The Investigation Of G-CSF Induces Neurogenesis And Improves The Cognition In APP Transgenic Mouse Model Of Aizheimer’s Diseae

BackgroundAlzheimer’s disease (AD) is an insidious-onset neurodegenetative disease of central nervous system (CNS), characterized by progressive memory loss, cognitive dysfunction and behavior disorder. AD occupies more than half of aging dysmnesia. With the increasing proportion of the elderly in the population, its incidence increases year by year. The working ability and life quality of the patients are damaged significantly and this disease places a heavy burden on the family and society. With its pathogenesis still veiled, AD can not be prevented and treated effectively.The main pathological changes of AD include senile plaque (SP) and neurofibrillary tangle (NFT) in the cerebral cortex, hippocampus and subcortical nucleus, as well as neuron loss, astrocytosis, and the abnormality of neurons and synapsis. The outstanding symptom of AD is progressive cognitive dysfunction, which is closely related to the loss of glutamatergic and cholinergic neurons. Therefore, reducing neuron loss and increasing neuroregeneration is an important trend of AD treatment.Granulocyte colony-stimulating factor (G-CSF), a hematopoietic growth factor produced by monocyte, fibroblast and endotheliocyte, can bind with specific receptors on the cellular surface, promote the proliferation and differentiation of granulocyte hemopoietic progenitor, protect the neutrophils from apoptosis and intensify their function. G-CSF has been widely used in the treatment of granulocytopenia caused by various reasons. Several recent researches indicated that G-CSF could exert multiple effects on CNS, including inducing marrow-derived stem cells to migrate to the impaired brain area, anti-apoptosis, promoting neurogenesis. Neural stem cells exist in certain areas within the brain, especially subventricular zone area (ependymal), olfactory bulb and hippocampus, where G-CSF and G-CSFR are expressed. G-CSF plays such a neuroprotective role that it promotes the recovery of nervous tissue both functionally and morphologically. Recently, researches on adult animal models of cerebral ischemia showed that G-CSF is an effective drug. Kawada and his team found that the G-CSF treatment for the animal model of ischemic stroke could improve the recovery of the ischemic brain function by mobilizing bone marrow stem cells. The treatment of G-CSF for adult ischemic stroke has entered PhaseⅠandⅡclinical trials abroad.Based on previous researches, G-CSF might be able to improve cognitive function by promoting neurogenesis. In this research, G-CSF was administrated to APPV7171 transgenic mouse model of AD in order to determine whether G-CSF could induce neuroregeneration and improve the cognition. This research consisted of 3 parts, which would be described as follows.Part 1. The influence of G-CSF on the cognition of AD micePurpose:To study the changes of biological behavior of AD mice after the administration of G-CSF, Morris water maze was used to test their study and memory functions, which was in order to discuss whether G-CSF treatment could improve the cognition of AD mice.Method:1.Divide 10-month old APPV7171 transgenic AD mice and wild type group randomly into the G-CSF group, the control group and the wild type group. G-CSF group:Subcutaneous injection of G-CSF (50μg/kg·d) for 7 days in succession. Control group and wild type group:Subcutaneous injection of PBS for 7 days in succession. According to the time after the G-CSF treatment, each group was divided into 3 sub-groups:7-day subgroup,14-day subgroup and 28-day subgroup.2. Each group of mice was under Water maze orientation navigation experiments before and after injection.3. Analyze the data of Water maze orientation navigation experiments with statistics software. Observe the influence that G-CSF exerts on the AD mice.Results:Results of Water maze orientation navigation experiments of each group: Comparing to the wild type group, the escape response latency and swimming distance of the AD mice were significantly prolonged; those data of the G-CSF group were shortened significantly and similar with the wild type group. Among mice of the same group, the shortening of the escape response latency and swimming distance increased as the training time extended (P<0.01). There was no significant difference between the swimming speeds of each group. Difference caused by individual variation could be excluded.Conclusions:The cognitive function of AD mice was improved after the G-CSF treatment.Part 2 The influence of G-CSF on the CD 34+/CD 45+ratio of AD micePurpose:With flow cytometry, we investigated the difference of CD 34+/CD 45+ ratio between the G-CSF group and the control group, which aimed to discuss the influence of G-CSF on the hematopoietic stem cells of AD mice.Method:1. Divide 10-month old APPV7171 transgenic AD mice randomly into the G-CSF group and the control group. G-CSF group:Subcutaneous injection of G-CSF(50μg/kg·d) for 7 days in succession. Control group:Subcutaneous injection of PBS for 7 days in succession.2. On the 14th day after the injection, take 0.5ml of peripheral blood from postorbital vein plexus of mice after Morris water maze test and observe the peripheral CD 34+/CD 45+ ration with flow cytometry. Results:The peripheral CD 34+/CD45+ ratio of G-CSF group was significantly increased compared to the control group (P<0.01), which was about 3 times as large as that of the control group.Conclusions:1. G-CSF elevated CD 34+/CD 45+ ratio in the peripheral blood of AD mice.2. G-CSF could promote the proliferation of CD 34+ hematopoietic stem cell (HSC) and mobilize those cells into peripheral blood.Part 3 The influence of G-CSF on the neurogenesis of AD micePurpose:By performing the immunohistochemical and immunofluorescence double-labeled detection tests on the G-CSF group and the control group (immunofluorescence staining of CD 34+, immunofluorescence double-labeling of Nestin+/BrdU+ and MAP-2+/BrdU+), discuss whether G-CSF could influence the neurogenesis.Method:1. G-CSF group:Subcutaneous injection of G-CSF (50μg/kg·d) for 7 days in succession. Control group:Subcutaneous injection of PBS for 7 days in succession. During the period, both groups were given intraperitoneal injection of BrdU (50 mg/kg·d) for 10 days in succession2. On the 14th day after the injection, remove the mice brain of both groups by paraformaldehyde perfusion, and frozen on dry ice for the specimen. Then conduct the immunofluorescence staining of CD34+, and immunofluorescence double-labeling of Nestin+/BrdU+, MAP-2+/BrdU+.Results:1. On the 14th day after the injection, red-stained CD34+ cells were found in subventricular zone area, olfactory bulb and hippocampus of the G-CSF group.2. BrdU+ cells could be detected in subventricular zone area, olfactory bulb and hippocampus of both groups. Compared to the control group, the number of BrdU+ cells of the G-CSF group was significantly increased (p<0.05)3. With immunofluorescence double-labeled detection tests,Nestin+/BrdU+、MAP-2+/BrdU+ double positive cells could be detected in subventricular zone area, olfactory bulb and hippocampus in both groups. Compared to the control group, the number of Nestin+/BrdU+、MAP-2+/BrdU+ double positive cells in the G-CSF group was significantly increased (p<0.05)Conclusions:1. G-CSF could induce the migration of HSC into brain.2. G-CSF could promote the proliferation of cells in the brain of AD mice significantly.3. G-CSF could induce the differentiation of neural stem cells into neurons in the brain of AD mice.SignificanceThis research indicated that subcutaneous injection of G-CSF could improve the cognition in APP transgenic mouse model of Alzheimer’s disease. The mechanisms were that G-CSF could mobile the proliferation and differentiation of peripheral HSC and induce the migration into the brain; it also could induce the proliferation and the differentiation of neural stem cells into neurons. In those ways, the loss of neurons could be substituted and the lesions could be repaired. It provided new experimental evidence for the treatment of AD.