Chung Shi Yang

Sustained intraspinal delivery of neurotrophic factor encapsulated in biodegradable nanoparticles following contusive spinal cord injury

Glial cell line derived neurotrophic factor (GDNF) induces neuronal survival and tissue repair after spinal cord injury (SCI). A continuous GDNF supply is believed to gain greater efficacy in the neural restoration of the injured spinal cord. Accordingly, nanovehicle formulation for their efficient delivery and sustained release in injured spinal cord was examined. We first used fluorescence-labeled bovine serum albumin (FBSA) loaded in biodegradable poly(lactic acid-co-glycolic acid) (PLGA) for intraspinal administration after SCI and for in vitro study. Our results showed that the preservation of PLGA-FBSA was observed in the injured spinal cord at 24h, and PLGA-FBSA nanoparticles were well absorbed by neurons and glia, indicating that PLGA as a considerable nanovehicle for the delivery of neuroprotective polypeptide into injured spinal cord. Furthermore, intraspinal injection of GDNF encapsulated in PLGA (PLGA-GDNF) nanoparticles into the injured spinal cord proximal to the lesion center had no effect on gliosis when compared to that observed in SCI rats receiving PLGA injection. However, local administration of PLGA-GDNF effectively preserved neuronal fibers and led to the hindlimb locomotor recovery in rats with SCI, providing a potential strategy for the use of PLGA-GDNF in the treatment of SCI.

Inhibition of cadmium‐induced oxidative injury in rat primary astrocytes by the addition of antioxidants and the reduction of intracellular calcium

Exposure of the brain to cadmium ions (Cd2+) is believed to lead to neurological disorders of the central nervous system (CNS). In this study, we tested the hypothesis that astrocytes, the major CNS‐supporting cells, are resistant to Cd2+‐induced injury compared with cortical neurons and microglia (CNS macrophages). However, treatment with CdCl2 for 24 h at concentrations higher than 20 µM substantially induced astrocytic cytotoxicity, which also resulted from long‐term exposure to 5 µM of CdCl2. Intracellular calcium levels were found to rapidly increase after the addition of CdCl2 into astrocytes, which led to a rise in reactive oxygen species (ROS) and to mitochondrial impairment. In accordance, preexposure to the extracellular calcium chelator EGTA effectively reduced ROS production and increased survival of Cd2+‐treated astrocytes. Adenovirus‐mediated transfer of superoxide dismutase (SOD) or glutathione peroxidase (GPx) genes increased survival of Cd2+‐exposed astrocytes. In addition, increased ROS generation and astrocytic cell death due to Cd2+ exposure was inhibited when astrocytes were treated with the polyphenolic compound ellagic acid (EA). Taken together, Cd2+‐induced astrocytic cell death resulted from disrupted calcium homeostasis and an increase in ROS. Moreover, our findings demonstrate that enhancement of the activity of intracellular antioxidant enzymes and supplementation with a phenolic compound, a natural antioxidant, improves survival of Cd2+‐primed astrocytes. This information provides a useful approach for treating Cd2+‐induced CNS neurological disorders. J. Cell. Biochem. 103: 825–834, 2008.

Microglial phagocytosis attenuated by short-term exposure to exogenous ATP through P2X7 receptor action

Microglia, the CNS resident macrophages responsible for the clearance of degenerating cellular fragments, are essential to tissue remodeling and repair after CNS injury. ATP can be released in large amounts after CNS injury and may mediate microglial activity through the ionotropic P2X and the metabotropic P2Y receptors. This study indicates that exposure to a high concentration of ATP for 30u2003min rapidly induces changes of the microglial cytoskeleton, and significantly attenuates microglial phagocytosis. A pharmacological approach showed that ATP‐induced inhibition of microglial phagocytotic activity was due to P2X7R activation, rather than that of P2YR. Activation of P2X7R by its agonist, 2′‐3′‐O‐(4‐benzoyl)benzoyl‐ATP (BzATP), produced a Ca2+‐independent reduction in microglial phagocytotic activity. In addition, the knockdown of P2X7R expression by lentiviral‐mediated shRNA interference or the blockade of P2X7R activation by the specific antagonists, oxidized ATP (oxATP) and brilliant blue G, has efficiently restored the phagocytotic activity of ATP and BzATP‐treated microglia. Our results reveal that P2X7R activation may induce the formation of a Ca2+‐independent signaling complex, which results in the reduction of microglial phagocytosis. This suggests that exposure to ATP for a short‐term period may cause insufficient clearance of tissue debris by microglia through P2X7R activation after CNS injury, and that blockade of this receptor may preserve the phagocytosis of microglia and facilitate CNS tissue repair.

