This work was in part supported by a grant from the National Cancer Institute, grant number U24CA264298. We thank Dawn Holley, Kim Halbert, and Mehdi Khalighi from the PET/MRI Metabolic Service Center for their assistance with the acquisition of PET/MRI scans at the Lucas Research Center at Stanford. We thank the members of the Daldrup-Link lab for valuable input and discussions regarding this project.

This protocol has been generated for a prospective clinical trial and co-clinical research. The study was compliant with the Health Insurance Portability and Accountability Act (HIPAA) and approved by the Stanford University institutional review board (IRB). All patients or their legally authorized representative signed a written informed consent, and all children between 7 and 18 years of age signed an assent form.
1. Installing and starting the T2 Fit Map plugin
<ol>
	<li>Start the Osirix software. Install the T2 Fit Map plugin from the plugin manager and restart the software.
	<ol>
		<li>On the menu bar, click the Plugins button. Click on the drop-down menu and select Install a plugin package (Figure 1).</li>
		<li>Once the plugin manager is loaded, select the available plugins from the drop-down menu and then T2 Fit Map (Figure 2).</li>
		<li>Click download &#38; install. Close the plugin manager and restart the software.</li>
	</ol>
	</li>
	<li>Load the multi-echo gradient echo sequence images as DICOM files into the software.</li>
	<li>Change the mouse button function to draw a Region of Interest (ROI) (Figure 3).</li>
	<li>Using this mouse button function, define a shape for the required ROI: select Oval or Closed Polygon, or the desired shape from the drop-down menu (Figure 4).</li>
	<li>Draw ROIs in the required images with different echo times (TEs).</li>
	<li>Select the ROIs in all the images with different TEs for which the T2* map is required.</li>
	<li>Click the Plugins button, select Image filters from the drop-down menu, and then select T2 Fit Map.</li>
	<li>Click on T2 Fit Map. A dialog box will open; click on Generate Map (which is located at the bottom of the dialog box) (Figure 5). 
	NOTE: A fitting curve is generated with minimum, mean, and maximum T2* values for the selected ROIs with various TEs (ms). The mean T2* value is calculated and displayed below the curve (Figure 6).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64773/64773fig01.jpg" /> 
Figure 1: Selecting &#39;Install a plugin package&#39; from the drop-down menu. <a href="https://www.jove.com/files/ftp_upload/64773/64773fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64773/64773fig02.jpg" /> 
Figure 2: Selecting &#39;T2 Fit Map&#39; from the available plugins. <a href="https://www.jove.com/files/ftp_upload/64773/64773fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64773/64773fig03.jpg" /> 
Figure 3: Screenshot demonstrating how to change the mouse button function to draw a region of interest (ROI). <a href="https://www.jove.com/files/ftp_upload/64773/64773fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64773/64773fig04.jpg" /> 
Figure 4: Screenshot showing how to select different shapes for the ROI. <a href="https://www.jove.com/files/ftp_upload/64773/64773fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64773/64773fig05.jpg" /> 
Figure 5: Screenshot showing how to select &#39;Generate Map&#39; after selecting &#39;T2 Fit Map&#39;. <a href="https://www.jove.com/files/ftp_upload/64773/64773fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64773/64773fig06.jpg" /> 
Figure 6: Screenshot showing the generation of a fitting curve for T2* values. <a href="https://www.jove.com/files/ftp_upload/64773/64773fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Exclusion of noise using the definition of mask
NOTE: To define a mask on the first echo of the T2map_MSME data used to calculate the parametric maps, set a lower first-echo signal threshold for T2* value calculation (the T2* map volume will now exclude the pixels with signals too low for accurate calculation at the first multi-spin multi-echo (MSME) echo. The threshold can be increased to exclude more pixels, or decreased to include more pixels).
<ol>
