The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme [FP7/2007-2013] / ERC grant agreement [337333]. The research of SvV and GJB is supported by a VIDI grant [91711384] from ZonMw, the Netherlands Organization for Health Research and Development.

The use of the autopsy samples and in vivo MR images for this protocol was in accordance with local regulations and approved by the local institutional review board of the University Medical Center Utrecht (UMCU).
1. MR-guided Histopathological Validation of Cortical Microinfarcts
<ol>
	<li>Ex vivo MRI
	<ol>
		<li>When handling brain tissue, always wear gloves and suitable protective clothing.</li>
		<li>Based on the research question, select appropriate, preferably 10-mm thick, formalin-fixed brain slabs. The brain slabs for this paper were derived from the neuropathology department of the UMCU, and VU University Medical Centre (VUMC), based on known Alzheimer pathology.
		<ol>
			<li>Formalin-fix whole brains for at least 3-4 weeks by immersion in 10% formalin, prior to cutting. Cut the brains into coronal slabs, containing both hemispheres.</li>
			<li>For post-mortem scanning, select for example three brain slabs per brain, taken from the frontal, temporo-parietal, and occipital regions of the brain. The current protocol is optimized for the use of three coronal brain slabs, containing both hemispheres, in one scan session.</li>
		</ol>
		</li>
		<li>Take photographs of the brain slabs at both sides (dorsal and caudal), and take careful notes (or make sketches) of the orientation of the slabs in the container and in the scanner, for later co-localization of histology with MRI.</li>
		<li>Fill a purpose-built container (Figure 1) &#8211; in this case one that fits within the MR head coil &#8211; with 10% fresh formalin at RT. If MRI signal from the fluid is undesired, use a perfluoropolyether (PFPE) lubricant with a suitable density instead of formalin (such as Fomblin or Galden PFPE). Ensure to use a flow cabinet when handling formalin.</li>
		<li>When placing the brain slabs in the container, make sure to avoid air bubbles. Remove the majority of air bubbles by gently shaking the tissue, either by hand, or using a shaker or ultrasound bath.</li>
		<li>Make sure the slabs cannot move within the container and limit the amount of needed fluid, by using a smaller container to keep the slabs in place (Figure 1).</li>
		<li>Cover the container with plastic or parafilm, to prevent evaporation and to protect the MR (head) coil from potential contamination (Figure 2).</li>
		<li>Use a whole-body 7T MRI scanner with an appropriate coil. In this protocol a dual transmit and 32-channel receive head coil is used.</li>
		<li>Place the container in the head coil, wrapped in a towel or surgical underpad, to prevent potential spilling of fluid. Make sure the container cannot move, and that the slabs remain in horizontal position (Figure 2).</li>
		<li>Run a survey scan that can be used for the planning of the high resolution scans, correct B0 inhomogeneity by using an appropriate shimming tool, and calibrate the RF power to obtain the correct flip angles (the few slabs require less power compared to in vivo scanning of a whole head) according to manufacturer&#8217;s protocol.</li>
		<li>Plan the high resolution acquisitions on the survey scan, to ensure the brain slabs are fully included in the field-of-view. Scan the brain slabs O/N with the high resolution acquisitions shown in Table 1, which are optimized for ex vivo imaging. The acquisition protocol presented here includes a 3D FLAIR, T2, and T1 weighted image with an isotropic resolution of 0.4 mm, and a T2* weighted image with an isotropic resolution of 0.18 mm.</li>
		<li>Identify automatic software processes that may interrupt scanning, such as automated up-dates that run O/N, or warnings for peripheral nerve stimulation, and make sure the scanner procedures will not be interrupted by these.</li>
		<li>Monitor the scanner O/N for possible confirmation pop-ups that may interrupt scanning, by using for example a VPN connection.</li>
		<li>Return the following morning (after a total scan time of approximately 12 hr in the current protocol). Store the brain slabs in formalin, clean up.</li>
		<li>Save the images to an external hard disk.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/53125/53125fig1.jpg" /> 
Figure 1. Preparation of formalin-fixed brain slabs for post-mortem scanning at 7T MRI. A purpose-built Perspex container is filled with either 10% formalin or a perfluoropolyether (PFPE) lubricant if MRI signal from the fluid is undesired. Three 10-mm thick formalin-fixed coronal brain slabs are placed in the container. A smaller container is used to keep the slabs in place. Tape the second container to the first one, to prevent movement.
<img alt="Figure 2" src="/files/ftp_upload/53125/53125fig2.jpg" /> 
Figure 2. Placement of purpose-built container in 7T head coil. Cover the container with plastic or parafilm to prevent evaporation of the formalin. Place the container, enclosed in a towel or surgical underpad, in the head coil of a 7T MR scanner. Make sure the container cannot move, and that the slabs remain in horizontal position.
<ol start="2">
	<li>Histopathology
	<ol>
		<li>Identify possible cortical CMIs - or other lesions of interest - on the acquired images. These lesions are the targets for histological analysis. Watch out for artifacts, such as post-mortem tissue damage (which sometimes appear at the surface of brain slabs due to cuts) or long-term formalin storage artifacts (e.g., coarse MRI hypointensities representing neuropil changes8). 
		Note: Different histopathological subtypes of cortical CMIs have different MR characteristics. For further details on CMI subtypes, the reader is referred to a recent ex vivo study7.</li>
		<li>After identifying possible cortical CMIs on the MR images, sample the region of interest for histopathological validation. Make sure to cut out a region, containing anatomical landmarks, for later matching of the MRI with histopathology. Perform standard histopathology, as followed (but other approaches might also apply).</li>
