Tohoku J. Exper. Med., 1961, 76, 374-387

On the Adipose Tissue in Visceroptosis

By

Susumu Shibata

From the Department of Surgery (Prof. T. M a k i and Prof. K. 0 h u c h i) and from the Department of Anatomy of Prof. V. Mizuhira, School of Medicine, Hirosaki University, Hirosaki (Received for publication, December 5, 1961)

INTRODUCTION Visceroptosismay be usually characterized by the ptosis of major abdominal viscera such as the stomach and intestine, although this term is used to indicate all abdominalvisceral sagging. Still the weakeningin supporting tissues of these organs is generally consideredto be one of the main etiologicalfactors. On this point of view, elucidation of the changes of the supporting tissues in this disease seems essential in understanding its pathophysiology. However, little work has been carried out on this problem. The author has selected the subcutaneous adipose tissue of visceroptotic patients and examined its changesspecially in fat cells and connective tissue fibers for the purpose to clarify the peculiarity of the supportingtissues in this disease. The results obtained in author's examina tion are reported in this paper.

MATERIALS Thirty-one patients with visceroptosis, comprising 15 males and 16 females, who had been diagnosed and admitted to our surgical clinic during the period between June, 1960 andFebruary, 1961, were examined. The patients ranged from 16 to 54 years in age, the majoritywere in their thirties. The abdominal subcutaneous adipose tissue of the visceroptotic patients biopsised at the median and right or left pararectal incisions for abdominoplastyl1), right hemicolectomy and sigmoidectomy, etc. were used for histological examinations. The same tissue biopsised at laparotomies for acute , diseases and gastric diseases in 30 non-ptotic patients comprising 15 males, and 15females, who were in good nutritional conditions served as the control.

EXPERIMENTAL

Macroscopically the thickness of the subcutaneous fat was measured by the

柴 田晋 374 Adipose Tissue in Visceroptosis 375 skinfold method. Microscopically the size of the fat cells and connective tissue fibers in the adipose tissue and the staining reactions of lipids in the fat cells were examined. As the collagen fibers in the adipose tissue show remarkably various forms intermediate between the extreme changes in severely ptotic cases and almost normal appearance in slight ptotic cases, the typical extreme changes in visceroptotic cases was chiefly compared with the control. 1) The thickness of the subcutaneous fat To estimate the relative body fatness, the skinfold thickness was measured at three points, which have been described as the most distinguished indicators of total body fatness ;2)3) (1) the point 1 cm horizontally to the right from the umbilicus (), (2) the mid point on the stretch side between the right acromion and the olecranon (Arm), (3) the point on the back at the inferior scapular angle (Back). Key's skinfold calipers2) with round contact plates of 25 mm2, setting the spring tension to10 mg/mm2 were used. The contact plates were placed at about 1 cm from the point at which skinfold is held up by the thumb and the other fingers longitudinally to the body axis. Tables ‡T and ‡U represent the double skin-plus-fat thickness in these points of visceroptotic patients and control cases. In visceroptotic patients, the value ranged from 3.5 to 7.5 mm with an average of 5.2 mm at the abdomen, from 2.5 to 6.0 mm with an average of 4.2 mm at the arm, and from 4.0 to 8.5 mm with an average of 6.0 mm at the back in the male and in the female from 6.0 to 11.0 mm with an average of

8.7 mm at the abdomen, from 7.0 to 12.5 mm with an average of 9.0 mm at the arm, from 6.5 to 13.5 mm with an average of 9.2 mm at the back. In control

TABLE ‡T. Skinfold Thickness in Visceroptotic Patients (mm.) 376 S. Shibata

TABLE ‡U. Skinfold Thickness in Control Cases (mm.)

cases, the value ranged from 4.5 to 10.5 mm with an average of 7.5 mm at the abdomen, from 4.0 to 9.5 mm with an average of 6.7 mm at the arm, from 5.0 to 10.0 mm with an average of 7.3 mm at the back in the male and female from 10.0 to 17.0 mm with an average of 14.3 mm at the abdomen, from 9.0 to 16.5 mm with an average of 13.1 mm at the arm, from 9.5 to 15.0mm with an average of 12.7 mm at the back. In comparison of these values with visceroptotic patients and control cases, significant decreases were noted in all three points in visceroptotic cases as shown Fig. 1. Further comparison of the values for visceroptotic patients and for

