Valproic acid ameliorates inflammation in experimental autoimmune encephalomyelitis rats.

Valproic acid (VPA) is a short-chain branched fatty acid with anti-inflammatory, neuro-protective and axon remodeling effects. Here we have studied effects of VPA in gpMBP(68-84)-induced experimental autoimmune encephalomyelitis (EAE). Both preventive (from Day 0 to Day 18) and therapeutic (from Day 7 to Day 18 or from Day 9 to Day 19) VPA (500 mg/kg, intra-gastric) administration to EAE rats once daily greatly reduced the severity and duration of EAE, and suppressed mRNA levels of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-17, matrix metalloproteinase 9 (MMP9), inducible nitric oxide synthase (iNOS) and transcription factor T-bet, but increased levels of IL-4 mRNA in EAE spinal cords. Furthermore, preventive VPA treatment greatly attenuated accumulation of macrophages and lymphocytes in EAE spinal cords. VPA treatment altered the cytokine milieu of lymph nodes, modulating the Th profile from Th1 and Th17 to a profile of Th2 and regulatory T cells. In addition, in vitro study showed that VPA inhibited non-specific lymphocyte proliferation in a dose-dependent manner. In summary, our data demonstrated that VPA could suppress systemic and local inflammation to improve outcome of EAE, suggesting that VPA might be a candidate for treatment of multiple sclerosis.

Aptamers: selection, modification and application to nervous system diseases.

Aptamers are nonnaturally occurring oligonucleotides generated by the SELEX (Systematic Evolution of Ligands by Exponential enrichment) process. Due to their unique three-dimensional structures, aptamers can bind to various targets, ranging from small compounds to cells and tissues, with high affinity and specificity. While first reported in 1990, aptamers have become useful tools in the biomedical field because of their unique characteristics, such as easy and quick preparation, cost-effectiveness, small size, versatility, et al. Recently various chemical modifications have been introduced to enhance aptamers' stability in the body fluids and their bioavailability in animals, which have pushed aptamer closer to therapeutic and diagnostic application. This review provides an overview of the aptamer modifications and their application in the nervous system disorders.

Histone acetylation patterns of typical and atypical pituitary adenomas indicate epigenetic shift of these tumours.

Pituitary adenomas are benign endocrine tumours of the anterior pituitary that are subclassified as typical (conventional) or atypical adenomas, with uncertain prognosis based on histopathological features. Clarifying epigenetic alterations of pituitary tumours, as well as the mechanisms underlying them, will hopefully open new windows to treatment and the classification of these tumours and maybe even prediction of patient survival. In the present study, using immunohistochemistry, we investigated the acetylation pattern of histone 3 lysine 9 (H3K9), an epigenetic marker of active chromatin state and gene transcription, in typical and atypical pituitary adenomas and the normal pituitary. We observed a significant increase in H3K9 acetylation from the normal pituitary to typical and atypical pituitary adenomas, which was associated with significant hyperacetylation of H3K9 in atypical adenomas (P < 0.0001). MIB-1 (Ki-67) overexpression was also highly associated with increased acetylation of H3K9, correlating prositively with tumour severity (P < 0.0001). p53 overexpression had a contributing effect on altered global H3K9 acetylation of atypical pituitary adenomas (P < 0.05). These data suggests that H3K9 acetylation status might serve as a relevant additional biomarker of tumour severity in pituitary adenomas, and also as a proper target for epigenetic-based therapies.

Sphingosine 1-phosphate receptor modulator FTY720 suppresses rat experimental autoimmune prostatitis.

Experimental autoimmune prostatitis (EAP) is an autoimmune inflammatory disease of male sex accessory glands and is characterized by a cellular and humoral prostate-specific autoimmune response associated with inflammatory mononuclear cell (MNC) infiltration. EAP shares important clinical and immunological features with human chronic prostatitis and chronic pelvic pain syndrome. Using immunohistochemistry, we determined the pattern of pathological changes during rat EAP development, regarding the cellular responses and infiltration into the prostate gland, and therapeutic effect of FTY720, a modulator of sphingosine-1-phosphate receptors which has shown promising protective effects in animal models and clinical trials of several autoimmune diseases. Significant accumulations of total MNCs, pan-T cells and CD8(+) cells were observed in prostatic stroma as early as 11 days after autoimmune induction. However, accumulation of reactive macrophages became significant 4 days later. After reaching the maximal level at Day 16, the accumulations of all the different cell populations fell back rapidly and returned to normal level by Day 35. Suppressive FTY720 significantly reduced inflammatory infiltration of different immune cell populations and tissue disruption in prostate of EAP rats. Our results therefore suggest that FTY720 might be a potential candidate for treatment of inflammatory prostatitis.

