David Sindram

Protective effects of ischemic preconditioning for liver resection performed under inflow occlusion in humans.

ObjectiveTo determine whether ischemic preconditioning protects the human liver against a subsequent period of ischemia in patients undergoing hemihepatectomy, and to identify possible underlying protective mechanisms of ischemic preconditioning, such as inhibition of hepatocellular apoptosis. Summary Background DataIschemic preconditioning is a short period of ischemia followed by a brief period of reperfusion before a sustained ischemic insult. Recent studies in rodents suggest that ischemic preconditioning is a simple and powerful protective modality against ischemic injury of the liver. The underlying mechanisms are thought to be related to downregulation of the apoptotic pathway. MethodsTwenty-four patients undergoing hemihepatectomy for various reasons alternatively received ischemic preconditioning (10 minutes of ischemia and 10 minutes of reperfusion) before transection of the liver performed under inflow occlusion for exactly 30 minutes. Liver wedge and Tru-cut biopsy samples were obtained at the opening of the abdomen and 30 minutes after the end of the hepatectomy. Serum levels of aspartate transferase, alanine transferase, bilirubin and prothrombin time were determined daily until discharge. Hepatocellular apoptosis was evaluated by in situ terminal deoxynucleotidyl transferase mediated d-UTP nick end-labeling (TUNEL) assay and electron microscopy. Caspase 3 and 8 activities were measured in tissue using specific fluorometric assays. ResultsSerum levels of aspartate transferase and alanine transferase were reduced by more than twofold in patients subjected to ischemic preconditioning versus controls. The analysis of a subgroup of patients with mild to moderate steatosis indicated possible increased protective effects of ischemic preconditioning. In situ TUNEL staining demonstrated a dramatic reduction in the number of apoptotic sinusoidal lining cells in the ischemic preconditioning group. Electron microscopy confirmed features of apoptosis present in control but not in ischemic preconditioning patients. There was no significant difference in caspase 3 and 8 activity when patients with ischemic preconditioning were compared with controls. ConclusionsIschemic preconditioning is a simple and effective modality protecting the liver against subsequent prolonged periods of ischemia. This strategy may be a more attractive technique than intermittent inflow occlusion, which is associated with increased blood loss during each period of reperfusion.

Platelets induce sinusoidal endothelial cell apoptosis upon reperfusion of the cold ischemic rat liver

BACKGROUND & AIMSnSinusoidal endothelial cell (SEC) apoptosis is a central feature of reperfusion injury in liver transplantation. Platelet sequestration occurs after transplantation with possible deleterious effects. We tested the hypothesis that platelets mediate SEC apoptosis.nnnMETHODSnLivers were perfused after 24 hours of cold preservation in University of Wisconsin solution in an isolated perfused rat liver model. The perfusate contained isolated syngeneic red blood cells and purified platelets. Effects of inhibiting platelet adhesion on SEC apoptosis was tested using sialyl Lewis-X oligosaccharide (sLe(x)), a natural ligand of selectin adhesion molecules. Reperfusion injury was assessed by established markers of injury. Apoptosis was determined by TUNEL and electron microscopy.nnnRESULTSnA third of the circulating platelets was rapidly sequestered in the liver after reperfusion. This was associated with increased graft injury. Single platelets were adherent to sinusoidal lining without morphological or dynamic evidence of impairment of microcirculation. TUNEL staining revealed a 6-fold increase in the number of apoptotic SECs at 1 hour of reperfusion. No hepatocyte death or evidence of necrosis was detected up to 3 hours of reperfusion. Addition of sLe(x) inhibited adhesion and significantly reduced SEC apoptosis.nnnCONCLUSIONSnPlatelets cause SEC apoptosis upon reperfusion of liver grafts. Prevention of adhesion is protective.

Comparison of Ischemic Preconditioning and Intermittent and Continuous Inflow Occlusion in the Murine Liver

