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 and under standardized conditions of 24 to 26 &#176;C and about 60% relative humidity, with steady access to water and food ad libitum.</li>
	<li>Keep up to five male animals in one cage. Provide bedding and enrichment material during the housing of the animals for their well-being.</li>
</ol>
2. Prearrangement of the Animals
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Put the mouse back into the cage until the onset of anesthesia. To verify sufficient anesthesia, pinch the tail with forceps.</li>
	<li>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.</li>
	<li>Place the anesthetized mouse on a heating plate in the facedown position. Adjust the heating plate to 37 &#176;C.</li>
	<li>Put eye ointment on the cornea of the mouse. Disinfect the skin and use sterile instruments.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Translocate the animal on the platform under the operation stereomicroscope. Use 16X magnification.</li>
</ol>
3. Preparation of the Left Jugular Vein and Injection of FITC-Dextran
Note: For microscopy of the right ear, prepare the left jugular vein.
<ol>
	<li>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.</li>
	<li>Free the vein from its adventitia using microforceps and microscissors without touching the vessel.</li>
	<li>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.</li>
	<li>Stop the bleeding after withdrawing the syringe using cotton swabs. Avoid blood and dye contamination of the ear.</li>
</ol>
4. Positioning of the Right Ear for Intravital Fluorescence Microscopy
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Tape the sutures to the acrylglass to fix the position of the ear.</li>
	<li>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.</li>
</ol>
5. Intravital Fluorescence Microscopy and Thrombus Induction of the Right Ear
<ol>
	<li>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.</li>
	<li>Transfer the animal on the acrylglass containing the heating plate with the fixed abducted ear to the desk of the intravital fluorescence microscope.</li>
	<li>Using 20X magnification (20X/0.95 numeric aperture) and 20% light intensity, search for a venous vessel 50 - 60 &#181;m in diameter and with an anterograde blood flow of 400 - 600 &#181;m/s.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Start thrombus induction 5 min after the injection of FITC-dextran. For this purpose, raise the light intensity to 100%.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Follow-up Activities
<ol>
	<li>Perform a wound closure of the neck using transcutaneous polypropylene 6/0 sutures.</li>
	<li>During recovery from anesthesia, put the mouse back into the cage and warm the animal using infrared light.</li>
	<li>Transfer the recorded data from the DVD recorder to software allowing the measurement of the diameter of the vessels and velocity of blood flow.</li>
</ol>
7. Examination of the Left Ear
<ol>
	<li>Let the animal recover and eliminate all injected FITC-dextran for 48 h.</li>
	<li>Rerun the steps described above, this time preparing the right jugular vein and the left ear.</li>
</ol>
8. Tissue Asservation
<ol>
	<li>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.</li>
	<li>After blood sampling, sacrifice the animal by injecting 500 mg/kg bw ketamine into the tail vein.</li>
	<li>Count the blood cells using a hematology analyzer for a quantitative assessment of leukocytes, erythrocytes, thrombocytes, hemoglobin, and hematocrit.</li>
	<li>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.</li>
	<li>Using scissors, cut the auricles and fix them in 4% formaldehyde for histological examination.</li>
</ol>

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The authors have nothing to disclose.

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.