		<li>Cut out a region of approximately 30 x 20 x 5 mm3 containing a possible cortical CMI.</li>
		<li>To obtain accurate sampling, estimate the lesion location by the slice thickness of the MR images, and tissue architecture. Manually cut the tissue slightly above the estimated lesion location to limit the amount of serial sectioning (after paraffin embedding) that is needed for targeting the lesion.</li>
		<li>Make sure the sampled tissue fits a tissue cassette. Place the surface to be cut face-down in the cassette.</li>
		<li>Keep all tissue cassettes in 10% formalin, until processing of the tissue.</li>
		<li>Process the tissue for paraffin embedding. This usually involves an automated procedure of dehydrating the tissue, through a series of graded alcohol (e.g., 70% to 95% to 100%) baths, and clearing of the tissue in xylene.</li>
		<li>Embed the tissue in paraffin wax blocks. Ensure the surface to be cut faces up after embedding.</li>
		<li>Cut 4-6 &#181;m serial sections with a microtome, until the targeted lesion is retrieved.</li>
		<li>Float the sections on the surface of a 37 &#176;C water bath. Mount the sections on glass slides. Place the slides on a warming block to bond the tissue to the glass. Store slides O/N at RT.</li>
		<li>Perform an appropriate staining (e.g., H&#38;E staining) on the first sections, keep adjacent blank sections for further use (e.g., immunohistochemistry).</li>
		<li>Coverslip the H&#38;E stained sections, using a drop of mounting medium of choice. Gently lower the slip, avoiding air bubbles.</li>
		<li>Study the sections using a light microscope, at an appropriate magnification. Compare sections to the previously obtained MR images.</li>
	</ol>
	</li>
</ol>
2. Assessing Cortical Microinfarcts on In Vivo 7T MRI
<ol>
	<li>Perform 7T MRI in the patient population of your interest, using the in vivo MRI protocol (which includes at least a 3D FLAIR) as described in6.</li>
	<li>Assess cortical CMIs on the in vivo 7T MR images as detailed in the steps below, using the following 7T rating criteria for CMIs: cortical CMIs are hyperintense on FLAIR (with or without a hypointense center), hyperintense on T2, hypointense on T1, detectable on at least two views of the brain (e.g., sagittal and transversal), restricted to the cortex, distinct from perivascular spaces, with a greatest dimension &#8804;4 mm6,7.</li>
	<li>Use an interface with three image viewers, to simultaneously view FLAIR, T1, and T2 images, e.g., MeVisLab (Figure 3). This platform allows to incorporate multiple viewers and to place markers on possible lesion locations.</li>
	<li>First assess one hemisphere on FLAIR in sagittal view. Screen the whole cortex for hyperintense lesions. Any hyperintens lesion &#8804;4 mm is a possible CMI. Place markers by clicking on each possible CMI.</li>
	<li>Repeat for the other hemisphere.</li>
	<li>Verify all marked locations on T1 and T2. Discard a location if it is not hypointense on T1 or hyperintense on T2.</li>
	<li>Assess transversal view, on FLAIR, T1, and T2. Discard a location if it is not visible. Check the coronal view in case of doubt.</li>
	<li>Watch out for MRI artifacts and anatomical variations (especially sulcal edges).</li>
	<li>Save markers.</li>
</ol>
<img alt="Figure 3" src="/files/ftp_upload/53125/53125fig3.jpg" /> 
Figure 3. Example image viewing platform for the assessment of cortical microinfarcts. An interface is used, integrated in MeVisLab. This program allows to incorporate multiple viewers simultaneously, to switch easily between sagittal / transversal / coronal orientation, and to place and save markers on possible lesion locations. (Different markers can be chosen for different types of lesions).
3. Assessing Cortical Microinfarcts on In Vivo 3T MRI
<ol>
	<li>Acquire 3T MR images of the patient population of your interest. Existing data can also be used as long as the MR imaging protocol contained at least a 3D T1, and a FLAIR and T2.</li>
	<li>Assess cortical CMIs on the in vivo 3T MR images as detailed in the steps below, using the following 3T rating criteria for CMIs: cortical CMIs are hypointense on T1 (isointense with CSF), detectable on at least two views of the brain (e.g. sagittal and transversal), restricted to the cortex, distinct from perivascular spaces, with a greatest dimension &#8804;4 mm.
	<ol>
		<li>Explore the location of a hypointense cortical lesion found on T1 on FLAIR and T2 weighted images. Rate the lesion as a probable cortical CMI if the location is hyperintense or isointense (with the grey matter) on FLAIR and T2. Discard the lesion if at the same location a hypointense signal is found on T2, indicating the T1 hypointense lesion is either due to a hemorrhagic lesion, a vessel, or an artifact. In case of doubt, check the location on a T2* weighted image9.</li>
	</ol>
	</li>
	<li>Use the same interface as described above.</li>
	<li>First assess one hemisphere on T1 in sagittal view. Screen the whole cortex for focal hypointense lesions. Any hypointense lesion &#8804;4 mm is a possible CMI. Place markers by clicking on each possible CMI.</li>
	<li>Repeat for the other hemisphere.</li>
	<li>Assess transversal T1, and simultaneously verify all marked locations on transversal FLAIR and T2. Regard the location as a probable CMI if it is hyperintense or isointense on FLAIR and T2. Discard a location if it seems to be an artifact or anatomical variation. Discard a location if it is hypointense on T2.</li>
	<li>Watch out for artifacts that look like CMIs on T1 weighted images, especially ringing artifacts at the &#8216;edges&#8217; of the brain which will appear at several adjacent gyri, watch out for edges of sulci, watch out for large vessels in the temporal lobes (at the poles). Finally, it is recommended to discard possible cortical CMIs in tissue in close proximity to a larger cortical infarct.</li>
	<li>Save markers.</li>
</ol>

