Name: evaluation of injury-induced senescence and in vivo reprogramming in the skeletal muscle
Uploaded: 2017-10-26T14:00:00.000+00:00
Duration: 9 min 14 s
Description: Watch this Scientific Journal Video about Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle at JoVE.com

We are indebted to Clemire Cimper for her excellent technical support. Work in the laboratory of H.L. was funded by Institut Pasteur, Centre National pour la Recherche Scientific, and the Agence Nationale de la Recherche (Laboratoire d&#39;Excellence Revive, Investissement d&#39;Avenir; ANR-10-LABX- 73), the Agence Nationale de la Recherche (ANR-16-CE13-0017-01) and Fondation ARC (PJA 20161205028). C.C. and A.C. are funded by the Ph.D. and postdoctoral fellowships from the Revive Consortium.

Animals were handled as per European Community guidelines and the ethics committee of the Institut Pasteur (CETEA) approved protocols.
1. Preparations of the Stock Solutions
<ol>
	<li>Prepare the materials for muscle sample fixation.Dissolve 0.5 g of tragacanth gum with 20 mL water at RT to make the freezing-embedding medium for muscle fixation.</li>
	<li>Prepare the solutions for SA-&#946;-Gal staining.
	<ol>
		<li>Prepare the stock solutions of K3Fe(CN)6 (100 mM), K4Fe(CN)6 (100 mM), MgCl2 (1M) by dissolving the respective powders in distilled water.</li>
		<li>Prepare the stock solution of 0.2% C20H6Br4Na2O5 (Eosin) by diluting it in water.</li>
		<li>Prepare the stock solution of C14H15BrClNO6(X-gal) (50 mg/mL) by dissolving the X-gal powder in C3H7NO (dimethylformamide, DMF).</li>
		<li>Store K3Fe(CN)6 and K4Fe(CN)6 solutions at 4 &#176;C, and MgCl2 at RT. 
		NOTE: X-gal can be stored in aliquot at -20 &#176;C, up to 6 months. Eosin solution can be kept at RT and reused after filtering if necessary. K3Fe(CN)6 and K4Fe(CN)6 solutions are protectedfromthe light. X-gal solution is not stable in water and has to be protected from light.</li>
	</ol>
	</li>
	<li>Preparation of the solutions for Nanog staining: the permeabilization solution contains 0.1% Na3C6H5O7 (trisodium citrate), 0.1% C14H22O(C2H4O)n(n = 9-10) (Triton X-100) in distilled water, which should be stored at 4 &#176;C. Prepare the blocking solution containing 5% fetal bovine serum (FBS) in phosphate-buffered saline (PBS), which should be stored at RT.</li>
</ol>
2. SA-&#946;-Gal Staining on Frozen TA Muscle Section
<ol>
	<li>Prepare the fixation material for the TA muscle by placing a small amount of the tragacanth gum on a slice of cork.</li>
	<li>Injured mice of both sexes (2 month old, C57/B6) were injured 10 days before with cardiotoxin (CDX) as described previously13. Use non-injured (PBS injection) TA from the same mouse as the negative control. If in vivo reprogramming is desired, treat each mouse with Dox (1 mg/mL) in the drinking water on the same day (protected from the light), right after CTX injury. 
	NOTE: The Dox solution needs to be changed every 3 days for a total treatment duration of 7 days.
	<ol>
		<li>Isolate both TA muscles (injured and control) from mice as described previously (Figure 1A)13. To ensure the transverse sections, insert the distal tendon of the TA muscle into the tragacanth gum and leave roughly &#190; part of the muscle outside (Figure 1B) and freeze directly in liquid nitrogen cooled isopentane for &#60;1 min. 
		NOTE: Make sure the TA muscle is in a perpendicular position and in the center of the cork. Samples can be stored at -80&#176;C or directly cryosectioned in 10 &#181;m sections.</li>
	</ol>
	</li>
	<li>Process the TA muscles as described in the Figure 1B.
	<ol>
		<li>Distribute the 1st-10th sections in the correct order onto ten different slides at the upper left position of each slide. Place the 11th section adjacent to the 1st section on the first slide; the 12th section will follow the same order to be placed right besides the 2nd section on the second slide. Repeat this process till 10 sections/slide are obtained for ten slides (100 sections in total) to ensure minimum 1 mm distance between the first section and the last section.</li>
	</ol>
	</li>
	<li>Fix the sections for 4 min in PBS containing 1% paraformaldehyde and 0.2% glutaraldehyde. Wash with PBS, 2x&#160;10 min. Next, incubate the sections in PBS (pH = 5.5) for 30 min. Perform all the steps at RT. 
	NOTE: The fixation has to be mild to maintain enzymatic activity. Perform this step under the hood. The pH of the PBS is critical and the X-gal solution must be protected from light.</li>
	<li>Incubate sections in the X-gal solution containing: 4 mM K3Fe(CN)6, 4 mM K4Fe(CN)6, 2 mM MgCl2, and 400 &#181;g/mL X-Gal in PBS, pH = 5.5. Incubate in the dark at 37 &#176;C for a minimum of 24 h. Wash the slides with PBS, 3x 10 min, at RT. 
	NOTE: The incubation requires a minimum of 24 hours and can last for 48 hours to maximize the SA-&#946;-Gal signal. The solution needs to be changed after 24 hours of incubation. The slides should be protected from light. If only SA-&#946;-Gal staining is desired, continue with step 2.5. If co-staining with Nanog is desired, please skip forward to step 3.1.</li>
	<li>Fix slides in 1% paraformaldehyde in PBS for 30 min. Wash slides with PBS, 3x 10 min. Counterstain with 0.2% eosin. Immerse the slides in the eosin solution for 1 min and rinse them with distilled water briefly. Finally, mount the slides with aqueous non-fluorescing mounting medium (see Table of Materials). 
	NOTE: It is essential to perform the post-fixation. Perform this step under the hood. All steps are performed at RT.</li>
