Name: A Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots (Video) | JoVE Journal
Uploaded: 2023-07-21T13:30:00.000+00:00
Duration: 6 min 12 s
Description: Learn a novel method for analyzing cell components of cerebral blood clots using fibrinolytic enzyme treatment and routine blood examination. This technique releases intact cells from fibrin for quantitative and qualitative analysis.

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5 minutes

This research was funded by the Science and Technology Bureau of Xiamen City (3502Z20227197), and the Science and Technology Bureau of Fujian Province (No. 2019J01070, No.2021Y0027).

The research was performed in compliance with the institutional guidelines of the Medical Ethics Committee of Huaqiao University. The cerebral blood clots were surgically removed and collected at Quanzhou First Hospital, affiliated to Fujian Medical University, with informed consent from the patients.
1. Blood clot pretreatment
<ol>
	<li>Place the clots on a clean dish, add 5 mL of physiological saline with a tweezer, shake the dish gently, and remove the saline with a pipette. 
	NOTE: Repeat the rinse three times.</li>
	<li>Cut the clots into smaller pieces (smaller than 5 mm x 5 mm x 5 mm) with a scissor. Add 5 mL of physiological saline, shake the dish gently, and remove the saline with a pipette. 
	NOTE: Repeat the rinse three times. Ensure no clot pieces are aspirated during the rinse.</li>
	<li>Transfer the clots to another clean U-plate. 
	&#8203;NOTE: The protocol can be paused here (store the plate at 2-8 &#176;C) and restarted later.</li>
</ol>
2. Thrombolysis
<ol>
	<li>Prepare the sFE working solution by adjusting the concentration of sFE to 2000 U/mL with physiological saline. 
	NOTE: The sFE was prepared according to the well-established protocols of Tang Lab15.</li>
	<li>Add 300 &#181;L of sFE working solution into the pretreated clots.</li>
	<li>Perform the first round of degradation.
	<ol>
		<li>Incubate the mixture of sFE and clots at 37 &#176;C for 0.5 h. 
		NOTE: The blot treated with physiological saline was set as negative control. Do not rotate the sample on the shaker.</li>
		<li>Mix the sample gently. Transfer the liquid part to another clean tube with a clean pipette. 
		NOTE: The remaining clots were used for another round of degradation.</li>
		<li>Centrifuge the liquid part at 200 &#215; g for 5 min at 4 &#176;C.</li>
		<li>Transfer the supernatant to another clean tube with a pipette to obtain the recovered sFE solution.</li>
		<li>Mix the cell precipitation with 50 &#181;L of physiological saline gently. 
		NOTE: The cell mixture was stored 2-8 &#176;C for later use.</li>
	</ol>
	</li>
	<li>Perform subsequent degradation.
	<ol>
		<li>Degrade the remaining clots (step 2.3.2) with the recovered sFE solution (step 2.3.4) at 37 &#176;C for 0.5 h. 
		NOTE: The rest steps were the same as the first degradation round. The degradation process was finished until the whole clots were degraded.</li>
	</ol>
	</li>
	<li>Perform cell collection.
	<ol>
		<li>Prepare the blood cell sample by collecting the cell mixture of each round of degradation.</li>
	</ol>
	</li>
</ol>
3. Wright&#39;s staining
<ol>
	<li>Perform cell smear.
	<ol>
		<li>Adjust cells to a density of 1 x 106 cells/mL.</li>
		<li>Add 5 &#181;L of the cells to the poly-L lysine-coated glass slide, then smear them using the cover slip.</li>
		<li>Evaporate the liquid at room temperature.</li>
	</ol>
	</li>
	<li>Perform the staining.
	<ol>
		<li>Add 200 &#181;L of Wright&#39;s dye (see Table of Materials) to the cell smear gently, and add 600 &#181;L of Wright&#39;s dye B to mix evenly.&#160;&#160;Stain for 10 min at room temperature.</li>
		<li>Rinse the dye gently with clean water. 
		NOTE: The stained cell smear can be stored at room temperature for later use.</li>
	</ol>
	</li>
	<li>Perform microscopic imaging.
	<ol>
		<li>Examine the stained cells using a light microscope (see Table of Materials).</li>
	</ol>
	</li>
</ol>
4. Routine blood examination
<ol>
	<li>Adjust cells to a density of 1 x 106 cells/mL.</li>
	<li>Analyze the cell components such as platelets, erythrocytes, white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, basophils, and immature granulocytes using an autohematology analyzer according to the manufacturer&#39;s instructions (see Table of Materials).</li>
</ol>

