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 induction of deep anesthesia. Test for adequate sedation with a pain stimulus, such as a toe pinch, and verify the absence of a reaction.</li>
	<li>Carefully shave the neck hair of the mouse with a razor, clean it with 70% ethanol followed by betadine/chlorhexidine, and apply 1% lidocaine on the skin surface for local pain control.</li>
	<li>Place the mouse in a supine position. Use tape to fix the limbs and tail, gently stretching the skin of the neck to the opposite side of the surgery. Simultaneously, elevate the neck slightly.</li>
	<li>Use ophthalmic ointment (e.g., 5% dexpanthenol) to prevent dehydration of the eyes during the operation.</li>
</ol>
2. SAH induction
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63150/63150fig01.jpg" /> 
Figure 1: Step-by-step images of surgical technique. (A) Depiction of the exposed right carotid artery anatomy: the CCA and its bifurcation into ICA and ECA are identified, as well as the small branches of the ECA (OA and STA). (B) The ECA is mobilized from the surrounding tissue and ligated with two sutures before cutting it. A third ligation needs to be placed loosely near the bifurcation without occluding it. (C) The ICA and CCA are occluded temporarily (with either ligation or clips) to prevent excessive bleeding when the ECA is carefully incised. (D) The filament is inserted into the ECA and advanced into the CCA. The prearranged ligation must be tightened carefully so that no blood effusion occurs but advancing the filament remains possible. (E) The ICA and CCA are reopened, and the ECA stump needs to be adjusted to a cranial direction. By pushing the filament ~9 mm forward into the ICA, the ACA-MCA bifurcation will be reached, and the vessel is then perforated by pushing the filament ~3mm further. (F) The filament is withdrawn after ensuring a temporal re-ligation of the CCA. The prearranged ligation of the ECA is quickly occluded, and the CCA is reopened to allow reperfusion. Abbreviations: ACA = anterior cerebral artery, CCA = common carotid artery, ECA = external carotid artery, MCA = middle cerebral artery, ICA = internal carotid artery, OA = occipital artery, PPA = pterygopalatine artery, STA = superior thyroid artery. Scale bar = 2 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/63150/63150fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol>
	<li>Open the neck skin with a sterile scalpel, from the chin to the upper edge of the breastbone (1.5 cm), and bluntly separate salivary glands from their surrounding connective tissue.</li>
	<li>Separate the muscle group along one side [in this case, the right side] of the trachea, exposing the common carotid artery (CCA) sheath covered with nourishing blood vessels and venules. The CCA and the vagal nerve are located in close proximity to each other.</li>
	<li>Dissociate the CCA and leave a free 8-0 silk suture around the CCA without ligating it in advance. Pay attention to the protection of the vagal nerve, as it is easily damaged (Figure 1A).</li>
	<li>A triple bifurcation of the CCA, the ICA, and the ECA is visible along the lower posterior third of the diastasis. Dissect the distal end of the ECA and ligate the vessel twice as far distaally as possible.</li>
	<li>Disconnect the ECA at the midpoint of the twice ligated segment, creating a vessel stump.</li>
	<li>Prearrange one ligation for the filament around the ECA stump, do not close it until successful filament insertion.</li>
	<li>Use a suture or micro clip to occlude the ICA and CCA temporarily (Figure 1B).</li>
