Minimally invasive surgical approach for three-vessel occlusion as a model of vascular dementia in the rat-brain bioenergetics assay.

Brain energy disorders and oxidative stress due to chronic hypoperfusion are considered to be major risk factors in the pathogenesis of dementia. The aim of our study was to evaluate changes of the brain creatine kinase (BB-CK) reaction and mitochondrial respiratory chain function in male Wistar rats exposed to chronic cerebral hypoperfusion. Three-vessel occlusion (3-VO) was accomplished without thoracotomy using a minimally-invasive surgical approach for the occlusion of the brachiocephalic trunk and the left common carotid artery (CCA). The forward rate constant of creatine kinase (k(for)) was measured in vivo by saturation transfer of (31)P magnetic resonance spectroscopy (MRS) at 2 and 10 weeks of permanent 3-VO. The function of the mitochondrial respiratory chain in vitro was assessed polarographically at 10 weeks after 3-VO. When compared to the controls, the significant 42% reduction of k(for) at 2 resp. 10 weeks indicated disorders in brain energy metabolism, which is in agreement with the 12% decrease of the oxidative phosphorylation coefficient (ADP:O) and with the 14% decrease of the oxidative phosphorylation rate (OPR) measured in isolated mitochondria. Oxidative modification of the creatine kinase system (inactivation of enzymes) and metabolic disorders due to chronic 3-VO, thus, may participate in vascular cognitive impairment and neuronal degeneration.

Effects of sizofiran on endotoxin-enhanced cold ischemia-reperfusion injury of the rat liver.

Kupffer cells (KC), resident macrophages of the liver, have been strongly implicated in lipopolysaccharide (LPS)-induced liver graft injury. However, our recent study showed that sizofiran (schizophyllan glucan) (SPG), which activates KC, did not influence cold ischemia-reperfusion liver injury of LPS-exposed rats. Here we investigated some mechanisms by which SPG does not aggravate LPS-enhanced cold ischemia-reperfusion rat liver injury. Control and SPG-treated rats were exposed to LPS for 2 h prior to hepatectomy. The livers were cold-preserved in University of Wisconsin solution followed by reperfusion with Krebs-Henseleit buffer. We found that SPG dramatically inhibited LPS-induced increases of tumor necrosis factor-alpha (TNF-alpha) in the plasma and bile in vivo. Moreover, LPS-induced TNF- release into the washout solution after cold ischemia was also abrogated by SPG pretreatment. However, SPG increased TNF- release into the perfusate after reperfusion. On the other hand, SPG completely abolished expression of c-myc protooncogene, which is known to sensitize cells to TNF-alpha cytotoxicity. In conclusion, inhibition of both TNF- release after LPS challenge and c-myc expression may explain why activation of KC with SPG does not aggravate endotoxin-enhanced cold ischemia-reperfusion liver injury.

Improvement of rat liver function by energy repletion after the preservation period: implications for hepatic graft management.

We very recently showed (using a blood-free perfusion model) that cold preservation sensitized rat hepatocyte functions to rewarming ischemic injury and that the injury can be prevented by repleting high-energy adenylates in the liver by short-term oxygenated warm reperfusion. Here we investigated whether short-term reperfusion after the preservation period can improve hepatic graft function in a blood reperfusion model. Eighteen-hour cold-preserved rat livers either untreated (Group A) or pretreated by 30-min oxygenated warm reperfusion after preservation (Group B) were subjected to 20-min ischemic rewarming and then reperfused with blood. Livers in Group B compared to Group A exhibited approx. three times increased bile production and bromosulfophthalein excretion, nearly 7-fold decreased swelling, and 1.2-fold improved blood flow. These results suggest that repletion of the energy by short-term oxygenated reperfusion after prolonged preservation may improve markedly initial hepatic graft function.

A rapid, simple, and cost-effective method for screening liver preservation solutions in the rat.