MS-275, an histone deacetylase inhibitor, reduces the inflammatory reaction in rat experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN) is a T cell-mediated autoimmune inflammatory demyelinating disease of the peripheral nervous system and serves as the animal model of human inflammatory demyelinating polyradiculoneuropathies. MS-275, a potent histone deacetylase inhibitor currently undergoing clinical investigations for various malignancies, has been reported to demonstrate promising anti-inflammatory activities. In our present study, MS-275 administration (3.5 mg/kg i.p.) to EAN rats once daily from the appearance of first neurological signs greatly reduced the severity and duration of EAN and attenuated local accumulation of macrophages, T cells and B cells, and demyelination of sciatic nerves. Further, significant reduction of mRNA levels of pro-inflammatory interleukin-1beta, interferon-gamma, interleukine-17, inducible nitric oxide synthase and matrix metalloproteinase-9 was observed in sciatic nerves of MS-275 treated EAN rats. In lymph nodes, MS-275 depressed pro-inflammatory cytokines as well, but increased expression of anti-inflammatory cytokine interleukine-10 and of foxhead box protein3 (Foxp3), a unique transcription factor of regulatory T cells. In addition, MS-275 treatment increased proportion of infiltrated Foxp3(+) cells and anti-inflammatory M2 macrophages in sciatic nerves of EAN rats. In summary, our data demonstrated that MS-275 could effectively suppress inflammation in EAN, through suppressing inflammatory T cells, macrophages and cytokines, and inducing anti-inflammatory immune cells and molecules, suggesting MS-275 as a potent candidate for treatment of autoimmune neuropathies.

FTY720 attenuates lesional interleukin-17(+) cell accumulation in rat experimental autoimmune neuritis.

AIMS: Experimental autoimmune neuritis (EAN) is a well-known animal model of human demyelinating polyneuropathies. Here we have studied the spatiotemporal accumulation of interleukin (IL)-17(+) cells in sciatic nerves of EAN rats and effects of FTY720, an agonist of sphingosine-1-phosphate (S1P) receptors. METHODS: In this study, we examined the spatiotemporal expression of IL-17 using immunohistochemistry and RT-PCR, and analysed the IL-17(+) cell proportion in blood and lymph nodes using flow cytometry. RESULTS: In sciatic nerves of EAN rats, IL-17(+) cells were mainly found to concentrate around blood vessels and IL-17(+) cell accumulation was temporally correlated with severity of neurological signs. FTY720, which has been shown to reduce severity of EAN, attenuated accumulation of IL-17(+) cells in sciatic nerves, decreased IL-17(+) cell proportion in peripheral blood, but increased IL-17(+) cell proportion in lymph nodes, suggesting the involvement of S1P signal pathway in regulating IL-17(+) cell trafficking. CONCLUSIONS: our data are consistent with the possibility that IL-17(+) cells might contribute to the pathogenesis of EAN and the S1P signal pathway may be involved in the in vivo trafficking of IL-17(+) cells.

Toll-like receptor-2, CD14 and heat-shock protein 70 in inflammatory lesions of rat experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN) is a well-known animal model of Guillain-Barré syndrome (GBS) characterized by inflammation and demyelination in the peripheral nervous system. Toll-like receptors (TLRs) together with their co-receptors form the first line of the self-defense, and play important roles in innate immune responses and inflammation. TLRs can be activated by endogenous ligands, like heat shock protein 70 (HSP70). In this study, we examined the spatiotemporal expressions of TLR2, CD14 and Hsp70 in EAN rats using immunohistochemistry and RT-PCR. A significant up-regulation of TLR2, CD14 and Hsp70 was seen in sciatic nerves of EAN rats and correlated with disease severity. Furthermore, activated macrophages were the main cellular resource of TLR2, CD14 and Hsp70 in EAN. Our results suggest that TLR2-, CD14- or Hsp70-based immunomodulation might have potential in the control of unwanted innate immune system activation in inflammatory neuropathies.

