This study received significant support and modeling assistance from the R&#38;D department of Beijing Intelligent Entropy Science &#38; Technology Co Ltd., Beijing, China.

This study was approved by the Institutional Review Board of Beijing Dongzhimen Hospital, Beijing, China. An MRI scanner was used with pulsed ASL (PASL) sequence based on turbo gradient spin echo (TGSE) for 3D arterial spin labeling (3D-ASL) with the following parameters: TR 4000 ms, TE 25 ms, bolus duration 700 ms, inversion time 1990 ms. The software tools used in this research are listed in the Table of Materials.
1. Data collection and preparation
NOTE: The variance in parameters remains unaffected by the research approach. Both DICOM and NIFTI formats are used for storing digital medical imaging data, with DICOM being the usual output from clinical imaging devices. However, the NIFTI format is often preferred for computational convenience in research activities. Converting from DICOM to NIFTI is a straightforward and common practice17. In this study, authentic DICOM data were acquired and converted into NIFTI format. The data was acquired using a 1.5 Tesla MRI scanner. In the cross-image registration process of this study, the fluid-attenuated inversion recovery (FLAIR) sequence was mainly used and fused with CBF images. The CBF Atlas tool (Table of Materials) used in this study is commercial software.
<ol>
	<li>Copy data to the designated working directory.
	<ol>
		<li>Copy all NIFTI data to a customized working directory. 
		NOTE: The working directory is the same in the operating system and MATLAB. The research follows the Right Anterior Superior (RAS) orientation standard.</li>
		<li>Go to the directory housing the data within MATLAB&#39;s current working directory, and use the niftiread function to load FLAIR data into the workspace. Use the size function to check the dimensions of the FLAIR sequence. Call the Flair_Slice command to view the FLAIR sequence (as shown in Figure 1). Use the specific commands as follows: 
		FLAIR_XLF = niftiread(&#39;FLAIR_XFL.nii&#39;); 
		size(FLAIR_XLF) 
		&#8203;Flair_Slice(FLAIR_XLF);</li>
		<li>Refer to Figure 1 for an image of the interactive graphic user interface (GUI) for the FLAIR sequence. Use the bottom scroll bar to quickly browse the different sequences.</li>
	</ol>
	</li>
	<li>Quickly check images of CBF.
	<ol>
		<li>Use the niftiread function to load CBF data into the workspace. Use the size function to check the dimensions of the CBF sequence. Call the CBF_Slice command to view the CBF sequence (as shown in Figure 2). Use the specific commands as follows: 
		CBF_XLF = niftiread(&#39;CBF_XFL.nii&#39;); 
		size(CBF_XLF) 
		CBF_Slice(CBF_XLF);</li>
		<li>Refer to Figure 2 for an interactive GUI screenshot of the CBF sequence. Use the bottom scroll bar to quickly browse the different sequences. 
		NOTE: In Figure 2, the CBF value range is usually 0-120 mL/100 g/min. Figure 2 uses a color map to represent different CBF levels in different colors.</li>
	</ol>
	</li>
</ol>
2. Segmentation of cerebral functional regions from FLAIR sequence
NOTE: The FLAIR sequence serves as both structural imaging and provides excellent pathological diagnostic capabilities. Therefore, fusing FLAIR with CBF holds important diagnostic value in clinics. This study segments major cerebral functional regions from the FLAIR sequence.
<ol>
	<li>In the workspace, call the FLAIR_Segment function and run the pretrained 3-D U-Net-based image segmentation program to automatically generate triplanar views of cerebral functional regions segmentation, as shown in Figure 3. Each color in Figure 3 represents a distinct functional region.</li>
	<li>For real-time inspection of different cerebral functional regions, use the crosshair interaction (Figure 3). Click and drag the center of the crosshair for an arbitrary 3D examination of the reconstructed brain anatomy. 
	NOTE: The GUI in Figure 3 also enables adjustment of the grayscale intensity range, contrast, and brightness of the triplanar views.</li>
	<li>Press and drag the left mouse button over any region of the images for real-time modification of brightness and contrast levels. Release the mouse button to confirm and finalize the adjustments.</li>
</ol>
3. Triplanar views of CBF distribution across cerebral functional regions
NOTE: Examining CBF distribution across different cerebral functional regions facilitates precise clinical judgments of patient conditions. Under the functional region framework from Figure 3, incorporating the exact CBF values from the CBF sequence and presenting them in triplanar views allows comprehensive physician inspection.
<ol>
	<li>Call the CBF_triplanar function to generate the triplanar GUI view shown in Figure 4, displaying CBF spatial distribution across functional regions. Move the crosshair to allow examination of CBF distribution in regions of interest.</li>
	<li>Click the Data Tips button on the top right corner of the GUI to display the CBF values at any position.</li>
	<li>Press and drag the left mouse button over any region of the images for real-time modification of brightness and contrast levels. Release the mouse button to confirm and finalize the adjustments.</li>
</ol>
4. CBF atlas across major cerebral functional regions
NOTE: Normalizing the CBF probability distributions across different functional regions generates the Cerebral Functional Regions CBF Atlas, expressing the CBF levels across cerebral functional regions of the subject.
<ol>
	<li>Call the CBF_Atlas function to convert the CBF spatial distribution shown in Figure 4 into a CBF Atlas (as shown in Figure 5). 
	NOTE: In Figure 5, the x-axis represents different cerebral functional regions, and the y-axis represents different CBF levels; the different colors denote different probability levels (the redder the color, the more voxels present).</li>
	<li>Click the Zoom In/Out button on the top right of the GUI shown in Figure 5 to scale partial images. 
	NOTE: The curve in Figure 5 connects the average CBF across regions.</li>
</ol>
5. Significant differences in CBF_Atlas between healthy subjects and CCI patients
NOTE: Using the same process described in sections 1-4, the average CBF values across different cerebral functional regions can be obtained for CCI patients.
<ol>
	<li>Utilize the CBF_Compare function to generate a comparative plot of the CBF curve from a healthy subject versus a CCI patient (Figure 6).</li>
	<li>Observe the significant differences between the patient&#39;s CBF curve (colored in black) and the healthy curve (colored in red) (Figure 6). Identify the functional regions with more pronounced CBF drops in the patient. Integrating step 3.3, re-examine the regions where the patient performed worse compared to the other regions. 
	NOTE: Step 3.3 allows for the viewing of the patient&#39;s CBF levels in three-dimensional space, meaning doctors can utilize the triplanar GUI view shown in Figure 4 to examine the CBF at any position of the patient. Here, the degree of CBF decline varies in different locations for CCI patients. Doctors can revisit Figure 4 to focus on brain regions with significant CBF decline.</li>
	<li>Use the icons in the GUI&#39;s upper right corner (Figure 5) to access functions such as zooming out, zooming in, returning to the global view, and marking the coordinates of the selected pixel. 
	NOTE: Using the same principle, pre-treatment and post-treatment results from the same patient can also be compared using the approach in Figure 6 to evaluate clinical efficacy over time.</li>
</ol>

