Combined contrast-enhanced ultrasound and rt-PA treatment is safe and improves impaired microcirculation after reperfusion of middle cerebral artery occlusion

Abstract

In monitoring of recanalization and in sonothrombolysis, contrast-enhanced ultrasound (CEUS) is applied in extended time protocols. As extended use may increase the probability of unwanted effects, careful safety evaluation is required. We investigated the safety profile and beneficial effects of CEUS in a reperfusion model. Wistar rats were subjected to filament occlusion of the right middle cerebral artery (MCA). Reperfusion was established after 90 minutes, followed by recombinant tissue-type plasminogen activator (rt-PA) treatment and randomization to additional CEUS (contrast agent: SonoVue; 60 minutes). Blinded outcome evaluation consisted of magnetic resonance imaging (MRI), neurologic assessment, and histology and, in separate experiments, quantitative 3D nano-computed tomography (CT) angiography (900 nm(3) voxel size). Nano-CT revealed severely compromised microcirculation in untreated animals after MCA reperfusion. The rt-PA partially improved hemispheric perfusion. Impairment was completely reversed in animals receiving rt-PA and CEUS. This combination was more effective than treatment with either CEUS without rt-PA or rt-PA and ultrasound or ultrasound alone. In MRI experiments, CEUS and rt-PA treatment resulted in a significantly reduced ischemic lesion volume and edema formation. No unwanted effects were detected on MRI, histology, and intracranial temperature assessment. This study shows that CEUS and rt-PA is safe in the situation of reperfusion and displays beneficial effects on the level of the microvasculature.

Figure 1

Ultrasound setup. Distance from probe surface to skull was 4 cm, the sample volume placed in midbrain, resulting in a penetration depth of 5 cm. Continuous arrows denote reflection at rat skull and dotted arrows denote lateral boundary of skull.

Figure 2

Maximum-intensity projection (MIP) using micro- and nano-computed tomography (CT) in exemplary animals. (A–D) (Upper row) Axial view on micro-CT scans. For nano-CT analysis, regions of interest (boxes) were cut and rescanned. MIPs from nano-CT in coronal view are shown in (a1/2–d1/2): preparation directly after reperfusion (A, a1/2); 60 minutes after reperfusion (B, b1/2); after recombinant tissue-type plasminogen activator (rt-PA) (C, c1/2), and after rt-PA and contrast-enhanced ultrasound (CEUS) (D, d1/2). The nonoccluded left hemisphere is presented from a1 to d1. Persisting impairment of microcirculation (a2/b2) is partially improved after rt-PA treatment (c2) and completely reversed with rt-PA and CEUS (d2).

Figure 3

Maximum-intensity projection (MIP) using micro-computed tomography (CT) (A–C) and nano-CT (a1/2–c1/2) in exemplary animals after treatment with ultrasound alone (A, a1/2), contrast-enhanced ultrasound (CEUS) without recombinant tissue-type plasminogen activator (rt-PA) (B, b1/2), and rt-PA with ultrasound (without contrast enhancer) (C, c1/2). The nonoccluded hemisphere is presented from a1 to c1. The figure illustrates persisting impairment of microcirculation after ultrasound treatment (a2) and partial improvement after CEUS treatment and treatment with rt-PA and ultrasound (b2/c2).

Figure 4

Nano-computed tomography quantification. (A) Significant differences in the total vascular volume fraction between the right and left hemisphere are found in animals after reperfusion and animals treated with recombinant tissue-type plasminogen activator (rt-PA). No significant differences are present after treatment with rt-PA and contrast-enhanced ultrasound (CEUS). (B) No treatment effects were seen in animals treated with ultrasound alone. The CEUS without rt-PA and ultrasound with rt-PA (without contrast enhancer) showed partial improvement inferior to the effects seen after treatment with rt-PA and CEUS.

Figure 5

Hemorrhagic transformation was found in one animal treated with contrast-enhanced ultrasound (CEUS) and recombinant tissue-type plasminogen activator (rt-PA). (A) Hematoxylin and eosin staining of hemorrhage (arrow). Magnification of hemorrhage is shown in the upper right corner (frame). (B) Corresponding T2^* magnetic resonance imaging of hemorrhage (circle). The bottom pictures show examples of ischemic demarcation after treatment with CEUS and rt-PA (C) and after treatment with rt-PA (D). V, ventricle.

Figure 6

Intracerebral temperature changes during 1 hour insonation with contrast-enhanced transcranial color-coded duplex sonography. All animals additionally received recombinant tissue-type plasminogen activator. The temperature probe was placed into the territory of the right reperfused middle cerebral artery (A, n=6) and in the nonischemic contralateral hemisphere (B, n=6). Maximum temperature increase of single values was 0.4°C in (A) and 0.9°C in (B).