Dann-Ying Liou

Recovery of neurological function of ischemic stroke by application of conditioned medium of bone marrow mesenchymal stem cells derived from normal and cerebral ischemia rats

BackgroundSeveral lines of evidence have demonstrated that bone marrow-derived mesenchymal stem cells (BM-MSC) release bioactive factors and provide neuroprotection for CNS injury. However, it remains elusive whether BM-MSC derived from healthy donors or stroke patients provides equal therapeutic potential. The present work aims to characterize BM-MSC prepared from normal healthy rats (NormBM-MSC) and cerebral ischemia rats (IschBM-MSC), and examine the effects of their conditioned medium (Cm) on ischemic stroke animal model.ResultsIsolated NormBM-MSC or IschBM-MSC formed fibroblastic like morphology and expressed CD29, CD90 and CD44 but failed to express the hematopoietic marker CD34. The number of colony formation of BM-MSC was more abundant in IschBM-MSC than in NormBM-MSC. This is in contrast to the amount of Ficoll-fractionated mononuclear cells from normal donor and ischemic rats. The effect of cm of BM-MSC was further examined in cultures and in middle cerebral artery occlusion (MCAo) animal model. Both NormBM-MSC Cm and IschBM-MSC Cm effectively increased neuronal connection and survival in mixed neuron-glial cultures. In vivo, intravenous infusion of NormBM-MSC Cm and IschBM-MSC Cm after stroke onset remarkably improved functional recovery. Furthermore, NormBM-MSC Cm and IschBM-MSC Cm increased neurogenesis and attenuated microglia/ macrophage infiltration in MCAo rat brains.ConclusionsOur data suggest equal effectiveness of BM-MSC Cm derived from ischemic animals or from a normal population. Our results thus revealed the potential of BM-MSC Cm on treatment of ischemic stroke.

Dual effect of adenovirus‐mediated transfer of BMP7 in mixed neuron‐glial cultures: Neuroprotection and cellular differentiation

Bone morphogenetic proteins (BMPs), members of the TGF‐β superfamily, have been implicated in nervous system development and in response to injury. Previous studies have shown that recombinant BMP7 can enhance dendritic growth and protect cultured neurons from oxidative stress. Because of the presence of extracellular BMP antagonists, BMP7 seems to act locally. Therefore, the present study uses BMP7 overexpression using adenovirus (Ad)‐mediated gene transfer to examine its effect in mixed neuronal cultures. Enhanced BMP7 expression selectively induces neuronal CGRP expression in a time‐dependent manner. BMP7 overexpression not only significantly protects cultures from H2O2 toxicity but reduces lipopolysaccharide (LPS) stimulation. Concurrently, it profoundly reduces microglial numbers, but increases oligodendroglial and endothelial cells. Together, low‐dose and continuously expressed BMP7 is both neuroprotective and differentiation‐inductive.

Adenoviral gene transfer of bone morphogenetic protein-7 enhances functional recovery after sciatic nerve injury in rats

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-β subfamily, function as instructive signals for neuronal lineage commitment and promote neuronal differentiation. However, the mechanism of BMP7 action in vivo after peripheral nerve injury is poorly understood. This study examines the efficacy of gene transfer of adenoviral (Ad) BMP7 on peripheral neuropathy. Transgene expression was found in both Ad-infected sciatic nerves and their respective remote neurons, indicating Ad transduction by a retrograde transport. After AdBMP7 infection to nerves, the sciatic nerves were crushed or transected. Hind limb functional behavior, including rotarod test and sciatic functional index, were conducted in rats weekly after nerve injury. Interestingly, enhanced BMP7 expression significantly improved hind limb functional recovery in AdBMP7-transduced rats when compared with AdGFP-transduced nerve-crushed or transected rats. Furthermore, AdBMP7 transduction reduced injury-induced macrophage activation, nerve demyelination and axonal degeneration. By contrast, AdBMP7 infection did not affect the hyperalgesia paw-withdrawal latency after nerve injury. We further examined the effect of AdBMP7 infection on sciatic nerve explant and Schwann cell cultures. Enhanced cell proliferation was significantly increased by AdBMP7 transduction in both cultures. Taken together, BMP7 overexpression by Ad gene transfer was beneficial in both nerves and Schwann cells on functional recovery after sciatic nerve injury in rats.

Acidic FGF promotes neurite outgrowth of cortical neurons and improves neuroprotective effect in a cerebral ischemic rat model

Acidic fibroblast growth factor (aFGF) is a neurotrophic factor which is a powerful neuroprotective and neuroregenerative factor of the nervous system. Prior study had shown that levels of FGFs significantly increase following ischemic injury, reflecting a physiological protection mechanism. However, few reports demonstrated the efficacy of applying aFGF in cerebral ischemia. A recent report showed that the intranasal aFGF treatment improved neurological functional recovery; however, it did not significantly reduce the lesion size in ischemic rats. The present study examines the neuroprotective effect of aFGF on cortical neuron-glial cultures under oxygen glucose deprivation (OGD)-induced cell damage and investigates whether epidural application of slow-released aFGF could improve benefit on ischemic stroke injury in conscious rats. We used a topical application of aFGF mixed in fibrin glue, a slow-release carrier, over the peri-ischemic cortex and examined such treatment on cerebral infarction and behavioral impairments of rats subjected to focal cerebral ischemia (FCI). Results demonstrate that aFGF effectively protected cortical neuron-glial cultures from OGD-induced neuronal damage. Neurite extension from cortical neurons was significantly enhanced by aFGF, mediated through activation of AKT and ERK. In addition, topical application of fibrin glue-mixed aFGF dose-dependently reduced ischemia-induced brain infarction and improved functional restoration in ischemic stroke rats. Slow-released aFGF not only protected hippocampal and cortical cell loss but reduced microglial infiltration in FCI rats. Our results suggest that aFGF mixed in fibrin glue could prolong the protective/regenerative efficacy of aFGF to the damaged brain tissue and thus improve the functional restorative effect of aFGF.

