| BackgroundDemyelination disease is a global public health problem. An estimated3-4million persons worldwide are suffered with different kinds of demyelination disease, the incidence of which was6%-10%of neurological disease.A demyelinating disease is any disease of the nervous system in which the myelin sheath of neurons is damaged. This damage impairs the conduction of signals in the affected nerves. In turn, the reduction in conduction ability causes deficiency in sensation, movement, cognition, or other functions depending on which nerves are involved. Some demyelinating diseases are caused by genetics, some by infectious agents, some by autoimmune reactions, and some by unknown factors. Organophosphates, a class of chemicals which are the active ingredients in commercial insecticides such as sheep dip, weed-killers, and flea treatment preparations for pets, etc., will also demyelinate nerves. Neuroleptics can also cause demyelination.Lysophosphatidylcholine causes demyelination and is in unnaturally high amounts in foods with lecithin treated with the enzyme phospholipase (enzyme-modified foods) and as lysolecithin in products such as make up and personal care products. The precise mechanism of demyelination is not clearly understood but there is good evidence that the body’s own immune system is at least partially responsible. Acquired immune system cells called T-cells are known to be present at the site of lesions. Other immune system cells called Macrophages also contribute to the damage.Normally acute demyelination injury is followed by remyelination.Remyelination is the process of propagating oligodendrocyte precursor cells to form oligodendrocytes to create new myelin sheaths on demyelinated axons in the CNS. This is a process naturally regulated in the body and tends to be very efficient in a healthy CNS The process creates a thinner myelin sheath than normal, but it helps to protect the axon from further damage, from overall degeneration, and proves to increase conductance once again. Demyelinating diseases, such as Multiple Sclerosis, have been of utmost interest within the last couple of decades. Recent research is uncovering some of the many unknown pathways involved with remyelination in hopes of battling demyelinating diseases like MS which can ultimately cripple a person. While no treatment exists yet in preventing remyelination failure in the chronic stages of these diseases, future research may yet prove to unlock key pathways that can be targeted.Myelinating cell includes oligodendrocyte which mainly performs CNS myelination and Schwann cell primarily for myeinating in PNS. In preliminary experiment, we found that ependymocyre lining up the central canal in spinal cord could be labeled by Sox2antibody which is considered to be a stem cell marker,which strongly suggested that the ependymocyte might have stem cell potential.Based on the observation above, we recruited fate-mapping to mark the foxj1gene which had been reported to be a reliable and specific marker for ependymocyte and proceeded a series of experiments to assess if ependymocyte could get involve in CNS remyelination after demyelination. But in our experiment we didn’t find any GFP+cells in spinal cord dorsol white matter demyelinated area which didn’t prove the hypothesis.However,during the experiment,we unexpectedly found amount of cells in spinal roots was labeled by GFP staining and could migrate into spinal cord ventral white matter demyelinated lesion,which was identified as Schwann cell by immunostaining.AimsHere, three fate-mapping transgetic animals recruited experiments combined with immunostaining,western-blot and DNA sequencing provide a structured framework for the development,function of fibroblast-resemble cell in peripheral nerves provide an opportunity to analyze the relationship between fibroblast and neuro crest deprived Schwann cell lineage. This reflects, fibroblast in PNS could be a potential resourse for mylinating Schwann cell in CNS remyelination.Experimental Procedures1. SubjectsThree Cre-loxP rcombination transgenic animals are recruited in our project.In the field of genetics, Cre-Lox recombination is known as a site-specific recombinase technology, and is widely used to carry out deletions, insertions, translocations and inversions at specific sites in the DNA of cells. It allows the DNA modification to be targeted to a specific cell type or be triggered by a specific external stimulus. It is implemented both in eukaryotic and prokaryotic systems.The system consists of a single enzyme, Cre recombinase, that recombines a pair of short target sequences called the Lox sequences. This system can be implemented without inserting any extra supporting proteins or sequences. The Cre enzyme and the original Lox site called the LoxP sequence are derived from bacteriophage P1.Placing Lox sequences appropriately allows genes to be activated, repressed, or exchanged for other genes. At a DNA level many types of manipulations can be carried out. The activity of the Cre enzyme can be controlled so that it is expressed