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).

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.