Upregulation of Mineralocorticoid Receptor in the Hypothalamus Associated with a High Anxiety-like Level in Apolipoprotein E4 Transgenic Mice.

Anxiety symptoms occur in a large portion of Alzheimer's disease (AD) patients. ApolipoproteinE-4 (ApoE ε4 allele), a risk factor for AD, has been recognized as an important contributor to psychiatric disorders. In the present study, we aimed to investigate the corticosterone level in relation to anxiety-like behavior changes in transgenic male mice with different glial fibrillary acidic protein (GFAP)-ApoE isoforms. GFAP-ApoE4 transgenic mice aged 3 months showed higher anxiety-like behavior in open field, light-dark box and elevated plus maze tasks compared with that of age-matched GFAP-ApoE3 mice. However, corticotropin releasing factor levels in the hypothalamus and plasma corticosterone secretion were similar in GFAP-ApoE3 and GFAP-ApoE4 transgenic male mice. Additionally, increased expression of the mineralocorticoid receptor (MR) and unchanged expression of the glucocorticoid receptor were observed in the hypothalamus of GFAP-ApoE4 mice. However, no significant differences were found in the expression levels of the MR in GFAP-ApoE3 and GFAP-ApoE4 mice at postnatal day 2. In conclusion, we found that MR upregulation rather than corticosterone level changes in the early stage of adulthood was associated with the higher anxiety-like level measured in GFAP-ApoE4 mice.

Chronic retinoic acid treatment suppresses adult hippocampal neurogenesis, in close correlation with depressive-like behavior.

Clinical studies have highlighted an association between retinoid treatment and depressive symptoms. As we had shown before that chronic application of all-trans retinoic acid (RA) potently activated the hypothalamus-pituitary-adrenal (HPA) stress axis, we here questioned whether RA also induced changes in adult hippocampal neurogenesis, a form of structural plasticity sensitive to stress and implicated in aspects of depression and hippocampal function. RA was applied intracerebroventricularly (i.c.v.) to adult rats for 19 days after which animals were subjected to tests for depressive-like behavior (sucrose preference) and spatial learning and memory (water maze) performance. On day 27, adult hippocampal neurogenesis and astrogliosis was quantified using BrdU (newborn cell survival), PCNA (proliferation), doublecortin (DCX; neuronal differentiation), and GFAP (astrocytes) as markers. RA was found to increase retinoic acid receptor-α (RAR-α) protein expression in the hippocampus, suggesting an activation of RA-induced signaling mechanisms. RA further potently suppressed cell proliferation, newborn cell survival as well as neurogenesis, but not astrogliosis. These structural plasticity changes were significantly correlated with scores for anhedonia, a core symptom of depression, but not with water maze performance. Our results suggest that RA-induced impairments in hippocampal neurogenesis correlate with depression-like symptoms but not with spatial learning and memory in this design. Thus, manipulations aimed to enhance neurogenesis may help ameliorate emotional aspects of RA-associated mood disorders. © 2016 Wiley Periodicals, Inc.

The influence of chronic stress on anxiety-like behavior and cognitive function in different human GFAP-ApoE transgenic adult male mice.

The apolipoprotein E (ApoE) ɛ4 allele (ApoE4) is an important genetic risk factor for the pathogenesis of Alzheimer's disease (AD). In addition to genetic factors, environmental factors such as stress may play a critical role in AD pathogenesis. This study was designed to investigate the anxiety-like behavioral and cognitive changes in different human glial fibrillary acidic protein (GFAP)-ApoE transgenic adult male mice under chronic stress conditions. On the open field test, anxiety-like behavior was increased in the non-stressed GFAP-ApoE4 transgenic mice relative to the corresponding GFAP-ApoE3 (ApoE ɛ3 allele) mice. Anxiety-like behavior was increased in the stressed GFAP-ApoE3 mice relative to non-stressed GFAP-ApoE3 mice, but was unexpectedly decreased in the stressed GFAP-ApoE4 mice relative to non-stressed GFAP-ApoE4 mice. On the novel object recognition task, both GFAP-ApoE4 and GFAP-ApoE3 mice exhibited long-term non-spatial memory impairment after chronic stress. Interestingly, short-term non-spatial memory impairment (based on the novel object recognition task) was observed only in the stressed GFAP-ApoE4 male mice relative to non-stressed GFAP-ApoE4 transgenic mice. In addition, short-term spatial memory impairment was observed in the stressed GFAP-ApoE3 transgenic male mice relative to non-stressed GFAP-ApoE3 transgenic male mice; however, short-term spatial memory performance of GFAP-ApoE4 transgenic male mice was not reduced compared to non-stressed control mice based on the Y-maze task. In conclusion, our findings suggested that chronic stress affects anxiety-like behavior and spatial and non-spatial memory in GFAP-ApoE transgenic mice in an ApoE isoform-dependent manner.

