The Experimental Study About Mesenchymal Stem Cells Promoting Remyelination In Cuprizone-induced Demyelination Mice

Background:Multiple sclerosis (MS) is an inflammatory demyelinating disorder of the central nervesystem(CNS) and is one of the most devastating CNS diseases. Spontaneous remyelinationand functional recovery are possible in early stages, but eventually fail. Current therapeuticinterventions for multiple sclerosis predominantly modulate the immune system and reducethe inflammatory insult by general, non-specific mechanisms but have little effect on theneurodegenerative component of the disease.Mesenchyma stem cells (MSCs) have a multipotent stem cell capacity whichcharacterized by differentiate into cells and tissue of the mesenchymal lineage. MSCs areattractive for cell-based therapies for autoimmune diseases such as MS, as they havesignificant immunomodulatory functions and promote neural repair. What’s more, in vitrostudies demonstrated the effect of soluble human MSCs-derived cues to modulate the fateof neural stem cells; in co-culture with MSCs, neural stem cells are stimulated to migratelong distances and generate neurons and oligodendrocytes at the expense of astrocytes;several studies also have suggested that MSCs may be capable of transdifferentiation intoneural precursors.Studies in experimental allergic encephalomyelitis (EAE) models of demyelinationhave indicated that remyelination is performed by MSCs, this improvement was correlatedwith reduced pathology in EAE animal, including axonal sparing, smaller demyelinatedlesions and reduced infiltration of immune cells. While there is little doubt that MSCsameliorate EAE signs, the mechanisms by which they achieve this outcome continue to bedebated. One hypothesis is that MSCs modulate the immune system in such a way as tolimit the potentially autoreactive responses that would cause tissue damage in the CNS, thishypothesis has garnered the most support from experimental results; the other hypothesis isthat MSCs secrete neuroprotective factors, and stimulate endogenous repair mechanisms in the CNS that counteract ongoing tissue degeneration; the third hypothesis is thatMSCs transdifferentiate into brain cells leading to cell replacement, nevertheless,most of the current evidence indicates that this is not a robust phenomenon. However, EAEis a mainly lymphocyte driven model, which makes it difficult to distinguish between thedirect effects of MSCs on remyelination and indirects as a result of MSCsimmunomodulation. Therefore, to explor the direct effect of MSCs besidesimmunomodulation, we used the toxic, non-autoimmune driven cuprizone mouse model,which induces a highly reproducible demyelination of distinct brain regions, among thecorpus callosum(CC).MScs have an established history of clinical efficacy in reducing the burden ofimmune-mediated diseases, and are currently being tested in a variety of regenerativesettings. It seems likely that the information gained from these diverse approaches willprovide critical insights into how to best utilize MSCs in the treatment of demyelinatingdiseases such as MS. Success in this area would likely expand rapidly to other targetdiseases and CNS injuries, for example, classical neurodegenerative diseases such asAlzheimer’s disease, Huntington’s disease, Parkinson’s disease; non-immune-mediateddiseases such as stroke, spinal cord injury.Part one： The research of pathology and behavior of Cuprizone induceddemyelination model.Method:8weeks male mice were random separated normal group and CPZ modelgroup, model groups include acute demyelination group and chronic demyelination group.Acute group was fed with0.2%Cuprizone mixed diet for6weeks, and get the brain tissueevery week; chronic group was fed Cuprizone mixed diet for12weeks, and at the12thweeks get the brain tissue; normal group was fed with normal diet. We tested thedemyelination through lucas fast blue, used IHC and Western blot to test the expression ofMBP and GFAP in corpus callosum area, at last we combined the behavior test (rotation testand open filed) analyse the character of the model in each stage.Results：1、The IHC of MBP and LFB found that the myelin sheath in corpus callosum was lost as time goes on, the third week was a point time(P<0.01), after3weeks the myelin sheathwas lost faster, the4、5、6week was disparation compared with normal group(P<0.05，P<0.01，P<0.01).2、We test the astrocyte activation by GFAP, the expression of GFAP was higher astimes(P<0.05)；the fourth week was point, in the fourth week GFAP increased faster, andGFAP was increased double untile5week, but the6week has no difference with5week(P>0.05).3、Rotation test found that the motor function was decreased in the first week, the stainshowed a low level(P<0.01); Open filed test found, the auto behavor of CPZ model wasreduced in the fourth week compared with normal group(23.65±0.30vs28.64±0.24)(P<0.01).Conclusion: The myelin sheath of Cuprizone induced demyelination model wasdecreased, astrocytes were increased and motor function was lost as time going on; the3-4thweek was a pathology and behavior point time, it will affect the function of anaphase; the3-4thweek can imitate the acute phase of diease vividly, giving an intervening measure inthis phase can explore the effect of the therapy for function.Part two：The experimental study about mesenchymal stem cells promotingremyelination in cuprizone-induced demyelination model.Method:8week mice were randomly separated into normal group(n=10) andexperiment group(n=20), experiment group included MSCs injection group and mediainjection group,10mise each group, behavior and every target protein (GFAP、Olig-2、MBPand CC1)were test after2weeks injection.Results：Compared with normal group, LFB and transmission electron microscopefound the myelin sheath in corpus callosum of media group was lost heavily(0.13±0.01)vs(0.06±0.02)(P<0.01), but in MSCs group was repaired(0.10±0.02)(P<0.05), the expressionof GFAP was higher in media group and lower in MSCs group(50±5)vs (74±10)(P<0.05);After MSCs injection, the olig-2expression was more than media group(48±7)vs (23±3)(P<0.05); the same with CC1expression(26±3) vs (19±4)(P<0.05).Conclusion：The MSCs injection can improve the motor function of model mouse and decrease oligdendrocyte die in corpus callosum, this maybe have the relationship withthe high olig-2expression and decrease the activation of astrocyte.