1. The Changes Of TH Expression And The Mechanism Of Its Transcriptional Regulation In Locus Coeruleus And Adrenal Medulla After Traumatic Brain Injury 2. The Effect Of Activated Notch Signaling Pathway On Proliferation And Ensheathment Of Olfactory Enshe

Topic One THE CHANGES OF TH EXPRESSION AND THE MECHANISM OF ITS TRANSCRIPTIONAL REGULATION IN LOCUS COERULEUS AND ADRENAL MEDULLA AFTER TRAUMATIC BRAIN INJURYObjectiveThe present study was aiming at investigating the changes of tyrosine hydroxylase (TH) and its transcription factors, c-fos and activator protein 2α(AP-2α) expression in locus coeruleus(LC) and adrenal medulla in the brain injury rat models, so as to explore the mechanism of transcriptional regulation of TH expression after brain injury stress.Methods1. One hundred and five Wistar rats were randomly divided into control group and traumatic brain injury groups. The rat models of traumatic brain injury were established with Feeney method.2. The behavioral scores were assessed at different intervals after traumatic brain injury.3. The rats were perfused at various intervals post-injury, then the brain stems and adrenals were properly removed, sliced and stained with Nissl staining, HE staining to determine the morphological changes of LC and adrenal medulla;4. Immunohistochemical, double immunofluorescent stainings were used to detect the changes of TH, AP-2α, c-fos expression in LC and adrenal medulla;5. Western blot and RT-PCR were performed to determine the changes of TH, AP-2α, c-fos and their mRNA expression in LC and adrenal medulla of rats with brain injury.6. Computing-image analysis system was used to determine the changes of staining intensity and the number of TH, AP-2α, c-fos-positive neurons in injury groups, the number and fluorescence intensity of positive cells were detected by fluorescent microscope and Confocal microscope respectively.7. The obtained data were statistically analyzed with SPSS software.Results1. The behavioral and morphological changes of rats with brain injuryThe mean behavioral scores of the injury group at different intervals after brain injury were significantly lower than the controls.There were no obvious morphological changes in LC and adrenal medulla of rats with brain injury by HE staining. However, Nissl staining showed increased number of Nissl bodies and intenser staining in the neurons of LC.2. Expression of TH/AP-2αin LC The number and intensity of TH&AP-2α-positive cells were notably higher in LC in brain injury group compared to the controls by double immunofluorescence(P<0.05). The expression pattern of TH was the samilar to that of AP-2α. TH and AP-2αexpression became intenser in LC 1h after injury with the highest peaks 3h later, and kept remarkably higher 72h later than the controls. In addition, TH and AP-2αhad the overlapping expression areas in LC. AP-2αexpression was found in most TH-positive cells.3. Expression of TH/c-fos in LCImmunofluorescence showed the fluorescence intensity of TH in LC was significantly higher in brain injury group compared to the controls within 24 hours (P<0.05). The expression of TH became intenser 1h after injury, reached the highest level 3h later, and it decreased gradually with time passing, which was still higher 72h later than the control(P<0.05). Moreover, c-fos was also overexpressed 1h following injury, the highest expression was found 3h later, However, c-fos returned to the baseline level 24h post-injury. In addition, TH and c-fos showed the similar expression areas in LC. C-fos expression was found in most TH-positive cells.4. Expression of TH/AP-2αand TH/c-fos in adrenal medullaImmunohistochemical and double immunofluorescent stainings showed the number and staining intensity of TH-positive cells in adrenal medulla were notably higher in brain injury group at various intervals compared to the controls(P<0.05). The expression of TH became intenser 1h after injury, reached the climax 3h after injury, and decreased gradually with time passing. Higher expression of TH was observed 72h following injury than the control. The result of AP-2αexpression was consistant with that of TH in adrenal medulla. Moreover, the number and staining intensity of c-fos-positive cells increased 1h after injury, reached the climax 3h later, However, c-fos had returned to the baseline level 24h after injury. In addition, TH, c-fos and AP-2αshowed the overlapping expression areas in adrenal medulla at various intervals after injury. The expression of c-fos and AP-2αwere found in most TH-positive cells.5. The similar results of TH, AP-2αand c-fos expression were found