Anikó Pósa

Reduction of experimental colitis in the rat by inhibitors of glycogen synthase kinase‐3β

The effects of the inhibitors of glycogen synthase kinase‐3β (GSK‐3β), TDZD‐8 and SB 415286, which can substantially reduce the systemic inflammation associated with endotoxic shock in vivo, have now been investigated on the acute colitis provoked by trinitrobenzene sulphonic acid (TNBS) in the rat. Administration of the GSK‐3β inhibitor TDZD‐8 (0.1, 0.33 or 1.0 mg kg−1, s.c., b.i.d., for 3 days) caused a dose‐dependent reduction in the colonic inflammation induced by intracolonic TNBS assessed after 3 days, both as the area of macroscopic involvement and as a score using 0–10 scale. Likewise, following administration of the GSK‐3β inhibitor SB 415286 (0.1, 0.33 or 1.0 mg kg−1, s.c., b.i.d., for 3 days), the extent and degree of the TNBS‐provoked colonic inflammation was reduced. Administration of either TDZD‐8 or SB 415286 reduced the fall in body weight following challenge with TNBS at each dose level studied. The increase in myeloperoxidase activity, an index of neutrophil infiltration into the TNBS‐induced inflamed colon, was significantly inhibited by both TDZD‐8 and SB 415286 at each dose level. The increase in the levels of the proinflammatory cytokine, TNF‐α, in the inflamed colon was also significantly inhibited by either compound at the highest doses evaluated. The elevated levels of the transcription factor NF‐κB subunit p65, as determined by Western blot in the nuclear extracts from the TNBS‐provoked inflamed colonic tissue, were dose‐dependently reduced by TDZD‐8 or SB 415286 treatment. These findings demonstrate that two chemically distinct selective inhibitors of the activity of GSK‐3β reduce the inflammation and tissue injury in a rat model of acute colitis. The mechanisms underlying this anti‐inflammatory action may be related to downregulation of NF‐κB activity, involved in the generation of proinflammatory mediators.

Optimization of drug-eluting balloon use for safety and efficacy: Evaluation of the 2nd generation paclitaxel-eluting DIOR-balloon in porcine coronary arteries

Objectives: The aim of this preclinical study was to optimize the use of drug‐eluting balloon (DEB) DIOR2nd generation by measurements of tissue and plasma paclitaxel concentrations in porcine coronary artery overstretch and prove efficacy in inhibition of neointimal growth without complementary use of stent. Background: The usually recommended DEB 60 sec inflation time causes prolonged ischemia and arterial injury. Methods: Tissue, plasma, and balloon surface concentrations of paclitaxel were measured in pigs 45 min and 12 hr after balloon inflation times of 15, 20, 30, 45, and 60 sec. Extent of neointimal hyperplasia was compared using DIOR2nd generation or noncoated balloon at two‐week follow‐up. Paclitaxel was replaced by fluorescent paclitaxel derivative in DEB and DES to demonstrate the distribution of the drug in arterial wall. Results: DIOR2nd generation DEB provided 29 ± 3 μM/L, 52 ± 6 μM/L, 196 ± 44 μM/L, 202 ± 36 μM/L, and 184 ± 59 μM/L paclitaxel to the vessel wall after 15, 20, 30, 45, and 60 sec of dilation, reaching plateau at 30 sec inflation time. Paclitaxel penetrated up to 2 mm tissue deepness. Measurable plasma paclitaxel level (45 ± 28 ng/mL) was found only after 60 sec balloon inflation time. At follow‐up, the dilated arterial segment neointimal area and maximal neointimal thickness were significantly smaller with DIOR vs. uncoated balloon use. Fluorescence images of DIOR showed a homogenous distribution of the drug on the vessel, in contrast with DES. Conclusion: Using the DIOR2nd generation DEB, a maximal balloon inflation time of 30–45 sec is optimal, reducing effectively the neointimal hyperplasia.

