Contusion Spinal Cord Injury via a Microsurgical Laminectomy in the Regenerative Axolotl

Summary

This manuscript presents protocols for surgically inflicting controlled blunt and sharp spinal cord injuries to a regenerative axolotl (Ambystoma mexicanum).

Abstract

The purpose of this study is to establish a standardized and reproducible regenerative blunt spinal cord injury model in the axolotl (Ambystoma mexicanum). Most clinical spinal cord injuries occur as high energy blunt traumas, inducing contusion injuries. However, most studies in the axolotl spinal cord have been conducted with sharp traumas. Hence, this study aims to produce a more clinically relevant regenerative model. Due to their impressive ability to regenerate almost any tissue, axolotls are widely used as models in regenerative studies and have been used extensively in spinal cord injury (SCI) studies. In this protocol, the axolotls are anesthetized by submersion in a benzocaine solution. Under the microscope, an angular incision is made bilaterally at a level just caudal to the hind limbs. From this incision, it is possible to dissect and expose the spinous processes. Using forceps and scissors, a two-level laminectomy is performed, exposing the spinal cord. A custom trauma device consisting of a falling rod in a cylinder is constructed, and this device is used to induce a contusion injury to the spinal cord. The incisions are then sutured, and the animal recovers from anesthesia. The surgical approach is successful in exposing the spinal cord. The trauma mechanism can produce contusion injuries to the spinal cord, as confirmed by histology, MRI, and neurological examination. Finally, the spinal cord regenerates from the injury. The critical step of the protocol is removing the spinous processes without inflicting damage to the spinal cord. This step requires training to ensure a safe procedure. Furthermore, wound closure is highly dependent on not inflicting unnecessary damage to the skin during incision. The protocol was performed in a randomized study of 12 animals.

Introduction

The overall goal of this study was to establish a controlled and reproducible microsurgical method for inflicting blunt and sharp SCI to the axolotl (Ambystoma mexicanum), producing a regenerative spinal cord injury model.

SCI is a severe condition that, depending on the level and extent, inflicts neurological disability to the extremities along with impaired bladder and bowel control^1,2,3. Most SCI are the result of high energy blunt trauma such as traffic accidents and falls^4,5. ....

Protocol

All applicable institutional and governmental regulations concerning the ethical use of animals were followed during this study. The study was conducted under the approval id: 2015-15-0201-0061 by the Danish Animal Experiment Inspectorate. Animals were Mexican axolotls (Ambystoma mexicanum, mean body mass ± STD: 12.12 g ± 1.25 g).

1. Preparation

1. Prepare axolotl for anesthesia.
   1. Use high quality non-chemically treated tap water. If unav.......

Discussion

Because risk of injury to the spinal cord is significant, the critical steps of the protocol are removing the spinous processes and widening of the bony access to the spinal canal if needed. As mentioned in the protocol, removing the most cranial process first is highly recommended. This will mean that the more caudal processes protect the spinal cord from being hit by the scissors. It is recommended to ensure enough surgical access, meaning to not make too small a primary incision. Also, when grasping anything with forc.......

Materials

Name	Company	Catalog Number	Comments
25 g custom falling rod	custom home made
30 mm PVC pipe	custom home made
Acetone	Sigma-Aldrich	67-64-1	Propanone
Axolotl (Ambystoma mexicanum)	Exoterra GmbH	N/A	12-22 cm and 10 g - 80 g, All strains (wildtype, melanoid, white, albino, transgenic white with GFP)
Benzocain	Sigma-Aldrich	94-09-7	ethyl 4-aminobenzoate
electromaget	custom home made
Excel 2010	Microsoft	N/A	Excel 2010 or newer
ImageJ	National Institutes of Health		ImageJ 1.5e or newer. Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997-2016.
kimwipes
microsurgical instruments	N/A	N/A	Forceps and scissors
MS550s	Fujifilm, Visualsonics	MS550s	40 MHz center frequency, transducer
MS700	Fujifilm, Visualsonics	MS700	50 MHz center frequency, transducer
Petri dish	any maker
Soft cloth	N/A	N/A	Any piece of soft cloth measuring appromixately 70 x 55 cm^2 e.g. a dish towel
Stereo microscope
Vevo 2100	Fujifilm, Visualsonics	Vevo 2100	High frequency ultrasound system