Name: a stably established two-point injection of lysophosphatidylcholine-induced focal demyelination model in mice
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This work was supported by the National Natural Science Foundation of China (Grants: 82071380, 81873743).

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 &#176;C temperature and relative humidity of 55%-60%.
1. LPC solution preparation
<ol>
	<li>Dissolve 25 mg of LPC powder (see Table of Materials) with 250 &#181;L of chloroform and methanol mixed solution (1:1) to make a 10% LPC solution and transfer it to a 500 &#181;L 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.</li>
	<li>Divide the solution into 3 &#181;L/tube and store at &#8722;80 &#176;C. 
	NOTE: The solution can be stored for ~2 years.</li>
	<li>Before surgery, dilute the solution (step 1.2.) with 27 &#181;L of 0.9% NaCl solution, and keep the solution in a constant temperature water bath at 37 &#176;C. 
	&#8203;NOTE: Prepare the solution just before starting the injection.</li>
</ol>
2. Surgical preparation
<ol>
	<li>Use a 32 G, 2 in needle to connect to a 5 &#181;L syringe (see Table of Materials). Ensure that the microliter syringe is unobstructed. Withdraw 5 &#181;L of LPC solution for preparation of the injection.</li>
	<li>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.</li>
	<li>Confirm the depth of anesthesia by the lack of toe-pinch reflex while the breathing is smooth.</li>
	<li>Shave the head of the mouse between the ears using an&#160;electric shaver. Apply lubricating eye drops to prevent corneal dryness during the procedure.</li>
	<li>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. 
	&#8203;NOTE: Isoflurane concentration can be adjusted according to the respiratory status of the mice.</li>
</ol>
3. Surgical procedure
NOTE: Animals are placed on a heating pad during all procedures.
<ol>
	<li>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.</li>
	<li>Disinfect the skin on the head&#160;by wiping several times with iodophor followed by alcohol in circular motion. Then use a scalpel&#160;to cut a small incision of about 1.5 cm along the midline of the scalp to expose the skull.</li>
	<li>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.</li>
	<li>Ensure horizontal positioning of the animal&#39;s head (both front and rear and left and right).
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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, &#8722;[X + 2.4]), and the second injection site is (-1, 0.6, &#8722;[Y + 2.1]).</li>
	<li>Once the injection site is determined, make a mark with a sterile marker on the skull and record the coordinates.</li>
	<li>Gently drill the marked site with a skull drill (see Table of Materials). Take care to avoid any bleeding.</li>
	<li>Slowly move the needle to the given coordinates and start the injection. To induce corpus callosum demyelination, inject 2 &#181;L of LPC solution (step 1.) at each injection site (step 3.5.) at a rate of 0.4 &#181;L/min.</li>
	<li>After injection, keep the needle in each site for an additional 10 min. 
	&#8203;NOTE: Ensure that the interval of two injections is no more than 20 min.</li>
	<li>Suture the skin with a 4-0 suture and wait for the animal to wake up within 10 min. Administer analgesics&#160;postoperatively in accordance with institutional animal care regulations. 
	NOTE: The recommended time for euthanasia can be determined according to the purpose of the experiment.&#160;</li>
</ol>
4. Sample extraction for focal demyelination
NOTE: For details regarding this step, please see the previously published report14.
<ol>
	<li>Dissolve neutral red dye (see Table of Materials) in a 1% solution in phosphate-buffered saline (PBS).</li>
	<li>hours before sacrificing the mice (for details, see previous reference10), inject 500 &#181;L of 1% neutral red dye in PBS by intraperitoneal injection for each mouse.</li>
	<li>Perform cardiac perfusion12 with 30 mL of 0.1% PBS precooled at 4 &#176;C.</li>
	<li>Slice the brain into 1 mm with a brain mold (see Table of Materials).</li>
	<li>Visualize the lesion stained with neutral red under the microscope and dissociate the lesion. 
	&#8203;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.</li>
</ol>
5. Histological staining and immunofluorescence
<ol>
	<li>For histologic staining and immunofluorescence12, after cardiac perfusion (step 4.3.), remove the brain10, fix in 4% PFA overnight (at 4 &#176;C), and completely dehydrate in 30% sucrose.</li>
	<li>Cut into 20 mm coronal brain sections using a frozen slicer10 at constant temperature (&#8722;20 &#176;C).</li>
	<li>Use the slices for Luxol fast blue (LFB) staining, immunofluorescence, and western blot10.</li>
</ol>

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The authors have nothing to disclose.

