From the editor

The Genome Resource Bank (GRB) is a repository of frozen biological material, including semen and embryos. Cryo­banking is used in combination with modern reproductive technologies such as rederivation, in vitro culture and embryo transfer. Thirteen mouse and rat strains have been re-derived and 32 are kept frozen in the cryostorage at the Institute of Cytology and Genetics, Novosibirsk. Some other laboratory animal species have been cryopreserved as well. Embryos of two hamster species (Djungarian and Campbell’s) in the genus Phodopus were cryopreserved and the viability of thawed embryos was proved by their successful development in vitro and in vivo (by transfer to a recipient). A positive effect of the granulocyte-macrophage colony-stimulating factor (GM-CSF) was demonstrated with both these Phodopus species. Furthermore, semen of Djungarian (Phodopus sungorus) and Campbell’s (Phodopus campbelli) hamsters, domestic cat (Felis catus), amur cat (Prionailurus bengalensis euptilurus) and bobcat (Lynx rufus) was frozen and cryopreserved. Double staining by SYBR Green/PI and subsequent confocal microscopy demonstrated that more than 40 % of amur cat semen retained viability after cryopreservation. This is the world’s first reported successful freezing of semen of this wild felid (Prionailurus bengalensis euptilurus). This article reviews the results and discusses prospects of using reproductive technologies for conservation of laboratory species.

In vitro culture of preimplantation embryos of ICR, HT1AN/Icgn, HT1AC/Icgn and C57BL/6J-Ay mouse strains as well as in OXYS/Icgn rat strain in media containing granulocyte-macrophage colony stimulating factor (GM-CSF) or epidermal growth factor (EGF) has been studied. Both mouse and rat embryos were first frozen in a programmable freezer after a standard protocol using a mixture of glycerol and sucrose as cryoprotectants, thawed and cultured in vitro in R1ECM (rat one-cell embryo culture medium) for 24 hours (mice) and 72 hours (rats). For the in vitro culture experiments with these growth factors, 8-cell frozen-thawed mouse embryos and 2–4-cell frozen-thawed rat embryos were used. Supplementation of the culture medium with GM-CSF improved the rate of embryonic development in HT1AC/Icgn and C57BL/6J-Ay strain mice, while EGF had no effect. The reverse was true of the rats. Supplementation of the culture medium with EGF increased the percentage of deve­loping blastocysts in OXYS/Icgn rat strain, while GM-CSF had no effect. Co-culture of four-cell embryos of HT1AN/ Icgn strain mice with more advanced embryonic stages (morulas) of a different strain ICR led to the facilitation preimplantation embryo development. Experimental results presented here reveal the species-specific effects of growth factors on mouse and rat embryos and indicate that co-culture of different stages of embryo development have stimulatory effects on earlier stages.

The proper choice of cryoprotectant and thawing method affects cryopreservation efficiency. A freezing-thawing method for sparing embryonic cells was evaluated in experiments with ICR mice. Cleavage-stage embryos of ICR mice, GC rats, and OXYS rats were collected on Day 3 of pregnancy and frozen in plastic straws according to a standard protocol. Permeating (ethylene glycol and glycerol) and nonpermeating (sucrose) cryoprotectants and their combinations were compared during the freezing of ICR mouse embryos. With these mice, two thawing methods were compared: rapid (water bath, 10 s, 37 °С) and slow (40 s, room temperature; 40 s, 30 °С). Embryo viability in mice and rats was evaluated by their in vitro culturing after thawing. Our data on mice indicate that slow thawing is more suitable for sparing the integrity of embryonic cells; moreover, supplementation of the main cryoprotectant (either ethylene glycol or glycerol) with sucrose is beneficial for subsequent in vitro culture, especially in the case of glycerol. This freezing-thawing protocol (with glycerol and sucrose as cryoprotectant agents and slow thawing) was applied to rats of the GC and OXYS strains; the survival rate after cryopreservation was 68–83.3 %, and the rate of in vitro development was 64.7–66.6 %.

