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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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. 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H., 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=A+Novel+antithrombotic+protease+from+marine+worm+Sipunculus+Nudus.">A Novel antithrombotic protease from marine worm Sipunculus Nudus.</a> International Journal Of Molecular Sciences. 19 (10), 3023 (2018).</li><li>Talukder, M. A., Menyuk, C. R., Kostov, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinguishing+between+whole+cells+and+cell+debris+using+surface+plasmon+coupled+emission.">Distinguishing between whole cells and cell debris using surface plasmon coupled emission.</a> Biomedical Optics Express. 9 (4), 1977-1991 (2018).</li><li>Shapiro, D. J., Hicks, L. A., Pavia, A. T., Hersh, A. 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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 released cells were not affected significantly after sFE treatment. Thus, a unique, simple, and effective method was created for the first time for analyzing the cell components of cerebral blood clots.
Because the released cells are not as stable as within the clots, the degradation conditions need to be optimized to decrease the broken ratio of released cells, such as temperature, the dose of sFE, speed of rotation, and degradation time. Wright's staining results showed that the background of the 10 h degradation was more cloudy than that of 5 h. These differences are probably due to cell debris and other cell fragments. Moreover, the flow cytometry data indicated that the cell debris increased with the degradation time (Figure 2B). The cell debris rate of 10 h degradation (32.5%) was significantly higher than that of 5 h degradation (11.2%). Therefore, it was critical to separate the released cells from the working solution as soon as possible. So, in this study, the cells were collected every 0.5 h. Another key point was the rotation, which could accelerate degradation but is bad for keeping the intact cell structure of the released cells.
Although the degradation conditions have been optimized, some cells may be lysed in this process. Cell debris may be adverse to cell microscopy and routine blood examination17. Therefore, separating those cell debris from the intact cells is recommended. Another limitation of this technique is the possibility of the microbe contamination. The underlying reason for this phenomenon is probably the long degradation time in couple with the nonsterile operation. So, some suitable antibiotics should be added in future studies18. It is well known that different blood clots share similar clots structures. Thus, the protocol reported here will be suitable for cell component analysis of other blood clots. Moreover, with the aid of this technique, not only the cell components but also the plasma with the clots could be analyzed qualitatively and quantitatively.

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

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 incubation (Figure 1). These results showed that the blood cells enclosed in the clots were released successfully by this protocol.
After Wright&#39;s stain, the cell components were examined clearly under a light microscope (Figure 2A). Among those released blood cells, a large number of mature red blood cells with slightly concave and disc-shaped surfaces could be observed. A great quantity of irregularly shaped small purple-red particles was observed on the glass slide, which were the platelets released from the blood clots. The white blood cells exhibited larger plasma and condensed nuclear. Several types of granulocytes, such as mature neutrophil granulocyte, eosinophil, and monocyte, were also observed.
With the aid of auto hematology analyzer, the cell components were analyzed quantitatively. Among the detected cells, platelets and erythrocytes accounted for 51.25% and 45.24%, respectively. These data are consistent with the results of Wright&#39;s stain. Other blood cells, such as white blood cells, lymphocytes, neutrophils, monocytes, eosinophils, basophils, and immature granulocytes, were also detected (Table 1). Thus, following this protocol, the cell components of a cerebral thrombus can be studied not only qualitatively but also quantitatively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65791/65791fig01.jpg" /> 
Figure 1: Dissolving process. The cerebral thrombus was dissolved in the saline (NC) and the sFE, respectively. Photography was taken at 0 h, 5 h, and 10 h. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65791/65791fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65791/65791fig02.jpg" /> 
Figure 2:&#160;Microscopy and flow cytometry of the released cells. (A) The cells under the microscope after Wright&#39;s staining. Scale bar = 20 &#181;m. (B) Flow cytometry results of the released cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65791/65791fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="3">Routine Blood Test</td>
		</tr>
		<tr>
			<td>Parameters</td>
			<td>Results</td>
			<td>Unit</td>
		</tr>
		<tr>
			<td>Immature granulocyte absolute number (IG#)</td>
			<td>1</td>
			<td>109/L</td>
		</tr>
		<tr>
			<td>Immature granulocyte percentage (IG%)</td>
			<td>3.7</td>
			<td>%</td>
		</tr>
		<tr>
			<td>Erythrocyte Count (RBC)</td>
			<td>1.52</td>
			<td>1012/L</td>
		</tr>
		<tr>
			<td>Red blood cell specific volume(HCT)</td>
			<td>13.7</td>
			<td>%</td>
		</tr>
		<tr>
			<td>Mean corpuscular volume (MCV)</td>
			<td>90.1</td>
			<td>f1</td>
		</tr>
		<tr>
			<td>White blood cell number (WBC)</td>
			<td>26.98</td>
			<td>109/L</td>
		</tr>
		<tr>
			<td>Lymphocytes number (LYM)</td>
			<td>1.41</td>
			<td>109/L</td>
		</tr>
		<tr>
			<td>Lymphocytes percentage (LYM%)</td>
			<td>5.2</td>
			<td>%</td>
		</tr>
		<tr>
			<td>Neutrophils number (Neu#)</td>
			<td>24.08</td>
			<td>109/L</td>
		</tr>
		<tr>
			<td>Neutrophils percentage (Neu%)</td>
			<td>89.2</td>
			<td>%</td>
		</tr>
		<tr>
			<td>Monocytes number (Mon#)</td>
			<td>1.02</td>
			<td>109/L</td>
		</tr>
		<tr>
			<td>Monocytes percentage (Mon%)</td>
			<td>3.8</td>
			<td>%</td>
		</tr>
		<tr>
			<td>Eosinophils number (Eos#)</td>
			<td>0.34</td>
			<td>109/L</td>
		</tr>
		<tr>
			<td>Eosinophils percentage (Eos%)</td>
			<td>1.3</td>
			<td>%</td>
		</tr>
		<tr>
			<td>Basophils number (Bas#)</td>
			<td>0.13</td>
			<td>109/L</td>
		</tr>
		<tr>
			<td>Basophils percentage (Bas%)</td>
			<td>0.5</td>
			<td>%</td>
		</tr>
		<tr>
			<td>platelet count (PLT)</td>
			<td>1722</td>
			<td>109/L</td>
		</tr>
		<tr>
			<td>Mean platelet volume (MPV)</td>
			<td>11.6</td>
			<td>f1</td>
		</tr>
	</tbody>
</table>
Table 1: Routine blood examination.

