Journal of Neuropathology and Experimental Neurology
Copyright q 2004 by the American Association of Neuropathologists
Vol. 63, No. 6
June, 2004
pp. 610 617
Apoptosis of Hippocampal Neurons in Organotypic Slice Culture Models: Direct Effect of
Bacteria Revisited
CHRISTIAN GIANINAZZI, PHD, DENIS GRANDGIRARD, PHD, FRANZISKA SIMON, HANS IMBODEN, PHD, PHILIPP JOSS,
MARTIN G. TÄUBER, MD, AND STEPHEN L. LEIB, MD
Abstract. Neurons of the hippocampal dentate gyrus selectively undergo programmed cell death in patients suffering from
bacterial meningitis and in experimental models of pneumococcal meningitis in infant rats. In the present study, a membranebased organotypic slice culture system of rat hippocampus was used to test whether this selective vulnerability of neurons of
the dentate gyrus could be reproduced in vitro. Apoptosis was assessed by nuclear morphology (condensed and fragmented
nuclei), by immunochemistry for active caspase-3 and DC-APP, and by proteolytic caspase-3 activity. Co-incubation of the
cultures with live pneumococci did not induce neuronal apoptosis unless cultures were kept in partially nutrient-deprived
medium. Complete nutrient deprivation alone and staurosporine independently induced significant apoptosis, the latter in a
dose-response way. In all experimental settings, apoptosis occurred preferentially in the dentate gyrus. Our data demonstrate
that factors released by pneumococci per se failed to induce significant apoptosis in vitro. Thus, these factors appear to
contribute to a multifactorial pathway, which ultimately leads to neuronal apoptosis in bacterial meningitis.
Key Words:
Brain slice culture; Cell death; Hippocampus; Nutrient deprivation; Staurosporine; Streptococcus pneumoniae.
INTRODUCTION
In bacterial meningitis (BM), cells of the hippocampal
dentate gyrus selectively undergo apoptosis (1–3), which,
in experimental meningitis, is associated with impairment
of learning and memory (3). In contrast to ischemic brain
injury, where the cornus ammoni (CA) region undergoes
apoptotic damage (1, 2), this region remains largely unaffected in BM. Recently, we identified the vulnerable
cells in the dentate gyrus as immature neurons that were
recently generated from stem cells (3, 4). In humans suffering from BM and in corresponding animal models, the
active form of caspase-3, a key enzyme for the execution
of the apoptotic program (5), was localized to the dentate
gyrus (6). A causal role of caspase-3 in the apoptosis
machinery was shown by inhibition studies (3, 7).
Although the extent of apoptosis in BM can be modulated by the use of adjunctive therapies (4, 8, 9), detailed
knowledge about the exact molecular pathways and mediators is still lacking. Recent studies have suggested that
pathogen-derived factors (10, 11) and the inflammatory
response of the host (7, 12) may contribute to apoptotic
cell death in the dentate gyrus.
In the present study, we used membrane-based organotypic slice cultures from rat hippocampus to explore
whether the selective vulnerability of neurons in the dentate gyrus observed in vivo can be replicated in vitro (13).
From Institute for Infectious Diseases (CG, DG, FS, PJ, MGT, SLL)
and Institute of Cell Biology (HI), University of Bern, Bern, Switzerland.
Correspondence to: Dr. Stephen L. Leib, Institute for Infectious Diseases, University of Bern, Friedbühlstrasse 51, 3010 Bern, Switzerland.
E-mail: [email protected]
Grant sponsor: Swiss National Science Foundation (Grant number:
632-66057.01); NIH (Grant number: 2 P50 NS35902); Meningitis Research Foundation (Grant number: 14/00).
Compared to primary dissociated cell cultures, this organotypic culture system offers the advantages of preserved morphology and tissue specific connections (14).
Furthermore, the system allows for exposure of the brain
tissue to bacterial products, thus avoiding direct contact
between bacteria and cells. This system reproduces more
closely the situation in vivo, where intact bacteria accumulate in the CSF space but generally do not penetrate
into the hippocampal parenchyma (15).
Initiation of bactericidal therapy in patients with meningitis causes rapid lysis of bacteria and an associated
brisk release of bacterial cell wall products, which in turn
triggers a burst of the inflammatory response (16, 17). In
order to mimic this pathophysiologic process in vitro, we
challenged the cultures for 3 hours (h) with multiplying
bacteria and then induced bacterial killing and lysis by
adding penicillin and streptomycin.