Modern standards of Laboratory Animal Science include working with laboratory animals of high quality, in particular, with specific pathogen free (SPF) mice and rats. On the other hand, assisted reproductive technologies (ART) are widely used in modern medicine for human infertility treatment as well as for genome resource banking. In the present study, a comparison of body weight, blood pressure (BP) and behavior in the «elevated plus maze» (EPM) test was made between three groups of ISIAH (inherited stress induced arterial hypertension) rats: a group of animals that were born and raised in a conventional animal facility and two groups from an SPF animal facility (one with animals born naturally and another with animals resulting from ART). There were no changes in BP between the groups, but the behavior of ISIAH differed depending on rearing conditions. In particular, grooming time, as well as the number of defecations and the number of urinations during the test were decreased in both groups of ISIAH rats born in the SPF animal facility as compared to ISIAH rats born in the conventional animal facility. The behavior of the ISIAH rat offspring resulting from ART was different from that of the naturally born group: the EPM test revealed reduced anxiety in the former. The results of the present study indicate that the rearing conditions as well as reproductive technologies affect some behavioral characteristics in adult ISIAH rats, although they develop arterial hypertension in all the conditions used in this study.

The water Morris maze is the basic test to study the spatial ability to learn as well as spatial memory in laboratory rodents. It is a part of a series of tests necessary for behavioral phenotyping of mutant and transgenic mice. At the same time, conducting this test in SPF conditions must comply with very strict regulations concerning pathogen control. A white animal on the surface of whitened water is low contrast and this does not allow the animal to be traced automatically, which represents yet another major problem. A unique installation based on EthoStudio has been developed at the Institute of Cytology and Genetics SB RAS and the Institute of Automation and Electrometry SB RAS. This installation automates the process of tracing mice of any coat color in SPF conditions. This includes a setup to install a plastic water reservoir (110×40 cm), a digital camera and a light source. Water to fill the reservoir was sterilized using a Van Erp Blue Lagoon UV-C Tech 15000 ultraviolet decontaminator. The image of an animal was processed in a frame-by-frame fashion using the EthoStudio program, with the following parameters calculated: latent release time, route covered, cumulative distance to the platform and the time spent in the reservoir sectors. With this installation, we were able to study the spatial ability to learn and spatial memory in mice of the C57BL/6 strain and in mice of the C57BL/6/ Kaiso strain developed on the C57BL/6 background, with the gene encoding the methyl-DNA binding Kaiso protein knocked-out. It has been demonstrated that mice of these strains are able to learn to find the platform in the water Morris maze and have the location of the platform in their memory for at least the next four days.

The tumor necrosis factor (TNF) is a cytokine exerting both homeostatic and pathophysiological roles in the central nervous system (CNS). It has been demonstrated that TNF plays roles in such diseases as Alzheimer’s disease, Parkinson’s disease and multiple sclerosis. Nevertheless, the role of TNF in the CNS under normal physiological conditions is poorly studied. A novel mouse strain with TNF deficiency (TNFKO) was developed in S.A. Nedospa­sov’s labora­tory of Engelhardt Institute of Molecular Bio­logy of the Russian Academy of Sciences on the C57Bl/6 (WT) background. In our study, we compared the behavior of TNFKO and WT adult mice in a battery of tests: open-field, elevated plus-maze and the forced-swim test. We showed that TNF deficiency had no effect on locomotor activity or exploration in the openfield test. At the same time, in this test, TNFKO mice spent more time in the center of the arena, but had a higher level of defecation and lower rearing duration. This result indicates that, in the openfield conditions, TNFKO mice show disorientation rather than anxiety-like behavior. There were no differences between TNFKO and WT in anxiety level in the elevated plus-maze test or in depressive-like behavior in the forcedswim test. These data suggest that TNF deficiency leads to changes in neurofunctional interactions that alter the mouse response to mild stress in the open-field test.

