Demetris, AJ

Ishii, M

Mori, S

Oguma, S

Starzl, TE

Takahas, T

Takemura, M

Name not available

Prebiliary vascular diseases in rejected livers; computer-aided three-dimensional reconstruction and morphometry

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Peri biliary Vascular Diseases in Rejected Livers; Computer-Aided Three-Dimensional Reconstruction and Morphometry M. Takemura, S. Oguma, S. Mori, M. Ishii, T.E. Starzl, A.J. Demetris. and T. Takahasi I N recent years more and more attention has been paid to the so-called vanishing bile duct syndrome, a progres-sive loss of intrahepatic bile ducts observed in various liver diseases, including chronic rejection of allografts.I-5 As yet, the factors responsible for causing this condition still remain mostly unknown. In a series of analysis on trans-planted livers,6.7 we found that in failed grafts. vascular lesions particularly of hepatic arteries occur and underlie the bile duct damages, suggesting ischemia to be an important causative factor. However, little is unknown about the normal vascular supply of small bile ducts; the anatomical observations made so far only dealt with peri-ductal vessels of rodents,8-11 not of man. Not disclosed have been the nature and the predisposed site of such vascular injuries and their anatomical correlation with the ductular diseases. In the present report, we describe our recent studies on the 3-D microstructure of normal peribiliary small vessels and how and to what extent they could undergo obstruc-tive changes in chronic rejection, on which basis to shed light on the blood-vessel-dependent aspect of bile duct injuries in transplanted livers. Bile duct loss will be shown closely related with diseases of supplying vessels in the peribiliary plexus, reflecting the significance of ischemia as a co-factor underlying chronic rejection. Analysis was further extended to hepatic lesions in patients with primary biliary cirrhosis (PBC); this is to understand what this condition has in common with chronic rejection. since there appear to be similarities between the two diseases, as long as the microscopic appearance of the li\'er is con-cerned. MATERiAlS AND METHODS Studied were four normal autopsy livers and five surgical speci-mens of liver, the laller having been obtained from two patients with failed allografts and three with PBC. Clinical data of these patients are summarized in Table I. Two of the four normal livers were fixed by perfusing ",ith 2.5% glutaraldehyde via the hepatic artery and simultaneously via the portal vein: the other specimens. including the remaining two autopsy livers. were simply fixed by immersion in 1O'k formalde-hyde. All the specimens were embedded in celloidin-paraffin. Three hundred to six hundred serial sections were prepared from each block at 3 JLm thickness and stained with Elastica-Goldner stain. Three-D reconstruction of small bile ducts and supplying ves-sels was performed with the aid of a computer system (PAS-310) specially designed for tbe study of 3-D micrOSlnJcture at the Department of Pathology, the Institute for Tuberculosis and Cancer, Tohoku Universily.12 Using a profile projector (Reichert. model Visopan), a portal tract in the first section from a set of Tab6e 1. Cases Examined -Material Case Age Sex 19 F Chrcnc Rejection Transplanted for Cirrhosis 2 27 M Chronic Rejection Transplanted for CAH 3 58 F PBC (Stage I + II) 4 49 F PBC (Stage I + II) 5 49 F PBC (Stage II) 6 19 M Normal liver (autopsy) 7 40 F NormaJ liver (autopsy) 8 59 M Normal liver (autopsy) 9 61 M Normal liver (autopsy) PBC. primary biliary cirrhcss: CAH. chronic active heoatitis. serial sections was pfl:!eCted onto a sheet of tracing paper at x380. and the contoun of bile ducts and blood vessels were delineated as faithfully as possible. The picture was placed on a digitizer. and the contoors of bile ducts and blood vessels were input into the computer ,y tnKing with a cursor. The process was repeated one after anroer until the last s~"tion was input; then the computer was read:- to integrate in its display a 3-D picture rotated at any assigned mgle around the x. )'. and z axes. Different structures such as bile jucts. hepatic arteries. portal veins. and periductal small vessels .... ere expressed in different colors. In the 3·D graphics. the los5 of intrahepatic ducts was anatomically corrected with that of ;'O!riductal small vessels. This correlation was further substantia:~ by morphometr). The serial sections from the failed liver all...-wafts were entirely scanned to show the difference in the dens!,- of periductaI bkxxl vessels between injured and uninjured d::cts. by counting their number in each step of serial sections. The-><: results were classified into three groups (D < 40 JLm. 40 lUll < 0 < 80 p.m. and 80 lUll < D) based on the sizes of the bile ducts subjected to observation. From the Departmert of Pathology (M.T _ T.T.), The Research Institute for Tuberculoss and Cancer. Tohoku University. Second Department of Surger) (M.T., 5.0., S.M.), Third Department of Medicine (M.I.). TohoIc.. University Medical School, Sendai, Japan; and Department of ~ (T.E.S.) and Department of Pathology (A-J.D.), University of Pittsburgh. Pittsburgh. Pennsylvania. Address reprint reQl,eStS 10 M. Takemlr.l, Department of Pa-thology, The Researctl Instilute for Tuberculosis and Cancer, Tohuku University. 