Cervical canal stenosis and adjacent segment degeneration after anterior cervical arthrodesis.

PURPOSE: Adjacent segment degeneration (ASD) is known to occur after anterior cervical arthrodesis. However, it is not known whether cervical canal stenosis enhances the risk of ASD. The purpose of this study was to explore whether congenital stenosis could be used as a predictor of ASD after anterior cervical decompression and fusion (ACDF). METHODS: We enrolled 141 patients who had undergone ACDF for cervical myelopathy and/or radiculopathy, and had at least 6 years of follow-up. In standard radiographs of cervical spine in lateral view, bony congenital stenosis was evaluated and all patients were divided into two groups: stenosis (n = 63) and non-stenosis (n = 78). Radiographic ASD was assessed according to the criteria of Kellgren and Lawrence and correlated with symptomatic ASD. Clinical and radiological parameters were compared between the groups. The primary outcome was the rate of radiographic ASD after initial ACDF. The incidence of symptomatic ASD was assessed by Kaplan-Meier method. RESULTS: Radiographic ASD and symptomatic ASD developed in 46.8 % and 18.4 % of all patients, respectively. There was a significant association between congenital stenosis and radiographic ASD. The area under the receiver operating characteristic curve of preoperative anteroposterior (AP) diameter of cervical canal for predicting radiographic ASD was 0.756. 13.0 mm was the cutoff value of preoperative AP diameter of cervical canal predicting radiographic ASD. Kaplan-Meier analysis predicted a disease-free survival rate of symptomatic ASD in 97.2 % of patients at 5 years and 78.0 % at 10 years after ACDF. There was no significant difference in survival rates of the adjacent segment between the two groups via log-rank analysis (P = 0.102). CONCLUSION: Congenital stenosis can increase the rate of radiographic ASD after initial ACDF. The cutoff value of 13.0 mm for preoperative AP diameter of cervical canal had the highest validity for predicting radiographic ASD.

Predictors of surgical outcome in cervical spondylotic myelopathy: focusing on the quantitative signal intensity.

PURPOSE: The association between intramedullary increased signal intensity (ISI) on T2-weighted magnetic resonance imaging and surgical outcome in cervical spondylotic myelopathy (CSM) remains controversial. The purpose of this study is to assess the impact of quantitative signal change ratio (SCR) on the surgical outcome for CSM. METHODS: The prospective study included 108 consecutive patients who underwent surgical treatment for CSM. The Japanese Orthopaedic Association (JOA) score and recovery rate were used to evaluate clinical outcomes. JOA recovery rate less than 50% was defined as a poor clinical result. The SCR was defined as the signal intensity at the level of ISI or severely compressed cord (in cases with no ISI) divided by the signal intensity at the C7-T1 disc level. Age, sex, body mass index, duration of symptoms, surgical technique, preoperative JOA score, levels of compression, preoperative SCR, preoperative C2-7 angle, preoperative C2-7 range of motion were assessed. RESULTS: Forty patients (37.0%) had a recovery rate of less than 50%. Multivariate logistic regression analysis revealed that a higher preoperative SCR and a longer duration of symptoms were significant risk factors for a poor clinical outcome. Receiver operating characteristic (ROC) curve analysis showed that the optimal preoperative SCR cutoff value as a predictor of poor clinical result was 1.46. The area under the ROC curve of preoperative SCR for predicting a poor surgical outcome was 0.844. CONCLUSIONS: Preoperative SCR significantly reflected the surgical outcome in patients with CSM. Patients with SCR greater than or equal to 1.46 can experience poor recovery after surgery.

Beneficial effects of enriched environment on behaviors were correlated with decreased estrogen and increased BDNF in the hippocampus of male mice.

