Dynamically changed HSP70 after reperfusion following cerebral infarction in human and rats: correlation with p38 MAPK.

We aimed to clarify the correlation between dynamic change of blood HSP70 and the prognosis of thrombolysis in human and rats, so as to explain the neuroprotection and early warning role of HSP70 in cerebral ischemia-reperfusion. Forty-two patients with acute ischemic stroke were divided into two groups according to the time from onset to thrombolytic therapy: 0 h-3 h (27 patients) and 3-4.5 h group (15 patients). The level of HSP70 in serum before and after thrombolysis was detected by ELISA. Furthermore, a rat model was also used to mimic the ischemic stroke and reperfusion. Peripheral blood of rat samples was collected to detect the level of HSP70 using Elisa. Several signal proteins from MAPK signaling pathway including JNK, p38, ERK (p42/44) were detected at different time points by Western blot of brain tissue. Patients who underwent thrombolytic therapy within 0-3 h had the highest HSP70 level at 1 h after thrombolysis. The higher HSP70 after thrombolysis, the better the patient prognosis. NIHSS scores showed HSP70 was positively correlated with cerebral ischemia. The levels of ERK family (p42/44 MAPK) and p-JNK were decreased gradually along with the time suffering cerebral ischemia. P-ERK, JNK, p-p38 had dynamic changes with increased ischemic time in the middle cerebral artery occlusion model. Dynamic change of HSP70 level in blood may be a biological index that reflects the functional condition of cell survival for cerebral ischemia and estimating the prognostic conditions. Importantly, HSP70 levels in blood were positively correlated with the p38 MAPK pathway in brain tissue.

Integrated Analysis of Chromatin and Transcriptomic Profiling Identifies PU.1 as a Core Regulatory Factor in Microglial Activation Induced by Chronic Cerebral Hypoperfusion.

In addition to causing white matter lesions, chronic cerebral hypoperfusion (CCH) can also cause damage to gray matter, but the underlying molecular mechanisms remain largely unknown. In order to obtain a better understanding of the relationship between gene expression and transcriptional regulation alterations, novel upstream regulators could be identified using integration analysis of the transcriptome and epigenetic approaches. Here, a bilateral common carotid artery stenosis (BCAS) model was established for inducing CCH in mice. The spatial cognitive function of mice was evaluated, and changes in cortical microglia morphology were observed. RNA-sequencing (RNA-seq) and the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) were performed on isolated mouse cortical brain tissue. Then, a systematic joint analysis of BCAS hypoperfusion-induced cortex-specific RNA-seq and ATAC-seq was conducted in order to assess the extent of the correlation between the two, and PU.1 was found to be greatly enriched through motif analysis and transcription factor annotation. Also, the core regulatory factor PU.1 induced by BCAS hypoperfusion was shown to be colocalized with microglia. Based on the above analysis, PU.1 plays a key regulatory role in microglial activation induced by CCH. And the transcriptome and epigenomic data presented in this study can help identify potential targets for future research exploring chronic hypoperfusion-induced brain injury.

Transcriptome Profiling of Hippocampus After Cerebral Hypoperfusion in Mice.

Chronic cerebral hypoperfusion (CCH) is considered to be one of the major mechanism in the pathogenesis of vascular cognitive impairment (VCI). Increased inflammatory cells, particularly microglia, often parallel hypoperfusion-induced gray matter damage such as hippocampal lesions, but the exact mechanism remains largely unknown. To understand the pathological mechanisms, we analyzed hippocampus-specific transcriptome profiles after cerebral hypoperfusion. The mouse hypoperfusion model was induced by employing the 0.16/0.18 mm bilateral common carotid artery stenosis (BCAS) procedure. Cerebral blood flow (CBF) was assessed after 3-week hypoperfusion. Pathological changes were evaluated via hematoxylin staining and immunofluorescence staining. RNA-sequencing (RNA-seq) was performed using RNA samples of sham- or BCAS-operated mice, followed by quantitative real-time PCR (qRT-PCR) validation. We found that the 0.16/0.18 mm BCAS induced decreased CBF, hippocampal neuronal loss, and microglial activation. Furthermore, GSEA between sham and BCAS mice showed activation of interferon-beta signaling along with inflammatory immune responses. In addition, integrative analysis with published single-cell RNA-seq revealed that up-regulated differentially expressed genes (DEGs) were enriched in a distinct cell type of "microglia," and down-regulated DEGs were enriched in "CA1 pyramidal," not in "interneurons" or "S1 pyramidal." This database of transcriptomic profiles of BCAS-hypoperfusion will be useful for future studies to explore potential targets for vascular cognitive dysfunction.

