The current research is funded by the Solorz-Zak Research Foundation.

All surgical procedures were subjected to ethical review and approved by the government of Upper Bavaria (reference number: 55.2-1-54-2532.3-13-13 and -2532-136-11). Animals are male C57BL/6 mice with a body weight of approximately 25 g.
1. Animal Preparation
<ol>
	<li>Induce anesthesia by putting the mouse into a chamber. Flush the chamber with 5% isoflurane until the animal loses consciousness.</li>
	<li>Inject premixed anesthetics intraperitoneally: fentanyl (0.05 mg/kg), midazolam (5 mg/kg) and medetomidine (0.5 mg/kg). Check reflexes before and regularly during the procedure. Reinject one third of the initial amount hourly to maintain anesthesia.</li>
	<li>Intubate orotracheally with a tube made from a 20 G venous catheter11. For intubation fix the animal on a slanted platform (30&#176;), retract the tongue with bent forceps, visualize the vocal cords under an operation microscope and insert the tube into the trachea during inspiration.</li>
	<li>Place the mouse in a prone position and check the correct placement of the tube with a piece of cotton or a microcapnograph.</li>
	<li>Connect the intubation tube to the respirator. Ventilate the mouse with room air supplemented with 25% oxygen with a frequency of 180-220 breaths/min and a stroke volume of 200-250 &#956;l.</li>
	<li>Connect the intubation tube to the microcapnograph. Maintain the end-expiratory pCO2 at 30 mmHg by adjusting the ventilation frequency.</li>
	<li>Insert a rectal temperature probe and place the animal on a heating pad in order to maintain 37 &#176;C core body temperature.</li>
	<li>Apply the annular pulsoximeter sensor on the right hind paw.</li>
	<li>Open the skin above the skull with a pair of scissors. The incision should be approximately 0.5 cm long and between ear and eye.</li>
	<li>Dissect the left temporal muscle with a scalpel from the temporal bone.</li>
	<li>Glue the laser Doppler flowmeter (LDF) probe on the left temporal bone. Hold the probe in a fixed position until the glue hardens.</li>
	<li>Drill a hole of approximately 1.5 mm diameter with a dental drill into the left temporal bone. Cool the bone with saline to prevent heat damage.</li>
	<li>Insert the ICP probe into the cranial cavity. Push forward as dorsally as possible to prevent brain tissue damage and bleeding.</li>
	<li>If the probe is at the right position, fix, and seal it with cement. Let the cement dry for 5 min.</li>
	<li>Turn the mouse carefully to a supine position.</li>
	<li>For continuous blood pressure monitoring, catheterize the left femoral artery.</li>
	<li>Connect the femoral catheter to the blood pressure monitoring device.</li>
</ol>
2. SAH Induction
<ol>
	<li>Open the skin with a pair of scissors from sternum to chin (2 cm). Dissect the connective tissue bluntly and push the salivary glands aside.</li>
	<li>Expose the left common carotid artery (CCA) and mobilize it. Preserve the vagal nerve, which runs in the same connective tissue sheath as the CCA. Move cranial and expose and mobilize the ICA and the ECA using the same technique.</li>
	<li>Ligate the ECA as far cranially as possible.</li>
	<li>Prearrange two more ligations for the filament around the ECA.</li>
	<li>Occlude the CCA and the ICA temporarily with microclips. Position the microclips with a microclip applicator. Make sure the clips are applied correctly by gently pulling them back.</li>
	<li>Cut a hole for the filament insertion into the ECA with a vessel scissors.</li>
	<li>Insert a Prolene 5-0 filament with 12 mm length into the ECA.</li>
	<li>Close the insertion site with one prearranged ligation.</li>
	<li>Remove the microclips with a microclip applicator from the CCA and the ICA.</li>
	<li>Advance the filament with a forceps into the ICA until the ICP rises. A sudden rise of the ICP indicates bleeding induction.</li>
	<li>Withdraw the filament immediately and ligate the ECA by closing both prearranged ligations consecutively. This prevents bleeding out of the insertion site.</li>
	<li>Suture the skin wound.</li>
	<li>Monitor the physiologic parameters of the animal for another 20 min.</li>
	<li>Remove ICP and LDF probes and suture the skin wound.</li>
</ol>
3. End of Experiment
<ol>
	<li>Perfuse the animal transcardially with 20 ml of saline (room temperature) followed by 20 ml of 4% PFA in PBS (4 &#176;C).</li>
	<li>Dissect the brain out of the skull. Cut the skull in the midline and between the orbital cavities. Then peel the bone from the brain.</li>
	<li>Evaluate the blood distribution in the subarachnoid space.</li>
</ol>
4.&#160;Considerations in Case of Survival Surgery (not shown in the video)
<ol>
	<li>Inject Carprofen (4 mg/kg subcutaneously) for postoperative analgesia directly after anesthesia induction. During the postoperative observation period Carprofen (4 mg/kg subcutaneously) is injected every 24 hr.</li>
	<li>Instead of invasive blood pressure measurement make use of a noninvasive blood pressure monitoring system.</li>
	<li>For termination of anesthesia inject antagonizing agents subcutaneously: naloxone (1.2 mg/kg), flumazenil (0.5 mg/kg) and atipamezole (2.5 mg/kg).</li>
	<li>Extubate the animal.</li>
	<li>After regaining of reflexes put the animal into a preheated chamber. Keep the animal at approximately 32 &#176;C for 24 hr.</li>
	<li>Animals are checked regularly for spontaneous breathing and their general condition during the first hours after surgery. If the brain stem is affected animals have breathing problems and should be euthanized.&#160;</li>
	<li>Animals are checked daily for their general condition and body weight as well as for neurological and sensory deficits15.</li>
</ol>

