Name: intravital microscopy and thrombus induction in the earlobe of a hairless mouse
Uploaded: 2017-04-02T14:00:00
Description: Watch this Scientific Journal Video about Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse at JoVE.com

The authors have no acknowledgements.

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
<ol>
	<li>Perform the experiments with male SKH1-Hrhr mice aged 4 to 6 weeks. Use animals with a weight between 20 and 25 g.</li>
	<li>Keep the animals in a pathogen-free facility an...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+aggregation+induced+in+the+hamster+cheek+pouch+by+a+photochemical+process+with+excited+fluorescein+isothiocyanate-dextran.">Platelet aggregation induced in the hamster cheek pouch by a photochemical process with excited fluorescein isothiocyanate-dextran.</a> Microvasc Res. 26 (2), 238-249 (1983).</li><li>Rumbaut, R. E., Slaff, D. W., Burns, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+thrombosis+models+in+venules+and+arterioles+in+vivo.">Microvascular thrombosis models in venules and arterioles in vivo.</a> Microcirculation. 12 (3), 259-274 (2005).</li><li>Lee, W. M., Lee, K. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+noninvasive+vascular+injury+models+in+mice+generate+platelet-+and+coagulation-dependent+thrombi.">Laser-induced noninvasive vascular injury models in mice generate platelet- and coagulation-dependent thrombi.</a> Am J Pathol. 158 (5), 1613-1622 (2001).</li><li>Agero, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+mutalysin+II+on+vascular+recanalization+after+thrombosis+induction+in+the+ear+of+the+hairless+mice+model.">Effect of mutalysin II on vascular recanalization after thrombosis induction in the ear of the hairless mice model.</a> Toxicon. 50 (5), 698-706 (2007).</li><li>Menger, M. 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There are several critical steps for the successful thrombus induction in the earlobe of SKH1-Hrhr 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...

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-Hrhr 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 &#181;m. 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 &#181;m (basal) and 10 &#181;m (apical). Close-meshed capillaries surround the empty hair follicles2. The anatomy of the hairless SKH1-Hrhr mouse makes the auricle a powerful and cost-effective model for thrombosis research.

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			<td style="width:231px;">XNEH7470</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">Prolene 6-0</td>
			<td style="width:161px;">Ethicon</td>
			<td style="width:231px;">XN8706.P33</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">electrocautery</td>
			<td style="width:161px;">Servoprax</td>
			<td style="width:231px;">H40140</td>
			<td>can be purchased from other vendors&#160;</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:216px;">acrylglass pad</td>
			<td style="width:161px;"></td>
			<td style="width:231px;"></td>
			<td>integrated heating, 0,5 cm high plane&#160;</td>
		</tr>
	</tbody>
</table>

intravital microscopy and thrombus induction in the earlobe of a hairless mouse

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.

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</s...

Watch this Scientific Journal Video about Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse at JoVE.com

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

The ear model of the hairless SKH1-Hrhr 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.

Thrombotic complications of vascular diseases are one leading cause of morbidity and mortality in industrial nations. Due to the complex interactions ...

