Accelerated sonothrombolysis with Definity in a xenographic porcine cerebral thromboembolism model.

Adjuvant ultrasound at 2 MHz with or without an ultrasound contrast agent improves the rate of thrombus resolution by recombinant tissue plasminogen activator (rt-PA) in laboratory and clinical studies. A sub-megahertz approach can further expand this therapy to a subset of patients with an insufficient temporal bone window, improving efficacy in unselected patient populations. The aim of this study was to determine if a clinical ultrasound contrast agent (UCA), Definity, and 220 kHz pulsed ultrasound accelerated rt-PA thrombolysis in a preclinical animal model of vascular occlusion. The effect of Definity and ultrasound on thrombus clearance was first investigated in vitro and subsequently tested in a xenographic porcine cerebral thromboembolism model in vivo. Two different microcatheter designs (end-hole, multi-side-hole) were used to infuse rt-PA and Definity at the proximal edge or directly into clots, respectively. Sonothrombolysis with Definity increased clot mass loss relative to saline or rt-PA alone in vitro, only when rt-PA was administered directly into clots via a multi-side-hole microcatheter. Combined treatment with rt-PA, Definity, and ultrasound in vivo increased the rate of reperfusion up to 45 min faster than clots treated with rt-PA or saline. In this porcine cerebral thromboembolism model employing retracted human clots, 220 kHz ultrasound, in conjunction with Definity increased the probability of early successful reperfusion with rt-PA.

In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke.

The development of adjuvant techniques to improve thrombolytic efficacy is important for advancing ischemic stroke therapy. We characterized octafluoropropane and recombinant tissue plasminogen activator (rt-PA)-loaded echogenic liposomes (OFP t-ELIP) using differential interference and fluorescence microscopy, attenuation spectroscopy, and electrozone sensing. The loading of rt-PA in OFP t-ELIP was assessed using spectrophotometry. Further, it was tested whether the agent shields rt-PA against degradation by plasminogen activator inhibitor-1 (PAI-1). An in vitro system was used to assess whether ultrasound (US) combined with either Definity or OFP t-ELIP enhances rt-PA thrombolysis. Human whole blood clots were mounted in a flow system and visualized using an inverted microscope. The perfusate consisted of either (1) plasma alone, (2) rt-PA, (3) OFP t-ELIP, (4) rt-PA and US, (5) OFP t-ELIP and US, (6) Definity and US, or (7) rt-PA, Definity, and US (n = 16 clots per group). An intermittent US insonation scheme was employed (220 kHz frequency, and 0.44 MPa peak-to-peak pressures) for 30 min. Microscopic imaging revealed that OFP t-ELIP included a variety of structures such as liposomes (with and without gas) and lipid-shelled microbubbles. OFP t-ELIP preserved up to 76% of rt-PA activity in the presence of PAI-1, whereas only 24% activity was preserved for unencapsulated rt-PA. The use of US with rt-PA and Definity enhanced lytic efficacy (p < 0.05) relative to rt-PA alone. US combined with OFP t-ELIP enhanced lysis over OFP t-ELIP alone (p < 0.01). These results demonstrate that ultrasound combined with Definity or OFP t-ELIP can enhance the lytic activity relative to rt-PA or OFP t-ELIP alone, respectively.

Cervicocerebral quantitative arterial tortuosity: a biomarker of arteriopathy in children with intracranial aneurysms.

OBJECTIVE: Although intracranial arterial aneurysms (IAAs) of childhood are usually idiopathic, it is possible that underlying arteriopathy escapes detection when using conventional diagnostic tools. Quantitative arterial tortuosity (QAT) has been studied as a biomarker of arteriopathy. The authors analyzed cervicocerebral QAT in children with idiopathic IAAs to assess the possibility of arteriopathy. METHODS: Cases were identified by text-string searches of imaging reports spanning the period January 1993 through June 2017. QAT of cervicocerebral arterial segments was measured from cross-sectional studies using image-processing software. Other imaging and clinical data were confirmed by retrospective electronic record review. Children with idiopathic IAAs and positive case controls, with congenital arteriopathy differentiated according to aneurysm status (with and without an aneurysm), were compared to each other and to healthy controls without vascular risk factors. RESULTS: Cervicocerebral QAT was measured in 314 children: 24 with idiopathic IAAs, 163 with congenital arteriopathy (including 14 arteriopathic IAAs), and 127 healthy controls. QAT of all vertebrobasilar segments was larger in children with IAAs (idiopathic and arteriopathic forms) (p < 0.05). In children with congenital arteriopathy without an aneurysm, QAT was decreased for the distal cervical vertebral arteries and increased for the supraspinal vertebral artery relative to healthy children. QAT of specific cervicocerebral segments correlated with IAA size and rupture status. CONCLUSIONS: Cervicocerebral QAT is a biomarker of arteriopathy in children with IAA, even in the absence of other disease markers. Additional findings suggest a correlation of cervicocerebral QAT with IAA size and rupture status and with the presence of IAA in children with congenital arteriopathy.

