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Jan 2020

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Intravenous and Non-invasive Drug Delivery to the Mouse Basal Forebrain Using MRI-guided Focused Ultrasound
MRI引导下聚焦超声对小鼠基底前脑静脉及无创给药影响的研究   

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Abstract

Basal forebrain cholinergic neurons (BFCNs) regulate circuit dynamics underlying cognitive processing, including attention, memory, and cognitive flexibility. In Alzheimer’s disease and related neurodegenerative conditions, the degeneration of BFCNs has long been considered a key player in cognitive decline. The cholinergic system thus represents a key therapeutic target. A long-standing obstacle for the development of effective cholinergic-based therapies is not only the production of biologically active compounds but also a platform for safe and efficient drug delivery to the basal forebrain. The blood-brain barrier (BBB) presents a significant challenge for drug delivery to the brain, excluding approximately 98% of small-molecule biologics and nearly 100% of large-molecule therapeutic agents from entry into the brain parenchyma. Current modalities to achieve effective drug delivery to deep brain structures, such as the basal forebrain, are particularly limited. Direct intracranial injection via a needle or catheter carries risks associated with invasive neurosurgery. Intra-arterial injection of hyperosmotic solutions or therapeutics modified to penetrate the BBB using endogenous transport systems lack regional specificity, which may not always be desirable. Intranasal, intrathecal, and intraventricular administration have limited drug distribution beyond the brain surface. Here, we present a protocol for non-invasively, locally, and transiently increasing BBB permeability using MRI-guided focused ultrasound (MRIgFUS) in the murine basal forebrain for delivery of therapeutic agents targeting the cholinergic system. Ongoing work in preclinical models and clinical trials supports the safety and feasibility of MRIgFUS-mediated BBB modulation as a promising drug delivery modality for the treatment of debilitating neurological diseases.

Keywords: Basal forebrain (基底前脑), Medial septum (内侧隔核), Nucleus basalis (基底核), Focused ultrasound (聚焦超声), Blood-brain barrier (血脑屏障), Brain drug delivery (大脑的药物输送)

Background

The majority of cholinergic afferents in cortical and limbic structures involved in cognition originate from long-range projection neurons located in the basal forebrain (Hampel et al., 2018). Basal forebrain cholinergic neurons (BFCNs) are highly vulnerable in Alzheimer’s disease (AD) and other neurodegenerative diseases, including vascular dementia, Lewy body dementia, frontotemporal dementia, amyotrophic lateral sclerosis, and Parkinson’s disease, and contribute significantly to cognitive decline (Cykowski et al., 2014; Grothe et al., 2014; Hampel et al., 2018; Ray et al., 2018; Convery et al., 2020), validating BFCNs as an important therapeutic target. Although current clinically approved cholinergic agents for the treatment of AD have shown only modest benefits on cognition, recent studies suggest that they may also modify the course of disease with continued use (Dubois et al., 2015; Cavedo et al., 2016 and 2017). It is possible that the modest impact on cognition may reflect the drug dose that can be achieved; higher doses, which could be of greater benefit, cannot be tolerated due to peripheral side effects (Birks and Harvey, 2018). Other putative cholinergic agents, such as trophic factors and related gene therapies, which have the potential to simulate cholinergic function and prevent cholinergic degeneration, do not cross the blood-brain barrier (BBB) (Chen and Mobley, 2019; Xhima and Aubert, 2021). Thus, a major obstacle for the development of cholinergic-targeted agents, and brain-targeted therapeutics in general, is not only the production of biologically active compounds but a way to deliver them safely and efficiently to the brain.


Approximately 98% of small molecules and nearly 100% of large molecule drugs, including recombinant proteins, antibodies, and gene-related therapeutics, are unable to cross the BBB when administered systemically (Pardridge et al., 2020). Current approaches that accomplish direct drug delivery involve highly invasive intracranial procedures with multiple injections to reach the basal forebrain; these procedures carry substantial risk for surgical complications, and control of drug distribution can be difficult because drug concentration decreases exponentially from the injection site (Honig et al., 2018; Castle et al., 2020; Pardridge et al., 2020). Other approaches include: (1) intra-arterial injection of hyperosmotic solutions, such as mannitol, to increase BBB permeability, coupled with peripheral drug delivery; (2) systemic administration of therapeutics linked to protein vector delivery systems that can cross the BBB; (3) intraocular delivery; and (4) intranasal administration. However, even if therapeutics reach the brain in adequate concentrations with these methods, drug biodistribution will be non-targeted and may cause indiscriminate actions on all cholinergic synapses in the brain rather than BFCNs.


Transcranial focused ultrasound (FUS) in combination with intravenously injected microbubbles (i.e., an air/perfluorocarbon gas core stabilized by a phospholipid/protein/polymer shell) can transiently enhance BBB permeability in the targeted volume (Hynynen et al., 2001). FUS offers a minimally invasive avenue to deliver a variety of therapeutic agents to the brain with the advantages of being able to temporarily permeabilize the BBB in specific brain regions without widespread drug exposure elsewhere in the brain and to potentially reduce the peripheral dose required to achieve the desired bioeffects in the brain (Meng et al., 2020). Additionally, FUS targeting systems can be co-registered to the spatial coordinates of MRI scanners, allowing for highly precise BBB modulation in specific neuroanatomical locations, such as the basal forebrain (Xhima et al., 2020). Gadolinium-based MRI contrast agents, excluded by the intact BBB, offer the added advantage of confirming the site of therapeutic delivery following FUS exposure. In a recent study, we established the feasibility of MRI-guided FUS (MRIgFUS)-induced BBB modulation in the basal forebrain for drug delivery in an AD mouse model (Xhima et al., 2020). Here, we present a detailed protocol for using MRIgFUS to non-invasively and locally deliver intravenously administered therapeutic agents to the murine basal forebrain for preclinical drug development.


Materials and Reagents

  1. Cotton tipped applicators (AMG Medical Inc., catalog number: 018-450)

  2. Non-woven sponges (Covidien, catalog number: 9022)

  3. Medical tape (3M, catalog number: 1527-1)

  4. 70% isopropyl alcohol pads (Alliance, catalog number: 211-MM-05507)

  5. Circulating water warming pad (Stryker, catalog number: TP700)

  6. 26 G angiocatheter (Venisystems Abbocath-T, catalog number: G944-A01)

  7. Catheter injection cap (SAI Infusion Technologies, catalog number: IC)

  8. 1 ml insulin syringes (BD Biosciences, catalog number: BD329654)

  9. 27 G needles (BD Biosciences, catalog number: BD305109)

  10. 18 G blunt-fill needles (BD Biosciences, catalog number: BD305180)

  11. Wavelength clear ultrasound gel (National Therapy Products Inc., catalog number: NTPC502X)

  12. Saline bags heated by immersion in a water bath at 37°C

  13. Adult mice, 20-50 g

  14. Therapeutic agent of interest

  15. Sterile normal saline (Baxter, catalog number: 2B1307)

  16. Sterile heparinized saline (Baxter, catalog number: AHB0953U)

  17. Depilatory cream (e.g., Veet or Nair)

  18. Lubricant eye ointment (Refresh Lacri-Lube, Allergan, catalog number: 00210889)

  19. Definity microbubbles (Lantheus Medical Imaging, catalog number: DE4). Storage: 4°C

  20. Gadovist (Bayer, catalog number: 02241089). Storage: room temperature

  21. Isoflurane (Fresenius Kabi, catalog number: M60303)

Equipment

  1. Scissors (Fine Science Tools, catalog number: 14088-10)

  2. Small animal electric hair trimmer (Wahl Clipper, catalog number: 58112)

  3. Forced-air warming unit with blanket (Medtronic, catalog number: WT 6000)

  4. Small animal weigh scale

  5. Vialmix mechanical shaking device (Lantheus Medical Imaging, catalog number: VMIX)

  6. Small animal anesthesia machine connected to an induction chamber and nose cone

  7. Plastic tubing to connect the anesthesia machine to the animal positioned on the FUS system and MR scanner

  8. Class IIA2 biosafety cabinet for tail vein catheterization

  9. Small animal 7T MRI (Bruker, 7T horizontal bore Avance BioSpec 70/30 USR scanner)

  10. MRI body coil (Bruker, model: 86 mm Quad SN37)

  11. MRI surface coil (Bruker, model: 86 mm Quad Receive)

  12. LP100 focused ultrasound system (FUS Instruments, Inc.) including:

    1. 1.68 MHz spherically curved transducer (0.8 focal number, 75 mm external diameter, 20 mm internal diameter)

    2. Polyvinylidene difluoride or lead zirconate titanate hydrophone

    3. Function generator

    4. 14-bit scope card

    5. 50 W RF power amplifier

    6. RF power meter

    7. External matching circuit

    8. Motorized positioning system in all three orthogonal axes

    9. Degassed and deionized water tank

    10. MRI-compatible sled equipped with an anesthesia nose cone and a fixture that consists of two Kapton polyimide membranes containing degassed and deionized water

    11. MR-compatible focus finding marker

  13. Oxygen supply

  14. Medical air supply

Software

  1. ParaVision 5.0 (Bruker)

  2. Software for controlling and monitoring FUS exposure (FUS Instruments, Inc.)

Procedure

  1. Obtain approval for the animal use protocol from the affiliated institution

    The animal experiments must follow guidelines and policies set forth by the facility Animal Care Committee in accordance with relevant government legislation.


