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 lig...

<ol>
	<li>Gaire, B. P., Choi, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+roles+of+lysophospholipid+receptors+in+activation+of+neuroglia+and+their+neuroinflammatory+responses.">Critical roles of lysophospholipid receptors in activation of neuroglia and their neuroinflammatory responses.</a> International Journal of Molecular Sciences. 22 (15), 7864 (2021).</li><li>Compston, A., Coles, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis.">Multiple sclerosis.</a> Lancet. 372 (9648), 1502-1517 (2008).</li><li>Dobson, R., Giovannoni, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis+-+a+review.">Multiple sclerosis - a review.</a> European Journal of Neurology. 26 (1), 27-40 (2019).</li><li>Mahad, D. H., Trapp, B. D., Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathological+mechanisms+in+progressive+multiple+sclerosis.">Pathological mechanisms in progressive multiple sclerosis.</a> The Lancet Neurology. 14 (2), 183-193 (2015).</li><li>Filippi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis.">Multiple sclerosis.</a> Nature Reviews Disease Primers. 4 (1), 43 (2018).</li><li>Villoslada, P., Steinman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+targets+and+therapeutics+for+neuroprotection,+remyelination+and+repair+in+multiple+sclerosis.">New targets and therapeutics for neuroprotection, remyelination and repair in multiple sclerosis.</a> Expert Opinion on Investigational Drugs. 29 (5), 443-459 (2020).</li><li>Lassmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis+pathology.">Multiple sclerosis pathology.</a> Cold Spring Harbor Perspectives in Medicine. 8 (3), 028936 (2018).</li><li>Franklin, R. J., Ffrench-Constant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remyelination+in+the+CNS:+From+biology+to+therapy.">Remyelination in the CNS: From biology to therapy.</a> Nature Reviews Neuroscience. 9 (11), 839-855 (2008).</li><li>Gentile, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+exercise+in+experimental+multiple+sclerosis.">Immunomodulatory effects of exercise in experimental multiple sclerosis.</a> Frontiers in Immunology. 10, 2197 (2019).</li><li>Chen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+of+microglial+Hv1+channel+is+associated+with+activation+of+autophagic+pathway+and+ROS+production+in+LPC-induced+demyelination+mouse+model.">Deficiency of microglial Hv1 channel is associated with activation of autophagic pathway and ROS production in LPC-induced demyelination mouse model.</a> Journal of Neuroinflammation. 17 (1), 333 (2020).</li><li>Luo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stable+and+easily+reproducible+model+of+focal+white+matter+demyelination.">A stable and easily reproducible model of focal white matter demyelination.</a> Journal of Neuroscience Methods. 307, 230-239 (2018).</li><li>Blakemore, W. F., Franklin, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remyelination+in+experimental+models+of+toxin-induced+demyelination.">Remyelination in experimental models of toxin-induced demyelination.</a> Current Topics in Microbiology and Immunology. 318, 193-212 (2008).</li><li>Degaonkar, M. N., Raghunathan, P., Jayasundar, R., Jagannathan, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+relaxation+characteristics+during+preacute+stage+of+lysophosphatidyl+choline-induced+demyelinating+lesion+in+rat+brain:+An+animal+model+of+multiple+sclerosis.">Determination of relaxation characteristics during preacute stage of lysophosphatidyl choline-induced demyelinating lesion in rat brain: An animal model of multiple sclerosis.</a> Magnetic Resonance Imaging. 23 (1), 69-73 (2005).</li><li>Baydyuk, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+the+evolution+of+CNS+remyelinating+lesion+in+mice+with+neutral+red+dye.">Tracking the evolution of CNS remyelinating lesion in mice with neutral red dye.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (28), 14290-14299 (2019).</li><li>Plemel, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+lysophosphatidylcholine-induced+demyelination:+A+primary+lipid+disrupting+myelinopathy.">Mechanisms of lysophosphatidylcholine-induced demyelination: A primary lipid disrupting myelinopathy.</a> Glia. 66 (2), 327-347 (2018).</li><li>Nave, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelination+and+support+of+axonal+integrity+by+glia.">Myelination and support of axonal integrity by glia.</a> Nature. 468 (7321), 244-252 (2010).