Ischemia With Preconditioning in Wistar Rats Maintains Mitochondrial Respiration, Even With Mild Hepatocellular Disturbance.

INTRODUCTION: In hepatectomy or liver transplantation, preconditioning is a procedure indicated to protect the organ from ischemia-reperfusion injury (I-R). OBJECTIVE: Evaluate the effect of preconditioning after hepatic I-R in Wistar rats, through mitochondrial respiration, liver histology, and profile. METHOD: Twenty male Wistar rats, weighing on average 307.1 g, were anesthetized with sodium thiopental (25 mg/kg) intravenously and xylazine hydrochloride (30 mg/kg) intramuscularly. The animals were divided into 2 groups: the preconditioning group (PCG), which contained 10 animals, and the hepatic pedicle was isolated and submitted to clamping with microvascular clamp (10 minutes of ischemia and 10 minutes of reperfusion, followed by 30 minutes of ischemia and 30 minutes of reperfusion); and the simulated operation group (SOG), which contained 10 animals submitted to manipulation of the hepatic pedicle and observation for the same length of time, with blood collected for transaminase dosage measurements, and liver biopsy for evaluation of mitochondrial respiration and histologic liver analysis and after sacrificed under anesthesia. The project was approved by the Ethics Committee on Animal Experimentation CEEA/UNICAMP under protocol number 3905-1. RESULT: The PCG mitochondria showed the same respiration level as the SOG, when stimulated with the addition of adenosine diphosphate or carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the respiratory control ratio and resting of velocity of respiration the groups behaved in a similar way. The PCG presented high aspartate and alanine transaminases (P < .03) and about 60% of sinusoidal congestion and venous congestion in the histologic analysis when compared with SOG. CONCLUSION: We found that ischemia with preconditioning in Wistar rats can lead to mild histologic and biochemical dysfunction without leading to impairment of mitochondrial respiration.

Later Evaluation of Ischemia and Reperfusion by the Pringle Maneuver in Wistar Rats, Demonstrating That Hepatic Lesions Can Be Reversible.

OBJECTIVE: There has been much research on hepatic ischemia and reperfusion by means of short or longer interruption of the portal triad. The aim of this work was to evaluate the mitochondrial respiratory activity and liver histology at 2 different times after the Pringle maneuver. METHODS: Twenty-eight male Wistar rats, weighing ∼308 g, with histologic and mitochondrial study: immediate ischemic group (IIG; 40 minutes; 9 animals) and late ischemic group (LIG; 28 days; 9 animals). The rats were anesthetized and underwent a U-incision in the abdomen. In a simulated operation, manipulation of the hepatic pedicle was performed (5 animals immediate [ISG] and 5 late [LSG]). The hepatic pedicle was clamped for 20 minutes of ischemia foloowed by 20 minutes of reperfusion. The animals were killed under anesthesia. RESULTS: Mitochondria when stimulated by adenosine diphosphate or carbonylcyanide p-trifluoromethoxyphenylhydrazone had a significant respiratory reduction (P < .001). The respiratory control ratio in the LIG was altered (P < .02) compared with IIG. In the resting state, there was no change in the velocity of respiration between ischemic groups. Histopathologic findings showed 55.5% sinusoidal dilatation in IIG and 66.6% in LIG; 77.7% ballooning in IIG and 55.5% in LIG; and 11.1% focal necrosis in both IIG and LIG. CONCLUSIONS: The oxidative phosphorylation system recovered with improvement in mitochondrial respiration; however, morphologic recovery was associated with the type and intensity of injury.

Long-Time Choledochal Clamping in Wistar Rats Causes Biliary Obstruction Progressing to Hepatic Fibrosis.

