Indian Journal of Experimental Biology

Vol. 51, August 2013, pp. 606-614

Differential microglial and astrocytic response to bacterial and viral infection in

the developing hippocampus of neonatal rats
Nisha Patro1, Kavita Singh1 & Ishan Patro1,2,*
Schools of Studies in 1Neuroscience and 2Zoology, Jiwaji University, Gwalior 474 011, India

Received 8 January 2013; revised 25 April 2013

Polyinosinic:polycytidylic acid (Poly I:C; 5 mg/kg body weight, ip) and lipopolysaccharide (LPS; 0.3 mg/kg
body weight, ip) induced microglial and astrocytic activation in Sprague Dawley rats. Higher microglial and astrocytic
activities were noticed in Poly I:C infused rats throughout the hippocampus till postnatal day 21 with a comparatively
weaker response in LPS group. However, LPS induced inflammation persisted even after postnatal day 21, indicating
thereby, that the Poly I:C (viral mimic) produces an acute inflammation, while LPS (bacterial endotoxin) produces chronic
inflammation when exposed during early neonatal life.

Keywords: Astrocytes, Infections, Lipopolysaccharide, Microglia, Poly I:C

Early life exposures to infections and related
neuroinflammatory experiences may influence neural
development and thus critically influence cognitive
abilities and neuroinflammatory responses at
adulthood and senility. Neuroinflammation is a
complex host defense response to injury, infections
and diseases, including neurodegenerative diseases
like multiple sclerosis, Alzheimers disease,
Parkinsons disease, narcolepsy and even autism1-5.
Inflammation in the CNS is driven by the activation
of resident microglia, astrocytes and infiltrating
peripheral macrophages. These cells release a battery
of anti- and pro-inflammatory cytokines, chemokines,
neurotransmitters and reactive oxygen and nitrogen
species6.
Lipopolysaccharide (LPS), is a heat stable and non-
proteinaceous endotoxin consisting of highly variable
conserved segments7,8. This is an outer membrane
molecule essential for virtually all gram-negative
bacteria. Intracerebral LPS injection has been reported
to induce chronic brain inflammation including
morphological activation of microglia, neutrophil
infiltration and mRNA/protein expression of
inflammatory mediators within 1-3 days in the various
brain regions9. Such chronic brain inflammation
aggravates brain damage and may cause
neurodegenerative diseases including Parkinsons
disease9,10.
Polyinosinic:polycytidylic acid (Poly I:C) is a
synthetic double stranded RNA (dsRNA) that elicits
immune response similar to what is observed during
viral infection11,12. Poly I:C mediated microglial
activation has been reported to directly affect motor
coordination and spontaneous locomotor activity13.
Poly I:C is a potent interferon (IFN)- inducer in
rodents. Toll like receptor 3 (TLR3), a member of
the TLR family of proteins has been identified as
receptors for dsRNA14,15. Microglia recognizes
dsRNA through TLR3 and associated signaling
molecules and thus is considered to be the key sensors
of dsRNA producing viruses that may invade the
CNS16. In CNS, both microglia and astrocytes express
toll like receptors (TLRs) specific for the recognition
of pathogen associated molecular patterns (PAMPS)
from diverse organisms like bacteria, viruses, yeasts,
fungi and other parasites and thus have the ability to
contribute to the innate immunity in CNS17-20. It is
also believed that such early life experiences may
influence on the individuals response to challenges
later in life.
It is now believed that neuroinflammation acts as
an important factor in neuronal repair and adult
neurogenesis21-24. How such challenge would

