Effect of Prolactin on Biochemical and Morphological Parameters of Rabbit Liver in Warm Ischemia.

BACKGROUND: The aim of the study was to assess the degree of liver damage in a rabbit perfused with histidine-tryptophan-ketoglutarate (HTK [Custodiol]) solution with and without the presence of prolactin (PRL) based on biochemical studies in perfundate and ultrastructural analysis of hepatocytes. MATERIALS AND METHODS: The experiment was carried out on rabbits. Liver ischemia was used in the study, based on Pringle's maneuver. About 70% of the rabbit liver lobes were perfused with HTK with or without the addition of PRL (2.5µg/g liver/h) under ischemic conditions for 2 hours. In the perfundate, the activity of enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), γ-glutamyl transpeptidase (GGT), and lactate concentration were determined. Liver biopsies were collected for histopathologic evaluation under an electron microscope. RESULTS: The addition of PRL to the HTK significantly reduced the leakage of enzymes from the liver to perfundate compared with the control group without PRL. The activity of ALT, AST, LDH, and GGT in the perfundates obtained after 2-hour perfusion with HTK-PRL solution was lower when compared with activity of the same parameters determined in perfundates with liver perfused with HTK without PRL. The area under the curve (AUC0-2h) calculated for GGT, LDH, and lactates was significantly higher after perfusion with the HTK than with HTK with the addition of PRL. In the study group, bile was secreted throughout the whole experiment. The morphological confirmation of these results was obtained by means of transmission microscopy. CONCLUSION: PRL added to the preservation solution significantly inhibits the process of liver cell cytolysis, which may suggest its hepatoprotective effect.

Comparison of Vascular Endothelial Growth Factor Concentration in Porcine Kidneys Removed From Living Donors After Cardiac and Brain Death.

The aim of this paper was to describe the differences in vascular endothelial growth factor (VEGF) concentration in porcine kidneys removed from living donors (group I), donors after prior induction of brain death by brain herniation (group II), and donors after cardiopulmonary arrest (group III). The groups consisted of 6 animals which underwent dual renal removal procedures; kidneys were rinsed, stored for 24 hours at 4°C and rinsed again. Renal specimens (4g) were collected before and after perfusion (time 0 and 1), after 12 hours (time 2), and after reperfusion (time 3). A Western blot was used to evaluate VEGF concentration in collected tissues homogenates. Additionally, the levels of VEGF, interleukin 1ß, tumor necrosis factor α, and endothelial nitric oxide synthase (eNOS) were detected with enzyme-linked immunosorbent assays. Directly after the removal procedure, no significant differences in VEGF levels (IOD) were observed depending on the donor (moderate levels were observed in all groups: 1.51 in group I, 1.48 in group II, and 1.35 in group III). As a consequence of perfusion and 12 hours of storage, a stable concentration in groups I and III was observed with a gradual increase of VEGF levels in group II (1.23, 2.08, and 1.67 in the respective groups at time 1; 1.49, 2.12, and 1.63 in the respective groups at time 2). After the following 12 hours, a statistically significant (P < .05) higher level of VEGF was observed in group II (2.34) in comparison to groups I and III (1.58 and 1.81, respectively). In group I, a correlation between VEGF concentration and IL-1ß was observed, while in group II there was correlation between VEGF and eNOS levels.

Effect of Recombinant Human Prolactin Addition to Biolasol Solution on Biochemical Indicators in Perfundates of Porcine Kidneys.

