This work was supported by Charley&#39;s Fund and NIH R01NS055099.

All methods described here have been approved by the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center under protocol numbers (031-2019; 025-2019). Wear proper PPE equipment to handle animals and adhere to IACUC guidelines for all animal work.
1. In vivo surface EIM
<ol>
	<li>Place the animal in an anesthesia box to induce anesthesia. 
	NOTE: For rats, 1.5%-3.5% isoflurane and 2 O2 L&#183;min-1 were used, ...

<ol>
	<li>Rutkove, S. B., Sanchez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+impedance+methods+in+neuromuscular+assessment:+An+overview.">Electrical impedance methods in neuromuscular assessment: An overview.</a> Cold Spring Harbor Perspectives in Medicine. 9 (10), 034405 (2019).</li><li>Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+impedance+myography:+Background,+Current+State,+and+Future+Directions.">Electrical impedance myography: Background, Current State, and Future Directions.</a> Muscle &amp; Nerve. 40, 936-946 (2009).</li><li>Sanchez, B., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Present+uses,+future+applications,+and+technical+underpinnings+of+electrical+impedance+myography.">Present uses, future applications, and technical underpinnings of electrical impedance myography.</a> Current Neurology and Neuroscience Reports. 17 (11), 86 (2017).</li><li>Garmirian, L. P., Chin, A. B., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discriminating+neurogenic+from+myopathic+disease+via+measurement+of+muscle+anisotropy.">Discriminating neurogenic from myopathic disease via measurement of muscle anisotropy.</a> Muscle &amp; Nerve. 39 (1), 16-24 (2009).</li><li>Rutkove, S. B., 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=Loss+of+electrical+anisotropy+is+an+unrecognized+feature+of+dystrophic+muscle+that+may+serve+as+a+convenient+index+of+disease+status.">Loss of electrical anisotropy is an unrecognized feature of dystrophic muscle that may serve as a convenient index of disease status.</a> Clinical Neurophysiology. 127 (12), 3546-3551 (2016).</li><li>Wang, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+alterations+in+the+electrical+impedance+of+muscle+after+experimental+nerve+injury+via+finite-element+analysis.">Assessment of alterations in the electrical impedance of muscle after experimental nerve injury via finite-element analysis.</a> IEEE Transactions on Biomedical Engineering. 58 (6), 1585-1591 (2011).</li><li>Katirji, B., Ruff, R. L., Kaminski, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuromuscular Disorders in Clinical Practice. , (2014).</li><li>Rutkove, S. B., 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=Electrical+impedance+myography+for+assessment+of+Duchenne+muscular+dystrophy.">Electrical impedance myography for assessment of Duchenne muscular dystrophy.</a> Annals of Neurology. 81 (5), 622-632 (2017).</li><li>Semple, C., 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=Using+electrical+impedance+myography+as+a+biomarker+of+muscle+deconditioning+in+rats+exposed+to+micro-+and+partial-gravity+analogs.">Using electrical impedance myography as a biomarker of muscle deconditioning in rats exposed to micro- and partial-gravity analogs.</a> Frontiers in Physiology. 11, 557796 (2020).</li><li>Kortman, H. G. J., Wilder, S. C., Geisbush, T. R., Narayanaswami, P., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-+and+gender-associated+differences+in+electrical+impedance+values+of+skeletal+muscle.">Age- and gender-associated differences in electrical impedance values of skeletal muscle.</a> Physiological Measurement. 34 (12), 1611-1622 (2013).</li><li>Clark, B. C., Rutkove, S., Lupton, E. C., Padilla, C. J., Arnold, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+utility+of+electrical+impedance+myography+in+evaluating+age-related+skeletal+muscle+function+deficits.">Potential utility of electrical impedance myography in evaluating age-related skeletal muscle function deficits.</a> Frontiers in Physiology. 12, 666964 (2021).</li><li>Kapur, 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=Predicting+myofiber+size+with+electrical+impedance+myography:+A+study+in+immature+mice.">Predicting myofiber size with electrical impedance myography: A study in immature mice.</a> Muscle and Nerve. 58 (1), 106-113 (2018).</li><li>Kapur, K., Nagy, J. A., Taylor, R. S., Sanchez, B., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+myofiber+size+with+electrical+impedance+myography:+a+study+in+amyotrophic+lateral+sclerosis+mice.">Estimating myofiber size with electrical impedance myography: a study in amyotrophic lateral sclerosis mice.</a> Muscle and Nerve. 58 (5), 713-717 (2018).</li><li>Mortreux, M., Semple, C., Riveros, D., Nagy, J. A., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+impedance+myography+for+the+detection+of+muscle+inflammation+induced+by+&#955;-carrageenan.">Electrical impedance myography for the detection of muscle inflammation induced by &#955;-carrageenan.</a> PLoS ONE. 14 (10), 0223265 (2019).</li><li>Pandeya, S. 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=Predicting+myofiber+cross-sectional+area+and+triglyceride+content+with+electrical+impedance+myography:+A+study+in+db/db+mice.">Predicting myofiber cross-sectional area and triglyceride content with electrical impedance myography: A study in db/db mice.</a> Muscle and Nerve. 63 (1), 127-140 (2021).</li><li>Pandeya, S. 