Josiane de Oliveira Gonçalves

Dynamic expression of desmin, α-SMA and TGF-β1 during hepatic fibrogenesis induced by selective bile duct ligation in young rats

We previously described a selective bile duct ligation model to elucidate the process of hepatic fibrogenesis in children with biliary atresia or intrahepatic biliary stenosis. Using this model, we identified changes in the expression of alpha smooth muscle actin (α-SMA) both in the obstructed parenchyma and in the hepatic parenchyma adjacent to the obstruction. However, the expression profiles of desmin and TGF-β1, molecules known to be involved in hepatic fibrogenesis, were unchanged when analyzed by semiquantitative polymerase chain reaction (RT-PCR). Thus, the molecular mechanisms involved in the modulation of liver fibrosis in this experimental model are not fully understood. This study aimed to evaluate the molecular changes in an experimental model of selective bile duct ligation and to compare the gene expression changes observed in RT-PCR and in real-time quantitative PCR (qRT‐PCR). Twenty-eight Wistar rats of both sexes and weaning age (21-23 days old) were used. The rats were separated into groups that were assessed 7 or 60 days after selective biliary duct ligation. The expression of desmin, α-SMA and TGF-β1 was examined in tissue from hepatic parenchyma with biliary obstruction (BO) and in hepatic parenchyma without biliary obstruction (WBO), using RT-PCR and qRT‐PCR. The results obtained in this study using these two methods were significantly different. The BO parenchyma had a more severe fibrogenic reaction, with increased α-SMA and TGF-β1 expression after 7 days. The WBO parenchyma presented a later, fibrotic response, with increased desmin expression 7 days after surgery and increased α-SMA 60 days after surgery. The qRT‐PCR technique was more sensitive to expression changes than the semiquantitative method.

Effects of ischemic preconditioning in a pig model of large-for-size liver transplantation

OBJECTIVE: In most cases of pediatric liver transplantation, the clinical scenario of large-for-size transplants can lead to hepatic dysfunction and a decreased blood supply to the liver graft. The objective of the present experimental investigation was to evaluate the effects of ischemic preconditioning on this clinical entity. METHODS: Eighteen pigs were divided into three groups and underwent liver transplantation: a control group, in which the weights of the donors were similar to those of the recipients, a large-for-size group, and a large-for-size + ischemic preconditioning group. Blood samples were collected from the recipients to evaluate the pH and the sodium, potassium, aspartate aminotransferase and alanine aminotransferase levels. In addition, hepatic tissue was sampled from the recipients for histological evaluation, immunohistochemical analyses to detect hepatocyte apoptosis and proliferation and molecular analyses to evaluate the gene expression of Bax (pro-apoptotic), Bcl-XL (anti-apoptotic), c-Fos and c-Jun (immediate-early genes), ischemia-reperfusion-related inflammatory cytokines (IL-1, TNF-alpha and IL-6, which is also a stimulator of hepatocyte regeneration), intracellular adhesion molecule, endothelial nitric oxide synthase (a mediator of the protective effect of ischemic preconditioning) and TGF-beta (a pro-fibrogenic cytokine). RESULTS: All animals developed acidosis. At 1 hour and 3 hours after reperfusion, the animals in the large-for-size and large-for-size + ischemic preconditioning groups had decreased serum levels of Na and increased serum levels of K and aspartate aminotransferase compared with the control group. The molecular analysis revealed higher expression of the Bax, TNF-alpha, I-CAM and TGF-beta genes in the large-for-size group compared with the control and large-for-size + ischemic preconditioning groups. Ischemic preconditioning was responsible for an increase in c-Fos, IL-1, IL-6 and e-NOS gene expression. CONCLUSION: Ischemia-reperfusion injury in this model of large-for-size liver transplantation could be partially attenuated by ischemic preconditioning.

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 β1 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.

Large-for-size liver transplantation: a flowmetry study in pigs.

BACKGROUND Ischemia-reperfusion injury is partly responsible for morbidity in pediatric liver transplantation. Large-for-size (LFS) liver transplantation has not been fully studied in the pediatric population, and the effects of reperfusion injury may be underestimated. MATERIALS AND METHODS Thirteen Landrace-Large white pigs weighing 23 kg (range, 17-38 kg) underwent orthotopic liver transplantation. They were divided into two groups according to the size of the donor body: LFS and control (CTRL). After transplantation, the abdominal cavity of the recipient was kept open and portal venous flow (PVF) was measured after 1 h. The ratio of recipient PVF (PVFr) to donor PVF was used to establish correlations with ischemia and reperfusion parameters. Liver biopsies were taken 1 h after transplantation to assess ischemia and reperfusion and to quantify the gene expression of endothelial nitric oxide synthase, interleukin 6, BAX, and BCL. RESULTS Recipient weight, total ischemia time, and warm ischemia time were similar between groups. Among hemodynamic and metabolic analyses, pH, central arteriovenous PCO2 difference, and AST were statistically worse in the LFS group than in the CTRL group. The same was found with endothelial nitric oxide synthase (0.41 ± 0.18 versus 1.56 ± 0.78; P = 0.02) and interleukin 6 (4.66 ± 4.61 versus 16.21 ± 8.25; P = 0.02). In the LFS group, a significant decay in the PVFr was observed in comparison with the CTRL group (0.93 ± 0.08 and 0.52 ± 0.11, respectively; P < 0.001). CONCLUSIONS The implantation of a graft was responsible for poor hemodynamic status of the recipient 1 h after transplantation. Furthermore, the LFS group demonstrated markers of ischemia and reperfusion that were worse when compared with the CTRL group and exhibited a more significant decrease in PVF from donor to recipient.