ObjectiveTo compare protection of the liver by ischemic preconditioning and intermittent inflow occlusion in a mouse model of prolonged periods of ischemia. Summary Background DataPreconditioning (short ischemic stress prior to a prolonged period of ischemia) and intermittent inflow occlusion protect the liver against reperfusion injury. This is the first study comparing these two modalities with continuous inflow occlusion (control). MethodsMice were subjected to 75 or 120 minutes of 70% hepatic ischemia and 3 hours of reperfusion. Each ischemic period was evaluated using three different protocols: continuous ischemia (control), preconditioning (10 minutes ischemia and 15 minutes reperfusion) prior to the prolonged ischemic insult, and intermittent clamping (cycles of 15 minutes ischemia and 5 minutes reperfusion). Organ injury was evaluated using serum levels of aspartate aminotransferase (AST), hematoxylin and eosin staining, and specific markers of apoptosis (cytochrome C release, caspase 3 activity, and TUNEL staining). Animal survival was determined using a model of total hepatic ischemia. ResultsIntermittent inflow occlusion and ischemic preconditioning were both protective against ischemic insults of 75 and 120 minutes compared with controls (continuous ischemia only). Protection against 75 minutes of ischemia was comparable in the intermittent clamping and the ischemic preconditioning group, whereas intermittent clamping was superior at 120 minutes of ischemia. One hundred percent animal survival was observed after 75 minutes of total hepatic ischemia using both protective protocols, whereas all animals subjected to continuous ischemia died after surgery. After 120 minutes of ischemia, intermittent inflow occlusion was associated with better animal survival (71%) compared with preconditioning (14%). ConclusionsPreconditioning and intermittent clamping are both protective against prolonged periods of ischemia. In the clinical setting, preconditioning is superior for ischemic periods of up to 75 minutes because it is not associated with blood loss during transection of the liver. However, for prolonged ischemic insults exceeding 75 minutes, intermittent clamping is superior to preconditioning.

Synergism between platelets and leukocytes in inducing endothelial cell apoptosis in the cold ischemic rat liver: a Kupffer cell-mediated injury.

Sinusoidal endothelial cell (SEC) apoptosis is the hallmark of early reperfusion injury in liver transplantation. Platelet sequestration occurs after transplantation and causes SEC apoptosis. Leukocytes also adhere to the liver upon reperfusion. Their role and interaction with platelets in inducing SEC apoptosis are unknown. Kupffer cells possibly interact with circulating cells; therefore, they could be involved in inducing SEC apoptosis. We tested the hypothesis that platelets and leukocytes synergistically induce SEC apoptosis through a Kupffer‐cell‐dependent mechanism. Livers were preserved for 24 h in cold University of Wisconsin solution and were reperfused in an isolated perfused rat liver model with perfusate containing red blood cells and either platelets, leukocytes, or both. In some experiments, Kupffer cells were inhibited by either gadolinium chloride or pentoxifylline. Apoptosis was determined after reperfusion by terminal deoxynucleotidyl transferase‐mediated nick‐end labeling and electron microscopy. Leukocytes and platelets were rapidly sequestered into the liver and induced SEC apoptosis. The presence of both platelet and leukocyte adhesion was associated with a dramatic increase in SEC apoptosis compared with liver reperfused with the use of one population of circulating elements or in absence of both platelets and leukocytes. In liver reperfused with platelets and leukocytes, SEC apoptosis was completely abrogated by either modality of Kupffer cell inhibition. Platelets and leukocytes induce SEC apoptosis upon reperfusion of cold preserved liver through a Kupffer‐cell‐dependent mechanism.

Transgenic mice overexpressing human Bcl-2 are resistant to hepatic ischemia and reperfusion

BACKGROUND/AIMSnApoptosis is a key mechanism of reperfusion injury in the ischemic liver. The apoptotic pathway is highly regulated by anti-apoptotic factors, such as Bcl-2. We evaluated the effect of Bcl-2 overexpression on apoptosis and the activation of the apoptotic cascade after hepatic ischemia and reperfusion.nnnMETHODSnNinety minutes of ischemia and reperfusion was performed in Bcl-2 transgenic and non-transgenic mice. Bcl-2 overexpression was determined by immunohistochemistry and Western blot. Liver injury was determined by aspartate aminotransferase (AST), Tunel test and the activation of the apoptotic cascade and animal survival.nnnRESULTSnBcl-2 overexpression was present in all hepatocytes and non-parenchymal liver cells in transgenic mice. Bcl-2 overexpression resulted in significant decreased AST levels after ischemic injury, and complete inhibition of apoptosis. After 90 min of total hepatic ischemia all control mice died, while four transgenic mice survived permanently. Bcl-2 overexpression was associated with inhibition of caspase 3 activation after reperfusion and increased baseline levels of cytoplasmic cytochrome c, caspase 3, and a reduction of Bcl-x(L) production.nnnCONCLUSIONSnBcl-2 overexpression protects against ischemic injury by inhibiting apoptosis. Extensive overproduction of Bcl-2 is associated with a compensatory increase of baseline levels of cytoplasmic cytochrome c and caspase 3, and a deletion of Bcl-x(L).

