Morphometric analysis of the phrenic nerve in male and female Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)

Ventilatory differences between rat strains and genders have been described but the morphology of the phrenic nerve has not been investigated in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. A descriptive and morphometric study of the phrenic nerves of male (N = 8) and female (N = 9) SHR, and male (N = 5) and female (N = 6) WKY is presented. After arterial pressure and heart rate recordings, the phrenic nerves of 20-week-old animals were prepared for epoxy resin embedding and light microscopy. Morphometric analysis performed with the aid of computer software that took into consideration the fascicle area and diameter, as well as myelinated fiber profile and Schwann cell nucleus number per area. Phrenic nerves were generally larger in males than in females on both strains but larger in WKY compared to SHR for both genders. Myelinated fiber numbers (male SHR = 228 ± 13; female SHR = 258 ± 4; male WKY = 382 ± 23; female WKY = 442 ± 11 for proximal right segments) and density (N/mm²; male SHR = 7048 ± 537; female SHR = 10355 ± 359; male WKY = 9457 ± 1437; female WKY = 14351 ± 1448) for proximal right segments) were significantly larger in females of both groups and remarkably larger in WKY than SHR for both genders. Strain and gender differences in phrenic nerve myelinated fiber number are described for the first time in this experimental model of hypertension, indicating the need for thorough functional studies of this nerve in male and female SHR.

Transcriptome analysis of nicotine-exposed cells from the brainstem of neonate spontaneously hypertensive and Wistar Kyoto rats

In this study, the effects of nicotine on global gene expression of cultured cells from the brainstem of spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY) rats were evaluated using whole-genome oligoarrays. We found that nicotine may act differentially on the gene expression profiles of SHR and WKY. The influence of strain was present in 321 genes that were differentially expressed in SHR as compared with WKY brainstem cells independently of the nicotine treatment. A total of 146 genes had their expression altered in both strains after nicotine exposure. Interaction between nicotine treatment and the strain was observed to affect the expression of 229 genes that participate in cellular pathways related to neurotransmitter secretion, intracellular trafficking and cell communication, and are possibly involved in the phenotypic differentiation between SHR and WKY rats, including hypertension. Further characterization of their function in hypertension development is warranted. The Pharmacogenomics Journal (2010) 10, 134-160; doi:10.1038/tpj.2009.42; published online 15 September 2009

Gene Expression Profiling of Cultured Cells From Brainstem of Newborn Spontaneously Hypertensive and Wistar Kyoto Rats

The spontaneously hypertensive rat (SHR) is a good model to study several diseases such as the attention-deficit hyperactivity disorder, cardiopulmonary impairment, nephropathy, as well as hypertension, which is a multifactor disease that possibly involves alterations in gene expression in hypertensive relative to normotensive subjects. In this study, we used high-density oligoarrays to compare gene expression profiles in cultured neurons and glia from brainstem of newborn normotensive Wistar Kyoto (WKY) and SHR rats. We found 376 genes differentially expressed between SHR and WKY brainstem cells that preferentially map to 17 metabolic/signaling pathways. Some of the pathways and regulated genes identified herein are obviously related to cardiovascular regulation; in addition there are several genes differentially expressed in SHR not yet associated to hypertension, which may be attributed to other differences between SHR and WKY strains. This constitute a rich resource for the identification and characterization of novel genes associated to phenotypic differences observed in SHR relative to WKY, including hypertension. In conclusion, this study describes for the first time the gene profiling pattern of brainstem cells from SHR and WKY rats, which opens up new possibilities and strategies of investigation and possible therapeutics to hypertension, as well as for the understanding of the brain contribution to phenotypic differences between SHR and WKY rats.

Comparative mechanical study of isolated papillary muscle from Wistar-Kyoto and Wistar rats

Isolated papillary muscles have often been used in myocardial mechanical function studies. The objective of the present study was to compare the mechanical function of papillary muscle isolated from left ventricle between Wistar (W) and Wistar-Kyoto (WKY) rats of different ages (1, 3, 6 and 12 months), in order to examine whether there is a difference in intrinsic mechanical properties of muscle between the two rat strains. Muscles were perfused with Krebs-Henseleit solution at 28°C and studied isometrically and isotonically at a stimulation rate of 0.2 Hz. The W and WKY showed statistically significant differences during both isometric and isotonic contractions. During isometric contraction? (l) the peak developed tension (DT) and + dT/dt were lower in WKY rats in the 1 mo groups, (2) the resting tension (RT) was greater in WKY at 3, 6 and 12 mo. (3) time to peak tension (TPT) was greater in WKY at 3 and 12 mo, (4) time for tension to fall from peak to 50% of peak tension (RT 1/2) was greater in WKY at 3 mo and (5) - dT/dt was lower in WKY at 1 and 3 mo. During isotonic contraction, (1) the peak shortening (PS) and -dL/dt were lower in WKY at 12 mo, (2) the time to peak shortening (TPS) was greater in WKY at 3 and 12 mo; (3) + dL/dt was lower in WKY at 3, 6, and 12 mo and (4) the relative variation of length (Lmax-PS)/Lmax was greater in WKY at 6 and 12 mo. These data showed a difference in mechanical behaviour of the papillary muscle between Wistar and Wistar-Kyoto rats of different age.

