Curcumin promotes microglial M2 polarization and suppresses chronic constriction: Injury-induced neuropathic pain in a rat model of peripheral neuropathy.

OBJECTIVES: Glia (i.e., astrocyte and microglia) activation in the central nervous system plays a critical role in developing neuropathic pain. Microglia can be activated into proinflammatory (M1) and anti-inflammatory (M2) phenotypes. Switching microglial polarization from M1 to M2 phenotypes represents a novel therapeutic strategy for neuropathic pain. Curcumin has been widely used for its anti-inflammatory and immunomodulatory effects. This study investigated effects of curcumin on astrocyte activation and microglia polarization in the cuneate nucleus (CN) and development of neuropathic pain behavior after chronic constriction injury (CCI) of the median nerve. METHODS: Rats were fed with curcumin once daily at a dose of 40, 80, or 120 mg/kg 30 min before and until 7 d after median nerve CCI. Subsequently, mechanical allodynia and thermal hyperalgesia were evaluated using von Frey filaments and plantar tests, respectively. The levels of astrocyte marker, monoclonal glial fibrillary acidic protein; microglia marker, ionized calcium-binding adapter molecule 1; M1 marker, CD86; and M2 marker, CD206 in the cuneate nucleus were determined. Enzyme-linked immunosorbent assay was applied to measure cytokine concentrations. RESULTS: Curcumin administration dose-dependently reduced mechanical allodynia and thermal hyperalgesia and decreased monoclonal glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1 immunoreactivity in the ipsilateral cuneate nucleus after CCI. On ultrastructural observation, curcumin treatment was associated with fewer features of activated astrocytes and microglia. Furthermore, CCI rats given curcumin exhibited a decline in CD86 immunoreactivity and proinflammatory cytokine levels but an increase in CD206 immunoreactivity and release of anti-inflammatory cytokines. CONCLUSIONS: In our findings, curcumin switches microglial phenotypes from M1 to M2 by suppressing astrocytic activation, reducing proinflammatory cytokine release, promoting anti-inflammatory cytokine production, and contributing to relief of neuropathic pain.

Electroacupuncture-Regulated miR-34a-3p/PDCD6 Axis Promotes Post-Spinal Cord Injury Recovery in Both In Vitro and In Vivo Settings.

Electroacupuncture (EA) could enhance neuroregeneration and posttraumatic conditions; however, the underlying regulatory mechanisms remain ambiguous. PDCD6 (programmed cell death 6) is an established proapoptotic regulator which is responsible for motoneuronal death. However, its potential regulatory role in post-spinal cord injury (SCI) regeneration has remained largely unknown. Further investigations are warranted to clarify the involvement of PDCD6 post-SCI recovery and the underlying mechanisms. In our study, based on bioinformatics prediction, we found that miR-34a-3p might be an upstream regulator miRNA for PDCD6, which was subsequently validated through combined utilization of the qRT-PCR, western blot, and dual-luciferase reporter system. Our in vitro results showed that miR-34a-3p might promote the in vitro differentiation of neural stem cell (NSC) through suppressing PDCD6 and regulating other important neural markers such as fibroblast growth factor receptor 1 (FGFR1), MAP1/2 (MAP kinase kinases 1/2), myelin basic protein (MBP), ßIII-tubulin Class III ß-tubulin (ßIII tubulin), and glial fibrillary acidic protein (GFAP). Notably, in the post-SCI rat model, exogenous miR-34a-3p agomir obviously inhibited the expression of PDCD6 at the protein level and promoted neuronal proliferation, motoneurons regeneration, and axonal myelination. The restorations at cellular level might contribute to the improved hindlimbs functions of post-SCI rats, which was manifested by the Basso-Beattie-Bresnahan (BBB) locomotor test. The impact of miR-34a-3p was further promoted by EA treatment in vivo. Conclusively, this paper argues that a miR-34a-3p/PDCD6 axis might be a candidate therapeutic target for treating SCI and that the therapeutic effect of EA is driven through this pathway.

