Su-Lan Liao

Transplantation of bone marrow stromal cells for peripheral nerve repair

Cell transplantation using bone marrow stromal cells (BMSCs) to alleviate neurological deficits has recently become the focus of research in regenerative medicine. Evidence suggests that secretion of various growth-promoting substances likely plays an important role in functional recovery against neurological diseases. In an attempt to identify a possible mechanism underlying the regenerative potential of BMSCs, this study investigated the production and possible contribution of neurotrophic factors by transected sciatic nerve defect in a rat model with a 15 mm gap. Cultured BMSCs became morphologically homogeneous with fibroblast-like shape after ex vivo expansion. We provided several pieces of evidence for the beneficial effects of implanted fibroblast-like BMSCs on sciatic nerve regeneration. When compared to silicone tube control animals, this treatment led to (i) improved walking behavior as measured by footprint analysis, (ii) reduced loss of gastrocnemius muscle weight and EMG magnitude, and (iii) greater number of regenerating axons within the tube. Cultured fibroblast-like BMSCs constitutively expressed trophic factors and supporting substances, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), collagen, fibronectin, and laminin. The progression of the regenerative process after BMSC implantation was accompanied by elevated expression of neurotrophic factors at both early and later phases. These results taken together, in addition to documented Schwann cell-like differentiation, provide evidence indicating the strong association of neurotrophic factor production and the regenerative potential of implanted BMSCs.

Gliotoxic Action of Glutamate on Cultured Astrocytes

Abstract: Because of the well‐documented importance of glutamate clearance by astrocytes in protecting neurons against excitotoxicity, it was interesting to examine whether L‐glutamate exerts a toxic action on cultured astrocytes. Cell damage was evaluated by measuring activity of lactate dehydrogenase (LDH) released into the culture medium. Exposure of astrocyte cultures of the neonatal rat cerebral cortex to L‐glutamate resulted in a concentration‐ and time‐dependent increase in the release of LDH. L‐Glutamate‐induced gliotoxicity appeared to be mediated predominantly by the increase of oxidative stress because the reduced glutathione content and its effects were almost completely blocked by vitamin E and pyrrolidinedithiocarbamate. To support this notion further, the supplementation or depletion of intracellular reduced glutathione content attenuated or worsened L‐glutamate toxicity, respectively. Activation of the glutamate transporter mimicked the action of L‐glutamate on astrocytes. In addition, degrees of cell damage were not directly correlated to the levels of glutamate uptake. Moreover, the mechanism of this toxicity required energy and macromolecular synthesis. Taken together, brief exposure to L‐glutamate resulted in glutamate uptake and cell swelling, whereas sustained exposure injured astrocytes via oxidative stress instead of the excitatory mechanism.

Neuroprotection by tetramethylpyrazine against ischemic brain injury in rats

In traditional Chinese medicine, Ligusticum wallichii Franchat (Chuan Xiong) and its active ingredient tetramethylpyrazine (TMP) have been used to treat cardiovascular diseases and to relieve various neurological symptoms such as ischemic deficits. However, scientific evidence related to their effectiveness or precise modes of neuroprotective action is largely unclear. In the current study, we elicited the neuroprotective mechanisms of TMP after focal cerebral ischemic/reperfusion (I/R) by common carotid arteries and middle cerebral artery occlusion model in rats. TMP was administrated 60 min before occlusion via intraperitoneal injection. TMP concentration-dependently exhibited significant neuroprotective effect against ischemic deficits by reduction of behavioral disturbance. Neuronal loss and brain infarction in the ischemic side of rats were markedly lowered by treatment with TMP. Cerebral I/R-induced internucleosomal DNA fragmentation, caspase-8, caspase-9, and caspase-3 activation, and cytochrome c release were reduced by TMP treatment. Western blot analysis revealed the down-regulation of Bcl-2 and Bcl-xL and the up-regulation of Bax and Bad by cerebral I/R insult. Among them, only the alteration in Bcl-xL expression was reversed by TMP treatment. Moreover, the activation of microglia and/or recruitment of inflammatory cells within the ischemic side and the consequent production of monocyte chemoattractant protein 1 (MCP-1) were suppressed by TMP pre-treatment. Our findings suggest that TMP might provide neuroprotection against ischemic brain injury, in part, through suppression of inflammatory reaction, reduction of neuronal apoptosis, and prevention of neuronal loss.

