Name: real-time monitoring and modulation of blood pressure in a rabbit model of ischemic stroke
Uploaded: 2023-02-10T13:15:00
Description: Watch this Scientific Journal Video about Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke at JoVE.com

The research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers UL1TR002538 and KL2TR002539 and by Transformational Grant 19TPA34910194 from the American Heart Association.

This protocol is approved by the Institutional Animal Care and Use Committee (University of Utah IACUC Protocol Number 21-09021). Mature New Zealand White rabbits are obtained from commercial vendors.
1. Animal acquisition
<ol>
	<li>Acclimate the animals for the required duration after arrival according to institutional protocol, housing animals socially in a vivarium with standard chow diets. The acclimation period at our institution is 2 weeks.</li>
</ol>
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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=A+novel+model+of+large+vessel+ischemic+stroke+in+rabbits:+microcatheter+occlusion+of+the+posterior+cerebral+artery.">A novel model of large vessel ischemic stroke in rabbits: microcatheter occlusion of the posterior cerebral artery.</a> Journal of Neurointerventional Surgery. 7 (5), 363-366 (2015).</li><li>Peng, T. J., Ortega-Guti&#233;rrez, S., de Havenon, A., Petersen, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+pressure+management+after+endovascular+thrombectomy.">Blood pressure management after endovascular thrombectomy.</a> Frontiers in Neurology. 12, 723461 (2021).</li><li>Nepal, G., Shrestha, G. S., Shing, Y. 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Substantial progress has been made in the management of IS, particularly considering advances in acute intervention and secondary prevention strategies. However, more work can be done to improve care for IS patients. Limited progress in other aspects of IS treatment, particularly in the realm of neuroprotection, likely results from the limitations in pathophysiological understanding of mechanistic processes at the tissue and molecular level. Impactful data from humans is unrealistic and likely impossible to acquire. In s...

Ischemic stroke (IS) is a leading cause of death and long-term disability worldwide, and its prevalence is projected to increase as society ages1. While substantial advances have been made in acute interventions and secondary prevention strategies, adjunctive neuroprotective treatments have not followed apace2,3,4,5,6,7. Further research is needed into stroke pathobiology because mechanisms by which therapies may or may not prove effective are poorly understood. This is largely due to the heterogeneous nature of the stroke patient population, many of whom have numerous comorbidities that confound analysis1. One driver of limitations in research is the absence of tissue-level data-the gold standard in biomedical research-due to the prohibitive morbidity of sampling tissue from the human central nervous system. Specifically, vascular tissue harvesting in a living human would cause a stroke, so vascular tissue is typically only obtained at autopsy, which is under-representative of the general population and skews toward more advanced disease in elderly patients with concomitant diagnoses.
In such cases, when sufficient human data cannot be utilized, animal models can bridge the data gaps. Large animal models of stroke are limited as most large animals used in research are ungulates having a rete mirabile that prevents direct endovascular access to the cerebral arteries8,9,10,11,12,13,14,15,16,17. Rabbits have a long history of use for the investigation of cardiovascular disease, including intracranial pathologies8,9,10,11,12,13,14,15,16,17. Rabbits present an ideal model for cerebrovascular diseases because they are large enough for endovascular catheterization and lack the rete mirabile that precludes intracranial access in other large mammals9,15,16,17. They have been previously utilized specifically for the investigation of IS through precise and well-controlled occlusion of an intracranial artery with a microcatheter18.
Blood pressure (BP) control, both through modulation of absolute BP or BP variability (BPV), the degree to which arterial BP fluctuates around a mean BP, is an emerging potential therapeutic target for IS patients after reports of worse outcomes in those with poorly controlled BP or BPV19,20,21,22. Mechanistic investigation into how changes lead to poor outcomes in IS patients is lacking. This is partly due to the difficulty in obtaining tissue-level data and performing well-controlled analyses in humans. To test interventions that modulate BP or BPV, animal models must be utilized to overcome these limitations. This report describes the successful pairing of a previously validated rabbit model of IS using controlled occlusion of the posterior cerebral artery in conjunction with continuous intra-arterial measurement of BP18. The method presented here improves on the previous approaches to stroke pathophysiology by applying a validated and reproducible stroke model to a system in which precise measurement and control of BP can be achieved. In this refined model, infarct burden can be assessed with post-procedural histopathologic staining of the harvested brain, which is also amenable to various stains and more advanced analyses such as spatial transcriptomics. Additionally, the occluded posterior circulation artery can also be chosen to be evaluated for morbidity analysis following survival procedures.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-0 Silk Suture</td>
			<td>Ethicon</td>
			<td>A184H</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Sigma-Aldrich</td>
			<td>B9275</td>
			<td></td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>Terumo</td>
			<td>CG415</td>
			<td>4F glide catheter</td>
		</tr>
		<tr>
			<td>Endovascular Pressure Sensor</td>
			<td>Millar</td>
			<td>SPR-524</td>
			<td></td>
		</tr>
		<tr>
			<td>Euthasol</td>
			<td>Virbac</td>
			<td>PVS111</td>
			<td></td>
		</tr>
		<tr>
			<td>Guidewire</td>
			<td>Terumo</td>
			<td>GR1804</td>
			<td></td>
		</tr>
		<tr>
			<td>Iohexol</td>
			<td>ThermoFisher</td>
			<td>466651000</td>
			<td>Iodinated Contrast</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Biorbyt</td>
			<td>orb61131</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart Software</td>
			<td>ADInstruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lidocaine</td>
			<td>Spectrum</td>
			<td>LI102</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcatheter</td>
			<td>Medtronic</td>
			<td>EV3 105-5056</td>
			<td>Marathon Microcatheter</td>
		</tr>
		<tr>
			<td>Microwire</td>
			<td>Medtronic</td>
			<td>EV3 103-0608</td>
			<td>Mirage Microwire</td>
		</tr>
		<tr>
			<td>PowerLab&#160;</td>
			<td>ADInstruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit Brain 2mm Coronal Cutting Matrix</td>
			<td>Ted Pella</td>
			<td>15026</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>FisherScientific</td>
			<td>23-535435</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheath</td>
			<td>Merit Medical</td>
			<td>PSI-5F-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine&#160;</td>
			<td>ThermoFisher</td>
			<td>J61430.14</td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time monitoring and modulation of blood pressure in a rabbit model of ischemic stroke