Cortex-specific transcriptome profiling reveals upregulation of interferon-regulated genes after deeper cerebral hypoperfusion in mice.

Background: Chronic cerebral hypoperfusion (CCH) is commonly accompanied by brain injury and glial activation. In addition to white matter lesions, the intensity of CCH greatly affects the degree of gray matter damage. However, little is understood about the underlying molecular mechanisms related to cortical lesions and glial activation following hypoperfusion. Efforts to investigate the relationship between neuropathological alternations and gene expression changes support a role for identifying novel molecular pathways by transcriptomic mechanisms. Methods: Chronic cerebral ischemic injury model was induced by the bilateral carotid artery stenosis (BCAS) using 0.16/0.18 mm microcoils. Cerebral blood flow (CBF) was evaluated using laser speckle contrast imaging (LSCI) system. Spatial learning and memory were assessed by Morris water maze test. Histological changes were evaluated by Hematoxylin staining. Microglial activation and neuronal loss were further examined by immunofluorescence staining. Cortex-specific gene expression profiling analysis was performed in sham and BCAS mice, and then validated by quantitative RT-PCR and immunohistochemistry (IHC). Results: In our study, compared with the sham group, the right hemisphere CBF of BCAS mice decreased to 69% and the cognitive function became impaired at 4 weeks postoperation. Besides, the BCAS mice displayed profound gray matter damage, including atrophy and thinning of the cortex, accompanied by neuronal loss and increased activated microglia. Gene set enrichment analysis (GSEA) revealed that hypoperfusion-induced upregulated genes were significantly enriched in the pathways of interferon (IFN)-regulated signaling along with neuroinflammation signaling. Ingenuity pathway analysis (IPA) predicted the importance of type I IFN signaling in regulating the CCH gene network. The obtained RNA-seq data were validated by qRT-PCR in cerebral cortex, showing consistency with the RNA-seq results. Also, IHC staining revealed elevated expression of IFN-inducible protein in cerebral cortex following BCAS-hypoperfusion. Conclusion: Overall, the activation of IFN-mediated signaling enhanced our understanding of the neuroimmune responses induced by CCH. The upregulation of IFN-regulated genes (IRGs) might exert a critical impact on the progression of cerebral hypoperfusion. Our improved understanding of cortex-specific transcriptional profiles will be helpful to explore potential targets for CCH.

Assessment of Irinotecan Loading and Releasing Profiles of a Novel Drug-Eluting Microsphere (CalliSpheres) In Vitro.

Background: This study investigated irinotecan loading efficiency and release profiles of CalliSpheres in vitro. Materials and Methods: CalliSpheres with size of 50-150, 100-300, and 300-500 µm and irinotecan at different amounts (20, 40, 80, and 100 mg) and concentrations (5 and 10 mg/mL) were prepared for experiments. Dynamic light scattering and Agilent 1260 high-performance liquid chromatography system were used to quantify bead diameters and the efficiency of irinotecan loading and releasing properties, respectively. Results: The diameters of CalliSpheres with all sizes were reduced after being loaded with irinotecan compared with unloaded ones with shrinkage rate ranging from 8.5% to 16.2%. Above 80% irinotecan was incorporated with CalliSpheres with all sizes when being loaded with irinotecan 20, 40, and 80 mg, while loading efficiencies were 70%-80% when being loaded with irinotecan 100 mg. Besides, elevated loading efficiency was observed at a higher concentration of irinotecan solutions (10 mg/mL) compared with a lower concentration (5 mg/mL) for CalliSpheres with all sizes. As to release profiles, irinotecan was released from CalliSpheres very quickly, and irinotecan release rate was elevated in CalliSpheres with smaller size than CalliSpheres with larger size within the first 12 h, whereas it was similar among CalliSpheres with different sizes at 24 and 48 h with maximum release rate ∼100%. In addition, fetal bovine serum seemed to have an effect on the accelerating irinotecan release. Conclusion: CalliSpheres exhibits good physical characteristics, satisfied irinotecan loading efficiency, and acceptable releasing profiles.