	<li>To define the mask ROI on an image series outside the parametric data, open the desired series (e.g., first echo of the T2map_MSME with TE = 15) and select a slice.</li>
	<li>At bottom of the ROI drop-down menu, select Grow Region&#8230;.</li>
	<li>Select the 3D Growing Region&#8230; radio button (Figure 7).</li>
	<li>In the Algorithm drop-down menu, select Threshold (lower/upper bounds).</li>
	<li>Set the lower and upper thresholds to 0 and X% of the contralateral calf muscle signal, respectively (e.g., set the threshold to the value that will mask out the most speckle in the tumor ROI and will leave the most evaluable tumor T2(*)s).</li>
	<li>Set the ROI name as desired.</li>
	<li>Click on the image to place a seed for ROI growing.</li>
	<li>Click the Compute button.</li>
	<li>In the ROI menu, select Save All ROIs of this Series&#8230; (Figure 8). 
	NOTE: Now, the ROI defining the area to mask out of the parametric maps has been saved and can be applied to the parametric data.</li>
	<li>Open the Parametric dataset in the 4D viewer.</li>
	<li>In the ROI menu, select Import ROI(s).... 
	NOTE: The mask ROI is now in the first parametric series.</li>
	<li>Check that the ROIs are in the first 3D volume and not 4D.</li>
	<li>Apply mask to map the data. To do so, at the bottom of the ROI drop-down menu, select Set Pixel Values To. Then, select apply to: ROIs with same name&#8230; (Figure 9).</li>
	<li>Check the Propagate to 4D series box.</li>
	<li>Set pixels that are Inside ROIs.</li>
	<li>Set To this new value: as 0.</li>
</ol>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/64773/64773fig07.jpg" /> 
Figure 7: Screenshot showing the segmentation parameters. <a href="https://www.jove.com/files/ftp_upload/64773/64773fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/64773/64773fig08.jpg" /> 
Figure 8: Screenshot showing how to select &#34;Save All ROIs of this Series&#8230;&#34;. <a href="https://www.jove.com/files/ftp_upload/64773/64773fig08large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/64773/64773fig09.jpg" /> 
Figure 9: Screenshot showing the values to be inputted in &#34;Set Pixel Values To&#34;. <a href="https://www.jove.com/files/ftp_upload/64773/64773fig09large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle-based+magnetic+resonance+imaging+on+tumor-associated+macrophages+and+inflammation.">Nanoparticle-based magnetic resonance imaging on tumor-associated macrophages and inflammation.</a> Frontiers in Immunology. 8, 590 (2017).</li><li>Chen, X., Qiu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pilot+study+of+short+T2*+measurements+with+ultrashort+echo+time+imaging+at+0.35+T.">A pilot study of short T2* measurements with ultrashort echo time imaging at 0.35 T.</a> BioMedical Engineering OnLine. 17 (1), 70 (2018).</li><li>Yi, J., Lee, Y. H., Song, H. -. T., Suh, J. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+T2*+between+regular+echo+time+and+ultrashort+echo+time+with+3D+cones+at+3+tesla+for+knee+meniscus.">Comparison of T2* between regular echo time and ultrashort echo time with 3D cones at 3 tesla for knee meniscus.</a> Medicine. 97 (48), e13443 (2018).</li><li>Weishaupt, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> How Does MRI Work?: An Introduction to the Physics and Function of Magnetic Resonance Imaging. , (2006).</li><li>Wood, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+quantifying+iron+overload.">Guidelines for quantifying iron overload.</a> Hematology. American Society of Hematology. 2014 (1), 210-215 (2014).</li><li>Branisso, P. P. F., 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=Non-invasive+methods+for+iron+overload+evaluation+in+dysmetabolic+patients.">Non-invasive methods for iron overload evaluation in dysmetabolic patients.</a> Annals of Hepatology. 27 (4), 100707 (2022).</li><li>Schaefer, B., Meindl, E., Wagner, S., Tilg, H., Zoller, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+iron+supplementation+therapy.">Intravenous iron supplementation therapy.</a> Molecular Aspects of Medicine. 75, 100862 (2020).</li><li>Haacke, E. M., Mittal, S., Wu, Z., Neelavalli, J., Cheng, Y. -. C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Susceptibility-weighted+imaging:+technical+aspects+and+clinical+applications,+part+1.">Susceptibility-weighted imaging: technical aspects and clinical applications, part 1.