The authors gratefully acknowledge financial support from the following sources. The development and testing of various neuroimaging analyses was supported by several grants, most notably from the Canadian Institutes of Health Research (MOP#13129), the Alzheimer Society of Canada and Alzheimer Association (US), the Heart and Stroke Foundation Canadian Partnership for Stroke Recovery (HSFCPSR), and the LC Campbell Foundation. JR receives salary support from the Alzheimer Society of Canada; SEB from the Sunnybrook Research Institute and the Departments of Medicine at Sunnybrook and U of T, including the Brill Chair in Neurology. Authors also receive salary support from the HSFCPSR.

<ol>
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Q., Kovacevic, N., Roy, P., Rockel, C., O'Toole, C. M., Lobaugh, N. J., Feinstein, A., Levine, B., Black, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semiautomatic+brain+region+extraction:+a+method+of+parcellating+brain+regions+from+structural+magnetic+resonance+images.">Semiautomatic brain region extraction: a method of parcellating brain regions from structural magnetic resonance images.</a> Neuroimage. 22 (4), 1492-1502 (2004).</li><li>Shrout, P. E., Fleiss, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraclass+correlations:+uses+in+assessing+rater+reliability.">Intraclass correlations: uses in assessing rater reliability.</a> Psychol. Bull. 86, 420-428 (2008).</li><li>Zijdenbos, A. P., Dawant, B. M., Margolin, R. A., Palmer, A. 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The authors have nothing to disclose.

The LE segmentation and parcellation procedure was developed specifically to obtain regional volumetrics from MRI of AD and normal elderly. While there are numerous fully automatic pipelines which apply complex computational algorithms to perform these operations, these tools tend to lack the individualized accuracy and precision that LE&#8217;s semi-automatic pipeline produces. The trade-off with semi-automatic processes are the resources required to properly train operators with the anatomical knowledge and computational skills needed to apply such a comprehensive pipeline. However, one of the primary benefits of an individualized imaging pipeline is the ability to obtain quantitative volumetrics from moderate to severe cases of neurodegeneration when automatic pipelines fail.
As the LE pipeline has been previously evaluated and applied to various elderly and demented populations1,2,13,14,19,22,23, the main issues that are typically encountered by trained operators have been well-documented and are summarized below.
The manual checking and editing required with the Brain-Sizer component includes the TIV extraction masking procedure, vCSF reassignment and manual removal of the brain stem, cerebellum and other subtentorial structures. For brain extraction, the automatic TIV output is generally a decent mask provided that the original PD/T2 images are good quality. However, due to the relative intensity values of vascular and nerve tissue medial to the inferior temporal poles, proximal to the carotid arteries, this region typically requires some editing. Additionally, mucous in the nasal cavity tends to affect regional intensity histograms, skewing intensity cut-offs values in the anterior frontal regions, which tend to require additional manual editing of the automatic TIVauto mask. Finally, additional manual editing is typically required in the most superior regions, where global atrophy tends to result in an increase in the volume of subarachnoid CSF just below the dura mater. Alternatively, atrophy associated with ventricular enlargement tends to minimize operator interventions required with vCSF reassignment. Another benefit of having a tri-feature coregistration approach is the ability to identify cystic fluid-filled infarcts proximal to the ventricles, potentially due to periventricular venous vasculopathy5,24-26, which are identifiable due to their relative intensity on PD and T1 (hyperintense on PD, hypointense on T1). These hypointensities can be delineated from vCSF using manual limits drawn in ITK-SNAP_sb prior to floodfilling operations. Since vCSF reassignment is performed in T1-acquisition space, in cases where alignment deviates far from the ACPC plane, a limit may be required for the 3rd ventricle and the quadrigeminal cistern, if the PC is not fully visible. Although the tentorium is a relatively easy structure to differentiate, several anatomy-based rules assist in guiding manual removal of the brain stem and subtentorial structures, particularly when locating the separation of the cerebral peduncles from the medial temporal lobe.
SABRE landmarking is a stereotaxic-based procedure performed in standard ACPC aligned images, allowing for moderately predictable localization of particular anatomical landmarks. Exceptions to this are cases with extreme atrophy and normal variability due to individual differences in neuroanatomy. Brain atrophy results in an overall loss of parenchyma, increasing CSF along the midline surrounding the falx cerebri, which increases the difficulty of choosing appropriate points to place landmarks. Rule-based protocols are required, identifying cases where exceptions to the general rule are required. Normal variations in anatomy, particularly in the relative location of the central sulcus and the parieto-occipital sulcus, also increase the difficulty of manual delineation of these structures. However, the graphical user interface used by SABRE allows for real-time rotation of surface rendered images, which significantly assists in the decision-making process for visualization of these particular landmarks. Finally, some rule-based protocol have been integrated programmatically into the software to prevent operator violation e.g. central sulcus delineation is forced to move posteriorly (line tracing is prevented from going back onto itself).
The Lesion-Seg component&#8217;s manual checking procedure requires expertise in visual identification of relevant hyperintensities, a visual perception skill that is only acquired after exposure to scans with varying degrees of SH. False-positive minimization algorithms assist with the removal of most errors in the initial segmentation. However, differentiation between dilated perivascular spaces (Virchow-Robin spaces: VRS) in the lentiform nucleus and relevant SH in the external capsule, claustrum, extreme capsule, and subinsular regions can be difficult. This is particularly difficult in cases with VRS in the basal ganglia. A recent paper outlining STandards for ReportIng Vascular changes on nEuroimaging (STRIVE), recommended a size criterion to differentiate VRS from lacunes, and describe VRS to be more linear and CSF intensity on MRI. To address these issues with VRS identification, LE has adopted: a) an anatomy-based rule which prevents operators from selecting any hyperintensity that falls within the lentiform nucleus, b) a size criterion to exclude hyperintensities less than 5mm in diameter, and c) a relative intensity rule for additional exclusion due to the relative CSF intensity on PD, T2 and T127. Additionally, normal hyperintense signal can be found along the midline and falx cerebri, particularly on FLAIR imaging, which can be difficult to differentiate between relevant SH along the corpus callosum. In cases of such overlap, anatomy-based rules are implemented where only SH which extend out into the periventricular regions are accepted.
In conclusion, it is important to appreciate that this written component is meant to supplement a video-guided, standardized protocol publication in JoVE (<a href="http://www.jove.com">http://www.jove.com</a>). While traditional static figures assist in explaining some concepts, video-based tutorials are more efficient at communicating the complex methodological processes involved with a comprehensive neuroimaging pipeline such as Lesion Explorer.

