Knockdown of NADPH oxidase 4 reduces mitochondrial oxidative stress and neuronal pyroptosis following intracerebral hemorrhage.

Intracerebral hemorrhage is often accompanied by oxidative stress induced by reactive oxygen species, which causes abnormal mitochondrial function and secondary reactive oxygen species generation. This creates a vicious cycle leading to reactive oxygen species accumulation, resulting in progression of the pathological process. Therefore, breaking the cycle to inhibit reactive oxygen species accumulation is critical for reducing neuronal death after intracerebral hemorrhage. Our previous study found that increased expression of nicotinamide adenine dinucleotide phosphate oxidase 4 (NADPH oxidase 4, NOX4) led to neuronal apoptosis and damage to the blood-brain barrier after intracerebral hemorrhage. The purpose of this study was to investigate the role of NOX4 in the circle involving the neuronal tolerance to oxidative stress, mitochondrial reactive oxygen species and modes of neuronal death other than apoptosis after intracerebral hemorrhage. We found that NOX4 knockdown by adeno-associated virus (AAV-NOX4) in rats enhanced neuronal tolerance to oxidative stress, enabling them to better resist the oxidative stress caused by intracerebral hemorrhage. Knockdown of NOX4 also reduced the production of reactive oxygen species in the mitochondria, relieved mitochondrial damage, prevented secondary reactive oxygen species accumulation, reduced neuronal pyroptosis and contributed to relieving secondary brain injury after intracerebral hemorrhage in rats. Finally, we used a mitochondria-targeted superoxide dismutase mimetic to explore the relationship between reactive oxygen species and NOX4. The mitochondria-targeted superoxide dismutase mimetic inhibited the expression of NOX4 and neuronal pyroptosis, which is similar to the effect of AAV-NOX4. This indicates that NOX4 is likely to be an important target for inhibiting mitochondrial reactive oxygen species production, and NOX4 inhibitors can be used to alleviate oxidative stress response induced by intracerebral hemorrhage.

The synthesis and luminescence of europium (III) complex based on deprotonated 1-(4-ethyl-4H-thieno[3,2-b]indol-6-yl)-4,4,4-trifluorobutane-1,3-dionate and 1,10-phenanthroline.

A new ß-diketone ligand, 1-(4-ethyl-4H-thieno[3,2-b]indol-6-yl)-4,4,4-trifluoro-butane-1,3-dione(HL) was synthesized by four steps reaction (Suzuki-Miyaura cross-coupling, Cadogan cyclization, N-ethylation and Claisen condensation reaction) from 1-(4-bromo-3-nitrophenyl)ethanone and thiophen-2-ylboronic acid. Deprotonated ligand (L(-1)) and 1,10-phenanthroline (phen) coordinated to Eu(3+) to obtain a new europium (III) complex, EuL(3)(phen). The complex was characterized by elementary analysis, IR, (1)H NMR, UV-Visible absorption spectroscopy, thermogravimetric analysis (TGA) and photoluminescence (PL) measurements in detail. TGA shows that the decomposition temperature of the complex is up to 320 °C. PL measurement results indicate that the Eu(III) complex exhibit intense red-emission with the characteristic of europium ion. Red LED device was successfully fabricated by employing the complex onto 380 nm-emitting InGaN chip, which shows that the complex can act as red phosphor in combination with 380 nm-emitting chips.

Effects of differentiated versus undifferentiated adipose tissue-derived stromal cell grafts on functional recovery after spinal cord contusion.

Controversies exist concerning the need for mesenchymal stromal cells (MSCs) to be transdifferentiated prior to their transplantation. In the present study, we compared the results of grafting into the rat contused spinal cord undifferentiated, adipose tissue-derived stromal cells (uADSCs) versus ADSCs induced by two different protocols to form differentiated nervous tissue. Using Basso, Beattie, and Bresnahan scores and grid tests, we found that three cell-treated groups, including uADSCs-treated, dADSCs induced by Protocol 1 (dADSC-P1)-treated, and dADSCs induced by Protocol 2 (dADSC-P2)-treated groups, significantly improved locomotor functional recovery in SCI rats, compared with the saline-treated group. Furthermore, functional recovery was better in the uADSC-treated and dADSC-P2-treated groups than in the dADSC-P1-treated group at week 12 postinjury (P < 0.05 for dADSC-P1 group vs. uADSCs or dADSC-P2 groups). Although both protocols could induce high percentages of cells expressing neural markers in vitro, few BrdU-labeled cells survived at the injury sites in the three cell-treated groups, and only a small percentage of BrdU-positive cells expressed neural markers. On the other hand, the number of NF200-positive axons in the uADSC-treated and dADSC-P2-treated groups was significantly larger than those in the dADSC-P1-treated and saline-treated control groups. Our results indicate that ADSCs are able to differentiate into neural-like cells in vitro and in vivo. However, neural differentiated ADSCs did not result in better functional recovery than undifferentiated ones, following SCI. In vitro neural transdifferentiation of ADSCs might therefore not be a necessary pretransplantation step. Furthermore, cellular replacement or integration might not contribute to the functional recovery of the injured spinal cord.

