Daniel Hinkerohe

Microglia activation influences dye coupling and Cx43 expression of the astrocytic network

Under inflammatory conditions, activated microglia are capable of producing proinflammatory cytokines that are reported to influence cell‐to‐cell communication. The present study was performed to evaluate the influence of microglial activation on the coupling efficiency of the astroglial network. Primary astrocyte cultures of newborn rats were cocultured with either 5% (M5) or 30% (M30) microglia. Microglial activation (rounded phagocytotic phenotype) was investigated using the monoclonal anti‐ED1 antibody, and immunofluorescence with a polyclonal anti‐Cx43 antibody was used to study astroglial Cx43 expression and distribution. Functional coupling of astrocytes was evaluated by monitoring the transfer of microinjected Lucifer yellow into neighboring cells. The data obtained can be summarized as follows: astroglia/M30 cocultures contained significantly fewer resting microglia and significantly more activated microglia than the M5 cocultures; significantly reduced astroglial Cx43 staining was found in M30 cocultures concurrently with a reduced number of dye coupled astrocytes; and the positive correlation of percent activated microglia with reduced astroglial Cx43 expression was highly significant, indicating that the degree of intercellular communication in the astroglial network may be modulated by the activation of microglia under in vitro conditions. GLIA 42:101–108, 2003.

Effects of Cytokines on Microglial Phenotypes and Astroglial Coupling in an Inflammatory Coculture Model

Cytokines play an important role in the onset, regulation, and propagation of immune and inflammatory responses within the central nervous system (CNS). The main source of cytokines in the CNS are microglial cells. Under inflammatory conditions, microglial cells are capable of producing pro‐ and antiinflammatory cytokines, which convey essential impact on the glial and neuronal environment. One paramount functional feature of astrocytes is their ability to form a functionally coupled syncytium. The structural link, which is responsible for the syncytial behavior of astrocytes, is provided by gap junctions. The present study was performed to evaluate the influence of inflammation related cytokines on an astroglial/microglial inflammatory model. Primary astrocytic cultures of newborn rats were cocultured with either 5% (M5) or 30% (M30) microglial cells and were incubated with the following proinflammatory cytokines: tumor necrosis factor‐α (TNF‐α), interleukin‐1β (IL‐1β), interleukin‐6 (IL‐6), interferon‐γ (IFN‐γ), and the antiinflammatory cytokines transforming growth factor‐β1 (TGF‐β1) and IFN‐β. Under these conditions, i.e., incubation with the inflammatory cytokines and the high fraction of microglia (M30), microglial cells revealed a significant increase of activated round phagocytotic cells accompanied by a reduction of astroglial connexin 43 (Cx43) expression, a reduced functional coupling together with depolarization of the membrane resting potential (MRP). When the antiinflammatory mediator TGF‐β1 was added to proinflammatory altered M30 cocultures, a reversion of microglial activation and reconstitution of functional coupling together with recovery of the astroglial MRP was achieved. Finally IFN‐β, added to M5 cocultures was able to prevent the effects of the proinflammatory cytokines TNF‐α, IL‐1β, and IFN‐γ.

Implications of Antiinflammatory Properties of the Anticonvulsant Drug Levetiracetam in Astrocytes

There is accumulating evidence that epileptic activity is accompanied by inflammatory processes. In the present study, we evaluated the effect of levetiracetam (Keppra), an anticonvulsant drug with decisive antiepileptic features, with regard to its putative antiinflammatory potential. We previously established an in vitro cell culture model to mimic inflammatory conditions: Primary astrocytic cultures of newborn rats were cocultured with 30% (M30) microglial cells. Alternatively, cocultures containing 5% microglia (M5) were incubated with the proinflammatory mediator, the cytokine interleukin‐1β (IL‐1β), and lipopolysaccharide (LPS), a potent bacterial activator of the immune system. For the M30 cocultures, we observed reduced expression of connexin 43 (Cx43), the predominant gap junction protein. Impaired functional dye coupling and depolarized membrane resting potential (MRP) were monitored in M30 cocultures as well as in M5 cocultures treated with IL‐1β and LPS. We could show that the Cx43 expression, the coupling property, and the membrane resting potential on which we focused our inflammatory coculture model were normalized to noninflammatory level under treatment with levetiracetam (Keppra). Cumulatively, our results provide evidence for antiinflammatory properties of levetiracetam in seizure treatment.

Anti-inflammatory effects of the anticonvulsant drug levetiracetam on electrophysiological properties of astroglia are mediated via TGFβ1 regulation

BACKGROUND AND PURPOSE The involvement of astrocytes as immune‐competent players in inflammation and the pathogenesis of epilepsy and seizure‐induced brain damage has recently been recognized. In clinical trials and practice, levetiracetam (LEV) has proven to be an effective antiepileptic drug (AED) in various forms of epileptic seizures, when applied as mono‐ or added therapy. Little is known about the mechanism(s) of action of LEV. Evidence so far suggests a mode of action different from that of classical AEDs. We have shown that LEV restored functional gap junction coupling and basic membrane properties in an astrocytic inflammatory model in vitro.

