[Restraint Stress-Induced Expression of Fos and Several Related Genes in the Hypothalamus of Hypertensive ISIAH Rats].

Stress can play a significant role in arterial hypertension and many other complications of cardiovascular diseases. Considerable attention is paid to the study of the molecular mechanisms involved in the body response to stressful influences, but there are still many blank spots in understanding the details. ISIAH rats model the stress-sensitive form of arterial hypertension. ISIAH rats are characterized by genetically determined enhanced activities of the hypothalamic-pituitary-adrenocortical and sympathetic-adrenomedullary systems, suggesting a functional state of increased stress reactivity. For the first time, the temporal expression patterns of Fos and several related genes were studied in the hypothalamus of adult male hypertensive ISIAH rats after a single exposure to restraint stress for 30, 60, or 120 min. Fos transcription was activated and peaked 1 h after the start of restraint stress. The time course of Fos activation coincided with that of blood pressure increase after stress. Activation of hypothalamic neurons also alters the transcription levels of several transcription factor genes (Jun, Nr4a3, Jdp2, and Ppargc1a), which are associated with the development of cardiovascular diseases. Because Fos induction is a marker of brain neuron activation, activation of hypothalamic neurons and an increase in blood pressure were concluded to accompany increased stress reactivity of the hypothalamic-pituitary-adrenocortical and sympathoadrenal systems in hypertensive ISIAH rats during short-term restraint.

[Genes Associated with Increased Stress Sensitivity in Hypertensive ISIAH Rats].

Inherited stress-induced arterial hypertension (ISIAH) rats are characterized by increased stress reactivity of the hypothalamic-pituitary-adrenal and sympathoadrenal systems. The genetic basis of increased susceptibility to stress was studied in hypertensive ISIAH rats. Adrenal transcriptomes were sequenced in hypertensive ISIAH and normotensive WAG rats, and nine differentially expressed genes (DEGs) were found in the X-chromosome locus that was previously associated with mild emotional stress-induced increases in blood pressure and plasma corticosterone and an increased adrenal weight in ISIAH rats. An analysis of the functions performed by DEG-encoded proteins suggested the Sms (spermine synthase) gene to be the most likely candidate gene in the X-chromosome locus associated with an elevated stress susceptibility in ISIAH rats.

RatDEGdb: a knowledge base of differentially expressed genes in the rat as a model object in biomedical research.

The animal models used in biomedical research cover virtually every human disease. RatDEGdb, a knowledge base of the differentially expressed genes (DEGs) of the rat as a model object in biomedical research is a collection of published data on gene expression in rat strains simulating arterial hypertension, age-related diseases, psychopathological conditions and other human afflictions. The current release contains information on 25,101 DEGs representing 14,320 unique rat genes that change transcription levels in 21 tissues of 10 genetic rat strains used as models of 11 human diseases based on 45 original scientific papers. RatDEGdb is novel in that, unlike any other biomedical database, it offers the manually curated annotations of DEGs in model rats with the use of independent clinical data on equal changes in the expression of homologous genes revealed in people with pathologies. The rat DEGs put in RatDEGdb were annotated with equal changes in the expression of their human homologs in affected people. In its current release, RatDEGdb contains 94,873 such annotations for 321 human genes in 836 diseases based on 959 original scientific papers found in the current PubMed. RatDEGdb may be interesting first of all to human geneticists, molecular biologists, clinical physicians, genetic advisors as well as experts in biopharmaceutics, bioinformatics and personalized genomics. RatDEGdb is publicly available at https://www.sysbio.ru/RatDEGdb.

[Differentially expressed genes in the locus associated with relative kidney weight and resting blood pressure in hypertensive rats of the ISIAH strain].

The comparative full-genome sequencing of transcriptomes of the renal cortex and medulla from hypertensive ISIAH rats and normotensive WAG rats revealed the differential expression of genes in the locus of chromosome 11 associated to the traits of resting blood pressure and relative kidney weight. Six differentially expressed genes (Kcne1, Rcan1, Mx1, Mx2, Tmprss2, and RGD1559516) were identified in the renal cortex, and three genes (Rcan1, Mx2, and Tmprss2) were identified in the renal medulla. An analysis of the functions of these genes pointed at the Rcan1 gene as the most relevant candidate gene associated with both the traits of resting blood pressure and relative kidney weight in ISIAH rats. The elevation of the transcription levels of the Mx1 and Mx2 genes in hypertensive ISIAH rats may represent an adaptation that contributes to the alleviation of inflammatory processes in the kidneys.

