Journal of Neuroinflammation

BioMed Central

Research

Open AccessA refined in vitro model to study inflammatory responses inorganotypic membrane culture of postnatal rat hippocampal slices
Jari Huuskonen*1, Tiina Suuronen1, Riitta Miettinen1,3, Thomas van Groen2,3
and Antero Salminen1,3
Address: 1Department of Neuroscience and Neurology, University of Kuopio, PO Box 1627, FIN-70211 Kuopio, Finland, 2Department of Cell
Biology, University of Alabama at Birmingham, Birmingham, AL 35294-0006, USA and 3Department of Neurology, University Hospital of Kuopio,
PO Box 1627, FIN-70211 Kuopio, Finland
Email: Jari Huuskonen* - Jari.Huuskonen@uku.fi; Tiina Suuronen - Tiina.Suuronen@uku.fi; Riitta Miettinen - Riitta.Miettinen@uku.fi;
Thomas van Groen - vangroen@uab.edu; Antero Salminen - Antero.Salminen@uku.fi* Corresponding author

Published: 15 November 2005
Journal of Neuroinflammation 2005, 2:25

doi:10.1186/1742-2094-2-25

Received: 04 November 2005
Accepted: 15 November 2005

This article is available from: http://www.jneuroinflammation.com/content/2/1/25© 2005 Huuskonen et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Propagated tissue degeneration, especially during aging, has been shown to beenhanced through potentiation of innate immune responses. Neurodegenerative diseases and awide variety of inflammatory conditions are linked together and several anti-inflammatorycompounds 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.
Methods: The responses of the slices to pro- and anti-inflammatory stimuli were monitored atvarious time points by measuring the leakage of lactate dehydrogenase (LDH) and the release ofcytokines interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) and nitric oxide (NO) fromthe 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.
Results: 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 byquantitative biochemical assays and morphological characterizations.
Conclusion: The present report outlines the techniques for studying immune responses using aserum-free slice culture model. Statistically powerful data under controlled culture conditions andwith ethically justified use of animals can be obtained as soon as after 4–5 DIV. The model is mostprobably suitable also for studies of chronic inflammation.

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BackgroundThe discovery of upstream sensors, the Toll-like receptors(TLRs) [1,2], greatly multiplied our understanding ofinnate 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 [5]. These cells, which can present antigen and are
responsible for the production and release of a variety ofcytokines and chemokines, interact with immune cells
and are intimately involved in immunoregulation within
the CNS [6]. Whereas the role of microglia in the brain has
been studied extensively [7-11], most progress on theunderstanding 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 thecomposition 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 [12].
In the slice culture systems, whether supplemented withserum 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 phe-

A
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IL-6

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Figure 1 recovery of P7 hippocampus slices after explantation
Biochemical
Biochemical recovery of P7 hippocampus slices after explantation. Culture medium was collected at 24 h intervals during the 7
DIV culture period. Values are means ± SD (n = 6 in each group).

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Live/dead-assay
Figure 2
showing the viability of the P7 hippocampus slice cultured for 4 weeks in serum-free media
Live/dead-assay showing the viability of the P7 hippocampus slice cultured for 4 weeks in serum-free media. Green fluorescence (A) is an indicator of live cells and red (B) indicates the dead-cell population. An overlay (C) shows that mainly live cells
are present. Scale bar 200 µm.
notype of microglia would have reduced functional status
but a recent in vivo study by Nimmerjahn and co-workers[19] convincingly demonstrates how microglia cells constantly monitor their immediate environment by extending and retracting their projections in a minute-to-minutetime 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 arecapable of complex behaviour and therefore it is of crucial
importance to pay attention to the factors that contributeto the consistency of in vitro models used to mimic in vivo
situations.
In the present study, we used hippocampus tissue slices inserum-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 invitro culture of postnatal brain provide a model where thecytoarchitecture and connectivity of different anatomical
regions, as well as the functional relationships and interactions with neighbouring cell types (i.e., neurons andastrocytes) are preserved [22,23]. Organotypic culturesoffer also the advantage of controlled manipulations inliving tissue and thus they might represent an analogously
feasible intermediate between simpler cell lines and invivo 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,

