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

Summary

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).

Abstract

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.

Introduction

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 bleeding^2,3. 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 mice^{5,6,7,8,9,10,11,12,13}. 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 practice^{14,15,16,17,18}, 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.

Protocol

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 either SAH induction or sham surgery, which has been described in detail elsewhere^10,12,13.

1. Preparation of materials

1. Switch on the ultrasound machine and enter the animal ID.
2. Warm the heating plate of the ultrasound system to 37 °C. Ensure that the rectal temperature probe is ready for use.
3. Use a water bath to heat the ultrasound gel to 37 °C. Prepare hair removal cream, contact cream for the electrodes, and eye ointment.

2. Anesthesia

1. Induce anesthesia by putting the mouse in a chamber flushed with 4% isoflurane and 40% O₂ for 1 min. Protect the eyes with eye ointment. Continue only after a sufficiently deep anesthesia has been reached (absence of reactions to pain stimuli).
2. Maintain anesthesia with 1.5% isoflurane and 40% O₂ using an anesthesia mask throughout the entire procedure.

3. Determination of blood flow velocities of the intracranial internal carotid arteries with transcranial high-frequency Duplex sonography

1. Place the mouse in the prone position on the heating plate of the ultrasound system to maintain a body temperature of 37 °C.
2. Coat the four extremities of the animal with conductive paste and fix them with tape on the ECG electrodes embedded in the board. Check if the physiological parameters (ECG, respiration signal) are displayed correctly on the screen of the imaging system (e.g., Vevo3100). If necessary, adjust the level of anesthesia to obtain target heart rate of 400-500 beats per minute (bpm).
3. Place lube on a rectal temperature probe and carefully insert it to monitor the body temperature. Use an additional warming lamp if necessary.
4. Before the first exam, remove the fur at the occiput chemically using hair removal cream. Use a cotton swab to spread and rub the cream for 2 min until the hairs start to fall out.
   1. After an additional 2 min, remove the cream and hairs with a spatula and disinfect the skin with an alcoholic skin antiseptic. Coat it with ultrasound gel warmed to 37 °C.
5. Use a 38 MHz linear array transducer and a frame rate above 200 frames/s to acquire ultrasound images and fixate the probe in the mechanical arm. Place the transducer on the occiput tilted back by 30°.
6. Use Brightness-(B)-mode and Color-wave-(CW) Doppler-mode to visualize the right intracranial internal carotid artery and move the transducer with the control unit back and forward, until the maximal flow of the arteries is found.
7. To collect anatomical information, use the traditional B-Mode and CW-Doppler-mode and start acquisition by clicking on the Acquire button.
   1. To record information on the flow characteristics of the intracranial vessels click on the Pulse-Wave (PW) Doppler button, place the sample volume in the center of the vessel, and acquire a cine loop longer than 3 s.
8. Proceed identically with the left side.
9. Proceed with the extracranial carotid arteries.

4. Determination of blood flow velocities of the extracranial internal carotid arteries with high frequency Duplex sonography

1. Place the mouse in the supine position on the heating plate of the ultrasound system to maintain a body temperature of 37 °C.
2. Coat the four extremities of the animal with conductive paste and fix them with tape on the ECG electrodes embedded in the board. Check again for the correct display of the physiological parameters on the screen.
3. Before the first exam, remove the hair at the front neck chemically by using hair removal cream as described above. Coat the front neck with ultrasound gel warmed to 37 °C.
4. Use a 38 MHz linear array transducer and a frame rate above 200 frames/s to acquire ultrasound images. Place the transducer parallel to the animal and adjust the position in order to obtain longitudinal images of the right carotid artery.
5. Use Brightness-(B)-mode and Color-wave-(CW) Doppler-mode to visualize the right carotid artery. The image should contain the right common carotid artery (RCC), the right internal carotid artery (RICA) and the right external carotid artery (RECA).
6. To collect anatomical information, use the traditional B-Mode and CW-Doppler-mode and start acquisition by clicking on the Acquire button.
   1. To record information on the flow characteristics of the extracranial carotid artery click on the Pulse-Wave (PW) 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 3 s.
7. Proceed identically with the left side.
8. Terminate anesthesia and remove the animal from the warming plate. Return the animal to a cage placed in an incubator heated to 37 °C for 1 hour to prevent hypothermia and check for full recovery.

5. Processing of ultrasonography data

1. Use an 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.
2. Open the exported ultrasound study. Select one animal and open the PW-Doppler cine loop of the intracranial carotid artery. In this protocol typically 7 to 8 heartbeats and corresponding flow-velocity curves are recorded.
3. 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 (PSV). 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.
4. Now choose RICA EDV to measure the enddiastolic velocity (EDV). Click left on 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.
5. Choose RICA VTI to measure the velocity time integral (VTI). Click left at the beginning of a velocity curve and follow the curve with the mouse until the end of the diastolic plateau. Then click right again to determine the measurement.
6. 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.
7. Use the same approach to measure PSV, EDV and VTI of the right extracranial internal carotid arteries and export the data accordingly.
8. Proceed identically with the left side.

Results

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 °C¹⁹.

Discussion

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 known^3,15. Several clinical studies have shown that elevated blood flow velocities of the large intracrania...

Materials

List of materials used in this article

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