A Fibrinolytic Enzyme-Based Method to Study Cerebral Thrombus Cell Components

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<li>Devasagayam, S., Wyatt, B., Leyden, J., Kleinig, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cerebral+venous+sinus+thrombosis+incidence+is+higher+than+previously+thought%3A+a+retrospective+population-based+study%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">Cerebral venous sinus thrombosis incidence is higher than previously thought: a retrospective population-based study.</a> Stroke. 47 (9), 2180-2182 (2016).</li>
<li>Sacco, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+updated+definition+of+stroke+for+the+21st+century%3A+a+statement+for+healthcare+professionals+from+the+American+Heart+Association%2FAmerican+Stroke+Association%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">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/pubmed?term=((Neutrophil+extracellular+traps%3A+villains+and+targets+in+arterial%2C+venous%2C+and+cancer-associated+thrombosis%5BTitle%5D)+AND+%22Arteriosclerosis+Thrombosis+and+Vascular+Biology%22%5BJournal%5D)">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/pubmed?term=((Diverse+novel+functions+of+neutrophils+in+immunity%2C+inflammation%2C+and+beyond%5BTitle%5D)+AND+%22Journal+of+Experimental+Medicine%22%5BJournal%5D)">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/pubmed?term=((PKM2+promotes+neutrophil+activation+and+cerebral+thromboinflammation%3A+therapeutic+implications+for+ischemic+stroke%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">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/pubmed?term=((Thrombus+neutrophil+extracellular+traps+content+impair+tpa-induced+thrombolysis+in+acute+ischemic+stroke%5BTitle%5D)+AND+%22Stroke%22%5BJournal%5D)">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/pubmed?term=((Assessing+the+methodology+for+calculating+platelet+contribution+to+clot+strength+%28platelet+component%29+in+thromboelastometry+and+thrombelastography%5BTitle%5D)+AND+%22Anesthesia+and+Analgesia%22%5BJournal%5D)">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/pubmed?term=((Automated+fiber+diameter+and+porosity+measurements+of+plasma+clots+in+scanning+electron+microscopy+images%5BTitle%5D)+AND+%22Biomolecules%22%5BJournal%5D)">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/pubmed?term=((Diverse+thrombus+composition+in+thrombectomy+stroke+patients+with+longer+time+to+recanalization%5BTitle%5D)+AND+%22Thrombosis+Research%22%5BJournal%5D)">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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Concepts+of+clot+lysis%5BTitle%5D)+AND+%22Annual+Review+of+Medicine%22%5BJournal%5D)">Concepts of clot lysis.</a> Annual Review of Medicine. 37 (1), 187-204 (1986).</li>
<li>Preparation and application of natural fibrinolytic enzyme from peanut worm. , https://patents.google.com/patent/CN109295042A/en (2019).</li>
<li>Fotso Fotso, A., Drancourt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laboratory+Diagnosis+of+tick-borne+african+relapsing+fevers%3A+latest+developments%5BTitle%5D)+AND+%22Front+Public+Health%22%5BJournal%5D)">Laboratory Diagnosis of tick-borne african relapsing fevers: latest developments.</a> Front Public Health. 3, 254(2015).</li>
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<li>Tang, M., Lin, H., Hu, C., Yan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Affinity+purification+of+a+fibrinolytic+enzyme+from+Sipunculus+nudus%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Affinity purification of a fibrinolytic enzyme from Sipunculus nudus.</a> Journal of Visualized Experiments. 196, e65631(2023).</li>
<li>Ge, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel+antithrombotic+protease+from+marine+worm+Sipunculus+Nudus%5BTitle%5D)+AND+%22International+Journal+Of+Molecular+Sciences%22%5BJournal%5D)">A Novel antithrombotic protease from marine worm Sipunculus Nudus.</a> International Journal Of Molecular Sciences. 19 (10), 3023(2018).</li>
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The authors have no conflicts of interest to disclose.

