This work was supported by the Hebei Province Introducing Foreign Intelligence Project in 2023. We thank Lisa Kreiner, PhD, from Liwen Bianji, (Edanz) (www.liwenbianji.cn), for editing the English text of a draft of this manuscript.

The experimental procedure received approval from the Institutional Animal Care and Use Committee at Yantai University (Approval Number: YTDX20230410) and adhered to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats weighing between 300-350 g were utilized for this study. A comprehensive list of the animals, reagents, and equipment used is provided in the Table of Materials.
1. Animal preparation
<ol>
	<li>House the animals in an environment maintaining a 12 h light/dark cycle, controlled temperature, and humidity. Ensure they have unrestricted access to food and water.</li>
</ol>
2. Anesthesia
<ol>
	<li>Anesthetize the rats via intraperitoneal injection with a mixture containing ketamine (80 mg/kg) and xylazine (20 mg/kg). 
	NOTE: Tilt the head and body of rats downward. The injection site is in the animal&#8217;s lower right quadrant of the abdomen. Pull back on the plunger before injecting. The average injection volume is 0.4 mL.</li>
	<li>Confirm the induction of anesthesia by assessing the withdrawal reflex of the hind paw and the blinking reflex. If adequate depth of anesthesia is not achieved, supplement with isoflurane during the surgical procedure.</li>
</ol>
3. Ischemic modeling procedure
<ol>
	<li>Sterilize the surgical tools using an autoclave. Disinfect the operating tables and related equipment with 70% ethanol.</li>
	<li>Prepare the surgical site by shaving the fur around the anticipated incision site. Disinfect the skin at the surgical site three times with betadine and 70% ethanol.</li>
	<li>Make a midline incision on the ventral side, approximately 1.5 cm long, and open the superficial fascia.</li>
	<li>Perform sharp and blunt dissection to expose the external carotid artery (ECA), internal carotid artery (ICA), and common carotid artery (CCA) as per the previously described method7.</li>
	<li>Place three 4-0 silk sutures around the CCA and another silk suture through the ECA near the ICA bifurcation. Tie a releasable slip knot on the proximal CCA. Tighten the suture on the distal CCA using a hemostat to block blood flow. Loosely tie another slip knot around the CCA during suturing to ensure the vessel is not occluded. 
	NOTE: Ensure that the slip knot is not overly tight, only sufficient to block blood flow, as it will need to be released during vascular suturing.</li>
	<li>Make a 0.5 mm incision between the proximal silk suture and the middle suture on the CCA using scissors. 
	NOTE: Ensure that the incision allows the filament (silicon-coated tip) to enter the CCA.</li>
	<li>Introduce a 3 cm length of 4-0 filament (silicon-coated tip) into the CCA. Tighten the silk sutures around the CCA to secure the intraluminal filament and prevent bleeding; then, remove the suture from the distal CCA.</li>
	<li>Continue to insert the filament into the lumen of the MCA, controlling the sutures around the ECA to ensure the filament does not enter the ECA. The insertion length is typically 18 mm to 20 mm.</li>
