Name: analysis of cerebral vasospasm in a murine model of subarachnoid hemorrhage with high frequency transcranial duplex ultrasound
Uploaded: 2021-06-03T14:00:00
Description: Watch this Scientific Journal Video about Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound at JoVE.com

The authors would like to thank Stefan Kindel for preparation of the illustrations in the video. PW, MM and SHK were supported by the German Federal Ministry for Education and Research (BMBF 01EO1503). The work was supported by a Large Instrumentation Grant of the German Research Foundation (DFG INST 371/47-1 FUGG). MM was supported by a grant from the Else Kr&#246;ner-Fresenius-Stiftung (2020_EKEA.144).

The animal experiments were approved by the responsible animal care committee (Landesuntersuchungsamt Rheinland-Pfalz) and conducted in accordance with the German Animal Welfare Act (TierSchG). All applicable international, national, and institutional guidelines for the care and use of animals were followed. In this study, we performed measurements of blood flow velocities of intracranial and extracranial arteries in female C57BL/6N mice aged 11-12 weeks with a body weight between 19-21 g. The mice were subjected to eith...

<ol>
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F., Nornes, H., Bakke, S. J., Sorteberg, W., Nakstad, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+vasospasm+diagnosis+by+means+of+angiography+and+blood+velocity+measurements.">Cerebral vasospasm diagnosis by means of angiography and blood velocity measurements.</a> Acta Neurochirurgica. 100 (1-2), 12-24 (1989).</li><li>Cassia, G. S., Faingold, R., Bernard, C., Sant'Anna, G. 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To the best of our knowledge, this study is the first to present a protocol for monitoring of cerebral vasospasm in a murine model of SAH with high frequency transcranial color-coded Duplex ultrasound. We show that this method can measure an increase in intracranial blood flow velocities after SAH induction in mice. In human medicine this phenomenon is well known3,15. Several clinical studies have shown that elevated blood flow velocities of the large intracrania...

Spontaneous subarachnoid hemorrhage (SAH) is a form of hemorrhagic stroke mostly caused by the rupture of an intracranial aneurysm1. The neurological outcome is mainly influenced by two factors: early brain injury (EBI), which is caused by the effects of the bleeding and the associated transient global cerebral ischemia, and delayed cerebral ischemia (DCI), which occurs during the weeks following the bleeding2,3. DCI was reported to affect up to 30% of SAH patients2. The pathophysiology of DCI involves angiographic cerebral vasospasm, a disturbed microcirculation caused by microvasospasms and microthrombosis, cortical spreading depressions, and effects triggered by inflammation4. Unfortunately, the exact pathophysiology remains unclear and there is no treatment available that effectively prevents DCI3. Therefore, DCI is investigated in many clinical and experimental studies.
Nowadays, most experimental studies on SAH use small animal models, especially in mice5,6,7,8,9,10,11,12,13. In such studies, cerebral vasospasm is frequently investigated as an endpoint. It is common to determine the degree of vasospasm ex vivo. This is because noninvasive methods for in vivo examination of cerebral vasospasm requiring short anesthesia time and imposing only little distress on the animals are lacking. However, examination of cerebral vasospasm in vivo would be advantageous. This is because it would allow longitudinal in vivo studies on vasospasm in mice (i.e., imaging of cerebral vasospasm at different time points during the days after induction of SAH). This would enhance the comparability of data acquired at different time points. Furthermore, using a longitudinal study design is a strategy to reduce animal numbers.
Here we demonstrate the use of high frequency transcranial ultrasound to determine the blood flow in cerebral arteries in mice. We show that, similar to transcranial Doppler sonography (TCD) or transcranial color-coded Duplex sonography (TCCD) in clinical practice14,15,16,17,18, this method can be used to monitor cerebral vasospasm by measuring the blood flow velocities of the intracranial arteries after SAH induction in the murine model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Balea hair removal creme</td>
			<td>Balea; Germany</td>
			<td>ASIN B0759XM39V</td>
			<td>hair removal creme</td>
		</tr>
		<tr>
			<td>C57BL/6N mice</td>
			<td>Janvier; Saint-Berthevin Cedex, France</td>
			<td>n.a.</td>
			<td>mice</td>
		</tr>
		<tr>
			<td>Corneregel</td>
			<td>Bausch&#38;Lomb; Rochester, NY, USA</td>
			<td>REF 81552983</td>
			<td>eye ointment, lube</td>
		</tr>
		<tr>
			<td>cotton swabs</td>
			<td>Hecht Assistent; Sondenheim vor der R&#246;hn, Germany</td>
			<td>REF 44302010</td>
			<td>cotton swabs</td>
		</tr>
		<tr>
			<td>Ecco-XS razor</td>
			<td>Tondeo; Soligen, Germany</td>
			<td>DE 28693396</td>
			<td>razor</td>
		</tr>
		<tr>
			<td>Electrode cream</td>
			<td>GE; Boston, MA, USA</td>
			<td>REF 21708318</td>
			<td>conductive paste</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Medax; Kiel, Germany</td>
			<td>2005-205-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Abvie; Wiesbaden, Germany</td>
			<td>n.a.</td>
			<td>volatile anesthetic</td>
		</tr>
		<tr>
			<td>Leukofix</td>
			<td>BSN medical; Hamburg, Germany</td>
			<td>REF 02137-00</td>
			<td>tape</td>
		</tr>
		<tr>
			<td>Mechanical arm + micromanipulator</td>
			<td>VisualSonics; FujiFilm, Toronto, CA</td>
			<td>P/N 11277</td>
			<td></td>
		</tr>
		<tr>
			<td>Microbac tissues</td>
			<td>Paul Hartmann AG; Hamburg, Germany</td>
			<td>REF 981387</td>
			<td>antimicrobial tissues</td>
		</tr>
		<tr>
			<td>MZ400, 38 MHz linear array transducer</td>
			<td>VisualSonics; FujiFilm, Toronto, CA</td>
			<td>REF 51068-30</td>
			<td>ultrasound transducer</td>
		</tr>
		<tr>
			<td>Sonosid</td>
			<td>ASID Bonz GmbH; Herrenberg, Germany</td>
			<td>REF 782010</td>
			<td>ultrasonography gel</td>
		</tr>
		<tr>
			<td>Ultrasound platform with heating plate and ECG-recording</td>
			<td>VisualSonics; FujiFilm, Toronto, CA</td>
			<td>P/N 11179</td>
			<td></td>
		</tr>
		<tr>
			<td>UniVet-Porta</td>
			<td>Groppler; Oberperasberg, Germany</td>
			<td>S/N BKGM0437</td>
			<td>isoflurane vaporizer</td>
		</tr>
		<tr>
			<td>Vevo3100</td>
			<td>VisualSonics; FujiFilm, Toronto, CA</td>
			<td>REF 51073-45</td>
			<td>ultrasonography device</td>
		</tr>
		<tr>
			<td>VevoLab software</td>
			<td>VisualSonics; FujiFilm, Toronto, CA</td>
			<td>n.a.</td>
			<td>evaluation software</td>
		</tr>
	</tbody>
</table>