Immune depression syndrome following human spinal cord injury (SCI): a pilot study.

Experimental spinal cord injury (SCI) has been identified to trigger a systemic, neurogenic immune depression syndrome. Here, we have analyzed fluctuations of immune cell populations following human SCI by FACS analysis. In humans, a rapid and drastic decrease of CD14+ monocytes (<50% of control level), CD3+ T-lymphocytes (<20%, P<0.0001) and CD19+ B-lymphocytes (<30%, P=0.0009) and MHC class II (HLA-DR)+ cells (<30%, P<0.0001) is evident within 24 h after spinal cord injury reaching minimum levels within the first week. CD15+ granulocytes were the only leukocyte subpopulation not decreasing after SCI. A contributing, worsening effect of high dose methylprednisolone cannot be excluded with this pilot study. We demonstrate that spinal cord injury is associated with an early onset of immune suppression and secondary immune deficiency syndrome (SCI-IDS). Identification of patients suffering spinal cord injury as immune compromised is a clinically relevant, yet widely underappreciated finding.

Valproic acid attenuates inflammation in experimental autoimmune neuritis.

Valproic acid (VPA) is a short-chain branched fatty with anti-inflammatory, neuro-protective and axon-remodeling effects. We investigated the effects of VPA in rats in which experimental autoimmune neuritis (EAN) had been induced (EAN rats). VPA (300 mg/kg, intraperitoneally) administration to EAN rats once daily immediately following immunization significantly suppressed mRNA levels of interferon-gamma, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-4, IL-6 and IL-17 in the lymph nodes of EAN rats. In peripheral blood and sciatic nerves of EAN rats, Foxp3(+) cells were increased but IL-17(+) cells were decreased during VPA treatment. Furthermore, suppressive and therapeutic treatment with VPA greatly attenuated both accumulation of macrophages, T cells and B cells, and demyelination in sciatic nerves, and greatly reduced the severity and duration of EAN. In summary, our data demonstrated that VPA could effectively suppress inflammation in EAN, suggesting that VPA could be a potent candidate for treatment of autoimmune neuropathies.

In vivo and in vitro differences between leukocytic uptake of oligodeoxynucleotides.

Development and application of therapeutic oligonucleotides rely on proper analysis of binding and uptake. We have used several model oligodeoxynucleotides (ODNs) to analyze binding/uptake by rat and human leukocytes. Here we describe: (1) differences between in vivo and in vitro uptake of ODNs to rat leukocytes, (2) differences after injection of lipopolysaccharide (LPS), (3) large in vitro differences between primary mononuclear cells in PBS, plasma and blood, and (4) differences of ODN uptake between rat and human leukocytes. Our data show that ODN uptake by primary blood cells was different in PBS, plasma and blood. In addition, LPS treatment increased ODN uptake by leukocytes in blood, indicating that pathological conditions may influence ODN uptake. Furthermore, ODN uptake in rat and human blood is also different, suggesting that preclinical ODN uptake data from rat blood cannot easily be extrapolated to the human condition.

Mechanical allodynia and spinal up-regulation of P2X4 receptor in experimental autoimmune neuritis rats.

Experimental autoimmune neuritis (EAN) is the animal model of acute inflammatory demyelinating polyradiculoneuropathy (AIDP) that is the most common subtype of Guillain-Barre syndrome (GBS). While neuropathic pain is a common symptom of GBS, its underlying mechanisms remain elusive. Central sensitization, particularly spinal glia (microglia and astrocytes) activation, is important for the initiation and maintenance of neuropathic pain. P2X(4) receptor (P2X(4)R) is an ATP-gated ion channel and its spinal up-regulation has been found to be crucial for the development of neuropathic pain following peripheral nerve injury. The initiation of mechanical allodynia in rat EAN was observed at day 9 before the onset of neurological signs. Maximal level of mechanical allodynia was observed from days 17-19 and then a slow recovery, long after the cessation of typical neurological signs of EAN, until day 37 was observed. Expression of P2X(4)R in lumbar spinal cords was studied by immunohistochemistry. P2X(4)R immunoreactivity (IR) was mainly seen in gray matter, particularly in the dorsal horn. Accumulation of P2X(4)R(+) cells in the lumbar dorsal horn was observed at day 9, reached the maximal level at day 17 and remained elevated until day 37 after immunization. Furthermore, a negative correlation between the density of P2X(4)R(+) cells in the lumbar dorsal horn with mean hind-paw withdrawal threshold in EAN rats was seen, indicating that P2X(4)R might contribute to EAN mechanical allodynia. Double staining revealed that almost all P2X(4)R(+) cells co-expressed CD68, a marker for reactive microglia, but not the astrocyte marker, glial fibrillary acidic protein (GFAP). Our data demonstrate that EAN induces mechanical allodynia and P2X(4)R expression in spinal microglia, suggesting that EAN is a good animal model for neuropathic pain in polyneuropathy and spinal microglia activation might participate in EAN-induced neuropathic pain.