	<li>Start a timer and record the time when occlusion begins. Suture the neck incision with 3-0 silk sutures and carefully place the animal in a recovery cage. Administer 0.5 mL of normal saline intraperitoneally.</li>
</ol>
4. Microsurgical suturing of the CCA
<ol>
	<li>Re-anesthetize the rat 1.5 h post-ischemia. Disinfect the incision site three times with 70% ethanol and reopen the incision.</li>
	<li>Gently withdraw the occluding filament until the white tip is clearly visible near the CCA incision. Place a micro clamp horizontally close to the secured intraluminal suture knot near the CCA bifurcation. 
	NOTE: Avoid clamping the white filament tip to prevent silicone detachment and subsequent thrombosis.</li>
	<li>Release the secured intraluminal suture knot and completely remove the filament.</li>
	<li>Place another micro clamp near the heart-end side of the proximal CCA knot. Subsequently, remove the suture knot around the proximal CCA. 
	NOTE: Proper placement of clamps is crucial for elevating fixed vessels in later steps to facilitate microvascular suturing. Place clamps horizontally on surrounding muscles to prevent slippage.</li>
	<li>Rinse the incision site with normal saline and dry the area with a sterile cotton ball to remove the blood and achieve a clear surgical field.</li>
	<li>Under a microscope (4x magnification), suture the CCA incision using 11-0 microsurgical sutures in an interrupted pattern, typically requiring two stitches depending on the incision size.</li>
	<li>After suturing, remove the micro clamps from both ends of the CCA to restore blood flow.</li>
	<li>Apply pressure with a sterile cotton ball to the CCA suture site for 30 s. Observe the CCA for 2 min to ensure no bleeding occurs. Suture the rat&#39;s neck incision and place the animal in a 35 &#176;C care box for anesthesia recovery before returning it to the cage. 
	NOTE: Administer intraperitoneally with meloxicam at a dose of 2 mg/kg for post-operative analgesia.</li>
</ol>
5. Staining with 2,3,5-triphenyltetrazolium chloride (TTC)
<ol>
	<li>Three days after the ischemia-reperfusion,&#160;euthanize the animals under deep anesthesia with 4% isoflurane and cervical dislocation.</li>
	<li>Remove the brains and cut them into 2 mm-thick coronal sections using a scalpel blade8.</li>
	<li>Incubate the brain sections with 2% TTC for 20 min at 37 &#176;C.</li>
	<li>Rinse the brain sections twice with 10% formalin, then fix them with 10% formalin for 30 min at room temperature.</li>
	<li>Capture digital photographs of consecutive slices and quantitatively measure the infarction areas using Image J software.</li>
</ol>
6. Neurological function test
NOTE: Neurological function was assessed using the Garcia test, following a previous report9.
<ol>
	<li>Assess spontaneous activity, symmetry in the movement of all four limbs, forepaw outstretching, climbing, body proprioception, and response to vibrissae touch. Assign scores based on the Garcia test criteria, with a maximum score of 18 points indicating normal neurological function.</li>
</ol>
7. Statistical analysis
<ol>
	<li>Utilize GraphPad Prism to conduct statistical analysis using Student&#39;s t-test. Express the data as the mean &#177; standard deviation. Consider differences between means statistically significant if P &#60; 0.05.</li>
</ol>

