenated hemoglobin concentration indicating an increase of blood volume associated with sexual arousal in healthy men. An average response delay of 4 seconds was observed in the hemodynamic changes between the brain and genital organ. CONCLUSION: In this preliminary study, we presented a NIRS system capable not only of detecting cerebral hemodynamic changes related to sexual arousal but also the simultaneous measurement of penile hemodynamics. We believe the NIRS system can be a potential technique to supplement the field of sexual medicine and can be expanded further to diagnose erectile dysfunction. Kim E, Kim S, Zephaniah PV, et al. Simultaneous Monitoring of Hemodynamic Response in the Pre-Frontal Cortex and Genital Organ During Sexual Arousal Using Near-Infrared Spectroscopy. Sex Med 2018;6:234-238.

Monitoring cerebral hemodynamic change during transcranial ultrasound stimulation using optical intrinsic signal imaging.

Transcranial ultrasound stimulation (tUS) is a promising non-invasive approach to modulate brain circuits. The application is gaining popularity, however the full effect of ultrasound stimulation is still unclear and further investigation is needed. This study aims to apply optical intrinsic signal imaging (OISI) for the first time, to simultaneously monitor the wide-field cerebral hemodynamic change during tUS on awake animal with high spatial and temporal resolution. Three stimulation paradigms were delivered using a single-element focused transducer operating at 425 kHz in pulsed mode having the same intensity (ISPPA = 1.84 W/cm2, ISPTA = 129 mW/cm2) but varying pulse repetition frequencies (PRF). The results indicate a concurrent hemodynamic change occurring with all actual tUS but not under a sham stimulation. The stimulation initiated the increase of oxygenated hemoglobin (HbO) and decrease of deoxygenated hemoglobin (RHb). A statistically significant difference (p < 0.05) was found in the amplitude change of hemodynamics evoked by varying PRF. Moreover, the acoustic stimulation was able to trigger a global as well as local cerebral hemodynamic alteration in the mouse cortex. Thus, the implementation of OISI offers the possibility of directly investigating brain response in an awake animal during tUS through cerebral hemodynamic change.

A discrepancy of penile hemodynamics during visual sexual stimulation observed by near-infrared spectroscopy.

BACKGROUND: In this paper, we observed a discrepancy of penile hemodynamics dependent on location by using near infrared spectroscopy (NIRS) sensor, and showcase NIRS as a potentially suitable sensor in supplementing the diagnosis and treatment of erectile dysfunction. METHODS: To observe the effect that location has on penile hemodynamics, the NIRS sensor was placed on the top and the side of genital organ, and oxy- (HbO), deoxy-(RHb), and total (HbT) hemoglobin concentration changes were acquired. Our results from 6 healthy subjects show that hemodynamic changes vary depending on where the probe was placed. To observe a statistical difference between the signals, a Wilcoxon signed-rank test was performed. RESULTS: The result shows a significant difference (p < 0.05) between concentration changes of RHb and HbT depending on the probes' location. Moreover, the sensor placed on the top of the organ shows a rise of HbO and HbT concentration while RHb concentration decreased. However, hemodynamics from the side of the organ showed that RHb concentration increased along with HbO. CONCLUSIONS: The outcomes demonstrates an ability of NIRS to be sensitive enough to detect the different hemodynamic changes in various locations of a healthy male genital organ during visual sexual stimulation. The results also show the importance of sensor location on the genital organ for the resulting hemodynamic changes. We can foresee our results as a way for clinicians to obtain more accurate hemodynamic measurements from the penis, and also show the likelihood for NIRS enhanced diagnosis tool of male erectile dysfunction over the current standards.