Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification

Summary

Here, we present a protocol of adult mouse terminal phalanx amputation to investigate mammalian blastema formation and intramembranous ossification, analyzed by fluorescent immunohistochemistry and sequential in-vivo microcomputed tomography.

Abstract

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic regeneration, which involves blastema formation and intramembranous ossification analyzed by fluorescence immunohistochemistry and sequential in-vivo microcomputed tomography (μCT). Mammalian regeneration is restricted to amputations transecting the distal region of the terminal phalanx (P3); digits amputated at more proximal levels fail to regenerate and undergo fibrotic healing and scar formation. The regeneration response is mediated by the formation of a proliferative blastema, followed by bone regeneration via intramembranous ossification to restore the amputated skeletal length. P3 amputation is a preclinical model to investigate epimorphic regeneration in mammals, and is a powerful tool for the design of therapeutic strategies to replace fibrotic healing with a successful regenerative response. Our protocol uses fluorescence immunohistochemistry to 1) identify early-and-late blastema cell populations, 2) study revascularization in the context of regeneration, and 3) investigate intramembranous ossification without the need for complex bone stabilization devices. We also demonstrate the use of sequential in vivo μCT to create high resolution images to examine morphological changes after amputation, as well as quantify volume and length changes in the same digit over the course of regeneration. We believe this protocol offers tremendous utility to investigate both epimorphic and tissue regenerative responses in mammals.

Introduction

Mammals, including humans and mice, have the capacity to regenerate the tips of their digits after distal amputation of the terminal phalanx (P3)^1,2,3. In mice, the regeneration response is amputation-level-dependent; increasingly proximal digit amputations display a progressively attenuated regenerative response until complete regenerative failure at amputations transecting and proximal to the P3 nail matrix^4,5,6,7,8

Protocol

All animal use and techniques were in compliance with the standard operating procedures of the Institutional Animal Care and Use Committee of Texas A&M University.

1. Adult Mouse Hind Limb Distal P3 Amputation

1. Anesthetize an 8 to 12 week-old CD-1 mouse (Table of Materials) using isoflurane gas in oxygen; initially anesthetize at 3% in a chamber, followed by 2% isoflurane supplied by a nosecone over the duration of the surgery. Apply ophthalmic ointment on eyes to prevent dryness while under anesthesia.
   NOTE: The adult P3 amputation studies standardized in our lab are performed on 8 to....

Results

Adult mouse regenerating P3 digits at 6/7 DPA (Figure 2A-D), 9 DPA (Figure 2E-H), and 10 DPA (Figure 2I-L) were immunostained with antibodies to Runx2, OSX, and PCNA to visualize intramembranous bone regeneration, and immunostained with antibodies to CXCR4 and vWF to visualize blastema formation. Representative μCT renderings of digits scanned prior to amputation and at various time.......

Discussion

This protocol describes a standardized procedure of adult mouse distal P3 amputation, fluorescent immunohistochemical staining to visualize and investigate blastema formation and intramembranous ossification, and sequential in-vivo µCT scanning to identify bone morphological, volume, and length changes post amputation. P3 amputation is a unique, procedurally simple, and reproducible model to analyze a pro-regenerative wound environment that triggers blastema formation. Furthermore, the P3 digit model offers numerous.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Protein Block Serum Free	DAKO	X0909	Ready to use
Mouse anti-PCNA antibody	Abcam	ab29	1:2000 dilution
Rat anti-CXCR4 antibody	R&D Systems	MAB21651	1:500 dilution
Rabbit anti-human vWF XIII antibody	DAKO	A0082	1:800 dilution
Rabbit anti-osterix, SP7 antibody	Abcam	ab22552	1:400 dilution
Rabbit anti-Runx2 antibody	Sigma-Aldrich Co.	HPA022040	1:250 dilution
Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L)	Invitrogen	A21235	1:500 dilution
Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L)	Invitrogen	A11008	1:500 dilution
Alexa Fluor 568-conjugated goat anti-rat IgG (H+L)	Invitrogen	A11077	1:500 dilution
Prolong Gold antifade reagent	Invitrogen	P36930	Ready to use
Surgipath Decalicifier 1	Leica Biosystems	3800400	Ready to use
Z-Fix, Aqueous buffered zinc formalin fixative	Anatech LTD	174	Ready to use
CD-1 Female Mouse	Envigo	ICR(CD-1)	8-12-weeks-old
vivaCT 40	SCANCO Medical