	<li>Make a small incision (approximately half of the ECA diameter) in the ECA using microvascular scissors. Insert a 5-0 (alternatively 4-0) prolene filament into the ECA and advance it into the CCA.</li>
	<li>Close the ligature on the ECA slightly while loosening the micro clip on the ICA and CCA (Figure 1C).</li>
	<li>Gently pull back on the filament and adjust the ECA stump in the cranial direction, invaginating the filament through the bifurcation into the ICA (Figure 1D).</li>
	<li>Point the filament tip medially at an angle of ~30&#176; to the tracheal midline and ~30&#176; to the horizontal plane. Push the filament forward inside the ICA. After reaching the ACA-MCA bifurcation, resistance is encountered (~9 mm).</li>
	<li>Advance the filament 3 mm further, perforating the right ACA. Promptly withdraw the filament to the ECA stump, allowing blood flow into the subarachnoid space.</li>
	<li>Keep the filament in this position for about 10 s (Figure 1E). The presence of muscle tremors, ipsilateral miosis, gasping for breath, altered heart rhythm, and urinary incontinence can be supporting evidence of successful surgery.</li>
	<li>Temporarily close the CCA to avoid excess blood loss. Pull out the filament instantly and ligate the ECA with the prearranged suture. Reopen the CCA and allow reperfusion and further effusion of blood into the subarachnoid space (Figure 1F).</li>
	<li>After checking for bleeding leakage, disinfect the skin surrounding the wound to prevent postoperative skin infections, and suture the wound with a non-absorbable 4-0 polyester fiber suture.</li>
	<li>Place the mouse in a thermal box until consciousness is regained. Wait until the animal is fully awake and ensure it has regained sufficient consciousness to maintain sternal recumbency. Do not return animals to the company of other mice until fully recovered.</li>
	<li>Administer 200-300 mg/kg body weight&#160;paracetamol for postoperative pain relief.</li>
	<li>Check on the mice daily after surgery.</li>
</ol>
3. MRI measurement
<ol>
	<li>24 h after surgery, perform MRI using a rodent scanner (Table of Materials) and a dedicated mouse head resonator- here, a 20 mm transmit/receive quadrature volume resonator was used.</li>
	<li>Place the mouse on a heated circulating water blanket to ensure a constant body temperature of ~37 &#176;C. Induce anesthesia with 2.5 % isoflurane in an O2/N2O mixture (30%/70%) and maintain with 1.5-2 % isoflurane via facemask under continuous ventilation monitoring.</li>
	<li>First perform a fast reference scan acquiring 3 orthogonal slice packages (Tri-Pilot-Multi, FLASH with repetition time TR/echo time TE = 200 ms/3 ms, 1 average, flip angle FA = 30&#176;, field of view FOV = 28 mm x 28 mm, matrix MTX = 256 x 256, slice thickness 1 mm, total acquisition time TA =30 s).</li>
	<li>Then use a high resolution T2-weighted 2D turbo spin-echo sequence for imaging (imaging parameters TR/TE = 5505 ms/36 ms, RARE factor 8, 6 averages, 46 contiguous axial slices with a slice thickness of 0.35 mm to cover the whole brain, FOV = 25.6 mm x 25.6 mm, MTX = 256 x 256, TA = 13 min).</li>
	<li>If the result is unclear, use an additional respiration triggered T2*weighted gradient echo sequence with the same isodistance as the T2w scan (2D FLASH, TR/TE = 600 ms/6.3 ms, FA = 30&#176;, 1 average, 20 axial slices with 0.35 mm thickness, FOV and MTX identical to T2w, TA = 5-10 min depending on the respiration rate).</li>
	<li>Transfer the data into the DICOM image format and use ImageJ software for SAH grading and volumetry of blood clots. Details on the quantification are listed as a step-by-step guide in the supplementary material (Supplementary Figure 1).</li>
</ol>