Aluminium exposure leads to neurodegeneration and alters the expression of marker genes involved to parkinsonism in zebrafish brain.

Aluminium, despite being extremely widespread in the world, is a non-essential metal to human metabolism. This metal is known to have toxic effects on a variety of organs including the brain and is considered an etiological factor in neurodegenerative diseases. However, the molecular mechanisms by which aluminium exerts neurotoxic effects are not yet completely understood. Zebrafish is an animal model also used to study neurodegenerative diseases since the overall anatomical organization of the central nervous system is relatively conserved and similar to mammals. Adult zebrafish were exposed to 11 mg/L of Al for 10, 15, and 20 days and the neurotoxic effects of aluminium were analysed by histological, biochemical, and molecular evaluations. Histological stainings allowed to evaluation of the morphology of the brain parenchyma, the alteration of myelin and the activation of neurodegenerative processes. The expression of the Glial Fibrillary Acidic Protein, a marker of glial cells, was evaluated to observe the quantitative alteration of this important protein for the nervous system. In addition, the poly(ADP-ribose) polymerase activity was measured to verify a possible oxidative DNA damage caused by exposure to this metal. Finally, the evaluation of the markers involved in Parkinsonism was assessed by Real-Time PCR to better understand the role of aluminium in the regulation of genes related to Parkinson's neurodegenerative disease. Data showed that aluminium significantly affected the histology of cerebral tissue especially in the first periods of exposure, 10 and 15 days. This trend was also followed by the expression of GFAP. At longer exposure times, there was an improvement/stabilization of the overall neurological conditions and decrease in PARP activity. In addition, aluminium is involved in the deregulation of the expression of genes closely related to Parkinsonism. Overall, the data confirm the neurotoxicity induced by aluminium and shed a light on its involvement in neurodegenerative processes.

Chronic intraperitoneal injection of polyethylene glycol 200 in mice induces hippocampal neuroinflammation.

In vivo treatment of hydrophobic substances requires the use of organic solvents, which are often toxic. Consequently, polyethylene glycols (PEGs), which are considered as nontoxic, have been widely used for many years in chemistry and biology. We used PEG 200, which was administrated by intraperitoneal (i.p.) injection once a week to mice. After 4 months of injections, at the dose of 1.67 mL/kg, a surprising increase in expression of GFAP (glial fibrillary acidic protein) and IBA1 (ionized calcium binding adaptor molecule 1), glial markers of astrocytes and microglia respectively, was observed in the mice's hippocampus. These results were associated with a dramatic increase in pro-inflammatory cytokine interleukin-1ß (IL-1ß) expression, all together suggesting an inflammatory process. It is important to communicate these results to the scientific community to provide awareness of this potential effect when PEG 200 is used under similar conditions as a vehicle in mice.

Evaluation of Traumatic Spinal Cord Injury in a Rat Model Using 99mTc-GA-5 as a Potential In Vivo Tracer.

Spinal cord injury (SCI) refers to the damage suffered in the spinal cord by any trauma or pathology. The purpose of this work was to determine whether 99mTc-GA-5, a radiotracer targeting Glial Fibrillary Acidic Protein (GFAP), can reveal in vivo the reactivation of astrocytes in a murine model with SCI. A method for the 99mTc radiolabeling of the mouse anti-GFAP monoclonal antibody GA-5 was implemented. Radiochemical characterization was performed, and radioimmunohistochemistry assays were used to evaluate the integrity of 99mTc-GA-5. MicroSPECT/CT was used for in vivo imaging to trace SCI in the rats. No alterations in the GA-5's recognition/specificity ability were observed after the radiolabeling. The GA-5's radiolabeling procedure implemented in this work offers a practical method to allow the in vivo following of this monoclonal antibody to evaluate its biodistribution and specificity for GFAP receptors using SPECT/CT molecular imaging.