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=Estimating+myofiber+cross-sectional+area+and+connective+tissue+deposition+with+electrical+impedance+myography:+A+study+in+D2-mdx+mice.">Estimating myofiber cross-sectional area and connective tissue deposition with electrical impedance myography: A study in D2-mdx mice.</a> Muscle &amp; Nerve. 63 (6), 941-950 (2021).</li><li>St&#229;lberg, E., 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=Standards+for+quantification+of+EMG+and+neurography.">Standards for quantification of EMG and neurography.</a> Clinical Neurophysiology. 130 (9), 1688-1729 (2019).</li><li>Theodorou, D. J., Theodorou, S. J., Kakitsubata, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+disease:+Patterns+of+MRI+appearances.">Skeletal muscle disease: Patterns of MRI appearances.</a> British Journal of Radiology. 85 (1020), 1298-1308 (2012).</li><li>Simon, N. G., Noto, Y., Zaidman, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+imaging+in+neuromuscular+disease.">Skeletal muscle imaging in neuromuscular disease.</a> Journal of Clinical Neuroscience. 33, 1-10 (2016).</li><li>Kwon, H., Rutkove, S. B., Sanchez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+characteristics+of+electrical+impedance+myography+needle+electrodes.">Recording characteristics of electrical impedance myography needle electrodes.</a> Physiological Measurement. 38 (9), 1748-1765 (2017).</li><li>Kwon, H., Di Cristina, J. F., Rutkove, S. B., Sanchez, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+characteristics+of+electrical+impedance-electromyography+needle+electrodes.">Recording characteristics of electrical impedance-electromyography needle electrodes.</a> Physiological Measurement. 39 (5), 055005 (2018).</li><li>Rutkove, S. B., 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=Characterizing+spinal+muscular+atrophy+with+electrical+impedance+myography.">Characterizing spinal muscular atrophy with electrical impedance myography.</a> Muscle and Nerve. 42 (6), 915-921 (2010).</li><li>Schwartz, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+electrical+impedance+myography+measurements+by+using+a+multifrequency+ratio:+A+study+in+Duchenne+muscular+dystrophy.">Optimizing electrical impedance myography measurements by using a multifrequency ratio: A study in Duchenne muscular dystrophy.</a> Clinical Neurophysiology. 126 (1), 202-208 (2015).</li><li>Li, J., Pacheck, A., Sanchez, B., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+and+modeled+multifrequency+electrical+impedance+myography+parameters+and+their+relationship+to+force+production+in+the+ALS+SOD1G93A+mouse.">Single and modeled multifrequency electrical impedance myography parameters and their relationship to force production in the ALS SOD1G93A mouse.</a> Amyotrophic Lateral Sclerosis and Frontotemporal Degeneration. 17 (5-6), 397-403 (2016).</li><li>Hu, N., 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=Antisense+oligonucleotide+and+adjuvant+exercise+therapy+reverse+fatigue+in+old+mice+with+myotonic+dystrophy.">Antisense oligonucleotide and adjuvant exercise therapy reverse fatigue in old mice with myotonic dystrophy.</a> Molecular Therapy - Nucleic Acids. 23, 393-405 (2021).</li><li>Sanchez, B., 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+assessment+of+muscle+injury+in+healthy+and+dystrophic+animals+with+electrical+impedance+myography.">Non-invasive assessment of muscle injury in healthy and dystrophic animals with electrical impedance myography.</a> Muscle &amp; Nerve. 56 (6), 85-94 (2017).</li><li>Sanchez, B., Li, J., Bragos, R., Rutkove, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+the+intracellular+structure+of+slow-+versus+fast-twitch+muscle+fibers+through+evaluation+of+the+dielectric+properties+of+tissue.">Differentiation of the intracellular structure of slow- versus fast-twitch muscle fibers through evaluation of the dielectric properties of tissue.</a> Physics in Medicine and Biology. 59 (10), 2369-2380 (2014).</li><li>Shefner, J. 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=Assessing+ALS+progression+with+a+dedicated+electrical+impedance+myography+system.">Assessing ALS progression with a dedicated electrical impedance myography system.</a> Amyotrophic Lateral Sclerosis &amp; Frontotemporal Degeneration. 19 (7-8), 555-561 (2018).</li><li>Lungu, C., 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=Quantifying+muscle+asymmetries+in+cervical+dystonia+with+electrical+impedance:+a+preliminary+assessment.">Quantifying muscle asymmetries in cervical dystonia with electrical impedance: a preliminary assessment.</a> Clinical Neurophysiology. 122 (5), 1027-1031 (2011).</li><li>Wang, 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=Electrical+impedance+myography+for+assessing+paraspinal+muscles+of+patients+with+low+back+pain.">Electrical impedance myography for assessing paraspinal muscles of patients with low back pain.</a> Journal of Electrical Bioimpedance. 10 (1), 103-109 (2019).</li><li>Leitner, M. 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This article provides the basic methods for performing EIM in rodents, both in vivo and ex vivo. To acquire reliable measurements, it is critical to perform a series of steps. First, one needs to properly identify the muscle of interest, as each muscle will have different responses to diseases, treatment, and pathology. One must be mindful that the data acquired on one muscle (e.g., gastrocnemius) will not provide the same information as on another muscle (e.g., tibialis anterior). Second, one needs to ...

