Name: Auteur in de schijnwerpers: een nieuwe methode voor uitgebreide analyse van celcomponenten van cerebrale bloedstolsels
Uploaded: 2023-07-21T13:30:00
Description: Watch this Scientific Journal Video about A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots at JoVE.com

Dit onderzoek werd gefinancierd door het Science and Technology Bureau van de stad Xiamen (3502Z20227197) en het Science and Technology Bureau van de provincie Fujian (nr. 2019J01070, nr. 2021Y0027).

Het onderzoek is uitgevoerd in overeenstemming met de institutionele richtlijnen van de Commissie Medische Ethiek van de Huaqiao University. De cerebrale bloedstolsels werden operatief verwijderd en verzameld in het Quanzhou First Hospital, verbonden aan de Fujian Medical University, met geïnformeerde toestemming van de patiënten.
1. Voorbehandeling van bloedstolsels
<ol><li>Leg de stolsels op een schone schaal, voeg 5 ml fysiologische zoutoplossing toe met een pincet, sch...

Een op fibrinolytische enzymen gebaseerde methode om cerebrale trombuscelcomponenten te bestuderen

<ol>
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L., 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=An+updated+definition+of+stroke+for+the+21st+century:+a+statement+for+healthcare+professionals+from+the+American+Heart+Association/American+Stroke+Association.">An updated definition of stroke for the 21st century: a statement for healthcare professionals from the American Heart Association/American Stroke Association.</a> Stroke. 44 (7), 2064-2089 (2013).</li><li>Thalin, C., Hisada, Y., Lundstrom, S., Mackman, N., Wallen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutrophil+extracellular+traps:+villains+and+targets+in+arterial,+venous,+and+cancer-associated+thrombosis.">Neutrophil extracellular traps: villains and targets in arterial, venous, and cancer-associated thrombosis.</a> Arteriosclerosis Thrombosis and Vascular Biology. 39 (9), 1724-1738 (2019).</li><li>Mocsai, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+novel+functions+of+neutrophils+in+immunity,+inflammation,+and+beyond.">Diverse novel functions of neutrophils in immunity, inflammation, and beyond.</a> Journal of Experimental Medicine. 210 (7), 1283-1299 (2013).</li><li>Dhanesha, N., 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=PKM2+promotes+neutrophil+activation+and+cerebral+thromboinflammation:+therapeutic+implications+for+ischemic+stroke.">PKM2 promotes neutrophil activation and cerebral thromboinflammation: therapeutic implications for ischemic stroke.</a> Blood. 139 (8), 1234-1245 (2022).</li><li>Ducroux, C., 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=Thrombus+neutrophil+extracellular+traps+content+impair+tpa-induced+thrombolysis+in+acute+ischemic+stroke.">Thrombus neutrophil extracellular traps content impair tpa-induced thrombolysis in acute ischemic stroke.</a> Stroke. 49 (3), 754-757 (2018).</li><li>Solomon, C., Ranucci, M., Hochleitner, G., Schochl, H., Schlimp, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+the+methodology+for+calculating+platelet+contribution+to+clot+strength+(platelet+component)+in+thromboelastometry+and+thrombelastography.">Assessing the methodology for calculating platelet contribution to clot strength (platelet component) in thromboelastometry and thrombelastography.</a> Anesthesia and Analgesia. 121 (4), 868-878 (2015).</li><li>Daraei, A., 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=Automated+fiber+diameter+and+porosity+measurements+of+plasma+clots+in+scanning+electron+microscopy+images.">Automated fiber diameter and porosity measurements of plasma clots in scanning electron microscopy images.</a> Biomolecules. 11 (10), 1536 (2021).</li><li>Abbasi, M., 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=Diverse+thrombus+composition+in+thrombectomy+stroke+patients+with+longer+time+to+recanalization.">Diverse thrombus composition in thrombectomy stroke patients with longer time to recanalization.</a> Thrombosis Research. 209, 99-104 (2022).</li><li>C W Francis, a., Marder, V. 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sFE is een fibrinolytisch middel dat het fibrine direct en effectief kan afbreken12,16. Hier werd sFE gebruikt om het verknoopte fibrine van de cerebrale bloedstolsels af te breken, de ingesloten cellen in de stolsels vrij te maken en de celcomponenten van de stolsels kwalitatief en kwantitatief te analyseren. De microscopiegegevens en routinematig bloedonderzoek gaven aan dat de ingesloten cellen uit de bloedstolsels waren vrijgekomen. Bovendien werden de celtyp...