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LPS (µg/ml)

Figureof3the dose of LPS on the inflammatory response in P7 hippocampus slices
EffectEffect of the dose of LPS on the inflammatory response in P7 hippocampus slices. LPS was added to the medium after 4 DIV
and the exposure time was 24 h. LDH secretion remained close to control levels in all LPS groups (A). NO and IL-6 secretion
increased prominently already at the dose of 0.1 µg/ml but there was no significant dose-dependent difference in any treatmentgroup. Values are means ± SD (n = 6 in each group).
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.

Methods
Preparation of hippocampal slice cultures
Organotypic slice cultures from hippocampus were prepared using the modified interface culture method
described by Stoppini et al. [24]. Postnatal day 7–8 (P7–P8) Wistar rat pups were decapitated, and the brains wererapidly dissected and placed in a petri dish in ice-cold 1 ×
Dulbecco's Phosphate Buffered Saline without calcium
and magnesium (Biowhittaker™, Belgium). The hippoc-

ampi of both sides were isolated and sectioned into 400µm 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 (bothfrom 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, inacti-

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alin [26] at a concentration of 0.5 µM. TSA was purchasedfrom Sigma and helenalin from BIOMOL Research Laboratories (Plymouth Meeting, PA, USA). We also preexposed 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 atleast three times.

IL-6

4000

LPS 5 µg/ml

3000

2000

1000

7

14

21

DIV

FigureEffectIL-6
responseof4culture time prior to LPS exposure on LPS-induced
Effect of culture time prior to LPS exposure on LPS-induced
IL-6 response. LPS was added to the medium after 7, 14 and
21 DIV; exposure time was 24 h. LPS evoked extensive
secretion of IL-6 at all of the time points. Values are means ±SD (n = 6 in each group).

vated fetal bovine serum (FBSi, Gibco) was added to theculture medium at a concentration of 10%. On the nextday, the culture medium was replaced with fresh medium
without serum and from then on serum-free media waschanged twice a week.
Animals were obtained from National Laboratory Animal
Center (NLAC), University of Kuopio, Finland. The experiments were conducted according to the Council ofEurope (Directive 86/609) and Finnish guidelines, andapproved 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 [25] and as an anti-inflammatory stimulus we used a known NF-κB inhibitor helen-

Histological methods
For revealing microglia, hippocampal slices were stainedwith 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 TBST, 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 antimouse, 1:1000, Serotec) and 2 h incubation (room temperature) with avidin peroxidase (1:1000 ExtrAvidin E2886, 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 andthorough 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) followedby 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 andresponses
First, some hippocampi were rapidly sliced at 400 µm andimmediately fixed and stained for microglia, astrocytes

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48 hours

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Figureof5LPS exposure time at 4 DIV and 7 DIV
EffectEffect of LPS exposure time at 4 DIV and 7 DIV. Medium was collected after 3, 6, 12, 24 and 48 h of exposure to LPS. Values
are means ± SD (n = 6 in each group).

and neurons with IB4, GFAP and DCX, respectively, toreveal the morphological status at the beginning of theculture. Then, the culture medium was collected and tissues fixed at 24 h intervals during the 7 days in vitro (7DIV) culture period. Cytokines IL-6 and TNF-α, NO andLDH 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 andbiochemical 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 theGriess 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 incubationfor 10 min. The concentrations of cytokines IL-6 andPage 6 of 15(page number not for citation purposes)

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A
ng/ml

B

IL-6
IL

µM

*

40

NO
*
*

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30

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LPS+TSA+HELE

LPS+TSALPS+HELE

CONTROL

LPS+TSA+HELE

LPS+TSA2

LPS+HELE

10

LPS

4

CONTROL

20

LPS

*

Figure 6 and downregulation of LPS-induced inflammatory response by TSA and helenalin
Potentiation
Potentiation and downregulation of LPS-induced inflammatory response by TSA and helenalin. Exposure time was 24 h at 4
DIV. LPS concentration was 5 µg/ml, TSA concentration was 20 nM and that of helenalin 0.5 µM. Values are means ± SD (n =6 in each group). * p < 0.05 (Mann-Whitney U test).

tumour necrosis factor (TNF)-a released into the medium
were measured by an enzyme linked immunosorbent
assay (ELISA) using OptEIA™ kits or sets obtained fromPharmingen (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 tothe 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, yetappropriate 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 tosix per group and the significance level to 5% to see theeffect 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.

