Name: ferric chloride-induced arterial thrombosis and sample collection for 3d electron microscopy analysis
Uploaded: 2023-03-17T13:45:00
Description: Watch this Scientific Journal Video about Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis at JoVE.com

Forfatterne takker medlemmerne af Whiteheart Laboratory for deres omhyggelige gennemlæsning af dette manuskript. Arbejdet blev støttet af tilskud fra NIH, NHLBI (HL56652, HL138179 og HL150818) og en Department of Veterans Affairs Merit Award til S.W.W., R01 HL 155519 til BS og NIBIB intramural programtilskud til RDL.

Alle eksperimenter, der diskuteres her, blev gennemgået og godkendt af Institutional Animal Care and Use Committee (IACUC) ved University of Kentucky.
BEMÆRK: Kirurgiske instrumenter er anført i figur 1 og materialetabellen. C57BL/6J mus, 8-10 uger gamle, han/hun eller relevante genetisk manipulerede (Knockout eller Knockin) stammer blev anvendt.
1. FeCl 3-induceret halspulsåreskade
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Den topiske anvendelse af FeCl3 til vaskulaturen for at inducere trombose er en meget anvendt teknik og har været medvirkende til at etablere roller for forskellige blodpladereceptorer, ligandsignalveje og deres hæmmere20,21,22,23. Mekanismen, hvorigennemFeCl3 forårsager trombose, er mangefacetteret; Tidligere blev endoteldenudation betragtet som en årsag til trombose...

Trombose er dannelsen af en blodprop, der helt eller delvis blokerer et blodkar, hvilket hæmmer blodets naturlige strømning. Dette fører til alvorlige og dødelige kardiovaskulære hændelser, såsom iskæmisk hjertesygdom og slagtilfælde. Hjerte-kar-sygdomme er den hyppigste årsag til sygelighed og dødelighed og forårsager et ud af fire dødsfald på verdensplan 1,2,3. Selvom trombose manifesteres som en funktionsfejl i vaskulærsystemet, kan det være et resultat af en underliggende mikrobiel eller viral infektion, immunforstyrrelse, malignitet eller metabolisk tilstand. Blodstrømmen opretholdes af den komplekse interaktion mellem forskellige komponenter i vaskulærsystemet, herunder endotelceller, røde / hvide blodlegemer, blodplader og koagulationsfaktorer4. Ved vaskulær skade interagerer blodplader med klæbende proteiner på subendotelmatrixen og frigiver deres granulære indhold, som rekrutterer flere blodplader5. Samtidig aktiveres koagulationskaskaden, hvilket fører til fibrindannelse og aflejring. I sidste ende dannes en blodprop, der indeholder blodplader og røde blodlegemer fanget i et fibrinnet6. Selvom antiplatelet og antikoagulerende lægemidler er tilgængelige for at modulere trombose, er falsk blødning fortsat et stort problem med disse terapier, hvilket kræver finjustering af doser og kombinationer af disse lægemidler. Der er således stadig et presserende behov for at opdage nye antitrombotiske lægemidler7.
Trombose undersøges ved hjælp af flere metoder til at påføre vaskulær skade: mekanisk (karligering), termisk (laserskade) og kemisk skade (FeCl3 / Rose Bengal applikation). Trombosens art varierer afhængigt af placeringen (arteriel vs. venøs), metode eller omfang af skaden. Blandt alle disse typer er FeCl 3-induceret vaskulær skade den mest anvendte metode. Det har været anvendt i mus, rotter, kaniner, marsvin og hunde 8,9,10,11,12. Metoden er relativt enkel, nem at bruge, og hvis vigtige parametre er standardiserede, er den følsom og reproducerbar i forskellige vaskulære systemer (fx arterier [carotis og lårben], vener [jugular] og arterioler [cremaster og mesenteric]) (supplerende tabel 1).
Denne model kan også bruges til at fremme vores forståelse af koagulationsdannelsens mekanik og morfologi. Denne teknik giver unikt fordelen ved at stoppe trombose ved forskellige strømningshastighedspunkter for at studere de mellemliggende stadier af processen, før den bliver okklusiv. Nylige fremskridt inden for tromboseforskning har brugt denne model til at fokusere opmærksomheden på ikke-farmakologiske metoder til trombolyse13 eller ikke-invasiv levering af antitrombotiske og / eller fibrinolytiske midler14,15. Flere grupper har vist, at når blodplademembraner er belagt med disse behandlinger, kan lægemidlerne aktiveres ved termisk stimulering for at målrette blodpropper16. De teknikker, der er beskrevet her, kan være nyttige for sådanne undersøgelser som validering af deres resultater på enkelt blodpladeniveau. I dette manuskript beskriver protokol 1 den grundlæggende FeCl3-medierede vaskulære skadeprocedure, mens protokol 2 beskriver metoden til indsamling og fiksering af den vaskulære skadeprøve til yderligere analyse ved elektronmikroskopi.