<ol>
	<li>Vander Kolk, A. G., Hendrikse, J., Zwanenburg, J. J., Visser, F., Luijten, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+applications+of+7+T+MRI+in+the+brain.">Clinical applications of 7 T MRI in the brain.</a> Eur J Radiol. 82 (5), 708-718 (2013).</li><li>Pantoni, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+small+vessel+disease:+from+pathogenesis+and+clinical+characteristics+to+therapeutic+challenges.">Cerebral small vessel disease: from pathogenesis and clinical characteristics to therapeutic challenges.</a> Lancet Neurol. 9, 689-701 (2010).</li><li>Gouw, A. 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=Heterogeneity+of+small+vessel+disease:+a+systematic+review+of+MRI+and+histopathology+correlations.">Heterogeneity of small vessel disease: a systematic review of MRI and histopathology correlations.</a> J Neurol Neurosurg Psychiatry. 82 (2), 126-135 (2011).</li><li>Smith, E. E., Schneider, J. A., Wardlaw, J. M., Greenberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+microinfarcts:+the+invisible+lesions.">Cerebral microinfarcts: the invisible lesions.</a> Lancet Neurol. 11, 272-282 (2012).</li><li>Brundel, M., de Bresser, J., van Dillen, J. J., Kappelle, L. J., Biessels, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+microinfarcts:+a+systematic+review+of+neuropathological+studies.">Cerebral microinfarcts: a systematic review of neuropathological studies.</a> J. Cereb. Blood Flow Metab. 32 (3), 425-436 (2012).</li><li>Van Veluw, S. J., 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=In+vivo+detection+of+cerebral+cortical+microinfarcts+with+high-resolution+7T+MRI.">In vivo detection of cerebral cortical microinfarcts with high-resolution 7T MRI.</a> J Cereb Blood Flow Metab. 33 (3), 322-329 (2013).</li><li>Van Veluw, S. J., 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+spectrum+of+MR+detectable+cortical+microinfarcts;+a+classification+study+with+7+tesla+post-mortem+MRI+and+histopathology.">The spectrum of MR detectable cortical microinfarcts; a classification study with 7 tesla post-mortem MRI and histopathology.</a> J Cereb Blood Floow Metab. Jan. 21, 10-1038 (2015).</li><li>Van Duijn, 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=MRI+artifacts+in+human+brain+tissue+after+prolonged+formalin+storage.">MRI artifacts in human brain tissue after prolonged formalin storage.</a> Magn Reson Med. 65 (6), 1750-1758 (2011).</li><li>Van Veluw, S. J., 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=Cortical+microinfarcts+on+3T+MRI:+clinical+correlates+in+memory-clinic+patients.">Cortical microinfarcts on 3T MRI: clinical correlates in memory-clinic patients.</a> Alzheimers Dement. 5, 10-1016 (2015).</li><li>Van Dalen, J. W., 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=Cortical+microinfarcts+detected+in+vivo+on+3+Tesla+MRI:+clinical+and+radiological+correlates.">Cortical microinfarcts detected in vivo on 3 Tesla MRI: clinical and radiological correlates.</a> Stroke. 46 (1), 255-257 (2015).</li><li>Vander Kolk, A. 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=Imaging+the+intracranial+atherosclerotic+vessel+wall+using+7T+MRI:+initial+comparison+with+histopathology.">Imaging the intracranial atherosclerotic vessel wall using 7T MRI: initial comparison with histopathology.</a> AJNR Am J Neuroradiol. 36 (4), 694-701 (2015).</li><li>Visser, F., Zwanenburg, J. J., Hoogduin, J. M., Luijten, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+magnetization-prepared+3D-FLAIR+imaging+at+7.0+Tesla.">High-resolution magnetization-prepared 3D-FLAIR imaging at 7.0 Tesla.</a> Magn Reson Med. 64 (1), 194-202 (2010).</li><li>Brundel, M., 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=High+prevalence+of+cerebral+microbleeds+at+7Tesla+MRI+in+patients+with+early+Alzheimer&#8217;s+disease.">High prevalence of cerebral microbleeds at 7Tesla MRI in patients with early Alzheimer&#8217;s disease.</a> J Alzheimers Dis. 31 (2), 259-263 (2012).</li><li>Kuijf, H. J., 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=Detecting+cortical+cerebral+microinfarcts+in+7.0+T+MR+images.">Detecting cortical cerebral microinfarcts in 7.0 T MR images.</a> IEEE International Symposium on Biomedical Imaging. , 982-985 (2013).</li></ol>

