Histological Changes in the Spleen Consequent on. Department of Surgery (Prof. T. Maki), Tohoku University School of Medicine, Sendai

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1 Tohoku J. exp. Med., 1968, 96, Histological Changes in the Spleen Consequent on Circulatory Disturbance* Toshio Sato, Yasuo Suda, Kenji Koyama Kenichi Watanabe and Shunichi Kimura Department of Surgery (Prof. T. Maki), Tohoku University School of Medicine, Sendai Histological changes produced in the spleen of adult dogs by disturbed blood flow were described. Constriction of the splenic vein did not cause fibrosis of tile splenic cord even after a long period. On the other hand, clamping of splenic artery produced thickening and proliferation of reticulin fibers of the pulp cord. When venous constriction was applied in addition to arterial clamping, a relative increase and dilatation of the splenic sinus were observed. These changes gave a histological feature simulating that of the Banti spleen, but there was no absolute increase in splenic pulp. Consequently, it was not concluded that splenomegaly equivalent to the Banti spleen could be produced by this experimental proce dure. In Europe and. America, the view most generally accepted on the pathogenesis of splenomegaly with portal hypertension is that the splenic tumor is produced by congestion of the spleen due to portal hypertension. This concept, however, has not hitherto attained a unanimous agreement, because experimentally produced portal hypertension does not cause pronounced chronic splenomegaly. As reported previously,' our attempt to produce chronic splenomegaly by means of various experimental procedures causing circulatory disturbance of the spleen was unsuccessful. The histological study of the spleens, however, revealed a series of findings, which seemed interesting in comparison with those of the Banti spleen. MATERIALS AND METHODS As described in a previous report,1 a total of 118 adult dogs, weighing 10 to 30kg were subjected to the following manipulations. 1) Production of splenic congestion by means of constriction of the splenic vein. 2) Production of ischemic spleen by means of clamping of the splenic artery. 3) Constriction of splenic vein in addition to arterial clamping. At various intervals following one of these procedures, the spleen was removed and submitted to histological examination. In order to facilitate the demonstration of the fine structure of the spleen, the extirpated organ was out in halves and one of them was irrigated with 1,000ml of normal saline solution injected into the splenic artery under a Received for publication, July 13, * Attempt at Experimental Production of Splenomegaly, Report II. 281

2 282 T. Sato et al. pressure of about 100mm H2O. Both irrigated and non-irrigated parts were sliced in pieces, cleared of excessive water and blood, and fixed in formalin. Paraffin blocks were made thereof and stained with hematoxylin-eosin and silver impregnation for microscopic study. 1) Histology of the control spleen RESULTS The canine spleen appears different from the human spleen when stained by the silver impregnation method. There is more trabecular and capsular tissue in the former than in the latter. Herrath2 performed quantitative study of the splenic tissue and found that the canine spleen had trabecular and capsular tissues two to three times as much as those of the human spleen. Furthermore, the canine red pulp occupies a larger proportion of the splenic tissue than. the human. In the spleen of adult dogs, the structure of 'Grenzfaser',3 which constitutes the central core of the pulp cord in the human spleen is not distinctly recognizable, and reticular networks of reticulin fibers of approximately uniform thickness are arranged around the sinuses (Fig. 1). These findings were seen in 6 of 10 control dogs. In 2 of the control dogs, there was fibrosis of the pulp cord with periar terial fibrosis, while in 2 others, slight thickening and fibrosis of the pulp cord. The variety in the findings seen in the 'normal' dogs is probably due to that mongrel dogs were used in the experiment. There were also some differences in the histological features of the capsule, trabecle, follicle and sinuses among individual dogs. 2) Histology of the spleen in dogs subjected to venous constriction In the dogs sacrificed or dead shortly after constriction of the splenic vein, the spleen showed diffuse congestion of the pulp cord and fragile capsule. In 9 dogs that were kept alive for a tong period of time from 23 to 230 days (78 days on the average), splenic capsule was coated densely with perisplenitic fibro sis and proliferation of connective tissue was evident in histological examination. However, extension of fibrous tissue from the trabecle or an increase in fibrous tissue of the pulp cord was not observed. In one dog, the finding suggestive of sinufication of the perifollicular area was observed. Considering the special struc ture of this area, it was difficult to assert that this finding represented new forma tion of the sinus, especially because the cellular elements lining of the sinus wall were absent. In tissue slices examined after splenic perfusion, a reticular structure made of circular and longitudinal fibers was evident in the sinus wall (Fig. 2). Although the structure was occasionally demonstrated even in the normal spleen, it appeared to represent an expansion of reticular mesh of the sinus wall due to increased venous pressure. 3) Histology of post-ischemic spleen The spleens from the dogs which had been subjected to the clamping of the