Electrical impedance myography (EIM) provides a powerful method to assess muscle condition, potentially enabling the diagnosis of neuromuscular disorders, tracking of disease progression, and assessment of response to therapy1,2,3. It can be applied analogously to animal disease models and humans, allowing for relatively seamless translation from preclinical to clinical studies. EIM measurements are easily obtained using four linearly-placed electrodes, with the two outer ones applying a painless, weak electrical current across a range of frequencies (generally between 1 kHz and approximately 2 MHz), and the two inner ones recording the resulting voltages1. From these voltages, the impedance characteristics of the tissue can be obtained, including the resistance (R), a measure of how difficult it is for current to pass through the tissue, and the reactance (X) or &#34;chargeability&#34; of the tissue, a measure related to the tissue&#39;s ability to store charge (capacitance). From the reactance and resistance, the phase angle (&#952;) is calculated via the following equation: <img alt="Equation 1" src="/files/ftp_upload/63513/63513eq01.jpg" style="margin: auto;" />, providing a single summative impedance measure. Such measurements can be obtained using any multifrequency bioimpedance device. As myofibers are essentially long cylinders, muscle tissue is also highly anisotropic, with current flowing more easily along fibers than across them4,5. Thus, EIM is often performed in two directions: with the array placed along the fibers such that current runs parallel to them, and across the muscle such that the current flows perpendicular to them. Additionally, in ex vivo measurements, where a known volume of tissue is measured in an impedance measuring cell, the inherent electrical properties of the muscle (i.e. the conductivity and relative permittivity), can be derived6.
The term &#34;neuromuscular disorders&#34; defines a wide range of primary and secondary diseases that lead to structural muscle alteration and dysfunction. This includes amyotrophic lateral sclerosis and various forms of muscular dystrophy, as well as simpler changes related to aging (e.g., sarcopenia), disuse atrophy (e.g., due to prolonged bedrest or microgravity) or even injury7. While the causes are plentiful and can originate from the motor neuron, nerves, neuromuscular junctions, or the muscle itself, EIM can be used to detect early alterations in muscle due to many of these processes and to track progression or response to therapy. For example, in patients with Duchenne muscular dystrophy (DMD), EIM has been shown to detect disease progression and response to corticosteroids8. Recent work has also shown EIM to be sensitive to varying disuse states, including fractional gravity9, as would be experienced on the Moon or Mars, and the effects of aging10,11. Finally, by applying predictive and machine learning algorithms to the data set obtained with each measurement (multifrequency and directionally dependent data), it becomes possible to infer histological aspects of the tissue, including myofiber size12,13, inflammatory changes and edema14, and connective tissue and fat content15,16.
Several other noninvasive or minimally invasive methods are also used to evaluate muscle health in humans and animals, including needle electromyography17 and imaging technologies such as magnetic resonance imaging, computerized tomography, and ultrasound18,19. However, EIM demonstrates distinct benefits compared with these technologies. For example, electromyography records only the active electrical properties of the myofiber membranes and not the passive properties, and thus cannot provide a true assessment of muscle composition or structure. In a certain respect, imaging methods are more closely related to EIM, as they too provide information about the structure and composition of tissue. But in some sense, they provide too much data, requiring detailed image segmentation and expert analysis rather than just providing a quantitative output. Moreover, given their complexities, imaging techniques are also greatly impacted by the specifics of both the hardware and software being used, ideally requiring the use of identical systems so that data sets can be compared. In contrast, the fact that EIM is much simpler means that it is less impacted by these technical issues and does not require any form of image processing or expert analysis.
The following protocol demonstrates how to perform in vivo EIM in rats and mice, using both noninvasive (surface array) and minimally invasive (subdermal needle array) techniques, as well as ex vivo EIM on freshly excised muscle.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Printer</td>
