高效液相色谱法测定犬尿氨和 Kynurenic 酸: 对大鼠认知和睡眠的相关生物化学研究
Summary
犬尿氨通路 (KP) neuroactive 代谢物的改变与精神疾病有牵连。调查啮齿类动物体内改变的犬尿氨通路代谢的功能性结果可能有助于阐明新的治疗方法。目前的协议结合了生物化学和行为学的方法来研究急性犬尿氨挑战对大鼠的影响。
Abstract
色氨酸降解的犬尿氨通路 (KP) 与精神疾病有牵连。具体地说, 星形胶质细胞衍生代谢物 kynurenic 酸 (KYNA) 是n-甲基-d-天门冬氨酸 (NMDA) 和α7烟碱乙酰胆碱 (α7nACh) 受体的拮抗剂, 与健康和疾病的认知过程有牵连。随着精神分裂症患者大脑中 KYNA 水平的升高, 谷氨酸和胆碱能受体的故障被认为是与认知功能障碍相关的因果关系, 这是疾病精神病理学的核心领域。KYNA 可能在精神分裂症的个体中扮演 pathophysiologically 重要的角色。通过直接 bioprecursor 犬尿氨对动物进行治疗, 可以提高啮齿动物脑内内源性 KYNA, 大鼠的临床前研究表明, KYNA 的急性海拔可能会影响他们的学习和记忆过程。本协议详细描述了这一实验方法, 并结合 a) 对血液犬尿氨水平和脑 KYNA 形成 (使用高效液相色谱) 的生化分析, b) 行为测试, 以探讨与海马相关的上下文记忆 (被动回避范式) 和 c) 对睡眠唤醒行为的评估 [将脑电图 (EEG) 和肌电图 (肌电信号) 信号结合在一起的遥测记录] 在大鼠中。结合起来, 研究了高 KYNA、睡眠和认知之间的关系, 该协议详细描述了一种实验方法来了解大鼠体内犬尿氨海拔和 KYNA 形成的功能结果。通过这一协议的变化得到的结果将测试的假说, KP 和 KYNA 在调节睡眠和认知的健康和疾病状态的关键作用。
Introduction
金伯利进程负责降解近95% 的必需氨基酸色氨酸1。在哺乳动物的大脑中, 犬尿氨被带入星形胶质细胞代谢成 neuroactive 小分子 KYNA 主要由酶犬尿氨转氨酶 (KAT) II2。KYNA 作为拮抗剂在 NMDA 和α7nACh 受体在大脑2,3,4, 也靶向信号受体包括芳烃受体 (AHR) 和 G 蛋白耦合受体 35 (GPR35)5 ,6。在实验动物中, 大脑 KYNA 的海拔在行为分析的2、7、8、9、10中被证明会损害他们的认知能力。.一个新的假说表明, KYNA 在调节认知功能方面起着不可或缺的作用, 它影响睡眠唤醒行为11, 从而进一步支持星形胶质细胞衍生分子在调节睡眠神经生物学方面的作用, 并认知12。
临床上, KYNA 的海拔在脑脊液和验尸后的脑组织中发现, 精神分裂症13,14,15,16, 一个衰弱的精神紊乱以认知障碍为特征的。精神分裂症患者也经常被睡眠紊乱所困扰, 这可能使病情恶化17。了解 KP 代谢和 KYNA 在调节睡眠与认知之间的关系, 特别是学习和记忆之间的作用, 可能会导致新的治疗精神分裂症和其他一些不良结局的治疗方法的发展。精神疾病。
一个可靠和一致的方法来测量金伯利的代谢物, 是重要的, 以确保从各个机构的研究可以融入到科学的理解, kp 生物学。目前, 我们描述了用高效液相色谱法测定大鼠脑血浆和 KYNA 中犬尿氨的方法。本议定书在锌2 +的存在下使用荧光检测, 最初是由柴田18和最近适应和优化到 derivatize 以500毫米醋酸锌作为后柱试剂, 从而使在脑内内源性、nanomolar 量 KYNA 的检测11。
为刺激本议定书所述的 KYNA 内源性生产, 直接 bioprecursor 犬尿氨在大鼠体内注入腹腔 (ip)。结合生化评估来确定 KYNA 生产的程度, 犬尿氨挑战对海马依赖性记忆 (被动回避范式) 和睡眠唤醒体系 (EEG 和肌电信号) 的影响也是调查11。这些技术的结合允许研究犬尿氨挑战在大鼠体内的生物化学和功能影响。
Protocol
我们的实验性议定书得到了马里兰大学动物保育和使用委员会的批准, 并遵循了国家卫生研究院的护理和使用实验动物指南。 注: 所有实验均采用成年雄性大鼠 (250–350 g)。不同的动物群被用于生化分析、行为实验和睡眠唤醒记录。这些动物被安置在一个温度控制的设施在马里兰精神病学研究中心。他们被保持在一个12/12 小时的光黑暗的周期, 与灯在授时时间 (ZT) 0 和熄灯在 ZT …
Representative Results
为了验证采用腹腔犬尿氨注射液作为提高脑 KYNA 的方法, 对组织进行了 HPLC 分析。标准曲线 (图 1) 是使用相关软件构造的, 并允许对组织样本进行量化。犬尿氨和 KYNA 的代表性图谱如图 2所示。犬尿氨在6分钟的保留时间内观察到, KYNA 的保留时间为11分钟。为了观察 KP 动力学, 在本协议中管理了100毫克/千克的犬尿氨, 并在注射后立…
Discussion
为了可靠地评估周围犬尿氨管理后的脑 KYNA, 必须结合和解释生化和功能性实验。在这里, 我们提出了一个详细的协议, 允许新用户建立有效的方法来测量血浆犬尿氨和脑 KYNA 的大鼠。血浆中犬尿氨的测定证实了准确的注射和代谢产物的测定 KYNA 证实了大脑中的从头合成。所描述的荧光液相色谱法有以下几个优点: 从柴田18到 derivatize KYNA, 在锌2 +的存在下, 包括在 nanom…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
本研究部分由国立卫生研究院 (R01 NS102209) 资助, 并由克莱尔. 福布斯信托基金捐赠。
Materials
| Wistar rats | Charles River Laboratories | adult male, 250-350 g | |
| L-kynurenine sulfate | Sai Advantium | ||
| ReproSil-Pur C18 column (4 x 150 mm) | Dr. Maisch GmbH | ||
| EZ Clips | Stoelting Co. | 59022 | |
| Mounting materials screws | PlasticsOne | 00-96 X 1/16 | |
| Nonabsorbable Sutures | MedRep Express | 699B | CP Medical Monomid Black Nylon Sutures, 4-0, P-3, 18", BOX of 12 |
| Absorbable Sutures | Ethicon | J310H | 4-0 Coated Vicryl Violet 1X27'' SH-1 |
| Dental Cement | Stoelting Co. | 51458 | |
| Drill Bit | Stoelting Co. | 514551 | 0.45 mm |
| Name | Company | Catalog Number | Comments |
| Alliance HPLC system | |||
