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>
 <li>A straight 1 cm midline incision is taken on the ventral aspect of the tail</li>
 <li>The dissection is extended until the tail artery is identified and isolated. </li>
 <li>Using a sterile 26-gauge catheter, the tail artery is cannulated and 0.15 ml of arterial blood is withdrawn into a syringe.</li>
 <li>A sterile gauze is wrapped around the incision to ensure hemostasis before application of the vetbond to seal the incision. </li>
 <li>The rat is turned prone on the table and the shaved area over the sub-occipital region is swabbed with betadine.</li>
 <li>Using a vertical midline incision access is gained to the cisterna magna. </li>
 <li>Once identified a 25 gauge needle is inserted into the cisterna magna and 0.15 ml of CSF is withdrawn into a syringe to avoid increased intracranial pressures with injection of autologous blood volume. </li>
 <li>Now, the 0.15 ml of blood extracted from the tail artery is injected slowly into the cisterna magna. </li>
 <li>The needle is left in place for 30 sec to ensure clotting in the subarachnoid space and then carefully withdrawn. </li>
 <li>Hemostasis is ensured and the incision is closed using a stapling device.</li>
 <li>The animal is now placed prone on a warming surface with a 20 &deg; head down position for 20 min to allow blood to congeal in the cisterns around the basilar artery. </li>
 <li>Steps 1.1 to 1.15 are repeated during the second surgery 48 hr apart. </li>
</ol>
2.	Rat Surgery for SAH Subject Injected with 0.15 ml Saline
<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>
 <li>A straight 1 cm midline incision is taken on the ventral aspect of the tail </li>
 <li>The dissection is extended until the tail artery is identified and isolated. </li>
 <li>Using a sterile 26-gauge catheter, the tail artery is cannulated and 0.15 ml of arterial blood is withdrawn into a syringe. </li>
 <li>A sterile gauze is wrapped around the incision to ensure hemostasis before application of the vetbond to seal the incision. </li>
 <li>The rat is turned prone on the table and the shaved area over the sub-occipital region is painted with betadine. </li>
 <li>Using a vertical midline incision access is gained to the cisterna magna.</li>
 <li>Once identified a 25 gauge needle is inserted into the cisterna magna and 0.15 ml of CSF is withdrawn into a syringe and the sample is stored. </li>
 <li>Now, the 0.15 ml of normal saline (37 &deg;C) is injected slowly into the cisterna magna. </li>
 <li>The needle is left in place for 30 sec and carefully withdrawn. </li>
 <li>Hemostasis is ensured and the incision is closed using a stapling device. </li>
 <li>The animal is now placed prone on a warming surface with a 20 &deg; head down position for 20 min. </li>
 <li>Steps 2.1 to 2.15 are repeated during the second surgery 48 hr apart. </li>
</ol>
3. Rat Sacrifice
<ol>
 <li>On day 5 after the second surgery, the rats are sacrificed by cardiac perfusion. </li>
 <li>The rat is given a fatal dose (0.2 ml/kg) of Fatal Plus (VORTECH PHARMACEUTICALS LTD., DEARBORN, MI) </li>
 <li>With a vertical midline incision, the abdominal cavity is approached and the peritoneum is opened. </li>
 <li>An anterior thoracotomy is performed and the heart is exposed.</li>
 <li>Using a 26-gauge catheter connected to a phosphate buffer solution (PBS pH 7.4 and at 37 &deg;) the animal is drained of blood and is then perfused with 4% paraformaldehyde.</li>
 <li>After ensuring adequate perfusion, the perfusion is stopped and the rat is brought over to the decapitation table. </li>
 <li>After decapitation, a bone rongeur is used to remove the cranium for brain removal. </li>
 <li>The brain and brainstem are carefully extracted from the cranial vault and placed into a 4% paraformaldehyde solution and stored at 4 &deg;C for 48 hr. </li>
</ol>
4.	Creating Sections to Assess Vasospasm
<ol>
 <li>The rat brain that has now been submerged in 30% sucrose for 4 days is brought to the cryostat for sectioning. </li>
 <li>Once cryoprotected, 12 &mu;M sections are created using the cryostat, with the Anterior Inferior Cerebellar artery (AICA) as the starting point to ensure inter-subject consistency. </li>
 <li>20 sections are created for each animal, ending at the Superior Cerebellar artery (SCA). </li>
 <li>The sections are placed onto a glass slide and assessed for vasospasm using histological methodology</li>
</ol>

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We have nothing to disclose pertaining to this study.

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...

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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) ...

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14.2K Views.  SUNY Upstate Medical University. 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 th...