Hierarchically clustering to 1,033 genes differentially expressed in mouse superior colliculus in the courses of optic nerve development and injury.

Tempo spatially specific expression of many development-related genes is the molecular basis for the formation of the central nervous system (CNS), especially those genes regulating the proliferation, differentiation, migration, axon growth, and orientation of nerve cells. The development-related genes are usually prominent during the embryonic and newborn stages, but rarely express during the adulthood. These genes are believed to be suitable target genes for promoting CNS regeneration, despite majority of which remains unknown. Hence, the aim of this study was to screen development-related genes which might contribute to CNS regeneration. In this study, 1,033 differentially-expressed genes of superior colliculus in the courses of mouse optic nerve development and injury, as previously identified by cDNA microarrays, were hierarchically clustered to display expression pattern of each gene and reveal the relationships among these genes, and infer the functions of some unknown genes based on function-identified genes with the similar expression patterns. Consequently, the expression patterns of 1,033 candidate genes were revealed at eight time points during optic nerve development or injury. According to the similarity among gene expression patterns, 1,033 genes were divided into seven groups. The potential function of genes in each group was inferred on the basis of the dynamic trend for mean gene expression values. Moreover, the expression patterns of six function-unidentified genes were extremely similar to that of the ptn gene which could promote and guide axonal extension. Therefore, these six genes are temporally regarded as candidate genes related to axon growth and guidance. The results may help to better understand the roles of function-identified genes in the stages of CNS development and injury, and offer useful clues to evaluate the functions of hundreds of unidentified genes.

[Association between high sensitivity C-reactive protein and contrast induced acute kidney injury in patients with acute coronary syndrome undergoing percutaneous coronary intervention: impact of atorvastatin].

OBJECTIVE: To observe the association between preprocedural high sensitivity C-reactive protein (hs-CRP) level and incidence of contrast induced acute kidney injury (CI-AKI) in acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) and the impact of atorvastatin pretreatment on CI-AKI. METHODS: According to the level of preprocedural hs-CRP, 270 ACS patients were divided into three groups: high hs-CRP group (hs-CRP ≥ 3 mg/L, n = 176), moderate hs-CRP group (hs-CRP 1-3 mg/L, n = 60) and normal hs-CRP group (hs-CRP < 1 mg/L, n = 34). According to the dosage of preprocedural atorvastatin, the high hs-CRP group was further divided into 10 mg group (n = 49), 20 mg group (n = 66) and 40 mg group (n = 61). Serum creatinine (Scr), blood urea nitrogen (BUN), cystatin C (Cys C), hs-CRP were measured at before and 24 hours, 48 hours after PCI. CCr and GFR were calculated according to Scr and Cys C. Risk factors for CI-AKI were determined by multivariate logistic regression analysis. RESULTS: (1) Cys C was significantly increased and GFR after PCI significantly reduced in high and moderate hs-CRP groups compared with normal hs-CRP group (P < 0.05). (2) Incidence of CI-AKI was 43.18%, 38.33%, 20.59% in high, moderate and normal hs-CRP groups, respectively (P < 0.05). (3) In high hs-CRP group, postprocedural GFR was significantly higher while postprocedural Cys C and hs-CRP were significantly lower in 40 mg statin subgroup than 10 mg and 20 mg statin subgroups (P < 0.05), similar trends were documented when comparing 20 mg statin subgroup with 10 mg statin subgroup (P < 0.05). (4) Multivariate logistic regression analysis showed that pretreatment with high dose atorvastatin was a protective factor for post CI-AKI (20 mg atorvastatin: OR = 0.15, 95%CI 0.06 - 0.33, P = 0.001; 40 mg atorvastatin: OR = 0.10, 95%CI 0.04 - 0.23, P = 0.001), while high levels of preprocedural hs-CRP (OR = 2.06, 95%CI 1.01 - 4.23, P = 0.048), diabetes mellitus (OR = 10.71, 95%CI 5.29 - 21.70, P = 0.001), advanced age (OR = 2.64, 95%CI 1.05 - 6.63, P = 0.038) and renal failure (OR = 5.14, 95%CI 1.13 - 23.39, P = 0.034) were independent risk factors of CI-AKI. CONCLUSION: High hs-CRP level is linked with the development of CI-AKI in ACS patients undergoing PCI and pretreatment with 40 mg atorvastatin is associated with lower incidence CI-AKI, possibly by reducing the postprocedural inflammation responses.

Downregulation of P2X7 receptor expression in rat oligodendrocyte precursor cells after hypoxia ischemia.