sFE is a fibrinolytic agent that can degrade the fibrin directly and effectively12,16. Here, sFE was employed to degrade the cross-linked fibrin of the cerebral blood clots, release the enclosed cells within the clots, and analyze the cell components of the clots qualitatively and quantitatively. The microscopy data and routine blood examination indicated that the enclosed cells were released from the blood clots. Furthermore, the cell types and structures of the...

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.

<table><tbody><tr><td>Agglutination Reaction Plate</td><td>ROTEST</td><td>RTB-4003</td><td></td></tr><tr><td>Auto Hematology Analyzer</td><td>SYSMEX</td><td>XNB2</td><td></td></tr><tr><td>Automatic Vertical Pressure Steam Sterilizer&amp;nbsp;</td><td>SANYO</td><td>MLS-3750</td><td></td></tr><tr><td>Centrifuge Tube (1.5 mL)</td><td>Biosharp</td><td>BS-15-M</td><td></td></tr><tr><td>Clean bench</td><td>AIRTECH</td><td>BLB-1600</td><td></td></tr><tr><td>Constant Temperature Incubator</td><td>JINGHONG</td><td>JHS-400</td><td></td></tr><tr><td>Culture Dish (100 mm)</td><td>NEST</td><td>704001</td><td></td></tr><tr><td>DHG Series Heating and Drying Oven&amp;nbsp;</td><td>SENXIN</td><td>DGG-9140AD</td><td></td></tr><tr><td>Electronic Analytical Balance</td><td>DENVER</td><td>TP-213</td><td></td></tr><tr><td>Filter Membrane (0.22 &amp;micro;m)</td><td>Millex GP</td><td>SLGP033NK</td><td></td></tr><tr><td>Micro Refrigerated Centrifuge&amp;nbsp;</td><td>Cence</td><td>H1650-W</td><td></td></tr><tr><td>Microscope Slides</td><td>CITOGLAS</td><td>01-30253-50</td><td></td></tr><tr><td>Milli-Q Reference</td><td>Millipore</td><td>Z00QSV0CN</td><td></td></tr><tr><td>Normal Saline</td><td>CISEN</td><td>H37022337</td><td></td></tr><tr><td>Optical Microscope</td><td>Nikon</td><td>ECLIPSE E100</td><td></td></tr><tr><td>Parafilm</td><td>Bemis</td><td>PM-996</td><td></td></tr><tr><td>Phosphate-Buffered Saline</td><td>Beyotime</td><td>C0221A</td><td></td></tr><tr><td>Pipette Tip (1 mL )</td><td>Axygene</td><td>T-1000XT-C</td><td></td></tr><tr><td>Pipette Tip (200 &amp;micro;L)</td><td>Axygene</td><td>T-200XT-C</td><td></td></tr><tr><td>Pipettor (1 mL)</td><td>Thermo Fisher Scientific</td><td>ZY18723</td><td></td></tr><tr><td>Pipettor (200 &amp;micro;L)</td><td>Thermo Fisher Scientific</td><td>ZY20280</td><td></td></tr><tr><td>Scalpel</td><td>MARTOR</td><td>23111</td><td></td></tr><tr><td>Small-sized Vortex Oscillator</td><td>Kylin-Bell</td><td>VORTEX KB3</td><td></td></tr><tr><td>Tweezer</td><td>Hystic</td><td>HKQS-180</td><td></td></tr><tr><td>Wright Staining Solution</td><td>Beyotime</td><td>C0135-500ml</td><td></td></tr></tbody></table>

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

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.