The authors have nothing to disclose.

CMIs have attracted increasing attention over the last few years. A growing body of evidence derived from autopsy studies has identified CMIs as important contributors to age-related cognitive decline and dementia4,5. CMIs are now detectable on 7T and also 3T MRI. Optimization and standardization of assessment protocols for these lesions will support rapid implementation of robust and valid CMI detection in cohort studies throughout the world. This will enable a widespread evaluation of the clinical relevance ...

The application of ultra-high field 7 Tesla (T) MRI in patient studies is rapidly progressing1. This paper introduces a representative application of 7T MRI in the context of cerebrovascular disease in the aging human brain. Cerebrovascular disease is a major cause of cognitive decline and dementia. This vascular contribution to dementia frequently involves the small vessels of the brain, such as arterioles, small veins, and capillaries. Hence, it is referred to as cerebral small vessel disease (SVD)2. Because the cerebral small vessels are too small to capture with conventional MRI, only the consequences of SVD - i.e., the resulting tissue injury - can be visualized. This includes white matter hyperintensities, cerebral microbleeds, and lacunar infarcts3.
Other important manifestations of SVD are cerebral microinfarcts (CMIs)4. Autopsy studies report high prevalence of CMIs in vascular dementia, and Alzheimer&#8217;s disease5. However, due to their small sizes (ranging from 50 &#181;m to a few mm) they escape detection on conventional MRI4,5. 7T MRI provides high resolution images with improved signal-to-noise-ratio and contrast, which enables the detection of certain structures and lesions beyond the detection limit of conventional MRI. This technique was therefore applied to detect CMIs. To identify possible CMIs, many in vivo 7T MRI scans were previously screened for lesions with sizes &#60;5 mm and imaging characteristics consistent with ischemic properties. Such lesions could reliably be identified in the cortex. These focal elongated lesions were hyperintense on 7T FLAIR (0.8 mm isotropic voxels), restricted to the cortex and seemed to extend from the cortical surface, hyperintense on T2 (0.7 mm isotropic voxels), and hypointense on T1 (1.0 mm isotropic voxels). It was confirmed that these lesions were cortical CMIs using an MR-guided histopathology approach in post-mortem human brain tissue6,7.
Here, the ex vivo MRI protocol is presented that was used in previous studies for the histopathological validation of cortical CMIs. Secondly, guidelines are provided for the assessment of cortical CMIs on in vivo 7T MRI. Finally, the assessment of cortical CMIs on 7T has been translated to more widely available 3T MRI, and guidelines are provided how to identify cortical CMIs on 3T MRI.

