Histogenesis of the rabbit liver (pars hepatica) with particular reference to the portal area

1. The hepatic bud developed at the 10^th day of gestation in rabbits. 
2. The hepatoblasts' proliferation began on the 12^th day of gestation. 
3. The hepatocyte differentiation began on the 18^th day of gestation.
4. Portal area formation began on the 20^th day, and its complete differentiation occurred on the 26^th day of gestation. 

Introduction 

Rabbits (Oryctolagus-Cuniculus) are small mammals in the family Leporidae order Lagomorpha found in several parts of the world. They are very docile, non-aggressive, and easy to be handled and observed. Rabbits have short vital cycles (gestation, lactation, and puberty) (1).Rabbits are phylogenetically closer to primates than rodents and are standard laboratory animals in biomedical research. The rabbits are animal models for various human diseases(2). The liver is the largest internal organ, providing essential metabolic, exocrine, and endocrine functions (3, 4).Hepatocytes are the primary cell type in the liver, accounting for 70% of the mass of the adult organ. Hepatocytes, along with biliary epithelial cells, were of endodermal origin. At the same time, the stromal cells, stellate cells, Küpffer cells, and blood vessels were of mesodermal origin. The hepatoblasts proliferated on the 12^th day of embryonic life in domestic rabbits, and mitotic division occurred. At this stage, the primordial hepatoblasts were arranged as short hepatic cords (4). The liver developed as a hollow ventral diverticulum from the caudal region of the foregut. This diverticulum is divided into the hepatic cranial part (Pars hepatica), which forms hepatic parenchyma, and the caudal cystic part (Pars cystic), which forms the gall bladder. The endodermal epithelial cells of the hepatic portion proliferated and formed Figures of hepatocytes. Liver cords differentiated into the parenchyma (liver cells) and formed the lining of the biliary ducts (5-8). The relatively large endodermal cells are differentiated into hepatocytes and arranged in rods or plates with intervening sinusoids. Later, the hepatocyte Figures became oriented radially around the central vein. Even later, the connective tissue became organized around the lobules. The liver proliferated and expanded to occupy most of the abdominal cavity, creating the liver prominence on the outside of the embryo. Hence, with the caudal growth of the liver, the organ gradually became almost detached from the Septum transversum. During its growth, the liver was of great significance as a blood-forming structure (7). After the specification of the liver, hematopoietic cells moved into this organ and emitted signals, which induced the maturation of hepatocytes (9).

A superior understanding of liver development would provide effective treatment for liver diseases. So, this work aimed to explain the liver histogenesis in rabbits to understand fetal and neonatal liver histology better. 

Materials and methods 

The present study applied on ten white New Zealand pregnant women (1.5-3 years old, and 2-4.5 kg, weight). They were collected from the Faculty of Veterinary Medicine and Faculty of Agriculture, Zagazig University farms. Thirty embryos and fetuses at different periods and 10 neonatal kittens were used for histogenesis and embryogenesis of the liver. The pregnant does were anesthetized with an intramuscular injection of 35 mg/kg ketamine Hcl (KETALARKETALAR, 100 mg/ml, Pfizer, NY) and five mg/kg xylazine (Xylaject, 20 mg/ml, ADWIAADWIA, Egypt) according to Enoka (10) and Mahmood (11). For the histological study, the following steps were applied on the 10^th, 12^th, 14^th, 16^th, 18^th, 20^th, 22^nd, 26^th, and 30^th day's old embryos and fetuses and neonatal kittens. After the laparotomy of pregnant does, the fetuses were pulled out from the fetal sacs. Also, the liver samples were extracted from the neonatal kittens' bodies. Then, they were fixed with 10% neutral buffered formalin for 24-72 hours and Bouin’s solution for 6-8 hours, followed by 70% ethyl alcohol (12). All embryos, fetuses, and neonatal liver samples were subjected to dehydration, clearing, embedding, blocking, cutting, and staining with different stains, including Harris' H and E and Masson's trichrome stain, according to (13). The stained sections were examined with a standard light microscope (Objectives Xs 4, 10, 40) and a stereomicroscope (Objectives Xs 10, 20) and photographed with a Moticam X3 plus camera (a compact Wi-Fi camera with a resolution of four megapixels) according to Drury and Wallington (14). The CVR length of fetuses used in our study was clarified in figure 1 (15-17). The nomenclature used in this work was adopted (18-20). 

Ethical approve

The research protocol was reviewed by the Institutional Animal Care and Use Committee, Zagazig University (No, ZU-IACUC/2/F/13/2021). All surgical approaches were performed under general anesthesia, and all efforts were made to minimize animal pain.

Figure 1:The CVR length of the embryos and fetuses (in cm at 12^th (A), 14^th (B), 16^th (C), 18^th (D), 20^th (E), 22^nd (F), 26^th (G), and 30^th (H) days of gestation. 