MRI-Arterial Spin Labeling Integration to Detect and Monitor Cerebral Blood Flow 

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R., 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=Cognition+and+cerebral+blood+flow+after+extracranial+carotid+revascularization+for+carotid+atherosclerosis:+A+systematic+review.">Cognition and cerebral blood flow after extracranial carotid revascularization for carotid atherosclerosis: A systematic review.</a> Clin Ther. 45 (11), 1069-1076 (2023).</li><li>Togao, O., 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=Arterial+spin+labeling-based+MR+angiography+for+cerebrovascular+diseases:+Principles+and+clinical+applications.">Arterial spin labeling-based MR angiography for cerebrovascular diseases: Principles and clinical applications.</a> J Magn Reson Imaging. , (2023).</li><li>Haidar, H., Majzoub, R. E., Hajeer, S., Abbas, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+spin+labeling+(ASL-MRI)+versus+fluorodeoxyglucose-PET+(FDG-PET)+in+diagnosing+dementia:+a+systematic+review+and+meta-analysis.">Arterial spin labeling (ASL-MRI) versus fluorodeoxyglucose-PET (FDG-PET) in diagnosing dementia: a systematic review and meta-analysis.</a> BMC Neurol. 23 (1), 385 (2023).</li><li>Xu, Y., Tan, G., Chen, D., Liu, J., Zhou, Z., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+spin+labeling+perfusion+MRI+applications+in+drug-resistant+epilepsy+and+epileptic+emergency.">Arterial spin labeling perfusion MRI applications in drug-resistant epilepsy and epileptic emergency.</a> Acta Epileptol. 5, 23 (2023).</li><li>Xie, 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=Abnormalities+of+cerebral+blood+flow+and+the+regional+brain+function+in+Parkinson's+disease:+a+systematic+review+and+multimodal+neuroimaging+meta-analysis.">Abnormalities of cerebral blood flow and the regional brain function in Parkinson's disease: a systematic review and multimodal neuroimaging meta-analysis.</a> Front Neurol. 14, 1289934 (2023).</li><li>Tenberg, 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=Dysautonomia+and+activity+in+the+early+stroke+recovery+period.">Dysautonomia and activity in the early stroke recovery period.</a> Neurol Sci. 45 (6), 2505-2521 (2024).</li><li>Joshi, D., Prasad, S., Saini, J., Ingalhalikar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+arterial+spin+labeling+(ASL)+images+in+Parkinson's+disease+(PD):+A+systematic+review.">Role of arterial spin labeling (ASL) images in Parkinson's disease (PD): A systematic review.</a> Acad Radiol. 30 (8), 1695-1708 (2023).</li><li>Mohamed, A. Z., Kwiatek, R., Fante, P. D., Calhoun, V. D., Lagopoulos, J., Shan, Z. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+MRI+of+the+brainstem+for+assessing+its+autonomic+functions:+From+imaging+parameters+and+analysis+to+functional+atlas.">Functional MRI of the brainstem for assessing its autonomic functions: From imaging parameters and analysis to functional atlas.</a> J Magn Reson Imaging. , (2024).</li><li>Vanherle, L., Matuskova, H., Don-Doncow, N., Uhl, F. E., Meissner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+cerebrovascular+function+to+increase+neuronal+recovery+in+neurodegeneration+associated+to+cardiovascular+disease.">Improving cerebrovascular function to increase neuronal recovery in neurodegeneration associated to cardiovascular disease.</a> Front Cell Dev Biol. 8, 53 (2020).</li><li>Fujimura, M., Tominaga, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+cerebral+blood+flow+analysis+in+the+acute+stage+after+revascularization+surgery+for+Moyamoya+disease.">Significance of cerebral blood flow analysis in the acute stage after revascularization surgery for Moyamoya disease.