Influence of Antioxidant SkQ1 on Accumulation of Mitochondrial DNA Deletions in the Hippocampus of Senescence-Accelerated OXYS Rats.

Reduction of efficiency of oxidative phosphorylation associated with aging and the development of neurodegenerative diseases including Alzheimer's disease is thought to be linked to the accumulation of deletions in mitochondrial DNA (ΔmtDNA), which are seen as a marker of oxidative damage. Recently, we have shown that mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl)decyltriphenylphosphonium) can slow the development of signs of Alzheimer's disease in senescence-accelerated OXYS rats. The purpose of this study was to explore the relationship between the development of neurodegenerative changes in the brain of OXYS rats and changes in the amount of mtDNA and the 4834-bp mitochondrial DNA deletion (ΔmtDNA4834) as well as the effect of SkQ1. We studied the relative amount of mtDNA and ΔmtDNA4834 in the hippocampus of OXYS and Wistar (control) rats at ages of 1, 2, 6, 10, and 20 days and 3, 6, and 24 months. During the period crucial for manifestation of the signs of accelerated aging of OXYS rats (from 1.5 to 3 months of age), we evaluated the effects of administration of SkQ1 (250 nmol/kg) and vitamin E (670 mmol/kg, reference treatment) on the amount of mtDNA and ΔmtDNA4834 and on the formation of the behavioral feature of accelerated senescence in OXYS rats - passive type of behavior in the open field test. In OXYS rats, the level of ΔmtDNA4834 in the hippocampus is increased compared to the Wistar rats, especially at the stage of completion of brain development in the postnatal period. This level remains elevated not only at the stages preceding the manifestation of the signs of accelerated brain aging and the development of pathological changes linked to Alzheimer's disease, but also during their progression. However, at age of 24 months, there were no detectable differences between the two strains. SkQ1 treatment reduced the level of ΔmtDNA4834 in the hippocampus of Wistar and OXYS rats and slowed the formation of passive behavior in OXYS rats. These results support the possible use of SkQ1 for prophylaxis of brain aging.

MS2 phage ribonucleoproteins as exogenous internal control for RT-qPCR data normalization in gene expression study of developing rat brain.

The most popular strategy for normalization of RT-qPCR data involves presenting them in comparison with expression of "housekeeping" genes. However, the required stable expression of the control genes is not always achievable. As an alternative, we used ribonucleoprotein phage particles as an exogenous internal control and demonstrated that this type of normalization provides a simple and reliable method for quantification in RT-qPCR experiments. Using phage-based normalization, we analyzed mRNA levels of three popular housekeeping genes coding ß-actin, glyceraldehyde-3-phosphate dehydrogenase, and ribosomal protein L30 and showed high variability in their expression patterns during rat brain development, indicating that they should not be used as controls in gene expression studies of the developing brain either individually or in combination. Using phage-based controls, we showed interstrain differences and age-related changes in the expression of genes involved in proteoglycan biosynthesis and degradation in developing brain of senescence-accelerated OXYS rats and control Wistar rats.

Expression of renin-angiotensin system genes in brain structures of ISIAH rats with stress-induced arterial hypertension.

We studied the expression of genes encoding angiotensinogen (Agt), renin (Ren), angiotensin-converting enzyme (ACE), and type 1 angiotensin receptor (AT1A) in the hypothalamus and medulla oblongata of hypertensive ISIAH rats and normotensive WAG rats. The amount of Agt mRNA in the hypothalamus of young ISIAH rats was increased by 30% compared to WAG controls. In the medulla oblongata of young ISIAH rats, the levels of mRNA of Agt and AT1A receptor were enhanced by 60% and 24%, respectively, compared to WAG rats. In adult animals, the expression of the studied genes did not differ from the control. It is concluded that changes in gene expression of the renin-angiotensin system in brain structures of ISIAH rats may contribute to the development of hypertension.

Expression of Ext1, Ext2, and heparanase genes in brain of senescence-accelerated OXYS rats in early ontogenesis and during development of neurodegenerative changes.