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			<td>Dodenyl Succinic Anhydride/ DDSA</td>
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			<td>13700</td>
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			<td>Electron microscopy Services&#160;</td>
			<td>14900</td>
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			<td>Ethyl Alcohol, anhydrous 200 proof&#160;</td>
			<td>Electron microscopy Services&#160;</td>
			<td>15055</td>
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			<td>Eye Dressing Forceps, 4&#34; Full Curved, Standard, 0.8mm Wide Tips</td>
			<td>Integra Miltex</td>
			<td>18-784</td>
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			<td>VWR</td>
			<td>28310-106</td>
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			<td>Fine Scissors - Sharp-Blunt</td>
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			<td>Finger Loop Ear Punches&#160;</td>
			<td>Fine Science Tools&#160;</td>
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			<td>16120</td>
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			<td>Knife Handle Miltex&#174; Extra Fine Stainless Steel Size 3</td>
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			<td>L-aspartic acid</td>
			<td>Sigma&#160;Fisher</td>
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			<td>L-aspartic acid</td>
			<td>Fisher Scientific&#160;</td>
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			<td>LEICA S8AP0 Microscope Stand&#160;</td>
			<td>LEICA</td>
			<td>10447255</td>
			<td>No longer available from the company</td>
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			<td>Light-Duty Tissue Wipers&#160;</td>
			<td>VWR</td>
			<td>82003-822</td>
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			<td>Micro Dissecting Forceps; 1x2 Teeth, Full Curve; 0.8 mm Tip Width; 4&#34; Length</td>
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			<td>Osmium Tetroxide 4% aqueous solution&#160;</td>
			<td>Electron microscopy Services&#160;</td>
			<td>19150</td>
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			<td>Paraformaldehyde (16% solution)</td>
			<td>Electron microscopy Services&#160;</td>
			<td>15710</td>
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			<td>Potassium ferricyanide</td>
			<td>SIGMA-ALDRICH</td>
			<td>P-8131</td>
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			<td>Propylene Oxide, ACS reagent&#160;</td>
			<td>Electron microscopy Services&#160;</td>
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			<td>Rainin Classic Pipette PR-10</td>
			<td>Rainin</td>
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			<td>Research Flowmeter&#160;</td>
			<td>Transonic Systems Inc.</td>
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			<td>Model: T402</td>
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			<td>Scotch Magic Invisible Tape, 3/4&#34; x 1000&#34;, Clear</td>
			<td>Scotch&#160;</td>
			<td>305289</td>
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			<td>Small Animal Heated Pad</td>
			<td>K&#38;H Manufacturing Inc.</td>
			<td>Model: HM10</td>
			<td></td>
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			<td>Sodium Cacodylate Buffer 0.2M, pH7.4</td>
			<td>Electron microscopy Services&#160;</td>
			<td>11623</td>
			<td></td>
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			<td>Sterile Cotton Tipped Applicators&#160;</td>
			<td>Puritan Medical Products&#160;</td>
			<td>25-806 1WC</td>
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			<td>Steromaster Illuminator&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>12-562-21</td>
			<td>No longer available from the company</td>
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			<td>Surgical Dumont #7 Forceps&#160;</td>
			<td>Fine Science Tools&#160;</td>
			<td>11271-30</td>
			<td></td>
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			<td>Thiocarbohydrazide (TCH)</td>
			<td>SIGMA-ALDRICH</td>
			<td>88535</td>
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			<td>Universal Low Retention Pipet Tip Reloads (0.1-10 &#181;L)</td>
			<td>VWR</td>
			<td>76323-394</td>
			<td></td>
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			<td>Uranyl Acetate</td>
			<td>Electron microscopy Services&#160;</td>
			<td>22400</td>
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			<td>Reckitt Benckiser</td>
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			<td>White Antistatic Hexagonal Weigh Boats, Medium, 64 x 15 x 19 mm</td>
			<td>Fisher Scientific&#160;</td>
			<td>S38975</td>
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			<td>WinDAQ/100 Software for Windows</td>
			<td>DATAQ Instruments, Inc.</td>
			<td>Version 3.38</td>
			<td>Freely available to download. https://www.dataq.com/products/windaq/</td>
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			<td>ZEISS</td>
			<td>57615</td>
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</table>

ferric chloride-induced arterial thrombosis and sample collection for 3d electron microscopy analysis

