Intact Animals Research Articles

(046) THE ROLE OF ADENOSINE IN THE RELAXANT MECHANISM OF RAT DISTAL VAGINA

Abstract Introduction Adenosine (ADO) is a purinergic neuromodulator that in the male exerts a positive effect on penile erection by controlling smooth muscle (SM) relaxation. However, its role in the relaxant/contractile pathways in the vagina is yet to be investigated. Objective The aim of this study was to characterize the effect of ADO stimulation on SM activity in distal vagina, evaluating its possible effect on the regulation of nitric oxide (NO) pathway using a validated animal model of female Sprague-Dawley rats. Methods The immunolocalization of ADORA2A and ADORA2B in distal vagina tissues was detected by immunohistological staining. In vitro contractility studies on noradrenaline (NA)-precontracted vaginal strips from intact Sprague-Dawley rats were performed; rat smooth muscle cells (rvSMCs) from distal vagina of intact animals were stimulated with ADO for cyclic adenosine monophosphate (cAMP) quantification. mRNA (messenger ribonucleic acid) isolated from vaginal tissue was analyzed by semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction. Results All ADORAs mRNA were expressed in the vaginal tissue, with a significant prevalence of ADORA2B; furthermore, ADORA2A and ADORA2B immunolocalization highlighted that both receptors are highly expressed in the epithelium, in blood capillaries of the vascular bed and in the smooth muscle bundles of vagina wall. In vitro ADO stimulation induced a dose-dependent relaxation (IC50=31.9±2.38 μM), which was significantly reduced by the administration of NO-synthase inhibitor L-NAME; similar effects were observed after the mechanical ablation of endothelium and in vitro administration of the soluble guanylate cyclase inhibitor ODQ, highlighting the pivotal contribution of NO signaling to the ADO relaxant activity. In vitro stimulation with increasing doses of the selective ADO receptor ADORA2A agonist CGS2-1680 induced a dose-dependent relaxation (IC50=132.6±3.47 nM); a similar but less potent relaxant effect was observed with ADORA2B selective agonist BAY60-6583. The relaxing effect induced by ADO on NA-precontracted vaginal strips was completely counteracted by the stimulation with the re-spectively selective ADORA2A and ADORA2B antagonists SHC-442416 and PSB603; a similar effect was induced by ADORA1 selective antagonist SLV320, but not by ADORA3 selective antagonists VUF5574. The mRNA relative expression of enzymes regulating the ADO turnover [adenosine deami-nase (ADA), 5′-nucleotidase (5NTD), AMP deaminase type 1 and type 2 (AMPD1, AMPD2) and adenosine kinase (ADK)] showed a higher expression of genes regulating the de novo ADO/cAMP syn-thesis (ADK, 5NTD), compared to those acting on ADO degradation (ADA, AMPD1, AMPD2). Finally, stimulation of rvSMCs with increasing doses of ADO induced a significant production of intracellular cAMP after 3 minutes (1 and 10 μM). Conclusions These data show that ADO acts as an important relaxant modulator of the vagina through the activation of its receptors, specifically ADORA2A, and the consequent cAMP-dependent adenylate cyclase re-sponse. However, in vitro studies show that ADO also acts trough the NO-dependent pathway. These results therefore demonstrate for the first time a new relaxant pathway in distal vagina, ADO signaling, that results deeply intertwined with the well-known NO pathway. This finding unravels new biological mechanisms underlying vagina physiology that need to be further studied in future research. Disclosure No.

Histopathological Changes in a Rat Model of Diabetic Neuropathy

This study was done to investigate the histopathological Changes in a Rat Model of Diabetic Neuropathy. The 24 male rats used in this investigation were split into two groups of 12 animals. The first group (G1), which consisted of healthy individuals, was given standard saline. Normal saline was administered after G2 (STZ group) received STZ at a dosage of 60 mg/kg. After rats were starved overnight, a single intraperitoneal injection of 60 mg/kg of freshly produced STZ was given to induce diabetes. (0.1 M, pH 4.5) Sodium Citrate Buffer was used to dissolve STZ. On the third day after the injection of STZ, a digital glucometer was used to measure the blood glucose levels of 12-hour fasting rats; hyperglycemia was defined as values greater than 250 mg/dl. In order to assess neuropathic pain, cold allodynia was calculated by counting the number of times the foot withdrew in response to cold stimuli applied to the back of the hand. The sciatic nerve was carefully removed after the sacrifice (after 2 months) and preserved in 10% formalin for histopathological study. Normal blood glucose levels were maintained in the control rats throughout the study. However, G2 exhibited much greater FBG levels than healthy animals. In the 2-month study, the G2 maintained FBG levels above 250 mg/dl. The score for severity of STZ on sciatic nerves showed a significant difference between treated group as compared with G1. No paw reactions were seen when intact animals were subjected to plantar surface acetone. The experimental groups did not show any notable reactions to acetone on the tenth day after the diagnosis of diabetes. On days 20, 30, 40, 50 and 60 after diabetes confirmation, there were significant differences in paw withdrawal frequency between the G1 and G2 (p<0.001). Within the control group, nerve fibres were found to be regularly arranged and there was no evidence of axonal swelling. However, in the G2 group, nerve fibres were found to be significantly disorganised, and there was a notable increase in the number of Schwann cells in the sciatic nerves, suggesting damage, in comparison to the control group. In conclusion, potentially via antinociceptive, antihyperglycemic, antioxidant, and neuro-regeneration boosting pathways in STZ-induced diabetic peripheral disease.