in a particular cell type or triggered by an external stimulus like a chemical signal or a heat shock. These targeted DNA changes are useful in cell lineage tracing and when mutants are lethal if expressed globally.Fate mapping is a method of understanding the embryonic origin of various tissues in the adult organism by establishing the correspondence between individual cells (or groups of cells)) at one stage of development, and their progeny at later stages of development. When carried out at single-cell resolution, this process is termed cell lineage tracing.The details of the transgenic animals are listed below:●FOXJ1-CreERT2;Rosa26R-GFPForkhead box protein J1or foxj1for short encodes a member of the forkhead family of transcription factors. Similar genes in zebrafish and mouse have been shown to regulate the transcription of genes that control the production of motile cilia. The mouse ortholog also functions in the determination of left-right asymmetry. It generally accepted that the foxj1could be specific gene to identify ependymocyte in neuro system.The animal we recruited had been inserted Cre recombinase driven by foxj1gene which recombines a pair of short target sequences called the Lox sequences that could control the expression of GFP and the the expression of Cre enzyme was triggered by tamoxifen injecting,which means after tamoxifen injection, all of the foxj1expressed cell in the transgenic animals could be labeled by GFP nad papers reported only ependymocyte could be marked in spinal cord.●Fgfr3-icreERT2:Rosa26-YFP Fgfr3-icreERT2:Rosa26-YFP mice, in which astrocytes and ependymalcyte cells are YFP labeled after tamoxifen administration (Young et al.,2010). Examination of lysolecithin-induced dorsal and ventral lesions at21dpl revealed that96%±3%of YFP+cells expressing AQP4+(which comprises virtually all AQP4+cells within the lesion area) and93%±5%of those cells expressing GFAP+throughout the repaired area, indicating that the majority of astrocytes associatedwith lesion repair were derived from Fgfr3-expressing cells. However, we did not find any YFP+cells that also immunolabelled for OLIG2, CC1, or Periaxin, so it appears that Fgfr3+cells do not generate remyelinating oligodendrocytes or Schwann cells.●Wnt1-Cre:Rosa26-YFPThe close correlation between fibroblast and Schwann cell prompt that the fibroblast have neurogenic potential, shown to be derived from neural crest Therefore, we tested the hypothesis that fibroblast might be a neural crest-derived population in adult peripheral nerve. The Wnt/beta-catenin signaling pathway is essential to the development of the neural crest and Wnt-1-Cre animals crossed with floxed RosaR26R-stop-YFP reporter mice have been successfully used in lineage tracing analyses to establish that a specific population is derived from neural crest lineage, as cells in which Wntl has been activated are indelibly marked and can be traced by the presence of the YFP. Mice were housed under standard laboratory conditions on a12hr light/dark cycle with constant access to food and water. Homozygous or heterozygous Cre mice were crossed with homozygous Rosa26-YFP reporters to generate double-heterozygous offspring for analysis. Demyelination experiments were performed on female mice aged11-13weeks (nominally postnatal day85,P85) according to the principles of laboratory animal care approved by the UK Home Office.2.Tamoxifen Induction Cre recombination was induced by administering tamoxifen (Sigma,40mg/ml), dissolved in corn oil by sonication for45min at30C. Adult mice were given300mg per kg of body weight by oral gavage on4consecutive days, then allowed to recover for7days prior to inducing demyelination. In spinal cords of Foxj1-creERT2: Rosa26-GFP mice examined1day after the final dose of tamoxifen.In Fgfr3-icreERT2:Rosa26-YFP mice subjected to a similar regimen, practically all ependymal cells of the central canal and a high proportion (>95%) of Fgfr3-expressing astrocytes in the parenchyma labeled for YFP. In Wntl-creERT2mice,83%±2%of cells in the ventral and dorsal roots became YFP labeled.3.Surgical ProceduresSeven days after tamoxifen induction, mice underwent a spinal cord white matter demyelination. In brief, mice received subcutaneous buprenorphine (30mg per kg of body weight) as analgesic treatment and were anesthetized with isoflurane. Demyelination was induced by injection of1ml of1%La-lysophosphatidylcholine (lysolecithin, SIGMA) into the ventral or dorsal white matter funiculus at the level of T12as previously described in detail (Arnett et al.,2004). The position of T13was identified and the epaxial musculature was cleared from the immediate area. The space between T12and T13was exposed and carefully cleared, the central vein was identified, and the dura was pierced with a dental needle lateral to the vein. A three-way manipulator was then used to position the needle for stereotaxic injection of lysolecithin. Hamilton needle with a fine glass tip was advanced through the pierced dura at an angle appropriate for ventrolateral or dorsal funiculus injection. Injection was