Name: imaging vital and non-vital brain pericytes in brain slices following subarachnoid hemorrhage
Uploaded: 2023-08-18T15:00:00.000+00:00
Duration: 5 min 11 s
Description: Watch this Scientific Journal Video about Imaging Pericytes Post-Subarachnoid Hemorrhaging in Rodents at JoVE.com

The study was supported by grants from the National Natural Science Foundation of China (81960226,81760223); the Natural Science Foundation of Yunnan Province (202001AS070045,202301AY070001-011)

The experimental protocol was approved by the Animal Ethics and Use Committee of Kunming Medical University (kmmu20220945). Sprague-Dawley (SD) rats of both sexes, 300-350 g, were used for the present study.
1. Inducing the SAH model
<ol>
	<li>Anesthetize the rats using 2% isoflurane and 100% oxygen. Maintain anesthesia by supplying continuous inhalation anesthesia with isoflurane (1%-3%). Secure the rat&#39;s head using a stereotaxic apparatus (see Table of Materials).</li>
	<li>Create the SAH model following the steps below.
	<ol>
		<li>Insert a microinjection needle into the suprasellar cistern. Then, inject autologous blood from the rat&#39;s tail artery (without anticoagulant) into the suprasellar cistern using a syringe pump (see Table of Materials).</li>
		<li>Implant a microinjection needle into the right lateral cerebral ventricle using the mentioned coordinates: bregma, &#8722;0.8 mm; lateral, 1.4 mm; depth, 4 mm9. For SAH groups, inject 0.2 mL of rat tail artery blood. For sham groups, administer the same volume of isotonic saline (Figure 1A).</li>
	</ol>
	</li>
</ol>
2. Brain slice preparation and stabilization
<ol>
	<li>Deeply anesthetize the rats using 2% isoflurane inhalation. After decapitation, extract the entire brains and immerse them in ice-cold artificial cerebrospinal fluid (ACSF). 
	NOTE: Use freshly prepared ACSF solutions with the following composition: 134 mM NaCl, 2.8 mM KCl, 29 mM NaHCO3, 1.1 mM NaH2PO4, 1.5 mM MgSO4, 2.5 mM CaCl2, and 10.11 mM D-Glucose (see Table of Materials). Maintain a pH between 7.3 and 7.4.</li>
	<li>Use a vibratome (see Table of Materials) to prepare acute brain slices with a thickness of 200 &#181;m from the SD rats in ice-cold ACSF that&#39;s aerated with 5% CO2 and 95% O2 (Figure 2).</li>
	<li>Place the 200 &#181;m-thick acute brain slices in a storage chamber on a nylon mesh submerged in ACSF. Equilibrate the ACSF solution with 95% O2 and 5% CO2, maintaining a temperature range of 35-37 &#176;C. Allow brain slices to stabilize for 2 h, giving them time to adjust (Figure 3).</li>
</ol>
3. Labeling pericytes in acute brain slices with TO-PRO-3
NOTE: Pericytes from acute brain slices were fluorescently labeled using the tracer TO-PRO-310.
<ol>
	<li>Add the fluorescent dye TO-PRO-3 to the ACSF, achieving a final concentration of 1 &#181;M. Incubate the acute brain slices at room temperature in a dark environment with the TO-PRO-3-containing ACSF for 20 min. 
	NOTE: Remember to wear latex gloves while handling TO-PRO-3 for protection.</li>