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

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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.

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

Evaluation of the Cognitive Performance of Hypertensive Patients with Silent Cerebrovascular Lesions

Evidence accumulated from the last decade has proven that silent cerebrovascular lesions (SCLs) and their underlying pathogenic processes contribute to cognitive decline in the elderly. However, the distinct effects of each type of the lesions on cognitive performance remain unclear. Moreover, research data from Chinese elderly with SCLs is scarce. In this study, 398 otherwise healthy hypertensive elderly subjects (median age 72 years) were included and assessed. All participates were required to complete a battery of structured neuropsychological assessment, including forward and backward digit span tests, symbol digit modalities test, Stroop test, verbal fluency test and Montreal Cognitive Assessment. These tests were used to assess attention, executive function, information processing speed, language, memory and visuospatial function. A multi-sequence 3T MRI scanning was arranged within one month of the neuropsychological assessment to evaluate the burden of SCLs. SCLs were rated visually. Cerebral microbleeds (CMBs) and silent lacunes (SLs) were identified as strictly lobar CMBs and SLs or deep CMBs and SLs according to their locations, respectively. Similarly, white matter hyperintensities (WMHs) were separated into periventricular WMHs (PVHs) and deep WMHs (DWMHs). A series of linear regression models were used to assess the correlation between each type of SCLs and individual cognitive function domain. The results showed that CMBs tend to impair language-related cognition. Deep SLs affect executive function, but this association disappeared after controlling for other types of SCLs. PVHs, rather than DWMHs, are associated with cognitive decline, especially in executive function and processing speed. It is concluded that different aspects of SCLs have differential impact on cognitive performance in hypertensive elderly Chinese.

Here, we present a protocol to assess whether various types of silent cerebrovascular lesions are differentially associated with deficits in certain cognitive domains in a cohort of 398 hypertensive elderly Chinese, using a combination of neuropsychological tests and multi-sequence 3T MRI scanning.

Evidence accumulated from the last decade has proven that silent cerebrovascular lesions (SCLs) and their underlying pathogenic processes contribute to ...

Behavior

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

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

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.

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Medicine

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

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

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.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Immunology and Infection

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes.
Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. 
The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro

Here, we describe the method for establishing a triple cell culture model of the blood-brain barrier based on primary human brain microvascular endothelial cells, astrocytes, and pericytes. This multicellular model is suitable for studies of neurovascular unit dysfunction during ischemic stroke in vitro or for the screening of drug candidates.

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is ...

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

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of stroke than relying on indirect measurements, and will be a potent and robust vascular research tool. We use an optical imaging approach that labels thrombi with a molecular imaging thrombus marker&#160;&#8212; a Cy5.5 near-infrared fluorescent (NIRF) probe that is covalently linked to the fibrin strands of the thrombus by the fibrin-crosslinking enzymatic action of activated coagulation factor XIIIa during the process of clot maturation. A micro-computed tomography (microCT)-based approach uses thrombus-seeking gold nanoparticles (AuNPs) functionalized to target the major component of the clot: fibrin. This paper describes a detailed protocol for the combined in vivo microCT and ex vivo NIRF imaging of thromboemboli in a mouse model of embolic stroke. We show that in vivo microCT and fibrin-targeted glycol-chitosan AuNPs (fib-GC-AuNPs) can be used for visualizing both in situ thrombi and cerebral embolic thrombi. We also describe the use of in vivo microCT-based direct thrombus imaging to serially monitor the therapeutic effects of tissue plasminogen activator-mediated thrombolysis. After the last imaging session, we demonstrate by ex vivo NIRF imaging the extent and the distribution of residual thromboemboli in the brain. Finally, we describe quantitative image analyses of microCT and NIRF imaging data. The combined technique of direct thrombus imaging allows two independent methods of thrombus visualization to be compared: the area of thrombus-related fluorescent signal on ex vivo NIRF imaging vs. the volume of hyperdense microCT thrombi in vivo.

