Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea

Summary

The planarian Schmidtea mediterranea is an excellent model for studying stem cells and tissue regeneration. This publication describes a method to selectively remove one organ, the pharynx, by exposing animals to the chemical sodium azide. This protocol also outlines methods for monitoring pharynx regeneration.

Abstract

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call "chemical amputation." Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Introduction

Regeneration is a phenomenon that occurs throughout the animal kingdom, with regenerative capacities ranging from full body regeneration in certain invertebrates to more restricted abilities in vertebrates¹. Replacement of functional tissues is a complex process and often entails the simultaneous restoration of multiple cell types. For example, to regenerate the salamander limb, osteoblasts, chondrocytes, neurons, muscles, and epithelial cells need to be replaced². These newly generated cell types also need to be organized properly to facilitate new limb function. Understanding these complex processes requires techniques....

Protocol

1. Preparation

1. Preparation of planaria water¹⁷
   1. Maintain planarians in a 1X Montjuïc salt solution. To prepare planarian water, make individual stock solutions of 1 M CaCl₂, 1 M MgSO₄, 1 M MgCl₂, 1 M KCl and 5 M NaCl in ultrapure water. Filter-sterilize with a 0.2 µm bottle-top filterfor long-term storage.
      Note: Use only ultrapure deionized water (with a resistivity of 18.2 MΩ at 25 °C) to prepare Montjuïc salts.
   2. To prepare a 1 L stock of 5X salt solution, combine 5 mL of 1 M CaCl₂, 5 mL of 1 M MgSO

Results

Exposure to sodium azide disrupts the normal motility of planarians, causing animals to stretch and writhe. These movements force the pharynx to emerge from the ventral side of the animal, and after approximately 6 min in sodium azide solution, the white tip of the pharynx can be seen (Figure 1B-left panel). A few minutes later, animals actively contract and fully extend the pharynx by forcefully pushing it out of the body. (

Discussion

This protocol describes a method of selective ablation of the pharynx using sodium azide. Other targeted ablation studies in planarians have used modified surgery to remove photoreceptors²¹ or pharmacological treatment to ablate dopaminergic neurons²². One significant advantage of chemical amputation over existing methods is that it does not require surgery. The rigid structure of the pharynx compared to the rest of the planarian body facilitates its complete removal from t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Calcium Chloride Dihydrate (CaCl2)	Fisher Chemical	C79-3	Montjuïc salt solution
Magnesium Sulfate Anhydrous (MgSO4)	Fisher Chemical	M65-3	Montjuïc salt solution
Magnesium Chloride Hexahydrate (MgCl2)	Acros/VWR	41341-5000	Montjuïc salt solution
Potassium Chloride (KCl)	Acros Organics/VWR	196770010	Montjuïc salt solution
Sodium Chloride (NaCl)	Acros Organics/VWR	207790050	Montjuïc salt solution
Sodium Bicarbonate (NaHCO3)	Acros Organics/VWR	123360010	Montjuïc salt solution
Nalgene autoclavable polypropylene copolymer lowboy with spigot	ThermoFisher Scientific/VWR	2324-0015	Storing planaria water
Instant Ocean Sea Salt	Spectrum Brands	Amazon	Planaria water
Gentamicin Sulfate	Gemini Bio-products	400-100P	Planaria water
Razor blades	Electron Microscopy Sciences	71970	Mincing liver
Disposable pastry bags-16”, 12 pack	Wilton	Amazon	Liver aliquots
5 mL syringes	BD/VWR	309647	Liver aliquots
Petri dishes-35mm/60mm	Greiner Bio-One/VWR	82050-536/82050-544
Plastic containers (various sizes)	Ziploc	Amazon	Housing planarians in static culture
Sodium Azide	Sigma	S2002
Transfer pipette	Globe Scientific	138030
Forceps - Dumont Tweezer, Style 5	Electron Microscopy Sciences	72700-D (0203-5-PO)
Triton X-100	Sigma	T8787
DAPI	ThermoFisher	62247
Streptavidin, Alexa Fluor 488 conjugate	ThermoFisher	S11223
Glycerol	Fisher BioReagents	BP229-1