Tetramethylpyrazine reduces ischemic brain injury in rats

Tetramethylpyrazine (TMP), which is widely used in the treatment of ischemic stroke by Chinese herbalists, is one of the most important active ingredients of the traditional Chinese herbal medicine, Ligusticum wallichii Franchat (Chung Xiong). However, the mechanism by which TMP protects the brain is still not clear. We examined neuroprotective effects of TMP after transient focal cerebral ischemia using common carotid artery and middle cerebral artery occlusion model in rats and evaluated the involvement of anti-inflammation. TMP administrated intraperitoneally significantly protected the brain against ischemic insult as evidenced by the reduction in infarction volume, preservation of neurons, and decrease in brain edema. TMP markedly reduced cerebral ischemia/reperfusion-induced inflammatory cell activation and proinflammatory mediator production. Moreover, TMP suppressed lipopolysaccharide/interferon-gamma-induced inflammation and prostaglandin E(2) production in cultured glial cells. Our findings suggest that one of neuroprotective effects of TMP against ischemic brain injury might involve its anti-inflammatory potential.

Upregulation of RANTES gene expression in neuroglia by Japanese encephalitis virus infection.

ABSTRACT Infection with Japanese encephalitis virus (JEV) causes cerebral inflammation and stimulates inflammatory cytokine expression. Glial cells orchestrate immunocyte recruitment to focal sites of viral infection within the central nervous system (CNS) and synchronize immune cell functions through a regulated network of cytokines and chemokines. Since immune cell infiltration is prominent, we investigated the production of a responding chemoattractant, RANTES (regulated upon activation, normal T-cell expressed and secreted), in response to JEV infection of glial cells. Infection with JEV was found to elicit the production of RANTES from primary neurons/glia, mixed glia, microglia, and astrocytes but not from neuron cultures. The production of RANTES did not seem to be directly responsible for JEV-induced neuronal death but instead contributed to the recruitment of immune cells. RANTES expression required viral replication and the activation of extracellular signal-regulated kinase (ERK) as well as transcription factors, including nuclear factor kappa B (NF-κB) and nuclear factor IL-6 (NF-IL-6). The induction of RANTES expression by JEV infection in glial cells needed the coordinate activation of NF-κB and NF-IL-6. Using enzymatic inhibitors, we demonstrated a strong correlation between the ERK signaling pathway and RANTES expression. However, JEV replication was not dependent on the activation of ERK, NF-κB, and NF-IL-6. Altogether, these results demonstrated that infection of glial cells by JEV provided the early ERK-, NF-κB-, and NF-IL-6-mediated signals that directly activated RANTES expression, which might be involved in the initiation and amplification of inflammatory responses in the CNS.

Glial activation involvement in neuronal death by Japanese encephalitis virus infection.

Japanese encephalitis is characterized by profound neuronal destruction/dysfunction and concomitant microgliosis/astrogliosis. Although substantial activation of glia is observed in Japanese encephalitis virus (JEV)-induced Japanese encephalitis, the inflammatory responses and consequences of astrocytes and microglial activation after JEV infection are not fully understood. In this study, infection of cultured neurons/glia with JEV caused neuronal death and glial activation, as evidenced by morphological transformation, increased cell proliferation and elevated tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6 and RANTES (regulated upon activation, normal T-cell expressed and secreted) production. Replication-competent JEV caused all glial responses and neurotoxicity. However, replication-incompetent JEV lost these abilities, except for the ability to change microglial morphology. The bystander damage caused by activated glia also contributed to JEV-associated neurotoxicity. Microglia underwent morphological changes, increased cell proliferation and elevated TNF-alpha, IL-1beta, IL-6 and RANTES expression in response to JEV infection. In contrast, IL-6 and RANTES expression, but no apparent morphological changes, proliferation or TNF-alpha/IL-1beta expression, was demonstrated in JEV-infected astrocytes. Supernatants of JEV-infected microglia, but not JEV-infected astrocytes, induced glial activation and triggered neuronal death. Antibody neutralization studies revealed that TNF-alpha and IL-1beta, but not RANTES or IL-6, released by activated microglia appeared to play roles in JEV-associated neurotoxicity. In conclusion, following JEV infection, neuronal death was accompanied by concomitant microgliosis and astrogliosis, and neurotoxic mediators released by JEV-activated microglia, rather than by JEV-activated astrocytes, had the ability to amplify the microglial response and cause neuronal death.

Association of serum estrogen level and ischemic neuroprotection in female rats.