</ol>
3. Immunohistochemistry Using Anti-Nanog Antibody
<ol>
	<li>Fix the slides with PBS containing 4% paraformaldehyde for 10 min. Wash with PBS, 2x 10 min. Add 200 &#181;L of the permeabilization solution directly onto the slides and incubate for 5 min.
	<ol>
		<li>Wash slides with PBS, 2x 5 min and in the last wash, use 200 &#181;L PBS containing 0.25% BSA directly on the slides. Perform all these steps at RT. 
		Caution: Perform the fixation step under the hood.</li>
	</ol>
	</li>
	<li>Incubate slides with the primary anti-Nanog antibody (final concentration: 1.25 ug/mL) overnight at 4 &#176;C in PBS containing 5% FBS. Wash slides with PBS, 2x 10 min and in the last wash, use 200 &#181;L PBS containing 0.25% BSA for 5 min. Perform all the washing steps at RT. 
	NOTE: Incubate slides in a box with wet paper towel to prevent evaporation.</li>
	<li>Incubate slides with 100 &#181;L of rAb-HRP secondary antibody from a ready to use kit (see Table of Materials) for 45 min. Wash slides with PBS, 3x 5 min, to remove secondary antibody. Perform all the steps at RT with protection from light.</li>
	<li>Visualization
	<ol>
		<li>First, dilute the 3,3&#39;-diaminobenzidine (C12H14N4C12H18Cl4N4(4HCl), DAB) in the substrate buffer provided by the ready to use kit (20 &#181;L of DAB for 1 mL of substrate buffer solution). Add 100 &#181;L of DAB solution directly onto each slide and incubate for a maximum of 10 min at RT. 
		NOTE: The diluted DAB solution should be prepared freshly and can be stored for up to one week at 4 &#176;C. The incubation time can be adjusted to minimize the background signal but must be kept the same for all slides.</li>
		<li>Remove the DAB solution by rinsing with water. Counterstain the slides with fast red solution (ready to use, see the Table of Materials) for 20 min. Wash the slides with water again briefly.</li>
		<li>Dehydrate them with 95% ethanol for 5 min followed by 100% ethanol, 2x 5 min. Finally, mount the slides with quick-hardening mounting medium (see Table of Materials).</li>
		<li>Observe the slides under the microscope in bright field at 20X to avoid background. 
		NOTE: Fast red solution can be kept at RT and re-used after filtering.</li>
	</ol>
	</li>
</ol>
4. Analysis and Quantification
<ol>
	<li>SA-&#946;-Gal quantification
	<ol>
		<li>Scan the slides and choose the best sections on each slide based on the acquired images. Use at least 2 sections/TA for the quantification. Judge the section&#39;s quality based on tissue integrity, quality of the staining, and counterstaining. For the quantification, choose the two highest quality sections with the maximum possible distance in between samples, which allows better representation of SA-&#946;-Gal quantification throughout the whole TA muscle.</li>
		<li>Quantification of SA-&#946;-Gal positive cells (Figure 2).
		<ol>
			<li>Determine the rank of the pixel size by manually selecting the smallest and the largest positive SA-&#946;-Gal cells.
			<ol>
				<li>Open the digital image of the scanned slide using ImageJ software.</li>
				<li>In the interface, click &#39;Analyze&#39; &#62; &#39;Tools&#39; &#62; &#39;ROI Manager&#39; &#62; &#39;Analyze&#39; &#62; &#39;set measurement&#39; &#62; &#39;select Area&#39;. Use the selection tool and surround the smallest and biggest positive SA-&#946;-Gal cell and add it to the ROI manager by clicking on &#39;Add&#39;. Measure the sizes using the &#39;Measure&#39; button and save the values for later use.</li>
			</ol>
			</li>
			<li>Adjust the threshold parameter to ensure all the visible positive cells are selected.
			<ol>
				<li>Convert the image to gray scale by clicking &#39;Image&#39; &#62; &#39;type&#39; &#62; &#39;8-bit&#39; (Figure 2A). Next, go to &#39;Image&#39; &#62; &#39;Adjust&#39; &#62; &#39;Threshold&#39;, move the second cursor until all the positive SA-&#946;-Gal cells are covered in red (Figure 2B), then click &#39;Apply&#39;.</li>
				<li>Analyze particles by clicking &#39;Analyze&#39; &#62; &#39;Analyze Particles&#39; &#62; &#39;Size (pixel^2)&#39;, and apply the value defined in step 4.1.2.1, enter &#39;Circularity&#39;: &#39;0.00-1.00&#39;, click &#39;ok&#39; ; a summary of all the counted particles is shown in the ROI manager (Figure 2D).</li>
			</ol>
			</li>
			<li>Transfer all the selected particles to the original image. Adjust the selection manually to ensure accurate quantification. Add positive cells (green arrow, Figure 2E) and/or remove false positive cells (red arrow, Figure 2E). Finally, measure the area by outlining the section (Figure 2D) using the &#39;selection tool&#39;. Click &#39;ROI manager&#39; &#62; &#39;Add&#39; &#62; &#39;Measure&#39;.</li>
			<li>Importantly, normalize the number of positive cells by the area of the section.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Nanog quantification
	<ol>
		<li>Count the Nanog positive cells under the microscope in the bright field manually at 20X magnification. 
		NOTE: The fast red counterstaining allows good evaluation of the morphology of the TA muscle. However, the staining is very light and lacks a clear outline of the section. The scanner often fails to identify the boundary of the sections and cannot focus correctly. It is possible to use marker to circle the edges of the sections before scanning or use a more sensitive scanner to detect the section. For the current protocol, it is most efficient to count the Nanog positive cells manually.</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