</a> AJNR: American Journal of Neuroradiology. 30 (1), 19-30 (2009).</li><li>. T2 Mapping Slicer Extension Available from: <a target="_blank" href="https://github.com/gattia/Slicer-T2mapping">https://github.com/gattia/Slicer-T2mapping</a> (2021)</li><li>3D Slicer image computing platform. 3D Slicer Available from: <a target="_blank" href="https://slicer.org/">https://slicer.org/</a> (2023)</li><li>Messroghli, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open-source+software+tool+for+the+generation+of+relaxation+time+maps+in+magnetic+resonance+imaging.">An open-source software tool for the generation of relaxation time maps in magnetic resonance imaging.</a> BMC Medical Imaging. 10, 16 (2010).</li><li>. GNU Octave Available from: <a target="_blank" href="https://octave.org/">https://octave.org/</a> (2023)</li><li>Chavhan, G. B., Babyn, P., Thomas, B., Shroff, M., Haacke, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles,+techniques,+and+applications+of+T2*-based+MR+imaging+and+its+special+applications.">Principles, techniques, and applications of T2*-based MR imaging and its special applications.</a> Radiographics. 29 (5), 1433-1449 (2009).</li><li>Aghighi, 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=Magnetic+resonance+imaging+of+tumor+associated+macrophages:+clinical+translation.">Magnetic resonance imaging of tumor associated macrophages: clinical translation.</a> Clinical Cancer Research. 24 (17), 4110-4118 (2018).</li><li>Trujillo-Alonso, V., 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=FDA-approved+ferumoxytol+displays+anti-leukaemia+efficacy+against+cells+with+low+ferroportin+levels.">FDA-approved ferumoxytol displays anti-leukaemia efficacy against cells with low ferroportin levels.</a> Nature Nanotechnology. 14 (6), 616-622 (2019).</li><li>Ishiyama, K., 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=Tumor-liver+contrast+and+subjective+tumor+conspicuity+of+respiratory-triggered+T2-weighted+fast+spin-echo+sequence+compared+with+T2*-weighted+gradient+recalled-echo+sequence+for+ferucarbotran-enhanced+magnetic+resonance+imaging+of+hepatic+malignant+tumors.">Tumor-liver contrast and subjective tumor conspicuity of respiratory-triggered T2-weighted fast spin-echo sequence compared with T2*-weighted gradient recalled-echo sequence for ferucarbotran-enhanced magnetic resonance imaging of hepatic malignant tumors.</a> Journal of Magnetic Resonance Imaging. 27 (6), 1322-1326 (2008).</li><li>Hirokawa, Y., 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=Hepatic+lesions:+improved+image+quality+and+detection+with+the+periodically+rotated+overlapping+parallel+lines+with+enhanced+reconstruction+technique-evaluation+of+SPIO-enhanced+T2-weighted+MR+images.">Hepatic lesions: improved image quality and detection with the periodically rotated overlapping parallel lines with enhanced reconstruction technique-evaluation of SPIO-enhanced T2-weighted MR images.</a> Radiology. 251 (2), 388-397 (2009).</li><li>Tonan, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+small+(&#8804;2cm)+dysplastic+nodules+and+well-differentiated+hepatocellular+carcinomas+with+ferucarbotran-enhanced+MRI+in+a+1.0-T+MRI+unit:+Utility+of+T2*-weighted+gradient+echo+sequences+with+an+intermediate-echo+time.">Evaluation of small (&#8804;2cm) dysplastic nodules and well-differentiated hepatocellular carcinomas with ferucarbotran-enhanced MRI in a 1.0-T MRI unit: Utility of T2*-weighted gradient echo sequences with an intermediate-echo time.</a> European Journal of Radiology. 64 (1), 133-139 (2007).</li><li>Rief, 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=Detection+of+focal+liver+lesions+in+unenhanced+and+ferucarbotran-enhanced+magnetic+resonance+imaging:+a+comparison+of+T2-weighted+breath-hold+and+respiratory-triggered+sequences.">Detection of focal liver lesions in unenhanced and ferucarbotran-enhanced magnetic resonance imaging: a comparison of T2-weighted breath-hold and respiratory-triggered sequences.</a> Magnetic Resonance Imaging. 27 (9), 1223-1229 (2009).</li></ol>