Oligodendrocyte precursor cells (OPCs) are the predominant oligodendrocyte-lineage stage in the cerebral hemispheres of neonatal rat. Prior studies have shown that OPCs are highly vulnerable to hypoxic-ischemic injury, yet the mechanisms are not well understood. P2X(7) receptor (P2X(7)R) is an ATP-gated ion channel that has unusual properties and plays very complex roles in a variety of neuropathologic conditions. However, little is known about the involvement of P2X(7)R in OPCs development and injury. The present study was aimed at examining the presence of P2X(7)R in OPCs and evaluating the change of the receptor expression after hypoxia ischemia. Using Immunofluorescence, RT-PCR, and western blot analysis, we demonstrated that OPCs expressed P2X(7)R in vitro and in vivo. Activation of P2X(7)R in OPCs in response to 3'-O-(4-benzoyl) benzoyl-ATP (BzATP) led to an increased mobilization of intracellular calcium [Ca(2+)]i, formation of large pores and cell death. These functional responses were sensitive to pretreatment of cells with the P2X(7)R antagonist, Brilliant Blue G (BBG, 100 nM), which was a selective antagonist for P2X(7)R in nanomole range. A decrease in P2X(7)R expression was observed in cultured OPCs after exposure to oxygen-glucose deprivation (OGD) for 2 h in vitro. Using a neonatal hypoxic-ischemic injury model in postnatal 3 rats, the similar downregulation was also detected in ischemic cerebral cortex, subcortical white matter and hippocampus compared with sham operation controls. In conclusion, the present data demonstrated that OPCs expressed functional P2X(7)R. The post-ischemic downregulation of P2X(7)R suggested a role for this receptor in the pathophysiology of hypoxic-ischemic brain injury.

[Transplantation of mesenchymal stem cells transfected with hepatocyte growth factor gene improves heart function in rats with heart failure].

AIM: To investigate the effects of hepatocyte growth factor gene transfected MSCs transplantation on cardiac function and fibrosis in rats heart failure model induced by adriamycin. METHODS: MSCs were isolated from SD rats by density gradient centrifugation, purified, and transfected with Ad-hHGF. ELISA were used to detect the protein expression of hHGF in these MSCs. Forty SD rats underwent intraperitoneal injection with adriamycin to induce heart failure model. 8 healthy rats served as control, 24 survival rats were randomly divided into 3 groups (n = 8): Rats in Ad-hHGF transfected MSCs group were injected with Ad-hHGF transfected MSCs 2 weeks after the establishment of the model, rats in MSC group injected with suspension of MSCs only, and model group was injected with cold culture fluid. Heart function was evaluated by a physiological recorder 4 weeks after cell transplantation. Myocardial cell morphology and interstitial collagen were studied by electron microscope and were stained by Sirus red. TGF-beta1 was detected by immunohistochemical method. RESULTS: (1) MSCs could be transfected efficiently by Ad-hHGF, manifested by a higher level of expression in vitro, persisting 14 days at least. (2) Four weeks after the cells transplantion, cardiac necrosis in MSC-hHGF rats was improved when compared with those in the MSCs (P < 0.05) and Model group (P < 0.01). The heart function of the MSC-hHGF rats was greatly improved with an significant increase in LVSP and + dp/dt(max), although LVEDP still highter than that of normal rats. (3) MSC-Ad-hHGF decreased Myocardial collagen content and the level of TGFbeta1 compaired with MSCs transplanted rats (P < 0.01). CONCLUSION: Transplantation of HGF gene transfected MSCs improved heart function, decreased myocardial collagen and the level of TGFbeta1.

A morphological and electrophysiological study on the postnatal development of oligodendrocyte precursor cells in the rat brain.

A widespread population of cells in CNS is identified by specific expression of the NG2 chondroitin sulphate proteoglycan and named as oligodendrocyte precursor cell (OPC). OPCs may possess stem cell-like characteristics, including multipotentiality in vitro and in vivo. It was proposed that OPCs in the CNS parenchyma comprise a unique population of glia, distinct from oligodendrocytes and astrocytes. This study confirmed that NG2 immunoreactive OPCs were continuously distributed in cerebral cortex and hippocampus during different postnatal developmental stages. These cells rapidly increased in number over the postnatal 7 days and migrate extensively to populate with abundant processes both in developing cortex and hippocampus. The morphology of OPCs exhibited extremely complex changes with the distribution of long distance primary process gradually increased from neonatal to adult CNS. Immunohistochemical studies showed that OPCs exhibited the morphological properties that can be distinguished from astrocytes. The electrophysiological properties showed that OPCs expressed a small amount of inward Na(+) currents which was distinguished from Na(+) currents in neurons owing to their lower Na-to-K conductance ratio and higher command voltage step depolarized maximum Na(+) current amplitude. These observations suggest that OPCs can be identified as the third type of macroglia because of their distribution in the CNS, the morphological development in process diversity and the electrophysiological difference from astrocyte.