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.

In 6 mice, in 3 of which SAH was induced using the endovascular filament perforation model while 3 obtained sham surgery, the blood flow velocities of the intracranial internal carotid artery (ICA) and of the extracranial ICA were determined one day before surgery, and 1, 3, and 7 days after surgery. The measurements were performed as part of the echocardiography examinations of another study under anesthesia with isoflurane while maintaining the body temperature at 37 &#176;C19.
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Watch this Scientific Journal Video about Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound at JoVE.com

Analysis of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage with High Frequency Transcranial Duplex Ultrasound

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 ...

The overall goal of this procedure is to monitor cerebral vasospasm after subarachnoid hemorrhage in mice in vivo. This is accomplished by the application of high-frequency color-coded duplex sonography under anesthesia with isoflurane. The imaging data is processed to determine the blood flow velocities in the intra and extracranial internal carotid arteries.An accelerated intracranial blood flow velocity indicates cerebral vasospasm. In this study, we performed measurements of blood flow velocities of intracranial and extracranial arteries in female, C57BL/6N mice aged 11 to 12 weeks. The mice were subjected to either SAH induction or sham surgery which has been described in detail elsewhere.To prepare the ultrasonography exam switch on the ultrasound machine and enter the animal ID.Warm the heating plate of the ultrasound system to 37 degrees Celsius. Ensure that the rectal temperature probe is ready for use. Use a warm water bath to heat the ultrasound gel to 37 degrees Celsius.Prepare the hair removal cream, contact cream for the electrodes and eye ointment. Make sure that the transducer is properly mounted on the mechanical arm and ensure that the chamber of for anesthesia induction is flushed with 4%isoflurane and 40%oxygen. Induce anesthesia by putting the mouse in the chamber for one minute.Protect the eyes with ointment. Continue only after a sufficiently deep anesthesia has been reached. Maintain anesthesia with 1.5%isoflurane and 40%oxygen using an anesthesia mask throughout the entire procedure.In the first step, the blood flow velocities of the intracranial internal carotid arteries are determined with transcranial high-frequency duplex sonography. Place the mouse in the prone position on the heating plate of the ultrasound system to maintain a body temperature 37 degrees Celsius. Apply eye ointment on both sides.Before the first exam shorten the fur at the occiput with an electric shaver. Then remove remaining hair chemically using hair removal cream. Use a cotton swab to spread and rub the cream for two minutes until the hair start to fall out.After an additional two minutes remove the cream and hair with a spatula and disinfect the skin with an alcoholic skin antiseptic. Coat the four extremities of the animal with conductive paste and fix them with tape on the ECG electrodes embedded in the board. Place lube on a rectal temperature probe and carefully insert it to monitor the body temperature, using additional warming lamp if necessary.Check, if the physiological parameters, ECG and respiration signal are displayed correctly on the screen of the ultrasound system. If necessary, the level of anesthesia can be adjusted to obtain the target heart rate of 4 to 500 beats per minute. Coat it with ultrasound gel.Use a linear array transducer and a frame rate above 200 frames per second to acquire ultrasound images and mount it on the mechanical arm. Place the transducer on the occiput tilted back by 30 degrees. Use B-mode and CW-Doppler mode to visualize the right intracranial internal carotid artery and move the transducer with the control unit back and forth until we find the maximum flow of the arteries.To collect anatomical information use the traditional B-mode and CW-Doppler mode and start acquisition by clicking on the acquire button. To record information on the flow characteristics of the intracranial vessels click on the pulse wave doppler button, place a sample volume in the center of the vessel and acquire a cine loop longer than three seconds. Proceed identically with the left side.In the next step the blood flow velocities of the extracranial internal carotid arteries are determined with high-frequency duplex sonography. Place the mouse in the supine position on the heating plate of the ultrasound system to maintain a body temperature of 37 degrees Celsius. Before the first exam remove the hair of the front of the neck with an electric shaver.Then remove remaining hair chemically using hair removal cream as described previously. Coat the four extremities of the animal with conductive paste and fix them with tape on the ECG electrodes embedded in the board. Place