Results

The results revealed that the hepatic bud was developed from the ventral part of the foregut on the 10^th day of embryonic life ±0.54cm, figure (2A). The hepatic bud invaded Septum transversum and formed from two types of cells: endodermal cells and Septum transversum mesenchymal cells. The endodermal cells of the foregut formed from cuboidal cells. They had large rounded vesicular nuclei, which showed mitotic division and pale cytoplasm. The mesenchymal tissue was undifferentiated c.t with fibroblasts and had large and intensely stained nuclei (Figure 2B, C).

The hepatic diverticulum developed on the 12^th day ±0.9 cm, and the hepatoblasts invaded Septum transversum mesenchyme (Figure 2E). The hepatoblasts are arranged as irregular aggregations of hepatic masses separated by blood spaces (Figure 2E, F). The hepatoblasts at this stage showed high mitotic division, and the liver revealed high hematopoietic activities (Figure 2E, F). The hepatoblasts appeared as large, rounded, pyramidal cells with heterochromatic, highly vesicular nuclei. The nucleus contained multiple basophilic nucleoli with pale eosinophilic cytoplasm (Figure 2F). The blood sinusoids were formed at this stage with a wide diameter (Figure 2E, F). They contained both nucleated erythroblasts and Küpffer cells. This indicated the beginning of hematopoietic activities in the liver (Figure 2F, G). For the first time, we observed scattered Küpffer cells on the margin of sinusoids in the 12^th week. Megakaryoblasts were first detected at this stage and increased in number until the end of gestation (Figure 2G).

On the 14^th day of gestation ±1.3 cm, the size of the liver expanded considerably and inhabited a significant part of the cranial half of the abdominal cavity (Figure 2H). Intrahepatic portal vein ramification was developed at this stage (Figure 6A). The central vein appeared within the liver parenchyma at this stage (Figure 2J). The liver, at this stage, became the essential blood-forming organ at gestation. Hence, the hematopoietic activities increased, and most hematopoietic cells were nucleated erythroblasts. A group of hepatoblasts organized themselves in the periportal area. These smaller cells formed ductal plates that later formed the cholangioblasts (Figure 6A). So, both hepatocytes and cholangioblasts originated from the hepatoblasts.

On the 16^th day of gestation ±2 cm, the hepatic parenchyma consisted of highly mitotic divided hepatoblasts and blood sinusoids filled with nucleated erythroblasts. Some erythroblasts began to extrude their nuclei on reaching maturity (Figure 3B, C, D). On the 18^th day of gestation ±2.5 cm, the sinusoid endothelium became more organized as a thin layer of endothelial cells (Figure 3G). At this age, a mixed population of both hepatoblasts and hepatocytes coexisted (Figure 3H). The fetal hepatocytes were oval or rounded and smaller than the hepatoblasts. They had a large rounded heterochromatic nucleus with one or two nucleoli.

On the 20^th day of gestation ±3.2 cm, the blood sinusoids began to converge around the central veins (Figure 3J). Fatty vacuoles were detected within the hepatocyte’s cytoplasm at this stage (Figure 3K). These vacuoles increase in number with the advancement of the fetal age till they reach their maximum at the 30^th day of gestation (Figure 5C). On the 22^nd and 26^th days ±4.5cm and ±5.8cm, respectively), the hepatic parenchyma showed crowded hepatic cell masses with intervening blood sinusoids. Central veins were randomly distributed inside the parenchyma. The hepatocytes were the primary cells accounting for the hepatic parenchyma (Figure 4). 

Granulopoiesis was detected in the livers of the 30^th-day fetus ±7.5 cm and neonatal kittens (Figure 5C, H). The neonatal liver showed the same liver architecture as the adult liver tissue (Figure 5E-I). No lobular pattern was detected at the parenchyma of the liver in rabbits, even in neonatal liver. The central veins and portal area were randomly distributed within the parenchyma (Figures 4E, 5E).

Figure 2:Histogenesis of the liver on the 10^th day (A, B, and C), 12^th day (D, E, F, and G), and 14^th day (H, I, J, and K) of old rabbit embryos, showing vitelline veins (Vv), coelomic cavity (cc), blood spaces (blue stars), central vein (CV), hepatoblasts (dotted red arrow), mitotic division of hepatoblasts nuclei (blue circle), foregut (F), endodermal epithelium lining of foregut (double yellow arrow), nucleated erythroblasts (green arrowhead), hepatic bud (Hb), hepatic masses (Hm), mesenchyme (m), megakaryoblasts (orange arrow), endothelial lining (black arrowhead), septum transversum (red arrowhead), küpffer cells (blue arrow), H and E stain, Xs 4 (A, D, H), 10 (B, C, E, I, J), and 40 (F, G, K). 

