A refined in vitro model to study inflammatory responses in organotypic membrane culture of postnatal rat hippocampal slices
© Huuskonen et al; licensee BioMed Central Ltd. 2005
Received: 04 November 2005
Accepted: 15 November 2005
Published: 15 November 2005
Propagated tissue degeneration, especially during aging, has been shown to be enhanced through potentiation of innate immune responses. Neurodegenerative diseases and a wide variety of inflammatory conditions are linked together and several anti-inflammatory compounds considered as having therapeutic potential for example in Alzheimer's disease (AD). In vitro brain slice techniques have been widely used to unravel the complexity of neuroinflammation, but rarely, has the power of the model itself been reported. Our aim was to gain a more detailed insight and understanding of the behaviour of hippocampus tissue slices in serum-free, interface culture per se and after exposure to different pro- and anti-inflammatory compounds.
The responses of the slices to pro- and anti-inflammatory stimuli were monitored at various time points by measuring the leakage of lactate dehydrogenase (LDH) and the release of cytokines interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) and nitric oxide (NO) from the culture media. Histological methods were applied to reveal the morphological status after exposure to stimuli and during the time course of the culture period. Statistical power analysis were made with nQuery Advisor®, version 5.0, (Statistical Solutions, Saugus, MA) computer program for Wilcoxon (Mann-Whitney) rank-sum test.
By using the interface membrane culture technique, the hippocampal slices largely recover from the trauma caused by cutting after 4–5 days in vitro. Furthermore, the cultures remain stable and retain their responsiveness to inflammatory stimuli for at least 3 weeks. During this time period, cultures are susceptible to modification by inflammatory stimuli as assessed by quantitative biochemical assays and morphological characterizations.
The present report outlines the techniques for studying immune responses using a serum-free slice culture model. Statistically powerful data under controlled culture conditions and with ethically justified use of animals can be obtained as soon as after 4–5 DIV. The model is most probably suitable also for studies of chronic inflammation.
The discovery of upstream sensors, the Toll-like receptors (TLRs) [1, 2], greatly multiplied our understanding of innate and adaptive immune interactions and responses. Downstream, a well known family of transcription factors, the nuclear factor kappa B (NF-κB), is one of the key players in the regulation of inflammatory responses [3, 4]. Recent studies have revealed that this unique interplay also exists in the brain macrophages, i.e., the microglial cells . These cells, which can present antigen and are responsible for the production and release of a variety of cytokines and chemokines, interact with immune cells and are intimately involved in immunoregulation within the CNS . Whereas the role of microglia in the brain has been studied extensively [7–11], most progress on the understanding of the role of microglia in inflammation has come from cell culture and slice culture studies. The behaviour of microglia in different culture models has been shown to be affected by the culture time and the composition of culture media [12–14]. It has been emphasized that the presence of serum in the culture media potentiates the LPS-induced microglial response [15, 16]. On the other hand, even though they exhibit amoeboid, "active" morphology under serum-free culture conditions, microglia are suggested to be functionally in an "inactive" or "resting" state .
In the slice culture systems, whether supplemented with serum or not, microglial cells revert to a "resting", ramified phenotype after a prolonged culture time [17, 18]. This morphological transformation starts at around 4 DIV and from approximately 10 DIV on, the overall population of microglia appear for the most part as a ramified type. It has been assumed that this "resting", ramified phenotype of microglia would have reduced functional status but a recent in vivo study by Nimmerjahn and co-workers  convincingly demonstrates how microglia cells constantly monitor their immediate environment by extending and retracting their projections in a minute-to-minute time scale. Furthermore, time-lapse imaging of live hippocampal slices [20, 21] have also revealed the capacity of microglia to undergo highly dynamic behaviour.
As these observations demonstrate, the microglia are capable of complex behaviour and therefore it is of crucial importance to pay attention to the factors that contribute to the consistency of in vitro models used to mimic in vivo situations.
In the present study, we used hippocampus tissue slices in serum-free culture conditions to examine the behaviour of microglia per se and to investigate how these slices respond to pro- and anti-inflammatory stimuli. This in vitro culture of postnatal brain provide a model where the cytoarchitecture and connectivity of different anatomical regions, as well as the functional relationships and interactions with neighbouring cell types (i.e., neurons and astrocytes) are preserved [22, 23]. Organotypic cultures offer also the advantage of controlled manipulations in living tissue and thus they might represent an analogously feasible intermediate between simpler cell lines and in vivo models.
