Experimental Studies on Etiology of Portal Hypertension in Allergic Aspect Minoru NAKAO, Taiichiro NA~AlSm, Masakatsu INOUE, Hidekazu KI~ACm and Shigemasa KOOA A B S T R A C T : T o elucidate the mechanism of portal hypertension seen as a symptom of so-called Banti's syndrome (idiopathic portal hypertension), observation was made of rising of the portal pressure experimentally induced in sensitized rabbits. Intraintestinal injection of the same antigen as used for the sensitization resulted in elevation of the portal pressure. This phenomenon appears to be attributable to antigen-antibody reaction caused by the injected antigen absorbed from the intestine and entered thereby into the portal system while maintaining its antigenicity. From the phenomenon also, the site of the antigen-antibody reaction is estimated to be limited at least to the hepatic level. The portal pressure-rising phenomenon observed following intraintesfinal introduction of antigen may suggest the possibility of entrance of the orally introduced antigen to the portal system, emphasizes importance of alimentary factors in the genesis of this syndrome. KEY-WORDS: phenomenon.

Banti's syndrome, portal hypertension, pressure-rising

INTRODUCTION

A s an etiologic agent of Banti's syndrome, allergic factors have been suggested to deeply participate in the syndrome in our previous studies. As for etiology of portal hypertension in this syndrome, the underlying mechanism has been investigated, yet many problems including the introducing pathway of antigens remain uncertain. To clarify the probable involvement of allergic factors in portal hypertension of Banti's syndrome, the oral pathway of antigens was hypothesized in the first place. After rabbits were sensitized with ovalbumin, it was administered as antigen into the intestine to examine the change of portal pressure in this condition. This paper summarizes the experimental procedures and the results obtained. MATERIALS AND METHODS

Animals used were healthy adult rabbits of 2 to 3 kg in weight, selected irrespectively of sex. They were intragluteally injected with 3 ml of 1 per cent ovalbumin suspension, the sensitizer, at three-day intervals (max duration of sensitization: 200 days). Three days after the final injection, they were subjected to experiments through 12 to 24 hr fasting. When experiments were completed, they were exsanguinated by via the posterior vena cava to death, and the weights of the spleen and liver measured. Sensitized animals were

From the Department of Surgery (Director: Prof. S. Koga), Tottori University School of Medicine, Yonago~ Tottori Prefecture,Japan. JAPANESEJOURNAL OF SURO~RY, VOL. 7, No. 4, pp. 269-278, 1977

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270 T a b l e 1.

Group Group Group Group Group

Jap. J. Surg. Dec. 1977

Classification by days of sensitization

N A B C D

nonsensitized control group, 10 cases less than 50 days of sensitization, 6 cases 51 to 100 days, 10 cases 101 to 150 days, 5 cases more than 151 days, 7 cases

Total case number 38

divided into four groups according to periods of sensitization (Table 1) for comparison of the degree of the spleen and liver enlargement and hypertension of the portal pressure. In the experiments, a Universal dual-beam oscilloscope VC-7 and a continuous recording camera PC-2B ( N I H O N Koden Industry Co.) were used in combination along with Type-SS film oscillograph paper. The portal pressure was continuously recorded with an integrating digital voltmeter. For electrocardiographic recording to observe the effect on cardiac functions during the experiment, electrodes were mounted. Experimental procedures: Under anesthesia with 25 to 35 mg/kg of thiamylal sodium injected intramuscularly, the rabbit was laparotomized along the midline of the upper abdomen. A cutdown tube of 2-mm in outer diameter was immediately passed into the superior mesenteric vein distributed in the ileocecal region via its branch. After confirming the tip of the tube reaching in the prehepatic portal vein, the cutdown tube was restrained. The peripheral end of the tube was connected to the integrating digital voltmeter/Universal dual-beam oscilloscope complex via an electronic manometer. To observe changes in E C G and in blood flow volume of the splenic vein, a flow probe of an electromagnetic blood flowmeter microflow was placed and restrained in a branch of the splenic vein. Subsequently, a 30 to 40-cm segment of the upper part of the small bowel (jejunum) was blocked, and contents in the blocked portion were washed out with 40 to 60 ml of sterile physiological saline and vacated. The portal pressure was measured thereafter. After a definite time (2-3 minutes) had passed, various antigen suspensions were administered in a volume of 30 ml into the blocked bowel to observe changes in portal pressure. RESULTS