Brain image analysis is an emerging field of neuroscience requiring skilled operators with a high degree of computational and neuroanatomical competency. In order to obtain quantitative information from magnetic resonance imaging (MRI), a trained operator is often required to implement, monitor, and edit, computer-generated imaging outputs generated from raw MRIs. While many &#8216;fully automatic' imaging tools are freely available via the internet, accuracy, and reliability is questionable when applied by a novice operator lacking knowledge, training and familiarity with the downloaded tool. Although on-site training is the most preferable teaching approach, the presentation of a video-guided, standardized protocol is a viable alternative, particularly if accompanied by a training set of images. Additionally, the training set of images may be used for quality control measures, such as an off-site inter-rater reliability test.
The challenges of developing an image processing pipeline, particularly when studying aging and Alzheimer's disease (AD), include a wide range of technical and biological issues. Although some technical issues are addressed with post-processing correction algorithms3, variability due to individual differences and pathological processes introduce more complex obstacles. Brain atrophy and ventricular enlargement can reduce the viability of registration warping and template-matching approaches. The presence of age-related white matter changes4 and small vessel disease5,6, observed as subcortical hyperintensities (SH)7,8, cystic fluid-filled lacunar-like infarcts9,10, and dilated perivascular spaces11,12, further complicate segmentation algorithms. In cases of significant white matter disease, a single T1 segmentation could result in overestimation of gray matter (GM)13, which can only be corrected with an additional segmentation using proton density (PD), T2-weighted (T2), or fluid-attenuated inversion recovery (FLAIR) imaging. In light of these challenges, the Lesion Explorer (LE) image processing pipeline implements a semi-automatic tri-feature (T1, PD, T2) approach, utilizing trained operators at particular stages when human intervention is preferable1,2.
Brain extraction (or skull stripping) is typically one of the first operations performed in neuroimaging. Given this, the accuracy of the total intracranial vault (TIV) extraction process greatly influences subsequent operations further down the pipeline. Significant over-erosion, resulting in loss of brain, may lead to over-estimation of brain atrophy. Alternatively, significant under-erosion, resulting in inclusion of dura and other nonbrain matter, may lead to inflation of brain volumes. LE's Brain-Sizer component addresses many of these issues by using a tri-feature (T1, T2, and PD) approach to generate a TIV mask, which yields superior results compared to single-feature methods1. Additionally, the automatically generated TIV mask is manually checked and edited using standardized protocol which identifies regions susceptible to skull stripping errors. After brain extraction, segmentation is performed on the skull-stripped T1, where each brain voxel is assigned to 1 of 3 labels: GM, white matter (WM), or cerebrospinal fluid (CSF). Segmentation is accomplished automatically using a robust curve-fitting algorithm applied to global and local intensity histograms; a technique developed to address intensity nonuniformity artifact and a decreased separation between GM and WM intensity amplitude in AD cases14.
The Brain-Sizer component also includes procedures for manual designation of ventricles and removal of subtentorial structures. Segmentation of ventricular CSF (vCSF) is particularly important as ventricle size is a commonly used biomarker for AD dementia15. Additionally, delineation of ventricles and choroid plexus is imperative for proper identification of periventricular hyperintensities (pvSH), which are believed to reflect a form of small vessel disease characterized by venous collagenosis5,16,17. Using T1 for reference, manual relabeling of CSF voxels to vCSF is accomplished with manual floodfill operations on the segmented image. Typically, the lateral ventricles are easier to differentiate from sulcal CSF. For this reason, it is recommended to begin floodfilling in axial view, starting from superior slices and moving inferiorly. The medial parts of the ventricular system, particularly the 3rd ventricle, is more difficult to delineate and is given special anatomy-based rules which are outlined in the manual. Brain-Sizer's final step includes removal of the brain stem, cerebellum, and other subtentorial structures, using manual tracing procedures described in an additional set of anatomy-based standardized protocols.
The Semi-Automated Brain Region Extraction (SABRE) component is the pipeline's parcellation procedure. This stage requires trained operators to identify the following anatomical landmarks: anterior and posterior commissure (AC, PC); posterior brain edge; central canal; mid-sagittal plane; preoccipital notch; occipito-parietal sulcus; central sulcus, and; Sylvian fissure. Based on these landmark coordinates, a Talairach-like18 grid is automatically generated and regional parcellation is accomplished19. Landmarks are easily identified on ACPC aligned images, which are generated automatically and manually checked prior to SABRE landmarking procedures.
The Lesion-Seg component is the final stage of the pipeline where SH identification and quantification is accomplished. The initial automatic SH segmentation implements a complex algorithm which includes PD/T2-based SH segmentation, fuzzy c-means masking, and ventricular dilatation. These operations result in an automatically generated lesion segmentation mask that is manually checked and edited for false positives and other errors. As hyperintense signal on MRI may result from nonpathological sources (e.g. motion artifact, normal biology), proper training is required for accurate identification of relevant SH.
The final result of the LE pipeline is a comprehensive volumetric profile containing 8 different tissue and lesion volumetrics which are parcellated into 26 SABRE brain regions. To obtain an individual operator's inter-rater reliability test off-site, it is recommended to execute the full LE pipeline on the training set provided with the software (http://sabre.brainlab.ca). Using the volumetric results, inter-class correlation coefficient (ICC)20 statistics can be calculated for each tissue class (GM/WM/CSF) in each SABRE region. Using the segmentation images, Similarity Index (SI)21 statistics can be calculated to evaluate the degree of spatial congruence. Additionally, intra-rater reliability can be assessed on the same operator's results, after a brief period of time has passed between the operator's 1st and 2nd segmentation edits. Provided that the off-site operator adheres to the file naming conventions outlined in the LE manual, reliability statistics can be calculated off-site using most basic statistical software packages. Given these quality control and video-guided standardized protocol, off-site operators can have greater confidence that the LE pipeline is applied accurately and reliably.