controlled at1ml per minute and the needle remained in the injection site for2min to allow maximal diffusion of toxin. For sciatic nerve crush, an incision was made over the length of the right hip and, after exposing the sciatic nerve, haemostatic forceps were used for45s to induce a crush injury of the sciatic nerve. Lesions in adult rat cerebellar peduncle and spinal cord were as previously described.4.Tissue PreparationMice were anesthetized with pentobarbitone and perfused through the ascending aorta with4%(w/v) paraformaldehyde (PFA, Sigma) in phosphate-buffered saline (PBS)(pH7.4). Unfixed tissue was used for reverse transcriptase-PCR. The tissue surrounding the injection site was dissected, postfixed in4%PFA at4C overnight, cryo-preserved in30%(w/v) sucrose for12-24hr at4C, embedded in OCT, frozen on dry ice, and sectioned in a cryotome (12mm). Coronal and longitudinal cryo-sections were thawmounted onto Poly-L-lysine-coated slides and stored at80C.5.ImmunohistochemistryFrozen sections, after several rinses in PBS, were permeabilized and blocked with PBS containing0.3%(v/v)Triton X-100and10%(v/v) donkey serum in PBS for1hr at room temperature (RT,20-25C), then incubated for12hr at4C with primary antibodies followed by incubation with fluorophore-conjugated secondary antibodies for1hr at RT. Primary and secondary antibodies were diluted in PBS containing0.1%Triton X-100. For double or triple labeling, the above procedure was repeated sequentially with primary antibodies from different animal species and distinguishable fluorophore-conjugated secondary antibodies.Slides were coverslipped in mounting medium containing DAPI dye (Dako) and examined under the fluorescence microscope.6.MicroscopyFor cell imaging and counting, micrographs were acquired with a203objective on a Zeiss Axioplan fluorescent microscope under an appropriate filter with a digital camera. Composite images were assembled with Adobe Photoshop and defined areas were measured with Carl Zeiss AxioVision4.5. The lesion area was defined by the density of DAPI-stained nuclei present in the injured white matter, a feature of inflammatory response to demyelination, or by solochrome cyanine staining on adjacent sections to those used for immunohistochemistry. Sections from at least five mice were analyzed for each data point. A Leica SP2confocal laser scanning microscope with Leica Lite software was used for most of the micrography. The optical slice thickness was0.5—1mm. Images were processed with Image J (NIH) software.Results1.Changes of ependymalcyte after spinal cord white matter demyelination The foxjl expressed profile in unlesioned spinal cord and peripheral nerves FOXJ1CreER2T;Rosa26R-GFP transgenic mice triggered by tamoxifen was perfused with4%PFA in7days,take the spinal cord and siatic nerves sectioned in a cryotome (12mm)and immunostained with GPF antibody.found that practically all ependymal cells of the central canal labeled for GFP which matched what the papers reported:foxj1could be the reliable marker for ependymalcyte.But we also found in peripheral nerves there are many cell could be labeled by GFP antibody consistently which hasn’t been described before.The foxjl expressed profile in lesioned spinal cord dorsol white matter FOXJ1CreER2T;Rosa26R-GFP transgenic mice triggered by tamoxifen, received demyelination induced by injection of1ml of1%La-lysophosphatidylcholine (lysolecithin, SIGMA) into the dorsal white matter funiculus at the level of T12and perfused with4%PFA in5days or14days,take the spinal cord and siatic nerves sectioned in a cryotome (12mm)and immunostained with GPF antibody combined with olig2or GFAP shown there was no GFP+cell migrated into lesion at neither5dpl nor14dpl,either did any cells could be labeled by GFP antibody and Olig2or GFAP. The foxjl expressed profile in lesioned spinal cord dorsl and ventral white matter FOXJ1CreER2T;Rosa26R-GFP transgenic mice triggered by tamoxifen, received demyelination induced by injection of1ml of1%lysolecithin into the dorsal white matter funiculus at the level of T12and perfused with4%PFA in5days or14days,take the spinal cord and siatic nerves sectioned in a cryotome (12mm)and immunostained with GPF antibody combined with olig2or GFAP shown there was no GFP+cell migrated into dorsol lesion at neither5dpl nor14dpl,either did any cells could be labeled by GFP antibody and Olig2or GFAP.However, in foxj1mice spinal cord of14dpl ventral lesion area,we found lots of GFP+cell located on the edge of the spinal cord lesion,which couldn’t be co-localized by Olig2or GFAP either.2. Test cell type and function of foxj1expressed cells in peripheral nevre and spinal cordVerify the expression profile of foxj1in peripheral nerve Because the gene that encodes Cre recombinase only target on a part of foxj1gene,there is a possibility that other gene happen to have the target gene segment.The siatic nerve of foxj1transgenic mice triggered by tamoxifen was taken in14days,the spinal cord also was collected as positive control,verified if there was any cell could express foxj1in siatic nerve from