A simplified experimental model of large-for-size liver transplantation in pigs

OBJECTIVE: The ideal ratio between liver graft mass and recipient body weight for liver transplantation in small infants is unknown; however, if this ratio is over 4%, a condition called large-for-size may occur. Experimental models of large-for-size liver transplants have not been described in the literature. In addition, orthotopic liver transplantation is marked by high morbidity and mortality rates in animals due to the clamping of the venous splanchnic system. Therefore, the objective of this study was to create a porcine model of large-for-size liver transplantation with clamping of the supraceliac aorta during the anhepatic phase as an alternative to venovenous bypass. METHOD: Fourteen pigs underwent liver transplantation with whole-liver grafts without venovenous bypass and were divided into two experimental groups: the control group, in which the weights of the donors were similar to the weights of the recipients; and the large-for-size group, in which the weights of the donors were nearly 2 times the weights of the recipients. Hemodynamic data, the results of serum biochemical analyses and histological examination of the transplanted livers were collected. RESULTS: The mortality rate in both groups was 16.5% (1/7). The animals in the large-for-size group had increased serum levels of potassium, sodium, aspartate aminotransferase and alanine aminotransferase after graft reperfusion. The histological analyses revealed that there were no significant differences between the groups. CONCLUSION: This transplant method is a feasible experimental model of large-for-size liver transplantation.

Immediate expression of c-fos and c-jun mRNA in a model of intestinal autotransplantation and ischemia-reperfusion in situ

OBJECTIVE: Intestinal ischemia-reperfusion injury occurs in several clinical conditions and after intestinal transplantation. The aim of the present study was to investigate the phenomena of apoptosis and cell proliferation in a previously described intestinal ischemia-reperfusion injury autograft model using immunohistochemical markers. The molecular mechanisms involved in ischemia-reperfusion injury repair were also investigated by measuring the expression of the early activation genes c-fos and c-jun, which induce apoptosis and cell proliferation. MATERIALS AND METHODS: Thirty adult male Wistar rats were subjected to surgery for a previously described ischemia-reperfusion model that preserved the small intestine, the cecum and the ascending colon. Following reperfusion, the cecum was harvested at different time points as a representative segment of the intestine. The rats were allocated to the following four subgroups according to the reperfusion time: subgroup 1: 5 min; subgroup 2: 15 min; subgroup 3: 30 min; and subgroup 4: 60 min. A control group of cecum samples was also collected. The expression of c-fos, c-jun and immunohistochemical markers of cell proliferation and apoptosis (Ki67 and TUNEL, respectively) was studied. RESULTS: The expression of both c-fos and c-jun in the cecum was increased beginning at 5 min after ischemia-reperfusion compared with the control. The expression of c-fos began to increase at 5 min, peaked at 30 min, and exhibited a declining tendency at 60 min after reperfusion. A progressive increase in c-jun expression was observed. Immunohistochemical analyses confirmed these observations. CONCLUSION: The early activation of the c-fos and c-jun genes occurred after intestinal ischemia-reperfusion injury, and these genes can act together to trigger cell proliferation and apoptosis.

Analysis of the reversibility of biliary cirrhosis in young rats submitted to biliary obstruction

BACKGROUND/PURPOSE Biliary atresia and other liver biliary obstructions are relevant conditions in pediatric surgery due to their progression to biliary cirrhosis and indication for liver transplantation. It is known that the period during which biliary obstruction persists determines the development of cirrhosis and its reversibility after a biliary drainage procedure. However, no time or histological markers of biliary cirrhosis reversibility have been established. MATERIALS AND METHODS One hundred and twenty-nine young Wistar rats underwent surgery for ligation of the common bile duct and were maintained until 8weeks. A part of these animals was submitted to biliary drainage surgery at 2, 3, 4, 5, or 6weeks after the initial procedure. After cyst formation at the site of obstruction, cyst-jejunal anastomosis was performed to restore bile flow. After biliary obstruction and drainage, liver samples were collected for histological and molecular analysis of the genes responsible for collagen deposition and fibrosis. RESULTS The mortality rates were 39.8% and 56.7% after the first and second procedures, respectively. Ductular proliferation (p=0.001) and collagen deposition increased according to the period under obstruction (p=0.0001), and both alterations were partially reduced after biliary drainage. There were no significant differences in the values of desmin and α-actin according to the period during which the animal remained with biliary obstruction (p=0.09 and p=0.3, respectively), although increased values of transforming growth factor beta 1 (TGFβ1) occurred after 8weeks (p=0.000). Desmin levels decreased, and α-actin and TGFβ1 levels increased according to the period under obstruction. The molecular alterations were partially reversed after biliary drainage. CONCLUSIONS The histologic and molecular changes in the liver parenchyma promoted by biliary obstruction in the young animal can be partially reversed by a biliary drainage procedure.