<ol>
	<li>Cahill, J., 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=Subarachnoid+hemorrhage:+is+it+time+for+a+new+direction.">Subarachnoid hemorrhage: is it time for a new direction.</a> Stroke. 40, 86-87 (2009).</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> Lancet. 369, 306-318 (2007).</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> J. Neurosci. Methods. 123, 89-97 (2003).</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> J. Neurosci. Methods. 183, 136-140 (2009).</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> J. Neurosci. Methods. 190, 164-170 (2010).</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, 1086-1091 (1995).</li><li>Veelken, J. A., Laing, R. J., 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, 1279-1283 (1995).</li><li>Kamii, 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=Amelioration+of+vasospasm+after+subarachnoid+hemorrhage+in+transgenic+mice+overexpressing+CuZn-superoxide+dismutase.">Amelioration of vasospasm after subarachnoid hemorrhage in transgenic mice overexpressing CuZn-superoxide dismutase.</a> Stroke. 30, 867-871 (1999).</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> Neurol. Res. 24, 510-516 (2002).</li><li>Broderick, J. P., Brott, T. G., Duldner, J. E., Tomsick, T., Leach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+and+recurrent+bleeding+are+the+major+causes+of+death+following+subarachnoid+hemorrhage.">Initial and recurrent bleeding are the major causes of death following subarachnoid hemorrhage.</a> Stroke. 25, 1342-1347 (1994).</li><li>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=Non-invasive+intraoperative+monitoring+of+blood+pressure+and+arterial+pCO2+during+surgical+anesthesia+in+mice.">Non-invasive intraoperative monitoring of blood pressure and arterial pCO2 during surgical anesthesia in mice.</a> J. Neurosci. Methods. 159, 261-267 (2007).</li><li>Hockel, K., Trabold, R., Scholler, K., Torok, E., 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=Impact+of+anesthesia+on+pathophysiology+and+mortality+following+subarachnoid+hemorrhage+in+rats.">Impact of anesthesia on pathophysiology and mortality following subarachnoid hemorrhage in rats.</a> Exp. Transl. Stroke Med. 4, 5 (2012).</li><li>Wang, Z., Schuler, B., Vogel, O., Arras, M., Vogel, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+optimal+anesthetic+protocol+for+measurements+of+cerebral+autoregulation+in+spontaneously+breathing+mice?.">What is the optimal anesthetic protocol for measurements of cerebral autoregulation in spontaneously breathing mice?.</a> Exp. Brain Res. 207, 249-258 (2010).</li><li>Schwartz, A. Y., Masago, A., Sehba, F. A., Bederson, J. B. <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+in+the+rat:+a+refinement+of+the+endovascular+filament+model.">Experimental models of subarachnoid hemorrhage in the rat: a refinement of the endovascular filament model.</a> J. Neurosci. Methods. 96, 161-167 (2000).</li><li>Feiler, S., Plesnila, N., Thal, S. C., Zausinger, S., Scholler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+matrix+metalloproteinase-9+to+cerebral+edema+and+functional+outcome+following+experimental+subarachnoid+hemorrhage.">Contribution of matrix metalloproteinase-9 to cerebral edema and functional outcome following experimental subarachnoid hemorrhage.</a> Cerebrovasc. Dis. 32, 289-295 (2011).</li></ol>

Authors have nothing to disclose.