Fig. 1. Skinfold thickness of visceroptotic patients and control cases. Shaded rectangles: Visceroptotic patients. White recutangles: Controls, Adipose Tissue in Visceroptosis 377 normal subjects reported by Aso,3) (in male, abdomen 11.2 mm arm 7.5 mm, back 11.3 mm, in female, abdomen 13.8 mm, arm 14.8 mm, back 15.8mm) which were measured by the preceding method, marked decreases in skinfold thickness ranging from 40 to 50 per cent were revealed invisceroptotic patients at the three points. As Aso's values concerns the people of Kyushu, the south end of Japan, further comparison was done with the values reported by Takamatsu4) for the people living in the Hirosaki district (in male, abdomen 6.54 mm, arm 6.20 mm, back 8.87 mm, and in female abdomen 12.42 mm, arm 15.36 mm, back 11. 97 mm), the north end of Japan, using the esthesiometer. In this comparison, also, slight to moderate decreases in the skinfold thickness of visceroptotic cases are noted in the three points, though there are some differences in age construction and measuring technique. Skinfold thickness is also larger in female than in males in visceroptotic subjects, as generally accepted in normal persons. 2) The diameter of fat cells The method of measuring was essentially similarto that used by Reh,5) a small piece of adipose tissue fixed in 10 per cent neutral formalin solution was cut into smaller pieces on a slide-glass using two dissecting needles under microscopic observation until the fat cells were laid as a thin film. A cover-glass was placed on the film of the cells and sealed with glycerin to prevent drying of the specimen. The diameters of 100 successive fat cells were measured with an ocular micrometer.

The value obtained from the fat cells of the visceroptotic patients were in the range of 42.lƒÊ to 91.2 ƒÊ with an average of 63.2 ƒÊ as shown in Table ‡V.

In control cases, the diameters of the fat cells ranged from 59.3ƒÊ to 124.6,ƒÊ with an average of 92.5ƒÊ as given in Table ‡W, showing a significant difference compared with the value of the visceroptotic patients. According to Mori and others6) the diameters of normal fat cells is about 50-80ƒÊ; while Seki7) reported that the diameter is usually over 100ƒÊ, and Schaffer,8) between 40 to 120ƒÊ. Reh5) who measured with the precedingmethod stated that the normal value ranged between 70 to 110ƒÊ. Though the value differs among the authors, in the comparison ofvisceroptotic patients and Reh's value measured with the same

TABLE ‡V. Diameters of Fat Cells of Visceroptotic Patients (ƒÊ)

Average 63.2 ƒÊ 378 S. Shibata

TABLE ‡W. Diameters of Fat Cells of Control Cases (ƒÊ)

Average 92.5 ƒÊ

method, the diameters of fat cells of visceroptotic patients were generally smaller than the normal cells reported by Reh, and muchsmaller cells with diameters around 40ƒÊ were also observed. Only in one case, the fat cells was larger than

the normal mean of 90ƒÊ and the average was below the normal lower limit. Of

course, attention must be paid to that Reh's value was obtained in German

subjects and our value in Japanese patients.

If we compare the volume of the fat cells, the volume of average cell with a diameter of 63.2ƒÊwas calculated as 127,398.5ƒÊ3 while that of the cells of the

control cases with diameters of 92.5ƒÊ, 4l3,709.5ƒÊ3. In other words, the fat

cells of patients with visceroptosis have the cubic volume approximately equal to one-third of the fat cells of the control cases. 3) Connective tissue fibers in the adipose tissue Biopsised adipose tissue was fixed in 10 per cent formalin and kept for two weeks or more in 10 per cent neutral formalin containingresorcin in the rate of one per cent. To demonstrate the connective tissue fibers, these were embedded in paraffin, cut into serial sections of the thickness between 10 and 15 microns and then stained with Lillie's and Pap's silver method. In brief it may be stated that adipose tissue is divided into many lobules by the interlobular connective tissue which is abundant in the collagen fibers containing blood vessels and nerves, and each fat cells, furthermore were wrapped with a network of fine argyrophil fibers. The difference in these fibers between ptotic patients and control cases were as follows : In control cases the collagen fibers in the interlobular connective tissue were abundant and intensely blackened by this method, while in visceroptotic cases the collagen fibers in the interlobular connective tissue were scarce and less blackened and the interlobular boundaries appear obscure. In some visceroptotic cases the vessels and nerves werelocated between the lobules almost nakedly without being covered with connective tissue fibers. The membrane around the fat cells of Adipose Tissue in Visceroptosis 379 visceroptotic patients is thin, weakly blackened and easily rupturable, while that of the normal fat cell is thick and markedly blackened by this method. The network of argylophile fibers around the fat cells of visceroptotic patients is coarser and argylophile fibers are thinner than that of thecontrol cases. Fur thermore, the control cases are characterized with relative thick fibers running in the network branching off at various sites. These changes in the connective tissue fibers in the visceroptotic patients areschematized in Fig. 2 ; the fibrous component of interlobular connective tissue is scarce, the membrane covering the fat cells arethin, and the network of argylophile fibers around the fat cell is coarse and thin.

