Ischemic Preconditioning Prevents Free Radical Production and Mitochondrial Depolarization in Small-for-Size Rat Liver Grafts

Abstract

Background. Ischemic preconditioning (IP) renders tissues more tolerant to subsequent longer episodes of ischemia. This study tested whether IP attenuates injury of small-for-size liver grafts by preventing free radical production and mitochondrial dysfunction. Methods. IP was induced by clamping the portal vein and hepatic artery for 9 min. Livers were harvested 5 min after releasing the clamp. Mitochondrial polarization and cell death were assessed by intravital confocal/multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide. Free radicals were trapped with ␣-(4-pyridyl 1-oxide)-Ntert-butylnitrone and measured using electron spin resonance. Results. After quarter-size liver transplantation, alanine aminotransferase, serum bilirubin, necrosis, and apoptosis all increased. IP blocked these increases by more than 58%. 5-Bromo-2Ј-deoxyuridine labeling and increases of graft weight were only ϳ3% and 0.2% in quarter-size grafts without IP, respectively, but increased to 32% and 60% in ischemic-preconditioned grafts, indicating better liver regeneration. Eighteen hours after implantation, viable cells with depolarized mitochondria in quarter-size grafts were 15 per high power field, and dead cells were less than 1 per high power field, indicating that depolarization preceded necrosis. A free radical adduct signal was detected in bile from quarter-size grafts. IP decreased this free radical formation and prevented mitochondrial depolarization. IP did not increase heat shock proteins 10,27,32,60,70,72, 75 and Cu/Zn-superoxide dismutase (SOD) but increased heat shock protein-90, a chaperone that facilitates protein import into mitochondria, and mitochondrial Mn-SOD. Conclusion. Taken together, IP decreases injury and improves regeneration of small-for-size liver grafts, possibly by increasing mitochondrial Mn-SOD, thus protecting against free radical production and mitochondrial dysfunction.

Adenosine receptors: regulatory players in the preservation of mitochondrial function induced by ischemic preconditioning of rat liver

“…Therefore, modulation of mitochondria has emerged as a critical survival strategy for the prevention of I/R injury. IPC-triggered signal transduction appears to directly preserve several cell functions including intracellular energy state, pH, and redox system [2,3,14,17,24]. Our data show that PC (both ischemic and pharmacological) was capable to decrease the plasma activities of ALT, AST, and LDH, indicating a clear hepatoprotective effect against warm I/R injury.…”

“…Furthermore, IPC decreases liver injury after both warm and cold I/R [2,4,9,10]. Several mechanisms have been proposed for protection by IPC, including activation of adenosine receptors [11][12][13][14], increased nitric oxide production [15], activation of protein kinase C [16], up-regulation of heat shock proteins, and increased antioxidant capacity [17,18]. Probably, the process better established to be associated with decrease tolerance to I/R injury is the inability to restore energetic balance following I/R [1,19,20].…”

“…Accordingly, IPC can prevent I/R-induced mitochondrial dysfunction suggesting that PC protects the integrity of mitochondrial oxidative phosphorylation (OXPHOS) and increases the resistance to the induction of the mitochondrial permeability transition [21][22][23]. The precise mechanism of PC-induced hepatoprotection is unknown, but it is likely to be a receptor-mediated process, related with preservation of energy metabolism [2,3,14,17,24]. However, little is known about the coupling of extracellular signals with intracellular adaptive mechanisms involved in preconditioning.…”

Quantitative intravital microscopy of hepatic transport

“…Accordingly, intravital microscopy has been applied to a wide range of studies of the liver. Intravital microscopy has been almost routinely used to characterize microvascular flow in the liver, [11][12][13] but has also been used to assay hepatocyte cell death [14][15][16][17][18][19][20] apoptosis, 21 inflammatory cell infiltration, 22-24 microvascular permeability, [25][26][27] mitochondrial function, 14,15,[17][18][19]28,29 steatosis, 30,31 tumor angiogenesis 32 and fibrosis. 33 Fluorescent transport substrates cultured cell systems, it is more difficult to achieve in intravital microscopy, especially in the liver, whose proximity to the diaphragm makes it particularly susceptible to respiration-induced motion.…”

Mitochondrial Glutathione, a Key Survival Antioxidant

“…Thus, ROS generation may play a dual role in I=R injury. Although preventing ROS overgeneration by antioxidants may be of therapeutic value, the early counteraction of ROS generation during ischemic preconditioning was shown to cancel the protective mechanisms triggered by preconditioning, including MnSOD overexpression (120,125).…”