[Recent advances on the study between steroids and brain function].

It is vital for steroid hormones synthesized in the gonad gland, adrenal gland and placenta to the development and physiological metabolism of the body. In particular, its regulatory effect on brain functions has attracted more and more attention, as nearly all the steroid hormones can be synthesized de novo in the brain. This means steroids derived from brain and peripheral gland can modulate the physiology and pathology of the brain reciprocally and importantly on many aspects such as learning and memory, synaptic transmission, neuroprotection, neurodegenerative diseases (especially Alzheimer's disease), emotion, stress, and menstrual-cycle-linked disorders. In the present review, some new advances are summarized.

Poly-lactic acid and agarose gelatin play an active role in the recovery of spinal cord injury.

Objective To investigate the role of poly-lactic acid and agarose gelatin in promoting the functional recovery of the injured spinal cord. Methods Poly-lactic acid (PLA) or agarose was embedded in the space between two stumps of the hemisectioned spinal cord. Immunohistochemistry was used to show astroglia proliferation and the infiltration of RhoA-positive cells. Locomotor activity recovery was evaluated by testing the function of hindlimbs. Results Astroglias and RhoA labeled non-neuronal cells accumulated in the area adjacent to the implant, while the number of RhoA-positive cells was decreased dramatically in the absence of implant. Animals implanted with agarose gelatin recovered more quickly than those with PLA, concomitant with a higher survival rate of the neurons. Conclusion Both PLA and agarose gelatin benefited the recovery of spinal cord after injury by providing a scaffold for astroglia processes. Modulation of the rigidity, pore size and inner structure of PLA and agarose gelatin might make these biodegradable materials more effective in the regeneration of the central nervous system (CNS).

Repair of spinal cord injury by neural stem cells modified with BDNF gene in rats.

Objective To explore repair of spinal cord injury by neural stem cells (NSCs) modified with brain derived neurotrophic factor (BDNF) gene (BDNF-NSCs) in rats. Methods Neural stem cells modified with BDNF gene were transplanted into the complete transection site of spinal cord at the lumbar 4 (L4) level in rats. Motor function of rats' hind limbs was observed and HE and X-gal immunocytochemical staining, in situ hybridization, and retrograde HRP tracing were also performed. Results BDNF-NSCs survived and integrated well with host spinal cord. In the transplant group, some X-gal positive, NF-200 positive, GFAP positive, BDNF positive, and BDNF mRNA positive cells, and many NF-200 positive nerve fibers were observed in the injury site. Retrograde HRP tracing through sciatic nerve showed some HRP positive cells and nerve fibers near the rostral side of the injury one month after transplant and with time, they increased in number. Examinations on rats' motor function and behavior demonstrated that motor function of rats' hind limbs improved better in the transplant group than the injury group. Conclusion BDNF-NSCs can survive, differentiate, and partially integrate with host spinal cord, and they significantly ameliorate rats ' motor function of hind limbs, indicating their promising role in repairing spinal cord injury.

SVZa neural stem cells differentiate into distinct lineages in response to BMP4.

Neural stem cells (NSCs) reside in the anterior portion of the forebrain subventricular zone (SVZa) and generate the progenitors which will differentiate into neurons, and via a tangential migratory pathway, known as the rostral migratory stream (RMS), migrate to the olfactory bulbs (OB). Bone morphogenetic proteins (BMPs) play significant roles in neural development at different stages and locations, but their roles have not been determined in the SVZa. To explore possible roles of BMPs in SVZa NSCs, BMP4 at various concentrations were tested for their capacity to induce SVZa NSCs. The expression of BMP4 was also examined in living cells using a reportor vector, in which the BMP4 promotor was conjugated with red fluorescent protein (RFP). In the meantime, the differentiation of SVZa NSCs was dynamically monitored by using reportor vectors of the Nestin enhancer and the promoters of TH and GFAP. In the OB, high expression of BMP4 was found using both promoter activity analysis and in situ hybridization. However, low BMP4 expression was found in the RMS and only moderate expression of BMP4 was displayed in the SVZa. The results also demonstrated that low concentrations (1-5 ng/ml) of BMP4 promoted the proliferation of SVZa NSCs but high concentrations (10-100 ng/ml) of BMP4 inhibited this proliferation. BMP4 enhanced neuron commitment before 4 days but inhibited it after 4 days. As the antagonist of BMP4, Noggin almost completely blocked all these BMP4 responses. Thus, our findings indicate that BMP4 promotes the exit from the cell cycle and triggers the differentiation of neuron progenitors in the OB. BMP4 also promotes the proliferation of the committed neuron progenitors in the RMS, but in the SVZa, BMP4 may facilitate the commitment of NSCs into astrocytes.

