Yuan Ji

Small-for-Size Syndrome in the Rat: Does Size or Technique Matter?

BACKGROUNDnThe incidence of the small-for-size syndrome (SFSS) is inversely correlated to the size of the remnant liver or the partial graft. The relevance of factors besides the absolute liver mass is discussed controversially. It is the aim of this study to test the effect of two different mass ligation techniques in comparison with our newly developed parenchyma-preserving vessel-oriented liver resection technique on the induction of a SFSS after extended 90% liver resection in the rat.nnnMATERIALS AND METHODSnNinety percent liver resections were performed using three surgical techniques, two mass ligation techniques, and a vessel oriented technique. Diagnosis of SFSS was based on the combination of biochemical and morphological criteria on the first postoperative day and was related to the outcome on postoperative day 7 and the regenerative capacity of the liver.nnnRESULTSnOnly the use of mass ligation techniques was associated with a low 1-wk survival rate (<40%), more pronounced histomorphological signs of liver damage at 24 h postoperatively, and a delayed onset of hepatocyte proliferation. Histological analysis revealed an extended stump necrosis in the paracaval liver and signs of sinusoidal damage in the remaining caudate lobes as morphological correlates of a putative outflow obstruction as the possible underlying reason. The lesions added up to a high small-for-size score in rats operated according to mass ligation techniques.nnnCONCLUSIONSnThese results indicate that preservation of functional liver mass and prevention of an outflow obstruction by delicate surgery is essential to prevent a SFSS in a size-reduced liver.

G-CSF Administration in a Small-for-Size Liver Model

Background: Extended liver resection is applied in the treatment of liver tumors and during living-related liver transplantation. This procedure can lead to transient, in some patients even lethal liver failure. Administration of G-CSF is associated with an increased survival after toxic liver damage. It is the aim of this study to test the effect of G-CSF administration in the rat 90% extended liver resection model. Materials: Rats with and without G-CSF treatment were subjected to 90% partial hepatectomy using a mass ligation technique. Animals were sacrificed 6 hr, 24 hr, and 7 days after resection. Read-out parameters were liver damage in terms of liver enzymes and histomorphological alterations. Hepatic regeneration was determined by measuring liver weight recovery and calculating the BrdU proliferation index of hepatocytes. G-CSF-receptor expression was visualized in both groups by immunohistochemistry. Expression of lipopolysaccharide-binding-protein (LBP) mRNA was evaluated by quantitative PCR. Results: Survival rate was increased in the G-CSF-treatment group. Full liver weight recovery within one week was only achieved in the treatment group and was accompanied by reduced liver damage. G-CSF-receptor upregulation subsequent to administration of G-CSF may indicate a direct receptor mediated effect of G-CSF on the liver. Upregulation of LBP mRNA in the liver of G-CSF treated animals—reported to be associated with an increased host defense—could demonstrate a mode of action of G-CSF.

Impact of donor gender on male rat recipients of small-for-size liver grafts

The aim of this study was to assess the impact of donor gender on small‐for‐size (SFS) liver transplantation in male recipients using a rat model. Adult female or male Lewis rats were used as donors and male Lewis rats as recipients. Size‐matched (SM) and SFS liver grafts from either male or female donors were transplanted into male recipients. Animals receiving SFS grafts were sacrificed at postoperative week 1, week 4, and week 12, respectively (n = 6‐8 per group), those receiving SM grafts after 3 months. The cumulative survival rate (SVR) in the female‐to‐male (F‐M) SFS group was significantly lower (62%; 13 of 21) compared with the male‐to‐male (M‐M) group (90%; 18 of 20) (P < 0.05). Spontaneous death occurred in the F‐M SFS combination either in the early postoperative period (<3 weeks) in animals with confluent hepatic necrosis or in the late postoperative period (>8 weeks) in animals with biliary obstruction. In contrast, no death was observed in the early posttransplantation period after M‐M liver transplantation. The relative graft size in the SM F‐M group was significantly higher (graft‐to‐recipient weight ratio [GRWR] 2.40% ± 0.8%) than in the SFS M‐M group (GRWR 1.35% ± 0.2%; P < 0.001). Regardless of graft size, the outcome was worse in terms of SVR as well as regarding the incidence and severity of biliary complications in F‐M compared with M‐M liver transplantation. In conclusion, male recipients of female livers had a less favorable outcome irrespective of graft size. Confluent hepatic necrosis as well as biliary obstruction were perceived as consequence of a severe perfusion problem in F‐M liver transplantation, which was possibly related to an enhancement of ischemia‐reperfusion (I/R) injury by the lack of estrogen in male recipients of female grafts. (Liver Transpl 2005;11:669–678.)

Influence of stem cell mobilization and liver regeneration on hepatic parenchymal chimerism in the rat.