BACKGROUND: Rat liver transplantation models or isolated liver perfusion models are currently used for assessing efficacy of liver preservation methods. We tested the hypothesis that hepatocellular enzymes released into the washout solution after preservation may predict hepatic function during reperfusion and could thus be alternatively used for evaluating efficiency of liver preservation solutions. Furthermore, we applied this approach for assessing the role of Kupffer cells (KC) in preservation-induced liver damage. METHODS: After preservation in University of Wisconsin (UW) or Euro-Collins (EC) solution, rat livers were washed with Ringer-lactate solution. Correlations between enzymes released into the washout solution and hepatocyte functional parameters determined during reperfusion on using a blood-free perfusion model were investigated. RESULTS: In UW-preserved livers, acid phosphatase (ACP) activity correlated negatively with bile flow (R = -0.904), taurocholate intrinsic clearance (R = -0.841), and bromosulfophthalein excretion (R = -0.831). Both alanine transaminase and aspartate transaminase activities correlated with the functional parameters investigated. In EC-stored livers, correlation was also found between ACP activity and bile flow (R = -0.666). Livers stored in UW solution exhibited approximately 3 times lower washout activities of enzymes studied than livers stored in EC solution. Mitochondria isolated from UW-stored livers exhibited significantly better function than those isolated from EC-stored livers. Blockade of KC did not influence enzyme release into the washout solution. CONCLUSIONS: Determination of ACP, alanine transaminase, and aspartate transaminase activities in the washout solution can be used as a rapid, simple, and cost-effective way for screening liver preservation solutions. The results also suggest that KC were not involved in preservation-induced liver damage.

Direct high-performance liquid chromatographic determination of (R)- and (S)-propranolol in rat microdialysate using on-line column switching procedures.

Two different column-switching HPLC systems (CSWs), employing restricted access material for initial pretreatment of biological samples, were developed for the determination of propranolol enantiomers in microdialysate. CSW 1 was a single-pump set-up based on an initial sample clean-up step with a RP-18 ADS precolumn coupled with an ovomucoid analytical column for direct drug enantioseparation. For the two-pump column set-up (CSW 2), a teicoplanin analytical column was applied for the enantioselective assay after initial sample pretreatment using a RP-8 ADS precolumn. The inter-day precision of the CSW 1 ranged from 0.5 to 5.1% for (R)-propranolol and from 5.1 to 10.5% for (S)-propranolol. The limit of detection (LOD) was set at 10 ng/ml and 15 ng/ml for (R)- and (S)-propranolol, respectively. Inter-day relative standard deviation values of the CSW 2 ranged from 1.1 to 9.9% for (R)-propranolol and from 1.3 to 9.6% for (S)-propranolol. The LOD of the method was 3.0 ng/ml for (R)-propranolol and 2.5 ng/ml for (S)-propranolol. Both approaches were successfully applied for stereoselective monitoring of unbound propranolol levels in rat microdialysates.

Endotoxin-induced aggravation of preservation-reperfusion injury of rat liver and its modulation.

BACKGROUND/AIMS: In clinical transplantation, exposure of donors to gut-derived endotoxin occurs frequently and may adversely affect liver transplantation therapy. The aim of this study was to investigate: 1) whether brief exposure of rats to endotoxin before liver procurement aggravates the early phase of reperfusion injury of hepatic explants; and if so 2) whether Kupffer cell activation is a contributing factor to liver injury; and 3) whether heparin and pentoxifylline could minimize this effect. METHODS: Male Wistar rats were injected with 0.2-4.0 mg/kg of Escherichia coli lipopolysaccharide 2 h prior to liver harvest. After preservation in University of Wisconsin cold-storage solution, the livers were reperfused using a blood-free perfusion model. To inactivate Kupffer cells, some rats were pretreated with gadolinium chloride or liposome-encapsulated dichloromethylene-diphosphonate before lipopolysaccharide administration. The other rats received lipopolysaccharide with heparin or pentoxifylline. RESULTS: In a dose-independent fashion, lipopolysaccharide impaired portal flow during graft reperfusion. In a dose-dependent way, lipopolysaccharide increased lactate dehydrogenase release into the perfusate and decreased bile flow and bromosulfophthalein excretion. Gadolinium chloride, liposomal dichloromethylene-diphosphonate, heparin, and pentoxifylline reduced lactate dehydrogenase release by 34%, 43%, 59%, and 64%, respectively, and improved functional parameters of the liver. A 52-fold increased neutrophil infiltration in the liver sinusoids after lipopolysaccharide exposure was not affected significantly by the drugs studied; however, heparin reduced markedly neutrophil activation. CONCLUSIONS: The results of this investigation provide direct evidence that aggravation of preservation-reperfusion injury of rat liver by endotoxin is mediated by Kupffer cell-dependent mechanism(s) and it can be minimized by heparin and pentoxifylline.