Glia and epilepsy: Experimental investigation of antiepileptic drugs in an astroglia/microglia co-culture model of inflammation

The contribution of glial cells, mainly astrocytes and microglia, to the pathophysiology of epilepsy is increasingly appreciated. Glia play a pivotal role in the initiation and maintenance of the central nervous system (CNS) immune response and neuronal metabolic and trophic supply. Recent clinical and experimental evidence suggests a direct relationship between epileptic activity and CNS inflammation, which is characterized by accumulation, activation, and proliferation of microglia and astrocytes. Concomitant glia‐mediated mechanisms of action of several antiepileptic drugs (AEDs) have been proposed. However, their direct effects on glial cells have been rarely investigated. We aimed to investigate the effect of commonly used AEDs on glial viability, the gap junctional network, the microglial activation, and cytokine expression in an in vitro astroglia/microglia co‐culture model.

Dexamethasone prevents LPS-induced microglial activation and astroglial impairment in an experimental bacterial meningitis co-culture model

We analyzed the effect of dexamethasone on gram-negative bacteria derived lipopolysaccharide (LPS) induced inflammation in astroglial/microglial co-cultures. At the cellular level the microglial phenotype converted to an activated type after LPS incubation. Furthermore, LPS compromised functional astroglial properties like membrane resting potential, intracellular coupling and connexin 43 (Cx43) expression. This change in Cx43 expression was not due to a downregulation of Cx43 mRNA expression. Morphological and functional changes were accompanied by a time-dependent release of inflammation related cytokines. Co-incubation of dexamethasone with LPS prevented these LPS-induced changes within our glial co-culture model. The ability of dexamethasone to reconstitute astrocytic properties and to decrease microglial activation in vitro could be one possible explanation for the beneficial effects of dexamethasone in the treatment of acute bacterial meningitis in vivo.

Intracellular application of TNF-alpha impairs cell to cell communication via gap junctions in glioma cells

Human gliomas are the most common class of brain neoplasm. In order to better characterize their response to inflammation, we evaluated the influence of tumor necrosis factor alpha (TNF-α) on the coupling behaviour and the membrane resting potential (MRP) of glioma cells (F98 glioma cell line) compared to primary astrocytes. In contrast to cultured primary astrocytes which exhibited a profound inhibition of gap junction mediated intercellular communication (GJIC), extracellular exposure of TNF-α to F98 glioma cells gained no effect on the functional coupling. Whereas, intracellular application of TNF-α into the glioma cells elicited similar effects as those found in primary astrocytes indicating a compromised accessability of the TNF-α receptor in F98 cells. Western blotting, immunocytochemical staining and real time RT PCR analysis revealed a differential expression and distribution of TNF-α receptor 1 (TNFR1) in the glioma cells. Connexin 43 (Cx43) is the major astrocytic gap junction protein which when phosphorylated has been shown to reveal altered gating properties. Here we show that TNF-α increases the level of phosphorylated Cx43 in primary astrocytes but not in the F98 glioma cells. Our observations could account for the decreased regulatory effects of TNF-α on GJIC of F98 glioma cells.

Dexamethasone and levetiracetam reduce hetero-cellular gap-junctional coupling between F98 glioma cells and glial cells in vitro

Gap junctions (GJs) in astrocytes and glioma cells are important channels for cell-to-cell communication that contribute to homo- and heterocellular coupling. According to recent studies, heterocellular gap-junctional communication (H-GJC) between glioma cells and their surrounding environment enhances glioma progression. Therefore, we developed a new in vitro model to examine H-GJC between glioma cells, astrocytes and microglia. Consequently, F98 rat glioma cells were double-labeled with GJ-impermeable (CM-DiI) and GJ-permeable dye (calcein AM) and were seeded on unlabeled astrocyte-microglia co-cultures. Dual whole cell voltage clamp recordings were carried out on selected cell pairs to characterize the functional properties of H-GJC in vitro. The expression of four types of connexins (Cxs), including Cx32, Cx36, Cx43 and Cx45, and microglial phenotypes were analyzed by immunocytochemistry. The H-GJC between glioma cells and astrocytes/microglia increased after a longer incubation period with a higher number of glioma cells. We provided evidence for the direct GJ coupling of microglia and glioma cells under native in vitro conditions. In addition, we exploited this model to evaluate H-GJC after incubation with levetiracetam (LEV) and/or dexamethasone (DEX). Previous in vitro studies suggest that LEV and DEX are frequently used to control seizure and edema in glioma. Our findings showed that LEV and/or DEX decrease the number of heterocellular coupled cells significantly. In conclusion, our newly developed model demonstrated H-GJC between glioma cells and both astrocytes and microglia. The reduced H-GJC by LEV and DEX suggests a potential effect of both drugs on glioma progression.

Resection of an intramedullary high cervical metastasis from a malignant mixed Muellerian tumour

We present a 66-year-old female patient with a high cervical intramedullary metastasis from a malignant mixed Muellerian tumour (MMMT; carcinosarcoma) with concomitant syringomyelia. She was admitted to our clinic with symptoms of cervical myelopathy. MRI revealed an intramedullary tumour of 2.6cm×1.2cm at the cervical vertebral body C2. We performed a laminectomy on C2 followed by a dorsal median myelotomy from C1 to C3 to resect the tumour. The surgical intervention removed the tumour completely and resolved the syringomyelia. During the 36months of follow-up, the patient presented in a stable condition with no evidence of tumour recurrence. To our knowledge, this is the first report of an intramedullary metastasis of a MMMT.