Hyperosmolar blood-brain barrier opening using intra-arterial injection of hyperosmotic mannitol in mice under real-time MRI guidance

Abstract

The blood-brain barrier (BBB) is the main obstacle to the effective delivery of therapeutic agents to the brain, compromising treatment efficacy for a variety of neurological disorders. Intra-arterial (IA) injection of hyperosmotic mannitol has been used to permeabilize the BBB and improve parenchymal entry of therapeutic agents following IA delivery in preclinical and clinical studies. However, the reproducibility of IA BBB manipulation is low and therapeutic outcomes are variable. We demonstrated that this variability could be highly reduced or eliminated when the procedure of osmotic BBB opening is performed under the guidance of interventional MRI. Studies have reported the utility and applicability of this technique in several species. Here we describe a protocol to open the BBB by IA injection of hyperosmotic mannitol under the guidance of MRI in mice. The procedures (from preoperative preparation to postoperative care) can be completed within ~1.5 h, and the skill level required is on par with the induction of middle cerebral artery occlusion in small animals. This MRI-guided BBB opening technique in mice can be utilized to study the biology of the BBB and improve the delivery of various therapeutic agents to the brain.

Fig. 1 |. BBBO under real-time MRI guidance in mice.

a, Schematic illustration of the protocol. b, Procedural steps. The step numbers correspond to the Procedure. BBBO, blood–brain barrier opening; CCA, common carotid artery; ECA, external carotid artery; ICA, internal carotid artery; MCA, middle cerebral artery; MRI, magnetic resonance imaging; OA, occipital artery; PPA, pterygopalatine artery.

Fig. 2 |. Schematic illustration of BBBO under real-time MRI guidance in large animals and humans.

Following anesthesia, a microcatheter is positioned via a transfemoral access for selectively catheterizing the artery in the brain under guidance of x-ray fluoroscopy. MRI contrast agent (Feraheme) is infused via the microcatheter for visualization and optimization of a desired parenchymal perfusion territory and the subsequent BBBO.

Fig. 3 |. Schematic diagram of the catheter assembly.

a, Prepare a 1– 1.5-m-long catheter for MRI. The distal end is cut with a 45° angle; the proximal end is connected with a 30 gauge half-inch needle. b, Apply a droplet of Krazy Glue to the distal end of the catheter. c, Place the catheter, suspended on a clean trestle, and allow it to dry for 12 h. The bead displays an almost round shape. d, The portion between the tip and bead is ~5 mm long.

Fig. 4 |. Schematic diagram of the preoperative preparation of the mouse.

The mouse is placed in a chamber with 4% isoflurane to induce anesthesia, and 1.5–2% isoflurane is used to maintain the anesthesia. The hair in the operative area is removed using depilation cream (Nair). 0.5% iodophor and 70% ethanol are subsequently applied for disinfection of the operative area.

Fig. 5 |. Surgical procedures before catheter insertion.

a, Skin cut along the midline of the neck (Step 14). b, Dissection of superficial adipose tissue and the sternohyoid muscles to expose trachea (Step 15). c, Isolation of CCA (Step 16). d, Isolation of ECA (Step 17). e, Temporary ligation of ECA and exposure of OA and ICA (Step 17). f, The anatomical position of the arteries. g, Isolation of OA and subsequent cauterization (Step 17). h, Isolation of PPA (Step 18). i, Temporary ligation of PPA (Step 18).The IACUC at the Johns Hopkins University approved all experimental procedures shown in this figure.

Fig. 6 |. Schematic diagram of catheter cannulation.

Before arteriotomy and catheter insertion (lefthand picture), the proximal CCA is permanently ligated with a suture to block the blood flow and another suture around the distal CCA is temporarily ligated. A loose suture is then placed in between. Once the catheter is inserted (middle picture), the loose suture is tightened to secure the catheter, and at that point the temporary ligation is released. When the catheter is advanced into ICA (righthand picture), the suture released previously is tightened around the catheter to provide better stability. Finally, the suture around the proximal CCA is used to again tie the catheter. In total, the catheter is held by three sutures.

Fig. 7 |. Mouse MRI setup.

The mouse with a secured catheter connected to a syringe is transferred to a MRI scanner room and placed in a small MRI animal bed in a prone position. The syringe is connected to the MRI-compatible pump, which can be controlled outside the MRI scanner room. On the console, the images are acquired using a computer with installed Bruker ParaVision 6.0.1 system and the respiration is monitored.

Fig. 8 |. Use of real-time MRI to ensure an effective infusion rate via IA injection to predict perfusion territory in a mouse brain.

a, Injecting an MRI contrast (SPIO) at 0.15 ml/min results in cerebral perfusion, as marked by a decrease in pixel intensity on T2*-weighted scans (red square indicates region of interest (ROI), quantified in b), with no distinct difference in pixel intensity in untreated hemisphere as a baseline (blue square indicates ROI, quantified in b). b, Signal intensity is normalized by setting the maximal value of ROIs as 1. Start represents the beginning of IA infusion. Stop represents the end of the infusion. The IACUC at the Johns Hopkins University approved all experimental procedures shown in this figure. Figure adapted with permission from ref. .

Fig. 9 |. Variability of cortical involvement during IA infusion of a contrast agent in the mouse brain.

a,b, Representative T2* images during injection of a contrast agent (Gd) at a rate of 0.15 ml/min in which the cortex was (a) or was not (b) perfused as outlined by the red box indicating ROI. c, The constituent ratio of cortical involvement (% of mice this was seen in, n = 26 mice). ‘Cortex⁺’ represents contrast perfusion in the cortex, and ‘Cortex⁻’ represents the lack of perfusion. The IACUC at the Johns Hopkins University approved all experimental procedures. Figure adapted with permission from ref. .

Fig. 10 |. Prediction of mannitol-induced BBBO territory.

a, Signal change map after contrast perfusion. b, Histogram analysis of pixel intensities in a, showing two Gaussian distributions (red lines). The blue arrow indicates the point where a cutoff of −53.9% was used to separate the two distributions. c, Segmented map shows the area where the relative signal change was smaller than −53.9%. d–f, Signal change map (d); histogram analysis (e); and segmented map (f; △S% > 31.4) at 5 min after i.p. injection of Gd. g,h, Bar graph (g) and correlation analysis (h) of the BBBO territory predicted by the contrast perfusion and that assessed using Gd (n = 8). Data shown as mean ± SD were compared by paired two-tailed t-test. The MRI images were processed by custom-written scripts in MATLAB (Supplementary Data 1 and Supplementary Methods). Figure adapted with permission from refs. ^,.

Fig. 11 |. MRI and histological assessment post-BBBO.

a, T2 and T2* images 3 and 7 d after BBBO showing no indication of brain damage. No Gd enhancement on T1-weighted images was observed in the brain, suggesting that the BBB was resealed. b, Fluorescent staining of the BBBO region and corresponding contralateral region with IBA-1, GFAP and NeuN. Scale bar 50 μm. c, Quantification of histological assessments showing comparable cell density for IBA1 (n = 8), GFAP (n = 8) and NeuN (n = 5) immunostaining between the ipsilateral and the contralateral hemisphere, indicating no inflammation and no neuronal loss after BBBO. Data shown as mean ± SD were compared by paired two-tailed t-test. Antibodies: GFAP (1:250, Dako), IBA-1 (1:250, Wako), NueN (1:100, Cell Signaling Technology). The detailed immunostaining protocol and analysis can be found in Supplementary Methods. The IACUC at the Johns Hopkins University approved all experimental procedures shown in the figure. Figure adapted with permission from refs. ^,.