Effect Of ChABC On Glial Scar: A Preliminary Experiments Study In Vitro

Background:Central nervous system (CNS) is the most important part of the nervous system, including brain and spinal cord. The main functions of CNS include to conduct, store and process information, resulting in various mental and behavior activities. However, the treatment of CNS injury is the difficulty until now, because some of inhibit factors including inhibit factors, glial scar, etc.Following injury of the mammalian CNS, those axons that attempt to regenerate face several inhibitory challenges. The result is the failure of regeneration or the limited local sprouting. The formation of a glial scar around the site of injury is a key event in the CNS injury response, and the scar tissue is an important inhibitor of axon regeneration. It is made up of reactive astrocytes, oligodendrocyte precursors, microglia/macrophages, meningeal cells and vascular endothelial cells. Generally, the scar tissue contains several types of axon growth inhibitory molecules, of which chondroitin sulphate proteoglycans (CSPGs) are the most important. Several types of CSPG are rapidly up-regulated after injury, contributing to regenerative failure. In many tissues, CSPGs are up-regulated after injury, probably helping to seal off the injury and to prevent the bacterial invasion. Various lines of evidences demonstrated that much of the axon growth inhibitory activity of CSPGs came from their glycosaminoglycan (GAG) chains. However, the removal of the GAG side chains by chondroitinase-ABC (chABC), could decrease the inhibitory characteristic of CSPGs to axon growth. Therefore, the application of chABC in the injured mammalian brain and spinal cord in vivo, can lead the regeneration of the severed nerve fibres, also the recovery of function. Otherwise, the mechanism of ChABC for decreasing glial scars has not been clear until now.In this study, we try to assess the effects of different concentrations of ChABC on glia scar in vitro, and to explore the mechanism and feasibility of ChABC for decreasing scars.Part I Primary culture, purification, and identification of cerebral astrocytes and meningeal fibroblasts.Objective:To isolate, passage, and identify the cerebral astrocytes and meningeal fibroblasts in order to carry out the study further.Method 1:Primary culture, purification, passage and maturation culture of astrocyte.1.1 Primary culture of astrocyte. Two of Wistar rats (age as 1-3 days old) were used in the experiment. After they were immerged into 75% ethanol for 3-5min, the brain tissues were removed and then rinsed 3 times with cool D-Hanks. The dissected cortices were then digested with 0.125% trypsin-EDTA 5ml for 10-15min at room temperature. The cells were then re-suspended by DMEM/F12, centrifuged at 1000rpm for 5min. Following to dilute the cellular suspension in DMEM/F12-10% FBS,5×105 cells/ml were plated in 75cm2-type culture flask, total volume as 15 mL, then cultured at 37℃,5% CO2 incubator for 10days.. The medium was changed three times a week.1.2 Purification and passage of astrosytes. Mixed glia cells were cultured in 75cm2-type flasks for 10 days. Each of flask (with 10 ml of fresh medium) was placed into an incubator and allowed to equilibrate with the CO2 air atmosphere for 2h. The flasks were then shaken at 250 rpm,37℃for 15-18h. Following the shaking, the suspended cells were discarded from all flasks. The culture flasks then contained a nearly intact layer of astrocytes and a few oligodendrocytes scattered across the surface of the suspension. The purity of the astroglial cells was increased beyond 95% by two successive steps as bellow. First, the flasks were vigorously shaken by hand. When all oligodendrocytes showed to detach from the astrocyte layer which just localed under the oligodendrocyte-layer, the medium was removed and the pellets were rinsed 3 times with fresh medium. Second, the astrocytes were replated at about 1:3 of their confluent density. The astrocytes were then suspended by adding 1.5ml of 0.25% trypsin-EDTA, with gently shaking. Then the cellular pellets were collected, by centrifugation at 1000rmp for 8min, and then plating at about 1:3, following resuspending in fresh medium. The purification rate of the astroglial cells was identified by Immuno-fluorescence as over 95%.1.3 Maturation culture of astrocytes. After the astrocytes were cultured for much more than 4 weeks in vitro, their identification was performed by Immuno-fluorescence and their growth and amplification were evaluated by