			<td>Formlabs Inc.</td>
			<td>Form 2 Desktop</td>
			<td>3D printer</td>
		</tr>
		<tr>
			<td>3D Printer</td>
			<td>Shenzhen Creality 3D Technology Co. LTD</td>
			<td>Creality Ender 3 V2</td>
			<td>3D printer</td>
		</tr>
		<tr>
			<td>3M Micropore surgical tape</td>
			<td>Fisher</td>
			<td>19-027761 and 19-061655</td>
			<td>models 1530-0 and 1530-1</td>
		</tr>
		<tr>
			<td>3M TRANSPORE surgical tape</td>
			<td>Fisher</td>
			<td>18-999-380 and 18-999-381</td>
			<td>models 1527-0 and 1527-1</td>
		</tr>
		<tr>
			<td>Connector header vertical 10 POS 1 mm spacing</td>
			<td>Digi-Key (Sullins connector solution)</td>
			<td>S9214-ND (SMH100-LPSE-S10-ST-BK)</td>
			<td>Plastic spacer 1 mm holes for the rat in vivo array displayed in Figure 2A</td>
		</tr>
		<tr>
			<td>Cotton-tipped applicators</td>
			<td>Fisher</td>
			<td>22-363-172</td>
			<td></td>
		</tr>
		<tr>
			<td>Dental Wax</td>
			<td>Fisher</td>
			<td>NC9377103</td>
			<td></td>
		</tr>
		<tr>
			<td>Depilatory agent</td>
			<td>NAIR</td>
			<td>NA</td>
			<td>hair remover lotion with softening baby oil</td>
		</tr>
		<tr>
			<td>Dumont #7b Forceps</td>
			<td>Fine Science Tools</td>
			<td>No. 11270-20</td>
			<td>Used for dissection, Style: #7b, Tip Shape: Curved, Tips: Standard, Tip Dimensions: 0.17 mm x 0.1 mm, Alloy/Material: Inox, Length: 11 cm</td>
		</tr>
		<tr>
			<td>Electronic Digital Caliper</td>
			<td>Fisher</td>
			<td>14-648-17</td>
			<td>Used to measure out the dimensions of the Gastrocnemius muscle</td>
		</tr>
		<tr>
			<td>Epoxy adhesive dual cartridge 4 min work life</td>
			<td>Devcon</td>
			<td>series 14265, model 2217</td>
			<td>Glue used in the rat in vivo array displayed in Figure 2A</td>
		</tr>
		<tr>
			<td>Ex vivo dielectric impedance cell</td>
			<td>Custom</td>
			<td>NA</td>
			<td>Dielectric cells were 3D printed in the Rutkove laboratory</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>Fine Science Tools</td>
			<td>No. 11051-10</td>
			<td>Used for muscle to place and adjust, Length: 10 cm, Tip Shape: Curved, Tips: Serrated, Tip Width: 0.8 mm, Tip Dimensions: 0.8 mm x 0.7 mm, Alloy/Material</td>
		</tr>
		<tr>
			<td>Hair clipper</td>
			<td>Amazon</td>
			<td>NA</td>
			<td>Wahl professional animal BravMini+</td>
		</tr>
		<tr>
			<td>Impedance Animal Device</td>
			<td>Myolex</td>
			<td>EIM1103</td>
			<td>mView system - investigational electrical impedance myography device for use in animal research</td>
		</tr>
		<tr>
			<td>In vivo needle arrays</td>
			<td>Custom</td>
			<td>NA</td>
			<td>Custom arrays using 27 G subdermal needles from Ambu. The construction was finalized using a 3D printer in the Rutkove laboratory</td>
		</tr>
		<tr>
			<td>In vivo surface array</td>
			<td>Custom</td>
			<td>NA</td>
			<td>The in vivo surface array was printed and assembled in the Rutkove laboratory</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Patterson Veterinary Supplies</td>
			<td>07-893-8441 (NDC: 46066-755-04)</td>
			<td>Pivetal - 250 mL bottle</td>
		</tr>
		<tr>
			<td>Non-woven gauze</td>
			<td>Fisher</td>
			<td>22-028-559</td>
			<td>2 x 2 inch</td>
		</tr>
		<tr>
			<td>Polystyrene Weighing Dishes</td>
			<td>Fisher</td>
			<td>S67090A</td>
			<td>Dimensions (L x W x H): 88.9 mm x 88.9 mm x 25.4 mm</td>
		</tr>
		<tr>
			<td>Razor Blades</td>
			<td>Fisher</td>
			<td>12-640</td>
			<td>Used to cut muscle to right dimensions, Single-edge carbon steel blades</td>
		</tr>
		<tr>
			<td>Student Fine Scissors</td>
			<td>Fine Science Tools</td>
			<td>No. 91460-11</td>
			<td>Used for dissection, Tips: Sharp-Sharp, Alloy/Material: Student Stainless Steel, Serrated: No, Tip Shape: Straight, Cutting Edge: 20 mm, Length: 11.5 cm, Feature: Student Quality</td>
		</tr>
		<tr>
			<td>Subdermal needles 27 G Neuroline</td>
			<td>Ambu</td>
			<td>745 12-50/24</td>
			<td>Needles used in the rat in vivo array displayed in Figure 2A</td>
		</tr>
		<tr>
			<td>Surgical Scissors - Sharp</td>
			<td>Fine Science Tools</td>
			<td>No. 14002-13</td>
			<td>Used to cut skin, Tips: Sharp-Sharp, Alloy/Material: Stainless Steel, Serrated: No, Tip Shape: Straight, Cutting Edge: 42 mm, Length: 13 cm</td>
		</tr>
		<tr>
			<td>TECA ELITE monopolar needle electrodes</td>
			<td>Natus</td>
			<td>902-DMG50-S</td>
			<td>0.46 mm diameter (26 G). Blue hub</td>
		</tr>
		<tr>
			<td>Teknova 0.9% saline solution</td>
			<td>Fisher</td>
			<td>S5815</td>
			<td>1000 mL sterile</td>
		</tr>
	</tbody>
</table>