1. Pathological Findings of the Liver and Spleen in Sensitized Rabbits a. Splenic and hepatic weights (Fig. 1) : The spleen of control rabbits 2 to 3 kg in weight was 3 to 5 crn in length, 0.5 to 1.8 cm in the max. width and 0.3 to 0.8 crn in thickness, generally in a flat shape. In sensitized cases, in contrast, the shape of the spleen was classifiable into three types, i.e., the flat, thick and intermediate, accounting respectively for 42, 35 and 21 per cent. Generally in the early stage of sensitization, the flat type was frequent, whereas in the late stage, the swollen spleens were frequently of the thick type. When viewed with respect to splenic weight, cases less than 3 g were mostly of the flat type, while those more than 5 g often had the shape of the thick type. The degree of splenic enlargement was assessed according to weight of the organ isolated. The splenic weight was 0.9• g in the control group. In sensitized groups A, B, C and D, it was 3.0~z0.91, 5.9• 5.2~2.04 and 5.0-+-2.34 g, respectively. The so-called splenomegaly more than I0 g in weight were encountered in three sensitized cases~ all being in group B of 51 to 100 days of sensitization.

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140

m•220I 180t

:~100 o"2

~ 140 50 I

I

I

Sensitized days

100 I

I

I

Sensitized days

I

I

I

t

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Sensitized days

Fig. 1.

Mean and standard derivation of splenic and hepatic weights and portal pressure.

Fig. 2.

Histology of the spleen in the early stage of sensitization (HE-stain) : multiple formation of lymphfollicles.

The liver also showed a slight swelling tendency. The hepatic weight in the control and sensitized groups A, B, C and D averaged 84.2!22.78, 84.4• 97.9• 115.9~15.57 and 98.4~20.39 g, respectively. Significant differences from the control group were observed in groups C and D. b. Histological findings of the spleen and liver: The spleen showed medullary hypermia, cellular infiltration and neoformation of multiple follicles up to about 50 days of sensitization. In the late stage of sensitization, the spleen tended to be atrophied and came to show perivascular, peritrabecular and perifollicular fibroplasia (Figs. 2 and 3). The liver rarely showed morbid findings in the early stage. But in the later stage, liver cells presented hypochromasia, swelling and anisocytosis due to glycogen deposition~

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Fig. 3.

Histology of the spleen in the later stage of sensitization (silver impregnation stain: SIS) perivaseular and perifollicular fibrosis.

Fig. 4.

Histology of the liver in the early stage of sensitization (SIS): slight fibrosis of Glisson's capsule.

hepatic cords became irregular and the Glisson's capsule showed a conspicuous trend to fibrosis (Figs. 4 and 5). 2. Changes in Portal Pressure a. Portal pressure after sensitization (Fig. 1): Portal pressure at laparotomy, when assessed according to the criteria of portal hypertension by Suzuki et al. (Table 2), was slightly elevated in 53 per cent of cases, moderately increased in 21 per cent and markedly

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Histology of the liver in the later stage of sensitization (SIS) : marked fibrosis of Glisson's capsule and linkage to adherent ones. Table 2.

Classification of portal hypertension in rabbit

144 mmH20 145 - - 174 mmH20 175 - - 199 mmH20 200 mmH20 -

-

normal pressure slight hypertension moderate hypertension marked hypertension

elevated in 7 per cent; hence portal hypertension in approximately 80 per cent of the sensitized cases. By periods of sensitization, the pressure was 149.2~31.11, 175.7i36.27, 158.6-t=49.3 and 1 6 1 . 4 i 8 . 4 6 m m H 2 0 respectively in groups A, B, C and D, compared to 103• 19.28 m m H 2 0 in the control group. Sensitized groups were all significantly higher in portal pressure than the control, but no significant difference was noted among the four sensitized groups. There were two advanced cases showing more than 200 m m H 2 0 , both in group B (51-100 days). b. Experiments on elevation of portal pressure: Portal pressure change following injection of 30 ml of antigen suspension, observed using the aforementioned apparatus, was all upward. Since the respiratory fluctuation of the portal pressure averages 5 mmH~O, not exceeding 10 m m H z O generally. Therefore, when pressure rising over 11 m m H 2 0 was recorded, it was regarded as representing an experimentally induced pressure-rising phenomenon. The portal pressure remained consistently unchanged after a 30 ml ovalbumin injection into the intestine of nonsensitized rabbits. However, the portal pressure-rising phenomenon was produced in about 70 per cent of sensitized cases for an average of 30 minutes from several min. after intraintestinal injection of 30 ml suspension of the same ovalbumin antigen as used for sensitization. Mean max. portal pressure and mean pressure elevation were as follows: group A, 174 and 24.8 m m H 2 0 ; group B, 194 and 18.3; group C, 174 and 14.1; and group D, 180 and 18.6, and these did not differ significantly among the four groups. Experimental elevation of the portal pressure was less than 30

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250

200

150

100

0- . . . . . . . . . . . . . . . . . . . .