transcriptional activation to translational protein by PCR and western-blot Idetify the cell type of GFP+cells in unlesioned peripheral nerves To indetify the cell type of GFP+cells in unlesioned peripheral nerves, we recruited different typical markers for all cell types in peripheral nerves.First we need to sort out how many types of cell in peripheral nerves, what they are and what kinds of markers we used for labeling them..Mature adult’s peripheral nerves,usually contain myelinationg Schwann cells,non-myelinating Schwann cells,fibrablast,pericyte and endothelial cell and we picked periaxin,S100,p0for marking myelinating and non-myelinating schanwann cell;Fn,HSP47,SMA and P4HB for marking fibroblast;PDGFR β for marking pericyte;CD31for marking endothelial cells.Double immunostained each of the antibodys mention above with GFP antibody,we found in transgenic mice unleison siatic nerves,majority of GFP+cells could be labeled by Fn,SMA,HSP47and P4HB considered to be fibroblast’s marker,a few of them was stained by CD31which is endothelial cell’s antibody,No cell co-locolized by GFP and periaxin,S100,p0or PDGFR β was observed.Changes of GFP+cell in peripheral nerves after siatic nerves crushed lesion compared with unlesioned nerves,GFP+cell in the crushed siatic nerves from foxj1transgenic mice14dpl was are obviously increasing, some GFP+cells were observed double labeling by myelinating Schwann cell marker periaxin antibody which manisfest that lesion could be the trigger to make GFP+cells start to express periaxin。The changing of GFP+cells in the lesion of spinal cord ventral white matter FOXJ1CreER2T;Rosa26R-GFP transgenic mice triggered by tamoxifen, received demyelination induced by injection of1ml of1%lysolecithin into the ventral white matter funiculus at the level of T12and perfused with4%PFA in14days,take the spinal cord sectioned in a cryotome (12mm)and double immunostained with GPF antibody and Olig2,GFAP,Ibal,we found there is no cell in lesion area could be double stained,but on the edge of ventral lesion,there are many GFP+cells could be labeled by periaxin,which means like siatic nerves,lesion might be the reason that make GFP+cells start to express Schwann cell’s marker.We did the lesion on same location in FGFR3transgenic mice which turned out there is no cells in lesion area could be labeled by GFP and periaxin, which suggest the FGFR3expressed cells including ependymalecyte didn’t give the rise of Schwann cells in lesion area. Identify cell type in vitro for convicing that the GFP+cells which could differentiate into Schwann cells, we tissue cultured the fibroblast in siatic nerves of foxj1 transgenic mice. The cells were purified by fibroblast culture medium.After fixation,the cells were stained using the typical different cells antibody shown the same results as in vivo staining.3.The origin and development of GFP+cells in pheripheral nerves Immunostaining on unlesioned sciatic of foxj1transgenic mice received tamoxifen form the first after birth Cre recombination was induced by administering tamoxifen (Sigma,40mg/ml), dissolved in corn oil by sonication for45min at30C. Mice were given300mg per kg of body weight by oral gavage on4consecutive days from the first day after birth, then allowed to recover for1month prior to perfusion.Immunostaing unlesioned siactic nerves with GFP and periaxin or sox10which was considered to be a marker for Schwann cell lineage.which showed there are lots of GFP+cell also positive to sox10antibody,there is no GFP cells could be labeled by periaxin.The changing of spinal cord of wnt-1transgenic mice after ventral white matter demyelination lesion Adult transgenic wnt-1mice received demyelination induced by injection of1ml of1%lysolecithin into the ventral white matter funiculus at the level of T12and perfused with4%PFA in14days,take the spinal cord sectioned in a cryotome (12mm)and double immunostained with YPF antibody and periaxin,which show there is no double labeling cell could be observed in lesion area.Conclusion1. Ependymal cells from central canal do not contribute to remyelination in spinal cord.As our experiment demonstrated,we couldn’t observe any GFP+cells in dorsol white matter lesion area at5dpl,14dpl or21dpl in foxj1transgenic mice,which comfirmed by FGFR3trangic animals recruited.2. Foxj1+cells from peripheral nerve have potential to make remyelinating Schwann cells following injury.We found GFP+cells could express Schwann cell’s marker like periaxin and S100,some of them are positive to PO,the Schwann cell generating myelin’s marker,which means they could differentiate into schwann cells and mylinate axons.3. Foxj1labelled cells in peripheral nerve resemble fibroblasts.Even though we still couldn’t claim the GFP+cells are fibroblast,all of the evidence we got point that they could express many fibroblast marker in vivo and in vitro.These GFP+cell could not be labeled by Sox10which suggest they may not deprived from Schwann cell lineage but on contrast,wnt-1cell doesn’t get involve in remyelination. |
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