Ischemic preconditioning protects against cold ischemic injury through an oxidative stress dependent mechanism

BACKGROUND/AIMSnIschemic injury in cold preserved livers is characterized by sinusoidal endothelial cell (SEC) detachment and matrix metalloproteinase activity. Upon reperfusion reversible ischemic injury becomes permanent with SEC rapidly undergoing apoptosis. Ischemic preconditioning prevents reperfusion injury after normothermic ischemia. We hypothesized that ischemic preconditioning, through an oxygen free radical burst, protects against injury during cold preservation and reperfusion.nnnMETHODSnIschemic preconditioning was achieved in rats by clamping blood supply to the left and median lobes for 10 min followed by 15 min of reperfusion prior to preservation in cold University of Wisconsin solution for 30 h. In a second set of experiments, rats were pretreated with N-acetyl-cysteine (NAC). SEC apoptosis upon reperfusion was assessed in an isolated perfused rat liver (IPRL) model.nnnRESULTSnSEC detachment and activities of matrix metalloproteinase were significantly reduced in preconditioned livers. A decrease of SEC apoptosis after 1h of reperfusion in the IPRL was noted in preconditioned livers compared to controls. Pretreatment with NAC reversed the beneficial effects of ischemic preconditioning on SEC detachment and apoptosis.nnnCONCLUSIONSnIschemic preconditioning is an effective strategy to prevent injury during cold preservation and after reperfusion. The protective effect is possibly mediated by oxygen free radicals.

Calpain inhibition prevents sinusoidal endothelial cell apoptosis in the cold ischemic rat liver.

BACKGROUNDnCold preservation of the liver followed by reperfusion results in sinusoidal endothelial cell (SEC) apoptosis. Calpain-like activity is dramatically increased during reperfusion and inhibition of calpains results in lower graft injury and longer survival. Recently, calpains have been implicated in inducing apoptosis. Our aim was to determine the effect of calpain inhibition on SEC apoptosis.nnnMETHODSnLivers were stored in the University of Wisconsin solution for 24 hr (survival conditions) and 40 hr (nonsurvival conditions) and ex vivo reperfused for 1 hr at 37 degrees C. Calpain-like activity was inhibited in some experiments using an i.p. injection of a selective inhibitor 2 hr before explantation. Apoptosis was quantified using the terminal deoxynucleotidyl trans. ferase-mediated dUTP nick end-labeling assay. Cross-inhibition by the inhibitor was determined for caspases 1 and 3.nnnRESULTSnApoptosis of exclusively the SEC was a key feature of reperfusion injury after both storage periods in University of Wisconsin solution after 1 hr normothermic reperfusion. Inhibition of calpain activity with Cbz-Val-Phe methyl ester resulted in a 50% reduction of apoptotic SEC in the 40-hr preserved liver, and an almost complete abrogation of SEC apoptosis after 24 hr preservation. Only minimal cross-inhibition of caspases was determined at high concentrations in vitro by the calpain inhibitor.nnnCONCLUSIONnApoptosis of exclusively SEC is a key feature of reperfusion injury partially mediated through calpain-dependent processes. Calpain inhibition reduces the number of apoptotic SEC. Based on these data and our previous work, calpain inhibition may prove to be useful in clinical transplantation.

Hepatic Tumor Ablation

Ablation of liver tumors is part of a multimodality liver-directed strategy in the treatment of various tumors. The goal of ablation is complete tumor destruction, and ultimately improvement of quality and quantity of life for the patient. Technology is evolving rapidly, with important improvements in efficacy. The current state of ablation technology and indications for ablation are described in this review.

Invasive biliary mucinous cystic neoplasm: a review

OBJECTIVESnBiliary mucinous cystic neoplasms (BMCNs) are recently redefined rare liver tumours in which insufficient recognition frequently leads to an incorrect initial or delayed diagnosis. A concise review of the subtle, sometimes non-specific, clinical, serologic and radiographic features will allow for a heightened awareness and more comprehensive understanding of these entities.nnnMETHODSnLiterature relating to the presentation, diagnosis, treatment, pathology and outcomes of BMCNs and published prior to March 2012 was reviewed.nnnRESULTSnBiliary mucinous cystic neoplasms most commonly occur in females (≥60%) in the fifth decade of life. Clinical symptoms, serologic markers and imaging modalities are unreliable for diagnosis of BMCNs, which leads to misdiagnosis in 55-100% of patients. Perioperative cyst aspiration is not recommended as invasive BMCNs can only be differentiated from non-invasive BMCNs by microscopic evaluation for the presence of ovarian-type stroma. Intraoperative biopsy and frozen section(s) are essential to differentiate BMCNs from other cystic liver lesions. The treatment of choice is complete excision and can result in excellent survival with initial correct diagnosis.nnnCONCLUSIONSnA low threshold for considering BMCN in the differential diagnosis of cystic liver lesions and increased attentiveness to its subtle diagnostic characteristics are imperative. The complete surgical resection of BMCNs and the use of appropriate nomenclature are necessary to improve outcomes and accurately define prognosis.

Bile duct injury after single incision laparoscopic cholecystectomy.

This study notes that the development of single-incision laparoscopic surgery is not without risk and that obtaining the critical view in appropriately selected patients is essential for safe single-incision laparoscopic surgery.