  2. Experimental setup and co-registration of MRI and the FUS system

    1. The experimental setup of the FUS system is illustrated in Figure 1A to 1D.

    2. Mount the ultrasound transducer to the arm of the motorized positioning system and immerse it into the degassed and deionized water tank.

    3. Establish connections between the components of the FUS system (see Figure 1A).

    4. Place the MR-compatible sled to be spatially registered on the plate over the water bath.

    5. Position the transducer underneath the polyimide membrane fixture. Sonicate using continuous mode at 0.5 W and position the transducer focus at the water surface tank to produce a small cone-shaped fountain of water.

    6. Tape the focus finding marker at the transducer focus. Turn off the output of the function generator.

    7. Place the sled into the MRI scanner and perform a three-axis localizer scan of the focus finding marker. Using the FUS Instruments software, input the MRI spatial coordinates of the marker to co-register the transducer positioning system with the imaging system. In Xhima et al. (2020), a FLASH tripilot sequence with TE/TR = 3 ms/200 ms, slice thickness = 1 mm, and in-plane resolution = 0.23 × 0.23 mm was performed to acquire MR images.



      Figure 1. LP100 setup for FUS exposure. (A) Schematic of system components for FUS-mediated BBB permeability enhancement. From Xhima et al. (2020). © The Authors, some rights reserved, exclusive licensee AAAS. Distributed under a CC BY-NC 4.0 License. (B) Side view and (C-D) top views of the experimental setup.


  3. Mouse preparation

    1. Administer 0.5 ml of saline subcutaneously the day prior to the experiment and approximately 45 min pre-sonication to ensure adequate hydration for tail vein catheter placement.

    2. The mouse must be warmed to dilate the tail vein for successful catheterization. Pre-warm the home cage under a forced-air warming blanket at 38°C for approximately 30 min prior to anesthesia and keep the mouse on a warm water pad while anesthetized during catheterization.

    3. Use aseptic technique for tail vein catheterization in a Class IIA2 biosafety cabinet.

      1. Place the mouse in an induction chamber and anesthetize using isoflurane (5%) with oxygen carrier gas (flow rate = 1 L/min).

      2. When the animal is anesthetized, remove it from the induction chamber and gently fit the nose cone. Set the isoflurane vaporizer to 2% (or dose to effect).

      3. Apply lubricant eye ointment to prevent the eyes from overdrying. Re-apply as needed.

      4. Clean the tail with an alcohol prep pad.

      5. Prime a 26 G angiocatheter with heparinized saline, then insert it into the tail vein. Remove the guiding needle from the catheter and secure the catheter injection cap.

      6. Flush heparinized saline through the catheter to confirm correct placement within the tail vein. Back pressure indicates improper placement.

      7. Add structural support to the catheter by securing a rigid structure along the long axis of the tail (e.g., the narrow wooden dowel of a cotton-tipped applicator) with medical tape.

    4. Remove fur on the dorsal surface of the head (from the eye line to the neck) with an electric hair trimmer and depilatory cream.

    5. Wash the depilatory cream with mild detergent and warm water. Wipe the skin with gauze and an alcohol swab.

    6. Determine the body weight of the mouse to calculate the volume of injectables.


  4. MRIgFUS-mediated BBB permeability in the basal forebrain

    1. An experimental timeline of the procedure is illustrated in Figure 2A.

    2. Apply ultrasound gel to the Kapton polyimide window on the MR-compatible sled and position the anesthetized mouse in the supine position on the sled. Ensure there is an unobstructed path for the ultrasound to propagate from the transducer, through the Kapton polyimide window, to the murine brain. Attach the nose cone to plastic tubing connected to the anesthesia machine using isoflurane (1-1.5% or dose to effect) with medical air carrier gas (flow rate = 1 L/min). Secure the head to the MR-compatible sled with medical tape to minimize movement during the procedure. Movement of the head during the procedure will reduce the accuracy of FUS targeting.

    3. Cover the body of the mouse with a heated saline bag to maintain core temperature during the procedure (see Figure 1D).

    4. Place the mouse secured on the sled into the MRI scanner and acquire images of the brain for target planning (tripilot, axial T1-weighted, and axial T2-weighted sequences; see Figure 2B and 2C). In Xhima et al. (2020), RARE T1-weighted images were obtained with TE/TR = 10 ms/500 ms, RARE factor = 2, averages = 3, slice thickness = 1.5 mm, number of slices = 5, and in-plane resolution = 0.25 × 0.25 mm. RARE T2-weighted images were obtained with TE/TR = 75 ms/4,000 ms, RARE factor = 10, averages = 4, slice thickness = 1.5 mm, number of slices = 5, and in-plane resolution = 0.25 × 0.25 mm.

    5. In the FUS Instruments software, select target spots in the basal forebrain from the T2-weighted scan (Figure 2B). The medial septum/vertical diagonal band of Broca (MS/VDB) are targeted with one focal spot. The nucleus basalis/horizontal band of Broca (NBM/HDB) are targeted with two focal spots. The lateral ventricle is used as the MRI-visible anatomical landmark to position the medial focal spot on the appropriate axial MR image. The lateral foci are positioned according to the stereotaxic coordinates from a mouse neuroanatomical atlas (Paxinos and Franklin, 2012).

    6. Position the sled on the FUS system. Perform the sonication using the following FUS parameters: 1.68 MHz frequency, 10 ms burst length, 1 Hz pulse repetition frequency, 120 s duration, and 1% duty cycle. The parameters for passive cavitation detection are 20 MHz sampling rate and 800 mV input range. An acoustic feedback control algorithm, similar to the one described by O’Reilly and Hynynen (2012), is used to calibrate peak negative pressure (PNP) based on the in vivo microbubble response. In brief, starting PNP is set to 250 kPa (measured in water without skull attenuation) and increased incrementally every second by 25 kPa. Definity microbubbles are delivered 10 s following the start of sonication to collect baseline acoustic emissions. Once the magnitude of acoustic emissions at 0.5ƒ passes 3.5 times the baseline level at each target, the sonicating pressure is dropped by 75% and maintained at this level for the remainder of the sonication.

    7. To prepare Definity microbubbles, allow the vial to reach room temperature prior to activation. Shake the Definity vial in the Vialmix device for 45 s. Use a blunt-fill 18 G needle with a 1 ml syringe to slowly draw 0.5 ml of microbubbles from the vial. Immediately before sonication, add 0.02 ml of microbubbles to 0.98 ml saline in a 1 ml syringe and gently combine until evenly mixed. Use a blunt-fill 18 G needle to slowly inject 1 ml/kg of the microbubble dilution through the tail vein catheter 10 s after the sonication begins, then inject a 0.15 ml saline flush.

    8. Inject the desired therapeutic agent, followed by a 0.15 ml saline flush through the catheter immediately after the burst that triggers subharmonic emissions so that the first pass of the drug enters the focal volume following BBB permeability enhancement.

    9. Prepare a dilution of Gadovist with 0.1 ml of Gadovist in 0.9 ml of saline in a 1 ml syringe. Inject 1ml/kg of the diluted solution, followed by a 0.15 ml saline flush.

