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 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 effects7,8,9,10. Additionally, due to bioprospecting, the earthworm is an alternative source of fibrinolytic, anti-coagulative, anti-microbial, and anti-cancer molecules11,12; to the point that a team in 1991 extracted and purified lumbricine from the earthworm skin and placed on mammary tumors of SHN mice, which led to tumor growth inhibition13. The earthworm is also a pedagogically useful animal model, as it can be used to expose students to surgery and to understanding the anatomy of a specimen; from studying blood circulation to electrophysiology14,15.
In our own research we have examined the response of the vessels of live earthworms to high intensity ultrasound18. We found that vessel rupture in the worm occurred under conditions similar to those that we associated in rupture damage in human micro-vessels. Our ongoing work involves injection of microbubbles into the earthworm vasculature. Microbubbles are composed of a heavy gas encased by a lipid, albumin or polymer shell, these agents can be used as image contrast agents as well as vehicles for targeted drug delivery.
This novel protocol is relevant to any study that would benefit from intravenous (IV) injection of a compound that could utilize the earthworm's natural bioindicators. The approach is based around IV microinjection into one of several possible entry points, including any of the earthworm's five-pair pseudo hearts, the dorsal vessel, and the ventral vessel. The procedure involves an elaborate surgical incision to expose the vessels, followed by a micro-positioner-controlled injection. This is achieved using custom micropipettes constructed specifically for earthworm vascular microinjection. These micropipettes allow precision targeting of vessels as small as a 90 µm diameter ventral vessel.
This protocol is designed to improve upon earlier micro-pipetting techniques, including a 1948 study for the extraction of earthworm blood and urine16. As seen in Figure S1, the setup for this extraction can be difficult, and, as stated by the author, can take up to one hour or longer. A similar method was developed in 1970, but the author experienced multiple broken tips while injecting fluids into the giant fibers of the earthworm17. In the present method described below, extraction of blood is a matter of seconds to minutes and is relevant to both the injection of compounds and extraction of earthworm fluids. In this specific case, we injected contrast agents, microbubbles.
Protocol
Representative Results
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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).
Materials
| 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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