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.

Direct thrombus imaging visualizes the root cause of thromboembolic infarction. Being able to image thrombus directly allows far better investigation of ...

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

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

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.

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Neuroscience

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

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The endothelium is the primary source of many of the major hemostatic molecules that control platelet aggregation, coagulation, and fibrinolysis. Although the mechanism of thrombosis has been investigated for decades, in vitro studies mainly focus on situations of vascular damage where the subendothelial matrix gets exposed, or on interactions between cells with single blood components. Our method allows studying interactions between whole blood and an intact, confluent vascular cell network.
By utilizing primary human endothelial cells, this protocol provides the unique opportunity to study the influence of endothelial cells on thrombus dynamics and gives&#160;valuable insights into the pathophysiology of thrombotic disease. The use of custom-made microfluidic flow channels allows application of disease-specific vascular geometries and model specific morphological vascular changes. The development of a thrombus is recorded in real-time and quantitatively characterized by platelet adhesion and fibrin deposition. The effect of endothelial function in altered thrombus dynamics is determined by postanalysis through immunofluorescence staining of specific molecules.
The representative results describe the experimental setup, data collection, and data analysis. Depending on the research question, parameters for every section can be adjusted including cell type, shear rates, channel geometry, drug therapy, and postanalysis procedures. The protocol is validated by quantifying thrombus formation on the pulmonary artery endothelium of patients with chronic thromboembolic disease.

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.

The formation of blood clots involves complex interactions between endothelial cells, their underlying matrix, various blood cells, and proteins. The ...

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

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke involves middle cerebral artery occlusion (MCAO). Infarct zone, brain edema and blood-brain barrier (BBB) breakdown are measured as parameters that reflect the extent of brain injury after MCAO. A significant limitation to this method is that these measurements are normally obtained in different rat brain samples, leading to ethical and financial burdens due to the large number of rats that need to be euthanized for an appropriate sample size. Here we present a method to accurately assess brain injury following MCAO by measuring infarct zone, brain edema and BBB permeability in the same set of rat brains. This novel technique provides a more efficient way to evaluate the pathophysiology of stroke.

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.

One of the most common causes of morbidity and mortality worldwide is ischemic stroke. Historically, an animal model used to stimulate ischemic stroke ...

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

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional deficits. Numerous studies by our group and others have shown that mTBI stimulates cytokine expression and activates microglia, decreases cerebral blood flow and metabolism, and impairs cerebrovascular reactivity. Moreover, several works have reported an association between derangements in these neuroinflammatory and hemodynamic markers and cognitive impairments. Herein we detail methods to characterize the neuroinflammatory and hemodynamic tissue response to mTBI in mice. Specifically, we describe how to perform a weight-drop model of mTBI, how to longitudinally measure cerebral blood flow using a non-invasive optical technique called diffuse correlation spectroscopy, and how to perform a Luminex multiplexed immunoassay on brain tissue samples to quantify cytokines and immunomodulatory phospho-proteins (e.g., within the MAPK and NF&#954;B pathways) that respond to and regulate activity of microglia and other neural immune cells. Finally, we detail how to integrate these data using a multivariate systems analysis approach to understand the relationships between all of these variables. Understanding the relationships between these physiologic and molecular variables will ultimately enable us to identify mechanisms responsible for mTBI.

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.

Mild traumatic brain injuries (mTBIs) are a significant public health problem. Repeated exposure to mTBI can lead to cumulative, long-lasting functional ...

A Fibrin-Enriched and tPA-Sensitive Photothrombotic Stroke Model

An ideal thromboembolic stroke model requires certain properties, including relatively simple surgical procedures with low mortality, a consistent infarction size and location, precipitation of platelet:fibrin intermixed blood clots similar to those in patients, and an adequate sensitivity to fibrinolytic treatment. The rose bengal (RB) dye-based photothrombotic stroke model meets the first two requirements but is highly refractory to tPA-mediated lytic treatment, presumably due to its platelet-rich, but fibrin-poor clot composition. We reason that combination of RB dye (50 mg/kg) and a sub-thrombotic dose of thrombin (80 U/kg) for photoactivation aimed at the proximal branch of middle cerebral artery (MCA) may produce fibrin-enriched and tPA-sensitive clots. Indeed, the thrombin and RB (T+RB)-combined photothrombosis model triggered mixed platelet:fibrin blood clots, as shown by immunostaining and immunoblots, and maintained consistent infarct sizes and locations plus low mortality. Moreover, intravenous injection of tPA (Alteplase, 10 mg/kg) within 2 h post-photoactivation significantly decreased the infarct size in T+RB photothrombosis. Thus, the thrombin-enhanced photothrombotic stroke model may be a useful experimental model to test novel thrombolytic therapies.

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 Comprehensive Approach to Analyze the Cell Components of Cerebral Blood Clots

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