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 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 disease20,25. 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 days26,27. 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 encephalomyelitis29,30. 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.

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.

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

a stably established two-point injection of lysophosphatidylcholine-induced focal demyelination model in mice

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.

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-&#960; is the marker of OPC differentiation maturation16. After 10 days of LPC injection, it can be seen by immunofluorescence that the Gst-&#960; 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.&#160;Each mouse received training beginning on day 2 or day 20.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64059/64059fig01v2.jpg" /> 
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 &#181;m). (E)&#160;Quantification of the demyelination area at different time points. Data are represented as mean &#177; SD, one-way ANOVA followed by Bonferroni&#39;s multiple comparison tests. ****p &#60; 0.0001, n = 6 per group. (F)&#160;Representative images of dMBP immunostaining in the corpus callosum (scale bar = 100 &#181;m). 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. &#946;-actin was used as the loading control to measure baseline expression. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64059/64059fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64059/64059fig02.jpg" /> 
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 &#181;m). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64059/64059fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64059/64059fig03.jpg" /> 
Figure 3: Two-point injection influenced the maturation and proliferation of OPCs. (A) Representative images of GST-&#960; in the corpus callosum of sham and LPC-injected mice (scale bar = 20 &#181;m). The red signifies GST-&#960;. (B) Representative images of Olig2 and Ki67 co-localization (scale bar = 20 &#181;m). 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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64059/64059fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64059/64059fig04.jpg" /> 
Figure 4: Spatial memory impairment induced by LPC injection. (A) Representative images of the mice&#39;s swimming path in each group in the platform&#39;s presence (learning phase). (B) Representative images of the mice&#39;s swimming paths in each group after removing the platform (memory phase). The number of used animals is listed in Table 1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64059/64059fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Group</td>
			<td>Sham</td>
			<td>LPC-Group</td>
		</tr>
		<tr>
			<td>LFB staining</td>
			<td>6</td>
			<td>12 (10 dpi, 6; 28 dpi, 6)</td>
		</tr>
		<tr>
			<td>Electron microscopy</td>
			<td>4</td>
			<td>4</td>
		</tr>
		<tr>
			<td>IF</td>
			<td>6</td>
			<td>6</td>
		</tr>
		<tr>
			<td>Western Blot</td>
			<td>4</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Morrris Water Maze</td>
			<td>12</td>
			<td>12</td>
		</tr>
	</tbody>
</table>
Table 1: The number of animals used for the different tests.

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

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

Receptor-mediated lysophospholipid signaling contributes to the pathophysiology of diverse neurological diseases, especially multiple sclerosis (MS). ...

a-stably-established-two-point-injection-lysophosphatidylcholine

Nanyang Technological University

Research

JoVE Journal

Neuroscience

3.7K Views.  Huazhong University of Science and Technology. Ons model is zeer relevant voor demyelinisatieziekten, met name multiple sclerose. En het kan bijdragen aan het gerelateerde voortschrijdende klinisch relevante onderzoek. Het is een gedetailleerd protocol dat direct ernstige demyelinisatie snel en stabiel kan veroorzaken bij muizen door chirurgische ingreep.Terwijl u deze techniek voor de eerste keer probeert, moet u geduldig zijn en ervoor zorgen dat de coördinaten nauwkeurig zijn. Begin met het aansluiten van een naald van 32 gauge...

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

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

Immunology and Infection

Determining Immune System Suppression versus CNS Protection for Pharmacological Interventions in Autoimmune Demyelination

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in myelin destruction, which not only slows conduction of nerve impulses, but causes axonal injury resulting in motor and cognitive decline. Current treatments for MS focus on attenuating immune cell infiltration into the central nervous system (CNS). These treatments decrease the number of relapses, improving quality of life, but do not completely eliminate relapses so long-term disability is not improved. Therefore, therapeutic agents that protect the CNS are warranted. In both animal models as well as human patients with MS, T cell entry into the CNS is generally considered the initiating inflammatory event. In order to assess if a drug protects the CNS, any potential effects on immune cell infiltration or proliferation in the periphery must be ruled out. This protocol describes how to determine whether CNS protection observed after drug intervention is a consequence of attenuating CNS-infiltrating immune cells or blocking death of CNS cells during inflammatory insults. The ability to examine MS treatments that are protective to the CNS during inflammatory insults is highly critical for the advancement of therapeutic strategies since current treatments reduce, but do not completely eliminate, relapses (i.e., immune cell infiltration), leaving the CNS vulnerable to degeneration.