* influence development of the hippocampus needs

Correspondent author
Telephone: +91 751 2442789 attention. The present study proposes the hypothesis
E-mail: ishanpatro@gmail.com that early neonatal infection by bacterial and viral
 PATRO et al.: GLIAL RESPONSE TO NEONATAL BACTERIAL AND VIRAL INFECTION
agents induce inflammatory response as indicated by
glial activation and associated degeneration in the
developing hippocampus that persists to cause
developmental damage during early life (up to
postnatal day 21 in rats). Further, early fetal period is
quite distinct from later developmental time points in
terms of cytokines expression20. The postnatal days
0-21 are equivalent to the last trimester of human
pregnancy and brain development. Thus this time
point has been used to study the influence of
infections on brain development when major and
distinct brain development occurs in terms of
cytogenesis, morphogenesis, histogenesis and
synaptogenesis.
Materials and Methods
Neonatal pups born to healthy Sprague Dawley
females were randomly allocated to following 3
groups: control, LPS or Poly I:C group. The day of
parturition was designated as postnatal day (PND) 0.
On PND-3 and PND-5 pups of both LPS and Poly I:C
groups were briefly removed from their home cages
and administered intraperitoneally either with LPS
(0.3 mg/kg body weight; Escherichia coli 011 : B4,
Sigma-Aldrich Chemicals Co., USA) or Poly I:C
(5 mg/kg body weight; Sigma-Aldrich Chemicals Co.,
USA) and then returned to their mothers. Age
matched pups were used as controls and were injected
with equal volume of the vehicle intraperitoneally.
These pups from each group, i.e., control, LPS and
Poly I:C were perfused transcardially with phosphate
buffer saline to clear out the blood vessels followed
by 2% paraformaldehyde as fixative on PND-7, -12,
-21 and -30 and their brains were dissected.
Paraformaldehyde (2%) was used as mild fixative
for better accessibility of the desired antigens for
immunohistochemical procedures. Sections (15 m
thick) were cut through the hippocampus to study the
response to astrocytes and microglia in developing
hippocampus. All the protocols were pre-approved
by the Institutional Animal Ethics Committee.
Immunohistochemical localization of astrocytes
and microgliaRandomly selected sections were air
dried for one hour at 37 C and washed in phosphate
buffer saline (PBS, 3 changes of 5 min each). The
sections were then treated with 1% Triton X-100
(Sigma) in phosphate buffer for 15 min at 37 C to
increase the permeability of the membrane. Sections
were further washed with PBS (3 changes of 5 min
each) and treated with 1% H2O2 for 20 min to block
607
endogenous peroxidase activity. Non-specific proteins
were blocked by incubating sections in 1% normal
serum (Vector ABC Kit, PK 6200) in PBS in a wet
chamber at room temperature for 90 min. The sections
were then incubated with primary antibodies
anti-GFAP (1:3000 Rabbit Polyclonal, DAKO) or
anti-Iba 1 (1:1000 Rabbit polyclonal, Wako Japan) in
a humid chamber. Next morning the sections were
brought to room temperature and washed thrice in
PBS (3 changes of 5 min each). The sections were
incubated with secondary antibody (Vector ABC Kit,
PK6200) diluted in 1% BSA in PBS at a titer of 1:100
for 90 min at room temperature. The sections were
again washed in PBS thrice and then sections were
treated with 3,3 diaminobenzidine tetrahydrochloride
(DAB) solution (25 mg in 100 mL of PBS + 60 L
H2O2) for 20 min. The sections were washed in
running tap water followed by distilled water and then
air dried for 60 min. The sections were quickly
dehydrated in absolute alcohol, cleared in xylene and
mounted in DPX. The sections were visualized with
Leica DM 600 microscope and the light microscopic
images were obtained with a DFC 420 digital camera
and Leica Application Suite (LAS) Software.
Results
Response of microglia to LPS and Poly I:C
Microglia are the immunocompetent cells in the brain
and act as a first line of defense in the central nervous
system. In the normal brain, microglial cells are
present in the resting state having small cell body and
highly ramified constantly moving processes to scan
the CNS for damaged cells and infectious agents.
Any injury or insult of the brain causes microglia to
retract their processes, turn into activated/reactive
states or may transform into phagocytes. In
hippocampal sections of control animals microglia
presenting various morphologies were found scattered
throughout the hippocampus. Most of these microglia
were in their normal resting ramified morphology.
However, a large number of amoeboid microglia
were also noted and were found evenly distributed
throughout the hippocampal subfields. These amoeboid
microglia were more prevalent in the early postnatal
life, i.e., PND-7 and -12 day mainly because of the
developmental events (Fig. 1A, D and 2A). However,
by PND-21 when most of the developmental processes
were complete, the amoeboid microglia also started
disappearing (Fig. 1G and 2D). The intensity of Iba 1
staining also diminished linearly from PND-7 to -30,
608 INDIAN J EXP BIOL, AUGUST 2013

Fig. 1Light microscopic images of Iba 1 immunolabelled sections of the developing hippocampus showing microglial activation
(arrows, clusters encircled) following LPS (B, E, H and K) or Poly IC (C, F and I) treatment as compared to their age-matched controls.
Microglial activation persists till 30 days of age after LPS treatment (K) while is attenuated by 21 and 30 PND folowing Poly IC exposure
(I, L). Bar=50 m. All the pictures are of same magnification.