Kaiso is a methyl DNA-binding protein, which participates in the epigenetic regulation of gene expression. It binds methylated DNA with its zinc-finger domain and recruits repressive protein complexes to the methylated DNA fragments by the interaction of the BTB/POZ domain with the complex of NCoR corepressor and histone deacetylase, thereby performing transcription repression. A Kaiso-deficient mouse strain (KO) with the C57BL/6 strain background has been bred. Here we compare the behavior of KO mice and wild-type control C57BL/6 mice (WT) in the classic battery of behavioral tests, including the open field, elevated plus maze, and forced swim tests. We have shown that knockout of the Kaiso gene increases the locomotory and exploratory activities of KO mice in the open field test. Kaiso-deficient mice spend more time in the center of the open field than WT mice. No effect of Kaiso gene knockout on anxietyrelated behavior has been observed in the elevated plus-maze. However, Kaiso gene deficiency produces a pronounced antidepressant-like effect in the forced swim test: Unlike WT mice, KO mice do not show any depressive-like freezing in this test. These results are the first piece of experimental evidence for the involvement of Kaiso protein in the regulation of brain functioning and beha­vior. The Kaiso-deficient strain is a new and promising model of the genetic, molecular, and neuronal mechanisms mediating the epigenetic regulation of brain functions and behavior.

TNF is a multifunctional cytokine that, at physiological concentrations, maintains the balance between apoptosis and survival of male germ cells and, at higher concentrations, has adverse effects on various stages of the reproductive process. Although ant-cytokine therapies have been used in millions of patients, the consequences of cytokine deficiency for reproductive functions are poorly understood and need attention. In this work, we have studied behavioral interactions between males and females, spermatogenesis, male fertility, and embryonic developmental characteristics of the progeny in TNFα knockout mice (TNF-/-). We have demonstrated that TNF is involved in the regulation of sexual behavior, spermatogenesis, pre- and postimplantation development. Complete TNF deficiency led to decreased reproductive efficiency: a lower number of viable embryos were observed in TNF-/- mice than in wild-type mice. The decrease in fertility was caused by preimplantation embryo loss in TNF-/- mice. Preimplantation loss in females might be caused by asospermia in TNF-/- males. Additionally, the sensitivity of reproductive functions to female stimuli was different between TNF-/- mice and wild-type mice, while interactions with females increased the concentrations of sper­matozoids in both TNF-/- and wild-type mice. Still higher levels were observed in knockout animals, which led to increase in the number of immature spermatozoids in epididymides.

Inflammatory processes in the gut lead to abnormal­ities in various systems of the body, in particular, to changes in the activity of the central nervous system. Although the mechanisms of these effects are not yet known, it has been demonstrated that intestinal inflammation is associated with anxiety and depression. In this work, we used an animal model of intestinal inflammation, which might result in behavioral changes. The animals used were knock-out mice with double mutations in the Kaiso and Mucin-2 genes. The Kaiso gene encodes a transcription factor that is expressed both in the brain and in the intestine. The Mucin-2 gene encodes a protein that serves as a scaffold for the synthesis of intestinal proteoglycan. Mucin-2 is a major proteoglycan of the intestinal mucus layer and performs multiple functions, including barrier and defensive ones. We used knock-out animals with a mutation in the trans­cription factor Kaiso in tests assessing social behavior, but did not observe any difference between test subjects and wild-type animals. By contrast, double knock-out animals that additionally had a mutation in Mucin-2, a major gene for intestinal proteoglycan, displayed significant changes in social behavior: lower aggression rates and higher rates of courtship behavior toward a male intruder. These results suggest that intestinal homeostasis might have a strong impact on the nervous system of the animals. It remains unclear whether the influence of the two genes is synergistic or the knock-out of the Mucin-2 gene alone determines this behavior in mice. Further investigations will help clarify the matter.