Background. Despite the newly discovered plasticity of hematopoietic stem cells, their capacity to “transdifferentiate” into parenchymal cells and the regulation of this process is not yet elucidated. The present study was designed to investigate the effect of stem cell mobilization and liver regeneration on this process using a syngeneic rat female-to-male liver transplantation model. Methods. Rate and frequency of Y-chromosome containing hepatocytes was determined by fluorescence-in situ hybridization (FISH) using a rat Y-chromosome specific probe. Rats were subjected to full-size or partial liver transplantation with and without stem cell mobilization using granulocyte colony stimulating factor (G-CSF). The effect of stem cell mobilization was confirmed by assessing the white blood count and by evaluating the migration of granulopoietic precursor cells to the liver. Results. Treatment with G-CSF induced a twofold increase of peripheral white blood cells and a strong influx of granulopoietic precursor cells into the liver. Transplantation of a partial liver graft was followed by a 90% recovery of the original liver weight within a week, demonstrating the strong regenerative stimulus. Irrespective of the experimental model, the incidence and rate of Y-chromosome containing hepatocytes never exceeded 0.77%. Conclusion. Neither stem cell mobilization nor liver regeneration enhanced the incidence or rate of stem cell derived hepatocytes in a liver graft with unimpaired regenerative potential.

Induction of Rejection After Small-for-Size Liver Transplantation: Size Matters

Background: Reduced-size liver transplantation is associated with liver regeneration. This study was designed to analyze the influence of graft size on liver rejection and liver regeneration. Methods: Reduced-size liver transplantations were performed in the rejecting ACI to Lewis and the graft acceptance BN to Lewis strain combination. The BN to Lewis control group was treated with the immunosuppressive drug FK506. Results: An accelerated liver rejection in the ACI to Lewis strain combination was found in small-for-size partial liver grafts. Graft weight to recipient liver weight ratio (GW/RLW) showed a positive correlation with survival time. In the BN to Lewis strain combination, lethal rejection was seen in small-for-size partial liver grafts. A critical immunologic GW/RLW of 33% was calculated. In rats dying from lethal rejection, GW/RLW and survival time showed a positive correlation. However, GW/RLW showed a negative correlation with hepatocellular proliferation. In regenerating livers, MHC II upregulation was also observed in the control group. All control animals survived small-for-size liver transplantation. Conclusions: The relative graft size seems to be a decisive factor influencing the kinetic of liver rejection and the induction of liver rejection. Relative critical immunologic liver mass was determined to be 33%.

Probe production for in situ hybridization by PCR and subsequent covalent labeling with fluorescent dyes.

A simple procedure for fluorescent labeling of probes just before in situ hybridization is provided. Aminoallyl-dUTP is introduced during probe production by polymerase chain reaction (PCR). The aminoallyl-dUTP functions as a reactive site for subsequent labeling of the probe. Activated fluorescent dyes such as fluorescein are covalently attached to the probe through the formation of a stable amide bond. Labeled probes are purified by size-exclusion gel chromatography to remove unincorporated dye. Target genes used to demonstrate the efficacy of this technique with in situ hybridization are rat Y-chromosome and rat granulocyte colony-stimulating factor receptor. PCR amplicons containing aminoallyl-dUTP were produced in high yield. Probes obtained after labeling with activated fluorophores demonstrated high intrinsic activity within in situ hybridizations. The introduction of aminoallyl-dUTP into the PCR reaction enables the production of “unlabeled” probes by PCR having a shelf life, which is not limited by the storage and stability challenges of fluorophore-labeled probes. Subsequent labeling of the probes with activated fluorescent dyes just before use allows one step in situ hybridization with high activity and minimal background staining.

Preliminary investigation of mitoxantrone coating on stent-grafts to inhibit neointimal proliferation.

Purpose: To investigate the inhibition of neointimal proliferation induced by a stent-graft loaded with mitoxantrone. Methods: Stent-grafts with and without mitoxantrone loading (150 μg per device) were inserted into the carotid artery of 7 and 6 rabbits, respectively. After an observation period of 28 days, the animals were sacrificed, and a detailed morphological and morphometric workup of the stented vessels was performed. Results: Uncoated stent-grafts induced a thick neointima (median diameter 97±99 μm), whereas no neointima formed in mitoxantrone-loaded stent-grafts. However, the loaded devices were not fully covered by an endothelial layer. The underlying media was significantly thinner (31.8±5.6 versus 48.6±3.3 μm, p=0.002) and showed a widespread loss of smooth muscle cells. Conclusions: Mitoxantrone loading of a stent-graft inhibited the formation of a neointima. However, important regenerative processes were prevented as well, indicating a local overdose. More experiments using lower doses are warranted.