PAG neuronal NMDARs activation mediated morphine-induced hyperalgesia by HMGB1-TLR4 dependent microglial inflammation.

Morphine is one of the most effective and widely used analgesic drugs. However, chronic morphine use caused opioid-induced hyperalgesia (OIH). The development of OIH limits the use of morphine. The mechanisms of OIH are not fully understood. Toll-like receptor4 (TLR4) and glutamate receptors in the periaqueductal gray (PAG) are critical in OIH, however, the association between TLR4 and N-methyl-D-aspartate Receptors (NMDARs) activation in PAG remains unclear. Microglia activation, increased TLR4/p65 nuclear factor-kappa B (p65 NF-κB) and proinflammatory cytokines in microglia, and phosphorylation of NMDAR1 subunit (NR1) and NMDAR2B subunit (NR2B) in neurons were observed in PAG of OIH mice. Up-regulations of TLR4/p65 NF-κB and proinflammatory cytokines (IL-1ß, IL-6, TNF-α) in BV2 cells were prevented by inhibiting and knocking down TLR4. By inhibiting myeloid differentiation factor 2 (MD2) and knocking down the High-mobility group box 1 (HMGB1), we found that morphine activated TLR4 by HMGB1 but not MD2. We co-cultured Neuro-2a (N2A) with BV2 microglial cell line and found that instead of directly phosphorylating NMDAR subunits, morphine increased the phosphorylation of NR1 and NR2B by inducing TLR4-mediated microglia inflammation. Knocking TLR4 out of PAG by Lentivirus-GFP-TLR4 shRNA reversed these changes and relieved OIH. Our findings suggested that the secretion of HMGB1 induced by morphine-activated TLR4 in microglia, and the proinflammatory factors released by activated microglia phosphorylated NR1 and NR2B of adjacent neurons, induced increased neuronal excitability. In conclusion, TLR4/NMDARs in PAG were involved in the development and maintenance of OIH and supported novel strategies for OIH treatment.

Identification of potential biomarkers for artificial cold exposure-induced hypertensive stroke by proteomic analysis.

BACKGROUND: The effect of changing temperature on an individual's cerebrovascular risk is both biologically plausible and supported by epidemiologic evidence. We used a global proteomic-based approach to analyze the expression alterations of proteins in artificial cold exposure (ACE)-induced hypertensive stroke in renovascular hypertensive rats (RHR) and to identify the biomarker of ACE-induced hypertensive stroke. METHODS: The RHR models were established by 2 kidney 2 clip methods. ACE treatment was achieved using an intelligent artificial climate cabinet. Blood pressure and neurologic symptoms were observed before and after ACE treatment. Hemorrhagic condition and infarction survey were examined using 2,3,5-triphenyltetrazolium chloride staining. The total number of proteins derived from the cerebral tissue of the RHR models were analyzed with 2-dimensional gel electrophoresis (2-DE), ImageMaster 2D Platinum software, and mass spectrometry. Significantly regulated proteins selected for further functional studies using the Search Tool for the Retrieval of Interacting Genes/Proteins system were verified by Western blot. RESULTS: ACE-induced stroke in the RHR group (31.25%, 25 of 80 vs. 16.25%, 13 of 80; P < .05) but not in the sham-operated group. Following ACE treatment, we identified 37 differentially expressed proteins and 28 were unique. Two of the upregulated proteins, Syt1 and Idh3a, were obtained by bioinformatics analysis and verified by Western blot. CONCLUSIONS: The rate of morbidity as a result of stroke in RHR was obviously elevated after ACE treatment. ACE might affect protein expression profile in cerebral tissues of RHR. Syt1 and Idh3a may play a vital role in ACE-induced hypertensive stroke.

OX26/CTX-conjugated PEGylated liposome as a dual-targeting gene delivery system for brain glioma.