	<li>Transfer the nylon mesh strainer, carrying the brain slices, from the loading chamber to a 6-well plate rinsing chamber (see Table of Materials). Allow the brain slices to stay in the rinsing chamber for 10 min (Figure 4D). 
	NOTE: This rinsing step terminates the staining process, decreases background labeling, and prevents nonspecific staining.</li>
	<li>Rinse the preparations for a total of 15 min using ACSF solution that&#39;s equilibrated with a mixture of 5% CO2 and 95% O2. This rinsing step halts dye uptake and minimizes background labeling (Figure 4C).</li>
</ol>
4. Staining non-vital pericytes of cerebral microcirculation in acute brain slices
<ol>
	<li>Incubate brain slices preloaded with TO-PRO-3 at 37 &#176;C with isolectin B4 conjugated to Alexa Fluor 488 (FITC-ISOB4; 5 &#181;g/mL, see Table of Materials) for 30 min in a dark environment. After incubation, rinse the brain slices for 15 min in ACSF (Figure 4E).</li>
	<li>Incubate the brain slices in ACSF gassed with 5% CO2 and 95% O2 as usual. Add propidium iodide (PI) at a concentration of 37 &#181;M to both solutions at 37 &#176;C. This will label non-vital brain pericytes. Incubate the preparations in this ACSF solution for 60 min in the dark (Figure 4F).</li>
	<li>Next, rinse the preparations for 15 min with ACSF solutions to halt dye uptake and minimize background labeling.</li>
</ol>
5. Imaging of vital and non-vital brain pericytes in acute brain slices
<ol>
	<li>Gently transfer brain slices to glass bottom confocal dishes (see Table of Materials) using plastic Pasteur pipettes. Fill the dishes with ACSF solution previously equilibrated with 5% CO2 and 95% O2. Use an iron mesh to secure rat brain slices in place.</li>
	<li>Transfer the glass bottom confocal dishes to the stage of a confocal microscope. Position the acute brain slice at the bottom of the dish and perfuse9 it with ACSF solution equilibrated with a mixture of 95% O2 and 5% CO2 during confocal microscopy imaging (Figure 5A and Figure 5Ab&#39;).</li>
	<li>Visualize the wall cells of the cerebral cortical microvasculature using a 40x objective. Capture image stacks using compatible software (see Table of Materials). Use appropriate filters for IB4 (excitation/emission 460 nm/520 nm), PI (excitation/emission 545 nm/595 nm), and TO-PRO-3 (excitation/emission 606 nm/666 nm) (Figure 5D). 
	NOTE: Capture images with the following details: 40&#215; DIC N1 objective lens, 3 &#215; 12 bit: 512 &#215; 512 pixels (0.22 &#215; 0.22 mm), calibration: 0.42 &#181;m/px.</li>
	<li>Identify cerebral microvasculature and pericytes based on their network and morphology. Locate a specific region containing a cerebral microvasculature network on each brain slice and capture images.</li>
	<li>Carefully note the imaging location to ensure consistency in subsequent captures (Figure 5B,C). For further processing and analysis, use image analysis software (ImageJ) and photo editing software (see Table of Materials).</li>
</ol>