<table border="0" cellpadding="0" cellspacing="0" width="815">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Magnetic resonance imaging machine (1.5 Tesla)</td>
			<td style="width:91px;">General Electric</td>
			<td style="width:119px;"></td>
			<td style="width:273px;">See Table 1 for acquisition parameters</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:333px;">Magnetic resonance imaging machine (3 Tesla)</td>
			<td style="width:91px;">General Electric</td>
			<td style="width:119px;"></td>
			<td>See Table 2 for acquisition parameters</td>
		</tr>
	</tbody>
</table>

lesion explorer: a video-guided, standardized protocol for accurate and reliable mri-derived volumetrics in alzheimer's disease and normal elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are exacerbated when significant brain atrophy and age-related white matter changes (e.g. Leukoaraiosis) are present. Lesion Explorer (LE) is an accurate and reliable neuroimaging pipeline specifically developed to address such issues commonly observed on MRI of Alzheimer's disease and normal elderly. The pipeline is a complex set of semi-automatic procedures which has been previously validated in a series of internal and external reliability tests1,2. However, LE's accuracy and reliability is highly dependent on properly trained manual operators to execute commands, identify distinct anatomical landmarks, and manually edit/verify various computer-generated segmentation outputs.
LE can be divided into 3 main components, each requiring a set of commands and manual operations: 1) Brain-Sizer, 2) SABRE, and 3) Lesion-Seg. Brain-Sizer's manual operations involve editing of the automatic skull-stripped total intracranial vault (TIV) extraction mask, designation of ventricular cerebrospinal fluid (vCSF), and removal of subtentorial structures. The SABRE component requires checking of image alignment along the anterior and posterior commissure (ACPC) plane, and identification of several anatomical landmarks required for regional parcellation. Finally, the Lesion-Seg component involves manual checking of the automatic lesion segmentation of subcortical hyperintensities (SH) for false positive errors.
While on-site training of the LE pipeline is preferable, readily available visual teaching tools with interactive training images are a viable alternative. Developed to ensure a high degree of accuracy and reliability, the following is a step-by-step, video-guided, standardized protocol for LE's manual procedures.