Nanosilver: a nanoproduct in medical application.

Nanotechnology is a most promising field for generating new applications in medicine. However, only few nanoproducts are currently in use for medical purposes. A most prominent nanoproduct is nanosilver. Nanosilver particles are generally smaller than 100nm and contain 20-15,000 silver atoms. At nanoscale, silver exhibits remarkably unusual physical, chemical and biological properties. Due to its strong antibacterial activity, nanosilver coatings are used on various textiles but as well as coatings on certain implants. Further, nanosilver is used for treatment of wounds and burns or as a contraceptive and marketed as a water disinfectant and room spray. Thus, use of nanosilver is becoming more and more widespread in medicine and related applications and due to increasing exposure toxicological and environmental issues need to be raised. In sharp contrast to the attention paid to new applications of nanosilver, few studies provide only scant insights into the interaction of nanosilver particle with the human body after entering via different portals. Biodistribution, organ accumulation, degradation, possible adverse effects and toxicity are only slowly recognized and this review is focusing on major questions associated with the increased medical use of nanosilver and related nanomaterials.

Early attenuation of lesional interleukin-16 up-regulation by dexamethasone and FTY720 in experimental traumatic brain injury.

AIMS: Interleukin-16 (IL16) is an immunomodulatory cytokine, which induces lymphocyte migration, expression of proinflammatory IL1 beta, IL6 and tumour necrosis factor-alpha, and modulates apoptosis. IL16 expression has been observed in several central nervous system diseases and may play a role in promoting inflammatory responses. Inflammation contributes considerably to secondary injury following traumatic brain injury (TBI). The aim of this study was to investigate early IL16 expression following experimental TBI and the effects of dexamethasone and FTY720 on early expression of IL16 in TBI rats. METHODS: Rat TBI was induced using an open-skull weight-drop model. IL16 expression was studied by immunohistochemistry. TBI rats received an intraperitoneal injection of dexamethasone (1 mg/kg in 1 ml saline), FTY720 (1 mg/kg in 1 ml saline) or saline (1 ml) on Day 0 and Day 2 immediately after surgery. RESULTS: Significant up-regulation of IL16 was seen as early as 24 h post TBI. Double-staining experiments, together with morphological classification, revealed a multicellular origin of IL16, including activated microglia/macrophages (about 85%), astrocytes (about 8%), neurones (about 5%) and granulocytes. Following peripheral administration of dexamethasone and FTY720, attenuated numbers of IL16(+) cells were observed on Days 1 and 2 but not on Day 4 post TBI for dexamethasone and on Day 4 but not earlier for FTY720 respectively. CONCLUSIONS: Our observations reveal that dexamethasone and FTY720 have different but complementary effects on reduction of early IL16 expression following TBI.

Lesional accumulation of RhoA(+) cells in brains of experimental autoimmune encephalomyelitis and multiple sclerosis.

