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Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Divulgaciones
Acknowledgements
Materials
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Neurociencias

自由移动小鼠中肠道来源的微生物代谢物的脑室内递送

Published: June 02, 2022
doi:
10.3791/63972
, ,
1Institute of Basic Medical Sciences, College of Medicine,National Cheng Kung University, 2Department of Physiology, College of Medicine,National Cheng Kung University

Summary

肠道来源的微生物代谢物具有多方面的作用，导致动物的复杂行为。我们的目标是提供一种循序渐进的方法，通过引导套管通过脑室内输送来描述肠道来源的微生物代谢物在大脑中的作用。

Abstract

肠道微生物群及其代谢物对宿主生理和行为的影响在这十年中得到了广泛的研究。大量研究表明，肠道微生物群衍生的代谢物通过宿主中复杂的肠脑通路调节大脑介导的生理功能。短链脂肪酸（SCFA）是肠道微生物组在膳食纤维发酵过程中产生的主要细菌衍生代谢物。来自肠道的分泌的SCFA可以在外周的多个部位起作用，由于SCFAs受体的广泛分布，影响免疫，内分泌和神经反应。因此，通过口服和腹膜内给药来区分SCFAs的中枢效应和外周效应具有挑战性。本文提出了一种基于视频的方法，通过引导套管在自由移动的小鼠中询问SCFAs 在 大脑中的功能作用。大脑中SCFA的数量和类型可以通过控制输注量和速率来调节。这种方法可以为科学家提供一种方法来了解肠道衍生代谢物在大脑中的作用。

Introduction

人体胃肠道含有影响宿主的各种微生物1，2，3。这些肠道细菌在利用宿主消耗的膳食成分时可以分泌肠道衍生的代谢物4，5。有趣的是，未在外周代谢的肠道代谢物可以通过循环转运到其他器官6。值得注意的是，这些分泌的代谢物可以作为肠脑轴的介质，定义为中枢神经系统和肠道之间的双向通信7。先前的研究表明，肠道衍生的代谢物可以调节动物的复杂行为和情绪8，9，10，11。

短链脂肪酸（SCFAs）是肠道微生物群在膳食纤维和难消化碳水化合物发酵过程中产生的主要代谢物6。乙酸盐、丙酸盐和丁酸盐是肠道中含量最高的 SCFAs12。SCFA是胃肠道细胞的能量来源。肠道中未代谢的SCFA可以通过门静脉运输到大脑，从而调节大脑和行为6，12。先前的研究表明，SCFA可能在神经精神疾病中起关键作用6，12。例如，在自闭症谱系障碍（ASD）的动物模型BTBR T+ Itpr3tf / J（BTBR）小鼠中腹腔注射丁酸盐挽救了他们的社会缺陷13。接受来自抑郁受试者微生物群的抗生素处理的大鼠显示出焦虑样行为和粪便SCFAs的增加14。临床上，与通常的对照组相比，在ASD患者中观察到粪便SCFAs水平的变化15，16。抑郁症患者的粪便SCFAs水平低于健康受试者17，18。这些研究表明，SCFAs可以通过各种途径改变动物和人类的行为。

微生物代谢物对体内多个部位产生多种影响，影响宿主生理和行为4，19，包括胃肠道、迷走神经和交感神经。当通过外周途径施用代谢物时，很难确定肠道来源代谢物在大脑中的确切作用。本文提出了一种基于视频的方案，用于研究肠道衍生代谢物对自由移动的小鼠大脑的影响（图1）。我们发现，在行为测试期间，可以通过引导套管急性给予SCFA。代谢物的类型、体积和输注速率可以根据目的进行修改。可以调整插管部位，以探索肠道代谢物在特定大脑区域的影响。我们的目标是为科学家提供一种方法来探索肠道来源的微生物代谢物对大脑和行为的潜在影响。

Protocol

所有实验方案和动物护理均已获得国立成功大学（NCKU）机构动物护理和使用委员会（IACUC）的批准。 1.实验动物的准备 从供应商处获得6-8周龄的野生型C57BL / 6JNarl雄性小鼠。 将小鼠饲养在标准小鼠笼中，用标准老鼠食物和 随意消毒的水。注意：NCKU实验动物中心的外壳条件为22±1°C温度，55%±10%湿度和13小时/ 11小时亮/暗循环。 <p cla…

Representative Results

在引导套管植入恢复后1周向小鼠注入SCFAs，以评估新型笼子中的运动活动。将小鼠放置在一个新的笼子中，并在前5分钟（输送速率为7nL / s）通过植入大脑侧脑室的商业引导套管向大脑注入2，100nL SCFAs或ACSF。输注后再记录30分钟新笼中的运动活动。在SCFAs和ACSF输注之间的新笼子中的运动活动没有观察到差异（图6）（每组n = 2只小鼠;数据显示为平均±s.e.m.并通过双向方差分析?…

Discussion

肠道来源的代谢物与脑介导的疾病有关，没有太多精确的机制，部分原因是它们在体内的多个结合位点6，12，24。先前的报告表明，SCFAs可以作为G蛋白偶联受体的配体，表观遗传调节因子和体内多个部位的能量产生来源6，12。为了绕过源自外周的混杂因素（如免疫细胞、激素和自主?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

