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 either SAH induction or sham surgery, which has been described in detail elsewhere10,12,13.
1. Preparation of materials
<ol>
	<li>Switch on the ultrasound machine and enter the animal ID.</li>
	<li>Warm the heating plate of the ultrasound system to 37 &#176;C. Ensure that the rectal temperature probe is ready for use.</li>
	<li>Use a water bath to heat the ultrasound gel to 37 &#176;C. Prepare hair removal cream, contact cream for the electrodes, and eye ointment.</li>
</ol>
2. Anesthesia
<ol>
	<li>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).</li>
	<li>Maintain anesthesia with 1.5% isoflurane and 40% O2 using an anesthesia mask throughout the entire procedure.</li>
</ol>
3. Determination of blood flow velocities of the intracranial internal carotid arteries with transcranial high-frequency Duplex sonography
<ol>
	<li>Place the mouse in the prone position on the heating plate of the ultrasound system to maintain a body temperature of 37 &#176;C.</li>
	<li>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).</li>
	<li>Place lube on a rectal temperature probe and carefully insert it to monitor the body temperature. Use an additional warming lamp if necessary.</li>
	<li>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.
	<ol>
		<li>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 &#176;C.</li>
	</ol>
	</li>
	<li>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&#176;.</li>
	<li>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.</li>
	<li>To collect anatomical information, use the traditional B-Mode and CW-Doppler-mode and start acquisition by clicking on the Acquire button.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Proceed identically with the left side.</li>
	<li>Proceed with the extracranial carotid arteries.</li>
</ol>
4. Determination of blood flow velocities of the extracranial internal carotid arteries with high frequency Duplex sonography
<ol>
	<li>Place the mouse in the supine position on the heating plate of the ultrasound system to maintain a body temperature of 37 &#176;C.</li>
	<li>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.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>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).</li>
	<li>To collect anatomical information, use the traditional B-Mode and CW-Doppler-mode and start acquisition by clicking on the Acquire button.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Proceed identically with the left side.</li>
	<li>Terminate anesthesia and remove the animal from the warming plate. Return the animal to a cage placed in an incubator heated to 37 &#176;C for 1 hour to prevent hypothermia and check for full recovery.</li>
</ol>
5. Processing of ultrasonography data
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Use the same approach to measure PSV, EDV and VTI of the right extracranial internal carotid arteries and export the data accordingly.</li>
	<li>Proceed identically with the left side.</li>
</ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+delayed+cerebral+ischemia+after+subarachnoid+hemorrhage.">Management of delayed cerebral ischemia after subarachnoid hemorrhage.</a> Critical Care. 20 (1), 277 (2016).</li><li>van Lieshout, J. H., 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=An+introduction+to+the+pathophysiology+of+aneurysmal+subarachnoid+hemorrhage.">An introduction to the pathophysiology of aneurysmal subarachnoid hemorrhage.</a> Neurosurgical Review. , (2017).</li><li>Altay, T., 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+method+for+subarachnoid+hemorrhage+to+induce+vasospasm+in+mice.">A novel method for subarachnoid hemorrhage to induce vasospasm in mice.</a> J Neurosci Methods. 183 (2), 136-140 (2009).</li><li>Momin, E. 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Detection and monitoring of vasospasm and delayed cerebral ischemia: a review and assessment of the literature.</a> NeuroCritical Care. 15 (2), 312-317 (2011).</li><li>Greke, C., 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=Image-guided+transcranial+Doppler+sonography+for+monitoring+of+defined+segments+of+intracranial+arteries.">Image-guided transcranial Doppler sonography for monitoring of defined segments of intracranial arteries.</a> Journal of Neurosurgical Anesthesiology. 25 (1), 55-61 (2013).</li><li>Neulen, A., Prokesch, E., Stein, M., Konig, J., Giese, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided+transcranial+Doppler+sonography+for+monitoring+of+vasospasm+after+subarachnoid+hemorrhage.">Image-guided transcranial Doppler sonography for monitoring of vasospasm after subarachnoid hemorrhage.</a> Clinical Neurology and Neurosurgery. 145, 14-18 (2016).</li><li>Neulen, A., 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=Image-Guided+Transcranial+Doppler+Ultrasound+for+Monitoring+Posthemorrhagic+Vasospasms+of+Infratentorial+Arteries:+A+Feasibility+Study.">Image-Guided Transcranial Doppler Ultrasound for Monitoring Posthemorrhagic Vasospasms of Infratentorial Arteries: A Feasibility Study.</a> World Neurosurgery. 134, 284-291 (2020).</li><li>Neulen, A., 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=Correlation+of+cardiac+function+and+cerebral+perfusion+in+a+murine+model+of+subarachnoid+hemorrhage.">Correlation of cardiac function and cerebral perfusion in a murine model of subarachnoid hemorrhage.</a> Scientific Reports. 11 (1), 3317 (2021).</li><li>Neulen, A., 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+segmentation-based+volumetric+approach+to+localize+and+quantify+cerebral+vasospasm+based+on+tomographic+imaging+data.">A segmentation-based volumetric approach to localize and quantify cerebral vasospasm based on tomographic imaging data.</a> PLoS One. 12 (2), 0172010 (2017).</li><li>Marbacher, S., 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=Systematic+Review+of+In+Vivo+Animal+Models+of+Subarachnoid+Hemorrhage:+Species,+Standard+Parameters,+and+Outcomes.">Systematic Review of In Vivo Animal Models of Subarachnoid Hemorrhage: Species, Standard Parameters, and Outcomes.</a> Translational Stroke Research. , (2018).</li><li>Figueiredo, G., 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=Comparison+of+digital+subtraction+angiography,+micro-computed+tomography+angiography+and+magnetic+resonance+angiography+in+the+assessment+of+the+cerebrovascular+system+in+live+mice.">Comparison of digital subtraction angiography, micro-computed tomography angiography and magnetic resonance angiography in the assessment of the cerebrovascular system in live mice.</a> Clinical Neuroradiology. 22 (1), 21-28 (2012).</li><li>Lindegaard, K. 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The authors declare no competing interests.

