Identification of α7 nicotinic acetylcholine receptor on hippocampal astrocytes cultured in vitro and its role on inflammatory mediator secretion ★

The present study found expressions of α7 nicotinic acetylcholine receptor on hippocampal slices and hippocampal astrocytes using double immunofluorescence stainings. Expression of glial fibrillary acidic protein in the cultured hippocampal slices and hippocampal astrocytes significantly increased, and levels of macrophage inflammatory protein 1α, RANTES, interleukin-1β, interleukin-6, and tumor necrosis factor-α increased in the supernatant of cultured astrocytes following exposure to 200 nM amyloid β protein 1 - 42. Preconditioning of 10 μM nicotine, a nicotinic acetylcholine receptor agonist, could attenuate the influence of amyloid β protein 1 - 42 in inflammatory mediator secretion of cultured astrocytes. Experimental findings indicated that α7 nicotinic acetylcholine receptor was expressed on the surface of hippocampal astrocytes, and activated α7 nicotinic acetylcholine receptor was shown to inhibit inflammation induced by amyloid β protein 1 - 42.


INTRODUCTION
Previous studies have shown that cholinergic neurotransmitters released from the peripheral vagus nerve activate the α7 nicotinic acetylcholine receptor in macrophages, as well as inhibit synthesis and secretion of inflammatory mediators of macrophages, thereby resulting in decreased peripheral inflammation [1][2] . Alzheimer's disease (AD) is associated with cerebral chronic inflammation caused by genetic and environmental factors [3] . Astrocytes and microglia, which exhibit similar characteristics to peripheral macrophages, are predominant in chronic inflammation of the central nervous system. It is difficult to ascertain the role of α7 nicotinic acetylcholine receptor on astrocytes in vivo, because of the variety of cells. Therefore, in vitro studies are extremely helpful. Although cell culture is considered to be the best model for analyzing www.nrronline.org cellular functions, the relationships between cells are different in cultured cells compared with those in the organisms. Therefore, the use of brain slice organotypic cultures has become a powerful tool for the analysis of neural mechanisms, allowing for close-to-normal relationships between neurons, glia, and other membrane structures [4] . However, the localization of α7 nicotinic acetylcholine receptor on astrocytes in organotypically cultured hippocampal slices remains poorly understood.
Previous results have shown that cholinergic drugs delay AD development [5] . However, the role of astrocytes in this process remains unclear. The present study analyzed the localization of α7 nicotinic acetylcholine receptor in rat hippocampal astrocytes to determine the role of α7 nicotinic acetylcholine receptor in inflammatory factor secretion induced by amyloid β protein 1-42 (Aβ 1-42 ).

Expression of α7 nicotinic acetylcholine receptor on astrocytes in cultured hippocampal slices
Expression of α7 nicotinic acetylcholine receptor on astrocytes in cultured hippocampal slices was observed using double immunofluorescence. As shown in Figure  1A, a large number of α7 nicotinic acetylcholine receptor-expressing cells were present in cultured hippocampal slices, as evident by fluorescence staining. Antibodies specific to glial fibrillary acidic protein (GFAP) were used to detect astrocytes, and Figure 1B shows astrocyte distribution. Both GFAP and α7 nicotinic acetylcholine receptor were expressed in the cells ( Figure 1C).

Expression of α7 nicotinic acetylcholine receptor in cultured hippocampal astrocytes
Expression of α7 nicotinic acetylcholine receptor on astrocytes, which were purified and cultured in vitro, was detected by double immunofluorescence. Green fluorescence represented α7 nicotinic acetylcholine receptor (Figure 2A), and astrocytes exhibited red fluorescence ( Figure 2B). Superimposed images revealed a co-localization of astrocytes and α7 nicotinic acetylcholine receptor ( Figure 2C).

