Summary: A three-dimensional (3-D) computer assisted reconstruction of the biliary tract was performed in human and rat embryos at Carnegie stage 23 to des-.
Surgical Radiologic Anatomy
Surg Radiol Anat (1999) 21:55-58
Journal of Clinical Anatomy
© Springer-Verlag I999
A new method of three-dimensional computer assisted reconstruction of the developing biliary tract M. Pmdhomme 1, R. Gaubert-Cristol 2, M. Jaeger 3, P. De Reffye3 and G. Godlewski I i Laboratoire d'Anatomie Exp6rimentale, Facult6 de M6decine, Montpellier-Nirnes, Universit6 Montpellier I, F-30900 N~mes, France 2 Facultd d'Odontologie, Universit6 Montpellier I, F-34000 Montpellier, France 3 CIRAD Unite de Moddlisation, BP 5035, F-34032 Montpellier Cedex 1, France
Summary: A three-dimensional (3-D) computer assisted reconstruction of the biliary tract was performed in human and rat embryos at Carnegie stage 23 to describe and compare the biliary structures and to point out the anatomic relations between the structures of the hepatic pedicle. Light micrograph images from consecutive serial sagittal sections (diameter 7 mm) of one human and 16 rat embryos were directly digitalized with a CCD camera. The serial views were aligned automatically by software. The data were analysed following segmentation and thresholding, allowing automatic reconstruction. The main bile ducts ascended in the mesoderm of the hepatoduodenal ligament. The extrahepatic bile ducts: common bile duct (CD), cystic duct and gallbladder in the human, formed a compound system which could not be shown so clearly in histologic sections. The hepato-pancreatic ampulla was studied as visualised through the duodenum. The course of the CD was like a chicane. The gallbladder diameter and length were similar to those of the CD. Computer-assisted reconstruction permitted easy acquisition of the data by direct examination of the sections through the microscope. This method showed the relationships between the different structures of the hepatic pedide and allowed estimation of the volume of the bile duct.
Correspondence to: M. Prudhomme
These findings were not obvious in twodimensional (2-D) views from histologic sections. Each embryonic stage could be rebuilt in 3-D, which could introduce the time as a fourth dinaension, fundamental for the study of organogenesis.
Nouvelle m6thode de reconstruction tri-dimensionnelle assist~e par ordinateur de la voie biliaire chez l'embryon Rfisumd : Une reconstruction tridimensionnelle assist6e par ordinateur de l'arbre biliaire a 6t6 r6atis6e chez des embryons de rats et un embryon humain pour d6crire et comparer les structures biliaires et pour mettre en 6vidence les rapports anatomiques des diff6rents 616ments du p6dicule h6patique. Des coupes sagittales (6paisseur de 7 pm) d ' u n e m b r y o n humain et de t6 embryons de rat ont 6t6 directement acquises par ordinateur. Les coupes s6ri6es ont 6t6 automatiquement align6es. Les donn6es ont 6t6 analysdes en utilisant une segmentation et un seuillage permettant une reconstruction automatique. Les voies biliaires principales avaient un trajet ascendant dans te m6soderme du ligament h6pato-duod6nal. Les voies biliaires extra-h6patiques : conduit chol6doque (CD), conduit cystique et la v6sicule biliaire chez l'humain, 6taient en continuit6. L'ampoule h6pato-pancr6atique a 6t6 explor6e par transparence ~ travers le duod6num. Le CD avait un trajet en chicane. Le diam~tre et la longueur de la v6sicule biliaire 6taient identiques
celles du CD. La reconstruction assistde par ordinateur permet une acquisition ais6e des donn6es par l'examen direct des coupes histologiques ~ travers un microscope. Cette mdthode determine clairement les relations entre les diff6rents 616ments du p6dicule h6patique et leur volume, ce qui n'6tait pas 6vident g l'examen en 2 dimensions des coupes histologiques. Chaque stade embryonnaire pourrait ~tre ainsi reconsmait en 3-D, introduisant ainsi le temps comme quatri~me dimension, ceci 6tant fondamentat pour l'6tude de l'organog6n~se.
Key words: Three-dimensional computer assisted reconstruction - - Biliary tract - - Human embryo - - Carnegie stage 23
In current practice, anatomic atlases are based on a collection of planar images presented in books or, more recently, stored on digital media. These data do not give any idea of volume or relations between the different structures. Threedimensional presentations provide novel information about such structures [1, 9, 10, 13, 14]. In this study we have used a histologic computer analysing system to obtain three-dimensional reconstruction (3-D) views of the biliary tract in human and rat embryos at Carnegie stage 23. This technique allowed us to perform morphological studies of developing organs as well as their ultimate 3-D. At the embryonic period of Carnegie stage 23, 90% of the anatomic structures were
56 individualized [9] and sufficiently differentiated to be analysed [7]. The aim of this study was to perform a three-dimensional computer-assisted reconstruction of the biliary tract in human and rat embryos at Carnegie stage 23 and to compare the anatomic relationships of the different components of the hepatic pedicle. Material and methods
Human embryos Nine specimens of human embryos between 30 and 60 post ovulatory days were provided by the Department of Pathology. They were derived from spontaneous abortions and from abortions performed on strictly medical indications. This group was composed of one specimen per stage through Carnegie. stages 15 to 23. Embryos at stage 23 were only considered for this study because of their wellindividualized structures, favouring the adjustment of the 3-D reconstructions.