<table border="0">
	<tbody>
		<tr>
			<td>Fomblin / Galden PFPE</td>
			<td>Solvay Solexis, Bollate, Italy</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>7T MR system</td>
			<td>Philips Healthcare, Cleveland, OH, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>32-channel receive head coil</td>
			<td>Nova Medical, Wilmington, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MeVisLab</td>
			<td>MeVis Medical Solutions AG, Bremen, Germany</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

assessing cortical cerebral microinfarcts on high resolution mr images

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes it is challenging to study them on clinical MRI scans. It was recently demonstrated that cortical microinfarcts can be depicted with MRI scanners using high magnetic field strengths (7T). Based on this experience, a proportion of these lesions is also visible on lower resolution 3T MRI. These findings were corroborated with ex vivo imaging of post-mortem human brain tissue, accompanied by histopathological verification of possible cortical microinfarcts.
Here an ex vivo imaging protocol is presented, for the purpose of validating MR observed cerebral microvascular pathology with histological evaluation. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on both in vivo 7T and 3T MR images. These guidelines provide researchers with a tool to rate cortical microinfarcts on in vivo images of larger patient samples, to further unravel their clinical relevance in cognitive decline and dementia, and establish these lesions as a novel biomarker of cerebral small vessel disease.

An impression of the high resolution and high image quality of an ex vivo sequence acquired at 7T is provided <a href="https://www.jove.com/files/ftp_upload/53125/53125fig4.mp4" target="_blank">here (Figure 4)</a>. This is a 3D T2* weighted ex vivo scan, with an isotropic resolution of 0.18 mm. Tissue was derived from an 84-year old demented female with pathologically proven Alzheimer&#8217;s disease and severe cerebral amyloid angiopathy (CAA). The detail of the image allows the identification of corti...

Watch this Scientific Journal Video about Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images at JoVE.com

Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images

A high resolution ex vivo 7T MR imaging protocol is presented, to perform MR-guided histopathological validation of microvascular pathology in post-mortem human brain tissue. Furthermore, guidelines are provided for the assessment of cortical microinfarcts on in vivo 7T as well as 3T MR images.

Cerebral microinfarcts are frequent findings in the post-mortem human brain, and are related to cognitive decline and dementia. Due to their small sizes ...

assessing-cortical-cerebral-microinfarcts-on-high-resolution-mr-images

Research

JoVE Journal

Medicine

13.4K Views.  University Medical Center Utrecht.  Высокое разрешение экс естественных условиях протокол изображения 7Т МР представлены, чтобы выполнить МР-руководствуясь гистопатологическое проверку микрососудов патологии в посмертном человека мозговой ткани. Кроме того, руководящие принципы предн?...

Video: Assessing Cortical Cerebral Microinfarcts on High Resolution MR Images

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

How to Measure Cortical Folding from MR Images: a Step-by-Step Tutorial to Compute Local Gyrification Index

