Wen Ta Chiu

The effect of the Taiwan motorcycle helmet use law on head injuries.

OBJECTIVES This study evaluated the effect of the motorcycle helmet law implemented in Taiwan on June 1, 1997. METHODS Collecting data on 8795 cases of motorcycle-related head injuries from 56 major Taiwanese hospitals, we compared the situation 1 year before and after implementation of the helmet law. RESULTS After implementation of the law, the number of motorcycle-related head injuries decreased by 33%, from 5260 to 3535. Decreases in length of hospital stay and in severity of injury and better outcome were also seen. The likelihood ratio chi 2 test showed that severity decreased after the laws implementation (P < .001). Full helmets were found to be safer than half-shell helmets. CONCLUSION The helmet law effectively decreased the mortality and morbidity from motorcycle-related head injuries.

In vitro stage-specific chondrogenesis of mesenchymal stem cells committed to chondrocytes

OBJECTIVE Osteoarthritis is characterized by an imbalance in cartilage homeostasis, which could potentially be corrected by mesenchymal stem cell (MSC)-based therapies. However, in vivo implantation of undifferentiated MSCs has led to unexpected results. This study was undertaken to establish a model for preconditioning of MSCs toward chondrogenesis as a more effective clinical tool for cartilage regeneration. METHODS A coculture preconditioning system was used to improve the chondrogenic potential of human MSCs and to study the detailed stages of chondrogenesis of MSCs, using a human MSC line, Kp-hMSC, in commitment cocultures with a human chondrocyte line, hPi (labeled with green fluorescent protein [GFP]). In addition, committed MSCs were seeded into a collagen scaffold and analyzed for their neocartilage-forming ability. RESULTS Coculture of hPi-GFP chondrocytes with Kp-hMSCs induced chondrogenesis, as indicated by the increased expression of chondrogenic genes and accumulation of chondrogenic matrix, but with no effect on osteogenic markers. The chondrogenic process of committed MSCs was initiated with highly activated chondrogenic adhesion molecules and stimulated cartilage developmental growth factors, including members of the transforming growth factor beta superfamily and their downstream regulators, the Smads, as well as endothelial growth factor, fibroblast growth factor, insulin-like growth factor, and vascular endothelial growth factor. Furthermore, committed Kp-hMSCs acquired neocartilage-forming potential within the collagen scaffold. CONCLUSION These findings help define the molecular markers of chondrogenesis and more accurately delineate the stages of chondrogenesis during chondrocytic differentiation of human MSCs. The results indicate that human MSCs committed to the chondroprogenitor stage of chondrocytic differentiation undergo detailed chondrogenic changes. This model of in vitro chondrogenesis of human MSCs represents an advance in cell-based transplantation for future clinical use.

Nitric oxide from both exogenous and endogenous sources activates mitochondria-dependent events and induces insults to human chondrocytes

During inflammation, overproduction of nitric oxide (NO) can damage chondrocytes. In this study, we separately evaluated the toxic effects of exogenous and endogenous NO on human chondrocytes and their possible mechanisms. Human chondrocytes were exposed to sodium nitroprusside (SNP), an NO donor, or a combination of lipopolysaccharide (LPS) and interferon‐γ (IFN‐γ) as the exogenous and endogenous sources of NO, respectively. Administration of SNP or a combination of LPS and IFN‐γ in human chondrocytes increased cellular NO levels but decreased cell viability. Exposure to exogenous or endogenous NO significantly induced apoptosis of human chondrocytes. When treated with exogenous or endogenous NO, the mitochondrial membrane potential time‐dependently decreased. Exposure to exogenous or endogenous NO significantly enhanced cellular reactive oxygen species (ROS) and cytochrome c (Cyt c) levels. Administration of exogenous or endogenous NO increased caspase‐3 activity and consequently induced DNA fragmentation. Suppression of caspase‐3 activation by Z‐DEVD‐FMK decreased NO‐induced DNA fragmentation and cell apoptosis. Similar to SNP, exposure of human chondrocytes to S‐nitrosoglutathione (GSNO), another NO donor, caused significant increases in Cyt c levels, caspase‐3 activity, and DNA fragmentation, and induced cell apoptosis. Pretreatment with N‐monomethyl arginine (NMMA), an inhibitor of NO synthase, significantly decreased cellular NO levels, and lowered endogenous NO‐induced alterations in cellular Cyt c amounts, caspase‐3 activity, DNA fragmentation, and cell apoptosis. Results of this study show that NO from exogenous and endogenous sources can induce apoptotic insults to human chondrocytes via a mitochondria‐dependent mechanism. J. Cell. Biochem. 101: 1520–1531, 2007.