in the injury group with Western blot and PCR. The expression of TH was consistant with that of c-fos within 24h following brain injury. Meanwhile, the expression of TH was consistant with that of AP-2αfrom 1h to 72 h after injury.Conclusions1. The present study demonstrated TH was overexpressed in LC and adrenal medulla in different time periods after brain injury stress. This indicates that a lot of catecholamine may be released from LC and adrenal medulla in response to the injury stress. The released catecholamine can affect the related receptors and/or targeted organs to adapt to the reactions of acute stress through regulating the functions of nervous system, cardiovascular system and metabolism. 2. C-fos expression increased in LC and adrenal medulla within 24 hours after brain injury, and c-fos and TH coexisted in the same cells; In addition, c-fos had the binding site on TH promoter, so it might be involved in the regulation of TH expression during the early period of trauma.3. The number and staining intensity of AP-2α-positive neurons in LC and adrenal medulla are higher at various intervals after brain injury; coexpression of AP-2αand TH was found in the same cells; Moreover, AP-2αhad the binding site on TH promoter. The results have showed that AP-2αmay regulate TH transcription in the stress of brain injury.4. It is firstly reported that c-fos and AP-2αsimultaneously participate in TH transcription in LC and adrenal medulla during the brain injury stress . The present study provides a theoretical base to study the molecular mechanisms of brain injury stress. TOPIC TWO THE EFFECT OF ACTIVATED NOTCH SIGNALING PATHWAY ON PROLIFERATION AND ENSHEATHMENT OF OLFACTORY ENSHEATHING CELLSThe effect of activated Notch signaling pathway on the proliferation of olfactory ensheathing cellsObjectiveThe aim of present study was to investigate the effect of activated Notch signaling pathway on the proliferation of olfactory ensheathing cells (OECs), so as to explore the mechanisms of the enhancement of OECs proliferation induced by Notch signaling pathway.Methods1. OECs were dissected from the outer nerve and glomerular layers of the newborn rat olfactory bulb, and cultured for 7d in DMEM medium including 10% fetal calf serum (FCS), and then transfected with the active Notch plasmid (Notch^ICV, c-myc as a reporter gene),inactive Notch plasmid (Notch^VK, c-myc as a reporter gene) and empty plasmid (control group). The results were observed at 3d after transfection; 2. HE staining was performed to determine the cell density and the morphological changes of OECs in Notch^ICV group, Notch^VK group and control group; the expression of GFAP and P75 were determined with immunocytochemistry;3. Immunocytochemical staining was used to detect the expression of c-myc (a report gene of transfection) in OECs; moreover, c-myc positive cells were counted in Notch^ICV group and Notch^VK group, respectively;4. Double immunofluorescent staining was used to detect the changes of glial fibrillary acidic protein (GFAP) and c-myc expression in OECs at 3d after transfection, the differences of the GFAP expression were compared among these three groups;5. BrdU was used to label the proliferation of OECs at 12 hours before fixation of the cells; Immunocytochemical staining were used to check the expression of BrdU in OECs; BrdU positive cells were counted at these three groups, respectively;6. Computing-image analysis system was used to determine the changes of staining intensity, the number and fluorescence intensity of positive cells were detected by fluorescent microscope and confocal microscope, respectively;7. The obtained data were statistically analyzed with SPSS software.Results1. GFAP and P75 positive cells were found in most of the cultured cells. The result showed that the high purity of OECs was obtained with the present method. HE staining showed that OECs were fusiform or triangular in shape, no obviously morphological changes were found in OECs following transfection. The cell density in Notch^ICV group was signicifantly increased compared with Notch^VK group (P<0.05), and there was no difference between Notch^VK group and control group;2. The expression of c-myc was observed in both Notch^ICV group and Notch^VK group, which indicated transfection of the plasmids was successful. The number of c-myc-positive cells in Notch^ICV group was significantly increased than that in Notch^VK group (P<0.05), no c-myc-positive cells were found in control group;3. Immunocytochemical staining showed the number of BrdU-positive cells in Notch^ICV group notably inceased compared