Metabolic disturbances are characteristic of BM and
lead to energy and glucose deprivation of brain tissue (18,
19). To mimic the in vivo situation, we exposed organotypic cultures to live pneumococci in combination with
partially or completely nutrient-deprived medium. As an
additional pro-apoptotic stimulus we used staurosporine
(STS), a compound that has been used to induce neuronal
apoptosis in primary cell cultures (20–22). We examined
the localization and extent of apoptosis by nuclear morphology, immunostaining for active caspase-3 and amyloid-beta precursor protein (DC-APP) (23), and by proteolytic activity of caspase-3 (3).
MATERIALS AND METHODS
Organotypic Cultures
The culturing technique used in this study is a modification
of the procedure described by Stoppini et al (13). Briefly, a
total of 12 nursing Wistar rat pups were killed on postnatal day
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NEURONAL APOPTOSIS IN HIPPOCAMPAL SLICE CULTURES
Fig. 1. Schematic representation of the organotypic culture
model used. In contrast to the roller-tube technique, membranebased organotypic cultures allow indirect exposure of the hippocampal formation to the pathogen. This enables bacteria-released molecules to diffuse to the hippocampal slice without
direct contact of the pathogen with the neuronal cells, representing the situation found in vivo.
7 by an overdose of pentobarbital (100 mg/kg i.p. Nembutal,
Abbott Laboratories, North Chicago, IL). The brain was removed and gently submerged in cooled Tris-buffered dissection
medium (MEM, 10 mM Tris) containing penicillin and streptomycin. After separation of both hemispheres, each hippocampus was carefully removed by separating it from the neighboring thalamus and basal ganglia and severing the
septo-hippocampal connection with a scalpel. Both hippocampi
were placed on the Teflon plate of a McIlwain tissue chopper
(Mickle Laboratory, Guildford, UK), aligned perpendicularly to
the chopper blade, and cut into 400-mm-thick slices.
The slices were transferred into fresh dissection medium and
selected for clear hippocampal morphology (intact CA regions
and dentate gyrus). The slices were then transferred onto the
porous (0.4 mm) membrane of the Transwellt inserts (Corning
Inc., Corning, NY). Per animal, up to 32 slices could be obtained for culture and 5 slices were placed per membrane. The
inserts were placed into the tissue culture 6-well plate with
equilibrated serum-free Neurobasaly medium (Life Technologies, Basel, Switzerland), supplemented with B27-Supplement
(Life Technologies). The slices were cultured at 378C in 5%
CO2-enriched atmosphere. The culture medium was replaced
with fresh equilibrated medium on days 1, 4, 7, and 9. Experiments were performed on day 11 (Fig. 1).
Stimuli
Medium: The following media were used: i) Neurobasaly
medium supplemented with B27 (NBM); ii) Hank’s buffered
with HEPES (10 mM) and NaHCO3 (1,000 mg/l) containing
25% of B27-supplemented Neurobasaly medium, set to a pH
of 7.45, (i.e. partially nutrient-deprived medium [Hank’s/
NBM]); and iii) Hank’s buffered with HEPES (10 mM) and
NaHCO3 (1,000 mg/l) (i.e. completely nutrient-deprived medium). All experiments were performed at 378C with 5% CO2.
Exposure to Staurosporine (STS): A 1-mM stock solution of
STS was diluted in 1 ml of NBM to final concentrations of 1,
2.5, and 10 mM (n 5 4 for each concentration). Exposure time
was 8 h.
