Egypt. J. Histol. Vol. 33, No. 4, Dec.

, 2010: 709 - 721

(ISSN: 1110 - 0559)

Original Article

Histological and Immunohistochemical Study on Rat Spleen in

Experimentally Induced Liver Cirrhosis
Dina M. Radwan, Hanan A. Amin and Ashraf M. F. Kamel
Histology Department, Faculty of Medicine, Cairo University

ABSTRACT
Introduction: The present study aimed to examine the histological changes in the spleen of rats with liver cirrhosis,
and to determine the immunohistochemical expression of endothelial nitric oxide synthase (e-NOS), and its upstream
effectors; tumor necrosis factor (TNF-) and vascular endothelial growth factor (VEGF).
Materials and Methods: Twenty male adult albino rats were divided into two equal groups. The first was control.
In the second group, liver cirrhosis was induced by intraperitoneal (ip) injection of thioacetamide 200 mg/kg twice
weekly for 12 weeks. Splenic index (spleen weight / body weight) was determined and the spleens of rats which
developed liver cirrhosis were subjected to the following stains: hematoxylin and eosin (H & E), silver impregnation,
and immunostaining with specific antibodies for e-NOS, TNF- and VEGF. Quantitative assessments were carried out
using image analyzer with statistical analysis of the results.
Results: Splenic sections of cirrhotic rats showed in addition to congestion of venous sinuses, significant increase in
reticular fibers in capsule and trabeculae as well as throughout the red pulp. The percentages of red pulp and fibrous
trabeculae areas were significantly higher in cirrhotic rats, while the percentage of the white pulp areas was significantly
smaller. Immunohistochemical staining of both e-NOS and TNF- in spleen sections of group II rats were significantly
lower than control, while VEGF immunostaining was significantly higher.
Conclusion: Splenomegaly in liver cirrhosis was not only congestive but there was also significant increase of reticular
fibers, red pulp area and angiogenesis. Moreover, nitric oxide (NO) reduction resulting from suppression of e-NOS and
TNF- seen in this study contributed to the increased volume of the spleen.

Key Words: cirrhosis; thioacetamide; spleen; e-NOS;

TNF, VEGF; silver impregnation.

Corresponding author: Ashraf M F Kamel

Tel.: 0111765111

E-mail: ashrafkamel@cu.edu.eg

INTRODUCTION
Splenomegaly is often detected in patients with liver
cirrhosis and portal hypertension with a prevalence of
60-65%1,2. Splenic congestion due to portal hypertension
has been reported to play a part in this increase in spleen
size3. However, splenomegaly in cirrhosis cannot be
considered as a mere consequence in the rise in portal vein
pressure (PVP). Such congestion cannot be considered as
the only cause of the enlarged spleen in liver cirrhosis,
since no relationship was found between the spleen size
and PVP4-7 or the degree of oesophageal varices8.

that have been implicated as potential mediators of

portal hypertension. Among them, nitric oxide (NO)
has received the greatest attention13. NO is a potent
vasodilator and there are three isoforms of NO synthese
(NOS): Inducible NOS (iNOS), endothelial NOS
(e-NOS) and neuronal NOS (nNOS)14,15. The respective
roles of iNOS and e-NOS, in intrahepatic vasoregulation
have been well studied in cirrhotic liver13.

After liver transplantation, only a slight decrease in

spleen size is detected despite the dramatic decrease in
portal vein pressure probably due to a recovery of the
congestive component only9-12. This confirmed that in
cirrhosis splenic hemodynamics is characterized both
by congestion and tissue hyperplasia, the later probably
being at least partially, irreversible3.

Alterations in eNOS-derived NO synthesis in the

intrahepatic microcirculation and in the splanchnic
and systemic vasculature are strikingly opposite. Liver
cirrhosis is associated with endothelial dysfunction
and deficiency of endothelial NO release resulting in
enhanced vasoconstriction, increased intrinsic vascular
tone16. In contrast, splanchnic and systemic vasculature
exhibit endothelial NO overproduction and arterial
vasodilation leading to hyperdynamic circulation17-23.

Recently in liver cirrhosis, it has been shown that

the spleen is a source of several vasoactive substances

This alteration of e-NOS activity has been

reported to be mediated by abnormalities in TNF-

63 (1228-2010)
709

Dina M. Radwan et al.

and VEGF expression via the Akt signaling pathway

in portal hypertension24-27. To our knowledge, e-NOS
immunohistochemical expression in the enlarged spleen
of liver cirrhosis has not been reported.