The authors have nothing to disclose.

Our protocol allows us to measure tumor T2* relaxation times based on multi-echo gradient-echo sequences, an external software, and a plugin for creating T2* maps. The critical steps within the protocol are the inclusion of the multi-echo gradient-echo sequence with very short TEs into the scanning protocol, and the monoexponential fit of the multi-echo gradient-echo images using external software. It is important to arrange the input multi-echo gradient-echo images according to their acquisition times. This can be achie...

Noninvasive quantification of tumor T2* relaxation times in various tissues of the body with magnetic resonance imaging (MRI) is widely established1. The rationale for this article is to provide a protocol for the measurement of tumor T2* relaxation times which is independent of scanner software like Osirix2. This will allow uniform analyses of imaging data from different centers, different scanners, and different vendors. Indeed, thousands of users could potentially use the same approach, thereby increasing the standardization of tumor T2* measurements. T2* measurements are used for different purposes by neuroradiologists, cardiac imaging experts, and abdominal imaging experts, among others. MRI pulse sequences for measurements of tissue T2* relaxation times have been applied and optimized for the assessment of intracranial bleeds3, hepatic iron content1,4, and cardiac iron content5,6, among others. Other investigators have used T2* measurements to generate quantitative estimates of iron oxide nanoparticle accumulations in malignant tumors7,8. However, many of these previous approaches utilized institutional software or specific scanner software, which would be limited to use at a specific institution or for processing data obtained on a specific scanner. Here, we describe a universally applicable approach for generating tumor T2* maps and tumor T2* relaxation times based on preclinical or clinical MRI data from any scanner that can generate multi-echo gradient echo images. The required gradient echo sequence should have very short first echo times and close inter-echo spacing9,10. The multi-echo gradient echo images are then fed into the external software, tumor T2* maps are calculated, and tumor T2* relaxation times are measured. The T2 Fit Map plugin in the external models&#39; T2* decay curves as a monoexponential fit to S(t) = Soe-t/T2* 11 where S(t) represents the signal or process value at a given time t; S0 is the initial value of the signal or process at t = 0; t denotes time; T2*, also known as the apparent transverse relaxation time, characterizes the decay rate of the signal or process; and e is the base of the natural logarithm (approximately equal to 2.71828). The equation describes an exponential decay, where the signal or process decreases over time as a function of the decay rate T2*. The larger the value of T2*, the slower the decay rate, and vice versa. The same software can also be used to input multi-echo spin echo images and generate tumor T2 values by fitting the T2 decay curve to S(t) = Soe-t/T2. The curve fitting was performed using external software, without incorporating a constant offset. Both decay curves exhibit single exponential behavior, with T2* demonstrating a shorter duration compared to T2.
In patients with hemosiderosis and hemochromatosis, the quantification of liver iron content by tissue biopsy is the gold standard, whereas noninvasive MR imaging is the point of care for establishing baseline values and monitoring changes over time noninvasively12,13. While generating T2* maps for liver iron quantification is well established4, there is no standardized protocol to measure tumor T2* relaxation times. While T2* maps can also be generated by scanner software, it is limited to a specific scanner and vendor. In the field of oncology, serial imaging studies of a given patient often occur on different scanners, and multicenter MRI data are acquired based on imaging studies from different scanners and different vendors. In addition, co-clinical imaging research is being increasingly implemented and requires the comparison of MRI data of patients and mouse models that simulate their tumor. The purpose of this protocol is to provide a protocol for the measurement of tumor T2* relaxation times that are independent of the scanner software. This will allow uniform analysis of imaging data from different centers and different scanners. Indeed, thousands of users could potentially use the same approach, thereby increasing the standardization and reproducibility of tumor T2* measurements. Our protocol utilizes external software, which can be downloaded from the internet. Multi-echo gradient echo images are fed into the software and fit to a formula for monoexponential decay to generate a T2* map, on which tumor T2* relaxation times can be measured using operator-defined regions of interest (ROIs)5. Iron oxide nanoparticles can be infused at different doses14, In our study, the patient received a Ferumoxytol injection (30 mg/mL) containing 510 mg of elemental iron in a 17 mL volume, at a dosage of 5 mg elemental iron per kg body weight. Subsequently multi-echo gradient echo sequences were obtained15 using set sequence parameters for data acquisition.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>OsiriX</td>
			<td>Pixmeo SARL</td>
			<td></td>
			<td>https://www.osirix-viewer.com/</td>
		</tr>
		<tr>
			<td>3T GE MR 750</td>
			<td>GE Healthcare, Chicago, IL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FERAHEME (ferumoxytol injection)</td>
			<td>AMAG Pharmaceuticals, Inc. 1100 Winter Street Waltham, MA 02451</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

measurement of tumor t2* relaxation times after iron oxide nanoparticle administration