</a> Neurol Med Chir (Tokyo). 55 (10), 775-781 (2015).</li><li>&#216;stergaard, L., 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=The+role+of+the+microcirculation+in+delayed+cerebral+ischemia+and+chronic+degenerative+changes+after+subarachnoid+hemorrhage.">The role of the microcirculation in delayed cerebral ischemia and chronic degenerative changes after subarachnoid hemorrhage.</a> J Cereb Blood Flow Metab. 33 (12), 1825-1837 (2013).</li><li>Mankoo, 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=The+role+of+the+autonomic+nervous+system+in+cerebral+blood+flow+regulation+in+stroke:+A+review.">The role of the autonomic nervous system in cerebral blood flow regulation in stroke: A review.</a> Auton Neurosci. 246, 103082 (2023).</li><li>Yang, X., Qiang, Q., Li, N., Feng, P., Wei, W., H&#246;lscher, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroprotective+mechanisms+of+glucagon-like+peptide-1-based+therapies+in+ischemic+stroke:+An+update+based+on+preclinical+research.">Neuroprotective mechanisms of glucagon-like peptide-1-based therapies in ischemic stroke: An update based on preclinical research.</a> Front Neurol. 13, 844697 (2022).</li><li>Ho, M. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arterial+spin+labeling:+Clinical+applications.">Arterial spin labeling: Clinical applications.</a> J Neuroradiol. 45 (5), 276-289 (2018).</li><li>Sforza, M., Bianchini, E., Alivernini, D., Salvetti, M., Pontieri, F. E., Sette, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+cerebral+vasomotor+reactivity+on+cerebrovascular+diseases+and+cognitive+impairment.">The impact of cerebral vasomotor reactivity on cerebrovascular diseases and cognitive impairment.</a> J Neural Transm (Vienna). 129 (11), 1321-1330 (2022).</li><li>Elhadad, A., Jamjoom, M., Abulkasim, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+NIFTI+files+storage+and+compression+to+facilitate+telemedicine+services+based+on+quantization+hiding+of+downsampling+approach.">Reduction of NIFTI files storage and compression to facilitate telemedicine services based on quantization hiding of downsampling approach.</a> Sci Rep. 14 (1), 5168 (2024).</li><li>Gonzalez-Ortiz, F., Kac, P. R., Brum, W. S., Zetterberg, H., Blennow, K., Karikari, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+phospho-tau+in+Alzheimer's+disease:+towards+diagnostic+and+therapeutic+trial+applications.">Plasma phospho-tau in Alzheimer's disease: towards diagnostic and therapeutic trial applications.</a> Mol Neurodegener. 18 (1), 18 (2023).</li><li>Magliozzi, R., 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=CSF+parvalbumin+levels+reflect+interneuron+loss+linked+with+cortical+pathology+in+multiple+sclerosis.">CSF parvalbumin levels reflect interneuron loss linked with cortical pathology in multiple sclerosis.</a> Ann Clin Transl Neurol. 8 (5), 534-547 (2021).</li><li>Kawano, 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=Aging+and+sex+differences+in+brain+volume+and+cerebral+blood+flow.">Aging and sex differences in brain volume and cerebral blood flow.</a> Aging Dis. , (2023).</li><li>Jia, R., Sol&#233;-Guardia, G., Kiliaan, A. 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The software tool CBF Atlas V1.0 is a product of Beijing Intelligent Entropy Science &amp; Technology Co., Ltd. The intellectual property rights of this software tool belong to the company. The authors have no conflicts of interest to declare.