4-' Seiryomac:hi. Aobaku. Sendai, Japan 980. e 1991 by AppIetor & Lange 0041-13451911$3.00 ,,0 Transplantation Proceedings, Vol 23, No 1 (FebfUary), 1991: pp 1409-1412 1409 ",,.--- ------- ----1410 a b c Fig 1. Computer-aided 3-D pictures of normal perfbiliary mi-crovasculature showing (a) the alterent system; (b) the draining system; (c) a schema showing the basic pattern of periductal vasculature. BD, bile duct; AA, accompanying artery; CA, commu-nicating arteriole; HA. larger hepatic artery; PV, portal vein; DV, draining venule. TAKEMURA, OGUMA, MORI ET AL RESULTS Normal Microvasculature Supplying the Intrahepatic Bile Ducts Fig la and Ib are computer-assisted 3-D pictures from a normal liver. showing a septal bile duct, about 80 JLm in diameter. and peribiliary blood vessels. We confirmed that the basic pattern of vasculature is the same in more peripheral ducts. including the interlobular ducts. In Fig la. portal veins were omitted from the graphic presenta-tion for the sake of better perspective of arterioles and capillaries. A dense capillary plexus. the peribiliary capil-lary network. is demonstrated surrounding a duct. Basi-cally. this network is supplied by arterioles branching from the arteries that run parallel with the ducts. We call the latter the accompanying arteries (AA). and we call the former the communicating arterioles (CAl. Usually. other arterioles are found dividing from AA and directly leading to sinusoids, without having any contact with the peribil-iary network. In Fig I b. the peri biliary network is shown drained into sub-branches of portal veins that surround the duct at a distance; these correspond to what has been called the "internal roots" of the portal system. The network also has a small number of venules that directly lead to sinusoids independently of the "internal roots." In Fig 1 c. the above pattern of vascular supply for a septal (or interlobular) duct is schematically presented. Probably this may be the first demonstration of the peribiliary vascula-ture of man. of which previous knowledge has been more or less partial. Of the result, special weight should be given 10 the finding thaI il is the communicating arterioles (CA) that finally supply the peribiJiary network. Changes of Microvasculature and Their Relation to Ductal Injuries In the failed allografts (Casel. 2), severe obstructive arte-riopathy proved to involve various levels of small arteries. including not only the accompanying arteries (AA) but the communicating arterioles (CA) around and in the peribil-iary network (Fig 2a.b). In these, besides intimal thicken-ing due to proliferating foam cells, the media were shown destroyed with infiltrating macrophages. Fig 3 is a 3-D Fig 2. Severe obstructive arte-riopathies in (a) an accompany-ing artery and (b) a prOximal part of a corrunmicating arteriole. Elastica·Goldner stain, X 260. PERIBILIARY VASCULAR DISEASES Fig 3. A 3-~ picture of peribiliary vessels from a failed graft (Case 1). The interlobular duct has undergone mar1led atrophy in the upper '.6. where CAs are destroyed and periductal capillaries are mostly missing. picture from the failed graft (Casel) visualizing the changes of small ducts and peribiliary microvasculature. The upper one third of the interlobular duct has undergone marked atrophy. where communicating arterioles (CA) are no longer found leading to the peribiliary plexus. Correspond-ingly, the plexus in this region has been severely destroyed with significantly reduced density of capillaries. The con-crete histologic appearances of its phenomenon are shown in Fig 4a-<:. Therefore. it appears likely that the severer the arteriolar injuries and loss of peribiliary capillaries. the more marked the ductal damage. Also in the livers of PBC. there was a similar relation-ship between vascular and ductal injuries in the portal tracts where loss of CAs and capillaries was remarkable around the inflamed ducts. The vascular injuries appeared more remarkable when there was granulomatous reaction of epithelioid cells. However. there was an unmistakable difference from the allografts. In PBC. the AAs remained uninjured. even in the areas with fulminant ductal lesions. It appears that in this disease, the vessels are involved Fig 4. (8) An intertobulN duct with signs of marked atrophy (b) and (c) A communicating arteri-ole in b dwindles and disappears (c) after several steps of serial sections. 