The overall goal of this experiment is to study the influence of potential vasoactive drugs on microvascular thrombus formation in vivo. This method can help answer key questions in thrombus formation such as identifying new antithrombotic substances that could help to prevent microvascular thrombus formation, which is one of the main complications in sepsis and during tissue transplantation. The main advantage of this technique is that the ear model of the hairless mouse enables intravital fluorescence microscopy and phototoxic thrombus induction without prior surgical preparation in the examined microvascular bed.Begin by weighing and recording the weight of the mouse. Then load the appropriate volume of the test substance into an insulin syringe. Administer the drug 30 minutes prior to thrombus induction.After anesthetizing the mouse 15 minutes prior to thrombus induction, place the mouse back into the cage until the onset of anesthesia. To verify sufficient anesthesia, pinch the tail with forceps and note the absence of a reaction. Next, 0.05 ml of defrosted 50 Dextran into an insulin syringe.After filling the syringe, ensure that no air bubbles remain, because even small intravenously administered air bubbles can be lethal for the animal. Place the anesthetized mouse on an acroglass pad with an integrated heating plate in the face-down position. Adjust the heating plate to 37 degress Celsius.Cover the mouse eye corneas with ophthalmic ointment. Then 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.Shift the mouse to a dorsal position. Fix all legs to the platform using adhesive bandage. Hook a suture under the front teeth and position the head in dorsiflexion by affixing the suture to the platform with adhesive bandage.Place the animal on the platform under the stereo microscope. Use 10X magnification. For microscopy of the right ear, prepare the left jugular vein by first creating a 5 mm incision in the skin of the neck in a craniocaudal direction.Then use microforceps and microscissors to dissect the subcutaneous tissue. Then, free the vein from its adventia without touching the vessel. Now, use the previously prepared insulin syringe for the injection of the fluoroscent dye.Carefully grab the vessel wall with the microforceps without perforating the vein. Penetrate the distended vessel wall with the syringe needle and inject 50 Dextran intravenously. Withdraw the needle and stop the bleeding using cotton swabs.Avoid blood and dye contamination of the ear. Transfer the animal on the heating plate to an acro glass construction with a slat for the hearing plate and 0.5 cm high plane for positioning the ear. Fix the animal in a prone position on the heating plate using adhesive bandage.Place the convex cartilage at the base of the ear beside the plane for the ear so that the apical part of the ear can be positioned flat on the plane. Add one drop of room temperature aqua to the acro glass plate. Using cotton swabs, absorb the drop of aqua, and let capillary forces attach the ear plane to the acro glass.The auricle must be positioned as flat as possible, even though the cartilage gives the auricle a convex shape. So only the more flexible distal part of the auricle should be positioned on the acrylic glass plate. To avoid tissue damage and extravasation of fluids and dye, touch the ear as little as possible with the forceps.Next, add drop of aqua to the convex dorsal side of the ear. Carefully put one cover slip on the ear without compressing the basal vessels entering the ear. Use cotton swabs to remove as much aqua as possible from under the cover slip To minimize the distance between the cover slip and the ear target vessels.Begin by adjusting the intravital fluorescence microscope for 50 Dextran visualization. Tranfer the prepared animal to the deck of the intravital fluorescence microscope. Using 5X, 10X and 20X magnification, and 20%light intensity, search for a venous vessel of 50 to 60 microns in diameter and with an anterograde blood flow.Add one drop of room-temperature water to the cover slip for water immersion of the 63X magnification objective. Immediately after the application of the water drop, begin recording the vessel for 20 seconds for the baseline assessment of the diameter and blood flow. Start thrombus induction five minutes after the injection of 50 Dextran.For this purpose, raise the light intensity to 100%During phototoxic thrombus induction, close the aperture of the microscope for two seconds within a 30-second period to check for occlusion of blood flow. If blood flow persists, open the aperture again. The vessel is classified as occluded if the flow stands still for 30 seconds or more, or if the blood flow is retrograde.Select and occlude five vessels per ear within a one hour period after injection of 50 Dextran. Here, animals were treated with vehicle or with a cannabinoids, anandamide, WIN 155, 212-2 or cannabidiol before injection of 50 Dextan prior to thrombus induction. Anandamide, but not CBD, and WIN 155, 212-2 significantly accelerated thrombus growth after one-time treatment as compared to vehicle control.In another setting, anandamide was combined with indomethacin to assess the impact of cyclooxygenase-dependent products on thrombus formation via anandamide. Co-administration with indomethacin neutralized the pro-thrombotic effect of anandamide. Therefore, acute exposure to anandamide elicits a cyclooxygenase-dependent pro-thrombotic effect in vivo.Once mastered, thrombus induction in tight vessels can be performed in 90 minutes, including preparation. While attempting this procedure, it's important to handle the auricle very carefully every time it is touched to avoid extravasation of the fluids and dye. Though this method can provide insight into thrombus formation, it can also be applied to other systems, such as wound healing, flap failure, and angiogenesis.After watching this video, you should have a good understanding of intravenous fluorescence dye application, position of the auricle, and illumination of specific vessels in order to perform intravital phototoxic thrombus induction.

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