Intervertebral disc regeneration in an ex vivo culture system using mesenchymal stem cells and platelet-rich plasma

An ex vivo degenerative intervertebral disc (IVD) organ culture system was established for the screening of disc regeneration agents. Its application was demonstrated by a stem cell and growth factor-based therapeutic approach for the amelioration of IVD. An ex vivo culture system using chymopapain to partially digest nucleus proposus tissue was established to mimic human IVD degeneration. This system was then used for the evaluation of different therapeutic regimens including: mesenchymal stem cell derived from eGFP-transgenic porcine (MSC-GFP), platelet-rich plasma (PRP) and MSC-GFP/PRP combined treatment, and confirmed in in vivo animal model. Chondrogenic-specific gene products including Col II and aggrecan were found upregulated and chondrogenic matrix deposition increased, as evident by sustained fluorescent signals over 4 weeks, in the MSC-GFP implanted group. Previously, we demonstrated in vitro stage-specific chondrogenesis of MSC by chondrocytic commitment. These same molecules upregulated for chondrogenesis were also observed in MSC-GFP group. PRP that has been shown to promote nucleus pulposus (NP) regeneration also resulted in significant increased levels of mRNA involved in chondrogenesis and matrices accumulation. The ex vivo IVD regeneration results were repeated and supported by in vivo porcine degenerative system. Moreover, the disc height index (DHI) was significantly increased in both in vivo MSC-GFP and PRP regeneration groups. Unexpectedly, the MSC-GFP/PRP combined therapy demonstrated an inclination towards osteogenesis in ex vivo system. The ex vivo degenerative IVD culture system described in this study could serve as an alternative and more accessible model over large animal model. This system also provides a high-throughput platform for screening therapeutic agents for IVD regeneration.

Lipoteichoic acid induces nuclear factor-κB activation and nitric oxide synthase expression via phosphatidylinositol 3-kinase, Akt, and p38 MAPK in RAW 264.7 macrophages

We previously demonstrated that lipoteichoic acid (LTA) might activate phosphatidylcholine‐phospholipase C (PC‐PLC) and phosphatidylinositol‐phospholipase C (PI‐PLC) to induce protein kinase C activation, which in turn initiates nuclear factor‐κB (NF‐κB) activation and finally induces inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) release in RAW 264.7 macrophages. In this study, we further investigated the roles of tyrosine kinase, phosphatidylinositiol 3‐kinase (PI3K)/Akt, and p38 mitogen‐activated protein kinase (MAPK) in LTA‐induced iNOS expression and NO release in RAW 264.7 macrophages. Tyrosine kinase inhibitors (genistein and tyrphostin AG126), PI3K inhibitors (wortmannin and LY 294002), and a p38 MAPK inhibitor (SB 203580) attenuated LTA‐induced iNOS expression and NO release in concentration‐dependent manners. Treatment of RAW 264.7 macrophages with LTA caused time‐dependent activations of Akt and p38 MAPK. The LTA‐induced Akt activation was inhibited by wortmannin, LY 294002, genistein, and tyrphostin AG126. The LTA‐induced p38 MAPK activation was inhibited by genistein, tyrphostin AG126, wortmannin, LY 294002, and SB 203580. The LTA‐induced formation of an NF‐κB‐specific DNA–protein complex in the nucleus was inhibited by wortmannin, LY 294002, genistein, tyrphostin AG126, and SB 203580. Treatment of macrophages with LTA caused an increase in κB‐luciferase activity, and this effect was inhibited by tyrphostin AG126, wortmannin, LY 294002, the Akt dominant negative mutant (AktDN), and SB 203580. Based on those findings, we suggest that LTA might activate the PI3K/Akt pathway through tyrosine kinase to induce p38 MAPK activation, which in turn initiates NF‐κB activation, and ultimately induces iNOS expression and NO release in RAW 264.7 macrophages.