Inter-rater reliability can be assessed using several metrics. Using the training set provided online (<a href="http://sabre.brainlab.ca">http://sabre.brainlab.ca</a>), the following steps are recommended to assess inter-rater reliability for each of the processing stages after completion of LE.
Brain-Sizer: 
To assess inter-rater reliability of the brain extraction procedures, generate volumetrics for each TIV-E masks, &#60;name&#62;_TIVedit, using the &#60;img_count&#62; command. Enter these volumetrics into a statistical software package (e.g. SPSS), along with the TIVedit volumetrics provided for each of the training set (see Excel/csv file provided online) and calculate the inter-rater correlation coefficient (ICC). Whole brain volumetrics for in-house trained raters obtain reported ICC=0.99, p&#60;0.0001 1,2. Additionally, evaluation of the spatial agreement for the TIV masking can be assessed using the SI21. MATLAB code is provided online to calculate SI values between two raters.
To assess ventricular reassignment, generate vCSF volumes using the &#60;img_count&#62; command for each of the segmentation files with the vCSF voxels reassigned, i.e. &#60;name&#62;_ seg_vcsf. The vCSF volume is the value beside row &#8216;7&#8217; under the column titled &#8216;volume&#8217;. Using the same procedures to evaluate TIV inter-rater reliability, calculate ICC and SI for vCSF. 
Removal of brain stem, cerebellum and subtentorial structures can be assessed similarly by running the &#60;img_count&#62; command on &#60;name&#62;_seg_vcsf_st. The volumes used for this segmentation mask are shown at the second last row titled &#8216;total count of nonzero voxels:&#8217; under &#8216;volume&#8217; (the last column on the right). Using the same procedures to evaluate TIV and vCSF, calculate ICC and SI for this masking procedure using the volumetrics in the excel file provided and the &#60;name&#62;_seg_vcsf_st files.
SABRE: 
While Brain-Sizer&#8217;s manual procedures can easily be assessed using standard metrics, ACPC alignment is slightly more difficult. For this reason, matrix files are provided to compare visually for training of off-site operators. After completion of ACPC alignment, open a new ITK-SNAP_sb window, load the T1 image, then load the matrix for the training case provided online, &#60;name&#62;_T1_IHCpre_toACPC.mat, and visually compare the pitch, roll, yaw, and ACPC slice between the two images.
To evaluate SABRE landmarking procedures, run &#60;img_count&#62; on the parcellated mask, &#60;name&#62;_SABREparcel_inACPC for each training case. Enter the volumetrics for each region (3-28). SABRE region codes are provided online. Using the same procedures to evaluate TIV and vCSF, calculate ICC for each SABRE brain region. SABRE parcellated regional volumetrics for in-house trained raters obtain reported mean ICCs=0.98, p&#60;0.01, with ICC values ranging from 0.91-0.99 1,2.
Lesion-Seg: 
As this component is the final stage of the LE pipeline, reliability and accuracy will depend on the prior stages. 
Inter-rater reliability of SH segmentation is accomplished using regional ICC of SH volumes and spatial agreement of the SH masks. To evaluate regional SH volumes, run &#60;SH_volumetrics&#62;, entering both the lobmask file in T1-acquisition space, &#60;name&#62;_SABREparcel and the final edited lesion segmentation file, &#60;name&#62;_LEedit. Using the same procedures to evaluate SABRE volumetrics, calculate ICC for lesion volumes within each SABRE brain region. Using the same procedures to evaluate spatial agreement of the TIV masking process, calculate SI for the final edited lesion masks, &#60;name&#62;_LEedit (or FLEXedit). The same reliability tests can be performed on both PD/T2-based segmentation and FLAIR-based segmentation.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>3D T1</td>
			<td>PD/T2</td>
		</tr>
		<tr>
			<td>Imaging Parameters</td>
			<td>Axial Volume SAT (S1) SPGR</td>
			<td>Axial Spin Echo FC VEMP VB (interleave)</td>
		</tr>
		<tr>
			<td>Pulse Timing</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TE (msec)</td>
			<td>5</td>
			<td>30/80</td>
		</tr>
		<tr>
			<td>TR (msec)</td>
			<td>35</td>
			<td>3,000</td>
		</tr>
		<tr>
			<td>Flip Angle (&#176;)</td>
			<td>35</td>
			<td>90</td>
		</tr>
		<tr>
			<td>TI (msec)</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
		<tr>
			<td>Scan Range</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FOV (cm)</td>
			<td>22</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Slice thickness (mm)</td>
			<td>1.2/0</td>
			<td>3/0</td>
