title: Complete spinal cord injury and brain dissection protocol for subsequent wholemount in situ hybridization in larval sea lamprey.
abstract: After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16°C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 °C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.
authors.affiliation: Centre for Neuroregeneration, School of Biomedical Sciences, University of Edinburgh; anton.barreiro@gmail.com.
authors.forename: Antón
authors.initials: A
authors.lastname: Barreiro-Iglesias
authors.affiliation: Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), Temple University School of Medicine.
authors.forename: Guixin
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), Temple University School of Medicine; Department of Neurology, Temple University School of Medicine.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Selzer
authors.affiliation: Shriners Hospitals Pediatric Research Center (Center for Neural Repair and Rehabilitation), Temple University School of Medicine.
authors.forename: Michael I
authors.initials: MI
authors.lastname: Shifman
mesh_terms: D000818:Animals; D001921:Brain / Q000502:physiology* / Q000601:surgery*; D004210:Dissection; D017403:In Situ Hybridization / Q000379:methods*; D023421:Models, Animal; D009416:Nerve Regeneration; D048428:Petromyzon / Q000502:physiology*; D013119:Spinal Cord Injuries / Q000473:pathology* / Q000503:physiopathology; D058630:Spinal Cord Regeneration