<ol>
	<li>Macdonald, R. L., Schweizer, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+subarachnoid+haemorrhage.">Spontaneous subarachnoid haemorrhage.</a> The Lancet. 389 (10069), 655-666 (2017).</li><li>van Gijn, J., Kerr, R. S., Rinkel, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subarachnoid+haemorrhage.">Subarachnoid haemorrhage.</a> The Lancet. 369 (9558), 306-318 (2007).</li><li>Abraham, M. K., Chang, W. -. T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subarachnoid+hemorrhage.">Subarachnoid hemorrhage.</a> Emergency Medicine Clinics of North America. 34 (4), 901-916 (2016).</li><li>Schertz, M., Mehdaoui, H., Hamlat, A., Piotin, M., Banydeen, R., Mejdoubi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incidence+and+mortality+of+spontaneous+subarachnoid+hemorrhage+in+martinique.">Incidence and mortality of spontaneous subarachnoid hemorrhage in martinique.</a> PLOS ONE. 11 (5), 0155945 (2016).</li><li>Okazaki, T., Kuroda, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneurysmal+subarachnoid+hemorrhage:+intensive+care+for+improving+neurological+outcome.">Aneurysmal subarachnoid hemorrhage: intensive care for improving neurological outcome.</a> Journal of Intensive Care. 6 (1), 28 (2018).</li><li>Kilbourn, K. J., Levy, S., Staff, I., Kureshi, I., McCullough, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+characteristics+and+outcomes+of+neurogenic+stress+cadiomyopathy+in+aneurysmal+subarachnoid+hemorrhage.">Clinical characteristics and outcomes of neurogenic stress cadiomyopathy in aneurysmal subarachnoid hemorrhage.</a> Clinical Neurology and Neurosurgery. 115 (7), 909-914 (2013).</li><li>de Oliveira Manoel, A. 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=The+critical+care+management+of+spontaneous+intracranial+hemorrhage:+a+contemporary+review.">The critical care management of spontaneous intracranial hemorrhage: a contemporary review.</a> Critical Care. 20 (1), 272 (2016).</li><li>Schneider, U. 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=Microglia+inflict+delayed+brain+injury+after+subarachnoid+hemorrhage.">Microglia inflict delayed brain injury after subarachnoid hemorrhage.</a> Acta Neuropathologica. 130 (2), 215-231 (2015).</li><li>Delgado, T. J., Brismar, J., Svendgaard, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subarachnoid+haemorrhage+in+the+rat:+angiography+and+fluorescence+microscopy+of+the+major+cerebral+arteries.">Subarachnoid haemorrhage in the rat: angiography and fluorescence microscopy of the major cerebral arteries.</a> Stroke. 16 (4), 595-602 (1985).</li><li>Piepgras, A., Thome&#769;, C., Schmiedek, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+anterior+circulation+rat+subarachnoid+hemorrhage+model.">Characterization of an anterior circulation rat subarachnoid hemorrhage model.</a> Stroke. 26 (12), 2347-2352 (1995).</li><li>Suzuki, 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=Heme+oxygenase-1+gene+induction+as+an+intrinsic+regulation+against+delayed+cerebral+vasospasm+in+rats.">Heme oxygenase-1 gene induction as an intrinsic regulation against delayed cerebral vasospasm in rats.</a> Journal of Clinical Investigation. 104 (1), 59-66 (1999).</li><li>Dudhani, R. V., Kyle, M., Dedeo, C., Riordan, M., Deshaies, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Low+mortality+rat+model+to+assess+delayed+cerebral+vasospasm+after+experimental+subarachnoid+hemorrhage.">A Low mortality rat model to assess delayed cerebral vasospasm after experimental subarachnoid hemorrhage.</a> Journal of Visualized Experiments: JoVE. (71), e4157 (2013).</li><li>Iuliano, B. A., Pluta, R. M., Jung, C., Oldfield, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+dysfunction+in+a+primate+model+of+cerebral+vasospasm.">Endothelial dysfunction in a primate model of cerebral vasospasm.</a> Journal of Neurosurgery. 100 (2), 287-294 (2004).</li><li>Barry, K. J., Gogjian, M. A., Stein, B. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+intracranial+in+vivo+animal+models+of+delayed+cerebral+vasospasm.">Standard intracranial in vivo animal models of delayed cerebral vasospasm.</a> British Journal of Neurosurgery. 24 (4), 415-434 (2010).</li><li>Thompson, J. W., 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=In+vivo+cerebral+aneurysm+models.">In vivo cerebral aneurysm models.</a> Neurosurgical Focus. 47 (1), 1-8 (2019).</li><li>Frontera, J. A., 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=Prediction+of+symptomatic+vasospasm+after+subarachnoid+hemorrhage:+The+modified+fisher+scale.">Prediction of symptomatic vasospasm after subarachnoid hemorrhage: The modified fisher scale.</a> Neurosurgery. 59 (1), 21-26 (2006).</li><li>Fisher, C. M., Kistler, J. P., Davis, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relation+of+cerebral+vasospasm+to+subarachnoid+hemorrhage+visualized+by+computerized+tomographic+scanning.">Relation of cerebral vasospasm to subarachnoid hemorrhage visualized by computerized tomographic scanning.</a> Neurosurgery. 6 (1), 1-9 (1980).</li><li>Wilson, D. A., 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+simple+and+quantitative+method+to+predict+symptomatic+vasospasm+after+subarachnoid+hemorrhage+based+on+computed+tomography:+Beyond+the+fisher+scale.">A simple and quantitative method to predict symptomatic vasospasm after subarachnoid hemorrhage based on computed tomography: Beyond the fisher scale.</a> Neurosurgery. 71 (4), 869-875 (2012).</li><li>Sch&#252;ller, K., B&#252;hler, D., Plesnila, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+subarachnoid+hemorrhage.">A murine model of subarachnoid hemorrhage.</a> Journal of Visualized Experiments: JoVE. (81), e50845 (2013).</li></ol>