Hjerte-kar-sygdomme er en førende årsag til dødelighed og sygelighed på verdensplan. Afvigende trombose er et fælles træk ved systemiske tilstande som diabetes og fedme og kroniske inflammatoriske sygdomme som aterosklerose, kræft og autoimmune sygdomme. Ved vaskulær skade virker normalt koagulationssystemet, blodpladerne og endotelet på en orkestreret måde for at forhindre blødning ved at danne en blodprop på skadestedet. Abnormiteter i denne proces fører til enten overdreven blødning eller ukontrolleret trombose/utilstrækkelig antitrombotisk aktivitet, hvilket udmønter sig i karokklusion og dens følgevirkninger. FeCl 3-induceret carotisskademodel er et værdifuldt værktøj til at undersøge, hvordan trombose initierer og udvikler sig in vivo. Denne model involverer endotelskader / denudation og efterfølgende koagulationsdannelse på det skadede sted. Det giver et meget følsomt, kvantitativt assay til overvågning af vaskulær skade og koagulationsdannelse som reaktion på forskellige grader af vaskulær skade. Når den er optimeret, kan denne standardteknik bruges til at studere de molekylære mekanismer, der ligger til grund for trombose, såvel som de ultrastrukturelle ændringer i blodplader i en voksende trombose. Dette assay er også nyttigt til at studere effekten af antitrombotiske og antiplatelet midler. Denne artikel forklarer, hvordan man initierer og overvåger FeCl 3-induceret arteriel trombose, og hvordan man indsamler prøver til analyse ved elektronmikroskopi.

Dataene præsenteres generelt som tid til okklusion eller tid, der kræves for at danne en fuldt okklusiv trombose. Disse data kan plottes som en Kaplan-Meier-overlevelseskurve (figur 4A)19, et prikplot med søjler, der viser den terminale blodgennemstrømning på tidspunktet for enten ophør af blodgennemstrømningen eller afslutning af et eksperiment (figur 4B) eller som et linjediagram (figur 4C). Trombose...

Watch this Scientific Journal Video about Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis at JoVE.com

Ferric Chloride-Induced Arterial Thrombosis and Sample Collection for 3D Electron Microscopy Analysis

Denne protokol beskriver, hvordan man bruger en FeCl3-medieret skade til at inducere arteriel trombose, og hvordan man indsamler og forbereder arterielle skadeprøver på forskellige stadier af trombose til elektronmikroskopianalyse.

Ferrichlorid-induceret arteriel trombose og prøveindsamling til 3D-elektronmikroskopianalyse

Cardiovascular diseases are a leading cause of mortality and morbidity worldwide. Aberrant thrombosis is a common feature of systemic conditions like ...

