Name: endovascular perforation model for subarachnoid hemorrhage combined with magnetic resonance imaging (mri)
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Description: Watch this Scientific Journal Video about Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging MRI at JoVE.com

SL was supported by the Chinese Scholarship Council. KT was supported by the BIH-MD scholarship of the Berlin Institute of Health and the Sonnenfeld-Stiftung. RX is supported by the BIH-Charit&#233; Clinician Scientist Program, funded by the Charit&#233; -Universit&#228;tsmedizin Berlin and the Berlin Institute of Health. We acknowledge support from the German Research Foundation (DFG) and the Open Access Publication Fund of Charite&#769; - Universita&#776;tsmedizin Berlin.

The experiments were performed in accordance with the guidelines and regulations set forth by Landesamt fuer Gesundheit und Soziales (LaGeSo), Berlin, Germany (G0063/18). In this study, C57Bl/6J male (8-12 weeks old) mice with a weight of 25 &#177; 0.286 g (average &#177; s.e.m.) were used.
1. Animal preparation
<ol>
	<li>Induce anesthesia by injecting ketamine (70 mg/kg) and xylazine (16 mg/kg) intraperitoneally. Maintain normal body temperature, contributing to quick induc...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+model+for+investigation+of+subarachnoid+hemorrhage+and+cerebral+vasospasm.">Small animal model for investigation of subarachnoid hemorrhage and cerebral vasospasm.</a> Stroke. 10 (5), 538-541 (1979).</li><li>Bederson, J. B., Germano, I. M., Guarino, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+blood+flow+and+cerebral+perfusion+pressure+in+a+new+noncraniotomy+model+of+subarachnoid+hemorrhage+in+the+rat.">Cortical blood flow and cerebral perfusion pressure in a new noncraniotomy model of subarachnoid hemorrhage in the rat.</a> Stroke. 26 (6), 1086-1092 (1995).</li><li>Veelken, J. A., Laing, R. J. C., Jakubowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Sheffield+model+of+subarachnoid+hemorrhage+in+rats.">The Sheffield model of subarachnoid hemorrhage in rats.</a> Stroke. 26 (7), 1279-1284 (1995).</li><li>Sugawara, T., Ayer, R., Jadhav, V., Zhang, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+grading+system+evaluating+bleeding+scale+in+filament+perforation+subarachnoid+hemorrhage+rat+model.">A new grading system evaluating bleeding scale in filament perforation subarachnoid hemorrhage rat model.</a> Journal of Neuroscience Methods. 167 (2), 327-334 (2008).</li><li>Egashira, Y., Shishido, H., Hua, Y., Keep, R. F., Xi, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+grading+system+based+on+magnetic+resonance+imaging+in+a+mouse+model+of+subarachnoid+hemorrhage.">New grading system based on magnetic resonance imaging in a mouse model of subarachnoid hemorrhage.</a> Stroke. 46 (2), 582-584 (2015).</li><li>Mutoh, T., Mutoh, T., Sasaki, K., Nakamura, K., Taki, Y., Ishikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Value+of+three-dimensional+maximum+intensity+projection+display+to+assist+in+magnetic+resonance+imaging+(MRI)-based+grading+in+a+mouse+model+of+subarachnoid+hemorrhage.">Value of three-dimensional maximum intensity projection display to assist in magnetic resonance imaging (MRI)-based grading in a mouse model of subarachnoid hemorrhage.</a> Medical Science Monitor. 22, 2050-2055 (2016).</li><li>Kothari, R. U., 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+ABCs+of+measuring+intracerebral+hemorrhage+volumes.">The ABCs of measuring intracerebral hemorrhage volumes.</a> Stroke. 27 (8), 1304-1305 (1996).</li><li>Leclerc, J. 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=A+comparison+of+pathophysiology+in+humans+and+rodent+models+of+subarachnoid+hemorrhage.">A comparison of pathophysiology in humans and rodent models of subarachnoid hemorrhage.</a> Frontiers in Molecular Neuroscience. 11, 71 (2018).</li><li>Titova, E., Ostrowski, R. P., Zhang, J. H., Tang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+subarachnoid+hemorrhage+for+studies+of+cerebral+vasospasm.">Experimental models of subarachnoid hemorrhage for studies of cerebral vasospasm.</a> Neurological Research. 31 (6), 568-581 (2009).</li><li>Marbacher, S., 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=Systematic+review+of+in+vivo+animal+models+of+subarachnoid+hemorrhage:+Species,+standard+parameters,+and+outcomes.">Systematic review of in vivo animal models of subarachnoid hemorrhage: Species, standard parameters, and outcomes.</a> Translational Stroke Research. 10 (3), 250-258 (2019).</li><li>Marbacher, S., Fandino, J., Kitchen, N. 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In summary, a standardized SAH mouse model induced by endovascular filament perforation operation is presented with minor invasion, short operative time, and acceptable mortality rates. MRI is conducted 24 h postoperatively to ensure the correct bleeding site and the exclusion of other relevant intracranial pathologies. Furthermore, we classified different SAH bleeding grades and measured bleeding volumes, allowing further subgroup analyses based on bleeding grade.
Adequate positioning of the ...