Here, we present a method to detect both senescent and pluripotent stem cells in the skeletal muscle of reprogrammable mice. This method could be used to evaluate and quantify both senescence and induce cellular plasticity in vivo, and examine the role of senescence in tissue repair and regeneration.
In the current protocol, the senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is used to detect in vivo senescent cells in the skeletal muscle. This assay detects the increased lysosomal &#946;-galactosidase activity at suboptimum pH (6.0 or 5.5), associated specifically with senescent cells, while this enzyme&#39;s activity is typically measured at acidic pH 4.516,17. Therefore, it is important to adjust the pH (pH = 6 or 5.5) to ensure a specific detection of senescence-associated activity. In addition, counter staining with eosin is essential for the automatic quantification of SA-&#946;-Gal+ cells, where the weak and diffuse signals are not counted. To avoid potential variability, it is preferable that the same person performs the entire counting procedure.
Although the SA-&#946;-Gal assay is the most widely used and accepted biomarker for senescent cells, it&#160;is not an exclusive marker for senescence. It has been suggested that over-confluent cells in culture might cause false positivity for SA-&#946;-Gal18. The sensitivity of the assay can be cell type and tissue type dependent in vivo19. Therefore, it is necessary to use other independent canonical markers, such as lack of proliferation, increased expression of senescence mediators (p16, ARF, p53, p21, and p27), and the secretion of various SASP&#160;factors, to further confirm and characterize senescence in vivo. Moreover, proper negative controls are indispensable for interpreting results, especially for in vivo study.
Despite the fact that SA-&#946;-Gal assay is not perfect, it does provide particularly valuable information for in vivo study. It permits the detection of senescent cells in their resident environment with intact tissue architecture, providing critical information facilitating the further understanding of the senescent cells&#39; role in different physiological and pathological contexts. Moreover, it can be coupled with the immunostaining of other markers, such as cell surface markers to determine the cellular identity of senescent cells; or stemness markers to examine the potential involvement of senescence in regeneration and tumorigenesis. Previously, we performed the SA-&#946;-Gal assay with immunohistochemical staining of Nanog in the same section or in close proximity to investigate the potential link between senescence and in vivo reprogramming8. While this protocol is focused on skeletal muscle, it can be certainly extended to other tissues.
Recently, cellular senescence has been implicated in tissue repair and regeneration, most likely via SASPs7,8,9. Understanding the mechanisms of how senescence contributes to tissue repair and regeneration will certainly have a tremendous impact on regenerative medicine. This assay provides an important and valuable tool to facilitate the identification and quantification of senescent cells in vivo.