4

9 . . . . . . . . . . . . . . . . . . . . .

-0

9

Preinieetion 9 .....

-Fig. 6.

4

|

Postinjeetion (maximum)

Control

~ Sensitized group

Changes of portal pressure following intraintestinal injection of 1% ovalbumin.

m m H 2 0 in about 60 per cent of elevated cases, and a pronounced elevation over 51 m m H 2 0 was noted only in one case. The degree of experimental portal pressure-rising was found to be scarcely related to the duration of sensitization at all, as can be seen in Fig. 3. Furthermore, the relationship between the preinjection portal pressure and the experimental pressure elevation in sensitized groups was investigated. Even in normotensive cases the rising phenomenon was experimentally inducible, whereas the experimental pressure-rising phenomenon did not occur in some cases of portal hypertension irrespective of the degree of portal hypertension (Figs. 6 and 7, Table 3). Injection of 3 ml ovalbumin suspension, however, into such nonresponders via the parotid vein was observed to give rise to a marked pressure elevation after several sec. When other foreign protein was injected intraintestinally in ovalbumin-sensitized rabbits, the portal pressurerising phenomenon was not observed at all, as well as in non-sensitized rabbits. To determine at what site the mechanism of action might operate for such experimental pressure-rising phenomenon in sensitized rabbits, the following experiments were carried out. The superior mesenteric vein was temporarily blocked at its junction with the portal vein. Immediately after blocking, the portal pressure declined by an average of

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70 E

60

fi0 io

40

30

00

~e

20 9

.l0

...................

9 ..........

9

Fig.

9

9 .......................

o--

o9

I

I

100

150

;

I

20 250 Portal pressure (mmH20)

Relationship between portal pressure and level of experimental portal elevation in sensitized rabbits.

7.

T a b l e 3.

Group

Level of experimental elevation of portal pressure

- - 10

N

--20

mmH~O --30

--40

--50

51--

no changes

A

1

2

2

--

--

1

B

3

4

2

--

1

--

C D

2 2

2 1

l 3

-1

---

---

8/28.6

9/32.1

8/28.6

1/3.6

1/3.6

1/3,6

Total/%

Pressure elevation was determined in the case of more than 11 mmH20.

33.7 m m H 2 0 in six sensitized a n i m a l s a n d b y a n average of 22.6 m m H ~ O in five controls. A slight recovery of pressure occurred after several minutes, a n d the pressure became stabilized at levels still lowered by 13.7 a n d 12.6 m m H 2 0 , respectively. U n d e r this condition a 3 ml suspension of the same a n t i g e n was injected into the vein 6f the blocked area, which did not cause a n y significant change in portal pressure in these groups. W h e n the blocking was u n d o n e , however, only sensitized a n i m a l s showed a m a r k e d portal pressure elevation (Fig. g).

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:

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: Sensitized group ....... - Control

? / /

// 150

100 ""

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.-~

Fig. 8.

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After block

C h a n g e s of p o r t a l pressure before a n d after b l o c k i n g of t h e s u p e r i o r m e s e n t e r i c vein. I n t r a v e n o u s i n j e c t i o n of 1% o v a l b u m i n t o w a r d b l o c k e d area. 200

:

:- Sensitized group ....... 9 Control

150

i00

...... ...... :111;;5:

X

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Injection I

.~

'

O'

,

3'

,

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After block

Fig. 9.

I

10--15'

!

,~ ~='~

Changes of portal pressure before and after blocking of the superior mesenteric vein. I n t r a i n t e s t i n a l i n j e c t i o n of 1 % o v a l b u m i n t o w a r d b l o c k e d area.

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220 200

3 E ~ Sensitized group 150

....

Control

100

i~- . . . . . . . . . . . . . .

.4

I

Preinjection

Postinjection (maximum)

Fig. I0. Changes of portal pressure by injection of 1~o ovalbumin into the superior mesenteric vein. When 30 ml of the same suspension was injected into the blocked segment of the small intestine, the portal pressure remained unchanged in both groups. But after unblocking, the portal pressure showed a rise again only in sensitized animals (Fig. 9). In nonblocked cases, 3 ml suspension of the same antigen was administered via a branch of the superior mesenteric vein. This injection brought about a marked portal pressure increase in a few seconds only in sensitized rabbits (Fig. 10). DISCUSSION On etiology of Banti's syndrome, many experimental studies have been published, reporting that Banti findings were not obtained by any mechanical procedures applied to the portal system.l-3,9A 0 But intravenous injection of foreign protein into animals produced splenomegaly, as Rich 6 reported. Suzuki 7,s demonstrated that splenomegaly and portal hypertension were induced by long-term intravenous injection of ovalbumin in rabbits, and thereby advocated that the mechanism of portal hypertension might be attributed to pressure elevation arising from antigen-antibody reaction. At this department, Iguchi 3 and Kojima 4 using rabbits and Maeda 5 using guinea pigs experimentally produced splenomegaly by the method of Suzuki et al. and examined Malpighian bodies in splenomegaly pathohistologically in detail, with the results demonstrating findings strikingly similar to Banti spleens, hence suggesting possible involvement of allergic factors contributing to etiology of the disease. Although sensitization with foreign protein has been proven to bring about splenomegaly and portal hypertension, there is no theory