Moreover, by carefully planning the experimental set-up, it should be possible to carry out slice culture studies where a minimum number of animals need to be sacrificed and yet to gain sufficient and reliable data to eliminate the risk of performing unsuccessful, more expensive, preclinical in vivo experiments. Therefore, we also wanted to study the possibility of refining the model itself by taking into the account the experimental design involving appropriate sample size and statistical power analysis.
Preparation of hippocampal slice cultures
Organotypic slice cultures from hippocampus were prepared using the modified interface culture method described by Stoppini et al. . Postnatal day 7–8 (P7–P8) Wistar rat pups were decapitated, and the brains were rapidly dissected and placed in a petri dish in ice-cold 1 × Dulbecco's Phosphate Buffered Saline without calcium and magnesium (Biowhittaker™, Belgium). The hippocampi of both sides were isolated and sectioned into 400-μm transverse slices with a McIlwain tissue chopper (Mickle Laboratory Engineering Co. Ltd, Goose Green, UK). The slices were then carefully separated and transferred on to porous membrane inserts (one slice per insert) of 12-well culture plates (Transwell TR 3462; Costar, Corning, NY, USA). To reach the level of insert membrane, some 600 μL culture medium, consisting of Neurobasal medium with 1 × B27-supplement (both from Gibco, Rockville, MD, USA), 1 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin, was added to the lower compartment of each well and the culture plates were then placed in a 37°C humified incubator enriched with 5% CO2. On the first day of culture, inactivated fetal bovine serum (FBSi, Gibco) was added to the culture medium at a concentration of 10%. On the next day, the culture medium was replaced with fresh medium without serum and from then on serum-free media was changed twice a week.
Animals were obtained from National Laboratory Animal Center (NLAC), University of Kuopio, Finland. The experiments were conducted according to the Council of Europe (Directive 86/609) and Finnish guidelines, and approved by the State Provincial Office of Eastern Finland.
Exposure of slices to different stimuli
A series of experiments was carried out to extend the culture time up to 1 month. First, to induce inflammation, we exposed the slices to 5 μg/ml of lipopolysaccharide (LPS from E.coli 055:B5, L6529, Sigma, St Louis, USA) for 24 h at 4 DIV, 7 DIV, 14 DIV and 21 DIV. Then we performed a concentration series study in order to determine whether the amount of LPS had any effect on cytokine production and LDH leakage at the 4 DIV time point. Next, the slices were exposed either to proinflammatory or anti-inflammatory compounds together with or without LPS induction at 4 DIV. As a proinflammatory stimulus we used trichostatin A (TSA, 20 nM), a well characterised histone deacetylase inhibitor  and as an anti-inflammatory stimulus we used a known NF-κB inhibitor helenalin  at a concentration of 0.5 μM. TSA was purchased from Sigma and helenalin from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA). We also pre-exposed slices to 5 μg/ml of LPS for 3 h, 6 h, 12 h, 24 h and 48 h to determine the possible time dependent effects either at 4 DIV or 7 DIV. In all experiments, the results were collected from parallel cultures with six individual slices per treatment group. Each study was replicated at least three times.
For revealing microglia, hippocampal slices were stained with Alexa Fluor 488 conjugated fluorescent Griffonia simplicifolia isolectin IB4 (Molecular Probes, Eugene, OR). Prior to staining slices were fixed with 4% paraformaldehyde for 1–2 h and rinsed three times with 0.05 M TBS-T, pH 7.6 (Tris-buffered saline + 0.1% Triton X-100). IB4 was applied at a concentration of 0.5 μg/ml in TBS-T and slices were incubated overnight at 4°C on a shaker. Before visualization, samples were rinsed with 0.1 M phosphate buffer and mounted on slides. We also performed immunocytochemical staining with a microglia marker OX-42 (against CD11b surface Ag, MCA 275R, Serotec, Oxford, UK), an antibody that recognizes type 3 complement receptors CR3 on mononuclear phagocytes. Primary antibody was diluted 1:20 000 with 0.05 M TBS-T, pH 7.6, slices were incubated for 1 week at 4°C on a shaker, rinsed thoroughly, followed by overnight incubation (4°C) with biotinylated secondary Ab (sheep anti-mouse, 1:1000, Serotec) and 2 h incubation (room temperature) with avidin peroxidase (1:1000 ExtrAvidin E-2886, Sigma). The immunoreactive product was visualised with 3,3'-diaminobenzidine tetrahydrochloride dihydrate (DAB, 0.5 mg/ml, Sigma) in a nickel solution containing hydrogen peroxidase (25 μg/ml).
