Environmental factors in the development and progression of late-onset Alzheimer＇s disease

Late-onset Alzheimer＇s disease （LOAD） is an age-related neurodegenerative disorder characterized by gradual loss of synapses and neurons, but its pathogenesis remains to be clarified. Neurons live in an environment constituted by neurons themselves and glial cells. In this review, we propose that the neuronal degeneration in the AD brain is partially caused by diverse environmental factors. We first discuss various environmental stresses and the corresponding responses at different levels. Then we propose some mechanisms underlying the specific pathological changes, in particular, hypothalamic-pituitary adrenal axis dysfunction at the systemic level; cerebrovascular dysfunction, metal toxicity, glial activation, and Aβ toxicity at the intercellular level; and kinase-phosphatase imbalance and epigenetic modification at the intracellular level. Finally, we discuss the possibility of developing new strategies for the prevention and treatment of LOAD from the perspective of environmental stress. We conclude that environmental factors play a significant role in the development of LOAD through multiple pathological mechanisms.     

高血压脑出血的显微外科治疗进展

高血压脑出血（Hypertensive intrac-rebral hemorrhage,HICH）是具有高发病率、高病死率、高致残率的急性脑血管疾病,占所有脑卒中患者的10%-20%,早期病死率可高达49.4%。随着人口老龄化,其发病率逐年提高;而外科手术的干预,使其病死率有所下降,但致残率居高不下。如何提高手术疗效和患者生存质量,一直是神经外科医师努力的方向。微侵袭血肿清除术因其手术创伤小,恢复快,是目前国内治疗高血压脑出血的重要手段。     

神经内镜联合亚低温治疗高血压基底节区脑出血

目的 探讨神经内镜联合亚低温在治疗高血压基底节区脑出血中的临床应用价值.方法 回顾性分析我院神经内镜治疗高血压基底节区脑出血患者40例的临床资料,并对治疗结果进行分析.结果 神经内镜治疗组22例(甲组),神经内镜联合亚低温治疗组18例(乙组),术后3个月根据GCS评分,甲组恢复良好1例,中残4例,重残6例,植物生存6例,死亡5例;乙组恢复良好4例,中残8例,重残3例,植物生存1例,死亡2例,两组比较差异有统计学意义(P＜0.05).两组颅内压比较第1天两者差异不明显,但第2、3天亚低温组颅内压明显降低.结论 神经内镜是治疗高血压基底节区脑出血较为有效的手术方式,联合亚低温治疗能有效降低颅内压,改善术后神经功能恢复,具有较好的临床应用价值.     

Effects of folic acid on in vitro astrocytic differentiation of neural stem cells from neonatal rats

BACKGROUND： Folic acid is essential for normal functioning of the nervous system. Previous studies have focused on the effects of folic acid on astrocyte proliferation. OBJECTIVE： To explore the effects of folic acid on astrocyte differentiation of neural stem cells （NSCs） and the related mechanisms in vitro. DESIGN, TIME AND SETTING： A randomized, controlled, grouping experiment was performed in Tianjin Medical University between August 2007 and October 2008. MATERIALS： Folic acid and 5-bromo-2-deoxyuridine （BrdU） were obtained from Sigma, MO, USA. Primary antibodies [rabbit anti-rat nestin, β-tubulin-Ⅲ, glial fibrillary acidic protein, and neurogeninl （Ngnl）; mouse anti-rat BrdU and β-actin monoclonal antibodies] were purchased from Santa Cruz Biotechnology, USA. METHODS： At 6 days of NSC proliferation from 24-hour-old neonatal rats, BrdU incorporation assay was performed. Seven days after primary culture, NSCs were induced to differentiate with medium containing 5% fetal bovine serum. Cultured NSCs were assigned to three groups： control, low-dose （liquid media with 8 mg/L folic acid）, and high-dose folic acid （liquid media with 44 mg/L folic acid）. MAIN OUTCOME MEASURES： At day 7 after primary culture, the cells were identified as NSCs by immunocytochemical methods. Double-label immunofluorescence technique for glial fibrillary acidic protein/BrdU detected differentiated cells 7 days after induction. Western blot was used to analyze expression of Ngnl protein in NSCs. RESULTS： In serum-free suspension medium, neurospheres comprised a large number of Nestin-, glial fibrillary acidic protein-, β-tubulin-Ⅲ-, and BrdU-positive cells. Compared with the control group, high-dose folic acid supplementation led to an marked increase in the percentage of glial fibrillary acidic protein/BrdU-positive cells （P 〈 0.05）, and significantly decreased Ngnl protein expression （P 〈 0.05）. CONCLUSION： Folic acid promotes astrocytic differentiation of NSCs, which might be related to downregulation of Ngnl protein expression.     

Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson’s disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.     

Dysfunctional autophagy in Alzheimer＇s disease： pathogenic roles and therapeutic implications

Neuronal autophagy is essential for neuronal survival and the maintenance of neuronal homeostasis. Increasing evidence has implicated autophagic dysfunction in the pathogenesis of Alzheimer＇s disease （AD）. The mechanisms underlying autophagic failure in AD involve several steps, from autophagosome formation to degradation. The effect of modulating autophagy is context-dependent. Stimulation of autophagy is not always beneficial. During the implementation of therapies that modulate autophagy, the nature of the autophagic defect, the timing of intervention, and the optimal level and duration of modulation should be fully considered.     

Oxidative stress in Alzheimer＇s disease

Oxidative stress plays a significant role in the pathogenesis of Alzheimer＇s disease （AD）, a devastating disease of the elderly. The brain is more vulnerable than other organs to oxidative stress, and most of the components of neurons （lipids, proteins, and nucleic acids） can be oxidized in AD due to mitochondrial dysfunction, increased metal levels, inflammation, and β-amyloid （Aβ） peptides. Oxidative stress participates in the development of AD by promoting Aβ deposition, tau hyperphosphorylation, and the subsequent loss of synapses and neurons. The relationship between oxidative stress and AD suggests that oxidative stress is an essential part of the pathological process, and antioxidants may be useful for AD treatment.     

蝙蝠葛酚性碱与褪黑素对缺血再灌注损伤神经元的作用比较

BACKGROUND： Recent studies have demonstrated that phenolic alkaloids from Menispermum dauricum （PAMD） can protect the heart and brain from ischemia/reperfusion injury, and promote neuron survival by inhibiting neuronal Bax and upregulating Bcl-2 expression following ischemia/reperfusion.
OBJECTIVE： To investigate the neuroprotective effects of PAMD versus exogenous melatonin against ischemia/reperfusion injury.
DESIGN, TIME AND SETTING： Observation and comparison experiments at a cellular level were performed at the Department of Biochemistry and Molecular Biology, Tongji Medical College of Huazhong University of Science and Technology between February 2007 and February 2008.
MATERIALS： PAMD （95% purity） was provided by Kunming Institute of Botany, Chinese Academy of Sciences; melatonin was provided by Sigma, USA.
METHODS： N2a mouse neuroblastoma cells were cultured in vitro deprived of glucose, serum and oxygen for 90 minutes, then cultured in normal medium containing different concentrations of PAMD （0.1, 1.0, 10 mg/L） or melatonin （1, 10, and 100 μmol/L）. Cells cultured in normal conditions served as a control.
MAIN OUTCOME MEASURES： The culture solution was collected to determine the content of ex- citatory neurotransmitters such as glutamic acid and aspartic acid; cell viability was detected by MTT methods; reactive oxygen species production was determined by fluorescence spectroscopy; mito- chondrial transmembrane potential （？Ψm） was detected by laser confocal scanning; cytochrome C was measured by western blotting; and caspase-3 activity was determined by visible spectropho- tometry.
RESULTS： Melatonin and PAMD both promoted oxygen-glucose-serum deprivation-mediated N2a cell survival （P 〈 0.01） and inhibited glutamic acid release （P 〈 0.01）, but melatonin did not inhibit aspartic acid production. The protective effects were the strongest using melatonin 100 μmol/L and PAMD 10 mg/L, so subsequent experiments were the performed at those doses. Although PAMD could no longer maintain mitochondrial transmembrane potential 6 hours after reperfusion, its in- hibitory effects on cytochrome C release from mitochondria and scavengers of reactive oxygen species were stronger than those of melatonin （P 〈 0.01）. However, its inhibitory effect on caspase-3 activity was weaker than that of melatonin： PAMD could inhibit caspase-3 activity 12 hours after reperfusion （P 〈 0.01）, but melatonin inhibited caspase-3 activity 28 hours after reperfusion （P 〈 0.01）.
CONCLUSION： The results show that melatonin and PAMD have neuroprotective effects, but that the mechanisms are varied. Melatonin can maintain mitochondrial transmembrane potential, but its inhibitory effects on cytochrome C release, caspase-3 activity, and reactive oxygen species scav-enging are different from those of PAMD.     

