Name: measuring cerebral vasospasm after subarachnoid hemorrhage in mice
Uploaded: 2022-02-28T17:01:03
Description: Subarachnoid hemorrhage (SAH) is a devastating illness, and patients who survive are still at risk for long-term neurological deficits. Cerebral vasospasm ...

This work was supported with funding from the following grants and organizations: National Institutes of Health (R01-NS110710 to BLH), The Brain Aneurysm Foundation (BAF2021-1483561969 to WSD and BLH), the James and Brigitte Marino Family Professorship Endowment, the Christine Desmond Fund, the Eblen Research Endowment, and the St. George Family Fund.

The present protocol was approved and performed in compliance with the institutional policy and guidelines of the University of Florida Institutional Animal Care and Use Committee (#201910613). The representative experiment was conducted consistently with ARRIVE guidelines21. Female C57BL/6J mice, 12-15-week-old, were used for the experiments; however, mice of any age, strain, or sex can be used. Mice were housed in ventilated cages on a 12 h/12 h light-dark cycle with ad libitum access t...

<ol>
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R., Testai, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delayed+cerebral+ischemia+after+subarachnoid+hemorrhage:+beyond+vasospasm+and+towards+a+multifactorial+pathophysiology.">Delayed cerebral ischemia after subarachnoid hemorrhage: beyond vasospasm and towards a multifactorial pathophysiology.</a> Current Atherosclerosis Reports. 19 (12), 50 (2017).</li><li>Budohoski, K. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathophysiology+and+treatment+of+delayed+cerebral+ischaemia+following+subarachnoid+haemorrhage.">The pathophysiology and treatment of delayed cerebral ischaemia following subarachnoid haemorrhage.</a> Journal of Neurology, Neurosurgery &amp; Psychiatry. 85 (12), 1343-1353 (2014).</li><li>Dodd, W. 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This protocol is a two-phase procedure for inducing SAH in mice and measuring cerebral vasospasm. This procedure is rigorous and reproducible while also maintaining time and resource efficiency. There are several critical steps in this procedure that must be adhered to closely. First, the coordinates of blood injections are of vital importance. Even a slight error in the location of the burr hole can lead to excessive bleeding from the dural venous sinuses and/or unintended injury to the cerebral vessels when the needle ...

