自由运动大鼠脑电图记录过程中兴奋氨基酸的微透析
Summary
在这里, 我们描述了一种方法, 在体内微透析分析天冬氨酸和谷氨酸释放在腹侧海马的癫痫和非癫痫大鼠, 结合脑电图记录。天冬氨酸和谷氨酸的细胞外浓度可能与疾病的不同阶段有关。
Abstract
微透析是一种成熟的神经科学技术, 它将扩散到大脑间质空间的神经质活性物质的变化与病理的行为和/或病理的具体结果 (如癫痫发作)联系起来。为癫痫)。在研究癫痫时, 微透析技术通常与短期甚至长期的视频脑电图 (eeg) 监测相结合, 以评估自发发作的频率、严重程度、进展和聚类。联合微透析-脑电图是在使用多种方法和仪器的基础上进行的。在这里, 我们进行了体内微透析和连续视频脑电图记录, 以监测谷氨酸和天冬氨酸流出随着时间的推移, 在不同阶段的自然历史癫痫在大鼠模型。这种组合方法允许将神经递质释放的变化与疾病发展和进展的特定阶段配对。采用液相色谱法测定透析液中氨基酸的浓度。在这里, 我们描述了方法, 并概述了一个人在体内微透析-脑电图应采取的主要预防措施, 特别注意立体定向手术, 基础和高钾刺激在微透析, 深度电极脑电图记录和高效液相色谱分析天冬氨酸和谷氨酸在透析液中。这种方法可以用来测试各种药物或疾病引起的大脑中天冬氨酸和谷氨酸的生理浓度的变化。根据适当的分析分析分析的可用性, 它可以进一步用于测试不同的可溶性分子时, 同时使用脑电图记录。
Introduction
为了深入了解谷氨酸介导的兴奋性和 gabap 抑制神经传递导致颞叶癫痫 (tle) 自发性癫痫发作的功能损害, 我们系统地监测了细胞外的浓度。gaba 1 及更高版本的谷氨酸和天冬氨酸2的水平, 通过微透析在大鼠的腹侧海马在疾病的自然过程的不同时间点,即在癫痫的发展和进展。我们利用大鼠的 tle 皮尔卡平模型, 在行为、电生理和组织病理学变化方面非常准确地模仿了这种疾病, 并将氨基的透析液浓度相关酸到其不同的阶段: 急性阶段后的癫痫侮辱, 潜伏期阶段, 第一次自发性发作的时间和慢性药物5,6, 7.通过长期的视频脑电图监测、精确的脑电图和自发性癫痫的临床特征, 确定了疾病阶段的框架。微透析技术与长期视频脑电图监测相关的应用使我们能够提出 tle 神经病理学的机械假设。总之, 本手稿中描述的技术允许将大脑区域内的神经化学改变与动物模型中癫痫的发展和进展配对。
配对装置由与微透析插管并列的深度电极组成, 经常用于癫痫研究, 在这些研究中, 神经递质、其代谢物或能量底物的变化应与神经元活动相关。在绝大多数情况下, 它被用于行为自由的动物, 但它也可以在人类以类似的方式进行,例如, 在手术前接受深度电极调查的药物耐药癫痫患者8。脑电图记录和透析液收集都可以单独进行 (例如, 将电极植入一个半球, 将微透析探针植入另一个半球, 甚至在一组动物中进行微透析在另一组动物中唯一的脑电图)。然而, 将电极耦合到探针可能有多重优点: 它简化了立体定向手术, 将组织损伤限制在一个半球 (同时保持另一个完整, 作为组织学研究的控制), 并将结果同质为这些是从相同的大脑区域和相同的动物中获得的。
另一方面, 耦合微透析探针电极装置的制备如果是自制的, 则需要技能和时间。如果从市场上购买, 人们可以花相对较高的钱。此外, 当微透析探针 (探针尖端通常直径 200-400μm, 7-12 毫米长)9和 eeg 电极 (电极尖端通常直径为 300-500μm, 长度足以达到感兴趣的大脑结构10时) 10 是耦合, 安装的设备代表一个笨重和相对沉重的物体在头部的一侧, 这是麻烦的动物和容易丢失, 特别是当它连接到透析泵和硬线脑电图记录系统。这一方面更相关的癫痫动物是困难的处理和较少适应微透析过程。适当的手术技术和适当的术后护理可以导致持久的植入物, 导致最小的动物不适, 应追求组合微透析-脑电图实验10,11, 12岁
微透析技术的优点和局限性已被许多神经科学家详细地综述。它比其他体内灌注技术 (例如, 快速流动推拉或皮质杯灌注) 的主要优势是探针的小直径, 涵盖了一个相对精确的感兴趣的区域13,14, 15岁第二, 微透析膜在组织和胚芽之间形成物理屏障;因此, 高分子量物质不交叉, 也不干扰分析16,17。此外, 组织被保护免受湍流流动的香水18。另一个重要的优点是有可能改变灌注流, 以最大限度地提高在热液中的分析物浓度 (即, 微透析的过程可以很好地定义数学, 并可以修改到产生高产量样品中分析物的浓度)19。最后, 该技术可用于将药物或药理活性物质注入感兴趣的组织, 并确定其在干预部位20的效果.另一方面, 与电化学或生物传感器相比, 微透析的解决时间有限 (由于采集样品所需的时间, 通常超过 1分钟);它是一种侵入性技术, 导致组织损伤;它破坏了膜周围空间内的神经化学平衡, 因为所有可溶性物质的连续浓度梯度与感兴趣的分析物一起进入香水。最后, 微透析技术受到用于对材料 9、21、22、23进行定量的分析技术的限制的高度影响..用邻苯二甲酯衍生后的高效液相色谱法 (hplc) 对生物样品中的谷氨酸和天冬氨酸进行分析, 得到了24,25,26的良好验证。,27及其广泛的讨论超出了本手稿的范围, 但将详细描述使用这种方法产生的数据。
当正确地进行, 而不修改的香水成分, 微透析可以提供可靠的信息, 神经递质释放的基础水平。基础水平的最大部分很可能是突触9的发射机溢出的结果.因为在许多情况下, 在额外的突触空间中对神经递质进行简单的取样不足以实现调查的目标, 所以微透析技术也可以用来刺激神经元或剥夺它们的重要作用生理离子, 如 k+或 ca2 +, 以唤起或防止神经递质的释放。
高 k+刺激经常用于神经生物学, 以刺激神经元的活动, 不仅在清醒的动物, 但也在初级和有机类型培养。健康的中枢神经系统暴露在高浓度 k+ (40-100 mm) 的溶液中, 会唤起神经递质28的流出.这种神经元在应对高 k+时提供额外释放的能力可能会在癫痫动物1和其他神经退行性疾病 29、30 中受到损害。同样, ca2 +剥夺 (通过灌注 ca2 +游离溶液获得) 被用来建立钙依赖释放的大多数神经递质测量微透析。一般认为 ca2 +依赖释放是神经元的起源, 而 ca2 +独立释放源于胶质细胞, 但许多研究对 ca2+敏感测量的含义引发了争议。谷氨酸或 gaba9: 因此, 如果可能的话, 最好支持微透析研究与微传感器研究, 因为后者具有较高的空间分辨率, 电极允许接近突触 31.