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+oligodendrocyte+differentiation+by+Olig2+and+Sox10:+evidence+for+reciprocal+interactions+and+dosage-dependent+mechanisms.">Induction of oligodendrocyte differentiation by Olig2 and Sox10: evidence for reciprocal interactions and dosage-dependent mechanisms.</a> Developmental Biology. 302 (2), 683-693 (2007).</li><li>Kassis, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+deacetylase+expression+in+white+matter+oligodendrocytes+after+stroke.">Histone deacetylase expression in white matter oligodendrocytes after stroke.</a> Neurochemistry International. 77, 17-23 (2014).</li><li>Vorhees, C. V., Williams, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morris+water+maze:+Procedures+for+assessing+spatial+and+related+forms+of+learning+and+memory.">Morris water maze: Procedures for assessing spatial and related forms of learning and memory.</a> Nature Protocols. 1 (2), 848-858 (2006).</li><li>Merkler, D., Ernsting, T., Kerschensteiner, M., Bruck, W., Stadelmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+focal+EAE+model+of+cortical+demyelination:+multiple+sclerosis-like+lesions+with+rapid+resolution+of+inflammation+and+extensive+remyelination.">A new focal EAE model of cortical demyelination: multiple sclerosis-like lesions with rapid resolution of inflammation and extensive remyelination.</a> Brain. 129, 1972-1983 (2006).</li><li>Karussis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+diagnosis+of+multiple+sclerosis+and+the+various+related+demyelinating+syndromes:+A+critical+review.">The diagnosis of multiple sclerosis and the various related demyelinating syndromes: A critical review.</a> Journal of Autoimmunity. 48-49, 134-142 (2014).</li><li>Kamma, E., Lasisi, W., Libner, C., Ng, H. S., Plemel, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+macrophages+in+progressive+multiple+sclerosis:+Relationship+to+neurodegeneration+and+therapeutics.">Central nervous system macrophages in progressive multiple sclerosis: Relationship to neurodegeneration and therapeutics.</a> Journal of Neuroinflammation. 19 (1), 45 (2022).</li><li>Kutzelnigg, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+demyelination+and+diffuse+white+matter+injury+in+multiple+sclerosis.">Cortical demyelination and diffuse white matter injury in multiple sclerosis.</a> Brain. 128, 2705-2712 (2005).</li><li>Lassmann, H., Bradl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+sclerosis:+Experimental+models+and+reality).">Multiple sclerosis: Experimental models and reality).</a> Acta Neuropathologica. 133 (2), 223-244 (2017).</li><li>Lamport, A. C., Chedrawe, M., Nichols, M., Robertson, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+autoimmune+encephalomyelitis+accelerates+remyelination+after+lysophosphatidylcholine-induced+demyelination+in+the+corpus+callosum.">Experimental autoimmune encephalomyelitis accelerates remyelination after lysophosphatidylcholine-induced demyelination in the corpus callosum.</a> Journal of Neuroimmunology. 334, 576995 (2019).</li><li>Torkildsen, O., Brunborg, L. A., Myhr, K. M., Bo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuprizone+model+for+demyelination.">The cuprizone model for demyelination.</a> Acta Neurologica Scandinavica. Supplementum. 188, 72-76 (2008).</li><li>Zhan, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cuprizone+model:+Dos+and+do+nots.">The cuprizone model: Dos and do nots.</a> Cells. 9 (4), 843 (2020).</li><li>Torre-Fuentes, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+demyelination+and+remyelination.">Experimental models of demyelination and remyelination.</a> Neurologia (Barcelona, Spain). 35 (1), 32-39 (2020).</li><li>Merkler, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myelin+oligodendrocyte+glycoprotein-induced+experimental+autoimmune+encephalomyelitis+in+the+common+marmoset+reflects+the+immunopathology+of+pattern+II+multiple+sclerosis+lesions.">Myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in the common marmoset reflects the immunopathology of pattern II multiple sclerosis lesions.</a> Multiple Sclerosis Journal. 12 (4), 369-374 (2006).</li><li>Ucal, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Widespread+cortical+demyelination+of+both+hemispheres+can+be+induced+by+injection+of+pro-inflammatory+cytokines+via+an+implanted+catheter+in+the+cortex+of+MOG-immunized+rats.">Widespread cortical demyelination of both hemispheres can be induced by injection of pro-inflammatory cytokines via an implanted catheter in the cortex of MOG-immunized rats.</a> Experimental Neurology. 294, 32-44 (2017).</li></ol>

