The Moleculer Mechanism Of Transplanted Olfactory Ensheathing Cells Reduced The Gliotic Response Of Müller Cells In RCS Rats

Retinitis pigmentosa (RP) is a type of inherited retinal degenerative disease leading toblindness.At present,there are no effective therapies to improve or maintain visualfunction.Müller glial cells could be activated during this progress,which begins to undergohypertrophy and proliferation,and eventually reside at subretina to form glial seal togetherwith outer segment debris.When scars are present in the subretinal space,the blood-retinalbarrier breaks down and the retinal vascularity and choroidal capillaries become poorlyperfused secondary to low oxygen pressure within the tissues,which trigger apoptosis ofphotoreceptors.It has been demonstrated that olfactory ensheathing cells (OECs),a uniquetype of glia cell,have the capacity to modify the characteristics of the constituent cells ofthe glial scar and to promote the regeneration of central nervous system (CNS) axons.A recent study from our laboratory showed that:1.the cultured syngeneic adult ratOECs migrated into surrounding space and every layer of intra retina after they weretransplanted into the subretina space of pigmented RCS-P+rats.2.The overexpressed GFAPin RCS rats’ retinas transplanted with OECs was downregulated.3.The b-wave of the ERGwas maintained after OECs were transplanted into RCS rats via the subretinal space.Theseresults suggested that the glial seal of RCS rats were inhibited after OECs weredelivered.However,how could the transplanted OECs degrade the outer segment debris andmigrate into various layers of retina?Is there an interactive relationship between OECs andMüller cells?If so,how do OECs affect Müller cells? Until now,there hasn’t such kind ofreport.We used the Royal College of Surgeons rats as our research object which had a diseaseprocess vrey similar to RP of human to study the mechanism of the OECs reducing the glialseal of Müller cells after subretinal transplantation.In the study of CNS regeneration,secreted matrix metalloproteinase (MMPs) were important for OECs migration.The MMPs family is a kind of proteolytic enzymes thatcleave matrix,and have ability to degrade extracellular macromolecules(ECM).Physiologically,MMPs are important in tissue remodeling,angiogenesis,andneurogenesis,especially in tumor cell infiltration and metastasis.So,we hypothesized thatthe transplanted OECs could enter various layers of retina through secreting MMPs.Notch signaling is an old and highly conserved signaling pathway in biologicevolution processes,which have significance functions.Notch gene encodes transmembranereceptors that bind to transmembrane ligands (Delta,Jagged) on the surface of adjacentcells.Its signal pathway could realize signal transduction in the cytoplasm and transcriptionin the nuclear through interactions of adjacent cells,and thus regulate the differentiation ofseveral lineages of cells and tissues precisely.After OECs transplantation,will the glial fatesof OECs and Müller cells,which both have gliacyte characteristics,be inhibited each otherthrough Notch-Delta signaling pathway?Till now,there are no relative reports availablefocusing particularly on this question.According to the above researches,we propose hypotheses that the transplanted OECsin the retina may migrate and contact with Müller cells through secreting MMPs andsuppress the Müller cell gliosis by inhibiting activated Notch-Delta signaling pathway.Based on the above hypothesis,this study focused on:1.Olfactory bulbs for OECs culture were harvested from adult RCS-rdy+-P+rats.Immunocytochemistry and ELISA were used to detect the expression and secretion ofMMPs in vitro and in vivo,and the change of MMPs in different observing time points.2.The subretinal transplantation model of RCS rats was established to be used to detectthe expression change of GFAP,which was the special marker of retinal gliosis.Theconfocal microscopy was used to observe the migration and patterns of interaction betweenOECs and Müller cells in recipients at various time points.3.Immunocytochemistry and RTFQ-PCR was used to examine the expression changesof Notch receptors and their ligands in OECs and Müller cells of RCS rats.We also detectthe expression change of some molecules involved in Notch signaling pathway in recipientsafter transplantation of OECs.The main results1.The OECs expressed and secreted MMP-2and MMP-3in vitro and in recipients.In order to determine whether OECs expressed and secreted MMPs in vitro and inrecipients,the immunofluorescence staining and ELISA was performed.Analysis showedthat:(1)NGFRp75and MMP-2or MMP-3co-expressed in OECs harvested from olfactorybulbs of adult rats.