(A) histological and histochemical study of the structural and functional unit of the liver of the mouse
As the structural unit of the hepatic tissue, Wepfer(1664) and Malpighi (1666) termed the parenchymal units attached to the intrahepatic portal venules hepatic acini by the macroscopic dissection.
Kiernan (1883) associated the three-dimensional structural unit of the liver with its secretory function.
Brissaud and Sabourin (1886) reported the area of the hepatic parenchymal tissue draining into a bile duct in the portal trigone as the secretory unit of the liver.
Mall (1906) proposed that the columnar structure around the portal field was the portal unit; he was supported by Arey (1932) and Opie(1944).
Rappaport et al. (1954) defined the small irregular morular parenchymal tissue mass attached to the portal terminal venules accompanying with the intrahepatic arteries and bile duct in the livers of dogs and house rabbits as the structural and functional unit of the liver, and called it the simple liver acinus. Rappaport(1958) termed the hepatic parenchymal tissue consisting of the simple liver acini around the preterminal portal vessel in man the complex acinus and the larger parenchymal tisssue of the simple liver and complex acini formed around the portal stem the acinar agglomerate.
Recently the studies of the liver concerning the function of it in the histological and histochemical fields were carried out by many authors. Padykula(1953) reported that the patterns of distribution of hepatic mitochondria, Golgi apparatus and glycogen in various mammals, and the secretion of bile were
associated with enzyme activity of hepatic succinic dehydrogenase in the histochemical way. Additionally he described the fact that his result was similar with them presented by Deane (1944), and Seligman and Rutenberg(1951).
The technique, as presented by Wachstein and Meisel (1957), outlines bile canaliculi sharply and completely with the histochemical way of adenosine triphosphatase (ATP-ase) and diphosphatase (ADP-ase), as well as bile ducts, endothelium, and fibrous tissue, and can thus be effectively used as special stain
for canaliculi, as well as an indicator of liver cell function.
Smith and Coote (1863) studied the imparirment of succinic dehydrogenase and other enzymes in the rat's liver given carbon tetrachloride in their histochemical study.
Schumacher (1957) studied the action of respiratory enzymes; succinic dehydrogenase, cytochrome oxidase, triphosphopyridine nucleotide diaphorase and diphosphopyridine diaphorase in the livers of the horse, pig and cattle, and observed that the distribution was identical with the earlier reports on the former
enzymes, but that it was in direct contrast to the former 2 enzymes in that the latter 2 enzymes were generally distributed to the pericentral area around the central vein.
The author has attempted this experiment in order to study the hepatic structural and function unit of the mouse by observing the histological and histochemical or functional characters of the hepatic cells in different parts of hepatic lobule in the points; the distribution of mitochondria (2) the enzyme activity of succinic dehydrogenase (3) the enzyme activities of adenosine triphosphatase and diphosphatase, (4) the enzyme activities of diphosphopyridine nucleotide (NADH^^2) diaphorase and triphosphopyridine nucletide (NADPH^^2) diaphorase, to accomplish
the transfer of hydrogen from the reduced coenzymes to the ytochrome system and to other metabolic processes, and (5) the enzyme change of succinic dehydrogenase and histochemical change of hepatic glycogen of the mouse given carbon tetrachloride.
Materials and Methods
The livers of mice consisting of 70 (44 males, 26 females with average weight of about 20 gm.) were freshly obtained after decapitation from the intact and experimental animals.
For the histological procedure to demonstrate mitochondria, small pieces of liver tissue were fixed in Helly's and Regaud's fixatives and the were cut serially 5 micra thick after embedding in paraffin. The sections were stained with Heidenhain's iron hematoxylin or with hematoxylin and eosin.
For the histochemical procedure to demonstrate succinic dehydrogeenase, fresh frozen sections of about 10 micra were made. They were incubated according to the method of Nachlas et al. (1957) using Nitro-BT and then they were mounted with glycerojelly and examined. For control an incubating medium without sodium succinate was used in order to demonstrate if any endogenous substrate was present in the liver tissue and such control preparations gave negative reactions.
For the demonstration of the enzyme activities of ATP-ase and ADP-ase, the method presented by Wachstein and Meisel (1957) was used. For control the incubating media without adenosine triphosphate and diphosphate were used to determine if any
artificial product was present in the liver and such control preparations gave negative reactions.
For the localization of NADH^^2 diaphorase and NADPH^^2 diaphorase the unfixed frozen sections of about 10 micra were incubated according to the method presented by Wolfe and Cohen (1963) and then they were mounted with glycerojelly and examined. For control the incubating media without both reduced coenzymes, were used to determine if any artifical product was present in the liver sections and such control preparations gave negative reactions.
For the observation of succinic dehydrogenase activity and glycogen in the animals given 0.1ml of 40% carbon tetrachloride solution dissolved in oliver oil by the peroral catheterization of polyethylene tube into stomach directly, the method of Nachlas et al. (1957) and the periodic acid Schiff method of Hotchkiss (1948) accompanying with the hematoxyline stain after the fixation in cold Rossman's fluid were used. The experimental animals were sacrificed and divided into the folowings; 8 mice for the 24 hours group, 7 mice for the 3 days group, 5 mice for the 6 days group, and 10 mice for the 14 days group.
