Summary

脑回路非侵入性断开的靶向神经元损伤

Published: September 27, 2020
doi:

Summary

该协议的目标是提供一种在大脑中产生非侵入性神经元病变的方法。该方法利用磁共振引导聚焦超声(MRgFUS)以瞬态和焦焦的方式打开血脑屏障,为脑帕伦奇瘤提供循环神经毒素。

Abstract

手术干预可以相当有效地治疗某些类型的医学上难以解决的神经系统疾病。这种方法对于可识别神经元回路发挥关键作用的疾病特别有用,例如癫痫和运动障碍。目前可用的手术方式虽然有效,但通常涉及侵入性外科手术,这可能导致非目标组织的手术损伤。因此,扩大手术方法的范围,包括非侵入性和神经毒性技术,将是有价值的。

在这里,提出了一种方法,以非侵入性的方式产生大脑中的焦点,神经元病变。这种方法利用低强度聚焦超声和静脉注射微泡来暂时和聚焦地打开血脑屏障(BBB)。然后利用瞬态 BBB 开放期,将系统管理的神经毒素定向到目标大脑区域。神经毒素奎诺林酸 (QA) 通常是 BBB 不透水, 在内向或静脉注射时耐受性良好。然而,当QA获得直接访问脑组织,它是有毒的神经元。这种方法已被用于大鼠和小鼠的特定大脑区域。在 MRgFUS 之后,使用对比度增强型 T1 加权成像确认 BBB 的成功打开。程序后,T2成像显示损伤仅限于大脑的目标区域,并且利用组织学技术在死后可以确认目标区域神经元的丧失。值得注意的是,注射盐水而不是QA的动物确实表现出BBB的开放性,但点不表现出损伤或神经元损失。这种方法,即精确脑内非侵入性导导手术(PING),可为治疗神经回路障碍相关的神经系统疾病提供非侵入性方法。

Introduction

这种方法的目的是提供一种在大脑目标区域产生非侵入性神经元病变的方法。开发这种方法的理由是断开导致神经紊乱的神经元回路。例如,手术可以相当有效地治疗某些医学上难以解决的神经紊乱,如耐药癫痫(DRE)1。然而,每种可用的手术方式在对大脑产生不良的附带损害方面都有局限性。传统的切除手术可能具有高度侵入性,有出血、感染、血栓、中风、癫痫发作、脑肿胀和神经损伤的风险。微创或非侵入性手术的替代方案包括激光间散热治疗和放射外科,这也证明在抑制DRE癫痫发作方面是有效的。最近,高强度聚焦超声(HIFU)产生的热病变在减少癫痫发作方面显示出了希望。HIFU 是非侵入性的;然而,其治疗窗口目前仅限于大脑的更中心区域,因为头骨附近的非目标组织有热损伤的风险。尽管有这些限制,手术的好处往往大于潜在的风险。例如,虽然DRE手术可以产生附带的脑损伤,但它在抑制癫痫发作和提高生活质量方面通常优先于手术风险。

本文描述的方法,精确脑内非侵入性导导手术(PING),是为了断开神经回路,同时限制附带的脑损伤而开发的。该方法利用低强度聚焦超声与静脉注射微泡相结合打开BBB,以传递神经毒素。,这种方法不会产生热病变大脑3,4,5,6,7,4,5,6和BBB开放期,可以利用,以提供BBB不渗透化合物到大脑帕伦奇玛。37BBB 的开口是瞬态的,可以使用磁共振成像制导以有针对性的方式生产。在我们的研究中,BBB开放期已被利用来传递循环神经毒素到大鼠和小鼠脑帕伦奇玛的目标区域,8,9。奎诺林酸是一种神经毒素,在静脉注射10、静脉内10或内腹酮,8、9、11时耐受性良好8QA毒性的缺乏是由于其不良的BBB渗透性,据报道,这是微不足道的10。相比之下,直接注射QA到脑帕伦奇玛产生神经元病变,使相邻的轴子12,13。12,因此,当循环QA获得进入BBB开放靶区的大脑帕伦奇马时,神经元死亡产生8,8,9。因此,目前的方法以精确针对性和非侵入性的方式产生焦点神经元损失。

