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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 based on the rupture of atherosclerotic plaques is involved in heart attacks, mesenteric infarctions, and apoplexy. Every surgery exposes subendothelial structures to blood components, changes the dynamics of blood flow, and immobilizes the patient. In endoprosthetic surgery of the lower limb, organ transplantation and flap surgery thrombosis are frequent causes of complications. Microvascular thrombosis in particular frequently causes irreversible damage, due to the lack of clinical symptoms. Likewise, microvascular thrombosis plays a crucial rule in several diseases, including thrombotic thrombocytopenic purpura, sepsis, disseminated intravascular coagulation, antiphospholipid syndrome, and chronic venous insufficiency, among others.

Several new drugs for the therapy and prevention of thrombosis were developed in recent years, but antiplatelet drugs and anticoagulants still have side effects, lack antagonists, and feature long duration effects. These deficiencies lead to problems in emergency medical care. Thus, more research is needed to uncover the complex processes that occur during thrombosis, which can hardly be simulated in vitro.

The hairless SKH1-Hr^hr mouse was discovered 1926 in a zoo in London. Due to a gene defect on chromosome 14, the animal loses its fur after postnatal day 10. This makes the well-vascularized auricle accessible to intravital microscopy of the vessels. The average thickness of the ear is 300 µm. It consists of two layers of dermis, which are separated by cartilage. On the convex dorsal side of the cartilage, 3 vascular bundles enter the earlobe. Apical vascular arcs and basal shunts connect the three bundles. The venules have diameters between 200 µm (basal) and 10 µm (apical). Close-meshed capillaries surround the empty hair follicles². The anatomy of the hairless SKH1-Hr^hr mouse makes the auricle a powerful and cost-effective model for thrombosis research.

Protokół

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.
3. Keep up to five male animals in one cage. Provide bedding and enrichment material during the housing of the animals for their well-being.

2. Prearrangement of the Animals

1. Weigh a mouse and load the respective drug (e.g., the cannabinoid, 5 mg/kg bodyweight (bw)) into an insulin syringe. Administer the drug 30 min prior to thrombus induction.
2. By holding the neck of the mouse between the thumb and the index finger and the tail of the mouse with the little finger, stretch the animal and inject the drug intraperitoneally (ip) into the bottom left quadrant of the abdomen. Put the animal back into the cage for 15 min.
3. Prepare anesthesia with ketamine (90 mg/kg bw) and xylazine (25 mg/kg bw). 15 min prior to thrombus induction, anesthetize the mouse. Put the mouse in the cage, pull the tail slightly, and inject the anesthetics ip with an insulin syringe.
4. Put the mouse back into the cage until the onset of anesthesia. To verify sufficient anesthesia, pinch the tail with forceps.
5. Load 0.05 mL of defrosted fluorescein isothiocyanate-labeled dextran (FITC-dextran; 5%, 150 kDa) into an insulin syringe. While filling the syringe, ensure that no air bubbles remain, because even small intravenously (iv)-administered air bubbles can be lethal for the animal.
6. Place the anesthetized mouse on a heating plate in the facedown position. Adjust the heating plate to 37 °C.
7. Put eye ointment on the cornea of the mouse. Disinfect the skin and use sterile instruments.
8. Stitch two sutures of polypropylene 7/0 into the cranial and caudal edge of the right ear. Place the stitches as close to the edge and as proximal to the base as possible (Figure 1B).
9. Shift the mouse to dorsal position. Fix all legs to the acrylglass platform using adhesive strips. Hook a suture under the front teeth and position the head in dorsiflexion by sticking the suture to the acrylglass with adhesive strips.
10. Translocate the animal on the platform under the operation stereomicroscope. Use 16X magnification.

3. Preparation of the Left Jugular Vein and Injection of FITC-Dextran

Note: For microscopy of the right ear, prepare the left jugular vein.

1. Using a scalpel, create a 5-mm incision in the skin on the left side of the neck in a cranio-caudal direction. Dissect the subcutaneous tissue with a microforceps and microscissors. Either ligate crossing vessels with polyester 8/0 sutures or with electrocoagulation.
2. Free the vein from its adventitia using microforceps and microscissors without touching the vessel.
3. Use the prepared insulin syringe for the injection of the fluorescent dye. Carefully grab the vessel wall with the microforceps, without perforating the vein. Penetrate the distended vessel wall with the syringe and inject FITC-dextran iv.
4. Stop the bleeding after withdrawing the syringe using cotton swabs. Avoid blood and dye contamination of the ear.