T2* relaxometry is one of the established methods to measure the effect of superparamagnetic iron oxide nanoparticles on tumor tissues with magnetic resonance imaging (MRI). Iron oxide nanoparticles shorten the T1, T2, and T2* relaxation times of tumors. While the T1 effect is variable based on the size and composition of the nanoparticles, the T2 and T2* effects are usually predominant, and T2* measurements are the most time-efficient in a clinical context. Here, we present our approach to measuring tumor T2* relaxation times, using multi-echo gradient echo sequences, external software, and a standardized protocol for creating a T2* map with scanner-independent software. This facilitates the comparison of imaging data from different clinical scanners, different vendors, and co-clinical research work (i.e., tumor T2* data obtained in mouse models and patients). Once the software is installed, the T2 Fit Map plugin needs to be installed from the plugin manager. This protocol provides step-by-step procedural details, from importing the multi-echo gradient echo sequences into the software, to creating color-coded T2* maps and measuring tumor T2* relaxation times. The protocol can be applied to solid tumors in any body part and has been validated based on preclinical imaging data and clinical data in patients. This could facilitate tumor T2* measurements for multi-center clinical trials and improve the standardization and reproducibility of tumor T2* measurements in co-clinical and multi-center data analyses.

<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/64773/64773fig10.jpg" /> 
Figure 10: The T2* map with an ROI overlaid on the metastatic osteosarcoma lesion which shows the mean and standard deviation T2* value. <a href="https://www.jove.com/files/ftp_upload/64773/64773fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<p class="jove_content biglegend" fo:keep-together....

Watch this Scientific Journal Video about Measurement of Tumor T2* Relaxation Times after Iron Oxide Nanoparticle Administration at JoVE.com

Measurement of Tumor T2* Relaxation Times after Iron Oxide Nanoparticle Administration

We present a standardized protocol for the quantification of T2* relaxation times of tumors using external software. Multi-echo gradient echo images are acquired and fed into the software to create tumor T2* maps and measure tumor T2* relaxation times.

T2* relaxometry is one of the established methods to measure the effect of superparamagnetic iron oxide nanoparticles on tumor tissues with magnetic ...

measurement-tumor-t2-relaxation-times-after-iron-oxide-nanoparticle

Research

JoVE Journal

Medicine

1.1K Views.  Stanford University, School of Medicine. We present a standardized protocol for the quantification of T2* relaxation times of tumors using external software. Multi-echo gradient echo images are acquired and fed into the software to create tumor T2* maps and measure tumor T2* relaxation times.

Video: Measurement of Tumor T2* Relaxation Times after Iron Oxide Nanoparticle Administration

Manual Muscle Testing:  A Method of Measuring Extremity Muscle Strength Applied to Critically Ill Patients

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, and persistent nerve and muscle impairments after hospital discharge.1-6 Using an explicit protocol with a structured approach to training and quality assurance of research staff, manual muscle testing (MMT) is a highly reliable method for assessing strength, using a standardized clinical examination, for patients following ARDS, and can be completed with mechanically ventilated patients who can tolerate sitting upright in bed and are able to follow two-step commands. 7, 8 
This video demonstrates a protocol for MMT, which has been taught to &ge;43 research staff who have performed &gt;800 assessments on &gt;280 ARDS survivors. Modifications for the bedridden patient are included. Each muscle is tested with specific techniques for positioning, stabilization, resistance, and palpation for each score of the 6-point ordinal Medical Research Council scale.7,9-11 Three upper and three lower extremity muscles are graded in this protocol: shoulder abduction, elbow flexion, wrist extension, hip flexion, knee extension, and ankle dorsiflexion. These muscles were chosen based on the standard approach for evaluating patients for ICU-acquired weakness used in prior publications. 1,2.

Survivors of acute respiratory distress syndrome (ARDS) and critical illness frequently develop long-lasting muscle weakness. Manual muscle testing (MMT) is a standardized clinical examination commonly used to measure strength of peripheral skeletal muscle groups. This video demonstrates MMT using the 6-point Medical Research Council scale.

Survivors of acute respiratory distress syndrome (ARDS) and other causes of critical illness often have generalized weakness, reduced exercise tolerance, ...

Mesenteric Artery Contraction and Relaxation Studies Using Automated Wire Myography

