| Traumatic brain injury (TBI) can cause severe brain damage and is accompanied by serious symptoms, such as seizures, chronic headache, and executive dysfunction While most research primarily focuses on traumatic brain injury caused by accidents, some modern clinical circumstances can also cause traumatic brain injury. Insertion or implantation of appliances such as electrodes, needles, and probes can also damage the central nervous system. These can result in Stab wound injury (SWI) which a kind of TBI. Unfortunately,these repeated and minor insults tend to be neglected. Thus, we aimed to determine an effective approach to treating traumatic brain damage from clinical interventions. The needle insertion was adopted because it is highly reproducible and quantifiable. To imitate the actual conditions of clinically induced injury, we keep a 0.7 mm diameter metallic needle in the brain for 20 minutes, and employed a digital force gauge to monitor force when we were building the model. Of course, under the control of autostereotaxic frame is used for building the model, we can quantify the speed of the insertion and the deep of the vulnus.Osthole is a natural derivative from Cnidium monnieri (L.), recently known for its therapeutic effect on central nervous system diseases such as Alzheimer's disease,autoimmune encephalomyelitis, and cognitive impairment. According to previous research performed by our lab and other researchers, all found that osthole is effective in the treatment of traumatic brain injury. No matter in vivo or vitro osthole exhibited a potent neuroprotective effect against neurodegeneration. These effective functions mainly owe to the speciality of osthole in anti-inflammatory, anti-oxidative and anti-apoptotic. However, the effects of osthole have some limitations. Damaged tissue restores itself slowly because dead neurons cannot renew and proliferate by themselves. Thus, we considered another method to support the single therapeutic method.Stem cells can differentiate into neurons, astrocytes, and oligodendrocytes . The pluripotent characteristic make those cells a candidate for cell replacement therapy, not only applied in TBI but also in other types of neurodegenerative diseases. These diseases include stroke, Alzheimer disease, Huntington's disease and Parkinson's disease. Not only the stem cells have the ability to replace the apoptotic cells but also the stem cells can release some neurotrophic factors like glial-cell-line-derived neurotrophic factor (BDNF), neurotrophin-3(NT-3) and insulin-like growth factor-1 (IGF-1) those all can help to restore the damage tissue. Moreover, stem cells therapy also has the effect of anti-inflammation. According these researches stem cells therapy can suppress inflammation caused by some pro-inflammatory cytokines. Despite the versatility, the stem cells have insufficient in cure some nervous system diseases. For example, the stem cells therapy only plays an inessential role in anti-inflammatory of experimental autoimmune encephalomyelitis mice. So the stem cells therapy always by the aid of other methods to obtain a satisfactory outcome.As mentioned above,we propose combining the two methods to establish a better way to treat brain injuries.The first chapter Osthol is used to enhance the effect of neural stem cells on Taumatic brain injury by antiinflammaOBJECTIVE: To establish a model of mechanical brain injury caused by simulated brain endoscopic surgery. To compare the therapeutic effect of various treatment regimens on this kind of brain injury and to select the best treatment method.Methods:1. To determine the modeling conditions and the simulation of brain endoscopic surgery caused by mechanical brain injury model evaluationMechanical brain injury model was established by using automatic stereotaxic apparatus.HE staining method was used to measure the size of wound area caused by four different conditions, namely (1) 0.7mm diameter flat head acupuncture, immediately needle group. (2)1.2mm diameter flat head real needles, immediately pull the needle group. (3) 0.7mm diameter flat head acupuncture, Acupuncture brain tissue 20 minutes group. (4) 1,3mm diameter flat head acupuncture, Acupuncture brain tissue 20 minutes group.Neurological Severity Scores (NSS) tests were performed on the mice 24 hours after brain injury. Determine whether model building is available for experimentation.2. Extraction, Culture and Identification of Neural Stem CellsBMSCs were extracted from GFP transgenic mice and induced to become neural stem cells and their differentiation ability was identified by immunohistochemistry3. Experimental group and dosing regimenThe experimental group contained (1) Sham group (sham operation group) (2) Osthole group (Osthole treatment group), (3) Stem Cell group (stem cell transplantation group), (4)Osthole + Stem Cell group (stem cell transplantation combined with osthole treatment group).Thirty mice per group. Administration was administered at a dose of 30 mg / kg. The first time was given 30 minutes before surgery and then at the same time every day.4. Immunohistochemical method was used to compare the effects of different treatment methods on