Heparan sulfate (HS) and heparan sulfate proteoglycans (HSPG) play a significant role in brain development, and their structural and quantitative changes are revealed during aging and in neurodegenerative disorders. The mechanism of these changes is not clear, but is likely to be associated with alteration in the expression and/or activity of enzymes responsible for HSPG biosynthesis and degradation. The contents of mRNAs of the genes Ext1 and Ext2 encoding polymerization enzymes and of gene Hpse of heparanase degrading HS were determined in the brain of prematurely aging OXYS rats during early postnatal development and during appearance of signs of brain accelerated aging (at age of 1, 7, 14, 30, 60, and 420 days). Wistar rats of the same age were used as controls. Expression levels of the genes Ext1, Ext2, and Hpse in the brain of rats of both strains were maximal during the two first weeks of life, and the contents of mRNAs of all genes in the brain of newborn and 7-day-old OXYS rats were significantly higher than in Wistar rats. By the 14th day of life the differences leveled, but at the age of 30 days on the background of a decrease in the contents of mRNAs of Ext1, Ext2, and Hpse in OXYS rats they became more pronounced (three-, four-, and twofold, respectively). Differences between the strains were absent at the age of 60 days and 14 months, and expression of all the genes was significantly lower than in the newborn animals. A strong positive correlation was found between contents of mRNAs of all the studied genes, and this suggested that heparanase should be involved in HSPG metabolism together with Ext1 and Ext2. Based on these and earlier findings, we conclude that development of the OXYS rat brain occurs on the background of significant alterations in HSPG metabolism that precede the development of neurodegenerative manifestations recently detected by magnetic resonance imaging.

[Renin-angiotensin system gene expression in the kidney and in the heart in hypertensive ISIAH rats].

The content of mRNA of renin-angiotensin system (RAS) genes in the kidney and heart of hypertensive ISIAH and normotensive WAG rats was measured by the real-time PCR. Statistically significant decrease of RAS gene mRNA was registered in the kidney of ISIAH rats, including Ren (by 45%), Ace (43%), AT1A (34%), COX-2 (50%). In the myocardium AT1A mRNA expression decreased by 28% while Ace mRNA expression increased by 80%. These results demonstrate the reduction of renal RAS basal activity in the hypertensive ISIAH rats, and this allows us to consider the ISIAH rat, as a low-renin hypertensive strain. In support of this viewpoint, in the ISIAH rats, a two-fold increase in the connective tissue sodium concentration as well as statistically significant plasma sodium increase (from 136 +/- 0,25 micromol/l in WAG to 139 +/- 0,3 micromol/l in the ISIAH rats) were found. Our conclusion backed by a tendency of the ISIAH plasma aldosterone level decrease giving in sum a classical picture of a low-renin hypertensive state in the ISIAH rats. It was suggested that the formation of low-renin arterial hypertension in the ISIAH rats may depend on changes in kidney ion channels function. In addition, renal NO system alterations could be also involved in the pathogenesis of arterial hypertension in the ISIAH rats.

Renin system of the kidney in ISIAH rats with inherited stress-induced arterial hypertension.

The renal renin system was studied in ISIAH rats with inherited stress-induced arterial hypertension. The expression of genes for renin (Ren1) and cyclooxygenase (Cox-2) was evaluated in renal tissue of ISIAH and WAG rats (normotensive control). Basal gene expression for Ren1 and Cox-2 in ISIAH rats was much lower than in WAG rats. Water deprivation for 11 h was followed by a 4-fold increase in Cox-2 gene expression in ISIAH rats. The increase in gene expression was insignificant in WAG rats (by 30%). Renin gene expression in renal tissue of ISIAH and WAG rats remained practically unchanged after water deprivation. We conclude that a change in Cox-2 gene expression after short-term water deprivation serves as a reliable criterion for functional strain of the renal renin system in hypertensive ISIAH rats.

Gene expression for the renin system in the myocardium of hypertensive ISIAH rats.

The concentration of mRNA for angiotensin-converting enzyme (ACE) and angiotensin type 1A receptor (AT1A) in the myocardium of hypertensive ISIAH rats and normotensive WAG rats was measured by real-time PCR. Gene expression of the angiotensin type 1A receptor in the myocardium of 4-month-old ISIAH rats was lower than in WAG rats. The content of mRNA for angiotensin-converting enzyme in the myocardium of adult ISIAH rats was elevated by 80%. Therefore, the development of myocardial hypertrophy anticipates the increase in enzyme expression in the myocardium. Water deprivation (17 h) was accompanied by a decrease in the concentration of mRNA for angiotensin-converting enzyme in the myocardium of ISIAH rats, which did not differ from that in normotensive animals. Our results suggest that the decrease in cardiac preload and increase in plasma renin activity during dehydration reduce requirements for hyperactivity of the local cardiac renin-angiotensin system.