Пубертатний стрес спричиняє статевоспецифічні репродуктивні ефекти у дорослих щурів

Adolescence is one of the critical periods of individual development, which is highly sensitive to changes caused by stress factors. Stress-related hormonal imbalances can negatively affect puberty and reproductive function. The aim of the study was to investigate the long-lasting reproductive effects of chronic pubertal stress in rats. Animals were subjected to immobilization stress daily from 30 to 45 postnatal day by placing them for 1 h in cylinders 4.5 cm in diameter and 10 cm in long, equipped with breathing holes. The timing of puberty in animals was studied and their somatic development was assessed. Further studies were conducted in 6-month-old sexually mature rats. The results were compared with the corresponding parameters in intact animals. Pubertal stress delayed sexual maturation in females and decreased weight gain in males. In adult animals of both sexes, pubertal stress did not change testosterone levels or the weight and morphology of the gonads significantly, except for minor vacuolation of the spermatogenic epithelium. The spermatogenesis index in the experimental group was significantly lower than in the control, due to a decrease in the number of late spermatids. Pubertal stress led to a 25.9% decrease in the number of spermatozoa in epididymal washes, an increase in the number of their pathological forms and a 2.4-fold slowdown in oxidative-reductive processes in spermatozoa. The content of malonic dialdehyde increased in the testes and ovaries, which indicates the activation of lipid peroxidation. Thus, chronic pubertal stress led to a decrease in the reproductive potential of male rats. Females are less susceptible to the negative effects of pubertal stress.

The Wnt/β-catenin/TCF/Sp5/Zic4 Gene Network That Regulates Head Organizer Activity in Hydra Is Differentially Regulated in Epidermis and Gastrodermis.

Hydra head formation depends on an organizing center in which Wnt/β-catenin signaling, that plays an inductive role, positively regulates Sp5 and Zic4, with Sp5 limiting Wnt3/β-catenin expression and Zic4 triggering tentacle formation. Using transgenic lines in which the HySp5 promoter drives eGFP expression in either the epidermis or gastrodermis, we show that Sp5 promoter activity is differentially regulated in each epithelial layer. In intact animals, epidermal HySp5:GFP activity is strong apically and weak along the body column, while in the gastrodermis, it is maximal in the tentacle ring region and maintained at a high level along the upper body column. During apical regeneration, HySp5:GFP is activated early in the gastrodermis and later in the epidermis. Alsterpaullone treatment induces a shift in apical HySp5:GFP expression towards the body column where it forms transient circular figures in the epidermis. Upon β-catenin(RNAi), HySp5:GFP activity is down-regulated in the epidermis while bud-like structures expressing HySp5:GFP in the gastrodermis develop. Sp5(RNAi) reveals a negative Sp5 autoregulation in the epidermis, but not in the gastrodermis. These differential regulations in the epidermis and gastrodermis highlight the distinct architectures of the Wnt/β-catenin/TCF/Sp5/Zic4 network in the hypostome, tentacle base and body column of intact animals, as well as in the buds and apical and basal regenerating tips.

CORRECTION OF PATHOLOGICAL CHANGES IN SALIVARY GLANDS OF ANIMALS WITH PACLITAXEL-INDUCED NEUROPATHY

Background. Paclitaxel is a highly effective chemotherapeutic agent used to treat breast, ovarian, and other cancers. At the same time, paclitaxel causes peripheral neuropathy as a side effect in 45%—70% of patients. Aim. The aim of the study was to investigate the effect of paclitaxel-induced peripheral neuropathy on the development of pathological changes in the salivary glands of animals and to explore the possibility of correction of the identified changes with vitamin B/ATP complex. Materials and Methods. To simulate toxic neuropathy, animals were injected i/p with paclitaxel 2 mg/kg for 4 days. In order to correct the identified changes, rats were injected i/m with vitamin B/ATP complex (1 mg/ kg) for 9 days. In the homogenate of the submandibular salivary glands, α-amylase activity, total proteolytic activity, total antitryptic activity, the content of medium mass molecules, thiobarbituric acid reactive substances (TBARS), oxidatively modified proteins, and catalase activity were determined. Results. A significant increase in the content of oxidatively modified proteins, medium mass molecules, and the content of TBARS and significant decrease in the activity of catalase and amylase were determined in the salivary glands of animals with toxic neuropathy compared to these parameters in intact animals. Administration of vitamin B/ATP complex for 9 days against the background of paclitaxel-induced neuropathy led to normalization of antitryptic activity and amylase activity, a significant decrease in the content of oxidatively modified proteins, medium mass molecules, and TBARS along with a significant increase in catalase activity in the salivary glands of animals compared to the untreated rats with neuropathy. Conclusion. Paclitaxel-induced neuropathy caused the development of pathological changes in the salivary glands of rats, which was evidenced by a carbonyl- oxidative stress and impaired protein synthetic function. The correction with vitamin B/ATP complex restored the protein-synthetic function and the proteinase-inhibitor balance, suppressed the oxidative stress and normalized free radical processes in the salivary glands of rats.