Fig. 2Light microscopic images of Iba 1 immunolabelled sections of the developing hippocampus showing microglial activation
(arrows) following LPS (B and E) or Poly IC (C and F) treatment as compared to their age-matched controls. Bar=50 m. All the pictures
are of same magnification.
 PATRO et al.: GLIAL RESPONSE TO NEONATAL BACTERIAL AND VIRAL INFECTION
indicating that the microglia transformed back to their
quiescent states (Figs 1 A, D, G, J, 2A and D).
Following exposure to LPS, a clear response of
microglia in terms of their activation was visible in
7 day-old pups (Fig. 1B). An increased number of
microglia were evident throughout the hippocampus,
forming clusters (circled for example) in dentate
gyrus, CA3 and subventricular zone. Such clusters
comprised of highly activated microglia with
activated morphology (arrows) over expressing Iba 1
(Fig. 1B). An intense activation of microglia was also
evident in PND-7 pups following Poly I:C exposure.
The response was much higher as compared to the
LPS exposed pups (Fig. 1C). Hyperplasia and
hypertrophy of the microglia was clearly apparent
with intense Iba 1 expression. Further, the processes
were very short and tortuous indicating their
functional involvement as antigen presenting cells and
phagocytosis. Clustering of activated microglia was
clearly seen in dentate gyrus and CA3 regions
(Fig. 1C) indicating that the Poly I:C induced
inflammation resulted in formation of large amount of
cellular debris.
In both, 12 day old LPS and Poly I:C exposed
pups, the activation of the microglia was further
pronounced. Clustering of amoeboid shaped microglia
was observed throughout the dentate gyrus (Figs 1E
and 2B; 1F and 2C, respectively), CA4 and CA3
subfields. In CA1 and CA2 regions as well the highly
activated microglia either with rod shaped or
amoeboid morphology were observed. However,
these microglia were not in clusters, rather found
scattered throughout the regions. The most heightened
response of Poly I:C exposure was seen at this age,
i.e., PND-12 with a burst of microglial activity
throughout the hippocampus (Fig. 1F and 2C).
By postnatal day 21 the response of LPS exposure
continued to be high in the hippocampus in terms of
microglial presence and activation. The microglia
still persisted in their activated morphologies with
intense Iba 1 expression and dense population, but
clustering was very rare (Fig. 1H) rather were evenly
scattered throughout the hippocampus. However,
the microglial activation was much less in Poly
I:C exposed 21 day old pups as compared to
age-matched LPS exposed pups (Fig. 2E and F).
Most of the microglia receded from the hippocampal
areas, leaving only a few microglia with their
activated morphology indicating the recovery from
this immune response (Fig. 1I).
609
By postnatal day 30, although the microglial
response receded significantly and linearly following
early neonatal exposure to LPS, small clusters
activated amoeboid microglia were still clearly seen
in the DG region (Fig. 1K). Normal resting ramified
microglia typical of controls were very few in
number. In age matched Poly I:C treated pups,
most of the microglia were regaining their normal
ramified morphology with a very few activated
microglia (Fig. 1L).
Response of astrocytes to LPS and Poly I:CGlial
fibrillary acidic protein (GFAP) is a cell specific
intermediate filament protein in astrocytes and is
widely used as a specific biomolecular marker for
astrocytes. Increased synthesis of GFAP is considered
as a definite feature of activated astroglia.
In PND-7 controls, both resting and reactive
astrocytes were observed. However, the density of the
astrocytes was very less and most of the astrocytes
were found scattered except in the DG region where
small clusters of astrocytes were recorded (Fig. 3A).
Following LPS exposure clusters of highly activated
astrocytes were very clearly seen in the DG, CA3 and
CA1 region adjacent to subiculum. Most of these
astrocytes presented highly activated hypertrophied
morphology with intense GFAP expression (Fig. 3B).
Astrocytes responded robustly to the Poly I:C
exposure and increased greatly in number. Thus a
dense population of activated astrocytes surrounded
the entire hippocampal subfields. Most of these
astrocytes were hypertrophied with few thick
processes showing intense GFAP immunopositivity.
At few sites even necrotic areas surrounded by highly
activated astrocytes were also visible (Fig. 3C).
In 12 day old control hippocampus, most of the
astrocytes presented their normal star-shaped
morphology (Figs 3D and 4A). Their number also
receded and now more even distribution was visible.
Following LPS exposure astrocytic activation was
further enhanced compared to the PND-7 pups. In
these preparations activated astrocytes were found
densely distributed throughout the hippocampus.
The astrocytes remained highly active both
phenotypically and in GFAP expression (Figs 3E
and 4B). Similar to the PND-7 pups the PND-12 pups
as well presented burst of astrocytic activation
after Poly I:C exposure (Figs 3F and 4C) with huge
number of activated astrocytes in all the hippocampal
subfields, indicating the persistence of the immune
activation.
610 INDIAN J EXP BIOL, AUGUST 2013