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains challenging to identify the mechanisms that lead to poor outcomes or evaluate measures by which these effects can be mitigated because of the prohibitive limitations inherent to human data. In such cases, animal models can be utilized to conduct rigorous and reproducible evaluations of diseases. Here we report refinement of a previously described model of ischemic stroke in rabbits that is augmented with continuous blood pressure recording to assess the impacts of modulation on blood pressure. Under general anesthesia, femoral arteries are exposed through surgical cutdowns to place arterial sheaths bilaterally. Under fluoroscopic visualization and roadmap guidance, a microcatheter is advanced into an artery of the posterior circulation of the brain. An angiogram is performed by injecting the contralateral vertebral artery to confirm occlusion of the target artery. With the occlusive catheter remaining in position for a fixed duration, blood pressure is continuously recorded to allow for tight titration of blood pressure manipulations, whether through mechanical or pharmacological means. At the completion of the occlusion interval, the microcatheter is removed, and the animal is maintained under general anesthesia for a prescribed length of reperfusion. For acute studies, the animal is then euthanized and decapitated. The brain is harvested and processed to measure the infarct volume under light microscopy and further assessed with various histopathological stains or spatial transcriptomic analysis. This protocol provides a reproducible model that can be utilized for more thorough preclinical studies on the effects of blood pressure parameters during ischemic stroke. It also facilitates effective preclinical evaluation of novel neuroprotective interventions that might improve care for ischemic stroke patients.

In the initial experiments with this model, our group successfully achieved the desired outcome of a posterior cerebral or superior cerebellar artery occlusion in 12 out of 14 animals (85.7%). For the experiment, seven males and seven females were studied. The mean animal weight was 3.6 kg (&#177; 0.46 kg). In the two animals in which success was not achieved, profound catheter-induced vasospasm precluded safe access to the intracranial circulation. In one rabbit, intracranial access could not be obtained due to occlusiv...

Watch this Scientific Journal Video about Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke at JoVE.com

Real-Time Monitoring and Modulation of Blood Pressure in a Rabbit Model of Ischemic Stroke

Continuous arterial blood pressure recording allows the investigation of impacts of various hemodynamic parameters. This report demonstrates the application of continuous arterial blood pressure monitoring in a large animal model of ischemic stroke for determination of stroke pathophysiology, impact of different hemodynamic factors, and the assessment of novel treatment approaches.

Control of blood pressure, in terms of both absolute values and its variability, affects outcomes in ischemic stroke patients. However, it remains ...