Treatment options after SAH are scarce and mostly inefficacious. Therefore the pathophysiology of post-hemorrhagic brain damage needs to be further understood in order to identify new therapeutic targets and develop novel therapeutic approaches. Standardized and well reproducible animal models in genetically modified animals, i.e. mice, are crucial for such investigations. The CWp model has become a widely used model for SAH as it resembles the pathophysiology in humans closely; however, its use in mice is hampe...

SAH is the stroke subtype with the least beneficial outcome for patients: 40% of the patients die within a month after the bleeding1 and survivors rarely have a clinically favorable outcome.
The large majority of spontaneous SAHs (80%) are caused by rupture of intracranial aneurysms which are mostly located along the anterior and posterior communicating artery, the basilar artery, and middle cerebral artery (MCA)2.
Such aneurysms are difficult to model in animals and therefore animal models of SAH are either performed by injection of blood into the subarachnoid space/cerebral ventricles or by endovascular perforation of a subarachnoid vessel.
Autologous blood injection into the cisterna magna is easy to perform and reproducible as the blood volume can be directly controlled3. Unfortunately some aspects of the SAH pathophysiology, e.g. the vessel injury, cannot be modeled by this procedure. Another technical approach for induction of SAH is the opening of an intracisternal vein4.
However, the intraluminal CWp at the MCA branch appears to be the procedure that models the pathophysiology in humans most closely5. The method was developed and first described in rats by Bederson and colleagues and at the same time by Veelken and colleagues6,7. Later the intraluminal perforation model was adapted to mice8,9. A filament is inserted into the external carotid artery (ECA) and advanced to the skull base via the internal carotid artery (ICA). At the branching point of the MCA the filament perforates the vessel and induces a bleeding into the subarachnoid space at the skull base. The blood then distributes into the remaining subarachnoid space along fissures and blood vessels. Bleeding is stopped by clot formation at the site of perforation, but rebleedings, which are often detrimental in patients10, can occur. Accordingly, the endovascular filament model became a widely used SAH model during the past few years. The most frequently mentioned drawback of the filament perforation model is that bleeding volume cannot be directly controlled and may therefore be variable. This variability can significantly be reduced by tight control of animal physiology and post-hemorrhagic ICP.
Mice have the great advantage that a large number of genetically modified strains are available. However, due to their small size surgical procedures tend to be more complex than in larger species, e.g. rats or rabbits. Therefore the downscaling of techniques developed for rats to mice often does not lead to the desired results, e.g. as mice have a very limited body weight and blood volume noninvasive techniques for blood pressure and blood gas analysis as well as for hemoglobin saturation and heart rate monitoring have to be applied whenever possible. Accordingly, the aim of the current publication is to describe the filament perforation model for SAH in mice and to demonstrate how this model can be performed in a standardized and highly reproducible manner.

<table><tbody><tr><td colspan="4">&lt;strong&gt;Equipment&lt;/strong&gt;</td></tr><tr><td>operation microscope</td><td>Leica</td><td>KL2500</td><td></td></tr><tr><td>isoflurane vaporizer</td><td>Harvard Instruments</td><td>Continuous Flow Vaporizer</td><td></td></tr><tr><td>respirator</td><td>Hugo Sachs</td><td>Minivent 845</td><td></td></tr><tr><td>microcapnograph</td><td>Hugo Sachs</td><td>Type 340</td><td></td></tr><tr><td>temperature controller</td><td>FHC</td><td>DC Temperature Controller</td><td></td></tr><tr><td>dental drill</td><td>Paggen</td><td>Labset- N</td><td></td></tr><tr><td>ICP monitor</td><td>Codman</td><td>ICP monitor</td><td></td></tr><tr><td>blood pressure monitor</td><td>AD Instruments</td><td>Bridge Amp FE221</td><td></td></tr><tr><td>syringe pump</td><td>World Precision Instruments</td><td>SP101IZ</td><td></td></tr><tr><td>pulsoximeter</td><td>Kent Scientific</td><td>MouseSTAT</td><td></td></tr><tr><td>LDF</td><td>Perimed</td><td>Periflux 5000</td><td></td></tr><tr><td>analog data monitor</td><td>AD Instruments</td><td>Power Lab 16/35</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Material&lt;/strong&gt;</td></tr><tr><td>cement for ICP probe fixation</td><td>Speiko</td><td>Carboxylate cement</td><td></td></tr><tr><td>glue for LDF probe fixation</td><td>Bob Smith Industries</td><td>Cyanoacrylate glue (Maxi Cure and Insta Set)</td><td></td></tr><tr><td>venous catheter</td><td>Johnson Johnson</td><td>Jelco winged i.v. catheter; REF 4076</td><td>modified intubation tube</td></tr><tr><td>tubing for femoral catheter</td><td>Smiths Medical</td><td>Fine Bore Polythene Tubing; ID 0.28 mm OD 0.61 mm; REF 800/100/100</td><td>cut to 30 cm length</td></tr><tr><td>filament for vessel perforation</td><td>Ethicon</td><td>Prolene 5-0</td><td>cut to 12 mm length</td></tr><tr><td>surgical equipment</td><td>Fine Scientific Instruments</td><td>forceps medical #5, vessel scissors 8 cm, microclip 4 mm jaw</td><td></td></tr></tbody></table>

