重组胶质细胞源性神经营养因子腺病毒保护培养的原代脊髓运动神经元

目的:观察胶质细胞源性神经营养因子(glia cell line-derived neurotrophic factor,GDNF)重组腺病毒(AdCMVgdnf)对培养的原代脊髓运动神经元的保护作用.方法:原代培养的脊髓运动神经元转染不同滴度AdCMVgdnf不同时间后计数,并计算凋亡的运动神经元的百分率.结果:104～107 pfu*ml-1 (pfu,plaque form unit) AdCMVgdnf的保护作用中, 以107 pfu*ml-1作用最显著,腺病毒的滴度过大或过小,保护作用都减弱;观察1～9 d不同转染时间的效果时,在第3～9天均可观察到保护作用,以第9天最明显,存活的运动神经元数比对照组增多280%,同时凋亡神经元的百分比较对照组减少12.9%.结论:在一定的时间(9天)和合适的腺病毒滴度(107 pfu*ml-1)条件下,腺病毒介导的GDNF对体外培养的脊髓运动神经元起到了保护作用.     

Post-injury repeated administrations of minocycline improve the antinociceptive effect of morphine in chronic constriction injury model of neuropathic pain in rat

It is confirmed that pharmacological attenuation of glial cells can alleviate neuropathic pain by lowering proinflammatory cytokine expression. The present study tries to confirm that post-injury administration of glia inhibitor, minocycline, can attenuate the neuropathic pain symptoms and improves the efficacy of morphine anti-nociception in chronic constriction injury (CCI). Male Wistar rats (230-270 g) underwent surgery for induction CCI model of neuropathy. For assessment of the thermal hyperalgesia and mechanical allodynia after CCI induction, morphine (2.5, 5, 7.5, 10 and 15 mg/kg; s.c.) and saline were administered on post-operative days (PODs) 0, 6 and 14. Hargreaves and Von-Frey tests were performed before and 30 min after morphine administration, respectively. The results showed significant decrease in antinociceptive effect of morphine on POD 6 compared to POD 0 only at the dose of 5 mg/kg. On the other hand, on POD 14 the antinociceptive effect of morphine (5, 7.5, 10 and 15 mg/kg) significantly decreased in comparison with POD 0. In another set of experiments, animals received minocycline (10, 20 and 40 mg/kg; i.p.) for eight days from POD 6 to 13 and then the antinociceptive effect of single dose of morphine 5 mg/kg was tested on POD 14. Behavioral tests showed that minocycline (40 mg/kg) could effectively attenuate the thermal hyperalgesia and mechanical allodynia on POD 13. Moreover, minocycline (40, 20 mg/kg) improved the anti-hyperalgesic, and minocycline (40 mg/kg) improved the anti-allodynic effects of morphine 5 mg/kg on POD 14. It seems that the reduction of antinociceptive effect of morphine after CCI may be mediated through glia activation. Modulation of glial activity by minocycline can attenuate CCI-induced neuropathic pain. It is also shown that repeated post-injury administration of minocycline improves the antinociceptive effect of morphine in neuropathic pain.     

Increased hippocampal glycine uptake and cognitive dysfunction after peripheral nerve injury

Patients with chronic pain often have accompanying cognitive deficiency, which may reduce their quality of life and hamper efficient medical treatment. Alteration of extracellular glycine concentration may affect cognitive function and spinal pain signaling. In the present study, we assessed recognition memory by novel-object recognition and found that mice developing mechanical hypersensitivity after peripheral nerve injury exhibited impaired recognition ability for novelty, which was never observed in mice provided the selective glycine transporter 1 (GlyT1) inhibitor N-[3-(4′-fluorophenyl)-3-(4′-phenylphenoxy)propyl]sarcosine (NFPS) systemically. Although systemic NFPS generated analgesia via inhibitory effects of glycine in the spinal cord, the cognitive impairment in neuropathic mice was not restored upon relief of pain alone by intrathecal injection of NFPS. Whole-cell recordings were then made from hippocampal CA1 pyramidal neurons, and the effect of exogenously applied glycine or its endogenous increase by blockade of GlyT1 with NFPS on N-methyl-d-aspartate receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) was investigated in slices prepared from neuropathic mice and mice subjected to sham treatment. In slices from neuropathic mice, NMDA-EPSCs were less potentiated by glycine, whereas they were augmented by NFPS even at lower concentrations. After treating the slices with either NFPS or the glial-selective metabolic blocker fluoroacetate, glycine potentiated NMDA-EPSCs equally in slices from neuropathic and sham-treated mice. These findings imply that chronic pain has a crucial influence on hippocampal plasticity related to cognitive function, and strongly suggest that increasing the extracellular level of glycine via blockade of GlyT1 is a potential therapeutic approach for chronic pain with memory impairment.     