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between 100-400 &mu;m in diameter function primarily in directing blood flow to various organs according to the overall requirements of the body. The rat mesenteric artery has a diameter greater than 100 &mu;m. The myography technique, first described by Mulvay and Halpern1, was based on the method proposed by Bevan and Osher2. The technique provides information about small vessels under isometric conditions, where substantial shortening of the muscle preparation is prevented. Since force production and sensitivity of vessels to different agonists is dependent on the extent of stretch, according to active tension-length relation, it is essential to conduct contraction studies under isometric conditions to prevent compliance of the mounting wires. Stainless steel wires are preferred to tungsten wires because of oxidation of the latter, which affects recorded responses3.The technique allows for the comparison of agonist-induced contractions of mounted vessels to obtain evidence for normal function of vascular smooth muscle cell receptors.
We have shown in several studies that isolated mesenteric arteries that are contracted with phenylyephrine relax upon addition of cumulative concentrations of extracellular calcium (Ca2+e). The findings led us to conclude that perivascular sensory nerves, which express the G protein-coupled Ca2+-sensing receptor (CaR), mediate this vasorelaxation response. Using an automated wire myography method, we show here that mesenteric arteries from Wistar, Dahl salt-sensitive(DS) and Dahl salt-resistant (DR) rats respond differently to Ca2+e. Tissues from Wistar rats showed higher Ca2+-sensitivity compared to those from DR and DS. Reduced CaR expression in mesenteric arteries from DS rats correlates with reduced Ca2+e-induced relaxation of isolated, pre-contracted arteries. The data suggest that the CaR is required for relaxation of mesenteric arteries under increased adrenergic tone, as occurs in hypertension, and indicate an inherent defect in the CaR signaling pathway in Dahl animals, which is much more severe in DS.
The method is useful in determining vascular reactivity ex vivo in mesenteric resistance arteries and similar small blood vessels and comparisons between different agonists and/or antagonists can be easily and consistently assessed side-by-side6,7,8.

An automated myography method for force measurements in isolated mesenteric arteries is described. It employs a Mulvany-Halpern Auto Dual Wire Myograph 510A to determine responses to phenylephrine and extracellular calcium. The method allows consistent determination of isometric responses to agonists in small vessels of diameters of 60 - 300 &mu;m, independently.

Proximal resistance vessels, such as the mesenteric arteries, contribute substantially to the peripheral resistance. These small vessels of between ...

Labeling Stem Cells with Ferumoxytol, an FDA-Approved Iron Oxide Nanoparticle

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo kinetics of transplanted cells. A non-invasive method to repetitively monitor transplanted stem cells in vivo would allow investigators to directly monitor stem cell transplants and identify successful or unsuccessful engraftment outcomes.
A wide range of stem cells continues to be investigated for countless applications. This protocol focuses on 3 different stem cell populations: human embryonic kidney 293 (HEK293) cells, human mesenchymal stem cells (hMSC) and induced pluripotent stem (iPS) cells. HEK 293 cells are derived from human embryonic kidney cells grown in culture with sheared adenovirus 5 DNA. These cells are widely used in research because they are easily cultured, grow quickly and are easily transfected. hMSCs are found in adult marrow. These cells can be replicated as undifferentiated cells while maintaining multipotency or the potential to differentiate into a limited number of cell fates. hMSCs can differentiate to lineages of mesenchymal tissues, including osteoblasts, adipocytes, chondrocytes, tendon, muscle, and marrow stroma. iPS cells are genetically reprogrammed adult cells that have been modified to express genes and factors similar to defining properties of embryonic stem cells. These cells are pluripotent meaning they have the capacity to differentiate into all cell lineages 1. Both hMSCs and iPS cells have demonstrated tissue regenerative capacity in-vivo.
Magnetic resonance (MR) imaging together with the use of superparamagnetic iron oxide (SPIO) nanoparticle cell labels have proven effective for in vivo tracking of stem cells due to the near microscopic anatomical resolution, a longer blood half-life that permits longitudinal imaging and the high sensitivity for cell detection provided by MR imaging of SPIO nanoparticles 2-4. In addition, MR imaging with the use of SPIOs is clinically translatable. SPIOs are composed of an iron oxide core with a dextran, carboxydextran or starch surface coat that serves to contain the bioreactive iron core from plasma components. These agents create local magnetic field inhomogeneities that lead to a decreased signal on T2-weighted MR images 5. Unfortunately, SPIOs are no longer being manufactured. Second generation, ultrasmall SPIOs (USPIO), however, offer a viable alternative. Ferumoxytol (FerahemeTM) is one USPIO composed of a non-stoichiometric magnetite core surrounded by a polyglucose sorbitol carboxymethylether coat. The colloidal, particle size of ferumoxytol is 17-30 nm as determined by light scattering. The molecular weight is 750 kDa, and the relaxivity constant at 2T MRI field is 58.609 mM-1 sec-1 strength4. Ferumoxytol was recently FDA-approved as an iron supplement for treatment of iron deficiency in patients with renal failure 6. Our group has applied this agent in an &ldquo;off label&rdquo; use for cell labeling applications. Our technique demonstrates efficient labeling of stem cells with ferumoxytol that leads to significant MR signal effects of labeled cells on MR images. This technique may be applied for non-invasive monitoring of stem cell therapies in pre-clinical and clinical settings.

