Parts of this study are part of the doctoral thesis of T. Pantel, presented to the Medical Faculty of the Johannes Gutenberg-University of Mainz. The study was supported by the Friedhelm Frees Stiftung and by the Stiftung Neurochirurgische Forschung (grants to A.N.).

The animal experiments were approved by the responsible animal care committee (Landesuntersuchungsamt Rheinland-Pfalz) and carried out in accordance with the German Animal Welfare Act (TierSchG). All applicable international, national, and institutional guidelines for the care and use of animals were followed.
In this study, male C57BL6 mice (age 10-12 weeks) were used. In brief, subarachnoid hemorrhage was induced by endovascular filament perforation under anesthesia with isoflurane. The left external carotid artery was prepared surgically. Then, a filament was inserted into the external carotid artery and advanced intracranially through the internal carotid artery which was perforated at the carotid T, inducing a subarachnoid hemorrhage. A rise in intracranial pressure was taken as an indicator of successful endovascular&#160;perforation. A detailed protocol of an endovascular filament perforation model of SAH in mice has been published by others8,14.
1. Perfusion and Endovascular Casting
<ol>
	<li>In this study, perfusion was performed 72 hours after induction of SAH. Induce anesthesia by intraperitoneally injecting 5 &#181;g/g body weight (bw) midazolam, 30 ng/g bw fentanyl, and 0.5 &#181;g/g bw medetomidin. Continue only after a sufficient anesthesia level has been reached, which is confirmed by the absence of reactions to pain stimuli.</li>
	<li>Open the thorax, puncture the left ventricle with a 21G cannula, open the right atrium, and clamp the descending aorta as described elsewhere15.</li>
	<li>Perform a transcardiac perfusion using the following solutions: (i) Dulbecco&#39;s Phosphate Buffered Saline containing MgCl2 and CaCl2 at pH 7.4 with 1 g/L glucose, and (ii) 4% paraformaldehyde solution.
	<ol>
		<li>Start the perfusion with solution (i) for 2 minutes and continue with solution (ii) for 4 minutes.</li>
		<li>Infuse the solutions at a temperature of 37 &#176;C and using a pressure-controlled pump with variable perfusion rate to perfuse with a constant pressure of 70 mmHg, which was found to be the optimal perfusion pressure to analyze vasospasm in mice16. Avoid a loss of pressure when switching from solution (i) to solution (ii).</li>
	</ol>
	</li>
	<li>After perfusion with solutions (i) and (ii), continue perfusion for 20 minutes at room temperature with radiopaque casting agent (see Table of Materials) at a constant rate of 0.2 ml/min.</li>
	<li>Allow for curing of the radiopaque casting material at 4 &#176;C overnight. Then remove the brain from the skull as previously described17, transfer the sample to 4% PFA solution, and store the sample at 4 &#176;C until micro CT scanning.</li>
</ol>
2. Micro Computed Tomography
<ol>
	<li>Place the brain in the center of a plastic tube with a blunt anatomical forceps. Choose a tube with a slightly larger diameter than the sample to ensure that the object does not move during image acquisition. Use gauze to close the tube.</li>
	<li>Attach the plastic tube to the micro-stepping motor on the computer-navigated-control (CNC) positioning system in the X-ray cabin, in which the object is rotated around its horizontal axis.</li>
	<li>Align the sample in the field-of-view under X-ray radiography. To achieve the maximal magnification, place the object as close as possible to the x-ray source and maximize the distance to the detector as far as possible.</li>
	<li>Use a step-and-shoot image acquisition protocol with the following scan parameters: set exposure time to 1 s for each projection to optimize the signal-to-noise ratio (SNR), tube voltage 80 kV (current 38 &#181;A), 360&#176; rotation resulting in 1,000 projections.</li>
	<li>For reconstruction of RAW-Data use a filtered back projection algorithm applying the Shepp-Logan filter with a matrix of 1024 x 1024 x 1024 voxels using reconstruction software (see Table of Materials). For further analysis import the resulting DICOM data into 3D visualization software (see Table of Materials).</li>
</ol>
3. 3-dimensional Reconstruction of the Intracranial Vascular Tree and Determination of Vessel Volumes
Note: Background information on the functions of the visualization software can be found using the function help.
<ol>
	<li>Import Dicom data into visualization software using the function Import.</li>
	<li>Visualize the vessel tree with the function Volren. Choose the visualization threshold so that the large cerebral arteries are depicted in sharp outlines. It is important to use the same visualization threshold for all data sets belonging to the experimental series.</li>
	<li>Virtually dissect the basal cerebral arteries (Circle of Willis) with the function VolumeEdit by surrounding the vessels with the cursor. Then virtually dissect the vessel segment to be analyzed. Therefore, rotate the 3-dimensional model of the vascular tree in order to precisely separate all small branches from the main artery. It is essential for the further analysis to delete all vessels except the vessel segment to be analyzed.</li>
	<li>Apply the function Autoskeleton with threshold set to visualization threshold, which generates a center-line-based SpatialGraph.</li>
	<li>Then, apply the function SpatialGraphToLineSet to create a line set. Divide the line set into its single subsegments by manually choosing the single subsegments with the cursor and clicking on &#34;split&#34;. This step is crucial in order to calculate the volumes of the single subsegments.</li>
	<li>Use the function LineSetToSpatialGraph to create a Spatial Graph again.</li>
	<li>Use the function SpatialGraphStatistics to determine length, volume, and diameter of each subsegment. For color-coded visualization representing the course of the vessel diameter, use the function SpatialGraphView. Set segment coloring to &#34;thickness&#34;, which correlates with the vessel diameter. It is important to choose the same color map for all datasets belonging to the experimental series.</li>
	<li>Add the lengths of the subsegments to determine which subsegments are to be included in the further analysis. In the present study we evaluated a vessel segment consisting of 1 mm of the internal carotid artery proximal of the carotid T and 2.5 mm of the middle cerebral artery distal of the carotid T. Then add the volumes to determine the vessel volume of the defined vessel segment.</li>
</ol>