Subarachnoid hemorrhage (SAH) is caused by the rupture of an intracranial aneurysm and poses a life-threatening emergency, associated with substantial morbidity and mortality, accounting for approx. 5% of strokes1,2. SAH patients present with severe headaches, neurological dysfunction, and progressive disturbance of consciousness3. Around 30% of SAH patients die within the first 30 days after the initial bleeding event4. Clinically, 50% of patients experience delayed brain injury (DBI) after early brain injury. DBI is characterized by delayed cerebral ischemia and delayed neurological deficits. Current studies have shown that the synergistic effects of several different factors lead to the loss of neurological function, including the destruction of the blood-brain barrier, the contraction of small arteries, microcirculatory dysfunction, and thrombosis5,6.
One unique aspect of SAH is that the pathogenesis originates from an extraparenchymal location but then leads to detrimental cascades inside the parenchyma: the pathology begins with the accumulation of blood in the subarachnoid space, triggering a multitude of intraparenchymal effects, such as neuroinflammation, neuronal and endothelial cell apoptosis, cortical spreading depolarization, and brain edema formation7,8.
Clinical research is limited by several factors, making the animal model a critical element in consistently and accurately mimicking the pathomechanistic changes of the disease. Different SAH model protocols have been proposed, e.g., autologous blood injection into the cisterna magna (ACM). Also, a modified method with a double injection of autologous blood into the cisterna magna and optic chiasm cistern (APC) respectively9,10. While autologous blood injection is a simple way to simulate the pathological process of vasospasm and inflammatory reactions after subarachnoid hemorrhage, the following rise of intracranial pressure (ICP) is relatively slow, and no noteworthy changes in the permeability of the blood-brain barrier are induced11,12. Another method, the periarterial blood placement, usually used in large SAH models (e.g., monkeys and dogs), involves placing anticoagulated autologous blood or comparable blood products around the vessel. The diameter changes of the artery can be observed with a microscope, serving as an indicator for cerebral vasospasm after SAH13.
Barry et al. first described an endovascular perforation model in 1979 in which the basilar artery is exposed after removing the skull; the artery is then punctured with tungsten microelectrodes, using a microscopic stereotactic technique14. In 1995, Bederson and Veelken modified the Zea-Longa model of cerebral ischemia and established the endovascular perforation, which has been continuously improved ever since15,16. This method is based on the fact that mice and humans share a similar intracranial vascular network, known as the circle of Willis.
For postoperative evaluation and grading of SAH in the mouse model, different approaches have been proposed. Sugawara et al. developed a grading scale that has been widely used since 200817. This method assesses the severity of SAH based on morphological changes. However, for this method, the mouse&#39;s brain tissue morphology must be examined under direct vision, and therefore, the mouse must be sacrificed for assessment. Furthermore, several methods for determining SAH severity in vivo have been established. Approaches range from simple neurological scoring to monitoring of intracranial pressure (ICP) to various radiological imaging techniques. Furthermore, MRI grading has been shown as a new, non-invasive tool to grade SAH severity, correlating to neurological score18,19.
Here, a protocol for an SAH model caused by endovascular perforation is presented, combined with postoperative MRI. In an attempt to establish a system to objectify the amount of bleeding in an in vivo setting, we also developed a system for SAH grading and quantification of total blood volume based on 7.0 T high-resolution T2-weighted MRI. This approach ensures the correct induction of SAH and exclusion of other pathologies such as stroke, hydrocephalus, or intracerebral hemorrhage (ICH) and complications.