Disrupted structural and functional brain connectomes in mild cognitive impairment and Alzheimer＇s disease

Alzheimer＇s disease （AD） is the most common type of dementia, comprising an estimated 60-80% of all dementia cases. It is clinically characterized by impairments of memory and other cognitive functions. Previous studies have demonstrated that these impairments are associated with abnormal structural and functional connections among brain regions, leading to a disconnection concept of AD. With the advent of a combination of non-invasive neuroimaging （structural magnetic resonance imaging （MRI）, diffusion MRI, and functional MRI） and neurophysiological techniques （electroencephalography and magnetoencephaJography） with graph theoretical analysis, recent studies have shown that patients with AD and mild cognitive impairment （MCI）, the prodromal stage of AD, exhibit disrupted topological organization in large-scale brain networks （i.e., connectomics） and that this disruption is significantly correlated with the decline of cognitive functions. In this review, we summarize the recent progress of brain connectomics in AD and MCI, focusing on the changes in the topological organization of large-scale structural and functional brain networks using graph theoretical approaches. Based on the two different perspectives of information segregation and integration, the literature reviewed here suggests that AD and MCI are associated with disrupted segregation and integration in brain networks. Thus, these connectomics studies open up a new window for understanding the pathophysiological mechanisms of AD and demonstrate the potential to uncover imaging biomarkers for clinical diagnosis and treatment evaluation for this disease.     

Effects of p75 neurotrophin receptor knockout on axonal regeneration in a mouse model of facial nerve injury

BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.     

墨蝶呤还原酶基因与精神分裂症相关性的研究进展

墨蝶呤还原酶（SPR）催化四氢生物蝶呤（BH4）从头合成途径的最后一步反应。SPR基因遗传缺陷或突变可导致BH。的合成紊乱,影响单胺类神经递质（如多巴胺、5-羟色胺及谷氨酸等）的合成或释放,进而参与包括精神分裂症在内的多种神经精神系统疾病的发生发展过程。此外,SPR基因敲除小鼠表现出持续增强的自主活动等类精神分裂症症状,说明该基因在精神分裂症的发病中扮演重要的角色。进一步研究SPR基因及其单核苷酸多态性的功能,可为阐明精神分裂症的发病机制提供重要的线索,也为新一代抗精神病药物的研制及开发开拓新的视野。现对SPR基因与精神分裂症的相关研究做一综述。     

Edema and neuronal apoptosis in the hippocampus and cortex of elderly rats following transient cerebral ischemia/reperfusion injury

BACKGROUND： Previous studies of cerebral ischemia have used young animals, with an ischemic time greater than 5 minutes （safe time limit）. Despite an increased understanding of neuronal apoptosis, it remains uncertain whether brief cerebral ischemic events of 5 minutes or less damage brain tissue in elderly rodents. OBJECTIVE： To investigate the effects of transient cerebral ischemia （5 minutes）/reperfusion injury on brain cortical and hippocampal edema, aquaporin-4 （AQP-4） expression, and neuronal apoptosis in aged rats, and to compare ischemic sensitivity between cortex and hippocampus. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Institute of Cerebrovascular Disease, Qingdao University Medical School from April 2008 to March 2009. MATERIALS： Rabbit anti-AQP-4 polyclonal antibody, TUNEL kit, and SABC immunohistochemistry kit were purchased from Wuhan Boster Bioengineering, China. METHODS： A total of 160 healthy, male, aged 19-21 months, Wistar rats were randomly assigned to 4 groups： sham-surgery, and ischemia 1-, 3-, and 5-minute groups, with 40 rats in each group. The global cerebral ischemia model was established using the Pusinelli four-vessel occlusion, and the three cerebral ischemia groups were subdivided into reperfusion 12-hour, 1-, 2-, 3-, and 7-day subgroups, with 8 rats in each subgroup. The sham-surgery group was subjected to exposure of the first cervical bilateral alar foramina and bilateral common carotid arteries. MAIN OUTCOME MEASURES： The dry-wet weight assay was used to measure brain water content and histopathology of the cortex and hippocampus was observed following hematoxylin-eosin staining. In addition, cortical and hippocampal AQP-4 expression was detected by streptavidin-biotin complex immunohistochemistry, and neuronal apoptosis was detected by the TUNEL method. RESULTS： There was no significant difference in brain water content or AQP-4 expression in the cortex and hippocampus between ischemia 1- and 3-minute groups and the sham-surgery group or brain water content or AQP-4 expression in the cortex between ischemia 5-minute group and sham-surgery group （P 〉 0.05）. However, brain water content and AQP-4 expression in the hippocampus after 5 minutes of cerebral ischemia were significantly increased compared with the sham-surgery group （P 〈 0.05 or P 〈 0.01）. Several TUNEL-positive cells were observed in the cortex and hippocampus of the sham-surgery group and ischemia 1-minute group, as well as in the cortex of the ischemia 3-minute group. In addition, the number of apoptotic neurons in the hippocampus of ischemia 3-minute group and in the cortex and hippocampus of ischemia 5-minute group was significantly increased （P 〈 0.05 or P 〈 0.01 ）. Neuronal apoptosis was increased after 12 hours of ischemia/reperfusion, and it reached a peak by 2 days （P 〈 0.01）. CONCLUSION： Transient cerebral ischemia （5 minutes） resulted in increased hippocampal edema, AQP-4 expression, and neuronal apoptosis. Moreover, cerebral ischemia had a greater effect on neuronal apoptosis than brain edema or AQP-4 expression, and the hippocampus was more sensitive than the cortex.     

