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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

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

Published: April 1st, 2021

DOI:

10.3791/62449

1Bioinnovation PhD Program, Tulane University, 2Biology Department, Virginia Commonwealth University, 3Mechanical Engineering Department, Montgomery College, 4Biomedical Engineering Department, Tulane University, 5US Food and Drug Administration

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.

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.

The earthworm has been used as an important bioindicator and bioassay for previous scientific applications1,2,3,4,5,6; 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 effects7,8,9,.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

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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 .......

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The following representative results are based on a set of specific parameters that include the settings used to pull the glass pipette, the pipette opening size formed from a given beveling angle, and the pressure and time of the microinjections. In Figure 1, a schematic of the flow is displayed representing the process from start to finish.

Based on the pipette puller parameters selected, the following tip would emerge from the pull (Figure .......

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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.......

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This work was funded by the NSF-FDA Scholar-in-Residence Fellowship (NSF-FDA SIR, #1641221), US Food and Drug Administration Office Chief Scientist Challenge Grant (FDA OCS), National Science Foundation Integrative Graduate Education and Research Traineeship (NSF IGERT, #1144646) and supported by the Office of Science and Engineering Laboratories (OSEL) at the US Food and Drug Administration (FDA).

....

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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

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