James A. Hewett

Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes

Cultures of astrocytes can be readily established and are widely used to study the biological functions of these glial cells in isolation. Unfortunately, contamination by microglia can confound results from such studies. Herein, a simple and highly effective modification of a common procedure to remove microglia from astrocyte cultures is described. After becoming confluent, astrocytes were exposed to a mitotic inhibitor for 5-6 days then treated with 50-75 mM l-leucine methyl ester (LME) for 60-90 min. Unlike previous protocols that employed lower LME concentrations on subconfluent cultures or during passage of astrocytes, this protocol effectively depleted microglia from high-density astrocyte monolayers. This was evidenced by the selective depletion of microglial-specific markers. Purified monolayers appeared morphologically normal 24h after LME treatment and expressed nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2) proteins upon stimulation with LPS plus IFNgamma, albeit to a lower level than unpurified monolayers. This difference could be attributed to removal of contaminating microglia from monolayers and not to astrocyte dysfunction, since LME treatment did not alter global protein synthesis and a reactive phenotype could be induced in the purified monolayers. Thus, this modified protocol selectively depletes microglia from high-density primary astrocyte monolayers without compromising their functional integrity.

Determinants of regional and local diversity within the astroglial lineage of the normal central nervous system

Astrocytes are a major component of the resident non‐neuronal glial cell population of the CNS. They are ubiquitously distributed throughout the brain and spinal cord, where they were initially thought to function in both structural and homeostatic capacities, providing the framework and environment in which neurons performed their parenchymal duties. However, this stroma‐like view of astrocytes is no longer satisfactory. Mounting evidence particularly within the last decade indicates that astrocytes do not simply support neuronal activity but directly contribute to it. Congruent with this evolving view of astrocyte function in information processing is the emergent notion that these glial cells are not a homogeneous population of cells. Thus, astrocytes in various anatomically distinct regions of the normal CNS possess unique phenotypic characteristics that may directly influence the particular neuronal activities that define these regions. Remarkably, regional populations of astrocytes appear to exhibit local heterogeneity as well. Many phenotypic traits of the astrocyte lineage are responsive to local environmental cues (i.e., are adaptable), suggesting that plasticity contributes to this diversity. However, compelling evidence suggests that astrocytes arise from multiple distinct progenitor pools in the developing CNS, raising the intriguing possibility that some astrocyte heterogeneity may result from intrinsic differences between these progenitors. The purpose of this review is to explore the evidence for and mechanistic determinants of regional and local astrocyte diversity.

Regulation of system xc− activity and expression in astrocytes by interleukin-1β: implications for hypoxic neuronal injury

We recently demonstrated that interleukin-1β (IL-1β) increases system x(c)(-) (cystine/glutamate antiporter) activity in mixed cortical cell cultures, resulting in an increase in hypoxic neuronal injury when glutamate clearance is impaired. Herein, we demonstrate that neurons, astrocytes, and microglia all express system x(c)(-) subunits (xCT, 4F2hc, RBAT) and are capable of cystine import. However, IL-1β stimulation increases mRNA for xCT--the light chain that confers substrate specificity--in astrocytes only; an effect blocked by the transcriptional inhibitor actinomycin D. Additionally, only astrocytes show an increase in cystine uptake following IL-1β exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL-1β is lacking in astrocytes derived from mice harboring a mutation in Slc7a11 (sut gene), which encodes for xCT, and in wild-type astrocytes treated with the protein synthesis inhibitor cycloheximide. IL-1β does not regulate the light chain of the amino acid transporter, LAT2, or the expression and function of astrocytic excitatory amino acid transporters (EAATs), demonstrating some target selectivity. Finally, the enhanced neuronal vulnerability to hypoxia that followed IL-1β treatment in our mixed culture system was not observed in chimeric cultures consisting of wild-type neurons plated on top of sut astrocytes. Nor was it observed in wild-type cultures treated with a system x(c)(-) inhibitor or an NMDA receptor antagonist. Overall, our data demonstrate that IL-1β selectively regulates system x(c)(-) activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL-1β found under hypoxic conditions.We recently demonstrated that interleukin‐1β (IL‐1β) increases system xc− (cystine/glutamate antiporter) activity in mixed cortical cell cultures, resulting in an increase in hypoxic neuronal injury when glutamate clearance is impaired. Herein, we demonstrate that neurons, astrocytes, and microglia all express system xc− subunits (xCT, 4F2hc, RBAT) and are capable of cystine import. However, IL‐1β stimulation increases mRNA for xCT—the light chain that confers substrate specificity—in astrocytes only; an effect blocked by the transcriptional inhibitor actinomycin D. Additionally, only astrocytes show an increase in cystine uptake following IL‐1β exposure; an effect associated with a change in xCT protein. The increase in cystine uptake that follows IL‐1β is lacking in astrocytes derived from mice harboring a mutation in Slc7a11 (sut gene), which encodes for xCT, and in wild‐type astrocytes treated with the protein synthesis inhibitor cycloheximide. IL‐1β does not regulate the light chain of the amino acid transporter, LAT2, or the expression and function of astrocytic excitatory amino acid transporters (EAATs), demonstrating some target selectivity. Finally, the enhanced neuronal vulnerability to hypoxia that followed IL‐1β treatment in our mixed culture system was not observed in chimeric cultures consisting of wild‐type neurons plated on top of sut astrocytes. Nor was it observed in wild‐type cultures treated with a system xc− inhibitor or an NMDA receptor antagonist. Overall, our data demonstrate that IL‐1β selectively regulates system xc− activity in astrocytes and that this change is specifically responsible for the deleterious, excitotoxic effects of IL‐1β found under hypoxic conditions.