In the initial stage of the degradation process, it was found that the blood clots had a red compact structure, and the working solution was colorless. After incubation for 30 min, the working solution turned light red, which indicated the crossed blood cells were released into the working solution. Most clots were dissolved when lengthening the incubation time to 5 h, and the working solution became light red. On the contrary, there was no significant change in the physiological saline group (NC) even after 10 h incubat...

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 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 ...

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Source: Kim, D., et. al. Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli. J. Vis. Exp. (2016)This video demonstrates visualization of cerebral thrombus in mice using micro-computed tomography (micro-CT) imaging. A cerebral thrombus mouse model is injected with fibrin-targeted gold nanoparticles acting as a contrasting agent. The mouse is placed on the bed of a micro-CT machine, and images are acquired from multiple angles.

Visualization of a Cerebral Thrombus in a Mouse Using Micro-Computed Tomography Imaging

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

This protocol describes the application of combined near-infrared fluorescent (NIRF) imaging and micro-computed tomography (microCT) for visualizing cerebral thromboemboli. This technique allows the quantification of thrombus burden and evolution. The NIRF imaging technique visualizes fluorescently labeled thrombus in excised brain, while the microCT technique visualizes thrombus inside living animals using gold-nanoparticles.

Combined Near-infrared Fluorescent Imaging and Micro-computed Tomography for Directly Visualizing Cerebral Thromboemboli

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

We present an in vitro vascular disease model to investigate whole blood interactions with patient-derived endothelium. This system allows the study of thrombogenic properties of primary endothelial cells under various circumstances. The method is especially suited to evaluate in situ thrombogenicity and anticoagulation therapy during different phases of coagulation.

In Vitro Microfluidic Disease Model to Study Whole Blood-Endothelial Interactions and Blood Clot Dynamics in Real-Time

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

Measuring Post-Stroke Cerebral Edema, Infarct Zone and Blood-Brain Barrier Breakdown in a Single Set of Rodent Brain Samples

This protocol presents methods to characterize the neuroinflammatory and hemodynamic response to mild traumatic brain injury and to integrate these data as part of a multivariate systems analysis using partial least squares regression.

Systems Analysis of the Neuroinflammatory and Hemodynamic Response to Traumatic Brain Injury

Fibrin is responsible for clot formation during hemostasis and thrombosis. Turbidity assays and thromboelastograhy (TEG) can be utilized as synergistic tools that provide complementary assessment of a clot. These two techniques together can give more insight into how clotting conditions affect fibrin clot formation.

Leveraging Turbidity and Thromboelastography for Complementary Clot Characterization

Traditional photothrombotic stroke (PTS) models mainly induce dense platelet aggregates of a high resistance to tissue plasminogen activator (tPA)-lytic treatment. Here a modified murine PTS model is introduced by co-injecting thrombin and photosensitive dye for photoactivation. The thrombin-enhanced PTS model produces mixed platelet:fibrin clots and is highly sensitive to tPA-thrombolysis.

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

This work describes a microfluidic assay that uses shear stress to trigger thrombus formation in blood and characterizes the thrombus by multiple dimensions of readout. The assay can measure the prothrombotic tendency of blood samples, and is thus useful in disease diagnosis, drug discovery, as well as basic mechanistic studies related to thrombosis.

Thrombus Profiling Assay: A Microfluidics-Based Platform for Comprehensively Characterizing Biomechanical Thrombogenesis

Learn a novel method for analyzing cell components of cerebral blood clots using fibrinolytic enzyme treatment and routine blood examination. This technique releases intact cells from fibrin for quantitative and qualitative analysis.

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

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