Colony-forming potential of murine bone marrow-derived progenitor cells in experimental neurodegenerative pathology and under the impact of melatonin in vivo or in vitro

Cell therapy is a promising direction in the treatment of Parkinson’s disease/parkinsonism and multiple sclerosis, the effectiveness of which can be increased with the help of biologically active factors, in particular melatonin. The purpose of the study: to investigate the colony-forming ability of stromal and hematopoietic cells-progenitors of the bone marrow of mice with experimentally induced neurodegenerative pathology and reveal the possibility to change it under the influence of melatonin in vivo or in vitro. Materials and methods: In adult (6-7 months) male FVB/N (haplotype H-2q) and 129/Sv (haplotype H-2b) mice, the parkinsonism model was reproduced by a single subcutaneous injection of the neurotoxin 1-methyl-4-phenyl -1,2,3,6-tetrahydropyridine (MPTP) at a dose of 30 mg/kg 129/Sv mice also received the neurotoxin cuprizone daily with food (at the rate of 0.2 % of the mass of the pure substance from the daily feed) for three weeks (multiple sclerosis model). Melatonin was administered to the 129/Sv mice intraperitoneally at a dose of 1 mg/kg, daily, at 17.00, from the 8th day of receiving cuprizone, or was added in vitro to the culture of bone marrow cells of mice of both strains with parkinsonism at a dose of 0.1 μg/0.1 mL. In the bone marrow, the absolute number of nucleated cells was counted, the relative content of CD4+ lymphocytes was determined by flow cytometry, and the number of colony-forming precursor cells for fibroblasts (CFU-F) and for granulocyte-macrophages (CFU-GM) in culture in vitro was estimated. Results. In the bone marrow of 129/Sv mice with a model of multiple sclerosis, the number of nucleated cells, CFU-GM and CD4+ cells decrease, while under the influence of exogenous melatonin applied in vivo, the values of these indicators increase to the level of intact animals. After the administration of MPTP in the bone marrow of FVB/N mice, in contrast to 129/Sv mice, the number of nucleated cells and CFU-F decreases. After the incubation with melatonin of bone marrow cells of 129/Sv mice with a model of parkinsonism, an increase in the amount of CFU-F was observed compared to control values, and there were no changes in the indicator values in FVB/N mice. Conclusions. In the bone marrow of mice with the MPTP-model of parkinsonism, a decrease in the absolute amount of CFU-F was found, and in mice with the cuprizone model of multiple sclerosis – CFU-GM. The application of melatonin in vitro increases the colony-forming potential of progenitor cells for fibroblasts in the bone marrow of mice with the MPTP model of parkinsonism. The use of melatonin in vivo increases the colony-forming ability of progenitor cells for granulocyte-macrophages and the relative content of CD4+ cells in the bone marrow of mice with the cuprizone model of multiple sclerosis. Changes in the amount of CFU-F in the bone marrow in parkinsonism and under the influence of melatonin in vitro depend on the H-2 haplotype mice, which can be the basis for the development of personalized cell therapy for this pathology.

SOURCES OF SUPEROXIDE ANION RADICAL IN SMALL INTESTINE TISSUES IN RATS SUBJECTED TO SURGICAL TRAUMA SIMULATED UNDER EXPERIMENTAL MODEL OF POSTTRAUMATIC STRESS DISORDER

The sources of superoxide anion radical (.О ) in the tissues of the small intestine in rats subjected to surgical trauma simulated under an experimental model of posttraumatic stress disorder (PTSD) were investigated. The study involved 42 white Wistar rats weighing 210-230 g, divided into 6 groups: group 1 comprised intact animals, group 2 included animals with the PTSD induced through single-prolonged stress (SPS), group 3 consisted of rats subjected to a sham surgical operation, group 4 comprised animals undergoing laparotomy, group 5 involved rats undergoing a sham surgical operation following SPS, and group 6 included animals undergoing laparotomy under modeled SPS. The rate of .О generation in the small intestine homogenate was measured spectrophotometrically using the nitroblue tetrazolium test. The .О production by NADPH-dependent (microsomal and NO synthase) electron transport chains (ETC), NADH-dependent (mitochondrial) ETC and leukocyte NADPH oxidase was assessed. The findings obtained have demonstrated that experimental SPS modeling leads to an increase in oxidative stress in the small intestine tissues of rats. This is manifested by an increase in the rate of .О formation with the participation of microsomes, mitochondria and leukocyte NADPH oxidase. On the 7th day after laparotomy under the experimental PTSD model, the .О production in the tissues of the small intestine by different sources (microsomes, mitochondria and leukocyte NADPH oxidase) exceeds their values obtained after a single laparotomy or after performing a sham operation under single long-term stress.

