status of proinflammatory and anti-inflammatory cytokines in different brain regions of a rat model...

ORIGINAL RESEARCH PAPER

Status of proinflammatory and anti-inflammatory cytokinesin different brain regions of a rat model of Japanese encephalitis

Ruchi Srivastava • Jayantee Kalita •

Mohammad Yahiya Khan • Usha Kant Misra

Received: 6 August 2011 / Revised: 19 December 2011 / Accepted: 19 December 2011 / Published online: 30 December 2011

� Springer Basel AG 2011

Abstract

Objectives This study evaluated cytokines and chemokine

in different regions of the rat brain at different time points

following Japanese encephalitis virus (JEV) infection.

Design Twelve-day-old Wistar rats were infected by

intracerebral inoculation of 3 9 106 plaque-forming units

of JEV 78668A strain. Expression of cytokines and che-

mokine was assayed using cytokine bead array in different

regions of the brain at 3, 6, 10 and 20 days post-inoculation

(dpi). Pathological changes including immunohistochem-

istry for JEV antigen were observed.

Results The cytokine levels were increased in the acute

stage and declined on follow-up. In the acute stage (6 dpi), the

levels of TNF-a, IFN-c, IL-6 and IL-10 were maximum in the

cortex, whereas the level of IL-4 was maximum in the stria-

tum. Lower levels of these cytokines were observed in

mid-brain and thalamus compared to other regions studied.

Maximum expression of JEV antigen and histopathological

changes as well as cytokines and chemokine were observed at

6 dpi in all the brain regions studied, but declined thereafter.

Conclusion This experimental study revealed maximum

expression of cytokines and chemokine at 6 dpi of JEV

infection which corresponded with histopathological

changes in different brain regions.

Keywords Viral encephalitis � JEV � Cytokine �Animal � Brain regions � Acute stage � Inflammation �Immunopathogenesis

Introduction

Japanese encephalitis (JE) is an important endemic

encephalitis prevalent in South-East Asia. About 50,000 JE

patients present annually; 15,000 of them die and half the

survivors live with major neurological sequelae [1]. The

clinical picture of JE is characterized by fever and altered

sensorium with or without seizure or focal neurological

deficit. The clinical manifestations of viral encephalitis are

determined by the location and severity of central nervous

system injury. Computed tomography scan and magnetic

resonance imaging study in 56 patients with JE revealed a

high frequency of involvement of thalamus (83%), basal

ganglia (46%), brain stem (35%) and cerebral cortex (23%)

[2]. These results are similar to autopsy studies [3, 4].

Inflammatory response associated with cell damage is

responsible for the clinical and histopathological changes

in viral encephalitis. Following JEV infection, overpro-

duction of free radicals by neurons in the acute phase of

illness has been reported [5]. Free radicals are general

mediators of signal transduction pathways, which are

responsible for cytokine production [6–8]. Cytokine levels

are surrogate markers of the inflammatory cascade in the

brain. However, an association between cytokine produc-

tion and free radical generation has not been reported in JE.

The role of inflammatory markers has been studied in viral

encephalitis patients and high levels of tumor necrosis factor

alpha (TNF-a) and interleukin (IL)-6 have been reported in

serum and cerebrospinal fluid [9–11]. In these studies,

however, the cytokine profile in different brain regions was

Responsible Editor: Graham Wallace.

R. Srivastava � J. Kalita � U. K. Misra (&)

Department of Neurology, Sanjay Gandhi Postgraduate Institute

of Medical Sciences, Raebareily Road, Lucknow 226014, India

e-mail: [email protected]; [email protected]

J. Kalita

e-mail: [email protected]