The key steps (sections 3 and 4) constitute the basis for constructing the CBF Atlas, quantifying CBF distribution across cerebral functional regions. Step 4.2 explicitly delineates CBF levels for each brain area, pioneering a new technique. This not only provides physicians with a global view of patient CBF but also quantitative measurements of individual functional regions. Step 5.1&#160;demonstrates that the CBF Atlas holds substantial clinical diagnostic utility distinguishing CCI from healthy controls.
<p class=...

In the realm of neuroimaging, the quest for precise, noninvasive tools to assess cerebral function and pathology remains paramount. Among these, cerebral blood flow (CBF) stands as a vital indicator, reflecting the metabolic demands and health status of brain tissue1. Traditional approaches often entail empirical assessments, relying heavily on the expertise of clinicians to interpret images and discern pathological changes2. However, advancements in magnetic resonance imaging (MRI) techniques, particularly arterial spin labeling (ASL)3, offer a promising avenue for quantifying CBF with greater accuracy and objectivity4,5.
This study presents a pioneering methodology that integrates three-dimensional ASL (3D-ASL) with structural MRI to construct a comprehensive CBF atlas across cerebral functional regions6. By leveraging this novel approach, clinicians can not only obtain a global perspective of CBF but also delve into specific functional areas, allowing for a nuanced understanding of cerebral perfusion patterns7,8. This improvement in resolution is a direct result of technological progress in imaging equipment rather than the use of interpolated voxels. It is worth noting that the majority of mainstream MRI devices available on the market today typically offer imaging precision better than 1.5 mm9. These advancements in imaging technology have paved the way for more detailed and accurate CBF assessments. This represents a paradigm shift from conventional imaging, which often lacks the resolution to detect subtle changes in CBF associated with early-stage pathologies10.
The genesis of this methodology lies in the imperative to address the diagnostic challenges posed by cerebral conditions, including chronic cerebral ischemia (CCI) and other neurological disorders11,12. These conditions necessitate precise and timely assessments to guide therapeutic interventions effectively13,14. By comparing CBF atlases between healthy individuals and patients with CCI, this study unveils significant disparities in regional CBF distributions, offering insights into disease pathology and potential treatment avenues.
The utility of this MRI-ASL approach extends beyond diagnosis, encompassing therapeutic evaluation and monitoring of disease progression15. Longitudinal CBF atlases hold promise in tracking treatment responses and rehabilitation outcomes, providing clinicians with invaluable tools for personalized patient management. Moreover, the ability to discern subtle CBF changes may serve as an early biomarker for impending tissue abnormalities, enabling proactive interventions to mitigate neurological damage before it becomes irreversible16.
While this methodology represents an advanced tool, several avenues for refinement and expansion merit consideration. Standardizing scanning protocols, CBF normalization techniques, and constructing multi-subject healthy CBF atlases are crucial steps toward enhancing diagnostic accuracy and clinical utility. Collaborative efforts across diverse cerebral pathologies are essential to validate and refine this approach for widespread clinical adoption.
This study introduces a novel approach whereby MRI-derived CBF atlases offer clinicians deep insights into cerebral function and pathology. By bridging the gap between imaging group and clinical interpretation, this methodology has the potential to revolutionize the diagnosis and management of a myriad of neurological conditions, ushering in a future of precision medicine tailored to the unique needs of each patient.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>CBF Atlas</td>
			<td>Intelligent Entropy</td>
			<td>CBF Atlas V1.0</td>
			<td>Beijing Intelligent Entropy Science &#38; Technology Co Ltd. 
			Modeling for Thyroid Disease</td>
		</tr>
		<tr>
			<td>MATLAB</td>
			<td>MathWorks</td>
			<td>2023B</td>
			<td>Computing and visualization</td>
		</tr>
		<tr>
			<td>MRI Device</td>
			<td>Siemens</td>
			<td>Amria 1.5 T</td>
			<td>MRI scanner</td>
		</tr>
	</tbody>
</table>

construction and application of cerebral functional region-based cerebral blood flow atlas using magnetic resonance imaging-arterial spin labeling

