Name: a low mortality rat model to assess delayed cerebral vasospasm after experimental subarachnoid hemorrhage
Uploaded: 2013-01-17T12:00:00
Description: Watch this Scientific Journal Video about A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage at JoVE.com

We would like to acknowledge the efforts of Dr Mary-Lou Vallano, Department of Neuroscience and Physiology, for her valuable inputs in the write up for this manuscript.

1.	Rat Surgery for SAH Subject Injected With 0.15 ml Autologous Arterial Blood
<ol>
 <li>The rat is anesthetized using 0.1 mg/kg of Ketamine/Xylazine rodent cocktail and allowed to sit for 5 min.</li>
 <li>Adequate anesthesia is confirmed by reduction in hind limb reflex. </li>
 <li>Using an electronic shaver a neck to nose area of hair around the sub-occipital region is shaved. </li>
 <li>The animal is placed supine on the surgery table and the tail is swabbed with betadine to ensure a sterile incision. </li>
 <l...

<ol>
	<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>Cheng, G., Wei, L., Zhi-Dan, S., Shi-Guang, Z., Xiang-Zhen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atorvastatin+ameliorates+cerebral+vasospasm+and+early+brain+injury+after+subarachnoid+hemorrhage+and+inhibits+caspase-dependent+apoptosis+pathway.">Atorvastatin ameliorates cerebral vasospasm and early brain injury after subarachnoid hemorrhage and inhibits caspase-dependent apoptosis pathway.</a> BMC Neurosci. 10, 7-17 (2009).</li><li>Jackowski, A., Crockard, A., Burnstock, G., Russell, R. R., Kristek, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+time+course+of+intracranial+pathophysiological+changes+following+experimental+subarachnoid+hemorrhage+in+the+rat.">The time course of intracranial pathophysiological changes following experimental subarachnoid hemorrhage in the rat.</a> J. Cereb. Blood Flow Metab. 10, 835-849 (1990).</li><li>Kaoutzanis, M., Yokota, M., Sibilia, R., Peterson, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurologic+evaluation+in+a+canine+model+of+single+and+double+subarachnoid+hemorrhage.">Neurologic evaluation in a canine model of single and double subarachnoid hemorrhage.</a> J. Neurosci. Methods. 50, 301-307 (1993).</li><li>Karaoglan, A., Akdemir, O., Barut, S., Kokturk, S., Uzun, H., Tasyurekli, M., Colak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+resveratrol+on+vasospasm+after+experimental+subarachnoid+hemorrhage+in+rats.">The effects of resveratrol on vasospasm after experimental subarachnoid hemorrhage in rats.</a> Surg. Neurol. 70, 337-343 (2008).</li><li>Lee, J. Y., Huang, D. L., Keep, R., Sagher, O. <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+improved+double+hemorrhage+rat+model+for+the+study+of+delayed+cerebral+vasospasm.">Characterization of an improved double hemorrhage rat model for the study of delayed cerebral vasospasm.</a> J. Neurosci. Methods. 168, 358-366 (2008).</li><li>Lee, J. Y., Sagher, O., Keep, R., Hua, Y., 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=Comparison+of+experimental+rat+models+of+early+brain+injury+after+subarachnoid+hemorrhage.">Comparison of experimental rat models of early brain injury after subarachnoid hemorrhage.</a> Neurosurgery. 65 (2), 331-343 (2009).</li><li>Megyesi, J. F., Vollrath, B., Cook, D. A., Findlay, 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=In+vivo+animal+models+of+cerebral+vasospasm:+a+review.">In vivo animal models of cerebral vasospasm: a review.</a> Neurosurgery. 46, 448-460 (2000).</li><li>Prunell, G. F., Mathiesen, T., Diemer, N. H., 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=Experimental+subarachnoid+hemorrhage:+Subarachnoid+blood+volume,+mortality+rate,+neuronal+death,+cerebral+blood+flow,+and+perfusion+pressure+in+three+different+rat+models.">Experimental subarachnoid hemorrhage: Subarachnoid blood volume, mortality rate, neuronal death, cerebral blood flow, and perfusion pressure in three different rat models.</a> Neurosurgery. 52, 165-176 (2003).</li><li>Prunell, G. F., Mathiesen, T., 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=Experimental+subarachnoid+hemorrhage:+Cerebral+blood+flow+and+brain+metabolism+during+the+acute+phase+in+three+different+models+in+the+rat.">Experimental subarachnoid hemorrhage: Cerebral blood flow and brain metabolism during the acute phase in three different models in the rat.</a> Neurosurgery. 54, 426-436 (2004).</li><li>Ryba, M. S., Gordon-Krajcer, W., Walski, M., Chalimoniuk, M., Chrapusta, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxylamine+attenuates+the+effects+of+simulated+subarachnoid+hemorrhage:+implication+for+the+role+of+oxidative+stress+in+cerebral+vasospasm.">Hydroxylamine attenuates the effects of simulated subarachnoid hemorrhage: implication for the role of oxidative stress in cerebral vasospasm.</a> Neurol. Res. 31, 195-199 (1999).</li><li>Satoh, M., Parent, A. D., 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=Inhibitory+effect+with+antisense+mitogen-activated+protein+kinase+oligodeoxynucleotide+against+cerebral+vasospasm+in+rats.">Inhibitory effect with antisense mitogen-activated protein kinase oligodeoxynucleotide against cerebral vasospasm in rats.</a> Stroke. 33, 775-781 (2002).</li><li>Suzuki, H., Kanamaru, K., Tsunoda, H., Inada, H., Kuroki, M., Sun, H., Waga, S., Tanaka, T. <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> J. Clin. Invest. 104, 59-66 (1999).</li><li>Swift, D. M., Solomon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subarachnoid+hemorrhage+fails+to+produce+vasculopathy+or+chronic+blood+flow+changes+in+rats.">Subarachnoid hemorrhage fails to produce vasculopathy or chronic blood flow changes in rats.</a> Stroke. 19, 878-882 (1988).</li><li>Vatter, H., Weidauer, S., Konczalla, J., Dettmann, E., Zimmermann, M., Raabe, A., Preibisch, C., Zanella, F., Seifert, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time+course+in+the+development+of+cerebral+vasospasm+after+experimental+subarachnoid+hemorrhage:+clinical+and+neuroradiological+assessment+of+the+rat+double+hemorrhage+model.">Time course in the development of cerebral vasospasm after experimental subarachnoid hemorrhage: clinical and neuroradiological assessment of the rat double hemorrhage model.</a> Neurosurgery. 58, 1190-1197 (2006).</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>Zubkov, A. Y., Nanda, A., 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=Signal+transduction+pathways+in+cerebral+vasospasm.">Signal transduction pathways in cerebral vasospasm.</a> Pathophysiology. 9, 47-61 (2003).</li></ol>

