This study was supported in part by NIH/NIBIB R21EB008166 to DL and NIA AG028455 and NIA AG022385 for DJM. We thank Dr. James Olson at the Fred Hutchinson Cancer Research Center for providing their D282 tumor mice.

Part 1: Magnet Safety
Both MRI and MRS use a strong magnetic field that requires extreme caution. For example, the 4.7 T instrument that we used for the present work has a magnetic field approximately 90,000 times the earth magnetic field. The high magnetic field is always on even when the MR scanner is not being used. Any metallic object that comes into contact with such a high magnetic field will be strongly and rapidly attracted by the magnet. It is extremely dangerous if an experimental subject or operator is located in the projectile path of a metallic object flying into the magnet. Therefore, personnel who conduct MR experiments should be careful to remove any metallic objects from their clothing before entering the proximity of the instrument and also maintain the surrounding environment free from such objects. More detailed information on magnet safety appears in the literature1 and the following webpage: <a href="http://www.imrser.org/">http://www.imrser.org/</a> . The presence of metallic material can not only cause the aforementioned safety issue but interfere with experimental results by inducing imaging/spectroscopic artifacts. The metallic material present near or inside the imaging object can alter the magnetic field in the vicinity and so generate artifacts on acquired images or broaden line-width of spectra.
Accordingly, leave wallet, keys, pens, etc. outside the magnet if you handle animals for MR procedures.
Part 2: In Vivo MRI of Mouse in a Horizontal Bore Magnet
Animal Preparation for MRI
<ol>
 <li>All the animal procedures should be approved by the institutional animal care and use committee (IACUC) prior to executing any types of animal handling.</li>
 <li>We use vaporized isoflurane to anesthetize animals for MRI experiments. Anesthesia of animals can be achieved by other anesthetics such as avertin (2,2,2-tribromoethanol or TBE) and a cocktail of ketamine and xylazine. Dosage information of each anesthetic is found in Table 1. </li>
 <li>Line an induction chamber with plastic backed absorbent pad (blue pad or chuck). Place a mouse (or multiple mice for multi-mouse imaging) in the induction chamber. </li>
 <li>Adjust the flowmeter of the isoflurane vaporizer to 0.8 1.5 L/min. Then adjust the isoflurane vaporizer to 4 % for about 2 3 minutes.</li>
 <li>After reaching the surgical plane of anesthesia (i.e. no toe pinch response), place the mouse on an animal holder with its nose inserted into a nose cone (or mask). A head restrainer can be used for head imaging and a body holder can be used for body imaging. </li>
 <li>Animal holders are used to prevent potential motion: there are several types of commercial holders. Also, a custom designed holder can be fabricated to accommodate any special requirements of an experimental setup. For a custom designed holder, make sure to use only nonmagnetic material. </li>
 <li>During the imaging period, adjust the flowmeter to 0.4 0.8 mL/min and reduce the isoflurane vaporizer to 1.2 1.5 %. The expired gas coming from the mouse nose cone is collected by a pump and removed into an in-house vacuum.</li>
 <li>The eyes of the animal will be kept moist with a sterile eye lubricating ointment. The animal will be kept at 35-37&deg; C during the experiment within a warm water circulation system. Other types of heat source can be used such as heated pads and warm air blown into the radiofrequency (RF) coil.</li>
 <li>An animal monitoring system is in place to monitor body temperature, respiration/cardiac cycle, and synchronize respiration/cardiac gating with image acquisitions.</li>
 <li>A standard sample made of agarose is placed next to the animal to monitor abrupt signal change. This standard agar sample is particularly useful for multi-slice and multi-time point imaging. When an unexpected signal change is detected in a slice from the acquired images, the slice with the unexpected signal change can be eliminated. Also, its signal intensities can be adjusted based on the signal change of the standard sample during post-image analysis.</li>
 <li>After securely placing the animal and monitoring components on the animal holder, position the animal holder at the center of a RF coil.</li>
 <li>Move the RF coil to the magnet room and insert the RF coil into the warm water circulation system placed inside the magnet. Figure 2 displays several components of an MRI scanner observed from the front of the magnet bore. </li>
</ol>
MRI Experiment
<ol>
 <li>Tune the RF coil to the 1H resonance frequency and match the characteristic impedance of the coil to 50 Ohm using the tuning panel in the MR scanner. This is to achieve optimum conditions of signal reception. Most human MRI scanners do not require a separate process of tune/match except in MRS procedures. </li>
 <li>Conduct a shimming process using a single pulse sequence. An MR signal relies on the environmental magnetic field homogeneity. The shimming process enables the magnetic field in the region of interest to be as homogeneous as possible. Each MR scanner has its own way to perform the shimming process, including automatic fast shimming processes such as fast map and gradient shimming. </li>