Fig. 3Light microscopic images of GFAP immunolabelled sections of the developing hippocampus showing astroglial activation
(arrows, clusters encircled) following LPS (B, E, H and K) or Poly IC (C, F, I and L) treatment as compared to their age-matched
controls. Astrocytic activation persists till 30 days of age both after LPS (K) and Poly IC treatment (L). Bar=50 m. All the pictures are
of same magnification.

Fig. 4Light microscopic images of GFAP immunolabelled sections of the developing hippocampus showing astrocytic activation
(arrows) following LPS (B and E) or Poly IC (C and F) treatment as compared to their age-matched controls. Bar=50 m. All the pictures
are of same magnification.
 PATRO et al.: GLIAL RESPONSE TO NEONATAL BACTERIAL AND VIRAL INFECTION
By postnatal day 21 most of the astrocytes
presented their normal morphology and distribution
with normal hippocampal cytoarchitecture in controls
with only a very few activated astrocytes (arrows)
found scattered (Figs 3G and 4D). However, in both
LPS and poly I:C exposed pups the astrocytic
activation was still apparent at this age, throughout
the hippocampus, i.e., DG, CA1, CA2 and CA3
regions. Astrocytes maintained their hypertrophied
activated morphology and intense GFAP
immunoreactivity. Most of the astrocytes formed
dense clusters (circled areas as example) all over
the cortex (Figs 3H and 4E; 3I and 4F, respectively).
In PND-30 controls typical aster shaped
morphology of the astrocytes was attained, indicating
the normal development (Fig. 3J). However, in both
LPS and Poly I:C infected rat pups the astrocytic
reaction still persisted, more so in Poly I:C infected
neonates. The activated astrocytes in Poly I:C infected
pups were present in the networks indicating reticular
appearance (Fig. 2L). While in the hippocampus of
the LPS infused rat pups, the astrocytic reaction was
downregulated both in the terms of astrocytic
activation, retraction of the processes and activated
morphology as well as GFAP expression, however, at
few sites, the astrocytes were found stripping around
the neurons (Fig. 3K) indicating the neuronal
degeneration.
Discussion
The developing nervous system is susceptible to
adverse impacts of various neurotoxicants and
infectious agents largely due to the absence of the
blood brain barrier. The nature and severity of the
infection that disrupts the normal phenomenon of
brain development depend upon the type of the
infectious agent, the level and duration of exposure
and most importantly on the specific stages of brain
development during which the exposure occurs. In the
present study newborn rat pups were exposed to either
LPS or Poly I:C on postnatal day 3 with a booster
dose on postnatal day 5 to ensure infection. This is the
developmental window when hippocampal granule
cells of the dentate gyrus are generated and response
of glial cells to these infections was subsequently
studied in hippocampus on postnatal day 7, 12, 21,
and 30, when the formation and maturation of the
entire granule cell layer occurs. Both astrocytes and
microglia transformed to their reactive phenotypes
following LPS as well as Poly I:C challenge
611
indicating an immune response and remain activated
till postnatal day 30.
Perinatal immune activation following LPS
exposure has been reported to induce long term
functional alterations in the metabolic25, immune26,27,
behavioural28-30 and neuroendocrine systems31,32 in
adults. Recently Sominsky et al.33 have shown that
microglia activated in response to LPS modulates
the expression of certain behaviours and such
perinatally challenged pups exhibit behavioural and
neuroendocrinal changes and more specifically
increased anxiety-like behaviour in adulthood. Rats
challenged with LPS on postnatal days 3 and 5 have
been popularly used as a model of early life
bacterial driven immune activation34-36.
Activated microglia are hallmarks of neuro-
inflammatory process in many neurodegenerative
diseases and infections. Microglial activation using
immunohistochemical labeling of the ionized
calcium-binding adaptor molecule (Iba 1) protein is
an effective method for identifying changes in
microglia activation status37-40. In the present study an
increase in microglial activation was evident as
enhanced Iba 1 immunostaining and an increase in
microglial density associated with prolonged
activation and proliferation of microglia following
