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-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 research^10,11, 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 remyelination^12,13. 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

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 solution preparation

1. Dissolve 25 mg of LPC powder (see Table of Materials) with 250 µL of chloroform and methanol mixed solution (1:1) to make a 10% LPC solution and transfer it to a 500 µL centrifuge tube.
   NOTE: If the LPC is not completely dissolved, place the centrifuge tube in an ultrasonic cleaner and ultrasonicate at 40 kHz for ~1 h to obtain a uniform solution.
2. Divide the solution into 3 µL/tube and store at −80 °C.
   NOTE: The solution can be stored for ~2 years.
3. Before surgery, dilute the solution (step 1.2.) with 27 µL of 0.9% NaCl solution, and keep the solution in a constant temperature water bath at 37 °C.
   ​NOTE: Prepare the solution just before starting the injection.

2. Surgical preparation

1. Use a 32 G, 2 in needle to connect to a 5 µL syringe (see Table of Materials). Ensure that the microliter syringe is unobstructed. Withdraw 5 µL of LPC solution for preparation of the injection.
2. Anesthetize the mouse in an induction chamber connected to an isoflurane vaporizer with 3% isoflurane mixed with 100% oxygen at a rate of about 0.3 L/min.
3. Confirm the depth of anesthesia by the lack of toe-pinch reflex while the breathing is smooth.
4. Shave the head of the mouse between the ears using an electric shaver. Apply lubricating eye drops to prevent corneal dryness during the procedure.
5. Then, position the mouse in a stereotaxic frame (see Table of Materials) with the dorsal side up, and secure the head with a nose cone and tooth clamp. Maintain anesthesia with 1.2%-1.6% isoflurane through its nose.
   ​NOTE: Isoflurane concentration can be adjusted according to the respiratory status of the mice.

3. Surgical procedure

NOTE: Animals are placed on a heating pad during all procedures.

1. Fix the mouse to the stereotaxic apparatus with bilateral ear bars. Ensure that the ear bars are level and the head is horizontal and stable.
2. Disinfect the skin on the head by wiping several times with iodophor followed by alcohol in circular motion. Then use a scalpel to cut a small incision of about 1.5 cm along the midline of the scalp to expose the skull.
3. Wipe the skull with a cotton swab dipped in 1% hydrogen peroxide until the bregma, lambda, and posterior fontanelle are exposed. Place the syringe on the stereotaxic apparatus.
   NOTE: Use hydrogen peroxide with care and avoid touching the surrounding tissues.
4. Ensure horizontal positioning of the animal's head (both front and rear and left and right).
   1. Adjust the Z-axis knob of the stereotaxic frame so that the needle tip and the skull just touch without bending, then measure the Z-axis coordinate. Check the Z coordinates of bregma and posterior fontanelle.
   2. Adjust the ear bar so that the difference between the Z coordinates of bregma and posterior fontanelle is no more than 0.02 mm. Then, follow the same method to measure the Z coordinates of the corresponding positions on the left and right sides of the midline. Adjust the ear bar to ensure the left and right are at the same level.
5. Locate the corpus callosum. Set the XYZ origin to bregma.
   NOTE: The first injection site is 1.0 mm lateral to the bregma, 2.4 mm deep, and 1.1 mm anterior. The second injection site is 1.0 mm lateral to the bregma, 2.1 mm deep, and 0.6 mm anterior. For example, the coordinate of the bregma is (0,0,0). Measure the Z coordinate of the corresponding location marked as (-1, 1.1, X) and (-1, 0.6, Y). It can be determined the first injection site coordinate of the corpus callosum is (-1, 1.1, −[X + 2.4]), and the second injection site is (-1, 0.6, −[Y + 2.1]).
6. Once the injection site is determined, make a mark with a sterile marker on the skull and record the coordinates.
7. Gently drill the marked site with a skull drill (see Table of Materials). Take care to avoid any bleeding.
8. Slowly move the needle to the given coordinates and start the injection. To induce corpus callosum demyelination, inject 2 µL of LPC solution (step 1.) at each injection site (step 3.5.) at a rate of 0.4 µL/min.
9. After injection, keep the needle in each site for an additional 10 min.
   ​NOTE: Ensure that the interval of two injections is no more than 20 min.
10. Suture the skin with a 4-0 suture and wait for the animal to wake up within 10 min. Administer analgesics postoperatively in accordance with institutional animal care regulations.
    NOTE: The recommended time for euthanasia can be determined according to the purpose of the experiment. 

4. Sample extraction for focal demyelination

NOTE: For details regarding this step, please see the previously published report¹⁴.