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.
<p class="jov...

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 ...

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Research

JoVE Journal

Neuroscience

3.5K Views.  University Medical Center of the Johannes Gutenberg-University of Mainz. 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). 

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

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

There are many unanswered questions about cerebral lateralization. In particular, it remains unclear which aspects of language and nonverbal ability are lateralized, whether there are any disadvantages associated with atypical patterns of cerebral lateralization, and whether cerebral lateralization develops with age. In the past, researchers interested in these questions tended to use handedness as a proxy measure for cerebral lateralization, but this is unsatisfactory because handedness is only a weak and indirect indicator of laterality of cognitive functions1. Other methods, such as fMRI, are expensive for large-scale studies, and not always feasible with children2.
Here we will describe the use of functional transcranial Doppler ultrasound (fTCD) as a cost-effective, non-invasive and reliable method for assessing cerebral lateralization. The procedure involves measuring blood flow in the middle cerebral artery via an ultrasound probe placed just in front of the ear. Our work builds on work by Rune Aaslid, who co-introduced TCD in 1982, and Stefan Knecht, Michael Deppe and their colleagues at the University of M&uuml;nster, who pioneered the use of simultaneous measurements of left- and right middle cerebral artery blood flow, and devised a method of correcting for heart beat activity. This made it possible to see a clear increase in left-sided blood flow during language generation, with lateralization agreeing well with that obtained using other methods3.
The middle cerebral artery has a very wide vascular territory (see Figure 1) and the method does not provide useful information about localization within a hemisphere. Our experience suggests it is particularly sensitive to tasks that involve explicit or implicit speech production. The 'gold standard' task is a word generation task (e.g. think of as many words as you can that begin with the letter 'B') 4, but this is not suitable for young children and others with limited literacy skills. Compared with other brain imaging methods, fTCD is relatively unaffected by movement artefacts from speaking, and so we are able to get a reliable result from tasks that involve describing pictures aloud5,6. Accordingly, we have developed a child-friendly task that involves looking at video-clips that tell a story, and then describing what was seen.

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

Functional transcranial Doppler sonography (fTCD) is a simple and non-invasive ultrasound technique which can be used to assess the lateralization of cognitive functions, especially language, and is suitable for use with children.