Visualization of Brain Pericyte Contractility Post-Subarachnoid Hemorrhage

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M., Howarth, C., Mobbs, P., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+control+of+CNS+capillary+diameter+by+pericytes.">Bidirectional control of CNS capillary diameter by pericytes.</a> Nature. 443 (7112), 700-704 (2006).</li><li>Attwell, D., Mishra, A., Hall, C. N., O'Farrell, F. M., Dalkara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+a+pericyte.">What is a pericyte.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 36 (2), 451-455 (2016).</li><li>Yemisci, M., Gursoy-Ozdemir, Y., Vural, A., Can, A., Topalkara, K., Dalkara, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte+contraction+induced+by+oxidative-nitrative+stress+impairs+capillary+reflow+despite+successful+opening+of+an+occluded+cerebral+artery.">Pericyte contraction induced by oxidative-nitrative stress impairs capillary reflow despite successful opening of an occluded cerebral artery.</a> Nature Medicine. 15 (9), 1031-1037 (2009).</li><li>Korte, 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=Noradrenaline+released+from+locus+coeruleus+axons+contracts+cerebral+capillary+pericytes+via+&#945;2+adrenergic+receptors.">Noradrenaline released from locus coeruleus axons contracts cerebral capillary pericytes via &#945;2 adrenergic receptors.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. , (2023).</li><li>Hirunpattarasilp, C., Barkaway, A., Davis, H., Pfeiffer, T., Sethi, H., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperoxia+evokes+pericyte-mediated+capillary+constriction.">Hyperoxia evokes pericyte-mediated capillary constriction.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 42 (11), 2032-2047 (2022).</li><li>Neuhaus, A. A., Couch, Y., Sutherland, B. A., Buchan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+to+study+pericyte+contractility+and+responses+to+ischaemia+in+vitro+using+electrical+impedance.">Novel method to study pericyte contractility and responses to ischaemia in vitro using electrical impedance.</a> Journal of Cerebral Blood Flow and Metabolism: Official Journal of the International Society of Cerebral Blood Flow and Metabolism. 37 (6), 2013-2024 (2017).</li><li>Gong, Y., 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=Increased+TRPM4+Activity+in+cerebral+artery+myocytes+contributes+to+cerebral+blood+flow+reduction+after+subarachnoid+hemorrhage+in+rats.">Increased TRPM4 Activity in cerebral artery myocytes contributes to cerebral blood flow reduction after subarachnoid hemorrhage in rats.</a> Neurotherapeutics: The Journal of the American Society for Experimental NeuroTherapeutics. 16 (3), 901-911 (2019).</li><li>Mai-Morente, S. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericyte+mapping+in+cerebral+slices+with+the+far-red+fluorophore+TO-PRO-3.">Pericyte mapping in cerebral slices with the far-red fluorophore TO-PRO-3.</a> Bio-protocol. 11 (22), e4222 (2021).</li><li>Mai-Morente, S. P., Marset, V. M., Blanco, F., Isasi, E. E., Abudara, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nuclear+fluorescent+dye+identifies+pericytes+at+the+neurovascular+unit.">A nuclear fluorescent dye identifies pericytes at the neurovascular unit.</a> Journal of Neurochemistry. 157 (4), 1377-1391 (2021).</li><li>Zhao, 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=Rationale+for+the+real-time+and+dynamic+cell+death+assays+using+propidium+iodide.">Rationale for the real-time and dynamic cell death assays using propidium iodide.</a> Cytometry. Part A: The Journal of the International Society for Analytical Cytology. 77 (4), 399-405 (2010).</li><li>Van Hooijdonk, C. A., Glade, C. P., Van Erp, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TO-PRO-3+iodide:+A+novel+HeNe+laser-excitable+DNA+stain+as+an+alternative+for+propidium+iodide+in+multiparameter+flow+cytometry.">TO-PRO-3 iodide: A novel HeNe laser-excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry.</a> Cytometry. 17 (2), 185-189 (1994).</li><li>Lacar, B., Herman, P., Platel, J. C., Kubera, C., Hyder, F., Bordey, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+progenitor+cells+regulate+capillary+blood+flow+in+the+postnatal+subventricular+zone.">Neural progenitor cells regulate capillary blood flow in the postnatal subventricular zone.</a> The Journal of Neuroscience: The Official Journal of the Society for Neuroscience. 32 (46), 16435-16448 (2012).</li><li>Mai-Morente, S. P., Marset, V. M., Blanco, F., Isasi, E. E., Abudara, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nuclear+fluorescent+dye+identifies+pericytes+at+the+neurovascular+unit.">A nuclear fluorescent dye identifies pericytes at the neurovascular unit.</a> Journal of Neurochemistry. 157 (4), 1377-1391 (2021).</li><li>Hezel, M., Ebrahimi, F., Koch, M., Dehghani, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propidium+iodide+staining:+a+new+application+in+fluorescence+microscopy+for+analysis+of+cytoarchitecture+in+adult+and+developing+rodent+brain.">Propidium iodide staining: a new application in fluorescence microscopy for analysis of cytoarchitecture in adult and developing rodent brain.</a> Micron (Oxford, England). 43 (10), 1031-1038 (2012).</li><li>Mathiisen, T. M., Lehre, K. P., Danbolt, N. C., Ottersen, O. 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The authors declare that they have no competing financial interests.