1. Dissolve neutral red dye (see Table of Materials) in a 1% solution in phosphate-buffered saline (PBS).
2. hours before sacrificing the mice (for details, see previous reference¹⁰), inject 500 µL of 1% neutral red dye in PBS by intraperitoneal injection for each mouse.
3. Perform cardiac perfusion¹² with 30 mL of 0.1% PBS precooled at 4 °C.
4. Slice the brain into 1 mm with a brain mold (see Table of Materials).
5. Visualize the lesion stained with neutral red under the microscope and dissociate the lesion.
   ​NOTE: Remove as much surrounding normal tissue as possible to improve the accuracy of subsequent analysis. The lesion tissue can be examined with RT-PCR, electron microscopy, and western blot analyses.

5. Histological staining and immunofluorescence

1. For histologic staining and immunofluorescence¹², after cardiac perfusion (step 4.3.), remove the brain¹⁰, fix in 4% PFA overnight (at 4 °C), and completely dehydrate in 30% sucrose.
2. Cut into 20 mm coronal brain sections using a frozen slicer¹⁰ at constant temperature (−20 °C).
3. Use the slices for Luxol fast blue (LFB) staining, immunofluorescence, and western blot¹⁰.

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 on the 10 dpi compared to the sham group, showing that localized injection of LPC can successfully demyelinate the corpus callosum. A relatively high degree of demyelination still exists at 28 dpi, indicating persistent and stable demyelination due to the two-point LPC injection (Figure 1D-E).

To further evaluate the loss of myelin sheath, 10 days was selected as a key time point when the demyelination is relatively apparent. Through immunofluorescence staining degraded myelin basic protein (dMBP), a remarkable increase of dMBP was observed in the LPC-injected group at 10 dpi (Figure 1F), which represents myelin loss in the corpus callosum. Also, the protein of the lesion tissue obtained (step 4.) was used to analyze the expression of MBP by western blot. After modeling according to the protocol, MBP showed a significant loss (Figure 1G). These results were consistent with the LFB and the immunofluorescence staining.

The myelin morphology in the corpus callosum 10 days after LPC injection was observed with the electron microscope (the lesion tissue was extracted according to step 4.). The obvious demyelination verifies the success of the modeling (Figure 2).

Two-point LPC injection influenced myelin regeneration
The differentiation and maturation of oligodendrocytes (OLGs) play a crucial role in repairing myelin sheath in MS. Myelin regeneration occurs primarily by differentiating oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes. Thus, the myelin repair process will be influenced once the differentiation of OPCs into OLGs is blocked. Gst-π is the marker of OPC differentiation maturation¹⁶. After 10 days of LPC injection, it can be seen by immunofluorescence that the Gst-π decreased compared with the sham group (Figure 3A). At the same time, the proliferation ability of oligodendrocytes can be reflected by the ratio of Ki67 (oligodendrocytes proliferate) and Olig2 (total oligodendrocyte lineage cells)^17,18. The higher Ki67/ Olig2+ co-localization ratio represents more proliferation of oligodendrocytes after 10dpi (Figure 3B). These images suggest that the lesion area attempts to repair through the maturation and proliferation of OPCs after LPC injection.

Two-point LPC injection impaired the spatial memory of mice
To analyze the spatial memory ability of LPC injection in mice, the Morris water maze (WMM)¹⁹ was used and showed no difference in swimming speed between sham and LPC-injected mice. However, when the platform was removed in the Morris water maze, the latency to find the hidden platform was reduced (impaired spatial learning), and the time spent in the target quadrant increased (impaired memory retention) (Figure 4). The results suggest that the spatial memory of demyelinated mice is significantly impaired in the two-point LPC injection model. The number of animals used in different experiments is listed in Table 1. Each mouse received training beginning on day 2 or day 20.

Figure 1: Two-point injection of the LPC-induced demyelination. (A) Representation of the injection sites (1.0 mm lateral, 2.4 mm deep, and 1.1 mm anterior). (B) Representation of the injection sites (1.0 mm lateral, 2.1 mm deep, and 0.6 mm anterior). (C) Illustration of detection time points. (D) Detection of the white matter lesions via LFB staining. LFB staining at 10 days post-injection (dpi) and 28 dpi, showing persistent demyelination of the corpus callosum (scale bar = 200 µm). (E) Quantification of the demyelination area at different time points. Data are represented as mean ± SD, one-way ANOVA followed by Bonferroni's multiple comparison tests. ****p < 0.0001, n = 6 per group. (F) Representative images of dMBP immunostaining in the corpus callosum (scale bar = 100 µm). Compared with the sham, which had almost no dMBP, obvious dMBP could be seen after LPC injection. (G) Western blot analysis of MBP expression. MBP showed a significant loss after injection of LPC. β-actin was used as the loading control to measure baseline expression. Please click here to view a larger version of this figure.

Figure 2: Electron microscopy images confirm myelinated ultrastructure in the corpus callosum of sham compared to the demyelinated one in the LPC-injected mice (scale bar = 1 µm). Please click here to view a larger version of this figure.