OBJECTIVE: Previous studies reported that environmental enrichment might induce various beneficial effects in the central nervous system. However, the effect of environmental factors on endogenous estrogen level was not investigated. The present study was designed to examine the effect of enriched environment on endogenous estrogen in hippocampus and behavioral outcomes. METHODS: Behavioural measurements, including open field, elevated plus maze and Morris water maze, were used to evaluate anxiety and learning and memory of the male C57BL/6J mice that were housed in enriched environment for five months. In addition, the estrogen and brain-derived neurotrophic factor (BDNF) expression in the hippocampus were measured. RESULTS: We found that environmental enrichment decreased anxiety-like behaviors and facilitated spatial learning and memory in male C57BL/6J mice. In addition, the mice raised in enriched environment showed decreased endogenous estrogen levels both in the hippocampus and plasma compared to controls. Furthermore, our results indicated that environmental enrichment up-regulated BDNF mRNA expression level in the hippocampus. CONCLUSION: In conclusion, environmental enrichment decreased anxiety-like behaviors and facilitated spatial learning and memory in male C57BL/6J mice. Lastly, environmental enrichment up-regulated BDNF mRNA expression level in the hippocampus and decreased plasma estrogen level. The possible mechanism remained to be determined.

Serotoninergic and melatoninergic systems are expressed in mouse embryonic fibroblasts NIH3T3 cells.

OBJECTIVE: Melatonin not only plays an important role in regulating circadian rhythms, but is also involved in antioxidative defense and immunomodulation. Circulating melatonin levels are derived primarily from the pineal gland while other sources of melatonin have also been reported. Recently, we reported that cultured rat cortical astrocytes and glioma C6 cells synthesize melatonin. In addition, apolipoprotein E genotype influences melatonin biosynthesis by regulating NAT and MAOA expression in C6 cells. METHODS: Here, we investigated the expression of genes and enzymes that is responsible for the multistep conversion of tryptophan to serotonin and further to melatonin in mouse embryonic fibroblasts NIH3T3 cells by radioimmunoassay, Immunofluorescence staining, real-time PCR and Western blotting techniques. RESULTS: Our results showed that cultured NIH3T3 cells could synthesize melatonin and serotonin. Serotonin N-acetyltransferase (NAT), the key enzyme in the pathway of melatonin synthesis, was also detectable using both by western blot and PCR methods. In addition, two other key enzymes, tryptophan hydroxylase (TPH1 and TPH2) for serotonin synthesis and the metabolic enzyme monoamine oxidase A (MAOA) for 5-HT, were present in NIH3T3 cell line. CONCLUSIONS: In conclusion, we provided evidence that the NIH3T3 cells can synthesize intrinsic serotonin and melatonin and express key enzymes related biosynthetic pathways.

Increased hippocampal tau phosphorylation and axonal mitochondrial transport in a mouse model of chronic stress.

Corticotropin-releasing hormone (CRH) is considered the driving force of the hypothalamo-pituitary-adrenal (HPA) axis and plays an important role in mood regulation. The HPA axis is reported to be closely related to acute stress-induced tau phosphorylation in the rodent hippocampus. However, the relationship between the hyperactive HPA axis and tau phosphorylation in the hippocampus and hence the functional implications for chronic stress are not fully understood. In this study, we aimed to examine tau phosphorylation and the effect on axonal transport of mitochondria in the hippocampus of a chronic stress model. A mouse model was created by neonatal isolation before weaning, followed by chronic mild stress by social isolation after weaning. Behavioural tests showed that the model had a typical depression/anxiety-like behaviour accompanied by increased plasma corticosterone level and hypothalamic CRH mRNA expression. Phosphorylated tau increased significantly, accompanied by increased synaptosomal mitochondrial levels in hippocampus of the chronic stress model. CRH receptor 1 antagonist (CP154,526) treatment, not glucocorticoid receptor antagonist (RU486) treatment, decreased tau phosphorylation and synaptosomal mitochondrial levels in the hippocampus of the mouse model. Consistent with an in-vivo model, when hyperphosphorylated tau was inhibited by lithium in cultured primary hippocampal neurons, mitochondrial transport monitored by live imaging was also decreased. We show here for the first time that phosphorylated tau in the hippocampus of a chronic stress model, accompanied by increased mitochondrial transport, was mediated by CRH receptor 1, not by glucocorticoid receptors, which suggests that centrally derived CRH may be involved in the process of mitochondrial axon transport and hence play an important role in hippocampus of a chronic stress model.

Apolipoprotein E influences melatonin biosynthesis by regulating NAT and MAOA expression in C6 cells.

