Effects of selective bile duct ligation on liver parenchyma in young animals: histologic and molecular evaluations.

BACKGROUND/PURPOSE: The mechanisms of increased collagen production and liver parenchyma fibrosis are poorly understood. These phenomena are observed mainly in children with biliary obstruction (BO), and in a great number of patients, the evolution to biliary cirrhosis and hepatic failure leads to the need for liver transplantation before adolescence. However, pediatric liver transplantation presents with biliary complications in 20% to 30% of cases in the postoperative period. Intra- or extrahepatic stenosis of bile ducts is frequent and may lead to secondary biliary cirrhosis and the need for retransplantation. It is unknown whether biliary stenosis involving isolated segments or lobes may affect the adjacent nonobstructed lobes by paracrine or endocrine means, leading to fibrosis in this parenchyma. Therefore, the present study aimed to create an experimental model of selective biliary duct ligation in young animals with a subsequent evaluation of the histologic and molecular alterations in liver parenchyma of the obstructed and nonobstructed lobes. METHODS: After a pilot study to standardize the surgical procedures, weaning rats underwent ligation of the bile ducts of the median, left lateral, and caudate liver lobes. The bile duct of the right lateral lobe was kept intact. To avoid intrahepatic biliary duct collaterals neoformation, the parenchymal connection between the right lateral and median lobes was clamped. The animals were divided into groups according to the time of death: 1, 2, 3, 4, and 8 weeks after surgical procedure. After death, the median and left lateral lobes (with BO) and the right lateral lobe (without BO [NBO]) were harvested separately. A group of 8 healthy nonoperated on animals served as controls. Liver tissues were subjected to histologic evaluation and quantification of the ductular proliferation and of the portal fibrosis. The expressions of smooth muscle α-actin (α-SMA), desmin, and transforming growth factor ß1 genes were studied by molecular analyses (semiquantitative reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction, a quantitative method). RESULTS: Histologic analyses revealed the occurrence of ductular proliferation and collagen formation in the portal spaces of both BO and NBO lobes. These phenomena were observed later in NBO than BO. Bile duct density significantly increased 1 week after duct ligation; it decreased after 2 and 3 weeks and then increased again after 4 and 8 weeks in both BO and NBO lobes. The portal space collagen area increased after 2 weeks in both BO and NBO lobes. After 3 weeks, collagen deposition in BO was even higher, and in NBO, the collagen area started decreasing after 2 weeks. Molecular analyses revealed increased expression of the α-SMA gene in both BO and NBO lobes. The semiquantitative and quantitative methods showed concordant results. CONCLUSIONS: The ligation of a duct responsible for biliary drainage of the liver lobe promoted alterations in the parenchyma and in the adjacent nonobstructed parenchyma by paracrine and/or endocrine means. This was supported by histologic findings and increased expression of α-SMA, a protein related to hepatic fibrogenesis.

Studies of RET gene expression and acetylcholinesterase activity in a series of sporadic Hirschsprung's disease.

The purpose is to present the studies of RET gene expression and acetylcholinesterase activity in 23 patients operated for Hirschsprung's disease (HD). The patients underwent either transanal endorectal pull-through or Duhamell's procedure. Full-thickness intestinal samples from the three different segments (ganglionic, intermediate and aganglionic) were collected. Each tissue sample was divided in two portions, one for AChE histochemical staining and the other for examination of RET mRNA expression level. All patients had an uneventful postoperative recovery. In all patients, the AChE stainings demonstrated the absence of activity in the ganglionic area, the marked increase of positive fibers in the aganglionic area, and little increase of positive fibers in the intermediate area. In the ganglionic and intermediate areas, all patients (100%) showed significant RET gene expression. In the aganglionic area, 18 patients (78.3%) did not present gene expression and the other five patients (21.7%) presented gene expression that was similar to the ganglionic and intermediate areas. The results reinforce the conclusion that the method of AChE staining is effective for the diagnosis of intestinal aganglionosis and confirm the knowledge that genes beyond RET may be implicated in the genesis of sporadic cases of HD.

Study of acetylcholinesterase activity in rectal suction biopsy for diagnosis of intestinal dysganglionoses: 17-year experience of a single center.