 <li>Optimize the RF pulse by maximizing one dimensional image profile. RF pulse powers can be arrayed while keeping the pulse length constant and a long enough TR (recycle delay) that is approximately 3 - 5x the T1 of tissue. </li>
 <li>Acquire scout images along three orthogonal orientations to create axial, coronal and sagittal images. A fast image acquisition sequence (i.e. gradient echo or fast spin echo imaging sequence) can be used to acquire the scout images. The acquired images will be used to plan for actual imaging with determination of imaging planes. </li>
 <li>Change to spin echo sequence. Select proper sequence parameters: TR (recycle delay) should be three to five times the tissue T1 to acquire fully relaxed images such as proton density or T2 weighted images. TE (echo time) is the time duration between the first RF pulse and the center of echo signal. A TE value can be selected depending on image contrast as summarized in Table 2. Figure 2 shows in vivo images acquired with different relaxation effects of T1, T2 and T2* for a nude mouse with a xenograft tumor on its back.</li>
 <li>T2 measurements can be done either using multiecho imaging or single echo imaging with multiple TE values.</li>
 <li>Subsequent to the MRI/MRS in vivo experiment, animals should be monitored throughout the recovery process. After MR imaging, the animals will be taken out of the RF coil and monitored to assure full recovery when returned to the cage. Heat loss is rapid in anesthetized mice. Keep the animals warm by covering them with gauze pads or towels and/or providing a heat source until the animals are recovered from anesthesia.</li>
</ol>
Image Processing
<ol>
 <li>Review acquired images on the MR console and transfer selected data to a post-processing computer.</li>
 <li>We typically use ImageJ (<a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a>) for analyzing images. The image analysis includes image scaling/filtering, calculations of T1, T2 and diffusion, tumor volume measurements and segmentation of tumors.</li>
</ol>
Part 3. In Vivo MRS for Mouse Hindlimb Skeletal Muscle in a Vertical Bore Magnet
Construction of Cuff for Inducing Reversible Ischemia
<ol>
 <li>Start with a piece of PVC pipe that is approximately 5-7 mm wide with an i.d. of 12-15 mm. Drill a small hole through the wall of this piece and thread. </li>
 <li>Cut a piece of balloon so it is open on both ends (helium quality balloons work best). Insert this piece through the PVC piece and wrap back around and tape ends together on the outside wall of the PVC piece.</li>
 <li>Use shrink wrap to seal the balloon ends around the tube. You should have a cuff with a solid outer wall and an inflatable inner wall.</li>
 <li>Cut out an area of shrink wrap and balloon around the threaded hole, taking care to leave plenty of material between the hole and the edge of the PVC piece. Thread a 1.5 cm piece of non-ferrous metal (i.e. brass) into the hole in the PVC. This allows you to inflate the cuff. Seal the area with 5 minute epoxy.</li>
 <li>Fix this cuff in place next to the RF coil in the MRS probe and connect to an external sphygmomanometer.</li>
</ol>
Custom Respiration Monitor
<ol>
 <li>We use a custom made respiration monitor that is made to be compatible with the restricted space and access to the bore of the magnet. Several commercial models are also available.</li>
 <li>Tie a small balloon to the end of stretch resistant tubing fed into the probe.</li>
 <li>Attach the other end of the tubing to a pressure transducer.</li>
 <li>Be sure the line and balloon are free of air bubbles. The bubbles will attenuate the signal from compression of the balloon caused by movement of the mouse body due to respiration.</li>
</ol>
Mouse Positioning in MRS Probe
<ol>
 <li>Anesthetize the mouse with 5% Avertin (0.010 ml/g body weight).</li>
 <li>After reaching the surgical plane of anesthesia, position the mouse in the MRS probe by placing the mouse on its back on the mouse support. Place the fluid-filled balloon on the ventral side of the mouse and secure in place with mouse support straps.</li>
 <li>Position mouse and body support in the MRS probe.</li>
 <li>Pull one hindlimb through the ischemic cuff and MRS coil. The leg should be centered in the coil as much as possible. This arrangement allows the mouse body to be positioned horizontally in a vertical bore magnet (Figure 3). </li>
 <li>Fix leg in place by taping foot to rigid support.</li>
 <li>Place thermocouple subcutaneously in hindlimb outside the region sampled by the coil.</li>
 <li>Moisten the eyes with the eye lubricating ointment to prevent the eyes from drying. Cover mouse eyes and face to prevent rubbing or irritation from the wall of the probe.</li>
 <li>Other monitoring probes can be added depending on the specific needs of the experiment.</li>
</ol>
Setting up MRS Experiment
<ol>
 <li>Connect airflow to heating element on the MRS probe.</li>
 <li>Set the variable temperature control unit on VNMR software to maintain leg temperature at 35-37 &deg;C.</li>
 <li>Tune coil frequency and match impedance using the tuning panel in the MRS software for both 1H and 31P resonances.</li>
 <li>Adjust shimming circuits to optimize the homogeneity of the B1 magnetic field in the region of interest using 1H spectroscopy.</li>
 <li>Switch to 31P frequency to determine the RF pulse width to yield maximum signal from one free induction decay (FID) (90&deg; time).</li>
