Ultrasound-enhanced thrombolysis with tPA-loaded echogenic liposomes

Background and PurposeCurrently, the only FDA-approved therapy for acute ischemic stroke is the administration of recombinant tissue plasminogen activator (tPA). Echogenic liposomes (ELIP), phospholipid vesicles filled with gas and fluid, can be manufactured to incorporate tPA. Also, transcranial ultrasound-enhanced thrombolysis can increase the recanalization rate in stroke patients. However, there is little data on lytic efficacy of combining ultrasound, echogenic liposomes, and tPA treatment. In this study, we measure the effects of pulsed 120-kHz ultrasound on the lytic efficacy of tPA and tPA-incorporating ELIP (t-ELIP) in an in-vitro human clot model. It is hypothesized that t-ELIP exhibits similar lytic efficacy to that of rt-PA.MethodsBlood was drawn from 22 subjects after IRB approval. Clots were made in 20-µL pipettes, and placed in a water tank for microscopic visualization during ultrasound and drug treatment. Clots were exposed to combinations of [tPA] = 3.15 µg/ml, [t-ELIP] = 3.15 µg/ml, and 120-kHz ultrasound for 30 minutes at 37 °C in human plasma. At least 12 clots were used for each treatment. Clot lysis over time was imaged and clot diameter was measured over time, using previously developed imaging analysis algorithms. The fractional clot loss (FCL), which is the decrease in mean clot width at the end of lytic treatment, was used as a measure of lytic efficacy for the various treatment regimens.ResultsThe fractional clot loss FCL was 31% (95% CI: 26-37%) and 71% (56-86%) for clots exposed to tPA alone or tPA with 120 kHz ultrasound. Similarly, FCL was 48% (31-64%) and 89% (76-100%) for clots exposed to t-ELIP without or with ultrasound.ConclusionsThe lytic efficacy of tPA containing echogenic liposomes is comparable to that of tPA alone. The addition of 120 kHz ultrasound significantly enhanced lytic treatment efficacy for both tPA and t-ELIP. Liposomes loaded with tPA may be a useful adjunct in lytic treatment with tPA.     

Thrombolytic efficacy of tissue plasminogen activator-loaded echogenic liposomes in a rabbit thrombus model

Introduction

Ultrasound (US)-enhanced thrombolytic treatment protocols currently in clinical trials for stroke applications involve systemic administration of tissue plasminogen activator (tPA; Alteplase), which carries a risk of adverse bleeding events. The present study aimed to compare the thrombolytic efficacy of a tPA-loaded echogenic liposome (ELIP) formulation with insonification protocols causing rapid fragmentation or acoustically-driven diffusion.

Materials and Methods

Thrombi were induced in the abdominal aortas of male New Zealand white rabbits (2-3 kg) using thrombin and a sclerosing agent (sodium ricinoleate) after aortic denudation with a balloon catheter. Thrombolytic and cavitation nucleation agents (200 μg of tPA alone, tPA mixed with 50 μg of a microbubble contrast agent, or tPA-loaded ELIP) were bolus- injected proximal to the clot through a catheter introduced into the abdominal aorta from the carotid artery. Clots were exposed to transabdominal color Doppler US (6 MHz) for 30 minutes at a low mechanical index (MI = 0.2) to induce sustained bubble activity (acoustically-driven diffusion), or for 2 minutes at an MI of 0.4 to cause ELIP fragmentation. Degree of recanalization was determined by Doppler flow measurements distal to the clots.

Results

All treatments showed thrombolysis, but tPA-loaded ELIP was the most efficacious regimen. Both US treatment strategies enhanced thrombolytic activity over control conditions.

Conclusions

The thrombolytic efficacy of tPA-loaded ELIP is comparable to other clinically described effective treatment protocols, while offering the advantages of US monitoring and enhanced thrombolysis from a site-specific delivery agent.     