The morbidity and mortality caused by subarachnoid hemorrhage (SAH) are staggering despite decades of research and a plethora of disappointing candidate therapeutics1,2,3,4,5,6. Delayed cerebral ischemia (DCI) is a fundamental cause of poor outcomes among SAH patients, yet is an incompletely understood pathophysiologically7,8,9. There are many contributing factors to DCI, including microcirculatory dysfunction, early brain injury, cortical spreading depolarizations, and cerebral vasospasm10. Developing novel treatment strategies will require innovative basic science and translational investigation, including preclinical animal models. Mouse models are an appealing option due to their similar cerebrovascular anatomy compared to humans and the availability of genetically-modified strains11. The effectiveness of mouse models is dependent on the method of SAH induction and the selection of appropriate experimental endpoints. Critical evaluation and rigorous implementation of these variables are essential to improving SAH-related morbidity and mortality.
There are two primary variables in models of SAH induction; method of hemorrhage (endovascular puncture or transcranial blood injection) and location of hemorrhage (cisterna magna/posterior circulation or prechiasmatic cistern/anterior circulation)12. Endovascular puncture models have several strengths, including maintaining cranial integrity compared to transcranial procedures and the substantial severity of hemorrhage, allowing for more readily detectable neurological deficits. On the other hand, endovascular puncture methods are highly variable in the volume and location of hemorrhage. This variability can skew results if the study design is not adequately powered. Additionally, the perioperative mortality rate of endovascular puncture models is approximately 40%12,13, which can drastically increase time and cost. Overall, the transcranial blood injection model is much more consistent, allows precise control over the volume and location of hemorrhage, and is much lower in perioperative mortality12,14,15.
Transcranial blood injection was initially developed for injection in the cisterna magna via puncture of the atlanto-occipital membrane16; however, prechiasmatic injection through a cranial burr hole has now been reported as well14,17. The primary benefit of anterior circulation models is the similarity to human SAH, where most non-traumatic SAH/vasospasm occurs in the anterior circulation. Transcranial autologous blood injection targeting the prechiasmatic cistern is an ideal preclinical model for investigating SAH and cerebral vasospasm.
There are also critical methodologic variables to consider when measuring cerebral vasospasm. Many researchers perform hematoxylin and eosin (H&#38;E) staining on tissue sections to measure the inner and outer diameter, vessel wall thickness, and other histological parameters. This method has some critical limitations. First, cerebral vessels are rarely in a straight line; thus, tissue sections may be oblique for the ideal orthogonal orientation of a cross-section. This issue may be compounded when attempting to compare across samples. Second, histological analysis of vessels may limit the number of vessels to be studied. The sectioning angle needed to measure one cerebral artery can be quite different from the required angle to measure a nearby vessel. A gel-dye vascular casting method has been developed to solve these challenges18,19,20. In this method, gelatin casts the morphology of the cerebral vessels, and India ink dye allows for high contrast upon digital image analysis. This technique permits the measurement of many vessels along their natural anatomical course.
The purpose of this report is to demonstrate a beginning-to-end experimental procedure for inducing subarachnoid hemorrhage and measuring delayed cerebral vasospasm. This representative experiment compared vasospasm between sham and SAH groups. Mice were allocated to each group by block randomization of mouse cages. Carefully considering the variables in each step of the protocol ensures the results are rigorous and reproducible while maintaining cost and resource efficiency.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 qt plastic bags</td>
			<td>Ziploc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mm drill bit</td>
			<td>Dremel</td>
			<td>106</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL insulin syringes</td>
			<td>ThermoFisher Scientific</td>
			<td>BD 329461</td>
			<td></td>
		</tr>
		<tr>
			<td>23 G Luer-lock needle</td>
			<td>Beckton-Dickinson (BD)</td>
			<td>305145</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Elabscience</td>
			<td>E-IR-R113</td>
			<td></td>
		</tr>
		<tr>
			<td>5% Povidone-Iodine Solution</td>
			<td>PBS Animal Health</td>
			<td>11205</td>
			<td>Chlorhexadine may be used per institutional guidelines or investigator preference</td>
		</tr>
		<tr>
			<td>5-0 monofilament suture</td>
			<td>Ethicon</td>
			<td>698H</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent bench covering</td>
			<td>ThermoFisher Scientific</td>
			<td>22-131-401</td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable heating pad</td>
			<td>Thermotech</td>
			<td>S766D</td>
			<td></td>
		</tr>
		<tr>
			<td>Bone wax</td>
			<td>Braintree Scientific</td>
			<td>DYNJBW 26</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera microscope mount adapter</td>
			<td>AmScope</td>
			<td>CA-NIK-SLR</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Nikon</td>
			<td></td>
			<td>Any high resolution digital camera will be sufficient; specific model up to investigator preference</td>
		</tr>
		<tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>M60</td>
			<td></td>
		</tr>
		<tr>
			<td>Ear tags</td>
			<td>Kent Scientific</td>
			<td>INS1005-5LS</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric hair trimmer</td>
			<td>Kent Scientific</td>
			<td>CL7300-Kit</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Roboz Surgical</td>
			<td>RS-5136</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>ThermoFisher Scientific</td>
			<td>S25335</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe with 28 G needle</td>
			<td>Hamilton Company</td>
			<td>80630</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld rotary tool</td>
			<td>Dremel</td>
			<td>Model #4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>ImagePro</td>
			<td>10.0.04</td>
			<td>Other software platforms (e.g. ImageJ, Orbit) may be sufficient</td>
		</tr>
		<tr>
			<td>India ink dye</td>
			<td>ThermoFisher Scientific</td>
			<td>NC9903975</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Patterson Veterinary</td>
			<td>07-893-6763</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer-lock syringes (3, 5, &#38; 10 mL)</td>
			<td>Beckton-Dickinson (BD)</td>
			<td>309657, 309646, 309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Mice</td>
			<td>Charles River Laboratory</td>
			<td>C57BL/6</td>
			<td>Genetically-modfied strains may be used per study design</td>
		</tr>
		<tr>
			<td>Mouse brain frame matrix</td>
			<td>Harvard Apparatus</td>
			<td>51386</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle driver</td>
			<td>Roboz Surgical</td>
			<td>RS-7860</td>
			<td></td>
		</tr>
		<tr>
			<td>Opthalmic ointment</td>
			<td>Dechra</td>
			<td>17033-211-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax paper</td>
			<td>Stellar Scientific</td>
			<td>HS234526A</td>
			<td>Parafilm or equivalent</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>ThermoFisher Scientific</td>
			<td>50488886</td>
			<td></td>
		</tr>
		<tr>
			<td>Ruler or other measurement standard</td>
			<td>Harvard Apparatus</td>
			<td>51386</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel</td>
			<td>Roboz Surgical</td>
			<td>65-9843</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz Surgical</td>
			<td>RS-5882</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic frame with syringe holder</td>
			<td>Harvard Apparatus</td>
			<td>75-1822</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile cotton-tipped applicators</td>
			<td>Uline</td>
			<td>S-18991</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile gauze</td>
			<td>ThermoFisher Scientific</td>
			<td>19-090-729</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>Patterson Veterinary</td>
			<td>07-869-6657</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical gloves</td>
			<td>AD Surgical</td>
			<td>A5CS-LTX65</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile surgical gown</td>
			<td>AD Surgical</td>
			<td>GWN-D02-CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical microscope</td>
			<td>World Precision Instruments</td>
			<td>PSMB5N</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Patterson Veterinary</td>
			<td>07-893-8424</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring cerebral vasospasm after subarachnoid hemorrhage in mice

