The Expression Of NG2 And Reaction Of NG2 Positive Cells In The Facial Motor Nucleus After Facial Nerve Axotomy

[Background] Among the cranial nerves, facial nerve is the most liable to be damaged because of its specificity of anatomical position. When it goes out of intracalvarium, the pathway of facial nerve is superficial, complex and variant. Many kinds of craniomaxillofacial trauma or surgeries could cause its injury, leading to partial or complete facial paralysis which seriously affects facial appearance and function. Although recently great progress has been made in variety of nerve repair techniques, restoration of function after transection of the facial nerve is poor, and occurrence of postparalytic sequelae is inevitable. That's because axonal injury not only results in Wallerian degeneration of peripheral nerve but also is followed by substantial corresponding neuronal deactivation. The neurotrophic and guide role of microenvironment in the neuronal nucleus has important significance on the regeneration of damaged nerve. In central nervous system(CNS), glial cells are widely distributed, providing the physical support and nutrition to neurons, maintaining balance of fluid environment, forming myelin sheath, providing insulation protection, removing pathogens and dead neurons and so on. Therefore, knowing more about the glial microenvironment in facial nucleus after facial nerve injury could provide a theoretical basis for repair and regeneration of nerve. At present, a large number of studies have shown that microglia and astrocytes in facial nucleus could make a series of responses after facial nerve axotomy. However, as to the NG2 positive cells, recently discovered to be widespread in the CNS, showing a strong response in a variety of CNS diseases or injuries, there is little research in the facial nucleus both under physiological and pathological conditions. The real role of NG2 positive cells is still unknown, and there exists great controversy on the source and property of them. Therefore, in order to define their sources and properties, and explore the role of NG2 positive cells on damaged facial motor neurons, we intend to investigate NG2 protein expression and response of NG2 expressing cells in the facial nucleus after facial nerve injury.[Objective] (1) Establish rat facial nerve crush, axotomy and avulsion models; systematically compare the changes of facial motor neurons in different injury models and provide the basis for selection of animal models for different research aims; (2) Observe the changes of NG2 protein expression at different time points after facial nerve axotomy; compare the differences of responses among NG2 positive cells, microglia cells and astrocytes; (3) Explore the source and property of NG2 positive cells in the facial nucleus after facial nerve axotomy; (4) Explore the possible roles of proliferating NG2 positive cells towards to the damaged facial motor neurons.[Methods] (1) Establish facial nerve crush, axotomy and avulsion models of S-D rats; according to the different postoperative time points, divide into postoperative 1 day,2 days,7 days,14 days and 28 days groups. (2) At different time points, perfuse rats, fix brains later and take continuous frozen sections of brainstem containing facial nucleus; (3) Stain different frozen sections with Cresyl Violet; observe the morphological differences of neurons between the control side and injury side of facial nucleus; compare the differences of damaged neurons in different models. (4) Immunohistochemical stain different frozen sections with ChAT. Count the positive neurons on both sides of facial nucleus. The viable rate of neurons is calculated as L/R using one section for each animal (L, the number of ChAT-positive neurons in the left transected side; R, the number of ChAT-positive neurons in the right control side). Make statistic analysis accordingly. (5) Western Blot detection of apoptosis-related protein Caspase-3 expression in each group at different time points on both sides of facial nucleus; in accordance with the scanning density value, make semi-quantitative data analysis; all samples are obtained Caspase-3/β-actin values (mean±standard deviation); For both operation and control sides in each group at each time point, use the student-t test, P<0.05, considering statistically significance; (6) Western Blot detection of the NG2 protein expression in facial nucleus after facial nerve axotomy; use the above statistical methods to analyze the time-phase changes; (7) Through immunohistochemical method, observe the response of microglia, astrocytes, oligodendrocytes and NG2 positive cells in facial nucleus after facial nerve axotomy and compare the differences of their responses; (8) According to the BrdU proliferation assay and immunofluorescence double staining method, analyze the source, property and function of NG2 positive cells; (9) Establish rat needle-stick injury model, inject red fluorescent latex particles, and use the immunofluorescence staining method to observe the phagocytic function of NG2 positive cells after postoperative 1 day,2 days and 7 days.