1411 secondarily in the inflammatory process taking place in the surroundings, whereas in chronic rejection. arteriopathies are attributable not only to periportal inflammation but to other factors, including immune reaction against the small vessels themselves due to proximal arterial stenosis. Morphometry The number (Nv) of vessels around a duct, including capillaries and CAs. was counted at 60 ductular segments. These were sampled so as to contain each 30 segments with and without atrophy. In addition. another 30 ducts were sampled from PBC and control cases, respectively. In making statistical treatments of the data. the ducts were classified into three classes according to the external diameter D. The result is shown in Fig 5. In the failed allografts. Nvs were markedly low around segmental atro-phic ducts for the ranges of less than 40 JLm and between 40 JLIll and 80 JLm. On the other hand. non-atrophic ducts showed moderately reduced Nvs for the same ranges. A similar result was obtained from the PBC. The differences proved to be significant by Student's I test. implying a correlation between the ductal and vascular damages. DISCUSSION It has been known since the previous century II that the intrahepatic bile ducts are equipped with a peribiliary vascular network that is supplied by hepatic arterial twigs and drained into sinusoids by the .. internal roots," an alternative pathway of the terminal portal vein. This was followed by many studies. most of Which, however. were focused only on whether and to what degree the network functions as an arterio-portal shunt. Moreover, these stud-ies were done on rodent livers, not on human livers. except for the observations by Mitra8 and Murakami9 ; the former was performed on materials intravascularly injected with india ink, and the latter on corrosion cast of hepatic vessels using the SEM. However. these techniques only allow one to visualize the vascular contours, While the surrounding structures. particularly the bile ducts, are left unvisualized in the void spaces. In this respect. a computer-assisted 3-D 1412 0 t Group) Normal oorRI ·'.1· (0<40"",) ChronIc rejIcIon, non-atrophlc ··1-- . . ~ . ~ .. PBC, Iri"fnd .. ., · .,...., .. -Group • NonnaI oorRI . -... (40"",<0<80jll!1) ChronIc rejecIon, noo-atrophlc • ••• .. . ~ ...... PBC, Iri"fnd . . . · .,...., . ,. Grouplll NormaICOI'IIItII 180"",<0) CIvonIc rejeetion. non-atrophIc • . aIrophIc PBC, Iri"fnd · ~ •••• reconstruction appears to be a technique indispensable for the studies of microvasculature. The technique allowed us to establish an overall view of the vasculature supplying and draining the peribiliary network in an integrated schema. Worthy of special attention may be the presence of the communicating arterioles that have not been described in previous studies. These. periodically dividing off from the mother artery (AA) at intervals of 100 ~m to 200 ,.un, appear especially susceptible to injuries because of their fragile structure. thus bringing ischemia to ducts and ductules. Also. the draining system can be affected by chronically persisting periportal inftammation. The block-ade of venules by insults from within or without will further contribute to the impediment of peribiliary blood flow. The basic anatomy of the normal peribiliary vasculature. once established. greatly helps understand the mechanism of "vanishing bile duct syndrome." Oguma et alb., re-ported that in chronically rejected livers, there is a corre-lation of duct loss and the damage of hepatic arterial branches; the present report is an extension of that study where the observation was rather confined to AAs, nol covering the whole terminal vasculature. In the present 3·D reconstructions, obstructive vascular diseases were shown involving more peripheral arterioles as well. includ-ing the CAs. Moreover. the loss of ducts and peribiliary capillaries proved to be exactly in the areas supplied by the diseased CAs. as shown in both 3-D pictures and mor· phometry, which strongly suggests that. at least partially, ischemia is responsible for causing these injuries. On the other hand, no attention has been paid to the bile . • 1D . • • .... . . . . ••• ., . TAKEMURA. OGUMA, MORI ET AL Fig 5. The number (Nv) of small vessels around a duct. For explanation. see text. duct loss in terms of ischemia in PBC. The association of duct loss with the loss of CAs and peribiliary capillaries again raised the possibility of ischemia as a significant factor. However. in PBC, peribiliary vascular diseases appear to be secondary to immune responses against ducts. Much has yet to be studied about the significance of vascular factors involved in the rejection of transplanted organs. We think. however. that the present study has provided a possible breakthrough into the pathology of rejection of transplanted organs. We are looking forward to gaining more insight into this aspect of organ transplan-tation. REFERENCES I. Sherlock S: Lancet i :493. 1987 2. Ludwig J: Semin Liver Dis 7:293. 198'" 3. Snover DC. Freese OK. Sharp HL. Bloomer JR. Najarian JS. Ascher NL: Am J Pathol II: I. 1987 4. Vierling JM. Fennel RH. Baits KP. Peoons 10. Krom RAF: Hepatology 5:1076. 1985 5. Ludwig J. Wiesner RH. Baits KP. Perkins JO. Krom RAF: Hepatology 7:476. 1987 6. Oguma S. Zerbe T. Banner B. Belle S. Starzl TE. Demetris AJ: Transplant Proc 21:2203. 1989 7. Oguma S. Belle S. Starzl TE. Demetris AJ: Hepatology 9:204.1989 8. Milra SK: J Anal 100:651. 1966 9. Murakami T. Itoshima T. Shimada Y: Arch Histol Cytol 37:245. 1974 10. Yaegashi H. Takahashi T. Kawasaki M: J Microsc 146:55. 1987 II. Kiernan F: Philos Trans R Soc Lond 711, 1833 

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Prebiliary vascular diseases in rejected livers; computer-aided three-dimensional reconstruction and morphometry

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