comparative neuropathological study of sodium nitrite and nisin...

Egypt. J. Comp. Path &Clinic Path. Vol.26 No.2, 2013 ; 61- 71 ISSN 1110-7537

61

Comparative Neuropathological Study of Sodium

nitrite and Nisin Exposed Rats With Special Reference

to Expression of Glial Fibrillar Acidic Protein and

Vascular Endothelial Growth Factor

Faten. F. Mohammed

Department of Pathology, Faculty of Veterinary Medicine, Cairo University

ABSTRACT— The present study was performed to evaluate the neurotoxicity of

sodium nitrite (NaNO2) and nisin in cerebral cortex and hippocampus. A total of 27 adult female

rats were divided into three groups, control group, sodium nitrite and nisin treated groups with 9

rats per each. The rats of groups 2 and 3 were given orally 30 mg/kg body weight and 50 mg/kg

b.w daily of NaNO2 and nisin respectively for 2 months by gastric intubation while rats in

control group were given orally 0.9% saline. Histopathological examination of brain was

performed, in addition to immune histochemical staining for glial fibrillar acidic protein (GFAP)

and vascular endothelial growth factor (VEGF) were applied on brain sections of both treated

and control groups. Results revealed various histopathological alterations that were more evident

in deep cerebral grey matter and white matter including neuronal degeneration, astrogliosis and

vascular changes in addition to reduction in cellular mass in different regions of hippocampus

was detected. There was no recognized histopathological changes in nisin treated group. Increase

expression of GFAP & VEGF in NaNO2-treated group was evident compared with nisin and

control groups. Conclusion: sodium nitrite has neurotoxic potential via inducing

histopathological alterations that were evident in deep cerebral cortex and different region of

hippocampus with increased expression of GFAP &VEGF while the nisin is non neurotoxic

agent .

Key words: albino rats, sodium nitrite, , nisin, neurotoxicity, histopathology , GFAP,

VEGF , immunohistochemistry.

—————————— ——————————

INTRODUCTION

Sodium nitrite is one of synthetic

food preservatives that become popularly used

in various feed stuff in addition to biomedical

application as in post hemorrhagic cerebral

vasospasm (Pluta et al, 2005) and in

myocardial infarction (Webb et al, 2004).

Health hazards have encountered with sodium

nitrite consumption. Sodium nitrites cause

neurotoxicity via induction of brain hypoxia

(Zaidi, 2010), exerts oxidative stress and

retrogrades the endogenous antioxidants

(Hassan et al, 2010). In such concern role of

hypoxia and oxidative stress induced

neurotoxicity are well established.

The structural and functional

integrity of brain depends on regular oxygen

supply (Acker T & Acker H, 2004) although

the brain represents only about 2% of the

body’s weight, it utilizes about 20% of the

body’s oxygen. As a result, the brain is

especially sensitive to hypoxia. Symptoms of

mild cerebral hypoxia include inattentiveness,

poor judgment, memory loss, and a decrease in

EGYPT. J. COMP. PATH &CLINIC PATH. VOL.26 NO.2 2013 ; 61- 71

62

motor coordination. Brain tissue exposed to the

hypoxic insult responds by glycolysis,

angiogenesis, vasodilatation and erythropoiesis

(Harris, 2002). Vulnerability of brain tissue to

hypoxia is variable. Hypoxia is found to be a

potent inducer of vascular endothelial growth

factor (VEGF) expression in several organs

including the CNS (Minchenko et al, 1994).

Hypoxia is also activates and promotes

proliferation of resident astrocytes in vivo

(Ridet , 1997), a process often referred to as

astrogliosis or glial scarring. It is known that

astrocytes are more resistant to oxidative stress

than neurons due to compensation for a lack of

oxygen by switching to glycolytic metabolism

(Marrif H & Juurlink,1999) that allows the

constant ATP levels required to ensure cell

viability during stress (Hochachka &

Lutz,2001). The glial fibirillar acidic protein

(GFAP) is a sensitive and specific biomarker

for astrocytes (O'Callaghan, 1991).