Cerebral conditions often require precise diagnosis and monitoring, necessitating advanced imaging techniques. Current modalities may not adequately detect early signs of reversible tissue damage, underlining the need for innovative diagnostic tools that can quantify changes in cerebral blood flow (CBF) with high specificity and sensitivity.&#160;This study integrates three-dimensional arterial spin labeling (3D-ASL) with structural MRI to develop comprehensive CBF atlases that cover all main functional regions of the brain. This innovative magnetic resonance imaging- arterial spin labeling (MRI-ASL) methodology provides a rapid and noninvasive means of quantifying region-specific CBF, offering a detailed view of CBF levels across different functional regions.The comparison between chronic cerebral ischemia (CCI) patients and healthy subjects revealed significantly diminished CBF across the cerebral functional regions in the constructed CBF atlases for the former. This approach not only allows for the efficient identification of CCI by analyzing concurrent decreases in CBF across critical areas relative to healthy distributions but also enables the tracking of treatment responses and rehabilitation progress through longitudinal CBF atlases.The CBF atlas developed using the MRI-ASL technique represents a novel advancement in the field of cerebral diagnostics and patient care. By comparing regional CBF levels against normative standards, this method enhances diagnostic capabilities, enabling clinicians to provide personalized care to patients with cerebral conditions.

This investigation utilizes actual patient data acquired using a 1.5 T MRI scanner to validate the cerebral blood flow (CBF) quantification and atlas construction methodology. The preprocessing steps included FLAIR structural images (Figure 1), CBF images (Figure 2), and triplanar fused images (Figure 3 and Figure 4).
Cerebral functional regions were segmented from FLAIR scan...

Author Spotlight: Noninvasive Cerebral Blood Flow Determination in Human Functional Brain Region for Diagnosis of Neurological Disorders

This study integrated magnetic resonance imaging- arterial spin labeling images to derive cerebral blood flow (CBF) atlas for cerebral functional regions. Comparing typical healthy and chronic cerebral ischemia CBF atlases revealed significant differences in regional CBF distributions, enabling rapid, noninvasive assessments of functional CBF to assist in diagnosis and evaluate therapeutics.

Construction and Application of Cerebral Functional Region-Based Cerebral Blood Flow Atlas Using Magnetic Resonance Imaging-Arterial Spin Labeling

Cerebral conditions often require precise diagnosis and monitoring, necessitating advanced imaging techniques. Current modalities may not adequately ...

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401 Views.  Beijing University of Chinese Medicine. This study integrated magnetic resonance imaging- arterial spin labeling images to derive cerebral blood flow (CBF) atlas for cerebral functional regions. Comparing typical healthy and chronic cerebral ischemia CBF atlases revealed significant differences in regional CBF distributions, enabling rapid, noninvasive assessments of functional CBF to assist in diagnosis and evaluate therapeutics.

Video: Author Spotlight: Noninvasive Cerebral Blood Flow Determination in Human Functional Brain Region for Diagnosis of Neurological Disorders

The Application Of Permanent Middle Cerebral Artery Ligation in the Mouse

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to develop suitable animal models to study the events that unfold during ischemic insult. These techniques include transient or permanent, focal or global ischemia models using many different animal models, with the most common being rodents.
The permanent MCA ligation method which is also referred as pMCAo in the literature is used extensively as a focal ischemia model in rodents 1-6. This method was originally described for rats by Tamura et al. in 1981 7. In this protocol a craniotomy was used to access the MCA and the proximal regions were occluded by electrocoagulation. The infarcts involve mostly cortical and sometimes striatal regions depending on the location of the occlusion. This technique is now well established and used in many laboratories 8-13. Early use of this technique led to the definition and description of &#x201C;infarct core&#x201D; and &#x201C;penumbra&#x201D; 14-16, and it is often used to evaluate potential neuroprotective compounds 10, 12, 13, 17. Although the initial studies were performed in rats, permanent MCA ligation has been used successfully in mice with slight modifications 18-20 .
This model yields reproducible infarcts and increased post-survival rates. Approximately 80% of the ischemic strokes in humans happen in the MCA area 21 and thus this is a very relevant model for stroke studies. Currently, there is a paucity of effective treatments available to stroke patients, and thus there is a need for good models to test potential pharmacological compounds and evaluate physiological outcomes. This method can also be used for studying intracellular hypoxia response mechanisms in vivo. 
Here, we present the MCA ligation surgery in a C57/BL6 mouse. We describe the pre-surgical preparation, MCA ligation surgery and 2,3,5 Triphenyltetrazolium chloride (TTC) staining for quantification of infarct volumes.