Results
Slices recover from explantation by 4 DIVFirst, we wanted to know how well hippocampal slices
would recover from the trauma caused by the isolation
procedure. Therefore, we collected the medium after every
24 h during 7 DIV and used standardised protocols tomeasure the secretion of IL-6, TNF-α and the leakage of
LDH. The secreted level of IL-6 was highest at 48 h after
explantation and returned to the basal level by 4 DIV (Fig.
1A). The peak levels of LDH and TNF-α were recorded at
1 DIV and reverted to control levels by 3 DIV (Fig. 1B&1C). From that time, the levels of cytokines and LDH
exhibited some variability but remained at rather low levels. The Live/dead-assay showed that there were numerous dead/dying cells during the first days of culture (notPage 7 of 15(page number not for citation purposes)

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Table 1: 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 (MannWhitney) rank-sum test.

Test significance level, αC vs. LPS
LPS vs. LPS+TSA
LPS vs. LPS+Hele
LPS+TSA vs. LPS+TSA+Hele

Power (%)

n per group

Effect size, δ = µ1 - µ2/σ

IL-6

NO

IL-6

NO

IL-6

NO

IL-6

NO

0.05
0.05
0.05
0.05

0.05
0.05
0.05
0.05

85
78
53
77

85
4
57
80

6
6
6
6

6
6
6
6

-11.2
-2.4
1.4
2.3

-9.2
-0.2
1.5
2.7

shown) but gradually the number of dead/dying cells
decreased and at 4 weeks in vitro mostly live cells were visible (Fig. 2A–C)
Effect of LPS is not concentration dependent
It has been shown that microglia express Toll-like receptor
4 (TLR4) [27,28], which mediates the LPS induced intracellular NF-κB signaling pathway and evokes the release ofcytokines. To investigate whether the concentration of LPS
had any effect on the slices during the 24 h exposure, we
pre-exposed the cultures to 0.1, 0.5, 1, 5 and 10 µg/ml of
LPS at day 4. The LDH leakage did not differ significantly
at any of these LPS-concentration levels and the control
slices showed only minimally lower level of secretion
than LPS treated slices (Fig. 3A). The NO and IL-6 levels
were clearly higher compared to control slices but LPS did
not induce any prominent concentration-dependent
effect at any treatment groups (Fig. 3B &3C).
Effect of LPS is exposure but not culture time dependent
To address the question of whether the slice cultures
respond to LPS differently during the culture time, we
added LPS (5 µg/ml) to the medium for 24 h at days 7, 14
and 21 in vitro. LPS evoked extensive secretion of IL-6 at
all the time points (Fig. 4), thus reflecting the capability ofslices to respond to inflammatory stimuli with similarmanner as at 4 DIV.

