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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Neuroscience

Intracerebroventrikulær levering af tarmafledte mikrobielle metabolitter i frit bevægelige mus

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

Tarmafledte mikrobielle metabolitter har mangesidede virkninger, der fører til kompleks adfærd hos dyr. Vi sigter mod at give en trinvis metode til at afgrænse virkningerne af tarmafledte mikrobielle metabolitter i hjernen via intracerebroventrikulær levering via en guidekanyle.

Abstract

Virkningen af tarmmikrobiota og deres metabolitter på værtsfysiologi og adfærd er blevet grundigt undersøgt i dette årti. Talrige undersøgelser har afsløret, at tarmmikrobiota-afledte metabolitter modulerer hjernemedierede fysiologiske funktioner gennem indviklede tarm-hjerneveje i værten. Kortkædede fedtsyrer (SCFA’er) er de vigtigste bakterieafledte metabolitter, der produceres under kostfiberfermentering af tarmmikrobiomet. Udskillede SCFA’er fra tarmen kan virke på flere steder i periferien, hvilket påvirker immun-, endokrine og neurale reaktioner på grund af den store fordeling af SCFA-receptorer. Derfor er det udfordrende at differentiere de centrale og perifere virkninger af SCFA’er gennem oral og intraperitoneal administration af SCFA’er. Dette papir præsenterer en videobaseret metode til at forhøre SCFA’ers funktionelle rolle i hjernen via en guidekanyle i frit bevægelige mus. Mængden og typen af SCFA’er i hjernen kan justeres ved at kontrollere infusionsvolumen og -hastighed. Denne metode kan give forskere en måde at værdsætte den rolle, som tarmafledte metabolitter spiller i hjernen.

Introduction

Den menneskelige mave-tarmkanal har forskellige mikroorganismer, der påvirker værten 1,2,3. Disse tarmbakterier kan udskille tarmafledte metabolitter under deres udnyttelse af diætkomponenter, der forbruges af værten 4,5. Interessant nok kan tarmmetabolitterne, der ikke metaboliseres i periferien, transporteres til andre organer via cirkulation6. Bemærk, at disse udskillede metabolitter kan tjene som mediatorer for tarm-hjerneaksen, defineret som tovejskommunikationen mellem centralnervesystemet og tarmen7. Tidligere undersøgelser har vist, at tarmafledte metabolitter kan modulere kompleks adfærd og følelser hos dyr 8,9,10,11.

Kortkædede fedtsyrer (SCFA’er) er de vigtigste metabolitter, der produceres af tarmmikrobiota under fermentering af kostfibre og ufordøjelige kulhydrater6. Acetat, propionat og butyrat er de mest rigelige SCFA’er i tarmen12. SCFA’er tjener som energikilde for celler i mave-tarmkanalen. Umetaboliserede SCFA’er i tarmen kan transporteres til hjernen gennem portalvenen og dermed modulere hjerne og adfærd 6,12. Tidligere undersøgelser har antydet, at SCFA’er kan spille en kritisk rolle i neuropsykiatriske lidelser 6,12. For eksempel reddede intraperitoneal injektion af butyrat i BTBR T + Itpr3tf / J (BTBR) mus, en dyremodel for autismespektrumforstyrrelse (ASD), deres sociale underskud13. Antibiotikabehandlede rotter, der modtog mikrobiota fra depressive forsøgspersoner, viste en stigning i angstlignende adfærd og fækale SCFA’er14. Klinisk blev ændringer i fækale SCFA-niveauer observeret hos mennesker med ASD sammenlignet med typisk udviklende kontroller15,16. Mennesker med depression har lavere fækale SCFAs niveauer end raske forsøgspersoner17,18. Disse undersøgelser antydede, at SCFA’er kan ændre adfærd hos dyr og mennesker gennem forskellige ruter.

Mikrobielle metabolitter udøver forskellige virkninger på flere steder i kroppen, hvilket påvirker værtsfysiologi og adfærd 4,19, herunder mave-tarmkanalen, vagusnerven og sympatisk nerve. Det er vanskeligt at fastslå den præcise rolle, som tarmafledte metabolitter spiller i hjernen, når metabolitterne administreres via perifere veje. Dette papir præsenterer en videobaseret protokol til at undersøge virkningerne af tarmafledte metabolitter i hjernen hos en frit bevægende mus (figur 1). Vi viste, at SCFA’er kunne gives akut gennem guidekanylen under adfærdstest. Typen, volumen og infusionshastigheden af metabolitter kan ændres afhængigt af formålet. Stedet for kanylering kan justeres for at undersøge virkningen af tarmmetabolitter i en bestemt hjerneregion. Vi sigter mod at give forskere en metode til at udforske den potentielle indvirkning af tarmafledte mikrobielle metabolitter på hjernen og adfærd.

Protocol

Alle forsøgsprotokoller og dyrenes pleje blev godkendt af National Cheng Kung University (NCKU) Institutional Animal Care and Use Committee (IACUC). 1. Forberedelse til forsøgsdyret Få 6-8 uger gamle vildtype C57BL/6JNarl hanmus fra en leverandør. Hus musene i et standard musebur med standard musechow og steriliseret vand ad libitum.BEMÆRK: Staldforholdene for NCKU’s Laboratory Animal Center er 22 ± 1 ° C temperatur, 55% ± 10% fugtighed…

Representative Results

Musen blev infunderet med SCFA’er 1 uge efter genopretning fra guidekanyleimplantationen for at evaluere lokomotorisk aktivitet i et nyt bur. Musen blev anbragt i et nyt bur og infunderet med 2.100 nL SCFA’er eller ACSF i de første 5 minutter (leveringshastighed på 7 nL / s) i hjernen gennem den kommercielle guidekanyle implanteret i hjernens laterale ventrikel. Den lokomotoriske aktivitet i et nyt bur blev registreret i yderligere 30 minutter efter infusion. Der blev ikke observeret nogen forskel i den bevægelsesmæs…

Discussion

Tarmafledte metabolitter har været forbundet med hjernemedierede sygdomme uden meget præcis mekanisme, delvis på grund af deres flere bindingssteder i kroppen 6,12,24. Tidligere rapporter viste, at SCFA’er kunne tjene som ligander for G-proteinkoblede receptorer, epigenetiske regulatorer og kilder til energiproduktion på flere steder i kroppen 6,12. For at omgå de f…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Vi anerkender laboratoriet dyr center personale på National Cheng Kung University (NCKU) for at passe dyrene. Dette arbejde blev støttet af stipendiet fra Prof. Kun-Yen Huang Education Fund fra CHENG-HSING Medical Foundation til C.-W.L.; midlerne fra ministeriet for videnskab og teknologi (MOST) i Taiwan: (Undergraduate Research MOST 109-2813-C-006-095-B) til T.-H.Y.; (MEST 107-2320-B-006-072-MY3; 109-2314-B-006-046; 110-2314-B-006-114; 110-2320-B-006-018-MY3) til W.-L.W.; og Higher Education Sprout Project, Undervisningsministeriet til hovedkvarteret for University Advancement på NCKU til 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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Keywords: IntracerebroventricularGut MicrobiotaGut MetabolitesMouse BrainBehaviorStereotaxicGuide 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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