inflammatory cell aggregation after brain injury in miceImmunofluorescence histochemistry was used to detect the effect of each treatment on the infiltration of inflammatory cells (neutrophils and astrocytes) after brain injury in mice.5. FJB staining was used to compare the effects of various therapeutic methods on the number of neuronal apoptosis after brain injury in miceFJB staining was used to compare the number of apoptotic neurons after treatment.6. Effect of different treatment methods on recovery of blood-brain barrier in mice after brain injury by detecting the Evans blue spillage methodThe effect of each treatment on the repair of blood-brain barrier after trauma was evaluated by the method of detecting the Evansan spill.result:1. To determine the modeling conditionsThe modeling condition of simulated brain endoscopic surgery was 0.7mm diameter flat head acupuncture, and the needle was kept for 20min.The NSS score (0.75 ± 0.45) in the model group (4.83 ± 0.71) was higher than that in the normal group (P<0.05). The experimental results show that our model of brain injury can affect the neurological function of mice, the model can be applied.2. Successful cultivation of GFP-containing neural stem cells and transplantationThe GFP fluorescence gene of neural stem cells was detected by PCR. Under the microscope, neural stem cells also expressed GFP green fluorescent protein. Indicating that the transfection is successful, can be used for transplantation.3. Osthol can enhance the anti-inflammatory effect of stem cellsImmunohistochemistry showed that the number of neutrophils, microglia and astrocytes was significantly decreased (P<0.05) compared with other single treatment regimens.4. Osthol can enhance the ability of stem cells to inhibit neuronal apoptosisFJB staining showed that the method of single stem cell transplantation did not have satisfactory effects on the apoptosis of transplanted neurons. There was no significant difference compare with the sham group (P>0.05).The combination therapy was superior to osthol treatment and stem cell transplantation in inhibiting neuronal apoptosis (P <0.05) in 3, 5 and 7 days, however there are no singnificant difference at 9 day (P >0.05).5. Osthol can promote the repair of injured brain tissue by stem cellsAccording to the results of the combined treatment of blood-brain barrier damage after injury to be significantly better than other methods (P <0.05).Conclusion:Osthol can enhance the inhibitory effect of stem cells on inflammatory reaction and apoptosis, and improve the microenvironment to make the transplanted nerve stem better play its role in repairing damaged tissue.Chapter 2 Osthole up-regulates the expression of BDNF to promote neural regeneration of transplanted stem cellsPurpose: To investigate the survival and differentiation of transplanted neural stem cells(NSCs) in vivo and to verify the feasibility of osthol for promoting neural regeneration of transplanted neural stem cells.Methods:1. The differentiation of neural stem cells in vivo was detected by immunohistochemistryThree types of neural stem cells differentiated into neural stem cells, astrocytes, neurons and oligodendrocytes were detected by immunohistochemistry in brain injury model.2. Effect of osthole combined with stem cell Transplantation on the number of neurons in the injured Site by immunohistochemistryImmunohistochemistry was used to detect the effect of osthole combined with stem cell transplantation on the number of injured neurons.3. Mechanism of osthole to promote nerve regenerationWe detected the expression of BDNF protein by Western Blot.4. Water maze test cnidium on stem cell treatment of mice to restore the impact of Learning and memoryThe Learning and memory ability of mice was tested by water maze on days 5, 7 and 14 after injury.result:1. Transplanted neural stem cells in vivo survival and differentiationWe used immunohistochemical method to detect the survival of the transplanted neural stem cells on day 14 in mice and to differentiate into three types of astrocytes (GFAP),neurons (NF-M), oligodendrocytes (GALC).2. Osthol can increase the number of neurons in the lesion siteThe results showed that osthol could increase the number of neurons in injury site, and the neurons in injury site of combined treatment group were significantly higher than those of stem cell transplantation group (P <0.01).3. Osthol can increase the expression of brain BDNFThe results showed that osthol had a significant effect on the expression of BDNF in brain tissue. The expression of BDNF in brain tissue of combined treatment group was significantly higher than that of stem cell transplantation group (P <0.05).4. Osthol can improve the recovery of stem cells to the learning and memory memory of mice with brain injuryThe results showed that osthol could significantly improve the recovery of stem cells to the spatial memory ability of mice with brain injury, compared with the stem cell treatment group (P <0.05).Conclusion:Osthol can up - regulate the expression of BDNF and activate CREB pathway to promote neural regeneration of transplanted neural stem cells. |
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