This protocol describes how to determine whether pharmacological treatments for experimental autoimmune encephalomyelitis show CNS protection as a consequence of suppressing immune cell infiltration or are neuroprotective during the onslaught of immune cell infiltration.

A major hallmark of the autoimmune demyelinating disease multiple sclerosis (MS) is immune cell infiltration into the brain and spinal cord resulting in ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

Experimental Demyelination and Remyelination of Murine Spinal Cord by Focal Injection of Lysolecithin

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost oligodendrocytes, myelin, axons, and neurons. Remyelination is an endogenous repair mechanism whereby new myelin is produced subsequent to proliferation, recruitment, and differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes, and is necessary to protect axons from further damage. Currently, all therapeutics for the treatment of multiple sclerosis target the aberrant immune component of the disease, which reduce inflammatory relapses but do not prevent progression to irreversible neurological decline. It is therefore imperative that remyelination-promoting strategies be developed which may delay disease progression and perhaps reverse neurological symptoms. Several animal models of demyelination exist, including experimental autoimmune encephalomyelitis and curprizone; however, there are limitations in their use for studying remyelination. A more robust approach is the focal injection of toxins into the central nervous system, including the detergent lysolecithin into the spinal cord white matter of rodents. In this protocol, we demonstrate that the surgical procedure involved in injecting lysolecithin into the ventral white matter of mice is fast, cost-effective, and requires no additional materials than those commercially available. This procedure is important not only for studying the normal events involved in the remyelination process, but also as a pre-clinical tool for screening candidate remyelination-promoting therapeutics.

Demyelinating diseases can be modeled in animals by focal application of lysolecithin into the CNS. A single injection of lysolecithin into mouse spinal cord produces a lesion that spontaneously repairs over time. The goal is to study factors involved in de- and remyelination, and to test agents for enhancing repair.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system characterized by plaque formation containing lost ...

Rat Model of Widespread Cerebral Cortical Demyelination Induced by an Intracerebral Injection of Pro-Inflammatory Cytokines

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and death, caused by white matter lesions in the spinal cord and cerebellum, as well as by demyelination in grey matter. Whilst conventional models of experimental allergic encephalomyelitis are suitable for the investigation of the cell-mediated inflammation in the spinal and cerebellar white matter, they fail to address grey matter pathologies. Here, we present the experimental protocol for a novel rat model of cortical demyelination allowing the investigation of the pathological and molecular mechanisms leading to cortical lesions. The demyelination is induced by an immunization with low-dose myelin oligodendrocyte glycoprotein (MOG) in an incomplete Freund&#39;s adjuvant followed by a catheter-mediated intracerebral delivery of pro-inflammatory cytokines. The catheter, moreover, enables multiple rounds of demyelination without causing injection-induced trauma, as well as the intracerebral delivery of potential therapeutic drugs undergoing a preclinical investigation. The method is also ethically favorable as animal pain and distress or disability are controlled and relatively minimal. The expected timeframe for the implementation of the entire protocol is around 8 - 10 weeks.

The protocol presented here allows the reproduction of a widespread grey matter demyelination of both cortical hemispheres in adult male Dark Agouti rats. The method comprises of intracerebral implantation of a catheter, subclinical immunization against myelin oligodendrocyte glycoprotein, and intracerebral injection of a pro-inflammatory cytokine mixture through the implanted catheter.

Multiple sclerosis (MS) is the most common immune-mediated disease of the central nervous system (CNS) and progressively leads to physical disability and ...

Induction and Evaluation of Experimental Autoimmune Encephalomyelitis in Mouse

Modeling Multiple Sclerosis in the Two Sexes: MOG35-55-Induced Experimental Autoimmune Encephalomyelitis

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different prevalence in the sexes, affecting more women than men, and different outcomes, showing more aggressive forms in men than in women. Furthermore, MS is highly heterogeneous in terms of clinical aspects, radiological, and pathological features. Thus, it is necessary to take advantage of experimental animal models that allow the investigation of as many aspects of the pathology as possible. Experimental autoimmune encephalomyelitis (EAE) represents one of the most used models of MS in mice, modeling different disease features, from the activation of the immune system to CNS damage. Here we describe a protocol for the induction of EAE in both male and female C57BL/6J mice using myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55) immunization, which leads to the development of a chronic form of the disease. We also report the evaluation of the daily clinical score and motor performance of these mice for 28 days post immunization (28 dpi). Lastly, we illustrate some basic histological analysis at the CNS level, focusing on the spinal cord as the primary site of disease-induced damage.

