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

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Alla in vivo experiment (7221.3-1-006 / 15) genomfördes i enlighet med den tyska lagstiftningen om skydd av djur och NIH Guide för skötsel och användning av försöksdjur (Institute of Laboratory Animal Resources, National Research Council). 1. Allmän djurhållningen <ol><li> Utföra experimenten med manliga SKH1-hr hr möss åldern 4 till 6 veckor. Använda djur med en vikt mellan 20 och 25 g. </li><li> Hålla djuren i en patogenfri anläggning och under standardi...

<ol>
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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+coronary+atherosclerosis+in+swine+produced+by+combination+of+balloon-catheter+injury+and+cholesterol+feeding.">Advanced coronary atherosclerosis in swine produced by combination of balloon-catheter injury and cholesterol feeding.</a> Exp Mol Pathol. 23 (3), 491-499 (1975).</li><li>Callahan, A. B., Lutz, B. R., Fulton, G. P., Degelman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smooth+muscle+and+thrombus+thresholds+to+unipolar+stimulation+of+small+blood+vessels.">Smooth muscle and thrombus thresholds to unipolar stimulation of small blood vessels.</a> Angiology. 11, 35-39 (1960).</li><li>Rosen, E. 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. D., R&#246;sken, M., R&#252;cker, M., Seiffge, D., Vollmar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antithrombotic+and+thrombolytic+effectiveness+of+rhirudin+in+microvessels.">Antithrombotic and thrombolytic effectiveness of rhirudin in microvessels.</a> Langenbecks Arch Chir. 115 (1), 19-20 (1998).</li><li>Bilheiro, R. P., 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=The+thrombolytic+action+of+a+proteolytic+fraction+(P1G10)+from+Carica+candamarcensis.">The thrombolytic action of a proteolytic fraction (P1G10) from Carica candamarcensis.</a> Thromb Res. 131 (4), 175-182 (2013).</li><li>Kram, L., Grambow, E., Mueller-Graf, F., Sorg, H., Vollmar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anti-thrombotic+effect+of+hydrogen+sulfide+is+partly+mediated+by+an+upregulation+of+nitric+oxide+synthases.">The anti-thrombotic effect of hydrogen sulfide is partly mediated by an upregulation of nitric oxide synthases.</a> Thromb Res. 132 (2), 112-117 (2013).</li></ol>

Det finns flera viktiga steg för en framgångsrik tromb induktion i örsnibben av SKH1-hr hr möss. För felsökning är de respektive stegen i protokollet indikeras i parentes. Examination förhållandena är idealiska i unga djur vid en ålder av 4 - 6 veckor och med låg förhorning av epidermis. I äldre djur, är kvaliteten på visualisering av kärlen värre och mindre jämförbar grund av den högre avståndet mellan hudytan och målsekärlen (steg 1,1). <p class="jove...

Syftet med denna artikel är att beskriva den teknik för intravital mikroskopi appliceras på hjärtörat av den hårlösa musen för direkt observation och analys av mikrocirkulationen, trombbildning, och tromb evolution. Med en incidensen av en i 1000, är ​​venös trombos fortfarande en vanlig orsak till sjuklighet. Även diagnostik, förebyggande strategier och behandlingar har utvecklats under de senaste åren, en tredjedel av ventrombos manifesterar som en lungemboli 1. Arteriell trombos spelar en avgörande roll i hjärt- och kärlsjukdomar, som är den vanligaste dödsorsaken i industriländerna. Arteriell trombos baseras på bristning av aterosklerotiska plack är involverad i hjärtinfarkt, mesenteriska infarkter och slaganfall. Varje operation exponerar subendoteliala strukturer till blodkomponenter, ändrar dynamiken i blodflödet, och immobiliserar patienten. I endoprotesanordning kirurgi av den nedre extremiteten, organ transplantation och flikkirurgi trombos är vanliga orsaker till komplikationer. Mikrovaskulär trombos i synnerhet orsakar ofta obotliga skador på grund av bristen på kliniska symptom. Likaså spelar mikrovaskulär trombos en avgörande regel i flera sjukdomar, inklusive trombotisk trombocytopen purpura, sepsis, disseminerad intravaskulär koagulation, antifosfolipidsyndrom och kronisk venös insufficiens, bland annat. Flera nya läkemedel för behandling och förebyggande av blodpropp har utvecklats under de senaste åren, men trombocythämmande läkemedel och antikoagulantia har fortfarande biverkningar, brist antagonister och har långvariga effekter. Dessa brister leder till problem i akut medicinsk vård. Sålunda behövs mer forskning för att avslöja de komplexa processer som sker under trombos, vilket knappast kan simuleras in vitro. Den hårlösa SKH1-Hr HR mus upptäcktes 1926 i en djurpark i London.På grund av en gendefekt på kromosom 14, förlorar djuret pälsen efter postnatal dag 10. Detta gör den väl vaskulariserad ytteröra tillgänglig för intravital mikroskopi av kärlen. Den genomsnittliga tjockleken av örat är 300 um. Det består av två skikt av dermis, som skiljs åt av brosk. På den konvexa dorsala sidan av brosket, 3 kärlknippena in i örsnibben. Apikala kärl bågar och basala shuntar ansluta tre buntar. De venoler har diametrar mellan 200 ^ m (basal) och 10 | j, m (apikala). Finmaskigt kapillärer omger tomma hårsäckar 2. Anatomi hårlösa SKH1-Hr HR musen gör öronmusslan ett kraftfullt och kostnadseffektivt modell för trombos forskning.