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will be identifiable at any age. As recently reviewed by Mangin and colleagues2, several methods exist to quantify different characteristics of gyrification. For instance, sulcal morphometry can be used to measure shape descriptors such as the depth, length or indices of inter-hemispheric asymmetry3. These geometrical properties have the advantage of being easy to interpret. However, sulcal morphometry tightly relies on the accurate identification of a given set of sulci and hence provides a fragmented description of gyrification. A more fine-grained quantification of gyrification can be achieved with curvature-based measurements, where smoothed absolute mean curvature is typically computed at thousands of points over the cortical surface4. The curvature is however not straightforward to comprehend, as it remains unclear if there is any direct relationship between the curvedness and a biologically meaningful correlate such as cortical volume or surface. To address the diverse issues raised by the measurement of cortical folding, we previously developed an algorithm to quantify local gyrification with an exquisite spatial resolution and of simple interpretation. Our method is inspired of the Gyrification Index5, a method originally used in comparative neuroanatomy to evaluate the cortical folding differences across species. In our implementation, which we name local Gyrification Index (lGI1), we measure the amount of cortex buried within the sulcal folds as compared with the amount of visible cortex in circular regions of interest. Given that the cortex grows primarily through radial expansion6, our method was specifically designed to identify early defects of cortical development.
In this article, we detail the computation of local Gyrification Index, which is now freely distributed as a part of the FreeSurfer Software (<a href="http://surfer.nmr.mgh.harvard.edu/">http://surfer.nmr.mgh.harvard.edu/</a>, Martinos Center for Biomedical Imaging, Massachusetts General Hospital). FreeSurfer provides a set of automated reconstruction tools of the brain's cortical surface from structural MRI data. The cortical surface extracted in the native space of the images with sub-millimeter accuracy is then further used for the creation of an outer surface, which will serve as a basis for the lGI calculation. A circular region of interest is then delineated on the outer surface, and its corresponding region of interest on the cortical surface is identified using a matching algorithm as described in our validation study1. This process is repeatedly iterated with largely overlapping regions of interest, resulting in cortical maps of gyrification for subsequent statistical comparisons (Fig. 1). Of note, another measurement of local gyrification with a similar inspiration was proposed by Toro and colleagues7, where the folding index at each point is computed as the ratio of the cortical area contained in a sphere divided by the area of a disc with the same radius. The two implementations differ in that the one by Toro et al. is based on Euclidian distances and thus considers discontinuous patches of cortical area, whereas ours uses a strict geodesic algorithm and include only the continuous patch of cortical area opening at the brain surface in a circular region of interest.

Measuring gyrification (cortical folding) at any age represents a window into early brain development. Hence, we previously developed an algorithm to measure local gyrification at thousands of points over the hemisphere1. In this paper, we detail the computation of this local gyrification index.

Cortical folding (gyrification) is determined during the first months of life, so that adverse events occurring during this period leave traces that will ...

Automated Midline Shift and Intracranial Pressure Estimation based on Brain CT Images

In this paper we present an automated system based mainly on the computed tomography (CT) images consisting of two main components: the midline shift estimation and intracranial pressure (ICP) pre-screening system. To estimate the midline shift, first an estimation of the ideal midline is performed based on the symmetry of the skull and anatomical features in the brain CT scan. Then, segmentation of the ventricles from the CT scan is performed and used as a guide for the identification of the actual midline through shape matching. These processes mimic the measuring process by physicians and have shown promising results in the evaluation. In the second component, more features are extracted related to ICP, such as the texture information, blood amount from CT scans and other recorded features, such as age, injury severity score to estimate the ICP are also incorporated. Machine learning techniques including feature selection and classification, such as Support Vector Machines (SVMs), are employed to build the prediction model using RapidMiner. The evaluation of the prediction shows potential usefulness of the model. The estimated ideal midline shift and predicted ICP levels may be used as a fast pre-screening step for physicians to make decisions, so as to recommend for or against invasive ICP monitoring.

An automated midline shift estimation and intracranial pressure (ICP) pre-screening system based on computed tomography (CT) images for patients with traumatic brain injury (TBI) is proposed using image processing and machine learning techniques.

In this paper we present an automated system based mainly on the computed tomography (CT) images consisting of two main components: the midline shift ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

A Thrombotic Stroke Model Based On Transient Cerebral Hypoxia-ischemia

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA thrombolysis) rarely produces benefits in mechanical occlusion-based experimental models, which dominate preclinical stroke research. This split between the bench and bedside suggests the need to employ tPA-responsive models in preclinical stroke research. To this end, a simple and tPA-reactive thrombotic stroke model is invented and described here. This model consists of transient occlusion of the unilateral common carotid artery and delivery of 7.5% oxygen through a face mask in adult mice for 30 min, while maintaining the animal rectal temperature at 37.5 &#177; 0.5 &#176;C. Although reversible ligation of the unilateral carotid artery or hypoxia each suppressed cerebral blood flow only transiently, the combination of both insults caused lasting reperfusion deficits, fibrin and platelet deposition, and large infarct in the middle cerebral artery-supplied territory. Importantly, tail-vein injection of recombinant tPA at 0.5, 1, or 4 hr post-tHI (10 mg/kg) provided time-dependent reduction of the mortality rate and infarct size. This new stroke model is simple and can be standardized across laboratories to compare experimental results. Further, it induces thrombosis without craniectomy or introducing pre-formed emboli. Given these unique merits, the tHI model is a useful addition to the repertoire of preclinical stroke research.