Multicentre registration of wake-up stroke in China (MCRWUSC): a protocol for a prospective, multicentre, registry-based cohort study.

INTRODUCTION: Wake-up stroke (WUS) is a type of acute ischaemic stroke (AIS) that occurs during sleep with unknown time of symptom onset. The best treatment is usually not suitable for WUS, as thrombolysis is usually provided to patients who had a symptomatic AIS within a definite 4.5 hours, and WUS remains a therapeutic quandary. Efforts to explore the onset time characteristics of patients who had a WUS and the risk factors affecting poor prognosis support a role for providing new insights by performing multicentre cohort study. METHODS AND ANALYSIS: This multicentre, nationwide prospective registry will include 21 comprehensive stroke centres, with a goal of recruiting 550 patients who had a WUS in China. In this study, clinical data including patient's clinical characteristics, stroke onset time, imaging findings, therapeutic interventions and prognosis (the National Institutes of Health Stroke Scale Score and the modified Rankin Scale Score at different time points) will be used to develop prediction models for stroke onset time and prognostic evaluation using the fast-processing of ischemic stroke software. The purpose of this study is to identify risk factors influencing prognosis, to investigate the relationship between the time when the symptoms are found and the actual onset time and to establish an artificial intelligence-based model to predict the prognosis of patients who had a WUS. ETHICS AND DISSEMINATION: This study is approved by the ethics committee of Shanghai Pudong Hospital (Shanghai, China) and rest of all participating centres. The findings will be disseminated through peer-reviewed publications and conference presentations. PROSPERO REGISTRATION NUMBER: ChiCTR2100049133.

Exosomes from hypoxic pre-treated ADSCs attenuate acute ischemic stroke-induced brain injury via delivery of circ-Rps5 and promote M2 microglia/macrophage polarization.

BACKGROUND: Previous investigations have shown that exosome secretion from hypoxic pre-treated adipose-derived stem cells (ADSCs) affect ischemic injury treatment; however, the therapeutic effect relative to circRNA delivery is unclear. METHODS: In the present investigation inflammatory factors, nerve injury, and cognitive function were assessed using a middle cerebral artery occlusion mouse model. The isolated exosomes were identified using transmission electron microscopy and further tested by leveraging exosome particles in a nanoparticle tracking approach. Differences in circRNA expression between exosomes and hypoxic pre-treated ADSC exosomes were analyzed by high-throughput sequencing. The phenotypic transformation of microglia was detected by immunofluorescence. The circRNA and downstream target were analyzed by bioinformatics, RT-qPCR, and luciferase report. RESULTS: Exosomes from hypoxic pre-treated ADSCs improved cognitive function by reducing neuronal damage in the hippocampus after cerebral infarction. Exosomes from hypoxic pre-treated ADSCs improved cognitive function via delivery of circ-Rps5. SIRT7 and miR-124-3p were circ-Rps5 downstream targets, which was confirmed by luciferase report analysis. miR-124-3p overexpression or SIRT7 downregulation reversed the circ-Rps5-mediated M2 microglial shift under LPS conditions. Circ-Rps5-modified ADSC exosome improved cognitive function by decreasing neuronal damage and shifting microglia from an M1 to M2 phenotype in the hippocampus. CONCLUSION: The study showed that exosomes from hypoxic pre-treated ADSCs attenuated acute ischemic stroke-induced brain injury via delivery of circ-Rps5 and promoted M2 microglia/macrophage polarization.

Interrogating preclinical study of liposomes: The effect of mouse strain reexamined.