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			<td>Eye cream</td>
			<td>Bayer</td>
			<td>815529836</td>
			<td>Bepanthen</td>
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		<tr>
			<td>Images analysis software</td>
			<td>ImageJ</td>
			<td></td>
			<td>Bundled with Java 1.8.0_172</td>
		</tr>
		<tr>
			<td>Ligation suture (5-0)</td>
			<td>SMI</td>
			<td></td>
			<td>Silk black USP</td>
		</tr>
		<tr>
			<td>Light source for microscope</td>
			<td>Zeiss</td>
			<td>CL 6000 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>CP-pharma</td>
			<td>797-037</td>
			<td>100 mg/mL</td>
		</tr>
		<tr>
			<td>MRI</td>
			<td>Bruker</td>
			<td>Pharmascan 70/16</td>
			<td>&#160;7 Tesla</td>
		</tr>
		<tr>
			<td>MRI images acquired software</td>
			<td>Bruker</td>
			<td></td>
			<td>Bruker Paravision 5.1</td>
		</tr>
		<tr>
			<td>Paracetamol (40 mg/mL)</td>
			<td>bene Arzneimittel</td>
			<td>4993736</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolene filament (5-0)</td>
			<td>Erhicon</td>
			<td>EH7255</td>
			<td></td>
		</tr>
		<tr>
			<td>Razor</td>
			<td>Wella</td>
			<td>HS61</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (Fine Scissors)</td>
			<td>FST</td>
			<td>14060-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (forceps#1)</td>
			<td>AESCULAP</td>
			<td>FM001R</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (forceps#2)</td>
			<td>AESCULAP</td>
			<td>FD2855R</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (forceps#3)</td>
			<td>Hammacher</td>
			<td>HCS 082-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (Needle holder)</td>
			<td>FST</td>
			<td>91201-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical instrument (Vannas Spring Scissors)</td>
			<td>FST</td>
			<td>15000-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000 C</td>
			<td></td>
		</tr>
		<tr>
			<td>Ventilation monitoring</td>
			<td>Stony Brook</td>
			<td></td>
			<td>Small Animal Monitoring &#38; Gating System</td>
		</tr>
		<tr>
			<td>Wounding suture(4-0)</td>
			<td>Erhicon</td>
			<td>CB84D</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylavet</td>
			<td>CP-pharma</td>
			<td>797-062</td>
			<td>20 mg/mL</td>
		</tr>
	</tbody>
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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.

Mortality 
For this study, a total of 92 male C57Bl/6J mice aged between 8-12 weeks were subjected to SAH operation; in these, we observed an overall mortality rate of 11.9% (n = 12). Mortality occurred exclusively within the first 6-24 h after surgery, suggesting perioperative mortality as well as SAH bleeding itself as the most likely contributing factors.
SAH bleeding grade 
A total of 50 mice received MRI 24 h postoperatively to confirm SAH and e...

Watch this Scientific Journal Video about Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging MRI at JoVE.com

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.

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 ...

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