performing in vivo and ex vivo electrical impedance myography in rodents

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and disease. EIM is obtained by applying a low-intensity, directionally focused, electrical current to a muscle of interest across a range of frequencies (i.e., from 1 kHz to 10 MHz) and recording the resulting voltages. From these, several standard impedance components, including the reactance, resistance, and phase, are obtained. When performing ex vivo measurements on excised muscle, the inherent passive electrical properties of the tissue, namely the conductivity and relative permittivity, can also be calculated. EIM has been used extensively in animals and humans to diagnose and track muscle alterations in a variety of diseases, in relation to simple disuse atrophy, or as a measure of therapeutic intervention. Clinically, EIM offers the potential to track disease progression over time and to assess the impact of therapeutic interventions, thus offering the opportunity to shorten the clinical trial duration and reduce sample size requirements. Because it can be performed noninvasively or minimally invasively in living animal models as well as humans, EIM offers the potential to serve as a novel translational tool enabling both preclinical and clinical development. This article provides step-by-step instructions on how to perform in vivo and ex vivo EIM measurements in mice and rats, including approaches to adapt the techniques to specific conditions, such as for use in pups or obese animals.

EIM can be obtained in many conditions, including surface in vivo arrays (Figure 1), needle in vivo arrays (Figure 2A-F), and ex vivo dielectric cells (Figure 2G,H).
EIM provides a near-instantaneous snapshot of the muscle condition based on the measured impedance values. Measurements are acquired swiftly and result in a simple ...

Watch this Scientific Journal Video about Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents at JoVE.com

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

This article details how to perform in vivo (using surface and needle electrode arrays) and ex vivo (using a dielectric cell) electrical impedance myography on the rodent gastrocnemius muscle. It will demonstrate the technique in both mice and rats and detail the modifications available, (i.e., obese animals, pups).

Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

Electrical impedance myography (EIM) is a convenient technique that can be used in preclinical and clinical studies to assess muscle tissue health and ...

Electrical Impedance Myography is a none or minimally invasive technique for assessing muscle condition. It provides a quantitative index of muscle health serving as a diagnostic tool or biomarker. So technique is quantitative, rapid, and does not require detailed image analysis so it can serve as a convenient biomarker of disease status in a therapeutic study.Another useful aspect of Electrical Impedance Myography is that it is sensitive to most types of skeletal muscle pathology. Abnormal Electrical Impedance Myography values can reveal underlying histological alterations. For surface measurements, the skin needs to be prepared well.One has to become familiar with the appearance of contact impedance artifacts and remeasure if needed. To begin, place the mouse on the bench in a prone position after confirming the absence of response during the toe pinching test. Place the animal's leg to be analyzed at a 45 degree angle with the knee extended and secure the foot with medical tape.Trim the fur overlaying the gastrocnemius muscle using a hair clipper, and apply a thick layer of depilatory cream over the animal's skin for one minute. Remove the depilatory agent using the saline saturated gauze and repeat this process up to three times until all the fur overlaying the gastrocnemius muscle is removed. Connect the surface array to the Electrical Impedance Myography device and let the electrodes rest on a piece of gauze soaked in saline solution.Place the surface array directly on the skin, over the gastrocnemius muscle oriented longitudinally to the muscle fibers. Check for appropriate contact by checking that all the bars appear green on the software with the stability of the 50 kilohertz resistance, reactance, and phase values. Then acquire the Electrical Impedance Myography measurements.Rotate the surface array by 90 degrees and reposition it on the skin over the gastrocnemius to obtain the transverse measurements. Connect the needle array to the Electrical Impedance Myography device and let the electrode array rest in a weighing boat containing saline solution. Then check for connectivity and signal stability.Place the needle array in a longitudinal position compared to the my fibers, and press it firmly into the skin until all the needles penetrate the skin and the underlying muscle up to the plastic guard on the array. Acquire the data. Gently remove the array and reinsert it through the skin and into the muscle at a 90 degree angle relative to the first measurement in the transverse direction.Then acquire this data. For Ex Vivo Electrical Impedance Myography, prepare the ex vivo dielectric cell, add saline solution to the chamber and connect the cell to the Electrical Impedance Myography device to obtain the reference values. Place the gastrocnemius muscle on a sheet of dental wax and section it using a razor blade in a ruler to obtain a 10 millimeter by 10 millimeter section for rats.And five millimeter by five millimeter section for mice from the center of the gastrocnemius muscle. Gently place the gastrocnemius in the dialectric cells using tweezers, making sure the fibers are oriented longitudinally and the muscle is entirely in contact with the metal electrodes. Attach the top part of the dialectric cell and insert two monopolar needles of 26 gauge into the two holes.Connect the wires from the Electrical Impedance Myography device to the ex vivo cell in the order mentioned in the manuscript. Then acquire the longitudinal measurement. Open the dialectric cell and reorient the muscle in the transverse direction by rotating it 90 degrees.Reattach the top of the dialectric cell and acquire the transverse measurement. Electrical Impedance Myography can be obtained in many conditions with surface in vivo arrays, needle in vivo arrays, and ex vivo dialectric cells. Representative curves for surface E I M are shown for phase, reactance and resistance for longitudinal in transverse measurements.Some irregularities, which are typically related to contact issues can result in extreme values observed at low frequencies. A graph showing the reactance as a resistance function and longitudinal and transverse directions is displayed. Good skin preparation is crucial for surface measurements.There can be no fur remaining. Repeat fur removal, saline application and measurement if contact artifact is identified. Electrical Impedance Myography allows enhanced assessment of disease status and therapy effects in rodent models helping to speed the identification of promising new therapies for the treatment of neuromuscular diseases.