We also determined the temporal profile of LPS responseby measuring the levels of IL-6, TNF-α, NO and LDH after
3 h, 6 h, 12 h, 24 h and 48 h exposures to 5 µg/ml of LPS
at DIV 4 and 7. The secreted level of IL-6 was clearly
increased after 6 hours and the highest levels were foundafter 48 hours both at 4 DIV and 7 DIV (Fig. 5A). TNF-α
secretion was already prominently higher at 3 hours, continued rising at 6 hours and was highest after 12 hours of
exposure at 4 DIV. The pattern of TNF-α secretion at 7 DIV
was similar to that seen at 4 DIV, but the overall levels
were significantly lower and the value after 48 hours washigher than the value of 24 hour exposure (Fig. 5B). The
NO levels at 7 DIV increased by degrees at 3 h, 12 h and48 h time points but at 4 DIV ascended more evenly (Fig.
5D). At 7 DIV a similar elevation, as seen in NO levels,
was seen after 12 h when LDH was measured from thesame samples. The 4 DIV levels of LDH compared to thoseat 7 DIV were approximately three- to four-fold higher at
3 and 6 hour time points and two-fold higher after 12hour exposure to LPS (Fig. 5C).
Slices respond well to pro and anti-inflammatory stimuli
during the culture
Next, we wanted to see if either proinflammatory histone
deacetylase inhibitor TSA or anti-inflammatory NF-κB
inhibitor helenalin could influence the LPS-induced
response. Interestingly, TSA significantly potentiated theIL-6 response of LPS and helenalin seemed to downregulate the response to both LPS and LPS/TSA exposure after
24 hour exposure at DIV 4. Helenalin also downregulated
the nitric oxide levels when combined with LPS alone andtogether with LPS and TSA (Fig. 6A &6B).
Morphological changes of microglia, neurons andastrocytes during culture period
At the beginning of culture, immediately after sectioning,
microglia displayed an idiotypical "resting", ramified
morphology with small cell bodies and numerous
branching processes (Fig. 7A). After 1 DIV, the microglia
started to revert gradually into an intermediate, "reactive"
form with larger cell bodies and several thicker branches(Fig. 7B) and by 4–5 DIV a number of the IB4-positive
cells appeared as the characteristic rounded, "amoeboid"
phenotype, though also pleomorphic cells exhibiting projections were visible (Fig. 7C &7D). The vascular endothelium and the amoeboid cells with protruding filopodia
were also labeled by IB4. As the culture time extended, themorphological polymorphism of microglial-cell population continued, e.g., all shapes of microglia from "amoeboid" like to "resting", ramified were found in all of theIB4 labeled slices. The same phenomenon was seen when
the slices were stained with OX-42. Morphologically
active "phagocytic" cells were found in clusters mainly inthe cornu ammonis of hippocampus, and ramified cells

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B

A
ź

C

D

ź

FigureLabeling7of microglia with Alexa Fluor 488 conjugated lectin IB4
Labeling of microglia with Alexa Fluor 488 conjugated lectin IB4. (A) Hippocampus slice of P7 rat stained immediately after sectioning. Microglia demonstrate typical "resting", ramified morphology with small cell bodies and branching processes (arrows).
(B) After 1 DIV, the microglia started to revert to a more intermediate "reactive" form with larger cell bodies and thicker
branches (arrow) and by 4 DIV a number of cells appeared with a rounded "amoeboid" phenotype (C, arrows), but also more
pleomorphic cells with projections were visible (C&D, dashed arrows). Note also labeling of vascular endothelium (arrowheads in A and C). Scale bar equals 50 µm.
were scattered throughout the slice with no specific localisation. Despite the differences in cytokine secretion,
microglia exhibited a heterogenous morphology also in
all the experimental groups (Fig. 8A–B.)
GFAP staining revealed the characteristic star-shaped morphology of astrocytes immediately after the explantation.
In addition, the expansions of astrocytic processes, the"end-feet", enveloping the microvessels were clearly visible (Fig. 9A), thus mimicking the situation in vivo. After 2
DIV, GFAP-positive cells started to transform into fibrous

cells with long processes (Fig. 9B), eventually extending
throughout the whole slice by 7 DIV (Fig. 9C). As the culture time lengthened, astrocytes remained throughout theslice displaying the fibrous and star-shaped morphology(Fig. 10A–C). The dentate area remained prominently
covered with cells expressing doublecortin, showing thetypical morphology of neurons (Fig. 11A and 11B;
inserts). DCX-positive cells were found, though in lesser
numbers, also in the CA-areas. Regardless of LPS or LPS/TSA exposure either at 7 or 14 DIV, both astrocytes andneurons showed a similar staining pattern as control