 <li>Collect high signal to noise fully relaxed spectra (FRS) to determine the ratios of inorganic phosphate (Pi) and phosphocreatine (PCr) to ATP under fully relaxed conditions. These spectra are collected using the 90&deg; time with time between FID acquisitions (TR) of approximately 5x the T1 of PCr (20 sec. at 7 T). These will be used for quantification of PCr and Pi levels from MR spectra. </li>
</ol>
Ischemic Experiment
<ol>
 <li>A simple ischemic perturbation allows for the determination of resting and maximal mitochondrial ATP production by measuring changes in phosphocreatine during and immediately following ischemia.</li>
 <li>Set-up array to collect multiple spectra using a 45&deg; pulse width (i.e. 0.5 x the 90&deg; time) and a TR of 0.5x the T1 (~1.5 sec.). We typically collect 4 FIDs for every spectra (number of averaging (na) = 4 in VNMR software) for a time resolution of approximately 6 seconds. This time resolution is sufficient to accurately determine resting and maximal mitochondrial ATP production.</li>
 <li>Collect resting spectra for approximately 5 minutes.</li>
 <li>Induce ischemia by inflating the cuff to 270-300 mmHg for 10-12 minutes.</li>
 <li>Release the cuff and collect recovery spectra for 5 minutes.</li>
 <li>Remove the probe from magnet and the mouse from the probe. Allow the mouse to recover from anesthesia under appropriate conditions. Experiments can be repeated on subsequent days. Following the final spectroscopy experiment mouse leg muscles are removed and immediately freeze-clamped in liquid N2 for analysis of ATP concentrations by HPLC.</li>
</ol>
Data Analysis
<ol>
 <li>Data is analyzed offline using one of several spectral analysis programs for NMR spectra. Our laboratory typically uses fit to standard 2 and jMRUI (http://www.mrui.uab.es/mrui/mrui_Overview.shtml) for quantifying peak areas.</li>
<li>Initial PCr breakdown rates during ischemia are a measure of the mitochondrial ATP production under normoxic conditions as described in several papers 3-5. PCr recovery rates can be used to determine the maximal capacity for mitochondrial ATP production based on the approach described previously 4,6.</li>
</ol>
<img border="0" src="/files/ftp_upload/1592/1592fig1tmb.jpg" alt="Figure 1" /> Figure 1. In vivo MRI setup viewed from the front of the magnet bore. The setup comprises an RF coil, animal warming system (or warm water circulation system) and gradient insert. All these components are inserted into a horizontal magnet. Warm water is heated in a water reservoir outside the magnet room and introduced into the animal warming system through a Tygon tube (green tape). After circulating in the cylinder, water is pulled from the warming system back to the water reservoir to be heated. An isoflurane tube and vacuum tube are used to anesthetize mice during MRI experiments. Please <a href="https://www.jove.com/files/ftp_upload/1592/1592fig1.jpg">click here</a> to see a larger version of this figure.
<img border="0" src="/files/ftp_upload/1592/1592fig2tmb.jpg" alt="Figure 2" /> Figure 2. In vivo images for a nude mouse with xenograft tumor (D282 tumor) on its back (arrow) with different relaxation effects: A. T1 weighed image (TR/TE=500/14.2ms). B. T2 weighed image (TR/TE=2s/40ms). Both T1 and T2 weighted images were acquired by spin echo sequence. C. T2 map processed with 4 sets of images acquired by different TE values ranging from 20 to 80 ms. D. T2* weighed image (TR/TE=180/7.39ms, flip angle = 20 degrees) acquired by gradient echo sequence. Field of view of 35 x 35 mm2 is for all MR images. Please <a href="https://www.jove.com/files/ftp_upload/1592/1592fig2.jpg">click here</a> to see a larger version of this figure.
<img border="0" src="/files/ftp_upload/1592/1592fig3tmb.jpg" alt="Figure 3" /> Figure 3. Illustration of mouse positioned in horizontal body holder with leg secured in RF coil. Please <a href="https://www.jove.com/files/ftp_upload/1592/1592fig3.jpg">click here</a> to see a larger version of this figure.
<img border="0" src="/files/ftp_upload/1592/1592fig4tmb.jpg" alt="Figure 4" /> Figure 4. In vivo 31P spectra through one ischemia reperfusion cycle. Data were collected on a 7 T vertical bore magnet and line broadened with a 20 Hz exponential filter. TR=1, na=4, every 20 spectra are plotted. Please <a href="https://www.jove.com/files/ftp_upload/1592/1592fig4.jpg">click here</a> to see a larger version of this figure. 
Table 1. Dosage of anesthetic for mouse MRI/MRS.
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Animal 
 Species</td>
 <td>Anesthetic 
 Agent </td>
 <td>Dose 
 (mg/kg for injectables)</td>
 <td>Route</td>
 </tr>
 <tr>
 <td>Mouse</td>
 <td>Isoflurane gas</td>
 <td>4.0% for 2-3 min (induction), then 1.2 to 1.5% continuously (maintenance)</td>
 <td>Inhaled via nose cone</td>
 </tr>
 <tr>
 <td>Mouse</td>
 <td>Avertin</td>
 <td>5%, 10ml/kg body weight</td>
 <td>intraperitoneal (IP)</td>
 </tr>
 <tr>
 <td>Mouse</td>
 <td>Ketamine/Xylazine</td>
 <td>100 mg/kg and 10 mg/kg</td>
 <td>IP</td>
 </tr>
</tbody>
</table>
 Table 2. Image with relaxation weightings
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Image Weighting</td>
 <td>TR (recycle delay)</td>
 <td>TE (echo time)</td>
 </tr>
 <tr>
 <td>T1</td>
 <td>Short (Shorter than T1)</td>
 <td>Short (Shorter than T2)</td>
 </tr>
 <tr>
 <td>T2</td>
 <td>Long (3 ~ 5 times T1)</td>
 <td>Long (around T2)</td>
 </tr>
 <tr>
 <td>PD (proton density)</td>
 <td>Long (3 ~ 5 times T1)</td>
 <td>Short (shorter than T2)</td>
 </tr>
</tbody>
</table>
T1: spin lattice relaxation (or longitudinal relaxation) time 
 T2: spin spin relaxation (or transverse relaxation) time