BACKGROUND: The influence of recombinant human prolactin (rh-PRL) added to Biolasol solution (concentration 1 µg/L) on selected markers (pH, osmolarity, Na(I) and K(I) concentration) and enzymatic activity (alanine aminotransferase [ALT], aspartate aminotransferase [AST], and lactate dehydrogenase [LDH]) in perfundates was investigated during flushing and preservation of the isolated porcine kidneys. METHODS: The pH, osmolarity, concentration of K(I) and Na(I), and enzymatic activity were determined in perfundates collected after the 5th and 30th minutes of perfusion, after 24 hours of organ preservation, and in the 5th and 30th minutes of reperfusion. Kidneys had been flushed and stored in Biolasol (control group) and in Biolasol with rh-PRL (experimental group). Obtained results were compared with Biolasol solution. RESULTS: In the experimental group, the decrease in pH value in the 5th minute of reperfusion was noted. There was an increase in K(I) concentration, and Na(I) concentration decreased in the 5th and 30th minutes of reperfusion. ALT activity during perfusion and preservation increased, whereas at the 5th and 30th minutes of reperfusion it decreased. AST activity increased during perfusion and preservation and decreased in the 5th and 30th minutes of reperfusion. LDH activity was increased but decreased in the 5th minute of reperfusion. CONCLUSIONS: Addition of 1 µg/L rh-PRL to Biolasol solution decreases pH and osmolarity values; influences Na(I) and K(I) concentration; increases ALT, AST activity during perfusion and preservation of organs; and decreases ALT, AST activity during reperfusion.

Caspase-3 Expression and ALT, AST, and GGT Activity After 24 Hours of Porcine Liver Cold Storage, Depending on the Type of Transgenesis.

BACKGROUND: Because of an insufficient number of human organs for transplantation, xenotransplantation may become an effective alternative. We aimed to analyze if the type of transgenesis has an influence on the hepatic caspase-3 expression, the enzyme that executes apoptosis as well as ALT, AST, and GGT activity after 24 hours of cold storage. METHODS: The experiment was carried out on the 24 livers of Polish White Landrace pigs carrying human α1,2-fucosyltransferase and/or α-galactosidase (GAL) genes and livers without this genetic modification (control). Livers were perfused, stored for 24 hours in solution, and subsequently re-flushed. Hepatic concentration of the caspase-3 protein and its mRNA expression were measured just after the animal was killed as well as after 30 minutes of perfusion and after 24 hours of cold storage followed by 30 minutes of reperfusion. Caspase-3 mRNA level was detected with the RT-PCR method. Protein concentration (capsase-3 active and inactive) was assessed with the Western blotting technique. Kinetic methods were applied for the analysis of the ALT, AST, and GGT activity. RESULTS: The highest increase of the ALT activity after cold storage was observed in the group with GAL transgenesis, whereas the GGT activity was highest in the unmodified livers. There was no difference in the caspase-3 expression and AST activity after cold storage as compared with the respective initial results (P = .57 and P = .97, respectively). CONCLUSIONS: It appears that transgenesis does not aggravate ischemic injury of the liver.

Endothelial Nitric Oxide Synthase and Endothelin-1 Expression in the Early Post-Porcine Kidney Autotransplantation Period.

BACKGROUND: The aim of this study was the assessment of endothelial nitric oxide synthase (eNOS) and endothelin-1 (EDN-1) expression in porcine kidneys on the 14th and 30th days after the autotransplantation procedure. METHODS: The research was conducted on 12 animals that underwent a left renal transplantation procedure with further standardized rinsing and 24-hour storage in 4°C; subsequently, the kidneys were implanted in the right retroperitoneal space after right-sided nephrectomy. Removed kidneys were examined (group 0). Six randomly chosen animals (group 1) were under observation for 14 days and 6 others (group 2) for 30 days. RESULTS: After these observation periods, euthanasia was performed on the animals and 4-g samples were collected from the renal cortex and medulla. The Western blot technique was used to detect the eNOS and EDN-1 expression at the protein level. The obtained results are presented as absolute values of integrated optical density. Stable graft function was observed in all animals from the 2nd day after the procedure. eNOS in group 1 reached the mean value of 1.064 and was statistically significantly lower than in group 2 (2.085) or in the control group 0 (3.318). In the case of EDN-1 expression on 14th day after transplantation, the medium level was reported (0.248), which was similar to group 0 (0.216), whereas group 2 presented values 2 times higher (0.743). CONCLUSIONS: A lowered eNOS level in the organ was observed on the 14th day after autotransplantation of a pig kidney; further enzyme normalization is associated with increased EDN-1 expression.