[Liver steatosis and disorders of mitochondrial oxidative phosphorylation after experimental administration of simazine]. / Steatóza pecene a poruchy oxidacnej fosforylácie mitochondrií po aplikácii simazínu v experimente.

BACKGROUND: Triazine herbicides are widely used in extensive agricultural production, however, some ecological and health hazards occur due to water and food contamination. AIM: The aim of this study was to evaluate the effects of long-term simazine feeding on the steatosis development and the changes of liver bioenergetics in experimental animals. METHODS: A population of B6C3F1 mice were fed with simazine (2 g and 4 g/kg/day, respectively) for 35 weeks. The concentration of cholesterol and triacylglycerols were measured in liver tissue. Liver mitochondria were isolated and parameters of oxidative phosphorylation were assessed polarographically using Clark oxygen electrode with NAD glutamate and/or FAD succinate as substrates. RESULTS: Significant changes (p < 0.001) expressed as medians (with confidence intervals) against control animals were found in both experimental groups after simazine feeding. The concentration of triacylglycerols increased from 10.3 (8.8-10.9) to 20.1 (18.0-20.9) and 47.7 (23.8-56.0), respectively. The parameters of oxidative phosphorylation with NAD substrate glutamate decreased as follows: The index of respiratory control from 7.7 (6.4-9.0) to 4.8 (4.0-6.3) resp. 4.4 (3.9-4.6); the rate of oxygen consumption in the state 3 (with ADP) from 84.2 (82.0-92.3) to 65.4 (50.8-70.7) resp. 69.9 (65.0-78.4) nAtO.mg.prot-1.min-1; and phosporylation rate from 215.3 (204.4-232.2) to 166.3 (120.4-193.6) resp. 169.6 (155.3-176.9) nmolATP.mg.prot-1.min-1. Comparable changes were detected in oxidative phosphorylation with FAD succinate as substrate. CONCLUSIONS: Liver steatosis development and mitochondrial energetics inhibition were determined in mice after long-term simazine feeding, nevertheless, liver energy production was sufficient to satisfy the liver function and the needs of the whole organism. (Tab. 4, Fig. 6, Ref. 24.)

A study of creatine kinase reaction in rat brain under chronic pathological conditions-chronic ischemia and ethanol intoxication.

Creatine kinase reaction rates were measured by the magnetisation transfer technique in brains of healthy adult and aged rats and in rats with chronic cerebral ischemia and chronic ethanol intoxication. These measurements indicated that the rate constant of the creatine kinase reaction is significantly reduced in the case of severe chronic cerebral ischemia in aged rats. In the adult rats, during chronic ethanol intoxication after 3 weeks of administration of 3 ml of 30% ethanol once a day via a gastric tube, a significant decrease in the pseudo first-order rate constant k(for) of the creatine kinase reaction was also found. In contrast, mild chronic cerebral ischemia in adult rats produced an increase in the reaction rate 4 weeks after occlusion. At the same time, corresponding conventional phosphorus magnetic resonance spectra showed negligible changes in signal intensities.

Projections of the caudal ventrolateral medulla to the thoracic spinal cord in the rat.