LPS challenge on postnatal day 3 and 5. The loss or
deterioration of normal physiological microglial cell
function could be because of the influence of
inflammatory responses in the brain. Microglia being
an important player of the innate immune system of
the CNS play a crucial role in receiving and
propagating inflammatory signals in response to
activation of the peripheral immune system41 and
once activated, have macrophage like capabilities
including phagocytosis, inflammatory cytokine
production and antigen presentation42,43. In neonatal
hypoxia-ischemia (HI) as well, the earliest events to
occur is the appearance of abundant number of
activated microglia in an around ischemic white
matter sites44-46 and contribute to the white matter
damage in the preterm neonate. Thus, the activated
microglial cells after HI are associated with brain
injury in neonatal models and blocking microglial
activation using minocycline, a known microglial
inhibitor attenuates myelin loss in the neonatal rat
brain47-49. Thus, the persistent presence of activated
microglia following postnatal LPS challenge could be
a serious threat to the developing hippocampus and
invites further detailed studies.
612 INDIAN J EXP BIOL, AUGUST 2013
The astrocytic dysfunction is key player in the
pathogenesis of various viruses having double
stranded RNA genome including HIV, HHV and
HTLV. The main effect of these ultimately results in
the form of severe CNS disorders. Astrocytes serve as
a reservoir for the viruses and astrocyte dysfunction
is suspected to play a critical role in the
neuropathogenesis50. Astrocytic reactivity could be
easily tracked by the immunohistochemical studies
using antibodies against their intermediate filaments
or cytoplasmic proteins like GFAP. Expression of
GFAP has become the prototypical marker for
assessment of chemical identification of astrocytes.
In present study as well, the morphological stages
assumed by astrocytes during postnatal development
were examined using GFAP immunolabelling.
The cells displayed varying degrees of GFAP
immunoreactivity. In PND-7, most of the astrocytes
were immature in appearance and presented weak
GFAP expression. In contrast to this, the cells in
PND-12 and PND-21 of rat hippocampus were
numerous with strong GFAP positivity. Most of
the astrocytes were having typical star shaped
morphology. Many more GFAP positive cells are
present. The astrocytes of these mature stages showed
morphology of protoplasmic astrocytes.
However, following LPS and Poly I:C infusion,
clusters of highly activated astrocytes were seen
throughout the hippocampus. Such activated
astrocytes appeared hypertrophied with few thick
processes and expressing GFAP at high concentration.
Such astrocytic activation continued till PND-30 in
Poly I:C and LPS exposed rats, indicating
enhanced astrocytic reaction. Global increase in
GFAP expression and change in astrocytes is usually
assumed to contribute to the inflammation associated
neurotoxicity. Such reactive astrocytes are believed to
produce neurotoxic molecules such as NO, reactive
oxygen species and TNF- in in vitro situations2.
Toxic mediators produced by activated astrocytes are
considered to be involved in the pathogenesis of many
neurodegenerative disorders51,52. However, prolonged
activation of astrocytes as observed in the present
study could be detrimental to their survival and
function, leading to their elimination by apoptosis.
However, not much is known about the potential role
of such prolonged activation of astrocytes during early
postnatal life. The present study clearly depicts that
poly I:C, the viral infection mimic, produced acute
inflammation as opposed to the chronic inflammation
caused by LPS, the bacterial infection mimic. Such
comparative study stating the persistent astrocytic
activation following exposure to bacterial or viral
mimic opens up a plethora of questions to be pursued.
Acknowledgement
This work was presented as a poster at the Annual
Meeting of the Indian Academy of Neurosciences
2012 at Amritsar. Financial support from Department
of Biotechnology, New Delhi is thankfully
acknowledged. Ms. Kavita Singh was an M. Tech.
trainee from Madhav Institute of Technology &
Science, Gwalior. Facilities developed through
the Bioinformatics Infrastructural facility from
Department of Biotechnology, used in this study are
thankfully acknowledged.
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