Group Of Healthy Animals Research Articles

Improved uptake and therapeutic intervention of curcumin via designing binary lipid nanoparticulate formulation for oral delivery in inflammatory bowel disorder

This study was focussed on development of curcumin loaded solid binary lipid nanoparticles (C-SBLNs) to ameliorate stability, uptake and therapeutic potential of curcumin during inflammatory bowel disease (IBD). C-SBLNs with nano-size range (210.56 ± 41.22 nm) and high entrapment efficiency (83.12 ± 6.57%) were prepared by solvent emulsification evaporation method using binary lipids i.e. stearic acid and tristearin after optimizing various formulation and process variables. Physicochemical characterization of C-SBLNs by ATR-FTIR confirmed drug entrapment whereas thermal and pXRD study corroborated loss of crystallinity of drug into C-SBLNs. Lyophilized C-SBLNs were found to be spherical shaped with good gastrointestinal stability and prolonged drug release up to 24 h. Optimized C-SBLNs formulation displayed significantly enhanced cellular uptake and localization in inflamed tissues during IBD. Oral administration of C-SBLNs in DSS induced colitis model revealed significant reduction in leucocyte infiltration, oxidative stress, pro-inflammatory cytokine (TNF-α) secretion and maintenance of colonic structure similar to healthy animal group compared to curcumin. Thus, in vitro and preclinical findings of study clearly confirmed that C-SBLNs could be a stable and efficacious alternative platform for curcumin delivery with strong competence in IBD chemotherapy.

Theta burst stimulation influence the expression of BDNF in the spinal cord on the experimental autoimmune encephalomyelitis.

Repetitive transcranial magnetic stimulation (rTMS) induces changes in expression of proteins engaged in the activity of excitatory and inhibitory systems, restores these functions and suppresses the progression of disability in experimental autoimmune encephalitis (EAE). The structural type of TMS, the arrangement as theta burst stimulation (TBS) has been applied as intermittent TBS (iTBS) and continuous TBS (cTBS) protocols to female adult DA rats. The animals were randomly divided into experimental groups: control group (C), group treated with complete Freund's adjuvant (CFA), experimental autoimmune encephalomyelitis (EAE) group, group treated with iTBS post EAE immunization (EAE + iTBS), group treated with cTBS post EAE immunization (EAE + cTBS), group of healthy animals treated with iTBS or cTBS. Therapeutic protocols of iTBS or cTBS in all EAE groups of animals were performed starting from 14 days post immunization (dpi), for 10 days with time point decapitation at 24 dpi. After decapitation, spinal cords were analysed for BDNF and Ki67 expression. The results revealed reduced BDNF expression in the rat's spinal cord of EAE animals in the stage of remission, which was associated with increased Ki67 and GFAP expressions. Decreased Iba 1 and BDNF expression, contrary to increased Iba 1 and Ki67 expression, suggests clustered microglia in the resolution phase of EAE. Enhanced GABA expression in spinal cord sections indicates higher GABA metabolic turnover, and also GAD activity in astrocytes, or prominent activity of GABAergic neurons. Both TBS protocols induced advance BDNF expression; amongst iTBS application provoked elevating of BDNF and stabilizing of GFAP and Ki67 expressions.

Reduction of lung inflammation, oxidative stress and apoptosis by the PDE4 inhibitor roflumilast in experimental model of acute lung injury.

Damage of alveolar-capillary barrier, inflammation, oxidative injury, and lung cell apoptosis represent the key features of acute lung injury (ALI). This study evaluated if selective phosphodiesterase (PDE)-4 inhibitor roflumilast can reduce the mentioned changes in lavage-induced model of ALI. Rabbits with ALI were divided into 2 groups: ALI without therapy (A group) and ALI treated with roflumilast i.v. (1 mg/kg; A+R group). One group of healthy animals without ALI served as ventilated controls (C group). All animals were oxygen-ventilated for further 4 h. At the end of experiment, total and differential counts of cells in bronchoalveolar lavage fluid (BALF) and total and differential counts of white blood cells were estimated. Lung edema formation was assessed from determination of protein content in BALF. Pro-inflammatory cytokines (TNFalpha, IL-6 and IL-8) and markers of oxidation (3-nitrotyrosine, thiobarbituric-acid reactive substances) were detected in the lung tissue and plasma. Apoptosis of lung cells was investigated immunohistochemically. Treatment with roflumilast reduced leak of cells, particularly of neutrophils, into the lung, decreased concentrations of cytokines and oxidative products in the lung and plasma, and reduced lung cell apoptosis and edema formation. Concluding, PDE4 inhibitor roflumilast showed potent anti-inflammatory actions in this model of ALI.