Different Ipsi- and Contralateral Glial Responses to Anti-VEGF and Triamcinolone Intravitreal Injections in Rats.

PURPOSE: To investigate the glial response of the rat retina to single or repeated intravitreal injections (IVI). METHODS: Albino Sprague-Dawley rats received one or three (one every 7 days) IVI of anti-rat VEGF (5 µL; 0.015 µg/µL), triamcinolone (2.5 or 5 µL; 40 µg/µL; Trigón Depot), bevacizumab (5 µL; 25 µg/µL; Avastin), or their vehicles (PBS and balanced salt solution) and were processed 7 days after the last injection. Retinas were dissected as whole mounts and incubated with antibodies against: Iba1 (Ionized Calcium-Binding Adapter Molecule 1) to label retinal microglia, GFAP (Glial Fibrillary Acidic Protein) to label macroglial cells, and vimentin to label Müller cells. The retinas were examined with fluorescence and confocal microscopy, and the numbers of microglial cells in the inner retinal layers were quantified using a semiautomatic method. RESULTS: All the injected substances caused an important micro- and macroglial response locally at the injection site and all throughout the injected retina that was exacerbated by repeated injections. The microglial response was also observed but was milder in the contralateral noninjected eyes. The IVI of the humanized antibody bevacizumab caused a very strong microglial reaction in the ipsilateral retina. Two types of macroglial response were observed: astrocyte hypertrophy and Müller end-foot hypertrophy. While astrocyte hypertrophy was widespread throughout the injected retina, Müller end-foot hypertrophy was localized and more extensive with triamcinolone use or after repeated injections. CONCLUSIONS: Intravitreal injections cause micro- and macroglial responses that vary depending on the injected agent but increase with repeated injections. This inflammatory glial response may influence the effects of the injected substances on the retina.

Combination of basic fibroblast growth factor and epidermal growth factor enhances proliferation and neuronal/glial differential of postnatal human enteric neurosphere cells in vitro.

Human enteric neural stem cells (hENSCs) proliferate and differentiate into neurons and glial cells in response to a complex network of neurotrophic factors to form the enteric nervous system. The primary aim of this study was to determine the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on in-vitro expansion and differentiation of postnatal hENSCs-containing enteric neurosphere cells. Enteric neurosphere cells were isolated from rectal polyp specimens of 75 children (age, 1-13 years) and conditioned with bFGF, EGF, bFGF+EGF, or plain culture media. Proliferation of enteric neurosphere cells was examined using the methyl thiazolyl tetrazolium colorimetric assay over 7 days of culture. Fetal bovine serum (10%) was added to induce the differentiation of parental enteric neurosphere cells, and differentiated offspring cells were immunophenotyped against p75 neutrophin receptor (neural stem cells), peripherin (neuronal cells), and glial fibrillary acidic protein (glial cells). Combining bFGF and EGF significantly improved the proliferation of enteric neurosphere cells compared with bFGF or EGF alone (both P<0.01) throughout 7 days of culture. The addition of bFGF drove a significantly greater proportion of enteric neurosphere cells to differentiate into neuronal cells than that of EGF (P<0.01), whereas addition of EGF resulted in significantly more glial differentiation compared with addition of bFGF (P<0.01). Combining bFGF and EGF drove enteric neurosphere cells to differentiate into neuronal cells in a proportion similar to glial cells. Our results showed that the combination of bFGF and EGF significantly enhanced the proliferation and differentiation of postnatal hENSCs-containing enteric neurosphere cells in vitro.

Quantitative changes in gene transcription during induction of differentiation in porcine neural progenitor cells.