Concentration of Tumor Necrosis Factor-α and Interleukin-1ß in Isolated Porcine Liver Depending on Type of Transgenesis.

BACKGROUND: Transgenic animals may serve as organ donors in human organ transplantation. However, the number of the studies addressing all doubts related to this issue is currently insufficient for the clinical application of this approach. The aim of this study was to analyze the hepatic tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) synthesis during a 24-hour cold preservation of the transgenic pig liver, depending on the type of transgenesis. MATERIALS AND METHODS: The study was carried out on wild-type and transgenic pig livers with transferred human α1,2-fucosyltransferase (FUT) and/or α-galactosidase (GAL) gene (four groups; n = 6). Harvested livers were perfused for 30 minutes and stored for 24 hours in Biolasol (Biochefa) solution at 4°C with a subsequent 30-minute reperfusion (reflush). TNF-α and IL-1ß concentrations were analyzed with an enzyme-linked immunosorbent assay. Perfusates were collected during the initial perfusion as well as after 24 hours of preservation and during the reperfusion. Tissue samples were harvested just after animal sacrifice, and after organ perfusion and reperfusion. RESULTS: A decrease in TNF-α concentration in homogenates was noted after both perfusion and reperfusion in all experimental groups. In contrast, there was a significant decrease in IL-1ß concentration in the group with combined human FUT and GAL transgenes. Concurrently, increases in TNF-α and IL-1ß concentrations were observed in the reperfusion perfusates in all groups. CONCLUSION: This study shows that IL-1ß is synthesized in the ischemic livers of the transgenic animals with both human α1,2-fucosyltransferase and α-galactosidase transgenes. Further analysis is required to determine the importance of this observation.

Biolasol: novel perfusion and preservation solution for kidneys.

INTRODUCTION: Biolasol solution (Pharmaceutical Research and Production Plant "Biochefa," Sosnowiec, Poland) is a novel extracellular perfusion and ex vivo hypothermic kidney preservation solution. It ensures maintenance of homeostasis, reduces tissue edema, has low viscosity, and allows the graft to preserve structural and functional integrity. It minimizes ischemia-reperfusion damage. METHODS: Perfundates from control and transplanted kidneys flushed with Biolasol or ViaSpan solutions (Arkas, Warszawa, Poland) were analyzed. Parameters of serum and urine collected from 12 pigs after auto-transplantation were also analyzed. Renal medulla was investigated for structural alterations by analyzing hematoxylin and eosin-stained slides. The mean survival time of pigs after the auto-transplantation procedure was the measure for the novel Biolasol solution effectiveness. RESULTS: We observed a statistically significant decrease in marker enzyme levels alanine transaminase, aspartate transaminase, lactic dehydrogenase, and ions (Na and K) in pigs with grafts flushed with Biolasol. Histopathologic examination revealed that the renal cortex structure was not damaged after the use of Biolasol solution. CONCLUSION: Biolasol solution protects kidneys against ischemia damage and does not differ significantly from the "golden standard" ViaSpan solution.

Comparison of endothelial nitric oxide synthase and endothelin-1 levels in kidneys removed from living pigs after cardiac arrest and brain death.

OBJECTIVES: The aim of this paper was to describe differences between levels of endothelial nitric oxide synthase (NOS-3) and endothelin-1 (ET-1) in swine kidneys removed from living donors (group I) and after inducing brain death by brain herniation (group II) and cardiac arrest (group III). METHODS: Each group consisted of 3 animals who underwent dual renal removal procedure; kidneys were further rinsed according to standardized procedure with Biolasol perfusion liquid, stored for 24 hours (4°C), and rinsed again. Renal specimens of 4 g mass, including renal cortex and medulla, were collected before and after perfusion (times 0 and 1), after 12 hours (time 2), and after reperfusion (time 3). Enzyme-linked immunosorbent assay was used to describe levels of NOS-3 and ET-1 in collected tissues homogenates. Mann-Whitney U test was used to compare results in groups in relation to total protein content (ng/mg), and the correlation between the 2 substances was measured with the use of Spearman rho. RESULTS: Group I presented low and stable levels of NOS-3 in all time intervals (averages, 0.73, 0.99, 0.52, and 0.89, respectively). Level sof ET-1 were similar (0.87, 0.63, 0.69, and 0.86, respectively), and significant correlation between levels of the 2 substances was observed. Increased levels of NOS-3 (1.89 and 1.86) and ET-1 (1.38 and 1.49) were observed directly after removal in groups II and III and further maintained during organ storage. No correlation in group I was observed, and after perfusion significantly lower level of NOS-3 was observed in kidneys removed after brain death in relation to group III (1.77 vs 2.60). CONCLUSIONS: The lowest and stable levels of NOS-3 and ET1 during storage were observed in kidneys removed from living donors. Levels of analyzed substances in this group showed correlation in subsequent time intervals.