    10. Position the sled into the MRI scanner and acquire images with a T1-weighted sequence (Figure 2D).

    11. Return the mouse to the procedure room to remove the tail vein catheter. Press the puncture site with gauze until the bleeding stops. Depending on the experimental timeline, mice should be sacrificed immediately or recovered from anesthesia.



      Figure 2. Experimental setup of MRIgFUS targeted to the basal forebrain. (A) Timeline of the experimental procedure, including MRI, FUS exposure, and relevant injectables. (B) FUS target spots are selected in the basal forebrain from T2-weighted (T2w) MR images (red dots). (C) T1-weighted (T1w) MR images collected prior to FUS exposure. (D) Enhanced BBB permeability in FUS-targeted areas visualized on contrast-enhanced T1w (CE-T1w) MRI as regions of increased signal intensity (red circles). Scale bars (B-D), 5 mm.

Data analysis

Several analyses can be performed to assess MRIgFUS-induced BBB permeability enhancement and drug delivery in the basal forebrain. Considerations for the distribution and degree of BBB permeability across experimental conditions represent important controls for drug delivery; that is, FUS-mediated BBB modulation itself allows penetration of endogenous factors from the blood, which induce bioeffects that may impact the interpretation of outcome measures associated with drug safety and efficacy. We summarize common analysis methods for BBB permeability following MRIgFUS in this section.


  1. In vivo assessment of BBB modulation by contrast-enhanced MRI. BBB permeability in FUS-targeted foci can be assessed by voxel-wise intensity changes on contrast-enhanced T1-weighted MR images acquired following intravenous injection of a gadolinium-based MRI contrast agent. Regional-based analysis of MRI contrast enhancement following FUS treatment can reveal the degree of increased BBB permeability in FUS-targeted structures and adjacent, non-targeted brain areas. In Figure 3, we present an automated regional-based analysis of enhancement in a sample dataset using a computational pipeline that performs intensity-based and multi-scale image registration to the Allen Mouse Brain Reference Atlas (ARA) (https://miracl.readthedocs.io; Goubran et al., 2019). Due to the limited number of slices in the Z-dimension of the MRI scans, we perform a slice-level registration in lieu of whole-brain 3D mapping (Figure 3A). A manual initialization step is required whereby the corresponding section from the ARA is chosen to limit the target search of the registration algorithm (Figure 3B). Pre-processing steps include bias-field correction using the N4 algorithm (to eliminate inhomogeneities), cropping of the field of view, and skull stripping to extract the brain prior to the registration. The ARA 50 μm resolution template is employed for registration. To deal with the relatively lower resolution of the MRI scans, a lower depth version of Allen atlas labels is created for analysis by grouping labels based on the atlas ontology and hierarchy, creating ‘grand-parent’ labels (Figure 3B). The registration step consists of an intensity-based b-spline, three-stage registration with increasing degrees of freedom of their transformations, encompassing rigid, affine, and non-rigid (deformable) symmetric normalization stages, each consisting of a multi-resolution approach with four levels. We employ the mutual information similarity metric. The net product of the registration is a transformation that performs bidirectional warping of images to and from the MRI native space and to and from the ARA templates and labels (Figure 3C). We rank the regions by the highest voxel-wise maximum intensity (T1-weighted signal) using warped ARA labels in native MRI space (Figure 3D). This analysis quantitatively confirms FUS targeting and enhancement in the basal forebrain structures, as well as some off-target effects in the dorsal striatum with our single element transducer setup.



    Figure 3. Regional analysis of BBB permeability enhancement in the MRIgFUS-targeted basal forebrain. (A) Axial slices of pre-and post-FUS T1-weighted MR scans. (B) Left: Corresponding axial slice of the ARA 50 μm template used for registration; Right: ARA labels with the left hemisphere showing the original labels and the right hemisphere highlighting grouped ‘grand-parent’ labels at a lower depth used for MRI analysis. (C) ARA labels registered to the post-FUS MRI scans and warped to the native MRI space. (D) Mean MRI intensity within brain regions with contrast enhancement, including basal forebrain structures, dorsal striatum, and isocortex. Scale bars (A and B), 5 mm.


  2. Correlation between drug delivery and contrast-enhanced MRI. In Xhima et al. (2020), the increase in signal intensity on contrast-enhanced T1-weighted images following FUS was linearly correlated with the concentration of a small-molecule drug (~600 Da) delivered to the basal forebrain. However, specific drug properties (e.g., molecular weight, hydrophilicity, charge, and plasma half-life) may affect the degree of drug extravasation (Marty et al., 2012); thus, the correlation between contrast agent concentration and drug concentration must be estimated for each therapeutic. Dynamic contrast-enhanced (DCE) imaging, combined with pharmacokinetic modeling, has also been applied to capture the dynamics of BBB permeability and represents another predictive tool for estimating drug delivery following FUS (Park et al., 2017).

  3. Biochemical and/or histological evaluation of BBB permeability enhancement. Changes in BBB permeability can be assessed by examining the extravasation of blood proteins (i.e., albumin, fibrinogen, IgM, and IgG) or exogenous tracers introduced into the bloodstream in FUS-targeted brain tissue. In Xhima et al. (2020), Evans blue dye, which binds to serum albumin (66.5 kDa), was administered intravenously, and labeling in the brain was subsequently evaluated by immunohistochemistry (Figure 4). Please refer to Xhima et al. (2020) for tissue processing, immunohistochemistry, and image acquisition procedures related to the histological analyses in Figure 4. Evans blue dye extravasation can also be measured with an optical imaging assay (Yang et al., 2012). Dyes with a low binding affinity for serum proteins (e.g., dextran amines coupled to biotin or fluorophores) are smaller than endogenous tracers and may reveal more subtle changes in BBB permeability (Marty et al., 2012).



    Figure 4. Histological assessment of MRIgFUS-induced BBB permeability enhancement localized to the basal forebrain. (A-E) Extravasation of Evans blue-bound albumin (red) was detected in brain parenchyma of the FUS-targeted MS/VDB (A) and NBM/HDB (B) but absent in the adjacent brain regions of FUS-treated mice, including the hippocampus (C), cortex (D), and striatum (E) where Evans blue-bound albumin was restricted within blood vessels (white). (F-G) Similarly, in mice that received Evans blue (without FUS) intravenously, Evans blue-bound albumin was confined to the vasculature in the MS/VDB (F) and NBM/HDB (G). Yellow arrows in panels C-G indicate examples of Evans blue-bound albumin co-localized within blood vessels. Choline acetyltransferase (ChAT, green) was used to visualize cholinergic cell bodies. Scale bars (A, B, F, G), 20 μm and (C to E), 100 μm.


  4. Analysis of acoustic emissions data acquired during FUS. Oscillating microbubbles during FUS exert direct forces on the vascular walls that contribute to changes in BBB permeability. The spectral content of acoustic emissions originating from oscillating microbubble in vivo can thus provide insight into the nature of FUS-induced BBB permeabilization. Acoustic emissions during sonication can be assessed to ensure consistent BBB permeability enhancement across test subjects and to control for the bioeffects associated with FUS-induced BBB modulation that may affect outcome measures associated with drug safety and efficacy. In Figure 5, we present a typical analysis of acoustic emissions related to MRIgFUS-mediated delivery of a small molecule, D3, to the basal forebrain. The peak magnitude of 0.5ƒ, ƒ, 1.5ƒ, 2ƒ, and wideband emissions from a single burst during the sonication are presented in Figure 5. Analysis of the exposure-average magnitude of acoustic emissions may also be informative. Hydrophone signals were analyzed as previously described (McMahon et al., 2020). Additionally, subharmonic emissions collected during FUS exposure form the basis of the acoustic feedback control strategy used to calibrate PNP during the sonication. We direct the reader to Xhima et al. (2020) for an example of pressure analyses related to FUS-mediated delivery of compound D3 to the basal forebrain.