PURPOSE: Differentiation of neural stem/progenitor cells involves changes in the gene expression of these cells. Less clear is the extent to which incremental changes occur and the time course of such changes, particularly in non-rodents. METHODS: Using porcine genome microarrays, we analyzed changes in the expression of 23,256 genes in porcine neural progenitor cells (pNPCs) subject to two established differentiation protocols. In addition, we performed sequential quantitative assessment of a defined transcription profile consisting of 15 progenitor- and lineage-associated genes following exposure to the same treatment protocols, to examine the temporal dynamics of phenotypic changes following induction of differentiation. Immunocytochemistry was also used to examine the expression of seven of these phenotypically important genes at the protein level. Initial primary isolates were passaged four times in proliferation medium containing 20 ng/ml epidermal growth factor (EGF) and 20 ng/ml basic fibroblast growth factor (bFGF) before differentiation was induced. Differentiation was induced by medium without EGF or bFGF and containing either 10 ng/ml ciliary neurotrophic factor or 10% fetal bovine serum (FBS). Cultures were fed every two days and harvested on days 0, 1, 3, and 5 for quantitative real-time PCR. RESULTS: The microarray results illustrated and contrasted the global shifts in the porcine transcriptome associated with both treatment conditions. PCR confirmed dramatic upregulation of transcripts for myelin basic protein (up to 88 fold), claudin 11 (up to 32 fold), glial fibrillary acidic protein (GFAP; up to 26 fold), together with notable (>twofold) increases in message for microtubule associated protein 2 (MAP2) and C-X-C chemokine receptor type 4 (CXCR4), Janus kinase 1 (Jak1), signal transducer and activator of transcription 1 (STAT1), and signal transducer and activator of transcription 3 (STAT3). Transcripts for nestin and Krüppel-like factor 4 (KLF4) decreased sharply (>twofold). The specific dynamics of expression changes varied according to the transcript and treatment condition over the five days examined following induction. The magnitude of neuronal marker induction was greater for the ciliary neurotrophic factor condition while glial fibrillary acidic protein induction was greater for the FBS condition. CONCLUSIONS: The transient dynamic of CXCR4 expression during induction of differentiation, as well as the upregulation of several major histocompatibility complex (MHC) transcripts, has implications in terms of graft integration and tolerance, respectively. These data confirm and extend in the pig the findings previously reported with murine retinal progenitors and support the use of this large animal model for translational development of regenerative approaches to neurologic diseases.

Preferential vulnerability of mesencephalic dopamine neurons to glutamate transporter dysfunction.

Nigral depletion of the main brain antioxidant GSH is the earliest biochemical event involved in Parkinson's disease pathogenesis. Its causes are completely unknown but increasing number of evidence suggests that glutamate transporters [excitatory amino acid transporters (EAATs)] are the main route by which GSH precursors may enter the cell. In this study, we report that dopamine (DA) neurons, which express the excitatory amino acid carrier 1, are preferentially affected by EAAT dysfunction when compared with non-DA neurons. In rat embryonic mesencephalic cultures, l-trans-pyrrolidine-2,4-dicarboxylate, a substrate inhibitor of EAATs, is directly and preferentially toxic for DA neurons by decreasing the availability of GSH precursors and lowering their resistance threshold to glutamate excitotoxicity through NMDA-receptors. In adult rat, acute intranigral injection of l-trans-pyrrolidine-2,4-dicarboxylate induces a large regionally selective and dose-dependent loss of DA neurons and alpha-synuclein aggregate formation. These data highlight for the first time the importance of excitatory amino acid carrier 1 function for the maintenance of antioxidant defense in DA neurons and suggest its dysfunction as a candidate mechanism for the selective death of DA neurons such as occurring in Parkinson's disease.

The role of TNF-alpha and its receptors in the production of Src-suppressed C kinase substrate by rat primary type-2 astrocytes.