<ol>
	<li>Stecco, A., Saponaro, A., Carriero, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient+safety+issues+in+magnetic+resonance+imaging:+state+of+the+art.">Patient safety issues in magnetic resonance imaging: state of the art.</a> Radiol Med. 112, 491-491 (2007).</li><li>Heineman, F. W., Eng, J., Berkowitz, B. A., Balaban, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMR+spectral+analysis+of+kinetic+data+using+natural+lineshapes.">NMR spectral analysis of kinetic data using natural lineshapes.</a> Magn Reson Med. 13, 490-490 (1990).</li><li>Amara, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+function+in+vivo:+spectroscopy+provides+window+on+cellular+energetics.">Mitochondrial function in vivo: spectroscopy provides window on cellular energetics.</a> Methods. 46, 312-312 (2008).</li><li>Blei, M. L., Conley, K. E., Kushmerick, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separate+measures+of+ATP+utilization+and+recovery+in+human+skeletal+muscle.">Separate measures of ATP utilization and recovery in human skeletal muscle.</a> J Physiol. 465, 203-203 (1993).</li><li>Marcinek, D. J., Schenkman, K. A., Ciesielski, W. A., Conley, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+coupling+in+vivo+in+mouse+skeletal+muscle.">Mitochondrial coupling in vivo in mouse skeletal muscle.</a> Am J Physiol Cell Physiol. 286, C457-C457 (2004).</li><li>Paganini, A. T., Foley, J. M., Meyer, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linear+dependence+of+muscle+phosphocreatine+kinetics+on+oxidative+capacity.">Linear dependence of muscle phosphocreatine kinetics on oxidative capacity.</a> Am J Physiol. 272, 501-501 (1997).</li><li>Sun, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+MRI+detection+of+gliomas+by+chlorotoxin-conjugated+superparamagnetic+nanoprobes.">In vivo MRI detection of gliomas by chlorotoxin-conjugated superparamagnetic nanoprobes.</a> Small. 4, 495-495 (2008).</li><li>Marcinek, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+mitochondrial+coupling+in+vivo+alters+cellular+energetics+in+aged+mouse+skeletal+muscle.">Reduced mitochondrial coupling in vivo alters cellular energetics in aged mouse skeletal muscle.</a> J Physiol. 569, 467-467 (2005).</li></ol>