Author Spotlight: Creating a Versatile Experimental Autoimmune Encephalomyelitis Model Relevant for Both Male and Female Mice 

Experimental autoimmune encephalomyelitis is one of the most widely used murine models of multiple sclerosis. In the current protocol, C57BL/6J mice of both sexes are immunized with myelin oligodendrocyte glycoprotein peptide, resulting mainly in ascending paresis of the tail and limbs. Here we discuss the protocol of EAE induction and evaluation.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease affecting the central nervous system (CNS). It is characterized by different ...

Induction and Diverse Assessment Indicators of Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and axonal damage. Currently, there are no measures to cure MS completely, but multiple disease-modifying therapies (DMT) are available to control and mitigate disease progression. There are significant similarities between the CNS pathological features of experimental autoimmune encephalomyelitis (EAE) and MS patients. EAE has been widely used as a representative model to determine MS drugs' efficacy and explore the development of new therapies for MS disease. Active induction of EAE in mice has a stable and reproducible effect and is particularly suitable for studying the effects of drugs or genes on autoimmune neuroinflammation. The method of immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG35-55) and the daily assessment of disease symptoms using a clinical scoring system is mainly shared. Given the complex etiology of MS with diverse clinical manifestations, the existing clinical scoring system can't satisfy the assessment of disease treatment. To avoid the shortcomings of a single intervention, new indicators to assess EAE based on clinical manifestations of anxiety-like moods and osteoporosis in MS patients are created to provide a more comprehensive assessment of MS treatment.

The present protocol describes the induction of experimental autoimmune encephalomyelitis in a mouse model using myelin oligodendrocyte glycoprotein and monitoring the disease process using a clinical scoring system. Experimental autoimmune encephalomyelitis-related symptoms are analyzed using mouse femur micro-computed tomography analysis and open field test to assess the disease process comprehensively.

Multiple sclerosis (MS) is a typical autoimmune disease of the central nervous system (CNS) characterized by inflammatory infiltration, demyelination, and ...

Biology

Generation of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Tietz, S. M. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/59249">Visualizing Impairment of the Endothelial and Glial Barriers of the Neurovascular Unit during Experimental Autoimmune Encephalomyelitis In Vivo.</a> J. Vis. Exp.&#160; (2019)
This video demonstrates the method of inducing experimental autoimmune encephalomyelitis (EAE) by injecting myelin-derived peptides with an adjuvant to activate autoreactive T cells, which migrate to the brain. Pertussis toxin is administered to increase blood-brain barrier permeability, allowing these T cells and other immune cells to enter the brain. These immune cells cause demyelination, leading to impaired nerve transmission and development of EAE.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. EAE ...

JoVE Encyclopedia of Experiments

Neuropathology

Cerebrovascular Diseases

Induction of Experimental Autoimmune Encephalomyelitis in a Mouse Model

Source: Schmitz, K., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55321">Bioluminescence and Near-infrared Imaging of Optic Neuritis and Brain Inflammation in the EAE Model of Multiple Sclerosis in Mice.</a> J. Vis. Exp (2017).
In this video, a mouse model of multiple sclerosis is established using experimental autoimmune encephalomyelitis (EAE). The mouse is injected with an emulsion containing a neuronal peptide and an adjuvant, triggering an immune response that activates autoreactive T cells. This is followed by an injection of pertussis toxin (PTX) to increase blood-brain barrier permeability, allowing the autoreactive T-cells to infiltrate the brain. This infiltration initiates a signaling cascade that ultimately results in myelin damage, mimicking the demyelinating condition of multiple sclerosis.

1. EAE Induction in SJL/J Mice

	Mice
	
		Use 11-week-old female SJL/J mice and allow them to habituate to the experimental room for about 7 days. Use n = ...

Neuromuscular Disorders

Establishing a Focal Demyelination Mouse Model via a Two-Point Injection of Lysophosphatidylcholine

Source: Pang, X., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64059">A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice.</a> J. Vis. Exp. (2022)
This video demonstrates the generation of a focal demyelination mouse model using a two-point Lysophosphatidylcholine (LPC) injection. The skull of an anesthetized mouse is exposed. The injection coordinates are determined, and a hole is drilled to enable LPC injections at targeted sites. LPC induces focal demyelination of neurons via inflammation, while a two-point injection ensures its sustainability.

Source: Pang, X., et al. A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice. J. Vis. Exp. ...

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

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