THE INFLUENCE OF OZONE THERAPY ON THE MORPHOLOGIC CHANGES IN THE PATIENTS PRESENTING WITH PURULENT INFLAMMATION OF THE LUNGS IN THE EXPERIMENT

We have created the experimental model of chronic inflammation of lungs by means of prolonged mechanical irritation of the bronchi in 30 outbred rats and studied the morphological changes in the lung tissue of these animals rats in three series of experiments. Each rat was given an intraperitoneal injection of an ozonised saline solution produced by a «Binafsha» ozonator. The objective of the study was to compare a control group of healthy animals and the group of experimental animals with chronic purulent pneumonia. The prolonged irritation of the respiratory tract in experimental animals was found to induce the structural changes in the tissues of the lungs characteristic of chronic purulent inflammation. The course of treatment with the ozonised saline solution in healthy animals with experimentally modelled chronic purulent inflammation of the lungs did not have a negative impact on the general condition and the behaviour of the animals. The treatment of experimental chronic inflammation of lungs caused by prolonged mechanical irritation of the respiratory tract resulted in the improvement of the morphological status of the laboratory animals, but the purulent inflammation process failed to be completely resolved after the treatment which needs to be taken into consideration in the clinical practice.

Effect of original anticonvulsant meta-chloro-benzhydryl-urea on behavioral and immune parameters in mice with active and passive behavior types in experimental alcoholism

ObjectiveViolation of the functional activity of the nervous and immune systems is an essential link in the pathogenesis of chronic alcohol dependence. The search for new psychopharmacological agents whose action is directed to correction of neuroimmune interaction opens new perspectives for the treatment of alcohol dependence.Methods(CBAxC57Bl/6) F1 mice with active and passive behavioral types in a state of chronic alcohol dependence owing to 6 month 10% ethanol exposure were undergoing intragastric administration of original anticonvulsant meta-chloro-benzhydryl-urea. Animal's behavioral and immune parameters, brain cytokines synthesis before and after anticonvulsant receiving were estimated.ResultsIn the formation of experimental alcohol dependence in animals the most pronounced changes in motor and exploratory activities in open field test, brain cytokines synthesis and suppression of immune response were registered in mice with passive type of behavior. Daily consumption of ethanol solution in mice with chronic alcohol dependence decreased sharply starting from 2 days of anticonvulsant administration and led to the cessation of ethanol consumption by the 5 day. After anticonvulsant administration for 10 days behavioral parameters in mice were comparable with those in the control group of healthy animals. It also restored brain cytokines synthesis and significant stimulated humoral immune response, estimated by the relative number of antibody-forming spleen cells.ConclusionBehavior and immune changes following chronic ethanol exposure depended on the behavior status of animals; administration of the original anticonvulsant meta-chloro-benzhydryl-urea may correct both immune and behavior disorders in mice with chronic alcohol dependence, so it has promise in the treatment of alcoholism.Disclosure of interestThe authors have not supplied their declaration of competing interest.

MR Vascular Fingerprinting in Stroke and Brain Tumors Models.