		</tr>
		<tr>
			<td>No. Slices</td>
			<td>124</td>
			<td>62</td>
		</tr>
		<tr>
			<td>Acquisition</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix size</td>
			<td>256 x 192</td>
			<td>256 x 192</td>
		</tr>
		<tr>
			<td>Voxel size (mm)</td>
			<td>0.86 x 0.86 x 1.4</td>
			<td>0.78 x 0.78 x 3</td>
		</tr>
		<tr>
			<td>NEX</td>
			<td>1</td>
			<td>0.5</td>
		</tr>
		<tr>
			<td>Total Time (min)</td>
			<td>11:00</td>
			<td>12:00</td>
		</tr>
	</tbody>
</table>
Table 1. General Electric 1.5T Structural MRI Acquisition Parameters.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td></td>
			<td>3D T1</td>
			<td>PD/T2</td>
			<td>FLAIR</td>
		</tr>
		<tr>
			<td>Imaging Parameters</td>
			<td>Axial 3D FSPGR EDR IR Prep</td>
			<td>Axial 2D FSE-XL, EDR, FAST, fat sat</td>
			<td>Axial T2Flair, EDR, FAST</td>
		</tr>
		<tr>
			<td>Pulse Timing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>
			<h2>TE (ms)</h2>
			</td>
			<td>3.2</td>
			<td>11.1 / 90</td>
			<td>140</td>
		</tr>
		<tr>
			<td>TR (msec)</td>
			<td>8.1</td>
			<td>2,500</td>
			<td>9,700</td>
		</tr>
		<tr>
			<td>Flip Angle (&#176;)</td>
			<td>8&#176;</td>
			<td>90&#176;</td>
			<td>90&#176;</td>
		</tr>
		<tr>
			<td>TI (msec)</td>
			<td>650</td>
			<td>N/A</td>
			<td>2,200</td>
		</tr>
		<tr>
			<td>Scan Range</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FOV (cm)</td>
			<td>22</td>
			<td>22</td>
			<td>22</td>
		</tr>
		<tr>
			<td>Slice thickness (mm)</td>
			<td>1</td>
			<td>3</td>
			<td>3</td>
		</tr>
		<tr>
			<td>No. Slices</td>
			<td>186</td>
			<td>48</td>
			<td>48</td>
		</tr>
		<tr>
			<td>Acquisition</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrix size</td>
			<td>256 x 192</td>
			<td>256 x 192</td>
			<td>256 x 192</td>
		</tr>
		<tr>
			<td>Voxel size (mm)</td>
			<td>0.86 x 0.86 x 1</td>
			<td>0.86 x 0.86 x 3</td>
			<td>0.86 x 0.86 x 3</td>
		</tr>
		<tr>
			<td>NEX</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Total Time (min)</td>
			<td>7:20</td>
			<td>6:10</td>
			<td>7:20</td>
		</tr>
	</tbody>
</table>
Table 2. General Electric 3T Structural MRI Acquisition Parameters. 
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/50887/50887fig1highres.jpg" width="500" /> 
Figure 1. Axial T1 with unedited total intracranial vault (TIV) mask overlay (green). This is an example of the use of the closed polygon tool in ITK-SNAP_sb to remove nonbrain tissue as part of the manual editing procedure of the Brain-Sizer&#8217;s TIV extraction procedure.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/50887/50887fig2highres.jpg" width="500" /> 
Figure 2. Axial T1 with tissue segmentation overlay. Note that label colors are arbitrary and can be modified using the Label tool. Left image shows default colors. Middle image shows how CSF (5=purple) is reassigned to vCSF (7=magenta). Right image shows how the WM color can be modified without changing the tissue class label, i.e. Label 3=WM remains but color can be modified to blue.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/50887/50887fig3highres.jpg" width="500" /> 
Figure 3. Axial T1 with tissue segmentation overlay (left image, GM=yellow, WM=orange, CSF =purple) (left). Depicted is an example of manual removal of subtentorial structures using the closed polygon tool in ITK-SNAP_sb (middle) and final tissue segmentation after removal (right). As in Figure 2, right image shows how the WM color can be modified without changing the tissue class label, i.e. Label 3=WM remains but color can be modified to blue.
<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/50887/50887fig4highres.jpg" width="500" /> 
Figure 4. Axial T1 in acquisition space before (left), and after (right) AC-PC alignment is performed.
<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/50887/50887fig5highres.jpg" width="500" /> 
Figure 5. Two examples showing SABRE landmarking procedures. Axial AC-PC aligned T1 with AC (yellow), PC (blue), and posterior edge (pink) landmark placements (left). A 3D surface-rendered T1 (right) with Sylvian fissure (purple) and central sulcus (pink) delineation.
<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/50887/50887fig6highres.jpg" width="500" /> 
Figure 6. Axial PD (left) with automatically generated lesion overlay (center), and manually edited lesion (red) overlay (right).
<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/50887/50887fig7highres.jpg" width="500" /> 
Figure 7. Axial FLAIR (left), with automatically generated lesion overlay (center), and manually edited lesion (red) overlay (right).