Effects of Combinatorial Treatment with Pituitary Adenylate Cyclase Activating Peptide and Human Mesenchymal Stem Cells on Spinal Cord Tissue Repair

The aim of this study is to understand if human mesenchymal stem cells (hMSCs) and neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) have synergistic protective effect that promotes functional recovery in rats with severe spinal cord injury (SCI). To evaluate the effect of delayed combinatorial therapy of PACAP and hMSCs on spinal cord tissue repair, we used the immortalized hMSCs that retain their potential of neuronal differentiation under the stimulation of neurogenic factors and possess the properties for the production of several growth factors beneficial for neural cell survival. The results indicated that delayed treatment with PACAP and hMSCs at day 7 post SCI increased the remaining neuronal fibers in the injured spinal cord, leading to better locomotor functional recovery in SCI rats when compared to treatment only with PACAP or hMSCs. Western blotting also showed that the levels of antioxidant enzymes, Mn-superoxide dismutase (MnSOD) and peroxiredoxin-1/6 (Prx-1 and Prx-6), were increased at the lesion center 1 week after the delayed treatment with the combinatorial therapy when compared to that observed in the vehicle-treated control. Furthermore, in vitro studies showed that co-culture with hMSCs in the presence of PACAP not only increased a subpopulation of microglia expressing galectin-3, but also enhanced the ability of astrocytes to uptake extracellular glutamate. In summary, our in vivo and in vitro studies reveal that delayed transplantation of hMSCs combined with PACAP provides trophic molecules to promote neuronal cell survival, which also foster beneficial microenvironment for endogenous glia to increase their neuroprotective effect on the repair of injured spinal cord tissue.

Valproic acid attenuates microgliosis in injured spinal cord and purinergic P2X4 receptor expression in activated microglia

Peripheral injection with a high dose of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, into animals with mild or moderate spinal cord injury (SCI) for 1 week can reduce spinal cord tissue loss and promote hindlimb locomotor recovery. A purinergic adenosine triphosphate (ATP) receptor subtype, P2X4 receptor (P2X4R), has been considered as a potential target to diminish SCI‐associated inflammatory responses. In this study, using a minipump‐based infusion system, we found that intraspinal infusion with VPA for 3 days into injured spinal cord significantly improved hindlimb locomotion of rats with severe SCI induced by a 10‐g NYU impactor dropping from the height of 50 mm onto the spinal T9/10 segment. The neuronal fibers in the injured spinal cord tissues were significantly preserved in VPA‐treated rats compared with those observed in vehicle‐treated animals. Moreover, the accumulation of microglia/macrophages and astrocytes in the injured spinal cord was attenuated in the animal group receiving VPA infusion. VPA also significantly reduced P2X4R expression post‐SCI. Furthermore, in vitro study indicated that VPA, but not the other HDAC inhibitors, sodium butyrate and trichostatin A (TSA), caused downregulation of P2X4R in microglia activated with lipopolysaccharide (LPS). Moreover, p38 mitogen‐activated protein kinase (MAPK)‐triggered signaling was involved in the effect of VPA on the inhibition of P2X4R gene expression. In addition to the findings from others, our results also provide important evidence to show the inhibitory effect of VPA on P2X4R expression in activated microglia, which may contribute to reduction of SCI‐induced gliosis and subsequently preservation of spinal cord tissues.

Determination of extracellular glutathione in livers of anaesthetized rats by microdialysis with on-line high-performance liquid chromatography

An on-line analytical system for the continuous in vivo monitoring of extracellular glutathione (gamma-glutamyl-cysteinylglycine) (GSH) concentrations in the livers of anaesthetized rats was developed. Microdialysates perfused through implanted microdialysis probes were collected with a loading loop of an on-line injector for direct and automatic injection into a high-performance liquid chromatographic system equipped with an electrochemical detector with Au-Hg electrodes. This method shortened the analysis time and circumvented the sample preparation process which is essential for accurate determination of GSH levels in biological samples. Additionally, this method provided continuous and real-time monitoring of extracellular GSH levels. Basal extracellular GSH concentrations in the livers of anaesthetized rate were found to vary over a wide range (from 4.16 to 76.5 microM). The method was applied to study the effect of global liver ischaemia on extracellular GSH concentrations and it was found that extracellular GSH levels in livers increased immediately with the onset of ischaemia and remained elevated for the 30-min ischaemic period. Ensuing reperfusion did reduce the GSH increase; however, the GSH levels did not return to the basal value.

Oxidative stress-induced attenuation of thrombospondin-1 expression in primary rat Astrocytes

Astrocytes, the major glial population in the central nervous system (CNS), can secrete thrombospondin (TSP)‐1 that plays the role in synaptogenesis and axonal sprouting during CNS development and tissue repair. However, little is known about the regulation of TSP‐1 expression in astrocytes under oxidative stress condition. Here, a hypoxic mimetic reagent, cobalt chloride (CoCl2), was used to initiate hypoxia‐induced oxidative stress in primary rat astrocytes. CoCl2 at the concentration range of 0.1–0.5u2009mM was found to cause no significant cell death in primary rat astrocytes. However, CoCl2 at 0.2–0.5u2009mM increased intracellular reactive oxygen species (ROS) levels and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) gene expression that is known as a hallmark for oxidative damage. We further found that TSP‐1 mRNA expression in astrocytes was inhibited dose‐ and time‐dependently by CoCl2. TSP‐1 mRNA levels were increased in CoCl2‐exposed astrocytes in the presence of the inhibitors (U0126 and PD98059) of mitogen‐activated protein kinase/extracellular signal‐regulated kinases (MAPK/ERK), when compared to that detected in the culture only exposed to CoCl2. Moreover, the inhibition in TSP‐1 mRNA expression by CoCl2 was blocked by the addition of the potent antioxidant, N‐acetylcysteine (NAC). Thus, we conclude that CoCl2 inhibits TSP‐1 mRNA expression in astrocytes via a ROS mechanism possibly involving MAPK/ERK. This inhibition may occur after CNS injury and impair the supportive function of astrocytes on neurite growth in the injured CNS tissues. J. Cell. Biochem. 112: 59–70, 2011.