Recombinant tissue-plasminogen activator-induced thrombolysis after cerebral thromboembolism in mice

The effects of intracarotid thrombolysis with recombinant tissue-plasminogen activator (rt-PA) on cerebral laser Doppler flow (LDF) and the degree of ischemic injury, as revealed by triphenyltetrazolium chloride (TTC) staining, were studied 24 h following cerebral thromboembolism in mice. Thromboembolization with fibrin-rich clot material (0.28-μl clot volume) led to an LDF decline to about 20–30% of baseline in untreated mice, which resulted in reproducible infarcts of the middle cerebral artery (MCA) territory (122.8 ± 29.4 mm³). Administration of rt-PA (10 mg/kg) 15 min after clot injection induced a progressive LDF restoration to ≈ 115% of control values after 2 h, and brought about a significant reduction of the infarct volume (62.3 ± 42.4 mm³). Our results indicate that ischemic injury may be significantly attenuated by intracarotid thrombolysis. However, injury is not completely reversed, even if reperfusion is initiated as early as 15 min after embolization. Received: 12 October 1999 / Revised, accepted: 26 November 1999     

超声增强溶栓作用的体外实验研究

目的评估超声的溶栓作用和能否增强尿激酶的溶栓作用。方法采用体外实验方法,先制备血栓,置于37℃流动的缓冲液中,超声照射距离为4cm,脉冲式照射,时间为60、90、120、180min,观察同一频率(800kHz)、不同声强(0.2~0.8W/cm2)的超声和超声加尿激酶(0.17U/ml,0.34U/ml)对血栓失重的影响。所测的数据经统计学处理。结果从血栓失重的结果来看,与对照组相比,0.2W/cm2的超声,180min时的血栓失重,有统计学意义(P<0.05)。随着声强增大,有统计学意义的溶栓时间明显缩短,0.4W/cm2时为90min,而0.6W/cm2和0.8W/cm2时,60min即出现非常显著的差异(P<0.001)。超声(800kHz、0.6W/cm2)加尿激酶的溶栓结果,60min时即有非常显著溶栓作用(P<0.001),血栓重量,从起始的(508±5.51)mg减少至180min的(347.66±3.66)mg,减少了(161±1.85)mg,而单用尿激酶组只减少99.5±5.02mg。两组各个时段所测得的血栓失重相比均有明显差异(P<0.001)。结论本组所用的超声对血栓有显著的溶解作用,超声加尿激酶则有增强溶栓作用。     

Improvement of in vitro thrombolysis employing magnetically-guided microspheres

Significant shortcomings in clinical thrombolysis efficiencies and arterial recanalization rates still exist to date necessitating the development of additional thrombolysis-enhancing technologies. For example, to improve tPA-induced systemic clot lysis several supplementary treatment methods have been proposed, among them ultrasound-enhanced tissue plasminogen activator (tPA) thrombolysis which has already found some clinical applicability. The rationale of this study was to investigate whether biodegradable, magnetic spheres can be a useful adjuvant to currently existing tPA-induced thrombolysis and further enhance clot lysis results. Based on an envisioned, novel thrombolysis technology – magnetically-guided, tPA-loaded nanocarriers with triggered release of the shielded drug at an intravascular target site – we evaluated the lysis efficiencies of magnetically-guided, non-medicated magnetic spheres in various combinations with tPA and ultrasound. When tPA was used in conjunction with magnetic spheres and a magnetic field, the lysis efficiency under static, no-flow conditions improved by 1.7 and 2.7 fold for red and white clots, respectively. In dynamic lysis studies, the addition of ultrasound and magnetically-guided spheres to lytic tPA dosages resulted in both maximum clot lysis efficiency and shortest reperfusion time corresponding to a 2-fold increase in lysis and 7-fold reduction in recanalization time, respectively. Serial microscopic evaluations on histochemical sections reconfirmed that tPA penetration into and fragmentation of the clot increased with escalating exposure time to tPA and magnetic spheres/field. These results delineate the effectiveness of magnetic spheres as an adjuvant to tPA therapy accelerating in vitro lysis efficiencies beyond values found for tPA with and without ultrasound. We demonstrated that the supplementary use of magnetically-guided, non-medicated magnetic spheres significantly enhances in vitro static and dynamic lysis of red and white blood clots.     

Intra-arterial thrombolysis using double devices: mechanicomechanical or chemicomechanical techniques

Objective

To optimize the recanalization of acute cerebral stroke that were not effectively resolved by conventional intraarterial thrombolysis (IAT), we designed a double device technique to allow for rapid and effective reopening. In this article, we describe the feasibility and efficacy of this technique.

Methods

From January 2008 to September 2009, twenty patients with acute cerebral arterial occlusion (middle cerebral artery : n=12; internal carotid artery terminus : n=5; basilar artery : n=3) were treated by the double device technique. This technique was applied when conventional thrombolytic methods using drug, microwires, microcatheters and balloons did not result in recanalization. In the double device technique, two devices are simultaneously placed at the lesion (for example, one microcatheter and one balloon or two microcatheters). Chemicomechanical or mechanicomechanical thrombolysis was performed simultaneously using various combinations of two devices. Recanalization rates, procedural time, complications, and clinical outcomes were analyzed.

