Earthworm, Lumbricus Terrestris: A Novel Microinjection Vasculature In vivo Invertebrate Model

Summary

Earthworms are a novel invertebrate in vivo bench-top model for vasculature studies. We present techniques and equipment that allow efficient surgery and microinjection into the earthworm vasculature. Surgical protocols, microinjection techniques and the procedure for producing custom-made micropipettes are described.

Abstract

Although vertebrates are indispensable to biomedical research, studies are often limited by factors such as cost, lengthy internal review, and ethical considerations. We present the earthworm as an alternative, low-cost, invertebrate applicable to certain preliminary vasculature studies. Due to the surgical availability of the earthworm's dorsal vessels, ventral vessels, and five pairs of pseudo hearts, earthworms are readily accessible, offer low-cost maintenance, and require administration of only small doses of a given compound. The earthworm model provides a simple closed vascular circulatory system with a hemoglobin structure similar to human blood. A protocol is provided for anaesthetizing the earthworms and performing surgical incisions to expose relevant blood vessels. Micropipettes for compound administration are formed by heating and pulling glass with a pipette puller and using a beveling system to create a micron-scale fine needle tip. The tips are then used with a micropositioner and microinjector to inject arbitrary compounds into the vascular system of an earthworm, repeatably, with the availability of large sample sizes and small compound volumes. Details on the intricacies of injection procedure are provided. The small vessel size of the earthworm is challenging, particularly in the case of the ventral vessel; however, mastery of the techniques presented offers high repeatability as a low-cost solution, making studies of very large sample size practical.

Introduction

The earthworm has been used as an important bioindicator and bioassay for previous scientific applications^1,2,3,4,5,6; it is an ideal organism for assessing biological risks from hazardous and toxic waste in terrestrial environments for in situ and bioaccumulation studies, such as biocides (insecticides) in soil and adverse ecotoxicological effects^7,8,9,

Protocol

1. Micropipette preparation: pulling glass and beveling tip

1. Micropipette pulling
   1. Turn on the micropipette puller and select a program to enter specific parameters for earthworm microinjection.
   2. Set parameters to Pressure=500, Pull=75, Time=250, Heat=336, and Velocity=70. Results may vary puller to puller; therefore, experiment with parameters to achieve desired tip (size, sharpness, shape, etc.).
   3. Be sure to conduct a RAMP test to determine the heat value for the micropipettes .......

Discussion

While the earthworm is in 10% ethanol, particularly if the earthworm is of older age, there may be unwanted effects for exposure times greater than 30 minutes; the intestines will start to deteriorate, and when the earthworm is surgically opened, its internal intestines spread out. Therefore, it is encouraged to use young to mid-aged earthworms. During the process of cutting through the skin of the earthworm, it is imperative that a full scissor cut is not made, meaning the investigator must cut only halfway and keep pus.......

Materials

Name	Company	Catalog Number	Comments
3M Vetbond Tissue Adhesive	3M Vetbond	084-1469SB	3mL bottle vet adhesive - liquid band-aide
40x Stereo Microscope	Sutter Instrument Co.	BV-10-D	Not needed, can add on other scopes
500 Large Worms	Windsor Wholesale Bait	500 Large
Beveler pedestal oil	Sutter Instrument Co.	008
Blades	Ted Pella, Inc	121-2
Borosilicate Glass with Filament	Sutter Instrument Co.	BF150-86-10
Camera	AmScope	MU500
Camera	AmScope	MU1803-CK	8MP USB3.0 Microscope Digital Camera
Electrode Impedance Meter	Sutter Instrument Co.	BV-10-C
Ethanol	Sigma Aldrich	E7023-1L	Pure ethanol
Filament	Sutter Instrument Co.	FT315B	trough filament
Grinding Plate	Sutter Instrument Co.	104D	Fine Plate
Hospital Grade Saline	Baxter Healthcare Corporation	2F7124	0.9% Sodium Chloride Irrigation
Joystick Micromanipulator	Narishige	MN-151
KimWipes Kimtech Science	Kimberly-Clark Professional	34155
Leafgro	LeafGro	589252	1.5-cu. ft.
Metal Hub Needle	Hamilton	91024	Luer Lock Metal Needle
Micro Vessel Clips	WPI	501779-G
Microinjector	TriTech Research	MINJ-D
Micropiette Puller Model P-97	Sutter Instrument Co.	P-97
Micropipette Beveler	Sutter Instrument Co.	BV-10-B
Microscope	AmScope	SM-8TPW2-144S	3.5X-225X Simul-Focal Articulating Microcope
Needle Holder	TriTech Research	MINJ-4
NeverWet	Rust-Oleum	NeverWet
Pyrex Glass	Corning	08747A	Fisher Manufacturer
Stainless Micro-Ruler	Ted Pella, Inc	13635	Micro-Ruler mounted on a Handle, 10mm scale, with lines at 0.01mm intervals
Surgical Grips	Ted Pella, Inc	53073	Forceps, Hemostat
Surgical scissors	Ted Pella, Inc	1320	Fine Iris Scissors, Straight
U.S.P. Mineral Oil Lubricant Laxative	Swan	Mineral Oil