| BackgroundPeripheral nerve injury is one of the most common clinical diseases which cause disability. Once injured, it is very difficult to recover, and even causes permanent dysfunction. Therefore, it is a great challenge for us to fully explain the recovery mechanisms and to develop treatment method in neuroscience.Peripheral nerve consists of connective tissues and nerves. Connective tissue is closely associated with nerves. As the endplates are usually far from the neuron bodies, the axons become thin and long, which isolate each other and cluster together. There are three layers of connective tissues surrounding the axons, which are called endoneurium, perineurium and epineurium from the inside out. The perineurium envelops the axons, Schwann cells and endoneurium, forming nerve fascicles. Several nerve fascicles surrounded by the epineurium form a nerve.When injured, the first step for the damage repair of peripheral nerve will be the enlargement of the neuron body, disintegration of Nissl bodies, and peripheral translocation of nucleus and initiation of relative proteins. Once injured, the distal nerve will be faced with nutritional deficiency, the cytoplasm begins to condense and liquefy and the axons begin to demyelinate and sputtering, which presents the typical Wallerian degeneration of peripheral nerves.As the Wallerian degeneration initiated, the local Schwann cells will be activated to proliferate. At the same time, a great number of macrophages will be recruited at the injury site. The macrophages are recruited from the monocyte in the blood circulation. The local Schwann cells and macrophages can engulf the debris of axons and myelin sheath. The clearance of the debris is very important to the repair of nerves. Because there are inhibitors in the myelin sheath, which can inhibit the growth of axons, possibly impacting the nerve regeneration. When injured, the Schwann cells and macrophages will interact closely to promote the nerve regeneration.Wallerian degeneration causes the sputtering of distal axons and myelin sheath, while the Schwann cells will largely survive. In the regeneration process, the Schwann cells play a crucial role. It has been showed that Schwann cells not only can clear away the debris of Wallerian degeneration, but also can express various growth factors which can promote the growth of axons and impact on the adherence and chemotaxis of axons.Nerve growth factor (NGF) is one of the most important bioactive molecules in nervous system. When injured, the Schwann cells proliferate massively and secret NGF. The high concentration of NGF in the local injury site can promote the regeneration of nerve. Laminin is the main constituent of basement membrane of Schwann cells. It regulates the function and vitality of Schwann cells and guides the migration of Schwann cells from the high concentration to lower. In a word, the Schwann cells have multiple mechanisms impacting on the peripheral nerves after injury. Schwann cells form adequate microenvironment contributing to the nervous damage repair and regeneration.On the other hand, tissues and organs are dealing with various mechanical stimulations all the time. Loading of physical mechanics to the organs can influence their function in cellular and genetic levels. When stimulated by mechanics, cells can respond with biochemical, biomechanical and bioelectrical reactions, which in turn, impact on the tissues and organs in cellular level.It has been demonstrated that the mechanical stimulation can lead to various impact on cells, including directional differentiation, cell proliferation and migration, increase of extracellular matrix formation, change of cell arrangement and morphology, increase of signal transduction, etc.Similarly, the peripheral nerves are faced with multiple mechanical stimulations in different situation. Notably, in normal physiological state, when performing their normal function, nerves are under different tensile stress. For instance, to adjust to different positions and movements, nerves are tolerant of mechanical stimulations. Generally, the mechanical stimulations applied to nerves are sorted into three types, compressive, shear force and tensile. As the situations of compressive and shear force are complex, and the research model is immature, tension load is employed to nerve researches more often.At present, tensile loading is extensively researched in nerve functional rehabilitation, Ilizanov technique, etc. Therefore, researching on the biomechanical properties of normal and injured nerve, as well as the mechanical strain induced by daily activities is helpful to make diagnosis and guide the physicotherapeutics.Tensile load can be applied to nerves either parallel or perpendicular to the length of the nerve, causing longitudinal or transverse stress in the nerve, respectively. The deformation or change in nerve length caused by longitudinal tensile stress is called strain and is expressed as percent elongation. When elongated, the nerve cross-sectional area decreases. A theoretical model suggested that the connective tissue tube or sheath constraining the pressurized neural core contributes significantly to the biomechanical properties of a nerve loaded with tensile strain. Elongated, the increased pressure produced in the neural core will resist the transverse contraction. With the increase of tensile load, structural separation occurs first in the core-sheath interface, then in the axons and connective tissues of the endoneurial core, and finally in the cells and connective tissues of the perineurium and epineurium. The structural change of connective tissue can influence the compliance of nerves. There are a number of factors that impact on the nerve compliance, like fascicle number, cross-sectional area of extrafascicular connective tissues. In addition, the velocity of elongation, time of fixed elongation and repetition also contribute to the compliance change.The non-specific connective tissue all over the body is closely associated with nerves. They interact with nerves and form an information network permeating the whole body, controlling and regulating the life activities. Macrophages are important cell constituent in fascia connective tissue, which play an important role in the damage repair of peripheral nerves. Previous research of our lab has studied the co-culture model of macrophages and Schwann cells. However, it is established on static state. Our previous research demonstrated that tensile load can induce the remodeling of the fascia connective tissue and change at genetic level. In addition, there’s other research shows that cyclic tensile strain can impact the proliferation and genetic expression of Schwann cells. However, it is still unknown what effect will be induced when tensile