Middle cerebral artery (MCA) ligation is a technique to study focal cerebral ischemia in animal models. In this method, the middle cerebral artery is exposed by craniotomy and ligated by cauterization. This method gives highly reproducible infarct volumes and increased post-operative survival rates compared to other methods available.

Focal cerebral ischemia is among the most common type of stroke seen in patients. Due to the clinical significance there has been a prolonged effort to ...

Endothelin-1 Induced Middle Cerebral Artery Occlusion Model for Ischemic Stroke with Laser Doppler Flowmetry Guidance in Rat

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in 20091, 2. Several models of cerebral ischemia have been developed to mimic the human condition of stroke. It has been suggested that up to 80% of all strokes result from ischemic damage in the middle cerebral artery (MCA) area3. In the early 1990s, endothelin-1 (ET-1) 4 was used to induce ischemia by applying it directly adjacent to the surface of the MCA after craniotomy. Later, this model was modified 5 by using a stereotaxic injection of ET-1 adjacent to the MCA to produce focal cerebral ischemia. The main advantages of this model include the ability to perform the procedure quickly, the ability to control artery constriction by altering the dose of ET-1 delivered, no need to manipulate the extracranial vessels supplying blood to the brain as well as gradual reperfusion rates that more closely mimics the reperfusion in humans5-7. On the other hand, the ET-1 model has disadvantages that include the need for a craniotomy, as well as higher variability in stroke volume8. This variability can be reduced with the use of laser Doppler flowmetry (LDF) to verify cerebral ischemia during ET-1 infusion. Factors that affect stroke variability include precision of infusion and the batch of the ET-1 used6. Another important consideration is that although reperfusion is a common occurrence in human stroke, the duration of occlusion for ET-1 induced MCAO may not closely mimic that of human stroke where many patients have partial reperfusion over a period of hours to days following occlusion9, 10. This protocol will describe in detail the ET-1 induced MCAO model for ischemic stroke in rats. It will also draw attention to special considerations and potential drawbacks throughout the procedure.

Several animal models of cerebral ischemia have been developed to simulate the human condition of stroke. This protocol describes the endothelin-1 (ET-1) induced middle cerebral artery occlusion (MCAO) model for ischemic stroke in rats. In addition, important considerations, advantages, and shortcomings of this model are discussed.

Stroke is the number one cause of disability and third leading cause of death in the world, costing an estimated $70 billion in the United States in ...

Utilizing a Cranial Window to Visualize the Middle Cerebral Artery During Endothelin-1 Induced Middle Cerebral Artery Occlusion

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be performed in many locations overlying the rat cerebrum, but is most easily carried out by creating a craniectomy over the readily accessible frontal or parietal bones. Most frequently, we have used this technique in combination with the endothelin-1 middle cerebral artery occlusion model of ischemic stroke to quantify the changes in middle cerebral artery vessel diameter that occur with injection of endothelin-1 into the brain parenchyma adjacent to the proximal MCA4, 5. In order to visualize the proximal portion of the MCA during endothelin -1 induced MCAO, we use a technique to create a cranial window through the temporal bone on the lateral aspect of the rat skull (Figure 1). Cerebral arteries can be visualized either with the dura intact or with the dura incised and retracted. Most commonly, we leave the dura intact during visualization since endothelin-1 induced MCAO involves delivery of the vasoconstricting peptide into the brain parenchyma. This bypasses the need to incise the dura directly over the visualized vessels for drug delivery. This protocol will describe how to create a cranial window to visualize cerebral arteries in a step-wise fashion, as well as how to avoid many of the potential pitfalls pertaining to this method.

This article describes a method for visualizing rat cerebral arteries through a cranial window using temporal craniectomy in order to view proximal portions of the middle cerebral artery (Figure 1). This versatile method can be combined with various techniques of drug delivery to measure cerebral artery reactivity in vivo.

Creation of a cranial window is a method that allows direct visualization of structures on the cortical surface of the brain1-3. This technique can be ...

Optimized System for Cerebral Perfusion Monitoring in the Rat Stroke Model of Intraluminal Middle Cerebral Artery Occlusion

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal models of acute ischemic stroke allows to confirm successful arterial occlusion and exclude subarachnoid hemorrhage. Cerebral perfusion monitoring can also be used to study intracranial collateral circulation, which is emerging as a powerful determinant of stroke outcome and a possible therapeutic target. Despite a recognized role of Laser Doppler perfusion monitoring as part of the current guidelines for experimental cerebral ischemia, a number of technical difficulties exist that limit its widespread use. One of the major issues is obtaining a secure and prolonged attachment of a deep-penetration Laser Doppler probe to the animal skull. In this video, we show our optimized system for cerebral perfusion monitoring during transient middle cerebral artery occlusion by intraluminal filament in the rat. We developed in-house a simple method to obtain a custom made holder for twin-fibre (deep-penetration) Laser Doppler probes, which allow multi-site monitoring if needed. A continuous and prolonged monitoring of cerebral perfusion could easily be obtained over the intact skull.