Sexual behavior is one of the biologically highly relevant types of behavior. Sexual arousal, or an initial stage of sexual behavior, is of particular interest since it triggers all the following events but still remains the least known element of this behavior. Sexual dysfunctions are caused by aging, stress, or side effects of psychotropic drugs; they are symptoms of a variety of neurological and psychiatric disorders. Therefore, the study of sexual behavior appears to be an important step in modeling various animal pathologies and the effects of psychotropic drugs. We have performed phenotyping of animals with hereditary predisposition to catalepsy using our previous development, a model of male sexual arousal, and examined the relationship between catalepsy and sexual arousal. The main gene for a high predisposition to catalepsy was shown to be associated with the expression of sexual motivation, but not with the hormonal component of sexual arousal (increase in plasma testosterone levels following exposure to a receptive female). ASC (Antidepressant Sensitive Catalepsy) mice, proposed as a model of depression, had a decreased manifestation of sexual motivation, while male GC (Genetic Catalepsy) strain rats had enhanced sexual motivation. Noteworthy, highly excitable GC strain animals corresponding to the manic pole of bipolar disorders prevail at the current stage of breeding. Our results are in a good agreement with clinical data that indicate reduced libido in depressed patients and hypersexuality in people with bipolar disorder.

Olfactory perception plays the key role in the inter­action of animals with biotic factors of the species-specific econiche. Identification of odorants informs nocturnal animals about social environment, presence of predators, or infected food. Olfactory efficiency depends on physiological conditions; in particular, odor sensitivity can be changed by infection. This work considers use of fMRI in the study of the influence of innate immunity activation on neuronal response during perception and differentiation of socially significant (2.5-dimethylpyrazine, 2-heptanon) and socially insignificant (1-hexanol and isoprene) olfactory stimuli by CD-1 mice. We stimulated innate immunity by intraperitoneal injection of bacterial lipopolysaccharide (LPS) at the dose 500 µg/kg three hours before tomography. Urethane anesthesia was used during MRI trail. Odor stimulation was done with a lab-made metering unit for supplying standard doses of volatile organic compounds. The supply of olfactory stimuli induced activation of neurons in the primary perceptual center and the centers of secondary processing of olfactory information. Olfactory stimulus type affected neuronal response rate in an olfactory bulb but did not affect response parameters in other brain regions studied. This increase in neuronal activity is likely to be of adaptive significance as a mechanism supporting olfactory sensitivity increase, which plays the key role in the identification of potential sources of infection.

Hypertension is one of the most common human diseases. This disease leads to serious disturbances such as myocardial infarction and stroke. Due to the development of nuclear magnetic resonance spectroscopy (NMRS), a decrease in neuron viability in different parts of the brain in humans with hypertension has been shown. Translation of NMRS tools to the clinic requires the accumulation of empirical data about neurometabolic changes in a strictly controlled experiment. It is particularly interesting to compare the metabolic parameters of laboratory animals with normal and high blood pressure kept in standard conditions on exactly the same diet. In this study, cortex and hypothalamus metabolites of ISIAH and Wistar male rats at the age of 8–9 weeks were examined. Cortex and hypothalamus metabolites were measured in animals under isoflurane anesthesia using proton magnetic resonance spectroscopy (1Н MRS). Processing of primary data using Partial least squares Discriminant Analysis (PLS-DA) allowed us to identify the main discriminating axis (Y1), its variations reflecting the predominance of excitatory neurometabolites (glutamine and glutamate) over inhibitory ones (GABA and glycine). In the cortex, the values of the Y1-axis were lower in ISIAH than in Wistar rats. This fact indicates a decrease in cortical excitability in hypertensive animals. By contrast, in the hypothalamus, the values of the Y1-axis were higher in ISIAH than in Wistar rats and the predominance of excitatory neurometabolites positively correlated with the level of mean blood pressure, which agrees well with the view of caudal hypothalamic activation in hypertensive animal models.