Cellular senescence is a form of stress response characterized by a stable cell-cycle arrest. In the last decade, research has firmly established that senescence is associated with various biological and pathological processes including embryonic development, fibrosis, and organism ageing1,2. Cellular senescence was first identified in human fibroblasts at the end of their replicative lifespan triggered by telomere shortening3. Besides replicative stress, there are many other stimuli that can induce senescence, including DNA damage, oxidative stress, oncogenic signals, and genomic/epigenomic alterations, any of which may eventually activate the p53/p21 and/or pRB pathways to establish and reinforce the permanent growth arrest1. One of the important characteristics of senescent cells is that they remain metabolically active and robustly express a senescence-associated secretory phenotype (SASP): secretion of many inflammatory cytokines, growth factors, and extracellular matrix factors4. SASP factors have been proposed to play an important role in mediating and amplifying the senescence effect, due to their potent effects on attracting immune cells and altering local and systemic tissue milieus1. Interestingly, senescence has been recently proposed to be important for tissue repair and regeneration5,6. In addition, data from several labs, including ours, has suggested that tissue damage-induced senescence might enhance cellular plasticity, via SASPs, to promote regeneration7-9. Therefore, all the emerging data highlight the importance of studying senescence in vivo.
In the post induced pluripotent stem cell&#160;(iPSC) era, cellular plasticity is the capacity of a cell to acquire a new identity and to adopt an alternative fate when exposed to different stimuli both in culture and in vivo10. It is known that full reprogramming can be achieved in vivo11,12, where the expression of the the cassette containing four Yamanaka factors: Oct4, Sox2, Klf4, and c-Myc (OSKM) can be induced in vivo to promote teratomas formation in multiple organs. Therefore, a reprogrammable mouse model (i4F) can be used as a powerful system to identify critical regulators and pathways that are important for cellular plasticity11.
A suitable and sensitive in vivo system is essential to understand how cellular senescence regulates cellular plasticity in the context of tissue regeneration. Here, we present a robust system and a detailed protocol to evaluate the link between senescence and cellular plasticity in the context of tissue regeneration. The combination of cardiotoxin (CTX) induced muscle damage in the Tibialis Anterior (TA) muscle group, a well-established system to study tissue regeneration, and the i4F mouse model, allows the detection of both cellular senescence and in vivo reprogramming during muscle regeneration.
To evaluate the link between cellular plasticity and senescence, i4F mice are injured with CTX to induce acute muscle damage and treated with doxycycline (0.2 mg/mL) over 7 days to induce in vivo reprogramming. While a CTX induced acute muscle damage and regeneration protocol has been recently published13, for ethical reasons, this procedure will be omitted in the current protocol. TA muscle samples will be collected at 10 days post injury13, when the peak of senescent cells have been previously observed14. Here, this detailed protocol describes all the steps required to evaluate the level of senescence (via SA-&#946;-Gal) and reprogramming (via IHC staining of Nanog).
Senescence-associated beta-galactosidase (SA-&#946;-Gal) assay is the most commonly used assay to detect senescent cells both in culture and in vivo15. Compared to other assays, the SA-&#946;-Gal assay allows the identification of the senescent cells in their native environment with intact tissue architecture, which is particularly important for in vivo study. Moreover, it is possible to couple the SA-&#946;-Gal assay with other markers using IHC. However, the SA-&#946;-Gal assay does require fresh or frozen samples, which remains a major limitation. When fresh or frozen tissues are routinely available, such as frozen TA muscle samples, SA-&#946;-Gal is obviously the most suitable assay to detect senescent cells. Nanog is the marker used to detect reprogramed cells for two reasons: 1) it is an essential marker for pluripotency; 2) more importantly, its expression is not driven by doxycycline (dox), therefore it detects induced pluripotency rather than the forced expression of the Yamanaka cassette.
It is important to note, the staining protocols presented in this study can be conducted separately to simplify the quantification procedure, but can also be done in a co-staining procedure to visualize both senescent and pluripotent stem cells on the same section.

