Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

Summary

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

Abstract

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

Introduction

The human gastrointestinal tract harbors diverse microorganisms impacting the host^1,2,3. These gut bacteria can secrete gut-derived metabolites during their utilization of dietary components consumed by the host^4,5. Interestingly, the gut metabolites not metabolized in the periphery can be transported to other organs via circulation⁶. Of note, these secreted metabolites can serve as mediators for the gut-brain axis, defined as the bidirectional communication between the central nervous....

Protocol

All the experimental protocols and the animals' care were approved by the National Cheng Kung University (NCKU) Institutional Animal Care and Use Committee (IACUC).

1. Preparation for the experimental animal

1. Obtain 6-8-week-old wild-type C57BL/6JNarl male mice from a vendor.
2. House the mice in a standard mouse cage with standard mouse chow and sterilized water ad libitum.
   ​NOTE: The housing conditions for the Laboratory Animal Center of NCKU are 22 ± 1 °C temperature, 55% ± 10% humidity, and a 13 h/11 h light/dark cycle.

2....

Results

The mouse was infused with SCFAs 1 week after recovery from the guide cannula implantation to evaluate locomotor activity in a novel cage. The mouse was placed in a novel cage and infused with 2,100 nL of SCFAs or ACSF in the first 5 min (delivery rate of 7 nL/s) into the brain through the commercial guide cannula implanted in the lateral ventricle of the brain. The locomotor activity in a novel cage was recorded for an additional 30 min after infusion. No difference was observed in the locomotor activity in the novel ca.......

Discussion

Gut-derived metabolites have been associated with brain-mediated diseases without much precise mechanism, partially due to their multiple binding sites in the body^6,12,24. Previous reports indicated that SCFAs could serve as ligands for G protein-coupled receptors, epigenetic regulators, and sources for energy production at multiple sites in the body^6,12. To bypass the c.......

Materials

Name	Company	Catalog Number	Comments
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