Umbilical cord blood cell-derived neurospheres differentiate into Schwann-like cells.

In this study, we examined the phenotypic and bioassay characteristics of human umbilical cord blood-derived mesenchymal stromal cells (UCB-MSCs) differentiated along a Schwann cells lineage. Initially, we induced human UCB-MSCs into floating neurospheres, and then, neurospheres were induced to differentiate into Schwann-like cells using glia growth factors. Differentiated UCB-MSCs showed morphological changes similar to those of Schwann cells. Expression of the Schwann cell markers was determined by immunocytochemical staining and western blotting. Furthermore, differentiated UCB-MSCs could promote neurite outgrowth in coculture with dorsal root ganglia neurons. These results show that UCB-MSCs can be differentiated into cells that are Schwann-like in terms of morphology, phenotype, and function.

Adenoviral-mediated interleukin-18 expression in mesenchymal stem cells effectively suppresses the growth of glioma in rats.

Glioma is the most common primary intracranial malignant tumor. Despite advances in surgical techniques and adjuvant radio- and chemotherapies, the prognosis for patients with glioma remains poor. We have explored the effects of using genetically modified mesenchymal stem cells (MSCs) to treat malignant glioma in rats. Mesenchymal stem cells isolated from Sprague-Dawley rats can directly suppress the growth of C6 cells in vitro. MSCs transplanted intratumorally can also significantly inhibit the growth of glioma and prolong survival in C6 glioma-bearing models. MSCs producing Interleukin-18 infected by adenoviral vector inhibited glioma growth and prolonged the survival of glioma-bearing rats. Transplantation of IL-18 secreting MSCs was associated with enhanced T cell infiltration and long-term anti-tumor immunity. Thus, IL-18 may be an effective adoptive immunotherapy for malignant glioma. When used in conjunction with MSCs as targeting vehicles in vivo, IL-18 may offer a promising new treatment option for malignant glioma.

Temporal changes in the level of neurotrophins in the spinal cord and associated precentral gyrus following spinal hemisection in adult Rhesus monkeys.

Neurotrophins (NTs) appear to be crucial for the survival and potential regeneration of injured neurons. However, their temporal changes and remote regulations following spinal cord injury (SCI) have been only partially determined, especially in primates. In this study, ELISA was performed on the extracts of injured spinal cord and the associated precentral gyrus contralateral to the site of spinal cord hemisection to investigate the temporal changes in the levels of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4) in adult rhesus monkeys subjected to T8 spinal hemisection. Animals were allowed to survive 3, 7, 14, 30 and 90 days post-operation (dpo). In the spinal cord, the levels of NGF, BDNF and NT-3 sharply decreased between 3 and 7dpo. Thereafter, the levels of NGF and BDNF were transiently elevated while NT-3 level continuously increased and recovered to normal level at 30dpo. In the contralateral precentral gyrus (cPG), only the NT-3 level was altered and in fact elevated above the normal value. No obvious changes were observed in NT-4 level in any of the regions studied. Taken together, the present findings indicated that intrinsic NGF, BDNF and NT-3 may play a local role in the responses to the SCI in primates. Especially, the increase of NT-3 level occurred continuously in both the cPG and the spinal cord pointed to a possible transportation of NT-3 to the cord following SCI.

Immobilization of lipases on hydrophobilized zirconia nanoparticles: highly enantioselective and reusable biocatalysts.

Our study has demonstrated for the first time that zirconia nanoparticles modified by a simple carboxylic surfactant of a very long alkyl chain can significantly enhance the activity of the immobilized lipases for asymmetric synthesis in organic media. Zirconia nanoparticles of ca. 20 nm diameter were grafted with carboxylic surfactant modifiers from Tween 85 and erucic acid. The surface of nanoparticles was successfully changed from hydrophilic to hydrophobic. Lipases from Candida rugosa and Pseudomonas cepacia were immobilized on the modified zirconia nanoparticles by adsorption in aqueous solution. The immobilized lipases were used for the resolution of ( R, S)-ibuprofen and ( R, S)-1-phenylethanol through esterification and acylation, respectively, in isooctane organic solvent. When immobilized on erucic acid-modified zirconia, both lipases gave significantly higher activity and enantioselectivity compared with those from their corresponding crude lipase powders. The nanohybrid biocatalysts are stable and can be reused for eight cycles without loss in activity and selectivity. The interaction between the hydrophobic surface of zirconia support and lipases probably induces the conformational rearrangement of lipases into an active, stable form.