Nisin is a natural food preservative

produced by Lactococcus lactis. It is of wide

inhibition effect on Gram positive bacteria, and

is widely accepted as a safe biological

preservative. Nisin reduces chemical

preservatives concentration required

(Mobasseri et al, 2009). Also, it has

biomedical application as dental care products

(Turner et al, 2004) and shown to be effective

in the treatment of head and neck squamous

cell carcinoma (Ritchie et al, 2012). Previous

studies suggested that nisin did not affect the

normal physiology of body organs and had no

adverse effects on the reproductive

performance in nisin-treated male rats and their

offspring (Gupta et al.,2008 and Reddy et

al.,2012).The present study is designed to

provide an new point of view in explaining the

role of sodium nitrite induced neurotoxicity

regarding immunohistochemical expression

of glial fibrillar acidic protein and vascular

endothelial growth factor in cerebral cortex

and different region of hippocampus as well as

to establish a comparative histopathological

study between sodium nitrite and nisin used as

synthetic and natural food preservatives

respectively.

MATERIALS AND METHODS

Animals: A total of twenty seven

female albino rats weighing about 100-120 g

were used in the present study. They were

obtained from the animal house belong to

Ophthalmology research Institute, Giza, Egypt.

Animals were fed on basal diet and watered

ad-libitum. Rats were left for two weeks for

acclimatization before starting the experiment.

All experimental manipulations were

undertaken in accordance with the Institutional

Guidelines for the Care and Use of Laboratory

Animals.

Chemicals:

NaNO2 (El Gomhoria Company,Cairo,Egypt)

was applied as a freshly prepared solution.

Nisin from Lactococcus lactis (N5764-1G,P

code :101183002) (Sigma–Aldrich, St. Louis,

MO). That was dissolved in 0.9% saline.

Glia fibrillar acidic protein (GFAP) and

vascular endothelial growth factor (VEGF) (catalog MA1-166629,Thermo Scientific Co.,

UK)

Experimental design:

Rats were divided into three groups

with nine rats per each: G1-control groups that

were received standard diet without any

treatment and the orally given 0.9% saline. G2-

NaNO2-treated group; received standard diet

and given orally sodium nitrite at a dose of 30

mg/kg body weight daily for 2 months as

previously described by Isyaku and Joseph,

(2011) and G3- nisin treated group at dose of

50mg/kg b.w that was dissolved in 0.9 %

saline orally by gavages daily for 2 months as


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described previously by Reddy et al,(2012) .

At the end of the experimental period

animals were euthanized by cervical

dislocation and brains were dissected.

Histopathological and

immunohistochemical examination:-

Brain tissue specimens were collected

and preserved in 10% neutral buffered

formalin then routinely dehydrated in different

grades of alcohol, cleared in xylene, embedded

in paraffin, sectioned with microtome at 5µ

thickness and finally stained with hematoxylin

and eosin (H&E) according to Bancroft and

M. Gamble, (2008). On the other hand,

selected tissue sections were dewaxed in

xylene, rehydrated and pretreated with 3 %

hydrogen peroxide for blocking the activity of

endogenous peroxidase. Microwave as sited

antigen retrieval was done for 20 minutes.

Sections were incubated overnight at 40 C°

with primary antibody for Glia fibrillar acidic

protein (GFAP) and vascular endothelial

growth factor (VEGF) (catalog MA1-

166629,Thermo Scientific Co., UK) was

diluted with phosphate buffer saline (PBS)

(1:50) then washed with PBS and incubated

with biotinylated mouse secondary antibody

(Cat No.32230,ThermoScientific Co., UK) and

finally conjugated with streptavidin-

peroxidase. Sections were washed with PBS

and incubated with diaminobenzidid (DAB)

for 5 minutes.