Results

The initial median National Institute of Health Stroke Scale (NIHSS) was 16 (range 5-26). The double device technique was applied after conventional IAT methods failed. Recanalization was achieved in 18 patients (90%). Among them, 55% (11 cases) were complete (thrombolysis in cerebral infarction 2B, 3). The median thrombolytic procedural time including the conventional technique was 135±83.7 minutes (range 75-427). Major symptomatic hemorrhages (neurological deterioration ≥4 points in NIHSS) developed in two patients (10%). Good long term outcomes (modified Rankin Scale ≤2 at 90 days) occurred in 25% (n=5) of the cases. Mortality within 90 days developed in two cases (10%).

Conclusion

The double device technique is a feasible and effective technical option for large vessel occlusion refractory to conventional thrombolysis.     

Emergent stent placement following intra-arterial thrombolysis for the treatment of acute basilar artery occlusion

Acute basilar artery occlusion (BAO) is a condition producing high rates of morbidity and mortality. Intravenous thrombolysis or intra-arterial thrombolysis are therapeutic options; however, the clinical outcomes remain poor. The purpose of the present study was to evaluate feasibility, safety, and efficacy of emergency stent placement following intra-arterial thrombolysis for patients with acute BAO. Thirty-six consecutive patients were treated for acute BAO using intra-arterial therapy from September 2004 to October 2009. Nine patients, with a Glasgow Coma Scale (GCS) score ranging from 8 to 12, underwent emergency stent placement following inadequate revascularization after thrombolysis. Neurological status prior to treatment was evaluated using the GCS score. Modified Rankin Scale (mRS) scores at 90 days post-treatment were used to assess functional outcome and we reviewed clinical records for frequency of procedure-related complications. Stents were deployed at the target lesion in all patients. Successful revascularization was achieved in eight of nine (88.9%) patients (residual stenosis <50%). The median GCS score prior to thrombolysis was 9 (range: 6-12) and prior to stent placement was 10 (range: 8-12). Four patients (44.4%) achieved good outcomes as determined by the mRS scale (0-2 at 90 days). Mortality was 33.3% in all procedures with one patient (11.1%) experiencing acute intrastent thrombus formation. No patient developed symptomatic intracerebral hemorrhage. Data from our small case series demonstrates that emergency stent placement following intra-arterial thrombolysis is a feasible treatment for patients with acute BAO and may reduce mortality and prevent re-occlusion of the basilar artery.     

尿激酶静脉溶栓治疗脑梗死的TCD监测

目的 应用经颅多普勒(TCD)监测急性脑梗死静脉溶栓治疗前后的血流变化.方法 对20例急性脑梗死静脉溶栓的病人,在治疗前及溶栓治疗开始进行TCD监测并且持续2h,同时评价溶栓前后的NIHSS评分.结果 不同闭塞部位及不同血流信号级别患者,血管再通率有显著性差异,随血流信号的改善,NIHSS评分下降.结论 TCD在尿激酶静脉溶栓时能了解血管再通情况,其血流改善与患者临床情况的改善密切相关.     

Intra-arterial thrombolysis of a distal internal carotid artery occlusion in an adolescent

Introduction: This article describes the first reported case of an adolescent being treated with intra-arterial urokinase for a distal internal carotid artery occlusion. Methods: A 15-year-old male presenting with an acute ischemic stroke caused by a distal internal carotid artery occlusion was treated with intra-arterial urokinase at 5 hours and 45 minutes after symptom onset. Results: The artery completely recanalized, and the patient improved significantly from an admission National Institutes of Health Stroke Scale (NIHSS) score of 28 to a NIHSS score of 8 at a 2.5-month follow-up, despite an asymptomatic intraparenchymal hemorrhage. Conclusion: This article reviews the only two reported cases of intravenous thrombolysis and three cases of intra-arterial thrombolysis in children with ischemic stroke and suggests that thrombolytic therapy should be considered a treatment option in selected pediatric patients with stroke, especially in adolescents who are generally treated as young adults.     