Primates, having a more similar genetic composition and anatomical features to the human, more closely mimic the events of delayed cerebral vasospasm and can more easily undergo non-invasive imaging (MRI and angiography) to monitor arterial changes, than rodents 8. However, primate models are cost-prohibitive and associated with more complex care and ethical issues, than small animal models. Small animal SAH models that have been developed previously have focused on three methods of inducing SAH: 1) Endovascu...

<table border="1">
 <tbody>
 <tr>
 <td>Name of equipment / reagent</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 </tr>
 <tr>
 <td>Male SD rats (250-300 g)</td>
 <td>Taconic</td>
 <td>SD-M</td>
 </tr>
 <tr>
 <td>26 G Catheters</td>
 <td>Webster</td>
 <td>8416683</td>
 </tr>
 <tr>
 <td>25 G Needles</td>
 <td>Buffalo</td>
 <td>305122</td>
 </tr>
 <tr>
 <td>1 cc Syringes</td>
 <td>Central stores</td>
 <td>54245</td>
 </tr>
 <tr>
 <td>Ketamine/Xylazine cocktail</td>
 <td>Animal Care (SUNY)*</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Betadine</td>
 <td>Central stores</td>
 <td>51458</td>
 </tr>
 <tr>
 <td>Sucrose</td>
 <td>Sigma</td>
 <td>S9378-1kg</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Sigma</td>
 <td>P6148-500G</td>
 </tr>
 <tr>
 <td>Phosphate buffer solution</td>
 <td>Fisher</td>
 <td>BP-399-4</td>
 </tr>
 <tr>
 <td>Surgical Table</td>
 <td>Harvard</td>
 <td>PY2 72-2590</td>
 </tr>
 <tr>
 <td>OCT Compound (cryoprotection)</td>
 <td>VWR</td>
 <td>25608-930</td>
 </tr>
 <tr>
 <td>Superfrost Slides</td>
 <td>Fisher</td>
 <td>12-550-15</td>
 </tr>
 </tbody>
 </table>
* Synthesized at Department of Laboratory Animal Care, SUNY Upstate Medical University. Add 1 cc [100 mg/ml] of Xylazine to 10 ml [100 mg/ml] of Ketamine.

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.

Within the protocols described above, there are several steps that we believe require a better characterization of the model than what has been previously described in the literature. Here we focus on the steps that are essential in order to achieve a reproducible low mortality cerebral vasospasm small animal model and avoid potential pitfalls associated with this model if not done correctly. 
1. Autologous Blood Draw from the Tail Artery: 
Careful placement of the ang...

Watch this Scientific Journal Video about A Low Mortality Rat Model to Assess Delayed Cerebral Vasospasm After Experimental Subarachnoid Hemorrhage at JoVE.com

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

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) ...

Research

JoVE Journal

Medicine

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Surgical Procedures for a Rat Model of Partial Orthotopic Liver Transplantation with Hepatic Arterial Reconstruction

Orthotopic liver transplantation (OLT) in rats using a whole or partial graft is an indispensable experimental model for transplantation research, such as ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

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 ...

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 ...

Integrated Compensatory Responses in a Human Model of Hemorrhage

Hemorrhage is the leading cause of trauma-related deaths, partly because early diagnosis of the severity of blood loss is difficult. Assessment of ...

Transplantation of Adipose Tissue-Derived Stem Cell Sheet to Reduce Leakage After Partial Colectomy in A Rat Model

Anastomotic leakage is a disastrous complication after colorectal surgery. Although current methods for leakage prevention have different levels of ...

Implantation of hiPSC-derived Cardiac-muscle Patches after Myocardial Injury in a Guinea Pig Model

Due to the limited regeneration capacity of the heart in adult mammals, myocardial infarction results in an irreversible loss of cardiomyocytes. This loss ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...