Middle Cerebral Artery Occlusion in Rats with Preserved Vascular Anatomy

<ol>
	<li>Tu, W. J., 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=China+stroke+surveillance+report+2021.">China stroke surveillance report 2021.</a> Mil Med Res. 10 (1), 33 (2023).</li><li>Wu, 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=Targeting+oxidative+stress+and+inflammation+to+prevent+ischemia-reperfusion+injury.">Targeting oxidative stress and inflammation to prevent ischemia-reperfusion injury.</a> Front in Mol Neurosci. 13, 28 (2020).</li><li>Ringelstein, E. B., 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=Type+and+extent+of+hemispheric+brain+infarctions+and+clinical+outcome+in+early+and+delayed+middle+cerebral+artery+recanalization.">Type and extent of hemispheric brain infarctions and clinical outcome in early and delayed middle cerebral artery recanalization.</a> Neurology. 42 (2), 289-298 (1992).</li><li>Koizumi, J., Yoshida, Y., Nakazawa, T., Ooneda, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+studies+of+ischemic+brain+edema:+A+new+experimental+model+of+cerebral+embolism+in+rats+in+which+recirculation+can+be+introduced+in+the+ischemic+area.">Experimental studies of ischemic brain edema: A new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area.</a> Jap J Stroke. 8, 1-8 (1986).</li><li>Longa, E. Z., Weinstein, P. R., Carlson, S., Cummins, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+middle+cerebral+artery+occlusion+without+craniectomy+in+rats.">Reversible middle cerebral artery occlusion without craniectomy in rats.</a> Stroke. 20 (1), 84-91 (1989).</li><li>Trueman, R. 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=A+critical+re-examination+of+the+intraluminal+filament+MCAO+model:+Impact+of+external+carotid+artery+transection.">A critical re-examination of the intraluminal filament MCAO model: Impact of external carotid artery transection.</a> Transl Stroke Res. 2 (4), 651-661 (2011).</li><li>Ansari, S., Azari, H., McConnell, D. J., Afzal, A., Mocco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraluminal+middle+cerebral+artery+occlusion+(MCAO)+model+for+ischemic+stroke+with+laser+Doppler+flowmetry+guidance+in+mice.">Intraluminal middle cerebral artery occlusion (MCAO) model for ischemic stroke with laser Doppler flowmetry guidance in mice.</a> J Vis Exp. (51), e2879 (2011).</li><li>Yu, Z., 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=Icaritin+inhibits+neuroinflammation+in+a+rat+cerebral+ischemia+model+by+regulating+microglial+polarization+through+the+GPER-ERK-NF-&#954;B+signaling+pathway.">Icaritin inhibits neuroinflammation in a rat cerebral ischemia model by regulating microglial polarization through the GPER-ERK-NF-&#954;B signaling pathway.</a> Mol Med. 28 (1), 142 (2022).</li><li>Garcia, J. H., Wagner, S., Liu, K. F., Hu, X. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurological+Deficit+and+extent+of+neuronal+necrosis+attributable+to+middle+cerebral+artery+occlusion+in+rats.">Neurological Deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats.</a> Stroke. 26 (4), 627-634 (1995).</li><li>Bracko, O., 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=3-Nitropropionic+acid-induced+ischemia+tolerance+in+the+rat+brain+is+mediated+by+reduced+metabolic+activity+and+cerebral+blood+flow.">3-Nitropropionic acid-induced ischemia tolerance in the rat brain is mediated by reduced metabolic activity and cerebral blood flow.</a> J Cereb Blood Flow Metab. 34 (9), 1522-1530 (2014).</li><li>Goldmacher, G. V., 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=Tracking+transplanted+bone+marrow+stem+cells+and+their+effects+in+the+rat+MCAO+stroke+model.">Tracking transplanted bone marrow stem cells and their effects in the rat MCAO stroke model.</a> PLoS One. 8 (3), e60049 (2013).</li><li>Lee, H. J., 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=The+therapeutic+potential+of+human+umbilical+cord+blood-derived+mesenchymal+stem+cells+in+Alzheimer's+disease.">The therapeutic potential of human umbilical cord blood-derived mesenchymal stem cells in Alzheimer's disease.</a> Neurosci Lett. 481 (1), 30-35 (2010).</li><li>Connolly, E. S., Winfree, C. J., Solomon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedural+and+strain-related+variables+significantly+affect+outcome+in+a+murine+model+of+focal+cerebral+ischemia.">Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia.</a> Neurosurgery. 38 (3), 523-531 (1996).</li><li>Barone, F. C., Knudsen, D. J., Nelson, A. H., Feuerstein, G. Z., Willette, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+strain+differences+in+susceptibility+to+cerebral+ischemia+are+related+to+cerebral+vascular+anatomy.">Mouse strain differences in susceptibility to cerebral ischemia are related to cerebral vascular anatomy.</a> J Cereb Blood Flow Metab. 13 (4), 683-692 (1993).</li><li>Dittmar, M., Spruss, T., Schuierer, G., Horn, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+carotid+artery+territory+ischemia+impairs+outcome+in+the+endovascular+filament+model+of+middle+cerebral+artery+occlusion+in+rats.">External carotid artery territory ischemia impairs outcome in the endovascular filament model of middle cerebral artery occlusion in rats.</a> Stroke. 34 (9), 2252-2257 (2003).</li><li>Virtanen, T., Jolkkonen, J., Sivenius, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=External+carotid+artery+territory+ischemia+impairs+outcome+in+the+endovascular+filament+model+of+middle+cerebral+artery+occlusion+in+rats.">External carotid artery territory ischemia impairs outcome in the endovascular filament model of middle cerebral artery occlusion in rats.</a> Stroke. 34 (9), 2252-2257 (2004).</li></ol>

The authors do not have any potential conflicting interests to disclose.

MCAo is a widely used method to create animal models of stroke, effectively replicating the stroke process observed in patients by restoring blood flow after ischemia10,11,12. Our laboratory has developed an ischemic stroke model that enables reperfusion from the CCA without causing damage to the ECA. Following the induction of ischemia, microsurgical repair of the CCA is performed. Although direct measurement of CCA blood flow ...