In vitro culture and induced differentiation of adult rat neural stem cells from the corpus striatum.

OBJECTIVE: To investigate the in vitro multipotential differentiation of neural stem cells from adult rat corpus striatum. METHODS: The neural stem cells isolated from adult rat corpus striatum were cultured in serum-free medium to obtain cell suspension before monoclonal subculturing and differential induction. Immunocytochemical staining and reverse transcriptional PCR (RT-PCR) were performed to identify the properties of the differentiated cells. RESULTS: Numerous cell clusters were formed in the phase of monoclonal culture, and different types of cells were observed 3 d after induction with fetal bovine serum. The differentiated cells contained cells positive for nestin, neuron-specific enolase (NSE) positive cells, and glial fibrillary acidic protein (GFAP) positive cells. RT-PCR identified expressions of the transcripts for neural cell-associated genes including brain factor-1, gamma-aminobutyric acid alpha-receptor gamma-subunit, tyrosine hydroxylase and tryptophan hydroxylase. CONCLUSION: The cells separated from adult rat corpus striatum possess the ability of self-proliferation and multipotential differentiation, and are identified as the stem cells of the central nervous system.

[Rapid construction and identification of full-length cDNA library of human glioma tissues].

OBJECTIVE: To explore a method for rapid construction of a full-length cDNA library of human glioma tissues using switching mechanism at 5' end of RNA transcript (SMART). METHODS: The total RNA was extracted from several samples of human glioma tissues and the mRNA was subsequently separated. Multiple mRNA samples were mixed to be used as the template for the first-strand cDNA synthesis. The CDS /3' PCR primer (containing Sfi IB site) was used in the first-strand reaction, and the SMART IV Oligo(dT) (containing Sfi A site) served as the short, extended template at the 5' end of the mRNA. With the above two primers, the primer-extension step generated full-length double-strand cDNA, which was digested by Sfi I restriction enzyme and ligated to the Sfi I A & B -digested lambdaTriplEx2 vector. The ligated vector was then packaged by lambda packaging extract for the final construction of the cDNA library. RESULTS: The unamplified human glioma cDNA library consisted of 2.4x10(6) independent clones with a recombination rate of 100%. The titer of the amplified cDNA library was 4.5x10(9) pfu/ml, and the average exogenous inserts of the recombinants was 1.2 kb in length. CONCLUSION: A high-quality full-length cDNA library of human gliomas was constructed successfully, which may facilitate further study of the screening and cloning of new tumor suppressor genes and tissue-specific genes of human glioma.

[Relationship between aquaporin-4 expression in astrocytes and brain edema caused by glioma].

OBJECTIVE: To investigate the relationship between aquaporin-4 (AQP4) water channel expression in astrocytes and brain edema caused by glioma. METHODS: The changes of water transport in in vitro blood-brain barrier models were examined using high-performance liquid chromatography (HPLC), and the expression level of AQP4 was assayed by semiquantitative RT-PCR. RESULTS: The water transport of the in vitro blood-brain barrier model from the luminal side to basolateral side was increased after coculture with glioma cells, which induced significantly decreased expression level of AQP4 in the astrocytes. CONCLUSIONS: Glioma cells can promote water transport in in vitro blood-brain barrier model from the luminal side to abluminal side, and the brain edema induced by the glioma cells may not necessarily be the result of hyperpermeability of the blood-brain barrier to macromolecules in the plasma. The decreased expression level of AQP4 induced by glioma cells may be a molecular mechanism for neoplastic brain edema in patients with glioma.

[Effect of hyperthermia on tight junctions between endothelial cells of the blood-brain barrier model in vitro].

OBJECTIVE: To investigate the effect of hyperthermia in vitro on tight junctions between the endothelial cells of the blood-brain barrier. METHODS: An in vitro blood-brain barrier model was established by coculture of ECV304 cells with astrocytes. Transendothelial resistance (TER) of in vitro blood-brain barrier was determined by Millicell-ERS system, and the morphological change of tight junctions examined by silver staining. The expression level of zonula occluden-1(ZO-1) was analyzed by semi-quantitative reverse transcriptase-PCR. RESULTS: The integrity of tight junctions was disrupted and the expression level of ZO-1 decreased after treatment with hyperthermia. CONCLUSIONS: Tight junctions between the endothelial cells of the blood-brain barrier can be destroyed by hyperthermia, and decreased expression level of ZO-1 induced by hyperthermia is one of the most important molecular mechanisms.