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.

Mortality
Once the surgery technique is mastered the procedure does not elicit any intraoperative mortality. Also bleeding can be achieved in virtually all animals. Postoperative mortality is 30-40% with most animals dying on day 1 after surgery (Figure 5).
ICP values after SAH
The ICP before bleeding is around 4 mmHg. Bleeding results in a sharp increase of the ICP up to 120 mmHg. ICP values then stabilize within 5 min at appr...

Watch this Scientific Journal Video about A Murine Model of Subarachnoid Hemorrhage at JoVE.com

A Murine Model of Subarachnoid Hemorrhage

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 ...

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Research

JoVE Journal

Medicine

19.6K Views.  University of Munich Medical Center. 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.

Video: 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) ...

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 ...

Neuroscience

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 ...

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

Source: Liu, S., et al. <a href="https://app.jove.com/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.
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Neuropathology

Cerebrovascular Diseases

Establishing a Subarachnoid Hemorrhage Rat Model Induced by Autologous Blood Injection

To place the laser Doppler probe, make a 15-millimeter incision caudally in the midline, starting just anterior to the eyes. After removing the connective tissue and the muscles with forceps, use the end of a sterile cotton swab as a regime to identify the bregma and the coronal sutures.
After placing the armed retractor, place a 25-gauge spinal needle in the stereotaxic frame exactly on the bregma and note the position. Next, remove the needle from the bregma. Move the frame anteriorly by 6.5 millimeters, then replace the needle in the midline to mark the side of drilling.
Drill until the dura mater is identified below the bone. Using straight forceps, gently remove the bone fragments, then fill the cavity with bone wax. Next, 3 to 4 millimeters lateral to the right of the bregma and just anterior to the coronal suture for the laser Doppler, drill another hole taking care not to penetrate the dura mater.
Look for the vessels where the laser Doppler can measure the blood flow. Place the laser Doppler and check the values. A minimum value of 100 flux units is required. If the values remain acceptable after removing the microscope, add 1 drop of glue to fix the probe. Recheck to confirm whether the value is above 80 flux units.
To induce subarachnoid hemorrhage, insert the needle gently through the skull in the midline between the hemispheres until resistance is felt due to the base of the skull. Retract the needle by 1 millimeter to ensure correct placement just anteriorly to the optic chiasm. To ensure the most homogeneous result when injecting the blood, turn the needle clockwise by 90 degrees such that the needle tip points to the right. Then, remove the stiletto.
After a 15-minute equilibration, adjust the level of anesthesia to obtain a mean arterial blood pressure in the range 80 to 100 millimeters of mercury. Then, perform a blood gas analysis to confirm that the pH, partial pressure of carbon dioxide and partial pressure of oxygen are within physiological parameters.
Next, using a 1-milliliter syringe with a blunt 23-gauge needle, withdraw 500 microliters of blood from the tail catheter. To avoid injection of air, fill the dead space of the spinal needle chamber with blood. After adjusting the volume of the blood in the syringe to 300 microliters, connect the syringe to the spinal needle. Then, grasping firmly around the syringe, inject the blood manually to surpass mean arterial pressure. Observe a steep rise in the intracerebral pressure and a concurrent steep fall in the cerebral blood flow.