We have used the nervous system of the medicinal leech as a preparation to study the molecular basis of neural repair. The leech central nervous system, unlike mammalian CNS, can regenerate to restore function, and contains identified nerve cells of known function and connectivity. We have constructed subtractive cDNA probes from whole and regenerating ganglia of the ventral nerve cord and have used these to screen a serotonergic Retzius neuron library. This identifies genes that are regulated as a result of axotomy, and are expressed by the Retzius cell. This approach identifies many genes, both novel and known. Many of the known genes identified have homologues in vertebrates, including man. For example, genes encoding thioredoxin (TRX), Rough Endoplasmic Reticulum Protein 1 (RER-1) and ATP synthase are upregulated at 24 h postinjury in leech nerve cord. To investigate the functional role of regulated genes in neuron regrowth we are using microinjection of antisense oligonucleotides in combination with horseradish peroxidase to knock down expression of a chosen gene and to assess regeneration in single neurons in 3-D ganglion culture. As an example of this approach we describe experiments to microinject antisense oligonucleotide to a leech isoform of the structural protein, Protein 4.1. Our approach thus identifies genes regulated at different times after injury that may underpin the intrinsic ability of leech neurons to survive damage, to initiate regrowth programs and to remake functional connections. It enables us to determine the time course of gene expression in the regenerating nerve cord, and to study the effects of gene knockdown in identified neurons regenerating in defined conditions in culture.
Three-dimensional (3D) collagen gels provide a stable matrix in which isolated regenerating ganglia from leech and snail can be maintained for studies of the molecular and cellular mechanisms underlying the regenerative process. Segmental ganglia from leech, or supraoesophageal, suboesophageal or buccal ganglia from snail were maintained for up to 3 weeks in 3D matrices of mammalian Type I collagen. The collagen matrix supports the regenerative outgrowth of axon tracts as well as the migration of microglial cells, important elements in the repair process. Proteins or soluble factors or target tissue may be added to the basic collagen matrix to manipulate the environment of the regenerating tissue. We describe techniques for immunostaining of regenerating axons and microglial cells within the gel matrix in combination with staining of cell nuclei, and the use of intracellular labelling to distinguish axons of identified neurons within the regenerative outgrowth.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.