Induced interleukin-33 expression enhances the tumorigenic activity of rat glioma cells

BACKGROUNDnGlioma development is a multistep process associated with progressive genetic alterations but also regulated by cellular and noncellular components in a tumor-associated niche.nnnMETHODSnUsing 2 rat C6 glioma cell clones with different tumorigenesis, named C6-1 and C6-2, this study characterized genes associated with enhanced tumorigenic features of glioma cells by comparative cDNA microarray analysis combined with Q-PCR. Neurospehere formation and clonogenicity were examined to determine the growth of tumorigenic C6 glioma cells. The lentivirus-mediated gene knockdown approach was conducted to determine the role of interleukin-33 (IL-33) in glioma cell proliferation and migration. Transwell cell invasion assay was used to examine microglia migration induced by tumorigenic C6 cells.nnnRESULTSnThe functional analysis of gene ontology (GO) biological processes shows that the upregulated genes found in tumorigenic C6 (C6-1) cells are closely related to cell proliferation. Tumorigenic C6 cells expressed cytokines and chemokines abundantly. Among these genes, IL-33 was profoundly induced in tumorigenic C6 cells with the expression of IL-33 receptor ST2. Furthermore, the growth rate and colony formation of tumorigenic C6 cells were attenuated by the inhibition of IL-33 and ST2 gene expression. Moreover, IL-33 was involved in tumorigenic glioma cell migration and regulation of the expression of several glioma-associated growth factors and chemokines in tumorigenic C6 cells.nnnCONCLUSIONnAccordingly, we concluded that glioma cells with abundant production of IL-33 grow rapidly; moreover, the interactions of multiple cytokines/chemokines induced by glioma cells may develop a microenvironment that facilitates microglia/macrophage infiltration and fosters glioma growth in the brain.

Ca2+‐dependent reduction of glutamate aspartate transporter GLAST expression in astrocytes by P2X7 receptor‐mediated phosphoinositide 3‐kinase signaling

J. Neurochem. (2010) 10.1111/j.1471‐4159.2010.06589.x

Reduction in antioxidant enzyme expression and sustained inflammation enhance tissue damage in the subacute phase of spinal cord contusive injury.

BackgroundTraumatic spinal cord injury (SCI) forms a disadvantageous microenvironment for tissue repair at the lesion site. To consider an appropriate time window for giving a promising therapeutic treatment for subacute and chronic SCI, global changes of proteins in the injured center at the longer survival time points after SCI remains to be elucidated.MethodsThrough two-dimensional electrophoresis (2DE)-based proteome analysis and western blotting, we examined the differential expression of the soluble proteins isolated from the lesion center (LC) at day 1 (acute) and day 14 (subacute) after a severe contusive injury to the thoracic spinal cord at segment 10. In situ apoptotic analysis was used to examine cell apoptosis in injured spinal cord after adenoviral gene transfer of antioxidant enzymes. In addition, administration of chondroitinase ABC (chABC) was performed to analyze hindlimb locomotor recovery in rats with SCI using Basso, Beattie and Bresnahan (BBB) locomotor rating scale.ResultsOur results showed a decline in catalase (CAT) and Mn-superoxide dismutase (MnSOD) found at day 14 after SCI. Accordingly, gene transfer of SOD was introduced in the injured spinal cord and found to attenuate cell apoptosis. Galectin-3, β-actin, actin regulatory protein (CAPG), and F-actin-capping protein subunit β (CAPZB) at day 14 were increased when compared to that detected at day 1 after SCI or in sham-operated control. Indeed, the accumulation of β-actin+ immune cells was observed in the LC at day 14 post SCI, while most of reactive astrocytes were surrounding the lesion center. In addition, chondroitin sulfate proteoglycans (CSPG)-related proteins with 40-kDa was detected in the LC at day 3-14 post SCI. Delayed treatment with chondroitinase ABC (chABC) at day 3 post SCI improved the hindlimb locomotion in SCI rats.ConclusionsOur findings demonstrate that the differential expression in proteins related to signal transduction, oxidoreduction and stress contribute to extensive inflammation, causing time-dependent spread of tissue damage after severe SCI. The interventions by supplement of anti-oxidant enzymes right after SCI or delayed administration with chABC can facilitate spinal neural cell survival and tissue repair.