| E2695 separation module | Waters | 176269503 | |
| 2475 fluorescence detector | Waters | 186247500 | |
| post-column reagent manager | Waters | 725000556 | |
| Lenovo computer | Waters | 668000249 | |
| Empower software | Waters | 176706100 | |
| Name | Company | Catalog Number | Comments |
| Passive avoidance box for rat | |||
| Extra tall MDF sound attenuating cubicle | MedAssociates | ENV-018MD | Interior: 22"W x 22"H x 16"D |
| Center channel modulator shuttle box chamber | MedAssociates | ENV-010MC | |
| Stainless steel grid floor for rat | MedAssociates | ENV-010MB-GF | |
| Auto guillotine door | MedAssociates | ENV-010B-S | |
| Quick disconnect shuttle grid floor harness for rat | MedAssociates | ENV-010MB-QD | |
| Stimulus light, 1" white lens, mounted on modular panel | MedAssociates | ENV-221M | |
| Sonalert module with volume control for rat chamber | MedAssociates | ENV-223AM | |
| SmartCtrl 8 input/16 output package | MedAssociates | DIG-716P2 | |
| 8 Channel IR control for shuttle boxes | MedAssociates | ENV-253C | |
| Infrared source and dectector array strips | MedAssociates | ENV-256 | |
| Tabletop interface cabinet, 120 V 60 Hz | MedAssociates | SG-6080C | |
| Dual range constant current aversive stimulation module | MedAssociates | ENV-410B | |
| Solid state grid floor scrambler module | MedAssociates | ENV-412 | |
| Dual A/B shock control module | MedAssociates | ENV-415 | |
| 2' 3-Pin mini-molex extension | MedAssociates | SG-216A-2 | |
| 10' Shock output cable, DB-9 M/F | MedAssociates | SG-219G-10 | |
| Shuttle shock control cable 15', 6 | MedAssociates | SG-219SA | |
| Small tabletop cabinet and power supply, 120 V 60 Hz | MedAssociates | SG-6080D | |
| PCI interface package | MedAssociates | DIG-700P2-R2 | |
| Shuttle box avoidance utility package | MedAssociates | SOF-700RA-7 | |
| Name | Company | Catalog Number | Comments |
| Sleep-Wake Monitoring Equipment | |||
| Ponehmah software | Data Sciences International (DSI) | PNP-P3P-610 | |
| MX2 8 Source Acquisition interface | Data Sciences International (DSI) | PNM-P3P-MX204 | |
| Dell computer, Optiplex 7020, Windows 7, 64 bit | Data Sciences International (DSI) | 271-0112-013 | |
| Dell 19" computer monitor | Data Sciences International (DSI) | 271-0113-001 | |
| Receivers for plastic cages, 8x | Data Sciences International (DSI) | 272-6001-001 | |
| Cisco RV130 VPN router | Data Sciences International (DSI) | RV130 | |
| Matrix 2.0 | Data Sciences International (DSI) | 271-0119-001 | |
| Network switch | Data Sciences International (DSI) | SG200-08P | |
| Neuroscore software | Data Sciences International (DSI) | 271-0171-CFG | |
| Two biopotential channels transmitter, model TL11M2-F40-EET | Data Sciences International (DSI) | 270-0134-001 |
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Diesen Artikel zitieren
Baratta, A. M., Viechweg, S. S., Mong, J. A., Pocivavsek, A. A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats. J. Vis. Exp. (138), e58129, doi:10.3791/58129 (2018).