Stroke, the leading cause of death and long-term neurological dysfunction in adults worldwide, encompass various types, with ischemic stroke accounting for approximately 81.9% of cases, while cerebral hemorrhage and subarachnoid hemorrhage account for 14.9% and 3.1%, respectively1. Ischemic stroke often results from atherosclerosis or occlusion of the middle cerebral artery (MCA), leading to local cerebral blood flow reduction and subsequent brain damage. This reduction in nutrient supply to ischemic cells triggers energy depletion, causing membrane integrity loss, cell swelling, and eventual lysis. Additionally, factors like increased intracellular calcium, excitatory amino acid release, heightened free radical production, and inflammatory cell activation contribute to brain tissue injury post-stroke2.
The treatment goals for ischemic stroke are multifaceted: reduce ongoing neurological damage and lower mortality and long-term disability; prevent complications arising from immobility and neurological dysfunction; and minimize the risk of stroke recurrence. Given the complexity of ischemic stroke pathophysiology, a robust stroke model is essential for the research and development of treatments targeting ischemic stroke.
Currently, middle cerebral artery occlusion (MCAo) is a common animal model of ischemic stroke3. MCAo is typically performed by inserting a silicon-tipped or flame-blunted monofilament through the cervical vessels until it reaches the origin of the middle cerebral artery (MCA). In 1986, Koizumi and colleagues developed a technique involving the insertion of a silicon-tipped monofilament through an incision in the common carotid artery (CCA), allowing it to reach the entrance of the MCA and block its blood flow4. In 1989, Longa's group reported another MCAo method that involved introducing a monofilament through an incision in the external carotid artery (ECA). The monofilament then traverses the bifurcation of the internal carotid artery (ICA) and ECA before reaching the starting point of the MCA5.
The limitations of the two existing methods for inducing MCAo are evident. Koizumi's method necessitates the permanent blockage of the ipsilateral CCA, leading to reduced MCA perfusion since reperfusion solely relies on the circle of Willis4. Conversely, Longa's method involves cutting the ECA, resulting in damage to the ECA system and subsequent impairment of facial muscle function5. This damage can impact food and water intake, as well as cause sensory dysfunction of the face, affecting rehabilitation and the assessment of neurological function post-ischemic stroke6.
The current protocol seeks to address these limitations by developing a new method to induce an ischemic stroke model without damaging the anatomical structure of cerebral vessels. This novel approach utilizes microsurgical techniques to repair the CCA, thus avoiding the drawbacks associated with previous methods.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% formalin</td>
			<td>Beijing Labgic Technology Co.,Ltd.</td>
			<td>BL913A</td>
			<td></td>
		</tr>
		<tr>
			<td>11-0 microsurgical sutures</td>
			<td>Ningbo Medical Suture Needle Co., Ltd.</td>
			<td>3/8 1&#215;5</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3,5-triphenyltetrazolium chloride (TTC)</td>
			<td>SIGMA</td>
			<td>T8877</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 silk suture</td>
			<td>Ningbo Medical Suture Needle Co., Ltd.</td>
			<td>3-0</td>
			<td></td>
		</tr>
		<tr>
			<td>4% isoflurane&#160;&#160;</td>
			<td>Tianjin Ringpu Bio-Technology Co., Ltd.</td>
			<td>20221102</td>
			<td></td>
		</tr>
		<tr>
			<td>6-0 silk suture</td>
			<td>Ningbo Medical Suture Needle Co., Ltd.</td>
			<td>6-0</td>
			<td></td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Shandong Lierkang Medical Technology Co., Ltd.</td>
			<td>20230408</td>
			<td></td>
		</tr>
		<tr>
			<td>Filament</td>
			<td>Xinong Technologies Co Ltd.</td>
			<td>2838-A5</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine&#160;&#160;</td>
			<td>Jiangsu Hengrui Pharmaceutical Co., Ltd.</td>
			<td>230613BL</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Sprague Dawley rats</td>
			<td>Jinan Pengyue Experimental Animal Breeding Co., Ltd.</td>
			<td>SCXK (Lu) 20190003</td>
			<td></td>
		</tr>
		<tr>
			<td>Microclips</td>
			<td>Shanghai Jinzhong Medical Device Factory</td>
			<td>W40150</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEISS</td>
			<td>S100 OPMI pico</td>
			<td></td>
		</tr>
		<tr>
			<td>Nursing box</td>
			<td>Beijing Fuyi Electrical Appliance Co., Ltd.</td>
			<td>FYL-YS-100L</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>SIGMA</td>
			<td>PHR3263</td>
			<td></td>
		</tr>
	</tbody>
</table>

a rat model of middle cerebral artery occlusion/reperfusion without damaging the anatomical structure of cerebral vessels