Cerebral perfusion monitoring has been demonstrated to improve accuracy in ischemic stroke models. Technical difficulties often limit the use of this essential tool for cerebrovascular research. In this video, an optimized system is shown to obtain a single or multi-site hemodynamic monitoring during intraluminal middle cerebral artery occlusion in rats.

The translational potential of pre-clinical stroke research depends on the accuracy of experimental modeling. Cerebral perfusion monitoring in animal ...

Embolic Middle Cerebral Artery Occlusion (MCAO) for Ischemic Stroke with Homologous Blood Clots in Rats

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic stroke. However, the use of tPA is limited by its narrow therapeutic window and by increased risk of hemorrhagic transformation. There is an urgent need to develop suitable stroke models to study new thrombolytic agents and strategies for treatment of ischemic stroke. At present, two major types of ischemic stroke models have been developed in rats and mice: intraluminal suture MCAO and embolic MCAO. Although MCAO models via the intraluminal suture technique have been widely used in mechanism-driven stroke research, these suture models do not mimic the clinical situation and are not suitable for thrombolytic studies. Among these models, the embolic MCAO model closely mimics human ischemic stroke and is suitable for preclinical investigation of thrombolytic therapy. This embolic model was first developed in rats by Overgaard et al.1 in 1992 and further characterized by Zhang et al. in 19972. Although embolic MCAO has gained increasing attention, there are technical problems faced by many laboratories. To meet increasing needs for thrombolytic research, we present a highly reproducible model of embolic MCAO in the rat, which can develop a predictable infarct volume within the MCA territory. In brief, a modified PE-50 tube is gently advanced from the external carotid artery (ECA) into the lumen of the internal carotid artery (ICA) until the tip of the catheter reaches the origin of the MCA. Through the catheter, a single homologous blood clot is placed at the origin of the MCA. To identify the success of MCA occlusion, regional cerebral blood flow was monitored, neurological deficits and infarct volumes were measured. The techniques presented in this paper should help investigators to overcome technical problems for establishing this model for stroke research.

Embolic Middle Cerebral Artery Occlusion MCAO for Ischemic Stroke with Homologous Blood Clots in Rats

In this paper, we demonstrate a standard method for producing an embolic middle cerebral artery occlusion with homologous blood clots (fibrin-rich) in adult rat. This model closely mimics human ischemic stroke and is suitable for preclinical study of thrombolytic therapy for ischemic stroke.

Clinically, thrombolytic therapy with use of recombinant tissue plasminogen activator (tPA) remains the most effective treatment for acute ischemic ...

Stereological and Flow Cytometry Characterization of Leukocyte Subpopulations in Models of Transient or Permanent Cerebral Ischemia

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after stroke. These myeloid cell subpopulations can display different phenotypes and functions and need to be distinguished and characterized to study their regulation and contribution to tissue damage. This protocol provides two different methodologies for brain immune cell characterization: a precise stereological approach and a flow cytometric analysis. The stereological approach is based on the optical fractionator method, which calculates the total number of cells in an area of interest (infarcted brain) estimated by a systematic random sampling. The second characterization approach provides a simple way to isolate brain leukocyte suspensions and to characterize them by flow cytometry, allowing for the characterization of microglia, infiltrated monocytes and neutrophils of the ischemic tissue. In addition, it also details a cerebral ischemia model in mice that exclusively affects brain cortex, generating highly reproducible infarcts with a low rate of mortality, and the procedure for histological brain processing to characterize infarct volume by the Cavalieri method.

Brain myeloid cells characterization following stroke can be performed by stereology using the optical fractionator method, or by a flow cytometric analysis of brain leukocytes suspensions. Both are useful techniques to perform an accurate phenotypical distinction of the main myeloid cell subsets found in the ischemic brain.

Microglia activation, as well as extravasation of haematogenous macrophages and neutrophils, is believed to play a pivotal role in brain injury after ...