Src-suppressed C kinase substrate (SSeCKS), an in vivo and in vitro protein kinase C substrate, is a major lipopolysaccharide (LPS) response protein which markedly upregulated in several organs, including brain, lung, heart, kidney, etc., indicating a possible role of SSeCKS in inflammatory process. In the central nervous system (CNS), astrocytes play a pivotal role in immunity as immunocompetent cells by secreting cytokines and inflammatory mediators, there are two types of astrocytes. Type-1 astrocytes can secrete TNF-alpha when stimulated with lipopolysaccharide (LPS), while the responses of type-2 astrocytes during inflammation are unknown. So we examined the expression change of SSeCKS mRNA in type-2 astrocytes after exposure to TNF-alpha and LPS. Real-time PCR showed that TNF-alpha or LPS affected SSeCKS mRNA expression in a time- and dose-dependent manner. Now that LPS induces SSeCKS expression in type-2 astrocytes and type-1 astrocytes are well known to play a pivotal role in immunity, we compared SSeCKS mRNA expression in type-1 astrocytes with type-2 astrocytes after LPS stimulation. Real-time PCR showed that SSeCKS mRNA level was higher in normal untreated type-2 astrocytes than that in normal untreated type-1 astrocytes, increased significantly after 0.1-100 ng/ml LPS stimulation in type-2 astrocytes, but increased weakly after 10-100 ng/ml LPS stimulation in type-1 astrocytes. By using siRNA knockdown of SSeCKS expression, LPS-induced TNF-alpha synthesis and secretion in type-2 astrocytes were partly inhibited, which indicated that SSeCKS played a role in the TNF-alpha biosynthesis in type-2 astrocytes during the stimulation with LPS. RT-PCR analysis revealed that TNFR1 and TNFR2 were present in normal untreated type-2 astrocytes and that TNF-alpha, TNFR1 and TNFR2 increased in type-2 astrocytes after exposure to TNF-alpha or LPS. Immunocytochemistry showed that TNFR1 was expressed in the cytoplasm, nucleus and processes of normal untreated type-2 astrocytes and distributed mainly in the cytoplasm and processes after exposure to LPS. TNFR2 was mainly expressed in the nucleus of normal untreated type-2 astrocytes and distributed mainly in the processes of type-2 astrocytes after exposure to LPS. Both anti-TNFR1 and anti-TNFR2 antibodies suppressed SSeCKS mRNA expression induced by TNF-alpha or LPS. From these results, we conclude that TNF-alpha signaling via both TNFR1 and TNFR2 translocated from nucleus to cytoplasm or processes is sufficient to induce SSeCKS mRNA. In addition, we observed that not only exogenous TNF-alpha but also TNF-alpha produced by type-2 astrocytes affected SSeCKS mRNA production in type-2 astrocytes. These results suggest that an autocrine loop involving TNF-alpha contributes to the production of SSeCKS mRNA in response to inflammation. In addition, SSeCKS production was also drastically suppressed by U0126 (ERK inhibitor), SB203580 (p38 inhibitor), or SP600125 (SAPK/JNK inhibitor), which indicated that type-2 astrocytes which regulated SSeCKS expression after LPS stimulation were via ERK, SAPK/JNK, and P38MAP kinase signal pathway.

Transcriptionally mediated gene targeting of gas1 to glioma cells elicits growth arrest and apoptosis.

Induction of growth arrest-specific genes (gas1) prevents cell proliferation and/or leads to apoptosis in different cell types. In neurons, it has been recently reported that mild excitotoxic neuronal death is associated with gas1 induction, and that overexpression of Gas1 induces apoptosis in terminally differentiated neurons or in proliferating neuroblastoma cells. In the present study, we have analysed the effects of the transcriptionally mediated targeting of gas1 to C6 rat glioma cells. Expression of Gas1 decreased glial proliferation and induced C6 cell apoptosis. While the identity of the caspase(s) responsible for Gas1-induced apoptosis in neurons has remained elusive, in C6 glioma cells, overexpression of Gas1 reproducibly activated caspase-3. Our results support the concept of targeted expression of gas1 as a potentially useful gene therapy strategy in the treatment of human gliomas.