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have significantly contributed to our understanding of its pathological mechanisms and the development of potential treatments. Presently, there are two common methods involving filament (endovascular suture) techniques to induce animal models of cerebral ischemia. However, these methods have inherent limitations, such as reduced blood perfusion to the brain, damage to the external carotid artery system, impaired food and/or water intake, and sensory dysfunction of the face. This article introduces a new method for inducing a rat ischemic stroke model without compromising the cerebral vascular anatomy. In this study, the common carotid artery (CCA) of Sprague-Dawley rats was exposed, and an incision was made. A filament was then inserted through the incision into the internal carotid artery to occlude the middle cerebral artery. After 1.5 h of induced ischemia, the occluding filament was fully removed from both the internal carotid artery and the CCA. The incision in the CCA was subsequently sutured using 11-0 microsurgical sutures under a microscope (magnification 4x). Through the utilization of microsurgical techniques to repair the CCA, this study successfully developed a unique method to induce an ischemic stroke model in rats while preserving the anatomical integrity of cerebral blood vessels.

The process of microsuture 
The filament was withdrawn near the incision of the CCA until the white filament tip was visible (Figure 1A). Micro clamps were then placed on the CCA (Figure 1B). Under a microscope at 4x magnification, the incision of the CCA was sutured using microsurgical sutures in an interrupted pattern (Figure 1C). Subsequently, the microclamps were removed from the CCA, restoring the anatomic...

Read this Scientific Article Protocol about A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion without Damaging the Anatomical Structure of Cerebral Vessels at JoVE.com

A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

Ischemic stroke stands as the primary cause of long-term disability and mortality among adults worldwide. Animal models of ischemic stroke have ...

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364 Views.  The Third Hospital of Hebei Medical University. The current protocol describes a rat model of middle cerebral artery occlusion/reperfusion that preserves the anatomical structure of cerebral vessels without causing damage.

Video: A Rat Model of Middle Cerebral Artery Occlusion/Reperfusion Without Damaging the Anatomical Structure of Cerebral Vessels

Focal Cerebral Ischemia Model by Endovascular Suture Occlusion of the Middle Cerebral Artery in the Rat

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans2,3. In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO.

Surgical induction of ischemic brain damage in the rat is a widely used model for stroke research. Here we demonstrate the induction of focal cerebral ischemia by occlusion of the middle cerebral artery. Visualization of the resulting infarct by histological staining and magnetic resonance imaging is also shown.

Stroke is the leading cause of disability and the third leading cause of death in adults worldwide1. In human stroke, there exists a highly variable ...

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of motor and cognitive disabilities. Its high prevalence poses a serious health burden, as stroke is among the principal causes of long-term disability and death worldwide1. Recovery of neuronal function is, in most cases, only partial. So far, treatment options are very limited, in particular due to the narrow time window for thrombolysis2,3. Determining methods to accelerate recovery from stroke remains a prime medical goal; however, this has been hampered by insufficient mechanistic insights into the recovery process. Experimental stroke researchers frequently employ rodent models of focal cerebral ischemia. Beyond the acute phase, stroke research is increasingly focused on the sub-acute and chronic phase following cerebral ischemia. Most stroke researchers apply permanent or transient occlusion of the MCA in mice or rats. In patients, occlusions of the MCA are among the most frequent causes of ischemic stroke4. Besides proximal occlusion of the MCA using the filament model, surgical occlusion of the distal MCA is probably the most frequently used model in experimental stroke research5. Occlusion of a distal (to the branching of the lenticulo-striate arteries) MCA branch typically spares the striatum and primarily affects the neocortex. Vessel occlusion can be permanent or transient. High reproducibility of lesion volume and very low mortality rates with respect to the long-term outcome are the main advantages of this model. Here, we demonstrate how to perform a chronic cranial window (CW) preparation lateral to the sagittal sinus, and afterwards how to surgically induce a distal stroke underneath the window using a craniotomy approach. This approach can be applied for sequential imaging of acute and chronic changes following ischemia via epi-illuminating, confocal laser scanning, and two-photon intravital microscopy.