    Figure 5. Analysis of acoustic emissions acquired during FUS exposure. (A) For mice, the acoustic emissions feedback control algorithm is set at a starting pressure of 250 kPa and increased incrementally by 25 kPa with each subsequent burst until subharmonic emissions are detected. The applied pressure is then reduced to 25% of the pressure at which subharmonic emissions were detected and set at that level for the remainder of sonication. (B-F) Spectral analysis of mean acoustic emissions in FUS-treated controls (i.e., intravenous saline, MRIgFUS to the basal forebrain) and D3/FUS-treated mice (i.e., intravenous D3, MRIgFUS to the basal forebrain). D3 refers to the small-molecule TrkA agonist delivered with FUS as in Xhima et al., 2020. (G) The peak magnitude of 2ƒ0 emissions across sonication targets exhibited a strong linear correlation with drug delivery in the basal forebrain.


  5. Assessment of central and peripheral inflammation and/or tissue injury following FUS-mediated drug delivery. There is ongoing concern over the neuroinflammatory response and potential tissue damage triggered by BBB permeability enhancement from oscillating microbubbles (Kovacs et al., 2017; McMahon and Hynynen, 2017; McMahon et al., 2020), particularly in the disease state. Histological analysis of necrosis, apoptosis, hemorrhage, and inflammation is warranted after FUS-mediated drug delivery. Further potential peripheral side effects associated with intravenous drug delivery should be investigated as part of the overall drug safety profile.

Notes

  1. For long-term experimental endpoints after FUS-mediated drug delivery, follow institutional animal use protocols, guidelines, and regulations for animal recovery from anesthesia.

  2. FUS, in combination with intravenously injected microbubbles, can be used to deliver a wide range of therapeutic agents, including antibodies, proteins, nanoparticles, viral vectors, and cells, to targeted brain areas (Meng et al., 2020). Biologics can be injected into the bloodstream along with microbubbles or can be encapsulated within or linked to the microbubble shell to enhance delivery (Meng et al., 2020). When a therapeutic agent is administered in combination with FUS, interactions may take place that synergistically or antagonistically modify the effect of the given agent. Thus, for compounds that require repeated dosing, preliminary pharmacological experiments are encouraged, including a dose-response study with at least three dose levels and measurement of pharmacokinetic properties with FUS delivery.

  3. The maximum volume of all intravenous injectables (i.e., Definity, Gadovist, saline, and therapeutic agent) should not exceed 25 ml/kg of body weight during the sonication. To minimize the dosing variability resulting from injection through the catheter hub, the volume to be administered should be 20 μl or greater. Additionally, the solution properties of the therapeutic agent (e.g., tonicity and pH) should also be considered when determining the volume to be administered intravenously.

  4. The most suitable FUS parameters and regimes of acoustic cavitation, tuned to the properties of a given therapeutic agent, require individualized investigation. The FUS parameters and acoustic feedback control algorithm described in this protocol led to extravasation of large serum proteins, including Evans blue-bound albumin (66.5 kDa) and endogenous IgG (150 kDa) and IgM (970 kDa), albeit with different distributions around permeabilized vessels (Xhima et al., 2020).

  5. Monitoring systems to improve safety and to minimize variability in the bioeffects and treatment efficacy of BBB opening within and across test subjects represent a significant advance towards clinical translation. Here, we implement an acoustic feedback control strategy based on subharmonic emissions generated by oscillating microbubbles (O’Reilly and Hynynen, 2012). Alternative strategies for cavitation monitoring and real-time control of BBB modulation with FUS are also under investigation (Sun et al., 2017; Jones et al., 2018).

  6. In previous clinical trials, bilateral delivery of AAV2-NGF vectors to the NBM was achieved via six separate injections with limited therapeutic distribution from the individual sites of injection (Tuszynski et al., 2005 and 2015; Castle et al., 2020). Targeting the NBM with FUS would be a significantly faster procedure with all focal spots permeabilized in a single sonication. Additionally, relative to intracranial injections, increased drug distribution covering the entire basal forebrain can be accomplished using FUS by targeting multiple focal spots in the same sonication scheme.

  7. We use the LP100 (FUS Instruments, Inc.) in this protocol as described in Xhima et al. (2020). The LP100 can be operated outside the MRI room, as we have done here, or fitted in a clinical MRI scanner. The RK300 (FUS Instruments, Inc.) is designed to perform the sonication inside the small-bore MR system. The RK50 (FUS Instruments, Inc.) uses stereotaxic guidance, rather than MR imaging, for target selection.

  8. For safe and effective clinical translation of FUS-related drug delivery applications, it is important to control for the effects of FUS-induced BBB permeability enhancement in the absence of drug delivery as part of the overall experimental design. Secondary bioeffects linked to FUS-induced permeability of the BBB itself remain to be fully understood and are under active investigation. For instance, transient neuroinflammation, Aβ and tau clearance, alterations in neuronal activity, and elevated neurotrophin levels have all been reported following FUS-mediated BBB modulation (Jordão et al., 2013; Burgess et al., 2014; Leinenga and Götz, 2015; Kovacs et al., 2017; McMahon and Hynynen 2017; McMahon et al., 2020; Meng et al., 2020; Xhima et al., 2020). Thus, we recommend including a control group that receives a drug vehicle solution with identical FUS exposure.

  9. BBB dysfunction can occur following a stroke in cases of Alzheimer’s disease and related dementias, amyotrophic lateral sclerosis, and depression. Prior evidence also supports altered blood-brain transport mechanisms in aging (Yang et al., 2020). Additionally, an elevated degree of central and peripheral inflammation is characteristic of many brain disorders, which may further exacerbate BBB dysfunction (Menard et al., 2017; Abdullahi et al., 2018; Sweeney et al., 2018). In the presence of pathological changes to BBB integrity and inflammation, it is important to consider the resolution of the inflammatory and immune response triggered by FUS-mediated BBB modulation itself; this will ensure the tolerability and safety of the FUS treatment, especially in the context of repeated dosing paradigms.

  10. Experimental groups of mice should be balanced for sex and weight. It is important to consider whether there are sex-dependent differences in pathology and drug response. For instance, a 34% difference between the sexes in the number of cholinergic neurons in the NBM but not the MS/VDB and associated behaviors have been reported in preclinical models of AD (Kelley et al., 2014). In humans, sex-specific differences in the basal forebrain cholinergic system and the benefit of cholinergic-targeted agents have been demonstrated (Giacobini and Pepeu, 2018). Potential sex differences in response to FUS-induced BBB permeability remain to be thoroughly investigated. It is possible that the kinetics of BBB modulation may differ between males and females, particularly in disease states, and thus may require revisiting dosing regimens for drug delivery using FUS.

  11. While the atlas registration was performed on a slice-level due to the limited out-of-plane resolution of our current MRI protocol, a 3D whole-brain registration and subsequent analysis could be achieved using an updated imaging acquisition. Our MRI-ARA registration pipelines were optimized for 3D whole-brain registrations and have been validated on scans with a higher resolution in the Z-dimension (Goubran et al., 2019).

Acknowledgments

This protocol was adapted from Xhima et al. (2020). We thank Kristina Mikloska for MRIgFUS expertise and for acquiring photos of the MRIgFUS setup. This work was supported by the Canadian Institutes of Health Research (grants FRN 137064, 166184, 168906 to I.A., FRN 154272 awarded to K.H.), the Canada Research Chairs Program (I. A. Tier 1 Canada Research Chair in Brain Repair and Regeneration), the National Institute of Biomedical Imaging and Bioengineering of the National Institute of Health (RO1-EB003268 awarded to K.H.), and the Temerty Chair in Focused Ultrasound Research at Sunnybrook Health Sciences Centre (K.H.). Additional funding was received from the FDC Foundation, the WB Family Foundation, Gerald and Carla Connor, and the Weston Brain Institute (TR130117 to I.A.). K.X. was awarded a Frederick Banting and Charles Best Canada Graduate Scholarship (GSD 152271).

Competing interests

K.H. is a co-founder of FUS Instruments, a manufacturer of pre-clinical focused ultrasound devices, from which he receives non-study-related financial support. He is also an inventor on several pending and issued patents related to BBB modulation using ultrasound. The other authors declare no competing interests.

Ethics

All animal procedures were approved by the Sunnybrook Research Institute Animal Care Committee and conducted in accordance with the Canadian Council on Animal Care Policies & Guidelines and the Animals for Research Act of Ontario.