CCK8 Kit. The majority of Generation 5 of astrocytes grew slowly and expressed GFAP positive, which showed the mature phenotype of astrocytes. Thus, it implied that the most cells in this experiment were highly differentiated astrocytes.Method 2:Primary culture and passage of meningeal fibroblasts. Two of Wistar rats (1-3 days old) were needed. Following the anesthesia and cerebral specimen harvest, the meningeal was put into a-penicillin flasket containing 0.5ml DMEM/F12-10% FBS, and then plated on 25cm2-type flasks coated with poly-D-lysine, to culture in DMEM/F12 containing 10% fetal calf serum at 37℃in 5% CO2 incubator for 5 days. The medium was changed three times a week. The cells were then dissociated with 0.25% trypsin, resuspended with culture medium and passaged culture at 1:3. Before passage of the cultured cells, culture flasks were weakly shaken to detach more of adherent microglia/macrophages. The Passage2～5 of meningeal cells were used for next experiments.Counting of positive cells. Immunocytochemistry photographs were obtained by the CCD imaging system. The cells with each kind of fluorescence stain were respectively selected from the three samples. Every sample was then selected 5 non-overlapping of vision fields. The positive cells and nuclei were checked. Counting statistics, and was described as X±SD.Results:1. The majority of mixed glia cells adhered to the bottom of culture flasks at the 4th h after plating, reached 80% confluence at the 4th d after culture, and formed three layers at the 9th d. The underlayer was as astrocytes, the middle layer was as oligodendrocytes, and the upper layer was as the microglia. The astrocytes were identified by Immune-fluorescence and the IPP image analysis, as 95.9%±1.3% of purification.2. Meningeal fibroblasts. The purity of meningeal fibroblasts showed 98.5%±0.6% in this experiment.Discussion and Conclusion:The methods of culturing could successively obtained high purity of the astrocytes and meningeal fibroblasts, which could be used to next experiments.PartⅡIdentification of the glia scar in vitroObjective:To approach a method of making glia scar in vitro, in order to carry out further study next step.Method:There were four groups in this experiment:(1) normal astrocytes control (A-Ctrl), (2) astrocytes scratch (A-Scr), (3) astrocyte-meningeal fibroblasts co-culture (A+F), (4) astrocyte-meningeal fibroblasts-Scratch (A+F-Scr).The reactive astrogliosis was made by combining the mechanical scratch among the differentiated astroctyes with the addition of the meningeal fibroblasts. The glia scar model was identified by the expression of GFAP and CSPGs.1. Astrocyte-meningeal fibroblasts coculture. The highly differentiated astrocytes labeled with red colour were plated into 12-well culture flast coated by PDL, at 4.0×105/well, cultured at 37℃in 5% CO2 incubator for 5～7days. The medium was changed three times a week. Meningeal fibroblasts labeled with green colour were added to the astrocyte cultures for co-culture when the astrocytes showed long processes.2. Mechanical scratch. Astrocyte-meningeal fibroblasts co-culture 2d later, 10μl-type plastic micropipette was used to scratch "#". Then, the cells were incubated in the complete medium, at 37℃in 5% CO2 incubator for 24 hours, following the rinsing with D-hanks. To observe and identifiy at 0h,24h after mechanical scratch respectively.Results:1. Results of coculture. The meningeal fibroblasts caused astrocyte reactivity and increased the positive GFAP expression. In the normal astrocytes control (A-ctrl), the differentiated astrocytes showed the obvious processes with the positive GFAP, which also expressed at the perinuclear. At the 1st day after astrocyte-fibroblast cocultures (A+F), the astrocytes with elongated processes surrounded meningeal fibroblasts. Astrocytes contacting fibroblasts showed the brighter GFAP staining. At the 2nd day after coculture between the astrocytes and fibroblasts, "Reactive sites" contained much more strong GFAP-positive astrocytes which located around the fibroblasts. In the astrocyte-fibroblast coculture, lots of astrocytes formed the clusters with meningeal fibroblasts and their processes entered into the fibroblast territory. At the 3rd day after fibroblast addition, astrocytes contacting fibroblasts showed the much brighter GFAP staining.2. Results of mechanical scratch. The astrocytes entered the central of scratch rapidly. The growth of astrocytes processes could be found at the 4th hour after scratch. Some of processes of astrocytes began