Zoran Stanojkovic

The effect of ursodeoxycholic acid on oxidative stress level and DNase activity in rat liver after bile duct ligation

Accumulation of hydrophobic bile acids (BAs) during cholestasis plays an important role in apoptosis initiation as well as oxidative stress increase in liver cells. Ursodeoxycholic acid (UDCA) acts as a protector in BA-induced cell injury.The aim of the study was to evaluate the effect of UDCA on oxidative stress level and DNase I and II activity caused by liver injury in bile duct ligation (BDL) rats.Wistar rats were divided in four groups: group 1, control (sham-operated); group 2, sham-operated and injected with UDCA (30 mg/kg); group 3,animals with BDL; and group 4,UDCA-treatedcholestatic rats. Animals were sacrificed after 9 days. Malondialdehyde (MDA; lipid peroxidation end-product) level and protein-molecule oxidative modification (carbonyl group content) significantly increased in BDL rat liver. Catalase (CAT) activity in liver tissue was found to be decreased in BDL rats. In addition, xanthine oxidase (XO) activity, which is thought to be one of the key enzymes producing reactive oxygen species, was found to be increased in the cholestatic group. The apoptotic effect in cholestasis was probably triggered by the increased activation of DNase I and II. The protective effect of UDCA on liver tissue damage in BDL rats, in comparison to cholestatic liver, were 1) decrease of MDA levels, 2) increased CAT activity, 3) reduced XO activity, and 4) effect on terminal apoptotic reaction, shown as a decrease in DNase I and II activity.Therefore, UDCA may be useful in the preservation of liver function in cholestasis treatment.

The Effects of Melatonin on Oxidative Stress Parameters and DNA Fragmentation in Testicular Tissue of Rats Exposed to Microwave Radiation

BACKGROUND Microwaves from mobile phones are one of the environmental toxicants that are capable of compromising male fertility by inducing oxidative stress and apoptosis in the testes. Melatonin is a lipophilic tryptophan indole amine and a potent antioxidant. OBJECTIVES The aim of the study was to evaluate the effect of melatonin treatment on oxidative stress parameters and DNA fragmentation in the testicular tissue of rats exposed to microwave radiation (4 h/day). MATERIAL AND METHODS Adult Wistar rats were divided in 4 groups: I--treated with saline; II--treated with melatonin; III--exposed to microwaves; IV--exposed to microwaves and treated with melatonin. The melatonin (2 mg/kg ip) was administered daily. The animals were sacrificed after 20, 40 and 60 days. RESULTS Melatonin treatment prevented previously registered increases in malondialdehyde after only 20 days. Furthermore, it reversed the effects of microwave exposure on xanthine oxidase (after 40 days) and acid-DNase activity (after 20 days). However, neither protein carbonyl content nor catalase and alkaline Dnase activity were changed due to melatonin treatment. CONCLUSIONS Melatonin exerts potent antioxidant effects in the testes of rats exposed to microwaves by decreasing the intensity of oxidative stress; it also reduces DNA fragmentation.

Clinical efficacy of riboflavin and ultraviolet light inactivated fresh frozen plasma evaluated with INR-quantification

Treatment of blood products by riboflavin and ultraviolet (UV) light prevents of white blood cell (WBC) replication and inactivates of pathogens. The aim of this study was to determine the effects of the inactivation by riboflavin and UV light upon plasma clinical performance, based on effect on the pretransfusion international normalized ratio (INR). A prospective, controlled randomized study included 60 patients who received transfusion of plasma on the Clinic for hematology of Clinical Centre in Nis. Experimental group (EG; 30 patients) was treated with Mirasol-inactivated fresh frozen plasma (FFP) and control group (CG; 30 patients) was transfused with noninactivated FFP. Besides pretransfusion vs. posttransfusion INR, the improvement in INR patients plasma level per one FFP unit transfused was evaluated. Total of 68 units of FFP were transfused to patients of CG (2.24±0.83 units per patient). Patients of EG received 84 units of Mirasol-inactivated plasma (i.e. 2.80±1.19 units per patient). There was significant increase in number of FFP transfusions that normalized coagulation parameters in EG compared to CG (p=0.039). Also, there was a significant improvement of INR after every FFP unit application (p=0.046). We found a linear relationship between pretransfusion INR and improvement of INR (r=0.97; p<0.001). Plasma treated with riboflavin and UV light retains hemostatic competence and can be used efficiently in the therapy of congenital or acquired coagulopathies, but in larger quantity as compared to noninactivated FFP volume.