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established as to pathogenetic mechanism of portal hypertension as yet. In the present experimental study aimed to clarify the mechanism, some encouraging findings were obtained using sensitized rabbits. Sensitizing animals with egg albumin generated splenomegaly and portal hypertension in this experiment. Histologically, the spleen mainly showed perifollicular and perivascular fibroplasia and the liver presented fibroplastic findings in the Glisson's capsule. These data are mostly consistent with other reports. Portal pressure changes by administration of the same antigen as used for sensitization were investigated under such various conditions as injection into the intestine, directly into the superior mesenteric vein, or into the vein and intestine, blocked and unblocked at the junction of the superior mesenteric vein with the portal vein. Although different in degree by ways of infusion, portal pressure changes observed were all rising phenomena. These facts indicate that in cases of such a portal pressure-rising phenomena, the pressure-rising mechanism operates at sites central to the area of the superior mesenteric vein (probably in the hepatic level). Further, the pressure-rising phenomenon seems to arise from antigen-antibody reaction due to entrance of intraintestinally injected antigens into the portal system in the condition where the antigenicity is maintained. In this context, the view by Suzuki 7 that the pressure-rising mechanism is ascribable to contraction of intrahepatic portal capillaries, may provide an interesting suggestion on discussing the relationship of hepatic lesions and portal hypertension. To sum up, the portal pressure-rising phenomenon probably caused by the antigenantibody reaction throws light on elucidation of the mechanism whereby portal hypertension is produced. Moreover, by the possible entrance of antigens from the intestine to the portal system, as what may be referred to as a "trigger" to facilitate the establishment of this disease in the aspect of allergy, would stress importance of the involvement of alimentary factors. (Received for publication on April 28, 1977)

References 1. Douglass, T.C.: Attempt at the experimental production of portal hypertension, Arch. Surg. 62: 785-788, 1951. 2. Higgins, G.M. : Experimental pathology of the liver, Arch. Path. 14: 491~1~97, 1932. 3. Iguchi, M." Pathohistological studies on the etiology of Banff's disease, especially on the vascular plexuses in the splenic Malpighian body obtained from experimental splenomegalia of rabbits, Yonago Igaku Zasshi 10: 1733-1744, 1959 (in Japanese). 4. Kojima, T.: Three-dimensional approach to the structure of the splenomegalia obtained from sensitized rabbits with foreign proteins, with particular reference to the lymphatic tissues and arteries in the spleen, Yonago Igaku Zasshi 13: 290-306, 1962 (in Japanese). 5. Maeda, K.: Experimental studies on the etiology of Banff's disease, especially on the vascular plexuses in the splenic Malpighian body obtained from sensitized guinea pigs

6.

7.

8. 9.

10.

with foreign proteins, Yonago Igaku Zasshi 1 0 : 1843-1854, 1959 (in Japanese). Rich, A.R. : The activity of the lymphocyte in the body's reaction to foreign protein, as established by the identification of the acute splenic tumor cell, Bull. Johns Hopkins Hosp. 65: 311-327, 1939. Suzuki, T. : Etiology and pathology of portal hypertension. Nippon Gekagakkai Zasshi, 57: 987-1013, 1956 (in Japanese). Suzuki, T. : Banti's syndrome, Rinsho Kagaku 8 : 309-319, 1972 (in Japanese). Taniguchi, H. : The histo-pathological study of the congestive spleen; especially three-dimensional approach to the structure of the intrasplenic lymphfollicles and arteries, Yonago Igaku Zasshi 14: 427-459, 1963 (in Japanese). Warthin, A.S.: The relation of thrombophlebids of the portal and splenic veins to splenic anemia and Banff's disease, Int. Clin. 4: 189226~ 1940.

Experimental studies on etiology of portal hypertension in allergic aspect.

Experimental Studies on Etiology of Portal Hypertension in Allergic Aspect Minoru NAKAO, Taiichiro NA~AlSm, Masakatsu INOUE, Hidekazu KI~ACm and Shige...
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