Focused ultrasound-mediated intranasal brain drug delivery technique (FUSIN)

The blood-brain barrier (BBB) is the major obstacle for brain drug delivery and limits the treatment options for central nervous system diseases. To circumvent the BBB, we introduce focused ultrasound-mediated intranasal brain drug delivery (FUSIN). FUSIN utilizes the nasal route for direct nose-to-brain drug administration, bypassing the BBB and minimizing systemic exposure to the major organs, such as heart, lung, liver, and kidney [1]. It also uses transcranial ultrasound energy focused at a targeted brain region to induce microbubble cavitation, enhancing the transport of intranasally administered agents at the FUS-targeted brain location. FUSIN is unique because it can achieve noninvasive and localized brain drug delivery with minimized systemic toxicity to other major organs. The goal of this paper is to provide a detailed protocol for FUSIN delivery to the mouse brain.•FUSIN delivery utilizes the nose-to-brain pathway for brain drug delivery.•FUSIN utilizes FUS combined with microbubble to significantly enhance the delivery efficiency of intranasally administered drugs to the FUS targeted brain regions.•FUSIN achieves efficient brain delivery with minimized systemic exposure in the major organs

Focused ultrasound-enhanced delivery of intranasally administered anti-programmed cell death-ligand 1 antibody to an intracranial murine glioma model

Immune checkpoint inhibitors have great potential for the treatment of gliomas; however, their therapeutic efficacy has been partially limited by their inability to efficiently cross the blood-brain barrier (BBB). The objective of this study was to evaluate the capability of focused-ultrasound-mediated intranasal brain drug delivery (FUSIN) in achieving the locally enhanced delivery of anti-programmed cell death-ligand 1 antibody (aPD-L1) to the brain. Both non-tumor mice and mice transcranially implanted with GL261 glioma cells at the brainstem were used in this study. aPD-L1 was labeled with a near-infrared fluorescence dye (IRDye 800CW) and administered to mice through the nasal route to the brain, followed by focused ultrasound sonication in the presence of systemically injected microbubbles. FUSIN enhanced the accumulation of aPD-L1 at the FUS-targeted brainstem by an average of 4.03- and 3.74-fold compared with intranasal (IN) administration alone in the non-tumor mice and glioma mice, respectively. Immunohistochemistry staining found that aPD-L1 was mainly located within the perivascular spaces after IN delivery, while FUSIN further enhanced the penetration depth and delivery efficiency of aPD-L1 to the brain parenchyma. The delivered aPD-L1 was found to be colocalized with the tumor cells after FUSIN delivery to the brainstem glioma. These findings suggest that FUSIN is a promising technique to enhance the delivery of immune checkpoint inhibitors to gliomas

Incisionless targeted adeno-associated viral vector delivery to the brain by focused ultrasound-mediated intranasal administration

BACKGROUND: Adeno-associated viral (AAV) vectors are currently the leading platform for gene therapy with the potential to treat a variety of central nervous system (CNS) diseases. There are numerous methods for delivering AAVs to the CNS, such as direct intracranial injection (DI), intranasal delivery (IN), and intravenous injection with focused ultrasound-induced blood-brain barrier disruption (FUS-BBBD). However, non-invasive and efficient delivery of AAVs to the brain with minimal systemic toxicity remain the major challenge. This study aims to investigate the potential of focused ultrasound-mediated intranasal delivery (FUSIN) in AAV delivery to brain. METHODS: Mice were intranasally administered with AAV5 encoding enhanced green fluorescence protein (AAV5-EGFP) followed by FUS sonication in the presence of systemically injected microbubbles. Mouse brains and other major organs were harvested for immunohistological staining, PCR quantification, and in situ hybridization. The AAV delivery outcomes were compared with those of DI, FUS-BBBD, and IN delivery. FINDINGS: FUSIN achieved safe and efficient delivery of AAV5-EGFP to spatially targeted brain locations, including a superficial brain site (cortex) and a deep brain region (brainstem). FUSIN achieved comparable delivery outcomes as the established DI, and displayed 414.9-fold and 2073.7-fold higher delivery efficiency than FUS-BBBD and IN. FUSIN was associated with minimal biodistribution in peripheral organs, which was comparable to that of DI. INTERPRETATION: Our results suggest that FUSIN is a promising technique for non-invasive, efficient, safe, and spatially targeted AAV delivery to the brain. FUNDING: National Institutes of Health (NIH) grants R01EB027223, R01EB030102, R01MH116981, and UG3MH126861

Mechanically manipulating glymphatic transport by ultrasound combined with microbubbles

The glymphatic system is a perivascular fluid transport system for waste clearance. Glymphatic transport is believed to be driven by the perivascular pumping effect created by the pulsation of the arterial wall caused by the cardiac cycle. Ultrasound sonication of circulating microbubbles (MBs) in the cerebral vasculature induces volumetric expansion and contraction of MBs that push and pull on the vessel wall to generate a MB pumping effect. The objective of this study was to evaluate whether glymphatic transport can be mechanically manipulated by focused ultrasound (FUS) sonication of MBs. The glymphatic pathway in intact mouse brains was studied using intranasal administration of fluorescently labeled albumin as fluid tracers, followed by FUS sonication at a deep brain target (thalamus) in the presence of intravenously injected MBs. Intracisternal magna injection, the conventional technique used in studying glymphatic transport, was employed to provide a comparative reference. Three-dimensional confocal microscopy imaging of optically cleared brain tissue revealed that FUS sonication enhanced the transport of fluorescently labeled albumin tracer in the perivascular space (PVS) along microvessels, primarily the arterioles. We also obtained evidence of FUS-enhanced penetration of the albumin tracer from the PVS into the interstitial space. This study revealed that ultrasound combined with circulating MBs could mechanically enhance glymphatic transport in the brain