The pre-acquisition steps of tune/match and shimming are critical to acquire high spatial resolution and a high signal-to-noise ratio (SNR). It is also important to monitor animal conditions with an animal monitoring system to maintain a stable physiological state for animals during signal acquisitions to comply with humane animal handling and to prevent any potential artifactual measurements. The procedures explained in the protocol can be modified to acquire additional information including diffusion, perfusion and flo...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Inova 200 MR scanner/4.7 T</td>
<td>&nbsp;</td>
<td>Varian/Bruker</td>
<td>&nbsp;</td>
<td>Used for mouse MRI</td>
</tr>
<tr>
<td>Inova 300 NMR spectrometer/7 T</td>
<td>&nbsp;</td>
<td>Varian/Oxford</td>
<td>&nbsp;</td>
<td>Used for MRS of mouse skeletal muscle</td>
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noninvasive in vivo small animal mri and mrs: basic experimental procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Watch this Scientific Journal Video about Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures at JoVE.com

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

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16.0K Views.  University of Washington. This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

Video: Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are evident. A means to detect apoptosis in its earliest, reversible stages would afford a pre-clinical 'window' during which preventive or therapeutic measures could be taken to protect the heart from permanent damage. We present herein a simple and robust method to conjugate human Annexin V (ANX), which avidly binds to cells in the earliest, reversible stages of apoptosis, to superparamagnetic iron oxide (SPIO) nanoparticles, which serve as an MRI-detectable contrast agent. The conjugation method begins with an oxidation of the SPIO nanoparticles, which oxidizes carboxyl groups on the polysaccharide shell of SPIO. Purified ANX protein is then added in the setting of a sodium borate solution to facilitate covalent interaction of ANX with SPIO in a reducing buffer. A final reduction step with sodium borohydride is performed to complete the reduction, and then the reaction is quenched. Unconjugated ANX is removed from the mix by microcentrifuge filtration. The size and purity of the ANX-SPIO product is verified by dynamic light scattering (DLS). This method does not require addition to, or modification of, the polysaccharide SPIO shell, as opposed to cross-linked iron oxide particle conjugation methods or biotin-labeled nanoparticles. As a result, this method represents a simple, robust approach that may be extended to conjugation of other proteins of interest.