Understanding the pathophysiology of ischemic stroke is limited due to the absence of high quality tissue data in humans. Since such samples are impossible to acquire, well-controlled animal model studies can serve as a surrogate. The rabbit model used in this protocol provides consistent high-quality data for the interrogation of ischemic stroke.The technique described here allows for precise control and modulation of hemodynamic factors to assess their impact on ischemic stroke pathophysiology. A well-planned procedure can lead to high-quality results, particularly by employing strategies to mitigate vasospasm in the arteries, through a combination of pharmacologic prophylaxis and effective angiographic techniques. To begin, place the rabbit in a supine position on a fluoroscopy compatible operative table.Extend the head as it optimizes positioning for subsequent angiographic views. To mitigate vasospasm, place 0.5 inches of transdermal nitroglycerin on the inside of the ear after induction of anesthesia. After removing the fur from both inguinal regions using electric clippers, prepare the skin with scrubs of chlorhexidine and alcohol.Drape the skin in the usual sterile fashion, and palpate the bilateral femoral arterial pulses. Make a five-centimeter surgical incision with a number 10 blade at the site where lidocaine was injected. Use blunt dissection to expose the neurovascular bundle, and if needed, extend the incision to expose an arterial segment large enough for access.Upon isolation of the neurovascular bundle, drop several drops of 1%lidocaine on the artery to prevent vasospasm. Separate the artery gently from the vein and the adjacent nerve using forceps. Identify the artery by the characteristic appearance of its muscular wall compared to the thin walls of the vein.After isolating the artery, pass the right angle forceps under the vessel. Then grasp two vessel loops with the instrument and gently pass them under the artery. Situate one each at the upstream and downstream ends of the exposed vessel.Subject the artery to gentle traction by pulling the vessel loops, and inspect the vessel for any residual tissue to be removed with gentle dissection, increasing the chances of successful access. After dissecting the vessel and preparing the angiocatheter, drip lidocaine on the vessel again. The artery visibly dilates, increasing the chances of successful access and placement of a sheath using the Seldinger technique.Apply gentle traction to the downstream vessel loop to engorge the artery by reducing outflow and stabilizing the vessel for the access attempt. Next, slowly advance the needle of the angiocatheter into the middle of the exposed arterial segment. On seeing a flash of blood in the angiocatheter and the hub chamber, advance the catheter into the arterial lumen.On successfully placing the angiocatheter, advance a cope microwire through the angiocatheter lumen and into the aorta. Then remove the angiocatheter over the wire and replace it with a five French slim hydrophilic sheath. Confirm the return of the arterial blood through the sidearm tubing by opening the three-way valve.Lock the valve closed, and flush the sheath with 0.9%saline. Secure the sheath hub to the adjacent skin with an additional 3.0 silk suture, and repeat this process for the contralateral femoral artery. Under fluoroscopic visualization, advance a four French glide catheter over a 0.035-inch glide wire inserted through the left femoral sheath.Positioned the tip of the glide catheter in the proximal left vertebral artery. After removing the wire, flush the catheter with heparinized 0.9%saline. Perform angiography by injecting the left vertebral artery with iodinated contrast under lobe magnification to visualize the entire head and neck.For the left vertebral injection, inject 50%contrast, diluted in normal saline, with a gentle crescendo from a three CC syringe. Determine the injection amount by checking the reflux down the right vertebral artery and into the right subclavian artery. During this injection, also note the posterior cerebral artery or superior cerebral artery, one of which will be the target to occlude with the microcatheter.Prepare a 2.4 French flow-directed microcatheter with a 0.010 inch microwire, and make a C shape on the tip of the microwire. Under roadmap guidance, advance the microcatheter inside a four French glide catheter using the right femoral sheath and over the wire into the right vertebral artery. Advance the microcatheter through the cervical segment of the left vertebral artery.To pass the sharp turn as it's best from the V2 to V3 segment, advance the microcatheter alone while the microwire is back proximal to its tip. After passing the sharp turn from V2 to V3, the microcatheter often passes easily to the proximal basal or artery. At this point, advance the microwire and select the desired posterior cerebral and superior cerebellar arteries, as microcatheter injections are not advisable due to the fragile nature of the intracranial arteries.Further advance the microcatheter over the microwire into the target artery and choose a proximal position, as it is typically safest in the posterior to communicate due to its angulation at its origin. A deeper position is feasible in the superior cerebellar artery. Repeat the angiogram by injecting the left vertebral artery catheter with high magnification over the head to confirm occlusion of the target artery.For optimal imaging, inject full-strength contrast, typically no more than one CC, which is adequate for pacification of all intracranial arteries. Gently remove the microwire from the microcatheter under fluoroscopic visualization to confirm a stable position. Place a stopcock on the hub of the microcatheter and close the stopcock to prevent blood loss from retrograde blood flow.Remove the left vertebral catheter to make the left femoral access sheath available. During the ensuing occlusion period, acquire intermittent fluoroscopic images to confirm the stable position of the occlusive microcatheter. Remove the occlusive microcatheter after three hours.And then continue arterial blood pressure measurement and modulation for the additional desired period. Inflate and deflate the balloon catheter in the aorta to monitor the blood pressure variability. In acute procedures with immediate brain harvesting, remove the calvarium in a piecemeal fashion with rongeurs, starting at the occipital ridge and working anteriorly until the brain can be harvested intact.Place the brain in formalin or flash freeze, depending on the type of tissue analysis desired. In all the animals, the brain was successfully harvested and histopathological analysis was carried out, demonstrating infarction in the right cerebellum. The blood pressure was traced from a Fogarty balloon catheter placed in the infrarenal aorta, and the data from approximately one hour of monitoring demonstrates real-time arterial pressure changes with changes in balloon inflation.Short-term tracing of the blood pressure demonstrates the pressure changes throughout the cardiac cycle. Additionally, small rapid changes were noted from respiratory variability, which is physiologically normal. An immediate near-doubling of measured blood pressure was noted following the inflation of the balloon.While attempting this procedure, meticulous blunt dissection and dripping lidocaine to prevent vasospasm during access of the angiocatheter is critical. Also, angiography of the left vertebral artery provides a roadmap for efficient endovascular access to the targeted intracranial artery. Hemodynamics can be modulated in pharmacological ways or by inflating a balloon catheter in the aorta.The harvested brain specimens can be analyzed with a variety of techniques, such as histopathology or spatial transcriptomics.

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