RESULTS

Histopathological findings:

Histopathological examination of cerebral

cortex revealed individual necrosis of motor

neurons that comprising the deep cerebral grey

matter and congestion of blood vessel (Fig.1.a)

associated with glia cell proliferation

presumably astrocytes (astrogliosis) that

extended into cerebral white matter. The latter

showed diffuse gliosis (Fig.1.b). Gliosis was

specifically concentrated around blood

capillaries that showing hypertrophy of its

lining endothelium with elongation of the

blood vessel length (Fig1. c, d and e). In

addition, focal gliosis was observed in deep

white matter with perivascular glia cell

aggregation associated with congestion of

blood vessels (Fig.1f) compared with nisin

treated and control groups which showed

normal histological structure of motor neurons

that have abundant eosinophilic cytoplasm,

large centrally located nuclei and normal

distrubtion of glia cells in both cerebral grey

and white matter (Fig.1. g & h). In

hippocampus there was reduction in the

cellular thickness comprising the different

regional areas. In dentate gyrus region (DG)

there was reduction in the thickness of small

basophilic granular cells and cellular

vacuolation (Fig. 2a) associated with

individual neuronal necrosis within the

molecular layer and glia cell proliferation.

Partial loss of small and large pyramidal cells

comprising hippocampus cornu ammonis

region 1 (CA1) and cornu ammonis region

3(CA3) respectively were detected (Fig.2. c-e).

In comparison with nisin treated group, the

three hippocampus regions (Fig.2b,d and f)

showed normal histological cellular density .

Immunohistochemical findings:-

Immunohistochemical staining revealed

relative increased expression of glial fibrillar

acidic protein (GFAP) denoting positive

astrogliosis that was evident in cerebral white

matter (Fig.3.a & c) and DG hippocampus

region (Fig.3.e) than CA1 and CA3 region

compared with the nisin treated and control

group that showed faint expression of

astrocytic dendrites either in number or

size(Fig.3. b, d & f). While the VEGF


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expression showed the same pattern of

positivity expression in different areas of

cerebrum and hippocampus as previously

described in GFAP in sodium nitrite treated

group (Fig.4a, c) compared with nisin treated

group (Fig.4b, d).

Legends of Figures:- Fig.1) figures a-f illustrating sodium nitrite

treated rats and g, h illustrating nisin

treated rats

a-Cerebral grey matter showing necrosis with

neuronophagia of individual motor

neurons(H&EX400).

b-Cerebral white matter showing diffuse glia

cellproliferation(H&EX200).

c- higher magnification of the previous note

the focal and diffuse glia cell proliferation in

the neuropil(H&EX400).

d-Cerebral white matter showing elongation of

cerebral blood capillary with increased

perivascular aggregation of glia cells

(H&EX400).

e- Cerebral white matter showing perivascular

glial cell proliferation associated with

hypertrophy of capillary endothelium

(H&EX400).

f- Deep cerebral white matter showing

congetion of cerebral blood vessel associated

with intense perivascualr aggregation of glia

cells(H&EX400).

g- Cerebral grey matter showing normal motor

neurons morphology with relatively large cell

body and vacuolated nuclei containing

prominent nucleoli(H&EX400).

h- Cerebral white matter showing normal

distribution of glia cell in neuropil with normal

histological structure of capillary endothelium

(H&EX400).

Fig.2)figures a ,c,e illustrating hippocampus

of sodium nitrite treated rats while b,d,f

concerning nisin treated ones

a-DG area showing extensive vacuolation of

granular basophilic cells with necrosis and

shrinkage of neurons comprising the molecular

layer with glia cell proliferation(H&EX400).

b-DG area of nisin treated rat showing

compact granular basophilic cells with normal

morphology of pyramidal neurons comprising

the molecular layer with normal glia cell

distribution (H&EX400).

c-CA1 area of sodium nitrite treated rat

showing cellular reduction of small pyramidal

neurons comprising the CA1 region

(H&EX400).

d-CA1 of nisin treated rat showing normal

compact cellular density of small pyramidal

neurons (H&EX400).

e-CA3 region of sodium nitrite treated rat

showing shrinkage and loss of large pyramidal

neurons associated (H&EX400) .

f- CA3 region showing normal large

pyramidal neurons with vesicular nuclei

(H&EX400).

Fig.3) GFAP stained sections

a-Cerebral white matter of sodium nitrite

treated rat showing relative increased number

of positive stained astrocytes(X100).

b-Cerebral white matter of nisin treated rat

normal astrocytes distribution(X100).

c-higher magnification of (a) note the

increased positive staining density of

astrocytes processes and cytoplasm (X400).