Mice are arguably the most important tool in the preclinical evaluation of liposomes; however, the effects of inter-strain physiological variabilities on in vivo performance of liposomes have been seriously overlooked. The present study validated that plasma proteins (PPs) and the capability of mononuclear phagocyte system (MPS) (typically expressed by phagocytosis rate, K) were mice strain-dependent. Physiological variabilities in PPs and the phagocytosis rate jointly contributed to the inter-strain inconsistency of pharmacokinetic (PK) profiles of liposomes. For the PPs sensitive liposomes (such as plain PEGylated liposomes and folic acid functionalized PEGylated liposomes), inter-strain variabilities in PK profiles could be calibrated using the corrected phagocytic rate (KC = K×(c × Ig)/(alb×apo)), where c, Ig, alb and apo were respective the total content of complement proteins, immunoglobulins, albumin and apolipoproteins. While for the PPs insensitive liposomes (e.g., cRGD functionalized liposomes), phagocytic rate could be directly used to calibrate inter-strain difference of liposome PK profiles. Our data also warn that the reciprocal interaction between payloads and organisms would be much more complicated than that between liposomes and organisms, thus independent investigation should be conducted for each individual therapeutic agent.

Interrogation of Folic Acid-Functionalized Nanomedicines: The Regulatory Roles of Plasma Proteins Reexamined.

Folic acid (FA) has been extensively exploited to facilitate targeted delivery of nanomedicines by recognizing the folate receptor-α (FR-α) overexpressed in many human cancers. Unfortunately, none have been approved for clinical use yet. Here we reveal that FA functionalization induces heavy natural IgM absorption on the liposomal surface, depriving FA of receptor recognition and accelerating complement activation in vivo. FA functionalization does not enhance distribution of liposomes in FR-α-overexpressed tumors in comparison to plain liposomes (without FA), but leads to aggravated capture of liposomes by macrophages in the tumor, liver, and spleen. In addition, FA-functionalized polymeric nanoparticles are also vulnerable to natural IgM absorption. This work highlights the pivotal roles of natural IgM in regulating in vivo delivery of FA-functionalized nanomedicines. Due to the prevalent association of immune disorders and varying levels of immunoglobulins with cancer patients, extraordinary cautiousness is urged for clinical translation of FA-enabled targeted delivery systems.

Benign Paroxysmal Positional Vertigo and Occult Subarachnoid Hemorrhage Complicated after Head Trauma.

Benign paroxysmal positional vertigo (BPPV) is the most prevalent form of peripheral vertigo and is common in posttraumatic patients. Sometimes, posttraumatic BPPV and subarachnoid hemorrhage (SAH) exist together. How to effectively recognize SAH especially concealed bleeding before maneuver treatment for BPPV is worth paying attention by every clinician. Presently described is a case that when there are some clinical symptoms cannot be completely explained by simple BPPV, the combination of CT and FLAIR MRI sequences are needed in the early-stage detection of acute SAH.

RVG-modified exosomes derived from mesenchymal stem cells rescue memory deficits by regulating inflammatory responses in a mouse model of Alzheimer's disease.

BACKGROUND: Exosomes are lipid-bilayer enclosed nano-sized vesicles that transfer functional cellular proteins, mRNA and miRNAs. Mesenchymal stem cells (MSCs) derived exosomes have been demonstrated to prevent memory deficits in the animal model of Alzheimer's disease (AD). However, the intravenously injected exosomes could be abundantly tracked in other organs except for the targeted regions in the brain. Here, we proposed the use of central nervous system-specific rabies viral glycoprotein (RVG) peptide to target intravenously-infused exosomes derived from MSCs (MSC-Exo) to the brain of transgenic APP/PS1 mice. MSC-Exo were conjugated with RVG through a DOPE-NHS linker. RESULTS: RVG-tagged MSC-Exo exhibited improved targeting to the cortex and hippocampus after being administered intravenously. Compared with the group administered MSC-Exo, in the group administered RVG-conjugated MSC-Exo (MSC-RVG-Exo) plaque deposition and Aß levels were sharply decreased and activation of astrocytes was obviously reduced. The brain targeted exosomes derived from MSCs was better than unmodified exosomes to improve cognitive function in APP/PS1 mice according to Morris water maze test. Additionally, although MSC-Exo injected intravenously reduced the expression of pro-inflammatory mediators TNF-α, IL-ß, and IL-6, but the changes of anti-inflammatory factors IL-10 and IL-13 were not obvious. However, administration of MSC-RVG-Exo significantly reduced the levels of TNF-α, IL-ß, and IL-6 while significantly raised the levels of IL-10, IL-4 and IL-13. CONCLUSIONS: Taken together, our results demonstrated a novel method for increasing delivery of exosomes for treatment of AD. By targeting exosomes to the cortex and hippocampus of AD mouse, there was a significant improvement in learning and memory capabilities with reduced plaque deposition and Aß levels, and normalized levels of inflammatory cytokines.