with Notch^VK group (P<0.05), while there was no difference between Notch^VK group and control group;4. C-myc and GFAP proteins coexisted in OECs of both Notch^ICV group and Notch^VK group, which suggested the Notch gene was transfected into OECs successfully. Fluorescence intensity and Western blot showed the expression of GFAP became intenser in Notch^ICV group in comparison to Notch^VK group and control group(P<0.05), and there was no difference between the latter two groups.Conclusions 1. The results of BrdU and c-myc detections have demonstrated that activated Notch can promote the proliferation of OECs.2. Two mechanisms might be involved in Notch promoting OECs proliferation: first, the downstream effector Hes gene, whose transcription can be triggered by activated Notch, promotes the proliferation of OECs; second, activated Notch may regulate the transcription of GFAP through binding to GFAP promoter, the increased GFAP expression may induce the proliferation of OECs in a certain way.The primary study about the promotion of ensheathment of olfactory ensheathing cells by activated Notch signaling pathwayObjectiveThe aim of present study was to investigate the effect of activated Notch signaling pathway on the ability of ensheathment of OECs, so as to explore the roles of Notch signaling pathway on ensheathment of OECs and the possible mechanism.Methods1. The sensorimotor cortex were seperated from the brains of newborn rats, and then cultured in DMEM medium including 10% FCS. At the 3rd day of the culture, 10μmol/L arabinoside was added in the medium to inhibit the proliferation of glia cells. After 2 days arabinoside was removed, and then nerve filament (NF), as a marker of neurons, was detected to identify neurons by immunocytochemistry;2. The procedures of seperation, culture and identification of OECs were the same as above. OECs were transfected with the plasmids of Notch^ICV, Notch^VK and empty plasmid after seven-day culture. The reagent of transfection was Lipofectamine 2000. Nerve cell adhesion molecule (NCAM) and c-myc were determined by double immunofluorescence and Western blot 3d after transfection, the differences of NCAM expression were compared among the three groups;3. The Notch-transfected OECs were tranferred in the plate containing neurons, and were cocultured with neurons for 3 days. C-myc was determined by immunocytochemistry, the ditribution of c-myc-positive cells was observed near the nerve fibers;4. The number and fluorescence intensity of positive cells were detected by fluorescent microscope and confocal microscope, respectively. The obtained data were statistically analyzed with SPSS software.Results1. Immunocytochemical staining showed NF was expressed in most cells, and NF was distributed in the cytoplasm and process of neurons. High purity of neurons could be obtained with the way of adding arabinoside in the medium. HE staining demonstrated that each of the neurons under the cultured condition had a large cell body, with a long axon and many dendrites; 2. The expression of c-myc was observed in both Notch^ICV group and Notch^VK group, which indicated the transfection of plasmids was successful. The number of c-myc-positive cells in Notch^ICV group was significantly increased than that in Notch^VK group, there was no c-myc-positive cell found in control group;3. C-myc and NCAM were coexpressed in Notch^ICV and Notch^VK-transfected OECs by double immunofluorescence, which suggested the Notch gene was transfected into OECs successfully. Fluorescence intensity and Western blot showed the NCAM expression increased significantly in Notch^ICV group than Notch^VK group and control group, and there was no difference between the latter two groups;4. Many c-myc-positive cells adhered to, fused with and wrapped the nerve fibers in the co-culture of neurons with Notch^ICV-transfected OECs, and NCAM expression of these cells notably increased, whereas fewer c-myc-positive cells could adhere to the fibers in the co-culture of neurons with Notch^VK-transfected OECs.Conclusions1. The results have demonstrated that activated Notch pathway can induce NCAM expression in OECs.2. Two mechanisms might be involved in increased ability of ensheathment of OECs by Notch pathway: one is that activated Notch promoted the proliferation of OECs, the increased number of OECs could have more possibilities to contact with the nerve fibers; another is that activated Notch can upregulate NCAM expression which play an important role in adhesion and ensheathent of OECs; the increased NCAM expression may enhance the ability of ensheathent of of OECs.