MACSPI enables tissue-selective proteomic and interactomic analyses in multicellular organisms

Multicellular organisms are composed of many tissue types that have distinct morphologies and functions, which are largely driven by specialized proteomes and interactomes. To define the proteome and interactome of a specific type of tissue in an intact animal, we developed a localized proteomics approach called Methionine Analog-based Cell-Specific Proteomics and Interactomics (MACSPI). This method uses the tissue-specific expression of an engineered methionyl-tRNA synthetase to label proteins with a bifunctional amino acid 2-amino-5-diazirinylnonynoic acid in selected cells. We applied MACSPI in Caenorhabditis elegans, a model multicellular organism, to selectively label, capture, and profile the proteomes of the body wall muscle and the nervous system, which led to the identification of tissue-specific proteins. Using the photo-cross-linker, we successfully profiled HSP90 interactors in muscles and neurons and identified tissue-specific interactors and stress-related interactors. Our study demonstrates that MACSPI can be used to profile tissue-specific proteomes and interactomes in intact multicellular organisms.

Визначення білків гострої фази в крові щурів після імплантації поліпропіленових хірургічних сіток із покриттям на основі танталу та його похідних

At present, more than 30 types of polypropylene surgical meshes are available in the world, but the statistics regarding the secondary inflammatory process after implantation still remain disappointing. Since polypropylene surgical meshes do not decompose well in the body, they can stimulate the surrounding tissuesto develop an inflammatory process that causes adhesions. For the past 50 years, tantalum has been successfully used to produce biocompatible medical implants in surgery, orthopedics, and dentistry. Its excellent anti-inflammatory and antibacterial properties have been repeatedly noted, indicating the possibility of its use as a coating for mesh implants. In order to better predict the outcome after implantation of biomedical materials, it is important to determine the content of acute phase proteins, namely C-reactive protein, haptoglobin and ceruloplasmin. The aim of the study is to determine the content of acute phase proteins, namely C-reactive protein, haptoglobin and ceruloplasmin in the blood of rats after implantation of surgical meshes coated on the basis of tantalum, tantalum oxide and tantalum nitride. Materials and methods. The experimental group included 40 male rats of the WAG population. With the help of surgical intervention, a polypropylene surgical mesh measuring 15x15 mm was implanted between the abdominal wall and various sections of the colon. 28 days after surgery, the experimental animals were decapitated by cervical dislocation. Blood samples were used to determine the content of C-reactive protein, the content of haptoglobin and the content of ceruloplasmin. Results and discussion. The content of C-reactive protein in the blood of rats was statistically 260 % higher in the experimental group implanted with uncoated surgical mesh and 228.8 % higher in the experimental group implanted with polypropylene surgical mesh with tantalum nitride coating in compared with the results in the group of intact animals. Haptoglobin content was statistically 110.5 % higher and ceruloplasmin was statistically 52.6 % higher in the uncoated surgical mesh group compared to the results of the intact animal group. We obtained similar results in the experimental group, which was implanted with a polypropylene surgical mesh with a coating based on tantalum nitride. The content of haptoglobin was 130 % higher and the content of ceruloplasmin was 50.6 % higher statistically compared to the results of the group of intact animals. The C-reactive protein content was 113.3 % and 95.5 % higher, respectively, in the groups implanted with polypropylene surgical meshes with a coating based on tantalum and tantalum oxide compared to the results in the group of intact animals. The content of haptoglobin in the tantalum-based coating group was 83.8 % higher and the content of ceruloplasmin was 32.6 % higher compared to the results obtained in the group of intact animals. In the tantalum oxide- based group, the haptoglobin content was 60.1 % higher and the ceruloplasmin content was 29.3 % higher compared to the results obtained in the intact animal group. Conclusion. Based on the results of the study, it was established that polypropylene surgical meshes coated with tantalum and tantalum oxide significantly reduce the inflammatory reaction compared to uncoated meshes and meshes coated with tantalum nitride. This is supported by lower levels of key inflammatory markers, indicating improved biocompatibility and anti-inflammatory effects of tantalum and tantalum oxide coatings.