Urbanization and stroke prevalence in Taiwan: analysis of a nationwide survey.

This study aims to explore the prevalence of strokes among individuals and the association with urbanization levels. A total sample of 9,794 individuals was obtained from a nationwide survey on Taiwan for subsequent analysis in this study. After adjusting for gender, age, other risk factors for stroke and individual socioeconomic status, a multivariate logistic regression model was employed to investigate the relationships existing between the prevalence of strokes and the level of urbanization. This study finds that those living in areas at the highest level of urbanization (level 1) had the highest prevalence of strokes (2.49%). With decreasing urbanization level, there was a general decline in stroke prevalence. After adjusting for other factors, the multivariate logistic regression analyses showed that compared to participants living in the highest urbanization level, the respective odds ratios of suffering a stroke for those living in areas at the lowest levels of urbanization (levels 7 and 8), were 0.43 and 0.30. We conclude that after adjusting for other stroke risk factors, the level of urbanization is an important contributory factor to the overall prevalence of strokes in Taiwan.

MicroRNA-210 targets antiapoptotic Bcl-2 expression and mediates hypoxia-induced apoptosis of neuroblastoma cells

MicroRNAs (miRNAs) can regulate cell survival and death by targeting apoptosis-related gene expression. miR-210 is one of the most hypoxia-sensitive miRNAs. In this study, we evaluated the roles of miR-210 in hypoxia-induced insults to neural cells. Treatment of neuro-2a cells with oxygen/glucose deprivation (OGD) induced cell apoptosis in a time-dependent manner. In parallel, OGD time-dependently increased cellular miR-210 levels. Knocking down miR-210 expression using specific antisenses significantly attenuated OGD-induced neural apoptosis. Concurrently, OGD increased hypoxia-inducible factor (HIF)-1α mRNA and protein syntheses. Pretreatment with YC-1, an inhibitor of HIF-1α, reduced OGD-caused cell death. Sequentially, OGD specifically decreased antiapoptotic Bcl-2 mRNA and protein levels in neuro-2a cells. A search by a bioinformatic approach revealed that miR-210-specific binding elements exist in the 3′-untranslated region of Bcl-2 mRNA. Application of miR-210 antisenses simultaneously alleviated OGD-involved inhibition of Bcl-2 mRNA expression. In comparison, overexpression of miR-210 synergistically diminished OGD-caused inhibition of Bcl-2 mRNA expression and consequently induced greater cellular insults. Taken together, this study shows that OGD can induce miR-210 expression through activating HIF-1α. And miR-210 can mediate hypoxia-induced neural apoptosis by targeting Bcl-2.

Contribution of reactive oxygen species to migration/invasion of human glioblastoma cells U87 via ERK-dependent COX-2/PGE2 activation

In the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulation, an increase in the migration/invasion of U87 glioblastoma cells was detected by a wound healing assay, transwell analysis, and spheroid formation assay by inducing matrix metalloproteinase-9 (MMP-9) enzyme activity via a gelatin zymographic analysis. A dose- and time-dependent increase in cyclooxygenase-2 (COX-2) gene expression with elevated prostaglandin E(2) (PGE(2)) production was identified in TPA- but not in 4alpha-TPA (a respective inactive compound)-treated U87 cells TPA-induced migration/invasion was significantly blocked by adding the COX-2-specific inhibitor, NS398, through a reduction in PGE(2) production. Data from the pharmacological studies using specific chemical inhibitors showed that activation of protein kinase C (PKC) and extracellular signal-regulated kinases (ERKs) was involved in TPA-induced migration/invasion, COX-2 protein expression, and MMP-9 activation. Stimulation of intracellular peroxide production by TPA was detected by a DCHF-DA assay, and the addition of superoxide dismutase (SOD) or tempol significantly inhibited TPA-induced migration/invasion and COX-2 protein expression accompanied by a decrease in peroxide production. An increase in NADPH oxidase activity by TPA was examined, and TPA-induced migration/invasion was blocked by adding DPI, an NADPH oxidase inhibitor. Additionally, the natural flavonoids quercetin (QE), baicalein (BE), and myricetin (ME) effectively blocked TPA-induced migration/invasion while simultaneously inhibiting COX-2/PGE(2) production, MMP-9 enzyme activity, and peroxide production in U87 cells. The contribution of ROS production to the migration/invasion of U87 glioblastoma cells via ERK-activated COX-2/PGE(2) and MMP-9 induction was first investigated here, and agents such as QE, BE, and ME with the ability to block these events possess the potential to be developed for use against migration/invasion by glioblastomas.