Lateral Chronic Cranial Window Preparation Enables In Vivo Observation Following Distal Middle Cerebral Artery Occlusion in Mice

Surgical occlusion of a distal middle cerebral artery branch (MCAo) is a frequently used model in experimental stroke research. This manuscript describes the basic technique of permanent MCAo, combined with the insertion of a lateral cranial window, which offers the opportunity for longitudinal intravital microscopy in mice.

Focal cerebral ischemia (i.e., ischemic stroke) may cause major brain injury, leading to a severe loss of neuronal function and consequently to a host of ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

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 ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

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 ...

Modeling Stroke in Mice: Transient Middle Cerebral Artery Occlusion via the External Carotid Artery

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options are limited to a small proportion of stroke patients within the first hours after stroke. Novel therapeutic strategies are being extensively investigated, especially to prolong the therapeutic time window. These current investigations include the study of important pathophysiological pathways after stroke, such as post-stroke inflammation, angiogenesis, neuronal plasticity, and regeneration. Over the last decade, there has been increasing concern about the poor reproducibility of experimental results and scientific findings among independent research groups. To overcome the so-called &#8220;replication crisis&#8221;, detailed standardized models for all procedures are urgently needed. As an effort within the &#8220;ImmunoStroke&#8221; research consortium (https://immunostroke.de/), a standardized mouse model of transient middle cerebral artery occlusion (MCAo) is proposed. This model allows the complete restoration of the blood flow upon removal of the filament, simulating the therapeutic or spontaneous clot lysis that occurs in a large proportion of human strokes. The surgical procedure of this &#8220;filament&#8221; stroke model and tools for its functional analysis are demonstrated in the accompanying video.

Different models of middle cerebral artery occlusion (MCAo) are used in experimental stroke research. Here, an experimental stroke model of transient MCAo via the external carotid artery (ECA) is described, which aims to mimic human stroke, in which the cerebrovascular thrombus is removed due to spontaneous clot lysis or therapy.

Stroke is the third most common cause of mortality and the leading cause of acquired adult disability in developed countries. To date, therapeutic options ...

A Model for Encephalomyosynangiosis Treatment after Middle Cerebral Artery Occlusion-Induced Stroke in Mice

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The brain's ability to self-heal after ischemic stroke is limited by inadequate blood supply in the impacted area. Encephalomyosynangiosis (EMS) is a neurosurgical procedure that achieves angiogenesis in patients with moyamoya disease. It involves craniotomy with placement of a vascular temporalis muscle graft on the ischemic brain surface. EMS has never been studied in the setting of acute ischemic stroke in mice. The hypothesis driving this study is that EMS enhances cerebral angiogenesis at the cortical surface surrounding the muscle graft. The protocol shown here describes the procedure and provides initial data supporting the feasibility and efficacy of the EMS approach. In this protocol, after 60 min of transient middle cerebral artery occlusion (MCAo), mice were randomized to either MCAo or MCAo + EMS treatment. The EMS was performed 3-4 h after occlusion. The mice were sacrificed 7 or 21 days after MCAo or MCAo + EMS treatment. Temporalis graft viability was measured using nicotinamide adenine dinucleotide reduced-tetrazolium reductase assay. A mouse angiogenesis array quantified angiogenic and neuromodulating protein expression. Immunohistochemistry was used to visualize graft bonding with brain cortex and change in vessel density. The preliminary data here suggest that grafted muscle remained viable 21 days after EMS. Immunostaining showed successful graft implantation and increase in vessel density near the muscle graft, indicating increased angiogenesis. Data show that EMS increases fibroblast growth factor (FGF) and decreases osteopontin levels after stroke. Additionally, EMS after stroke did not increase mortality suggesting that protocol is safe and reliable. This novel procedure is effective and well-tolerated and has the potential to provide information of novel interventions for enhanced angiogenesis after acute ischemic stroke.

The protocol aims to provide methods for encephalomyosynangiosis-grafting of a vascular temporalis muscle flap on the pial surface of ischemic brain tissue-for the treatment of non-moyamoya acute ischemic stroke. The approach's efficacy in increasing angiogenesis is evaluated using a transient middle cerebral artery occlusion model in mice.

There is no effective treatment available for most patients suffering with ischemic stroke, making development of novel therapeutics imperative. The ...