Simultaneous PET/MRI Imaging During Mouse Cerebral Hypoxia-ischemia

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics disturbance in affected cells. Diffusion weighted magnetic resonance imaging (MRI) identifies regions that are damaged, potentially irreversibly, by hypoxia-ischemia. Alterations in glucose utilization in the affected tissue may be detectable by positron emission tomography (PET) imaging of 2-deoxy-2-(18F)fluoro-&#7429;-glucose ([18F]FDG) uptake. Due to the rapid and variable nature of injury in this animal model, acquisition of both modes of data must be performed simultaneously in order to meaningfully correlate PET and MRI data. In addition, inter-animal variability in the hypoxic-ischemic injury due to vascular differences limits the ability to analyze multi-modal data and observe changes to a group-wise approach if data is not acquired simultaneously in individual subjects. The method presented here allows one to acquire both diffusion-weighted MRI and [18F]FDG uptake data in the same animal before, during, and after the hypoxic challenge in order to interrogate immediate physiological changes.

The method presented here uses simultaneous positron emission tomography and magnetic resonance imaging. In the cerebral hypoxia-ischemia model, dynamic changes in diffusion and glucose metabolism occur during and after injury. The evolving and irreproducible damage in this model necessitates simultaneous acquisition if meaningful multi-modal imaging data are to be acquired.

Dynamic changes in tissue water diffusion and glucose metabolism occur during and after hypoxia in cerebral hypoxia-ischemia reflecting a bioenergetics ...

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of cerebral malaria can be difficult. We present an experimental mouse model of cerebral malaria that shares multiple features of the human disease, including edema and microvascular pathology. Using magnetic resonance imaging (MRI), we can detect and track the blood-brain barrier disruption, edema development, and subsequent brain swelling. We describe multiple MRI techniques that can visualize these pertinent pathological changes. Thus, we show that MRI represents a valuable tool to visualize and track pathological changes, such as edema, brain swelling, and microvascular pathology, in vivo.

In Vivo Tracking of Edema Development and Microvascular Pathology in a Model of Experimental Cerebral Malaria Using Magnetic Resonance Imaging

We describe a mouse model of experimental cerebral malaria and show how inflammatory and microvascular pathology can be tracked in vivo using magnetic resonance imaging.

Cerebral malaria is a sign of severe malarial disease and is often a harbinger of death. While aggressive management can be life-saving, the detection of ...

Application of an Amplitude-integrated EEG Monitor (Cerebral Function Monitor) to Neonates

Amplitude-integrated EEG (aEEG) is an easily accessible technique to monitor the electrocortical activity in preterm and term infants in neonatal intensive care units (NICUs). This method was first used to monitor newborns after asphyxia, providing information about future neurological outcomes. The aEEG is also helpful to select newborns who benefit from cooling. The aEEG monitoring of preterm infants is becoming more widespread, as various studies have shown that neurodevelopmental outcome is related to early aEEG tracings. Here, we demonstrate the application of the aEEG monitoring system and present typical patterns that depend upon gestational age and pathophysiological conditions. Furthermore, we mention pitfalls in the interpretation of the aEEG, as this method requires accurate fixation and localization of the electrodes. Additionally, the raw EEG can be used to detect neonatal seizures or to identify aEEG application problems. In conclusion, aEEG is a safe and generally well-tolerated method for the bedside monitoring of neonatal cerebral function; it can even provide information about long-term outcome.

Application of an Amplitude-integrated EEG Monitor Cerebral Function Monitor to Neonates

Here, we show how to apply amplitude-integrated electroencephalography to monitor cerebral function in neonates.

Amplitude-integrated EEG (aEEG) is an easily accessible technique to monitor the electrocortical activity in preterm and term infants in neonatal ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained using laser Doppler flowmetry (LDF). This paper demonstrates a closed skull preparation that allows cerebral blood flow to be assessed without penetrating the skull or installing a chamber or cerebral window. To evaluate autoregulatory mechanisms, a model of controlled blood pressure reduction via graded hemorrhage can be utilized while simultaneously employing LDF. This enables the real time tracking of the relative changes in the blood flow in response to reductions in arterial blood pressure produced by the withdrawal of circulating blood volume. This paradigm is a valuable approach to study cerebral blood flow autoregulation during reductions in arterial blood pressure and, with minor modifications in the protocol, is also valuable as an experimental model of hemorrhagic shock. In addition to evaluating autoregulatory responses, LDF can be used to monitor the cortical blood flow when investigating metabolic, myogenic, endothelial, humoral, or neural mechanisms that regulate cerebral blood flow and the impact of various experimental interventions and pathological conditions on cerebral blood flow.

This article demonstrates the use of laser Doppler flowmetry to evaluate the ability of the cerebral circulation to autoregulate its blood flow during reductions in arterial blood pressure.

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...