4. Positioning of the Right Ear for Intravital Fluorescence Microscopy

1. Transfer the animal on the heating plate to an acrylglass construction with a slot for the heating plate and a 0.5 cm-high plane for positioning the ear.
2. Fix the animal face down on the heating plate using adhesive strips. Place the relatively strong and convex cartilage at the base of the ear beside the 0.5 cm-high plane for the ear (Figure 1B) so that the apical part of the ear can be positioned flat on the plane.
3. Add one drop of room-temperature 0.9% NaCl to the acrylglass plane in order to position the ear. Place the right ear, with the prearranged sutures on its concave ventral side facing downwards, on the drop of 0.9% NaCl. Using cotton swabs, absorb the drop of NaCl and let capillary forces attach the ear plane to the acrylglass.
4. Tape the sutures to the acrylglass to fix the position of the ear.
5. Add one drop of 0.9% room temperature NaCl to the convex dorsal side of the ear. Carefully put one coverslip (0.5 cm diameter) on the ear without compressing the basal vessels entering the ear. Using cotton swabs, remove as much NaCl as possible from under the coverslip in order to minimize the distance between the coverslip and the ear target vessels.

5. Intravital Fluorescence Microscopy and Thrombus Induction of the Right Ear

1. Adjust the intravital fluorescence microscope for FITC-dextran visualization (450 - 490 nm; FT: 510; LP: 520). Use a variable 100-W mercury lamp as a light source. Connect a high-resolution, black-and-white CCD camera to a DVD recorder.
2. Transfer the animal on the acrylglass containing the heating plate with the fixed abducted ear to the desk of the intravital fluorescence microscope.
3. Using 20X magnification (20X/0.95 numeric aperture) and 20% light intensity, search for a venous vessel 50 - 60 µm in diameter and with an anterograde blood flow of 400 - 600 µm/s.
4. Add one drop of room-temperature water to the coverslip for water immersion of the 63x magnification objective (63X/0.95 numeric aperture). Use a syringe with a 1-mm diameter cannula and place the drop on the objective of the microscope. Add just enough water to contact the coverslip and the objective with the water drop.
5. Immediately after the application of the water drop, begin recording the vessel for 20 s with 20% light intensity for the offline measurement of the diameter and blood flow.
6. Start thrombus induction 5 min after the injection of FITC-dextran. For this purpose, raise the light intensity to 100%.
7. During thrombus induction, close the aperture of the microscope for 2 s within a period of 30-s to check the blood flow. In case of persisting blood flow, open the aperture again. In case of stopped blood flow, observe the vessel for 30 s.
   NOTE: The vessel is classified as occluded if the flow stands still for 30 s or more or if the blood flows retrogradely. If the orthograde blood flow starts again, completely open the aperture and continue the thrombus induction until vessel occlusion occurs as described above. During early thrombus induction, ensure that the times when the aperture was closed to check the blood flow are as short as possible in order to maintain almost continuous epi-illumination. Later, during thrombus growth, the vessel is perfused with less fluorescent dye, so it can be observed continuously.
8. Select and occlude 5 vessels per ear. Limit the time of thrombus induction under the microscope to approximately 1 h after the injection of FITC-dextran.

6. Follow-up Activities

1. Perform a wound closure of the neck using transcutaneous polypropylene 6/0 sutures.
2. During recovery from anesthesia, put the mouse back into the cage and warm the animal using infrared light.
3. Transfer the recorded data from the DVD recorder to software allowing the measurement of the diameter of the vessels and velocity of blood flow.

7. Examination of the Left Ear

1. Let the animal recover and eliminate all injected FITC-dextran for 48 h.
2. Rerun the steps described above, this time preparing the right jugular vein and the left ear.

8. Tissue Asservation

1. After intravital fluorescence microscopy of the left ear, sample 0.5 mL of blood from the retrobulbar vein plexus of the eye using a glass capillary. Carefully penetrate the inner palpebral angle with screwing movements, until venous blood flows through the capillary. Collect the probe in an ethylenediaminetetraacetic acid (EDTA) blood tube.
2. After blood sampling, sacrifice the animal by injecting 500 mg/kg bw ketamine into the tail vein.
3. Count the blood cells using a hematology analyzer for a quantitative assessment of leukocytes, erythrocytes, thrombocytes, hemoglobin, and hematocrit.
4. Centrifuge the remaining EDTA blood at 2,500 x g and room temperature for 10 min. Pipette and freeze the blood plasma for further investigations.
5. Using scissors, cut the auricles and fix them in 4% formaldehyde for histological examination.

Wyniki

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

Dyskusje

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...

Materiały

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