Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Summary

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

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.

Introduction

In mammals spinal cord injury (SCI) is a devastating condition that leads to permanent loss of function below the site of injury because injured axons do not regenerate through the trauma zone and reconnect to their appropriate targets. In contrast to mammals, lampreys recover locomotion after a complete spinal cord injury.¹ Interestingly, lampreys have a set of 36 spinal cord projecting neurons that are individually identifiable in whole-mount brain preparations because of their big size^2,3 (Figure 1). All of these spinal-projecting neurons are axotomized by a high-level complete spinal cord transection. Previous studies of our ....

Protocol

See Table 1 for all materials used in this protocol.

Experiments were approved by the Institutional Animal Care and Use Committee at Temple University.

1. Animals

1. Obtain wildtype larval sea lampreys (Petromyzon marinus L.), 10 – 14 cm in length (4 – 7 years old) from streams feeding Lake Michigan, from tributaries to the Delaware River (Pennsylvania) or streams in Maine.
2. In the laboratory, maintain larvae in groups of 50 - 100 animals in 50 gallon freshwater tanks at 16 °C. Line the tanks with an inch of gravel for the larvae to burrow in, and the w....

Results

As an example of the results that can be obtained when using this method, representative images of wholemounted brains showing the expression of the neogenin transcripts in identifiable spinal cord-projecting neurons of control and 2 weeks post lesion larval sea lampreys are shown in Figure 2. The readers are referred to a previous study⁶ reporting the relationship between the expression of neogenin after a complete spinal cord transection and the regenerative ability of the identifiable spina.......

Discussion

Here we present a detailed protocol to perform a complete spinal cord transection and posterior brain dissection in larval sea lampreys. This procedure allows analyzing differences in gene expression between identifiable spinal cord projecting neurons after spinal cord injury by means of a whole-mount brain in situ hybridization. The critical step in the procedure is the correct performance of a complete spinal cord transection, which can be controlled by observing the cut ends of the spinal cord under the stereomicrocop.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tricaine methane sulfonate	Spectrum	TR108	Benzocaine saturated solution in PBS for sacrifice
Scalpel #3	Fine Science Tools (FST)	10003-12
Blades for scalpel: #11	Fine Science Tools	10011-00
Castroviejo scissors #8	Fine Science Tools	15002-08
Forceps #4 & #5	Dumont, Switzerland	Roboz RS4955	#4 for dissection of Spinal cord; #5 for stripping menninges
Dissecting Microscope	Olympus	SZ51
Sylgard	Dow Corning Co.	184
Insect pins 0.15, 0.20 mm	Austerlitz	No catalogue #	0.15 mm for pinning brain and spinal cord; 0.20 mm for the body
7 ml HDPE Scintillation Tubes with Caps	Fisher Scientific	03-337-1
Paraformaldehyde 16%	Electron Microscopy Science (EMS)	19210	Dilute to 4% in PBS