Proportions of major neurometabolites with regard to their total amount in the dorsal region of the hippocampus were studied in adult male rats of populations selected for long for increase and absence of aggressivefearful response to humans and in unselected vivarium- kept rats by 1H magnetic resonance spectrometry. Tame and unselected males showed no significant differences in the proportions of any neurometabolites studied. Differences in the proportions of some neurometabolites were found in aggressive vs. tame and in aggressive vs. unselected animals. Tame animals showed higher pro­portions of GABA, N-acetylaspartate (NAA), and choline derivatives and a lower proportion of phosphoryl­ethanolamine than aggressive ones. It is likely that the elevated content of GABA, one of the main inhibitory neurotransmitters in the brain, lowers excita­tion intensity in tame pups in comparison to aggressive ones. In comparison to unselected animals, aggressive rats demonstrated higher proportions of glutamine, aspartate, phosphorylethanolamine, and lactate and lower proportions of NAA and creatinine+ phosphocreatinine. Aspartate is one of the main excitement transmitter, and its elevated proportion in the brain of aggressive rats may favor more intense excitation than in unselected rats. In contrast, the elevated proportion of glutamine in aggressive rats vs. tame rats may be indicative of (1) a metabolic disturbance in the glutamate–glutamine cycle, which links neural and glial cells, and (2) decrease in the activity of glutaminase, the enzyme converting glutamine to glutamate (GABA precursor). The reduced NAA proportion together with the elevated proportion of glutamine in aggressive rats point to impaired energy metabolism in comparison to unselected animals. The differences in neurometabolite patterns between hippocampi of male rats of the unselec­ted and aggressive populations suggest the existence of different neurobiological mechanisms governing aggression manifestation.

Air pollution by particulate matter (PM) has been associated with cardiopulmonary morbidity and mortality in many recent epidemiological studies. It has been shown that transition metal compounds, well- known toxic components of PM, are able to induce hypothermia following whole-body inhalation exposure. Low temperature appears to protect tissue against toxic effects of PM metal compounds in vivo and in vitro. To study the role of soluble and insoluble irritants in the induction of the hypothermic response, we analyz­ed the decrease in mouse body temperature (Δtbody) after intranasal administration of PtO nanoparticles or a K2[PtCl 4] solution. Between-strain differences in Δtbody after intranasal administration of the irritants were evaluated using 6 inbred (BALB/cJ, C57BL/6J, AKR/OlaHsd, DBA/2JRccHsd, C3H/HeNHsd, and SJL/J) and 2 outbred mouse strains (SCID and CD1). BALB/cJ and SCID mice showed the most pronounced effect of intranasal admini­stration of the xenobiotic on tbody. Thus, tbody was signi­ficantly lower after nasal administration the PtO nano­particles than after administration of the K2[PtCl 4] solution. To study the mechanism of this decrease, we compar­ed the respective values for Δtbody following intra­nasal, intravenous and peroral administration of PtO nanoparticles in Balb/c mice. Neither intravenous nor peroral administration had any effect on mouse body temperature. This fact together with data on the dynamics of the decrease in mouse body temperature following intranasal administration of PtO nanoparticles (max Δtbody ~ 80–100 min) allowed us to assume that this process is under nervous regulation. The correlation found between our data and some well-known phenotypic characteristics (phenome.jax.org) of the mouse strains used confirms this hypothesis.

Alzheimer’s disease (AD) is the most prevalent neuro­degenerative disease. It produces atrophic changes in the brain, which cause dementia. The incidence of AD is increasing with increasing life expectancy and gradual aging of the population in developed countries. There are no effective prophylactic inter­ventions because of insufficient understanding of the AD pathogenesis and the absence of adequate experimental models. Recently, we showed that senescence-accelerated OXYS rats represent a promis­ing model of AD; in these rats, accelerated aging of the brain is accompanied by the typical signs of AD: degenerative alterations and death of neurons, a de­crease in synaptic density, mitochondrial dysfunction, hyperphosphorylation of the tau protein, an increased level of amyloid β (Aβ1–42), and the formation of amyloid plaques. To elucidate how these signs develop, we used a nextgeneration RNA sequencing technique (RNA-Seq) to study the prefron­tal-cortex transcriptome of OXYS rats during the manifestation of AD signs (at an age of 5 months) and during their active progres­sion (at an age of 18 months), using age-matched Wistar rats (parental strain) as controls. At the age of 5 months, there were significant differences between OXYS and Wistar rats (p < 0.01) in the mRNA expression of more than 900 genes (> 2000 genes at the age of 18 months) in the prefrontal cortex. Most of these genes were related to neuronal plasticity, protein phosphorylation, Са2+ homeostasis, hypoxia, immune processes, and apoptosis. Between the ages of 5 and 18 months, there were changes in the expression of 499 genes in Wistar rats and changes in the expres­sion of 5500 genes in OXYS rats. Only 333 genes were common between these sets. This finding points to differences in the mechanisms and rates of age-related changes in the brain between normal aging and the period of development of AD-specific neuro­degene­rative processes.