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Figure 8
Hippocampus
slice stained with OX-42 at 8 DIV after 24 h exposure to 5 µg/ml of LPS
Hippocampus slice stained with OX-42 at 8 DIV after 24 h exposure to 5 µg/ml of LPS. The heterogenous morphology of
microglia continued despite the extensive culture time and LPS exposure (A-C, arrows). A, "resting", ramified microglia, B,"reactive", activated microglia, and C, "phagocytic", macrophage microglia. Scale bar equals 50 µm.

slices, as judged from the morphological point of view(Fig. 11A–D). Also the overall morphological integrity ofthe slices remained mostly well preserved (Fig. 12A–C),
i.e. the neuronal layers could be recognized despite thedifferent treatments.
Statistical power analysis and optimal sample size
Our results from parallel cultures indicate that with randomly chosen slices (one per well) and a 5% test significance level it is possible to obtain over 80% statisticalpower with a sample size of six, when control and LPS
groups are compared (Table 1). Likewise, the analysis
yielded a biologically satisfactory 77–80% power with thesame sample size for the TSA groups. The effect of helenalin without TSA (53% power for IL-6 and 57% for nitricoxide) was not statistically prominent and TSA clearly had

no effect on nitric oxide secretion when combined withLPS (4% power).

Discussion
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 hasreceived much attention since there are both ethical andtechnical 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 andcommitment among the regulatory authorities, in the

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Figure 9 in P7 hippocampus slice stained with a GFAP antibody
Astrocytes
Astrocytes in P7 hippocampus slice stained with a GFAP antibody. (A) Immediately after sectioning, astrocytes displayed a starshaped phenotype (arrows), also the "end-feet" enveloping microvessels were clearly visible (dashed arrows). (B) After 2 DIV
the GFAP-positive cells started to transform into fibrous cells with long processes and (C) by 5 DIV, astrocytes with long processes covered the whole slice, with the semblance of an astrocytic scar. Scale bars in A and B equal 50 µm, C = 100 µm.

European Union and the USA, to find ways to reduce thenumber of animals used in laboratories for experimental
and other scientific purposes to the minimum level [31].
We have previously observed that, in the presence ofserum, the LPS response is higher than without serum(Fig. 13). Also, our earlier studies have shown that the efficient concentrations of LPS are dependent on the type of
LPS product. In this study, especially to ensure the penetration of LPS throughout the whole slice, we used a saturation level of Sigma L6529 product since we have tested
the concentration dependence of that LPS product in our
different models (see e.g. [32]). As our results demon-

strate, the concentration used did not show any toxic
response (Fig. 3A) and did not prevent the pro- and antiinflammatory responses (Fig. 6A &6B). The vehicle effect(i.e. water) was also carefully excluded.
Our present findings demonstrate that slices cultured inserum-free medium still respond significantly and in asimilar manner to an inflammatory stimulus between theculture period from 4 DIV to 21 DIV. We demonstrate thatin our model the phenotype of the microglial-cell population remains heterogenous throughout the culture timeafter a recovery period of approximately 4–5 DIV. Despite
the nonuniform microglial appearance, the non-treatedPage 11 of 15(page number not for citation purposes)

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cytokine IL-6 and NO.

A

B

C

FigureP7
GFAP
hippocampus
antibody
10
for
slice
astrocytes
cultured for 14 DIV and stained with a
P7 hippocampus slice cultured for 14 DIV and stained with a
GFAP antibody for astrocytes. (A) GFAP-positive cells continued to extend throughout the whole tissue (B) displaying
the fibrous long processes and (C) star-shaped morphology
through the slice. Scale bars: A = 200 µm, B = 100 µm and C= 50 µm.

control slices seem to retain stability as what comes tolysis of cells (LDH leakage) and to their ability to release