No conflicts of interest

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.

<table><tbody><tr><td>Eye cream</td><td>Bayer</td><td>815529836</td><td>Bepanthen</td></tr><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>&amp;nbsp;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 &amp;amp; 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></table>

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...

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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 ...

endovascular-perforation-model-for-subarachnoid-hemorrhage-combined

Research

JoVE Journal

Neuroscience

3.5K Views.  Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, and Humboldt-Universität zu Berlin, and Berlin Institute of Health. Het endovasculaire perforatiemodel in combinatie met postoperatieve MRI maakt verificatie van de bloeding en uitsluiting van andere intercraniële pathologieën mogelijk. Een van de voordelen van deze aanpak is de mogelijkheid om het bloedingsvolume te kwantificeren voor verdere subgroepanalyse. Begin met het scheren van het nekhaar van de muis.Breng oogheelkundige zalf aan tijdens de procedure. Reinig de huid met 70% ethanol gevolgd door Betadine of chloorhexidine en breng 1% lidocaï...

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

A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) in rats, in order to identify the initiating events, pathophysiological changes and potential targets for treatment.
Methods: Twenty-eight male Sprague-Dawley rats (250 - 300 g) were arbitrarily assigned to one of two groups - SAH or saline control. Rat subarachnoid hemorrhage in the SAH group (n=15) was induced by double injection of autologous blood, 48 hr apart, into the cisterna magna. Similarly, normal saline (n=13) was injected into the cisterna magna of the saline control group. Rats were sacrificed on day five after the second blood injection and the brains were preserved for histological analysis. The degree of vasospasm was measured using sections of the basilar artery, by measuring the internal luminal cross sectional area using NIH Image-J software. The significance was tested using Tukey/Kramer's statistical analysis.
Results: After analysis of histological sections, basilar artery luminal cross sectional area were smaller in the SAH than in the saline group, consistent with cerebral vasospasm in the former group. In the SAH group, basilar artery internal area (.056 &mu;m &plusmn; 3) were significantly smaller from vasospasm five days after the second blood injection (seven days after the initial blood injection), compared to the saline control group with internal area (.069 &plusmn; 3; p=0.004). There were no mortalities from cerebral vasospasm.
Conclusion: The rat double SAH model induces a mild, survivable, basilar artery vasospasm that can be used to study the pathophysiological mechanisms of cerebral vasospasm in a small animal model. A low and acceptable mortality rate is a significant criterion to be satisfied for an ideal SAH animal model so that the mechanisms of vasospasm can be elucidated 7, 8. Further modifications of the model can be made to adjust for increased severity of vasospasm and neurological exams.

Aneurysmal subarachnoid hemorrhage (SAH) is bleeding that occurs into the subarachnoid space when an aneurysm ruptures. While the morbidity and mortality from this event has been on a decline due to improved treatment approaches, the risk of vasospasm after subarachnoid hemorrhage continues to be the same as it was several years ago. The importance of establishing a comprehensive and reproducible animal model to identify initiating events of cerebral vasospasm has been the focus of research since the first use of rats in an experimental vasospasm model in 1979 by Barry et al. Early work in rats demonstrated that a single injection of autologous blood into the cisterna magna led to acute (within minutes) but not delayed cerebral vasospasm 3, 6, 14. Here we characterize a low mortality SAH rat model that results in reproducible delayed vasospasm.