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			<td height="19" style="height:19px;width:216px;">SKH-1/hr mice</td>
			<td style="width:161px;">Charles River</td>
			<td style="width:231px;">477</td>
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			<td height="17" style="height:17px;width:216px;">standard laboratory food</td>
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			<td>V1594-0&#160;</td>
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			<td height="19" style="height:19px;width:216px;">intravital microscope</td>
			<td style="width:161px;">Zeiss</td>
			<td>Axiotech Vario 100&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
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			<td height="19" style="height:19px;width:216px;">objective (20x/0.95)&#160;</td>
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			<td>20x/0,50 W; Plan-NEOFLUAR&#160;</td>
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			<td>63x/0,95 W; ACHROPLAN&#160;</td>
			<td>can be purchased from other vendors&#160;</td>
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			<td style="width:231px;">5/12612055/1011</td>
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			<td style="width:231px;">604-A-1</td>
			<td>can be purchased from other vendors&#160;</td>
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			<td height="19" style="height:19px;width:216px;">infrared light</td>
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			<td>can be purchased from other vendors&#160;</td>
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			<td height="19" style="height:19px;width:216px;">single use synringe</td>
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			<td height="19" style="height:19px;width:216px;">disposable hypodermic needles</td>
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			<td height="19" style="height:19px;width:216px;">scissors 14,5 cm</td>
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			<td>integrated heating, 0,5 cm high plane&#160;</td>
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</table>

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

Trombotiska komplikationer av kärlsjukdomar är en ledande orsak till sjuklighet och dödlighet i industriländerna. På grund av att de komplexa interaktionerna mellan cellulära och icke-cellulära blodkomponenter under trombbildning, kan tillförlitliga studier av fysiologi och patofysiologi av trombos endast utföras in vivo. Därför denna artikel presenteras en öronmodell hårlösa möss och fokuserar på analys av mikrocirkulationen, blodproppsbildning och tromb evolution in vivo. Genom användning av intravital fluorescensmikroskopi och intravenös (iv) applicering av respektive fluorescerande färgämnen, kan lätt utföras en repetitiv analys av mikrocirkulationen i hjärtörat, utan behov av kirurgisk beredning. Dessutom kan denna modell anpassas för in vivo-studier av olika frågor, inklusive sårläkning, reperfusionsskada, eller angiogenes. Sammanfattningsvis är örat av hårlösa möss en idealmodell för in vivo studie av hud mikrocirkulation i fysiologiska eller patofysiologiska tillstånd och för utvärdering av dess reaktion på olika systemiska eller utvärtes behandlingar.

Effekter av Cannabinoid Behandling på trombogenes Efter injektion av 0,05 ml av FITC-dextran, leder fototoxisk tromb induktion till en endotel lesion och bildandet av en parietal plättplugg (fig 2 och 3). I föreliggande studie, tromb induktion efter ip-injektion av cannabinoider (5 mg / kg kroppsvikt) eller vehikel resulterade i en trombotisk kärlocklusion i alla venoler (Figur 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

Örat modell av den hårlösa SKH1-Hr hr mus möjliggör intravital fluorescensmikroskopi av mikrocirkulationen och fototoxisk tromb induktion utan föregående kirurgisk beredning i den undersökta mikrovaskulära bädden. Därför är örat av den hårlösa musen ett utmärkt in vivo-modell för att studera de komplexa interaktioner under mikrovaskulär trombbildning, tromb evolution och trombolys.

Intravital mikroskopi och trombos induktion i örsnibben av en hårlös mus

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.

Research

JoVE Journal

Medicine

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

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

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Cardiac Magnetic Resonance for the Evaluation of Suspected Cardiac Thrombus: Conventional and Emerging Techniques

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