Figure 3:Histogenesis of the liver on the 16^th day (A, B, C, and D), 18^th day (E, F, G, and H), and 20^th day's old rabbit fetuses (I, J, K, and L), showing central vein (CV), hepatoblasts (dotted red arrow), nucleated erythrocytes (green arrowhead), developing sinusoids (green arrow), megakaryoblasts (orange arrow), endothelial lining (black arrowhead), küpffer cells (blue arrow), shedding of erythrocytes nuclei (orange circle), lipid vacuoles (yellow arrowhead), H and E stain, light microscope, Xs 10 (B, F, J, K) and 40 (C, D, G, H, L) and stereo microscope, X 20 (A, E, I). 

Figure 4:Histogenesis of the liver on the 22^nd day (A, B, C, and D) and 26^th day's old rabbit fetuses (E, F, and G), showing central vein (CV), hepatic masses (Hm), hepatocytes (dotted red arrow), developing sinusoids (green arrow), megakaryoblasts (orange arrow), Kupffer cells (blue arrow), lipid vacuoles (yellow arrowhead), H and E stain, Xs 4 (A, E), 10 (B, F) and 40 (C, D, G). 

Figure 5:Histogenesis of the liver at 30^th day's old rabbit fetus (A, B, C, and D) and neonatal kitten (E, F, G, H, and I), showing central vein (CV), portal area (blue square), hepatocytes (dotted red arrow), developing sinusoids (green arrow), megakaryocytes (orange arrow), Kupffer cells (blue arrow), granulocytes (black double-headed arrow), bile canaliculi (green rectangle), lipid vacuoles (yellow arrowhead), H and E stain, Xs 4 (E), 10 (A, B, C, F), and 40 (D, G, H, I). 

The results of this study showed the sequential differentiation of portal area on the 20^th,22^nd, 26^th, and 30^th day's fetal and neonatal livers (Figures 6 and 7). On the 20^th day of gestation, the intrahepatic bile duct differentiation occurred within the portal mesenchyme. We found that the portal area on the 22^nd day consisted of only a tributary of the portal vein and one or two bile ducts with a scanty amount of collagen fibers (prominent by Masson's trichrome). No hepatic arteriole was detected in the portal area at this age due to delayed arterial vascular organization than the venous one. While at 26^th day, the portal area began to be more organized. It consisted of a portal vein tributary, a hepatic artery branch, and one or two bile ducts. At this stage, it contained a moderate amount of collagen fibers. On the 30^th day, we noticed increased collagen fibers in the portal area. 

Figure 6:Histogenesis of the portal area in rabbit liver at 14^th (A), 16^th (B),18^th (C), 20^th (D, E, F), 22^th (G, H), and 26^th (I, J, K) days of gestation in rabbit, showing bile ducts (BD), hepatic artery (HA), hepatic parenchyma (HP), portal vein (PV), beginning of portal area formation (green circle), developing ductal plate (blue circle, orange arrowhead), common bile ducts (yellow arrows), H and E and Masson's trichrome stains (H, J, K), Xs 4 (D, E), 10 (A, B, C, G, H, J), and 40 (F, I, K). 

Figure 7:Histogenesis of the portal area in rabbit liver at 30^th day of gestation in rabbit (A, B, C, D), and neonatal kitten (E, F), showing bile ducts (BD), hepatic artery (HA), portal vein (PV). H and E stain and Masson's trichrome stain (C, D), Xs 10 (A, B, C, D, E), and 40 (F). 

Discussion 

The present study aimed to clarify liver histogenesis at different gestational periods. The hepatic bud was developed from the foregut on the 10^th day of embryonic life. On the 12^th day, the hepatoblasts invaded Septum transversum mesenchyme. This result was in line with those found by (4) in the rabbit. In comparison, (21), (22), (23), and (24) in mice cleared that the liver diverticulum formed at 9^th-9.5^th days of gestation. However, (25) mice observed this diverticulum on the 8.5^th day. In humans, (26) showed it at the fourth week of fetal life, (27) at the 18^th day of gestation, and (23) at the third week of gestation. 

The hepatoblasts are arranged as irregular aggregations of hepatic masses. This result was agreed upon by (28) in rats. They saw a cluster of disorganized hepatoblasts surrounded by many nucleated blood precursor cells at 12.5^th-14.5^th days. Also, the result of this study came to an agreement with (29) humans, who confirmed that the parenchymal cells are arranged as irregular clumps and cords. At the same time, (4) and (30) in rabbits mentioned the hepatoblasts arranged as short hepatic cords. Also, (31) in rats revealed that hepatoblasts arranged as cords invaded Septum transversum at 9.5^th day, and (22) in mice showed this result at 10^th-10.5^th days. The hepatoblasts at this stage showed high mitotic division, and the liver revealed high hematopoietic activities. This result agreed with the results of (4) in rabbits. 