Aβ 1-42 increased GFAP expression in astrocytes
Cultured hippocampal slices and astrocytes were treated with Aβ 1-42 (200 nM) for 48 hours, and then the expression of GFAP was detected. Results showed increased GFAP expression in cultured organic astrocytes and cultured single astrocytes ( Figure 3). Astrocytes reacted to Aβ 1-42 , by increasing GFAP expression, which is the first stage of an inflammatory reaction in astrocytes [6] .

Effects of Aβ 1-42 on secretion of inflammatory mediators in astrocytes
Following exposure to 200 nM Aβ 1-42 , chemokine levels in the supernatants of cultured astrocytes were measured. Aβ 1-42 increased macrophage inflammatory protein 1α and RANTES release from astrocytes in a time-dependent manner ( Figure 4).
Concentrations of macrophage inflammatory protein 1α increased at 8 hours after treatment with Aβ 1-42 and reached 12-fold expression levels at 96 hours (F = 85.510, P < 0.01). Increased RANTES expression was less than macrophage inflammatory protein 1α; expression increased by 16 hours after treatment, and reached a 4.6-fold increase by 96 hours (F = 40.424, P < 0.01).

DISCUSSION
Data are expressed as means, from three independent experiments. Cognitive functional impairment in AD patients has been shown to be related to cholinergic deficiency in the central nervous system. However, nicotinic acetylcholine receptor excitomotors improve cognition in animals and humans [7][8] . In the clinic, cholinergic agents are the most effective and most generally used, although little is known about the mechanisms of action. It is thought that transmitters released by the vagus nerve, which activates α7 nicotinic acetylcholine receptor on macrophages, can also inhibit peripheral inflammation [2] . In contrast to macrophages, astrocytes are abundant in the central nervous system and play a crucial role in central inflammatory reaction. Therefore, α7 nicotinic acetylcholine receptor expression on hippocampal astrocytes cultured in vitro was analyzed, as well as the role of α7 nicotinic acetylcholine receptor in inducing secretion of inflammatory mediator. Double immunofluorescence was used to identify astrocytes and expression of α7 nicotinic acetylcholine receptor in cultured hippocampal slices. Results showed that cells other than astrocytes also expressed α7 nicotinic acetylcholine receptor, as previously reported [9] . In hippocampal cells, astrocytes are mixed with other cells in the hippocampal slice. Therefore, detailed functions of α7 nicotinic acetylcholine receptor on astrocytes remains poorly understood due to limitations with astrocyte purity. Therefore, it is important to study the mechanisms of astrocytes in astrocytic cultures. As described in a previous study [10] , pooled hippocampal cells were cultured for 8-10 days and then transferred to a new culture flask to purify astrocytes from microglia and oligodendroglia. Purified astrocytes were further confirmed by immunofluorescence with GFAP-specific antibody. Expression of α7 nicotinic acetylcholine receptor is influenced by many factors. In different encephalic regions, astrocytes can express different α7 nicotinic acetylcholine receptor expression patterns [11] . In addition, there is a significant difference in growth conditions between organotypic cultures and cultured single cells. Therefore, it is necessary to determine whether cultured single astrocytes express α7 nicotinic acetylcholine receptor. Double immunofluorescence staining was used in the present study, and results demonstrated that α7 nicotinic acetylcholine receptor was expressed on cultured single astrocytes. These results suggested that it is possible to analyze the function of α7 nicotinic acetylcholine receptor in astrocytes using cultured single astrocyte models.
In the brain, astrocytes sustain and nourish neurons, and they have been shown to play a role in intracephalic inflammatory reactions, with direct effects on the central nervous system [12] . Diffuse plaques, consisting of Aβ, results in the accumulation of astrocytes and microglia, as well as other pathological change. Previous studies have shown that Aβ directly injures neurons [13] , as well as induces cytokine secretion resulting in neuronal damage via the activation of astrocytes and microglia. To determine the effects of Aβ on astrocytes, GFAP expression was measured in cultured hippocampal slices following exposure to Aβ  . Results showed that Aβ 1-42 significantly increased GFAP expression. However, when combined with other cells, it becomes difficult to determine that the effect of Aβ 1-42 on astrocytes is direct or indirect. Therefore, Aβ 1-42 was added to cultured single astrocytes, demonstrating that Aβ 1-42 increased GFAP expression, enlarged astrocytic cell bodies, and created rough synapses. These results suggested that Aβ 1-42 directly activated astrocytes, resulting in increased GFAP expression.
Once activated, astrocytes evoked inflammatory reactions in the brain and secreted inflammatory mediators that induced neurotoxicity. The present study observed concentration changes of cytokines following treatment with Aβ 1-42 . All tested cytokines, with exception to interleukin-1α, exhibited expression changes in a time-dependent manner. Astrocytes have been shown to express tumor necrosis factor-α in response to an inflammatory response [14] . However, in the present study, expression increased by only 4.6 folds, compared with 8-fold increases in interleukin-6. Expression of chemotactic factors increased earlier and more dramatically than cytokines, and macrophage inflammatory protein 1α exhibited the most significant change. Chemotactic factors promote the migration and infiltration of other phagocytes, as well as enhance inflammatory reactions and prolong inflammatory processes, which can subsequently exacerbate the symptoms of AD [15] . The ion channel α7 nicotinic acetylcholine receptor exists in the peripheral and central nervous systems and exhibits a high affinity to nicotine [9] . To identify the influence of activated astrocytic α7 nicotinic acetylcholine receptor on secretion of inflammatory mediators mediated by Aβ 1-42, the cultured astrocytes were pretreated with nicotine for 60 minutes prior to Aβ 1-42 exposure. Results suggested that once astrocytes expressed α7 nicotinic acetylcholine receptor, action of Aβ 1-42 in inflammatory mediators increased. Levels of cytokines and chemotactic factors significantly decreased compared with non-activated α7 nicotinic acetylcholine receptor. Therefore, results suggested that activated astrocytic α7 nicotinic acetylcholine receptor decreased secretion of inflammatory mediators and chemotactic factors, suppressed migration and infiltration of other phagocyte, and decreased formation of a self-toxic circuit loop. α7 nicotinic acetylcholine receptor activation did not completely return cytokine or chemotactic factors to normal levels, which suggested that activated α7 nicotinic acetylcholine receptor only de-creased the inflammatory reaction of astrocytes. These mechanisms could help to explain why cholinergic treatment only improves AD symptoms and postpones the pathological processes of AD, but does not cure it.