BACKGROUND: The successful gene delivery into the brain is a major challenge due to the presence of the blood-brain barrier (BBB). In order to transport plasmid DNA across the BBB and target the brain glioma, the PEGylated liposomes (PLs) modified with OX26 and chlorotoxin (CTX) were developed as a dual-targeting gene delivery system, and the therapeutic efficacy of OX26/CTX-PL/pC27 against glioma was evaluated using in vitro and in vivo experimental models. METHODS: The PEGylated liposome complexes were prepared by the reverse phase evaporation method, and their physicochemical properties were examined. The transfection efficiency, intracellular distribution, in vitro effects of OX26/CTX-PL/pC27 were determined on C6, F98 and HEK293T cell lines. The dual-targeting therapeutic efficacy of OX26/CTX-PL/pC27 against glioma were assessed using the BMVECs/C6 cells co-culture model and the rat orthotopic glioma model. RESULTS: The OX26/CTX-PL/pDNA complexes exhibited a subglobose shape, and possessed notably low toxicities to HEK293T and C6 cells post 4 h incubation. In the in vitro transfection experiment, gene expressions of hTERTC27 from C6 and F98 cells were significantly improved by OX26 and CTX modification. Our in vitro results also showed that OX26 endowed the PLs with the transport ability across the BBB. Using the BMVECs/C6 cells co-culture model, the viability of C6 cells was decreased to 46.0% after OX26/CTX-PL/pC27 transfection. The OX26/CTX-PL/pC27 complexes exhibited enhanced therapeutic effects on C6 cells. Moreover, the dual-targeting therapeutic effects were further conformed with diminished tumor volumes (18.81 ± 6.15 mm³) and extended median survival time (46 days) in C6 glioma-bearing rats. Immunohistochemical analysis revealed the therapeutic effects derived from enhanced hTERTC27 expression in the tumor site. CONCLUSIONS: The PEGylated liposomes modified with OX26 and CTX are able to significantly promote cell transfection, increase the transport of plasmid DNA across the BBB and afterwards target the brain glioma cells in vitro and in vivo, exhibit the most significant therapeutic efficacy. The ligand OX26 plays a critical role in transporting the lipoplexes across the BBB, and CTX acts as a major role in targeting brain glioma cells. The results would encourage further developments for non-invasive targeting therapy of brain gliomas by intravenous injection.

Inhibition of hepatocellular carcinoma growth by adenovirus-mediated expression of human telomerase reverse transcriptase COOH-27 terminal polypeptide in mice.

A 27-kDa C-terminal fragment of human telomerase reverse transcriptase, hTERTC27, has previously been reported to inhibit the growth and tumorigenicity of HeLa human cervical cancer cells and U87-MG human glioblastoma multiforme cells. However, the antitumor effects of hTERTC27 in hepatoma and its underlying mechanisms are unclear. In the current study, the therapeutic effect of hTERTC27, mediated by recombinant adenovirus, in hepatocellular carcinoma (HCC) was explored in vitro and in vivo to investigate the possible mechanisms. The results indicated that recombinant adenovirus carrying hTERTC27 (rAdv-hTERTC27) effectively inhibited the growth and induced apoptosis of the Hepa 1-6 HCC cells. Dendritic cells transduced with rAdv-hTERTC27 were highly effective at inducing antigen-specific T cell proliferation and increasing the activated cytotoxicity of T cells against Hepa 1-6 cells. HCC was inhibited significantly when a single dose of 5×107 pfu rAdv-hTERTC27 was administered intravenously. In summary, the results of this study demonstrated that rAdv-hTERTC27 may serve as a reagent for intravenous administration when combined with telomerase-based gene therapy and immunotherapy for cancer.

A case of Hashimoto's encephalopathy misdiagnosed as viral encephalitis.