<ol>
	<li>Macdonald, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+neurological+deterioration+after+subarachnoid+haemorrhage.">Delayed neurological deterioration after subarachnoid haemorrhage.</a> Nature Reviews Neurology. 10 (1), 44-58 (2014).</li><li>Dorsch, 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+clinical+review+of+cerebral+vasospasm+and+delayed+ischaemia+following+aneurysm+rupture.">A clinical review of cerebral vasospasm and delayed ischaemia following aneurysm rupture.</a> Acta Neurochirurgica Supplement. 110 (Pt 1), 5-6 (2011).</li><li>Vergouwen, M. D., 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=Definition+of+delayed+cerebral+ischemia+after+aneurysmal+subarachnoid+hemorrhage+as+an+outcome+event+in+clinical+trials+and+observational+studies:+Proposal+of+a+multidisciplinary+research+group.">Definition of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage as an outcome event in clinical trials and observational studies: Proposal of a multidisciplinary research group.</a> Stroke. 41 (10), 2391-2395 (2010).</li><li>Friedrich, 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=CO2+has+no+therapeutic+effect+on+early+microvasospasm+after+experimental+subarachnoid+hemorrhage.">CO2 has no therapeutic effect on early microvasospasm after experimental subarachnoid hemorrhage.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 34 (8), e1-e6 (2014).</li><li>Friedrich, B., Muller, F., Feiler, S., Scholler, K., 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=Experimental+subarachnoid+hemorrhage+causes+early+and+long-lasting+microarterial+constriction+and+microthrombosis:+An+in+vivo+microscopy+study.">Experimental subarachnoid hemorrhage causes early and long-lasting microarterial constriction and microthrombosis: An in vivo microscopy study.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 32 (3), 447-455 (2012).</li><li>Terpolilli, N. 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=Nitric+oxide+inhalation+reduces+brain+damage,+prevents+mortality,+and+improves+neurological+outcome+after+subarachnoid+hemorrhage+by+resolving+early+pial+microvasospasms.">Nitric oxide inhalation reduces brain damage, prevents mortality, and improves neurological outcome after subarachnoid hemorrhage by resolving early pial microvasospasms.</a> Journal of Cerebral Blood Flow &amp; Metabolism. 36 (12), 2096-2107 (2016).</li><li>Neulen, 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+segmentation-based+volumetric+approach+to+localize+and+quantify+cerebral+vasospasm+based+on+tomographic+imaging+data.">A segmentation-based volumetric approach to localize and quantify cerebral vasospasm based on tomographic imaging data.</a> PLoS One. 12 (2), e0172010 (2017).</li><li>Schuller, K., Buhler, 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. (81), e50845 (2013).</li><li>Altay, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+subarachnoid+hemorrhage+to+induce+vasospasm+in+mice.">A novel method for subarachnoid hemorrhage to induce vasospasm in mice.</a> Journal of Neuroscience Methods. 183 (2), 136-140 (2009).</li><li>Momin, E. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+delivery+of+nitric+oxide+inhibits+leukocyte+migration+and+prevents+vasospasm+in+haptoglobin+2-2+mice+after+subarachnoid+hemorrhage.">Controlled delivery of nitric oxide inhibits leukocyte migration and prevents vasospasm in haptoglobin 2-2 mice after subarachnoid hemorrhage.