Dynamics of reactive oxygen intermediate production in human glioma: n-6 essential fatty acid effects.

BACKGROUND: Reactive oxygen intermediates (ROIs) are important signals controlling cell growth and cell death. Local essential fatty acid (EFA) deficiencies in tumour cells may limit tumour ROI generation. This deficiency may be rectified by the addition of exogenous EFA. MATERIALS AND METHODS: The n-6 EFA effects on tumour ROIs were analysed in terms of kinetics, dose-response and individual cell type responses using flow cytometry of intracellular 2',7'-dichlorofluorescin oxidation. ROI formation in 30 gliomas and five paired samples of normal brain tissue, > 500 000 cells per specimen, was analysed every 10 s for 0-25 min. RESULTS: Tumour cell basal ROI was lower than normal brain tissue ROI from the same subjects (P < 0.00002). Normal and tumour cell ROIs were stimulated by 4-40 micromol L-1 n-6 EFAs, arachidonic acid (AA) and gamma-linolenic acid (GLA). The stimulated ROI rate was exponential, with the maximum dependent on EFA concentration and tumour grade. CONCLUSIONS: EFAs stimulated tumour cells more than normal cells (P < 0.0000017, n = 71) and increased ROIs in glial fibrillary acidic protein-positive cells in tumours. This indicated high sensitivity of glioma cell ROIs to n-6 EFAs.

Neurosteroids modulate the reaction of astroglia to high extracellular potassium levels.

The extracellular concentration of potassium ([K+]o) in brain tissue is modified by neuronal activity and is increased under several pathological conditions. The influence of neurosteroids on the astroglia response to high [K+]o was assessed on cultured slices from rat hippocampus. Exposure to [K+]o above physiological (3 mM) levels resulted in the progressive appearance of cell processes immunoreactive for glial fibrillary acidic protein (GFAP). The maximal effect was observed at 50 mM [K+]o, and further increases of [K+]o did not increase the extension of GFAP-immunoreactive processes. The effect was observed as early as 10 min after increasing [K+]o, was independent of new protein synthesis, and was reversible, reaching control conditions by 15 h after resetting [K+]o to physiological levels. Gonadal hormones and neurosteroids had prominent and variable effects on the stimulatory influence of high [K+]o on astroglia morphology. At physiological [K+]o, 17beta-estradiol and pregnenolone, as well as its sulfate derivative, increased the extension of GFAP-immunoreactive processes. However, at high [K+]o, testosterone, pregnenolone, and dehydroepiandrosterone and its sulfate derivative decreased the extension of GFAP-immunoreactive processes. Effects of gonadal hormones and neurosteroids were blocked by the protein synthesis inhibitor cycloheximide. These results suggest that non-genomic effects of high [K+]o on glial cells interact with genomic effects of steroids to modulate astroglia morphology.

[Gliotoxic factor and multiple sclerosis]. / Un facteur gliotoxique et la sclérose en plaques.

Multiple sclerosis in a disease of the central nervous system characterized by perivascular and periventricular lesions of the myelin and immune cell infiltrates and increased permeability of the blood-brain barrier. We have found a cytotoxic factor of the cerebrospinal fluid (CSF) specific for multiple sclerosis patients which has 2 main characteristic effects in vitro on primary or immortalized astrocyte cultures: (1) disruption of the gliofilament network of the cells; and (2) apoptotic cell death induction. Moreover, in vivo, intraventricular injections of minute amounts of partially purified gliotoxic factor in adult rats have striking effects on both the morphology and general organization of astrocytes in the entire brain and the permeability characteristics of the blood brain barrier, which becomes leaky to immunoglobulins. These pathological effects are strongly similar to some of the neuropathological findings reported during the course of MS--They suggest an entirely new hypothesis to explain the active stage of the disease: the presence of a new factor of unknown extrinsic (viral) or intrinsic (cellular) origin, able to disorganize the glial cytoskeleton and glial cell differentiation. This factor is then able to provoke glial cell death. Such glial cell death may result in both demyelination and increased blood brain barrier permeability. Both in vitro and in vivo studies strongly support the idea that this gliotoxic factor plays a central role in the pathogenesis of MS, making its full identification a critical theme for MS research.