Antisense Noggin oligodeoxynucleotide administration decreases cell proliferation in the dentate gyrus of adult rats.

The dentate gyrus of the hippocampus is one of few regions in the adult mammalian brain characterized by ongoing neurogenesis. It has been demonstrated that Noggin antagonizes bone morphogenetic protein-4 (BMP4) to create a niche for subventricular zone neurogenesis. We previously demonstrated that Noggin and BMP4 showed strong expression in the proliferative subgranular layer of the dentate gyrus in adult rats. To examine the action of Noggin on cell proliferation in the dentate gyrus of adult rats, we administered antisense oligodeoxynucleotide (ASODN) to Noggin by continuous infusion into the lateral ventricle of rats. Antisense-infused rats displayed significant reduction in number of bromodeoxyuridine (BrdU) labeled cells in the dentate gyrus. This indicated that endogenous Noggin activity is important for naturally occurring cell proliferation in the dentate gyrus, and perhaps neurogenesis, and is one of the many factors involved in its regulation.

[Construction and expression of adeno-associated virus vectors of Smad 6 and Smad 7 genes in human renal tubule epithelial cells].

AIM: To construct the adeno-associated virus(AAV) vectors of Smad 6 and Smad 7 genes and observe their expressions in human renal tubule epithelial cells. METHODS: The plasmids pcDNA3-Smad 6/flag and pcDNA3-Smad 7/flag were digested with BamH I and Xho I. Then the Smad 6/flag and Smad 7/flag gene fragments were cloned into plasmid pAAV-MCS, respectively to construct the recombinant pAAV-Smad 6/flag and pAAV-Smad 7/flag plasmids. The recombinant expression plasmid or pAAV-LacZ plasmid were co-transfected into the HEK 293 cells with pHelper and pAAV-RC by calcium-phosphate precipitation method. Recombinant AAV-2 viral particles were prepared from infected HEK293 cells and then were used to infect human renal tubule epithelial cells (HKCs), The expressions of Smad 6 and Smad 7 in HKCs were demonstrated by immunocytochemical staining. RESULTS: The recombinant AAV vectors of Smad 6 or Smad 7 genes were constructed and expressed in the HKCs successfully. CONCLUSION: These results indicate that AAV can deliver Smad 6 and Smad 7 genes to renal cells in-vitro, suggesting the recombinant AAV can be used for gene therapy of renal fibrosis.

Immunolocalization of estrogen receptor beta in the hypothalamic paraventricular nucleus of female mice during pregnancy, lactation and postnatal development.

Previous studies have shown that estrogen receptor beta (ERbeta) is the predominant estrogen receptor in the hypothalamic paraventricular nucleus (PVN) of mouse, mediating estrogen regulation of the neuroendocrine activities of the PVN, but the exact roles that ERbeta plays in the PVN remain unclear. In this study, we used immunocytochemistry to investigate the expression of ERbeta in the maternal PVN of mice during pregnancy (pregnant days 8, 10, 12, 15 and 18), lactation (postpartum days 1, 4 and 8) as well as in the PVN of the females from postnatal days 1, 3, 5, 7, 9, 15, 30 and 70. We found out that ERbeta was predominantly localized in the magnocellular divisions of PVN. In the pregnant female brain, generally, the ERbeta was lower than that of the postnatal development, the lowest level was found at gestational days 10-12; then from gestational day 18 to postpartum day 1, it increased to higher levels, followed by a decrease from postpartum day 4. During the postnatal development, the highest level of ERbeta was found at early postnatal days (before postnatal day 15), thereafter, it decreased to a lower level. The above results indicate that circulating sex steroids may differentially regulate the expression of ERbeta in the PVN of mice. It also suggests that this receptor may play important roles in the regulation of parturition and in the development, food intake and body weight increases of the newborns by acting on the neuropeptides, which were also detected in the PVN.