Toll-like receptors 2 and 4 in pigs' kidneys early after autotransplantation procedure.

OBJECTIVES: The aim of this paper was to evaluate mRNA expression of Toll-like receptors 2 (TLR2) and 4 (TLR4) and the adaptor protein myeloid differentiation primary-response protein 88 (MyD88) in pigs' kidneys 14 and 30 days after autotransplantation. METHODS: The research was conducted on 12 animals that underwent left renal transplantation procedure with further standardized rinsing with Biolasol solution and 24 hours' storage in 4°C; subsequently the kidneys were implanted in the right retroperitoneal space after right-side nephrectomy. Six randomly chosen animals (group I) were under observation for 14 days, the other 6 (group II) for 30 days. After these observation periods, the animals were killed and 4-g samples were collected from the renal cortex and medulla. RESULTS: Expression of mRNA in homogenates of collected samples were determined with the use of reverse-transcription polymerase chain reaction analysis. Obtained results in both groups, presented in relation to GAPDH, were compared with the use of Mann-Whitney U test. Stable graft function was observed in all animals from the 2nd day after the procedure. TLR2 in group I reached the mean value of 3.64 and was statistically significantly higher than in group II (2.19). Inverse proportion was observed in case of mRNA for TLR4: group II presented 2 times higher value than group I (0.25 vs 0.11). Similarly, significant difference was observed in MyD88 (group I, 0.067; group II, 0.45). CONCLUSIONS: At 14 days after autotransplantation of a pig kidney, mRNA expression for TLR2 is dominant; later, expression increases for TLR4 and MyD88.

Cytochrome P450 3 A expression in pigs livers after 24-hour preservation in Biolasol solution depending on the type of transgenesis.

BACKGROUND: An insufficient number of organs for transplantation shows the need for the development of new technologies. Xenotransplantation might be the answer. OBJECTIVE: To determine if the type of transgenesis influences the level of CYP3A4, which takes an active part in xenobiotics metabolism in livers after 24-hour storage, depending on the kind of solution used for preservation. MATERIALS AND METHODS: The experiment was carried out on 30 livers of Polish White Landrace divided into 5 groups depending on transgene type. The following human genes were transferred: α1,2-fucosyltransferase (groups I and II), α-galactosidase (III), combined α1,2-fucosyltransferase/α-galactosidase transgene (IV), and livers without modification (V). The livers were perfused and subsequently stored for 24 hours in Ringer's solution (group I) or Biolasol solution (II-V). Reperfusion/reflush was performed. CYP3A29 isomer concentration was analyzed in liver specimens collected twice: 30 minutes after perfusion and 30 minutes after reperfusion/reflush. Expression of mRNA CYP3A29 was marked using RT-PCR analysis and of protein CYP3A29 using Western blotting technique. RESULTS: The most significant decrease in protein CYP3A29 expression after 24-hour preservation was observed in group I (55.88% decrease), while the least significant was observed in group IV (10.44% decrease). mRNA expression evaluation was similar: the most significant decrease was observed in group I (87.8% decrease) and the least significant in group III (4.6% decrease). CONCLUSION: α1,2-Fcosyltransferase transgene seems to influence mRNA and protein CYP3A expression in case of liver grafting and preservation for transplantation. CYP3A expression was also influenced by the kind of preservation solution used.

