Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse

Summary

The ear model of the hairless SKH1-Hr^hr mouse enables intravital fluorescence microscopy of microcirculation and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed. Therefore, the ear of the hairless mouse is an excellent in vivo model to study the complex interactions during microvascular thrombus formation, thrombus evolution, and thrombolysis.

Abstract

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions between cellular and non-cellular blood components during thrombus formation, reliable studies of the physiology and pathophysiology of thrombosis can only be performed in vivo. Therefore, this article presents an ear model in hairless mice and focuses on the in vivo analysis of microcirculation, thrombus formation, and thrombus evolution. By using intravital fluorescence microscopy and the intravenous (iv) application of the respective fluorescent dyes, a repetitive analysis of microcirculation in the auricle can easily be performed, without the need for surgical preparation. Furthermore, this model can be adapted for in vivo studies of different issues, including wound healing, reperfusion injury, or angiogenesis. In summary, the ear of hairless mice is an ideal model for the in vivo study of skin microcirculation in physiological or pathophysiological conditions and for the evaluation of its reaction to different systemic or topical treatments.

Introduction

The purpose of the present article is to describe the technique of intravital microscopy applied to the auricle of the hairless mouse for the direct observation and analysis of microcirculation, thrombus formation, and thrombus evolution. With an incidence rate of 1 in 1,000, venous thrombosis is still a common cause of morbidity. Although diagnostics, prevention strategies, and therapies have been developed in recent years, one-third of venous thrombosis manifests as a pulmonary embolism¹. Arterial thrombosis plays a critical role in cardiovascular diseases, which are the most common cause of death in industrial nations. Arterial thrombosis ba....

Protocol

All in vivo experiments (7221.3-1-006/15) were conducted in accordance with the German legislation on the protection of animals and the NIH Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Research Council).

1. General Keeping of the Animals

1. Perform the experiments with male SKH1-Hr^hr mice aged 4 to 6 weeks. Use animals with a weight between 20 and 25 g.
2. Keep the animals in a pathogen-free facility and under standardized conditions of 24 to 26 °C and about 60% relative humidity, with steady access to water and food ad libitum.
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Results

Effects of Cannabinoid Treatment on Thrombogenesis

Upon injection of 0.05 mL of FITC-dextran, phototoxic thrombus induction leads to an endothelial lesion and the formation of a parietal platelet plug (Figures 2 and 3). In the present study, thrombus induction after the ip injection of cannabinoids (5 mg/kg bw) or vehicle resulted in a thrombotic vessel occlusion in all venules (Figure 4

Discussion

There are several critical steps for the successful thrombus induction in the earlobe of SKH1-Hr^hr mice. For troubleshooting, the respective steps of the protocol are indicated in parenthesis.

Examination conditions are ideal in young animals at the age of 4 - 6 weeks and with low cornification of the epidermis. In older animals, the quality of visualization of the vessels is worse and less comparable due to the higher distance between the skin surface and the target vessels (step 1.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SKH-1/hr mice	Charles River	477	can be purchased from other vendors
standard laboratory food	ssniff Spezialdiaeten	V1594-0	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
formaldehyde 4%	Sigma	HT501128-4L	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
Adhesive strips	Leukosilk	4683400	can be purchased from other vendors
centrifuge	Beckman Coulter	CLGS 15	can be purchased from other vendors
hematology analyzer	Sysmex	KX-21 A6980	can be purchased from other vendors
EDTA-blood tube	Sarstedt	201,341	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 synringe	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
Prolene 6-0	Ethicon	XN8706.P33	can be purchased from other vendors
electrocautery	Servoprax	H40140	can be purchased from other vendors
acrylglass pad			integrated heating, 0.5 cm high plane