Developed are high-resolution confocal imaging techniques for visualizing vital brain pericytes, non-vital brain pericytes, and the microvasculature in brain slices. In acute rat brain slices, the process entails initial labeling of pericytes with TO-PRO-311, followed by microvascular endothelial cells with IB412; subsequently, identification of deceased pericytes is conducted using PI. This protocol is straightforward, reproducible, and highly applicable for functional res...

Brain pericytes, distinguished by their slender protuberances and protruding cell bodies, encircle the microcirculation1,2. While cerebral blood flow augmentation is predominantly driven by capillary dilation, smaller arteries exhibit slower rates of dilation3. Pericyte contractility exerts influence over capillary diameter and pericyte morphology, impacting vascular dynamics4. Contraction of brain pericytes leads to capillary constriction, and in pathological scenarios, excessive contraction may impede erythrocyte flow5. Various factors, including norepinephrine released from the locus coeruleus, can induce brain pericyte contraction within capillaries6. With a regulatory role in cerebral blood flow, pericytes exhibit 20-HETE synthesis, serving as an oxygen sensor during hyperoxia7. Oxidative-nitrative stress-triggered contraction of brain pericytes detrimentally affects capillaries5. Despite both in vivo and ex vivo investigations into brain pericyte contraction8, limited knowledge persists regarding the imaging of viable and non-viable brain pericytes within brain slices.
Crucially, post-tissue fixation imaging of brain pericytes compromises their vitality and subsequent contractility assessment. Moreover, in scenarios such as neurological disorders (e.g., subarachnoid hemorrhage - SAH), transgenic labeling of brain pericytes fails to differentiate between viable and non-viable pericytes, as confirmed by our preliminary SAH-induced brain pericyte death study9. 
To surmount these challenges, we employed TO-PRO-3 to label live pericytes, while deceased ones were stained with propidium iodide (PI). We used high-resolution confocal imaging technologies to visualize viable and non-viable brain pericytes in brain slices while preserving slice activity during imaging. This article aims to present a reproducible method for imaging viable and non-viable brain pericytes in brain slices, serving as a valuable tool to probe the impact of brain pericytes on cerebral microcirculation post SAH.

<table><tbody><tr><td>6-well plate</td><td>ABC biochemistry</td><td>ABC703006</td><td>RT</td></tr><tr><td>Adobe Photoshop</td><td>Adobe</td><td>Adobe Illustrator CS6 16.0.0</td><td>RT</td></tr><tr><td>Aluminium foil</td><td>MIAOJIE</td><td>225 mm x 273 mm</td><td>RT</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;&amp;middot;2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C3881</td><td>RT</td></tr><tr><td>Confocal imaging software</td><td>Nikon</td><td>NIS-Elements 4.10.00</td><td>RT</td></tr><tr><td>Confocal Laser Scanning Microscope</td><td>Nikon</td><td>N-SIM/C2si</td><td>RT</td></tr><tr><td>Gas tank (5% CO&lt;sub&gt;2&lt;/sub&gt;, 95% O&lt;sub&gt;2&lt;/sub&gt;)</td><td>PENGYIDA</td><td>40L</td><td>RT</td></tr><tr><td>Glass Bottom Confocal Dishes</td><td>Beyotime</td><td>FCFC020-10pcs</td><td>RT</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G5767</td><td>RT</td></tr><tr><td>Glue</td><td>EVOBOND</td><td>KH-502</td><td>RT</td></tr><tr><td>Ice machine</td><td>XUEKE</td><td>IMS-20</td><td>RT</td></tr><tr><td>Image analysis software</td><td>National Institutes of Health</td><td>Image J</td><td>RT</td></tr><tr><td>Inhalation anesthesia system</td><td>SCIENCE</td><td>QAF700</td><td>RT</td></tr><tr><td>Isolectin B 4-FITC</td><td>SIGMA</td><td>L2895&amp;ndash;2MG</td><td>Store aliquots at &amp;ndash;20 &amp;deg;C</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>7447&amp;ndash;40&amp;ndash;7</td><td>RT</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P0662</td><td>RT</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>M7506</td><td>RT</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>7647&amp;ndash;14&amp;ndash;5</td><td>RT</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;middot;H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>10049&amp;ndash;21&amp;ndash;5</td><td>RT</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S5761</td><td>RT</td></tr><tr><td>Pasteur pipette</td><td>NEST Biotechnology</td><td>318314</td><td>RT</td></tr><tr><td>Peristaltic Pump</td><td>Scientific Industries Inc</td><td>Model 203</td><td>RT</td></tr><tr><td>Propidium (Iodide)</td><td>Med Chem Express</td><td>HY-D0815/CS-7538</td><td>Store aliquots at &amp;ndash;20 &amp;deg;C</td></tr><tr><td>Stereotaxic apparatus</td><td>SCIENCE</td><td>QA</td><td>RT</td></tr><tr><td>Syringe pump</td><td>Harvard PUMP</td><td>PUMP 11 ELITE Nanomite</td><td>RT</td></tr><tr><td>Thermostatic water bath</td><td>OLABO</td><td>HH-2</td><td>RT</td></tr><tr><td>Vibrating microtome</td><td>Leica</td><td>VT1200</td><td>RT</td></tr></tbody></table>