In this study, we evaluated an MRI fingerprinting approach (MRvF) designed to provide high-resolution parametric maps of the microvascular architecture (i.e., blood volume fraction, vessel diameter) and function (blood oxygenation) simultaneously. The method was tested in rats (n = 115), divided in 3 models: brain tumors (9 L, C6, F98), permanent stroke, and a control group of healthy animals. We showed that fingerprinting can robustly distinguish between healthy and pathological brain tissues with different behaviors in tumor and stroke models. In particular, fingerprinting revealed that C6 and F98 glioma models have similar signatures while 9 L present a distinct evolution. We also showed that it is possible to improve the results of MRvF and obtain supplemental information by changing the numerical representation of the vascular network. Finally, good agreement was found between MRvF and conventional MR approaches in healthy tissues and in the C6, F98, and permanent stroke models. For the 9 L glioma model, fingerprinting showed blood oxygenation measurements that contradict results obtained with a quantitative BOLD approach. In conclusion, MR vascular fingerprinting seems to be an efficient technique to study microvascular properties in vivo. Multiple technical improvements are feasible and might improve diagnosis and management of brain diseases.

Clinical and subclinical endometritis induced alterations in bovine endometrial transcriptome and miRNome profile.

BackgroundClinical and subclinical endometritis are known to affect the fertility of dairy cows by inducing uterine inflammation. We hypothesized that clinical or subclinical endometritis could affect the fertility of cows by disturbing the molecular milieu of the uterine environment. Here we aimed to investigate the endometrial molecular signatures and pathways affected by clinical and subclinical endometritis. For this, Holstein Frisian cows at 42–60 days postpartum were classified as healthy (HE), subclinical endometritis (SE) or clinical endometritis (CE) based on veterinary clinical examination of the animals and histological evaluation the corresponding endometrial biopsies. Endometrial transcriptome and miRNome profile changes and associated molecular pathways induced by subclinical or clinical endometritis were then investigated using GeneChip® Bovine Genome Array and Exiqon microRNA PCR Human Panel arrays, respectively. The results were further validated in vitro using endometrial stromal and epithelial cells challenged with subclinical and clinical doses of lipopolysaccharide (LPS).ResultTranscriptome profile analysis revealed altered expression level of 203 genes in CE compared to HE animals. Of these, 92 genes including PTHLH, INHBA, DAPL1 and SERPINA1 were significantly upregulated, whereas the expression level of 111 genes including MAOB, CXCR4, HSD11B and, BOLA, were significantly downregulated in CE compared to the HE animal group. However, in SE group, the expression patterns of only 28 genes were found to be significantly altered, of which 26 genes including PTHLH, INHBA, DAPL1, MAOB, CXCR4 and TGIF1 were common to the CE group. Gene annotation analysis indicated the immune system processes; G-protein coupled receptor signaling pathway and chemotaxis to be among the affected functions in endometritis animal groups. In addition, miRNA expression analysis indicated the dysregulation of 35 miRNAs including miR-608, miR-526b* and miR-1265 in CE animals and 102 miRNAs including let-7 family (let-7a, let-7c, let-7d, let-7d*, let-7e, let-7f, let-7i) in SE animals. Interestingly, 14 miRNAs including let-7e, miR-92b, miR-337-3p, let-7f and miR-145 were affected in both SE and CE animal groups. Further in vitro analysis of selected differentially expressed genes and miRNAs in endometrial stroma and epithelial cells challenged with SE and CE doses of LPS showed similar results to that of the array data generated using samples collected from SE and CE animals.ConclusionThe results of this study unraveled endometrial transcriptome and miRNome profile alterations in cows affected by subclinical or clinical endometritis which may have a significant effect on the uterine homeostasis and uterine receptivity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2513-9) contains supplementary material, which is available to authorized users.

Mutant MMP-9 and HGF gene transfer enhance resolution of CCl4-induced liver fibrosis in rats: role of ASH1 and EZH2 methyltransferases repression.