Watch this Scientific Journal Video about Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly at JoVE.com

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer&#039;s Disease and Normal Elderly

Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Obtaining in vivo human brain tissue volumetrics from MRI is often complicated by various technical and biological issues. These challenges are ...

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39.5K Views.  Sunnybrook Health Sciences Centre. Lesion Explorer (LE) is a semi-automatic, image-processing pipeline developed to obtain regional brain tissue and subcortical hyperintensity lesion volumetrics from structural MRI of Alzheimer's disease and normal elderly. To ensure a high level of accuracy and reliability, the following is a video-guided, standardized protocol for LE's manual procedures.

Video: Lesion Explorer: A Video-guided, Standardized Protocol for Accurate and Reliable MRI-derived Volumetrics in Alzheimer's Disease and Normal Elderly

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&beta; using a Whole Blood Stimulation Assay

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues as well as altered cytokine homeostasis. The innate immune system plays a crucial role in recognizing pathogens and other endogenous danger stimuli. One of the major cytokines released by innate immune cells is Interleukin (IL)-1. Therefore, we utilize a whole blood stimulation assay in order to measure the secretion of inflammatory cytokines and specifically of the pro-inflammatory cytokine IL-1&beta; 1, 2, 3.
Patients with genetic dysfunctions of the innate immune system causing autoinflammatory syndromes show an exaggerated release of mature IL-1&beta; upon stimulation with LPS alone. In order to evaluate the innate immune component of patients who present with inflammatory-associated pathologies, we use a specific immunoassay to detect cellular immune responses to pathogen-associated molecular patterns (PAMPs), such as the gram-negative bacterial endotoxin, lipopolysaccharide (LPS). These PAMPs are recognized by pathogen recognition receptors (PRRs), which are found on the cells of the innate immune system 4, 5, 6, 7. A primary signal, LPS, in conjunction with a secondary signal, ATP, is necessary for the activation of the inflammasome, a multiprotein complex that processes pro-IL-1&beta; to its mature, bioactive form 4, 5, 6, 8, 9, 10.
The whole blood assay requires minimal sample manipulation to assess cytokine production when compared to other methods that require labor intensive isolation and culturing of specific cell populations. This method differs from other whole blood stimulation assays; rather than diluting samples with a ratio of RPMI media, we perform a white blood cell count directly from diluted whole blood and therefore, stimulate a known number of white blood cells in culture 2. The results of this particular whole blood assay demonstrate a novel technique useful in elucidating patient cohorts presenting with autoinflammatory pathophysiologies.

Accurate and Simple Measurement of the Pro-inflammatory Cytokine IL-1&amp;#946; using a Whole Blood Stimulation Assay

We describe a simple immunoassay to measure the production of pro-inflammatory cytokines, such as IL-1 beta production, in patients presenting with autoinflammatory phenotypes. By activating cells in whole blood cultures with pathogen-associated molecular patterns, specifically with lipopolysaccharide, cytokine secretion can be conveniently evaluated in whole blood supernatants.

Inflammatory processes resulting from the secretion of soluble mediators by immune cells, lead to various manifestations in skin, joints and other tissues ...

Isolation of Soluble and Insoluble PrP Oligomers in the Normal Human Brain

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform PrPSc 1. PrPC is detergent-soluble and sensitive to proteinase K (PK)-digestion, whereas PrPSc forms detergent-insoluble aggregates and is partially resistant to PK2-6. The conversion of PrPC to PrPSc is known to involve a conformational transition of &alpha;-helical to &beta;-sheet structures of the protein. However, the in vivo pathway is still poorly understood. A tentative endogenous PrPSc, intermediate PrP* or "silent prion", has yet to be identified in the uninfected brain7.
Using a combination of biophysical and biochemical approaches, we identified insoluble PrPC aggregates (designated iPrPC) from uninfected mammalian brains and cultured neuronal cells8, 9. Here, we describe detailed procedures of these methods, including ultracentrifugation in detergent buffer, sucrose step gradient sedimentation, size exclusion chromatography, iPrP enrichment by gene 5 protein (g5p) that specifically bind to structurally altered PrP forms10, and PK-treatment. The combination of these approaches isolates not only insoluble PrPSc and PrPC aggregates but also soluble PrPC oligomers from the normal human brain. Since the protocols described here have been used to isolate both PrPSc from infected brains and iPrPC from uninfected brains, they provide us with an opportunity to compare differences in physicochemical features, neurotoxicity, and infectivity between the two isoforms. Such a study will greatly improve our understanding of the infectious proteinaceous pathogens. The physiology and pathophysiology of iPrPC are unclear at present. Notably, in a newly-identified human prion disease termed variably protease-sensitive prionopathy, we found a new PrPSc that shares the immunoreactive behavior and fragmentation with iPrPC 11, 12. Moreover, we recently demonstrated that iPrPC is the main species that interacts with amyloid-&beta; protein in Alzheimer disease13. In the same study, these methods were used to isolate Abeta aggregates and oligomers in Alzheimer's disease13, suggesting their application to non-prion protein aggregates involved in other neurodegenerative disorders.

A new species of cellular prion protein (PrPC) has recently been identified in uninfected human brains using the methods described here. These methods can be used to isolate various PrP species, while some of them are also useful in isolating other misfolded protein aggregates from human brains.