Attainment of local drug delivery with paclitaxel-eluting balloon in porcine coronary arteries.

ObjectiveOur purpose was to confirm the local drug delivery of a paclitaxel-eluting balloon by percutaneous intervention of single arterial segments or bifurcations of porcine coronary arteries. MethodsEight domestic pigs were subjected to 2×30 s Dior balloon dilatation of the mid left anterior descending, left circumflex and proximal right coronary arteries. Bifurcation intervention was performed in six arteries. The dilated, and the distal and proximal reference segments were prepared for tissue paclitaxel concentration measurement. Tissue samples were harvested at mean 1.5, 12, 24 and 48 h after balloon dilatation and plasma samples were taken at various time points. ResultsThe tissue paclitaxel concentration of the single dilated segment was at 1.5 h postdilatation 1.82±1.60 μmol/l, which decreased significantly to 0.73±0.27 (P=0.032), 0.62±0.34 and 0.44±0.31 μmol/l at 12, 24 and 48 h. The bifurcation intervention resulted in 5.10±1.80 μmol/l tissue paclitaxel amount in the main branch, which at 12 h had diminished to 1.41±1.23 μmol/l (P=0.004). The bifurcation side contained 7.00±4.80 μmol/l paclitaxel at 1.5 h postdilatation, which lowered to 2.72±0.40 μmol/l (P=0.034). The mean paclitaxel concentration of the reference segments decreased gradually from 0.84±0.99 to 0.34±0.36 μmol/l (P=0.09), 0.28±0.16 and 0.19±0.18 μmol/l tissue at 1.5, 12, 24 and 48 h postdilatation, respectively. No paclitaxel was found in the peripheral blood at any time point. ConclusionShort exposure of the coronary artery to paclitaxel with a coated balloon is sufficient for the attainment of an adequate tissue concentration of paclitaxel, which is known to be efficient in inhibiting neointimal growth.

The involvement of heme oxygenase-1 activity in the therapeutic actions of 5-aminosalicylic acid in rat colitis

The mechanism of action of 5-aminosalicylic acid (5-ASA), the active therapeutic moiety of a number of clinically used anti-colitic agents, is unclear. The present study investigates whether the beneficial effects in vivo could involve induction of the heat shock protein, heme oxygenase-1 (HO-1), known to provide endogenous anti-oxidant and anti-inflammatory moieties which can modulate colonic inflammation. The effects of 5-ASA on the colonic expression and activity of HO-1 along with its effect on the inflammatory damage have been evaluated in the colitis provoked by instillation of trinitrobenzene sulphonic acid (TNBS) over 48 h in the rat. Intracolonic administration of 5-ASA (8, 25 and 75 mg/kg/day) dose-dependently reduced the TNBS-provoked macroscopic colonic inflammatory injury, myeloperoxidase (MPO) activity and TNF-alpha levels, while also dose-dependently increasing colonic heme oxygenase enzyme activity. Colonic HO-1 protein expression, determined by Western blot analysis in this colitis model, was likewise further induced by 5-ASA. Intracolonic administration of 5-ASA alone under unchallenged conditions also induced colonic HO-1 protein expression and stimulated heme oxygenase enzyme activity. Administration of zinc protoporphyrin (50 micromol/kg/day, s.c.), which prevented the increase in colonic heme oxygenase activity, abolished the anti-colitic effect of 5-ASA. These results suggest that 5-ASA may exert its colonic anti-oxidant and anti-inflammatory effects in vivo in part through the up-regulation of HO-1 enzyme expression and activity.

Attenuation of inflammation and cytokine production in rat colitis by a novel selective inhibitor of leukotriene A4 hydrolase

Leukotriene B4 (LTB4), formed by the sequential actions of the 5‐lipoxygenase (5‐LO) and leukotriene A4 hydrolase (LTA4H), is a pro‐inflammatory mediator implicated in the pathogenesis of inflammatory bowel disease. However, inhibitors of 5‐LO have not proved to be consistent in their therapeutic efficacy in colitis. Another approach to inhibiting LTB4 synthesis is through the use of inhibitors of LTA4H, such as the novel, potent and selective compound, JNJ 26993135.