<table><tbody><tr><td>K&lt;sub&gt;3&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>13746-66-2</td><td>For SA-&amp;beta; Gal staining solution</td></tr><tr><td>K&lt;sub&gt;4&lt;/sub&gt;Fe(CN)&lt;sub&gt;6&lt;/sub&gt;</td><td>Sigma</td><td>14459-95-1</td><td>For SA-&amp;beta; Gal staining solution</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma</td><td>7786-30-3</td><td>For SA-&amp;beta; Gal staining solution</td></tr><tr><td>X-Gal</td><td>Sigma</td><td>B4252</td><td>For SA-&amp;beta; Gal staining solution</td></tr><tr><td>Doxycycline</td><td>Sigma</td><td>D3447</td><td>For inducing in vivo reprogramming</td></tr><tr><td>Cardiotoxin</td><td>Lotaxan Valence, France</td><td>L8102</td><td>For muscle injury</td></tr><tr><td>Glutaraldehyde</td><td>Sigma</td><td>111-30-8</td><td>For Fixation solution</td></tr><tr><td>Paraformaldehyde</td><td>Electron microscopy science</td><td>50-980-487</td><td>For Fixation solution</td></tr><tr><td>NaCitrate : &lt;br/&gt;Sodium Citrate monobasic bioxtra, anhydre</td><td>Sigma</td><td>18996-35-5</td><td>For permeabilization solution</td></tr><tr><td>Triton</td><td>Sigma</td><td>93443</td><td>For permeabilization solution</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A3608</td><td>Washing solution</td></tr><tr><td>Antibody anti- Nanog</td><td>Cell signalling</td><td>8822S</td><td>Rabbit monoclonal antibody</td></tr><tr><td>EnVision+ Kits (HRP. Rabbit. DAB+)</td><td>Dako</td><td>K4010</td><td>For Nanog revelation</td></tr><tr><td>Eosin 1%</td><td>Leica</td><td>380159EOF</td><td>Counterstainning</td></tr><tr><td>Fast red</td><td>Vector Laboratories</td><td>H-3403</td><td>Counterstainning</td></tr><tr><td>Thermo Scientific Shandon Immu-Mount</td><td>Fisher scientific</td><td>9990402</td><td>Mounting solution</td></tr><tr><td>Quick-hardening mounting medium for microscopy : Eukitt&amp;reg;</td><td>Sigma</td><td>25608-33-7</td><td>Mounting solution</td></tr><tr><td>Microscope Phase Contrast Brightfield CKX41: 10X-20X-40X objectives</td><td>Olympus</td><td>CKX41</td><td>Microscope for Nanog quantification</td></tr><tr><td>Mouse: i4F-A</td><td>Abad et al., 2013</td><td>N/A</td><td>Reprogrammable mouse model</td></tr><tr><td>Skeletal muscle, Tibialis Anterior</td><td></td><td></td><td></td></tr><tr><td>Slide Scanner</td><td>Zeiss</td><td>Axio Scan Z1</td><td>slides scanning</td></tr></tbody></table>