Interleukin-6 concentration in the transgenic pig's liver preserved for 24 hours in Biolasol solution.

INTRODUCTION: Increasing the human lifespan contributes to a higher number of patients with end-stage organ failure, which in turn stimulates the search for alternative sources. Xenotransplantation seems to be a promising approach in this respect. OBJECTIVE: Analysis of changes in interleukin (IL)-6 concentration during 24-hour preservation of transgenic swine livers, depending on the kind of transgenesis and preservation solution used. MATERIALS AND METHODS: The experiment was carried out in swine livers with transferred human genes that were divided into 5 groups. The following human genes were transferred: α1,2-fucosyltransferase (group I and II), α-galactosidase (III), combined α1,2-fucosyltransferase/α-galactosidase transgene (IV), and livers without modification (V). The livers were perfused and subsequently stored for 24 hours in Ringer's (group I) or Biolasol solutions (II-V). Reflush was then performed. IL-6 concentration was analyzed in the solution samples collected at the beginning and end of perfusion, and after 24 hours of preservation. ELISA was used to evaluate IL-6 concentration. RESULTS: In liver homogenates from group I, IL-6 concentration after 24 hours of preservation increased by 8.24% compared to the levels observed after perfusion, whereas in the other groups IL-6 concentration decreased. The most significant decrease, 49.51%, was observed in group II; the least significant in group IV, 10.72%. In case of supernatants, a statistically significant increase of AUC0-30min level in relation to perfusion was observed in every group after 24-hour preservation and reperfusion. The highest values of AUC0-30min were observed in group I (α1,2-fucosyltransferase, Ringer's solution). CONCLUSION: The study indicates the hepatoprotective action of Biolasol solution.

The effect of HTK solution modification by addition of thyrotropin and corticotropin on biochemical indices reflecting ischemic damage to porcine kidney.

INTRODUCTION: The aim of our study was to evaluate the impact of perfusion with HTK (histidine-tryptophan-ketoglutarate, Custodiol®, Dr. Franz Kohler Chemie, Germany) solution, modified by the addition of porcine thyroid-stimulating hormone (TSH) and corticotropin (ACTH), on selected biochemical parameters of porcine renal damage within 24 and 48 hours after the onset of cold ischemia time. METHODS: Each study group consisted of 10 adult pigs. During harvesting the kidneys were rinsed with Ringer solution (group 1), HTK (group 2), HTK-TSH (1 µg/dL) or HTK-ACTH (1 µg/dL) in groups 3 and 4. The solutions were cooled to 4°C-6°C. Within 30 minutes of the first perfusion, the discharged fluid was clear and the kidneys cooled to 4°C. The levels of lactate dehydrogenase, asparagine and alanine aminotransferases, lactates, total protein, potassium, calcium, and pH were determined in the perfusate. After 24 and 48 hours the rinsing procedure and the above-mentioned tests were repeated. Differences between the means of 2 independent samples were tested with a nonparametric Mann-Whitney U test. RESULTS: As the result of hormone addition, in both time intervals it was possible to observe considerably lower protein concentrations (g/L) in perfusates compared with HTK solution, without an addition. At 24 hours, we measured following values: 36 ± 4, 8 ± 3 and 6 ± 1 versus 48 hours, 34 ± 1, 2 ± 1, and 4 ± 1 in groups 2, 3, and 4. A similar pattern was observed with LDH (U/L) at 48 hours: 662 ± 89, 374 ± 151, and 386 ± 111, respectively. Lactate concentrations (mmol/L) were then significantly higher: 1.4 ± 0.3 in the TSH group and 1.2 ± 0.5 in the ACTH group as opposed to 0.2 ± 0.1 in unmodified HTK group. CONCLUSION: We observed the possibility of cytoprotective actions of TSH and ACTH addition to the perfusion fluid during cold ischemia, positive effects that were especially visible upon prolonged 48-hour storage.