Med denne metode kan man bestemme, hvilke komponenter af blodplader og det hæmostatiske system der er vigtige for trombose. Derudover kan denne metode bestemme, hvordan trombosevækst og arkitektur påvirkes på mobilniveau. Den største fordel ved denne teknik er, at den tillader en reproducerbar måde at studere mange stadier af trombose på strukturelt niveau.En person, der er ny til denne teknik, bør undgå overhåndtering af vævet i det kirurgiske område. Dette kan føre til blødning og kan overkomplicere identifikationen af halspulsåren. Begynd at markere skadestedet efter at have udsat halspulsåren ved løst at omkranse regionen mellem to kirurgiske tråde med en størrelse på 0,1 millimeter.Placer sonden under arterien distal fra den nederste tråd. Indsæt plaststykket under arterien mellem de to tråde for at markere stedet for jernchloridskade. Tag flowmålinger, før du udfører skade for at notere basalstrømmen.Udfør skaden ved at placere jernchlorid-gennemblødt filterpapir på arterien i tre minutter. Fjern filterpapiret efter tre minutter for at forhindre det variable omfang af skaden og for at lette sondens flowtælling. Tilsæt saltvand på skadestedet for at fjerne overskydende resterende jernchlorid.Overvåg flowaflæsningen. Når strømmen falder til under 50% af den oprindelige værdi, fjernes sonden. Tør hurtigt området og tilsæt fikseringsmidlet for at fastgøre skadeområdet eksternt.Hold derefter straks arterien nær skadeområdet med tang. Skær nedstrøms for den nederste tråd og opstrøms for den øverste tråd. Sæt vævet på plastvævskulturskålen i samme retning som det blev opsamlet, og tilsæt et par dråber af fikseringsmidlet.Brug en skalpel til at rense det ekstra væv omkring arterien. Sørg for at registrere orienteringen af blodgennemstrømningen ved at skære den ene ende vandret og den anden tilspidset eller skråt. Anbring prøven i fikseringsmiddel i en time ved stuetemperatur, opbevar derefter prøven i 1% paraformaldehyd ved 4 grader Celsius, indtil den sendes ned våd is til elektronmikroskopianalyse.Dataene blev plottet som en Kaplan-Meier-overlevelseskurve, et prikplot med søjler, der viser den terminale blodgennemstrømning på tidspunktet for enten ophør af blodgennemstrømningen eller afslutningen af et eksperiment eller som en linjegraf. Blodgennemstrømningen steg pludseligt i nogle tilfælde efter et gradvist fald i et par minutter, hvilket indikerer delvis afgivelse af den voksende trombose og blev betragtet som en emboliseringshændelse. Okklusiv trombosemorfologi blev også undersøgt ved hjælp af denne metode.Den hurtige indsamling af prøven under tromboseprocessen er det vigtigste skridt, da forsinkelse i denne proces kan føre til fejlfortolkning af vores strukturelle analyse.

Research

JoVE Journal

Medicine

Quantitative Analysis and Characterization of Atherosclerotic Lesions in the Murine Aortic Sinus

Atherosclerosis is a disease of the large arteries and a major underlying cause of myocardial infarction and stroke. Several different mouse models have ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Ortotopisk Injektion af brystkræft celler i brystfedtpuden af ​​mus til at studere tumorvækst.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

A Multicenter MRI Protocol for the Evaluation and Quantification of Deep Vein Thrombosis

En multicenter MRI-protokollen for Vurdering og kvantificering af dyb venetrombose

We evaluated a magnetic resonance venography (MRV) approach with gadofosveset to quantify total thrombus volume changes as the principal criterion for ...

Ferric Chloride-induced Thrombosis Mouse Model on Carotid Artery and Mesentery Vessel

Ferric Chloride-induceret Trombose Mouse Model på Arteria carotis og mesenterium Vessel

Severe thrombosis and its ischemic consequences such as myocardial infarction, pulmonary embolism and stroke are major worldwide health issues. The ferric ...

Ferric Chloride-induced Murine Thrombosis Models

Ferrichlorid-induceret murin Trombose Modeller

Arterial thrombosis (blood clot) is a common complication of many systemic diseases associated with chronic inflammation, including atherosclerosis, ...

Quantitative Visualization of Leukocyte Infiltrate in a Murine Model of Fulminant Myocarditis by Light Sheet Microscopy

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Light-sheet fluorescence microscopy (LSFM), in combination with chemical clearing protocols, has become the gold standard for analyzing fluorescently ...

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

Preparation and In Vitro Characterization of Magnetized miR-modified Endothelial Cells

fremstilling og<em&gt; In vitro</em&gt; Karakterisering af Magnetized miR-modificerede endotelceller

To date, the available surgical and pharmacological treatments for cardiovascular diseases (CVD) are limited and often palliative. At the same time, gene ...

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

Intravital mikroskopi af monocyt målsøgende og Tumor-relaterede angiogenese i en Murine Model af perifer arteriel sygdom

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

Ferric Chloride-induced Canine Carotid Artery Thrombosis: A Large Animal Model of Vascular Injury

Ferrichloridopløsning-induceret Canine carotis arterie trombose: En stor dyr Model af vaskulære skade

Occlusive arterial thrombosis leading to cerebral ischemic stroke and myocardial infarction contributes to ~13 million deaths every year globally. Here, ...

Immunofluorescence Labelling of Human and Murine Neutrophil Extracellular Traps in Paraffin-Embedded Tissue

Immunofluorescens mærkning af humane og murin neutrofile ekstracellulære fælder i paraffin-indlejret væv

Neutrophil granulocytes, also called polymorphonuclear leukocytes (PMN) due to their lobulated nucleus, are the most abundant type of leukocytes. They ...