AIM: Infiltration of autoantigen-specific T cells and monocytes into the central nervous system is essential for the development of both experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS). RhoA is one of the best-known members of Rho GTPases, and inhibition of RhoA has been shown to attenuate the progression of EAE. The aim of this study was to investigate the expression of RhoA in brains of EAE rats and MS tissue. METHODS: EAE was induced by immunization with the synthetic peptide gpMBP68-84 in rats, and clinical severity was scored. RhoA expression pattern was investigated in brains of EAE rats at different time points and in different lesions of brain tissue specimens from six MS brains and five neuropathologically unaffected controls by immunohistochemistry. METHODS: In EAE rat brains, accumulation of RhoA(+) cells reached maximal levels around Day 13, correlating to the clinical severity of EAE, and up-regulation lasted until the recovery stage of the disease. Double-labelling experiments showed that the major cellular sources of RhoA were reactive macrophages/microglia. While RhoA(+) cells in normal human brain parenchyma were rarely observed, RhoA expression was found to be spatially associated with MS lesions, showing a marked decrease from active lesions via chronic stages to its near absence in normal-appearing white matter. In addition, major RhoA(+) cells in brain parenchyma of MS were identified to be activated macrophages/microglia. CONCLUSION: Our present data indicated that RhoA may play an important role during the effector phase of EAE and MS. Therefore, RhoA inhibitors might be a therapeutic option for MS patients.

Lesional expression of the endogenous angiogenesis inhibitor endostatin/collagen XVIII following traumatic brain injury (TBI).

We have analysed the expression of the endogenous angiogenesis inhibitor endostatin/collagen XVIII following stab wound injury and observed a highly significant (p<0.0001) lesional accumulation confined to areas of pan-necrotic injury and developing secondary damage. Maximal cell numbers were detected at Day 14, declining until Day 21 after injury. Further, endostatin/collagen XVIII(+) monocytic cells accumulated in Virchow-Robin spaces where they formed cell clusters. Besides being prevailingly localised to ED1(+) activated microglia/macrophages, endostatin/collagen XVIII expression was also detected by subendothelial surrounding vessels in the lesioned area. Late and prolonged accumulation of endostatin/collagen XVIII(+) microglia/macrophages and increased numbers of endostatin/collagen XVIII(+) subendothelial cells/vessels in areas of vascular pruning and regression, point to a role in the termination of the transient angiogenic response, linked to a "late" inflammatory milieu.

Dexamethasone transiently attenuates up-regulation of endostatin/collagen XVIII following traumatic brain injury.

Endostatin/collagen XVIII is a specific inhibitor of endothelial proliferation and migration in vitro. It has also been shown to have anti-angiogenic activity and tumor growth inhibitory activity in vivo and in vitro. Here we studied expression of endostatin/collagen XVIII in a rat traumatic brain injury (TBI) model, focusing on the early phase. A significant up-regulation of endostatin/collagen XVIII in TBI began as early as 24 h post-TBI. Double-staining experiment revealed that the major resource of endostatin/collagen XVIII(+) cells in our TBI rat model was a subpopulation of reactivated microglia/macrophages. Our data further showed that dexamethasone attenuated up-regulation of endostatin/collagen XVIII expression at days 1 and 2, but not at day 4, post-TBI, indicating that dexamethasone might possess an early and transient influence to the angiogenesis following TBI.

Reactive astrocytes and activated microglial cells express EAAT1, but not EAAT2, reflecting a neuroprotective potential following ischaemia.

AIMS: Glutamate receptor antagonists have failed clinical stroke trials and it has been proposed that the action of N-methyl D-aspartate receptors is necessary for neuronal survival. Thus, excitatory amino acid transporters (EAATs) might be a promising therapeutic target. The aim of this study was to investigate glial expression of EAATs following ischaemia. METHODS AND RESULTS: Expression of EAAT1 (GLAST) and EAAT2 (Glt-1) in 24 cases of ischaemia was examined by immunohistochemistry. Cortical expression of both EAATs in the lesion decreased within 24 h (P < 0.01, each). Whereas EAAT1+ white matter cells increased 18-fold (P < 0.05) within 24 h in the lesion and remained elevated for months in adjacent (469-fold, P < 0.01) and remote areas (20-fold, P < 0.05), EAAT2+ white matter cells were equivalent in ischaemia and controls. In the first week after stroke mainly activated (ramified and amoeboid) microglia expressed EAAT1, whereas monocytic cells in perivascular spaces and foamy macrophages lacked EAAT1. After more than 1 week, predominantly reactive astrocytes expressed EAAT1. CONCLUSIONS: Microglial EAAT1 expression is restricted to the early/intermediate stage of activation and blood-derived (perivascular) monocytes do not contribute to EAAT1+ cells following ischaemia. Whether a pharmacological increase in glial EAAT expression may compensate for loss of cortical EAAT expression and reduce neuronal damage following stroke requires investigation by further functional studies.