<table><tbody><tr><td>SKH-1/hr mice</td><td>Charles River</td><td>477</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>standard laboratory food</td><td>ssniff Spezialdiaeten</td><td>V1594-0&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>operation stereomicroscope</td><td>Leica&amp;nbsp;</td><td>M651/M655&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>intravital microscope</td><td>Zeiss</td><td>Axiotech Vario 100&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>objective (20X/0.95)&amp;nbsp;</td><td>Zeiss</td><td>20x/0,50 W; Plan-NEOFLUAR&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>objective (63X/0.95)</td><td>Zeiss</td><td>63x/0,95 W; ACHROPLAN&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>black and white CCD-camera&amp;nbsp;</td><td>Pieper&amp;nbsp;</td><td>FK 6990 IQ-S&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>DVD-recorder</td><td>Panasonic</td><td>DMR-EX99V&amp;nbsp;</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>sodium chloride</td><td>Braun</td><td>5/12612055/1011</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Ketamine 10%</td><td>Bela pharm</td><td>F3901-6</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Xylazine 2%</td><td>Bayer</td><td>6293841.00.00</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>FITC-dextran 5%</td><td>Sigma&amp;nbsp;</td><td>46945-100MG-F</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>dexapanthenol 5% eye ointment</td><td>Bayer</td><td>6029009.00.00</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>formaldehyde 4%</td><td>Sigma</td><td>HT501128-4L</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>DMSO</td><td>Sigma</td><td>472301</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>coverslips 5 x 5 x 1 mm</td><td>Menzel</td><td>L4339</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Adhesive strips</td><td>Leukosilk</td><td>4683400</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>centrifuge</td><td>Beckman Coulter</td><td>CLGS 15</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>hematology analyzer</td><td>Sysmex</td><td>KX-21 A6980</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>EDTA-blood tube</td><td>Sarstedt</td><td>201,341</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>cotton swabs</td><td>Sanyo</td><td>604-A-1</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>infrared light</td><td>Beurer</td><td>5/13855</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>single use synringe</td><td>Braun&amp;nbsp;</td><td>2020-08</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>insulin syringe</td><td>Braun</td><td>9161502</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>disposable hypodermic needles</td><td>Braun</td><td>465 7640</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>end-to-end capillary</td><td>Sarstedt</td><td>19,447</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>heating plate</td><td>Klaus Effenberg</td><td>OP-T 185/03</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>scissors 14.5 cm</td><td>Aesculap</td><td>BC259R</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>needle Holder</td><td>Aesculap</td><td>BM081R</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>microforceps</td><td>Aesculap</td><td>BD331R</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>microscissors</td><td>Aesculap</td><td>OC496R</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>scalpel 21</td><td>Dahlhausen</td><td>11.000.00.511</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Prolene 7-0</td><td>Ethicon</td><td>XNEH7470</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>Prolene 6-0</td><td>Ethicon</td><td>XN8706.P33</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>electrocautery</td><td>Servoprax</td><td>H40140</td><td>can be purchased from other vendors&amp;nbsp;</td></tr><tr><td>acrylglass pad</td><td></td><td></td><td>integrated heating, 0.5&amp;nbsp;cm high plane</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 ...

intravital-microscopy-thrombus-induction-earlobe-hairless

Research

JoVE Journal

Medicine

9.7K Views.  Rostock University Medical Center.  Het oor model van de kale SKH1-Hr uur muis maakt intravitale fluorescentiemicroscopie microcirculatie en fototoxische trombus inductie zonder voorafgaande chirurgische preparaten in de onderzochte microvasculaire bed. Daarom is het oor van de haarloze muis is een uitstekend in vivo model voor de complexe interacties te bestuderen tijdens microvasculaire trombusvorming, trombus evolutie, en trombolyse. 

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

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neuroscience

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

Immunology and Infection

Long-term Intravital Immunofluorescence Imaging of Tissue Matrix Components with Epifluorescence and Two-photon Microscopy

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue function during development and organ homeostasis. It does so by acting via biochemical, biomechanical, and biophysical signaling pathways, such as through the release of bioactive ECM protein fragments, regulating tissue tension, and providing pathways for cell migration. The extracellular matrix of the tumor microenvironment undergoes substantial remodeling, characterized by the degradation, deposition and organization of fibrillar and non-fibrillar matrix proteins. Stromal stiffening of the tumor microenvironment can promote tumor growth and invasion, and cause remodeling of blood and lymphatic vessels. Live imaging of matrix proteins, however, to this point is limited to fibrillar collagens that can be detected by second harmonic generation using multi-photon microscopy, leaving the majority of matrix components largely invisible. Here we describe procedures for tumor inoculation in the thin dorsal ear skin, immunolabeling of extracellular matrix proteins and intravital imaging of the exposed tissue in live mice using epifluorescence and two-photon microscopy. Our intravital imaging method allows for the direct detection of both fibrillar and non-fibrillar matrix proteins in the context of a growing dermal tumor. We show examples of vessel remodeling caused by local matrix contraction. We also found that fibrillar matrix of the tumor detected with the second harmonic generation is spatially distinct from newly deposited matrix components such as tenascin C. We also showed long-term (12 hours) imaging of T-cell interaction with tumor cells and tumor cells migration along the collagen IV of basement membrane. Taken together, this method uniquely allows for the simultaneous detection of tumor cells, their physical microenvironment and the endogenous tissue immune response over time, which may provide important insights into the mechanisms underlying tumor progression and ultimate success or resistance to therapy.