imaging vital and non-vital brain pericytes in brain slices following subarachnoid hemorrhage

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility adjustments. Conventionally, their contractility is gauged by observing morphological shifts and nearby capillary diameter changes under specific circumstances. Yet, post-tissue fixation, evaluating vitality and ensuing pericyte contractility of imaged brain pericytes becomes compromised. Similarly, genetically labeling brain pericytes falls short in distinguishing between viable and non-viable pericytes, particularly in neurologic conditions like subarachnoid hemorrhage (SAH), where our preliminary investigation validates brain pericyte demise. A reliable protocol has been devised to surmount these constraints, enabling simultaneous fluorescent tagging of both functional and non-functional brain pericytes in brain sections. This labeling method allows high-resolution confocal microscope visualization, concurrently marking the brain slice microvasculature. This innovative protocol offers a means to appraise brain pericyte contractility, its impact on capillary diameter, and pericyte structure. Investigating brain pericyte contractility within the SAH context yields insightful comprehension of its effects on cerebral microcirculation.

Under normal physiological conditions, brain pericytes generally do not undergo cell death. Figure 6 illustrates this phenomenon, with yellow indicating the presence of vital brain pericytes; brain pericytes show no staining with PI, indicating their viability. To further investigate whether pericytes remain attached to the microvasculature following cell death, methods were employed in a SAH rat model, and subsequent imaging was conducted.
Methods for imaging bot...

Watch this Scientific Journal Video about Imaging Pericytes Post-Subarachnoid Hemorrhaging in Rodents at JoVE.com

Author Spotlight: Imaging Pericytes Post-Subarachnoid Hemorrhaging in Rodents 

The preliminary inquiry confirms that subarachnoid hemorrhage (SAH) causes brain pericyte demise. Evaluating pericyte contractility post-SAH requires differentiation between viable and non-viable brain pericytes. Hence, a procedure has been developed to label viable and non-viable brain pericytes concurrently in brain sections, facilitating observation using a high-resolution confocal microscope.

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility ...

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Research

JoVE Journal

Neuroscience

1.0K Views.  The First Affiliated Hospital of Kunming Medical University. Onze studie toonde een significante toename van het aantal sterfgevallen door pericyten. Dus beginnend bij een ziek paard na SAH, toonde het een positieve correlatie aan tussen het aantal lage vitale parasieten en de tijd die verstreek na SAH. Het in beeld brengen van vitale en niet-vitale hersenparasieten in hersenplakjes volgens SAH en de parasietisolatietechnieken, hebben het onderzoek in mijn vakgebied vooruit geholpen.Momenteel is het vrij moeilijk om volledig onderscheid te maken...