The hepatoblasts appeared as large rounded or oval cells with heterochromatic, highly vesicular nuclei with multiple basophilic nucleoli. This result agrees with (28) in rats and (22) in mice in that blood sinusoids were formed at this stage with a wide diameter. This was achieved by (22) in mice on the 11.5^th day. They contained both nucleated in erythroblasts and Küpffer cells. This result was per (30) in rabbits and (28) in rats. Nevertheless, (32) in humans appeared at 6^th-8^th weeks of gestation. Megakaryoblasts were first detected at this stage and increased in number until the end of gestation. However, (30) rabbits said they appeared on the 15^th day of gestation.

On the 14^th day of gestation, the liver inhabited a significant part of the cranial half of the abdominal cavity. However, (22) in mice mentioned this result at 10.5^th-11.5^th days. Intrahepatic portal vein ramification was developed. (33) mice detected this result at 11^th-11.5^th days. Central vein appeared within the liver parenchyma at this stage, while (30) rabbits showed them first at the 18^th day of gestation and (29) humans showed it at the 17^th-20^th weeks of gestation. 

On the 14^th day, the liver became the most critical blood-forming organ at gestation. This finding concurred with results of (4) in rabbits. (24) in mice revealed this result at 10^th-15^th days. The ductal plates were formed at this stage. This result corresponded with results of (4) in rabbits, (31) in rats, (25) in mice, and (27) in humans. While (33) in mice revealed this result on the 15.5^th day. On the 18^th day of gestation, the sinusoid endothelium became more organized. However, (34) humans revealed that the sinusoid endothelium was detected in the 6.5 cm CRL fetuses.

On the 18^th day of gestation, both hepatoblasts and hepatocytes coexisted. While (28) rats showed this finding on the 14.5^th day. In humans, (34) mentioned that the fetal hepatocytes were appreciated from CRL of 6.5 cm. The fetal hepatocytes had a large rounded heterochromatic nucleus with one or two nucleoli. This result was found by (34) in humans, except that they said that the hepatocytes contained diffuse chromatin. 

On the 20^th day of gestation, the blood sinusoids began to converge around the central veins, while (28) rats revealed this result on 15.5^th days. However, (34) humans said that the sinusoids were dilated and seen to be open into a central vein and filled with hematopoietic cells during the late gestation period. While, (32) in humans said that the central vein was well formed with radiating cords of cells toward the periphery and portal triad at the 10^th week of gestation. Fatty vacuoles were detected within the hepatocyte’s cytoplasm at this stage. While (30) in rabbits showed them at the 22^nd day of embryonic life. These vacuoles increased until they reached their maximum at the 30^th day of gestation. This result follows those mentioned by (30) in rabbits.

On the 20^th day, the intrahepatic bile duct differentiation occurred within the portal mesenchyme. (33) mice saw this result on the 14^th day, and (26) humans at the 10^th week of gestation. At the same time, (30) rabbits showed portal veins surrounded by one or two bile ducts on the 18^th day of gestation. Delayed hepatic artery development than venous development was also achieved by (35).

 Granulopoiesis was detected in the liver of the 30^th-day fetus and the neonatal liver. While (30) in rabbits said that it began to be observed on the 26^th day and increased on the 30^th day of gestation. No lobular pattern was detected at the parenchyma of the liver in rabbits, even in the neonatal liver, and hepatic cells were arranged as irregular hepatic masses. This result was agreed by (30) in rabbits and (37) in mice. However, (36) in rats, (38) in humans, and (39) in rabbits showed that the hepatocytes were arranged as regular cords around the central vein. While (34) in humans said that the liver architecture showed an ill-defined lobular pattern of fetal hepatocytes of 10.4 cm CRLCRL embryo with a well-defined pattern noticed only in late gestation of 28 cm CRLCRL embryo.This study showed the sequential developmental stages of the developing portal area (the leading site of in-vivo localization of mesenchymal cells stem cells in liver tissue) as reported by the previous study (40). (41) in rats showed that in 20^th day's old fetus, the portal areas appeared containing branches of the portal vein, hepatic artery, lymphatic vessel, and bile duct. 

Conclusions 

This work supplied good data for hepatic parenchymal cell differentiation in different embryonic and fetal periods. This study concluded that liver histogenesis developed at the 10^th day of gestation. In contrast, hepatoblasts and hematopoietic cells proliferated on the 12^th day of fetal life. This study focused on the sequential developmental stages of the developing portal area. Its formation began on the 20^th day of gestation and wholly developed on the 26^th day. 

Conflict of interest

The authors declare no conflict of interest.

Acknowledgments 

The authors thank Anatomy and Embryology Department members, Zagazig University, for their cooperation.