TGF-β1 potentiates astrocytic nitric oxide production by expanding the population of astrocytes that express NOS-2

Both transforming growth factor‐β1 (TGF‐β1) and nitric oxide synthase‐2 (NOS‐2) are upregulated under various neuropathological states. Evidence suggests that TGF‐β1 can either attenuate or augment NOS‐2 expression, with the prevailing effect dependent on the experimental paradigm employed and the cell‐type under study. The purpose of the present study was to determine the effect of TGF‐β1 on astrocytic NOS‐2 expression. In purified astrocyte cultures, TGF‐β1 alone did not induce NOS‐2 or NO production. However, NO production induced by lipopolysaccharide (LPS) plus IFNγ was enhanced by TGF‐β1 in a concentration‐dependent manner between 10 and 1,000 pg/mL. The presence of IFNγ was not necessary for this effect to occur, as TGF‐β1 enhanced NO production induced by LPS in a similar fashion. In cultures stimulated with LPS plus IFNγ, the enhancement of NO production by TGF‐β1 was associated with a corresponding increase in NOS‐2 mRNA and protein expression. Interestingly, immunocytochemical assessment of NOS‐2 protein expression demonstrated that TGF‐β1 augmented astrocytic NO production, specifically by increasing the pool of astrocytes capable of expressing NOS‐2 induced by either LPS (∼threefold) or LPS plus IFNγ (∼sevenfold). In a broader sense, our results suggest that TGF‐β1 recruits a latent population of astrocytes to respond to stimulation by pro‐inflammatory mediators.

Inducible nitric oxide synthase expression in cultures enriched for mature oligodendrocytes is due to microglia

Expression of inducible nitric oxide (NO) synthase (NOS-2) occurs during inflammation in the central nervous system (CNS) and has been linked to demyelination accompanying certain CNS inflammatory diseases. Although astrocytes and microglia are thought to be the major sources of NOS-2 expression in the CNS in vivo, recent evidence suggested that the myelin-producing oligodendrocytes (OLs) themselves can express NOS-2 in culture. Given the potentially important pathological implications of this finding, the purpose of this study was to examine further the expression of NOS-2 by OLs in vitro. After exposure to lipopolysaccharide (LPS) and interferon-gamma (IFNgamma), primary cultures enriched for mature OLs released NO in a time-dependent manner, although the amount varied considerably between different culture preparations. Increased NO production was accompanied by expression of NOS-2 mRNA and protein, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Immunofluorescence analysis revealed that the cell-type expressing NOS-2 in these cultures was galactocerebroside (Gal C)-negative but CD11b-positive. Further, NO production could be attenuated in cultures treated with the microglial/macrophage toxin, leucine methyl ester, prior to LPS/IFNgamma stimulation. Thus, microglia were the source of NOS-2 catalytic activity in these cultures. The present results indicate that LPS and IFNgamma are not effective stimuli for induction of NOS-2 in OLs in primary cell culture.

Bile and bile salts potentiate superoxide anion release from activated, rat peritoneal neutrophils.

Certain bile salts cause hepatotoxicity as well as injury to extrahepatic organs when administered to animals. Activated neutrophils (PMNs) may cause tissue injury by releasing reactive oxygen species and other products. Since PMNs may come in contact with biliary components, such as bile salts, following chemical insult to the liver or during cholestasis, we examined the capacity of bile and bile salts to stimulate superoxide anion (O2-) release from rat peritoneal PMNs in vitro. Neither bile nor bile salts, with the exception of lithocholate, could by themselves stimulate O2- release from PMNs. Lithocholate (32 microM) caused small but statistically significant release of O2- from PMNs. When PMNs were primed with a barely suprathreshold concentration of 12-O-tetradecanoyl-phorbol-13-acetate (PMA), a classic stimulus for PMNs, the addition of bile and certain bile salts markedly enhanced O2- release from PMNs. The monohydroxy bile salt, lithocholate, had the greatest stimulatory activity toward PMA-primed PMNs, causing approximately an eightfold increase in O2- release. The enhancing effect of lithocholate was maximal between 10 and 32 microM, and it also occurred with PMNs isolated from rat blood. Dihydroxy bile salts, deoxycholate and chenodeoxycholate (100 microM), caused more modest enhancement of O2- release (two- to threefold) from primed PMNs. Cholate, a trihydroxy bile salt, was not active at these concentrations. Conjugation of either lithocholate or chenodeoxycholate with either glycine or taurine markedly reduced the ability of the bile salt to enhance O2- release from primed PMNs. Structural alterations on the hydrophilic side chain or within the planar, hydrophobic portion of the bile salt molecule reduced the capacity to enhance O2- release from PMA-primed PMNs. These results indicate that bile salts can potentiate the respiratory burst in PMNs and suggest a role for this interaction in toxicoses or disease states characterized by elevated serum bile salts.