Rat embryos 420 rat embryos were provided from the embryologic collection of the Department of Anatomy. They were collected from day 6 to day 21 of pregnancy in female OFA IOPS rats. Day-time and night-time periods were artificially induced and the coital age was fixed at 11 a.m. with a rate of error of +3 h. In the rat and human series, embryo identification was performed under an operative microscope using atraumatic microsurgical instruments. The crown-romp (CR) length was evaluated. The 57-day-old human embryo had a 31 mm CR length. Rat embryos of stage 23 (n = 16) measured 16 mm (+ 3 mm) in CR length and their age was the 16th postcoital day. The embryos were fixed in Bouin's fixative, dehydrated in methylbenzoate and embedded in paraffin. The sections (n = 48 for human and 64 + 4 for rots) were 7 pm thick and were cut in a sagittal plane. Serial sections were stained with hematoxylin-eosin and Heidenhain's azan. The biliary tract, including the gallbladder in humans, was microscopically identified and the sections of interest were selected for 3-D reconstruction. A 25-fold magnification was used for analysis of each embryo.
M+Prudhorrmae,et at.: Computerassistedreconstructionin embryo
3D computer assisted reconstruction technique A high-resolution video-camera (Gmndig CCD) was directly connected to the light micToscope (Leitz DRMB). The acquisition of the light micrograph images from consecutive serial sections was immediately digitalized and stored under TIFF image format on the hard disk of a Power Mac (Macintosh) by OPTILAB acquisition video software. Each section was translated into numerical data by this procedure. The data were represented in grey levels from 0 to 255. Before analysing the data, the serial sections were accurately aligned. The edges of the main structures - duodenum, pancreas and liver were automatically extracted from gradient images in each section, superimposed and then aligned by comparative edges [4]. Once completed, the images were processed like a classical set of serial slices The views were analysed using segmentation to isolate a particular structure (Fig. 1). To select structures of the same color intensity (e.g. liver) an image thresholding was performed altowing automatic selection (Fig. 1). Using the wire frame display only the organ outlines could be visualized on each section, giving transparency to the 3-D reconstructions. This method was used to show the course of the CD through the duodenal wall. Manual corrections were notably performed for the biliary structures. Colors were assigned to each sla'ucture (purple for the liver, green for the biliary tract, blue for the portal v., orange for the pancreas and yellow for the duodenum) Image alignment, filtering, labeling and analytic 3-D reconstructions were performed using the "CORPUS 2000" software developed by CIRAD, Unit6 de modtlisation, Montpellier, France". The 3-D reconstruction images could be rotated and examined in all planes. Results
The histologic sections were sufficiently differentiated and contrasted to be automatically individualized and reconstructed by computer. However, several contours such as the extrahepatic bile ducts and the pancreas had to be manually recognized because of their similar contrast.
Microscopic examination In humans, the liver was prominent, with a high density of hepatic cells organized in strands radiating from the hilum to the periphery. Blood sinusoids were numerous and interspersed between the hepatic strands. The continuity of the CD, the cystic duct and the gallbladder was difficult to determine; neither could the anatomic relationship between the CD and the pancreas be precisely visualised on histological sections (Fig. 2). In the rat, the liver was composed of irregular hepatic strands not yet radiating. The vascular sinusoids, compressed between the strands, were totally filled by blood cells. The hepatic duct connecting the liver to the duodenum was large in its inferior part, narrowing at the level of the hilum, No rudiment the gallbladder was found.