Correction of Nitrosative Stress During Reparative Regeneration of the Nasal Cavity Mucosa in an Experimental

Introduction. Nitric oxide metabolites play an important role in prolongation of the inflammatory reaction, disorders of epithelial regeneration in diseases of the upper respiratory tract. One of the promising approaches to regulating the bioactivity of nitric oxide is the use of antioxidants.The objective of the study is to evaluate the effectiveness of antioxidant therapy for the correction of nitrosative stress during reparative regeneration of the nasal mucosa in an experiment.Methods. A preclinical randomized experimental study is conducted on 160 rats, divided into 4 groups. The control group (n = 40) consists of intact animals. Animals in experimental group 1 (n = 40) do not receive treatment aſter injury; in group 2 (n = 40) anti-inflammatory treatment is prescribed. The third group consists of laboratory rats (n = 40), which are additionally injected into the nasal cavity with an antioxidant — a 15 % solution of 1,1-dimethyl-3-oxobutylphosphonic acid dimethyl ester. On days 2, 5, 10, and 14 of observation, the concentration of nitrates and nitrites and C-reactive protein in the blood of animals are determined.Results. In group 3, aſter an increase in the level of nitric oxide metabolites on day 2 aſter injury on day 5, a significant (p < 0.05) decrease was noted in comparison with other groups, and by day 10— normalization of the indicator. An increase in the level of C-reactive protein in blood serum correlated with the concentration of nitric oxide metabolites.Discussion. The optimal therapeutic effect when taking antioxidants develops when administered in the first hours aſter injury to reduce the excessive formation of reactive nitrogen radicals. However, the duration of administration should not exceed 4–5 days to avoid a significant decrease in the level of nitric oxide metabolites and deterioration of mucosal regeneration.Conclusions. Assessment of nitric oxide metabolitesin the blood at different times aſter injury is an important marker of inflammatory activity. The use of antioxidants helps to reduce the concentration of active nitrogen radicals.

The renal vasodilation from β-adrenergic activation in vivo in rats is not driven by KV7 and BKCa channels

Aim: The underlying mechanisms behind β-adrenergic renal vasodilation is not clarified. Several classes of K+ channels are potentially activated, so we tested the hypothesis that voltage-sensitive KV7 and Ca2+-activated BKCa channels contribute to the decrease in renal vascular tone in vivo and in vitro. Methods: Changes in renal blood flow (RBF) during β-adrenergic stimulation was measured in isoflurane-anesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional KV7.1 channels was examined in a wire-myograph. Results: The β-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of KV7 or BKCa channels reduced the β-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of KV7 channels significantly decreased the β-adrenergic vasorelaxation. However, inhibiting the BKCa channel was equally effective. The β-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking a functional KV7.1 channel. As opposed to rats, inhibition of KV7 channels did not affect the murine β-adrenergic vasorelaxation. Conclusion: Although inhibition and activation of KV7 and BKCa channels significantly changed baseline RBF in vivo, none of the treatments affected the β-adrenergic vasodilation. In segmental arteries however, inhibition of KV7 and BKCa channels significantly reduced the β-adrenergic vasorelaxation indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data stresses the importance of verifying data in intact animals. None. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of per os administration of fucoxanthin alcoholic and oil extracts to Balb/c mice on biochemical parameters and serum cytokine level

Microalgae are a source of various biologically active substances, including carotenoids. One of the carotenoids of diatom microalgae is fucoxanthin, which has antioxidant, antimicrobial, antitumour and anti-inflammatory properties that can be used in the treatment of a wide range of human and animal pathologies. The aim of the study was to investigate the effect of alcohol and oil extract of fucoxanthin on biochemical parameters and levels of serum cytokines in mice during intragastric administration. Material and methods. Fucoxanthin extracts from the microalga Nanofrustulum shiloi were used. Mice of Balb/c strain were intragastrically administrated with 0.5 ml of oil (500 μg/ml) or alcoholic extract of fucoxanthin (187.5 μg/ml), mice in the comparison group were given 0.5 ml of olive oil or 45% ethyl alcohol once a day within 5 days. On the 8th day, blood serum was obtained and the level of albumin, triglycerides, cholesterol, low (LDL) and high density lipoproteins (HDL), urea, uric acid, creatinine, aspartate (AST) and alanine aminotransferases (ALT), lactate, stable nitric oxide (NO) metabolites (nitrites), cytokines (IL-1β, IL-6, IL-10, TNF-α, IFN-γ) was determined using ELISA with photometric detection. Results and discussion. In the comparison group, ethyl alcohol promoted an increase in serum triglyceride, cholesterol, LDL, HDL, ALT, AST, nitrite, IL-10, IFN-γ and TNF-α content compared to intact animals. Fucoxanthin alcoholic extract administration increased the level of LDL, HDL, uric acid, TNF-α and decreased albumin, cholesterol, uric acid, creatinine, ALT, AST, IL-6 content compared to control. The olive oil group showed a decrease in serum triglycerides, cholesterol, urea, uric acid, ALT and AST content but an increase in LDL, HDL, nitrite level compared to the control. Administration of fucoxanthin oil extract to animals decreased albumin, triglycerides, cholesterol, HDL, uric acid, ALT, AST, lactate, IL-6, IL-10 compared to control. Conclusions. Thus, it was discovered that fucoxanthin in Balb/c mice contributes to a decrease in hepatic synthetic function, as evidenced by the change in the concentration of albumin, triglycerides, uric acid), influences energy metabolism (creatinine, lactate), cell membrane stability (cholesterol, LDL, HDL), serum transaminase activity, pro- and anti-inflammatory cytokines content.