3 Experimental Splenomegaly II 283 splenic artery were studied by the same technic as in the experiment of venous constriction. Thickening of the splenic capsule was evident in all the dogs, but there was no fibrosis or fine dissociation of the trabecle extending from the capsule into the parenchym (Table 1). In one dog in which arterial clamping for 60 minutes was performed twice and further twice at 60 days' interval, the spleen examined 70 days after the last arterial clamping showed extensive fibrosis of peritrabecular areas. Fibrous networks of the pulp cord were in connection with perivascular fibers, and TABLE 1. Histopathology of spleen following arterial clamping * Perfused with normal saline solution. more or less pronounced thickening and proliferation of pulp cord fibers were present in all of the post-ischemic spleens. It appeared, however, that fibrosis was more pronounced in the spleen after repeated clamping of the artery than after single clamping (Fig. 3). In dogs that survived a long time after clamping of the artery, there was no remarkable change in the cells of the pulp cord and sinus, and proliferation of reticulin fibers or new formation of the sinus was not present. In many dogs sacrificed 30 to 50 days after single clamping of the splenic artery, a large number of erythrocytes were seen in the pulp cord, but there was no remarkable proliferation of the fibrous tissue (Fig. 4). Extension of reticular meshes with accumulation of erythrocytes in pulp cord was also demonstrated in a dog which died one day after clamping of the artery. On the other hand, in the pulp cord of the spleen 30 days after intermittent arterial clampings, stagnation of ery throcytes and increase in fibrous tissue around the sinuses were observed (Fig. 5). 4) Histology of the spleen in. dogs subjected to constriction of splenic vein in addition to arterial clamping In dogs in which venous constriction was produced after a single clamping of the artery for 90 minutes, there was a slight increase of fibrous tissue of the pulp

4 284 T. Sato et al. cord, but there was no essential difference in the histological finding of the spleen whether or not venous constriction was produced after arterial clamping (Table 2). However, in 5 dogs to which venous constriction was applied in addition to repeated clampings of the artery (twice a day for 20 minutes initially and further twice for 20 or 30 minutes), increased fibrosis of the pulp cord was evident and there were areas in which the pulp cord assumed a sinus-like appearance (Fig. 6). Dogs to which venous constriction was applied after clamping of the artery 3 times for 30 minutes each presented fine dissociation of the pulp cord and accumulation of a large number of erythrocytes in the pulp cord. Venous constriction TABLE 2. Histopathology of spleen following arterial clamping and venous constriction * Perfused with normal saline solution. after intermittent clamping of the artery, 4 times for 20 minutes each, brought about relative increase of the sinus due to atrophy of the pulp cord and gave a picture comparable to that of the Banti spleen (Fig. 7). There was also fibrosis around siderotic areas. In some of the dogs of the same group, there was enlargement of pores in the sinus wall, and a direct communication between the pulp cord and sinus was clearly demonstrated (Fig. 8). DISCUSSION The fine vascular structure of the spleen has been studied by many workers. Using the method of vascular reconstruction, Jdger,4 Hueck5 and others observed arterioles terminating with open endings, while Knisely6 described closed connec tions between the terminal arterioles and the sinuses in living mammals. At the present stage, a generally accepted view is that there is no closed connection between the arterial and venous systems in the spleen, and the arterioles open in the pulp cord. We have studied serial sections of the human spleen and arrived at the same conclusion.7 It is also generally accepted that in most of the mammalian species, the arter ioles of the spleen are provided with ellipsoid tissues which consist of reticulum cells and reticulin fibers. There are, however, many different opinions in regard to the function of the ellipsoid tissue. Some investigators think that it serves as a filter, while others hold the opinion that it plays a role in regulating blood flow or