PATIENT: Male, 61 FINAL DIAGNOSIS: Hashimoto's encephalopathy Symptoms: Neuropsychiatric or neurological manifestations Medication: Steroids and immunoglobulins Clinical Procedure: Immunoglobulin combined with corticosteroid therapy Specialty: Neurology. OBJECTIVE: Mistake in diagnosis. BACKGROUND: Hashimoto's encephalopathy is a rare autoimmune syndrome characterized by various neuropsychiatric or neurological manifestations and associated with Hashimoto's thyroiditis, responsive to steroids. Until now, misdiagnosis and delay of treatment of Hashimoto's encephalopathy are very common because of the diversity of the symptoms. CASE REPORT: This recent case of a 61-year-old man presented with unconsciousness, spasms and a previous misdiagnosis as viral encephalitis. Response to anti-viral and steroid therapy was unsatisfactory, but treatment with immunoglobulin combined with corticosteroid therapy achieved rapid and complete recovery. CONCLUSIONS: Any patient presenting with acute or subacute unexplained encephalopathy should be considered Hashimoto's encephalopathy, even if the thyroid function is normal. Thyroid antibody testing should be performed because this may be the most important clue to diagnosis. As soon as the diagnosis is made, steroid therapy is the first choice. If the steroid therapy does not lead to immediate improvement, IVIG is an effective alternative treatment.

VEGF induces angiogenesis in a zebrafish embryo glioma model established by transplantation of human glioma cells.

Zebrafish (Danio rerio) is becoming an increasingly popular vertebrate cancer model. In this study, we established a xenotransplanted zebrafish embryo glioma model to further investigate the molecular mechanisms of tumor angiogenesis. We find that the glioma cell line U87 can survive, proliferate and induce additional SIV branches in zebrafish embryos. In addition, by the means of in situ hybridization and quantitive RT-PCR analyses we find that the transplanted U87 cells can induce the ectopic zebrafish vascular endothelial growth factor A (VEGF A) and its receptor VEGFR2/KDR mRNA expression and increase their expression levels, resulting in additional SIV branches.

Effective antitumor immunity against murine gliomas using dendritic cells transduced with hTERTC27 recombinant adenovirus.

hTERTC27, a 27-kDa hTERT C-terminal polypeptide has been demonstrated to cause hTERT-positive HeLa cell apoptosis and inhibits the growth of mouse melanoma. hTERTC27 has been associated with telomere dysfunction, regulation of gene-regulated apoptosis, the cell cycle and activation of natural killer (NK) cells, but its mechanism of action is not fully understood. Here, we report that dendritic cells (DCs) transduced with hTERTC27 can increase T-cell proliferation, and augment the concentration of interleukin-2 (IL-2) and interferon-γ (IFN-γ) in the supernatants of T cells. It can also induce antigen-specific cytotoxic T lymphocytes (CTL) against glioma cells in vitro. Moreover, hTERTC27 gene-transduced DCs exhibit a very potent cytotoxicity to glioma cells in vivo. It could prolong the survival time and inhibit the growth of glioma-bearing mice. These data suggest that hTERTC27 gene-transduced DCs can efficiently enhance immunity against gliomas in vitro and in vivo.

Hypoxic tissues are associated with microvessel density following brain ischemia-reperfusion.

Hypoxic tissue has been observed in the surrounding areas of the ischemic core following cerebral infarction. The underlying mechanisms for this potentially reversible ischemic region remain to be determined. In this study, we generated permanent brain ischemia (PI) and reperfusion after inducing ischemia for 1.5 h (ischemia-reperfusion or IR) in a rat model of middle cerebral artery occlusion. Using immunofluorescence, we observed hypoxic tissue in ischemic brains and assessed microvessel density in and surrounding the hypoxic tissue. We found that the hypoxic tissues were observed at 1 and 3 days in PI rats and at 1, 3, 7, and 14 days in IR rats. The hypoxic tissue gradually decreased over time. The microvessel density increased in a time-dependent manner in focal brain ischemic tissue in PI and IR rats. Furthermore, IR induced a significant increase in microvessel density when compared with PI rats (P < 0.05). Microvessel density surrounding hypoxic tissue was significantly higher when compared with within the hypoxic tissue (P < 0.05). These data demonstrate that hypoxic tissue may exist for a long period (14 days) following brain IR and indicate that hypoxic tissue usually existed with low microvessel density. Furthermore, the duration of hypoxic tissue was partially dependent on the degree of microvessel proliferation.

The preventive effect of oral EGCG in a fetal alcohol spectrum disorder mouse model.