References

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简介

[摘要]基底前脑胆碱能神经元 (BFCN) 调节认知处理的回路动力学,包括注意力、记忆力和认知灵活性。在阿尔茨海默病和相关的神经退行性疾病中,BFCN 的退化长期以来一直被认为是认知能力下降的关键因素。因此,胆碱能系统代表了一个关键的治疗目标。开发基于胆碱能的有效疗法的一个长期障碍不仅是生物活性化合物的生产,而且是将药物安全有效地输送到基底前脑的平台。血脑屏障 (BBB) 对向大脑输送药物提出了重大挑战,将大约 98% 的小分子生物制剂和近 100% 的大分子治疗药物排除在脑实质中。当前实现有效药物递送至深部大脑结构(例如基底前脑)的方式特别有限。通过针头或导管直接颅内注射存在与侵入性神经外科手术相关的风险。动脉内注射高渗溶液或治疗剂以使用内源性运输系统穿透 BBB 缺乏区域特异性,这可能并不总是可取的。鼻内、鞘内和脑室内给药限制了脑表面以外的药物分布。在这里,我们提出了一个协议,用于非侵入性地,局部地,使用MRI引导聚焦超声(和瞬时增加血脑屏障通透性MRIgFUS在前脑用于递送治疗剂的靶向胆碱能系统的鼠基础)。临床前模型和临床试验中正在进行的工作支持MRIgFUS介导的 BBB 调制作为治疗衰弱神经疾病的有前途的药物递送方式的安全性和可行性。


[背景]参与认知的皮层和边缘结构中的大多数胆碱能传入来自位于基底前脑的远程投射神经元(Hampel等,2018)。基底前脑胆碱能神经元(BFCNs)是在阿尔茨海默氏病(AD)和其它神经变性疾病,包括血管性痴呆,路易体痴呆,额颞高度脆弱的痴呆,肌萎缩性侧索硬化,帕金森氏病,以及认知能力下降(显著有助于Cykowski等人. ,2014;Grothe等人,2014 年;Hampel等人,2018 年;Ray等人,2018 年;Convery等人,2020 年),证实 BFCNs 是一个重要的治疗靶点。虽然AD的治疗目前临床批准胆碱能制剂仅显示适度的好处s ^认知,最近的研究表明,它们也可能会改变疾病的过程中持续使用(杜波依斯等人,2015年; Cavedo等人在2016年和2017 年)。对认知的适度影响可能反映了可以达到的药物剂量;由于外周副作用,无法耐受更高的剂量,这可能会带来更大的好处(Birks 和 Harvey,2018 年)。其他推定的胆碱能药物,如营养因子和相关基因疗法,具有模拟胆碱能功能和预防胆碱能变性的潜力,不会穿过血脑屏障 (BBB ) (Chen 和 Mobley,2019 年;Xhima和 Aubert,2021 年) )。因此,开发胆碱能靶向药物和脑靶向疗法的主要障碍不仅是生物活性化合物的生产,而且是将它们安全有效地输送到大脑的方法。

大约 98% 的小分子和近 100% 的大分子药物,包括重组蛋白、抗体和基因相关治疗剂,在全身给药时无法穿过 BBB(Pardridge等,2020)。目前实现直接给药的方法涉及高侵入性颅内手术,多次注射到达基底前脑;这些程序具有手术并发症的巨大风险,并且药物分布的控制可能很困难,因为药物浓度从注射部位呈指数下降(Honig等人,2018 年;Castle等人,2020 年;Pardridge等人,2020 年)。其他方法包括:(1)动脉内注射高渗溶液,如甘露醇,以增加 BBB 通透性,并结合外周药物递送;(2)与可穿过 BBB 的蛋白质载体递送系统相关的治疗剂的全身给药;(3)眼内熟食极;(4)鼻内给药。然而,即使使用这些方法以足够的浓度治疗药物到达大脑,药物的生物分布也将是非靶向的,并且可能对大脑中的所有胆碱能突触而不是 BFCN 造成不加区分的作用。

经颅聚焦超声(FUS)与静脉注射微泡(即,由磷脂/蛋白质/聚合物壳稳定的空气/全氟化碳气体核心)组合可以瞬时增强目标体积中的 BBB 渗透性(Hynynen等,2001)。FUS 提供了一种微创途径将各种治疗药物输送到大脑,其优点是能够暂时渗透特定大脑区域的 BBB,而不会在大脑其他地方广泛暴露药物,并有可能减少实现所需的外周剂量大脑中所需的生物效应(Meng等人,2020 年)。此外,FUS 靶向系统可以与MRI 扫描仪的空间坐标共同注册,从而允许在特定神经解剖位置(例如基底前脑)进行高精度 BBB 调制(Xhima等,2020)。由完整 BBB 排除的基于钆的 MRI 造影剂提供了额外的优势,即在 FUS 暴露后确认治疗递送部位。在最近的一项研究中,我们确定了 MRI 引导的 FUS ( MRIgFUS ) 诱导的基底前脑 BBB 调制在n AD小鼠模型中用于药物递送的可行性(Xhima等,2020)。在这里,我们提出了一个详细的协议,用于使用MRIgFUS以非侵入性和局部方式向小鼠基底前脑提供静脉内给药的治疗剂,用于临床前药物开发。

关键字:基底前脑, 内侧隔核, 基底核, 聚焦超声, 血脑屏障, 大脑的药物输送



材料和试剂

1.棉尖涂抹器(AMG Medical Inc.,目录号:018-450)      


2.无纺布海绵(Covidien,目录号:9022)      


3.医用胶带(3M,目录号:1527-1)      


4. 70% 异丙醇垫(Alliance,目录号:211-MM-05507)      


5.循环水暖垫(Stryker,目录号:TP700)       


6. 26 G血管导管(Venisystems Abbocath -T,目录号:G944-A01)      


7.导管注射帽(SAI Infusion Technologies,目录号:IC)      


8. 1 ml胰岛素注射器(BD Bioscie nces ,目录号:BD329654)      


9. 27 G 针(BD Bioscie nces ,目录号:BD305109)      


10. 18 G 钝针(BD Bioscie nces ,目录号:BD305180)   


11.波长Ç利尔ù ltrasound克EL(国家治疗产品公司,目录号:NTPC502X)   


12.盐水袋浸入 37 °C 水浴加热   


13.成年小鼠,20-50 克   


14.感兴趣的治疗剂   


15.无菌生理盐水(Baxter,目录号:2B1307)   


16.无菌肝素盐水(Baxter,目录号:AHB0953U)   


17.脱毛霜(例如,威特或奈尔)   


18.润滑剂眼膏(刷新Lacri -大号UBE ,眼力,目录号:00210889)   


19. Definity微泡(Lantheus Medical Imaging,目录号:DE4)。储存:4°C   


20. Gadovist (拜耳,目录号:02241089)。储存:室温   


21.异氟醚(Fresenius Kabi ,目录号:M60303)   

设备

1.剪刀(Fine Science Tools,目录号:14088-10)      


2.小动物电动修剪器(Wahl Clipper,目录号:58112)      


3.带毯子的强制空气加温装置(Medtronic,目录号:WT 6000)      


4.小动物体重秤      


5. Vialmix机械振动装置(Lantheus Medical Imaging,目录号:VMIX)      


6.连接感应室和鼻锥的小动物麻醉机      


7.将麻醉机连接到位于 FUS 系统和 MR 扫描仪上的动物的塑料管      


8.尾静脉置管用IIA2级生物安全柜      


9.小动物 7T MRI(布鲁克,7T 水平孔Avance BioSpec 70/30 USR 扫描仪)      


10. MRI 体线圈(Bruker,型号:86 mm Quad SN37)   


11. MRI 表面线圈(布鲁克,型号:86 mm Quad Receive)   


12. LP100 聚焦超声系统(FUS Instruments, Inc.)包括:   


1.68 MHz 球面弯曲换能器(0.8 焦数,75 毫米外径,20 毫米内径)
聚偏二氟乙烯或锆钛酸铅水听器
函数发生器
14 位示波器卡
50 W 射频功率放大器
射频功率计
外部匹配电路
所有三个正交轴的电动定位系统
脱气和去离子水箱
MR I兼容雪橇配备麻醉鼻锥和固定装置,固定装置由两个含有脱气和去离子水的 Kapton 聚酰亚胺膜组成
兼容 MR 的寻焦标记
13.氧气供应   


14.医用空气供应   

软件

ParaVision 5.0 (布鲁克)
控制软件玲和监控FUS曝光(FUS仪器公司)
 


程序

从附属机构获得动物使用协议的批准
动物实验必须遵循设施动物护理委员会根据相关政府立法制定的指导方针和政策。

MRI 和 FUS 系统的实验设置和联合配准
1.将FUS系统的实验设置在图1A中被示出为1 D.      