For epifluorescence immunodetection, primary antibodies to glial fibrillary acidic protein for astrocytes (GFAP: dilution 1:10 000; DAKO, Denmark) and doublecortin for neurons (DCX C-18: dilution 1:2500; Santa Cruz Laboratories, Santa Cruz, CA, USA) were used. After an overnight incubation with primary antibody at 4°C and thorough rinsing, 1:1000 diluted goat anti-rabbit Alexa Fluor 488 secondary antibody (Molecular Probes) to GFAP and 1:500 diluted biotinylated rabbit anti-goat secondary (Vector BA-5000; Burlingame, CA, USA) followed by tertiary antibody (goat anti-rabbit Alexa Fluor 488; 1:1000 dilution) to DCX were applied, respectively, also overnight at 4°C. All the antibodies were diluted with 0.05 M TBS-T, pH 7.6.
Monitoring morphological and biochemical recovery and responses
First, some hippocampi were rapidly sliced at 400 μm and immediately fixed and stained for microglia, astrocytes and neurons with IB4, GFAP and DCX, respectively, to reveal the morphological status at the beginning of the culture. Then, the culture medium was collected and tissues fixed at 24 h intervals during the 7 days in vitro (7 DIV) culture period. Cytokines IL-6 and TNF-α, NO and LDH were analysed from the media and tissue samples were stained as described before. Subsequently, samples of slices and culture medium were collected at appropriate time points to further monitor the morphological and biochemical status of cultures in the 1 month time period. To visualize the morphological integrity and both dead or dying cells and living cells, we used standard Nissl staining and the Live/dead-cytotoxicity kit L-3224 (Molecular Probes), respectively, according to the manufacturer's protocol.
LDH leakage to the culture medium was measured with a CytoTox 96 nonradioactive cytotoxicity assay kit obtained from Promega (Madison, WI, USA). The nitrite concentration in the medium was measured by the Griess reaction. Briefly, to a 100-μL of sample an equal amount of the Griess reagent (1:1 0.1% naphthylethylene diamide in H2O and 1% sulfanilamide in 5% concentrated H2PO4) was added and the optical density (OD) was measured at 550 nm using an ELISA microplate reader after incubation for 10 min. The concentrations of cytokines IL-6 and tumour necrosis factor (TNF)-a released into the medium were measured by an enzyme linked immunosorbent assay (ELISA) using OptEIA™ kits or sets obtained from Pharmingen (BD Biosciences, San Diego, CA, USA).
Pictures from stained slices were collected with an Olympus DP50 microscope digital camera system connected to an Olympus BX40 microscope (Olympus Optical Co, Ltd, Japan) with appropriate filters. Except for adjustment to the contrast and brightness levels, no other manipulations were done in any of the images.
Statistical power analysis and estimation of optimal sample size
To determine the minimum number of animals, yet appropriate sample sizes for our experiments, we undertook power analysis calculations. First, based on our previous results, we estimated the effect size among different experimental units. Thereafter, the sample size was set to six per group and the significance level to 5% to see the effect on statistical power. In order to reduce the within-group variation we used lognormal distribution and carried out the statistical power analysis calculations using nQuery Advisor®, version 5.0, (Statistical Solutions, Saugus, MA) computer program for Wilcoxon (Mann-Whitney) rank-sum test.
Slices recover from explantation by 4 DIV
Effect of LPS is not concentration dependent
Effect of LPS is exposure but not culture time dependent
Slices respond well to pro and anti-inflammatory stimuli during the culture
Morphological changes of microglia, neurons and astrocytes during culture period
Statistical power analysis and optimal sample size
Statistical power analysis showing the effect of sample size (n = 6) and test significance level (5%) on statistical power. The effect size was calculated as the difference between two population means (e.g. control and treatment groups) divided by the standard deviation of either population. The analysis was done with nQuery Advisor®, version 5.1, computer program for Wilcoxon (Mann-Whitney) rank-sum test.