lube on a rectal temperature probe and carefully insert it to monitor the body temperature.Using additional warming lamp if necessary. Check again for the correct display of the physiological parameters on the screen. Coat the front of the neck with ultrasound gel warmed to 37 degrees Celsius.Place the transducer parallel to the animal and adjust the position in order to obtain longitudinal images of the right carotid artery. Use B-mode CW-Doppler mode to visualize the right carotid artery. The image should contain the right common carotid artery, the right internal carotid artery and the right external carotid artery.To collect anatomical information use the traditional B-mode and CW-Doppler mode and start acquisition by clicking on the acquire button. To record information on the flow characteristics of the extracranial carotid artery click on the pulse wave doppler button, place the sample volume in the center of the middle of the common carotid artery the internal carotid artery and the external carotid artery and acquire a cine loop longer than three seconds. Proceed identically with the left side.Terminate anesthesia and remove the animal from the warming plate and put it in a cage, placed in an incubator heated to 36 degrees Celsius for one hour to prevent hypothermia and check for full recovery. The next step is processing the ultrasonography data. Using external workstation for post-processing of the high frequency ultrasound data.Export the B-mode, CW-Doppler mode and PW-Doppler mode images and cine loops to the Vevo LAB software. Open the exported ultrasound study. Select one animal and open the PW-Doppler cine loop of the intracranial carotid artery.In this protocol typically seven to eight heartbeats in corresponding flow velocity curves are recorded. Pause the cine loop and click on the measurement button. Choose the vascular package and click on RICA PSV to measure the peak systolic pressure of the right intracranial carotid artery.Now click left on the peak of a velocity curve and pull the straight line to the zero line. Determine the measurement by a click with the right mouse button. Now choose RICA EDV to measure the end diastolic velocity.Click left on the minimal rash of the velocity curve at the end of the diastole. Pull the line straight to the zero line and determine measurement by a click with the right mouse button. Choose RICA VTI to measure the velocity time integral.Click left at the beginning of a velocity curve and follow the curve with a mouse until the end of the diastolic plateau. And then click right again to determine the measurement. Export the data of the intracerebral internal carotid arteries by using the report button.Press export and save the data as a VSI report file. Use the same approach to measurement PSV, EDV and VTI of the right extracranial internal carotid arteries and export the data accordingly. Proceed identically with the left side and use LICA PSV, EDV and VTI.In five mice and three of which SAH was induced while two obtain sham surgery the blood flow velocities of the intracranial internal artery and of the extracranial internal carotid artery was determined one day before surgery and one, three and seven days after surgery. Before surgery, extra and intracranial blood flow velocities as well as the ratio of intra and extracranial blood flow were similar between the SAH and sham animals. On the first day after SAH induction there were no major changes in intra or extracranial blood flow velocities or the ratios between intra and extracranial blood flow.On days three and seven the intracranial blood flow velocities of the internal carotid artery increased markedly and two of the SAH animals indicating cerebral vasospasm after SAH. As the extracranial blood flow velocities remained nearly unchanged, the ratios of intra and extracranial blood flow velocities also increased significantly on day seven in the SAH animals indicating cerebral vasospasm. In conclusion high-frequency color-coded transcranial duplex sonography can be used to perform in vivo measurements of the velocities of intracranial blood flow in mice.Similar to the situation in human patients intracranial blood flow velocities accelerate in mice after SAH indicating vasospasm. The ultra sonographic method shown here is fast and of little invasiveness. It allows longitudinal studies of vasospasm in murine SAH models.

Research

JoVE Journal

Neuroscience

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound (fTCD)

Assessment of Cerebral Lateralization in Children using Functional Transcranial Doppler Ultrasound fTCD

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Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. ...

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Author Spotlight: Exploring the Potential of Massage Therapy in Cerebral Palsy Using Animal Models 

Cerebral palsy (CP) is a disease with a high disability rate and morbidity. The clinical symptoms of cerebral palsy are motor dysfunction and abnormal ...