Targeting 13-secretase with RNAi in neural stem cells for Alzheimer＇s disease therapy

There are several major pathological changes in Alzheimer＇s disease, including apoptosis of cho- linergic neurons, overactivity or overexpression of 13-site amyloid precursor protein cleaving enzyme 1 （BACE1） and inflammation. In this study, we synthesized a 19-nt oligonucleotide targeting BACE1, the key enzyme in amyloid beta protein （AI3） production, and introduced it into the pSilenCircle vector to construct a short hairpin （shRNA） expression plasmid against the BACE1 gene. We transfected this vector into C17.2 neural stem cells and primary neural stem cells, resulting in downregulation of the BACE1 gene, which in turn induced a considerable reduction in reducing AI3 protein production. We anticipate that this technique combining cell transplantation and gene ther- apy will open up novel therapeutic avenues for Alzheimer＇s disease, particularly because it can be used to simultaneously target several pathogenetic changes in the disease.     

在SK-N-SH细胞中抑制干预β-淀粉样前体蛋白裂解酶基因和淀粉样β前体蛋白基因

BACKGROUND： Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein （APP） at the mRNA level. In addition, the piperlonguminine （A） and dihydropiperlonguminine （B） components （1 ： 0.8）, which can be separated from Futokadsura stem, selectively inhibit expression of the APP at mRNA and protein levels. OBJECTIVE： Based on previous findings, the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme （BACE1） and APP genes on the production of β-amyloid peptide 42 （Aβ42） in human neuroblastoma cells （SK-N-SH cells） using small interfering RNAs （siRNAs） and A/B components separated from Futokadsura stem, respectively. DESIGN, TIME AND SETTING： A gene interference-based randomized, controlled, in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministries of Education and Public Health, and Institute of Pharmacologic Research, School of Pharmaceutical Science ＆ Department of Biochemistry, School of Medicine, Shandong University between July 2006 and December 2007. MATERIALS： SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; mouse anti-human BACE1 monoclonal antibody was purchased from R＆D Systems, USA; mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology, USA; and horseradish peroxidase （HRP）-conjugated goat anti-mouse IgG was provided by Sigma, USA. METHODS： The human BACE1 cDNA sequence was obtained from NCBI website （www.ncbi.nlm.nih.gov/sites/entrez）. Three pairs of siRNAs, specific to human BACE1 gene, were synthesized through the use of Silencer pre-designed siRNA specification, and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs （10-50 nmol/L） on SK-N-SH cells. Futokadsura stem was separated and purified with chemical methods, and the crystal was composed of A/B components, with an A to B ratio of 1：0.8. The A/B （1 ： 0.8） components were added to the SK-N-SH cells at different concentrations （13.13, 6.56, and 3.28 mg/mL）. MAIN OUTCOME MEASURES： Using RT-PCR and Western blot methods, BACE1 and APP expression at mRNA and protein levels was detected in SK-N-SH cells following treatment with different siRNAs and concentrations of Futokadsura stem-separated A/B components, respectively. Altered Aβ42 secretion by SK-N-SH cells was determined by ELISA. RESULTS： BACE1 mRNA and protein levels were significantly suppressed by 40 and 50 nmol/L siRNAs at 48 hours post-transfection. A/B components （1 ： 0.8）, which were separated from Futokadsura stem, selectively inhibited mRNA and protein expression of APP in SK-N-SH cells. Aβ42 secretion by SK-N-SH cells was significantly decreased following treatment with siRNAs or A/B components. CONCLUSION： Inhibition of BACE1 and APP genes by various materials and methods efficiently decreased production of Aβ42.     