The central event in the pathogenesis of prion diseases involves a conversion of the host-encoded cellular prion protein PrPC into its pathogenic isoform ...

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Fast and Accurate Exhaled Breath Ammonia Measurement

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that uses a quantum cascade based laser. The monitor is coupled to a sampler that measures mouth pressure and carbon dioxide. The system is temperature controlled and specifically designed to address the reactivity of this compound. The sampler provides immediate feedback to the subject and the technician on the quality of the breath effort. Together with the quick response time of the monitor, this system is capable of accurately measuring exhaled breath ammonia representative of deep lung systemic levels. 
Because the system is easy to use and produces real time results, it has enabled experiments to identify factors that influence measurements. For example, mouth rinse and oral pH reproducibly and significantly affect results and therefore must be controlled. Temperature and mode of breathing are other examples. As our understanding of these factors evolves, error is reduced, and clinical studies become more meaningful. This system is very reliable and individual measurements are inexpensive. 
The sampler is relatively inexpensive and quite portable, but the monitor is neither. This limits options for some clinical studies and provides rational for future innovations.

Ammonia is an important physiologic metabolite relevant to various disease and wellness states. It is also a difficult molecule to measure in breath, which demands particular precautions be taken to obtain accurate results. Not all factors influencing ammonia are known, but progress can be difficult without accounting for these factors.

This exhaled breath ammonia method uses a fast and highly sensitive spectroscopic method known as quartz enhanced photoacoustic spectroscopy (QEPAS) that ...

Experimental and Imaging Techniques for Examining Fibrin Clot Structures in Normal and Diseased States

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on fibrin in the past years to include the investigation of synthesis, structure-function, and lysis of clots. However, there is still much unknown about the morphological and structural features of clots that ensue from patients with disease. In this research study, experimental techniques are presented that allow for the examination of morphological differences of abnormal clot structures due to diseased states such as diabetes and sickle cell anemia. Our study focuses on the preparation and evaluation of fibrin clots in order to assess morphological differences using various experimental assays and confocal microscopy. In addition, a method is also described that allows for continuous, real-time calculation of lysis rates in fibrin clots. The techniques described herein are important for researchers and clinicians seeking to elucidate comorbid thrombotic pathologies such as myocardial infarctions, ischemic heart disease, and strokes in patients with diabetes or sickle cell disease.

In this manuscript, experimental techniques, including blood preparation, confocal microscopy, and lysis rate analysis, to examine the morphological differences between normal and abnormal clot structures due to diseased states are presented.

Fibrin is an extracellular matrix protein that is responsible for maintaining the structural integrity of blood clots. Much research has been done on ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer's Disease with Stroke

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk factor for AD, stroke has also been implicated as a risk factor and an exacerbating factor. The co-morbidity of stroke and AD results in worsened stroke-related motor control and AD-related cognitive deficits when compared to each condition alone. To model the combined condition of stroke and AD, a novel transgenic rat model of AD, with a mutated form of amyloid precursor protein (a key protein involved in the development of AD) incorporated into its DNA, is given a small unilateral striatal stroke.
For a model with the combination of both stroke and AD, behavioral tests that assess stroke-related motor control, locomotion and AD-related cognitive function must be implemented. The cylinder task involves a cost-efficient, multipurpose apparatus that assesses spontaneous forelimb motor use. In this task, a rat is placed in a cylindrical apparatus, where the rat will spontaneously rear and contact the wall of the cylinder with its forelimbs. These contacts are considered forelimb motor use and quantified during video analysis after testing. Another cost-efficient motor task implemented is the beam-walk task, which assesses forelimb control, hindlimb control and locomotion. This task involves a rat walking across a wooden beam allowing for the assessment of limb motor control through analysis of forelimb slips, hindlimb slips and falls. Assessment of learning and memory is completed with Morris water maze for this behavioral paradigm. The protocol starts with spatial learning, whereby the rat locates a stationary hidden platform. After spatial learning, the platform is removed and both short-term and long-term spatial reference memory is assessed. All three of these tasks are sensitive to behavioral differences and completed within 28 days for this model, making this paradigm time-efficient and cost-efficient.

Motor and Hippocampal Dependent Spatial Learning and Reference Memory Assessment in a Transgenic Rat Model of Alzheimer&#039;s Disease with Stroke

To investigate the co-morbid Alzheimer&#39;s disease (AD) and stroke condition in a novel model, three behavior tasks are described that assess both motor control and cognitive behaviors. These tasks include the beam-walk task, cylinder task and Morris water maze.

Alzheimer&#39;s disease (AD) is a debilitating neurodegenerative disease that results in neurodegeneration and memory loss. While age is a major risk ...