B. Tumor Necrosis Factor (TNF , Neo Markers,

Lab Vision Corporation).
C. Vascular Endothelial Growth Factor (VEGF, Neo
Markers, Lab Vision Corporation).

aim of the study

The immunohistochemical staining was done
following the avidin -biotin- peroxidase complex
(ABC) method. Diaminobenzidine (DAB) served as
the chromogen. The slides were counterstained with
hematoxylin. Negative control sections were prepared
by adding phosphate buffer solution instead of the
corresponding primary antibody with all other steps
taken in the same fashion31.

The aim of the present study was to investigate

the
histological
changes
and
examine
the
immunohistochemical expression of e-NOS, TNF- and
VEGF in the spleen of rats with liver cirrhosis.

MATERIALS AND METHODS

The study was conducted at the Animal House of
Kasr Al Aini School of Medicine in accordance with the
guidelines for the care and use of laboratory animals.

Quantitative Morphometric Study:

Images were processed and analysed using computerbased image analysis software (Leica QWin 500;
Imaging Systems, Cambridge UK) with to measure the
area percent of the following:

It included twenty adult male albino rats (180-200

grams). Animals were housed with free access to food
and water. They were randomly allocated to two equal
groups:

A. Red pulp.
B. White pulp.
C. Red pulp/white pulp (RP/WP) index calculated from
the values of both pulps32.
D. Fibrous trabeculae.
E. Reticular fibers.
F. Endothelial nitric oxide synthase immunoreactivity.
G. Tumor Necrosis Factor immunoreactivity.
H. Vascular
Endothelial
Growth
Factor
immunoreactivity.

Group I (Control Group): Received saline

(intraperitoneal injection) twice weekly for 12 weeks and
served as control.
Group II (Cirrhosis Group): Liver cirrhosis
was induced by intraperitoneal (i.p.) injection of
Thioacetamide (TAA) (200 mg/kg body weight) twice a
week for 12 weeks28.
Thioacetamide powder was purchased from Sigma
Chemical Company St. Louis, Mo., USA and dissolved
in 0.9% saline.

Ten randomly selected low power fields from each

slide were analysed and expressed as a mean area percent
of total area.

All rats were sacrificed after 12 weeks under

isoflurane anaesthesia and body weights were recorded.
Liver and spleen were excised.

Statistical Analysis:
Computer software package SPSS 15.0 was used
in the analysis. For quantitative variables, mean
(as a measure of central tendency), standard deviation
(as a measure of variability) were presented.

Spleen Index: The spleen was weighed by an

electronic balance and the spleen index was calculated
according to the following formula:
Spleen index =spleen weight (g)/ body weight (g)29.

Independent Samples T-test was used to estimate

differences in quantitative variables between the
control and experimental group. It was considered to be
statistically significant if a P value was less <0.05

Spleen specimens were fixed in 10% buffered

formalin, embedded in paraffin, cut into 5 m serial
sections and subjected to the following stains:

RESULTS

1. Hematoxylin and Eosin (H & E) stain for histological

analysis.
2. Silver impregnation for detection of reticular fibers
fibers30.
3. Immunohistochemical staining using a primary anti
serum to:

I-Histological and Immunohistochemical Results:

The liver histology of the group II rats confirmed the
occurrence of liver cirrhosis with disorganization of the
normal lobular pattern accompanied by the formation
of well-defined pseudolobules (Fig. 1-a). Thick fibrous
connective tissue septa were seen bridging the portal
areas and frequently extending into the lobules (Fig. 1-b).

A. Endothelial nitric oxide synthase (e-NOS, Neo

Markers, Lab Vision Corporation).

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Histological and Immunohistochemical Study on Rat Spleen in Experimentally Induced Liver Cirrhosis

H & E stained control spleen sections, consisted of

discrete lymphoid masses (white pulp) and embedded
in highly vascular matrix (red pulp). The white pulp
was subdivided into Peri-Arteriolar Lymphatic Sheath
(PALS), follicular and marginal zones. The PALS could be
identified as the dark region around the central arteriole.
Some lymph follicles exhibited germinal centers. The red
pulp was composed of meshwork of splenic cords and
several venous sinuses. Hemosiderinladen macrophages
were also present. Supporting trabeculae containing
smooth muscles were seen (Fig. 2). On the other hand,
examination of group II- sections, showed obvious
congestion of spleen tissues with markedly distended
venous sinuses. Thickening of splenic trabeculea was
also noted (Fig. 3).