Objective: To characterize and establish a reproducible model that demonstrates delayed cerebral vasospasm after aneurysmal subarachnoid hemorrhage (SAH) ...

Medicine

A Murine Model of Subarachnoid Hemorrhage

In this video publication a standardized mouse model of subarachnoid hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of Willis perforation (CWp) and proven by intracranial pressure (ICP) monitoring. Thereby a homogenous blood distribution in subarachnoid spaces surrounding the arterial circulation and cerebellar fissures is achieved. Animal physiology is maintained by intubation, mechanical ventilation, and continuous on-line monitoring of various physiological and cardiovascular parameters: body temperature, systemic blood pressure, heart rate, and hemoglobin saturation. Thereby the cerebral perfusion pressure can be tightly monitored resulting in a less variable volume of extravasated blood. This allows a better standardization of endovascular filament perforation in mice and makes the whole model highly reproducible. Thus it is readily available for pharmacological and pathophysiological studies in wild type and genetically altered mice.

A standardized mouse model of subarachnoid hemorrhage by intraluminal Circle of Willis perforation is described. Vessel perforation and subarachnoid bleeding are monitored by intracranial pressure monitoring. In addition various vital parameters are recorded and controlled to maintain physiologic conditions.

In this video publication a standardized mouse model of subarachnoid  hemorrhage (SAH) is presented. Bleeding is induced by endovascular Circle of  Willis ...

The Rabbit Blood-shunt Model for the Study of Acute and Late Sequelae of Subarachnoid Hemorrhage: Technical Aspects

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and controllable animal models that simulate both conditions are presently uncommon. Therefore, new models are needed in order to mimic human pathophysiological conditions resulting from SAH.
This report describes the technical nuances of a rabbit blood-shunt SAH model that enables control of intracerebral pressure (ICP). An extracorporeal shunt is placed between the arterial system and the subarachnoid space, which enables examiner-independent SAH in a closed cranium. Step-by-step procedural instructions and necessary equipment are described, as well as technical considerations to produce the model with minimal mortality and morbidity. Important details required for successful surgical creation of this robust, simple and consistent ICP-controlled SAH rabbit model are described.

The experimental intracranial pressure-controlled blood shunt subarachnoid hemorrhage (SAH) model in the rabbit combines the standard procedures &#8212; subclavian artery cannulation and transcutaneous cisterna magna puncture, which enables close mimicking of human pathophysiological conditions after SAH. We present step-by-step instructions and discuss key surgical points for successful experimental SAH creation.

Early brain injury and delayed cerebral vasospasm both contribute to unfavorable outcomes after subarachnoid hemorrhage (SAH). Reproducible and ...

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 ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

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 ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Visualization of Brain Pericyte Contractility Post-Subarachnoid Hemorrhage

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.

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.

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

Induction of a Subarachnoid Hemorrhage in a Mouse Model through Endovascular Perforation

Source: Liu, S., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63150">Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging (MRI).</a>&#160;J. Vis. Exp. (2021).
The video demonstrates the induction of subarachnoid hemorrhage via endovascular perforation in a mouse model. An incision is made in the neck region of the anesthetized mouse to access the common carotid artery and its bifurcation. A filament is inserted through the arteries to perforate an intracranial artery branch, inducing subarachnoid hemorrhage.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Animal ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

Establishing a Subarachnoid Hemorrhage Rat Model Induced by Autologous Blood Injection

Source: B&#246;mers, J. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62567">Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model.</a> J. Vis. Exp. (2021)
This video demonstrates the procedure for establishing a subarachnoid hemorrhage rat model. An anesthetized rat is implanted with probes to measure intracranial pressure and cerebral blood flow. The target site is identified, and the needle is inserted at a pre-chiasmatic position. Autologous blood is then injected into the subarachnoid space, increasing intracranial pressure and decreasing cerebral blood flow, establishing the model.

Source: B&#246;mers, J. et al. Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model. J. Vis. ...

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

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