The innate immunity of the central nervous system in chronic pain: the role of Toll-like receptors.

Toll-like receptors (TLRs) are a family of pattern recognition receptors that mediate innate immune responses to stimuli from pathogens or endogenous signals. Under various pathological conditions, the central nervous system (CNS) mounts a well-organized innate immune response, in which glial cells, in particular microglia, are activated. Further, the innate immune system has emerged as a promising target for therapeutic control of development and persistence of chronic pain. Especially, microglial cells respond to peripheral and central infection, injury, and other stressor signals arriving at the CNS and initiate a CNS immune activation that might contribute to chronic pain facilitation. In the orchestration of this limited immune reaction, TLRs on microglia appear to be most relevant in triggering and tailoring microglial activation, which might be a driving force of chronic pain. New therapeutic approaches targeting the CNS innate immune system may achieve the essential pharmacological control of chronic pain.

Expression of EAAT1 reflects a possible neuroprotective function of reactive astrocytes and activated microglia following human traumatic brain injury.

UNLABELLED: Glutamate-mediated excitotoxicity is known to cause secondary brain damage following stroke and traumatic brain injury (TBI). However, clinical trials using NMDA antagonists failed. Thus, glial excitatory amino acid transporters (EAATs) might be a promising target for therapeutic intervention. METHODS AND RESULTS: We examined expression of EAAT1 (GLAST) and EAAT2 (Glt-1) in 36 TBI cases by immunohistochemistry. Cortical expression of both EAATs decreased rapidly and widespread throughout the brain (in lesional, adjacent and remote areas) following TBI. In the white matter numbers of EAAT1+ parenchymal cells increased 39-fold within 24h (p<0.001) and remained markedly elevated till later stages in the lesion (90-fold, p<0.01) and in peri-lesional regions (86-fold, p<0.01). In contrast, EAAT2+ parenchymal cells and EAAT1+ or EAAT2+ perivascular cells did not increase significantly. Within the first days following TBI mainly activated microglia and thereafter mainly reactive astrocytes expressed EAAT1. Perivascular monocytes and foamy macrophages lacked EAAT1 immunoreactivity. We conclude that following TBI i) loss of cortical EAATs contributes to secondary brain damage, ii) glial EAAT1 expression reflects a potential neuroprotective function of microglia and astrocytes, iii) microglial EAAT1 expression is restricted to an early stage of activation, iv) blood-derived monocytes do not express EAAT1 and v) pharmacological modification of glial EAAT expression might further limit neuronal damage.

Upregulation of frizzled 9 in astrocytomas.

Wnt/frizzled (FZD) cascades play important roles in controlling cell fate, proliferation, migration, tissue architecture and organogenesis during embryonic development and in adult organisms. The potential involvement of this pathway in tumorigenesis has been established in several types of cancers. Frizzled 9 (FZD9) is expressed in brain and its aberrant expression in gastric cancer was observed. However, its association with astrocytomas remains unknown therefore we studied FZD9 expression in astrocytomas of different malignancy. In the present study, FZD9 expression in 25 astrocytomas was investigated using immunohistochemistry with specific antibodies. Further FZD9 expression in native human brain tissue and glioblastoma cell line were analysed using real-time reverse transcription polymerase chain reaction (RT-PCR). In human astrocytomas, FZD9 immunoreactivity (IR) was observed in both microvessels and neoplastic cells. The percentage of FZD9+ microvessels in relation to FZD9+ vessels was significantly higher in malignant astrocytomas than in low-grade astrocytomas and positively correlated with the astrocytoma World Health Organization (WHO) grading (r = 1, P = 0.04). Furthermore, the FZD9 IR scores positively correlated with astrocytoma WHO grading (r = 1, P = 0.04) and proliferating activity (r = 0.77, P < 0.001). Real-time RT-PCR data showed that FZD9 expression in human glioblastoma was significant higher than in normal brain (P < 0.05) but FZD9 expression was only slightly induced in cobalt chloride-treated human glioblastoma T98G cells compared with untreated cells (P > 0.05). FZD9 is upregulated in astrocytomas, suggesting that FZD9 could be important in the tumorigenesis of human astrocytomas.