In addition, the slice cultures seem to be able to switchfrom the immunologically resting state to activatedmodes and vice versa when exposed to pro- and antiinflammatory stimuli at different time points. As microglia possess the Toll-like receptor 4, and thus recognized
as the major LPS-responsive cell type [32], 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 adefence system that encounters the microbial attack andmake 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, wehypothesize that the microglia population, even thoughsubjected to severe stress by 5 µg/ml of LPS alone, still
possesses some reserve to counter an even more profoundstimulus. As our DCX immunostaining revealed, the TSA/LPS induction was not toxic to neurons, thus suggestingthat the slices, at least to some degree, have the capacity tocope 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 canaffect the sample size or statistical power [35]. Keeping
this and the inevitable inherent biological variability inmind, 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 tominimize 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 oferror 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 forparallel cultures indicate that the variation between different litters is minimal and has no relevance when isogenic
rat pups are used.

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A

B

C

D

Figure 11
Neurons
stained with DCX and astrocytes with GFAP
Neurons stained with DCX and astrocytes with GFAP. (A) DCX-positive cells in the dentate gyrus of P7 hippocampus slice
treated for 24 h with LPS (5 µg/ml) and (B) LPS and TSA (20 nM) at 7 DIV. The treatments had no visible effect on morphology(inserts in A & B, scale bars equal 50 µm), staining intensities or number of labeled neurons. (C) Astrocytes in the CA-area ofcontrol slice and (D) LPS treated slice after 14 DIV. Similar long processes were visible in both groups as at 5 DIV (Fig. 9C).
Scale bars: A and B = 100 µm, C and D = 50 µm.

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 respondto anti- and proinflammatory stimuli in a similar manneras they do later on. Also, based on the data from the temporal profile of LPS response, we conclude that NF-κBmediated 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 bestudied with this model.

Conclusion
In this report, we used inflammation as a model to illustrate the efficacy of in vitro hippocampal slice culture as anintermediate 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 theexperimental design involving the culture conditions andappropriate sample size. Our study highlights the poten-

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A

http://www.jneuroinflammation.com/content/2/1/25

pg/ml

IL-6

CA1
1500

CA3

DG
1000

500

B

C

CA1
CA3
DG

Figure
Nissl
cal
free
integrity
media
stained12 ofsections
the P7 hippocampus
demonstratingslices
the overall
culturedmorphologiin serumNissl stained sections demonstrating the overall morphological integrity of the P7 hippocampus slices cultured in serumfree media. (A) Control slice cultured for 5 DIV. (B) LPS (5µg/ml/24 h) treated slice at 5 DIV. (C) LPS (5 µg/ml/24 h) andTSA (20 nM/24 h) treated slice at 14 DIV. The neuronal layers could be recognized despite the different treatments. DG= dentate gyrus, CA3 = cornu ammonis 3 and CA1 = cornu
ammonis 1. Scale bars equal 200 µm.

LPS+SERUM -BIII

LPS+SERUM +DG

CONTROL SERUM -CA3

CONTROL SERUM +CA1

Figureof13
EffectmentedLPS-induced(Neurobasal
serum-freeIL-6 response(Neurobasal+ B27 + 10%+FBSi)
B27) culturevs. serum
media
suppleon
Effect of serum-free (Neurobasal + B27) vs. serum supplemented (Neurobasal + B27 + 10% FBSi) culture media on
LPS-induced IL-6 response. LPS (5 µg/ml) was added to theculture media at 4 DIV for 24 h. LPS response was enhancedwith serum supplementation. Values are means ± SD (n = 6
in each group).
tial 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.

Competing interests
The author(s) declare that they have no competing interests.

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Journal of Neuroinflammation 2005, 2:25

Authors' contributions
JH conceived the design of the study, carried out all experiments, performed data analysis and drafted the manuscript. TS aided in slice culture experiments, participatedin study design and gave critical analysis of the manuscript. RM aided in manuscript preparation, experimental
design and supervised the histological studies. TvG helped
especially in histology and provided the facilities, participated in study design and preparation of manuscript. AS
aided in study design, especially with regards to inflammatory experiments, supervised all experiments andhelped to draft the manuscript. All authors read andapproved the final manuscript.

Acknowledgements
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 Universityof Kuopio.

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