我们感谢国立成功大学（NCKU）实验动物中心的工作人员照顾动物。这项工作得到了正兴医学基金会黄坤彦教授教育基金的奖学金支持;台湾科技部（MOST）的资助：（本科生研究MOST 109-2813-C-006-095-B）到T.-H.Y.;（大多数 107-2320-B-006-072-MY3; 109-2314-B-006-046; 110-2314-B-006-114; 110-2320-B-006-018-MY3） 至 W.-L.W.;以及高等教育萌芽项目，教育部到NCKU大学发展总部到W.-L.W。

Materials

Material
Advil Liqui-Gels Solubilized Ibuprofen A2:D41 Pfizer n/a
Alexa Fluor 488 donkey anti-rabbit ThermoFisher Scientific A-21206
Anti-Fluorescent Gold (rabbit polyclonal) Millipore AB153-I
Bottle Top Vacuum Filter, 500 mL, 0.22 μm, PES, Sterile NEST 121921LA01
CaCl2  Sigma-Aldrich C1016 ACSF: 0.14 g/L
Chlorhexidine scrub 2% Phoenix NDC 57319-611-09
Chlorhexidine solution Phoenix NDC 57319-599-09
Commercial dummy RWD Life Science 62004 Single_OD 0.20 mm/ M3.5/G = 0.5 mm
Commercial guide cannul RWD Life Science 62104 Single_OD 0.41 mm-27G/ M3.5/C = 2.5 mm 
Commercial injector RWD Life Science 62204 Single_OD 0.21 mm-33G/ Mates with M3.5/C = 3.5 mm/G = 0.5 mm
D-(+)-Glucose Sigma-Aldrich G8270 ACSF: 0.61 g/L
Dental acrylic HYGENIC n/a
Fixing screws RWD Life Science 62521
Fluoroshield mounting medium with DAPI Abcam AB104139
Horse serum ThermoFisher Scientific 16050130
Insulin syringes BBraun XG-LBB-9151133S-1BX 1 mL
Isoflurane  Panion & BF biotech DG-4900-250D
KCl  Sigma-Aldrich P3911 ACSF: 0.19 g/L
Ketoprofen  Swiss Pharmaceutical n/a
Lidocaine  AstraZeneca n/a
Low melting point agarose Invitrogen 16520
MgCl2  Sigma-Aldrich M8266 ACSF: 0.19 g/L
Microscope cover slips MARIENFELD 101242
Microscope slides ThermoFisher Scientific 4951PLUS-001E
Mineral oil light, white NF Macron Fine Chemicals MA-6358-04
NaCl  Sigma-Aldrich S9888 ACSF: 7.46 g/L
NaH2PO4  Sigma-Aldrich S8282 ACSF: 0.18 g/L
NaHCO3  Sigma-Aldrich S5761 ACSF: 1.76 g/L
n-butyl cyanoacrylate adhesive (tissue adhesive glue) 3M 1469SB 3M Vetbond
Neural tracer  Santa Cruz SC-358883 FluoroGold
Paraformaldehyde Sigma-Aldrich P6148
Polyethylene tube RWD Life Science 62329 OD 1.50, I.D 0.50 mm and OD 1.09, I.D 0.38 mm
Puralube Vet (eye) Ointment Dechra  12920060
Sodium acetate  Sigma-Aldrich S2889 SCFAs: 13.5 mM
Sodium azide  Sigma-Aldrich S2002
Sodium butyrate  Sigma-Aldrich B5887 SCFAs: 8 mM
Sodium propionate  Sigma-Aldrich P1880 SCFAs: 5.18 mM
Stainless guide cannula Chun Ta stainless steel enterprise CO., LTD. n/a OD 0.63 mm; Local vendor
Stainless injector Chun Ta stainless steel enterprise CO., LTD. n/a OD 0.3 mm; dummy is made from injector; local vendor
Superglue Krazy Glue KG94548R
Triton X-100 Merck 1.08603.1000
Equipment
Cannula holder RWD Life Science B485-68217
Ceiling camera FOSCAM R2
Digital stereotaxic instruments Stoelting 51730D
Dissecting microscope INNOVIEW SEM-HT/TW
Glass Bead Sterilizer RWD Life Science RS1501
Heating pad Stoelting 53800M
Leica microscope  Leica DM2500
Micro Dissecting Forceps ROBOZ RS-5136 Serrated, Slight Curve; Extra Delicate; 0.5mm Tip Width; 4" Length 
Micro Dissecting Scissors ROBOZ RS-5918 4.5" Angled Sharp
Microinjection controller World Precision Instruments (WPI) MICRO2T SMARTouch Controller
Microinjection syringe pump World Precision Instruments (WPI) UMP3T-1 UltraMicroPump3  
Microliter syringe Hamilton 80014 10 µL
Optical Fiber Cold Light with double Fiber Step LGY-150 Local vendor
Pet trimmer WAHL 09962-2018
Vaporiser for Isoflurane Step AS-01 Local vendor
Vibratome Leica VT1000S
Software
Animal behavior video tracking software Noldus EthoVision Version: 15.0.1416
Leica Application Suite X software Leica LASX Version: 3.7.2.22383
DOWNLOAD MATERIALS LIST

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IntracerebroventricularGut MicrobiotaGut MetabolitesMouse BrainComportamientoStereotaxicGuide CannulaN-Butyl CyanoacrylateDental Acrylic

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Liou, C., Yao, T., Wu, W. Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice. J. Vis. Exp. (184), e63972, doi:10.3791/63972 (2022).
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