Name: adult mouse digit amputation and regeneration: a simple model to investigate mammalian blastema formation and intramembranous ossification
Uploaded: 2019-07-12T14:00:00
Description: Watch this Scientific Journal Video about Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification at JoVE.com

We thank members of the Muneoka Lab and the Texas Institute for Genomic Medicine (TIGM). This work was supported by Texas A&#38;M University.

All animal use and techniques were in compliance with the standard operating procedures of the Institutional Animal Care and Use Committee of Texas A&#38;M University.
1. Adult Mouse Hind Limb Distal P3 Amputation
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	<li>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 ...

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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 &#181;CT 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...

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 matrix4,5,6,7,8. P3 regeneration is mediated by the formation of a blastema, defined as a population of proliferating cells that undergo morphogenesis to regenerate the amputated structures9. The formation of a blastema to regenerate the structures lost by amputation, a process termed epimorphic regeneration, distinguishes the multi-tissue-level P3 regeneration response from traditional tissue repair after injury6,10. P3 regeneration is a reproducible and procedurally simple model to investigate complex regenerative processes including wound healing11,12, bone histolysis11,12, revascularization13, peripheral nerve regeneration14, and blastemal conversion to bone via intramembranous ossification15.
Previous studies using immunohistochemistry have demonstrated that the blastema is heterogeneous, avascular, hypoxic, and highly proliferative11,13,15,16. Following distal P3 amputation, the early blastema is initially associated with the P3 periosteum and endosteum and is characterized by robust proliferation and nascent osteogenesis adjacent to the bone surface15. Subsequent to bone degradation and wound closure, the heterogeneous blastema is formed by the merging of periosteal and endosteal-associated cells, followed by the differentiation of blastemal components including bone via intramembranous ossification15.
Bone repair in response to injury typically occurs by endochondral ossification, i.e. via an initial cartilaginous callus that forms a template for subsequent bone formation17,18. Long bone intramembranous ossification, i.e., bone formation without a cartilaginous intermediate, is commonly induced using complex distraction devices or surgical fixation19,20. The digit regeneration response is a pre-clinical model that offers advantages over conventional intramembranous ossification models: 1) it does not require external or internal fixation post injury to stimulate intramembranous ossification, 2) it is performed using 4 digits from each animal, thus maximizing samples while minimizing animal use, and 3) sequential in vivo microcomputed tomography (&#956;CT) analysis can be performed with ease and speed.
In the present study, we show the standardized P3 amputation plane to achieve a reproducible and robust regeneration response. Additionally, we demonstrate an optimized fluorescence immunohistochemistry protocol using paraffin sections to visualize blastema formation, revascularization in the context of regeneration, and blastemal conversion to bone via intramembranous ossification. We also demonstrate the use of sequential in-vivo &#956;CT to identify changes in bone morphology, volume, and length in the same digit over the course of regeneration. The goal of this protocol is to investigate mammalian blastema formation after amputation and to demonstrate 2 techniques, fluorescence immunohistochemistry and sequential in vivo &#956;CT, for the study of intramembranous bone regeneration.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Protein Block Serum Free</td>
			<td>DAKO</td>
			<td>X0909</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Mouse anti-PCNA antibody</td>
			<td>Abcam</td>
			<td>ab29</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr>
			<td>Rat anti-CXCR4 antibody</td>
			<td>R&#38;D Systems</td>
			<td>MAB21651</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-human vWF XIII antibody</td>
			<td>DAKO</td>
			<td>A0082</td>
			<td>1:800 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-osterix, SP7 antibody</td>
			<td>Abcam</td>
			<td>ab22552</td>
			<td>1:400 dilution</td>
		</tr>
		<tr>
			<td>Rabbit anti-Runx2 antibody</td>
			<td>Sigma-Aldrich Co.</td>
			<td>HPA022040</td>
			<td>1:250 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21235</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11008</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568-conjugated goat anti-rat IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A11077</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36930</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Surgipath Decalicifier 1</td>
			<td>Leica Biosystems</td>
			<td>3800400</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>Z-Fix, Aqueous buffered zinc formalin fixative</td>
			<td>Anatech LTD</td>
			<td>174</td>
			<td>Ready to use</td>
		</tr>
		<tr>
			<td>CD-1 Female Mouse</td>
			<td>Envigo</td>
			<td>ICR(CD-1)</td>
			<td>8-12-weeks-old</td>
		</tr>
		<tr>
			<td>vivaCT 40</td>
			<td>SCANCO Medical</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

adult mouse digit amputation and regeneration: a simple model to investigate mammalian blastema formation and intramembranous ossification

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 (&#956;CT). 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 &#956;CT 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.

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 &#956;CT renderings of digits scanned prior to amputation and at various...

Watch this Scientific Journal Video about Adult Mouse Digit Amputation and Regeneration: A Simple Model to Investigate Mammalian Blastema Formation and Intramembranous Ossification at JoVE.com

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

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.

Here, we present a protocol of adult mouse distal terminal phalanx (P3) amputation, a procedurally simple and reproducible mammalian model of epimorphic ...

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