A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice

Summary

The present protocol describes a two-point injection of lysophosphatidylcholine via a stereotaxic frame to generate a stable and reproducible demyelination model in mice.

Abstract

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). Lysophosphatidylcholine (LPC) is an endogenous lysophospholipid associated with inflammation, and it could induce rapid damage with toxicity to myelin lipids, leading to focal demyelination. Here, a detailed protocol is presented for stereotactic two-point LPC injection that could directly cause severe demyelination and replicate the experimental demyelination injury quickly and stably in mice by surgical procedure. Thus, this model is highly relevant to demyelination diseases, especially MS, and it can contribute to the related advancing clinically-relevant research. Also, immunofluorescence and Luxol fast blue staining methods were used to depict the time course of demyelination in the corpus callosum of mice injected with LPC. In addition, the behavioral method was used to evaluate the cognitive function of mice after modeling. Overall, the two-point injection of lysophosphatidylcholine via a stereotaxic frame is a stable and reproducible method to generate a demyelination model in mice for further study.

Introduction

Receptor-mediated lysophospholipid signaling involves diverse physiological processes of almost all organ systems¹. In the central nervous system (CNS), this signaling plays a critical role in the pathogenies of autoimmune neurological diseases such as multiple sclerosis (MS). Multiple sclerosis is a chronic immune-mediated disorder characterized by pathological demyelination and inflammatory response, causing neurologic dysfunction and cognitive impairment^2,3. After continuous relapsing and remitting during the early disease, most patients eventually progress to the secondary-progressi....

Protocol

All animal procedures were approved by the Institute of Animal Care Committee of Tongji Medical College, Huazhong University of Science and Technology, China. Adult C57BL/6 male and female mice (wild type, WT; 20-25 g; 8-10 weeks old) were used for the present study. The mice were obtained from commercial sources (see Table of Materials). Mice were housed in a specific pathogen-free (SPF) animal facility with water and food supplied ad libitum. They were kept in an alternating 12 h period of light and dark cycle in the standard conditions of 22 °C temperature and relative humidity of 55%-60%.

1. LPC solu....

Results

Two-point injection of the LPC resulted in a more durable demyelination
LPC mainly leads to rapid damage with toxicity to myelin and cleavage of the axon integrity¹⁵. The day of injection was regarded as day 0. Mice were kept for a period of 10-28 days (10 dpi and 28 dpi). Luxol fast blue (LFB) staining¹⁰ was used to evaluate the area of demyelination in mice at these time points. In the two-point injection model, there was significant demyelination on .......

Discussion

MS, a chronic demyelinating disease of the CNS, is one of the most common causes of neurological dysfunction in young adults²⁰. Clinically, approximately 60%-80% of MS patients experience the cycle of relapses and remissions before developing a secondary-progressive MS^21,22, and it eventually leads to cumulative movement impairments and cognitive deficits over time²³. Currently, no single experimental model covers t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
L-α-Lysophosphatidylcholine from egg yolk	Sigma-Aldrich	L4129-25MG
32 gauge Needle	HAMILTON	7762-05
10 μl syringe	HAMILTON	80014
high speed skull drill	strong,korea	strong204
drill	Hager & Meisinger, Germany	REF 500 104 001 001 005
Matrx Animal Aneathesia Ventilator	MIDMARK	VMR
Portable Stereotaxic Instrument for Mouse	Reward	68507
Micro syringe	Reward	KDS LEGATO 130
Isoflurane	VETEASY
Paraformaldehyde	Servicebio	G1101
Phosphate buffer	BOSTER	PYG0021
LuxoL fast bLue	Servicebio	G1030-100ML
Suture	FUSUNPHARMA	20152021225
Brain mold	Reward	68707
Electron microscope fixative	Servicebio	G1102-100ML
Neutral red (C.I. 50040), for microscopy Certistain	Sigma-Aldrich	1.01376
Anti-Myelin Basic Protein Antibody	Millipore	#AB5864
Anti-GST-P pAb	MBL	#311
Ki-67 Monoclonal Antibody (SolA15)	Thermo Fisher Scientific	14-5698-95
Beta Actin Monoclonal Antibody	Proteintech	66009-1-Ig
Myelin Basic Protein Polyclonal Antibody	Proteintech	10458-1-AP
OLIG2 Polyclonal Antibody	Proteintech	13999-1-AP
Alexa Fluor 488 AffiniPure Donkey anti-Rabbit IgG (H+L)	YEASEN	34206ES60
Alexa Fluor 594 AffiniPure Donkey Anti-Rat IgG (H+L)	YEASEN	34412ES60
Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L)	YEASEN	34212ES60
HRP Goat Anti-Rabbit IgG (H+L)	abclonal	AS014
HRP Goat Anti-Mouse IgG (H+L)	abclonal	AS003
Adult C57BL/6 male and female mice	Hunan SJA Laboratory Animal Co. Ltd