The effect of 220 kHz insonation scheme on rt-PA thrombolytic efficacy in vitro.

Ultrasound-enhanced recombinant tissue plasminogen activator (rt-PA) thrombolysis is under development as an adjuvant to ischemic stroke therapy. The goal of this study was to design a pulsed ultrasound (US) exposure scheme that reduced intracranial constructive interference and tissue heating, and maintained thrombolytic efficacy relative to continuous wave (CW) insonation. Three 220 kHz US schemes were evaluated, two pulsed insonation schemes (15 cycles, 68 µs pulse duration, 33% or 62.5% duty cycle) and an intermittent CW insonation scheme (50 s active, 30 s quiescent) over a 30-min treatment period. An in silico study using a finite-difference model of transcranial US propagation was performed to estimate the intracranial acoustic field and temperature rise in the skull for each insonation scheme. In vitro measurements with flow were performed to assess thrombolysis using time-lapse microscopy. Intracranial constructive interference was not reduced with pulsed US using a pulse length of 15 cycles compared to intermittent CW US. The 33.3% duty cycle pulsed US scheme reduced heating in the temporal bone as much as 60% relative to the intermittent CW scheme. All insonation schemes promoted sustained stable cavitation in vitro and augmented thrombolysis compared to rt-PA alone (p  < 0.05). Ultraharmonic (UH) and harmonic cumulative energy over a 30 min treatment period was significantly higher (p  < 0.05) for the intermittent CW US scheme compared to either pulsed US scheme. Despite the difference in cavitation emissions, no difference was observed in the clot lysis between the three US schemes. These findings demonstrate that a 33.3% duty cycle pulsed US scheme with a 15-cycle burst can reduce bone heating and achieve equivalent thrombolytic efficacy as an intermittent CW scheme.

Effect of Clot Stiffness on Recombinant Tissue Plasminogen Activator Lytic Susceptibility in Vitro.

The lytic recombinant tissue plasminogen activator (rt-PA) is the only drug approved by the Food and Drug Administration for treating ischemic stroke. Less than 40% of patients with large vessel occlusions who are treated with rt-PA have improved blood flow. However, up to 6% of all patients receiving rt-PA develop intracerebral hemorrhage. Predicting the efficacy of rt-PA treatment a priori could help guide therapeutic decision making, such that rt-PA is administered only to those individuals who would benefit from this treatment. Clot composition and structure affect the lytic efficacy of rt-PA and have an impact on elasticity. However, the relationship between clot elasticity and rt-PA lytic susceptibility has not been adequately investigated. The goal of this study was to quantify the relationship between clot elasticity and rt-PA susceptibility in vitro. Human and porcine highly retracted and mildly retracted clots were fabricated in glass pipettes. The rt-PA lytic susceptibility was evaluated in vitro using the percent clot mass loss. The Young's moduli of the clots were estimated using ultrasound-based single-track-location shear wave elasticity imaging. The percent mass loss in mildly retracted porcine and human clots (28.9 ± 6.1% and 45.2 ± 7.1%, respectively) was significantly higher (p < 0.05) than in highly retracted porcine and human clots (10.9 ± 2.1% and 25.5 ± 10.0%, respectively). Furthermore, the Young's moduli of highly retracted porcine and human clots (5.33 ± 0.92 and 3.21 ± 1.97 kPa, respectively) were significantly higher (p < 0.05) than those of mildly retracted porcine and human clots (2.66 ± 0.55 and 0.79 ± 0.21 kPa, respectively). The results revealed an inverse relationship between the percent clot mass loss and Young's modulus. These findings motivate continued investigation of ultrasound-based methods to assess clot stiffness in order to predict rt-PA thrombolytic efficacy.

Genetic Determinants of Cerebral Arterial Adaptation to Flow-loading.