  Previous studies have demonstrated that apolipoprotein E (ApoE) genotype and melatonin are closely associated with Alzheimer's disease (AD). However, the relationship between ApoE genotype and melatonin remains unclear. Recently, we reported that cultured rat cortical astrocytes and glioma C6 cells synthesize melatonin. In the current study, we investigated the effect of ApoE genotype on melatonin biosynthesis. C6 cells with stable expression of ApoE isoforms (ApoE 2, 3 and 4) were established. A higher level of melatonin was demonstrated in cultured ApoE4-C6 cells than that in ApoE3-C6 cells. In addition, we found that N-acetyltransferase (NAT) protein level was up-regulated in ApoE4-C6 cells compared with ApoE3-C6 cells. Further study suggested that mRNA expression of monoamine oxidase A (MAOA) and monoamine oxidase B (MAOB) decreased in ApoE4-C6 cells. In conclusion, the increased melatonin level in ApoE4-C6 cells results from up-regulation of NAT expression, a key enzyme for melatonin synthesis, and down-regulation of MAOA and MAOB expression, the metabolic enzyme for its precursor serotonin.

Azoarene activation for Schmidt-type reaction and mechanistic insights.

The Schmidt rearrangement, a reaction that enables C-C or C-H σ bond cleavage and nitrogen insertion across an aldehyde or ketone substrate, is one of the most important and widely used synthetic tools for the installation of amides and nitriles. However, such a reaction frequently requires volatile, potentially explosive, and highly toxic azide reagents as the nitrogen donor, thus limiting its application to some extent. Here, we show a Schmidt-type reaction where aryldiazonium salts act as the nitrogen precursor and in-situ-generated cyclopenta-1,4-dien-1-yl acetates serve as pronucleophiles from gold-catalyzed Nazarov cyclization of 1,3-enyne acetates. Noteworthy is that cycloketone-derived 1,3-enyne acetates enabled ring-expansion relay to access a series of 2-pyridone-containing fused heterocycles, in which nonsymmetric cycloketone-derived counterparts demonstrated high regioselectivity. Aside from investigating the scope of this Schmidt-type reaction, mechanistic details of this transformation are provided by performing systematic theoretical calculations.

SUMO negatively regulates BACE expression.

OBJECTIVE: The effect of SUMO on the promoter activity and mRNA expression of BACE gene was investigated to find the connection between sumoylation and APP processing. METHODS: The BACE promoter activity was measured by reporter gene analysis and BACE mRNA level was investigated using real-time RT-PCR method. RESULTS: BACE gene promoter activity was inhibited by the over-expression of SUMO proteins and was blocked by disrupting the SP1 site. Endogenous BACE mRNA level was also negatively regulated by the induction of SUMO proteins. Using a specific inhitor of SP1, BACE promoter activity was coordinately inhibited. CONCLUSIONS: SUMO negatively regulates the BACE expression and SP1 is involved in the process.

Preparation of uniform-sized PLA microcapsules by combining Shirasu porous glass membrane emulsification technique and multiple emulsion-solvent evaporation method.

Relatively Uniform-sized biodegradable poly(lactide) (PLA) microcapsules were successfully prepared by combining a Shirasu Porous Glass (SPG) membrane emulsification technique and multiple emulsion-solvent evaporation method. An aqueous phase containing lysozyme was used as the internal water phase (w1), and PLA and Arlacel 83 were dissolved in a mixture solvent of dichloromethane (DCM) and toluene which was used as the oil phase (o). These two solutions were emulsified by a homogenizer to form a w1/o primary emulsion. The primary emulsion was permeated through the uniform pores (5.25 microm) of an SPG membrane into the external water phase by the pressure of nitrogen gas to form the uniform w1/o/w2 droplets. Then, the solid polymer microcapsules were obtained by simply evaporating the solvent. It is necessary to avoid the phase separation of primary emulsion during the SPG membrane emulsification. It was found that when the density difference of the internal water phase and oil phase was reduced to nearly zero and Arlacel 83 was used as the oil emulsifier, the phase separation was not observed within 24 h. The w1/o/w2 emulsion with uniform diameter was obtained only when Arlaecl 83 concentration was limited below 2.5 wt.% based on oil phase. The drug encapsulation efficiency was found to be related to several factors including PLA molecular weight, additive type and its concentration in the internal water phase, the emulsifier type and concentration in the oil phase, the NaCl concentration and the pH value in the external water phase. Comparing with the stirring method, it was found that the size was more uniform and the drug encapsulation efficiency was much higher when the microcapsules were prepared by SPG membrane emulsification technique and the highest drug encapsulation efficiency of 92.20% was obtained. This is the first study to prepare PLA microcapsules by combining an SPG membrane emulsification technique and multiple emulsion-solvent evaporation method.