Exposure to Live and Heat-Inactivated Pneumococci: A clinical isolate of Streptococcus pneumoniae (SP) known to induce
neuronal apoptosis in a rat model of BM (3, 4) was used as
previously described. The strain has been shown to produce
611
H2O2 and pneumolysin (unpublished data), both factors proposed to contribute to neuronal apoptosis in BM (12). Additionally, we used the capsule-deficient R6 SP strain (in Hank’s/
NBM medium), which allows stimulation of cells by
inflammatory components of the cell wall (24, 25). Pneumococci were grown on blood agar plates, cultured overnight in
10 ml of brain heart infusion medium, diluted in fresh medium,
and grown for 4.5 h to logarithmic phase. The suspension was
centrifuged for 10 min at 5,000 3 g and resuspended to a concentration of log10 8.3 6 0.7 cfu/ml for both pathogens in NBM,
Hank’s/NBM, and Hank’s, respectively. The concentration of
bacteria used corresponds to the one found in the CSF of infected animals in acute disease (9, 12, 26). Heat-inactivated
pneumococci were prepared by heating the bacterial pellet obtained after the first centrifugation in 0.9% NaCl at 708C for 10
min. Heat-killed bacteria were pelleted, resuspended, and sonicated 5 3 10 s on ice using the Branson Sonifier 250 (Skan
AG, Basel, Switzerland). Organotypic cultures were exposed to
2 conditions. Either live pneumococci (SP) for 9 h, with penicillin and streptomycin added after 3 h to induce bacterial killing and lysis (n 5 8 in NBM; n 5 13 in Hank’s/NBM; n 5 10
in Hank’s). Alternatively, we used heat-inactivated, sonicated
bacteria for 9 h (n 5 15 Hank’s/NBM). The corresponding controls were exposed to NBM (n 5 6), Hank’s/NBM (n 5 15),
and Hank’s (n 5 5), respectively, containing penicillin and
streptomycin. In an additional set of experiments, cultures were
exposed to live and heat-inactivated R6 at a concentration between log10 5.8 and 7.8 cfu/ml for 24 to 48 h.
Evaluation of Apoptosis
Resectioning of the Slice Cultures: The experiment was
stopped by fixation of the slice cultures in 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) for 1.5 h at
48C. The cultures were then cryo-protected in 18% sucrose solution in PBS for 4 h at 48C. Tissue Freezing Mediumy (Leica
Microsystems, Glattbrugg, Switzerland) was put on a specimen
disk (Leica Microsystems), frozen at 2208C, and an even surface was cut using a Jung CM 1800 cryostat (Leica Microsystems). A drop of tissue freezing medium was applied on a piece
(1 3 1 cm) Parafilmt (American National Can, Greenwich,
CT), and 1 slice culture was carefully pushed into the freezing
medium using a fine brush. The film was then reversed, placed
onto the surface of the freezing medium on the specimen desk,
and frozen at 2208C for 1 min. The film was removed, and
each culture cut into 10 or 20 mm sections with the cryostat.
The sections were collected in cold PBS, transferred onto
chrome-alum-gelatin-coated glass slides (27), dried for 15 min,
and evaluated by immunohistochemistry or Nissl stain.
Quantitative Assessment of Apoptotic Damage: Per slice culture, 2 to 3 Nissl-stained sections displaying clear hippocampal
morphology were evaluated for apoptosis (Fig. 2A). The number of cells with apoptotic morphology (fragmented and condensed nuclei; Fig. 2B) was counted in 1 visual field containing
the dentate gyrus or the CA3 region of the hippocampus (Fig.
2A) at a magnification of 3400 and the count averaged per
slice culture.
Immunohistochemistry: The following primary antibodies
were used at the indicated dilution to stain various targets. For
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GIANINAZZI ET AL
Fig. 2.
Documentation of morphology (A) and apoptosis in organotypic hippocampal slice cultures after exposure to STS
in Neurobasal medium (B–F). A: Morphology of a 20-mm-thick section of an organotypic hippocampal slice culture displaying
the CA regions and the dentate gyrus (DG). The open circles correspond to the visual fields for evaluation of apoptosis in the
dentate gyrus (red circle) and the CA3 region (yellow circle), respectively (Nissl; Bar: 200 mm). B: Organotypic section after
exposure to 2.5 mM of STS for 8 h. Cells with apoptotic morphology (arrowheads) are located to the inner rim of the dentate
J Neuropathol Exp Neurol, Vol 63, June, 2004
NEURONAL APOPTOSIS IN HIPPOCAMPAL SLICE CULTURES
mature neurons: mNeuN, dilution 1:2,000 (Chemicon, Temecula, CA); for activated caspase-3: pCM 1, dilution 1:500 (Idun
Pharmaceuticals, La Jolla, CA); for amyloid-b precursor protein: paDCCsp-APP, dilution 1:500 (Merck Frosst Canada, Dorval, Quebec, Canada). Ten-mm-thick sections were rinsed in
PBS and incubated with the primary antibodies diluted in TBS
containing 0.5% bovine serum albumin for 1 h at 378C. After
washing, the sections were incubated with fluorescent-labeled
secondary antibodies for 45 min at room temperature, washed,
and Dapi-stained for 3 min. The following secondary antibodies
were used: Red: anti-mouse Cy3-labeled (m), 1:1,000; anti-rabbit Cy3-labeled (p), 1:1,000; Green: anti-rabbit Alexa 488 (p),
1:500 (Alexa 488 from Molecular Probes, Eugene, OR; all others from Jackson, West Grove, PA). The slides were then
mounted using the ProLongt Antifade Kit (Molecular Probes)
and coverslipped (Fig. 2).