2.安装超声换能器到机动定位系统的臂和沉浸它进入脱气和去离子水缸。      


3.在 FUS 系统的组件之间建立连接(见图 1A)。      


4.将与 MR 兼容的雪橇放置在水浴上方的板上进行空间注册。      


5.将换能器放置在聚酰亚胺薄膜固定装置下方。超声处理采用连续模式在0.5 W和位置在换能器焦点水表面罐中以产生少量的水锥形喷泉。      


6.在换能器焦点处贴上寻焦标记。关闭函数发生器的输出。      


7.将滑橇放入 MRI 扫描仪中,并对寻焦标记执行三轴定位器扫描。使用 FUS Instruments 软件, 输入标记的 MRI 空间坐标, 以将换能器定位系统与成像系统进行配准。在Xhima等人。(2020),一个FLASH tripilot用TE / TR = 3序列毫秒/ 200毫秒,切片厚度= 1毫米,并且在-平面内分辨率= 0.23 × 0.23毫米的执行以采集MR图像。      

 


图 1. FUS 曝光的 LP100 设置。(A) FUS 介导的 BBB 通透性增强系统组件示意图。来自Xhima等人。(2020)。© 作者,保留部分权利,独家被许可人 AAAS。在 CC BY-NC 4.0 许可下分发。(B)侧视和的(CD)的顶视图的实验装置。

小鼠准备
1.辖0.5毫升的生理盐水皮下注射前一天实验和大约45分钟的预超声处理以确保对尾静脉导管放置充分水化。       


2.小鼠必须加热以扩张尾静脉以成功插入导管。在麻醉前在 38 ° C的强制空气加温毯下预热家笼约 30 分钟,并在导管插入期间麻醉时将鼠标保持在温水垫上。       


3.在IIA2级生物安全柜中使用无菌技术进行尾静脉置管。       


将鼠标放在感应室中, 并使用异氟醚 (5%) 和氧气载气 (流速 = 1 升/分钟) 进行麻醉。
当动物被麻醉时,将其从感应室中取出并轻轻安装鼻锥。将异氟醚蒸发器设置为 2%(或有效剂量)。
涂抹润滑眼膏,防止眼睛过度干燥。重-根据需要申请。
用酒精准备垫清洁尾部。
用肝素盐水灌注 26 G血管导管,然后将其插入尾静脉。从导管中取出引导针并固定导管注射帽。
通过导管冲洗肝素盐水, 以确认尾静脉内的正确放置。背压表明放置不当。
通过沿尾部的长轴固定的刚性结构到导管增加结构支撑件(例如,一个狭窄的木制销钉棉-涂药器)用医用胶带。
4.用电动理发器和脱毛膏去除头部背面(从眼线到颈部)的毛发。       


5.用温和的清洁剂和温水清洗脱毛膏。用纱布和酒精棉签擦拭皮肤。       


6.确定小鼠的体重以计算注射量。       

MRIgFUS介导的基底前脑 BBB 通透性
该过程的实验时间表如图 2A 所示。
将超声凝胶应用于 MR 兼容雪橇上的 Kapton 聚酰亚胺窗口,并将麻醉鼠标置于雪橇上的仰卧位置。确保存在用于无阻碍路径的超声波从换能器传播,通过所述聚酰亚胺卡普顿窗口,与鼠脑。使用异氟醚 (1-1.5% 或剂量效应) 将鼻锥连接到连接到麻醉机的塑料管上, 使用医用空气载气 (流速 = 1 L/min)。用医用胶带将头部固定到 MR 兼容的雪橇上,以尽量减少手术过程中的移动。在手术过程中头部的移动会降低 FUS 瞄准的准确性。
覆盖用加热的生理盐水袋鼠标的主体到MAINT AIN在手术期间核心温度(参见图1D)。
将固定在雪橇上的鼠标放入 MRI 扫描仪中,并获取用于目标规划的大脑图像(tripilot 、轴向 T1 加权和轴向 T2 加权序列;参见图2 B 和2 C)。在Xhima等人。(2020),RARE T1加权IM用TE / TR = 10获得年龄毫秒/ 500毫秒,RARE因子= 2,平均值= 3,切片厚度= 1.5mm时,片的数目= 5 ,一个d在-平面内分辨率= 0.25 × 0.25 毫米。用TE得到RARE T2加权图像/ TR = 75毫秒/ 4000毫秒,RARE因子= 10,平均= 4,切片厚度= 1.5mm时,片= 5的数,和在-平面内分辨率= 0.25 × 0.25毫米。
在 FUS 仪器软件中,从 T2 加权扫描中选择基底前脑中的目标点(图 2B)。Broca 的内侧隔膜/垂直对角带 (MS/VDB) 以一个焦点为目标。Broca 基底核/水平带 (NBM/HDB) 的目标是两个焦点。侧脑室用作 MRI 可见的解剖标志,以在适当的轴向 MR 图像上定位内侧焦斑。根据来自小鼠神经解剖图谱的立体坐标定位侧向焦点(Paxinos和 Franklin,2012)。
将雪橇放在 FUS 系统上。使用以下 FUS 参数执行超声处理:1.68 MHz 频率、10毫秒突发长度、1 Hz 脉冲重复频率、120 秒持续时间和1% 占空比。所述p为无源空化检测arameters是20MHz的采样率和800毫伏的输入范围。一种类似于 O'Reilly 和Hynynen (2012)描述的声反馈控制算法,用于根据体内微泡响应校准峰值负压 (PNP) 。简而言之,起始 PNP 设置为 250 kPa(在没有颅骨衰减的水中测量)并每秒递增25 kPa。在超声处理开始后 10 秒内传送 Definity 微泡以收集基线声发射。一旦 0.5ƒ 处的声发射幅度超过每个目标的基线水平的 3.5 倍,超声处理压力将下降 75%,并在超声处理的剩余时间内保持在该水平。
要制备 Definity 微泡,请在激活前让小瓶达到室温。摇动Vialmix设备中的 Definity 小瓶45 秒。使用带有 1 ml注射器的钝填充 18 G 针头从小瓶中缓慢抽取 0.5 ml 微泡。在超声处理之前,将 0.02 毫升微泡加入 1 毫升注射器中的 0.98 毫升生理盐水中,轻轻混合直至混合均匀。超声开始 10 秒后,使用钝填充 18 G 针头通过尾静脉导管缓慢注入 1 ml/kg 的微泡稀释液,然后注入 0.15 ml 生理盐水冲洗。
注入所需的治疗剂,然后通过导管0.15毫升生理盐水冲洗突发之后立即触发分谐波的排放,使得第一通的药物进入以下BBB通透性增强聚焦体积。
在 1 毫升注射器中用0.9 毫升生理盐水中的0.1 毫升Gadovist制备Gadovist的稀释液。注射 1ml/kg 稀释溶液,然后用 0.15ml 生理盐水冲洗。
将雪橇放入 MRI 扫描仪中,并使用 T1 加权序列获取图像(图 2D)。
将鼠标返回手术室以取出尾静脉导管。用纱布按压穿刺部位直至出血停止。根据实验时间表,小鼠应立即处死或从麻醉中恢复。
 

图 2.针对基底前脑的MRIgFUS实验装置。(甲)的时间轴的实验程序,包括MRI,FUS曝光,和相关的注射剂。( B ) FUS 目标点是从 T2 加权 (T2w) MR 图像 (红点) 的基底前脑中选择的。( C )在 FUS 曝光之前收集的 T1 加权 (T1w) MR 图像。( D )在对比增强 T1w (CE-T1w) MRI 上可视化为信号强度增加的区域(红色圆圈)的FUS 靶向区域的 BBB 渗透性增强。比例尺 (BD),5 毫米。