Effect of iodine-enriched yeast supplementation of diet on performance of laying hens, egg traits, and egg iodine content.

The aim of the study was to evaluate the effect of iodine yeast (I-yeast) supplementation on the performance, egg traits, and iodine content of eggs of laying hens. The experiment was conducted as a completely randomized design. A total of 60 laying hens (Hy-Line Brown), 25 wk of age, was divided into 3 groups (4 replicates), and a feeding experiment was conducted for 12 wk. The concentrations and forms of iodine added to the basal diet were as follows: control group, 1 mg of iodine/kg of feed, Ca(IO(3))(2)•H(2)O; experimental groups E1 and E2, 1 and 2 mg of iodine per kilogram of feed, I-yeast, respectively. The iodine yeast did not significantly affect BW gain. Lower level of hen day egg production for groups E1 and E2 was not confirmed statistically; however, it was probably the consequence of low replication. Feed intake was the lowest in the E1 group and feed conversion rate was the highest in the E2 group. Furthermore, the egg and albumen weight was the highest in the group supplemented with 2 mg/kg of iodine from I-yeast (P < 0.05). The concentration of iodine in the egg yolk from groups E1 and E2 was respectively about 80 and 90% higher, compared with the control group. Eggshells from the group fed with 2 mg/kg of I-yeast contained almost 3 times more iodine than eggshells from the control group. The results suggest that iodine yeast supplementation in the diet of laying hens is an effective method for increasing iodine concentration in eggs and thus could contribute to elimination of iodine deficiency disorders in humans consuming iodine-enriched eggs.

Hepatoprotective effect of prolactin and cysteine contained in perfusion and preservation solutions on porcine liver stored in simple hypothermia.

INTRODUCTION: The aim of our study was to determine the results of histidine-tryptophan-ketoglutarate (HTK) solutions modified by the addition of the antioxidant cysteine (Cys), and of prolactin (PRL) on storage of isolated porcine livers. METHODS: We measured in the media of isolated livers stored for 24 hours in HTK (control group) or modified HTK+Cys (0.3 mmol/L)+PRL (3 IU/L study group) the amounts of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), lactic acid as well as Ca (II), Mg (II), Na (I) and K (I) ions during a 30-minute perfusion after 24 hours of storage. RESULTS: All tested markers were released more slowly into HTK+Cys+PRL with less release of K(I) and Mg(II) and greater of Na(I) and Ca(II) ions. CONCLUSIONS: Addition of the Cys and PRL to HTK positively affected 24-hour storage of isolated livers.

Evaluation of cysteine effect on redox potential of porcine liver preserved by simple hypothermia.

INTRODUCTION: Cysteine (cys), a thiol amino-acid, is involved in de novo glutathione (GSH) synthesis in the extra- and intracellular space. It is also probably involved in the anaerobic glycolysis process. Both these facts may affect the metabolic condition of the liver preserved by simple hypothermia for transplantation. The aim of the study was to verify whether cysteine addition to histidine-tryptophan-ketoglutarate (HTK) organ preservation solution showed a positive effect on liver redox potential after 12-hour preservation in simple hypothermia. MATERIALS AND METHODS: After collecting livers of Great White breed pigs that underwent 30 min of warm ischemia, before 30-min perfusion and cooling to 4°C with modified HTK solution containing cysteine prior to 12 h of preservation. Activity of glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) was determined in liver homogenates after perfusion and after the preservation period. The results were compared with pure HTK, Ringer's and reference University of Wisconsin (UW) solutions. RESULTS: 30 min of perfusion and 12 h of cold preservation (CIT) in the Ringer's solution markedly increased GPx, SOD, and GR activities in liver homogenates compared with the activity using other fluids. After 12-h CIT the activities of GR, GPx and SOD were significantly higher in cys-modified HTK solution than the control HTK solution. They were comparable to the values recorded for the UW group. CONCLUSIONS: Addition of cys to the HTK solution positively influenced the total pool of free radical scavengers in a liver undergoing 12-hour ischemia in the simple hypothermia, which was reflected in the elevated redox enzyme activity possibly due to cys participation in GSH synthesis.