</a> Neurosurgery. 65 (5), 937-945 (2009).</li><li>Froehler, M. T., 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=Vasospasm+after+subarachnoid+hemorrhage+in+haptoglobin+2-2+mice+can+be+prevented+with+a+glutathione+peroxidase+mimetic.">Vasospasm after subarachnoid hemorrhage in haptoglobin 2-2 mice can be prevented with a glutathione peroxidase mimetic.</a> Journal of Clinical Neurocience. 17 (9), 1169-1172 (2010).</li><li>Lin, C. 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+murine+model+of+subarachnoid+hemorrhage-induced+cerebral+vasospasm.">A murine model of subarachnoid hemorrhage-induced cerebral vasospasm.</a> Journal of Neuroscience Methods. 123 (1), 89-97 (2003).</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>Feiler, S., Friedrich, B., Scholler, K., Thal, S. C., 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=Standardized+induction+of+subarachnoid+hemorrhage+in+mice+by+intracranial+pressure+monitoring.">Standardized induction of subarachnoid hemorrhage in mice by intracranial pressure monitoring.</a> Journal of Neuroscience Methods. 190 (2), 164-170 (2010).</li><li>Ghanavati, S., Yu, L. X., Lerch, J. P., Sled, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perfusion+procedure+for+imaging+of+the+mouse+cerebral+vasculature+by+X-ray+micro-CT.">A perfusion procedure for imaging of the mouse cerebral vasculature by X-ray micro-CT.</a> Journal of Neuroscience Methods. 221, 70-77 (2014).</li><li>Parra, 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=Mouse+model+of+subarachnoid+hemorrhage+associated+cerebral+vasospasm:+methodological+analysis.">Mouse model of subarachnoid hemorrhage associated cerebral vasospasm: methodological analysis.</a> Neurological research. 24 (5), 510-516 (2002).</li><li>Boulay, A. C., Saubamea, B., Decleves, X., Cohen-Salmon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+Mouse+Brain+Vessels.">Purification of Mouse Brain Vessels.</a> Journal of Visualized Experiments. 105 (e53208), (2015).</li><li>Marjamaa, 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=Mice+with+a+deletion+in+the+first+intron+of+the+Col1a1+gene+develop+dissection+and+rupture+of+aorta+in+the+absence+of+aneurysms:+High-resolution+magnetic+resonance+imaging,+at+4.7+T,+of+the+aorta+and+cerebral+arteries.">Mice with a deletion in the first intron of the Col1a1 gene develop dissection and rupture of aorta in the absence of aneurysms: High-resolution magnetic resonance imaging, at 4.7 T, of the aorta and cerebral arteries.</a> Magnetic Resonance in Medicine. 55 (3), 592-597 (2006).</li><li>Schambach, S. 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=Ultrafast+high-resolution+in+vivo+volume-CTA+of+mice+cerebral+vessels.">Ultrafast high-resolution in vivo volume-CTA of mice cerebral vessels.</a> Stroke. 40 (4), 1444-1450 (2009).</li><li>Figueiredo, G., 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=Comparison+of+digital+subtraction+angiography,+micro-computed+tomography+angiography+and+magnetic+resonance+angiography+in+the+assessment+of+the+cerebrovascular+system+in+live+mice.">Comparison of digital subtraction angiography, micro-computed tomography angiography and magnetic resonance angiography in the assessment of the cerebrovascular system in live mice.</a> Clinical Neuroradiology. 22 (1), 21-28 (2012).</li></ol>