evaluation of injury-induced senescence and in vivo reprogramming in the skeletal muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Detecting muscle injury-induced cellular senescence
It has been recently demonstrated that muscle injury induces transient cellular senescence14. At 10 days post-injury (DPI), the majority of the damaged myofibers are undergoing regeneration with centrally located nuclei, a hallmark of regenerating myofibers, and the architecture of the muscle is re-established. The infiltrating inflammatory cells are dramatically reduced while remaining visible in certain regions. 10 DPI is a good time point to detect senescent cells by SA-&#946;-Gal, since there are fewer necrotic and inflammatory cells present in the muscle to interfere with the staining. To determine the specificity of the staining, TA injected with PBS from the same mouse (Figure 1A) is used as a critical negative control.
To ensure a better and more precise evaluation of the SA-&#946;-Gal positive cells, sections from different planes of the TA muscle are placed in the same slide (Figure 1B). Counter staining with eosin is important for the automatic quantification of the SA-&#946;-Gal positive cells by ImageJ software. Eosin counter staining outlines the section, which allows the digital scanner to detect the sections with the correct focus. It is important to carefully define the range and the threshold of the detection (Figure 2A-D). In addition, a manually curated process is essential to permit more accurate detection and quantification (Figure 2E).
Cellular senescence facilitates in vivo reprogramming in muscle
Reprogrammable mouse model (i4F) provides an ideal system to evaluate the impact of senescence on cellular plasticity and regeneration. Upon muscle injury, i4F mice are treated with dox to induce reprogramming in vivo. 7 days dox (1 mg/mL) treatment is sufficient to induce reprogramming on the cellular level, while still being well tolerated by mice (Figure 3A). Therefore, we harvest injured muscles at 10 DPIs from i4F mice treated with dox for 7 days.
Although it is possible to perform co-staining of SA-&#946;-Gal with Nanog, it is not recommended for quantification due to potential interfering staining. As mentioned above, counter staining is essential for digital scanner detection. The best counter staining for the SA-&#946;-Gal together with Nanog is fast red orhematoxylin. However, counter staining might mask over either the SA-&#946;-Gal or Nanog signal. Therefore, for more accurate quantification, it is better to perform them separately on consecutive slides (Figure 3B). By quantifying SA-&#946;-Gal positive and Nanog positive cells from adjacent slides, we established a positive correlation between them (Figure 3C), suggesting a potential involvement of senescence on cellular plasticity and regeneration.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56201/56201fig1.jpg" /> 
Figure 1: Evaluate senescence level after muscle injury. (A) Schematic representation of the muscle injury strategy used to induce senescence. (B) Schematic representation of the muscle section preparation. (C) Representative images of SA&#946;Gal staining counterstained with eosin. Arrows point to the SA-&#946;-Gal+ cells. Scale bars = 50 &#181;m. (D) Quantification of SA-&#946;-Gal+ cells in injured and non-injured TA-muscle. Each dot corresponds to an individual animal. Statistical significance was assessed by the two-tailed Student&#180;s t-test: ***p &#60;0.001. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56201/56201fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56201/56201fig2.jpg" /> 
Figure 2: Quantification of SA-&#946;-Gal+ cells by ImageJ software. (A-D) Screen shots of a muscle section in the ImageJ software interface. Screen shot of converting a muscle section image to gray scale (A); Selecting all the SA-&#946;-Gal+ cells in the section (B); Analyzing the selected particles (C); Summary of all the counted particals in the ROI manager (D). (E) Screen shot of the manual curation process. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56201/56201fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56201/56201fig3.jpg" /> 
Figure 3: Evaluation of in vivo reprogramming after muscle injury. (A) Schematic representation to evaluate in vivo reprogramming and senescence level after muscle injury. (B) Representative images of SA-&#946;-Gal and Nanog staining on frozen sections of damaged skeletal muscle. SA&#946;Gal staining counterstained with eosin (left); immunohistochemical staining of Nanog counterstained with fast red (right). Non-injured muscles are shown on the top and injured muscle below. Scale bars = 50 &#181;m. (C) Quantification and correlation of SA-&#946;-Gal + and Nanog+ cells in consecutive sections (n = 9 mice, value represents the average of 2 sections per mouse). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56201/56201fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Volume for 50 mL</td>
		</tr>
		<tr>
			<td>100 mM stock K3Fe(CN)6 solution</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>100 mM stock K4Fe(CN)6 solution</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>1 M MgCl2</td>
			<td>100 &#956;L</td>
		</tr>
		<tr>
			<td>50 mg/mL X-Gal</td>
			<td>400 &#956;L</td>
		</tr>
		<tr>
			<td>PBS (pH 5.5)</td>
			<td>45.50 mL</td>
		</tr>
	</tbody>
</table>
Table 1: Composition of 50 mL X-gal solution.