We describe a technique for labeling and tracking stem cells with FDA-approved, superparamagnetic iron oxide (SPIO), ferumoxytol (Feraheme). This cellular imaging technique that utilizes magnetic resonance (MR) imaging for visualization, is readily accessible for long-term monitoring and diagnosis of successful or unsuccessful stem cell engraftments in patients.

Stem cell based therapies offer significant potential for the field of regenerative medicine. However, much remains to be understood regarding the in vivo ...

Intranasal Administration of CNS Therapeutics to Awake Mice

Intranasal administration is a method of delivering therapeutic agents to the central nervous system (CNS). It is non-invasive and allows large molecules that do not cross the blood-brain barrier access to the CNS. Drugs are directly targeted to the CNS with intranasal delivery, reducing systemic exposure and thus unwanted systemic side effects1. Delivery from the nose to the CNS occurs within minutes along both the olfactory and trigeminal neural pathways via an extracellular route and does not require drug to bind to any receptor or axonal transport2. Intranasal delivery is a widely publicized method and is currently being used in human clinical trials3.
Intranasal delivery of drugs in animal models allows for initial evaluation of pharmacokinetic distribution and efficacy. With mice, it is possible to administer drugs to awake (non-anesthetized) animals on a regular basis using a specialized intranasal grip. Awake delivery is beneficial because it allows for long-term chronic dosing without anesthesia, it takes less time than with anesthesia, and can be learned and done by many people so that teams of technicians can dose large numbers of mice in short periods. Efficacy of therapeutics administered intranasally in this way to mice has been demonstrated in a number of studies including insulin in diabetic mouse models 4-6 and deferoxamine in Alzheimer's mouse models. 7,8
The intranasal grip for mice can be learned, but is not easy and requires practice, skill, and a precise grip to effectively deliver drug to the brain and avoid drainage to the lung and stomach. Mice are restrained by hand using a modified scruff in the non-dominant hand with the neck held parallel to the floor, while drug is delivered with a pipettor using the dominant hand. It usually takes 3-4 weeks of acclimating to handling before mice can be held with this grip without a stress response. We have prepared this JoVE video to make this intranasal delivery technique more accessible.

A method to intranasally administer drugs to awake mice for the purpose of targeting the brain is described. This method allows for repeat dosing over long periods using intranasal administration of drug without anesthesia, and nose-to-brain delivery with minimal systemic exposure.

Intranasal administration is a method of delivering  therapeutic agents to the central nervous system (CNS). It is  non-invasive and allows large ...

Synthesis, Characterization, and Application of Superparamagnetic Iron Oxide Nanoprobes for Extrapulmonary Tuberculosis Detection

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was synthesized to enhance imaging sensitivity for extrapulmonary tuberculosis (ETB). An SPIO nanoprobe was synthesized and conjugated with MtbsAb. The purified SPIO-MtbsAb nanoprobe was characterized using TEM and NMR. To determine the targeting ability of the probe, SPIO-MtbsAb nanoprobes were incubated with Mtb for in vitro imaging assays and injected into Mtb-inoculated mice for in vivo investigation with magnetic resonance (MR). The contrast enhancement reduction on magnetic resonance imaging (MRI) of Mtb and THP1 cells showed proportional to the SPIO-MtbsAb nanoprobe concentration. After 30 min of intravenous SPIO-MtbsAb nanoprobe injection into Mtb-infected mice, the signal intensity of the granulomatous site was enhanced by 14-fold in the T2-weighted MR images compared with that in mice receiving PBS injection. The MtbsAb nanoprobes can be used as a novel modality for ETB detection.

To improve serological diagnostic tests for Mycobacterium tuberculosis antigens, we developed superparamagnetic iron oxide nanoprobes to detect extrapulmonary tuberculosis.