Cerebrovasculaire ziekte is een van de drie belangrijkste ziekten die de menselijke gezondheid kunnen bedreigen, waaronder ischemische cerebrovasculaire ziekte meer dan 80%. Cerebrale trombose en cerebrale veneuze trombose zijn tegenwoordig de meest bezorgde ischemische cerebrovasculaire aandoeningen, voornamelijk veroorzaakt door cerebrale bloedstolsels 1,2. Als de behandeling niet goed wordt uitgevoerd, zal deze hoge invaliditeits- en sterftecijfers hebben en een hoog recidiefpercentage na ontslag3.
Onlangs heeft een groeiend aantal onderzoeken aangetoond dat de celcomponenten van cerebrale bloedstolsels nauw gecorreleerd zijn met de diagnose, behandeling en prognose van cerebrale trombose 4,5,6. Daarom is de beschikbaarheid van gegevens over de samenstelling van de trombus, met name de celcomponenten, belangrijk voor klinische diagnose en behandeling. Helaas kunnen de momenteel beschikbare methoden de bloedstolselcomponent niet kwantitatief en kwalitatief volledig analyseren. Op Martius Scarlett Blue gebaseerde in-situ kleuring kan bijvoorbeeld alleen de rood/witte bloedcellen van bepaalde plakjes van het stolsel7 bestuderen. Op immunohistochemie (IHC) gebaseerde in-situ kleuring kan slechts beperkte bloedcomponenten van bepaalde plakjes van het stolsel bestuderen met behulp van hun antilichamen8. De microscopische beeldgebaseerde methoden houden zich alleen bezig met de specifieke structuur van het stolsel9. Bovendien zijn al die methoden arbeidsintensief en tijdrovend10. Tot op heden zijn de procedures voor het kwantitatief en kwalitatief bestuderen van cerebrale trombicelcomponenten niet gerapporteerd. Het wordt algemeen erkend dat het verknoopte fibrine de bloedcellen stevig omhult in de stolsels11. Bijgevolg is de specifieke afbraak van het verknoopte fibrine en het vrijkomen van de intacte cellen van cruciaal belang voor de nauwkeurige analyse van celcomponenten.
Eerdere werken isoleerden een fibrinolytisch enzym uit Sipunculus nudus (sFE), dat het fibrine specifiek en snel kanafbreken12. Hierin werd een methode voorgesteld voor het analyseren van de celcomponenten van de cerebrale trombi op basis van de unieke activiteit van sFE. Dit protocol maakte gebruik van sFE om eerst het fibrine van stolsels af te breken en analyseerde vervolgens de celcomponenten door middel van Wright's kleuring en routinematig bloedonderzoek13,14. Volgens deze methode kunnen de celcomponenten van cerebrale trombi kwantitatief en kwalitatief worden bestudeerd. Dit eenvoudige en effectieve protocol kan worden toegepast voor de analyse van celcomponenten van andere bloedstolsels.

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a comprehensive approach to analyze the cell components of cerebral blood clots

Cerebrale trombose, een bloedstolsel in een hersenslagader of ader, is het meest voorkomende type herseninfarct. De studie van de celcomponenten van cerebrale bloedstolsels is belangrijk voor diagnose, behandeling en prognose. De huidige benaderingen voor het bestuderen van de celcomponenten van de stolsels zijn echter voornamelijk gebaseerd op in situ kleuring, die ongeschikt is voor de uitgebreide studie van de celcomponenten omdat cellen strak in de stolsels zijn gewikkeld. Eerdere studies hebben met succes een fibrinolytisch enzym (sFE) geïsoleerd uit Sipunculus nudus, dat het verknoopte fibrine direct kan afbreken, waardoor de celcomponenten vrijkomen. Deze studie heeft een uitgebreide methode vastgesteld op basis van de sFE om de celcomponenten van cerebrale trombus te bestuderen. Dit protocol omvat het oplossen van stolsels, het vrijgeven van cellen, celkleuring en routinematig bloedonderzoek. Volgens deze methode konden de celcomponenten kwantitatief en kwalitatief worden bestudeerd. De representatieve resultaten van experimenten met deze methode worden getoond.