Hepatocyte growth factor (HGF) gene transfer inhibits liver fibrosis by regulating aberrant cellular functions, while mutant matrix metalloproteinase-9 (mMMP-9) enhances matrix degradation by neutralizing the elevated tissue inhibitor of metalloproteinase-1 (TIMP-1). It was shown that ASH1 and EZH2 methyltransferases are involved in development of liver fibrosis; however, their role in the resolution phase of liver fibrosis has not been investigated. This study evaluated the role of ASH1 and EZH2 in two mechanistically different therapeutic modalities, HGF and mMMP-9 gene transfer in CCl4 induced rat liver fibrosis. Liver fibrosis was induced in rats with twice a week intraperitoneal injection of CCl4 for 8 weeks. Adenovirus vectors encoding mMMP-9 or HGF genes were injected through tail vein at weeks six and seven and were sacrificed one week after the second injection. A healthy animal group was likewise injected with saline to serve as a negative control. Rats treated with mMMP-9 showed significantly lower fibrosis score, less Sirius red stained collagen area, reduced hydroxyproline and ALT concentration, decreased transforming growth factor beta 1 (TGF-β1) mRNA and lower labeling indices of α smooth muscle actin (α-SMA) and proliferating cell nuclear antigen (PCNA) stained cells compared with HGF- or saline-treated rats. Furthermore, TIMP-1 protein expression in mMMP-9 group was markedly reduced compared with all fibrotic groups. ASH1 and EZH2 protein expression was significantly elevated in fibrotic liver and significantly decreased in mMMP-9- and HGF-treated compared to saline-treated fibrotic livers with further reduction in the mMMP-9 group. Conclusion: Gene transfer of mMMP-9 and HGF reduced liver fibrosis in rats. ASH1 and EZH2 methyltransferases are significantly reduced in mMMP-9 and HGF treated rats which underlines the central role of these enzymes during fibrogenesis. Future studies should evaluate the role of selective pharmacologic inhibitors of ASH1 and EZH2 in resolution of liver fibrosis.

Copper toxicity in a New Zealand dairy herd

Chronic copper toxicity was diagnosed in a Jersey herd in the Waikato region of New Zealand following an investigation into the deaths of six cattle from a herd of 250 dry cows. Clinical signs and post-mortem examination results were consistent with a hepatopathy, and high concentrations of copper in liver and blood samples of clinically affected animals confirmed copper toxicity. Liver copper concentrations and serum gamma-glutamyl transferase activities were both raised in a group of healthy animals sampled at random from the affected herd, indicating an ongoing risk to the remaining cattle; these animals all had serum copper concentrations within normal limits. Serum samples and liver biopsies were also collected and assayed for copper from animals within two other dairy herds on the same farm; combined results from all three herds showed poor correlation between serum and liver copper concentrations.To reduce liver copper concentrations the affected herd was drenched with 0.5 g ammonium molybdate and 1 g sodium sulphate per cow for five days, and the herd was given no supplementary feed or mineral supplements. Liver biopsies were repeated 44 days after the initial biopsies (approximately 1 month after the end of the drenching program); these showed a significant 37.3% decrease in liver copper concentrations (P <0.02). Also there were no further deaths after the start of the drenching program. Since there was no control group it is impossible to quantify the effect of the drenching program in this case, and dietary changes were also made that would have depleted liver copper stores.Historical analysis of the diet was difficult due to poor record keeping, but multiple sources of copper contributed to a long term copper over supplementation of the herd; the biggest source of copper was a mineral supplement. The farmer perceived this herd to have problems with copper deficiency prior to the diagnosis of copper toxicity, so this case demonstrates the importance of monitoring herd copper status regularly. Also the poor correlation between liver and serum copper concentrations in the three herds sampled demonstrates the importance of using liver copper concentration to assess herd copper status.

Нитрозирующий стресс и апоптоз нейронов са1-зоны гиппокампа в условиях моделирования хронической алкогольной интоксикации: нейропротективные эффекты тиоцетама