Thromboembolic stroke models are vital tools for optimizing the recanalization therapy. Here we report a murine thrombotic stroke model based on transient cerebral hypoxic-ischemic (tHI) insult, which triggers thrombosis and infarction, and responds favorably to tissue plasminogen activator (tPA)-mediated fibrinolysis in a therapeutic window similar to those in stroke patients.

Stroke research has endured many setbacks in translating neuroprotective therapies into clinical practice. In contrast, the real-world therapy (tPA ...

Performing Permanent Distal Middle Cerebral with Common Carotid Artery Occlusion in Aged Rats to Study Cortical Ischemia with Sustained Disability

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, obesity and diabetes. Accordingly, when evaluating therapies for stroke in animals, it is important to select a model with excellent face validity. Ischemic stroke accounts for 80% of all strokes, and the majority of these occur in the territory of the middle cerebral artery (MCA), often inducing infarcts that affect the sensorimotor cortex, causing persistent plegia or paresis on the contralateral side of the body. We demonstrate in this video a method for producing ischemic stroke in elderly rats, which causes sustained sensorimotor disability and substantial cortical infarcts. Specifically, we induce permanent distal middle cerebral artery occlusion (MCAO) in elderly female rats by using diathermy forceps to occlude a short segment of this artery. The carotid artery on the ipsilateral side to the lesion was then permanently occluded and the contralateral carotid artery was transiently occluded for 60 min. We measure the infarct size using structural T2-weighted magnetic resonance imaging (MRI) at 24 hr and 8 weeks after stroke. In this study, the mean infarct volume was 4.5% &#177; 2.0% (standard deviation) of the ipsilateral hemisphere at 24 hr (corrected for brain swelling using Gerriet&#8217;s equation, n = 5). This model is feasible and clinically relevant as it permits the induction of sustained sensorimotor deficits, which is important for the elucidation of pathophysiological mechanisms and novel treatments.

Here we present a protocol to produce permanent distal middle cerebral artery occlusion in elderly female rats with simultaneous occlusion of the carotid arteries to generate large cortical infarcts and sustained deficits. We show confirmation of the lesion size using structural MRI at 24 hr and 8 weeks after stroke.

Stroke typically occurs in elderly people with a range of comorbidities including carotid (or other arterial) atherosclerosis, high blood pressure, ...

Electrophysiological Assessment of Murine Atria with High-Resolution Optical Mapping

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and electrophysiological phenotype in the atria. That encourages us to utilize a genetically-engineered mouse model to elucidate the mechanism of AF. However, it is difficult to evaluate the electrophysiological properties in murine atria due to their small size. This protocol describes the electrophysiological evaluation of atria using an optical mapping system with a high temporal and spatial resolution in Langendorff perfused murine hearts. The optical mapping system is assembled with dual high-speed complementary metal oxide semiconductor cameras and high magnification objective lenses, to detect the fluorescence of a voltage-sensitive dye and Ca2+ indicator. To focus on the assessment of murine atria, optical mapping is performed with an area of 2 mm &#215; 2 mm or 10 mm x 10 mm, with a 100 &#215; 100 resolution (20 &#181;m/pixel or 100 &#181;m/pixel) and sampling rate of up to 10 kHz (0.1 ms) at maximum. A 1-French size quadripolar electrode pacing catheter is placed into the right atrium through the superior vena cava avoiding any mechanical damage to the atrium, and pacing stimulation is delivered through the catheter. An electrophysiological study is performed with programmed stimulation including constant pacing, burst pacing, and up to triple extrastimuli pacing. Under a spontaneous or pacing rhythm, the optical mapping recorded the action potential duration, activation map, conduction velocity, and Ca2+ transient individually in the right and left atria. In addition, the programmed stimulation also determines the inducibility of atrial tachyarrhythmias. Precise activation mapping is performed to identify the propagation of the excitation in the atrium during an induced atrial tachyarrhythmia. Optical mapping with a specialized setting enables a thorough electrophysiological evaluation of the atrium in murine pathological models.

This protocol describes the electrophysiological evaluation of murine atria utilizing an optical mapping system with a high temporal and spatial resolution, including dual recordings of the membrane voltage and Ca2+ transient under programmed stimulation through a specialized electrode catheter.

Recent genome-wide association studies targeting atrial fibrillation (AF) have indicated a strong association between the genotype and ...