Fig. 2. Schematic presentation of the connective tissue fibers in the adipose tissue.

4) The staining reactions of the lipids in fat cells

For the study of lipids biopsised adipose tissue fixed in Baker's calcium formalin were embedded in 10 per cent gelatin solution and sectioned into 15-20

microns by the freezing method and then stained with Sudan ‡V., Sudan black

B and Nile blue sulfate. Lipid droplets in the fat cells of both visceroptotic and

control cases took a dark blue color with SudanBlack B and an orange color

with Sudan ‡V. With Nile blue sulphate the lipid dropletsof fat cells of both

visceroptotic and control cases took a pink color which was regarded as specific for

neutral fats. Thus no remarkable difference between the fat cells of visceroptotic

patients and control cases was revealed by the preceding three staining methods. Still in observation in frozen sections the fat cells are smaller and the capsules of

the cells is thinner and more deformation is seen in the visceroptotic cases than

the control cases.

DISCUSSION AND CONCLUSION

In the adipose tissue, connective tissue fibersare abundantly seen and these 380 S. Shibata divide the adipose tissue into many fat lobules. The fat cells in the lobules are also surrounded with fine connective tissue fibers. According to Laubinger9) the functions of thesefibers in the adipose tissue is to connectthe fat cells with one another. In otherwords, the collagen fibers between the fatcells cross on the surface of the cells, tangle each other and produce close connection between the fat cells forming a dense network. Thus a gathering of the fat cells is changed into an independent tissue which functions as one of the supporting tissues of various organs. Any compression or pulling to the adipose tissue results in transformation of thefat lobules and fat cells. The round fat cells become flat and oval, but the change in the shape of cells does not reach to the extreme because of the shelter of the network of collagen fibers. With the removal of the external force the shape of the fat cell returns to the original by the elasticity of the collagen fiber network and fat cell membrane. The presence of the collagen fiber network keeps the relative location of each round fat cell to another constant in the adipose tissue, unlikely the balls in a ball-bearing. Thus the collagen fibers in the adipose tissue play an important role to perform the function of the adipose tissue as one of the supporting tissue in the body. The collagen fibers in the adipose tissue of patients with visceroptosis are thinner and fewer in number than normal both in the interlobular connective tissue and around the fat cells. The lack of the collagen fibers as well as the thinning of the cell membrane results in the loss of resistance of the adipose tissue against compression or pulling, and the loss of restorative capacity of the fattissue when the force is removed. This fact is also observed macroscopically at the operative wound of a visceroptotic patient undergoing laparotomies. Their adipose tissue at the operative wound appears to be less in amount, less elastic, and extremely asthenic compared with that of the control individuals. In addition, the volume of fat cells of visceroptotic patients is about one-third of the control. As a result, even if the collagen fibers and thickness of the cell membrane are assumed to be approximately equal to the control, the force given to the adipose tissue may cause more extensive deformation of the fatcells of visceroptosis than to the normal fat cells. The changes in collagen fibers, the decrease in the size of the cell and the thinning of the fat cell membrane all accumulate and weaken the resistance of the adipose tissue against exogenous forces, and accordingly the tissue loses elasticity and softness. The ability of the adipose tissue to fix or support abdominal organs is considered to be reduced markedly in this condition. The thickness of the subcutaneous fat tissue measured on the abdominal wall, upper arm. and back, which is thought to be good indicators ofthe amount of the fat tissue of the total body, is reduced markedly in the visceroptotic patients. So the amount of subcutaneous fat tissue on other parts of the body is considered to diminish parallel. According to Vierordt10) the amount of internal depot fat situated in the mesenterium, around the and kidney, which is Adipose Tissue in Visceroptosis 381 directly related with the fixation of abdominalorgans, is about one half of the totalfat in the body or about the same as that of the subcutaneous fat in man. Pitts11) investigated in detail the amounts of fat in guinea-pigs and reported that except for the immature individuals the fraction of the total body fat in subcutaneous and internal depcts isconstant at all levels of body fatness. Consequently, the decrease in internal depot fat in visceroptotic patients may be markedly proportional to the decrease of subcutaneous adipose tissue. Coffey12) reported that normal fat paddings of the abdominal wall, the psoas shelf, the kidney, the mesentery and the omentum aid in keeping the abdominal viscera in their normal positions, above psoas outlet, out of pelvis, and with the absorption of fat pad, the abdominal cavity and pelvic outlet enlarged, the viscera dropped into the pelvis. According to Bockus13), an abdominal wall of normal tonusoffers resistence to forward and downward displacement of the abdominal viscera and retroperitoneal mesenteric and omental fat undoubtedly assists in keeping the abdominal organs in their normal positions. Martin" described that defects in the development of fat layers and deposits are the strong factors in etiology of visceral prolapse. Other authors15)16) also reported that the changes inadipose tissue are one of the main etiologic factors of visceral ptosis and increasing of visceral mobility. The present results support the views of these authors; it may be said thatremarkable changes of adipose tissue in visceroptotic patients obviously involve the ptosis of abdominal viscera. Inquiring into the factors causing the remarkable change in connective tissue fibers of visceroptotic patients, it is a well known fact that connective tissue fibers is changed by the locality of the site as well as the general and constitutional conditions of the body. The collagen fibers at a site of the bodydevelop as a rule along the direction most advantageous to counteract the tensions acting to the site. For example, in the tendon in which the forces work in one definite direc tion, collagen fibers develop in parallel to a single direction. There is good development of the fibers when a proper amount of forces is acting. Electro myographic studies in our clinic") on the abdominal muscles of visceroptotic patients have revealed that the internal oblique muscle has an antigravity function which is important in visceral support. In severe cases of visceroptosis, a downward displacement of intestinal mass into the pelvic cavity is followed with a prolapse of the stomach and the colon which acts as a load on the lower part of the anterior abdominal wall and elicites antigravity discharge of action currents from the internal oblique muscle fibers. When a second diaphragm is produced at the height of the promontorium by abdominoplasty1) and the is raised to its original abdominal cavity, the antigravity discharge ac tion of theabdominal muscles is reduced. These facts suggests that the load on the muscles and othertissue of the abdominal wall caused by theprolapse of abdominal organs is much marked in the patients with visceroptosis. Such a 382 S. Shibata