There are many unanswered questions about cerebral lateralization.  In particular, it remains unclear which aspects of language and nonverbal ability are ...

A Rat Model of Mild Intrauterine Hypoperfusion with Microcoil Stenosis

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies of this phenomenon have been performed in either models with severe intrauterine ischemia or models with gradient degree of intrauterine hypoperfusion. No study has been performed in a model on uniform mild intrauterine hypoperfusion (MIUH). Two models have been used for studies of MIUH: a model based on suture ligation of either side of the arterial arcade formed with the uterine and ovarian arteries, and a transient model based on clipping the bilateral ovarian arteries and aorta having patency. Those two rodent models of MIUH have some limitations, e.g., not all fetuses are subjected to MIUH, depending on their position in the uterine horn. In our MIUH model, all fetuses are subjected to a comparable level of intrauterine hypoperfusion. MIUH was achieved by mild stenosis of all four arteries feeding the uterus, i.e., the bilateral uterine and ovarian arteries.
Arterial stenosis was induced by metal microcoils wrapped around the feeding arteries. Producing arterial stenosis with microcoils allowed us to control, optimize, and reproduce decreased blood flow with very little inter-animal variability and a low mortality rate, thus enabling accurate evaluation. When microcoils with an inner diameter of 0.24 mm were used, the blood flow in both the placenta and fetus was mildly decreased (approximately 30% from the pre-stenosis level in the placenta). The offspring of our MIUH model clearly demonstrates long-lasting alterations in neurological, neuroanatomical and behavioral test results.

Mild intrauterine hypoperfusion was produced by artery stenosis with metal microcoils wrapped around the uterine and ovarian arteries in rats at embryonic day 17. This procedure produced prenatal hypoperfusion and intrauterine growth restriction.

Intrauterine hypoperfusion/ischemia is one of the major causes of intrauterine/fetal growth restriction, preterm birth, and low birth weight. Most studies ...

A Volumetric Method for Quantification of Cerebral Vasospasm in a Murine Model of Subarachnoid Hemorrhage

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor determining patient outcome and is therefore frequently taken as a study endpoint. However, in small animal studies on SAH, quantification of cerebral vasospasm is a major challenge. Here, an ex vivo method is presented that allows quantification of volumes of entire vessel segments, which can be used as an objective measure to quantify cerebral vasospasm. In a first step, endovascular casting of the cerebral vasculature is performed using a radiopaque casting agent. Then, cross-sectional imaging data are acquired by micro computed tomography. The final step involves 3-dimensional reconstruction of the virtual vascular tree, followed by an algorithm to calculate center lines and volumes of the selected vessel segments. The method resulted in a highly accurate virtual reconstruction of the cerebrovascular tree shown by a diameter-based comparison of anatomical samples with their virtual reconstructions. Compared with vessel diameters alone, the vessel volumes highlight the differences between vasospastic and non-vasospastic vessels shown in a series of SAH and sham-operated mice.

The goal of this article is to present a method that allows a 3-dimensional reconstruction of the cerebrovascular tree in mice after micro computed tomography and determination of volumes of entire vessel segments that can be used to quantify cerebral vasospasm in murine models of subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a subtype of hemorrhagic stroke. Cerebral vasospasm that occurs in the aftermath of the bleeding is an important factor ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...

Pre-Chiasmatic, Single Injection of Autologous Blood to Induce Experimental Subarachnoid Hemorrhage in a Rat Model

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely afflicting a fairly young population. Several animal models of SAH have been developed to investigate the pathophysiological mechanisms behind SAH and to test pharmacological interventions. The pre-chiasmatic, single injection model in the rat presented in this article is an experimental model of SAH with a predetermined blood volume. Briefly, the animal is anesthetized, intubated, and kept under mechanical ventilation. Temperature is regulated with a heating pad. A catheter is placed in the tail artery, enabling continuous blood pressure measurement as well as blood sampling. The atlantooccipital membrane is incised and a catheter for pressure recording is placed in the cisterna magna to enable intracerebral pressure measurement. This catheter can also be used for intrathecal therapeutic interventions. The rat is placed in a stereotaxic frame, a burr hole is drilled anteriorly to the bregma, and a catheter is inserted through the burr hole and placed just anterior to the optic chiasm. Autologous blood (0.3 mL) is withdrawn from the tail catheter and manually injected. This results in a rise of intracerebral pressure and a decrease of cerebral blood flow. The animal is kept sedated for 30 min and given subcutaneous saline and analgesics. The animal is extubated and returned to its cage. The pre-chiasmatic model has a high reproducibility rate and limited variation between animals due to the pre-determined blood volume. It mimics SAH in humans making it a relevant model for SAH research.