Effect of metal nanoparticles usage on oxidative stress indicators and endotoxemia parameters under DMH-induced carcinogenesis

One of the properties of nanoparticles is their ability to correct manifestations of oxidative stress and endotoxemia, which are critical factors in cancer development. Therefore, the work aimed to investigate the effect of the usage of Au/Ag/Fe nanoparticles on oxidative stress indicators and endotoxemia parameters in experimental colon carcinogenesis. The study was performed on 90 white male rats kept in standard vivarium conditions. The division into groups: I – intact animals; II – intact animals with 21 days NPs administration; III – animals injected with N,N-dimethylhydrazine dihydrochloride for 30 weeks; ІV – animals to which Au/Ag/Fe nanoparticles were intragastrically administered daily for 21 days after induced adenocarcinoma. According­ to our results, the concentration of oxidative stress indicators significantly increases under DMH-induced carcinogenesis conditions. It was established that the 21-day intragastric administration of NP Au/Ag/Fe composition caused a significant (P < 0.001) decrease in the concentration of TBARS in the blood serum by 1.33 times, in the content of diene and triene conjugates by 1.63 and 1.98 times, respectively compared to the third experimental group. The introduction of NPs in the fourth experimental group reduces the concentration of the Schiff bases by 1.34 times (P < 0.001), decreases the content of POMP370 and POMP430 by 1.25 (P < 0.001) and 1.37 times (P < 0.001), respectively, compared to the third experimental group. We also observed the reduction of endotoxemia levels in a fourth experimental animal group based on a significant decrease in MMM indexis and EII percentage. Keywords: Au/Ag/Fe nanoparticles, endotoxemia, induced colon carcinogenesis, N‚N-dimethylhydrazine dihydrochloride, oxidative stress

Characteristic of a group of genes with low level of expression in the pancreas of rats under conditions of multi-day intermittent hypoxia influence

In modern medical science great attention is paid to the clarification of the molecular mechanisms, which are the basis of adaptation to environmental factors of unusual origin and/or extraordinary strength. The aim of the study is to determine the features of a group of genes with low expression level, associated with hypoxia in the pancreas of Wistar rats under conditions of intermittent hypoxia. Materials and methods. The study was conducted on 10 white, sexually mature Wistar rats, which were divided into 2 groups (5 animals in each). Animals of group 1 were part of the control (intact) group. The animals of the 2nd group were subjected to hypoxic training according to the following scheme: for 15 days, 6 hours daily, namely on days 1–5 they simulated an ascent to a height of one to five kilometers above sea level under the conditions of a barometer, and the last 10 days 6 km above the sea level. To analyze gene expression, we used the polymerase chain reaction method with real-time reverse transcription (PCR) CFX-96 Touch™ (Bio-Rad, USA) and the RT2 Profiler™ PCR Array Rat Hypoxia Signaling Pathway kit (QIAGEN, Germany), where 84 genes were the subject of research in experimental animals. Results. According to the results of the PCR study of genes in the pancreas samples of intact animals and animals exposed to hypoxic training, it was established that out of 84 genes associated with hypoxia, a group of 5 genes with a low expression level (∆∆Ct < 30) was found. This pattern includes Bhlhe40 genes, Ctsa, Hif1a, Lox, and Slc16a3, the expression of which is statistically reduced. Thus, compared to the level of their expression in intact animals, the expression of Bhlhe40 decreased by 2.59 times, Ctsa by 6.02 times, Hif1a by 3.85 times, Lox by 3.01 times, and Slc16a3 by 2.40 times. Conclusions. Intermittent hypoxia reduces the expression of the Bhlhe40 gene by 2.59 times, which can be considered as an element of adaptation of cells to a low level of oxygen and modulation of genetic programs. The decrease in Ctsa gene expression by 6.02, Hif1a by 3.85, and Lox by 3.01 times during intermittent hypoxia demonstrates, that these effects can be used as sanogenic factors in insulin resistance and type 2 diabetes. The 2.40-fold decreased expression level of Slc16a3 is probably an element of metabolic adaptation and adaptation of the metabolic pathway of cells to hypoxia conditions.

Immobilizing Second-Instar Drosophila Larvae for Imaging and Surgery Using the Larva Chip.

The simple body plan and semitranslucent cuticle of the Drosophila larva allow for imaging of structures close to the body wall within intact animals. These include sensory neurons, muscles, neuromuscular junctions, and some regions of the segmental nerve. However, imaging within an intact larva requires a strategy to immobilize the animal in a position that presents the structures within the working distance of the microscope objective. Although various methods have been implemented for Drosophila larvae, this protocol describes a simple and noninvasive method that makes use of the polydimethylsiloxane (PDMS) larva chip. This larva chip immobilizes animals without the use of anesthetics or changes in temperature, which alter neuronal physiology, making it suitable for calcium imaging of endogenous activity in live animals. The membrane is air-permeable. Animals robustly survive short periods of immobilization (up to 30 min) and can even survive longer time periods. Since animals recover well after the procedure, the same animal can be reimaged multiple times. This makes the method amenable to manipulations such as laser microsurgery, photobleaching, and photoconversion followed by imaging of outcomes of these manipulations over time.