Watch this Scientific Journal Video about Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle at JoVE.com

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

evaluation-injury-induced-senescence-vivo-reprogramming-skeletal

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.5K Views.  Institut Pasteur. Hier presenteren we een gedetailleerd protocol bij het detecteren van zowel verouderend en pluripotente stamcellen in de skeletspieren op letsel tijdens het inducerende in vivo herprogrammering. Deze methode is geschikt voor de evaluatie van de rol van cellulaire senescentie tijdens Weefselregeneratie en herprogrammering in vivo.

Video: Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs&#160;and their progeny&#160;&#8212; myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

This protocol describes a robust, reproducible and simple method of isolation and culture of myoblast progenitor cells from the skeletal muscle of adult and aged people. The muscles used here include foot and leg muscles. This approach enables the isolation of an enriched population of primary myoblasts for functional studies.

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

Genetics

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from traumatic injury, disease and various congenital, genetic and acquired conditions are quite common. Tissue engineering and regenerative medicine technologies have enormous potential to provide a therapeutic solution. However, utilization of biologically relevant animal models in combination with longitudinal assessments of pertinent functional measures are critical to the development of improved regenerative therapeutics for treatment of VML-like injuries. In that regard, a commercial muscle lever system can be used to measure length, tension, force and velocity parameters in skeletal muscle. We used this system, in conjunction with a high power, bi-phase stimulator, to measure in vivo force production in response to activation of the anterior crural compartment of the rat hindlimb. We have previously used this equipment to assess the functional impact of VML injury on the tibialis anterior (TA) muscle, as well as the extent of functional recovery following treatment of the injured TA muscle with our tissue engineered muscle repair (TEMR) technology. For such studies, the left foot of an anaesthetized rat is securely anchored to a footplate linked to a servomotor, and the common peroneal nerve is stimulated by two percutaneous needle electrodes to elicit muscle contraction and dorsiflexion of the foot. The peroneal nerve stimulation-induced muscle contraction is measured over a range of stimulation frequencies (1-200 Hz), to ensure an eventual plateau in force production that allows for an accurate determination of peak tetanic force. In addition to evaluation of the extent of VML injury as well as the degree of functional recovery following treatment, this methodology can be easily applied to study diverse aspects of muscle physiology and pathophysiology. Such an approach should assist with the more rational development of improved therapeutics for muscle repair and regeneration.

Applications of In Vivo Functional Testing of the Rat Tibialis Anterior for Evaluating Tissue Engineered Skeletal Muscle Repair

We describe an in vivo protocol to measure dorsiflexion of the foot following stimulation of the peroneal nerve and contraction of the anterior crural compartment of the rat hindlimb. Such measurements are an indispensable translational tool for evaluating skeletal muscle pathology and tissue engineering approaches to muscle repair and regeneration.

Despite the regenerative capacity of skeletal muscle, permanent functional and/or cosmetic deficits (e.g., volumetric muscle loss (VML) resulting from ...

Bioengineering

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

Medicine

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Biology

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Harvesting Mouse Skeletal Muscle to Culture Stem Cells and Their Niche

Unfractionated Bulk Culture of Mouse Skeletal Muscle to Recapitulate Niche and Stem Cell Quiescence

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and recovery from injuries depend on a multitude of cell types and on molecular signals between the core muscle cells (myofibers, muscle stem cells) and their niche. Most experimental settings do not preserve this complex physiological microenvironment, and neither do they allow the ex vivo study of muscle stem cells in quiescence, a cell state that is crucial for them. Here, a protocol is outlined for the ex vivo culture of muscle stem cells with cellular components of their niche. Through the mechanical and enzymatic breakdown of muscles, a mixture of cell types is obtained, which is put in 2D culture. Immunostaining shows that within 1 week, multiple niche cells are present in culture alongside myofibers and, importantly, Pax7-positive cells that display the characteristics of quiescent muscle stem cells. These unique properties make this protocol a powerful tool for cell amplification and the generation of quiescent-like stem cells that can be used to address fundamental and translational questions.