Subarachnoid hemorrhage continues to carry a high burden of mortality and morbidity in man. To facilitate further research into the condition and its pathophysiology, a pre-chiasmatic, single injection model is presented.

Despite advances in treatment over the last decades, subarachnoid hemorrhage (SAH) continues to carry a high burden of morbidity and mortality, largely ...

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging (MRI)

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high mortality rate as well as an uncontrollable volume of SAH and other intracranial complications such as stroke or intracranial hemorrhage. In this protocol, a standardized SAH mouse model is presented, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies. Briefly, C57BL/6J mice are anesthetized with an intraperitoneal ketamine/xylazine (70 mg/16 mg/kg body weight) injection and placed in a supine position. After midline neck incision, the common carotid artery (CCA) and carotid bifurcation are exposed, and a 5-0 non-absorbable monofilament polypropylene suture is inserted in a retrograde fashion into the external carotid artery (ECA) and advanced into the common carotid artery. Then, the filament is invaginated into the internal carotid artery (ICA) and pushed forward to perforate the anterior cerebral artery (ACA). After recovery from surgery, mice undergo a 7.0 T MRI 24 h later. The volume of bleeding can be quantified and graded via postoperative MRI, enabling a robust experimental SAH group with the option to perform further subgroup analyses based on blood quantity.

Endovascular Perforation Model for Subarachnoid Hemorrhage Combined with Magnetic Resonance Imaging MRI

Here we present a standardized SAH mouse model, induced by endovascular filament perforation, combined with magnetic resonance imaging (MRI) 24 h after operation to ensure the correct bleeding site and exclude other relevant intracranial pathologies.

The endovascular filament perforation model to mimic subarachnoid hemorrhage (SAH) is a commonly used model - however, the technique can cause a high ...

Modeling Neonatal Intraventricular Hemorrhage Through Intraventricular Injection of Hemoglobin

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and lifelong neurological deficits. While PHH can be treated by temporary and permanent cerebrospinal fluid (CSF) diversion procedures (ventricular reservoir and ventriculoperitoneal shunt, respectively), there are no pharmacological strategies to prevent or treat IVH-induced brain injury and hydrocephalus. Animal models are needed to better understand the pathophysiology of IVH and test pharmacological treatments. While there are existing models of neonatal IVH, those that reliably result in hydrocephalus are often limited by the necessity for large-volume injections, which may complicate modeling of the pathology or introduce variability in the clinical phenotype observed.
Recent clinical studies have implicated hemoglobin and ferritin in causing ventricular enlargement after IVH. Here, we develop a straightforward animal model that mimics the clinical phenotype of PHH utilizing small-volume intraventricular injections of the blood breakdown product hemoglobin. In addition to reliably inducing ventricular enlargement and hydrocephalus, this model results in white matter injury, inflammation, and immune cell infiltration in periventricular and white matter regions. This paper describes this clinically relevant, simple method for modeling IVH-PHH in neonatal rats using intraventricular injection and presents methods for quantifying ventricle size post injection.

We present a model of neonatal intraventricular hemorrhage using rat pups that mimics the pathology seen in humans.

Neonatal intraventricular hemorrhage (IVH) is a common consequence of premature birth and leads to brain injury, posthemorrhagic hydrocephalus (PHH), and ...