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d- higher magnification of (b) showing normal

astrocytes processes and cell body (X400).

e-Hippocampus of sodium nitrite treated rat

showing intense positive staining of astrocytes

processes and cell body (X400).

f- Hippocampus of nisin treated rat showing

normal stained astrocyte processes and body

(X400).

Fig.4) VEGF stained sections

a-Cerebral cortex of sodium nitrite treated rat

showing strong positive brown stained neurons

and glia cells (X400).

b- Cerebral cortex of nisin treated rat showing

negative stained neurons and glia cells (X400).

c- Hippocampus of sodium nitrite treated rat

showing relative positive brown stained

individual pyramidal neurons(X400).

d-Hippocampus of nisin treated rat showing

negative stained neurons and glia cells (X400).


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DISCUSSION

Nowadays contemporary addition of

preservatives in our food stuff become popular

but the trend for addition of synthetic food

preservatives is more likely occurred than the

natural ones. This may be due to cost issues.

However recent researchers studied the

deleterious hallmarks of synthetic food

additives .Sodium nitrite is one of the synthetic

food additives that is commonly used in cured

and processed meat products. In the present

study sodium nitrite as one of the synthetic

food additives and nisin as one of the natural

food additives were used to evaluate the

histopathological changes in cerebral cortex

and hippocampus with special reference to

GFAP and VEGF expression in brain sections.

Results showed that sodium nitrite

induced various histopathological alterations

involving the deep cerebral cortex and

different region of hippocampus that

collectively characterized by reduction in

hippocampal neuronal cellular density which

associated with increased expression of GFAP

& VEGF that was expressed in neurons,

astrocytes and vascular endothelium .Recent

studies have depicted the localization of VEGF

and its receptors on neurons and astrocytes

(Jeffrey and Janette, 2004).

The presented results in this work

proved the hypoxic mechanism of sodium

nitrite induced neurotoxicity because

expression of VEGF is up regulated by

hypoxia (Levy et al, 1996, Cobbs et al, 1998,

and Neufeld et al, 1999) which is one of the

potent inducers for VEGF expression in many

organs including CNS (Minchenko et al,

1994) .In the present study hypertrophy of

capillary endothelium was evident in cerebral

white matter and in medullary center of

hippocampus of sodium nitrite treated group

compared with nisin and control groups , a

result which agreed with Dombrowski et al,

(2008) who found that hypoxia elicited a

profound increase in vascular endothelial

growth factor receptor in hippocampus that

corresponded to increased blood vessel density

also VEGF has specific activity for endothelial

cell proliferation in vivo (Plate et al,1992).

Moreover , reduction in cellular density and

hippocampal neuronal loss that observed in

sodium nitrite treated group agreed with Zaidi

,(2010) also Biradar et al, (2013 ) who found

that memory impairment in sodium nitrite

induced neurohypoxia . In addition, it was

known that hippocampus is a structure heavily

implicated in memory consolidation

(Guacobini ,1990) . It has been reported that

during hypoxia the glutamate and aspartate

levels rise dramatically, which cause

excitotoxicity or disrupt the major excitatory

input to the hippocampus, that normally

protects the hippocampus from hypoxic

damage (Hagan & Beaughard, 1990), also

hippocampus contains high levels of

mineralocorticoid receptors, which make it

more vulnerable to stress compared to most

other brain areas (Plate et al, 1992).

In this study the encountered

astrogliosis of cerebral cortex and

hippocampus that showed strong expression

for GFAP may attributed in partly to neuronal

loss in different areas of cerebrum and

hippocampus as a part of reparative process to

form glial scar or as a consequence for

increased expression of VEGF as recorded by

Krum et al., (2002) who proved that VEGF is

considered as very potent mitogen for

astrocytes or astrogliosis may attributed to

hypoxia as referred by Ridet et al,(1997) who

noted that hypoxia and ischemia activate and

promote proliferation of resident astrocytes in

vivo.

Nisin treated group showing no

histopathological changes in both cerebral

cortex and hippocampus this agreed with

Delves-Broughton, (2005) who stated that

nisin is toxicologically safe. Also, Reddy et

al., (2012) confirmed suitability of nisin as a

safe and effective microbicide .

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