Sural nerve involvement in experimental hypertension: morphology and morphometry in male and female normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)

Background: The sural nerve has been widely investigated in experimental models of neuropathies but information about its involvement in hypertension was not yet explored. The aim of the present study was to compare the morphological and morphometric aspects of different segments of the sural nerve in male and female spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Rats aged 20 weeks (N = 6 in each group) were investigated. After arterial pressure and heart rate recordings in anesthetized animals, right and left sural nerves were removed and prepared for epoxy resin embedding and light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter. Results: Significant differences were observed for the myelinated fiber number and density, comparing different genders of WKY and SHR. Also, significant differences for the morphological (thickening of the endoneural blood vessel walls and lumen reduction) and morphometric (myelinated fibers diameter and G ratio) parameters of myelinated fibers were identified. Morphological exam of the myelinated fibers suggested the presence of a neuropathy due to hypertension in both SHR genders. Conclusions: These results indicate that hypertension altered important morphometric parameters related to nerve conduction of sural nerve in hypertensive animals. Moreover the comparison between males and females of WKY and SHR allows the conclusion that the morphological and morphometric parameters of sural nerve are not gender related. The morphometric approach confirmed the presence of neuropathy, mainly associated to the small myelinated fibers. In conclusion, the present study collected evidences that the high blood pressure in SHR is affecting the sural nerve myelinated fibers.

Intracellular free Ca2+ and basal Mn2+ influx in cultured aortic smooth muscle cells from spontaneously hypertensive and normotensive Wistar-Kyoto rats.

Numerous studies investigating the possible role of altered Ca2+ homeostasis in hypertension have compared resting and agonist-stimulated intracellular free Ca2+ ([Ca2+](i)) in cultured aortic smooth muscle cells from spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. However, such studies have not given consistent results. Differences in the method used to load cells with the Ca2+-sensitive indicator fura-2 have been investigated here as a possible source of variability between studies. We also describe the adaptation of a fluorescence technique for the assessment of basal Ca2+ permeability in SHR and WKY through the measurement of Mn2+ influx. The results are consistent with the hypothesis that basal Ca2+ influx is elevated in cultured aortic smooth muscle cells from SHR compared to those from WKY. However, this was not reflected as a significant difference between the two strains in basal or angiotensin II (200 nmol/L)stimulated [Ca2+](i). Furthermore, this result was not dependent on the protocol used to load cells with fura-2. Hence, measurement of bulk [Ca2+](i) does not appear to be the most sensitive parameter for altered Ca2+ homeostasis in SHR. Other compartments of the cell may better reflect altered Ca2+ fluxes in hypertension and are discussed in this work.

Functional characterization of the hypothalamic-pituitary-adrenal axis of the Wistar Audiogenic Rat (WAR) strain

The Wistar Audiogenic Rat (WAR) strain is a genetic model of sound-induced reflex epilepsy which was selected starting from audiogenic seizures susceptible Wistar rats. Wistar resistant rats were used as WAR`s control in this study. In the acute situation, audiogenic seizures (AS) in WARs mimic tonic-clonic seizures and, in the chronic protocol, mimic temporal lobe epilepsy. AS have been shown to evoke neuroendocrine responses; however, the hypothalamic-pituitary-adrenal activity in the WAR has not been established. The aim of this study was to evaluate the hypothalamic-pituitary-adrenal axis (HPA) responses to exogenous ACTH stimulation (8 ng/rat), fifteen minute restraint stress and circadian variation (8 am and 8 pm) under rest conditions in these animals through plasma measurements of ACTH and corticosterone concentrations. We also measured the body weight from birth to the 9th week of life and determined adrenal gland weight. We found that WARs are smaller than Wistar and presented a higher adrenal gland weight with a higher level of corticosterone release after intravenous ACTH injection. They also showed altered HPA axis circadian rhythms and responses to restraint stress. Our data indicate that, despite the lower body weight, WARs have increased adrenal gland weight associated with enhanced pituitary and adrenal responsiveness after HPA axis stimulation. Thus, we propose WARs as a model to study stress-epilepsy interactions and epilepsy-neuropsychiatry comorbidities. (C) 2011 Elsevier B.V. All rights reserved.