Evaluation of platelet activation in leukocyte-depleted platelet concentrates during storage

Structural and functional changes in platelets during storage can lead to the loss of platelet reactivity and response. Our aim was to evaluate leukocyte-depleted platelet concentrates on storage days 0, 3 and 5, obtained by in-line filtration. In non-filtered platelet concentrates (NF-PC) group, 180 whole blood units were collected with quadruple blood bags and then compared to another group of 180 whole blood units (leukocyte-depleted platelet concentrates [LD-PC]), collected in Imuflex Whole Blood Filter Saving Platelets (WB-SP) bags with an integrated leukoreduction filter, with regard to the platelet quality and characteristics. The efficacy of the two techniques for platelet concentrate preparation was evaluated by white blood cell (WBC) and platelet count on day 0. The partial pressure of oxygen (pO2), pH, platelets positive for P-selectin (CD62P), CD63, cluster of differentiation 42b (CD42b), phosphatidylserine (PS), and mitochondrial membrane potential (MMP) were analyzed during the storage in both groups. A significantly lower WBC count and higher platelet count was observed in LD-PC compared to NF-PC group, indicating the overall efficacy of the first technique. During the 5-day storage, pH and pO2 decreased in both groups. In LD-PC group, higher pH, increased pO2 and decreased platelet surface expression of CD62P, CD63 and PS were observed compared to NF-PC group. In both groups, the percentage of CD42b positive platelets and MMP did not change significantly during the 5-day period. The assessment of different markers of platelet activation may be an effective tool in evaluating the quality of platelets during storage. A better understanding of platelet activation may provide new insights for developing a novel therapeutic approach in the manipulation of platelet aggregation.

Radically reduced ex vivo cell activation by using "in-line" filtered whole blood as a source of platelet concentrate.