Wnt信号通路与骨髓间充质干细胞增殖及分化的关系

骨髓间充质干细胞（bonemarrow—derived mesenchymal stem cells,BMSCs）是骨髓中不同于造血干细胞的一类细胞,其来源丰富,取材简便,易分离、纯化、培养,在一定的条件下可以迅速体外扩增,具有多向分化潜能,可以通过不同的方法被诱导分化成骨细胞、软骨细胞、肌细胞、神经胶质细胞、神经元细胞等,而且它具有低免疫源性,向病变部位迁移的能力,     

癫痫与自杀

自杀而导致死亡被为是增加癫痫患者死亡率的最重要原因之一。国外许多研究报道都表明癫痫患者的自杀率比普通人群的自杀率高几倍到二十几倍。可能导致癫痫患者自杀的危险性因素是有多方面的,本文将从5-HT、抗癫痫药及癫痫手术治疗、精神病理等方面对癫痫患者可能存在自杀危险因素进行综述,并希望在癫痫的综合治疗中对这些危险因素能加以考虑。     

Correlation of hypoxia-inducible factor-1 alpha and erythropoietin protein and mRNA to cerebral ischemic tolerance in a focal ischemia/reperfusion model using the twice suture method

BACKGROUND： Numerous studies have shown that transient ischemic preconditioning induces cerebral ischemic tolerance. However, the underlying mechanisms of endogenous protection following ischemic preconditioning remain unclear. OBJECTIVE： To dynamically measure erythropoietin and hypoxia-inducible factor-1α （HIF-1α） mRNA and protein expression at various times following preconditioning, and to investigate effects of erythropoietin and HIF-1α on cerebral ischemic tolerance in a model of focal ischemia/reperfusion established using the twice suture method. DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Institute of Anatomy, Medical College, Qingdao University, China from March 2006 to March 2007. MATERIALS： Rabbit anti-rat HIF-1α monoclonal antibody and biotinylated goat anti-rabbit IgG （Boster, China）, rabbit anti-rat erythropoietin monoclonal antibody （Santa Cruz Biotechnology, USA）, and one-step RT-PCR kit （Qiagen, Germany） were used in this study. METHODS： A total of 99 healthy, male, Wistar rats were randomly assigned to three groups： sham surgery （n = 9）, non-ischemic preconditioning （n = 45）, and ischemic preconditioning （n = 45）. In the ischemic preconditioning group, rat models of pre-ischemia-reperfusion-ischemia-reperfusion were established by occluding the left middle cerebral artery using the twice suture method. In the non-ischemic preconditioning group, pre-ischemia was replaced by sham surgery. Subsequently, the ischemic preconditioning and non-ischemic preconditioning groups were equally divided into five subgroups according to time of first reperfusion, including 1-, 3-, 7-, 14-, and 21-day subgroups. The sham surgery group received the sham surgery twice. MAIN OUTCOME MEASURES： HIF-la and erythropoietin protein expression was measured in the cerebral cortex, corpus striatum, and hippocampus of the ischemic hemisphere. HIF-1α and erythropoietin mRNA expression were determined in the frontal and parietal cortex of the ischemic hemisphere. RESULTS： （1） Intergroup comparison： compared with the non-ischemic preconditioning group, HIF-1α protein expression significantly increased in the rat cerebral cortex, corpus striatum, and hippocampus in the ischemic hemisphere at 1,3, and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05 or P 〈 0.01）. Erythropoietin protein expression significantly increased in the cerebral cortex, corpus striatum, and hippocampus, as well as HIF-1α and erythropoietin mRNA expression in the frontal and parietal cortex in the ischemic hemisphere, at 3 and 7 days following reperfusion in the ischemic preconditioning group （P 〈 0.05）. （2） Temporal expression： HIF-1α protein expression in the rat cerebral cortex, corpus striatum, and hippocampus, as well as HIF-la mRNA expression in the frontal and parietal cortex, in the ischemic hemisphere increased at 3 days, and gradually decreased from 7 days following reperfusion in the ischemic preconditioning group. Temporal erythropoietin protein and mRNA expression was consistent with HIF-1α protein expression. （3） Correlation： erythropoietin mRNA expression positively correlated with HIF-1α mRNA expression （r= 0.737, P 〈 0.01）. CONCLUSION： Ischemic preconditioning induced cerebral ischemic tolerance. Pre-ischemiainduced increase in endogenous HIF-1αexpression, as well as its target gene erythropoietin, participated in the formation of cerebral ischemic tolerance.