The percentage of the areas occupied by the red pulp

was greater (P<0.05) in cirrhotic rats than in control rats
as was the case for both the fibrous trabeculae (P<0.005)
and the RP/WP index (P<0.005) (Table 2 Fig. 13).
Conversely, the percentage of the areas occupied by the
white pulp, was smaller in cirrhotic rats than in control
rats (P<0.005).
The area percent of silver stained reticular fibers was
greater (P<0.005) in cirrhotic rats than in control rats
(Table 3, Fig. 14-a).
The area percent of e-NOS immunoexpression
was significantly decreased (by 53%, P<0.005) in
cirrhotic rats compared with the area percent in control
rats (Table 3, Fig. 14-b). Similarly, the area percent of
TNF- immunostaining was significantly decreased
(by 45%, P<0.005) in cirrhotic rats than control rats
(Table 3, Fig. 14-c). In contrast, the area percent of VEGF
immunoreactivity was increased (by 30 %, P<0.005) in
cirrhotic rats than in control rats (Table3, Fig 14-d). Thus,
the increase in the area percent of VEGF immunostaining
in the cirrhotic rats was smaller than the decrease in the
area percent TNF- immunoreactivity.

Silver staining technique employed to demonstrate

the reticular architecture of the control spleen showed the
capsule and perivascular supporting tissue which provided
a framework ramifying throughout the section in the red
pulp. The reticular skeleton was almost absent in the
center of the white pulp nodules but was well developed
at their margins and around the central arteriole (Fig. 4).
With silver impregnation, an irregular fiber pattern was
visible in group II sections with increased reticular fibers
manifested as thickened capsule and trabeculae. Dense
meshwork was present throughout the red pulp (Fig. 5).

Table 1: Mean body weights, spleen weights and Spleen

weight / body weight indices of control and TAA treated rats:

Examination of e-NOS immunostained sections,

revealed that, red pulp had strong immunoreactivity
seen as brown cytoplasmic staining within sinus lining
endothelial cells with almost negatively stained white
pulp (Fig. 6). Compared to control sections, e-NOS
sections of group II showed weaker immunoractivity
within red pulp and in sinus lining cells. White pulp
exhibited negative reaction (Fig. 7).

Body
weight (g)

Spleen
weight (g)

Spleen weight
/ body weight
(%)

Control
(meanSD)

226.36.36

0.480.05

0.210.02

Cirrhosis
(meanSD)

1996*

1.260.05*

0.630.03*

* P<0.005 vs control rats which is highly statistically significant

Table 2: Area percent of white pulp, red pulp, index of red

pulp / white pulp and fibrous trabeculae of control and TAA
treated rats:

TNF- immunostained control sections showed

positive cytoplasmic reaction within mononuclear
cells in the red pulp with negatively stained white pulp
(Fig. 8). Decreased immunoreactivity was seen in group
II sections with almost similar distribution (Fig. 9).

Control
(meanSD)
Cirrhosis
(meanSD)

In VEGF-immunostained control sections, the positive

reaction was noted within red pulp only in endothelial
cells (Fig. 10). Examination of group II sections revealed
marked increase in immunoreactivity identified as brown
cytoplasmic staining within endothelial cells lining the
venous sinuses (Fig. 11).

White Pulp

Red Pulp

RP / WP
index

Fibrous
Trabeculae

21.463.67

71.444.44

3.450.85

7.111.70

11.052.13**

78.102.85*

7.381.82**

10.852.38**

* P<0.05 vs control rats which is statistically significant

** P<0.005 vs control rats which is highly statistically significant

Table 3: mean area percent of silver impregnation, e-NOS,

TNF- and VEGF in sections of rats of the control and TAA
treated groups:

II-Morphomertical and Statistical Results:

The body weights of cirrhotic rats were lower than
control rats (P<0.005) (Table 1, Fig. 12-a). On the other
hand, the spleen weights of cirrhotic rats were higher
than control rats (P<0.005) (Table 1), while the spleen
indices (Spleen weight / body weight) of cirrhotic
rats were higher than those of control rats (P<0.005)
(Table 1, Fig. 12-b).

Area %
Reticular
Fibers

Area %
e-NOS

Area %
TNF-

Area %
VEGF

Control
(meanSD)

30.378.58

12.234.08

16.834.05

8.171.21

Cirrhosis
(meanSD)

44.076.99*

5.741.51*

9.202.13*

11.822.96*

* P<0.005 vs their respective control rats which is highly statistically significant

711

Dina M. Radwan et al.

Fig. 1: Photomicrographs of a section of the liver of group II rats showing: Fig. 2-b: Hemosiderinladen macrophages (arrow head) are seen within
the red pulp (RP). Supporting trabeculae (T) containing smooth muscles
a- Disorganized lobular pattern with the formation of pseudolobules.

H & E x400
H & E x100 are also noted.