The extracellular matrix undergoes substantial remodeling during wound healing, inflammation and tumorigenesis. We present a novel intravital immunofluorescence microscopy approach to visualize the dynamics of fibrillar as well as mesh-like matrix components with high spatial and temporal resolution using epifluorescence or two-photon microscopy.

Besides being a physical scaffold to maintain tissue morphology, the extracellular matrix (ECM) is actively involved in regulating cell and tissue ...

Bioengineering

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Multiphoton Intravital Imaging for Monitoring Leukocyte Recruitment during Arteriogenesis in a Murine Hindlimb Model

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for analyzing collateral arteries and leukocytes in arteriogenesis is ex vivo (immuno-) histological methodology. However, this technique does not allow the measurement of dynamic processes such as blood flow, shear stress, cell-cell interactions, and particle velocity. This paper presents a protocol to monitor in vivo processes in growing collateral arteries during arteriogenesis utilizing intravital imaging. The method described here is a reliable tool for dynamics measurement and offers a high-contrast analysis with minimal photo-cytotoxicity, provided by multiphoton excitation microscopy. Prior to analyzing growing collateral arteries, arteriogenesis was induced in the adductor muscle of mice by unilateral ligation of the femoral artery.
After the ligation, the preexisting collateral arteries started to grow due to increased shear stress. Twenty-four hours after surgery, the skin and subcutaneous fat above the collateral arteries were removed, constructing a pocket for further analyses. To visualize blood flow and immune cells during in vivo imaging, CD41-fluorescein isothiocyanate (FITC) (platelets) and CD45-phycoerythrin (PE) (leukocytes) antibodies were injected intravenously (i.v.) via a catheter placed in the tail vein of a mouse. This article introduces intravital multiphoton imaging as an alternative or in vivo complementation to the commonly used static ex vivo (immuno-) histological analyses to study processes relevant for arteriogenesis. In summary, this paper describes a novel and dynamic in vivo method to investigate immune cell trafficking, blood flow, and shear stress in a hindlimb model of arteriogenesis, which enhances evaluation possibilities notably.

The recruitment of leukocytes and platelets constitutes an essential component necessary for the effective growth of collateral arteries during arteriogenesis. Multiphoton microscopy is an efficient tool for tracking cell dynamics with high spatio-temporal resolution in vivo and less photo-toxicity to study leukocyte recruitment and extravasation during arteriogenesis.

Arteriogenesis strongly depends on leukocyte and platelet recruitment to the perivascular space of growing collateral vessels. The standard approach for ...

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing.
Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

In Vivo Two-photon Imaging of Megakaryocytes and Proplatelets in the Mouse Skull Bone Marrow

We describe here the method for imaging megakaryocytes and proplatelets in the marrow of the skull bone of living mice using two-photon microscopy.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their ...

Biology

Intravital Microscopy to Study Platelet-Leukocyte-Endothelial Interactions in the Mouse Liver

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the end pathway for both inflammation and thrombosis, it is not capable of showing the temporal changes that occur throughout the time course of these processes. Intravital microscopy (IVM) is the use of live-animal imaging to gain temporal insight into physiologic processes in vivo. This method is particularly powerful when assessing cellular and protein interactions within the circulation due to the rapid and sequential events that are often necessary for these interactions to occur. While IVM is an extremely powerful imaging methodology capable of viewing complex processes in vivo, there are a number of methodological factors that are important to consider when planning an IVM study. This paper outlines the process of conducting intravital imaging of the liver, identifying important considerations and potential pitfalls that may arise. Thus, this paper describes the use of IVM to study platelet-leukocyte-endothelial interactions in liver sinusoids to study the relative contributions of each in different models of acute liver injury.

Intravital microscopy is a powerful tool that provides insight into both the temporal and spatial relationships of rapid and/or sequential processes. Herein, we describe a protocol to assess both protein-protein interactions and platelet-neutrophil-endothelial interactions in liver sinusoids in a murine model of experimental sepsis (endotoxemia).

Inflammation and thrombosis are complex processes that occur primarily in the microcirculation. Although standard histology may provide insight into the ...

Intravital Fluorescence Microscopy to Study Microvascular Thrombus Formation

Source: Str&#252;der, D., et al. <a href="https://app.jove.com/v/55174">Intravital Microscopy and Thrombus Induction in the Earlobe of a Hairless Mouse.</a> 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.

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

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