Sexual differentiation of cortical spreading depression propagation after acute and kindled audiogenic seizures in the Wistar audiogenic rat (WAR)

Brain excitability diseases like epilepsy constitute one factor that influences brain electrophysiological features. Cortical spreading depression (CSD) is a phenomenon that can be altered by changes in brain excitability. CSD propagation was presently characterized in adult mate and female rats from a normal Wistar strain and from a genetically audiogenic seizure-prone strain, the Wistar audiogenic rat (WAR), both previously submitted (RAS(+)), or not (RAS(-)), to repetitive acoustic stimulation, to provoke audiogenic kindling in the WAR-strain. A gender-specific change in CSD-propagation was found. Compared to seizure-resistant animals, in the RAS- condition, mate and female WARs, respectively, presented CSD-propagation impairment and facilitation, characterized, respectively, by lower and higher propagation velocities (P<0.05). In contraposition, in the RAS(+) condition, mate and female WARs displayed, respectively, higher and tower CSD-propagation rates, as compared to the corresponding controls. In some Wistar and WAR females, we determined estrous cycle status on the day of the CSD-recording as being either estrous or diestrous; no cycle-phase-related differences in CSD-propagation velocities were detected. In contrast to other epilepsy models, such as Status Epilepticus induced by pilocarpine, despite the CSD-velocity reduction, in no case was CSD propagation blocked in WARs. The results suggest a gender-related, estrous cycle-phase-independent modification in the CSD-susceptibility of WAR rats, both in the RAS(+) and RAS(-) situation. (C) 2008 Elsevier B.V. All rights reserved.

Intra-strain variations of baroreflex sensitivity in young Wistar-Kyoto rats

Purpose: To compare baroreflex sensitivity among conscious rats of the same strain. Methods: Male WKY rats (eight weeks old) were studied. Cannulas were inserted into the abdominal aortic artery through the right femoral artery to measure mean arterial pressure (MAP) and heart rate (HR). Baroreflex gain was calculated as the ratio between variation of HR in function of the MAP variation (Delta HR/Delta MAP) tested with a depressor dose of sodium nitroprusside (SNP, 50 mu g/kg, iv) and with a pressor dose of phenylephrine (PE, 8 mu g/kg, iv). We divided the rats into four groups: 1) Low bradycardic baroreflex (LB), BG between -1 and -2 bpm/mmHg tested with PE; 2) High bradycardic baroreflex (HB), BG < -2 bpm/mmHg tested with PE; 3) Low tachycardic baroreflex (LT), BG between -1 and -2 bpm/mmHg tested with SNP and; 4) High tachycardic baroreflex (HT), BG < -2 bpm/mmHg tested with SNP. Significant differences were considered for p<0.05. Results: Approximately 82% of the rats presented reduced bradycardic reflex while 22 showed attenuated tachycardic reflex. No alterations were noted regarding basal MAP and HR, tachycardic and bradycardic peak and HR range. Conclusions: There was alteration in baroreflex sensitivity among rats of the same strain. Care should be taken when interpreting studies employing WKY as a control for the SHR.

Adenosine receptor type 2a is differently modulated by nicotine in dorsal brainstem cells of Wistar Kyoto and spontaneously hypertensive rats

Hypertension can result from neuronal network imbalance in areas of central nervous system that control blood pressure, such as the nucleus tractus solitarius (NTS). There are several neurotransmitters and neuromodulatory substances within the NTS, such as adenosine, which acts on purinoreceptors A(2a) (A(2a)R). The A(2a)R modulates neurotransmission in the NTS where its activation may induce decrease in blood pressure by different mechanisms. Nicotine is a molecule that crosses the hematoencephalic barrier and acts in several areas of central nervous system including the NTS, where it may interact with some neurotransmitter systems and contributes to the development of hypertension in subjects with genetic predisposition to this disease. In this study we first determined A(2a)R binding, protein, and mRNA expression in dorsomedial medulla oblongata of neonate normotensive (WKY) and spontaneously hypertensive rats (SHR). Subsequently, we analyzed the modulatory effects of nicotine on A(2a)R in cell culture in order to evaluate its possible involvement in the development of hypertension. Data showed a decreased A(2a)R binding and increased protein and mRNA expression in tissue sample and culture of dorsal brainstem from SHR compared with those from WKY rats at basal conditions. Moreover, nicotine modulated A(2a)R binding, protein, and mRNA expression in cells from both strains. Interestingly, nicotine decreased A(2a)R binding and increased protein levels, as well as, induced a differential modulation in A(2a)R mRNA expression. Results give us a clue about the mechanisms involved in the modulatory effects of nicotine on A(2a)R as well as hypothesize its possible contribution to the development of hypertension. In conclusion, we demonstrated that A(2a)R of SHR cells which differ from WKY and nicotine differentially modulates A(2a)R in dorsal brainstem cells of SHR and WKY.