Are there differences in the growth adaptation processes of growing and mature organism models of short bowel syndrome

OBJECTIVES: The purpose of this study was to present an experimental model of short bowel syndrome (SBS) in weaning rats and to compare the adaptative mechanisms of the remaining bowel in weaning rats and adult animals by means of morphometric, histologic and molecular methods. METHODS: Twenty-four weaning rats were divided into 3 groups of 8 animals, one control group and two short bowel groups (euthanasia after 4 and 21 days), and were compared with similar adult groups. Morphometric evaluations of the animals and histopathological and molecular studies of the remaining bowel were performed. RESULTS: The weight of young rats increased after enterectomy, whereas that of adult rats decreased after enterectomy (p<0.0001). The ratio of intestinal length/body weight was significantly higher in weaning rats than in adults (p<0.002), showing that intestinal growth was more intense in weaning rats. Intestinal resection promoted increased thickness of the small bowel lamina propria (p=0.001) and reduced thickness of the colon lamina propria (p=0.04) in weaning rats relative to those in adults. In addition, intestinal resection promoted increased expression of the Bcl-xl gene (antiapoptotic) in adult animals compared with that in weaning rats (p=0.001). CONCLUSION: Morphometric, histological and molecular differences were shown in the adaptation processes of growing and mature organisms.

Does a meso-caval shunt have positive effects in a pig large-for-size liver transplantation model?

In pediatric liver transplantations with LFS grafts, higher incidences of graft dysfunction probably occur due to IRI. It was postulated that increasing the blood supply to the graft by means of a meso‐caval shunt could ameliorate the IRI. Eleven pigs underwent liver transplantation and were divided into two groups: LFS and LFS+SHUNT group. A series of flowmetric, metabolic, histologic, and molecular studies were performed. No significant metabolic differences were observed between the groups. One hour after reperfusion, portal flow was significantly lower in the recipients than in the donors, proving that the graft was maintained in low portal blood flow, although the shunt could promote a transient increase in the portal blood flow and a decrease in the arterial flow. Finally, it was verified that the shunt promoted a decrease in inflammation and steatosis scores and a decrease in the expression of the eNOS gene (responsible for the generation of nitric oxide in the vascular endothelium) and an increase in the expression of the proapoptotic gene BAX. The meso‐caval shunt was responsible for some positive effects, although other deleterious flowmetric and molecular alterations also occurred.

Is hematoxylin-eosin staining in rectal mucosal and submucosal biopsies still useful for the diagnosis of Hirschsprung disease?

BackgroundHematoxylin-eosin (HE) staining of a full-thickness rectal wall fragment is classically used for the diagnosis of Hirschsprung disease (HD). However, this technique requires large fragments for a better diagnosis. Additionally, the histochemical and immunohistochemical methods of staining small fragments of rectal mucosal and submucosal biopsies are not available in all centers. Therefore, the possibility of diagnosing HD through HE staining in these biopsies could be a valuable alternative for centers that do not have more specific techniques. The objectives of the current investigation were to evaluate the concordance of the results obtained by HE staining and the calretinin method with acetylcholinesterase (AChE) activity in fragments of mucosa and submucosa in the diagnosis of HD.MethodsFor this study, 50 cases from our laboratory were selected. The tissue material was embedded in paraffin. Sixty levels of each fragment were utilized for HE, and the other 3 levels were used for calretinin. These slides were analyzed under the microscope, photographed and classified as either positive for HD when no ganglion cells were found with nerve trunks present or as negative when ganglion cells were found. The results from reading the slides were compared with those of AChE.ResultsOf the 50 cases evaluated by the HE technique, only 5 contradicted the diagnosis based on AChE, with a Kappa value of 0.800 and an accuracy of 90%. In the comparison between calretinin and AChE, 8 cases were discordant, with a Kappa value of 0.676 and an accuracy of 84%.ConclusionsThe concordance of results from AChE and HE methods was satisfactory, allowing for the potential use of the HE method for fragments of mucosa and submucosa as a valid alternative in the diagnosis of HD. The immunohistochemical technique of calretinin did not show good agreement with the AChE activity in our study.