Naloxone-precipitated morphine withdrawal behavior and brain IL-1β expression: comparison of different mouse strains

The development of opioid dependence involves classical neuronal opioid receptor activation and is due in part to engagement of glia causing a proinflammatory response. Such opioid-induced glial activation occurs, at least in part, through a non-classical opioid mechanism involving Toll-like-receptor 4 (TLR4). Among the immune factors released following the opioid-glia-TLR4 interaction, interleukin-1β (IL-1β) plays a prominent role. Previous animal behavioral studies have demonstrated significant heterogeneity of chronic morphine-induced tolerance and dependence between different mouse strains. The aim of this study was to investigate whether the heterogeneity of chronic opioid-induced IL-1β expression contributes to differences in opioid tolerance and withdrawal behaviors. Chronic morphine-induced tolerance and dependence were assessed in 3 inbred wild-type mouse strains (Balb/c, CBA, and C57BL/6) and 2 knockout strains (TLR4 and MyD88). Analysis of brain nuclei (medial prefrontal cortex, cortex, brain stem, hippocampus, and midbrain and diencephalon regions combined) revealed that, of inbred wild-type mice, there are significant main effects of morphine treatment on IL-1β expression in the brain regions analyzed (p < 0.02 for all regions analyzed). A significant increase in hippocampal IL-1β expression was found in C57BL/6 mice after morphine treatment, whilst, a significant decrease was found in the mPFC region of wild-type Balb/c mice. Furthermore, the results of wild-type inbred strains demonstrated that the elevated hippocampal IL-1β expression is associated with withdrawal jumping behavior. Interestingly, knockout of TLR4, but not MyD88 protected against the development of analgesic tolerance. Gene sequence differences of IL - 1β and TLR4 genes alone did not explain the heterogeneity of dependence behavior between mouse strains. Together, these data further support the involvement of opioid-induced CNS immune signaling in dependence development. Moreover, this study demonstrated the advantages of utilizing multiple mouse strains and indicates that appropriate choice of mouse strains could enhance future research outcomes.     

Inhibitory RNA in epilepsy: Research tools and therapeutic perspectives

Since its discovery a decade ago, RNA interference (RNAi) has been developed not only into powerful experimental tools but also into promising novel therapeutics. In contrast to conventional antiepileptic drugs (AEDs) that target specific proteins such as ion channels or receptors, RNAi‐based therapeutics exploit an endogenous regulatory mechanism of gene expression and thereby are poised to prevent or reverse pathogenetic mechanisms involved in seizure development. Therapeutic RNAi has been widely explored for dominant targets involved in neurodegenerative diseases; however, their use for epilepsy therapy has received less attention. This review discusses potential RNAi‐based targets that are of interest for epilepsy therapy, including adenosine kinase (ADK), the key negative regulator of the brain’s endogenous anticonvulsant adenosine. Overexpression of ADK, and the resulting adenosine deficiency, are pathologic hallmarks of the sclerotic epileptic brain, and have been implicated in seizure generation. Therefore, RNAi‐strategies aimed at reducing ADK (and increasing adenosine) are based on a direct neurochemical rationale that has recently been explored experimentally using ex vivo and in vivo gene therapy approaches. Technical issues and challenges remain before those promising tools can be developed into future therapeutics for epilepsy.     