M. Y. Khan

Department of Biotechnology, Babasaheb Bhimrao Ambedkar

University, Lucknow, India

Inflamm. Res. (2012) 61:381–389

DOI 10.1007/s00011-011-0423-5 Inflammation Research

123

not possible. The severity of disease, its clinical manifesta-

tions and sequelae depend on the viral tropism. Such

information can only be obtained by autopsy or experimental

studies. Experimental studies in mice by Ghoshal and col-

leagues have reported microglial activation and expression

of a number of cytokines and free radicals which are

responsible for neuronal degeneration and death. They

reported changes in cytokine levels in cortex, hippocampus,

striatum and thalamus. In this study, involvement of the

midbrain, which is commonly involved in humans and is

responsible for various movement disorders, was not studied

[12]. The mouse model of JEV is short-lived, and therefore

the delayed effect of JEV cannot be studied. We have found a

rat model of JE to be more suitable for studying subacute and

chronic changes [5, 13]. Our study was undertaken to eval-

uate the cytokine and chemokine [monocyte chemotactic

protein-1 (MCP-1)] levels in different regions of the brain at

different time points and their relation to histopathological

changes following JEV infection.

Materials and methods

Animal

Twelve-day-old Wistar strain rats of either sex were used in

this study and were obtained from the animal house of the

Sanjay Gandhi Postgraduate Institute of Medical Sciences,

Lucknow, India. A maximum of five rats were kept in each

cage. The animals were maintained in an air-conditioned

room (25 ± 2�C) with 12-h light (7 a.m. to 7 p.m.) and dark

cycle. All the experiments were performed during the light

cycle, between 10 a.m. and 2 p.m., and were normalized in all

rats. Housing and procedures involving animals and their

care were conducted in accordance with the Principles of

Laboratory Animal Care (National Institutes of Health

publication number 86-23). The study was approved by the

local ethics committee, and all the experiments were carried

out in accordance with the institutional guidelines on the care

and use of experimental animals.

Virus

The JEV 78668A strain which was used was isolated from

the post-mortem brain tissue of a 38-year-old woman

patient who died during an epidemic of encephalitis in

Gorakhpur, India in 1978 [14]. The genome of GP78 is

10,976 nucleotides long. An open reading frame of 10,296

bases, capable of coding 3,432 amino acid polyproteins, is

flanked by 95- and 585-base-long 50- and 30-non-coding

regions, respectively [15]. The JEV was propagated in

3–4 days old suckling mice brain. After 4 days of infec-

tion, the rats were killed. Brain was removed aseptically,

homogenized in sterile phosphate-buffered saline (PBS)

and centrifuged at 160609g for 30 min at 8�C. The

supernatant was collected, aliquoted and stored at -70�C

until further use. The virus was plaque-purified twice on

monolayers of porcine stable kidney (PS) cells and the

isolate was subsequently referred to as the GP78 strain.

Infection of rats

The rats were divided into experimental and control groups. In

the experimental group, rats were inoculated intracerebrally

with 3 9 106 plaque-forming units of JEV according to a

fixed protocol [13]. The inoculation was done at the midpoint

of the line connecting the ears. Control rats were inoculated

with sterile PBS (19, Sigma). The rats were killed at 3, 6, 10,

and 20 days post-inoculation (dpi). Their brains were excised

aseptically and the regions of interest such as cortex, striatum,

thalamus and mid-brain were dissected using the standard

procedure, and cryopreserved for cytokine and chemokine

assay by cytometric bead array (CBA).

Histopathological studies

The rats were anaesthetized with ether and perfused with

150 ml of 0.1 M, pH 7.2, PBS followed by 250 ml of ice-

cold 4% paraformaldehyde in PBS for fixation of tissues.

The brains were removed and preserved in 10% parafor-

maldehyde. Serial coronal sections of 5-lm thickness were

cut on a freezing microtome (Slee Mainz Co., Germany).

The sections were then transferred to gelatinized slides and

stained with haematoxylin and eosin (H&E).

Immunohistochemical studies

The rats were anaesthetized with ether and perfused with

150 ml of 0.1 M, pH 7.2, PBS followed by 250 ml of ice-

cold 4% paraformaldehyde in PBS for fixation of tissues.