Successful intravenous thrombolysis for ischemic stroke as a complication of coronary intervention in patients with ticagrelor pretreatment

Ticagrelor is an antiplatelet agent used for treatment of coronary artery disease via inhibition of the P2Y12 receptor. Based on limited literature the safety of intravenous thrombolysis for ischemic stroke in patients with ticagrelor pretreatment is unknown. We present two patients established on ticagrelor treated with intravenous thrombolysis for acute ischemic stroke complicating coronary intervention.     

急性脑梗死（6h以内）静脉溶栓治疗

目的 评价尿激酶对急性脑梗死静脉溶栓的疗效及安全性。方法 凡符合人组标准的患者接受尿激酶50万U-150万U溶于生理盐水100-200ml内,30min内滴守,随后静脉滴注低分子右旋醣酐500ml每日1次共10d,溶栓后24h加用口服水溶性阿司匹林300mg每日1次共10d,然后改为100mg每日1次共80d,神经功能缺损评价采用欧洲卒中量表（ESS）。并记录在下列时间点;溶栓前,溶栓后2h,24h,3d,7d,14d,30d和90d。结果 符合入选标准者共409例,尿激酶平均用量为131万U,溶栓后ESS分值增加迅速,溶栓后24h有87.53%的患者ESS分值增加≥10分,溶栓后90d有46.6%的患者ESS分值达到≥95分,本组发生非症状性脑出血共19例（4.64%),发生症状性脑出血16例（3.91%),死亡率为12.22%(50/409),其中6.35%(26/409)死于大面积脑梗死,1.90%(8/409)死于脑实质内出血。结论 提示尿激酶静脉溶栓治疗急性脑梗死（6h以内）是有效的,如严格掌握时间窗及适应证,该疗法相对比较安全,并为第二阶段多中心,随机、双盲、安慰剂对照试验打下基础。     

Recruitment to trials of late thrombolysis: Lessons from the EXTEND study

To increase the percentage of acute stroke patients benefiting from thrombolysis, the utility of expanding the time window of treatment beyond 4.5 hours after stroke onset needs to be investigated. We aimed to identify the target population and the challenges of recruitment of patients for the time window beyond 4.5 hours. Extending the time for Thrombolysis in Emergency Neurological Deficits (EXTEND), a multicentre randomised controlled trial testing the efficacy of thrombolytic therapy in patients with clinically significant ischaemic penumbra between 4.5 to 9 hours after stroke onset, was used as a model to evaluate inclusion and exclusion criteria for late thrombolysis trials. Data from all stroke patients admitted to Austin Health over a 1 year period were retrospectively analysed. Case notes were examined to determine potential trial eligibility. Of 556 patients assessed, 95 (17%) presented during the EXTEND time window. Sixty-seven of these (70.5%) were wake-up strokes (WUS) and 28 (29.5%) arrived between 4.5 and 9 hours after symptoms onset. At least one exclusion criterion was found for 78 (82%) of them. Hence, 17 (3%) patients arrived within an appropriate time frame for the study without any exclusion criteria. Most of these (13) arrived outside routine MRI hours. The number of patients recruited would have increased more than three-fold if imaging had been available 24 hours, 7 days a week. A significant proportion (17%) of ischaemic stroke patients presented between 4.5 and 9 hours after stroke onset. The majority of these were WUS. The major challenge identified for patient recruitment was imaging availability.     

Saving the ischemic penumbra: Endovascular thrombolysis versus medical treatment

Endovascular thrombolysis may allow rapid arterial recanalization in patients with acute ischemic stroke. We present the first study to our knowledge comparing the ischemic penumbra saved with endovascular versus medical therapy. A retrospective review of 21 patients undergoing endovascular intervention for stroke from 2010 to 2011 was contrasted with 21 consecutive patients treated with antiplatelet agents alone. Immediate computed tomography perfusion (CTP) scan of the head and neck was obtained in all patients. Patients with lacunar and posterior circulation infarcts, and those who were medically unstable for MRI post-operatively were excluded. CTP and MRI underwent volumetric calculation. CTP penumbra was correlated with diffusion restriction volumes on MRI, and an assessment was made on the volume of ischemic burden saved with either endovascular treatment or antiplatelet agents. The median age was 70 years (interquartile range 62–80). Median National Institutes of Health Stroke Scale score was 18 and 14 in the control and endovascular groups, respectively. Intravenous tissue plasminogen activator was administered in 22 of 42 patients (52%). Median penumbra calculated was 32,808 mm³ in the control group and 46,255 mm³ in the endovascular group. Median penumbra spared was 9550 mm³ (4980–18,811) in the control group versus 38,155 mm³ in the endovascular group (p = 0.0001). Endovascular thrombolysis may be more efficient than medical therapy alone in saving ischemic penumbra. Future advances in recanalization techniques will further improve the efficacy of endovascular therapy.     