Changes in physicochemical parameters and alpha-crystallin expression in the lens during cataract development in OXYS rats. 5.

The pathogenesis of cataract is associated with oxidative stress and with altered crystallin expression but it is still understood incompletely. In this study, the senescence-accelerated OXYS rats were used as a model. The first biomicroscopic signs of cataract in OXYS rats were registered at the age of 1.5 months; at 3 months morbidity reached 90%, and at 6 months it reached 100%. Cataract manifestation progresses: at 24 months mature cataract was detected in 90% of eyes of OXYS rats, whereas in 80% of Wistar rat eyes only initial signs of this disease were detected. Analysis of lens redox-parameters has shown that in OXYS rats the intensity of tryptophan fluorescence is higher, the GSH content being higher at 2 months but during formation of mature cataract at 13, 18, and 24 months being lower than in Wistar rats. Decrease in solubility of OXYS rat lens proteins was observed at the age of 13 months. At the age of 3 months gene expression of alphaA-crystallin and alphaB-crystallin was 3-fold and 25% lower, respectively, than in Wistar rats. At the age of 14 months there was a 27-fold decrease in expression of alphaB-crystallin in OXYS rats and it became 21-fold lower than in control. Proteins are synthesized in lens epithelial cells and dystrophic changes in senile cataract result in decrease in structural protein expression. The changes observed in OXYS rats are evidently associated with the dystrophic changes in lens epithelium, which we have described earlier, and are consistent with the model of senile cataract.

Neuroendocrine profiling in inherited stress-induced arterial hypertension rat strain with stress-sensitive arterial hypertension.

The functions of the hypothalamic adrenal cortical and sympathetic adrenal medullary systems were studied in rats with inherited stress-induced arterial hypertension (ISIAH strain). A characteristic feature of the ISIAH strain is an increase in arterial blood pressure measured both under basal conditions and after restraint stress in particular. In the control ISIAH rats, the basal plasma ACTH concentration was slightly lower than that in the normotensive Wistar albino Glaxo (WAG) rats, and no differences were found in plasma corticosterone. However, the 0.5-h restraint stress produced higher activation of the adrenal cortex in the ISIAH rats. Gluco- and mineralocorticoid responses to the blood volume reduction stresses and ACTH and corticosterone responses to social stress were stronger in the ISIAH than in the control WAG rats. An increase in epinephrine content in adrenals in the basal state and enhanced response of the sympathetic adrenal medullary system to handling stress were observed in the ISIAH rats. Restraint stress produced significantly higher expression of genes encoding corticotropin-releasing hormone-mRNA in hypothalamus and proopiomelanocortin-mRNA in pituitary in the ISIAH than in the WAG rats. Restraint stress produced a decrease in glucocorticoid receptor (GR) gene expression (GR-mRNA) in hippocampus in the ISIAH, but not in the WAG rats. A persistent increase in tyrosine hydroxylase-mRNA in adrenals of the ISIAH rats was found. It is concluded that the ISIAH rat strain is an appropriate model of stress-sensitive hypertension with the predominant involvement of the hypothalamic adrenal cortical and sympathetic adrenal medullary systems in its pathogenesis.

[Characteristics of glucocorticoid receptor gene expression in spontaneously hypertensive rat strain]. / Osobennosti ékspressii gena gliukokortikoidnogo retseptora u gipertenzivnykh krys linii NISAG.

In ISIAH rat strain with stress-sensitive form of hypertension, the expression level of glucocorticoid receptor (GR) gene has been evaluated in hippocampus, hypothalamus and pituitary under basal and 2-hr restraint stress conditions. Corticosterone (CS) level in peripheral blood was also evaluated. Normotensive WAG strain was used as a control. Under basal condition, there were no interstrain differences in GR-mRNA level in any brain region under study. However, under stress condition, ISIAH rats demonstrated a significant fall of GR-mRNA in hippocampus and increase the pituitary gland as compared to basal level. On the contrary, no differences with basal level were found in stressed WAG rats. CS concentration in blood was nearly the same in nonstressed WAG and ISIAH rats. Stress influence led to a marked increase of CS in both strains. However CS level was significantly higher in stressed ISIAH rats than in stressed WAG group.