Proteolytic Caspase-3 Activity (DEVDase Activity): Five organotypic slices of the same experimental group were pooled
(n 5 7 for live SP-infected cultures; n 5 3 for STS-treated
cultures) and cytosols prepared by homogenization in 100 ml
of homogenization buffer (25 mM Hepes-KOH pH 7.4, 2 mM
EGTA, 2 mM MgCl2, 2 mM DTT, 100 mM PMSF, 10 mg/ml
leupeptin, 10 mg/ml pepstatin, and 10 mg/ml aprotinin). Homogenates were centrifuged at 100,000 3 g for 30 min at 48C
and supernatants referred to as cytosols. Protein concentration
was determined with the BCA kit (Pierce, Rockford, IL). Cytosols were incubated in the presence of 2 mM DEVD-AMC
(BioMol, Plymouth Meeting, PA) at 308C in assay buffer (100
mM Hepes-KOH pH 7.4, 2 mM DTT) and increasing fluorescence was measured kinetically with a SpectraMax Gemini
fluorometer (Molecular Devices, Sunnyvale, CA) at wavelengths of 380 nm (excitation) and 460 nm (emission). Results
were normalized to the protein concentration of each sample
and expressed as percent of activity in their respective control
cultures (100%).
Statistical Analysis: Data were analyzed by Prism 3.0
(GraphPad, San Diego, CA). Comparison between 2 groups was
performed by unpaired t-test for normally distributed variables.
Comparison of more than 2 groups was done by 1-way ANOVA followed by unpaired t-test for normally distributed variables and Kruskal-Wallis test followed by Mann-Whitney test
for variables that are not normally distributed. For all tests,
statistical significance was set at p , 0.05. Linear regression
was used for the STS experiment.
RESULTS
Organotypic Cultures
After 11 days in culture, more than 90% of the slice
cultures displayed the typical hippocampal organization
(Fig. 2A). The slices were found to flatten from 400 mm
613
Fig. 3. Assessment of apoptosis in the dentate gyrus (DG)
and the CA3 region of organotypic hippocampal cultures after
exposure to increasing concentrations of STS for 8 h in NBM
medium. In the DG, STS induced significant (*) apoptosis in a
dose-response fashion (r 5 0.973; p , 0.004 for 0 vs 2.5 mM,
p , 0.0001 for 0 vs 10 mM). The DG exhibited a discerning
sensitivity for STS compared to the CA3 region. High concentrations of STS also induced significant (*) apoptosis in the CA
region (p , 0.03 for 2.5 vs 10 mM). Data are presented as mean
6 SD.
(day 0 in culture) to approximately 160 mm (day 11 in
culture), as assessed by the number of 20-mm-thick cryosections generated per slice culture.
Quantification Apoptosis after Exposure to STS
In untreated control cultures, a low number of apoptotic cells were found in the dentate gyrus. Exposure to
STS led to an increase of apoptosis in the subgranular
zone of the dentate gyrus (Fig. 3) in a dose-dependent
manner (r 5 0.973). The 2 higher concentrations of STS
(2.5 and 10 mM), but not 1 mM, also induced apoptosis
in the CA3 region, although significantly less that in the
dentate gyrus (Fig. 3).