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JoVE Journal

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2.6K Views.  Harvard Medical School - Beth Israel Deaconess Medical Center. Electrical Impedance Myography is a none or minimally invasive technique for assessing muscle condition. It provides a quantitative index of muscle health serving as a diagnostic tool or biomarker. So technique is quantitative, rapid, and does not require detailed image analysis so it can serve as a convenient biomarker of disease status in a therapeutic study.Another useful aspect of Electrical Impedance Myography is that it is sensitive to most types of skeletal muscle pathology. Abn...

Video: Performing In Vivo and Ex Vivo Electrical Impedance Myography in Rodents

This article details how to perform in vivo (using surface and needle electrode arrays) and ex vivo (using a dielectric cell) electrical impedance myography on the rodent gastrocnemius muscle. It will demonstrate the technique in both mice and rats and detail the modifications available, (i.e., obese animals,...

ex vivo release of calcitonin gene-related peptide from the trigeminovascular system in rodents

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

The present protocol describes the ex vivo calcitonin gene-related peptide (CGRP) release model and the strategy to quantify the effect of pharmacological agents on the amount of CGRP released from the trigeminovascular system in...

Ex Vivo Release of Calcitonin Gene-Related Peptide from the Trigeminovascular System in Rodents

alignment of in vivo and ex vivo mouse retinal images

Alignment of In Vivo and Ex Vivo Mouse Retinal Images  

alignment of visible-light optical coherence tomography fibergrams with confocal images of the same mouse retina

Author Spotlight: Advancements in In Vivo and Ex Vivo Retinal Imaging for Improved Glaucoma Diagnosis and Treatment 

The present protocol outlines the steps for aligning in vivo visible-light optical coherence tomography fibergraphy (vis-OCTF) images with ex vivo confocal images of the same mouse retina for the purpose of verifying the observed retinal ganglion cell axon bundle morphology in the in vivo images.

Mouse Retina OCT Fibergram Alignment with Confocal Images

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transcranial electrical brain stimulation in alert rodents

Transcranial Electrical Brain Stimulation in Alert Rodents

This protocol describes a surgical set-up for a permanent epicranial electrode socket and an implanted chest electrode in rodents. By placing a second electrode into the socket, different types of transcranial electrical brain stimulation can be delivered to the motor system in alert animals through the intact...

in vivo and ex vivo imaging for nanoparticle tracking in murine glioblastoma models

In Vivo and Ex Vivo Imaging for Nanoparticle Tracking in Murine Glioblastoma Models

normothermic ex vivo liver machine perfusion in mouse

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

A normothermic ex vivo liver perfusion (NEVLP) system was created for mouse livers. This system requires experience in microsurgery but allows for reproducible perfusion results. The ability to utilize mouse livers facilitates the investigation of molecular pathways to identify novel perfusate additives and enables the execution of experiments focused on organ...

Normothermic Ex Vivo Liver Machine Perfusion in Mouse

ex vivo calcium imaging of flat-mounted rat retina upon electrical stimulation

Ex Vivo Calcium Imaging of Flat-Mounted Rat Retina upon Electrical Stimulation  

"avatar", a modified ex vivo work loop experiments using in vivo strain and activation

Author Spotlight: Bridging the Gap Between In Vivo and Ex Vivo Studies with the &quot;Avatar&quot; Technique to Advance Muscle Mechanics Research 

This article details the methodology for emulating in vivo muscle force production during ex vivo work loop experiments using an "avatar" muscle from a laboratory rodent to assess the contributions of strain transients and activation to the muscle force response.

