The Influencing Factors Of Neural Stem Cell Proliferation And Differentiation In The Dentate Gyrus Of Hippocampus

Brain injury including hypoxic-ischemic encephalopathy caused by perinatal asphyxia is one of the major diseases causing human death and disability and becoming a huge burden of the family and society. It had been thought that there was no regeneration in the brain, and therefor the research on brain injury focused on the mechanisms of neuronal cell death and possible methods to inhibit neuronal cell death. However, most of the neuroprotective methods from experimental studies are faield with clinical efficacy except the early hypothermia intervention and erythropoietin treatment, as well as tissue-type plasminogen activator in early ischemic stroke. After the discovery of neuronal regeneration in the adult brain in 1998 by the Swedish scientist Eriksson with the applications of 5-bromo-deoxyuridine (BrdU) labeling dividing cells, neural stem cell has become a hot research topic in both China and abroad because of the feature of proliferation and differentiation, as well as the potential advantages of being used for transplantation in the treatment of brain injury. Previous studies have found that neural stem cells exist at subgranular zone of hippocampal dentate gyrus and subependymal area of ventrical. The stem cells are activated and over proliferated for several weeks under the stimuli of physiological or pathological factors such as exercise or brain injury insult. The newborn cells can differentiate into neurons and migrated to the brain injury regions to replace the injuried neurons, which is closely related with the recovery of brain function. However, the recovery of cognitive function was delayed if the stem cells proliferation were inhibited after brain injury. It indicates the strong impact of neuronal regeneration on functional recovery after brain injury. In order to promote the rehabilitation and reconstruction of brain function after injury, stem cell transplantation has been investigated with brain injury models in both China and abroad. It was reported clinical efficacy of human umbilical stem cell transplantation on hypoxic-ischemic brain injury patients in China, but with much more debate. There are still lots of questions with stem cell transplantation, for example, directed differentiation of stem cells, migration and functional connection and controlling the over proliferation. But if we can stimulate or induce endogenous neural stem cells proliferation and differentiation to replace damaged neurons, we could avoid the potential problems of technical, ethical and other issues on neural stem cell transplantation, which will open a new avenue for the treatment of brain injury. However, endogenous stem cell proliferation and differentiation are influenced by many factors either negative or positive, especially some of the clinical diagnosis and treatment methods. In order to promote neural stem cell proliferation and the rehabilitation of brain function after injury, it is necessary to investigate the effect of clinical treatment methods on the neural stem cells proliferation and differentiation.Methods and ResultsThe progress of modern anesthesiology makes surgery to be possible during neonatal or fetal period. Isoflurane is the most widely used clinical anesthetic and it has been shown safe and neuroprotective effect. However, recent clinical studies showed that repeated exposure to anesthetics during childhood led to increase the incidence of multiple learning disabilities, which suggests that pediatric patients should be more careful with anesthetics. In order to clarify whether isoflurane would affect the brain, the rats or mice with different ages were exposed to isoflurane and the learning and memory function as well as the neural stem cell proliferation were evaluated at different recovery times after isoflurane inhalation. The results showed that object recognition and reversal learning ability were significantly impaired in isoflurane treated 14-day-old rats. The recognition index of P14-45 rats in anesthesia group (26.3%±10.1%) was significantly lower than that of control group (56.4%±9.4%), and even worse with longer recovery after anesthesia, whereas, there was no significant difference in the P60 adult rats between two groups. To confirm the effect of isoflurane on immature brain,14-day-old mice were exposed to isoflurane and the learning and memory function was evaluated by using unbiased IntelliCage. The results showed that the learning function was no difference between two groups, but the memory function was significantly impaired after isoflurane inhalation. No effects were observed on blood pressure, body temperature as well as new cell death in hippocampus with isoflurane inhalation, but the memory deficit was paralleled by a decrease of hippocampal stem cell pool and persistently reduced neurogenesis, subsequently a reduction in the number of dentate gyrus granule cell neurons. The cell proliferation indicated by BrdU and phosphorylated histone H3 labeling in 14-day-old rats reduced significantly at 24h after last isoflurane inhalation, which was more