The regulation of pro-inflammatory gene expression induced by pigment epithelium-derived factor in rat cultured microglial cells
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Abstract Background There is increasing interest in astrocyte biology because astrocytes have been demonstrated to play prominent roles in physiological and pathological conditions of the central nervous system, including neuroinflammation. To understand astrocyte biology, primary astrocyte cultures are most commonly used because of the direct accessibility of astrocytes in this system. However, this advantage can be hindered by microglial contamination. Although several authors have warned regarding microglial contamination in this system, complete microglial elimination has never been achieved. Methods The number and proliferative potential of contaminating microglia in primary astrocyte cultures were quantitatively assessed by immunocytologic and flow cytometric analyses. To examine the utility of clodronate for microglial elimination, primary astrocyte cultures or MG-5 cells were exposed to liposomal or free clodronate, and then immunocytologic, flow cytometric, and gene expression analyses were performed. The gene expression profiles of microglia-eliminated and microglia-contaminated cultures were compared after interleukin-6 (IL-6) stimulation. Results The percentage of contaminating microglia exceeded 15% and continued to increase because of their high proliferative activity in conventional primary astrocyte cultures. These contaminating microglia were selectively eliminated low concentration of liposomal clodronate. Although primary microglia and MG-5 cells were killed by both liposomal and free clodronate, free clodronate significantly affected the viability of astrocytes. In contrast, liposomal clodronate selectively eliminated microglia without affecting the viability, proliferation or activation of astrocytes. The efficacy of liposomal clodronate was much higher than that of previously reported methods used for decreasing microglial contamination. Furthermore, we observed rapid tumor necrosis factor-α and IL-1b gene induction in conventional primary astrocyte cultures after IL-6 stimulation, which was due to the activation of the Janus kinase/signal transducer and activator of the transcription pathway in contaminating microglia. Conclusions Because contaminating microglia could result in erroneous data regarding the pro-inflammatory properties of astrocytes, astrocyte biology should be studied in the absence of microglial contamination. Our simple method will be widely applicable to experimental studies of astrocyte biology and provide clues for understanding the role of astrocytes in neural development, function and disease.
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The neuroprotective effect of lithium has been attributed to its therapeutic action. However, the role of glial cells particularly astrocytes, and the possible interactions between neurons and astrocytes in neuroprotective effects of lithium have been disregarded. Thus, the aim of this study was to evaluate the direct effects of lithium on brain derived neurotrophic factor (BDNF) and glial cell line derived neurotrophic factor (GDNF) in rat primary neuronal, astrocytes, and mixed neuro-astroglial cultures to assess the possible effects of lithium on astrocytes and neuro-astroglia interactions.Rat primary astrocyte, neuronal and mixed neuro-astrocyte cultures were prepared from cortices of 18-day embryos. Cell cultures were exposed to lithium (1 mM) or vehicle for 1 day (acute) or 7 days (chronic). BDNF and GDNF mRNA and protein levels were determined by RT-PCR and ELISA, respectively.Chronic but not acute lithium treatment increased intracellular BDNF and GDNF protein levels in rat primary neuronal and astrocyte cultures, respectively (P<0.05). However, chronic lithium treatment had no significant effect on intracellular BDNF protein level in astrocyte and mixed neuron-astrocyte cultures or GDNF protein levels in mixed neuron-astrocyte culture. Furthermore, acute and chronic lithium treatment had no significant effect on mRNA and extracellular BDNF and GDNF protein levels in three studied cultures.Present study showed that chronic lithium treatment affected neurotrophins both in neurons and astrocytes in a cell-type specific manner with no effect on neuron-astrocyte interactions. The findings of this study also highlighted the importance of astrocytes as drug targets involved in the neuroprotective action of lithium.