Adrenaline and dopamine in porcine livers after cold preservation with University of Wisconsin histidine-tryptophan-ketoglutarate, prolactin-modified histidine-tryptophan-ketoglutarate solutions.

INTRODUCTION: Hepatic ischemia-reperfusion injury remains a significant factor influencing early liver graft function. The aim of this study was to assess the impact on hepatic ischemia as reflected by catecholamine concentrations of different methods of organ preservation. MATERIALS AND METHODS: Catecholamine levels were measured in 24 (n=6/group) pig livers, which underwent 30-minute warm ischemia followed by 30-minute perfusion and subsequent cold storage for 12 hours. For perfusion and preservation, we used University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), HTK-modified with prolactin (PRL) or Ringer's solutions. Dopamine (DO) and adrenaline (ADR) concentrations in liver venous effluents were assayed using a radioimmunological method after 30 minutes of perfusion and following 12 hours of preservation. RESULTS: DO and ADR levels were higher after 12 hours preservation compared to 30 minutes of perfusion. HTK produced an increase of over 100%. Addition of PRL (20 IU/L) did not affect DO and ADR levels after 30 minutes of perfusion, but significantly decreased their concentrations at 12 hours of preservation. After UW perfusion and preservation, we observed a 10% increase in catecholamine levels as compared with postperfusion values. Preservation with Ringer's solution demonstrated significantly higher DO and ADR levels compared with other solutions. CONCLUSION: Catecholamines are present in the liver after 30 minute of perfusion and 12 hours of cold storage. The increased levels after 12 hours of preservation may be due to their release from intracellular spaces (as a controlled process or as a result of necrosis). It may play a crucial role in reperfusion injury, which, in turn, may explain the mechanism of no-reflow phenomenon.

Evaluation of apoptosis in the liver preserved by simple hypothermia using histidine-tryptophan-ketoglutarate and prolactin-modified histidine-tryptophan-ketoglutarate solution.

INTRODUCTION: Organ ischemia is accompanied by cell death due to apoptosis. It occurs together with necrosis, which has more unfavorable consequences due to the release of cytokines that activate the inflammatory response cascade. The aim of this study was to assess the degree of apoptosis in porcine livers preserved by simple hypothermia for 12 hours using standard solutions (University of Wisconsin [UW] and histidine-tryptophan-glutarate [HTK]), and to evaluate the effect of prolactin (PRL) addition to the HTK solution. MATERIALS AND METHODS: The study was performed on the livers of Great White breed pigs, after inducing 30 minutes of warm ischemia (WIT30), followed by 30 minutes of perfusion-cooling to 4°C, and 12 hours of preservation. Livers were evaluated after preservation in Ringer's solution (control); UW (control reference fluid); HTK and HTK modified by the addition of prolactin (20 UI/L. Apoptosis was assessed in liver sections by the TdT-mediated dUTP nick-end labeling method after 12-hour preservation. We adopted a prevalence scale ranging from 0 to 3+, depending on the number of observed nuclei and apoptotic bodies (AB). RESULTS: Preservation in Ringer's solution yielded AB distribution at the 1+ level, with a lack of characteristic localization resulting from necrotic lesions. Analysis of the livers preserved in the UW solution showed high, 3+ level of AB presence. For the tested HTK solution, the observed ABs localization value was 3+, whereas in the PRL-modified group it was also 3+, but with a tendency to move from zone II to cluster III, which is important for liver metabolic functions. CONCLUSIONS: PRL improved the preservation properties of HTK for porcine livers by maintaining a high apoptosis level. It may stabilize cell membranes thus reducing the oncotic necrosis, promoting increased apoptosis during simple hypothermia.

Differential action of two prolactin isoforms on ischemia and re-perfusion-induced arrhythmias in rats in vivo.