Effect of antisense oligonucleotide of noggin on spatial learning and memory of rats.

AIM: To investigate the effect of antisense oligonucleotide (ASODN) of noggin on rat spatial learning and memory. METHODS: Expression of noggin mRNA was measured by in situ hybridization method and the ability to spatial learning and memory was tested with Morris water maze. RESULTS: Compared with control rats, noggin mRNA positive neurons in dentate gyrus (DG) and CA3 region of hippocampus were markedly increased after the Morris water maze training (P<0.01). The increase of noggin mRNA positive neurons in hippocampus following maze training could be significantly blocked by icv injection of antisense noggin ODN, and the injection also impaired the learning and memory formation as compared to that in control rats. But the sense oligonucleotide (SODN) had no effect. CONCLUSION: Noggin, as an embryonic gene expressed in adult hippocampus, plays an important role in the process of learning and memory formation.

[Relationship between level of sexual hormone in external blood and aromatase expression in cancer tissues of male patients with lung cancer].

BACKGROUND & OBJECTIVE: The conversion of androgen to estrogen is catalyzed by aromatase. Evidence to date indicates aromatase is the last rate-limited enzyme in this reaction. Previous studies have suggested that there is some imbalance and abnormality of sexual hormone of patients with lung cancer, but the reason is not clear yet. This study was designed to evaluate the relationship between level of sexual hormone of external blood and aromatase expression in cancer tissues of male patients with lung cancer. METHOD: The levels of estradiol(E2) and testosterone(T) in plasma of external blood of 25 patients with lung cancer and 30 healthy men were measured by Enzyme Immunoassay Magnetic Solid Phase (IEMA). The protein expression of aromatase in 25 cancer tissues, 25 juxtacancerous tissues, and 11 tissues with benign lung disease were detected by Immunohistochemistry. RESULTS: The level of plasma E2 in external blood was significantly increased (P < 0.01) while T was significantly decreased (P < 0.01) in male patients with lung cancer. The expression of aromatase in about 52% lung cancer tissue samples was positive, but no aromatase expression in the juxtacancerous tissues and tissues with benign lung disease were found. The level of E2 had positive correlation to the expression of aromatase in male lung cancer patients(r = 0.9308, P < 0.001). CONCLUSION: It is suggested that releasing of aromatase of lung cancer tissue, which catalyzes androgen to change into estrogen, into external blood might be the major reason of the increase of E2 and the decrease of T in external blood.

[Progress in the study of CCN family].

The last 5-6 years have seen the emergence of a new gene family that currently comprises CTGF, Cyr61, nov, elm1, HICP and WISP-3. The translational products of most CCN family members are secreted proteins that contain 343 to 381 amino-acid residues to compose 4 distinct structural modules, each having 38 conserved cysteine residues. These proteins have a variety of properties to affect the cellular behaviors such as growth, differentiation, adhesion and locomotion. They may play important roles in pregnancy, development and differentiation, angiogenesis, wound repair, fibrotic disorders, inflammation and tumor genesis.

Distribution and differences of estrogen receptor beta immunoreactivity in the brain of adult male and female rats.

Studies have shown that estrogen plays important roles in regulating neural structure and function in the brain, but the mechanism remains unclear. The actions of estrogen were thought to be mediated by a single estrogen receptor until the identification of another estrogen receptor, namely estrogen receptor-beta (ER-beta). Here we report a comprehensive study of the localization of ER-beta immunoreactivity and differences in the brains of adult male and female rats on the basis of a nickel ammonium sulfate-enhanced immunocytochemical method using a polyclonal antiserum sc-8974. The results of these studies revealed: (1) ER-beta immunoactive material was mainly localized in the neuronal nucleus, but it was also detectable in the cytoplasm and neuronal processes; (2) in both male and female rats, high levels of ER-beta immunopositive signals were detected in the anterior olfactory nucleus, cerebral cortex, Purkinje cells, vertical limb of the diagonal band, red nucleus, locus ceruleus, and motor trigeminal nucleus. Moderate levels were found in the medial septum, lateral amygdaloid nucleus, substantia nigra, and central gray. Weak signals were localized in other subregions of the hypothalamus and amygdaloid complex; (3) there was an obvious difference of ER-beta immunoreactivity between male and female rats, and its intracellular distribution also showed a sex difference. The above results provide the first detailed evidence that ER-beta protein is widely distributed in both male and female rat brains, but that distinctive sex differences also exist. Estrogen may exert its function in different brain regions in a gender-specific manner.