Prolylendopeptidase inhibitory activity of a glial fibrillary acidic protein fragment and other proline-rich peptides.

The brain prolylendopeptidase (PEP) inhibitory activity of synthetic peptides related to the bovine brain-derived PEP inhibitor, GFAP-(38-55) (MPPPLPARVDFSLAGALN), was investigated. Homologous peptides such as MPPPLPTRVDFSLAGALN (human type) and MTPPLPARVDFSLAGALN (mouse type) also inhibited PEP. Among various synthetic fragments of GFAP-(38-55), only MPPPLP had a Ki essentially the same as that of GFAP-(38-55). Similar synthetic proline-rich peptides such as synapsin fragments and SH3 domain-binding peptides had more or less inhibitory activity.

Redistribution and degeneration of retinal astrocytes in experimental murine cerebral malaria: relationship to disruption of the blood-retinal barrier.

To determine whether astrocytes play a critical role in the pathogenesis of experimental murine cerebral malaria (EMCM), we examined changes in astrocyte morphology and distribution, using retinal wholemounts, in three models: a fatal cerebral malaria (CM) model, in which mice die showing cerebral symptoms; a "resolving" model, in which mice exhibit mild cerebral symptoms, but then recover; and a non-CM model, in which cerebral symptoms are not seen. In the fatal model, retinal astrocytes lost their even distribution from day 3 post-inoculation (p.i.) with malaria parasites, progressing to gliosis (day 5 p.i.), well before the onset of cerebral symptoms on day 6-7 p.i. At the terminal stage of the disease there was a loss of astrocyte processes contacting retinal vessels, often along vessel segments containing adherent monocytes. These features occurred in a mild form in the resolving model and were absent in the non-CM models. To investigate the mechanisms underlying these astrocytic changes, we carried out two experimental manipulations. Firstly, since dexamethasone ameliorates cerebral complications in the fatal CM model, the astrocytic response was monitored after dexamethasone treatment on days 0 and 1 p.i., or days 3 and 4 p.i. Second, to determine whether increased blood-retinal barrier (BRB) permeability initiates the astrocyte changes, breakdown of the BRB was induced experimentally by intra-carotid injection of arabinose and astrocyte morphology and distribution were examined 12, 24, and 48 h later. Retinal astrocytes in both the dexamethasone- and the arabinose-treated groups showed loss of even astrocyte distribution but no loss of astrocyte ensheathment of vessels. It is concluded that: i) astrocytes are involved in the pathogenesis of EMCM, since these changes are only prominent in the fatal model and occur substantially before the onset of cerebral symptoms; ii) the initial changes in astrocyte distribution may be a consequence of the increase in BRB permeability; and iii) the immune response triggered by the malaria parasite may be responsible for the loss of astrocyte ensheathment of vessel segments.

Selective susceptibility of cultured striatal neurons to kainic acid.

Previous work has shown a selective sensitivity of striatal D2-receptor-containing neurons (D2 cells) to kainic acid (KA). In order to see whether this phenomenon exists in cultured cells, which could provide a very accessible model for subsequent mechanistic studies, we examined striatal cultures grown for up to 30 days and labeled with either [3H]spiperone, for D2 receptors, or with [3H]-SCH23390, for D1 receptors. Analysis of the cells was performed with a digital imaging system (RAS). The cultured cells were examined for ligand binding to receptors, cell size, and susceptibility to KA. The D2 cells showed an increased mortality over D1-receptor-containing neurons (D1 cells) in the presence of KA, of a magnitude similar to in vivo loss, with larger D2 cells showing the greatest vulnerability.