Video: Auteur in de schijnwerpers: Beeldvorming van pericyten na subarachnoïdale bloedingen bij knaagdieren 

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Medicine

Intravascular Perfusion of Carbon Black Ink Allows Reliable Visualization of Cerebral Vessels

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The cerebral vasculature dynamically responds to various pathophysiological states and it exhibits considerable differences between strains and under conditions of genetic manipulations. Essentially, a reliable technique for intracranial vessel staining is essential in order to study the pathogenesis of ischemic stroke. Until recently, a set of different techniques has been employed to visualize the cerebral vasculature including injection of low viscosity resin, araldite F, gelatin mixed with various dyes1 (i.e. carmine red, India ink) or latex with2 or without3 carbon black. Perfusion of white latex compound through the ascending aorta has been first reported by Coyle and Jokelainen3. Maeda et al.2 have modified the protocol by adding carbon black ink to the latex compound for improved contrast visualization of the vessels after saline perfusion of the brain. However, inefficient perfusion and inadequate filling of the vessels are frequently experienced due to high viscosity of the latex compound4. Therefore, we have described a simple and cost-effective technique using a mixture of two commercially available carbon black inks (CB1 and CB2) to visualize the cerebral vasculature in a reproducible manner5. We have shown that perfusion with CB1+CB2 in mice results in staining of significantly smaller cerebral vessels at a higher density in comparison to latex perfusion5. Here, we describe our protocol to identify the anastomotic points between the anterior (ACA) and middle cerebral arteries (MCA) to study vessel variations in mice with different genetic backgrounds. Finally, we demonstrate the feasibility of our technique in a transient focal cerebral ischemia model in mice by combining CB1+CB2-mediated vessel staining with TTC staining in various degrees of ischemic injuries.

Analysis of rodent cerebrovascular anatomy plays an important role in experimental stroke research. In this context, intravascular perfusion with colored latex has been considered as a standard tool for several years. However, this technique implies distinct technical limitations, which undermine its reproducibility. Here, we describe a simple method to visualize cerebral vessels in a reproducible manner. Injection of a mixture of two commercially available carbon black inks through the left myocardial ventricle results in adequate filling of cerebral vessels with high contrast visualization. We have successfully applied this technique to identify anastomotic points between cerebral vascular territories of mice with different genetic backgrounds. We finally give evidence that this novel and simple method for vessel staining can be combined with triphenyltetrazolium chloride (TTC) staining - a widely used tool to observe and analyze infarct volumes in mice.

The anatomical structure of cerebral vessels is a key determinant for brain hemodynamics as well as the severity of injury following ischemic insults. The ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Quantification of Neurovascular Protection Following Repetitive Hypoxic Preconditioning and Transient Middle Cerebral Artery Occlusion in Mice

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week protocol for repetitive hypoxic preconditioning (RHP) induces long-term protection against central nervous system (CNS) injury in a mouse model of focal ischemic stroke. RHP consists of 9 stochastic exposures to hypoxia that vary in both duration (2 or 4 hr) and intensity (8% and 11% O2). RHP reduces infarct volumes, blood-brain barrier (BBB) disruption, and the post-stroke inflammatory response for weeks following the last exposure to hypoxia, suggesting a long-term induction of an endogenous CNS-protective phenotype. The methodology for the dual quantification of infarct volume and BBB disruption is effective in assessing neurovascular protection in mice with RHP or other putative neuroprotectants. Adult male Swiss Webster mice were preconditioned by RHP or duration-equivalent exposures to 21% O2 (i.e. room air). A 60 min transient middle cerebral artery occlusion (tMCAo) was induced 2 weeks following the last hypoxic exposure. Both the occlusion and reperfusion were confirmed by transcranial laser Doppler flowmetry. Twenty-two hr after reperfusion, Evans Blue (EB) was intravenously administered through a tail vein injection. 2 hr later, animals were sacrificed by isoflurane overdose and brain sections were stained with 2,3,5- triphenyltetrazolium chloride (TTC). Infarcts volumes were then quantified. Next, EB was extracted from the tissue over 48 hr to determine BBB disruption after tMCAo. In summary, RHP is a simple protocol that can be replicated, with minimal cost, to induce long-term endogenous neurovascular protection from stroke injury in mice, with the translational potential for other CNS-based and systemic pro-inflammatory disease states.