Curcumin Provides Neuroprotection After Spinal Cord Injury

BACKGROUND Traumatic spinal cord injury (SCI) is a major cause of long-term disability. However, therapeutic agents targeting SCI are sorely lacking. The aim of this study was to investigate whether curcumin has neuroprotective effects after SCI in rats. MATERIALS AND METHODS Studies were performed in 39 male Sprague-Dawley rats after spinal cord hemisection. The animals were randomly divided into three groups: sham, vehicle, and curcumin. The Basso, Beattie, and Bresnahan (BBB) scale was used to evaluate functional outcome. Specimens were tested for histologic, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL), and immunohistochemical staining. Primary cultured astrocytes were used to test the inhibitory effect of curcumin on glial reactivation. RESULTS The BBB scores for the affected hindlimb after hemisection were significantly improved in the curcumin-treated group compared with the vehicle group (on d 3 and 7; P < 0.001). Immunohistochemistry of NeuN revealed remarkable neuronal loss in the vehicle group after hemisection. In comparison, curcumin significantly protected neurons after SCI (curcumin compared with vehicle; P < 0.001). Furthermore, curcumin significantly attenuated apoptosis after SCI (curcumin compared with vehicle; P < 0.001). RT-PCR demonstrated that the expression of glial fibrillary acidic protein (GFAP) was significantly inhibited by curcumin. CONCLUSIONS Curcumin inhibited apoptosis and neuron loss, quenched astrocyte activation, and significantly improved neurologic deficit 7 d after spinal cord hemisection. By down-regulating GFAP expression, curcumin seems to attenuate astrocyte reactivation, which may be beneficial for neuronal survival. This is the first report demonstrating the successful treatment of SCI by curcumin.

Prevention and repair of circulatory shock and cerebral ischemia/injury by various agents in experimental heatstroke.

The current report summarized animal models of heatstroke experimentation that advance our current knowledge of therapeutic effects on cerebrovascular dysfunction, hypercoagulable state and/or systemic inflammation with various agents in the setting of heatstroke. This was a narrative review of selected published primary basic literature from MEDLINE for 1973-2006. It was found that rodents shared with humans almost the same heatstroke reactions such as hyperpyrexia, hypotension, hyperventilation, pulmonary edema, hepatic and renal failure, hypercoagulable state, metabolic acidosis, systemic inflammation, and cerebral ischemia, injury and dysfunction. Therefore, the rodent model would allow testing of new therapeutic strategies for heatstroke. It was found that brain cooling produced by infusion of cold (4 degrees C) normal saline via the jugular vein or whole body cooling improved survival during heatstroke by reducing cerebrovascular dysfunction, multiple organ failure, systemic inflammation and hypercoagulable state. However, even under the absence of brain or whole body cooling, these heatstroke reactions still could be reversed by treating with the following agents: (1) free radical scavengers; (2) human recombinant protein C: (3) platonin; (4) hyperbaric oxygen; (5) hydroxyethyl starch, hypertonic solution, or human albumin; (6) glucocorticoids; (7) interleukin-1 receptor antagonists; (8) L-arginine; (9) estrogen; and (10) human umbilical cord blood cells or CD +34 cells. Before initiation of heat stress, prior manipulations with one of the following measures were found to be able to protect against heatstroke syndromes: (1) systemic delivery of inducible nitric oxide synthase inhibitors, mu-opioid receptor antagonists, endothelin-1A receptor antagonists, dopaminergic or serotoninergic nerve depletor or receptor antagonists, or glutamate receptor antagonists; or (2) heat shock protein 72 preconditioning.