Fulfilled researches showed that modeling of chronic alcohol intoxication by daily intragastric ethanol administration to non-pedigreed male white rats (first 10 days – 15% ethanol solution in dose 4 g/kg, next 10 days – 15% ethanol solution in dose 6 g/kg, then 10 days – 25% ethanol solution in dose 4 g/kg) leads to activation of nitrosorbidi stress reactions and initiation of neuro-apoptosis. Thus, as compared with healthy animals group, in rats’ brain after chronic ethanol administration it was revealed significant increase of nitro-tyrosine (nitrosorbidi stress marker) in cytosolic and mitochondrial fractions of brain homogenate against the background of revealing of apoptosis molecular markers in hippocampal CA1 zone neurons (fragmentation of neuron&amp;#180;s nuclei of hippocampal CA1 zone, increase of density and ratio of apoptotic- and destructive-changed cells) as well as deprivation of anti-apoptotic mechanisms (decrease of density of Bcl-2-positive neurons in hippocampal CA1 zone and decrease of Bcl-2 protein concentration in hippocampus). Subsequent 14-day intragastric administration of complex neuro-metabolic cerebro-protector Thiocetam in dose 100 mg/kg to the animals after chronic alcoholization showed significant protective effect on hippocampal CA1 zone neurons. Thus, Thiocetam administration resulted in decrease of nitrotyrosine levels in mitochondrion and in cytosol of brain of animals with chronic alcoholization as compared with untreated animals and with animals receiving Piracetam. Thiocetam administration to experimental animals led to significant decrease of density of apoptotic- and destructive-changed neurons of hippocampal CA1 zone and ratio of apoptotic-changed cells as compared with control group and animals receiving Piracetam. Course of treatment with Thiocetam increases significantly the density of Bcl-2-positive neurons in CA1 zone of hippocampus and significantly increases concentration of Bcl-2 in brain tissue of experimental animals as compared with control group and animals which received Piracetam according the same schedule. The increase of Bcl-2 protein expression in animals received Thiocetam testifies to the activation of anti-apoptotic defense of damaged neurons under the influence of this preparation. We suppose that one of the molecular mechanisms of neuro-protective effect of Thiocetam in chronic intoxication is the breaking of NO-depended mechanisms of neuro-apoptosis.

Bone marrow-derived mononuclear cells do not exert acute neuroprotection after stroke in spontaneously hypertensive rats

Bone marrow-derived mononuclear cells (BM-MNCs) were shown to improve the outcome in animal stroke models and clinical pilot studies on BM-MNCs for stroke patients were already conducted. However, relevant aspects of pre-clinical evaluation, such as the use of animals with comorbidities and dose-response studies, were not thoroughly addressed so far. We therefore investigated different BM-MNC doses in the clinical meaningful stroke model of spontaneously hypertensive (SH) rats. Three hours after the onset of transient middle cerebral artery occlusion (MCAO) animals received either one of three syngeneic BM-MNC doses or placebo intravenously. The primary endpoint was the infarct size. Secondary endpoints included functional outcome, mortality, inflammatory processes, and the dose-response relationship. In contrast to previous studies which used healthy animals no beneficial effect of BM-MNCs was found. Infarct volumes, mortality, behavioral outcomes, and the extent of the inflammatory response to cerebral ischemia were comparable in all groups. In conclusion, we could not demonstrate that early BM-MNC treatment improves the outcome after stroke in SH rats. Whether BM-MNCs improve neurological recovery after delayed treatment initiation was not investigated in the present study, but our data indicates that this should be determined in co-morbid animal stroke models before moving to large-scale clinical studies. Future preclinical stroke studies on co-morbid animals should also include groups of healthy animals in order to determine whether negative results can be attributed to the comorbid condition.

The Effect of Thiamine and Thiamine Pyrophosphate on Oxidative Liver Damage Induced in Rats with Cisplatin

The aim of this study was to investigate the effect of thiamine and thiamine pyrophosphate (TPP) on oxidative stress induced with cisplatin in liver tissue. Rats were divided into four groups; thiamine group (TG), TPP + cisplatin group (TPG), healthy animal group (HG), and cisplatin only group (CG). Oxidant and antioxidant parameters in liver tissue and AST, ALT, and LDH levels in rat sera were measured in all groups. Malondialdehyde levels in the CG, TG, TPG, and HG groups were 11 ± 1.4, 9 ± 0.5, 3 ± 0.5, and 2.2 ± 0.48 μmol/g protein, respectively. Total glutathione levels were 2 ± 0.7, 2.8 ± 0.4, 7 ± 0.8, and 9 ± 0.6 nmol/g protein, respectively. Levels of 8-OH/Gua, a product of DNA damage, were 2.7 ± 0.4 pmol/L, 2.5 ± 0.5, 1.1 ± 0.3, and 0.9 ± 0.3 pmol/L, respectively. A statistically significant difference was determined in oxidant/antioxidant parameters and AST, ALT, and LDH levels between the TPG and CG groups (P < 0.05). No significant difference was determined between the TG and CG groups (P > 0.05). In conclusion, cisplatin causes oxidative damage in liver tissue. TPP seems to have a preventive effect on oxidative stress in the liver caused by cisplatin.