Interleukin-1β potentiates neuronal injury in a variety of injury models involving energy deprivation

The purpose of this study was to develop a suitable in vitro model system to study the biochemical pathway(s) by which interleukin-1beta (IL-1beta) contributes to the pathogenesis of cerebral ischemia. Thus, the effect of IL-1beta on a number of injury paradigms associated with energy deprivation was investigated using murine mixed cortical cell cultures. While IL-1beta by itself was not neurotoxic, pre-treatment-but not concurrent or post-treatment-with this cytokine potentiated neuronal injury induced by depriving cultures of either oxygen, glucose, or both oxygen and glucose. Cytotoxicity was abolished by an IL-1beta-neutralizing antibody. Together, these results demonstrate the establishment of reliable and reproducible in vitro models that will now allow detailed investigation of the cellular and molecular mechanisms relating to IL-1beta-mediated neuronal cell death.

Oral Treatment with Rofecoxib Reduces Hippocampal Excitotoxic Neurodegeneration

The purpose of this study was to determine whether the selective cyclooxygenase-2 (COX-2) inhibitor rofecoxib [4-[4-(methylsulfonyl)phenyl]-3-phenyl-2(5H)-furanone] could effectively prevent hippocampal neuronal injury in an animal model of excitotoxic neurodegeneration. COX-2 protein levels increased between 3 and 6 h, peaked at 12 h, and declined to near baseline levels 24 h after injection of N-methyl-d-aspartate (NMDA; 18 nmol) into the CA1 region of the left hippocampus. Mice that were fed ad libitum a control rodent diet for 4 days before and 3 days after injection of NMDA demonstrated marked neuronal loss in the primary cell layers of the ipsilateral CA1, CA3, and dentate gyrus (50, 30, and 20% cell loss, respectively). This injury was potently and dose-dependently reduced by feeding animals a diet standardized to deliver 15 or 30 mg/kg rofecoxib per day. Neurodegeneration in the CA1 region was reduced by 30.1 ± 5.6 and 51.5 ± 9.0%, respectively; in the CA3 by 64.6 ± 12.4 and 69.0 ± 14.1%, respectively; and in the dentate gyrus by 47.8 ± 15.2 and 58.0 ± 18.2%, respectively. Moreover, rofecoxib chow slightly but significantly reduced injury-induced brain edema. These findings demonstrate that rofecoxib can ameliorate excitotoxic neuronal injury in vivo and, as such, may be a particularly promising pharmaceutical for the treatment of neurological diseases associated with overactivation of NMDA receptors.

Dieldrin activates rat neutrophils in vitro

Suppression of phagocytic cell function has been proposed as a possible mechanism for the enhanced sensitivity to certain infectious agents exhibited by animals exposed to the organochloride insecticide, dieldrin. In the present study, we examined the effects of dieldrin on superoxide production by glycogen-elicited peritoneal neutrophils (PMNs) from the rat. Dieldrin caused a concentration-dependent increase in superoxide production by PMNs incubated in vitro at 37 degrees C. Superoxide release was increased significantly with 10 microM dieldrin and reached a maximum of 17 nmol/10 min/2.0 X 10(6) PMNs at a dieldrin concentration of 35 microM. Preincubation of PMNs for 5 min at room temperature with a barely suprathreshold concentration of either phorbol 12-myristate 13-acetate (PMA) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) enhanced dieldrin-stimulated superoxide release by as much as ninefold or threefold, respectively. Maximum enhancement was obtained with 10 microM dieldrin for both PMA and FMLP. Time course studies with PMA-pretreated cells revealed that the rate of superoxide release was dependent on the concentration of dieldrin. Extracellular calcium played an important role in dieldrin-stimulated superoxide release, since PMNs treated with dieldrin in the absence of extracellular calcium did not release superoxide. Also, pretreatment with calcium ionophore A23187 greatly enhanced superoxide release from dieldrin-stimulated PMNs. These results show that dieldrin has a stimulatory effect on superoxide release from rat PMNs in vitro and that this stimulation is dependent on extracellular calcium.

Prophylactic, prandial rofecoxib treatment lacks efficacy against acute PTZ‐induced seizure generation and kindling acquisition

Purpose:  The goal of this study was to determine whether prophylactic prandial administration of rofecoxib, a selective cyclooxygenase‐2 (COX‐2) inhibitor, could alter seizure generation, kindling acquisition, and/or kindling maintenance in the mouse pentylenetetrazole (PTZ) epilepsy model.