This protocol describes repetitive hypoxic preconditioning, or brief exposures to systemic hypoxia that reduce infarct volumes and blood-brain barrier disruption following transient middle cerebral artery occlusion in mice. It also details dual quantification of infarct volume and blood-brain barrier disruption after stroke to assess the efficacy of neurovascular protection.

Experimental animal models of stroke are invaluable tools for understanding stroke pathology and developing more effective treatment strategies. A 2 week ...

Experimental Models to Study the Neuroprotection of Acidic Postconditioning Against Cerebral Ischemia

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for neuroprotection, postconditioning treatments have proven to be promising therapies against cerebral ischemia. However, complicated procedures and potential safety issues limit their clinical application. To overcome these disadvantages, we have developed acidic postconditioning (APC) as a therapy for experimental focal cerebral ischemia. APC refers to the mild acidosis treatment by inhaling CO2 during reperfusion following ischemia. Here we present two models to execute APC in vitro and in vivo, respectively. The oxygen-glucose deprivation (OGD) treatment of mice and the corticostriatal occlusion and middle cerebral artery occlusion (MCAO) of mice were employed to mimic cerebral ischemia. APC can be simply achieved by transferring brain slices to acidic buffer bubbled with 20% CO2, or by mice inhaling 20% CO2. APC showed significant protective effects against cerebral ischemia, as reflected by tissue viability and brain infarct volume.

Acidic postconditioning protects against cerebral ischemia. Here we present two models to execute APC. They are achieved respectively by transferring corticostriatal slices to acidic buffer after oxygen-glucose deprivation in vitro and by inhaling 20% CO2 after middle cerebral artery occlusion in vivo.

Stroke is one of the leading causes of mortality and disability worldwide, with limited therapeutic approaches. As an endogenous strategy for ...

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 ...

Quantification of Cerebral Perfusion using Laser Speckle Imaging and Infarct Volume using MRI in a Pre-clinical Model of Posterior Circulation Stroke

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical standard to diagnose ischemic stroke include computerized tomography (CT), CT angiography and perfusion and magnetic resonance imaging (MRI). Large animal pre-clinical models to accurately reflect the clinical disease as well as methods to assess stroke burden and evaluate treatments are lacking.
Methods: We describe a canine model of large vessel occlusion (LVO) stroke in the posterior circulation, and developed a laser speckle imaging (LSI) protocol to monitor perfusion changes in real time.&#160; We then utilized high b-value DWI (b=1800s/mm2) MRI to increase detection sensitivity. We also evaluated the ability of magnetic resonance angiography (MRA) to assess arterial occlusion and correlate with DSA. Finally, we verified infarct size from apparent diffusion coefficient (ADC) mapping with histology.&#160;
Results:&#160; Administration of thromboembolism occluded the basilar artery as tracked by DSA (n=7).&#160; &#160;LSI correlated with DSA, demonstrating a reduction in perfusion after stroke onset that persisted throughout the experiment, allowing us to monitor perfusion in real time.&#160; DWI with an optimized b-value for dogs illustrated the stroke volume and allowed us to derive ADC and magnetic resonance angiography (MRA) images. The MRA performed at the end of the experiment correlated with DSA performed after occlusion. Finally, stroke burden on MRI correlated with histology.
Conclusions: Our studies demonstrate real time perfusion imaging using LSI of a canine thromboembolic LVO model of posterior circulation stroke, which utilizes multimodal imaging important in the diagnosis and treatment of ischemic stroke.

A canine model of LVO stroke was utilized to develop laser speckle imaging to monitor cerebral perfusion in real-time.&#160; Diffusion-weighted MRI was optimized to image infarct volume utilizing a high b-value, enabling ADC and MRA, correlated with DSA at the time of stroke.&#160; Finally, ADC reconstructions correlated with histological findings.

Background: Basilar artery occlusion (BAO) is a subset of posterior circulation stroke that carries a mortality as high as 90%.&#160; The current clinical ...