(2)No obvious change of MMP-2/MMP-3in the culture medium wasobserved after OECs cultured for5days (P>0.05).The amount of MMP-2/MMP-3immunoreactivity observed in OECs culture medium was higher than that of general culturemedium supernatants from11thday to14th day (P<0.01).(3)For the experiment in vivo,thedifferences between the three groups were not significantly after OECs were injected intothe subretinal space of RCS rats at7thdays (P>0.05);The amount of MMP-2in OECstransplanted group was higher than PBS transplanted group at14thday (P<0.05);On the21thday and28thday,the amount of MMP-2/MMP-3in OECs transplanted group wassignificantly higher than the other two groups (P<0.01).No significant difference wasobserved between the PBS transplanted group and unoperation group (P>0.05).2.Overexpressed GAFP was downregulated in RCS rats’ retinas and direct interactionsbetween OECs and Müller cells of recipients were observed after OECs mixture cultureswere delivered into retinal subspace.To track the OECs after they were transplanted intosubretinal space of recipients,the cells were infected with Lentivirus-EGFP.The effects oftransplanted OECs on morphological changes of retinas in RCS rats after transplantation of30days and60days were observed,especially in GFAP expression.The GFAP expression inMüller cells was significantly downregulated at P30and P60in RCS rats’ retinas of OECstransplanted.At7thday after transplantation,most of LV-EGFP-OECs accumulated in theinjection point,only a small part of them migrated into the outer nuclear layer;at14thdayafter transplantation,some of LV-EGFP-OECs migrated into retina,most of them located inthe outer nuclear layer,a small part of them migrated into inner nuclear layer;at21th,some ofLV-EGFP-OECs migrated into peripheral subretinal space adjacent to the injectionpoint.Most of the LV-EGFP-OECs that had migrated into the retina located in the innerplexiform layer;at28th,most of LV-EGFP-OECs migrated into inner plexiform layer,andsome of them migrated into ganglion cell layer and contacted with ganglioncell.LV-EGFP-OECs that migrated into inner nuclear layer and inner plexiform layer had alot of ways contacting with Müller cells.3.The higher Notch-1expression levels were observed in RCS rat retinas at peak time of retinitis pigmentosa stage.The OECs may suppress Müller cells gliosis in RCS rats byinhibiting the expression of Notch receptors and their ligands.(1)Using RTFQ-PCR todetect the amount of Notch-1in RCS and normal rats at P30,P45,P60and P90.Usingimmunocytochemistry to observe the expression of Notch signaling pathway in OECs fromolfactory bulbs of adult rats and Müller cells in RCS rats.(2)Using RTFQ-PCR to test theNotch signaling mRNA expression level after OECs transplantation.The results showed that:(1)The amount of Notch1mRNA in RCS rat retinas increased markedly and reached peak atP30, and thereafter maintained a higher level as compared to control retinas.(2)Theimmunocytochemical resultes demonstrated that Notch-3presented positive signal incultured OECs,while Müller cells were positive of immunoreactive of Deltalike-1(DLL-1)/Delta like-4(DLL-4).(3)The mRNA expression levels of Notch-3and DLL4mRNA expression decreased from the P37to the P58in OECs transplanted group(P<0.01).There were no significant differences compared with other two controlgroups(P>0.05).There were no significant differences of other gene of Notch and theirligand family members among the three groups (P>0.05).Based on the results,we concluded:1.MMP-2/MMP-3were not only highly expressed in the cultured OECs of the outerolfactory bulb layer of adult rats,but also secreted and released by OECs into themedium.The transplanted OECs degraded the accumulating debris shed by rod and conecells and migrated into various layers of retina through secreting MMP-2/MMP-3.2.OECs were infected with the Lentivirus contained EGFP so that we can ribbon trailof transplanted OECs.The results showed that LV-EGFP-OECs migrated into INL and IPLfrom3weeks to4weeks after transplantation,and had several interacting patterns withMüller cells by contacting directly,such as body-body,body-processes,processes-processesand so on.These results suggested that transplanted OECs may suppress Müller cells gliosisby directly interacted with Müller cells of recipients.3.The higher Notch-1expression levels were observed in RCS rat retinas at peak timeof retinitis pigmentosa stage.After OECs,which highly expressed Notch-3,transplanted intothe subretinal space,the LV-EGFP-OECs migrated into INL and IPL of retina and began tocommunicate with Müller cells.When the physical contact of receptors and ligandsoccurs,lateral inhibition signaling started.The OECs may suppress Müller cells gliosis by inhibiting the expression of Notch-3and DLL-4.