Intravital Fluorescence Microscopy to Study Microvascular Thrombus Formation

Abstract

Source: Strüder, D., et al. Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse. J. Vis. Exp. (2017)

This video demonstrates the intravital fluorescence microscopy technique to study thrombus formation in a mouse model. The mouse is injected with a fluorophore-labeled polysaccharide to monitor blood circulation. Upon illumination with a high-intensity light, the phototoxic induction of an injury in the microvasculature of the ear leads to the formation of a thrombus, which is confirmed by visualizing the obstruction in the blood flow.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Prearrangement of the Animals Weigh a mouse and load the respective drug (e.g., the cannabinoid, 5 mg/kg body weight (bw)) into an insulin syringe. Administer the drug 30 min prior to thrombus induction. By holding the neck of the mouse between the thumb and the index finger and the tail of the mouse …

Representative Results

Figure 1. Preparation of the Jugular Vein (A) and Placement of the Ear for Intravital Microscopy (B). (A) Using the operation stereomicroscope, the right jugular vein is prepared. The sutures for the placement of the left ear are stitched before the injection of FITC-dextran. The wound from the prior dissection of the left jugular vein is closed with transcutaneous sutures (B). The left ear is fixed with polypro…

Materials

SKH-1/hr mice	Charles River	477	Can be purchased from other vendors
Operation stereomicroscope	Leica	M651/M655	Can be purchased from other vendors
Intravital microscope	Zeiss	Axiotech Vario 100	Can be purchased from other vendors
Objective (20x/0.95)	Zeiss	20x/0,50 W; Plan-NEOFLUAR	Can be purchased from other vendors
Objective (63x/0.95)	Zeiss	63x/0,95 W; ACHROPLAN	Can be purchased from other vendors
Black and white CCD-camera	Pieper	FK 6990 IQ-S	Can be purchased from other vendors
DVD-recorder	Panasonic	DMR-EX99V	Can be purchased from other vendors
Sodium chloride	Braun	5/12612055/1011	Can be purchased from other vendors
Ketamine 10%	Bela pharm	F3901-6	Can be purchased from other vendors
Xylazine 2%	Bayer	6293841.00.00	Can be purchased from other vendors
FITC-dextran 5%	Sigma	46945-100MG-F	Can be purchased from other vendors
Dexapanthenol 5% eye ointment	Bayer	6029009.00.00	Can be purchased from other vendors
DMSO	Sigma	472301	Can be purchased from other vendors
Coverslips 5 x 5 x 1 mm	Menzel	L4339	Can be purchased from other vendors
Plasters	Leukosilk	4683400	Can be purchased from other vendors
Cotton swabs	Sanyo	604-A-1	Can be purchased from other vendors
Infrared light	Beurer	5/13855	Can be purchased from other vendors
Single-use syringe	Braun	2020-08	Can be purchased from other vendors
Insulin syringe	Braun	9161502	Can be purchased from other vendors
Disposable hypodermic needles	Braun	465 7640	Can be purchased from other vendors
End-to-end capillary	Sarstedt	19,447	Can be purchased from other vendors
Heating plate	Klaus Effenberg	OP-T 185/03	Can be purchased from other vendors
Scissors 14.5 cm	Aesculap	BC259R	Can be purchased from other vendors
Needle Holder	Aesculap	BM081R	Can be purchased from other vendors
Microforceps	Aesculap	BD331R	Can be purchased from other vendors
Microscissors	Aesculap	OC496R	Can be purchased from other vendors
Scalpel 21	Dahlhausen	11.000.00.511	Can be purchased from other vendors
Prolene 7-0	Ethicon	XNEH7470	Can be purchased from other vendors
Electrocautery	Servoprax	H40140	Can be purchased from other vendors
Acrylglass pad			Integrated heating, 0.5 cm high plane