Establishing a Transcranial Middle Cerebral Artery Occlusion Mice Model

A Modified Transcranial Middle Cerebral Artery Occlusion Model to Study Stroke Outcomes in Aged Mice

In experimental stroke research, middle cerebral artery occlusion (MCAO) with an intraluminal filament is widely used to model ischemic stroke in mice. The filament MCAO model typically exhibits a massive cerebral infarction in C57Bl/6 mice that sometimes includes brain tissue in the territory supplied by the posterior cerebral artery, which is largely due to a high incidence of posterior communicating artery atresia. This phenomenon is considered a major contributor to the high mortality rate observed in C57Bl/6 mice during long-term stroke recovery after filament MCAO. Thus, many chronic stroke studies exploit distal MCAO models. However, these models usually produce infarction only in the cortex area, and consequently, the assessment of post-stroke neurologic deficits could be a challenge. This study has established a modified transcranial MCAO model in which the MCA at the trunk is partially occluded either permanently or transiently via a small cranial window. Since the occlusion location is relatively proximal to the origin of the MCA, this model generates brain damage in both the cortex and striatum. Extensive characterization of this model has demonstrated an excellent long-term survival rate, even in aged mice, as well as readily detectable neurologic deficits. Therefore, the MCAO mouse model described here represents a valuable tool for experimental stroke research.

Author Spotlight: Innovative Techniques and Future Directions in Stroke Research 

This protocol demonstrates a unique mouse stroke model with a medium-sized infarct and an excellent survival rate. This model allows preclinical stroke researchers to extend the ischemia duration, use aged mice, and assess long-term functional outcomes.

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

Chinese Massage Techniques: Enhancing Motor Function in Cerebral Palsy Rats

Abdominal Massage to Improve Motor Dysfunction in Rats with Cerebral Palsy

Cerebral palsy (CP) is a disease with a high disability rate and morbidity. The clinical symptoms of cerebral palsy are motor dysfunction and abnormal posture development, often accompanied by cognitive impairment. Massage, a traditional Chinese Medicine therapy, can coordinate Zang and Fu, regulate Qi and blood, make the viscera work more smoothly, and calm Yin and Yang. Furthermore, it has been an effective method for CP in clinical. This paper summarizes a set of simple and standardized manipulations of massage for young rats with CP, which is easy to follow. The procedure follows: first, massage of four limb acupoints, including Quchi (LI11) and Zusanli (ST36); second, massage of the abdomen acupoints Zhongwan (RN12), Tianshu (ST25), Guanyuan (CV4), and Qihai (CV6); and finally, massage of the abdomen of the rats. This set of massage methods considerably improved the motor function of young rats with CP and is simple, standardized, and easy to follow. We adapted this set of manipulation methods in animal models to promote the internationalization and standardization of massage.

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

Here, we present a set of massage manipulations in the rat model of cerebral palsy that can considerably improve the motor function of cerebral palsy rats.

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

Visualization of Brain Pericyte Contractility Post-Subarachnoid Hemorrhage

Imaging Vital and Non-vital Brain Pericytes in Brain Slices following Subarachnoid Hemorrhage

Pericytes are crucial mural cells situated within cerebral microcirculation, pivotal in actively modulating cerebral blood flow via contractility adjustments. Conventionally, their contractility is gauged by observing morphological shifts and nearby capillary diameter changes under specific circumstances. Yet, post-tissue fixation, evaluating vitality and ensuing pericyte contractility of imaged brain pericytes becomes compromised. Similarly, genetically labeling brain pericytes falls short in distinguishing between viable and non-viable pericytes, particularly in neurologic conditions like subarachnoid hemorrhage (SAH), where our preliminary investigation validates brain pericyte demise. A reliable protocol has been devised to surmount these constraints, enabling simultaneous fluorescent tagging of both functional and non-functional brain pericytes in brain sections. This labeling method allows high-resolution confocal microscope visualization, concurrently marking the brain slice microvasculature. This innovative protocol offers a means to appraise brain pericyte contractility, its impact on capillary diameter, and pericyte structure. Investigating brain pericyte contractility within the SAH context yields insightful comprehension of its effects on cerebral microcirculation.

Author Spotlight: Imaging Pericytes Post-Subarachnoid Hemorrhaging in Rodents 

The preliminary inquiry confirms that subarachnoid hemorrhage (SAH) causes brain pericyte demise. Evaluating pericyte contractility post-SAH requires differentiation between viable and non-viable brain pericytes. Hence, a procedure has been developed to label viable and non-viable brain pericytes concurrently in brain sections, facilitating observation using a high-resolution confocal microscope.