The non-coding RNA BC1 is down-regulated in the hippocampus of Wistar Audiogenic Rat (WAR) strain after audiogenic kindling

The aim of this study was to identify molecular pathways involved in audiogenic seizures in the epilepsy-prone Wistar Audiogenic Rat (WAR). For this, we used a suppression-subtractive hybridization (SSH) library from the hippocampus of WARs coupled to microarray comparative gene expression analysis, followed by Northern blot validation of individual genes. We discovered that the levels of the non-protein coding (npc) RNA BC1 were significantly reduced in the hippocampus of WARs submitted to repeated audiogenic seizures (audiogenic kindling) when compared to Wistar resistant rats and to both naive WARs and Wistars. By quantitative in situ hybridization, we verified lower levels of BC1 RNA in the GD-hilus and significant signal ratio reduction in the stratum radiatum and stratum pyramidale of hippocampal CA3 subfield of audiogenic kindled animals. Functional results recently obtained in a BC1-/- mouse model and our current data are supportive of a potential disruption in signaling pathways, upstream of BC1, associated with the seizure susceptibility of WARs. (C) 2010 Elsevier B.V. All rights reserved.

Increased Expression of GluR2-flip in the Hippocampus of the Wistar Audiogenic Rat Strain After Acute and Kindled Seizures

The Wistar Audiogenic Rat (WAR) is an epileptic-prone strain developed by genetic selection from a Wistar progenitor based on the pattern of behavioral response to sound stimulation. Chronic acoustic stimulation protocols of WARs (audiogenic kindling) generate limbic epileptogenesis, confirmed by ictal semiology, amygdale, and hippocampal EEG, accompanied by hippocampal and amygdala cell loss, as well as neurogenesis in the dentate gyrus (DG). In an effort to identify genes involved in molecular mechanisms underlying epileptic process, we used suppression-subtractive hybridization to construct normalized cDNA library enriched for transcripts expressed in the hippocampus of WARs. The most represented gene among the 133 clones sequenced was the ionotropic glutamate receptor subunit II (GluR2), a member of the a-amino-3-hydroxy-5-methyl-4-isoxazoleopropionic acid (AMPA) receptor. Although semiquantitative RT-PCR analysis shows that the hippocampal levels of the GluR2 subunits do not differ between naive WARs and their Wistar counterparts, we observed that the expression of the transcript encoding the splice-variant GluR2-flip is increased in the hippocampus of WARs submitted to both acute and kindled audiogenic seizures. Moreover, using in situ hybridization, we verified upregulation of GluR2-flip mainly in the CA1 region, among the hippocampal subfields of audiogenic kindled WARs. Our findings on differential upregulation of GluR2-flip isoform in the hippocampus of WARs displaying audiogenic seizures is original and agree with and extend previous immunohistochemical for GluR2 data obtained in the Chinese P77PMC audiogenic rat strain, reinforcing the association of limbic AMPA alterations with epileptic seizures. (C) 2009 Wiley-Liss, Inc.

Behavioral and EEG effects of GABAergic manipulation of the nigro-tectal pathway in the Wistar audiogenic rat (WAR) strain II: An EEG wavelet analysis and retrograde neuronal tracer approach

The role of the substantia nigra pars reticulata (SNPr) and superior colliculus (SC) network in rat strains susceptible to audiogenic seizures still remain underexplored in epileptology. In a previous study from our laboratory, the GABAergic drugs bicuculline (BIC) and muscimol (MUS) were microinjected into the deep layers of either the anterior SC (aSC) or the posterior SC (pSC) in animals of the Wistar audiogenic rat (WAR) strain submitted to acoustic stimulation, in which simultaneous electroencephalographic (EEG) recording of the aSC, pSC, SNPr and striatum was performed. Only MUS microinjected into the pSC blocked audiogenic seizures. In the present study, we expanded upon these previous results using the retrograde tracer Fluorogold (FG) microinjected into the aSC and pSC in conjunction with quantitative EEG analysis (wavelet transform), in the search for mechanisms associated with the susceptibility of this inbred strain to acoustic stimulation. Our hypothesis was that the WAR strain would have different connectivity between specific subareas of the superior colliculus and the SNPr when compared with resistant Wistar animals and that these connections would lead to altered behavior of this network during audiogenic seizures. Wavelet analysis showed that the only treatment with an anticonvulsant effect was MUS microinjected into the pSC region, and this treatment induced a sustained oscillation in the theta band only in the SNPr and in the pSC. These data suggest that in WAR animals, there are at least two subcortical loops and that the one involved in audiogenic seizure susceptibility appears to be the pSC-SNPr circuit. We also found that WARs presented an increase in the number of FG + projections from the posterior SNPr to both the aSC and pSC (primarily to the pSC), with both acting as proconvulsant nuclei when compared with Wistar rats. We concluded that these two different subcortical loops within the basal ganglia are probably a consequence of the WAR genetic background. (C) 2012 Elsevier Inc. All rights reserved.