Quantification Apoptosis after Exposure to
Pneumococci in Different Media
Challenge of the cultures with SP in nutrient-containing medium (NBM) had no statistically significant effect
on the number of apoptotic cells in the dentate gyrus
(5.64 6 3.23 vs 6.81 6 4.63; p . 0.6; data are number
←
gyrus (Nissl; Bar: 15 mm). C: Mature neurons (NeuN, red) are located in the outer rim of the dentate gyrus (full arrowheads)
and in the CA regions (asterisk, CA4/hilus is shown), but not in the subgranular zone of the dentate gyrus, where apoptosis
occurs (open arrowheads, Dapi, Bar: 100 mm). D: Apoptotic cells, as shown by staining for active caspase-3 (CM 1, green), do
not colocalize with NeuN (red)-positive neurons (Dapi; Bar: 25 mm). E, F: Colocalization of active caspase-3 (CM1, green) with
its cleaved specific substrate DC-APP (red); insets show colocalization of apoptotic morphology with active caspase-3 and DCAPP, respectively (Dapi; Bar: 25 mm).
J Neuropathol Exp Neurol, Vol 63, June, 2004
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GIANINAZZI ET AL
the dentate gyrus or the CA3 region (in Hank’s/NBM)
(Fig. 4). The capsule-deficient R6 strain failed to induce
apoptosis in both NBM and partially nutrient-deprived
Hank’s/NBM medium (Table 1). Complete removal of
nutrients by keeping the cultures in Hank’s buffer alone
for 9 h led to a significant increase (p , 0.0005) in apoptosis in the dentate gyrus (12.03 6 9.22) compared to
cultures kept in Hank’s/NBM (1.40 6 2.12), but no cell
death was observed in the CA regions. Exposure to SP
resuspended in Hank’s buffer alone did not change the
extent of apoptosis in the cultures compared to the corresponding control cultures kept in Hank’s (Table 1).
Immunostaining
Fig. 4. Apoptosis in the dentate gyrus (DG) and the CA3
region of the hippocampus in cultures challenged either with
live pneumococci for 9 h (inf) or heat-inactivated and sonicated
pneumococci (hi) in partially nutrient-deprived Hank’s/NBM
medium. A significant (*) increase in apoptosis occurred exclusively in the DG and only in cultures exposed to live pneumococci (p , 0.005 vs controls and hi). No significant changes
were observed in the CA3 after challenge with live or heatinactivated bacteria, respectively. Data are presented as mean
6 SD.
of apoptotic cells/visual field expressed as mean 6 SD).
A significant increase in apoptosis after exposure to SP
occurred if cultures were kept in partially nutrient-deprived Hank’s/NBM medium (Fig. 4), but the numbers
were significantly lower than after challenge with 2.5 and
10 mm STS (in NBM) (Fig. 3). Only a minimal, nonsignificant increase in apoptosis was found in the CA3
region after exposure to live bacteria compared to controls (in Hank’s/NBM) (Fig. 4). Cultures challenged with
heat-inactivated and sonicated bacteria did not show
more apoptosis than untreated control cultures in either
Immunostaining of 10-mm-thick sections revealed that
after exposure to STS, the cells of the inner layer of the
dentate gyrus with nuclear morphology of apoptosis were
consistently NeuN-negative (Fig. 2D). In the outer layer
of the dentate gyrus and the CA regions, mature NeuNpositive neurons were found exclusively (Fig. 2C). Active caspase-3 staining (Fig. 2E) colocalized with the
cleaved caspase-3 substrate DC-APP (Fig. 2F) in cells
with apoptotic morphology (Fig. 2E, F, insets), thus documenting the functional activity of caspase-3 in these
cells.
Immunostaining for active caspase-3 was less sensitive
in detecting apoptotic cells than morphological criteria in
Nissl- or Dapi-stained sections. In the presence and absence of SP, only a fraction of cells identified as apoptotic
by Nissl staining in control or nutrient-deprived medium
was immunopositive for caspase-3. Similar experiments
using colchicine as an apoptotic agent showed that not
all granule cells displaying a fragmented nucleus were
positive for active caspase-3 (28). This may be due to
temporal differences in the expression of apoptotic markers with active caspase-3 being detectable before fragmentation of the nucleus.
TABLE 1
Effect of Experimental Stimuli and Media on Apoptosis in the Dentate Gyrus of Organotypic Hippocampal Cultures
Challenges
Media
SP
SP
NBM
Hank’s/NBM
SP
R6
Hank’s
Hank’s/NBM
STS
NBM
Experimental
groups (n)
ctrl (6) vs inf (8)
ctrl (15) vs inf (13)
ctrl (15) vs hi (15)
inf (13) vs hi (15)
ctrl (5) vs inf (10)
ctrl (10) vs inf (10)
ctrl (10) vs hi (8)
inf (10) vs hi (8)
0 (4) vs 1 mM (4)
0 (4) vs 2.5 mM (4)
0 (4) vs 10 mM (4)
Fold change
in apoptosis
p value
6
6
6
6
6
6
6
6
6
6
6
n.s.