Mathematical modeling of transdermal delivery of topical drug formulations in a dynamic microfluidic diffusion chamber in health and disease.

Mathematical models of epidermal and dermal transport are essential for optimization and development of products for percutaneous delivery both for local and systemic indication and for evaluation of dermal exposure to chemicals for assessing their toxicity. These models often help directly by providing information on the rate of drug penetration through the skin and thus on the dermal or systemic concentration of drugs which is the base of their pharmacological effect. The simulations are also helpful in analyzing experimental data, reducing the number of experiments and translating the in vitro investigations to an in-vivo setting. In this study skin penetration of topically administered caffeine cream was investigated in a skin-on-a-chip microfluidic diffusion chamber at room temperature and at 32°C. Also the transdermal penetration of caffeine in healthy and diseased conditions was compared in mouse skins from intact, psoriatic and allergic animals. In the last experimental setup dexamethasone, indomethacin, piroxicam and diclofenac were examined as a cream formulation for absorption across the dermal barrier. All the measured data were used for making mathematical simulation in a three-compartmental model. The calculated and measured results showed a good match, which findings indicate that our mathematical model might be applied for prediction of drug delivery through the skin under different circumstances and for various drugs in the novel, miniaturized diffusion chamber.

Effects of Glyprolins on Lipid and Protein Peroxidation in the Hypothalamic Region in Experimental Hyperthyroidism

We investigate effects of glyproline-type neuropeptides on lipid and protein peroxidation in the hypothalamic brain region of rats under the conditions of experimental hyperthyroidism. The state of hyperthyroidism in animals was simulated by intragastric administration of sodium pentahydrate of L-thyroxine at a dose of 150 µg/kg for 21 days. The following experimental groups (n=10) were formed: 1) control group — intact animals (control); 2) animals treated with L-thyroxine sodium salt pentahydrate (hyperthyroidism); 3) animals treated with Thr-Lys-Pro-Arg-Pro-Gly-Pro (celank); 4) animals treated with Pro-GlyPro (doses of 87 and 33 µg/kg/day, respectively) intraperitoneally daily during 21 days starting one day after the last administration of sodium pentahydrate of L-thyroxine. The level of lipid peroxidation processes was assessed by the initial level of TBA-reactive products, spontaneous and ascorbate-dependent lipid peroxidation rates in hypothalamic tissue homogenate. Protein peroxidation products were determined by the reaction between oxidized amino acid residues of proteins and 2,4-dinitrophenylhydrazone. The enzymatic part of the antioxidant system of hypothalamic region was estimated by measuring the activity of superoxide dismutase and catalase. In the setting of Thr-Lys-Pro-Arg-Pro-Gly-Pro and Pro-Gly-Pro administration under experimental hyperthyroidism, the intensity of lipid and protein peroxidation processes was decreased, while the activity of antioxidant enzymes-superoxide dismutase and catalase was restored in the hypothalamic tissue. The experimental data obtained indicate that, under the conditions of experimental hyperthyroidism, Thr-Lys-Pro-Arg-Pro-Gly-Pro and Pro-Gly-Pro compounds exhibit an antioxidant and antiradical activity with respect to parameters of lipoperoxidation and oxidative modification of proteins, as well as with respect to enzymatic defense systems in the hypothalamic brain region of laboratory animals.

Study of Tildox AVZ effect on rat blood parameters in a subchronic experiment

The purpose of our research was to study the effect of Tildox AVZ on rat blood counts in a subchronic experiment. Tildox is a complex antibiotic in powder form. One gram of drug contains doxycycline hyclate – 100 mg and tylosin tartrate - 100 mg as active ingredients, sodium benzoate and sucrose as auxiliary substances. The experiment lasted 90 days. The study was conducted on 150 Wistar rats (75 males and 75 females). The body weight of the animals was 190-230 g. During the experiment 5 groups of 30 animals (15 males and 15 females) were formed, three of them were experimental groups, one - control and one – intact. Of the 150 rats involved in the experiment after its completion 100 individuals (50 males and 50 females) were killed for pathomorphological studies of internal organs.To identify the ability to recover and the appearance of resistance to toxic effects the remaining 50 animals (5 males and 5 females from each group) were monitored for 28 days after the experiment.An aqueous solution of the test sample of the drug was administered daily intragastrically using an atraumatic metal probe. The drug was administered to the first experimental group at a dose of 70 mg / kg, the second group - 265 mg / kg, the third group - at a dose of 1000 mg / kg of animal body weight. The control group was in equal conditions and received intragastric saline solution. Intact animals served as a reference. It was found that Tildox AVZ administered intragastrically at doses of 265 and 1000 mg / kg 1 time per day for 90 days has an effect on clinical and biochemical blood parameters. The drug administered at a dose of 70 mg / kg does not cause changes in blood parameters. However when examining the blood of rats 28 days after the recovery period there were no significant changes in clinical and biochemical parameters compared with the control and intact groups.From this it can be concluded that all blood changes had a dose-dependent effect and after stopping the use of the drug the body recovered which indicates the reversibility of the caused shifts in the blood picture.