Author Spotlight: Ex Vivo Protocol for Culturing Quiescent Muscle Stem Cells with Niche Components 

Skeletal muscle comprises multiple cell types, including resident stem cells, each with a special contribution to muscle homeostasis and regeneration. Here, the 2D culture of muscle stem cells and the muscle cell niche in an ex vivo setting that preserves many of the physiological, in vivo, and environmental characteristics are described.

Skeletal muscle is the largest tissue of the body and performs multiple functions, from locomotion to body temperature control. Its functionality and ...

Single-Cell CyTOF Analysis of Myogenic Progenitors in Muscle Regeneration

Identification and Analysis of Myogenic Progenitors In Vivo During Acute Skeletal Muscle Injury by High-Dimensional Single-Cell Mass Cytometry

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle stem cells become activated upon muscle injury. Following activation, they proliferate, and most of their progeny differentiate to generate fusion-competent muscle cells while the remaining self-renews to replenish the stem cell pool. While the identity of muscle stem cells was defined more than a decade ago, based on the co-expression of cell surface markers, myogenic progenitors were identified only recently using high-dimensional single-cell approaches. Here, we present a single-cell mass cytometry (cytometry by time of flight [CyTOF]) method to analyze stem cells and progenitor cells in acute muscle injury to resolve the cellular and molecular dynamics that unfold during muscle regeneration. This approach is based on the simultaneous detection of novel cell surface markers and key myogenic transcription factors whose dynamic expression enables the identification of activated stem cells and progenitor cell populations that represent landmarks of myogenesis. Importantly, a sorting strategy based on detecting cell surface markers CD9 and CD104 is described, enabling prospective isolation of muscle stem and progenitor cells using fluorescence-activated cell sorting (FACS) for in-depth studies of their function. Muscle progenitor cells provide a critical missing link to study the control of muscle stem cell fate, identify novel therapeutic targets for muscle diseases, and develop cell therapy applications for regenerative medicine. The approach presented here can be applied to study muscle stem and progenitor cells in vivo in response to perturbations, such as pharmacological interventions targeting specific signaling pathways. It can also be used to investigate the dynamics of muscle stem and progenitor cells in animal models of muscle diseases, advancing our understanding of stem cell diseases and accelerating the development of therapies.

Author Spotlight: Investigating Cellular and Molecular Dynamics During Muscle Regeneration Using Cutting-Edge Single-Cell Technologies

The protocol presented here enables the identification and high-dimensional analysis of muscle stem and progenitor cells by single-cell mass cytometry and their purification by FACS for in-depth studies of their function. This approach can be applied to study regeneration dynamics in disease models and test the efficacy of pharmacological interventions.

Skeletal muscle regeneration is a dynamic process driven by adult muscle stem cells and their progeny. Mostly quiescent at a steady state, adult muscle ...

Establishing an Orofacial Muscle Fibrosis Model in Mice via a Freezing Injury

Freezing Injury in Mouse Masseter Muscle to Establish an Orofacial Muscle Fibrosis Model

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived limb muscles, the orofacial muscles originate from the branchial arches, with exclusive contributions from the cranial neural crest. A recent study has revealed that regeneration is also different in the orofacial muscle group. However, the underlying regulatory mechanism remains to be uncovered. Current skeletal muscle regeneration models mainly focus on the limb and trunk muscle. In this protocol, dry ice was used to induce freezing injury in the mouse masseter muscle and tibialis anterior muscle to create an orofacial muscle fibrosis model. The temporal dynamics of muscle satellite cells and fibro-adipogenic progenitors were different between the two muscles, leading to impaired myofiber regeneration and excessive extracellular matrix deposition. With the help of this model, a deeper investigation into muscle regeneration in the orofacial area could be carried out to develop therapeutic approaches for patients with orofacial diseases.

Author Spotlight: Exploring Orofacial Muscle Regeneration &#8211; Insights and Innovations

The goal of this protocol is to establish an orofacial muscle fibrosis model. Comparison of the histology between mice masseter and tibialis anterior muscle after freezing injury confirmed masseter muscle fibrosis. This model will facilitate further investigation into the mechanism underlying orofacial muscle fibrosis.

Orofacial muscle constitutes a subset of skeletal muscle tissue, with a distinct evolutionary trajectory and development origin. Unlike the somite-derived ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

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