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

Summary

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.

Abstract

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 β-galactosidase (SA-β-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 β-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 (IHC). The major limitation of the SA-β-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-β-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use 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.

Introduction

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 ageing^1,2. 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/epige....

Protocol

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

1. 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.
2. Prepare the solutions for SA-β-Gal staining.
   1. 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.
   2. Prep....

Results

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 dramat.......

Discussion

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 β-galactosidase (SA-β-Gal) assay is used to detect in vivo senescent cells in the skeletal muscle. This assay detects.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
K3Fe(CN)6	Sigma	13746-66-2	For SA-β Gal staining solution
K4Fe(CN)6	Sigma	14459-95-1	For SA-β Gal staining solution
MgCl2	Sigma	7786-30-3	For SA-β Gal staining solution
X-Gal	Sigma	B4252	For SA-β Gal staining solution
Doxycycline	Sigma	D3447	For inducing in vivo reprogramming
Cardiotoxin	Lotaxan Valence, France	L8102	For muscle injury
Glutaraldehyde	Sigma	111-30-8	For Fixation solution
Paraformaldehyde	Electron microscopy science	50-980-487	For Fixation solution
NaCitrate : Sodium Citrate monobasic bioxtra, anhydre	Sigma	18996-35-5	For permeabilization solution
Triton	Sigma	93443	For permeabilization solution
Bovine Serum Albumin	Sigma	A3608	Washing solution
Antibody anti- Nanog	Cell signalling	8822S	Rabbit monoclonal antibody
EnVision+ Kits (HRP. Rabbit. DAB+)	Dako	K4010	For Nanog revelation
Eosin 1%	Leica	380159EOF	Counterstainning
Fast red	Vector Laboratories	H-3403	Counterstainning
Thermo Scientific Shandon Immu-Mount	Fisher scientific	9990402	Mounting solution
Quick-hardening mounting medium for microscopy : Eukitt®	Sigma	25608-33-7	Mounting solution
Microscope Phase Contrast Brightfield CKX41: 10X-20X-40X objectives	Olympus	CKX41	Microscope for Nanog quantification
Mouse: i4F-A	Abad et al., 2013	N/A	Reprogrammable mouse model
Skeletal muscle, Tibialis Anterior
Slide Scanner	Zeiss	Axio Scan Z1	slides scanning