W/O/W double emulsion technique using ethyl acetate as organic solvent: effects of its diffusion rate on the characteristics of microparticles.

Monomethoxypoly(ethylene glycol)-b-poly(DL-lactide) copolymer (PELA) microparticles loading lysozyme were prepared through a modified W/O/W double emulsion-solvent diffusion method using ethyl acetate (EA) as organic solvent. The modified process was divided into five steps: (1) primary emulsification (W1/O), (2) re-emulsification (W1/O/W2), (3) pre-solidification, (4) solidification and (5) purification. The pre-solidification step was carried out in the modified process to control the diffusion rate of EA from oil phase into outer aqueous phase, in order to prevent the wall polymer from precipitation, which usually occurred when the diffusion rate was too fast. The adequately rapid solidification of microparticle caused by controlled fast diffusion of EA and the use of amphiphilic copolymer PELA as wall material, facilitated a high protein entrapment (always above 94%) and full preservation of bioactivity of entrapped lysozyme. It was found that the volume of the outer aqueous phase in the re-emulsification step and the shear stress in the pre-solidification step had a significant effect on the diffusion rate of EA from the droplets into outer aqueous solution, and thereby on the characteristics of the resultant microparticles. With the volume or the shear stress increasing, the removal rate of EA increased, resulting in rapid solidification of the microparticles. This result led to a lower burst effect and a slower lysozyme release from the microparticles. This study suggests that the modified W/O/W double emulsion-solvent diffusion method with EA as organic solvent is a prospective technique to prepare biodegradable microparticles containing water-soluble sensitive agents.

[The preparation of an artificial red blood cell substitute by W/O/W double emulsion methods].

A method of preparing methoxypoly (ethylene glycol)-poly-DL-lactide (PELA) microcapsule containing bovine hemoglobin(BHb) by using W/O/W double emulsion-solvent diffusion process for use as artificial red cells was developed. The results suggested that preparation conditions such as solidification method, stirring rate, type of organic solvent had significant influence on the encapsulation efficiency and the activity of bovine hemoglobin. When ethyl acetate was used as the organic solvent and double emulsion-solvent diffusion process was used, there was no significant influence on the activity of hemoglobin if stirring rate was lower than 9000 r/min. High stirring rate, 12,000 r/min, decreased the P(50) and Hill coefficient of the hemoglobin encapsulated. Increasing the volume of solidification solution had an effect of improving the activity of hemoglobin to carry oxygen.

ApoE genotypes are associated with age at natural menopause in Chinese females.

Ages at natural menarche and menopause are influenced by several genetic factors. This study aimed to investigate the possible relationship between the apolipoprotein E (ApoE) genotype and the age at menarche and natural menopause in Chinese females. In the current study, 398 (elderly group, aged 47-80 years) and 825 (young group, aged 15-25 years) Chinese females were enrolled under informed content. Ages at natural menarche and menopause were obtained by questionnaires. ApoE genotypes were identified by restriction fragment length polymorphism analysis. In the elderly group, the number of pregnancies and live births and breastfeeding were associated with the age at menopause (P = 0.008, P = 0.002, and P = 0.023, respectively). One-way ANOVA analysis revealed that the ApoE genotype was significantly associated with age at natural menopause (ANM; P = 0.010). Compared with ApoE ε3/3 carriers, ApoE ε3/4 females showed a 1.8-year delay in ANM (P = 0.002). Single ApoE allele-positive/allele-negative analysis also showed that the age at menopause of ApoE ε4 carriers was delayed compared with those who were not carriers (P = 0.023). In the young group, no statistical difference was found in the age of menarche between the carriers of ApoE ε3/3 and ε3/4. Single ApoE allele-positive/allele-negative analysis showed that the age at menarche in ApoE ε4 carriers was slightly earlier than in those who were not carriers (P = 0.048). Meanwhile, univariate association analysis revealed that the ApoE genotypes were not significantly associated with the age at menarche using age as a covariate in the pooled group (young + elderly) (P = 0.143). We demonstrated that the ApoE genotype is significantly linked to the age at natural menopause.

Inhibition of oestrogen biosynthesis induces mild anxiety in C57BL/6J ovariectomized female mice.