Use of population pharmacokinetic modeling and Monte Carlo simulation to capture individual animal variability in the prediction of flunixin withdrawal times in cattle

The objective of this study was to develop a population pharmacokinetic (PK) model and predict tissue residues and the withdrawal interval (WDI) of flunixin in cattle. Data were pooled from published PK studies in which flunixin was administered through various dosage regimens to diverse populations of cattle. A set of liver data used to establish the regulatory label withdrawal time (WDT) also were used in this study. Compartmental models with first-order absorption and elimination were fitted to plasma and liver concentrations by a population PK modeling approach. Monte Carlo simulations were performed with the population mean and variabilities of PK parameters to predict liver concentrations of flunixin. The PK of flunixin was described best by a 3-compartment model with an extra liver compartment. The WDI estimated in this study with liver data only was the same as the label WDT. However, a longer WDI was estimated when both plasma and liver data were included in the population PK model. This study questions the use of small groups of healthy animals to determine WDTs for drugs intended for administration to large diverse populations. This may warrant a reevaluation of the current procedure for establishing WDT to prevent violative residues of flunixin.

Usefulness of Serum Cystatin C Measurement for Assessing Renal Function in Cats

Abstract The aim of the work was to determine the concentration of serum cystatin C and usability of its measurement in diagnostics of renal failure in cats. The reference point was the value of glomerular filtration rate (GFR), calculated on the basis of plasma clearance of iohexol (Pcio). Seventy cats were divided into two groups: group I - 24 healthy cats, group II - 46 cats affected with renal dysfunction. The measurement of serum cystatin C was done according to immunonefelometric method. In order to estimate Pcio, the following blood samples were collected: pre-sample - 0, and samples collected 120, 180, 240, and 300 min after intravenous administration of iohexol. Its concentration was determined using high performance liquid chromatography. Pcio was obtained using a one-compartment model, according to the method developed by Krutzen Back and Nilsson-Ehl. Serum concentration of cystatin C in cats with renal dysfunction was significantly statistically higher (1.3 ± 0.6 mg/L) as compared to the group of healthy animals (0.7 ± 0.2 mg/L). Statistical analysis of the data confirmed strong correlation between GFR and serum concentration of cystatin C (r = - 0.51). Data analysis on the basis of the receiver operating characteristic curve proved that cystatin C seems to be more reliable parameter than creatinine in the assessment of kidneys function in cats and the measurement of cystatin C in blood serum can be of a considerable meaning as far as early diagnosis of renal failure in cats is concerned.

Optical imaging of oral squamous cell carcinoma and cervical lymph node metastasis

In oral cancer surgery, intraoperative optical imaging could help the surgeon to determine adequate tumor-free margins. Tumor-specific near-infrared fluorescence agents targeting epidermal growth factor receptor (CW800 EGF) or glucose transporter system (CW800 2-DG) were administered to mice with tongue carcinoma and cervical lymph node metastases. Tumor growth was followed by bioluminescence imaging. Fluorescence signals were compared with a control group of healthy animals. Significantly higher fluorescence was found in tongue tumors and cervical lymph node metastases compared with that in control animals. Fluorescence correlated with histopathology. Tumor-to-background ratio of CW800 EGF in the tongue was 13.8 (SD = 6.1) and in the lymph nodes 15.7 (SD = 8.8). For CW800 2-DG, the tumor-to-background ratio in the tongue was 4.6 (SD = 2.1) and in the lymph nodes 33.9 (SD = 18.4). Optical imaging can be used to detect oral cancer and cervical lymph node metastases and could potentially improve complete surgical resection by real-time image-guided surgery.

