In vivo 2PFM monitoring of peripheral immune cell response to FUS+MB BBB treatments

All animal procedures were approved and conducted in compliance with the Animal Care Committee guidelines at Sunnybrook Research Institute, Canada. Male and female EGFP Wistar rats [Wistar-TgN(CAG-GFP)184ys] (310-630 g) (n = 15) were used in this study. Transgenic EGFP rats harbour the EGFP transgene and pCXN2 expression vector containing cytomegarovirus enhancer, chicken β-actin enhancer-promoter, and rabbit β-globin poly(A) signal 22. The GFP gene was designed to be expressed ubiquitously in this rat model, enabling monitoring of many different cell types 23.

Rats underwent cranial window surgeries for acute 2PFM experiments (Figure (Figure1).1). For the creation of cranial windows, rats were anesthetized with isofluorane and a core body temperature of 37°C was maintained using a heating pad with a rectal thermistor (TC-1000; CWE Inc., USA). The rat head was secured in a stereotaxic frame and a cranial window was created in the parietal bone by removing a rectangular (~ 3 × 4 mm) piece of bone using a dental drill. The dura was removed for optical clarity. Agarose gel was used to fill the removed portion of skull and dura for acoustic coupling, and a circular cover slip (5 mm diameter, no. 1 thickness; CS-5R, Warner Instruments, USA) was adhered to the cranial window using cyanoacrylate adhesive. The tail vein was cannulated with a 27 G catheter to enable intravenous injection of fluorescent dextrans and MBs.

2PFM FUS+MB BBB experiments. (A-C) Schematics of animal setup for monitoring FUS+MB BBB treatments using 2PFM (not to scale). (A) General workflow of hardware and animal setup. (B) Cranial window and ring transducer placement on skull. (C) Coronal view of experimental set up. (D) Maximum projection of 2PFM XYZ stack. Vasculature is visible from intravascular injection of fluorescent dextran (grey). FUS+MB induced enhanced BBB permeability is evident from the leakage of dextran into the extravascular space (arrows).

Imaging was performed on an FV1000MPE multiphoton laser scanning microscope (Olympus Corp., Japan) with an InSight DS tunable laser (Spectra-Physics, USA). A 25× water-immersion objective lens (XLPN25XWMP2, NA 1.05, WD 2 mm, Olympus Corp., Japan) was co-aligned with the ring transducer and water-coupled for imaging at lateral and axial resolutions of 0.994 μm/pixel and 2 μm/slice, respectively, and at an imaging speed of 2-8 μs/pixel for a maximum imaging duration of 4 h. Texas Red 70 kDa dextran (dissolved in PBS, 5 mg/kg; Invitrogen, Canada) was injected through a tail vein catheter for visualization of vasculature. EGFP cells and Texas Red dextran were excited at 900 nm. Fluorescent emissions were collected with photomultiplier tubes preceded by the following bandpass filters: 575-645 nm for Texas Red and 495-540 nm for EGFP.

After administration of Texas Red dextran, XYZT image stacks (XY: 0.994 μm/pixel, typically 512 × 512 pixels; Z: 2 μm/slice, 6-10 slices) were acquired to assess baseline immune cell dynamics. Regions-of-interest (ROIs) were centred on blood vessels that showed increased permeability following FUS+MB treatment. Imaging duration ranged from 2-4 h and was limited by increased noise from fluorescent cells reacting to the acute cranial window in the superficial layers of the cortex.

An in-house manufactured lead zirconate titanate (PZT-4) cylindrical transducer (10 mm diameter, 1.5 mm thickness, 1.1 mm height) was coupled to a circular glass coverslip with cyanoacrylate adhesive, matched to a 50 Ω impedance and 0° phase load with a custom matching circuit, and driven at 1 MHz in thickness mode, producing a circular focal spot 1 mm beneath the coverslip 24. The transducer was air-backed with a droplet of degassed deionized water in the centre of the transducer to allow for use with a water-immersion objective lens (Figure (Figure1).1). The transducer was driven by a computer-controlled function generator (Agilent, Palo Alto, USA), amplified with a 53 dB RF power amplifier (NP Technologies Inc., Newbury Park, USA), and transmitted through an in-house power meter and matching circuit prior to reaching the transducer. DefinityTM MB contrast agent (diluted 1:10 v/v in saline, 0.04 ml/kg; Lantheus Medical Imaging, MA) was administered intravenously through the tail vein catheter prior to sonication. Sonications were delivered to one location in 10 ms bursts, 1 Hz PRF, 120 s total sonication duration, with estimated in situ peak pressures of 0.28-0.55 MPa.

Two-photon fluorescence XYZT image stacks were analyzed in MATLAB (Mathworks, USA). The 575-645 nm channel (Texas Red dextran signal) in baseline images was corrected for GFP bleed-through, median filtered by 3 pixels in 3-dimensions, and contrast enhanced. Thresholding was then used to create a binarized vessel mask and vasculature was segmented.

Vessel leakage was assessed by applying the baseline mask to subsequent image stacks. Here, individual vessels with observable leakage were assessed by defining an ROI in 3D, such that the extent of leakage was captured without including adjacent vessels. For each ROI, fluorescence intensity in the intra- and extravascular regions was assessed over time and normalized to compartmental volume and baseline fluorescence, with the assumption that fluorescence intensity is proportional to concentration.

Spatiotemporal leakage was assessed by performing a Euclidean distance transform, creating a distance map from each extravascular pixel to the nearest vascular structure and removing boundary effects by truncating 20 pixels on all sides. Extravascular fluorescence was normalized to compartmental volume at each distance away from the nearest vessel, and to baseline fluorescence.

Vessel diameters were determined by taking the maximum length of the signal profile perpendicular to the local tangent. New vessels were defined whenever vessel branching occurred. Vessels were classified as arterioles, venules, or capillaries based on diameter, branching, and tortuosity patterns 25, 26.

Vascular leakage was classified as 'fast' or 'slow' based on the time it took for extravascular fluorescence intensity to plateau. 'Fast' responses rose within the first 2 min following exposure and plateaued within 10 min. 'Slow' leakage was characterized by reaching peak extravascular fluorescence after longer than 10 min, sometimes with a delay in BBB leakage onset following FUS+MB treatment.

Two-photon fluorescence microscopy image stacks were analyzed with Imaris (Bitplane, Belfast, UK). 3D reconstructions of blood vessels were created using the semi-automated Surface module. In images with high background noise, such as post-treatment images in which the intensity of dextran in the extravascular space was higher than that in the intravascular space, blood vessels were manually contoured. EGFP+ cells were detected using the semi-automated Surface module for XYZT images and Spots module for XYT images. Cell activity was automatically tracked using the Track module and manually corrected. For cell displacement measurements, a minimum of ten cells were tracked. Displacement was calculated as the distance between a tracked cells' first and last position.

To evaluate the proportion of blood vessels covered by fluorescent cells, the volume or area of blood vessels and cells were measured by creating a Surface in their respective channels.

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