Effects Of Erythropoietin On The Neurogenesis And Cognitive Function Of Vascular Dementia Rats

Background and objective:Vascular dementia (VD) is the damage of cognitive function. Cerebral infarction, low brain blood flow and hemorrhage are the reasons of the damage. Presently, VD is considered to be the second reason of dementia in aged people, next to Alzheimer disease (AD).The incidence rate of VD has been increasing, and been paid more attentions on, but an effective treating method has not been found yet. Therefore, it is very important to carry out the pathogenesis and the effective prevention and cure method of VD rats.Neurogenesis is the process of the neural stem cell proliferation, differentiation into neurons, and promoting neural function recover.More and more studies focused on neural stem cells and made a rapid progress recently.Researches showed that persistent neurogenesis occurs in discrete regions of the adult mammal brain,including the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) and the subventricular zone (SGZ) of the lateral ventricle,and that the human hippocampus retains an ability to generate neurons throughout lifetime.It is now expected that neurogenesis can be evoked by central nervous system disorders.But new neurons are insufficient to repair the damaged neural functions that were lost by central nervous system disorders.Thus, to study the stage of neurogenesis and find the way to stimulating neurogenesis,will make big progresses in the therapy of central neural system diseases such as cerebrovascular disease,dementia, and vegetative state.We have previously found that some neurotrophic factors could promote neural stem cells developing, regenerating, proliferating and differentiating. Erythropoietin (EPO) was first characterized as a hematopoietic growth factor and has been in clinical use of anemia.The research showed that EPO also has endogenous neuroprotection effect.The observed that EPO and its receptor are expressed in rodent and human brain tissue and that EPO had effects on neuronal cells. There was EPO secretion on the lack of blood and oxygen condition in the central nervous system, it is also approved that EPO secretion is the protective mechanism of brain tissue to the lack of blood and oxygen.EPO treatment can improve the cognitive abilities of experimental animals and the renal failure patients with dialysis, and promoting neurogenesis in some conditions.However, there were few researches on the effect of neurogenesis and cognitive abilities with EPO treatment in vascular dementia.Therefore, we have established VD animal model to estimate the effect of neurogenesis and cognitive dysfunction with EPO treatment from ethology and histomorphology. We adopted the computerized shuttle-training case to observe the learning-memory abilities, immunehistochemistry to observe the neurogenesis, western blot to observe the expression of cytokines BDNF and VEGF.MethodsPart I Effect of EPO on cognitive function and cytokines expression in VD rats1. Effect of EPO on cognitive function in VD ratsForty five Sprague-Dawlev (SD) rats were randomly divided into the sham control group, the VD group, and the therapy group. The therapy group was treated with EPO (5000IU/kg) daily for 7 days starting from the VD model was established, and the others were treated with saline. The learning-memory abilities were measured by using computerized shuttle-training case. The changes of learning-memory abilities were administered by active avoidance response (AAR) ratio.We test the AAR ratio at the times before the VD model was established and 1 week, 2 weeks and 4 weeks after the model was established.2. Effect of EPO on BDNF and VEGF expressing in VD ratsFifteen Sprague-Dawlev (SD) rats were randomly divided into the sham control group, the VD group, and the therapy group.Animals received EPO and saline as described in step 1. Western blot were used to observe the changes of the BDNF and VEGF expressing 2 weeks after the model was established.Part II Effect of EPO on neurogenesis in VD rats1. Effect of EPO on the proliferation of neural stem cells in VD ratsForty five Sprague-Dawlev (SD) rats were randomly divided into the sham control group, the VD group, and the therapy group. Animals received EPO as described in part 1.BrdU (50mg/kg, ip) was injected twice daily for 7 days after the model was established. Immunohistochemistry was processed to examine the BrdU positive cells at 1 week, 2 weeks and 4 weeks after the model was established.2. Effect of EPO on the differentiation of neural stem cells in VD ratsForty five Sprague-Dawlev (SD) rats were randomly divided into the sham control group, the VD group, and the therapy group. And animals received BrdU and EPO as described before. Immunofluorescence double labeling was processed to examine the phenotype of the newly generated cells.Results1. Effect of EPO on cognitive function in VD ratsBehavioral tests showed that the AAR ratio in each group had no significantly differences before the model was established. But after model was established, the AAR ratio of the VD group was significantly decreased compare with the sham control group.And the AAR ratio of the therapy group was lower than that of the sham control group, but higher than that of the VD group.2. Effect of EPO on BDNF, VEGF expressing in VD ratsWestern blot showed that the expressing of BDNF, VEGF in the VD group and the sham control group had no significantly differences. But the expressing in the therapy group was higher than that in the VD group and the sham control group.3. Effect of EPO on the proliferation of neural stem cells in VD ratsImmunohistochemical staining showed that 1 week after the model was established, the BrdU labeled cells were distributed in the subgranular zone. The number of BrdU labeled cell in the VD group is significantly higher than that in the sham control group, but lower than that in the therapy group. 4 weeks later, the number of BrdU labeled cells in the sham control group had no significantly difference, but the number of BrdU labeled cells in the other two groups were decreased significantly, especially in the VD group. The number of BrdU labeled cell in the therapy group is significantly higher than that in the other groups.4. Effect of EPO on the differentiation of neural stem cells in VD ratsImmunofluorescence staining showed that 1 week after model was established, the number of BrdU and doublecortin(DCX) double labeling cells in the therapy group is significantly higher than that in the VD group.4 weeks later, the number of BrdU labeled cell was decreased in each group, especially the VD group. And the number of BrdU and neuron-specific-enolase (NSE) double labeling cells in the therapy group is significantly higher than that in the VD group. But the percentage of BrdU-labeled cells colabeled with DCX/NSE is no significant differences.Conclusions:1. Behavioral tests show that the VD rats has a significantly cognitive dysfunction. But EPO treatment could improve the ability of cognitive function in VD rats.2. The BDNF and VEGF expressing in the VD group and the sham control group are no significantly differences. But EPO treatment can promote cytokine BDNF and VEGF secreting in VD rats.3. EPO treatment can enhance the proliferation of endogenous neural stem cells but no obvious influence on the differentiation of endogenous neural stem cells.