MATERIALS AND METHODS Design
A comparative, in vitro study.

Time and setting
The experiment was performed at the Department of Pharmacy, Zhujiang Hospital, Southern Medical University, China from August 2006 to May 2010.

Materials
Neonatal Sprague-Dawley rats (10 days old) and newborn rats (within 24 hours after birth) were obtained from the Experimental Animal Center of Southern Medical University, China (Certificate No. 2005A060). All experimental animal procedures complied with the Guidance Suggestions for the Care and Use of Laboratory Animals, issued by the Ministry of Science and Technology of China [16] .

Preparation of amyloid-beta peptide
Aβ 1-42 was prepared in fresh stock solution according to previously described methods, with slight modifications [17] . Briefly, Aβ 1-42 (Sigma, St. Louis, MO, USA) was dissolved in 35% acetonitrile and further diluted to 10 μM with incremental additions of PBS while vortexing between additions. The peptide solution was subsequently incubated at 37°C for 72 hours to promote fibrillization and aggregation, followed by storage at -20°C. The final working concentration used in the experiment was 200 nM Aβ 1-42 in culture medium [18] .

Preparation of hippocampal slices
The hippocampus was isolated from 10-day-old neonatal rats and sectioned for culture. The rinsed hippocampus slices were placed onto a membrane in a solution-air culture. The culture medium comprised 50% Eagle's-modified medium (Gibco, Carlsbad, CA, USA), 25% Earle's balanced salt solution, and 25% horse serum containing 6.5 mg/L D-glucose. In the first 3 days, the slices were cultured at 37°C with 5% CO 2 , and medium was replaced every day. Afterwards, the medium was replaced every 3 days, and culture temperatures were reduced to 33°C [19] .