5 Experimental Splenomegaly II 285 checking regurgitation of blood flow. Ebata8 studied the spleen in various pathological conditions and found atrophy of the ellipsoid tissue in atrophied spleen, fine dissociation and thickening with proliferation of reticulin fibers in con gestive spleen and extensive deposition of hemosiderin in the ellipsoid tissue in hemosiderosis. It would seem, therefore, that the ellipsoid tissue undergoes the same histological changes as those of the pulp tissue itself in circulatory disturbance of the spleen. In the present study, relaxation or fine dissociation of the structure of the splen ic cord with accumulation of erythrocytes was demonstrated in the dogs in which the artery bad been clamped once for 90 to 120 minutes. The finding indicates that ischemia lowers the tension of the connective tissue including the reticulin fiber and causes erythrocyte stagnation in the pulp cord. When the clamp is removed and blood flow is restored, a part of erythrocytes stagnating in the pulp cord invade the vascular wall or are trapped in thrombi in the ellipsoid tissues and reticulum cells and finally give rise to hemosiderosis. It is frequently observed in the post-ischemic spleen that fibrosis has developed in the area surrounding hemosiderin deposits, a similar finding is described by McMichael9 in the congestive spleen. It would seem, therefore, that development of fibrosis in the post-ischemic spleen is initiated by extravasation of blood components as a result of impairment of the capillary wall and reticuloendothelial cells, and by forma tion of microthrombi in the reticular mesh of the pulp. The circulatory disturb ance that diffusely involves the terminal vascular areas causes at first thicken ing and atrophy of the pulp cord which is followed by distortion of its structure. Some part of the pulp cord becomes dilated and gives a sinus-like appearance. On the repetition of ischemia, peripheral circulatory disturbance of the spleen is exaggerated and the above-mentioned changes are aggravated. Because of limited cellular proliferation, the post-ischemic spleen tends to un dergo atrophic changes without presenting the picture of sinus hyperplasia as seen in human Banti's spleen. On the other hand, our previous study1 has revealed that the morphological changes caused by transient ischemia increased splenic blood flow. It is therefore assumed that some disturbance in the peripheral circulation or a failure of regulatory function takes place. Although the disturbance of peripheral circulation can be assumed on the basis of the morphological changes of the pulp cord, the mechanism of regulatory function is still unknown. The results of Ebata's study8 and of ours suggest that the ellipsoid tissue or the structure at the arterial terminals in the pulp cord plays a role in regulating splenic peripheral circulation. In dogs in which venous constriction was applied in addition to arterial occlusion, a relative numerical increase and dilatation of the sinus were also demonstrated. There was not, however, an absolute increase in splenic tissue or proliferation of splenic cells. The procedure, therefore, induces morphological changes simultating that in human splenomegaly but does not make the human Banti spleen.