OBJECTIVE Letrozole, a next-generation aromatase inhibitor, has become a favored drug for the treatment of breast cancer in postmenopausal women. Although letrozole is generally well tolerated, its adverse effects on the central nervous system have been reported. The present study aimed to assess the behavioural outcomes of letrozole administration in mice to determine its side effects. METHODS C57BL/6J female ovariectomized mice received administration of letrozole (2.5 mg/kg per day) or vehicle by gavage for 3 weeks. Behavioural tasks were used to assess anxiety, depression, as well as learning and memory in mice. RESULTS Letrozole-treated mice showed an increased latency to enter the inner area of the chamber on the third day of the open field test, and traveled a shorter distance in the open arms of the elevated plus maze. No significant difference was found in the light-dark box or forced swimming task between letrozole-treated and vehicle-treated mice. Besides, letrozole did not change the spontaneous alternation behaviour of mice in the Y-maze. In the Morris water maze, mice administered with letrozole exhibited an improvement in spatial learning and memory compared with the vehicle-treated mice. CONCLUSION Our results indicate that the inhibition of oestrogen biosynthesis results in mild anxious behaviour, which may be a consideration in the treatment of breast cancer in postmenopausal women using aromatase inhibitors.

Acute hypoxia promote the phosphorylation of tau via ERK pathway.

Tau phosphorylation and hypoxia are both linked to the pathology of Alzheimer's disease. To find out the possible connection between hypoxia and tau phosphorylation, we performed this study to evaluate the level of phosphorylated tau under hypoxic or normal condition. We found in our study that hypoxia promoted the phosphorylation of tau protein via ERK pathway, which suggest hypoxia might be involved in the process of tau pathology.

[Influence of wall polymer and preparation process on the particle size and encapsulation of hemoglobin microcapsules].

Methoxypoly (ethylene glycol)- block-poly (DL-lactide) (PELA) microcapsules containing bovine hemoglobin (BHb) were prepared by a W/O/W double emulsion-solvent diffusion process. The P50 and Hill coeffcient were 3466 Pa and 2.4 respectively, which were near to the natural bioactivity of bovine hemoglobin. The results suggested that polymer composition had significant influence on encapsulation efficiency and particle size of microcapsules. The encapsulation efficiency could reach 90% and the particle size 3 - 5 microm when the PELA copolymer containing MPEG 2000 block was used. The encapsulation efficiency and particle size increased with the concentration of PELA. Increasing the concentrations of NaCl in outer aqueous solution resulted in the increase of encapsulation efficiency and the decrease of particle size. As the concentration of stabilizer in outer aqueous solution increased in the range of 10 g/L to 20 g/L, the particle size reduced while encapsulation efficiency was increased, further increase of the stabilizer concentration would decrease encapsulation efficiency. Increasing of primary emulsion stirring rate was advantageous to the improvement of encapsulation efficiency though it had little influence on the particle size. The influence of re-emulsion stirring rate was complicated, which was not apparent in the case of large volume of re-emulsion solution. When the wall polymer and primary emulsion stirring rate were fixed, the encapsulation efficiency decreased as the particle size reduced.

The preparation and characterization of monomethoxypoly(ethylene glycol)-b-poly-DL-lactide microcapsules containing bovine hemoglobin.

Methoxypoly(ethylene glycol)-b-poly-DL-lactide (PELA) microcapsules containing bovine hemoglobin (bHb) were prepared by a W/O/W double emulsion-solvent diffusion process. bHb solution was used as the internal aqueous phase, PELA/organic solvent as the oil phase, and polyvinyl alcohol (PVA) solution as the external aqueous phase. This W/O/W double emulsion was added into a large volume of water (solidification solution) to allow organic solvent to diffuse into water. The optimum preparative condition for PELA microcapsules loaded with bovine hemoglobin was investigated. It was found that homogenization rate, type of organic solvent, and volume of the solidification solution influenced the activity of bovine hemoglobin encapsulated. When the homogenization rate was lower than 9000 rpm and ethyl acetate was used as the organic solvent, there was no significant influence on the activity of hemoglobin. High homogenization rate as 12 000 rpm decreased the P50 and Hill coefficient. Increasing the volume of solidification solution had an effect of improving the activity of microencapsulated hemoglobin. The composition of the PELA had the most important influence on the success of encapsulation. Microcapsules fabricated by PELA with MPEG2k block (molecular weight of MPEG block: 2000) achieved a high entrapment efficiency of 90%, better than PL A homopolymer and PELA with MPEG5k blocks. Hemoglobin microcapsules with native loading oxygen activity (P50 = 26.0 mmHg, Hill coefficient = 2.4), mean size of about 10 microm, and high entrapment efficiency (ca. 93%) were obtained at the optimum condition.