to connect at the 24th hour following scratch, and some of them entered into the central of the scratch. The injuried astrocytes possessed the longer and thicker processes, with much more brighter GFAP-positive Immuno-fluorescence stain nearby the scratch than far away it.3. Identification of glia scar model. Immuno-fluorescence pictures of GFAP and CSPGs in different model groups were analyzed by IPP image process software. For the statistical analysis, all the date were described as "mean±tandard deviation(X±SD)", and analyzed by Spss (Version 13.0). The results of One-Way ANOWA analysis, were considered as significant difference, as soon as P<0.05.A-Ctrl:The GFAP-positive expression was the weaker than the one in treatment groups. Mean density was as 0.1746±0.0039. Immuno-fluorescence of CSPGs was weak also, and its mean density was as 0.0075±0.0007.A-Scr:Immuno-fluorescence of GFAP in the borderline of scratch was much brighter,and its mean density was as 0.2554±0.0061. Immuno-fluorescence of CSPGs in the scratch borderline was also up-regular, and its mean density was as 0.0111±0.0007.A+F:The expression of GFAP in astrocyte-meningeal fibroblasts coculture group was much brighter than it in Group A-Scr. Astrocytes contacting fibroblasts showed the brighter GFAP staining. And its mean density was as 0.4645±0.0049, while the expression of CSPGs was much brighter than it in the former two groups, the mean density was as 0.0398±0.0008. A+F-Scr:Group A+F-Scr showed the co-feature of" A-Scr " and " A+F ". In this group, the mean density of GFAP was as 0.5042±0.0051, and the mean density of CSPGs was as 0.0410±0.0006.Discussion and Conclusion:Both scratch and meninged fibroblasts could effectively stimulate the astrocytes, in which the meningeal fibroblasts was much more effective than scratch.Therefore, the co-culture between strocyte and meningeal fibroblasts combining mechanical scratch can be considered as an effective method to make glia scar model in vitro.PartⅢEffect of ChABC on decreasing glial scar:A preliminary experiments study in vitroObjective:To assess the different concentrations of ChABC on decreasing glial scar, and to explore the method of decreasing scars.Method:The experimental animals were divided into four groups. (1)Untreated control group, (2)1μU/mlChABC treatment group, (3)2.5μU/mlChABC treatment group, and (4)5μU/ml ChABC treatment group. Identifications of GFAP and CSPGs were performed by Immuno-fluorescence in the different groups, at the 24th hour after adding different concentrations of ChABC to the glia scar. ChBAC, a dissolved enzyme, was focus on digesting the GAG. Therefore, it was used to study the glia scar in this experiment.For the statistical analysis, we chose three wells for assaying each protein in different groups. Then immunocytochemistry photographs were obtained by the CCD Imaging system. Immuno-fluorescence pictures of GFAP and CSPGs in different treatment groups were respectively analyzed by IPP image process software. Data were described by "mean±standard deviation (X±SD)", and analysised by Spss (Version 13.0). The results of One-Way ANOVA were considered as significant difference as soon as P<0.05.Results:By treating with different concentrations of ChABC, the immuno-fluorescence of GFAP in the glia scar had no significant difference in the different groups, which all showed as brighter and uniformity. The intention of GFAP immuno-fluorescence was generally decrease after ChABC treatment with both low and high doses. Immuno-fluorescence pictures of GFAP and CSPGs in different treatment groups were also analyzed by IPP image process software, and the data were described by "mean±standard deviation (X±SD)". The detail date were showed as below.Untreated control group:mean density of GFAP:0.5061±0.0086; mean density of CSPGs:0.0416±0.0031.1μU/mLtreatment group:mean density of GFAP:0.5101±0.0071; mean density of CSPGs:0.0350±0.0022.2.5μU/mL treatment group:mean density of GFAP:0.5082±0.0097; mean density of CSPGs:0.0095±0.0018.5μU/mL treatment group:mean density of GFAP:0.5039±0.0087; mean density of CSPGs:0.0003±0.0003.There was no significant difference of GFAP among the four groups, (F= 1.463, P>0.05). There was significant difference of CSPGs, however, among the four groups (F=1334.634, P<0.05). And 5μU/mL treatment group was the best dose in the treatment of glia scar in vitro.Conclusion:ChABC is an effective method for treatment of glial scar, and 5μU ChABC/mL was the best dose in the treatment of glia scar in vitro in this experiment.