localized load naturally causes changes on the collagen fibers in the subcutaneous fat tissue of the abdominal wall. On the other hand, the influence of tension from the fat cell to the surrounding nets of collagen fibers must be taken into consideration. Muraiti18) reported that the collagen fibers around the fat cell became coarse when an animal was emaciated experimentally. If the tension exerted by the fat cell to the surrounding collagen fibers diminishes with the reduction of lipids in the cell, changes in the fibers such as atrophy or diminishment would inevitably ensue. Recent electron microscopic studies19)20)21) revealed that the membrane of the fat cell which appearsas a single layer under light microscopic observations consists of three layers : The dense plasma membrane, the light interspace and the less dense outer membranous layer and close contract with the outer membranous layer, fine fibrils measuring 200 to1,000A in width, lie on its outer surface. Such studies with the electron microscope will be necessary to clarify the ultrastructure of the membrane of the fat cells and collagen fibers in visceroptotic patients. At present our studies indicated that these changes in connective tissue fibers were the results of general and/or local affections of visceroptosis. The fat cell contains a large amount of lipids which is a nonconductor of heat, accordinglythe adipose tissue plays an important role in heat preservation mechanismof the body. In visceroptoticpatients, the reduction of subcutaneous and internal depot fat may cause lowering in intra-abdominal temperature. Salmon22)reported that hypothermia, local or systemic, reduced both amplitude and frequency of contraction, secretion and absorption of thebowel. Niijima23) reported also that hypothermia induced decreases the blood circulation of the mesenterium. The changes in adipose tissue may concern the pathophysiology of the abdominal organs in visceroptosis in lowering the intra-abdominal tem perature and decreasing intestinal motility and absorption, etc.

SUMMARY

The changes in the adipose tissue of the visceroptotic patients were examined and the following results were obtained . 1. The thickness of the subcutaneous adipose tissue of visceroptotic patients is markedly reduced compared with that of the control cases . 2. The diameter of the fat cells of visceroptotic patients is also smaller than that of the control cases. 3. The connective tissue fibers in the adipose tissue are thin and scarce in the interlobular connective tissue and around the fat cells in these nati ents. From these results is may be concluded that thefunction of the adip ose ti ssue as a supporting tissue of the abdominal organs in visceropt otic patients is decreased, and it contributes to the ptosis and displacement of th e adbominal organs. The quantitative reduction of adipose tissue may also ind uce lowering Adipose Tissue in Visceroptosis 383 in the intra-abdominal temperature and affect the pathophysiology of abdominal organs of visceroptotic patients .

