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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 impairment2,3. 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 lig.......

Representative 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 o.......

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 MS21,22, and it eventually leads to cumulative movement impairments and cognitive deficits over time23. Currently, no single experimental model covers t.......

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grants: 82071380, 81873743).

....

Materials

NameCompanyCatalog NumberComments
L-α-Lysophosphatidylcholine from egg yolkSigma-AldrichL4129-25MG
32 gauge NeedleHAMILTON7762-05
10 μl syringeHAMILTON80014
high speed skull drillstrong,koreastrong204
drillHager & Meisinger, Germany REF 500 104 001 001 005
Matrx Animal Aneathesia VentilatorMIDMARKVMR
Portable Stereotaxic Instrument for MouseReward68507
Micro syringeRewardKDS LEGATO 130
Isoflurane VETEASY
ParaformaldehydeServicebioG1101
Phosphate bufferBOSTERPYG0021
LuxoL fast bLueServicebioG1030-100ML
SutureFUSUNPHARMA20152021225
Brain moldReward68707
Electron microscope fixativeServicebioG1102-100ML
Neutral red (C.I. 50040), for microscopy CertistainSigma-Aldrich1.01376
Anti-Myelin Basic Protein Antibody Millipore#AB5864
Anti-GST-P pAbMBL#311
Ki-67 Monoclonal Antibody (SolA15)Thermo Fisher Scientific14-5698-95
Beta Actin Monoclonal AntibodyProteintech66009-1-Ig 
Myelin Basic Protein Polyclonal AntibodyProteintech10458-1-AP
OLIG2 Polyclonal AntibodyProteintech13999-1-AP
Alexa Fluor 488 AffiniPure Donkey anti-Rabbit IgG (H+L)YEASEN34206ES60
Alexa Fluor 594 AffiniPure Donkey Anti-Rat IgG (H+L) YEASEN34412ES60
Alexa Fluor 594 AffiniPure Donkey Anti-Rabbit IgG (H+L) YEASEN34212ES60
HRP Goat Anti-Rabbit IgG (H+L)abclonalAS014
HRP Goat Anti-Mouse IgG (H+L) abclonalAS003
Adult C57BL/6 male and female miceHunan SJA Laboratory Animal Co. Ltd

References

  1. Gaire, B. P., Choi, J. W. Critical roles of lysophospholipid receptors in activation of neuroglia and their neuroinflammatory responses. International Journal of Molecular Sciences. 22 (15), 7864 (2021).
  2. Compston, A., Coles, A.

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