Dear Sir, Factors affecting the quality of platelet concentrates, obtained from whole blood (WB) are: (i) the processing method used (originating from platelet-rich plasma or buffy coat-derived platelet concentrates [BC-PC]); (ii) the ex vivo manipulation applied (such as cryopreservation, pathogen reduction); (iii) cell storage conditions/duration; and (iv) quantity of residual white blood cells, particularly mononuclear cells1,2. As described previously3, higher mononuclear counts in platelet concentrates are associated with ex vivo cytokine release and cell/cytokine-mediated adverse events. Furthermore, mononuclear cells result in increased platelet surface antigen/marker expression and platelet activation ex vivo, followed by morphological and ultrastructural changes. Commonly, GP140/CD62p expression ≤40% correlates with acceptable clinical efficacy of platelet concentrates. However, discharge of alpha-granules and elevated GP140/CD62p expression (≥50–60%) can lead to decreased platelet recovery and impaired function. A high level of GP53/CD63 expression (due to the release of the contents of lysosomes) is typically detectable only following major platelet activation1,2,4. The aim of this research was to compare the quality of non-filtered platelet concentrates (NF-PC or control group) with “in-line-leucodepleted” platelet concentrates (LD-PC or study group) determined on the basis of residual mononuclear cell count and expression of activation markers (GP/140/CD62p and GP53/CD63), directly after collection and processing of whole blood (day 0), as well as on the 3rd and 5th days of storage of the platelet concentrates (day 3 and day 5). We assumed that the use of “in-line” filtered whole blood (as an optimised source of platelet concentrate) could radically prevent ex vivo platelet activation in LD-PC. For the preparation of NF-PC, whole blood (450±45 mL) from donors, non-reactive for infectious disease markers (hepatitis B and C, AIDS and syphilis) in serological screening tests (Biokit S.A, Llissa de Munt, Spain and Ortho Clinical Diagnostics, Raritan, JS, USA) and with normal clinical data, was collected in quadruple bags (Macopharma, Tourcoing, France). This bag system contains 63 mL citrate phosphate dextrose (CPD) and 100 mL saline-adenine-glucose-mannitol (SAGM) solutions. For the LD-PC, the same volume of whole blood from donors with normal clinical and laboratory findings was collected into Imuflex WB-SP bags (Terumo, Tokio, Japan) with CPD, SAGM, and an “in-line” filter that “saves” (not retains platelets)5. NF-PC were obtained from whole blood using a “standard buffy-coat” method. Non-filtered whole blood (total=180 units) was initially centrifuged with a “hard-spin” technique (3,890×g for 10 minutes, at 20±2 °C) in a Cryofuge 8500 (Heraeus, Langenselbold, Germany) and then processed using a T-ACE device (Terumo, Japan). NF-BC units were obtained from the separated buffy coat units were obtained following “light-spin” centrifugation (310×g for 7 minutes, at 20±2 °C) and second (manual) processing, as described previously2,3. Six NF-PC were pooled using a sterile connection system (TSCD, Terumo, Tokio, Japan) and stored on a shaker (Teknolabo Instruments Srl, Milan, Italy) at 22±2 °C for 5 days. In the same manner “in-line” filtered whole blood (total=180 units) underwent “hard-spin” centrifugation. The leucocyte-poor buffy-coat (platelet rich, but leucoreduced cell layer) was separated on a T-ACE device. The second (“light-spin”) centrifugation of these “leucocyte-poor buffy coats” was performed as described above. LD-PC were obtained by processing; the pooling and storage procedures for LD-PC were completed in the same manner as for the NF-PC. Standard haematological parameters were analysed on a Beckman-ACT device (Beckman-Coulter, Fullerton, CA, USA). The residual white blood cell count in LD-PC units was quantified manually in a Negeotte haemocytometer chamber, as described previously3. The platelet activation markers (GP140/CD62p and GP53/ CD63) were investigated using monoclonal antibodies (Becton Dickinson, San Jose, CA, USA) and flow cytometry. Briefly, samples of platelet concentrates (1×106 cells per tube) were incubated with fluorescein isothiocyanate-labelled monoclonal antibodies, in accordance with the manufacturer’s recommendations and instructions. Non-specific binding was detected using control cells, which were incubated with phosphate-buffered saline alone. After incubation, cells were washed and analysed on an Epics XL (Beckman-Coulter). The results were calculated and expressed as percentage of total cell number4. Differences between groups were analysed by Student’s t-test and were considered as statistically significant at p≤0.05. Our preclinical study confirms that the mononuclear cell count was lower in LD-PC (0.05±0.02×106/unit) than in NF-PC (5.32±1.7×106/unit) (t=6.693; p<0.001) (Table I). Table I The expression of platelet activation markers in NF-PC and LD-PC during 5 days of storage. The levels of GP140/CD62p and GP53/CD63 expression in both NF-PC and LD-PC over the storage period are shown in Table I. GP140/CD62p expression (calculated as a percentage) on day 0 was significantly higher in NF-PC than in LD-PC (t=10.642; p<0.001). Significant differences in GP140/CD62p expression between NF-PC and LD-PC (“intergroup” disparity) were also observed on day 3 (t=12.754; p<0.001) and day 5 (t=10.724; p<0.001). A marked linear increase of GP140/CD62p expression was confirmed in both investigated groups during the 5 days of storage (p<0.001). However, GP53/CD63 expression was significantly higher at each measurement (day 0, day 3, and day 5) in NF-PC than in LD-PC (p<0.001). Finally, GP53/CD63 expression increased significantly during the 5 days of storage in both NF-PC and LD-PC (p<0.001). As stated, the aim of this study was to determine the effect of leucodepletion on the expression of platelet activation markers in platelet concentrates. The results obtained showed that mononuclear cell count was significantly lower in LD-PC than in NF-PC (prepared by the “standard buffy-coat” method). This is consistent with research conducted by Paunovic et al., who showed that the “in-line” WB-SP filter (which “saves” platelets) achieves optimised, practical leucodepletion of blood products-packed (resuspended) red blood cells and platelet concentrates5. Our earlier research showed better quantitative and qualitative platelet recovery -that is, superior hypotonic shock response, aggregation ability, morphological score of platelets, ultrastructural properties (intact microtubules, minimal membrane changes, etc.)- in platelet concentrates treated by multiple, but optimised ex vivo manipulations2–4. The quality of platelet concentrates for transfusion is optimal when no activation markers are expressed, since activated platelets have inferior quantitative and qualitative recovery, and could promote prothrombotic and procoagulant events. Generally, platelet activation is accompanied by increased expression of GP140/CD62p and GP53/CD63 antigens on the platelet membrane. The expression of these antigens rises progressively during storage, and correlates with the degree of cell activation in platelet concentrates. The structural and adhesive glycoprotein GP140/CD62p (or P-selectin) is an integral constituent of the membrane of alpha-granules of “resting” platelets and Weibel-Palade bodies of endothelial cells. Its expression on the platelet surface upon alpha-granule exocytosis has been used as an ex vivo marker of platelet secretion and activation. In fact, GP140/CD62p expression is a marker of the platelet storage-lesion. Thus, GP140/CD62p expression is extensively used to evaluate platelet concentrate quality following whole blood processing, filtering, liquid-state storage or cryopreservation. Nevertheless, despite numerous investigations, the clinical relevance of GP140/CD62p expression levels or their value as an ex vivo measure of in vivo platelet activity or viability remains uncertain1,2,4. GP53/CD63 was the first identified marker of platelet activation that is elevated on the cell surface following releasing from granules2,4. Our recent study established that GP53/CD63 expression was really lower in LD-PC over the entire storage period. The expression of GP53/CD63 was also four times higher in NF-PC on day 5 than on day 0, which is in expected correlation. In summary, the use of optimised (with regards to timing) and effective (with regards to degree) “in-line” leucodepletion (Imuflex WB-SP system) undoubtedly reduces white blood cell and mononuclear cell counts. In the LD-PC investigated, GP140/CD62p and GP53/CD63 expression was less during the 5 days of storage, a finding strongly correlated with lower platelet activation. In accordance with our earlier research, we speculate that the use of LD-PC, with improved ex vivo cell quality, could have a better clinical effect.