Zerumbone exerts a beneficial effect on inflammatory parameters of cholecystokinin octapeptide-induced experimental pancreatitis but fails to improve histology.

Objective: Our experiments were designed to investigate the effects of zerumbone pretreatment on cholecystokinin octapeptide (CCK-8)-induced acute pancreatitis in rats. Methods: Male Wistar rats weighing 240 to 280 g were divided into a control group, a group treated with CCK-8, a group receiving 20 mg/kg zerumbone before CCK-8 administration, and a group treated with zerumbone only. Results: The serum amylase and lipase activities and the pancreatic weight-body weight ratio were significantly reduced by zerumbone pretreatment, but the drug failed to influence the histological parameters of pancreatitis. The anti-inflammatory effects of the drug were manifested in decreases in the cytosolic interleukin 6 and tumor necrosis factor &agr; concentrations and an elevation in the I-&kgr;B concentration, whereas the antioxidant ability of zerumbone was demonstrated by reductions in inducible nitric oxide synthase, Mn- and Cu/Zn-superoxide dismutase activities in the zerumbone-treated rats. Conclusion: Zerumbone ameliorated the changes of several parameters of acute pancreatitis probably by interfering with I-&kgr;B degradation, but in the applied dose, it failed to influence the histology of the disease.Abbreviations: CCK-8 - cholecystokinin octapeptide, DMSO - dimethyl sulfoxide, iNOS - inducible nitric oxide synthase, cNOS - constitutive nitric oxide synthase, TNF, tumor necrosis factor, IL-6 - interleukin 6, NF-&kgr;B - nuclear factor &kgr;B, SOD - superoxide dismutase, ASAT - aspartate aminotransferase

Differential effect of ischaemic preconditioning on mobilisation and recruitment of haematopoietic and mesenchymal stem cells in porcine myocardial ischaemia-reperfusion

Effects of ischaemic preconditioning (IP) on the mobilisation and recruitment of haematopoietic (HSCs) and mesenchymal stem (MSC) cells were determined in porcine coronary occlusion/reperfusion. Thirty-three pigs underwent percutaneous occlusion of the left anterior descending coronary artery (LAD) for 90 minutes (min), followed by 120 min reperfusion. IP was performed in 16 of the 33 pigs by two cycles of 5 min balloon occlusion/reperfusion prior to the 90 min occlusion (group IP vs. group C). Peripheral blood and myocardial tissue concentration of bone marrow origin HSCs (characterised by coexpression of CD31+, CD90+, CD45+) and MSCs (characterised by coexpression of CD44+, CD90+, CD45-) were measured by flow cytometry in the early phase of IP. Plasma/serum levels of stem cell mobilisation factors (stromal cell-derived factor-1a [SDF-1a], vascular endothelial growth factor [VEGF], tumour necrosis factor a[TNF-a] and interleukin-8 [IL-8]) were measured. IP led to a significant increase in circulating HSCs as compared with the group C (475 +/- 233 vs. 281 +/- 264 /ml, p=0.032) in the early phase of IP. In contrast, a rapid and prolonged decrease in level of circulating MSCs was observed in group IP as compared with group C (19 +/- 12 vs. 32 +/- 17 /ml, p=0.015). The recruitment of HSCs and MSCs in infarct and border zone was significantly greater in IP group, indicating a faster homing of MSCs as compared with the rate of mobilisation. Rapid increase in VEGF, TNF-a and IL-8 levels was induced by IP, which, however, was not correlated with the levels of circulating SCs. In conclusion, IP resulted in differential mobilisation and recruitment of HSCs and MSCs in the early phase of cardioprotection.