The authors have nothing to disclose.

Murine SAH models are an important tool for basic SAH research. Cerebral vasospasm is frequently used as an endpoint in experimental studies investigating the mechanisms leading to DCI after SAH9,11. However, quantification of cerebral vasospasm in mice or other small animal models of SAH is challenging. Commonly, vasospasm is quantified by ex vivo determination of vessel diameters at defined anatomical points after endovascular perfusion and casting7,9,12 or by determination of the circumference of defined vessels on histological sections10,11. However, these methods have some disadvantages: Vasospasm is evaluated only at defined anatomical points; vasospasm of neighboring vessel segments may escape evaluation. Histological artifacts present another source of errors. Furthermore, the evaluation can be rather subjective, because the exact position where the vessel diameter is measured is determined by the investigator. 
The goal was therefore to establish a method that quantifies cerebral vasospasm by calculating the vessel volume of entire cerebral vessel segments from cross-sectional imaging data7. The most important advantage of the volumetric method presented here is that entire vessel segments can be examined. This avoids the need of definition of a point where the vessel diameter is measured. A further advantage of the evaluation of whole vessel segments is that it presumably presents a more objective parameter to quantify vasospasm than determination of vessel diameters at defined points where vasospasm of the more proximal or distal vessel may escape evaluation. Digital representation of vessel diameters using a color code allows an intuitive estimation of the degree of vasospasm. Furthermore, volumetric evaluation leads to larger differences between vasospastic vessels compared to evaluation of vessel diameters as shown in the representative results. The virtual reconstruction achieved with the method presented here reflects the vascular anatomy accurately. This is shown by the evaluation of the representative series, in which vessel diameters measured microscopically and from the digital reconstructions were similar, reproducing the observations of a previous study7. However, despite its advantages, further studies are needed to evaluate whether or not the method presented here is superior to conventional methods of vasospasm analysis. 
A limitation of the method presented here is that it affords more time compared to microscopic analysis of casted brain samples or histological analysis (micro CT scanning time 90 minutes per brain sample, data processing 45 min per brain sample). Furthermore, the availability of micro CT scanners may limit its application. The number of animals examined here was sufficient to demonstrate the feasibility of the protocol described in this manuscript. However, if the protocol should be used in treatment studies, animal numbers would have to be calculated based on the expected effects on vessel volumes and diameters. Another limitation of this and other studies using murine SAH models is that vasospasm is determined ex vivo. This makes longitudinal studies impossible that investigate baseline values before SAH induction and vasospasm at different time points. Although studies have demonstrated that it is possible to depict the anatomy of the large intracranial vessels of mice in vivo using magnetic resonance tomography18, computed tomography angiography19, or digital subtraction angiography20, these methods, to our knowledge, have not yet been used to analyze cerebral vasospasm in murine SAH models in vivo. Of note, the digital reconstruction of the cerebral vasculature with subsequent volumetric evaluation of cerebral vasospasm presented here is not limited to the use on ex vivo micro CT data. If high resolution vascular cross sectional brain imaging in mice should become available in the future, it could be used to perform a volumetric analysis of vasospasm in vivo.

Aneurysmatic subarachnoid hemorrhage (SAH), a subtype of hemorrhagic stroke, is a common disease in neurointensive care units. Besides early brain injury (EBI), which comprises cerebral damage caused by the bleeding event itself, another important factor determining patient outcome is delayed cerebral ischemia (DCI), defined by clinical deterioration through impaired cerebral perfusion or cerebral infarction not associated with interventional or surgical procedures1,2,3. Important mechanisms contributing to DCI are vasospasms of large cerebral vessels on the one hand; on the other hand, microcirculatory dysfunction with vasospasm of microvessels and microthrombosis, and ischemia related to cortical spreading depressions play a role (reviewed in Madonald 20141). Therefore, diagnosis of vasospasm of the large cerebral vessels is crucial in clinical practice and displays an important endpoint in many clinical and experimental studies.
Despite the fact that the features of vasospasm in murine SAH models are not directly transferable to the human patient, murine models of SAH related vasospasm have been of growing significance in the last years. In these models SAH is induced by endovascular filament perforation4,5,6,7,8, transection of cisternal vessels9, or injection of blood into the CSF10,11,12. In contrast to large animal models of SAH which were traditionally designed to study vasospasm13, murine models have the great advantage that numerous transgenic mice strains are available. This makes them an excellent tool for studying molecular mechanisms leading to vasospasm and DCI. However, determination of cerebral vasospasm in mice is challenging. This is because in contrast to large animal models in which vasospasm can be examined using clinical imaging techniques, in vivo imaging to analyze cerebral vasospasm in mice is not yet available. Therefore, vasospasm is commonly determined using either histological sections10,11 or microscopically after casting of cerebral vessels7,9,12. However, these techniques have the disadvantage that vessel diameters are examined at defined points only.
Based on a previous study7, this manuscript presents a method for objective and reproducible analysis of vasospasm in a murine SAH model. The method is based on perfusion and casting of the cerebral vessels, ex vivo micro-CT scanning, digital reconstruction of the vessel tree, and subsequent evaluation of volumes of entire cerebral vessels.