关于癫痫动物的微透析研究, 重要的是要强调的是, 从它们中的大多数获得的数据依赖于对癫痫发作的视频或视频脑电图监测,即由于异常而短暂出现体征和症状大脑中过度或同步的神经元活动 32。在准备实验时, 应考虑皮卡平治疗动物的电图癫痫发作的一些具体问题。自发性发作后, 伴随着抑郁活动, 脑电图间尖峰频繁, 在33,34组发生。sham 操作的非癫痫动物可能表现出类似癫痫发作的活动35 , 因此脑电图记录评估的参数应标准化36 , 如果可能, 微透析的时间应明确界定。最后, 我们强烈建议遵循国际抗癫痫协会和美国癫痫学会专家在其最近的报告中概述的控制成年啮齿类动物的视频脑电图监测的原则和方法标准37 ,38。
本文介绍了谷氨酸和天冬氨酸的微透析与癫痫动物的长期视频脑电图记录以及它们在透析液中的高效液相色谱分析。我们将强调议定书的关键步骤, 一个人应该注意, 以取得最好的结果。
Protocol
所有实验程序都已由费拉拉大学动物护理和使用机构委员会和意大利卫生部根据欧洲共同体概述的准则批准 (授权: d. m. 246/ensen-b)1986年11月24日理事会指令 (86/609/EEC)。该方案是专门为谷氨酸和天冬氨酸的测定在脑电图控制下获得的癫痫大鼠和非癫痫大鼠微透析过程中的谷氨酸和天冬氨酸的测定而进行的。这里描述的许多材料可能很容易被你在实验室中用于脑电图记录或微透析的材料所取代。 <p c…
Representative Results
探头恢复 平均回收率 (即在小瓶溶液中的平均氨基酸含量占含量的百分比在小瓶溶液中的含量百分比) 为 15.49±0.42%, 流量为 2μl%, 在3μlp 的流量下为 6.32±0.64, 在流量率为2的情况下为14.89±0.36% 在使用铜叶膜探针时, 为天冬氨酸在3μl/min 时使用。如果使用聚丙烯腈膜探针, 平均回收率为 13.67±0.42%, 流量为 2μlmmin, 在3μlmin 下的平…
Discussion
在这项工作中, 我们展示了如何连续的视频脑电图记录与微透析相结合, 可以在一个实验模型的 tle。视频脑电图记录技术用于正确诊断动物疾病进展的不同阶段, 微透析技术用于描述及时发生的谷氨酸释放的变化 (没有发现任何变化)在先前发表的研究报告中的天冬氨酸 2)。出于简介中讨论的原因, 我们强烈建议使用单个设备植入物在每种动物中同时执行它们。
<p class="jove_content"…Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者希望感谢 anna binaschi、paolo roncon 和 eleonora palma 对优先出版的手稿的贡献。
Materials
| 3-channel two-twisted electrode | Invivo1, Plastic One, Roanoke, Virginia, USA | MS333/3-B/SPC | Material |
| guide cannula | Agn Tho's, Lindigö, Sweden | MAB 4.15.IC | Material |
| Resin KK2 Plastik | Elettra Sport, Lecco, Italy | KK2 | Material |
| Super Attack gel Loctite | Henkel Italia Srl, Milano, Italy | 2047420_71941 | Material |
| Imalgene-Ketamine | Merial, Toulouse, France | 221300288 (AIC) | Solution |
| Xylazine | Sigma, Milano, Italy | X1251 | Material |
| Isoflurane-Vet | Merial, Toulouse, France | 103120022 (AIC) | Solution |
| Altadol 50 mg/ ml – tramadol | Formevet, Milano, Italy | 103703017 (AIC) | Solution |
| Gentalyn 0.1% crm – gentamycine | MSD Italia, Roma, Italy | 20891077 (AIC) | Material |
| simplex rapid dental cement | Kemdent, Associated Dental Products Ltd, Swindon, United Kingdom | ACR811 | Material |
| GlasIonomer CX-Plus Cement | Shofu, Kyoto, Japan | PN1167 | Material |
| probe clip holder | Agn Tho's, Lindigö, Sweden | p/n 100 5001 | Equipment |
| Histoacryl® Blue Topical Skin Adhesive | TissueSeal, Ann Arbor, Michigan, USA | TS1050044FP | Material |
| Valium 10 mg/2 ml – diazepam | Roche, Monza, Italy | 019995063 (AIC) | Material |
| 1 mL syringe with 25G needle | Vetrotecnica, Padova, Italy | 11.3500.05 | Material |
| rat flexible feeding needle 17G | Agn Tho's, Lindigö Sweden | 7206 | Material |