Assessing Neuroprotective Effects of Glycyrrhizae Radix et Rhizoma Extract Using a Transient Middle Cerebral Artery Occlusion Mouse Model

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used animal model for studying stroke is the middle cerebral artery occlusion (MCAO) model. Previous research studies have reported different infarct sizes even when the same experimental animal species was used under similar MCAO conditions. Therefore, we developed an improved experimental method to address this discrepancy. Mice were subjected to MCAO using a filament as the occlusion material to mimic human stroke conditions and filament thickness was optimized to establish more reproducible infarction volume. Mice treated with a methanol extract of Glycyrrhizae Radix et Rhizome (GRex) following stroke induction showed a significantly decreased total infarction volume and increased number of surviving cells relative to the untreated control group. This modified experimental protocol successfully and reproducibly demonstrated the beneficial effect of GRex on ischemic stroke.

In this study, we modify an existing experimental method to obtain more reproducible results, by establishing a middle cerebral artery occlusion (MCAO) mouse model. Oral administration of Glycyrrhizae Radix et Rhizome (GR) methanol extract (GRex), following stroke induction, significantly decreased total infarction volume relative to the untreated control group.

Ischemia followed by reperfusion of cerebral blood flow after a stroke leads to the death of nerve cells and loss of brain tissue. The most commonly used ...

High-Resolution Cardiac Positron Emission Tomography/Computed Tomography for Small Animals

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding cardiac function and metabolism. In preclinical research, dedicated scanners with high sensitivity and high spatio-temporal resolution are employed, designed to cope with the demanding technological requirements posed by the small heart size and very high heart rates of mice and rats. In this paper, a bimodal cardiac PET/CT imaging protocol for experimental mouse and/or rat models of cardiac diseases is described, from animal preparation and image acquisition and reconstruction to image processing and visualization.
In particular, the 18F-labeled fluorodeoxyglucose ([18F]FDG)-PET scan allows for the measurement and visualization of glucose metabolism in the different segments of the left ventricle (LV). Polar maps are convenient tools to display this information. The CT part consists of a time-resolved 3D reconstruction of the entire heart (4D-CT) using retrospective gating without electrocardiography (ECG) leads, allowing the morphofunctional evaluation of the LV and the subsequent quantification of the most important cardiac function parameters, such as ejection fraction (EF) and stroke volume (SV). Using an integrated PET/CT scanner, this protocol can be executed within the same anesthesia induction without the need to reposition the animal between different scanners. Hence, PET/CT can be seen as a comprehensive tool for the morphofunctional and metabolic evaluation of the heart in several small animal models of cardiac diseases.

Here, we present an experimental imaging protocol for the quantification of cardiac function and morphology using high-resolution positron emission tomography/computed tomography for small animals. Both mice and rats are considered, discussing the different requirements of computed tomography contrast agents for the two species.

Positron emission tomography (PET) and computed tomography (CT) are among the most employed diagnostic imaging techniques, and both serve in understanding ...

Determining Respiration in Human Sperm Cells by High-Resolution Respirometry

High-Resolution Respirometry to Assess Mitochondrial Function in Human Spermatozoa

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm mitochondrial activity, so the measurement of sperm mitochondrial function is an indicator of sperm quality. High-resolution respirometry is a method of measuring the oxygen consumption of cells or tissues in a closed-chamber system. This technique can be implemented to measure respiration in human sperm and provides information about the quality and integrity of the sperm mitochondria. High-resolution respirometry allows the cells to move freely, which is an a priori advantage in the case of sperm. This technique can be applied with intact or permeabilized spermatozoa and allows for the study of intact sperm mitochondrial function and the activity of individual respiratory chain complexes. The high-resolution oxygraph instrument uses sensors to measure the oxygen concentration coupled with sensitive software to calculate the oxygen consumption. The data are used to calculate respiratory indices based on the oxygen consumption ratios. Consequently, the indices are the proportions of two oxygen consumption rates and are internally normalized to the cell number or protein mass. The respiratory indices are an indicator of sperm mitochondrial function and dysfunction.

Author Spotlight: Advancing Male Infertility Research by Unraveling Sperm Metabolism and Mitochondrial Function 

The analysis of sperm mitochondrial function by high-resolution respirometry permits the measurement of the oxygen consumption of freely moving spermatozoa in a closed-chamber system. The technique can be applied to measure respiration in human spermatozoa, which provides information on sperm mitochondrial characteristics and integrity.

Semen quality is often studied by routine semen analysis, which is descriptive and often inconclusive. Male infertility is associated with altered sperm ...