Although the utility of the acetylcholinesterase (AChE) histochemistry on rectal suction biopsy in diagnosing Hirschsprung's disease (HD) has been documented, few reports address a great number of biopsies and patients. Our aim is to present a 17-year experience on the method of rectal suction biopsy and AChE histochemical staining for diagnosis of intestinal dysganglionoses. Between August 1989 and July 2006, 297 children suspected of having HD were submitted to rectal suction biopsies that were evaluated by the same two surgeons. There were 18 complications (6.0%), namely one self-limited rectal bleeding and 17 (5.7%) inadequate procedures that were repeated. A total of 157 patients (52.8%) showed no increased AChE activity and the remaining patients (140-47.2.0%) presented patterns of increased AChE activity confirming the diagnosis of HD or neuronal intestinal dysplasia. Among the 140 cases suspected as having HD, in 131 children the diagnosis of HD was confirmed and they were operated on. The histological studies showed that 111 children presented the classic form of HD or a long spastic segment. Sixteen children presented total colonic aganglionosis and four children proved to have intestinal neuronal dysplasia, according to histological and radiological criteria. Nine (6.6%) newborns were identified as false-positives and no false-negative results were verified. The rectal suction biopsy combined with AChE staining is advantageous for the differentiation between normal bowel and intestinal dysganglionoses. The rectal suction method is simple and can easily be performed by experienced surgeons. The histological evaluation is very objective and can be performed by a non-pathologist.

Effect of the immunosuppressants on hepatocyte proliferation and apoptosis in a young animal model of liver regeneration: an immunohistochemical study using tissue microarrays.

Hepatocyte proliferation and apoptosis (programmed cell death) occur during the liver parenchyma regeneration and the liver size modeling is mainly controlled by hepatocyte apoptosis. The purpose of the present study was to verify the influence of immunosuppressant drugs on these phenomena by utilizing tissue microarray techniques. Thirty-six weaning rats (age 21-23 days, weight 30-50 g) were divided into six groups: control, sham, hepatectomy, hepatectomy plus solumedrol, hepatectomy plus CsA, and hepatectomy plus Tac. The animals were killed one day after hepatectomy, and the remnant livers were weighed and harvested for tissue microarray sections. Liver cell proliferation was evaluated by staining for PCNA and apoptosis was detected by the TUNEL method. It was verified that CsA promoted a decrease in the liver weight, Tac and CsA decreased the proliferation index of hepatocytes, and glucocorticoid had no significant effects. The apoptosis index was not altered by hepatectomy or immunosuppressants. Our data indicate that, in the growing rat, CsA and Tac have negative effects on hepatocyte proliferation and have no effect on the hepatocyte apoptosis.

Expression of interleukin 6 and apoptosis-related genes in suckling and weaning rat models of hepatectomy and liver regeneration.

BACKGROUND/PURPOSE: The most commonly used model to study the mechanisms of liver regeneration is the adult rat submitted to 70% to 80% hepatectomy. However, there are no studies using newborn or weaning rat models. The process of liver regeneration includes both the hypertrophy and hyperplasia of cells (processes regulated by growth factors and cytokines, mainly interleukin 6 [IL-6]) as well as apoptosis, or programmed cell death (a process regulated mainly by the Bcl-2 family of proteins). Proapoptotic proteins in this family include Bax and Bak. Conversely, Bcl-2 and Bcl-XL are antiapoptotic regulators. Therefore, to expand our understanding of liver regeneration, our study had 2 goals: first, to standardize 2 animal models of hepatectomy and liver regeneration using the newborn suckling and the weaning rat and second, to quantitate the expression levels of IL-6 and several members of the Bcl-2 gene family during the regeneration process. METHODS: To create the experimental models, newborn suckling rats (age, 5-7 days; weight, 6-10 g) and weaning rats (age, 21-23 days; weight, 30-50 g) underwent 70% hepatectomy. The animals were subsequently sacrificed at days 1, 2, 3, 4, and 7 after hepatectomy, and the remnant liver lobes were harvested for routine histologic examination. Groups of healthy animals not operated on served as controls. For the experimental study, 6 newborn rats and 6 weaning rats underwent hepatectomy. The animals were killed 1 day after liver resection and the remnant livers were harvested to assess gene expression by quantitative reverse transcription-polymerase chain reaction. The hepatectomized groups were compared with control and sham groups. RESULTS: During the creation of the experimental models, 70% of the suckling animals and all the weaning animals survived the hepatectomy. The decreased liver weight was completely restored to control levels by day 7 after hepatectomy. Histologically, the remnant livers of both hepatectomy groups exhibited steatosis, tumefaction of hepatocytes, and mitosis, which ceased at 7 days after the hepatectomy. The weaning rat model showed more robust gene expression responses. Specifically, expression levels of IL-6 gene were significantly increased after both surgical insult (sham group) and hepatectomy. However, this increase was significantly higher in the latter group. Furthermore, hepatectomy promoted a decrease in the expression levels of the proapoptotic genes and an increase in the expression levels of Bcl-2. CONCLUSIONS: Our data indicate that regulation of both IL-6 and genes involved in apoptosis are strongly implicated in the mechanisms of liver regeneration and that the weaning rat model represents an attractive model system for future investigations in this area.