Subarachnoid hemorrhage (SAH) is a devastating illness, and patients who survive are still at risk for long-term neurological deficits. Cerebral vasospasm is one of several contributing factors to morbidity and mortality after SAH. Preclinical animal models are essential resources to investigate pathophysiology and novel therapeutics. This protocol provides a two-phase method for both inducing SAH in mice and evaluating delayed cerebral vasospasm by measuring cerebral artery diameter. In the first step, an anterior circulation autologous blood injection method is used to reproduce the most common anatomical location of human non-traumatic SAH and reliably control the volume and distribution of hemorrhage. The duration of this procedure is approximately 20 min per animal. Next, on post-SAH day 5, the cerebral vasculature is fixed and casted using a gelatin-dye solution before removing the brain for imaging. Finally, the diameter of many cerebral arteries can be measured simultaneously using a variety of image analysis software platforms. These procedures are rigorous and reproducible while also offering a time- and resource-efficient method for studying the pathophysiology of SAH and cerebral vasospasm.

One of the primary advantages of this SAH model is the low mortality. Perioperative mortality is less than 10% for the SAH procedure, and perioperative deaths from the sham procedure are very rare; mortality rates during our development of this protocol were 6.7% and 0%, respectively (Table 1). Perioperative deaths in this SAH model, similar to other methods of SAH induction, are typically due to brain herniation secondary to increased intracranial pressure12.
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The present protocol provides a detailed procedure for inducing subarachnoid hemorrhage in mice via autologous blood injection to the anterior circulation and measuring delayed cerebral vasospasm by vascular gel casting.

Measuring Cerebral Vasospasm after Subarachnoid Hemorrhage in Mice

Subarachnoid hemorrhage (SAH) is a devastating illness, and patients who survive are still at risk for long-term neurological deficits. Cerebral vasospasm ...