The main views of the issue of stress and hypertension are briefly reviewed. It is well known that stress is one of the major risk factors for cardiovascular disease development. Increase in blood pressure is a typical manifestation of the acute stress response. This fact is the reason to hypothesize that chronic stress causes the development of hypertensive disease. An association of hypertension with psychological stress in humans was shown in several works. In addition, it was demonstrated that hypertension was accompanied by an increase in sympathetic tone. On the other hand, there were many population studies in which no association was found between different types of chronic stress and arterial hyper­tension. Thus, the question is far from being answered. Even in the cases when one managed to obtain a signi­ficant hypertensive effect in experimental studies with emotional stress, it was difficult to explain the mechanisms mediating the formation of stressinduced hypertension. To clarify the situation, one of the authors of this review decided to begin the breeding of a rat strain with increased blood pressure response to emotional stress. This breeding gave rise to inbred rats with persistent stress-induced arterial hypertension. A brief history of the development of the genetic model ofstress-induced arterial hypertension, the ISIAH rat strain, is given. A retrospective review of the studies performed with ISIAH rats is presented. The contribution of genotype changes in the neuroendocrine systems involved in stress and blood pressure regulation to the development ofstress-dependent hypertension in the ISIAH rat strain is shown.

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer with the average lifespan of patients about 9–12 months. The study of tumor formation and the evaluation of new therapies for GBM require accurate and reproducible experimental brain tumor animal models. In this study we used MRI for investigation of tumor morphology and growth dynamic in an orthopic xenotransplantation immunodeficient mouse model (SCID mouse line). Comparison of T1- and T2-weighed MRI scans preformed with a high-field MRI scanner (Bruker, BioSpec, 11,7 T) revealed insufficient tumor/normal tissue T1-contrast because of high longitudinal magnetization of the magnetic field in our scanner. Intravenous injection of paramagnetic manganese oxide (MnO) nanoparticles dramatically increased the tumor/normal tissue contrast in T1-weigthed MRI scans. The study of glioblastoma growth with T2-weighed images showed that a significant tumor development began not earlier than 3 weeks after cell culture intracranial injection and then the tumor grew exponentially. Thus, we developed a protocol of the characterization of glioblastoma U87 growth and morphology by T1- and T2-weighed and MnO-enhanced MRI in the orthopic xenotransplantation mouse model. The results demonstrate that this SCID model may be used as an in vivo preclinical model to test the efficacy and putative side effects of novel anticancer therapies.

The validity of experimental models of pathologies is one of the key challenges in translational medicine. Cholangiocarcinoma, or bile duct cancer, ranks second among oncological diseases of the liver. There is a strong association between bile duct cancer and parasitic infestation of the liver caused by trematodes in the family Opisthorchiidae. We have recently demonstrated that cholangiocarcinoma can develop in Syrian hamsters (Mesocricetus auratus) infected by Opisthorchis felineus and administered with dimethylnitrosamine. However, there is still no description of how this experimental model can possibly be used in translational research. The aim of this work was to study the morphological, functional and biochemical characteristics during cholangiocarcinoma development in Syrian hamsters infected by O. felineus and administered with dimethylnitrosamine. The experi­ment lasted 30 weeks with combined exposure to dimethylnitrosamine in drinking water at a dose of 12.5 ppm and a single injection of 50 metacercariae O. felineus. It was shown that the development of cholangiocarcinoma (18 weeks) increased the total number of basophils, eosinophils and monocytes, the relative number of granulocytes, the amount of total and direct bilirubin, and cholesterol and ALT levels, but reduced the relative number of lymphocytes. Based on pathological, morphometric and biochemical analyses, our model has characteristics similar to those in patients with opisthorchiasisassociated cholangiocarcinoma. Thus, this model can be used to test anticancer drugs, to study the mechanisms of cholangiocarcinogenesis and to search for molecular markers for early diagnosis of bile duct cancer.