BACKGROUND: In animal models, flow-loading is a necessary and sufficient hemodynamic factor to express the Cerebral Aneurysm (CA) phenotype. Using a rat model, this study characterizes the molecular events that comprise the cerebral arterial response to flow-loading and reveals their significance relating to the CA phenotype. OBJECTIVE: To characterize the molecular events that underlie expansive remodeling of cerebral arteries in two genetically distinct inbred rat strains with differential susceptibility to flow-dependent cerebrovascular pathology. METHODS: Thirty-two rats underwent bilateral common carotid artery ligation (BCL) (n=16) or Sham Surgery (SS) (n=16). Nineteen days later, vertebrobasilar arteries were harvested, histologically examined and analyzed for mRNA and protein expression. Flow-induced changes in histology, mRNA and protein expression were compared between BCL and SS rats. Differences between aneurysm-prone (Long Evans, LE) and resistant (Brown Norway, BN) strains were evaluated. RESULTS: Basilar Artery (BA) medial thickness/luminal diameter ratio was significantly reduced in BCL rats, without significant differences between LE (2.02 fold) and BN (1.94 fold) rats. BCL significantly altered BA expression of mRNA and protein but did not affect blood pressure. Eight genes showed similarly large flow-induced expression changes in LE and BN rats. Twenty-six flow responsive genes showed differences in flow-induced expression between LE and BN rats. The Cthrc1, Gsta3, Tgfb3, Ldha, Myo1d, Ermn, PTHrp, Rgs16 and TRCCP genes showed the strongest flow responsive expression, with the largest difference between LE and BN rats. CONCLUSIONS: Our study reveals specific molecular biological responses involved in flow-induced expansive remodeling of cerebral arteries that may influence differential expression of flowdependent cerebrovascular pathology.

Quantitative Arterial Tortuosity Suggests Arteriopathy in Children With Cryptogenic Stroke.

BACKGROUND AND PURPOSE: Quantitative arterial tortuosity (QAT) is a ratio of vessel length between 2 points to the shortest linear distance between same points. QAT has been reported as an imaging biomarker of arteriopathy in pediatric arterial ischemic stroke (AIS) because of dissection and transient cerebral arteriopathy. We sought to determine whether QAT abnormalities are present in other subtypes of pediatric AIS. METHODS: Children with AIS-absent conventional biomarkers of arteriopathy and case-controls who underwent magnetic resonance angiography were classified by stroke mechanism. The primary study population consisted of cryptogenic AIS cases. AIS with bow hunter physiology and cardiogenic emboli were also evaluated. AIS because of nontraumatic dissection served as positive controls. Patients without vascular risk factors served as negative controls. Segmental QAT of cervicocerebral arteries were measured using automated image processing and differences between groups analyzed. RESULTS: In negative controls, QAT showed significant age-related variability for most arterial segments. Positive controls showed significantly increased QAT of the distal extracranial vertebral arteries (VAs) and decreased QAT of the intracranial VA relative to negative controls. Cryptogenic stroke and bow hunter physiology cases were similar to positive controls showing increased QAT of the distal extracranial VA and decreased QAT of the intracranial VA relative to negative controls. Cardioembolic stroke cases were similar to negative controls showing decreased QAT of the distal extracranial VA and increased QAT of the intracranial VA relative to positive controls. CONCLUSIONS: Pediatric cryptogenic stroke is frequently associated with cervicocerebral arteriopathies expressing altered QAT. QAT may be a diagnostic biomarker of arteriopathy in pediatric AIS.

Hybrid stage I palliation in a 1.1 kg, 28-week preterm neonate with posterior malalignment ventricular septal defect, left ventricular outflow tract obstruction, and coarctation of the aorta.

The hybrid stage I procedure has emerged as a less-invasive alternative to the standard surgical Norwood procedure in the palliation of high-risk hypoplastic left heart syndrome and variants. This approach may also benefit patients requiring complex neonatal repair who have significant anatomic and/or perioperative risk factors that either prevent or complicate durable and robust biventricular circulation from being safely achieved. An extremely low-birth weight (1.1 kg) 28-week gestation preterm neonate with postnatal diagnosis of posterior malalignment ventricular septal defect, severe left ventricular outflow tract obstruction, aortic annular hypoplasia, and aortic arch obstruction underwent initial palliation with a hybrid stage I procedure. In this case, hybrid stage I palliation allowed for both somatic and left heart growth followed by ultimate uncomplicated biventricular repair.