Spatiotemporal expression of NDRG2 in the human fetal brain.

N-myc downstream-regulated gene 2 (NDRG2) has been implicated in the development of central nervous system and brain diseases such as brain tumors, ischemic stroke and neurodegenerative disorders. However, it remains unclear that the spatiotemporal distribution of NDRG2 in the human fetal brain. In this study, we examined the expression pattern of NDRG2 in different regions of human fetal brain at 16-28 gestational weeks (GWs) by using RT-PCR, western blot and immunohistochemistry. Firstly, RT-PCR revealed that mRNA of NDRG2 was detected in the human brain regions of fetuses at 16-28 GWs such as medulla oblongata (MdO), mesencephalon (MeE), cerebellum (Cbl), frontal lobe (Fr), ventricular (VZ)/subventricular zone (SVZ) and hippocampus (hip), and the expressions of NDRG2 mRNA in these human fetal brain regions were increased with gestational maturation. Furthermore, western blot and immunohistochemistry results revealed that at 28 GWs, the expression of NDRG2 protein was restricted to the MdO's olivary nucleus, MeE's aqueduct, cerebellar internal granular layers, cerebral cortex of the Fr, VZ/SVZ of lateral ventricle, and hippocampal dentate gyrus, and highest expression in the VZ/SVZ, and lowest in the MeE. Finally, double immunohistochemistry results showed that NDRG2 in the MdO, Cbl and VZ/SV at 28 GWS was mainly expressed in neurons (NeuN positive cells), and in some astrocytes (GFAP positive cells). Taken together, these results suggest that NDRG2 is mainly expressed in human fetal neurons of various brain regions during development, which may be involved in neuronal growth and maturation.

NMDA receptors inhibit axonal outgrowth by inactivating Akt and activating GSK-3ß via calcineurin in cultured immature hippocampal neurons.

Neurons are highly polarized cells with an axon and dendritic arbors. It is still not well studied that how formation and elaboration of axon and dendrites is controlled by diffusible signaling factors such as glutamate via specific receptors. We found that N-methyl-D-aspartate (NMDA) receptors were enriched (stage 2-3) but decreased expression (stage 4-5) at tip of axon of cultured hippocampal neurons during distinct development stages. Inhibition of NMDA receptor activity by competitive antagonist DL-2-amino-5-phosphonovalerate (APV) or channel blocker MK801 promoted axonal outgrowth at the early stages, whereas inhibited dendritic development in later stages. Meanwhile, knockdown of NMDA receptors also promoted axonal outgrowth and branch in immature neurons. Furthermore, GluN2B but not GluN2A subunit inhibited axonal outgrowth in immature hippocampal neurons. Finally, we found that NMDA receptors inhibited axonal outgrowth by inactivating Akt and activating GSK-3ß signaling in a calcineurin-dependent manner. Taken together, our results demonstrate that stabilization GSK-3ß activation in the axon growth cone by Ca2+ influx through NMDA receptors may be involved in regulation of axon formation in immature neurons at early stages.

DUSP1 alleviates cerebral ischaemia reperfusion injury via inactivating JNK-Mff pathways and repressing mitochondrial fission.