[Results] (1) Successfully established facial nerve crush, axotomy and avulsion injury models:animal behaviors in the early phase appeared as complete facial paralysis, followed by the recovery of facial nerve function within 28 days in the crush group. While the axotomy and avulsion groups showed a permanent facial paralysis. (2) Cresyl Violet staining showed that crush group only appeared the uneven staining of Nissl body in the early phase after surgery; Axotomy groups showed pro-apoptotic morphological changes in different extent, while the avulsion group appeared apoptotic neurons in postoperative 14 days and 28 days. (3) Immunohistochemical staining of ChAT showed that in the crush group, the capacity of synthesis and releasing neurotransmitter acetylcholine was reduced only around postoperative 7 days, then followed by a gradual recovery; However, in the axtomy and avulsion group, the rate of active neurons was gradually reduced and showed a significant difference compared with the crush group(P<0.05). (4) Western Blot of Caspase-3 showed that Caspase-3 expression in facial nucleus of crush group has no significant difference and maintained at a relatively low level; However, in the axotomy group after 7 days and avulsion group after 2 days, the expressing of Caspase-3 in the injury side showed a high expression compared with control side(P<0.05), and kept a gradual increase within 28 days. (5) Using Western Blot method, it was the first time to prove that NG2 protein expression existed in facial nucleus and NG2 protein in the injured nucleus has a regular time-phase change within 28 days after facial nerve axotomy.(gradual increased at postoperative two days, peaked at 7 days, then decreased gradually and turn down to the base level at 28 days). (6) Immunohistochemical staining showed that microglia, astrocytes and NG2 positive cells all undergone a marked response after facial nerve axotomy. The cell body of NG2 positive cells becomes hypertrophy. They showed thicker processes, increased the expressing of NG2 protein and gradually proliferated and migrated after two days. After 7 days, NG2 positive cells closely wrapped the damaged neurons, similarly to the response of microglia. However, this is significantly different from the rosette-like loose surrounding formed by astrocytes. (7) Using immunofluorescence double staining technique, we found that the NG2 positive cells in injuried facial nucleus after facial nerve axotomy were composed of two properties cells:1. the original NG2 positive cells in facial nucleus, these cells belong to the OPC. In this experiment, these cells showed NG2+/PDGF-α-R+/OX42-or NG2+/PDGF-α-R+/ Lectin-, but have no obvious response to the facial nerve axotomy; 2. activated microglia which was induced to express the NG2. This kind of cells showed up gradually at two days after facial nerve axotomy, and then further activated and proliferated. The number of these cells reached to the peak after 7 days and they formed tight encapsulations of injured neurons. (8) After a preliminary analysis of the function of induced NG2 expressing microglia, we found that their NG2 positive processes stretched into pre-and postsynaptic space and showed an extensive detachment of the Synapsin-I marked synaptic terminals. The expression of NG2 protein could regulate the cell proliferation, migration and adhesion. And further through the needle-stick model, we confirmed that this induced NG2 expressing microglia did not have the phagocytic capacity like the common activated microglia.[Conclusions] (1) Through the comparative study of three facial nerve injury models: crush, axotomy and avulsion, we suggest that despite the deactivation of a large number of neurons, they are not death and only show a series of changes of pro-apoptosis after facial nerve axotomy. They just enter into the "dormant" state of function within 28 days. This response is very likely relevant to the glial microenvironment of facial nucleus.(2) Further research on the glial microenvironment after facial nerve axotomy, we first found the regular time-phase changes of NG2 protein in facial nucleus; And confirmed that NG2 protein expression was not as a secreted protein involved in the formation of glial scar, but expressed on the surface of a special class of cell population, as a sign of its activation.(3) According to analysis of the source and property of activated NG2 positive cells, we suggested that the NG2 positive cells which showed response to the facial nerve axotomy were not the OPC in traditional sense, but the activated microglia cells which was induced to express NG2 protein. This induced NG2 expressing microglia showed up gradually at two days after facial nerve axotomy, then further activated, proliferated, migrated and formed tight encapsulations to the injured neurons.(4) The induced NG2 expressing microglia showed a wide range of "synaptic stripping" to the damaged neurons and made them in a completely closed resting state, thereby played a protective effect and help the functional recovery of neurons. However, this kind of cells did not have a phagocytic capacity like the common activated microglia. And their proliferation, migration and adhesion ability is relevant to the NG2 protein expression.