动脉内溶栓联合支架治疗基底动脉闭塞临床报道

目的探讨溶栓后即刻置入支架的方法治疗急性椎基底动脉系统卒中溶栓术后血管重新闭塞的有效性和安全性。方法回顾性分析北京宣武医院2003年7月-2004年12月采用动脉内溶栓加支架置入治疗的7例脑卒中患者的临床资料，采用尿激酶超选择动脉溶栓，溶栓后对血管狭窄行支架成形术。结果7例溶栓后均再通。基底动脉尾段狭窄1例，主干狭窄4例，头段狭窄2例．动脉狭窄率平均为85％．即刻置入冠脉支架。置入支架后造影显示血管形态良好．残留狭窄率小于20％。术后复查点片状脑出血2例。术后症状好转或消失6例，围手术期死亡1例。结论超选择动脉溶栓联合支架治疗能够防止血管再闭塞及卒中复发，改善病人预后。     

溶栓联合神经保护剂对大鼠脑梗死的保护作用

目的探讨高危儿早期干预降低脑性瘫痪发生率的效果。方法高危儿120例分为早期干预组(60例)和常规组(60例)。干预组除接受常规育儿指导外,还进行综合的康复训练。常规组仅接受常规的育儿指导。结果1岁时脑性瘫痪发生率干预组为3.33%(2/60),常规组为16.67%(10/60),2组比较差异有统计学意义(P<0.05)。结论高危儿早期干预可降低脑瘫的发生率。     

Gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain

Drug delivery to brain tumors has long posed a major challenge. Convection-enhanced delivery (CED) has been developed as a drug delivery strategy to overcome this difficulty. Ideally, direct visualization of the tissue distribution of drugs infused by CED would assure successful delivery of therapeutic agents to the brain tumor while minimizing exposure of the normal brain. We previously developed a magnetic resonance imaging (MRI)-based method to visualize the distribution of liposomal agents after CED in rodent brains. In the present study, CED of liposomes was further examined in the non-human primate brain (n = 6). Liposomes containing Gadoteridol, DiI-DS, and rhodamine were infused in corona radiata, putamen nucleus, and brain stem. Volume of distribution was analyzed for all delivery locations by histology and MR imaging. Real-time MRI monitoring of liposomes containing gadolinium allowed direct visualization of a robust distribution. MRI of liposomal gadolinium was highly accurate at determining tissue distribution, as confirmed by comparison with histological results from concomitant administration of fluorescent liposomes. Linear correlation for liposomal infusions between infusion volume and distribution volume was established in all targeted locations. We conclude that an integrated strategy combining liposome/nanoparticle technology, CED, and MRI may provide new opportunities for the treatment of brain tumors. Our ability to directly monitor and to control local delivery of liposomal drugs will most likely result in greater clinical efficacy when using CED in management of patients.     

An onion variety has natural antithrombotic effect as assessed by thrombosis/thrombolysis models in rodents

INTRODUCTION: Prevention of arterial thrombotic diseases has a high priority in developed countries. As inappropriate diet has been shown to be an important risk factor for thrombotic events, regular antithrombotic diet may offer a convenient and effective way of prevention. The aim of the present study was to test onion extracts for antithrombotic effect and to identify the effective varieties in Allium cepa. MATERIALS AND METHODS: A shear-induced platelet function test (haemostatometry) was used to screen for antithrombotic potential. Onion extracts showing significant antithrombotic activity in vitro were further assessed in vivo by using a laser-induced thrombosis test in mice. RESULTS AND CONCLUSIONS: An onion variety, Toyohira, showed significant antithrombotic activity both in vitro and in vivo. Toyohira showed thrombolytic activity in addition to the antiplatelet effect. Superkitamomiji, 2935A, and K83211 showed only thrombolytic activity. Quercetin, the richest flavonoid in onion, was measured, but no correlation was found between quercetin content and antithrombotic activity. It is concluded that onion A. cepa can be classified into varieties with or without antithrombotic and thrombolytic effects. This should be taken into account in future population studies on the antithrombotic effects of vegetables.