Biochemical and histological investigation of famotidine effect on postischemic reperfusion injury in the rat ovary

Background/Purpose In this study, an investigation was performed on the ovarian tissue of rats subjected to ischemia-reperfusion for the effect of famotidine on certain parameters of oxidation-antioxidation, cell DNA damage, and histological appearance. Methods The effects of famotidine on certain parameters of oxidation-antioxidation (total glutathione [tGSH], superoxide dismutase [SOD], malondialdehyde) and cellular DNA injury in the ovarian tissue of rats subjected to ischemia-reperfusion were investigated and underwent histological examination. Results The results show levels of 5.2 ± 0.6 nmol/g protein for tGSH, 8.3 ± 0.8 U/g for SOD activity, and 7.7 ± 0.9 μmol/g protein for malondialdehyde ( P < .0001 when compared with controls) in ovarian tissue subjected to ischemia-reperfusion following famotidine treatment. The tGSH levels in control rats and in a healthy animal group were, respectively, 1.76 ± 0.7 and 5.5 ± 0.3 nmol/g protein ( P < .0001). The SOD activity was 3.2 ± 0.9 U/g in control and 9.2 ± 0.6 U/g in healthy animal tissues. The differences between the values in the treatment and the control group, and between the healthy animal group and the control group were both highly significant ( P < .0001). It was also observed that famotidine prevented, to a significant extent, an increase in the level of 8-hydroxy-2-deoxyguanine/guanine, a DNA damage product, as compared with the control group. Conclusion These biochemical and histological results show that famotidine protects the ovarian tissue from ischemia-reperfusion injury.

Characterization of PRNP and SPRN coding regions from atypical scrapie cases diagnosed in Poland

Scrapie, a fatal transmissible spongiform encephalopathy (TSE) occurs in two phenotypes: classical and atypical. Many authors point out that the polymorphism of three codons (136, 154, 171) of the PRNP (PrP gene) is associated with a sheep susceptibility to classical scrapie. Until now, only one PRNP gene variant coding phenylalanine at codon 141 has been found to be associated with atypical scrapie. Another recently identified and interesting candidate gene for scrapie susceptibility in sheep is an SPRN gene coding for Shadoo protein (Sho). Sho is a highly interspecies conserved protein and an insertion/deletion (indel) found in a sheep Sho gene was associated with classical scrapie occurrence. Here we determined the polymorphism of PRNP and SPRN genes in nine atypical scrapie cases (six in native born sheep and three in imported sheep) and compared these results with a control group of healthy animals comprising six corresponding Polish sheep breeds. In atypical scrapie cases five PRNP diplotypes were identified: A(136)R(154)Q(171)/ARQ, AHQ/ARQ, ARR/ARQ, ARR/AHQ and AHQ/AHQ. The ARR/AHQ diplotype was found only in imported sheep. A previously unobserved SNP in PRNP (E224K) was also found in both atypical scrapie and in a few control animals. In the ORF of the SPRN gene, six SNPs and one indel were identified. None of these variations was exclusive for scrapie animals and they were probably, naturally occurring polymorphisms. Special attention was given to the 6-bp indel SPRN polymorphism which was previously associated with classical scrapie occurrence.

Modulatory effect of vitamins A, C and E mixtures against tefluthrin pesticide genotoxicity in rats

The present experiments to be reported were declared the effect of mixtures from the three antioxidant vitamins (A, C and E) on some hematologic parameters, liver and testes nucleic acid system, blood immune-system, chromosomal aberration and sperm-shape of tefluthrin (LD 50) intoxicated rats. A total of 90 male albino rats were used divided into six groups (15 rats each). Oral ingestion of technical tefluthrin significantly decreased the count of RBCs, Hb content and stimulated plasma LDH activity, also reduced the levels of blood immuno globulins (IgG, IgA and IgM). Under the effects of tefluthrin liver and tests nucleic acids (RNA and DNA) contents were decreased and the activities of nucleases (RNAase and DNAase) were also inhibited relative to these values of normal healthy control group. Chromosomal aberrations and sperm-shape abnormalities were significantly increased after ingestion of tefluthrin, compared to the control group. Mitotic index (MI) and sperm count was decreased significantly due to the potential cytotoxicity of tefluthrin. Treatment of intoxicated rats with antioxidant vitamins (A, C and E) mixtures reduced the harmful influences of tefluthrin. Blood IgG, IgA and IgM levels, RBCs count and Hb content were improved relative to normal healthy animal group. The frequency of chromosome aberrations in bone marrow cells of intoxicated rats treated with vitamins as well as the frequency of sperm abnormalities were decreased and readjusted near to that of the healthy control animals, also MI and sperm counts were increased significantly near to the control group after vitamins ingestion as treatments. It is interesting to note that, the ingestion of vitamins mixtures readjusted and normalized the hematologic parameters around those of normal healthy control. In case of liver and nucleic acids system, the disturbed effects of the pesticide induction of RNA and DNA contents as well as RNAase and DNAase activities were alleviated by the treatments with the present antioxidative vitamins mixtures, in which the nucleic acids contents and nucleases activities of liver and testes of tefluthrin intoxicated rats were ameliorated and normalized compared with those of normal control.

