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 impairment2,3. After continuous relapsing and remitting during the early disease, most patients eventually progress to the secondary-progressive stage, which could cause irreversible damage to the brain and resulting disability4. It is believed that the pathological hallmark of the secondary-progressive course is demyelinating plaques caused by inflammatory lesions5. Existing treatments for MS can significantly reduce the risk of relapse. However, there is still no effective therapy for long-term demyelinating damage caused by progressive MS6. Thus, a stably established and easily reproducible model is required to study preclinical therapeutics that focus on white matter degeneration.
Demyelination and remyelination are two major pathological processes in developing multiple sclerosis. Demyelination is the loss of myelin sheath around axons induced by microglia with pro-inflammatory phenotypes7, and it leads to slow conduction of nerve impulses and results in the loss of neurons and neurological disorders. Remyelination is an endogenous repair response mediated by oligodendrocytes, where disorders could lead to neurodegeneration and cognitive impairment8. The inflammatory response is crucial to the whole process, affecting both the degree of myelin damage and repair.
Therefore, a stable animal model of persistent inflammatory demyelination is meaningful for further exploration of therapeutic strategies for MS. Due to the complexity of MS, various types of animal models have been established to mimic demyelinating lesions in vivo, including experimental autoimmune encephalomyelitis (EAE), toxic-demyelinating models, cuprizone (CPZ), and lysophosphatidylcholine (LPC)9. LPC is an endogenous lysophospholipid associated with inflammation, and it could induce rapid damage with toxicity to myelin lipids, leading to focal demyelination. Based on previous reports and research10,11, a detailed protocol of two-point injection with some modifications is provided. Generally, the classic one-point LPC injection model only produces local demyelination at the injection site and is often accompanied by spontaneous remyelination12,13. However, the two-point injection LPC model can demonstrate that the LPC can directly induce demyelination in the mouse corpus callosum and cause more durable demyelination with little myelin regeneration.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grants: 82071380, 81873743).
Materials
| 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 |
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