References 1) Maki, T. , Operation (Jap.), 1957, 11, 1. 2) Keys, A. & Brozek, J., Physiol. Rev., 1953, 33, 245. 3) Aso, M., Acta Medica (Jan.). 1957, 27. 1609. 4) Takamatsu, I., Hirosaki Jed. J. (Jap.), 1955, 6, 277. 5) Reh, H., Virchows Arch., 1953, 324, 234. 6) Mori, 0., Hirasawa, K., Ogawa, T. & Mori, M., Anatomy ‡W (Jap.), Kanehara Shuppan, Tokyo, 1954. 7) Seki, S., Histology (Jap.), Kyorin Shoin, Tokyo, 1954. 8) Scaffer, J., Mollendorffs Handbuch der mikroskopischen Anatomic des Menschen, 11/2, Julius Springer, Berlin, 1930. 9) Laubinger, W., Morphol. Jb., 1938, 81, 230. 10) Vierordt, H., Anatomische physiologische and physikalische Daten and Tabellen zurn Gebrauche fiir Mediziner. Fischer, Jena, 1906, cited by Keys, A. & Brozek, J. 2). 11) Pitts. G.C.. Am. J. Phvsiol., 1956, 185, 41. 12) Coffe, R.C. , Gastroenteroptosis, New York,1923. cited by Speisman, M.G.15). 13) Bockus, H.L. et al., ‡V, W.B. Saunders, Philadelphia, 1953. 14) Martin, F.H. Surg. Gynec. & Obstetr., 1908,7, 638. 15) Spiesman, M.G., Rev. Gastroenterol., 1940, 7, 218. 16) Gray, H.T., Lancet, 1926, 210, 381. 17) Ono, K., Tohoku J. Exp. ill"ed., 1958, 68, 355. 18) Muraiti, Y., Bull. Tokyo Med. & Dent. Univ., 1960, 7, 73. 19) Wassermann, F. & McDonald, T.F., Zschr. Zellforsch. 1960, 52, 778. 20) Chase, W.H., Arch. Path., 1959, 67, 550. 21) Imaeda, T., Arch. histol. jap., 1959, 18, 57. 22) Salmon, P. A., Griffen, W.O. Jr., Jenson, C.B. & Wangensteen, O.H., Surgery, 1959, 46. 873. 23) Xiijima, S., Nihon Geka Gakkai Zassi (Jap.), 1961, 62, 102. 384 S. Shibata

Plate 1, 3. Adipose tissue of visceroptotic patient. Fibrous component of in terlobular connective tissue is scarce. Capillary is nakedly seen in interlobular space in Plate 3. Pap's silver staining. 10 X. Plate 2, 4. Similar tissue of control case. Fibrous component of interlobular connective tissue is abundant and markedly blackened. Pap's silver staining. 10 X. Adipose Tissue in Visceroptosis 385

Plate 5. Similar tissue of visceroptotic patient. Capsules of fat cells are thin and moredeformation of fat cells is seen. Pap's silver staining. 50X. Plate 6. Similar tissue of control case. Capsules of fat cells are thick and boundary of fat lobules are definite. Pap's silvier staining. 50X. Plate 7. Fat cells o visceroptotic patient. Argyrophil fibers around fat cells are thin and scarce. Pap's silver staining. 200X. Plate 8. Fat cells of control case. Argyrophil fibers around fat cells are abound ant and fat cells are connected with each other closely. Pap's silver staining. 200X. SRR S. Shibata

Plate 9. Fat cells of visceroptotic patient. Sudan ‡V, Staining. 50X. Plate 10. Fat cells of control case. Sudan ‡V, staining. 5OX. Plate 11. Fact cells of visceroptotic patient. Sudan black B. staining. 50X. Plate 12. Fat cells of control case. Sudan black B. staining. 50X. Adipose Tissue in Visceroptosis 387

Plate 13. Fat cells of visceroptotic patient. Nile blue sulfate staining. 50X. Plate 14. Fat cells of control case. Nile blue sulfate staining. 50X.

In plate 9-14, no remarkable difference in the staining reactions is revealed, still in these observations in frozen sections the fat cells are smaller and more deformation is seen in visceroptotic cases than the control.