The brains were removed and postfixed in the same fixative

overnight, followed by cryopreservation in 10, 20, and 30%

(w/v) sucrose in PBS. Five serial coronal sections of 20-lm

thickness were cut on a freezing microtome (Slee Mainz

Co., Germany). These tissue sections were treated with

0.3% H2O2–methanol to suppress endogenous peroxidase

activity followed by 10% normal goat serum and allowed

to react with primary antibody (anti-mouse JEV antibody,

1:100, obtained from National Institute of Virology, Pune,

India) at 4�C overnight. After removing the primary anti-

body, sections were washed three times with PBS and

incubated in biotinylated peroxidase-linked secondary

antibody (1:200) for 2 h at room temperature followed by

three washes with PBS. Color was developed for peroxi-

dase-linked antibody with 3,3-diaminobenzidine as

chromogen. Sections were dehydrated in a graded series of

382 R. Srivastava et al.

123

alcohol followed by xylene, mounted in DPX, cover-slip-

ped, and then examined under the microscope.

Protein extraction and quantitation

Snap-frozen brain tissues were homogenized in 19 PBS

containing proteinase inhibitor (10 ml ice-cold 19 PBS ?

5 ll proteinase inhibitor) chilled buffer using a glass

homogenizer under ice. Typically, each sample was

homogenized in 700 ll of buffer, followed by sonication

on ice for 30 s and centrifugation at 136809g for 30 min at

4�C [16]. Supernatant was collected, aliquoted and stored

at -80�C for further use in CBA after protein quantitation

by the standard Lowry assay [17].

Cytometric bead array

Cytometric bead array (CBA, rat flex kit; BD Biosciences,

Singapore) was used to quantitatively measure cytokine

expression levels in brain tissue lysates of different brain

regions (cortex, striatum, thalamus and mid-brain) of the

control and JEV-infected rats. The assay was performed

according to the manufacturer’s instructions and analyzed

on an FACS Calibur (Becton–Dickinson, UK). Analysis

was performed using CBA software (FCAP Array v1.01)

which allows the calculation of cytokine concentrations in

unknown samples [12].

Enzyme-linked immunosorbent assay (ELISA)

To examine the MCP-1 level in the tissue lysates of

different brain regions (cortex, striatum, thalamus and mid-

brain) of control and JEV-infected rats, ELISA was per-

formed. A commercially available kit from BD Biosciences

was used according to the manufacturer’s guidelines.

Statistical analysis

All the statistical analyses were done using GraphPad

Prism (v3.03) software. Mean differences in more than two

groups were analyzed using one-way analysis of variance

(ANOVA) followed by Newman–Keul’s multiple com-

parison test. The variable was considered significant if

p \ 0.05. Data were expressed as mean ± SD.

Results

Forty-five rats were included in the study group and six in

the control group.

Clinical signs

The clinical signs were monitored daily up to 20 dpi. The

clinical signs appeared from 4 dpi in the study group. All

the rats showed hypokinesia, pelvic elevation and hind

limb disability which was observed up to 20 dpi. However,

tremors and convulsions were not frequently observed.

Mortality was observed from 6 to 10 dpi; none of the rats

died after 10 dpi. The control group did not exhibit any of

these clinical symptoms (Table 1).

Histopathological findings

The histopathological features of JE such as inflammatory

cell infiltration, necrosis and neuronal shrinkage were

observed in the JEV-inoculated rats (Fig. 1). These

pathological changes were not present in the control rats.

Three rats in each group were used for the histopatholo-

gical study.

Immunohistochemical study

JEV antigen was present in different brain regions such as

thalamus, mid-brain, striatum and cortex (representative

photomicrographs are shown in Fig. 2). On the basis of

visual perception, the intensity of JEV antigen was noted

and is presented in Table 2. The intensity of JEV antigen

was maximum at 6 dpi in all the brain region and gradually

declined up to 20 dpi.

Table 1 Clinical signs and

symptoms and mortality in

12-day-old rats challenged with

3 9 106 pfu of JEV

Numbers in parenthesesindicate the number of rats

? denotes present; - denotes

absent; ± denotes poor or

inconsistent

pfu plaque-forming units,dpi days post-inoculation

Dpi Clinical signs Mortality

Hypokinesia Pelvic

elevation

Hind limb

disability

Tremor Convulsions

4 (45) ? (45) ± (45) ± (45) - - -

6 (41) ? (41) ? (41) ? (41) ? (7) ? (5) ? (4)

7 (35) ? (35) ? (35) ? (35) ? (7) ? (5) ? (6)

9 (32) ? (32) ? (32) ? (32) ? (4) ? (3) ? (3)

