Maria Cecília Mendonça Coelho

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.

Is procalcitonin useful to differentiate rejection from bacterial infection in the early post-operative period of liver transplantation in children?

Abstract:  PCT is a protein that is recognized as an acute marker of inflammation. Previous studies performed in adults who underwent liver or heart transplantation indicated that PCT plasmatic levels help to differentiate between rejection and infection. The objective of this study was to evaluate whether PCT has the same role in liver‐transplanted children. Thirty‐six patients were studied between the first and the thirtieth post‐operative days, and PCT determinations were prospectively performed according to the clinical status of the patient. In the non‐complicated patients, PCT measurements performed on the first and second post‐operative days revealed a median value of 1.60 ng/mL (mean 5.68 ± 7.05; range 0.69–18.30). After the fourth day of transplantation, PCT plasma concentrations decreased to a median value of 0.21 ng/mL (mean 0.47 ± 0.59; range 0.05–2.00; normal values are less than 0.5 ng/mL). In infected patients, PCT plasma levels demonstrated a significant increase, differing from the patients with acute liver rejection whose levels were similar to those of non‐complicated patients. In conclusion, we could demonstrate that in the early post‐operative period of liver transplantation in children, measuring PCT plasmatic levels might be a useful tool for differentiation between bacterial infection and acute liver rejection.

Experimental models of hepatectomy and liver regeneration using newborn and weaning rats

OBJECTIVES Liver regeneration is a complex process that has not been completely elucidated. The model most frequently used to study this phenomenon is 70% hepatectomy in adult rats; however, no papers have examined this effect in developing animals. The aims of the present study were: 1) to standardize two models of partial hepatectomy and liver regeneration in newborn suckling and weaning rats, and 2) to study the evolution of remnant liver weight and histological changes of hepatic parenchyma on the days that follow partial hepatectomy. METHODS Fifty newborn and forty-four weaning rats underwent 70% hepatectomy. After a midline incision, compression on both sides of the upper abdomen was performed to exteriorize the right medial, left medial and left lateral hepatic lobes, which were tied inferiorly and resected en bloc. The animals were sacrificed on days 0 (just after hepatectomy), 1, 2, 3, 4 and 7 after the operation. Body and liver weight were determined, and hepatic parenchyma was submitted to histological analysis. RESULTS Mortality rates of the newborn and weaning groups were 30% and 0%, respectively. There was a significant decrease in liver mass soon after partial hepatectomy, which completely recovered on the seventh day in both groups. Newborn rat regenerating liver showed marked steatosis on the second day. In the weaning rat liver, mitotic figures were observed earlier, and their amount was greater than in the newborn. CONCLUSIONS Suckling and weaning rat models of partial hepatectomy are feasible and can be used for studies of liver regeneration. Although similar, the process of hepatic regeneration in developing animals is different from adults.

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 Immunosuppressants on Hepatocyte Cell Mitosis During Liver Regeneration in Growing Animal Models of Partial Hepatectomy

OBJECTIVE We sought to evaluate the effects of immunosuppressant drugs (corticosteroid, cyclosporine [CsA], and tacrolimus [Tac]) on liver regeneration in growing animals submitted to 70% hepatectomy. MATERIALS AND METHODS Newborn and weaning rats were submitted to 70% hepatectomy receiving separately methylprednisolone, CsA, or Tac. All animals were sacrificed 24 hours after the procedure. The remnant liver lobes were subjected to histomorphometric analyses with determination of hepatocyte mitotic index. RESULTS Administration of immunosuppressants did not change the mitotic index of the regenerating liver in newborn animals. In weaning rats, methylprednisolone reduced the mitotic index (P = .01) and Tac caused a greater increase in this rate (P = .001). CsA had no effect on mitotic index. The number of hepatocyte mitoses in newborn animal livers was greater than that in weaning animal livers (P = .001). CONCLUSION In situations in which intense, fast processes of liver regeneration are crucial, the advantages of the use of Tac must be considered, such as in pediatric transplant patients.

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.

Effect of the immunosuppressants on hepatocyte cells proliferation and apoptosis during liver regeneration after hepatectomy – molecular studies

Abstract:  The regeneration and remodeling of the transplanted liver is the result of hepatocyte proliferation and apoptosis (programmed cell death). The purpose of this study was to verify the influence of immunosuppressants on the expression levels of genes: IL‐6 (regulator of hepatocyte proliferation), pro‐apoptotic (Bak and Bax) and anti‐apoptotic (Bcl‐Xl and Bcl‐2). 36 newborn suckling rats (age 5–7 days, weight 6–10 g) were divided into four groups: hepatectomy, hepatectomy plus methylprednisolone, hepatectomy plus CsA and hepatectomy plus Tac. The same experiments were performed in 24 weaning rats (age 21–23 days, weight 30–50 g). The animals were killed one day after the hepatectomy and the remnant livers were analyzed. The livers of all animals exhibited histological changes of liver regeneration. The immunosuppressants did not promote any alteration on IL‐6 gene expression levels. Methylprednisolone and CsA increased the expression levels of Bak gene in newborn rats. However, methylprednisolone and Tac promoted increased expression levels of Bcl‐2 in all groups. We hypothesize that these effects explain the efficacy of these drugs on the treatment of acute and chronic liver rejection as the expression of Bcl‐2 in cholangiocytes is decreased as a consequence of bile duct lesions.

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.

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

Abstract:  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.