"Avatar", a Modified Ex vivo Work Loop Experiments Using In vivo Strain and Activation

in vivo luminal measurement of distension-evoked urothelial atp release in rodents

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

This protocol describes the procedure for measuring ATP concentrations in the lumen of the bladder in an anesthetized...

In Vivo Luminal Measurement of Distension-Evoked Urothelial ATP Release in Rodents

ex vivo and in vivo animal models for mechanical and chemical injuries of corneal epithelium

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

simultaneous ex vivo functional testing of two retinas by in vivo electroretinogram system

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

ex vivo optogenetic dissection of fear circuits in brain slices

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

zebrafish scale regeneration in toto and ex vivo culture of scales

Author Spotlight: Use of Fish Scales for Bone Remodeling Research &amp;#8211; Advancements in Ex Vivo Imaging and Dissecting Cell Interactions

This protocol describes the harvesting and visualization of elasmoid scales of zebrafish during in vivo regeneration. In addition, the ex vivo culture of these scales for up to 7 days after harvest is...

Zebrafish Scale Regeneration In Toto and Ex Vivo Culture of Scales

&quot;Avatar&quot;- Optimized Ex Vivo Work Loop Experiment to Emulate In Vivo Muscle Force

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determination of mitochondrial respiration and glycolysis in ex vivo retinal tissue samples

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

fabrication and implantation of miniature dual-element strain gages for measuring in vivo gastrointestinal contractions in rodents.

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

The in vivo measurement of smooth muscle contractions along the gastrointestinal tract of laboratory animals remains a powerful, though underutilized, technique. Flexible, dual element strain gages are not commercially available and require fabrication. This protocol describes the construction of reliable, inexpensive strain gages for acute or chronic implantation in...

Fabrication and Implantation of Miniature Dual-element Strain Gages for Measuring In Vivo Gastrointestinal Contractions in Rodents.

"avatar" work loop technique using in vivo strain trajectories to replicate ex vivo muscle force

"Avatar" Work Loop Technique Using In Vivo Strain Trajectories to Replicate Ex Vivo Muscle Force

ex vivo assessment of contractility, fatigability and alternans in isolated skeletal muscles

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

validation of nanobody and antibody based in vivo tumor xenograft nirf-imaging experiments in mice using ex vivo flow cytometry and microscopy

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

This protocol outlines the steps required to perform ex vivo validation of in vivo near-infrared fluorescence xenograft imaging experiments in mice using fluorophore labelled nanobodies and conventional...

Validation of Nanobody and Antibody Based In Vivo Tumor Xenograft NIRF-imaging Experiments in Mice Using Ex Vivo Flow Cytometry and Microscopy

Ex vivo Mechanical Loading of Tendon

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace. Most are associated with repetitive, high force hand activities. The mechanisms of cellular and structural damage due to cyclical loading are not well known. The purpose of this video is to present a new system that can simultaneously load four tendons in tissue culture. The video describes the methods of sterile tissue harvest and how the tendons are loaded onto a clamping system that is subsequently immersed into media and maintained at 37&deg;C. One clamp is fixed while the other one is moved with a linear actuator. Tendon tensile force is monitored with a load cell in series with the mobile clamp. The actuators are controlled with a LabView program. The four tendons can be repetitively loaded with different patterns of loading, repetition rate, rate of loading, and duration. Loading can continue for a few minutes to 48 hours. At the end of loading, the tendons are removed and the mid-substance extracted for biochemical analyses. This system allows for the investigation of the effects of loading patterns on gene expression and structural changes in tendon. Ultimately, mechanisms of injury due to overuse can be studies with the findings applied to treatment and prevention.

A new in vitro system for simultaneously loading four tendons in culture is described.

Injuries to the tendon (e.g., wrist tendonitis, epicondyltis) due to overuse are common in sports activities and the workplace.  Most are associated with ...

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell differentiation, axon guidance, retinotopic organization and synapse formation[1]. One drawback is the inability to efficiently and reliably manipulate gene expression in RGCs in vivo, especially in the otherwise accessible murine visual pathways. Transgenic mice can be used to manipulate gene expression, but this approach is often expensive, time consuming, and can produce unwanted side effects. In chick, in ovo electroporation is used to manipulate gene expression in RGCs for examining retina and RGC development. Although similar electroporation techniques have been developed in neonatal mouse pups[2], adult rats[3], and embryonic murine retinae in vitro[4], none of these strategies allow full characterization of RGC development and axon projections in vivo. To this end, we have developed two applications of electroporation, one in utero and the other ex vivo, to specifically target embryonic murine RGCs[5, 6]. 
With in utero retinal electroporation, we can misexpress or downregulate specific genes in RGCs and follow their axon projections through the visual pathways in vivo, allowing examination of guidance decisions at intermediate targets, such as the optic chiasm, or at target regions, such as the lateral geniculate nucleus. Perturbing gene expression in a subset of RGCs in an otherwise wild-type background facilitates an understanding of gene function throughout the retinal pathway. Additionally, we have developed a companion technique for analyzing RGC axon growth in vitro. We electroporate embryonic heads ex vivo, collect and incubate the whole retina, then prepare explants from these retinae several days later. Retinal explants can be used in a variety of in vitro assays in order to examine the response of electroporated RGC axons to guidance cues or other factors. In sum, this set of techniques enhances our ability to misexpress or downregulate genes in RGCs and should greatly aid studies examining RGC development and axon projections.