Protocol

此处描述的所有方法都已获得弗吉尼亚大学动物护理和使用委员会的批准。 1. 试剂制备 手术当天,准备6.0 mL的可注射奎诺林酸(QA)。溶解 450 毫克 QA 在 4.0 mL 的 1.0 N NaOH.加入 0.6 mL 的 10x PBS,pH 到 7.4,并通过 0.22 μm 注射器过滤器将最终体积达到 6.0 mL,并带 dH2O. 过滤器。溶液在4°C下稳定2周。 通过探针从十氟丁烷气体中探出声子,在普通盐水中准备微…

Representative Results

本节介绍 PING 对位于新皮质发育不良的神经元的影响。组织发育不良是耐药癫痫患者大脑中的一个常见特征,手术切除癫痫原生障碍可以很好的控制癫痫发作。因此,定义 PING 对再生性脑组织的影响是一个重要的优先事项。遗传皮质发育不良的老鼠模型,蒂什大鼠,被选择研究这个问题,因为蒂什大脑表现出发育不良组织(下皮质异视)位于一个正常定位的新皮质(<strong class="x…

Discussion

PING 方法旨在产生非侵入性、靶向性神经元病变。,该方法源于聚焦超声,,3、4、5、6、7领域研究基础6的牢固和日益增长35通过BBB的短暂开放,提供对脑帕伦奇马特定区域的聚焦访问能力,为提供各种通常无法进入大脑的制剂创造了一条途径。这一机会在很大程度上是为中央?…

Declarações

The authors have nothing to disclose.

Acknowledgements

作者认可雷内·杰克·罗伊在核磁共振成像领域提供的出色技术支持。这项工作得到了国家卫生研究院(R01 NS102194至KSL和R01 CA217953-01至MW)、切斯特基金(KSL)和聚焦超声基金会(KSL和JW)的支持。

Materials

7T-ClinScan MRI System Bruker Biospin, Ettinglen, Germany MR Image Acquisition
Acoustic Gel Litho CLEAR 11-601 High Viscosity Accoustic Transmission Gel
DPX Mounting Medium Electron Microscopy Sciences 13512 Resin Based Cover Glass Mountant
Fluoro-Jade B EDM Millipore AG310 High Affinity Stain For Degenerating Neurons
Fluovac anesthetic adsorber Harvard Apparatus 34-0388 Organic Anaesthesia Scavenger
FUS System Image Guided Therapy, Pessac, France LabFUS MR Compatible Small Animal Focused Ultrasound System
Gadodiamide GE Healthcare AS, Oslo, Norway Omniscan MR Contrast Agent
Heparin SAGENT NDC2502140010 Anti-Coagulant
Hypodermic needle 30G x 1/2 Becton-Dickinson 26027 Tail Vein Catheterization
Insulin syringe 28G1/2 (1ml) EXEL 26027 Administration of Injectables to Tail Vein Catheter
Isofluorane atomizer SurgiVet VCT302 Anaesthesia Administration
Isoflurane Henry Schein NDC1169567762 Anaesthesia
KMnO4 Sigma 223468 Reagent Used in Fluoro-Jade B Staining
Microbubbles Produced internally: A. Klibanov 305106 Blood Brain Barrier Disrupting Agent
Microbubbles (commercial source) Lantheus Medical Imaging, North Billerica, MA Definity microbubbles Blood Brain Barrier Disrupting Agent
Monitoring & Gating System Small Animal Instruments Model 1030 Respiration Monitoring
Multisizer 3 Coulter counter Beckman-Coulter, Hialeah, FL Multisizer 3 Used to Determine Average Size of Microbubbles
Optixcare EYE LUBE CLC MEDICA, Ontario, Canada 11611 Corneal Protectant-Eye Lube
PE10 tubing Becton-Dickinson 427401 Tail Vein Catheter Component
Quinolinic Acid Santa Cruz Biotechnology, Dallas, TX CAS 89-00-9 Neurotoxin
Sprague-Dawley Rats Taconic Biosciences SD-M Rat Model
Syringe Pump Carnegie Medicin CMA 100 Controlled Delivery of Quinolinic Acid
Thermoguide Software Image Guided Therapy, Pessac, France Thermoguide Drives Lab FUS System
Tish Rats In-house colony Rat Model
Veet depilatory cream Reckitt Benckiser Removal of Scalp Hair

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Wang, W., Zhang, Y., Anzivino, M. J., Bertram, E. H., Woznak, J., Klibanov, A., Dumont, E., Wintermark, M., Lee, K. S. Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry. J. Vis. Exp. (163), e61271, doi:10.3791/61271 (2020).

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