pronounced with the extension of recovery time after isoflurane, while the cell proliferation had no apparent reduction in 60-day-old rats. The phenotypes of BrdU-labeled cells were determined by using BrdU-NeuN-GFAP triple immunofluorescent staining and found that neurogenesis was significantly lower, while the astrocyte was significantly higher in P14-45 rats after isoflurane inhalation. Further investigation with SOX-2 and GFAP double-staining showed that isoflurane anesthesia reduced the number of stem cells in the immature brain, but not in the P60 adult brain.Cranial irradiation is one of the effective method for treatment or prevention of brain tumors, but the long-term side effects of radiation to the children has been attracted much attention. To the best of our knowledge, there are no studies evaluating the long-lasting effects of irradiation to the immature brain on a subsequent ischemic insult to the developing brain. In this study 10-day-old mice were irradiated on the left cerebral hemisphere with a single dose of 8Gy. Hypoxic-ischemic brain injury was induced at 50 days after irradiation and brain injury as well as neural stem cell proliferation, differentiation and inflammatory response were evaluated at 30 days after HI. IR alone caused significant hemispheric tissue loss, or lack of growth (2.8±0.42 mm3, p<0.001). The infarct volume was (5.1±1.6 mm3) in the HI group, but nearly doubled if HI was preceded by IR (9.8±1.2 mm3, p<0.05). The brain tissue loss volume after HI (18.2±5.8 mm3) was synergistically increased if preceded by IR (32.0±3.5 mm3, p<0.05). Pathological scoring revealed that IR aggravated hippocampal, cortical and striatal, but not thalamic, injury. Hippocampal neurogenesis decreased>50%after IR but was unchanged by HI alone. The number of newly formed microglia was three times higher after IR+HI than after HI alone. In summary, IR to the immature brain produced long-lasting changes, including decreased hippocampal neurogenesis, subsequently rendering the adult brain more susceptible to HI, resulting in larger infarcts, increased hemispheric tissue loss and more inflammation than in non-irradiated brains.Buyang Huanwu decoction (BHD) consists of milkvetch root, Chinese angelica, red peony root, earthworm, peach seed, safflower and Szechwan Iovage rhizome and has been used for the treatment or promoting rehabilitation of ischemic brain injury. It has been shown effectively to improve cerebral blood flow and blood rheology, resists excitatory amino acid toxicity, but no study yet to evaluate the effect on cell proliferation and nerogenesis. In this study, the adult rat MCAO model was induced and treated with Nimodipine or different dose of BHD. The dysfunction score, water content of brain tissue as well as the stem cell proliferation and differentiation in dentate gyrus were evaluated. The results showed that BHD could inhibit cerebral tissue water content increased, reduce neurological disability score and the swelling of organelles. Histological examination showed BHD could significantly reduce the cerebral ischemia-reperfusion injury and much more pronounced in the middle dose group. The number of BrdU positive cells or BrdU/NeuN double-labeled positive cells was much higher in the BHD treated group. It indicates that BHD treatemtn reduced ischemic brain injury which was related at least partly with the stimulation of neural stem cell proliferation and differentiate.Acupuncture is now accepted as one of the most common complementary therapeutic techniques to cerebral injury rehabilitation by the World Health Organization. Acupuncture possesses many effects in the central nervous system, such as analgesia, promotion of homeostasis, improvement of brain circulation, and improvement of neuromodulatory function. In this study, rats were treated with electroacupuncture (EA) at postnatal day 14 (P14) at bilateral acupoints (Quchi, Waiguan, Huantiao, Zusanli) for 30 min daily for 7 successive days. The proliferation and differentiation of surviving cells were evaluated at 4 weeks after last stimulation. The proliferated and survival cells, indicated by BrdU labeling, increased significantly in the electroacupuncture group compared with control (P=0.0281), and more than 90% of the cells differentiated into neurons. The stimulatory effect of electroacupuncture on cell proliferation had a long-lasting effect, as indicated by the increased number of phosphor-histone H3-positive cells. Nonacupoint stimulation and ketamine anesthesia had no obvious effect on cell proliferation.Conclusions1. Isoflurane anesthesia of immature brains induced memory and recognition deficits by inhibiting stem cell proliferation and neural differentiation.2. Irradiation to the immature brain would produce long-lasting changes, including decreased hippocampal neurogenesis, subsequently rendering the adult brain more susceptible to hypoxic-ischemic injury.3. The actions of BHD against cerebral ischemia/reperfusion damage was related with increased stem cell proliferation and neuronal differentiation.4. Electroacupuncture stimulation could promote stem cell proliferation and neuronal differentiated in the young rats.