Fig. 1-b: Fibrous connective tissue septa (arrow) are noted bridging the Figure (3): Photomicrographs of splenic sections of group II showing:
portal areas and extending into the lobules. Masson trichrome stain x100 a- Marked congestion of red pulp (RP). Note the almost normal appearance
of the white pulp (WP) with central arteriole (CA).
H & E x200

Fig. 2: Photomicrographs of control splenic sections showing:

a- Lymphoid masses (white pulp) embedded in highly vascular matrix
(red pulp) [RP] with several venous sinuses (arrow head). The white
pulp has dark region around the central arteriole (CA) representing the Fig. 3-b: Markedly congested (C) and distended venous sinuses (arrow
peri-arteriolar lymphatic sheath (PALS). Germinal centers (GC) could be head) and thickened trabeculae (T).
H & E x 400
H & E x200
identified in some follicles.

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Histological and Immunohistochemical Study on Rat Spleen in Experimentally Induced Liver Cirrhosis

Fig. 4: Photomicrographs of control splenic sections showing:

Fig. 5: Photomicrographs of splenic sections of group II showing:
a- Capsule (arrow head) and perivascular supporting trabeculae (arrow).
a-
Irregular fiber pattern and thickened capsule (arrowhead) and
Silver impregnation x40 trabeculae (arrow).
Silver impregnation x40

Fig. 4-b: Reticular skeleton is almost absent in the center of the white pulp
nodules (WP) but well developed at their margins.
Silver impregnation x100

Fig. 5-b: Marked thickening of trabeculae (arrow) containing blood

vessels

Silver impregnation x100

Fig 4-c: Reticular framework is ramifying throughout the red pulp [RP]
(arrow head).
Silver impregnation x400

Fig. 5-c: Dense reticular meshwork is present throughout the red pulp
(RP) (arrow head)
Silver impregnation x400

713

Dina M. Radwan et al.

Fig. 6: Photomicrographs of control splenic sections, showing:

a- Red pulp (RP) with strong immunoreactivity with almost negatively
stained white pulp (WP).
e-NOS immunostaining x200

Fig. 7- b: obvious decrease in positive staining within sinus lining cells

(boxed areas)
e-NOS immunostaining x400

Fig. 6- b: Brown cytoplasmic staining within sinus lining endothelial

cells (boxed areas) with thickened trabeculae (arrow).
e-NOS immunostaining x400

Fig. 8: Photomicrographs of control splenic sections showing:

a-
positive brown staining within red pulp (arrow) while white
pulp (WP) is negatively stained.
TNF- immunostaining x200

Fig. 7: Photomicrographs of splenic sections of group II showing:

a- decreased immunoreactivity within red pulp (RP) with negatively
stained white pulp (WP).
e-NOS immunostaining x200

Fig. 8- b: positive brown reaction within mononuclear cells (arrow)

TNF- immunostaining x400
present in the red pulp (RP).

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Histological and Immunohistochemical Study on Rat Spleen in Experimentally Induced Liver Cirrhosis

Fig. 9: Photomicrographs of splenic sections of group II showing:

a- Decreased immunoreactivity within red pulp (RP) with negatively
stained white pulp (WP).
TNF- immunostaining x200

Fig. 10- b: positive brown reaction within endothelial cells lining venous
sinuses (arrow)
VEGF immunostaining x400

Fig. 11: Photomicrographs of splenic sections of group II showing:

a-
marked increase in immunoreactivity (arrow) within red pulp
Fig. 9- b: Obvious decrease in positive staining within mononuclear cells
(RP) with negatively stained white pulp (WP).
TNF- immunostaining x400
in the red pulp (RP).
VEGF immunostaining x200

Fig. 11- b: increased positive staining within endothelial cells lining

Fig. 10: Photomicrographs of control splenic sections showing:
positive brown staining (arrow) within the red pulp (RP) while venous sinuses (arrow) in the red pulp (RP).
a-
VEGF immunostaining x400
white pulp (WP) is negatively stained.
VEGF immunostaining x200

715

Dina M. Radwan et al.

Fig. 12-a: mean body weights of control and cirrhotic rats.

Fig. 14: Histograms comparing control and cirrhotic spleen sections as

regards: (a): mean area percent of silver stained reticular fibers.

Fig. 12-b: mean spleen indices (spleen weight / body weight) of control
and cirrhotic rats.

Fig. 14-b: mean area percent of e-NOS immunostaining,

Fig. 13: mean area percent occupied by the white pulp, red pulp and
fibrous trabeculae as well as the RP/WP index in spleen sections of both
cirrhotic and control rats.