Targeted delivery of therapeutic agents to the mouse brain using a stereotactic-guided focused ultrasound device

Existing protocols of focused ultrasound (FUS) combined with microbubble-mediated blood-brain barrier (BBB) opening (FUS-BBBO) in preclinical research require expensive ultrasound equipment and complex operating procedures. We developed a low-cost, easy-to-use, and precise FUS device for small animal models in preclinical research. Here, we provide a detailed protocol for building the FUS transducer, attaching the transducer to a stereotactic frame for precise brain targeting, applying the integrated FUS device to perform FUS-BBBO in mice, and evaluating the FUS-BBBO outcome. For complete details on the use and execution of this protocol, please refer to Hu et al. (2022)

Focused ultrasound-enabled brain tumor liquid biopsy

Abstract Although blood-based liquid biopsies have emerged as a promising non-invasive method to detect biomarkers in various cancers, limited progress has been made for brain tumors. One major obstacle is the blood-brain barrier (BBB), which hinders efficient passage of tumor biomarkers into the peripheral circulation. The objective of this study was to determine whether FUS in combination with microbubbles can enhance the release of biomarkers from the brain tumor to the blood circulation. Two glioblastoma tumor models (U87 and GL261), developed by intracranial injection of respective enhanced green fluorescent protein (eGFP)-transduced glioblastoma cells, were treated by FUS in the presence of systemically injected microbubbles. Effect of FUS on plasma eGFP mRNA levels was determined using quantitative polymerase chain reaction. eGFP mRNA were only detectable in the FUS-treated U87 mice and undetectable in the untreated U87 mice (maximum cycle number set to 40). This finding was replicated in GL261 mice across three different acoustic pressures. The circulating levels of eGFP mRNA were 1,500–4,800 fold higher in the FUS-treated GL261 mice than that of the untreated mice for the three acoustic pressures. This study demonstrated the feasibility of FUS-enabled brain tumor liquid biopsies in two different murine glioma models across different acoustic pressures

Leptomeningeal disease and tumor dissemination in a murine diffuse intrinsic pontine glioma model: Implications for the study of the tumor-cerebrospinal fluid-ependymal microenvironment

BACKGROUND: Leptomeningeal disease and hydrocephalus are present in up to 30% of patients with diffuse intrinsic pontine glioma (DIPG), however there are no animal models of cerebrospinal fluid (CSF) dissemination. As the tumor-CSF-ependymal microenvironment may play an important role in tumor pathogenesis, we identified characteristics of the Nestin-tumor virus A (Nestin-Tva) genetically engineered mouse model that make it ideal to study the interaction of tumor cells with the CSF and its associated pathways with implications for the development of treatment approaches to address CSF dissemination in DIPG. METHODS: A Nestin-Tva model of DIPG utilizing the 3 most common DIPG genetic alterations (H3.3K27M, PDGF-B, and p53) was used for this study. All mice underwent MR imaging and a subset underwent histopathologic analysis with H&E and immunostaining. RESULTS: Tumor dissemination within the CSF pathways (ventricles, leptomeninges) from the subependyma was present in 76% (25/33) of mice, with invasion of the choroid plexus, disruption of the ciliated ependyma and regional subependymal fluid accumulation. Ventricular enlargement consistent with hydrocephalus was present in 94% (31/33). Ventricle volume correlated with region-specific transependymal CSF flow (periventricular T2 signal), localized anterior to the lateral ventricles. CONCLUSIONS: This is the first study to report CSF pathway tumor dissemination associated with subependymal tumor in an animal model of DIPG and is representative of CSF dissemination seen clinically. Understanding the CSF-tumor-ependymal microenvironment has significant implications for treatment of DIPG through targeting mechanisms of tumor spread within the CSF pathways

Induction of a torpor-like hypothermic and hypometabolic state in rodents by ultrasound

Torpor is an energy-conserving state in which animals dramatically decrease their metabolic rate and body temperature to survive harsh environmental conditions. Here, we report the noninvasive, precise and safe induction of a torpor-like hypothermic and hypometabolic state in rodents by remote transcranial ultrasound stimulation at the hypothalamus preoptic area (POA). We achieve a long-lasting (\u3e24 h) torpor-like state in mice via closed-loop feedback control of ultrasound stimulation with automated detection of body temperature. Ultrasound-induced hypothermia and hypometabolism (UIH) is triggered by activation of POA neurons, involves the dorsomedial hypothalamus as a downstream brain region and subsequent inhibition of thermogenic brown adipose tissue. Single-nucleus RNA-sequencing of POA neurons reveals TRPM2 as an ultrasound-sensitive ion channel, the knockdown of which suppresses UIH. We also demonstrate that UIH is feasible in a non-torpid animal, the rat. Our findings establish UIH as a promising technology for the noninvasive and safe induction of a torpor-like state