Culture of hippocampal astrocytes
Hippocampi were dissected from the cerebrum of newborn rats within 24 hours after birth, and the tissue was digested with trypsin and centrifuged. Subsequently, the supernatant was discarded, and dissociated cells were plated in culture flasks. After 8-10 days, the culture flasks were shaken at 37°C for 4 hours in a rotator, and then the adherent cells were collected for further culture. To assess astrocytic purity, GFAP expression was detected [20] .

Identification of astrocytes
The hippocampal slices and cultured astrocytes were fixed in 4% paraformaldehyde (pH 7.2) for 20 minutes and washed with 0.01 M PBS. Following a blocking step in PBS containing 10% goat serum and 0.3% Triton X-100, the specimens were incubated with mouse anti-GFAP polyclonal antibody (1:200; Chemicon, Santa Cruz, CA, USA) at 4°C overnight. The sections were then washed three times with PBS for 10 minutes each and incubated with Texas Red-conjugated anti-mouse IgG secondary antibody (1:100; Chemicon) at room temperature for 1 hour. Then sections were washed three times with PBS, coverslipped, and examined under a fluorescence microscope (Olympus, Tokyo, Japan).

Double immunofluorescence of α7 nicotinic acetylcholine receptor in cultured hippocampal slices and cultured astrocytes
Double immunofluorescence was used to identify expression of astrocytic α7 nicotinic acetylcholine receptor in hippocampal organotypic-cultured hippocampal slices and cultured astrocytes. Sections and cultured astrocytes were fixed with 4% paraformaldehyde, and then incubated with 0.5% Triton X-100 for 30 minutes. Rinsed sections and cultured astrocytes were pre-blocked with goat serum prior to primary antibody incubation. Mouse anti-GFAP (1:200; Chemicon) and rabbit anti-α7 nicotinic acetylcholine receptor (1:200; Chemicon) polyclonal antibodies served as the primary antibodies at 4°C overnight, respectively. Normal rabbit serum was used as the negative control. Neuronal signals served as a putative positive control. After three washes with PBS for 5 minutes each, Texas Red-conjugated anti-mouse IgG (1:100; Chemicon) and FITC-labeled anti-rabbit IgG (1:100; Chemicon) were used as secondary antibodies, respectively. Then slides were washed three times with PBS and examined under a fluorescence microscope (BX51; Olympus).

Determination of inflammatory factor expression in supernatant of astrocytes using the liquid phase technique
Following treatment of cultured astrocytes with 200 nM Aβ 1-42 for 4, 8, 16, 32, 48, 72, and 96 hours, supernatants were collected to detect concentrations of cytokines and chemokines. To determine the role of α7 nicotinic acetylcholine receptor, the cells were pretreated with 10 μM nicotine (Sigma) for 1 hour prior to Aβ 1-42 administration. Control cultures were from the same culture set, but were treated with media only. Cytokines/ chemokines, including interleukin-1α, interleukin-1β, interleukin-6, tumor necrosis factor-α, macrophage inflammatory protein 1α, and RANTES in the culture supernatants of astrocytes were measured simultaneously using a LiquiChip work station, which employed bead-based xMAP (flexible multi-analyte profiling) technology, according to manufacture instructions [21] .

Statistical analysis
All statistical analyses were performed using SPSS 11.0 software (SPSS, Chicago, IL, USA). Data were expressed as mean ± SD or mean. One-way analysis of variance was used to compare means between groups, and multiple comparisons for further steps. With homogeneity of variance, least significant difference was adopted for each two-group comparison. Otherwise, the Welch method was utilized. Differences were considered statistically significant if the P value < 0.05.