10 (30) ? (30) ? (30) ? (30) - - ? (2)

15 (30) ? (30) ? (30) ? (30) - - -

20 (30) ? (30) ? (30) ? (30) - - -

Cytokines in different brain regions of JE infected rat 383

123

Cytokines and chemokine levels in different brain

regions

Levels of cytokines and MCP-1 in cortex

The cytokine and chemokine levels were highest at 6 dpi

and lowest at 20 dpi in the cortex of rat brain after JEV

infection. Levels of IFN-c reached maximum at 6 dpi but

gradually declined significantly at 10 dpi and further at

20 dpi when compared between the study groups

(Fig. 3a). Similar trends were observed for IL-6, IL-10,

TNF-a and MCP-1 (Fig. 3a–e). For IL-4, a different

pattern was observed. There was insignificant increase in

the level of IL-4 at 6 dpi compared to control. However,

there was a significant decrease in levels at 10 dpi and

20 dpi, like other cytokines, but the level at 20 dpi was

also significantly lower compared to control (p \ 0.001)

(Fig. 3b).

Fig. 1 Photomicrograph showing H&E staining in different brain

regions at different days post-inoculation in 12-day-old rats inocu-

lated with 3 9 106 pfu/ml of JEV. a control rat, b neuronal shrinkage

in cortical region (broken arrow shows shrunken neuron, arrowindicates normal neuron), c inflammatory cells infiltration with

necrosis in the cortex (arrow)

Fig. 2 Representative photomicrograph showing presence of JEV antigen in cortex, striatum, thalamus and mid-brain in 12-day-old rats

inoculated with 3 9 106 pfu/ml of JEV solution


123

Levels of cytokines and MCP-1 in striatum

The striatum of JEV-infected rat brain showed the maxi-

mum level of all cytokines and chemokine at 6 dpi. There

was a significant increase in IFN-c, IL-6, IL-10, TNF-a and

MCP-1 levels at 3, 6, 10 and 20 dpi compared to controls.

These levels declined at 10 dpi and further at 20 dpi when

compared between the groups. IL-4 levels, however, were

not significantly different between the experimental and the

control groups (Fig. 4).

Levels of cytokines and MCP-1 in thalamus

Expression patterns of different cytokines and chemokine

in the thalamus of JEV-infected rats were similar to the

expression patterns in the brain regions described above.

Levels of all cytokines and chemokine reached a maximum

at 6 dpi in the thalamus. These levels significantly declined

at 10 and 20 dpi when compared to 6 dpi. Interestingly, we

were not able to detect IFN-c in the thalamus of JEV-

infected rats at any time point (Fig. 5).

Table 2 Presence of JEV antigen in different brain regions in 12-day-old rats challenged with 3 9 106 pfu of JEV at 3 and 6 dpi

JEV staining 3 dpi 6 dpi 10 dpi 20 dpi

St Co MB Th St Co MB Th St Co MB Th St Co MB Th

? ? ? ? ??? ??? ??? ??? ? ? ? ? ± ± ± ±

? denotes low, ?? denotes moderate, ??? denotes high, ± denotes faint staining

pfu plaque-forming units, dpi days post-inoculation, St striatum, Co cortex, MB mid-brain, Th thalamus

Fig. 3 Bar graph showing

cytokines and chemokine levels

in cortex of 12-day-old rats

challenged with 3 9 106 pfu of

JEV. a IFN-c, b IL-4, c IL-6,

d IL-10, e TNF-a, f MCP-1.

Values are mean ± SD of 6 rats

in each of the 3, 6, 10, 20 dpi

and control groups. One way

ANOVA; p \ 0.05 is

considered significant

(*p \ 0.05, **p \ 0.01,

***p \ 0.001). dpi days

post-inoculation


123

Levels of cytokines and MCP-1 in mid-brain

The mid-brain of JEV-infected rats showed different

expression patterns for cytokines and chemokine compared

to patterns in cortex, striatum and thalamus. Levels of

IFN-c, IL-6, TNF-a and MCP-1 were found to be maxi-

mum at 6 dpi in the mid-brain; however, the level of IL-10

reached a maximum at 10 dpi. A significant increase in

levels of IL-6, IL-10, TNF-a and MCP-1 compared to

controls was noted at 3, 6, 10 and 20 dpi (p \ 0.001).