BACKGROUND: Biliary complications are important causes of morbidity and mortality in patients undergoing hepatic surgery. The aim of the study was to evaluate late liver alterations after a long period of choledochal clamping in Wistar rats. METHODS: Ten male Wistar rats, weighing 304 grams, anesthetized with sodium thiopental (25 mg/kg) and xylazine (10 mg/kg) intravenously, were distributed into 2 groups: the choledochal clamping group (CCG) and the operation sham group (OSG), with 5 animals each submitted to an abdominal incision. In the CCG, the choledochal was isolated, dissected, and clamped with a microvascular clamp for 40 minutes. After this occlusion time, the clamp was removed and the incision was closed. In the OSG the animals, under normal conditions, were submitted only to anesthesia and laparotomy for choledochal manipulation. In all animals, after the 31st day, a hepatic biopsy was carried out for histology and blood biochemical tests: total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyl transferase. The animals were euthanized under anesthesia. This research was approved by the Ethics Committee on Animal Use (CEUA, Unicamp, No. 2511-1). RESULTS: In the CCG, 100% of the animals showed bile duct dilatation, ductular proliferation, and portal inflammatory infiltrate; 60% showed regenerative nodule formation; and 80% had porta-porta septa and foci of necrosis, all of which were not found in the OSG. All CCG group biochemical tests had significant increases (P < .05) compared with OSG. CONCLUSIONS: Long-time choledochal clamping in Wistar rats caused hepatic dysfunction and biochemical and histological injuries with degrees of distortion to the hepatic architecture.

Erythrocyte oxidative stress is associated with cell deformability in patients with retinal vein occlusion.

Essentials Retinal vein occlusion (RVO), characterized by blood hyperviscosity, has an unclear pathogenesis. We aimed to find out if hemorheological profile is altered by oxidative stress in RVO patients. Red blood cell (RBC) oxidative stress is associated to whole blood viscosity and RBC deformability. Reactive oxygen species alter RBC membrane rigidity, playing a key role in RVO pathogenesis. SUMMARY: Background Retinal vein occlusion (RVO) is characterized by vision loss resulting from hypoperfusion and hypoxia of the retina. RVO pathogenesis is not yet fully understood, although blood hyperviscosity has been observed. Erythrocyte deformability plays a key role in determining blood viscosity, and it is critical to microvascular perfusion and oxygen delivery. It has been shown that oxidative stress-induced erythrocyte membrane fluidity alterations are linked to the progression of cardiovascular diseases. Objectives To determine whether erythrocytes from RVO patients show signs of oxidative stress, and whether this condition can modify the hemorheologic profile in these patients. Patients and Methods We analyzed the entire hemorheologic profile and erythrocyte oxidative stress - reactive oxygen species (ROS) production and membrane lipid peroxidation - in 128 RVO patients and 128 healthy subjects, matched for age and sex. Fluorescence anisotropy was used to evaluate the fluidity of erythrocyte membranes. Results In RVO patients, erythrocyte oxidative stress was present and positively correlated with whole blood viscosity and erythrocyte deformability. Multivariate linear regression analysis after adjustment for age, cardiovascular risk factors, medications, leukocyte number and mean corpuscular volume indicated that erythrocyte-derived ROS and erythrocyte lipid peroxidation were significantly and positively correlated with erythrocyte membrane viscosity and deformability. Moreover, in vitro experiments demonstrated that ROS have a key role in erythrocyte membrane fluidity. Conclusions Our findings indicate that erythrocyte oxidative stress plays a key role in the pathogenesis of RVO, and pave the way to new therapeutic interventions.

No protective function found in Wistar rats submitted to long ischemia time and reperfusion after intermittent clamping of the total hepatic pedicle.