Cosegregation analysis of natriuretic peptide genes and blood pressure in the spontaneously hypertensive rat

1. The natriuretic peptide precursor A (Nppa) and B (Nppb) genes are candidate genes for hypertension and cardiac hypertrophy in the spontaneously hypertensive rat (SHR). The purpose of the present study was to determine the role of the Nppa and Nppb genes in the development of hypertension in the SHR. 2. A cohort (n = 162) of F2 segregating intercross animals was established between strains of hypertensive SHR and normotensive Wistar-Kyoto rats. Blood pressure and heart weight were measured in each rat at 12-16 weeks of age. Rats were genotyped using 11 informative microsatellite markers, distributed in the vicinity of the Nppa marker on rat chromosome 5 including an Nppb marker. The phenotype values were compared with genotype using the computer package MAP-MAKER 3.0 (Whitehead Institute, Boston, MA, USA) to determine whether there was a link between the genetic variants of the natriuretic peptide family and blood pressure or cardiac hypertrophy. 3. A strong correlation was observed between the Nppa marker and blood pressure. A quantitative trait locus (QTL) for blood pressure on chromosome 5 was identified between the Nppa locus and the D5Mgh15 marker, less than 2 cM from the Nppa locus. The linkage score for the blood pressure QTL on chromosome 5 was 3.8 and the QTL accounted for 43% of the total variance of systolic blood pressure, 54% of diastolic blood pressure and 59% of mean blood pressure. No association was found between the Nppb gene and blood pressure. This is the first report of linkage between the Nppa marker and blood pressure in the rat. There was no correlation between the Nppa or Nppb genes or other markers in this region and either heart weight or left ventricular weight in F2 rats. 4. These findings suggest the existence of a blood pressure-dependent Nppa marker variant or a gene close to Nppa predisposing to spontaneous hypertension in the rat. It provides a strong foundation for further detailed genetic studies in congenic strains, which may help to narrow down the location of this gene and lead to positional cloning.

Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the developing male Wistar(Han) rat I: no decrease in epididymal sperm count after a single acute dose

It has been reported that fetal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes defects in the male reproductive system of the rat. We set out to replicate and extend these effects using a robust experimental design. Groups of 75 (control vehicle) or 55 (50, 200 or 1000 ng of TCDD kg-1 bodyweight) female Wistar(Han) rats were exposed to TCDD on Gestational Day (GD) 15, then allowed to litter. The high dose group dams showed no sustained weight loss compared to control, but four animals had total litter loss. Pups in the high dose group showed reduced body weight up till day 21, and pups in the medium dose group showed reduced body weight in the first week post partum. Balano-preputial separation (BPS) was significantly delayed in the high dose group male offspring. There were no significant effects of treatment when the offspring were subjected to a functional observational battery, or mated with females to assess reproductive capability. 25 males per group were killed on post natal day (PND) 70, and ~60 animals per group (~30 for the high dose group) on PND120 to assess seminology and other endpoints. At PND120, the two highest dose groups showed a statistically significant elevation of sperm counts, compared to control; however, this effect was small (~30%), within the normal range of sperm counts for this strain of rat, was not reflected in testicular spermatid counts nor PND70 data, and is therefore postulated to have no biological significance. Although there was an increase in the proportion of abnormal sperm at PND70, seminology parameters were otherwise unremarkable. Testis weights in the high dose group were slightly decreased at PND 70 and 120, and at PND120, brain weights were decreased in the high dose group, liver to body weight ratios were increased for all three dose groups, with an increase in inflammatory cell foci in the epididymis in the high dose group. These data show that TCDD is a potent developmental toxin after exposure of the developing fetus, but that acute developmental exposure to TCDD on GD15 caused no decrease in sperm counts.