In utero and ex vivo Electroporation for Gene Expression in Mouse Retinal Ganglion Cells

Here we present two techniques for manipulating gene expression in murine retinal ganglion cells (RGCs) by in utero and ex vivo electroporation. These techniques enable one to examine how alterations in gene expression affect RGC development, axon guidance, and functional properties.

The retina and its sole output neuron, the retinal ganglion cell (RGC), comprise an excellent model in which to examine biological questions such as cell ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, and membrane potential. All of these approaches depend on measurement of activity at one or multiple locations along the gut simultaneously which are then interpreted to provide a sense of overall motility patterns. Recently, the development of video recording and spatiotemporal mapping (STmap) techniques have made it possible to observe and analyze complex patterns in ex vivo whole segments of colon and intestine. Once recorded and digitized, video records can be converted to STmaps in which the luminal diameter is converted to grayscale or color [called diameter maps (Dmaps)]. STmaps can provide data on motility direction (i.e., stationary, peristaltic, antiperistaltic), velocity, duration, frequency and strength of contractile motility patterns. Advantages of this approach include: analysis of interaction or simultaneous development of different motility patterns in different regions of the same segment, visualization of motility pattern changes over time, and analysis of how activity in one region influences activity in another region. Video recordings can be replayed with different timescales and analysis parameters so that separate STmaps and motility patterns can be analyzed in more detail. This protocol specifically details the effects of intraluminal fluid distension and intraluminal stimuli that affect motility generation. The use of luminal receptor agonists and antagonists provides mechanistic information on how specific patterns are initiated and how one pattern can be converted into another pattern. The technique is limited by the ability to only measure motility that causes changes in luminal diameter, without providing data on intraluminal pressure changes or muscle tension, and by the generation of artifacts based upon experimental setup; although, analysis methods can account for these issues. When compared to previous techniques the video recording and STmap approach provides a more comprehensive understanding of gastrointestinal motility.

Spatiotemporal Mapping of Motility in Ex Vivo Preparations of the Intestines

Recently available video recording and spatiotemporal mapping (STmap) techniques make it possible to visualize and quantify both propagating and mixing patterns of intestinal motility. The goal of this protocol is to explain the generation and analysis of STmaps using the GastroIntestinal Motility Monitoring (GIMM) system.

Multiple approaches have been used to record and evaluate gastrointestinal motility including: recording changes in muscle tension, intraluminal pressure, ...

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, is expressed in neurons in response to various stimuli. The protein product can be readily detected with immunohistochemical techniques leading to the use of c-FOS detection to map groups of neurons that display changes in their activity. In this article, we focused on the identification of brainstem neuronal populations involved in the ventilatory adaptation to hypoxia or hypercapnia. Two approaches were described to identify involved neuronal populations in vivo in animals and ex vivo in deafferented brainstem preparations. In vivo, animals were exposed to hypercapnic or hypoxic gas mixtures. Ex vivo, deafferented preparations were superfused with hypoxic or hypercapnic artificial cerebrospinal fluid. In both cases, either control in vivo animals or ex vivo preparations were maintained under normoxic and normocapnic conditions. The comparison of these two approaches allows the determination of the origin of the neuronal activation i.e., peripheral and/or central. In vivo and ex vivo, brainstems were collected, fixed, and sliced into sections. Once sections were prepared, immunohistochemical detection of the c-FOS protein was made in order to identify the brainstem groups of cells activated by hypoxic or hypercapnic stimulations. Labeled cells were counted in brainstem respiratory structures. In comparison to the control condition, hypoxia or hypercapnia increased the number of c-FOS labeled cells in several specific brainstem sites that are thus constitutive of the neuronal pathways involved in the adaptation of the central respiratory drive.

The c-FOS Protein Immunohistological Detection: A Useful Tool As a Marker of Central Pathways Involved in Specific Physiological Responses In Vivo and Ex Vivo

Here, we present a protocol based on c-FOS protein immunohistological detection, a classical technique used for the identification of neuronal populations involved in specific physiological responses in vivo and ex vivo.

Many studies seek to identify and map the brain regions involved in specific physiological regulations. The proto-oncogene c-fos, an immediate early gene, ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...