Cerebrovascular disease is one of three major diseases that can threaten human health, among which ischemic cerebrovascular disease accounts for more than 80%. Cerebral thrombosis and cerebral vein thrombosis are the most concerned ischemic cerebrovascular diseases today, mainly caused by cerebral blood clots1,2. If the treatment is not done properly, it will have high disability and mortality rates and a high recurrence rate after discharge3.
Recently, a growing number of studies have shown that the cell components of cerebral blood clots are tightly correlated with the diagnosis, treatment, and prognosis of cerebral thrombosis4,5,6. Therefore, the availability of data on thrombus composition, especially the cell components, is important for clinical diagnosis and treatment. Unfortunately, the currently available methods cannot comprehensively analyze the blood clot component quantitatively and qualitatively. For example, Martius Scarlett Blue based in-situ staining can only study the red/white blood cells of certain slices of the clot7. Immunohistochemistry (IHC) based in-situ staining can only study limited blood components of certain slices of the clot using their antibodies8. The microscopic image-based methods are only concerned with the specific structure of the clot9. Moreover, all those methods are laborious and time-consuming10. To date, the procedures for quantitatively and qualitatively studying cerebral thrombi cell components have not been reported. It is widely acknowledged that the cross-linked fibrin tightly wraps the blood cells in the clots11. Consequently, the specific degradation of the cross-linked fibrin and release of the intact cells is critical for the accurate analysis of cell components.
Previous works isolated a fibrinolytic enzyme from Sipunculus nudus (sFE), which can degrade the fibrin specifically and quickly12. Herein, a method for analyzing the cell components of the cerebral thrombi based on the unique activity of sFE was proposed. This protocol utilized sFE to degrade the fibrin of clots first and then analyzed the cell components by Wright&#39;s Staining and routine blood examination13,14. According to this method, the cell components of cerebral thrombi can be quantitatively and qualitatively studied. This simple and effective protocol might be applied for the cell component analysis of other blood clots.

Auteur in de schijnwerpers: een nieuwe methode voor uitgebreide analyse van celcomponenten van cerebrale bloedstolsels 

Een alomvattende benadering om de celcomponenten van cerebrale bloedstolsels te analyseren

Our research mainly focuses on cerebral thrombosis. Many studies have shown that cerebral blood clot cell component correlate with the diagnosis, treatment, and prognosis of cerebral thrombosis. In this study, we established a novel method for the analysis of cell component of cerebral blood clots.This challenge in analyzing cell components of cerebral blood clots is the release of entire shaft from blood clots. This is because the clots leaked fibering tiry rash, the blood cells in the clots. Targeted approaches to starting the cell components of the clots are best done in C2 standing, which is unsuitable for the comprehensive starting of the cell components.On the contrary, our method enables both quantitative and qualitative enlightenings of the cerebral slope cell components. Our method utilizes fibrinolytic enzyme to delay the fibering in blood clots, releasing the intact cells. The data obtained from the analysis of the cell components will facilitate the study of the mechanisms of cerebral thrombosis at a molecular level.Our team will continue to focus on the study of fibrinolysis and coagulation system.

In de beginfase van het afbraakproces bleek dat de bloedstolsels een rode compacte structuur hadden en dat de werkoplossing kleurloos was. Na 30 minuten incubatie werd de werkoplossing lichtrood, wat aangaf dat de gekruiste bloedcellen in de werkoplossing waren vrijgegeven. De meeste stolsels werden opgelost bij het verlengen van de incubatietijd tot 5 uur en de werkoplossing werd lichtrood. Integendeel, er was geen significante verandering in de fysiologische zoutoplossinggroep (NC), zelfs niet na 10 uur incubatie (<str...

Watch this Scientific Journal Video about A Novel Method for Comprehensive Cell Component Analysis of Cerebral Blood Clots at JoVE.com

Deze studie beschrijft een snelle en effectieve methode voor de analyse van celcomponenten van cerebrale bloedstolsels door middel van stolseloplossing, celkleuring en routinematig bloedonderzoek.

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of ...