,0.002
n.s.
,0.002
n.s.
n.s.
n.s.
n.s.
n.s.
,0.004
,0.001
1.2
5.3
1.3
0.23
1.5
1.4
1.4
0.97
3.8
15.3
32.3
0.8
4.5
1.4
0.27
0.85
0.2
0.4
0.3
2.3
4.2
5.3
Abbreviations: SP 5 encapsulated Streptococccus pneumoniae strain; R6 5 capsule-deficient Streptococcus pneumoniae strain;
STS 5 staurosporine; NBM 5 Neurobasal medium; ctrl 5 controls; inf 5 infected; hi 5 heat-inactivated.
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NEURONAL APOPTOSIS IN HIPPOCAMPAL SLICE CULTURES
Double labeling for active caspase-3 and GFAP, a
marker for astroglial cells, did not show colocalization
(data not shown). GFAP staining was preferentially localized to astrocytic processes, while active caspase-3
stained cellular bodies. Furthermore, caspase-3-positive
cells were localized to the inner layer of the hippocampal
dentate gyrus, where GFAP staining was largely absent.
Together with clear colocalization of active caspase-3
with neuronal cell markers in animals with experimental
meningitis (author’s unpublished observation), these data
suggest that a majority of apoptotic cells are neurons.
Caspase-3 Activity
In homogenates of cultures exposed to 2.5 mM STS,
proteolytic caspase-3 activity significantly increased by
605.6% 6 83.9% compared to controls (p , 0.005).
Challenge of cultures with live pneumococci (in NBM/
Hank’s) failed to significantly increase the activity
(111.1% 6 49.2% of control cultures). This finding is in
agreement with the results of caspase-3 immunostaining
and the morphological assessment of apoptosis after exposure to STS and pneumococci. The lack of a significant
increase in caspase-3 activity after exposure to SP corroborates the notion that pneumococci alone are insufficient to induce significant apoptosis.
DISCUSSION
In organotypic brain slice cultures, a subpopulation of
neurons in the hippocampal dentate gyrus were selectively vulnerable to undergo apoptosis after exposure to STS
and, to a much lesser extent, after co-incubation with live
pneumococci under nutrient-deprived conditions. Staurosporine, a microbial alkaloid isolated from Streptomyces staurospores, has been used in neuronal and nonneuronal cells to induce programmed cell death (21, 22,
29). In the present study, STS induced apoptosis in a
dose-response manner, preferentially in the dentate gyrus
and to a lesser extent in the CA regions, as shown by
cell morphology, specific markers (active caspase-3, DCAPP), and increased proteolytic caspase-3 activity.
The subgranular zone of the dentate gyrus contains a
population of continuously dividing stem cells, the progenitors of which then migrate and differentiate into brain
cells, including neurons (30, 31). Our findings in the organotypic slice culture system are in agreement with results generated in the in vivo model of bacterial meningitis (3, 4). In both systems, NeuN-negative neurons in
the subgranular zone of the dentate gyrus were preferentially affected by apoptosis. The basis for this selective
vulnerability is currently unknown. It is tempting to speculate that the susceptibility of these immature cells and
the observation that STS induces apoptosis via cell cycle
arrest are related (32, 33).
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In vivo and in vitro data have produced evidence for
bacteria-derived H2O2 and pneumolysin as possible mediators of neuronal apoptosis in bacterial meningitis (an
H2O2 and pneumolysin producing strain was also used in
the present study) (10, 11). In addition, modulation of
host factors, such as inflammatory parameters (7, 12),
influences the extent of hippocampal apoptosis. In bacterial meningitis, metabolic abnormalities lead to cerebral
energy depletion with decreased glucose concentrations
(18, 19). Our data provide evidence for an environment
of nutrient deprivation as prerequisite for bacterial products to induce apoptosis in this system. Similar synergistic effects may play a role in vivo.