BACKGROUND: Fetal alcohol spectrum disorder (FASD) is a challenging public health problem. Previous studies have found an association between FASD and oxidative stress. In the present study, we assessed the role of oxidative stress in ethanol-induced embryonic damage and the effect of (-)-epigallocatechin-3-gallate (EGCG), a powerful antioxidant extracted from green tea, on the development of FASD in a murine model. METHODS: Pregnant female mice were given intraperitoneal ethanol (25%, 0.005 to 0.02 ml/g) on gestational day 8 (G8) to establish the FASD model. On G10.25, mice were sacrificed and embryos were collected and photographed to determine head length (HL), head width (HW), and crown rump length (CRL). For mice given EGCG, administration was through a feeding tube on G7 and G8 (dose: 200, 300, or 400 mg/kg/d, the total amount for a day was divided into 2 equal portions). G10.25 embryos were evaluated morphologically. Brain tissues of G9.25 embryos were used for RT-PCR and western blotting of neural marker genes and proteins and detection of oxidative stress indicators. RESULTS: Administration of ethanol to pregnant mice on G8 led to the retardation of embryonic growth and down-regulation of neural marker genes. In addition, administration of ethanol (0.02 ml/g) led to the elevation of oxidative stress indicators [hydrogen peroxide (H2O2) and malondialdehyde (MDA)]. Administration of EGCG on G7 and G8 along with ethanol on G8 ameliorated the ethanol-induced growth retardation. Mice given EGCG (400 mg/kg/d) along with ethanol had embryo sizes and neural marker genes expression similar to the normal controls. Furthermore, EGCG (400 mg/kg on G7 and G8) inhibited the increase in H2O2 and MDA. CONCLUSIONS: In a murine model, oxidative stress appears to play an important role in ethanol-induced embryonic growth retardation. EGCG can prevent some of the embryonic injuries caused by ethanol.

Relationship between cerebral hypoxic tissue volume and prognosis after stroke.

BACKGROUND/AIMS: Between 50 and 70% of stroke survivors suffer from severe disabilities such as paralysis and aphasia. Poor stroke outcome is a reflection of our incomplete understanding of the underlying mechanisms, and hence the capacity to implement appropriate treatment(s). We evaluated hypoxic tissue after stroke and patient condition severity and prognosis. METHODS: Hypoxic tissue volume was quantified within 14 days after stroke. Patients were classified as hypoxic positive or negative. Patients were evaluated at imaging and 21 days later. Prognosis was assessed at 30 and 90 days. RESULTS: Significant improvement was shown in hypoxia-positive (vs. hypoxia-negative) patients (p < 0.05). There were significant positive relationships between the volume of hypoxic tissue and the improvement in specialized test scores at 90 days (p < 0.05 for both). Presence of hypoxic tissue within 14 days after cerebral stroke was related to recovery at 3 weeks and prognosis at 90 days. CONCLUSIONS: The assessment of hypoxic tissue volume after stroke may be useful in predicting patient recovery.

Treatment of Parkinson disease with C17.2 neural stem cells overexpressing NURR1 with a recombined republic-deficit adenovirus containing the NURR1 gene.

To study the potential benefit of the NURR1 gene in Parkinson's disease (PD), we constructed a recombinant republic-deficit adenovirus containing the NURR1 gene (Ad-NURR1) and expressed it in transplanted neural stem cells (NSC). Ad-NURR1 was constructed, and NURR1 mRNA and protein expression were identified by in situ hybridization and western blot analysis, respectively. The identified NURR1 protein could directly or indirectly induce NSC differentiation into neurons. To identify a potential therapeutic use for the transfected NSCs, cells were transplanted into 6-hydroxydopamine lesioned rats. Histopathological and behavioral alterations were evaluated via immunohistochemistry and the ration test, respectively, in rats transplanted with NSCs with or without the Ad-NURR1 adenovirus. The Ad-NURR1 construct effectively expressed the NURR1 protein, which could directly or indirectly induce NSC differentiation into neurons. Both histopathological and behavioral alterations were seen in rats treated with NSCs with or without the Ad-NURR1 construct, although in the case of the latter, the benefits were more robust. These results suggest a potential therapeutic benefit for Ad-NURR1-expressing cells in the treatment of PD. The Ad-NURR1 modification induced NSC differentiation and therefore represents a potential therapy for PD.