AIMS: Evidence continues to accrue implicating mitochondrial fission in the aetiology of cerebral ischaemia reperfusion (IR) injury. Dual-specificity phosphatase-1 (DUSP1) has been found to be associated with mitochondrial protection in several diseases. We aimed to explore the functional role of DUSP1 in cerebral IR injury, focusing on its influence on mitochondrial fission. MAIN METHODS: WT mice and DUSP1 transgenic mice were subjected to cerebral IR in vivo. In vitro, the hypoxia-reoxygenation model was used with N2a cells to mimic cerebral IR injury. Mitochondrial function was monitored via western blotting and immunofluorescence. Pathway blocker was used to establish the role of the JNK-Mff signaling pathway in mitochondrial fission. KEY FINDINGS: DUSP1 expression is downregulated after cerebral IR injury, and overexpression of DUSP1 could significantly reduce the infarction area and attenuate neuronal death exerted by cerebral IR injury. In vitro, we found that HR injury induced neuronal mitochondrial damage via augmenting mitochondrial fission. DUSP1 overexpression inhibited mitochondrial fission, thereby preserving mitochondrial homeostasis against HR injury. Furthermore, our data illustrated that DUSP1 regulated mitochondrial fission via JNK-Mff pathways. Increased DUSP1 prevented JNK activation, upregulated Yap expression, and suppressed Mff phosphorylation, reducing the formation of mitochondrial fragmentation. However, re-activation of JNK-Mff pathways abrogated the inhibitory effect of DUSP1 overexpression on mitochondrial fission. SIGNIFICANCE: This finding first uncovers that DUSP1 downregulation could be considered the primary reason for cerebral IR injury via evoking deleterious mitochondrial fission and activating harmful JNK-Mff pathways.

MAPK8 mediates resistance to temozolomide and apoptosis of glioblastoma cells through MAPK signaling pathway.

OBJECTIVE: In this study, we aimed to evaluate the expression and functions of MAPK8 in temozolomide (TMZ) -resistant glioblastoma cells as well as to explore the mechanism of TMZ resistance in glioblastoma cells. METHODS: Gene Expression Omnibus (GEO) database was used for identifying the differentially expressed genes (DEGs) in TMZ resistant samples. The functional partner genes of TMZ were screened out by Gene-drug interaction network (STITCH) and the glioblastoma-related genes were selected by gene search engine with evidence sentences (Digsee). The interactions among identified DEGs and glioblastoma-related genes were detected by Search Tool for the Retrieval of Interacting Genes (STRING). The dysregulated pathways were identified by Gene set enrichment analysis (GSEA). qRT-PCR was performed to detect the expression level of MAPK8 in glioblastoma cells. Western blot was used to detect the expressions of MAPK8 and MAPK signaling pathway-related proteins. MTT assay was utilized to measure the cell viability of TMZ sensitive and resistant cells. Colony formation assay was performed to detect the clone ability and flow cytometry (FCM) assay was applied to identify the apoptosis rate of TMZ resistant glioblastoma cells. RESULTS: MAPK8 was one of the DEGs and was up-regulated in TMZ resistant glioblastoma cells. The MAPK signaling pathway was activated in TMZ resistant glioblastoma cells under the condition of over-expression of MAPK8. The inhibition of MAPK8 restrained the colony formation, inducing apoptosis of TMZ resistant glioblastoma cells and suppressed the MAPK signaling pathway. CONCLUSION: MAPK8 promoted the resistance to TMZ, accelerated cell proliferation and inhibited the apoptosis of glioblastoma cells via activating MAPK signaling pathway.

Combination of betulinic acid with diazen-1-ium-1,2-diolate nitric oxide moiety donating a novel anticancer candidate.

BACKGROUND: Betulinic acid (BA) is a complex lupane triterpenoid with unique antineoplastic activity. However, its antiproliferative activity is far from satisfaction. In order to improve its anticancer efficacy, betulinic acid was conjugated with a nitric oxide (NO)-releasing moiety to get a novel hybrid, BA-78. METHODS: The antiproliferative activity of BA-78 against 6 cell lines and the ability of releasing nitric oxide were determined. The pro-apoptosis mechanism of BA-78 was investigated as well. RESULTS: BA-78 exhibited time-dependent release of NO, and it displayed higher antiproliferative potential than BA through increasing apoptosis and inducing cell cycle arrest at G1 phase. Western blotting results showed that BA-78 increased the expression of Bax, Bid, Bad and cytochrome C and reduced the level of anti-apoptosis proteins including Bcl-2 and Bcl-xl. CONCLUSION: Our study revealed that novel compound BA-78, possessing betulinic acid and nitric oxide (NO)-releasing moiety, could be developed as an antitumor agent.

Visualization of Rostral Migratory Stream in the Developing Rat Brain by In Vivo Electroporation.