Ortho-aminoazotoluene (OAT) is a potent hepatocarcinogen for most strains of mice. It has previously been shown that OAT application activates the aryl hydrocarbon receptor (Ahr) and the constitutive androstane receptor (Car) in the mouse liver. Both of these receptors are directly involved in the process of hepatocarcinogenesis. In this study, we investigated the effect of chronic OAT administration on the mRNA expression levels of Ahr, Car and their target genes Cyp1a1 and Cyp2b10 in the liver of DD/He (DD) and CC57BR/Mv (BR) mouse strains contrasting in sensitivity to hepatocarcinogenesis. The inflammatory response of these strains was also studied. Male mice of both strains received OAT oil solution at the dose of 225 mg/kg body weight four times within two months. Control animals received the equivalent solvent amount. Mice were sacrificed on days 1 and 4 after the last OAT administration. Gene expression levels in the liver were determined by real-time PCR. The inflammatory response was evaluated by serum concentration of tumor necrosis factor alpha (TNF-alpha). In resistant BR mice, OAT induced a pronounced and prolonged increase in Cyp1a1 mRNA, showing primarily Ahr activation, while the DD strain displayed a more pronounced elevation of Cyp2b10 expression, indicative of Car activation. In addition, a strong inflammatory response to OAT was recorded in DD mice but not in BR. It is assumed that the prevalence of Ahr signaling pathway activation over Car signaling pathway activation is a factor of resistance to OAT-induced hepatocarcinogenesis.

Human carcinoma A431 cells were subcutaneously injected into nude mice at points remote from each other. One of the two xenografts developed after­wards was used for treatment with a recombinant vaccinia virus, while another served as an artificial metastasis. We used the attenuated recombinant vaccinia virus (VACV) VVdGF-GFP2 of the L-IVP strain (GenBank accession number KP233807), with deletion of two virulence genes: the virus growth factor and thymidine kinase, with the gene for the green fluorescent protein (GFP2) inserted in an area of the latter. Treatments were performed by a single intratumoral injection of the recombinant VACV at a dose of 107 PFU/mouse. VACV was detected in cells of the artificial metastasis as early as two days following infection, and after 8 days virus concentrations were com- parable with those in the infected tumor (~109 PFU/ml). Electron microscopy revealed selective replication of the recombinant in tumor cells. Targeted accumulation of GFP2 in both tumor and metastasis was shown in the UV-images of the mice obtained using the In-vivo Multispectral Imaging System (Bruker, Germany). Complete destruction of the tumor was registered after 12 days, and that of metastasis, after 20 days post injection of VVdGF-GFP2. The destruction process was accompanied by pronounced edema and leukocyte infiltration of tumor tissue. The recombinant virus induced a significant reduction in the sizes of the tumor and metastasis: by the end of the experiment (35 days) the xenografts in the control mice were 10 times larger than those in the treated mice (5000 vs. 500 mm3). Our study showed that the attenuated VACV administered by the peripheral route not only is able to destroy the primary tumor, but also has a distinct antimeta­static action.