Test significance level, α
n per group
Effect size, δ = μ1 - μ2/σ
C vs. LPS
LPS vs. LPS+TSA
LPS vs. LPS+Hele
LPS+TSA vs. LPS+TSA+Hele
Cell culture media have been routinely supplemented with animal serum as a source of nutrients. Over the past years the trend towards serum-free culture conditions has received much attention since there are both ethical and technical concerns related to the use of serum [29, 30]. Obtaining the serum from fetuses removed from pregnant cows at slaughter has raised questions about animal suffering. Moreover, the possibility of batch-to-batch variability in the undefined serum composition may add interference to the reproducibility of results between studies. At the same time, there is an evolving concern and commitment among the regulatory authorities, in the European Union and the USA, to find ways to reduce the number of animals used in laboratories for experimental and other scientific purposes to the minimum level .
Our present findings demonstrate that slices cultured in serum-free medium still respond significantly and in a similar manner to an inflammatory stimulus between the culture period from 4 DIV to 21 DIV. We demonstrate that in our model the phenotype of the microglial-cell population remains heterogenous throughout the culture time after a recovery period of approximately 4–5 DIV. Despite the nonuniform microglial appearance, the non-treated control slices seem to retain stability as what comes to lysis of cells (LDH leakage) and to their ability to release cytokine IL-6 and NO.
In addition, the slice cultures seem to be able to switch from the immunologically resting state to activated modes and vice versa when exposed to pro- and anti-inflammatory stimuli at different time points. As microglia possess the Toll-like receptor 4, and thus recognized as the major LPS-responsive cell type , we conclude that these responses are mediated mainly by microglia. A number of studies support the view that indeed the microglia and their cell surface receptors, the TLRs, form a defence system that encounters the microbial attack and make up the immune system of the CNS [27, 28, 33, 34].
Moreover, based on our finding that TSA together with LPS was able to enhance the inflammatory response, we hypothesize that the microglia population, even though subjected to severe stress by 5 μg/ml of LPS alone, still possesses some reserve to counter an even more profound stimulus. As our DCX immunostaining revealed, the TSA/LPS induction was not toxic to neurons, thus suggesting that the slices, at least to some degree, have the capacity to cope with the severe inflammation.
As new methods appear, we have the possibility to refine the design of animal experiments. With the aid of sophisticated computer programs it is nowadays relatively easy to explore how manipulation of different parameters can affect the sample size or statistical power . Keeping this and the inevitable inherent biological variability in mind, we estimated the power of the statistical tests related to defined sample sizes in order to provide biologically meaningful results (see Table 1.) If one wishes to minimize the number of animals to be sacrificed and, yet, to get enough statistical power for analysis, then we propose a "one slice per well/six slices per group" model. Hence the adequate sample size can be reached with an ethically and statistically justified number of animals. Our observations indicate that the distribution of data is often more skewed in the "treatment groups", this being probably due to the biological variation within these groups. Therefore we suggest that before applying an appropriate test (in our case the nonparametric Mann-Whitney U-test) the data should be carefully analysed.
We also argue that by applying this model it is possible to avoid the potential bias emerging from interactions between slices when a number of slices are placed in proximity on the same culture membranes. The possibility of error related to the use of littermates is a matter to be considered when deciding how many study replicates are required. Our replicated studies using different litters for parallel cultures indicate that the variation between different litters is minimal and has no relevance when isogenic rat pups are used.
We suggest that this model is well suited for revealing different biochemical immune responses and these studies can be carried out as soon as after 4–5 days in culture since the slices seem to have largely recovered from the trauma caused by dissection procedure and they seem to respond to anti- and proinflammatory stimuli in a similar manner as they do later on. Also, based on the data from the temporal profile of LPS response, we conclude that NF-κB-mediated inflammatory markers can be measured any time between 6 to 48 hour exposure time. On the other hand, since the slices remain stable for at least 4 weeks, also long term, more chronic exposures to stimuli can be studied with this model.
In this report, we used inflammation as a model to illustrate the efficacy of in vitro hippocampal slice culture as an intermediate model between single cell lines and in vivo models. Furthermore, we wanted to examine the possibility of refining the model by taking into the account the experimental design involving the culture conditions and appropriate sample size. Our study highlights the potential of the currently widely used organotypic hippocampal slice culture (OHSC) as a reliable, defined model to be used in preclinical drug studies involving immune reactions and inflammation.
We thank Dr Ewen MacDonald for checking the language of the manuscript and Mr Pasi Miettinen and Mrs Airi Boman for technical assistance. This study was financially supported by the Academy of Finland and University of Kuopio.
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