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A problem encountered in experimental studies using murine models of SAH is that methods for in vivo monitoring of cerebral vasospasm in mice are lacking. Here, we demonstrate the application of high frequency ultrasound to perform transcranial Duplex sonography examinations on mice. Using the method, the internal carotid arteries (ICA) could be identified. The blood flow velocities in the intracranial ICAs were accelerated significantly after induction of SAH, while blood flow velocities in the extracranial ICAs remained low, indicating cerebral vasospasm. In conclusion, the method demonstrated here allows functional, noninvasive in vivo monitoring of cerebral vasospasm in a murine SAH model.

The aim of this manuscript is to present a sonography-based method that allows in vivo imaging of blood flow in cerebral arteries in mice. We demonstrate its application to determine changes in blood flow velocities associated with vasospasm in murine models of subarachnoid hemorrhage (SAH).

Cerebral vasospasm that occurs in the weeks after subarachnoid hemorrhage, a type of hemorrhagic stroke, contributes to delayed cerebral ischemia. A ...

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging (MRI)

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high mortality rate as well as an uncontrollable volume of SAH and other intracranial complications such as stroke or intracranial hemorrhage. In this protocol, a standardized SAH mouse model is presented, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies. Briefly, C57BL/6J mice are anesthetized with an intraperitoneal ketamine/xylazine (70 mg/16 mg/kg body weight) injection and placed in a supine position. After midline neck incision, the common carotid artery (CCA) and carotid bifurcation are exposed, and a 5-0 non-absorbable monofilament polypropylene suture is inserted in a retrograde fashion into the external carotid artery (ECA) and advanced into the common carotid artery. Then, the filament is invaginated into the internal carotid artery (ICA) and pushed forward to perforate the anterior cerebral artery (ACA). After recovery from surgery, mice undergo a 7.0 T MRI 24 h later. The volume of bleeding can be quantified and graded via postoperative MRI, enabling a robust experimental SAH group with the option to perform further subgroup analyses based on blood quantity.

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging MRI

Here we present a standardized SAH mouse model, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies.

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high ...

Non-Invasive Integrated PAUSAT Whole-Brain Imaging of Ischemic Stroke

Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging technologies: photoacoustic, ultrasound, and angiographic tomography (PAUSAT). Combining these three modalities helps acquire multi-spectral photoacoustic tomography (PAT) of the brain blood oxygenation, high-frequency ultrasound imaging of the brain tissue, and acoustic angiography of the cerebral blood perfusion. The multi-modal imaging platform allows the study of cerebral perfusion and oxygenation changes in the whole mouse brain after stroke. Two commonly used ischemic stroke models were evaluated: the permanent middle cerebral artery occlusion (pMCAO) model and the photothrombotic (PT) model. PAUSAT was used to image the same mouse brains before and after a stroke and quantitatively analyze both stroke models. This imaging system was able to clearly show the brain vascular changes after ischemic stroke, including significantly reduced blood perfusion and oxygenation in the stroke infarct region (ipsilateral) compared to the uninjured tissue (contralateral). The results were confirmed by both laser speckle contrast imaging and triphenyltetrazolium chloride (TTC) staining. Furthermore, stroke infarct volume in both stroke models was measured and validated by TTC staining as the ground truth. Through this study, we have demonstrated that PAUSAT can be a powerful tool in noninvasive and longitudinal preclinical studies of ischemic stroke.

Author Spotlight: Integrated Photoacoustic, Ultrasound, and Angiographic Tomography (PAUSAT) for NonInvasive Whole-Brain Imaging of Ischemic Stroke  

This work demonstrates the use of a multimodal ultrasound-based imaging platform for noninvasive imaging of ischemic stroke. This system allows for the quantification of blood oxygenation through photoacoustic imaging and impaired perfusion in the brain through acoustic angiography.

Presented here is an experimental ischemic stroke study using our newly developed noninvasive imaging system that integrates three acoustic-based imaging ...

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage

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