数据分析

可以进行几种分析来评估MRIgFUS诱导的 BBB 通透性增强和基底前脑中的药物递送。对跨实验条件的 BBB 渗透性分布和程度的考虑代表了对药物递送的重要控制;也就是说,FUS 介导的 BBB 调节本身允许内源性因素从血液中渗透,这会引起生物效应,这可能会影响对与药物安全性和有效性相关的结果测量的解释。我们在本节中总结了MRIgFUS后BBB 通透性的常见分析方法。

1.对比增强 MRI 对 BBB 调制的体内评估。FUS 靶向病灶中的 BBB 通透性可以通过对比度的体素强度变化进行评估-在静脉注射基于钆的 MRI 造影剂后获得的增强的 T1 加权 MR 图像。FUS 治疗后 MRI 对比度增强的基于区域的分析可以揭示 FUS 靶向结构和相邻非靶向大脑区域中 BBB 通透性增加的程度。在图 3 中,我们使用计算管道对样本数据集中的增强进行了基于区域的自动分析,该计算管道对 Allen Mou se 脑参考图谱 (ARA) ( https://miracl )执行基于强度和多尺度的图像配准.readthedocs.io;Goubran等人,2019 年)。由于在MRI扫描的Z尺寸切片的有限数量,我们在整个代替的执行条带级登记-脑3D映射(图3A)。需要手动初始化步骤,从而选择来自 ARA 的相应部分来限制注册算法的目标搜索(图 3B)。预处理步骤包括使用N4算法(以消除不均匀性),视场的裁剪偏置场校正,和颅骨汽提以提取前的登记大脑。ARA 50 μm分辨率模板用于配准。为了应对MRI扫描的相对较低的分辨率,艾伦的较低深度版地图集标签由分组基于地图集标签分析创建本体和层次,打造“盛大-父”标签(图3B)。登记步骤包括基于强度的b样条,三阶段登记的与增加其变换的自由度,包含刚性,仿射,以及非刚性(变形的)对称归一化级小号,每个由一个多分辨率的有接近4倍的水平。我们采用互信息相似度度量。注册的净产物是一种变换图像和从MRI机的空间进行双向翘曲和向和从所述ARA模板和标签(图3C)。我们使用原始 MRI 空间中的扭曲 ARA 标签按最高体素最大强度(T1 加权信号)对区域进行排序(图 3D)。该分析定量地证实了基底前脑结构中的 FUS 靶向和增强,以及我们的单晶换能器设置在背侧纹状体中的一些脱靶效应。      

 


图 3. MRIgFUS靶向基底前脑中BBB 通透性增强的区域分析。( A )前后 FUS T1 加权 MR 扫描的轴向切片。( B )左:用于配准的ARA 50 μm模板的相应轴向切片;右:ARA标签与左半球展示了原标签和右半球突出分组“盛大-父母在用于MRI分析较低深度标签。( C ) ARA 标签注册到 FUS 后 MRI 扫描并扭曲到本地 MRI 空间。(d )平均MRI脑部区域内的对比度增强,包括基底前脑结构,背侧纹状体强度,和isocortex 。比例尺(A和B),5 毫米。

2.给药与对比增强MRI之间的相关性。在Xhima等人。(2020),FUS 后对比增强 T1 加权图像上信号强度的增加与递送至基底前脑的小分子药物 (~600 Da) 的浓度呈线性相关。然而,特定的药物特性(例如,分子量、亲水性、电荷和血浆半衰期)可能会影响药物外渗的程度(Marty等,2012);因此,必须针对每种治疗方法估计造影剂浓度和药物浓度之间的相关性。动态对比增强(DCE)成像,与药动学建模相结合,已也被应用到捕获的BBB渗透性的动力学,并且表示用于估计药物递送以下FUS另一个预测工具(公园等人,2017)。       


3. BBB 通透性增强的生化和/或组织学评价。在BBB渗透性变化可以通过检查血液中的蛋白质(外渗进行评估,即,白蛋白,纤维蛋白原,IgM抗体,和IgG)或外源的示踪剂引入FUS靶向脑组织血流。在Xhima等人。(2 020),与血清白蛋白 (66.5 kDa )结合的伊文思蓝染料静脉内给药,随后通过免疫组织化学评估大脑中的标记(图 4)。请参考Xhima等人。(2020)用于组织处理,免疫组化,并与在图的组织学分析的图像采集程序4的Evans蓝染料外渗也可以与光学成像测定来测量(杨等人,2012)。d是与一对血清蛋白低结合亲和力(例如,耦合到生物素或荧光团葡聚糖胺)比内源性示踪剂小,可以揭示BBB通透性更微妙的变化(马丁等人,2012)。      

 

图 4. MRIgFUS诱导的 BBB 通透性增强的组织学评估位于基底前脑。(A -E ) 在 FUS 靶向 MS/VDB (A)和 NBM/HDB (B) 的脑实质中检测到 Evans 蓝结合白蛋白(红色)的外渗,但在 FUS 治疗的小鼠的相邻脑区中不存在,包括海马(C) ,皮质(d) ,和纹状体(E)其中,伊文思蓝-白蛋白结合物的血管(白色)内的限制。(F -G ) 类似地,在静脉内接受伊文思蓝(无 FUS)的小鼠中,伊文思蓝结合白蛋白被限制在 MS/VDB (F)和 NBM/HDB (G)的脉管系统中。CG 中的黄色箭头表示在血管内共定位的Evans 蓝结合白蛋白的例子。胆碱乙酰转移酶(ChAT ,绿色)用于显示胆碱能细胞体。比例尺(A、B、F、G),20 μm和(C 到 E),100 μm 。

4. FUS 期间获得的声发射数据分析。FUS 期间O振荡微泡对血管壁施加直接力,导致 BBB 通透性的变化。从振荡微泡的声发射始发的频谱内容在体内可由此提供的洞察FUS诱导BBB通透性的性质。甲超声处理期间coustic排放可以进行评估,以确保整个测试对象,并用于与FUS诱导BBB调制可能影响药物安全性和有效性相关联的结果的措施相关联的生物效应对照一致BBB通透性增强。在图 5 中,我们展示了与MRIgFUS介导的小分子 D3 传递到基底前脑相关的声发射的典型分析。的0的峰值大小.5ƒ,ƒ,1.5ƒ,2ƒ ,和宽带排放从超声处理过程中的单个脉冲串在声学的曝光-平均幅度的图5.分析呈现排放也可以是信息性。如前所述对水听器信号进行分析(McMahon等,2020)。此外,在 FUS 暴露期间收集的次谐波发射构成了用于在超声期间校准PNP的声反馈控制策略的基础。我们将读者引向 Xhima等人。(2020 年)关于与FUS 介导的化合物 D3 向基底前脑递送相关的压力分析示例。      

 


图 5. FUS 暴露期间获得的声发射分析。(A) 对于小鼠,声发射反馈控制算法设置为 250 kPa 的起始压力,并在每次后续爆发时递增 25 kPa,直到检测到次谐波发射。然后将施加的压力降低到检测到次谐波发射时的压力的 25%,并在超声处理的其余部分设置为该水平。(B - F)在FUS处理的对照的平均声发射的光谱分析(即,静脉内盐水,MRIgFUS到基底前脑)和D3 / FUS处理的小鼠(即,静脉内D3,MRIgFUS到基底前脑)。D3 是指与Xhima等人中的FUS 一起递送的小分子TrkA激动剂。, 2020. (G)超声处理目标的 2ƒ 0排放的峰值幅度与基底前脑中的药物递送表现出很强的线性相关性。

5. FUS 介导的药物递送后中枢和外周炎症和/或组织损伤的评估。人们一直关注由振荡微泡引起的 BBB 通透性增强引发的神经炎症反应和潜在组织损伤(Kovacs等人,2017 年;McMahon 和Hynynen 2017 年;McMahon等人,2020 年),尤其是在疾病状态下。在 FUS 介导的药物递送后,需要对坏死、细胞凋亡、出血和炎症进行组织学分析。与静脉给药相关的其他潜在外周副作用应作为整体药物安全性的一部分进行调查。      