6 286 T. Sato et al. Other workers observed dilatation of the sinus, atrophy of the pulp cord and fibrous proliferation in their attempts at experimental production of splenomegaly. None of them, however, described an increase in splenic cells. Therefore, in order to produce splenomegaly with similar histological findings as those of the Banti spleen, certain preliminary conditions including an increase in splenic cells seem necessary. The histological change in the spleen without enlargement which occasionally observed in patients with liver cirrhosis appears to develop from the condition without splenic cellular proliferation. Extension of fibers in company with hemosiderosis of the pulp cord is often seen in the spleen after either venous constriction or arterial occlusion alone, and is a constant finding after combination of the two procedures. It seems, therefore, that hemosiderosis is a result of not only congestion and extravasation of blood cells, but also of active trapping and deposition of erythrocytes consequent on tissue damage due to ischemia. Although it is admitted that extravasation of erythrocytes around the arterial vessels leads to fibrosis, as has been pointed out by McMichael,9 it seems difficult to interpret all the splenic changes only as the consequence of portal hypertension. The histological study of the perfused spleen revealed that the sinus was surrounded by meshwork of longitudinal and circular fibers. The mesh is more clearly visualized in perfused congestive spleen. It seems likely that elongation of the sinus and enlargement of the pores in sinus wall exerts a direct influence upon the portal system. Furthermore, the circular fibers of the sinus have direct connections with the pulp mesh, and consequently, thickening and extension of reticulin fibers of the pulp cord seem to influence the expansibility of the sinus and pores in sinus wall. On the basis of these findings, it is assumed that the morphological changes characterizing the spleen of Banti's syndrome such as sinus hyperplasia are not brought about by portal hypertension alone but they are produced by combination of various other factors. For example, when there is a tendency to proliferation of reticuloendothelial cells, increase in blood flow or impairment of the regulatory function of splenic peripheral circulation would lead to widening of the pulp cord, which is later subdivided into sinus-like structures. References 1) Sato, T., Suda, Y., Koyama, K., Yamauchi, H. & Yamamoto, K. Attempt at experimental production of splenomegaly. I. Changes of weight and hemodynamics of the spleen consequent on circulatory disturbance. Tohoku J. exp. Med., 1968, 96, ) von Herrath, E. Bau and Funktion der normalen Milz. Walter Do Gruyter & Co. Berlin, ) Koboth, I. Uber das Gitterfasergerust der roter Milzpulpa. Beitr. path. Anat., 1939, 103, ) Jager, E. Milzbau and Kreislaufstorung. Toil II. Virchows Arch. path. Anat., 1937, 299, ) Hueck, W, Uber das Mesenchym, II. Teil. Beitr. path. Anat., 1930, 83,

7 Experimental Splenomegaly II 287 6) Knisely, M. H. Spleen studies I. Anat. Rec., 1936, 65, ) Yamauchi, H. Estimation of blood flow in the Banti spleen on anatomical basis. Tohoku J. exp. Med., 1968, 95, ) Ebata, T. Histological picture of Schweigger-Seidel ellipsoid of human spleen in various diseases. Fukuoka-lshi (Jap.), 1956, 47, ) McMichael, J. The pathology of hepatolienal fibrosis. J. Path. Bact., 1934, 39,

8 288 T. Sato et al. Fig. 1. Splenic histology of a control dog. Sinuses are seen scattered in the reticular meshes of the pulp cord. Perfused spleen with silver impregnation (10 ~25). Fig. 2. Splenic histology following venous constriction. Note the fine structure of the sinus wall. Perfused with saline. Silver impregnation and H. E. stain (10 ~25). Fig. 3. Splenic histology following arterial clamping (20min ~3). Thickening and proliferation of reticulin fibers of the pulp cord are seen with hemo siderin deposit. Perfused with saline. Silver impregnation and H. E. stain (10 ~25). Fig. 4. Splenic histology following arterial clamping (120min). Red cells are extravasating in the pulp cord with little tendency to fibrosis. Perfused with saline. Silver impregnation and H. E. stain (10 ~25).

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10 290 T. Sato et al. Fig. 5. Splenic histology following arterial clamping (20min, 3 times). Stagnation of red cells are seen among pulp cords with thickening and extension of reticulin fibers. Perfused spleen. Silver impregnation and H. E. stain (10 ~25). Fig. 6. Splenic histology following arterial clamping and venous constriction. Dilated spots are noticeable in the net work of pulp cord simulating the structure of sinuses. Perfused with saline. Silver impregnation and H. E. stain (10 ~25). Fig. 7. Splenic histology following repeated arterial clampings and venous constriction. Narrowing of pulp cord due to fibrosis is evident with dilatation of sinuses. Sinuses are seen in increasing number. Perfused with saline. Silver impregnation and H. E. stain (10 ~25). Fig. 8. Splenic histology following repeated arterial clampings and venous constriction. Direct communication between the sinus and the pulp cord is well noticeable. Per fused with saline. Silver impregnation and H. E. stain (10 ~25).

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