Interneurons in the olfactory bulb (OB) are generated from neuronal precursor cells migrating from anterior subventricular zone (SVZa) not only in the developing embryo but also throughout the postnatal life of mammals. In the present study, we established an in vivo electroporation assay to label SVZa cells of rat both at embryonic and postnatal ages, and traced SVZa progenitors and followed their migration pathway and differentiation. We found that labeled cells displayed high motility. Interestingly, the postnatal cells migrated faster than the embryonic cells after applying this assay at different ages of brain development. Furthermore, based on brain slice culture and time-lapse imaging, we analyzed the detail migratory properties of these labeled precursor neurons. Finally, tissue transplantation experiments revealed that cells already migrated in subependymal zone of OB were transplanted back into rostral migratory stream (RMS), and these cells could still migrate out tangentially along RMS to OB. Taken together, these findings provide an in vivo labeling assay to follow and trace migrating cells in the RMS, their maturation and integration into OB neuron network, and unrecognized phenomena that postnatal SVZa progenitor cells with higher motility than embryonic cells, and their migration was affected by extrinsic environments.

Proteomic analysis and comparison of intra­ and extracranial cerebral atherosclerosis responses to hyperlipidemia in rabbits.

The present study aimed to investigate protein expression levels of intra­ and extracranial atherosclerosis in rabbits following administration of a high­fat diet. Rabbits were randomly divided into control (group A; n=9) and high­fat diet (group B; n=9) groups. At week 12, tissues were sectioned from the common carotid artery (CCA) and middle cerebral artery (MCA). Pathological analysis was performed. Differential protein expression levels were examined by 2­D gel electrophoresis (2­DE) and mass spectrometry (MS) analysis and validated by western blotting. Serum lipid levels, the intima­media thickness (IMT) and degree of atherosclerosis of the CCA and MCA were increased at week 12 in the high­fat diet group compared with rabbits that received a normal diet. 2­DE and MS analysis of the protein extracted from CCA and MCA detected >439 different proteins; the expression of 25 proteins was altered, and 8 proteins [albumin A chain, tropomyosin α­1 chain (TPM1), heat shock protein 70 (HSP70), α­smooth muscle actin, ß­galactose binding agglutinin, TPM4 isoform 2, cell keratin 9, single octylic acid glyceride ß­2) demonstrated significant alterations in expression levels. Due to limited antibody sources, only three differentially expressed proteins (TPM1, HSP70 and α­smooth muscle actin) were examined by western blotting. The results of our previous study demonstrated that hyperlipidemia affected the IMT of intracranial and extracranial cerebral arteries. In the present study, protein expression levels of TPM1 and α­smooth muscle actin from extracranial cerebral arteries were significantly increased compared with intracranial cerebral arteries; however, protein expression levels of HSP70 from intracranial cerebral arteries was increased compared with extracranial cerebral arteries. The differences may be closely associated with cell proliferation and metastasis, and oxidoreduction, in intra­ and extracranial cerebral atherosclerosis. HSP70 may have protective properties against atherosclerosis via underlying anti­inflammatory mechanisms, furthermore, differential protein expression levels (TPM1, HSP70 and α­smooth muscle actin) between intra­ and extracranial cerebral arteries may facilitate the identification of novel biological markers for the diagnosis and treatment of cerebral arteriosclerosis.

MiR-146a suppresses hepatocellular carcinoma by downregulating TRAF6.

MicroRNAs have been proven to play important roles in many biological processes such as cellular growth and differentiation, apoptosis, and modulation of host response to viral infection. In the present study, we find that the expression of miR-146a was decreased in hepatocellular carcinoma (HCC) tissues compared with corresponding adjacent tissues, and the expression level in HCC cell lines was lower than in a normal liver cell. Over-expression suppressed the proliferation and invasion of HCC cells. In addition, luciferase reporter assays and western blotting confirmed that miR-146a directly target TRAF6 which attenuated the effect of miR-146a on cell proliferation and invasion in HepG2 and SMMC7721 cells. Meanwhile, lentivirus-mediated increased expression of miR146a repressed tumor formation in nude mice. Taken together, our findings demonstrate that miR-146a suppresses HCC by down-regulating TRAF6. We also discovered that miR-146a may represent a novel potential candidate of the HCC carcinoma diagnostic marker in the long term.