Although the culture system used here was optimized
to reproduce the in vivo situation of bacterial meningitis,
the extent of apoptosis induced by exposure to pneumococci in deprived medium was very low when compared
to the extent of apoptosis found in the infant rat model
of bacterial meningitis (in vitro 7.39 6 6.32 vs in vivo
25.65 6 30.27 apoptotic cells/visual field, normalized for
the quantitation system used [3, 4]). In addition, exposure
of organotypic slice cultures to live pneumococci for 12
and 18 h did not further increase the number of apoptotic
cells (data not shown). We conclude that, although addition of bacteria to membrane-based organotypic cultures allows soluble and secreted bacterial toxins to diffuse to the hippocampal neurons, other non-bacterial
factors are necessary for the induction of relevant neuronal apoptosis as observed in bacterial meningitis. Our
findings suggest that hippocampal apoptosis in bacterial
meningitis is a multifactorial event that includes energy
deprivation and bacterial products.
The complexity of the situation in vivo makes an accurate representation of the pathogenesis of neuronal injury in vitro difficult. For example, in an organotypic
model based on the roller-tube technique (34), cells undergoing apoptosis were in direct contact with the pathogen, i.e. a heat-inactivated capsule-deficient R6 Streptococcus pneumoniae strain (24, 25) (for a overview of
the methods and stimuli used in organotypic cultures of
hippocampi see Table 2). This situation is different from
the in vivo situation of bacterial meningitis, where intact
bacteria do not directly interact with neurons but are limited to the CSF space. Comparison of the present results
with the study by Schmidt et al (24) emphasizes that
differences in the in vitro culture systems are important,
since the same stimulus (R6 strain, 6 3 105 2 6 3 107
cfu/ml; 24 to 48 hours of exposure) led to significant cell
death in the roller tube model but not in the present study.
Intriguingly, the organotypic roller-tube cultures and primary brain cell cultures, where the cellular connections
between the different brain cell types is lost, showed different responses when challenged with heat-inactivated
pneumococci. Primary cell cultures were not damaged by
the heat-killed organism (35), whereas apoptosis was
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TABLE 2
Hippocampal Organotypic Cultures Used to Study Cell Death in Bacterial Meningitis
Technique/
animal
Stimuli
Hippocampal
apoptosis
Roller tube/rat
hiR6, LTA, PG, PDNA
yes
Roller tube/mouse
Roller tube/mouse
Pneumolysin
hiR6
yes
yes
Membrane/rat
SP, R6; hi and live
yes
Remarks
Necrosis in CA1 and CA3; apoptosis most
prominent with hiR6
Apoptosis selectively in the DG
Apoptosis selectively in DG and Pyramidal
cells; DG also affected by necrosis
Apoptosis selectively in the DG; apoptosis
observed only with live SP in combination with partial nutrient deprivation
References
(24)
(11)
(25)
this study
Abbreviations: hi R6 5 heat-inactivated R6; LTA 5 lipoteichoic acid; PG 5 peptidoglycan; PDNA 5 pneumococcal DNA;
DG 5 dentate gyrus; SP 5 Streptococcus pneumoniae.
found in the dentate gyrus of the organotypic cultures
kept in roller tubes (24). These results suggest that heatkilled organisms induce neuronal apoptosis if they come
in direct contact with brain cell cultures that include nonneuronal cells such as microglia. This is in keeping with
previous results in dissociated cell culture systems, where
bacterial cell wall induced neuronal injury only in the
presence of glial cells (36). The present model of organotypic culture models 2 important features of the situation
in vivo that are lost in the 2 other models. It retains the
hippocampal morphology and cellular context and allows
for indirect exposure of the brain tissue to live pathogens
with diffusion of mediators of cell death into the tissue.
In summary, our data suggest that diffusible pneumococcal factors alone are insufficient to induce relevant
apoptosis during bacterial meningitis. The present model,
which allows for exposure of the intact hippocampal formation to host factors and soluble bacterial products
without direct contact between the pathogen and brain
cells, provides a tool for further studies of factors from
the pathogen and the host as inducers of hippocampal
apoptosis during bacterial meningitis.
ACKNOWLEDGMENTS
We greatly acknowledge Dr. D. Nicholson (DC-APP antibody), Dr.
A. Srinivasan (CM 1 antibody), Dr. L. Stoppini for introducing us to
the culture system, and J. Zbären for optimizing immunohistochemistry.
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Received November 11, 2003
Revision received February 18, 2004
Accepted March 1, 2004
J Neuropathol Exp Neurol, Vol 63, June, 2004