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Abstract Objectives Diabetic retinopathy (DRP) is one of the most common microvascular complications of diabetes. The pigment epithelium-derived factor (PEDF) is a protein that is one of the most potent angiogenesis inhibitors. The effect of blood PEDF concentration on DRP formation remains unclear. The present study aimed to determine whether the plasma concentration of PEDF is effective on the appearance of DRP. Methods The present study consisted of 62 patients with diabetes mellitus and 20 healthy participants. The patient group included 28 patients with non-proliferative DRP, 13 with proliferative DRP, and 21 diabetic patients without DRP. The PEDF levels in patient serum samples were detected through the ELISA method. The body mass index of the participants was calculated. Results Serum PEDF levels of diabetic patients (1.533 ± 0.233 μg/mL) were found to be lower (2.163 ± 0.343 μg/mL) than healthy participants (p=0.002). The PEDF levels were similar in the DRP and non-DRP groups (p=0.337). The plasma PEDF level decreased along with the progression of DRP (p=0.001). Conclusions The PEDF concentration in the blood decreases along with the increase of DRP grade. Decreased blood concentration of PEDF may be important to predict microvascular complications . Agents containing PEDF may be used intraocularly/systemically for therapeutic purposes to prevent vascular complications of diabetes in the near future.
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Astrocytes, the most abundant type of glia in the brain, are considered to play a key role in Alzheimer's disease (AD) pathologies. We have previously shown that astroglial responses against Aβ occur before obvious neuronal damage can be detected. This finding suggests that the role of astrocytes during the early stages of AD pathology must be very important, implicating them as potential therapeutic targets for the treatment of AD. In the present study, we investigated how Aβ affects gene expression patterns in astrocytes during early stages of Aβ toxicity, and specifically focused our investigation on astrocyte–derived neurotrophic factors. We used rat primary cerebral cortical cultures and astrocyte cultures for investigating gene and protein expression patterns by using real–time PCR, ELISA and western blot analyses. We also used neuronally differentiated SH–SY5Y cells for investigating whether astrocyte–derived neurotrophic factors restore neuronal synapses after impairment was induced by Aβ treatment. For primary cortical and astrocyte cultures, Aβ peptides (5 μM) were added. For neuronally differentiated SH–SY5Y cells, Aβ peptides (10 μM) were added. We assessed the synapse–protective effect of astrocyte–derived neurotrophic factors by using western blot analyses and immunocytochemistry. Using real–time PCR and ELISA analyses, we found that Aβ induced astrocytes to produce brain–derived neurotrophic factor (BDNF) after 3 h of Aβ treatment. Western blot analyses showed that expression of the BDNF receptor TrkB increased in primary cerebral cortical cultures, the same time frame that expression of BDNF increased in astrocyte cultures. Expression of another BDNF receptor, p75NTR, however, significantly decreased during this time frame. These findings suggest that, relatively soon after exposure to Aβ, astrocytes produce BDNF, which in turn binds TrkB receptors in neurons, thereby upregulating neurotrophic signaling in neurons against Aβ. Moreover, BDNF treatment in neuronally differentiated human neuroblastoma cells rescued neurons from synaptic degeneration caused by Aβ toxicity. This is the first study to demonstrate that astrocytes are capable of increasing the production of a particular neurotrophic factor, BDNF, in response to Aβ. Our findings also identify BDNF as a potential therapeutic agent for preventing Aβ–related synaptic impairment.
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Microglia are resident immune cells of the central nervous system such as brain-specific macrophages and also known to regulate the innate immune functions of astrocytes through secretory molecules. This conversation plays an important role in brain functions and homeostasis as well as in neuropathologic disease. In this study, we aimed to elucidate whether astrocytes and microglia can cross-talk to induce microglial polarization and proliferation, which can be further regulated under the brain stroke-mimic microenvironment. Microglia in mixed glial culture increased their survival and proliferation and altered to the M2 microglia, whose role was provided by CD11b-GFAP+ astrocytes by showing approximately tenfold increase in microglia cell proliferation after the astrocyte reconstitution. Furthermore, GM-CSF stimulated microglial proliferation approximately tenfold and induced to CCR7+ M1 microglia, whose phenotype could be suppressed by anti-inflammatory cytokines such as IL-4, IL-10, and Substance-P. Also, astrocyte in the microglia co-culture revealed A2 phenotype, which could be activated to A1 astrocyte by TNFα and IFNγ under the stroke-mimic condition. Altogether, astrocyte in the mixed glial culture stimulated the microglia proliferation and M2 polarization possibly through its acquisition of A2 phenotype, both of which could be converted to M1 microglia and A1 astrocytes under the inflammatory stroke-mimic environment. This study demonstrated that microglia and astrocyte can be polarized to M2 microglia and A2 astrocytes respectively through the cross-talk in vitro and provided a system to explore how microglia and astrocyte may behave in the inflammatory disease milieu after in vivo transplantation.
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