Estrogen-related ischemic neuroprotection has been documented in male and ovariectomized female rats. The precise molecular mechanism underlying estrogens neuroprotective effect remains obscure. In the present study, we examined whether endogenous estrogen levels affect post-ischemic outcomes in normal cycling female rats. Occlusion of both the common carotid arteries and the right middle cerebral artery (1.5 h) followed by reperfusion (24 h) caused cortical infarction, increased neutrophil accumulation, and elevated antioxidant enzyme and lactate dehydrogenase activities. These post-ischemic changes varied in the female rats and were inversely correlated with circulating estrogen levels. More severe post-ischemic changes and injury accompanied the decline in circulating estrogen levels in normal cycling female rats, indicating that estrogen is probably the major hormonal player in female resistance to ischemia.

Inhibition of nitric oxide production by quercetin in endotoxin/cytokine-stimulated microglia

AIMS Flavonoids possess several biological and pharmacological activities. Quercetin, a naturally occurring flavonoid, has been shown to down-regulate inflammatory responses and provide neuroprotection. However, the mechanisms underlying the anti-inflammatory properties of quercetin are poorly understood. In the present study, we investigated the modulatory effect of quercetin against neuroinflammation. MAIN METHODS We herein describe a potential regulatory mechanism by which quercetin suppresses nitric oxide (NO) production by lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma)-stimulated BV-2 microglial cells. The underlying regulatory cascades were approached by biochemical and pharmacological strategies. KEY FINDINGS Quercetin produced an inhibitory effect on inducible nitric oxide synthase (iNOS) expression and NO production. Biochemical studies revealed that the anti-inflammatory effect of quercetin was accompanied by the down-regulation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38, Akt, Src, Janus kinase-1, Tyk2, signal transducer and activator of transcription-1, and NF-kappaB. In addition, quercetin scavenged free radicals and produced inhibitory effects on serine/threonine and tyrosine phosphatase activities. Intriguingly, the accumulation of lipid rafts, which is the critical step for signaling, was disrupted by quercetin. SIGNIFICANCE The data indicate that the anti-inflammatory action of quercetin may be attributable to its raft disrupting and anti-oxidant effects. These distinct mechanisms work in synergy to down-regulate iNOS expression and NO production.

Manganese modulates pro-inflammatory gene expression in activated glia.

Redox-active metals are of paramount importance for biological functions. Their impact and cellular activities participate in the physiological and pathophysiological processes of the central nervous system (CNS), including inflammatory responses. Manganese is an essential trace element and it is required for normal biological activities and ubiquitous enzymatic reactions. However, excessive chronic exposure to manganese results in neurobehavioral deficits. Recent evidence suggests that manganese neurotoxicity involves activation of microglia or astrocytes, representative CNS immune cells. In this study, we assessed the molecular basis of the effects of manganese on the modulation of pro-inflammatory cytokines and nitric oxide (NO) production in primary rat cortical glial cells. Cultured glial cells consisted of 85% of astrocytes and 15% of microglia. Within the assayed concentrations, manganese was unable to induce tumor necrosis factor alpha (TNF-alpha) and inducible nitric oxide synthase (iNOS) expression, whereas it potentiated iNOS and TNF-alpha gene expression by lipopolysaccharide/interferon-gamma-activated glial cells. The enhancement was accompanied by elevation of free manganese, generation of oxidative stress, activation of mitogen-activated protein kinases, and increased NF-kappaB and AP-1 binding activities. The potentiated degradation of inhibitory molecule IkappaB-alpha was one of underlying mechanisms for the increased activation of NF-kappaB by manganese. However, manganese decreased iNOS enzymatic activity possibly through the depletion of cofactor since exogenous tetrahydrobiopterin reversed manganeses action. These data indicate that manganese could modulate glial inflammation through variable strategies.

Prognostic and clinical implication of IL-6 expression in glioblastoma multiforme

Interleukin-6 (IL-6) is frequently produced in gliomas and has been implicated as a mediator of growth control in several human neoplasms. In this study, IL-6 expression was examined in 11 surgically resected glioblastomas and a cell line U87MG by immunohistochemical staining and quantitative real-time RT-PCR. The relationships between IL-6 expression level and clinical presentation, survival, imaging findings, age and preoperative Karnofsky performance status were analyzed. The median survival times were 16 months in patients with negative IL-6 expression and 7 months in those with positive IL-6 expression (P = 0.075). Three of these patients with a relatively longer survival time (> 1 year) did not express IL-6 in the tumor. Relatively more severe peri-focal edema on imaging was also noted in the glioblastomas with IL-6 expression. IL-6 was also found in the cytoplasm of endothelial cells of newly formed vessels and infiltrating inflammatory cells. These preliminary results implicate IL-6 expression as a possible prognostic indicator in glioblastoma. This cytokine may also play a role in tissue edema, angiogenesis and inflammation of this tumor, but whether IL-6 expression promotes malignancy is uncertain.