At present, there is no animal model for smallpox that reflects the weakened immune system in people and can therefore help assess the prophylactic (highly preventive) efficiency of antiviral drugs. To fill in the gap, we have explored the possibility of using outbred immunodeficient SCID mice as a model animal for smallpox with the aid of virolo­gical, histological and electron microscopic and sta­tistical methods. There was no clinical evidence of disease by intranasal infection of mice at a dose of 5.2 log10 PFU (plaque forming units). At the same time, the 50 % infective dose (ID50) of VARV estimated for animals by registering the presence of the virus in their lungs after 4 days post i.n. infection was 3.5 log10 PFU and was relatively similar to that in humans, theoretically determined by identification of the clinical picture of the disease. Virus replication was detected only in the respiratory organs of mice challenged i.n. with VARV at a dose of 5.2 log10 PFU (50 ID50). The values for its concentrations in the lungs and nose resembled those for affected people and well-known animal models (Macaca cynomolgus and ICR mice), respiratorily infected with VARV at similar doses. The existing model animals were not significantly different from SCID mice in the duration of viral presence in the lungs. Moreover, in SCID mice, as in humans and other animal models, similar pathomor- phological changes of inflammatory necrotic nature in the respiratory organs have been reported. Using SCID mice in assessing the prophylactic efficacy of the antiviral drugs NIOCH-14 and ST-246 demonstrated the adequacy of the results obtained to those described in the literature. This opens up the prospect of using SCID mice as an animal model for smallpox to develop antiviral drugs intended for people with severe immuno­suppressive states.

Abnormal synthesis of the main intestinal proteo­glycan mucin-2 is typical of ulcerative colitis and Crohn’s disease in humans. Those morphological changes of the mucus layer affect the diversity of the intestinal microflora. Antibiotics may be ineffective or even dangerous to humans or animals deficient for mucin-2 because of the risk of sepsis and chronic inflammation. In this study, we investigated the potential of antibiotics (clarithromycin, amoxicillin, and metronidazole) in elimination of patho­genic infection from Muc2 knockout mice (Muc2–/–). We assayed the population sizes of pathogens (Heli­co­bacter spp.) and symbiotic (E. coli) bacteria in the intestines of animals as a criterion of antibiotic efficacy. The damaging effect of antibacterial treatment on the host body was estimated from their survival rate. Three antibiotics were ineffective in the elimination of Helicobacter spp. from mucin-2-deficient mice. Moreover, the mortality of Muc2 knockout mice during the antibacterial treatment was 60 %. The survival of wild-type mice (C57BL/6J) during the treatment was 100 %. The weight of wild-type mice showed no decrease during the treatment. The Helico­bacter spp. pathogen was fully eradicated from wild-type mice. Thus, therapy of Helicobacter spp. infection in mucin-2 deficient animals is not only poorly efficient but even deadly. The high susceptibility to antibiotics allows Muc2 knockout mice to be used as a test model to evaluate the pharmacological safety of new antibiotics.

We have evaluated the efficiency of a metabonomic approach to metabolic phenotyping and detection of early metabolic changes under a toxic influence. For this purpose, a metabolic profiling of rat liver was performed with 1H NMR spectroscopy. Rat tissues from animals in three groups were analyzed. Group C consisted of control animals; animals in group A received alcohol repeatedly (15 % ethanol); and animals in group A+ R received alcohol in combination with a hepatoprotective herbal medicine (Reishi, Ganoderma lucidum) repeatedly. Noteworthy, alcohol consumption did not cause pathological changes, but stimulated hepatocyte proliferation. Our data suggest that changes in metabolite concentrations in A represent a typical metabolic response to alcohol consumption, namely decrease in glycine, leucine, isoleucine, valine, choline and lactate content, and increase in TMAO content. Treatment with Reishi (A+ R) had positive effects, in that it restored the levels of glycine, valine and TMAO. Furthermore, increase in NAD, ATP, UTP, succinate, pyranose, and acetate concentrations was observed in A+ R. A correlation was found between the valine, isoleucine, lactate, cho­line, and pyranose content and the num­ber of binuclear hepatocytes. Binuclear hepatocytes indicate proliferative activity, and the concentration of the metabolites participating in the formation of new hepatic cells decreases. Thus, the study of liver tissues by 1H NMR spectroscopy allows for detection of early changes in metabolite concentra­tions following chronic consumption of alcohol at insignificant doses. Consequently, 1H NMR spectro­scopy can serve as a promising approach to detecting alcohol-related liver pathologies and assessing the efficiency of the therapy used.