笔记

1.对于FUS介导的药物递送经过长期的实验端点,请按照机构动物使用协议,指导,并从麻醉动物复苏法规。      


2. FUS,与静脉内注射的微泡的组合,可以被用来提供广泛的治疗剂,包括抗体,蛋白质,纳米颗粒,病毒载体,以及细胞,有针对性的脑区域(孟等人,2020)。生物制剂可以与微泡一起注入血流中,也可以将其封装成薄薄的或与微泡壳相连以增强递送(Meng等人,2020 年)。当治疗剂与 FUS 联合给药时,可能会发生相互作用,协同或拮抗地改变给定药物的作用。Ť ħ我们,对于需要重复给药化合物,初步药理实验被鼓励,包括具有至少三个剂量水平和用FUS递送药代动力学性质的测定的剂量-响应研究。      


3.所有静脉内注射的最大体积(即,的Definity ,的Gadovist ,盐水,和治疗剂)不应在超声处理过程中超过25毫升/公斤的体重。为了最大限度地减少通过导管接头注射导致的剂量变化,要给药的体积应为20 μl或更大。此外,在确定静脉内给药的体积时,还应考虑治疗剂的溶液特性(例如,张力和pH)。      


4.最合适的 FUS 参数和声空化机制,根据给定治疗剂的特性进行调整,需要个性化研究。本协议中描述的 FUS 参数和声反馈控制算法导致大血清蛋白外渗,包括埃文斯蓝结合白蛋白 (66.5 kDa ) 和内源性 IgG (150 kDa ) 和 IgM (970 kDa ),尽管在周围分布不同透化血管(Xhima等人,2020 年)。      


5.监测系统以提高安全性并最大限度地减少测试对象内部和之间 BBB 开放的生物效应和治疗功效的变异性,代表了临床转化的重大进步。在这里,我们基于由振荡微泡产生的次谐波发射实施声反馈控制策略(O'Reilly 和Hynynen ,2012)。使用 FUS 进行空化监测和实时控制 BBB 调制的替代策略也在研究中(Sun等人,2017 年;Jones等人,2018 年)。      


6.在之前的临床试验中,AAV2-NGF 载体双侧递送至 NBM 是通过六次单独注射实现的,单个注射部位的治疗分布有限(Tuszynski等人,2005 年和2015 年;Castle等人,2020 年) . 使用 FUS 定位 NBM 将是一个明显更快的过程,所有焦点都在一次超声处理中渗透。此外,相对于颅内注射,使用 FUS 可以通过在相同的超声处理方案中瞄准多个焦点来增加覆盖整个基底前脑的药物分布。      


7.我们在本协议中使用 LP100(FUS Instruments, Inc.),如Xhima等人所述。(2020)。LP100 可以在 MR I房间外操作,就像我们在这里所做的那样,或者安装在临床 MRI 扫描仪中。RK300 (FUS Instruments, Inc.) 设计用于在小口径 MR 系统内执行超声处理。RK50 (FUS Instruments, Inc.) 使用立体定向引导而非 MR 成像进行目标选择。      


8.对于FUS-有关药物递送应用安全和有效的临床平移,但是控制重要对于FUS诱导的BBB通透性增强的在不存在药物递送的影响作为整体的实验设计的一部分。与 FUS 诱导的 BBB 通透性相关的二次生物效应仍有待充分了解并正在积极调查中。例如,短暂神经炎症,Aβ的d tau蛋白的间隙,在神经元活性的改变,和升高的神经营养因子的水平都有报道以下FUS介导的BBB调制(Jordão酒店等人,2013;伯吉斯等人,2014; Leinenga和格茨, 2015;Kovacs等人,2017 年;McMahon 和Hynynen 2017 年;McMahon等人,2020 年;Meng等人,2020 年;Xhima等人,2020 年)。因此,我们建议inclu丁接收具有相同FUS曝光的药物载体溶液对照组。      


9. BBB功能障碍可以发生以下一个在阿尔茨海默氏病和相关的痴呆,肌萎缩性侧索硬化,和抑郁症的情况下,行程。先前的证据也支持衰老过程中血脑转运机制的改变(Yang等,2020)。此外,中枢和外周炎症程度升高是许多脑部疾病的特征,这可能会进一步加剧 BBB 功能障碍(Menard等,2017;Abdullahi等,2018;Sweeney等,2018)。在BBB 完整性和炎症发生病理变化的情况下,重要的是要考虑由 FUS 介导的 BBB 调节本身引发的炎症和免疫反应的解决;这将确保的耐受性和安全性的FUS治疗,尤其是在R的情况下epeated计量范例。      


10.实验组小鼠应平衡性别和体重。重要的是要考虑在病理和药物反应方面是否存在性别依赖性差异。例如,在 AD 的临床前模型中报告了 NBM 中胆碱能神经元数量的性别差异,而不是 MS/VDB 和相关行为(Kelley等,2014)。在人类中,基底前脑胆碱能系统的性别特异性差异和胆碱能靶向药物的益处已得到证实(Giacobini和Pepeu ,2018 年)。对 FUS 诱导的 BBB 渗透性反应的潜在性别差异仍有待彻底研究。男性和女性的 BBB 调节动力学可能不同,特别是在疾病状态下,因此可能需要重新考虑使用 FUS 给药的给药方案。   


11.虽然由于我们当前 MRI 协议的平面外分辨率有限,图集配准是在切片级别上进行的,但可以使用更新的成像采集来实现 3D 全脑配准和后续分析。我们的 MRI-ARA 配准管道针对 3D 全脑配准进行了优化,并已在 Z 维更高分辨率的扫描中得到验证(Goubran等人,2019 年)。   

致谢

该协议是适于从Xhima等。(2020 年)。我们感谢克里斯蒂娜Mikloska为MRIgFUS的专业知识和对获取的照片MRIgFUS设置。这项工作是由健康研究加拿大学院的支持(授予FRN 137064 ,166184,168906 ,以IA,FRN 154272授予KH),加拿大研究主席小号计划(IA一级加拿大研究主席在大脑修复和再生)时,美国国立卫生研究院国家生物医学成像和生物工程研究所(RO1-EB003268 授予 KH),以及Sunnybrook 健康科学中心 (KH) 聚焦超声研究的Temerty主席。FDC 基金会、WB 家庭基金会、Gerald 和 Carla Connor 以及韦斯顿脑研究所(TR130117 至 IA)提供了额外资金。KX 获得了 Frederick Banting 和 Charles Best Canada 研究生奖学金 (GSD 152271)。

利益争夺

KH是FUS仪器,临床前聚焦超声设备制造商,从他接受非研究的创始人之一-相关财务小号upport。他还是多项与使用超声波调制 BBB 相关的待批和已发布专利的发明者。其他作者声明没有竞争利益。

伦理

所有的动物程序经森尼布鲁克研究所动物保护委员会的批准,并按照进行的加拿大动物保护协会政策规定和安大略省的研究行动的动物。

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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Xhima, K., McMahon, D., Ntiri, E., Goubran, M., Hynynen, K. and Aubert, I. (2021). Intravenous and Non-invasive Drug Delivery to the Mouse Basal Forebrain Using MRI-guided Focused Ultrasound. Bio-protocol 11(12): e4056. DOI: 10.21769/BioProtoc.4056.
  2. Xhima, K., Markham-Coultes, K., Nedev, H., Heinen, S., Saragovi, H. U., Hynynen, K., and Aubert, I. (2020). Focused ultrasound delivery of a selective TrkA agonist rescues cholinergic function in a mouse model of Alzheimer's disease. Sci Adv 6(4): eaax6646.
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Dan Predescu
Dan Predescu
Yes, the FUS coupled with microbubbles may open the BBB. However, the main problem remains: Is the opening restricted to the drug(s) administered or to all blood components? If all components pass into the brain, WHAT IS/ARE THE LOCAL REACTION(s) TO THE BLOOD COMPONENTS? Before jumping to conclusions, let put the basics of vascular permeability in the physiological context and not drug delivery without concern of the basics. So, as it is right now, the methodology using FUS with or without microbubbles is, at best: CRUDE and not even fully characterized. So, please do not take it to the humans before understanding it. After all, any published protocol should come after establishing the method that is not fully set apart.
6/23/2021 7:58:52 AM 回复