Changes in the Cellular Composition of the Rat Thymus 90 Days After Urethane Injection Against the Background of Selenium Intake

The aim of the study is to assess the cellular composition of rat thymus when isolated administration of selenium, urethane and combined exposure to two factors. Material and methods. The study was performed on 34 male Wistar rats, which were divided into 4 groups: 1 – intact, 2 – rats, which received selenium only for 1 month, 3 – animals, which were injected once intraperitoneal urethane, 4 – rats with combined selenium and urethane action. Histological preparations of the thymus were prepared according to generally accepted methods and further evaluated by the method of review microscopy, morphometry, immunohistochemical reactions, electron microscopy, statistical processing of data. Results. Ninety days after selenium, the rat thymus structure is slightly different from intact animals. The introduction of urethane leads to the development of excitation involution of the thymus, which is characterized by morphological changes, and manifests itself in a change in the shape of the organ lobes, in the expansion of the intercole gaps and in the fat tissue thereof, hyperplasia of the cortical substance of the thymus by increasing the number of mature lymphocytes, epithelial cells positive to pancytokeratin. At the ultramicroscopic level, the thymus lymphocyte decreases its nuclear-cytoplasmic index, and increases the number of mitochondria that have a light matrix with a small amount of crystal. Apoptotic bodies and a large number of electron-transparent vacuoles are visualized. When combined, the thymus structure differs from the intact group by an enlarged brain substance, but the number of epithelial cells that are positive to pancytokeratin and Ki67+-cells, as well as cells that express apoptosisregulating protein, decreases. Electron microscopy also indicates a large amount of mitochondria, but with welldefined crystals, when combined with selenium and urethane. There are a small number of electron-transparent vacuoles. Conclusion. The obtained data make it possible to draw a conclusion about the positive influence of the exchange rate of selenium on the T-cell link of immunity, at least for this period of study. In the course of the experiment, it was found that, against the background of the development of the adenoma of the lung caused by the introduction of urethane, at the stage of alveolar epithelium dysplasia signs of excitant thymus involution were detected. When selenium and urethane are combined, less pronounced changes are observed in the examined organ than in the isolated administration of urethane.

EVALUATION OF IMMUNOTROPIC PROPERTIES OF GLYPROLINE COMPOUNDS IN EXPERIMENTAL HYPERTHYROIDISM

Introduction. Thyroid disease is currently the most common among endocrine pathology. Hyperproduction of thyroid hormones is accompanied by stimulation of humoral reactions, contributing to the development of immune-mediated pathology under certain conditions, which actualizes the need to develop means for correcting neuroimmunoendocrine processes. Gliproline compounds analogous to neuropeptide protein molecules, among which are Thr-Lys-Pro-Arg-Pro-Gly-Pro and Pro-Gly-Pro, can be considered as such agents. The aim of the study. The aim of the study was to study the immunotropic properties of gliproline compounds Thr-Lys-Pro-Arg-Pro-Gly-Pro and Pro-Gly-Pro under experimental hyperthyroidism. Material and methods. The immunotropic properties of the gliproline compounds Thr-Lys-Pro-Arg-Pro-Gly-Pro and Pro-Gly-Pro were studied in white nonlinear male rats. Hyperthyroid status in animals was modeled by intragastric administration of L-thyroxine sodium salt pentahydrate («Sigma»; USA) at 150 μg/kg for 21 days. Laboratory animals were divided into groups (n=10): control group – intact animals (control); animals treated with L-thyroxine sodium salt pentahydrate (hyperthyroidism); rats receiving Thr-Lys-Pro-Arg-Pro-Gly-Pro (selanc) and rats receiving Pro-Gly-Pro intraperitoneally daily for 21 days at doses of 87 and 33 μg/kg/day. Results. The obtained results indicate the presence in gliproline compounds Thr-Lys-Pro-Arg-Pro-Gly-Pro and Pro-Gly-Pro of immune correcting activity manifested under the conditions of experimental hyperthyroidism, with the greatest impact on the humoral unit of immunity, which is confirmed by the presence of statistically significant changes in the cellular parameters of the spleen and titer antierythrocytic antibodies in a direct hemagglutination reaction. Conclusion. Thus, the study of the immunotropic properties of gliproline peptide compounds under experimental hyperthyroidism established the presence of immune-correcting activity in Thr-Lys-Pro-Arg-Pro-Gly-Pro and Pro-Gly-Pro substances.