Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Summary

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.

Abstract

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 & Eosin staining that is widely used for subsequent morphological and morphometric analysis.

Introduction

Mammalian adult skeletal muscles are formed by groups of fascicules of multinucleated muscle cells (myofibers) that are specialized for contraction. Each myofiber is an elongated syncytium, surrounded by the sarcolemma (plasmatic membrane) and containing myofibrils, which are made up of regularly and repeatedly organized contractile proteins (actin and myosin filaments). In adult life and in resting conditions, skeletal muscles have a very low turnover of their myonuclei¹; indeed, the myonuclei, which are located at the periphery of the myofiber, under the sarcolemma, are arrested in the G0 phase of the cell cycle and are unable to proliferate^1,2....

Protocol

All experiments were conducted in strict accordance with the institutional guidelines for animal research and approved by the Department of Public Health, Animal Health, Nutrition and Food Safety of the Italian Ministry of Health in accordance with the law on animal experimentation. Cervical dislocation procedures may vary from institution to institution based on IACUC or its equivalent requirements.

1. Cardiotoxin Injection in the Tibialis Anterior Muscle

1. Prepare a 10 μM working solution of cardiotoxin (CTX) by diluting the cardiotoxin stock solution in sterile Phosphate-Buffered Saline (PBS) or in water before starting the procedure.

Results

H&E staining allows for the evaluation of the morphology of the regeneration process at specific time points during skeletal muscle regeneration. Figure 3 shows the time course analysis performed on injured TA muscles of wild type mice. Muscles have been isolated at 3, 7, 15, and 30 days after CTX injection, as schematized in Figure 3A. Representative pictures of H&E-stained transverse sections show the dynamics of skeletal muscle repair over time.......

Discussion

Here, we describe a protocol to induce acute injury in skeletal muscle (i.e., the intramuscular injection of CTX). It is widely used as a powerful tool to study the dynamics of skeletal muscle regeneration in vivo. CTX injection induces the degeneration of muscle fibers, which is caused by the depolarization of the sarcolemma and the contraction of the fibers¹², and triggers the cascade of events that leads to muscle regeneration. Skeletal muscles are dissected at desired time points after th.......

Materials

Name	Company	Catalog Number	Comments
Cardiotoxin from Naja mossambica mossambica	SIGMA ALDRICH	C9759
Syringe For Insulin BD Micro-Fine+ Needle 30 G X 8 mm - Da 0,3 ml	BD	324826
Tragacanth Gum	MP BIOMEDICALS,LLC	104792
2-Methylbutane (Isopentane)	SIGMA ALDRICH	78-78-4.
OCT Killik Solution For Inclusion Cryostat	Bio-optica	05-9801
Feather Microtome Blade S35	Bio-optica	01-S35
Glass Slide Superfrost Plus	Menzel-Gläser	09-OPLUS
Dumon #5 Mirror Finish Forceps	2BIOLOGICAL INSTRUMENTS	11251-23
Scissors Straight Sharp/Sharp	2BIOLOGICAL INSTRUMENTS	15024-10
Scissors Noyes Straight	2BIOLOGICAL INSTRUMENTS	15012-12
Fine Iris Scissors Straight Sharp/Sharp 10,5 Cm	2BIOLOGICAL INSTRUMENTS	14094-11
Eukitt	Bio-optica	09-00100
Slide Coverslip	BIOSIGMA	VBS651
Xylene	SIGMA ALDRICH	214736
Ethanol 100%	sigma-Aldrich	02860-2.5L
Hematoxyline	J.T. BAKER	3873
Eosin	SIGMA ALDRICH	HT110116
Cryostat	LEICA	CM3050 S