BACKGROUND: Although the intermittent Pringle maneuver is used for major transplant surgery, traumas, and hepatic protection, long ischemia time and reperfusion may limit some protection in Wistar rats. The aim of the study was to evaluate the protection effects of intermittent clamping in the total hepatic pedicle after a long period of ischemia and reperfusion in Wistar rats. METHODS: Forty-two male Wistar rats, weighing ± 327.7 g, were anesthetized intravenously with sodium thiopental and given a U-shaped incision in the abdomen. The total hepatic pedicle was isolated and subjected to clamping with a microvascular clamp. Groups included were the continuous group (CG, n = 14, 40 minutes of ischemia/40 minutes of reperfusion); the intermittent group (IG, n = 14, 4 cycles a 10 minute ischemia/reperfusion 10 minutes); and the sham group (SG, n = 14, 80 minutes of observation time). Blood collection for transaminase dosage was carried out, and hepatic biopsy specimens were taken for mitochondrial respiration and histological evaluation. RESULTS: In groups CG and IG, aspartate aminotransferase and alanine aminotransferase enzymes were elevated in comparison to group SG (P < .008); mitochondrias, when stimulated by use of adenosine diphosphate or carbonylcyanide p-trifluoromethoxyphenylhydrazone, had a significant decrease in mitochondrial respiration (P < .05), and the respiratory control ratio in the ischemic groups was lower (P < .03) when compared with the GS. On histological examination, 100% of the GC had lesions: 33% focal hemorrhagic necrosis, 17% sinuzoidal congestion and/or vacuolization, and 50% venous congestion; in the IG, 100% had lesions: 43% sinusoidal congestion and/or vacuolization and 57% venous congestion. CONCLUSIONS: The intermittent total hepatic pedicle clamping for a long period of time in the Wistar rats had no efficacy in protection of liver injury.

Late biliary obstruction in Wistar rats after intermittent hepatic pedicle clamping.

INTRODUCTION: The temporary vascular occlusion of hepatic flow is one of the essential procedures in hepatic surgery. AIM: Evaluate the late liver alterations after intermittent pedicle hepatic clamping (IHPC) in Wistar rats. METHODS: Male Wistar rats (n = 14) with average weight of 281.1 g, were anesthetized with intraperitoneal ketamine 5%. The IHPC group (n = 7) was submitted to U-shaped abdominal incision; the hepatic pedicle was isolated and submitted to IHPC ischemia 4 times, 5 minutes each, followed by reperfusion 4 times, 5 minutes each. The simulated operation group (n = 7) was subjected to anesthesia, laparotomy, and manipulation of the hepatic pedicle. On day 35, after fasting for 12 hours, liver biopsies were collected and blood was tested for liver aminotransferases (aspartate aminotransferase/alanine aminotransferase). RESULTS: All the IHPC group animals had a dilated common bile duct and increased liver enzymes (P < .05 by Mann-Whitney test). Ductular proliferation (100% of cases), porta-porta septa (42.8%), formation of lumps (42.8%), foci of necrosis (14.2%), and bile plugs (14, 2%) were observed only in the IHPC group. CONCLUSION: In rats, IHPC caused morphologic features leading to biliary obstruction.

Biliary obstruction after short-time choledochal clamping in rats.

INTRODUCTION: Biliary complications after liver procedures can lead to morbidity and poor survival. AIM: The aim of this study was to evaluate the hepatic function after quick clamping of the common bile duct (BD) in Wistar rats. METHODS: Twelve male Wistar rats with a mean weight of 323.14 g were anesthetized with sodium thiopental intravenous (IV). The common BD Clamping Group (BDCG; n = 6) was submitted to an abdominal incision (2 cm); the BD was isolated, dissected, and underwent clamping for 10 minutes with a microvascular clamp. After this time, the clamp was removed and the incision closed. The Sham Operation Group (SOG; n = 6 rats), under normal conditions, were subjected only to anesthesia and laparotomy and later control tests. On the 28th day liver and choledoch biopsy and biochemical tests were performed on all animals: total bilirubin (TB), alkaline phosphatase (ALK-P), aminotransferases (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]), and gamma-glutamyl transferase (GGT). After the tests all the rats were humanely killed while still under anesthesia. RESULTS: In this study 83% of the animals in the BDCG had large dilatation of the common BD with ductular proliferation, formation of septae, as well as multiple foci of parenchymal necrosis including micro-abscess formation. We also observed alterations in biochemical tests (P < .05). CONCLUSION: Our study demonstrated that BD clamping even for a short time was sufficient to generate important morphological alterations in the liver and BD, as confirmed by enzymatic and histological analysis. Therefore, this technique can be used as a model of biliary obstruction for future studies.