Figure 3: Two-point injection influenced the maturation and proliferation of OPCs. (A) Representative images of GST-π in the corpus callosum of sham and LPC-injected mice (scale bar = 20 µm). The red signifies GST-π. (B) Representative images of Olig2 and Ki67 co-localization (scale bar = 20 µm). The red signifies Ki67, and the green signifies Olig2. The images were captured using a confocal microscopy system with 488 nm and 594 nm laser lines. Please click here to view a larger version of this figure.

Figure 4: Spatial memory impairment induced by LPC injection. (A) Representative images of the mice's swimming path in each group in the platform's presence (learning phase). (B) Representative images of the mice's swimming paths in each group after removing the platform (memory phase). The number of used animals is listed in Table 1. Please click here to view a larger version of this figure.

Group	Sham	LPC-Group
LFB staining	6	12 (10 dpi, 6; 28 dpi, 6)
Electron microscopy	4	4
IF	6	6
Western Blot	4	4
Morrris Water Maze	12	12

Table 1: The number of animals used for the different tests.

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 the entire variety of clinical, pathological, or immunological features of the disease²⁴. To simulate the pathological process and pathogenesis of MS at different stages, there are three common demyelinating animal models with their advantages and limitations, including the EAE model, feeding animals CPZ, and LPC-induced demyelination models.

In mice, EAE is induced by an immune response following injection of myelin antigens, resulting in microglial activation and infiltration of perivascular T and B lymphocytes, and the accompanying myelin damage is often associated with the recurrence of the disease^20,25. It better simulates the characteristics of the MS relapsing-remitting period. However, it also has several limitations. For instance, EAE is primarily a disease affecting the white matter of the spinal cord, whereas MS mainly causes demyelination of the cerebral cortex, and the location and timing of the lesions are random, making it difficult to obtain lesions accurately¹⁹.

CPZ and LPC are the most commonly used substances to induce toxic-demyelinating models. They are all capable of causing CNS demyelination after administration. In the CPZ model, feeding young mice the copper chelator cuprizone resulted in oligodendrocyte death and subsequent reversible demyelination. After the withdrawal of cuprizone, a spontaneous remyelination process was triggered within 4 days^26,27. CPZ mainly results in extensive demyelination, while LPC injection inclines toward focal demyelination. Due to the toxin and the inflammatory response, the classic one-point injection of LPC triggers a rapid and highly reproducible form of demyelination²⁸.

However, none of the above models can properly mimic the pathological process of progressive MS characterized by persistent demyelination. Therefore, the present protocol proposes a model for a two-point injection of LPC directly into the corpus callosum to induce long-term demyelination. Critical steps in the protocol include the horizontal adjusting of the animal's head and locating the injection coordinates. In step 3.5., one must ensure to adjust the bregma and posterior fontanelle level first and then adjust the left and right levels. At the same time, it needs to be noted that, after adjusting the left and right levels, the zero point must be repositioned before subsequent operations. This step is crucial because it is directly related to the accuracy of the positioning. In step 3.6., when determining the coordinates of the Z-axis, one must ensure that the end of the needle and the skull remain in exact contact, and the researcher can observe whether the needle is bent. In step 3.10., the needle stays in place for 10 min to prevent the drug from flowing out of the needle passage.

The present protocol has some limitations. Like other toxicity-induced demyelinating models, it lacks the modeling of immunological processes. Secondly, although the two-point injection model can maintain demyelination for a relatively long time, it is still inevitably accompanied by a slight degree of myelin regeneration as the disease progresses to a later stage. It may not be a more suitable model for simulating secondary progressive multiple sclerosis than myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis^29,30. The classic one-point LPC injection model produces only localized inflammatory demyelination accompanied by spontaneous remyelination. Previous research has shown that the infiltration of T cells, B cells, and macrophages after LPC injection is a key factor in myelin repair¹⁵. However, compared with the classic injection model, the two-point injection LPC model induces rapid focal demyelination of the corpus callosum and maintains the degree of demyelination for a longer period with little remyelination.

Due to the complexity of MS, different disease stages require different models to be better described. Two-point injection of LPC via a stereotaxic frame is a stable, efficient, and reproducible experimental mouse model. This model can lead to long-term demyelination, accompanied by less myelin repair over time. That means the two-point injection model can be used not only to study demyelination diseases but also to study myelin repair interventions. Besides, this model can easily and quickly evaluate the demyelination after the intervention through immunofluorescence and histological methods, and the cognitive dysfunction of MS can be reflected by the impaired spatial memory of demyelinated mice in the behavioral test. In conclusion, this model of stable demyelination could facilitate pathological and preclinical studies both on the secondary progression of MS and the relapsing-remitting MS.

Materials

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