Ons onderzoek richt zich vooral op cerebrale trombose. Veel onderzoeken hebben aangetoond dat de celcomponent van cerebrale bloedstolsels correleert met de diagnose, behandeling en prognose van cerebrale trombose. In deze studie hebben we een nieuwe methode ontwikkeld voor de analyse van de celcomponent van cerebrale bloedstolsels.Deze uitdaging bij het analyseren van celcomponenten van cerebrale bloedstolsels is het vrijkomen van de hele schacht uit bloedstolsels. Dit komt omdat de stolsels de bloedcellen in de stolsels lekten. Gerichte benaderingen voor het starten van de celcomponenten van de stolsels kunnen het beste worden gedaan in C2-stand, wat niet geschikt is voor het volledig starten van de celcomponenten.Integendeel, onze methode maakt zowel kwantitatieve als kwalitatieve verlichting van de celcomponenten van de cerebrale helling mogelijk. Onze methode maakt gebruik van fibrinolytisch enzym om de vezels in bloedstolsels te vertragen, waardoor de intacte cellen vrijkomen. De gegevens die worden verkregen uit de analyse van de celcomponenten zullen de studie van de mechanismen van cerebrale trombose op moleculair niveau vergemakkelijken.Ons team zal zich blijven richten op de studie van fibrinolyse en stollingssystemen.

a-comprehensive-approach-to-analyze-cell-components-cerebral-blood

Research

JoVE Journal

Biochemistry

A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

Cerebral thrombosis, a blood clot in a cerebral artery or vein, is the most common type of cerebral infarction. The study of the cell components of cerebral blood clots is important for diagnosis, treatment, and prognosis. However, the current approaches to studying the cell components of the clots are mainly based on in situ staining, which is unsuitable for the comprehensive study of the cell components because cells are tightly wrapped in the clots. Previous studies have successfully isolated a fibrinolytic enzyme (sFE) from Sipunculus nudus, which can degrade the cross-linked fibrin directly, releasing the cell components. This study established a comprehensive method based on the sFE to study the cell components of cerebral thrombus. This protocol includes clot dissolving, cell releasing, cell staining, and routine blood examination. According to this method, the cell components could be studied quantitatively and qualitatively. The representative results of experiments using this method are shown.

This study describes a fast and effective method for the cell component analysis of cerebral blood clots through clot dissolving, cell staining, and routine blood examination.

Cerebral Thrombus Clot Dissolution and Cell Staining for Cell Component Analyses

To begin, place the collected cerebral blood clot samples on a clean dish using tweezers. Then, using a pipette, add five milliliters of physiological saline to it and gently shake the dish. Remove the saline using a pipette.Using a pair of scissors, chop the clots into small pieces. Once again, add five milliliters of physiological saline to the clots and shake the dish gently before aspirating the saline with a pipette. Then, using tweezers, transfer the pieces of clots to a new, clean U-plate.Prepare the SFE working solution by adjusting the concentration of SFE to 2, 000 units per milliliter using physiological saline. Add 300 microliters of the SFE working solution to the pretreated clots. To initiate the first round of degradation, incubate the mixture of SFE and clots at 37 degrees Celsius for half an hour.After that, gently shake to mix the SFE treated sample. And using a clean pipette, transfer the liquid portion to a sterile tube. Set aside the remaining clot for another round of degradation.Centrifuge the collected liquid at 200 G for five minutes at four degrees Celsius. Next, using a pipette, transfer the supernatant or the recovered SFE solution to another clean tube. Resuspend the resultant cell pellet in 50 microliters of physiological saline by gentle mixing.After this, perform subsequent rounds of degradation on the remaining clots using the recovered SFE solution as demonstrated previously. Gather the cell mixture from each round of degradation to prepare the blood cell sample. Add five microliters of the obtained cell sample to the poly-L-lysine-coated glass slide.And using a cover slip, smear the cells. Allow the liquid to evaporate at room temperature. To perform cell staining, gently add 100 microliters of rights dye to the cell smear.Allow the cells to stain for 30 minutes at room temperature before gently rinsing the dye off with clean water. Finally, examine the stained cells using a light microscope. Compact red blood clots with a colorless working solution were observed during the early stage of degradation.Clot dissolution was observed after a 30-minute incubation and a prolonged incubation of up to five hours dissolved most clots, while there was no significant change in the negative control group, even after a 10-hour incubation. After a right staining, mature red blood cells, platelets, and various granulocytes were clearly identifiable under a light microscope. The cell components could be quantitatively analyzed with the aid of autohematology successfully.