BACKGROUND: The caudal ventrolateral medulla (CVLM), an area having cardiovascular and respiratory functions, contains neurons that project to the thoracic spinal cord. It has been presumed, although never demonstrated, that the CVLM thoracic projections terminate on intercostal and sympathetic motor neurons. Using a combination of tracers, the purpose of the present study was to describe a route through which the CVLM may directly influence sympathetic and intercostal motor neurons. METHODS: To label the medullospinal projections orthogradely, either biotinylated dextran amine or lectin conjugated horseradish peroxidase was injected into the CVLM. To label the intercostal and sympathetic motor neurons retrogradely, cholera toxin subunit B-horseradish peroxidase was injected into the intercostal muscles and superior cervical ganglion. Subsequently, projections from the CVLM were observed to terminate on, or in close proximity to, sympathetic and intercostal motor neurons. RESULTS: Projections to sympathetic neurons were largely ipsilateral, whereas projections to intercostal neurons were primarily contralateral. In retrograde labeling experiments, it was observed that many of the CVLM-thoracic projections originated from the nucleus retroambiguus. CONCLUSIONS: The finding that the CVLM projects to sympathetic and intercostal motor neurons supports its role in cardiovascular and respiratory functions. The authors suggest that CVLM influences upon sympathetic motor neurons may be inhibitory.

Creatine kinase reaction rates in rat brain during chronic ischemia.

Creatine kinase reaction rates were measured by magnetisation transfer technique in the brain of healthy adult and aged rats and in the rats with mild or severe chronic cerebral ischemia. These measurements indicated that the rate constant of the creatine kinase reaction is significantly reduced in the case of chronic brain ischemia in aged rats. In contrast, occlusion of both carotid arteries in adult rats produced a slight increase in the reaction rate 4 weeks after occlusion. At the same time, corresponding conventional phosphorus magnetic resonance spectra showed negligible changes in signal intensities.

Effects of blockade of Kupffer cells by gadolinium chloride on hepatobiliary function in cold ischemia-reperfusion injury of rat liver.

The mechanisms of liver injury from cold storage and reperfusion are not completely understood. The aim of the present study was to investigate: 1) whether the inactivation of Kupffer cells (KCs) by gadolinium chloride (GadCl) modulates cold ischemia-reperfusion injury of rat liver; and 2) whether cold storage of rat liver involves injury to biliary epithelial cells (BECs). Hepatobiliary function was assessed using an isolated perfused rat liver model. Compared with control livers, in livers subjected to cold storage at 4 degrees C in Euro-Collins solution (EC) for 18 hours or in University of Wisconsin solution (UW) for 48 hours, portal flow was lower and resistance significantly higher, taurocholate (TC) and bromosulfophthalein (BSP) elimination were markedly impaired, bile flow was reduced, and lactate dehydrogenase (LDH) leakage into the perfusate was increased. Pretreatment of rats with GadCl, a selective KC toxicant, abrogated disturbances of the microcirculation in both models, but it did not influence viability and functional parameters of the liver. Most of the parameters studied in livers stored in UW solution for 18 hours were not significantly different from those found in control livers. As to biliary activity of gamma-glutamyl transferase (GGT), as an index of BEC integrity, it was increased with increasing time of cold storage. The reabsorption of glucose from the bile decreased with longer storage time. The results suggest the following: 1) that cold ischemia-reperfusion injury of rat liver is mediated by KC-dependent (hepatic microcirculation) and -independent (parenchymal cell function) mechanisms; and 2) that cold storage of rat liver induces functional impairment of BECs.

[Atelocollagen/hydroxylapatite composite material as bone defect filler in an experiment on rats]. / Kompozit atelokolagén-hydroxylapatit ako výpln kostných defektov v pokuse na potkanoch.

Many types of biomaterials are used as skeletal bone fillers in reconstructive surgery. An attention is paid to hydroxyapatite due to its high biocompatibility with the surrounding tissue. The paper deals with the testing of new collagen/hydroxyapatite composite material applied to the bone defect on os parietale of rats. The composite material was prepared from the bovine atelocollagen dispersion and the dispersion of hydroxyapatite. Collagen serves as a matrix in which the particles of hydroxyapatite are anchored. The composite presents the advantage that after the saturation with physiological solution it is compact and can be shaped. The composite material was implanted in the form of plate into six male Wistar rats to the ground bed on the surface of os parietale. The implants were taken out after four moths. The macroscopic finding of soft tissue, bones and implants gave evidence about good healing without any undesirable reaction. This was also confirmed by the histological observations. Collagen was resorbed and the rest of the material strongly adhered to the bone. The marked osteocytes were present in the zone of the newly formed bone and the dividing line between new and old bone was clear. The experimental results give preconditions to the clinical use of this new composite implant material the structural improvement of which is in progress. (Fig. 2, Ref. 12.)