<table><tbody><tr><td>Medetomidin&amp;nbsp;</td><td>Pfizer, Karlsruhe, Germany</td><td>n.a.</td><td></td></tr><tr><td>Midazolam</td><td>Ratiopharm, Ulm, Germany</td><td>n.a.</td><td></td></tr><tr><td>Fentanyl&amp;nbsp;</td><td>Curamed, Karlsruhe, Germany</td><td>n.a.</td><td></td></tr><tr><td>Venofix 21G</td><td>B Braun Melsungen AG, Melsungen, Germany</td><td>n.a.</td><td>21G cannula&amp;nbsp;</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline containing MgCl2 and CaCl2, pH 7.4&amp;nbsp;</td><td>Sigma-Aldrich, Hamburg, Germany</td><td>D8662&amp;nbsp;</td><td></td></tr><tr><td>4% paraformaldehyde solution&amp;nbsp;</td><td>Sigma-Aldrich, Hamburg, Germany</td><td>100496</td><td></td></tr><tr><td>Microfil MV-122&amp;nbsp;</td><td>Flowtech Inc., Carver, MA, USA</td><td>n.a.</td><td>Radiopaque</td></tr><tr><td>Micro-CT system Y.Fox</td><td>Yxlon, Garbsen, Germany</td><td>n.a.</td><td></td></tr><tr><td>Reconstruction Studio software version 1.2.8.1</td><td>TeraRecon, Frankfurt am Main, Germany</td><td>n.a.</td><td>Reconstruction software</td></tr><tr><td>Amira software version 5.4.2&amp;nbsp;</td><td>FEI Visualization Sciences Group, Hillsboro, OR, USA</td><td>n.a.</td><td>Visualization software</td></tr><tr><td>PHD ultra syringe pump</td><td>Harvard Apparatus</td><td>70-3</td><td>Pressure controlled pump</td></tr><tr><td>anatomical forceps (blunt)</td><td>B Braun Melsungen AG, Melsungen, Germany</td><td>160323_v&amp;nbsp;</td><td></td></tr><tr><td>Infinity X-21</td><td>Deltapix, Maalov, Denmark</td><td>n.a.</td><td>high resolution camera</td></tr><tr><td>DeltaPix Insight software version 2.0.1</td><td>Deltapix, Maalov, Denmark</td><td>n.a.</td><td></td></tr><tr><td>C57BL6 mice</td><td>Charles River, Cologne, Germany</td><td></td><td></td></tr></tbody></table>

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.

