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. 2016 Apr 14;11(4):e0152898.
doi: 10.1371/journal.pone.0152898. eCollection 2016.

Sonothrombolysis with BR38 Microbubbles Improves Microvascular Patency in a Rat Model of Stroke

Nadine Schleicher  1   2   3 , Amelia J Tomkins  1   4 , Marian Kampschulte  5 , Jean-Marc Hyvelin  6 , Catherine Botteron  6 , Martin Juenemann  1   2 , Mesut Yeniguen  1   2 , Gabriele A Krombach  5 , Manfred Kaps  2 , Neil J Spratt  4   7 , Tibo Gerriets  1   2   8 , Max Nedelmann  9   10
Affiliations

Sonothrombolysis with BR38 Microbubbles Improves Microvascular Patency in a Rat Model of Stroke

Nadine Schleicher et al. PLoS One. .

Abstract

Background: Early recanalization of large cerebral vessels in ischemic stroke is associated with improved clinical outcome, however persisting hypoperfusion leads to poor clinical recovery despite large vessel recanalization. Limited experimental sonothrombolysis studies have shown that addition of microbubbles during treatment can improve microvascular patency. We aimed to determine the effect of two different microbubble formulations on microvascular patency in a rat stroke model.

Methods: We tested BR38 and SonoVue® microbubble-enhanced sonothrombolysis in Wistar rats submitted to 90-minute filament occlusion of the middle cerebral artery. Rats were randomized to treatment (n = 6/group): control, rt-PA, or rt-PA+3-MHz ultrasound insonation with BR38 or SonoVue® at full or 1/3 dose. Treatment duration was 60 minutes, beginning after withdrawal of the filament, and sacrifice was immediately after treatment. Vascular volumes were evaluated with microcomputed tomography.

Results: Total vascular volume of the ipsilateral hemisphere was reduced in control and rt-PA groups (p<0.05), but was not significantly different from the contralateral hemisphere in all microbubble-treated groups (p>0.1).

Conclusions: Microbubble-enhanced sonothrombolysis improves microvascular patency. This effect is not dose- or microbubble formulation-dependent suggesting a class effect of microbubbles promoting microvascular reopening. This study demonstrates that microbubble-enhanced sonothrombolysis may be a therapeutic strategy for patients with persistent hypoperfusion of the ischemic territory.

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Conflict of interest statement

Competing Interests: JMH and CB are employees of Bracco Suisse SA, part of Bracco Group. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Experimental timeline.
Microbubble enhanced sonothrombolysis was tested in a model of 90 minute MCA occlusion. Regional cerebral blood flow (rCBF) was measured by laser doppler flowmetry to confirm successful occlusion (post-occlusion). Randomization to treatment group occurred post-recanalization and immediately pre-treatment onset. rt-PA was administered every 5 minutes (dashed lines) in all treatment animals. Microbubbles (SonoVue or BR38) were administered at 15 minute intervals (▼). Control and rt-PA treatment groups received equal volumes of saline for rt-PA and microbubbles. Continuous ultrasound was applied for 60 minutes in conjunction with rt-PA and microbubble infusion.
Fig 2
Fig 2. Micro-CT scan for vascular volume.
Representative scans of the 3 mm slice encompassing the MCA territory used for micro-CT analysis from a control (saline treated) (panel A), a BR38 full dose (panel B) and a SonoVue full dose treated rat (panel C). Cortical and striatal regions of interest (ROI) for all animals were analyzed in each hemisphere to quantify vascular volume. Dashed line boxes in panel A have been drawn to demonstrate the ROI chosen for this animal. These scans demonstrate reduced vascular volume in both cortical and striatal regions of the ipsilateral hemisphere of the control rat, while demonstrating normal microvascular perfusion after BR38 or SonoVue microbubble-enhanced sonothrombolysis. Scale bar in each image = 800 μm.
Fig 3
Fig 3. Vascular volume of the ipsilateral hemisphere.
Total vascular volumes (VV) of the ipsilateral hemisphere quantified from micro-CT scans are presented as a percentage of the vascular volume of the contralateral (left) hemisphere. Control and rt-PA ipsilateral total VV were significantly reduced from contralateral total VV. * p<0.05.
Fig 4
Fig 4. Nano-CT scan of cortex.
Nano-CT images illustrating the “no-reflow phenomenon” in an untreated animal after the withdrawal of the MCA filament (A), and reperfusion of the microvasculature after BR38 (full dose) sonothrombolysis. Scale bar in each image = 100 μm
Fig 5
Fig 5. Volume of ischemic lesion.
The volume of acute ischemic changes was measured from 3 mm thick brain sections encompassing the MCA territory. Data indicate a reduction of lesion volume following treatment with BR38 full dose combined with rt-PA and ultrasound. *p = 0.044.
See this image and copyright information in PMC

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