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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >L-&#945;-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 &#956;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 &#38; Meisinger, Germany&#160;</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&#160;</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&#160;</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&#160;Scientific</td>
			<td >14-5698-95</td>
			<td></td>
		</tr>
		<tr >
			<td >Beta Actin Monoclonal Antibody</td>
			<td >Proteintech</td>
			<td >66009-1-Ig&#160;</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&#160;AffiniPure&#160;Donkey anti-Rabbit&#160;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)&#160;</td>
			<td >YEASEN</td>
			<td >34412ES60</td>
			<td></td>
		</tr>
		<tr >
			<td >Alexa Fluor 594&#160;AffiniPure Donkey Anti-Rabbit IgG (H+L)&#160;</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)&#160;</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 o...

Watch this Scientific Journal Video about A Stably Established Two-Point Injection of Lysophosphatidylcholine-Induced Focal Demyelination Model in Mice at JoVE.com

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). ...

Our model is highly relevant to demyelination diseases, especially multiple sclerosis. And it can contribute to the related advancing clinically relevant research. It's a detailed protocol that could directly cause severe demyelination quickly and stably in mice by surgical procedure.While trying this technique for the first time, be patient, and ensure that coordinates are accurate. Begin by connecting a 32 gauge needle to a 5 microliter syringe. Withdraw 5 microliters of LPC solution for the preparation of the injection.Next, confirm the depth of anesthesia of the mouse by the lack of toe pinch reflex while the breathing is smooth. Position the mouse in the stereotaxic frame with the dorsal side up, and secure the head with a nose cone and tooth clamp. 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. Disinfect the head skin by wiping with iodophor. Then use scalpel to cut a small incision to about 1.5 centimeters along the midline of the scalp to expose the skull.Wipe the skill 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. Ensure the horizontal positioning of the animal's head, and locate the corpus callosum.Once the injection site is determined, record the coordinates, and make a mark with a marker on the skull. Gently drill the marked site with a skull drill. Take care to avoid any bleeding.Slowly move the needle to the given coordinates. To induce corpus callosum demyelination, inject 2 microliters of LPC solution at each injection site at a rate of 0.4 microliters per minute. After injection, keep the needle in each site for an additional 10 minutes.Finally, suture the skin with a 4-0 suture, and wait for the animal to wake up within 10 minutes. The representative image shows the detection of the white matter legions via Luxol fast blue staining. From the results, stable demyelination of the corpus callosum can be seen at 10 and 28 days post injection.The quantification of the demyelination area at different time points is shown here. Through immunofluorescence staining degraded myelin basic protein, a remarkable increase of dMBP was observed in the LPC-injected group at 10 days post injection, which represents myelin loss in the corpus callosum. Western blot analysis of the myelin basic protein, or MBP, expression showed a significant loss of MBP after injection of LPC.Beta-actin was used as the loading control to measure the baseline expression. Electron microscopy images confirm the myelinated ultrastructure in the corpus callosum of sham group and LPC-injected group. GST-pi is the marker of oligodendrocyte precursor cell, or OPC, differentiation maturation.After 10 days of LPC injection, the GST-pi decreased compared with the sham group. At the same time, the proliferation ability of oligodendrocytes can be reflected by the ratio of oligodendrocytes proliferate and the total oligodendrocyte lineage cells. The higher ratio represents more proliferation of oligodendrocytes after 10 days post injection.The representative images show the mice's swimming path in each group, in the platform's presence and after removing the platform. The results suggest that the spatial memory of demyelinated mice is significantly impaired in the two-point LPC injection model. While attempting this procedure, make sure that the injection rate is accurate, and keep the needle in each site for an additional 10 minutes.

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JoVE Journal

Neuroscience

3.2K visualizações.  Huazhong University of Science and Technology. Nosso modelo é altamente relevante para doenças de desmielinização, especialmente a esclerose múltipla. E pode contribuir para o avanço relacionado à pesquisa clinicamente relevante. É um protocolo detalhado que pode causar desmielinização severa rapidamente e facada em camundongos por procedimento cirúrgico.Ao tentar essa técnica pela primeira vez, seja paciente e garanta que as coordenadas sejam precisas. Comece conectando uma agulha de calibre 32 a uma seringa de 5 micro...