Neural Stem Cell Transplantation in Experimental Contusive Model of Spinal Cord Injury

Summary

Spinal cord injury is a traumatic condition that causes severe morbidity and high mortality. In this work we describe in detail a contusion model of spinal cord injury in mice followed by a transplantation of neural stem cells.

Abstract

Spinal cord injury is a devastating clinical condition, characterized by a complex of neurological dysfunctions. Animal models of spinal cord injury can be used both to investigate the biological responses to injury and to test potential therapies. Contusion or compression injury delivered to the surgically exposed spinal cord are the most widely used models of the pathology. In this report the experimental contusion is performed by using the Infinite Horizon (IH) Impactor device, which allows the creation of a reproducible injury animal model through definition of specific injury parameters. Stem cell transplantation is commonly considered a potentially useful strategy for curing this debilitating condition. Numerous studies have evaluated the effects of transplanting a variety of stem cells. Here we demonstrate an adapted method for spinal cord injury followed by tail vein injection of cells in CD1 mice. In short, we provide procedures for: i) cell labeling with a vital tracer, ii) pre-operative care of mice, iii) execution of a contusive spinal cord injury, and iv) intravenous administration of post mortem neural precursors. This contusion model can be utilized to evaluate the efficacy and safety of stem cell transplantation in a regenerative medicine approach.

Introduction

A spinal cord injury (SCI) is the most common injury caused by high-energy trauma like motor vehicles accidents, falls, sports and violence ¹. In severe SCI, the injury force destroys or damages neural tissue, causing sudden loss of neurological function. Traumatic SCI occurs frequently in young adults between 10 and 40 years of age. It greatly affects the patient’s mental and physical condition and causes enormous economic impact to society ². The treatment approach in the acute phase is often limited to a high-dose of corticosteroid, surgical stabilization and decompression to possibly attenuate further damage ^3-4, but the role....

Protocol

NOTE: All the procedures were approved by the Review Committee of the University of Milan and met the Italian Guidelines for Laboratory Animals in compliance with European Communities Directive dated November 1986 (86/609/EEC).

1. Preparation of Cells for Transplantation

NOTE : Use neural stem cells between the 5th and the 9th passage in culture for these experiments; test the cultures for proliferation and differentiation ability before being labeled for transplantation. Determine the extent of differentiation by immunocytochemistry ¹².

1. Resuspend cells to a concentration of 1 x 10⁶

Results

The total number of transplanted cells is 1 x 10⁶ cells and was divided into three consecutive injections in the tail vein. We administered 3.3 x 10⁵ cells in 50 µl of phosphate buffer solution (PBS). The first injection was performed within 30 min after injury, the second 6 hr later and the last 18 hr after the lesion. The choice of a time limit of 18 hr after SCI for administering PM-NPCs was determined by the optimal permeability of the blood brain barrier at this time ¹⁴. To eval.......

Discussion

In this paper we described a method to obtain a reproducible model of traumatic spinal cord injury using an Infinite Horizon Impactor at a force of 70 kdyne (severe). Using a larger force paradigm (80 kdyne), we can cause a more severe injury that unfortunately is associated with higher mice mortality. In order to avoid this problem, we commonly choose a moderate force paradigm (70 kdyne) that is associated to a repeatable lesion with a gradual recovery of function and lower mortality. To produce such a stable injur.......

Materials

Name	Company	Catalog Number	Comments
Name of Material/ Equipment	Company	Catalog Number	Comments/Description
PKH26GL-1KT	Sigma	091M0973
Infinite horizon (IH) Impactor device	Precision Systems and Instrumentation, LLC	Model 0400 Serial 0171
Gentamycin 10mg/ml	Euroclone	ECM0011B	1mg/ml in sterile saline solution
Isoflurane-Vet 250ml	Merial	B142J12A
Blefarolin POM OFT 10g
Slide Warmer	2Biological Instruments	HB101-sm-402
Scalpel, size 10	Lance Paragon	26920
Small Graefe Forceps	2Biological Instruments	11023-14
Rongeur	Medicon Instruments	07 60 07
Micro scissors	2Biological Instruments	15000-00
Absorbable sutures (4/0)	Safil Quick	C0046203
Hemostat	2Biological Instruments	13014-14
Reflex 7 wound clip applicator	2Biological Instruments	12031-07
7mm Reflex wound clips	2Biological Instruments	12032-07
NGS	Euroclone	ECS0200D
Triton X 100	Merck Millipore	1086431000
Anti Microtubule Assocoated Protein  (MAP) 2	Millipore	AB5622
Alexa Fluor 488	Invitrogen	A11008
FluorSave Reagent	Calbiochem	345789
Neural stem cells medium			DMEM-F12 medium (Euroclone) containing 2 mm l-glutamine (Euroclone), 0.6% glucose (Sigma-Aldrich), 9.6 gm/ml putrescine (Sigma-Aldrich), 6.3 ng/ml progesterone (Sigma-Aldrich), 5.2 ng/ml sodium selenite (Sigma-Aldrich), 0.025 mg/ml insulin (Sigma-Aldrich), 0.1 mg/ml transferrin (Sigma-Aldrich), and 2 μg/ml heparin (sodium salt, grade II; Sigma-Aldrich), bFGF (human recombinant, 10 ng/mL; Life Technologies) and EGF (human recombinant, 20 ng/mL; Life Technologies)
DMEM-F12	Euroclone	ASM5002
l-glutamine	Euroclone	ECB3000D
glucose	Sigma-Aldrich	G8270-100G
putrescine	Sigma-Aldrich	P5780-25G
progesterone	Sigma-Aldrich	P6149-1MG
Sodium-selenite	Sigma-Aldrich	S9133-1MG
transferrin	Sigma-Aldrich	T 5391
Insulin	Sigma-Aldrich	I1882
Heparin sodium-salt	Sigma-Aldrich	H0200000
bFGF	Life Technology	PHG0024
h-EGF	Life Technology	PHG6045
Syringe 0.33cc 29G	Terumo	MYJECTOR
buprenorphine	Schering Plough SpA	TEMGESIC
eye gel	Bausch & Lomb	LIPOSIC