Effect of Enhanced Prostacyclin Synthesis by Adenovirus‐Mediated Transfer on Lipopolysaccharide Stimulation in Neuron‐Glia Cultures

Abstract: Prostacyclin (PGI2) is known as a short‐lived, potent vasodilator and platelet anti‐aggregatory eicosanoid. This work attempts to selectively augment PGI2 synthesis in neuron‐glia cultures by adenoviral (Ad) gene transfer of PGI synthase (PGIS) or bicistronic cyclooxygenase 1 (COX‐1)/PGIS and examines whether PGI2 confers protection against lipopolysaccharide (LPS) stimulation. Cultures released low levels of eicosanoids. Upon Ad‐PGIS or Ad‐COX‐1/PGIS infection, cultures selectively increased prostacyclin release. Both PGIS‐ and COX‐1/PGIS‐overexpressed cultures contained fewer microglial numbers. Further, they significantly attenuated LPS‐induced iNOS expression and lactate, nitric oxide, and TNF‐α production. Taken together, enhanced prostacyclin synthesis in neuron‐glial cultures reduced microglia number and suppressed LPS stimulation.

Enhanced Prostacyclin Synthesis by Adenoviral Gene Transfer Reduced Glial Activation and Ameliorated Dopaminergic Dysfunction in Hemiparkinsonian Rats

Prostacyclin (PGI2), a potent vasodilator and platelet antiaggregatory eicosanoid, is cytoprotective in cerebral circulation. It is synthesized from arachidonic acid (AA) by the sequential action of cyclooxygenase- (COX-) 1 or 2 and prostacyclin synthase (PGIS). Because prostacyclin is unstable in vivo, PGI2 analogs have been developed and demonstrated to protect against brain ischemia. This work attempts to selectively augment PGI2 synthesis in mixed glial culture or in a model of Parkinsons disease (PD) by direct adenoviral gene transfer of prostacyclin biosynthetic enzymes and examines whether it confers protection in cultures or in vivo. Confluent mixed glial cultures actively metabolized exogenous AA into PGE2 and PGD2. These PGs were largely NS398 sensitive and considered as COX-2 products. Gene transfer of AdPGIS to the cultures effectively shunted the AA catabolism to prostacyclin synthesis and concurrently reduced cell proliferation. Furthermore, PGIS overexpression significantly reduced LPS stimulation in cultures. In vivo, adenoviral gene transfer of bicistronic COX-1/PGIS to substantia nigra protected 6-OHDA- induced dopamine depletion and ameliorated behavioral deficits. Taken together, this study shows that enhanced prostacyclin synthesis reduced glial activation and ameliorated motor dysfunction in hemiparkinsonian rats. Prostacyclin may have a neuroprotective role in modulating the inflammatory response in degenerating nigra-striatal pathway.

Enhanced expression of glycine N-methyltransferase by adenovirus-mediated gene transfer in CNS culture is neuroprotective

Glycine N‐methyltransferase (GNMT) is the most abundant hepatic methyltransferase and plays important roles in regulating methyl group metabolism. In the central nervous system, GNMT expression is low and its function has not been revealed. The present study examines the effect of GNMT overexpression by adenovirus‐mediated transfer in cortical mixed neuron‐glial cultures. Infection of adenovirus encoding green fluorescence protein to cultures demonstrates high preference for non‐neuronal cells. Optimal GNMT overexpression in cultures by adenoviral GNMT (Ad‐GNMT) infection not only induces protein kinase C phosphorylation, but also increases neuronal/oligodendroglial survival. Furthermore, these Ad‐GNMT‐infected cultures are significantly resistant to H2O2 toxicity and lipopolysaccharide stimulation. Conditioned media from Ad‐GNMT‐infected microglia also significantly enhance neuronal survival. Taken together, enhanced GNMT expression in mixed neuronal‐glial cultures is neuroprotective, most likely mediated through non‐neuronal cells.

Cytoprotective and anti-inflammatory effects of PAL31 overexpression in glial cells

BackgroundAcute spinal cord injury (SCI) leads to a series of reactive changes and causes severe neurological deficits. A pronounced inflammation contributes to secondary pathology after SCI. Astroglia respond to SCI by proliferating, migrating, and altering phenotype. The impact of reactive gliosis on the pathogenesis of SCI is not fully understood. Our previous study has identified an inflammatory modulating protein, proliferation related acidic leucine-rich protein (PAL31) which is upregulated in the microglia/macrophage of injured cords. Because PAL31 participates in cell cycle progression and reactive astroglia often appears in the injured cord, we aim to examine whether PAL31 is involved in glial modulation after injury.ResultsEnhanced PAL31 expression was shown not only in microglia/macrophages but also in spinal astroglia after SCI. Cell culture study reveal that overexpression of PAL31 in mixed glial cells or in C6 astroglia significantly reduced LPS/IFNγ stimulation. Further, enhanced PAL31 expression in C6 astroglia protected cells from H2O2 toxicity; however, this did not affect its proliferative activity. The inhibiting effect of PAL31 on LPS/IFNγ stimulation was observed in glia or C6 after co-culture with neuronal cells. The results demonstrated that the overexpressed PAL31 in glial cells protected neuronal damages through inhibiting NF-kB signaling and iNOS.ConclusionsOur data suggest that PAL31upregulation might be beneficial after spinal cord injury. Reactive gliosis might become a good target for future therapeutic interventions.