A molecular imaging probe comprising superparamagnetic iron oxide (SPIO) nanoparticles and Mycobacterium tuberculosis surface antibody (MtbsAb) was ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Tracking Superparamagnetic Iron Oxide-labeled Mesenchymal Stem Cells using MRI after Intranasal Delivery in a Traumatic Brain Injury Murine Model

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical hurdles such as invasive cell delivery and tracking with low transplantation efficiency remain challenges in translational stem-based therapy. This article describes an emerging technique for stem cell labeling and tracking based on the labeling of the mesenchymal stem cells (MSCs) with superparamagnetic iron oxide (SPIO)&#160;nanoparticles, as well as intranasal delivery of the labeled MSCs. These nanoparticles are fluorescein isothiocyanate (FITC)-embedded and safe to label the MSCs, which are subsequently delivered to the brains of TBI-induced mice by the intranasal route. They are then tracked non-invasively in vivo by real-time magnetic resonance imaging (MRI). Important advantages of this technique that combines SPIO for cell labeling and intranasal delivery include (1) non-invasive, in vivo MSC tracking after delivery for long tracking periods, (2) the possibility of multiple dosing regimens due to the non-invasive route of MSC delivery, and (3) possible applications to humans, owing to the safety of SPIO, non-invasive nature of the cell-tracking method by MRI, and route of administration.

Presented here is a protocol for non-invasive mesenchymal stem cell (MSC) delivery and tracking in a mouse model of traumatic brain injury. Superparamagnetic iron oxide&#160;nanoparticles are employed as a magnetic resonance imaging (MRI) probe for MSC labeling and non-invasive in vivo tracking following intranasal delivery using real-time MRI.

Stem cell-based therapies for brain injuries, such as traumatic brain injury (TBI), are a promising approach for clinical trials. However, technical ...

Measurement of Natural Killer Cell-Mediated Cytotoxicity and Migration in the Context of Hepatic Tumor Cells

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by clearing pathogen-infected, malignant, and stressed cells. The ability of NK cells to eradicate cancer cells makes them an important tool in the fight against cancer. Several new immune-based therapies are under investigation for cancer treatment which rely either on enhancing NK cell activity or increasing the sensitivity of cancer cells to NK cell-mediated eradication. However, to effectively develop these therapeutic approaches, cost-effective in vitro assays to monitor NK cell-mediated cytotoxicity and migration are also needed. Here, we present two in vitro protocols that can reliably and reproducibly monitor the effect of NK-cell cytotoxicity on cancer cells (or other target cells). These protocols are non-radioactivity-based, simple to set up, and can be scaled up for high-throughput screening. We also present a flow cytometry-based protocol to quantitatively monitor NK cell migration, which can also be scaled up for high-throughput screening. Collectively, these three protocols can be used to monitor key aspects of NK cell activity that are necessary for the cells&#39; ability to eradicate dysfunctional target cells.

Evasion of natural killer (NK) cell-mediated eradication by cancer cells is important for cancer initiation and progression. Here, we present two non-radioactivity-based protocols to evaluate NK cell-mediated cytotoxicity toward hepatic tumor cells. Additionally, a third protocol is presented to analyze NK cell migration.

Natural killer (NK) cells are a subset of the cytotoxic lymphocyte population of the innate immune system and participate as a first line of defense by ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

Acetylcholine Re-Challenge After Intracoronary Nitroglycerine Administration

Coronary artery spasm (CAS) can be diagnosed in a large proportion of patients with recurrent angina with non-obstructive coronary artery disease (ANOCA) using acetylcholine (ACh) spasm provocation testing. CAS can further be divided into different subtypes (e.g., focal, diffuse epicardial, or microvascular spasm), each with different pathophysiological mechanisms that may require tailored drug treatment. The evidence behind the role of nitrates in the setting of each CAS subtype is lacking, and the effectivity can vary on a per-patient basis. In order to assess on a per-patient level whether nitroglycerine (NTG) can prevent inducible spasm, the vasospastic ACh dose can be readministered after NTG administration as part of the spasm provocation test. The preventive effect of NTG is assessed by evaluating improvements in the severity of induced symptoms, ischemic ECG changes, and by reassessing the site and mode of spasm on angiography. This technique can therefore be used to assess the nitrate responsiveness on a per-patient level and unmask co-existing microvascular spasm in patients with epicardial spasm that is prevented with NTG. The NTG rechallenge, therefore, allows to further guide targeted therapy for CAS and provide new insights into the pathophysiological mechanism behind vasospastic disorders.

This protocol presents the acetylcholine rechallenge after nitroglycerine as an add-on procedure to spasm provocation testing. The purpose of this technique is to unmask co-existing microvascular spasm&#160;in patients with epicardial spasm&#160;and to assess the protective efficacy of nitroglycerine on a per-patient level to guide medical therapy.

Coronary artery spasm (CAS) can be diagnosed in a large proportion of patients with recurrent angina with non-obstructive coronary artery disease (ANOCA) ...