Exogenous Irritant-Induced Airway Hyperreactivity and Inhibition of Soluble Guanylyl Cyclase

The majority of nitric oxide (NO) effects in the respiratory system are caused by stimulation of soluble guanylyl cyclase (sGC) with subsequent increase of cyclic guanosine monophosphate (cGMP) production. The importance of this mechanism of NO action in airway hyperreactivity (AHR) pathogenesis is unknown. Therefore, the aim of our experiment was to examine the changes of airway reactivity enhanced by toluene vapor exposure in the presence or inhibition of sGC activity in guinea pigs. Animals were treated with a nonspecific sGC inhibitor, methylene blue, in a dose of 50 or 100 mg/kg body weight, administered by intraperitoneal injection 30 min before or after exposure to toluene vapors. The toluene exposure lasted 2 hr in each of 3 consecutive days under in vivo conditions. Thereafter, the tracheal and lung tissue smooth muscle response to cumulative doses of mediators (histamine or acetylcholine) was recorded under in vitro conditions. The exposure to toluene vapors significantly increased the airway reactivity to both mediators in comparison with the healthy animal group. The administration of methylene blue decreased the amplitude of airway smooth muscle contraction in toluene-induced hyperreactivity. The decreases were dependent on the inhibitor doses, on a regimen of administration (before or after toluene inhalation), the level of the respiratory system (trachea, lung), and the bronchoconstrictor mediators. Our results suggest that the interaction between NO and sGC may be important for airway reactivity changes, but other mechanisms of NO action are important in AHR pathogenesis, too.

IGF-1 gene therapy to protect articular cartilage in a rat model of joint damage

An adeno-associated virus (AAV) derived vector in gene transfer model that induces IGF-1 expression could repair articular cartilage. Male Wistar rats, 150 and 200 g, and 7 weeks old, were used. Effectiveness of constructed vectors was assayed inoculating them in rat knees of control and damaged animals either mechanically or by collagen-induced arthritis. Inoculation was intra-articular with 50 microL of recombinant AAV-Luciferase (1.25 x 10(8) particles). The rats were killed after 1, 2, 4 and 8 weeks. IGF-I activity was analyzed by injecting 50 microL of recombinant AAV (1.25 x 10(8) particles) in animals with damaged knees. Final analysis was performed after 8 weeks. The activity of AAV vectors in vitro shows the presence of mRNA coding to IGF-I in cells infected with AAV-IGF and not in control cells without viral vectors and an increase in secreted IGF-I protein in culture medium. In vivo, AAV derived vectors induced protein expression in cartilage 2 months after inoculation. In the animals killed after 1 and 2 weeks, no significant increase in the reaction of luciferase was observed (P > 0.05). In the group of animals with no injury an increase was observed at 4 weeks, which was more marked and significant after 8 weeks (P = 0.029). The same behavior occurred in the animals with induced arthritis and in the mechanical injury group. In the levels of expression after 8 weeks, no significant differences were found between the two groups of injured animals and the group of healthy animals infected with the virus. The joints of the animals that were subjected to injuries in the cartilage and inoculated with AAV-IGF-I presented a similar appearance to those animals inoculated with saline solution. Autoimmune and mechanical lesions did not show improvement in the state of its cartilage after the treatment. The use of AAV vectors capable of inducing the expression of IGF-I in vitro is therefore not sufficient to protect the cartilage from the serious damage.