BACKGROUND: The different influences of one of the PRL isoforms (PRL I) on the cardiovascular system have been described in the past. AIM: Our goal was to establish an appropriate iv dose of 2 PRL isoforms (PRL I and PRL II) in intact rats. After establishing this dose, PRL I (0.01 mg/kg) or PRL II (0.001 mg/kg) was administered in bolus 10 min before left anterior descending coronary artery occlusion (7 min) followed by re-perfusion (15 min). We then aimed to study and compare the effects of these isoforms on ischemia- and re-perfusion-induced arrhythmias in the ischemia and re-perfusion-induced arrhythmias model in rats. MATERIALS AND METHODS: Mortality index, ventricular fibrillation and tachycardia (VF, VT) incidence and duration, systolic, diastolic, and mean arterial blood pressure, heart rate and myocardial index of oxygen consumption [pressure rate product (PRP)] were measured and calculated. RESULTS: Both PRL isoforms reduced animal mortality (from 50 to 18.75 and 25%, respectively). PRL II significantly reduced VF incidence (to 25%) as well as VT duration (18.21 ± 3.09) and these effects were markedly different from PRL I and from the control group (p<0.05). Both PRL reduced PRP in the recovery phase (p<0.05). CONCLUSIONS: We proved that supraphysiological doses of PRL isoforms administered in bolus could protect against sudden cardiac death as well as severe arrhythmias episodes during re-perfusion. Because of PRL's positive influence on the cardiovascular system and as an endogenous, well-tolerated substance, it might be of potential clinical use.

The effect of HTK solution modification by addition of prolactin on biochemical indices reflecting ischaemic damage to porcine kidney.

INTRODUCTION: The aim of our study was to evaluate the impact of perfusion with HTK solution, modified by the addition of prolactin (PRI), on selected biochemical parameters of porcine renal damage within 24 and 48 hours after the onset of cold ischemia time. METHODS: Each study group consisted of 10 adult pigs. During harvesting the kidneys were rinsed with Ringer's solution (group 1), HTK (group 2), and HTK+PRL in a dose of 0.2 mg/dL, 0.02 mg/dL, and 0.01 mg/dL in groups 3, 4 and 5, respectively. The levels of lactate dehydrogenase, asparagine (AST) and alanine aminotransferases, lactates, total protein, potassium and calcium were determined in the perfusate. After 24 and 48 hours the rinsing procedure and the abovementioned tests were repeated. RESULTS: After 24 hours of storage, in 4 groups, significantly lower levels of LDH (U/L) were recorded compared with HTK solution alone, namely 235 ± 93 versus 271 ± 125 (perfusion minute, 0), and 55 ± 21 versus 125 ± 94 (30th minute). Similar behavior pattern was presented by AST (U/L) and potassium (mmol/L), and the results were 31 ± 8 versus 35 ± 12 and 16 ± 10 versus 29 ± 14, and 12 ± 3 versus 16 ± 3 and 10 ± 1 versus 13 ± 1, respectively. The changes described above were not observed in the 48th hour of reperfusion. CONCLUSION: Our study results indicate the possibility of cytoprotective action of PRL after adding it to the fluid perfusing kidneys during cold ischemia. This effect, observed after 24 hours of storage, was to a considerable extent dose dependent. In our experiment the effect was pronounced only at 0.02 mg/dL supply of PRL.

Insulin availability from mucoadhesive tablets.

The widespread implementation of peptides as drugs encounters numerous obstacles, the main being invasive and inconvenient parenteral administration. Oral transmucosal administration is one of the possible alternatives, valuable for its noninvasiveness and easy accessibility. The aim of our study was to determine the implementation possibilities of mucoadhesive tablets prepared on a methylcellulose and sodium alginate basis with an addition of absorption-modifying hyaluronic acid, as carriers for peptides destined for oral transmucosal administration. Two series of 50 mg tablets containing 5mg of insulin were prepared for the study. The first series contained methylcellulose, hyaluronic acid and mannitol, while the second series' formulation included sodium alginate, hyaluronic acid and mannitol. Carried out study confirmed that insulin administration in the form of mucoadhesive tablets lowers blood glucose levels in rabbits. Better effects were reached in vivo in the case of MC-based tablets, for which stronger and longer glycemia lowering was achieved.