Determination of fibrin glue with antibiotics on collagen production in colon anastomosis

BACKGROUND/AIM Fibrin glue is used as a matrix for local application of antibiotics. The aim of this study was to determine whether application of fibrin glue in combination with antibiotics can strengthen collagen production, prevent dehiscence of colon anastomoses due to infection, and reduce frequency of mortality and morbidity comparing to the control group and the group with fibrin glue application. METHODS The adult male Wistar rats divided into three groups were used in the experiment. The group 1 was the control one (after partial colon resection, colonic anastomoses performed were not treated), while to the group 2 and the group 3 were applied fibrin glue and fibrin glue with antibiotics (clindamycin and ceftriaxon) on the site of anastomoses, respectively. Quality of colonic anastomoses were estimated by means of determination of collagen (L-hydroxyproline) amount in the collon wall with anastomoses and histological analysis of this colon segment using light and electronic microscope on the days 5, 7 and 13 postoperatively. RESULTS The highest morbidity rate was registered in the group 1 (30%), then in the group 2 (13.3%) and the lowest one in the group 3 (3.33%; p < 0.05 vs group 1). Mortality rate was significantly higher in the group 1 than in the group 3 (20% and 0%, respectively; p < 0.05). In the postoperative course, the highest concentrations of collagen in the colon wall on the site of anastomoses, which was confirmed by both light and electronic microscopy, were found in the group 3. CONCLUSION The application of fibrin glue with antibiotics on colon anastomoses reduces the number of dehiscence, provides good mechanical protection and shorten the time of anastomoses healing.