Virtual reconstruction of the 3-dimensional intracranial vascular tree
The 3-dimensionally reconstructed intracranial vascular trees provided a highly accurate vascular anatomy (Figure 1). To assess the accuracy, we performed a diameter-based comparison between vessel diameters determined microscopically and from the 3-dimensional virtual reconstructions at 2 anatomically defined points (1: left middle cerebral artery (MCA) 1 mm distal of the carotid T; 2: right MCA 1 mm distal of the carotid T). For microscopic determination of vessel diameters, a high-resolution camera (Infinity X-21, Deltapix) with DeltaPix Insight software version 2.0.1 calibrated to a micrometer scale was used. For this evaluation, 10 brain samples (5 SAH, 5 sham) were analyzed. These were from a series of 12 mice, in 7 of which an SAH was induced, while 5 underwent sham surgery (2 animals of the SAH group died on postoperative days 1 and 2, respectively). There were no significant differences between the diameters determined microscopically and virtually, indicating an accurate virtual reconstruction of the intracranial vascular anatomy (microscopic determination vs. virtual reconstruction, mean &#177; standard error: left MCA 150 &#177; 9 &#181;m vs. left MCA 150 &#177; 8 &#181;m; right MCA 153 &#177; 8 &#181;m vs.154 &#177; 9 &#181;m, see Figure 2). 
Quantification of cerebral vasospasm in mice with SAH
To quantify cerebral vasospasm, (i) the volume of a predefined representative 3.5 mm vessel segment, consisting of 1 mm internal carotid artery (ICA) and 2.5 mm MCA on the left, and (ii) the vessel diameters at 2 anatomically defined points (left and right MCA) were determined in brain samples from the SAH and sham-operated animals (n = 5). The vessel volume was significantly lower in SAH compared to sham (36 &#177; 4 nL vs. 71 &#177; 9 nL, p &#60; 0.05). Vessel diameters were lower in SAH compared to sham (left MCA: 140 &#177; 11 &#181;m vs. 160 &#177; 10 &#181;m, p = 0.11; right MCA: 130 &#177; 16 &#181;m vs. 158 &#177; 13 &#181;m, p &#60; 0.05; see Figure 3), while the level of significance was only reached for analysis of the right MCA.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57997/57997fig1.jpg" /> 
Figure 1. Virtual reconstruction of the intracranial vascular tree. (A) shows a representative brain sample; (B) shows the corresponding virtually reconstructed vascular tree. (C) Color-coded visualization of the diameter of the left MCA. <a href="https://www.jove.com/files/ftp_upload/57997/57997fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57997/57997fig2.jpg" /> 
Figure 2. Accuracy of the digital reconstruction of the vasculature. Mean diameters measured from 3D reconstructed brain samples compared with those determined microscopically. Data are shown as mean &#177; standard error of the mean. <a href="https://www.jove.com/files/ftp_upload/57997/57997fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57997/57997fig3.jpg" /> 
Figure 3. Vessel volume and vessel diameter after SAH. (A) Comparison of MCA diameters measured from 3D reconstructed vasculature in SAH and sham mice. (B) Vessel volume in SAH and sham mice. Data are shown as mean &#177; standard error of the mean. *p &#60; 0.05.&#160;<a href="https://www.jove.com/files/ftp_upload/57997/57997fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage at JoVE.com

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

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 ...

a-volumetric-method-for-quantification-cerebral-vasospasm-murine

Research

JoVE Journal

Neuroscience

8.0K Views.  Medical Center of the Johannes Gutenberg - University. Het doel van dit artikel is het presenteren van een methode waarmee een 3-dimensionale reconstructie van de cerebrovasculaire boom in muizen nadat micro berekende tomografie en bepaling van de hoeveelheden van hele schip segmenten die kunnen worden gebruikt voor het kwantificeren van cerebrale vasospasm in lymfkliertest modellen van subarachnoïdale bloeding.

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

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 ...

Modeling Stroke in Mice: Permanent Coagulation of the Distal Middle Cerebral Artery

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical options are available for a small proportion of stroke patients in the acute phase. Current research is intensively searching for novel therapeutic strategies and is increasingly focusing on the sub-acute and chronic phase after stroke because more patients might be eligible for therapeutic interventions in a prolonged time window. These delayed mechanisms include important pathophysiological pathways such as post-stroke inflammation, angiogenesis, neuronal plasticity and regeneration. In order to analyze these mechanisms and to subsequently evaluate novel drug targets, experimental stroke models with clinical relevance, low mortality and high reproducibility are sought after. Moreover, mice are the smallest mammals in which a focal stroke lesion can be induced and for which a broad spectrum of transgenic models are available. Therefore, we describe here the mouse model of transcranial, permanent coagulation of the middle cerebral artery via electrocoagulation distal of the lenticulostriatal arteries, the so-called &#8220;coagulation model&#8221;. The resulting infarct in this model is located mainly in the cortex; the relative infarct volume in relation to brain size corresponds to the majority of human strokes. Moreover, the model fulfills the above-mentioned criteria of reproducibility and low mortality. In this video we demonstrate the surgical methods of stroke induction in the &#8220;coagulation model&#8221; and report histological and functional analysis tools.

Various murine models of middle cerebral artery occlusion (MCAO) are widely used in experimental brain research. Here, we demonstrate the model of transcranial permanent distal MCAO which produces consistent cortical infarction of a size corresponding to damage imposed by the majority of human ischemic strokes.

Stroke is the third most common cause of death and a main cause of acquired adult disability in developed countries. Only very limited therapeutical ...

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

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

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

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

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

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

This protocol describes a novel technique of measuring the three most important parameters of ischemic brain injury on the same set of rodent brain samples. Using only one brain sample is highly advantageous in terms of ethical and economic costs.

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

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 ...

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

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

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

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

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

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

Samenvatting

Meer video's verkennen