Levels of IFN-c increased significantly compared to

controls only at 6 dpi (p \ 0.001). IL-4 levels were sig-

nificantly decreased at 3 dpi (p \ 0.05); however, no

significant changes were observed at 6, 10 and 20 dpi

compared to controls. IFN-c was increased significantly at

6 dpi compared to 3 dpi (p \ 0.01), and significantly

declined at 10 dpi compared to 6 dpi (p \ 0.001). TNF-aand MCP-1 levels were reduced significantly at 10 dpi

compared to 6 dpi (p \ 0.001). An insignificant increase

was noted in IL-10 level from 6 to 10 dpi (Fig. 6).

Discussion

In the present study, we observed increased levels of pro-

and anti-inflammatory cytokines in the different brain

regions of rats after JEV infection, compared to controls.

These cytokine levels varied from the early (3 to 10 dpi) to

the late (20 dpi) phases of infection. We used the rat model

of JE rather than the mouse model because the life span of

rats is longer; thereby, it is feasible to study the clinical

sequence and its underlying pathophysiological mecha-

nisms. We used younger rats because of greater

susceptibility and longer survival of younger rats has been

reported compared to older ones following intracerebral

inoculation of JEV [18].

In the acute stage (at 6 dpi), the maximum levels of

TNF-a, IFN-c, IL-6 and IL-10 were observed in the cortex.

However, the level of IL-4 was maximum in striatum.

Lower levels of these cytokines were observed in mid-

brain and thalamus compared to the other studied regions.

Our results are in agreement with those of Ghoshal et al.


cytokine levels in striatum of


3 9 106 pfu of JEV. a IFN-c,

b IL-4, c IL-6, d IL-10,

e TNF-a, f MCP-1. Values are

mean ± SD of 6 rats in each of

the 3, 6, 10, 20 dpi and control

groups. One way ANOVA;

p \ 0.05 is considered

significant (*p \ 0.05;

**p \ 0.01; ***p \ 0.001).

dpi days post-inoculation


123

[12] who reported increased expression of IL-6, TNF-a, IL-

1b and COX-2 after JEV infection in mice. On analyzing

the cytokine levels in different brain regions, the highest

expression of pro-inflammatory cytokines were found in

the hippocampus. The differential expression of cytokines

in the brain regions studied may be due to difference in

species (mice vs. rat), viral dose and route of inoculation.

In cortex, striatum and thalamus, the levels of IL-10, IL-4,

IL-6, TNF-a and IFN-c were increased significantly at

6 dpi; however, these levels declined at 10 and 20 dpi. In

mid-brain, the levels of IL-4 and IL-10 reached a maxi-

mum at 10 dpi. In another study, the expression of TNF-aand IFN-c in whole brain lysate was increased from 1 to

6 dpi. Levels of IL-4 and IL-10 were increased at 1 dpi and

started to decline at 3, 5 and 6 dpi in JEV-infected mice

inoculated intracerebrally [18]. After viral exposure, apart

from early innate immune response, activation of T cells

leads to the expression of a large number of cytokines that

facilitate both cell-mediated and antibody responses.

Cytokines contribute to cell death or cell dysfunction. A

major source of these cytokines could be the microglial

cells present in the central nervous system or infiltrating

lymphocytes which reach to the site of infection through

chemokine-mediated cell trafficking as well as due to the

breakdown of the blood–brain barrier [19]. Chemokines

cause the accumulation and activation of leukocytes in

tissues. Attraction of leukocytes to tissues is essential for

inflammation and the host response to infection. The

secretion of chemokines has been detected in a wide

variety of diseases [11, 20, 21]. In the present study, we

found a maximum level of MCP-1 at 6 dpi, which further

declined at 10 and 20 dpi in each brain region studied.

Taken together, there was an overall induction of cytokines

and chemokine (MCP-1) in the acute stage, which later

declined. It was observed that chemokines help in

recruiting the immune cells and enhancing the liberation of

various cytokines [21]. In viral meningitis patients, higher

levels of IL-10 and MCP-1 in cerebrospinal fluid have been

reported [22].

