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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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.

intravital-microscopy-thrombus-induction-earlobe-hairless

Research

JoVE Journal

Medicine

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Pharmacologic Induction of Epidermal Melanin and Protection Against Sunburn in a Humanized Mouse Model

Fairness of skin, UV sensitivity and skin cancer risk all correlate with the physiologic function of the melanocortin 1 receptor, a Gs-coupled signaling protein found on the surface of melanocytes. Mc1r stimulates adenylyl cyclase and cAMP production which, in turn, up-regulates melanocytic production of melanin in the skin. In order to study the mechanisms by which Mc1r signaling protects the skin against UV injury, this study relies on a mouse model with "humanized skin" based on epidermal expression of stem cell factor (Scf). K14-Scf transgenic mice retain melanocytes in the epidermis and therefore have the ability to deposit melanin in the epidermis. In this animal model, wild type Mc1r status results in robust deposition of black eumelanin pigment and a UV-protected phenotype. In contrast, K14-Scf animals with defective Mc1r signaling ability exhibit a red/blonde pigmentation, very little eumelanin in the skin and a UV-sensitive phenotype. Reasoning that eumelanin deposition might be enhanced by topical agents that mimic Mc1r signaling, we found that direct application of forskolin extract to the skin of Mc1r-defective fair-skinned mice resulted in robust eumelanin induction and UV protection 1. Here we describe the method for preparing and applying a forskolin-containing natural root extract to K14-Scf fair-skinned mice and report a method for measuring UV sensitivity by determining minimal erythematous dose (MED). Using this animal model, it is possible to study how epidermal cAMP induction and melanization of the skin affect physiologic responses to UV exposure.

Epidermal melanin is induced by topical application of forskolin in a murine model of the fair-skinned UV-sensitive human. Pharmacologic manipulation of cAMP levels in the skin and epidermal darkening strongly protect against UV-mediated inflammation (sunburn) as measured by the minimum erythematous dose (MED) assay.

Fairness of skin, UV sensitivity and skin cancer  risk all correlate with the physiologic function of the melanocortin 1  receptor, a Gs-coupled signaling ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

Combined Intravital Microscopy and Contrast-enhanced Ultrasonography of the Mouse Hindlimb to Study Insulin-induced Vasodilation and Muscle Perfusion

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate postprandial delivery of nutrients and hormones to insulin-sensitive tissues. We here describe a technique for combining intravital microscopy (IVM) and contrast-enhanced ultrasonography (CEUS) of the adductor compartment of the mouse hindlimb to simultaneously visualize muscle resistance arteries and perfusion of the microcirculation in vivo. Simultaneously assessing insulin's effect at multiple levels of the vascular tree is important to study relationships between insulin's multiple vasoactive effects and muscle perfusion. Experiments in this study were performed in mice. First, the tail vein cannula is inserted for the infusion of anesthesia, vasoactive compounds and ultrasound contrast agent (lipid-encapsulated microbubbles). Second, a small incision is made in the groin area to expose the arterial tree of the adductor muscle compartment. The ultrasound probe is then positioned at the contralateral upper hindlimb to view the muscles in cross-section. To assess baseline parameters, the arterial diameter is assessed and microbubbles are subsequently infused at a constant rate to estimate muscle blood flow and microvascular blood volume (MBV). When applied before and during a hyperinsulinemic-euglycemic clamp, combined IVM and CEUS allow assessment of insulin-induced changes of arterial diameter, microvascular muscle perfusion and whole-body insulin sensitivity. Moreover, the temporal relationship between responses of the microcirculation and the resistance arteries to insulin can be quantified. It is also possible to follow-up the mice longitudinally in time, making it a valuable tool to study changes in vascular and whole-body insulin sensitivity.

Insulin-induced vasodilation regulates muscle perfusion and increases the microvascular surface area (microvascular recruitment) available for solute exchange between blood and tissue interstitium. Combined intravital microscopy and contrast-enhanced ultrasonography is presented to simultaneously assess insulin&#39;s action at the larger vessels and the microcirculation in vivo.

It has been demonstrated that insulin's vascular actions contribute to regulation of insulin sensitivity. Insulin's effects on muscle perfusion regulate ...

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 ...

Intravital Microscopy of Monocyte Homing and Tumor-Related Angiogenesis in a Murine Model of Peripheral Arterial Disease

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic stenosis. Vascular surgery is a viable option in selected cases, but for patients without indications for surgery such as progression to rest pain, critical limb ischemia, or major disruptions to life or work, there are few possibilities for mitigating their disease. Cell therapy via monocyte-enhanced perfusion through the stimulation of collateral formation is one of a few non-invasive options.
Our group examines arteriogenesis after monocyte transplantation into mice using the hindlimb ischemia model. Previously, we have demonstrated improvement in hindlimb perfusion using tetanus-stimulated syngeneic monocyte transplantation. In addition to the effects on the collateral formation, tumor growth could be affected by this therapy as well. To investigate these effects, we use a basement membrane-like matrix mouse model by injecting the extracellular matrix of the Engelbreth-Holm-Swarm sarcoma into the flank of the mouse, after occlusion of the femoral artery.
After the artificial tumor studies, we use intravital microscopy to study in vivo tumor-angiogenesis and monocyte homing within collateral arteries. Previous studies have described the histological examination of animal models, which presupposes subsequent analysis to post-mortem artifacts. Our approach visualizes monocyte homing to areas of collateralization in real time sequences, is easy to perform, and investigates the process of arteriogenesis and tumor angiogenesis in vivo.

Monocytes are important mediators of arteriogenesis in the context of peripheral arterial disease. Using a basement membrane-like matrix and intravital microscopy, this protocol investigates monocyte homing and tumor-related angiogenesis after monocyte injection in the femoral artery ligation murine model.

The therapeutic goal for peripheral arterial disease and ischemic heart disease is to increase blood flow to ischemic areas caused by hemodynamic ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

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.