Attempt to classify glial cells by means of their process specialization using the rabbit retinal Müller cell as an example of cytotopographic specialization of glial cells

The rabbit retinal Müller cell is one of the most widely studied glial cell types, and it has all forms of contacts that a glial cell can express, viz. 1) to a (ventricular) fluid space, 2) to a mesenchymal borderline (basal lamina), and 3) to neuronal compartments. This cell demonstrates the local adaptation of cell processes to the microenvironment with which they are in contact. Summarizing available data on Müller cells and other glial cell types, it is concluded that the structure with which the process is in contact determines the type of glial cell process that develops. The type I process has microvilli, desmosome-like junctions, and high Na+,K+-ATPase activity; this type of process is in direct contact with a fluid such as cerebrospinal fluid. The type II endfoot-bearing process contains gliofilaments and has a high K+ conductivity; this type of process is covered by a basal lamina and is in contact with mesenchyme. The type III sheath-bearing process insulates neuronal compartments and expresses suitable membrane properties for glia-neuronal communication. Since structurally similar processes have been shown to have similar physiological properties, a new systematic classification of glial cells is proposed, based on the presence or absence of defined types of cell processes. This approach is believed to provide new insights into the function of neuroglia in both the central and peripheral nervous systems, in vertebrates and invertebrates, and even during ontogenetic development.     

From rodent glial precursor cell to human glial neoplasia in the oligodendrocyte-type-2-astrocyte lineage

With only a few exceptions, the precursor cells representing the normal counterparts of human tumours are unknown. The comparative lack of information about the lineages involved in tissue development, and difficulties in growing many human tumors in a manner suitable for cellular biological analysis, together often make it difficult to study the differences between normal and tumor and tumor cells and to develop many of the model systems that would be useful in the study of human cancer. By applying techniques previously utilized to study glial progenitor cells, we have isolated a human glioblastoma multiforme (GBM)-derived population that expresses many properties otherwise uniquely expressed by oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells. Hu-O-2A/Gb1 (for Human O-2A lineage Glioblastoma number 1) cells responded to similar mitogens and differentiation modulators as rodent O-2A progenitors, and generated cells with features of precursor cells, oligodendrocytes and astrocytes. Moreover, ¹H-NMR analysis of amino acid composition demonstrated a striking conservation of types and quantities of free amino acids between the human tumour cells and the rodent primary cells. Hu-O-2A/Gb1 cells represent the first human glioma-derived population for which unambiguous lineage assignment has been possible, and our results indicate that the human O-2A lineage can contribute to one of the most malignant of glial tumours. In addition, the highly diagnostic ¹H-NMR spectrum expressed by Hu-O-2A/Gb1 cells raises the possibility of eventual non-invasive identification of tumors of this lineage. © 1995 Wiley-Liss, Inc.     

Effects of colony stimulating factors on isolated microglia in vitro

Effects of colony stimulating factors (CSF), known regulators for cells in monocytic lineage, on isolated microglia were examined. Interleukin-3 (IL-3) induced only morphological changes in rod-shaped microglia, while granulocyte-macrophage CSF (GM-CSF) and CSF-1 induced both morphological changes and proliferation of microglia. CSF-1 also activated the enzyme activity of microglia. These observations indicated that, in terms of regulation by cytokines, microglia are similar to mature cells in monocytic lineage.Although astrocytes reportedly produce IL-3 and GM-CSF, the effects of astrocyte-conditioned medium (Ast-Sup) were different from those of either IL-3 or GM-CSF. Ast-Sup caused ameboid microglia to become ramified, and did not induce proliferation of microglia. Factors from astrocytes may have a role in the transformation of microglia which occurs in either normal developing brain or inflammation in the brain.     

Cytotopographical specialization of enzymatically isolated rabbit retinal Müller (glial) cells: K+ conductivity of the cell membrane

Müller (radial glial) cells were isolated from rabbit retinae by means of papaine and mechanical dissociation. Regional membrane properties of these cells were studied by intracellular microelectrode recordings of potential responses to local application of high K+ solutions. When different parts of the cell membrane were exposed to high K+, the amplitude of the depolarizing responses varied greatly, indicating a strong regional specialization of the membrane properties. Using morphometrical data of isolated rabbit Müller cells, and a simple circuit model, we calculated the endfoot membrane to constitute more than 80% of the total K+ conductance of the cell; the specific resistivity of the endfoot membrane was about 400 omega cm2, i.e., more than 40 times less than that of the membrane of the vitread process, which is immediately adjacent. This kind of regional membrane specialization seems to be optimized in respect to the Müller cells' ability to carry spatial buffering K+ currents.