In the immunohistochemical study, the JEV antigen was

localized in thalamus, mid-brain, striatum and cerebral

cortex. It was observed that JEV antigen was more pro-

nounced at 6 dpi in all the regions studied. In contrast, the

high levels of expression of cytokines in the present study


cytokine levels in thalamus of


3 9 106 pfu of JEV. a IL-4,

b IL-6, c IL-10, d TNF-a,

e MCP-1. Values are






**p \ 0.01; ***p \ 0.001).



123

was observed in those areas where the viral load and the

presence of viral antigen was low, suggesting the possible

role of cytokines in viral clearance. These findings may

suggest immune-mediated pathogenesis, i.e. the host

immune response may kill the infected cells in an effort to

clear the virus. In our earlier study, we reported the his-

topathological changes in thalamus, mid-brain, striatum

and cortex, in which maximum damage evidenced by

neuronal shrinkage, cellular infiltration and inflammatory

cells was observed at 6 dpi [13]. The extent of cellular

damage could be associated with the viral load and the

presence of JEV antigen in different brain regions.

In the present study, IFN-c was not detectable in thalamus.

The antiviral effect of IFN-c has been reported in West Nile

virus infection [23]. As part of innate immunity, IFN-c is

produced in response to viral infections. Instead of being

directly antiviral, it induces the production of effector pro-

teins in the cells, which inhibits various stages of viral

replication, assembly, or release. We hypothesize that the

absence of IFN-c might contribute to the vulnerability of the

thalamic region to viral infection. These observations are

supported by the high frequency of thalamic involvement in

JE as seen on magnetic resonance imaging [2].

Besides the proinflammatory cytokines, anti-inflamma-

tory cytokines are also expressed simultaneously to balance

the action of proinflammatory cytokines. The highest levels

of IL-10 and IL-4, which are immunosuppressive cyto-

kines, were noted at 6 dpi. It might be possible that the

expression of these cytokines would suppresses the

immune response and contributes to the recovery. The role

of the immune response in recovery from JEV infection is

poorly understood. The decline in cytokine levels at 10 and

20 dpi may be due to an associated immune response and

neutralizing antibodies. Early host defence against JEV

infections is mediated by phagocytic cells, followed by a

complex mechanism which involves B and T effector cells

[14, 24, 25]. The reciprocal relationship between cytokine

and virus load raises an important question of whether the

activation of cytokines results in viral proliferation and cell

damage, or absence of cytokines at a particular site results

in more viral load. We found different levels of cytokines

in different brain regions (cortex, striatum, thalamus and

mid-brain) of JEV-infected rat at different time points.

In our earlier study, we observed an increase in free

radical generation in the initial phase of JEV infection,

which declined at a later stage. Maximum levels of


cytokine levels in mid-brain of


3 9 106 pfu JEV. a IFN-c,

b IL-4, c IL-6, d IL-10,

e TNF-a, f MCP-1. Values are






**p \ 0.01; ***p \ 0.001).



123

neuronal reactive oxygen species and peroxinitrite were

observed at 6 dpi and declined significantly at 10 and

20 dpi as compared to control [5]. In the present study, we

noted the same pattern of expression of cytokines, which

peaked at 6 dpi and declined by 20 dpi. Many studies

reported an association of free radical generation with

cytokine production [26, 27]. Free radicals are general

mediators of signal transduction pathways which are able

to induce cytokine production [6–8]. In JE, free radicals

may also act as a signal for the release of different cyto-

kines. A detailed study, however, is needed to prove this

hypothesis.

This study highlights that the cytokines are expressed

maximally during the acute phase of JEV infection, which

is associated with histological changes, and decline sub-

sequently. Confirmation of these results may help in the

treatment of JE to reduce cellular damage.

Acknowledgments We acknowledge the Indian Council of Medical

Research for financial support to Ruchi Srivastava as Senior Research

Fellow, and Council of Science and Technology, Uttar Pradesh, India

for establishing the Encephalitis Research Center. We thank Mr.

Rakesh Kumar Nigam for secretarial help.

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