| Grass Technology apparatus | Grass Technologies, Natus Neurology Incorporated, Pleasanton, California, USA | M665G08 | Equipment (AS40 amplifier, head box, interconnecting cables, telefactor model RPSA S40) |
| modular data acquisition and analysis system MP150 | Biopac, Goleta, California, USA | MP150WSW | Equipment |
| digital video surveillance system | AverMedia Technologies, Fremont, California, USA | V4.7.0041FD | Equipment |
| microdialysis probe | Agn Tho's, Lindigö Sweden | MAB 4.15.1.Cu | Material |
| microdialysis probe | Synaptech, Colorado Springs, Colorado, USA | S-8010 | Material |
| block heater | Grant Instruments, Cambridge, England | QBD2 | Equipment |
| stirrer | Cecchinato A, Aparecchi Scientifici, Mestre, Italy | 711 | Equipment |
| infusion pump | Univentor, Zejtun, Malta | 864 | Equipment |
| fine bore polythene tubing | Smiths Medical International Ltd., Keene, New Hampshire, USA | 800/100/100/100 | Material |
| blue tubing adapters | Agn Tho's, Lindigö Sweden | 1002 | Material |
| red tubing adapters | Agn Tho's, Lindigö Sweden | 1003 | Material |
| 2.5 mL syringe with 22G needle | Chemil, Padova, Italy | S02G22 | Material |
| vial cap | Cronus, Labicom, Olomouc, Czech Republic | VCA-1004TB-100 | Material |
| septum | Thermo Scientific, Rockwoood, Tennessee, USA | National C4013-60 8 mm TEF/SIL septum | Material |
| glass insert with bottom spring | Supelco, Sigma, Milano, Italy | 27400-U | Material |
| autosampler vial | National Scientific, Thermo Fisher Scientific, Monza, Italy | C4013-2 | Material |
| Smartline manager 5000 system controller and degasser unit | Knauer, Berlin, Germany | V7602 | Equipment |
| Smartline 1000 quaternary gradient pump | Knauer, Berlin, Germany | V7603 | Equipment |
| spectrofluorometric detector | Shimadzu, Kyoto, Japan | RF-551 | Equipment |
| chromatogrphic column | Knauer, Berlin, Germany | 25EK181EBJ | Material |
| chromatogrphic pre-column | Knauer, Berlin, Germany | P5DK181EBJ | Material |
| mobile phase solution A | 0.1 M sodium phosphate buffer, pH 6.0 | Solution | |
| mobile phase solution B | 40% 0.1 M sodium phosphate buffer, 30% methanol, 30% acetonitrile, pH 6.5 | Solution | |
| Ringer solution | composition in mM: MgCl2 0.85, KCl 2.7, NaCl 148, CaCl2 1.2, 0.3% BSA | Solution | |
| modified Ringer solution | composition in mM: MgCl2 0.85, KCl 100, NaCl 50.7, CaCl2 1.2, 0.3% BSA | Solution | |
| saline | 0.9% NaCl, ph adjusted to 7.0 | Solution | |
| sucrose solution | 10% sucrose in distilled water | Solution |
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