Dose-ranging study of NG-nitro-L-arginine pharmacokinetics in rats after bolus intravenous administration.

1. NG-nitro-L-arginine (10, 30 and 100 mg/kg) was administered intravenously to the male Wistar rat. Plasma was collected over 48, 72 and 120 h and was analysed for the drug by hplc. Pharmacokinetic parameters were calculated using a non-compartmental method. 2. Drug concentration-time profiles of individual rats after all doses studied exhibited secondary peaks, while geometric mean concentration-time curves showed plateaus. 3. NG-nitro-L-arginine plasma concentrations divided by dose almost coincided. Pharmacokinetic parameters were not dose-dependent in the range of 10-30 mg/kg, but changed after 100 mg/kg of NG-nitro-L-arginine indicating some decline from linearity. 4. NG-nitro-L-arginine is a low-extracted drug in rat as the total clearance was low (0.05-0.07 l/h/kg). Half-life and mean residence time were found to be long (17-30 and 23-40 h, respectively). Despite its low lipophilicity, NG-nitro-L-arginine exhibited large steady-state and terminal volumes of distribution (1.4-2.2 l/kg and 1.4-2.4 l/kg, respectively). Together with the double peak phenomenon, these results may be explained by assuming NG-nitro-L-arginine is involved in a recirculation process in the body.

[Hemodynamics, lymphatic circulation and ultrastructure of the lungs during hemodilution with blood substitutes]. / Hemodynamické pomery, lymfatická cirkulácia a ultrastruktúra pl'úc pocas substitucnej hemodilúcie.

The effect of substitution hemodilution on transcapillary fluid exchange in the lung was studied in experiments on 30 dogs. After administration of Ringer's solution and of oncotic solutions (3.4% and 10% Rheodextran), the lymph flow through the right lymphatic trunk was measured and the protein content in the lymph was determined. The obtained results were compared with electron micrographs of the lung. Ringer's solution was found to escape rapidly from the vascular bed. A 3.9-fold increase in lymph flow was recorded and edematous changes were observed in the interstitial space. Administration of 10% Rheodextran induced an 8.3-fold increase of lymph flow and enhanced also the protein transport in the lymph. The permeability of pulmonary endothelial vascular walls was deranged. Isooncotic 3.4% Rheodextran provided adequate transcapillary exchange of fluids in the lungs and proved to be the suitable solution for substitution of blood losses. In the ultrastructure of the lungs no pathological changes were observed. (Fig. 8, Ref. 33).

[The physiologic significance of 2,3-diphosphoglycerate and changes in its erythrocyte levels in an experiment]. / Fyziologický význam 2,3-difosfoglycerátu a zmeny jeho obsahu v erytrocytoch pocas experimentu.

The regulatory effect of 2,3-DPG on oxygen transport and binding to hemoglobin was analyzed. Under conditions of substitution hemodilution by isooncotic 3.4% Rheodextran (Spofa), changes in the content of 2,3-DPG in arterial and venous blood were enzymatically determined over several days. Reference values of 2,3-DPG were obtained in the studied series of dogs (2.05 +/- 0.74 x 10(-6) mol.ml-1 in whole blood 4.69 +/- 1.52 x 10(-6) mol.g-1 of erythrocyte volume, and 13.39 +/- 2.82 x 10(-6) mol.ml-1 of hemoglobin). In anesthetized animals the content of 2,3-DPG in arterial blood was significantly higher (6.28 +/- 0.84 x 10(-6) mol.ml-1 of erythrocyte volume) than in venous blood (6.01 +/- 0.80 x 10(-6) mol.ml-1 of erythrocyte volume). At substitution hemodilution the 2,3-DPG content in erythrocytes of venous blood decreased from 5.46 +/- 0.67 to 4.97 +/- 1.31 x 10(-6) mol.ml-1 of erythrocyte volume. The subsequent increase to 6.04 +/- 0.71 x 10(-6) mol.ml-1 of erythrocyte volume was achieved by nonlinear increase over the following days, at persisting low hemoglobin content in blood.