Two types of Na(+)-currents in cultured rat optic nerve astrocytes: changes with time in culture and with age of culture derivation.

Na+ channel expression was studied in cultures of rat optic nerve astrocytes using whole-cell voltage-clamp recordings. Astrocytes from postnatal day 7 rat optic nerve (RON) expressed two distinct types of Na+ currents, which had significantly different h infinity curves. Stellate, GFAP+/A2B5+ astrocytes showed currents with h infinity curve midpoints close to -65 mV, similar to Na+ currents in most neurons. In contrast, flat fibroblast-like GFAP+/A2B5- astrocytes showed Na+ currents with h infinity midpoints around -85 mV, almost 20 mV more hyperpolarized than in neurons or A2B5+ astrocytes. Interestingly, Na+ current expression was maintained in A2B5+ astrocytes but began to decrease in A2B5- astrocytes after 6 days in vitro (DIV) and fell to or below the level of detection (i.e., 1 pA/pF) at 12 DIV. Astrocytes cultured from neonatal rats (P0) are almost exclusively GFAP+/A2B5-. These cells did not display measurable Na+ currents when studied at 2 DIV; however, Na+ current was observed after 5 DIV in A2B5- astrocytes from these neonatal (P0) cultures. These findings were substantiated by immunocytochemical experiments using 7493, an antibody raised against purified rat brain Na+ channels; in P0-derived astrocyte cultures 7493 antibody staining was initially lacking (up to 3 DIV), but it was prominent in cultures after 5 DIV, suggesting that Na+ current expression in RON astrocytes occurs postnatally.

Gangliosides alter morphology and growth of astrocytes and increase the activity of choline acetyltransferase in cultures of dissociated septal cells.

Administration of gangliosides has been reported to stimulate regeneration of motoneurons and of central dopaminergic and cholinergic neurons. To shed light on the mechanism by which gangliosides mediate the effects on cholinergic neurons, we studied their actions on cultures of cells dissociated from the septal area of fetal rat brains. These cultures contain cholinergic neurons, which, in vivo, give rise to the cholinergic septo-hippocampal pathway. Gangliosides produced prominent changes in the morphological appearance of the cultures. In contrast to control cultures, which contained many process-bearing cells and a confluent layer of flat cells, there were no flat cells in cultures grown in the presence of gangliosides (0.2 to 0.8 mg/ml of medium). Using immunocytochemical visualization of the astrocytic marker glial fibrillary acid protein, it was shown that all astrocytes in cultures grown in the presence of gangliosides exhibited the morphology of process-bearing cells, whereas in control cultures astrocytes represented the majority of the flat cells. Furthermore, gangliosides attenuated astrocytic proliferation. The effects of gangliosides apparently were not mediated by cAMP, since they could be differentiated from actions of forskolin, an activator of adenylate cyclase. Astrocytic growth and morphology were affected by ganglioside mixtures of various sources and composition and also by the pure gangliosides GM1 and GD1a, whereas lipid and carbohydrate components of gangliosides were ineffective. In contrast to the prominent effects on astrocytes, gangliosides failed to significantly alter survival or fiber growth of cholinergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Glial fibrillary acidic protein and differentiation of neonatal rat pituicytes in vitro.

The appearance and intracellular localisation of glial fibrillary acidic protein (GFAP) in pituicytes in neural lobe cultures of newborn rats aged 7 to 30 days were investigated by use of the indirect immunofluorescence method. GFAP-immunoreactive cells were observed mostly in the outgrowth zone. GFAP was localised in the perikaryal cytoplasm as well as in pituicyte processes. GFAP-positive pituicytes showed considerable morphological polymorphism. The presence of GFAP - astrocytic marker - in pituicytes in vitro and the evident morphological similarity to cultured astrocytes suggest the astroglial character of these cells.