3D r.econsmwtion In both series, the main bile ducts were clearly seen ascending in the mesoderm of the hepatoduodenal ligament. In human embryos the extrahepatic bite ducts were in continuity with the cystic duct and the gallbladder was perpendicularly implanted. This biliary tract was clearly depicted in 3-D reconstructions (Fig. 3). In the rat specimens the hepatic pedicles were packed under the liver with a short course of the CD (Fig. 4). The point of entry of the CD into the medial side of the duodenum was clearly identifiable. Reconsmlctive visuatisation of the duodenum by the wire frame technique (Fig. 3) allowed the transparent exploration of the terminal part of the CD and the hepato-pancreatic ampulla. The CD had a chicane-like course into the pancreas. Its upper part was situated behind and to the left of the pancreas, it then crossed into the gland from left to right more distally (Fig. 5). This particular course was not obvious in the histotogic sections (Fig. 2), The suprapancreatic part of the CD was in the vicinity of the anterior face of the portal v. (Fig. 5). In the rat, because of the existence of four well separated hepatic lobes, four portal branches were clearly identified behind the CD. In the human, the gallbladder was well developed with a diameter and length as large
M. Prudhomme, et al.: Computer assisted reconstruction in embryo
57
Fig, 1 Segmentation: The pancreas (P) and the common duct (CD) were selected into a circle at the first slice, they were identified and reconstructed for each following slice. Tbresholding: stuctures of the same intensity were determined and identified as the same structure as the liver (L) in this figure
Fig. 2 Histologic section of a humma embryo. The continuity and course of the biliary tract are not obvious on 2D image. PV, portal v.; GB, gallbladder; P, pancreas; CD, c o m m o n duct; HD, hepatic duct
Fig. 3 3-D reconstruction of the biliary tract in human embryo. The duodenum (yellow) was rebuilt using the wire frame technique. The biliary tract and hepato-pancreatic ampulla (green) are seen by transparency. The portal v. is blue, the pancreas orange and the liver
purple Fig. 5 Fig. 4 3-D reconstruction of the biliary tract in rat embryo. Note the hepatic pedicles packed down and the short course of the CD (green) throughout the pancreas (orange) and the duodenum (yellow)
3-D reconstruction of the biliary tract in human embryo. The course and anatomic relations of the CD (green) are well defined. Note the shape and angulation of the gallbladder (green). The portal v. is blue, the pancreas orange and the liver purple
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and long as the CD. In the comparison of fetuses and adults, the gallbladder and cystic duct were proportionally larger in the human embryo. The angle between the CD and the cystic duct was 90 °. The junction between the CD and the cystic duct was located high up in the hepatic pedicle (Figs. 3 and 5). Thus, the course of the hepatic duct itself was short. The intrahepatic biliary ductules and canaliculi were not well visualized and could not be reconstructed at the low magnification used in this work.
Discussion In our study, computer assisted reconstruction permitted easy direct acquisition of the data from direct examination of histologic sections through the microscope with a high resolution giving more precision to the reconstructions. Moreover, this new method offered an automatic and more precise alignment of serial histologic sections. This process is essential to prevent progressive shift of the stack of serial sections due to misalignment between sections [12]. Manual methods were more time-consuming and approximate, the contours being artificially smoothed according to the operator's wishes, leading to frequent changes or poor accuracy when compared to the original specimen [3, 6]. The reconstruction accuracy and the anatomic proportions depend on the number of sections and the distance between adjacent sections [2]. In our procedure, the histologic sections were cut at 7 ~am thickness, thus allowing a very precise reconstruction. The formation and development of the primordium of the human and rat liver and its vascular channels, as well as the development of the periportal bile ducts, occur between Carnegie stages 11 and 23 [7]. At stage 23, the anatomic structures and relationships between the different structures of the hepatic pedicte are established and can be analysed precisely [5]. Stage 23 in the rat and human emb~2cos are comparable for the assessment of hepatic development as well as for other organs as shown by GaubertCristol and Godlewski [5, 7]. In our study, comparison of the hepatic pedicte in human and rat
M. Prudhomane,et at.: Computer assisted reconstruction in embryo
embryos revealed a marked analogy. The difference of CR length between human and rat embryos (31 vs 16 ram) and the absence of a gallbladder explain the compact aspect of the rat hepatic pedicle. The similarity of the hepatic structures during embryonic development offers a good experimental model to evaluate the teratogenic effects of chemicals, drugs and radiation on the formation of the biliary tract during these different stages. Our method of 3-D computer-assisted reconstruction allowed to analyse the developing biliary tract more precisely. It determined the relationships between the different components of the hepatic pedMe with their volume (ie for the CD, d u o d e n u m and pancreas, and the portal v.) which were not obvious in 2examinations of histotogic sections. In addition, it permitted us to assess the particular course of the CD into the pancreas. The wire frame reconstruction technique allowed transparent visualisation of the duodenal medial wall. Moreover this technique permitted the study of the hepato-pancreatic ampulla through the duodenum and pancreas, which is an innovative and fascinating view of a "hidden" structure (Figs. 3 and 4). The great advantage of this method is that the hepatic pedicles can be observed in different planes, so that anatomic structures can be visualised far better in the anterior and lateral views in order to show the pancreatic course of the CD (Figs. 3 and 4). Volumes, surfaces, lengths and axes could be determined and modelized directly, providing instantaneous morphometry [8, 11, 13, 15]. It is possible to rebuild a 3D reconstruction of the hepatic pedicle of each embryonic stage from 1t to 23 and thus introduce a fourth dimension, time, which is fundamental for the concept of development. The use of 3-D reconstruction offers a better understanding of congenital abnormalities such as agenesia, atresia or cyst of the biliary tract and their morphogenesis. By this procedure surgical interventions can be simulated in order to perform more precise dissection and to avoid damaging vulnerable regions. Moreover, it offers a new tool for teaching, more attactive and with higher performance.
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Received June 24, 1998/Accepted in final form December 3, 1998