Synthesis of an In vivo MRI-detectable Apoptosis Probe

Early detection of apoptosis may identify at-risk cell populations in a variety of diseases. Here we demonstrate a method to link an early apoptosis-detection protein (Annexin V) to a MRI-detectable iron oxide nanoparticle (SPIO). This method may be extended to other proteins of interest to generate MRI-detectable molecular imaging probes.

Cellular apoptosis is a prominent feature of many diseases, and this programmed cell death typically occurs before clinical manifestations of disease are ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of central nervous system injuries in vivo, possibly by reducing oxidative stress. However, effects of R/NIR-LT on oxidative stress have been shown to vary depending on wavelength or intensity of irradiation. Studies comparing treatment parameters are lacking, due to absence of commercially available devices that deliver multiple wavelengths or intensities, suitable for high through-put in vitro optimization studies. This protocol describes a technique for delivery of light at a range of wavelengths and intensities to optimize therapeutic doses required for a given injury model. We hypothesized that a method of delivering light, in which wavelength and intensity parameters could easily be altered, could facilitate determination of an optimal dose of R/NIR-LT for reducing reactive oxygen species (ROS) in vitro.
Non-coherent Xenon light was filtered through narrow-band interference filters to deliver varying wavelengths (center wavelengths of 440, 550, 670 and 810nm) and fluences (8.5 x 10-3 to 3.8 x 10-1 J/cm2) of light to cultured cells. Light output from the apparatus was calibrated to emit therapeutically relevant, equal quantal doses of light at each wavelength. Reactive species were detected in glutamate stressed cells treated with the light, using DCFH-DA and H2O2 sensitive fluorescent dyes.&#160;
We successfully delivered light at a range of physiologically and therapeutically relevant wavelengths and intensities, to cultured cells exposed to glutamate as a model of CNS injury. While the fluences of R/NIR-LT used in the current study did not exert an effect on ROS generated by the cultured cells, the method of light delivery is applicable to other systems including isolated mitochondria or more physiologically relevant organotypic slice culture models, and could be used to assess effects on a range of outcome measures of oxidative metabolism.

Method for the Assessment of Effects of a Range of Wavelengths and Intensities of Red/near-infrared Light Therapy on Oxidative Stress In Vitro

Non-coherent Xenon light was passed through narrow-band interference and neutral density filters to deliver light of varying wavelength and intensity to cultured cells. This protocol was used to assess the effects of red/near-infrared light therapy on production of reactive species in vitro: no effects were observed using the tested parameters.

Red/near-infrared light therapy (R/NIR-LT), delivered by laser or light emitting diode (LED), improves functional and morphological outcomes in a range of ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka (Oryzias latipes)

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in all vertebrates by providing feedback to the brain and pituitary. Teleost fishes possess a higher degree of tissue plasticity and variation in reproductive&#160;strategies compared to mammals and appear to be useful models to investigate the role of sex steroids and the mechanisms by which they act. The removal of the main source of sex steroid production using gonadectomy together with blood sampling to measure steroid levels has been well-established and fairly feasible in bigger fish and is a powerful technique to investigate the role and effects of sex steroids. However, these techniques raise challenges when implemented in small size teleost models. Here, we describe the step-by-step procedures of gonadectomy in both males and female Japanese medaka followed by blood sampling. These protocols are shown to be highly feasible in medaka indicated by a high survival rate, safety for the life span and phenotype of the fish, and reproducibility in terms of sex steroid clearance. The use of these procedures combined with the other advantages of using this small teleost model will greatly improve the understanding of feedback mechanisms in the neuroendocrine control of reproduction and tissue plasticity provided by sex steroids in vertebrates.

Gonadectomy and Blood Sampling Procedures in the Small Size Teleost Model Japanese Medaka Oryzias latipes

The article describes a quick protocol to gonadectomize and sample blood from the small teleost fish, using Japanese medaka (Oryzias latipes) as a model, to investigate the role of sex steroids in animal physiology.

Sex steroids, produced by the gonads, play an essential role in brain and pituitary tissue plasticity and in the neuroendocrine control of reproduction in ...