Experimental Diabetes Mellitus in Different Animal Models

Animal models have historically played a critical role in the exploration and characterization of disease pathophysiology and target identification and in the evaluation of novel therapeutic agents and treatments in vivo. Diabetes mellitus disease, commonly known as diabetes, is a group of metabolic disorders characterized by high blood glucose levels for a prolonged time. To avoid late complications of diabetes and related costs, primary prevention and early treatment are therefore necessary. Due to its chronic symptoms, new treatment strategies need to be developed, because of the limited effectiveness of the current therapies. We overviewed the pathophysiological features of diabetes in relation to its complications in type 1 and type 2 mice along with rat models, including Zucker Diabetic Fatty (ZDF) rats, BB rats, LEW 1AR1/-iddm rats, Goto-Kakizaki rats, chemically induced diabetic models, and Nonobese Diabetic mouse, and Akita mice model. The advantages and disadvantages that these models comprise were also addressed in this review. This paper briefly reviews the wide pathophysiological and molecular mechanisms associated with type 1 and type 2 diabetes, particularly focusing on the challenges associated with the evaluation and predictive validation of these models as ideal animal models for preclinical assessments and discovering new drugs and therapeutic agents for translational application in humans.

Anti-Inflammatory Effect of Recreational Exercise in TNBS-Induced Colitis in Rats: Role of NOS/HO/MPO System

There are opposite views in the available literature: Whether physical exercise has a protective effect or not on the onset of inflammatory bowel disease (IBD). Therefore, we investigated the effects of recreational physical exercise before the induction of colitis. After 6 weeks of voluntary physical activity (running wheel), male Wistar rats were treated with TNBS (10 mg). 72 hrs after trinitrobenzene sulphonic acid (TNBS) challenge we measured colonic gene (TNF-α, IL-1β, CXCL1 and IL-10) and protein (TNF-α) expressions of various inflammatory mediators and enzyme activities of heme oxygenase (HO), nitric oxide synthase (NOS), and myeloperoxidase (MPO) enzymes. Wheel running significantly increased the activities of HO, constitutive NOS (cNOS) isoform. Furthermore, 6 weeks of running significantly decreased TNBS-induced inflammatory markers, including extent of lesions, severity of mucosal damage, and gene expression of IL-1β, CXCL1, and MPO activity, while IL-10 gene expression and cNOS activity were increased. iNOS activity decreased and the activity of HO enzyme increased, but not significantly, compared to the sedentary TNBS-treated group. In conclusion, recreational physical exercise can play an anti-inflammatory role by downregulating the gene expression of proinflammatory mediators, inducing anti-inflammatory mediators, and modulating the activities of HO and NOS enzymes in a rat model of colitis.

A novel method for the rapid determination of beta-amyloid toxicity on acute hippocampal slices using MTT and LDH assays

It is difficult task to measure precisely the toxic effect of beta-amyloid (Aβ 1-42) peptides and also the protective effect of novel drug candidates against Aβ-peptides. The widely used MTT-assay in cell lines or primary cell cultures could be insensitive against Aβ-peptides. We describe here an easy and relevant method for testing Aβ 1-42 toxicity on acute hippocampal slices derived from rat. Brain slice viability in different conditions was measured using MTT and LDH assays. The concomitant use of these two assays can give detailed and relevant results on the toxic effect of Aβ 1-42 in oxygen-glucose deprived (OGD) acute brain slice model. Both assays are capable of quantifying tissue viability by measuring optical density (OD). We found that simultaneous application of OGD and Aβ 1-42 treatment induced a more intensive decrease in hippocampal slice viability than their separate effects. The use of MTT and LDH assay for quantifying brain slice viability proved to be an easy ex vivo method for investigating Aβ toxicity. Testing brain slices is more relevant in Alzheimers Disease research than using in vitro cell cultures, due to maintenance of the three dimensional cellular network, the cell variability and intact cell connections.