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

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.