strength is applied to the co-cultured Schwann cells. In this study, we employed the co-culture model of Schwann cells and peritoneal macrophages to simulate the internal environment of the injured peripheral nerves. In this study, we will try to explore the effects on proliferation, migration, genetic expression of RSC96cells by applying cyclic tensile strain to co-culture model.ObjectiveThis study aims to explore the effects on proliferation, migration, genetic expression of RSC96cells by using peritoneal macrophage supernatant co-culture model and cyclic tensile strain.Methods1. To obtain the peritoneal macrophages, SD rats were decapitated and injected10ml of DMEM/F12into the peritoneal cavity.10min later, the cell suspension was collected from the peritoneal cavity and centrifuged for culture. The cells were purified by differential attachment method. Cells were marked with CD68and CD11b, and then identified by flow cytometry. The culture supernatant was collected every8hours and stored for co-culture.2. Cell grouping and treatment: RSC96cells were evenly planted on6-well BF-3001C Type I plates and4groups were treated as follows respectively:Group A: RSC96cell culture (control); Group B:co-culture; Group C: tensile load and Group D: co-culture and tensile load. Co-culture is for the co-culture of macrophage culture supernatant with RSC96cells. Tensile load is for the cyclic tensile strain. Flexcell FX-5000Tension system was employed for cyclic tensile strain. The cylinder tensile loading platform can provide biaxial stress controlled by the computer. The parameters were set as follows:sinusoidal wave,0.25Hz,5%strain, for1hour each time. The cells of tensile loading groups were treated every12hours for5times.3. To detect the proliferation of cells of all4groups, cells were collected on the3rd day of culture for MTT assay. The average optical density (OD) was counted at570nm. Wound healing assay was employed for cell migration test. Random fields of vision were obtained before and3days after treatment, and the cell-free area was analyzed by ImageJ software.4. To detect the mRNA levels of NGF and Laminin in all4groups after3days’ treatment, total RNA was obtained by Trizol method for qPCR experiment.5. To detect the protein levels of NGF and Laminin in all4groups after3days’ treatment, Western blotting method was employed. ImageJ software was used for gray value analysis.6. Experiments were repeated for3times. All results were analyzed by SPSS13.0software. One-way ANOVA was used to make comparisons. After normality and homogeneity of variance test, choose LSD or Dunnett’s T3for post comparisons alternatively. Significant level was a=0.05, and P<0.05was considered as significant difference.Results1. Cell cytometry results shows90%cells are CD68and/or CD11b positive, indicating successful obtaining of peritoneal macrophages.2. MTT assay shows the result in Group D is the highest, reduce progressively from Group C, B and A. Statistical analysis of group comparison shows significant difference (F=28.833, P=0.000). LSD test shows significant differences of other3groups compared with Group A(P=0.034,0.000,0.000, respectively). It demonstrates that the co-culture of macrophage culture supernatant and cyclic tensile strain loading with5%strain can both promote the proliferation of RSC96cells. Pairwise comparison shows significant difference between Group B and C (P=0.009), demonstrating that5%of cyclic tensile strain has greater effect on proliferation of RSC96cells than co-culture with macrophages. Similar results are obtained between Group B and D (P=0.000), Group C and D (P=0.026), demonstrating that the co-culture and cyclic tensile strain may have cooperative effect on promoting proliferation of RSC96cells.3. Wound healing assay shows that after3days’treatment, the cell-free area in all4groups decreases significantly compared with before treatment (P=0.000). B, C, D groups decreases significantly compared with area in Group A (P=0.000). It demonstrates that co-culture and tensile load can both promote the migration of RSC96cells. One-way ANOVA result suggests significant difference (F=571.670, P=0.000). LSD multiple comparisons show significant difference between each2groups (P=.000), except the comparison between Group B and C (P=0.063). It demonstrates that the co-culture and tensile load treatment may have cooperative effect on promoting migration of RSC96cells, and the causative effect of two treatments may have no significant difference.4. After3days’treatment, qPCR detection shows that the mRNA of NGF expresses significantly greater in B, C and D groups than that in Group A(P=0.002,0.006,0.002, respectively). Similar results are obtained with protein level of NGF by Western blotting (P=0.000). Both results demonstrate that co-culture and tensile load can both increase the NGF expression significantly. qPCR result shows no significant differences compared with Group B and C, B and D, C and D (P=0.463,0.948,0.502, respectively). While western blot results show significant differences between all the4groups (P=0.000). Group D expresses the highest, reduce progressively from Group C, B to Group A It demonstrates that the tensile load treatment may have greater effect on NGF expression of RSC96cells than co-culture treatment, and the two treatments may have cooperative effect on increasing NGF expression of RSC96cells. Ihe tensile loading may have better effect than co-culture on increasing NGF expression.5. After3days’treatment, qPCR detection shows that the mRNA of Laminin expresses significant greater in B, C and D groups than that in Group A (P=0.003,0.005,0.000, respectively). Similar results are obtained with protein level of Laminin by Western blotting (P=0.000). qPCR result shows that there is no significant difference between Group B and C (P=0.736). While there is significant difference of Laminin mRNA expression between B and D, C and D (P=0.049,0.028, respectively). It indicates that co-culture and tensile load can both increase Laminin expression of RSC96cells, and the two treatments may have cooperative effect on increasing Laminin expression of RSC96cells.Conclusions:1. Co-culture of peritoneal macrophage culture supernatant with RSC96cells can promote the cell proliferation and migration, as well as increasing the expression of NGF and Laminin.2. Cyclic tensile strain loading on RSC96cells can also promote the cell proliferation and migration, as well as increasing the expression of NGF and Laminin.3. Co-culture of peritoneal macrophage culture supernatant and cyclic tensile strain loading on RSC96cells may have cooperative effect on promoting cell proliferation and migration, as well as increasing the expression of NGF and Laminin. |
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