Results and Summary
1. On the distribution of mitochondria; In preparations of the mouse liver stained with hematoxylin and eosin, and iron
hematoxylin, the boundary of each hepatic lobule was not easily demonstrable because of scant interlobular connective tissue. According to the quantitative distribution of mitochondria in hepatic cells in different areas of the hepatic lobule, the area heavily deposited with mitochondria around the portal vessels
could easily be differentiated from the scantily contained area around the central vein. Mitochondria contained in hepatic cells became denser toward the periphery of the hepatic lobule. On observing the perivascular area around the portal stem which
contained to the preterminal portal branch, the hepatic cells composed of several layers around the portal stem contained many mitochondria which granular in shape, as a rule. Although the perivascular area of the preterminal portal branch revealed
a marked deposition of mitochondria in the hepatic cells, while the pericentral area adjacent to it showed less amount of mitochondria in the hepatic cells, while the pericentral area adjacent to it showed less amount of mitochondria in the
hepatic cells. In pursuing the course of the sublobular vein, the perivascular area of it showed almost the same result as the pericentral area. The author named the 3 different areas or geometrical structures around above mentioned intrahepatic
portal branches, which were formed and extended to 2 adjacent hepatic lobules together, as the functional and structural unit and found the geometrical structure formed around the terminal portal branch in the mice was corresponding to the simple liver acinus observed by Rappaport et al. (1954) in the livers of the dog and house rabbit.
2. On the distribution of succinic dehydrogenase; For demonstrating the distribution of succinic dehydrogenase, there were areas showing strong activity and areas showing weak enzyme activity in the hepatic parenchymal tissue surrounding the large blood vessel. The vessels sruurounded by strongly reacting areas were considered to be part of the portal stem, since branches were observed. The weak-reacting areas were considered to contain the intercalated vein or the sublobular vein. Although the author observed that the enzyme activity of the hepatic lobules was strong in the periportal area and showed lesser reaction toward the pericentral area around the central vein. In the hepatic structure of the mouse, 3 different geometrical areas were classified; the perivascular area of the portal stem, the area of preterminal portal vein, and the area of the terminal portal twig, this being considered to be the real functional and structural unit, which extends into the neighbouring hepatic lobules according to the pattern of distribution of it. The hepatic tissue, which showed strong enzyme activity of it, was considered to correspond with the heavily loaded parenchymal area with mitochondria.
3. On the distribution of adenosine triphosphatase and diphosphatase: Results obtained with both enzymatic activities were fairy similar. However, the result of ATP-ase was considerably more consistent and shorter incubation times were necessary. In fresh frozen sections the activities of both enzyme revealed in biliary canaliculi, hepatic sinusoids and hepatic cells in which granular staing and fiffuse reaction were observed. Although higher activities of both enzymes were easily recognized in the periportal area, the central zones of the unit and lower activities were observed in the peripheral portions of the hepatic lobule, the peripheral portions of the unit including the pericentral area. In fixed sections, however, zonal differences of both enzyme activities were less evident.
4. On the distribution of diphosphopyridine nucleotide (NADH^^2) diaphorase and triphosphopyridine nucleotide (NADPH^^2) diaphorase: In the preparations for demonstrating the distribution of NADH^^2 diaphorase, there were areas showing strong enzyme activity and areas showing weak enzyme activity in the hepatic lobule. The former areas were found around the central vein, intercalated or sublobular vein, and at the nodal point. The latter areas were found around the periportal parts of the hepatic lobule. About the enzyme activity of NADPH^^2 diaphorase, it showed a similar enzyme activity of the hepatic
lobule in comparing NADPH^^2 diaphorase with NADHP^62 diaphorase. The difference of NADHP^^2 diaphorase activity between both areas was, however, not so evident as NADH^^2 diaphorase. The difference of NADPH^^2 diaphorase activity between both areas ws, however, not so evident as NADH^^2 diaphorase showed. The author found these strong enzyme activities in the pericentral areas or the peripheral zones of the functional and structural unit and weak enzyme activites in the periportal areas of the same unit.
5. On histochemical changes of succinic dehydrogenase and glycogen in experimental animals given carbon tetrachloride: In an intact animal the enzyme activity of succinic dehydrogenase was present in the 2. In the intact animal the distribution of hepatic glycogen was almost uniform. However, some pericentral areas were showed less deposition of glycogen in the hepatic cells rather than in the periportal areas. In the 24 hours group the fatty metamorphosis and necrotic degeneration were
noticed in the pericentral area. On the enzyme activity of succinic dehydrogenase, the severe depletion of it was recognized in the periportal areas corresponding to the central part of the hepatic functional and structural unit. Although the activity of it was moderately decreased or absent in the pericentral areas
corresponding to the peripheral areas of the unit associated with the pericentral degenerative changes. In this stage the activity of it in both areas of the unit reached almost the least level.
On the 3 days group the depletion of glycogen was severe in the henatic cells of both areas and in some cases little glycogen was observed. In the 6 days group the enzyme activity was farily recovered in both areas of the unit and in the 14 days group there was no difference or slight difference between the normal and experimental groups. On the change of glycogen, the depletion of it was only occurred in the pericentral area in the 24 hours group. The depletion of it was reached the maximal state in the pericentral area, peripheral area of the unit in the 6 days group and in some places the depletion of it extended to the peripheral zone of the hepatic lobule or the peripheral zone of the unit. However, the periportal areas or the central areas of the unit showed the least depletion of it. In the 14 days group the deposition of glycogen was considerably recovered in the pericentral hepatic cells of the hepatic lobule.
The concepts of the structural units of the hepatic lobule presented by many authors since the 17th century, were controversial. By the observation of histochemical distribution of the enzymatic activities and of carbohydrate in the hepatic cells and the distribution of mitochondria in the hepatic lobules the auther deined 3 zones of the mice liver as the functional and
structural units; the terminal periportal unit, which is similar with the simple liver acinus presented by Rappaport, the preterminal periportal unit, and the periportal unit of the portal stem comparing with Rappaport's concepts.