A Step by Step Protocol for Subretinal Surgery in Rabbits

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in adults in industrially developed countries. RPE transplantation appears to be a promising therapy, as it may replace dysfunctional RPE, restore its function, and thereby vision.
Here we describe a method for transplanting a cultured RPE monolayer on a scaffold into the subretinal space (SRS) of rabbits. After vitrectomy xenotransplants were delivered into the SRS using a custom made shooter consisting of a 20-gauge metallic nozzle with a polytetrafluoroethylene (PTFE) coated plunger. The current technique evolved in over 150 rabbit surgeries over 6 years. Post-operative follow-up can be obtained using non-invasive and repetitive in vivo imaging such as spectral domain optical coherence tomography (SD-OCT) followed by perfusion-fixed histology.
The method has well-defined steps for easy learning and high success rate. Rabbits are considered a large eye animal model useful in preclinical studies for clinical translation. In this context rabbits are a cost-efficient and perhaps convenient alternative to other large eye animal models.

Retinal pigment epithelium (RPE) replacement strategies and gene-based therapy are considered for several retinal degenerative conditions. For clinical translation, large eye animal models are required to study surgical techniques applicable in patients. Here we present a rabbit model for subretinal surgery geared towards RPE transplantation, which is versatile and cost-efficient.

Age related macular degeneration (AMD), retinitis pigmentosa, and other RPE related diseases are the most common causes for irreversible loss of vision in ...

An Intramedullary Locking Nail for Standardized Fixation of Femur Osteotomies to Analyze Normal and Defective Bone Healing in Mice

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming more commonly used in trauma research. They offer a large number of mutant strains and antibodies for the analysis of the molecular mechanisms behind the highly differentiated process of bone healing. To control the biomechanical environment, standardized and well-characterized osteosynthesis techniques are mandatory in mice. Here, we report on the design and use of an intramedullary nail to stabilize open femur osteotomies in mice. The nail, made of medical-grade stainless steel, provides high axial and rotational stiffness. The implant further allows the creation of defined, constant osteotomy gap sizes from 0.00 mm to 2.00 mm. Intramedullary locking nail stabilization of femur osteotomies with gap sizes of 0.00 mm and 0.25 mm result in adequate bone healing through endochondral and intramembranous ossification. Stabilization of femur osteotomies with a gap size of 2.00 mm results in atrophic non-union. Thus, the intramedullary locking nail can be used in healing and non-healing models. A further advantage of the use of the nail compared to other open bone healing models is the possibility to adequately fix bone substitutes and scaffolds in order to study the process of osseous integration. A disadvantage of the use of the intramedullary nail is the more invasive surgical procedure, inherent to all open procedures compared to closed models. A further disadvantage may be the induction of some damage to the intramedullary cavity, inherent to all intramedullary stabilization techniques compared to extramedullary stabilization procedures.

This protocol describes an osteosynthesis technique using an intramedullary locking nail for standardized fixation of femur osteotomies, which can be used to analyze normal and defective bone healing in mice.

Bone healing models are essential to the development of new therapeutic strategies for clinical fracture treatment. Furthermore, mouse models are becoming ...

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer's Disease and Mild Cognitive Impairment

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior Cingulate Cortex (PCC) is a potential imaging marker for monitoring the progression of AD. Previous studies did not focus on the functional connectivity between the PCC and nodes in regions outside the DMN, but our study is an effort to explore these overlooked functional connections. For collecting data, we used functional Magnetic Resonance Imaging (fMRI) and Granger Causality Analysis (GCA). fMRI provides a non-invasive method for studying the dynamic interactions between the different brain regions. GCA is a statistical hypothesis test for determining whether one-time series is useful in forecasting another. In simple terms, it is judged by comparing the &#34;Known all the information on the last moment, the distribution of the probability of X at this time&#34; and the &#34;Known all the information on the last moment except Y, the distribution of the probability of X at this time&#34;, to determine whether there is a causal relationship between Y and X. This definition is based on the complete information source and stationary chronological sequence. The main step of this analysis is to use X and Y to establish the regression equation and draw a causal relationship by a hypothetical test. Since GCA can measure causal effects, we used it to investigate the anisotropy of the functional connectivity and explore the hub function of the PCC. Here, we screened 116 participants for MRI scanning, and after preprocessing the data obtained from neuroimaging, we used GCA to derive the causal relationship of each node. Finally, we concluded that the directed connection is significantly different between the Mild Cognitive Impairment (MCI) and AD groups, both from the PCC to the whole brain and from the whole brain to the PCC.

Application of Granger Causality Analysis of the Directed Functional Connection in Alzheimer&#039;s Disease and Mild Cognitive Impairment

Based on resting-state functional magnetic resonance imaging with Granger causality analysis, we investigated the alterations in the directed functional connectivity between the posterior cingulate cortex and whole brain in patients with Alzheimer&#39;s Disease (AD), patients with Mild Cognitive Impairment (MCI), and healthy controls.

Impaired functional connectivity in the Default Mode Network (DMN) may be involved in the progression of Alzheimer&#39;s Disease (AD). The Posterior ...