Fig. 14-c: mean area percent of TNF- immunostaining,

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Histological and Immunohistochemical Study on Rat Spleen in Experimentally Induced Liver Cirrhosis

This agreed with a previous study on spleens of 34

patients with advanced hepatosplenic schistosomiasis32.
However, other scientists found an increase
in the white pulp volume and thereby suggested a
possible immunologic involvement in the genesis of
splenomegaly37.
In our study the white pulp areas were found less
in cirrhotic rats than in control rats probably because it
was a percentage and not an absolute measurement and
perhaps the increase in red pulp and fibrous trabeculae
percentages was much more and masked any increase in
white pulp area that might have occurred.
Furthermore, the present results revealed significantly
increased VEGF immunoreactivity within endothelial
cells lining the venous sinuses in the spleen of cirrhotic rats.
Thus, angiogenesis and increased vascular permeability
may also be involved in the spleen enlargement. This
finding agreed with previous reports34,38,39.

Fig. 14-d: mean area percent of VEGF immunoreactivity.

DISCUSSION
Splenomegaly resulting from TAA-induced liver
cirrhosis was investigated in adult male albino rats. It
was found that animals in the cirrhotic group presented
a statistically significant smaller increase in BW than
control animals. The spleen indices of cirrhotic rats
were statistically higher than those of control rats. This
coincided with the results of a previous research33.

Since tissue hypoxia was an important stimulus for

VEGF expression40, another possible mechanism for
splenomegaly would be the hypoxic status with splenic
congestion. However, further studies are needed to fully
elucidate it.

The main finding in the present study was splenic

congestion with markedly distended venous sinuses in
the spleens of cirrhotic rats.

Therefore, even by only considering the histological

data, splenomegaly in cirrhosis cannot be simply
classified as congestive, but should be defined as
congestive-hyperplastic.
In the present study, sections of group II showed
significantly weaker e-NOS immunoractivity within red
pulp and in sinus lining cells and negative immunostaining
in the white pulp. These findings were in agreement with
a report that showed e-NOS reduction in the spleen by
Western blot34.

It was long established that spleen enlargement in liver

cirrhosis was considered to be congestive splenomegaly34.
However, such congestion could not be considered as the
only cause of the enlarged spleen in liver cirrhosis, since
no relationship was found between the spleen size and
PVP 4-7,34 and since complete resolution of splenomegaly
had never been reported after liver transplantation9-12.

NO generated by e-NOS was settled to be an important

regulator of the microcirculation at the sinusoidal level
by being a vaso-relaxing agent41-43.

In the present study by silver impregnation, the

reticular fibers significantly increased in group II splenic
sections manifested by thickening of the capsule and
trabeculae with dense reticular meshwork throughout the
red pulp.

Additionally, NO was found to have antiproliferative

and antifibrogenic activities44-47. Therefore, insufficient
production of NO could contribute to increased portal
pressure not only by increasing vascular tone but also by
increasing the fiber formation. This concept may have
therapeutic implications in portal hypertension. Recently,
NO received most attention in gene therapy for the
treatment of portal hypertension targeting its regulatory
system48.

These results were in agreement with previous studies

which had demonstrated that splenomegaly exhibited not
only pooling of blood in the red pulp, but also increased
reticular fibers that later evolved into diffuse fibrosis35,36.
Additionally, it was observed that the percentage of
the areas occupied by the red pulp and fibrous trabeculae
were significantly higher in cirrhotic rats compared to
control rats, while the percentage of the areas occupied
by the white pulp, was significantly smaller.

Moreover, NO derived from e-NOS was found

to contribute to angiogenesis, in addition to VEGF,
however, the exact roles of each remained to be defined49.

717

Dina M. Radwan et al.

In the present study, the splenic TNF-

immunoexpression was significantly decreased in
cirrhotic rats.
10.

TNF- is considered a mediator of NO release via

the PI 3-kinase Akt signaling pathway50-54. Inhibition of
TNF- synthesis was found to decrease NO synthesis55-57.
Thus, the findings of the present study indicated that
the decreased TNF- may be a direct contributor to the
decreased NO release in the spleen of cirrhotic rats as
previously suggested34.

11.

CONCLUSION
12.

In conclusion, splenomegaly in liver cirrhosis was

not totally congestive, but also involved hyperplasia of
reticular fibers and red pulp, as well as angiogenesis
verified by high VEGF expression. Moreover, suppression
of e-NOS and TNF- decreased NO. Studying the
role of NO and the factors influencing its level in the
hemodynamics of the spleen in cases of liver cirrhosis
may lead to a new therapeutic potential in the future.

13.

14.

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