Wistar-Kyoto Counterparts Research Articles

Three levels of enhanced inspired CO2 or fasted-state on stress response activation in behaviorally inhibited humans and rats

Behavioral inhibition (BI) is believed to be a genetically determined trait that affects roughly 33% of the human population. These individuals tend to respond to stressful situations differently than their non-behaviorally inhibited (NBI) counterparts. In the rodent population, a similar behavioral/stress model exists. The Sprague-Dawley (SD) strain often acts as a “control” among rat strains and can be considered to be the rat equivalent to NBI individuals, and the Wistar-Kyoto (WKY) strain is the rodent equivalent to BI individuals. The differences in the activation of the sympathetic nervous system between chronically stressed and non-stressed populations remain unknown. Our current study tested the hypothesis BI individuals and WKY rats will have increased stress responses. In humans, we tested physiological responses to increasing levels of carbon dioxide (CO2) through analyses of breathing, heart rate, and blood pressure. The Adult Measure of Behavioral Inhibition (AMBI) survey was used to determine the presence of BI. A 15-minute control period baseline was completed followed by an acquisition period of five minutes of each 3%, 5%, and 7% CO2, respectively. Preliminary results were analyzed through a CO2 sensitivity slope (inspired minute ventilation ((L/min) / end-tidal CO2) and show that BI individuals had increased, exaggerated responses to the respiratory stressors across the three levels of enhanced CO2. In the rodent model, signaled lever-press avoidance was used to examine the activation of stress responses. Rats were trained to avoid mild foot shock using a tone warning signal. As demonstrated previously, WKY rats acquired a higher level of avoidance responses. Rats were then food-deprived overnight and subsequently retested on signal lever-press avoidance. Food deprivation did not affect the high rate of avoidance seen in WKY rats; however, food deprivation increased the SD rate of avoidance to nearly the level of WKY rats. Our data suggest that WKY rats do not respond to food deprivation when paired with lever-press avoidance; however, SD rats do. Simply, the addition of a stressor causes SD rats to behave more similarly to their WKY counterparts. This is the full abstract presented at the American Physiology Summit 2023 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.

Short-term effect of ligature-induced periodontitis on cardiovascular variability and inflammatory response in spontaneously hypertensive rats

BackgroundWe previously reported that periodontal disease (PD) induces high arterial pressure variability (APV) consistent with sympathetic overactivity and elicits myocardial inflammation in Balb/c mice. However, it is unknown whether PD can change APV and heart rate variability (HRV) in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. This study aimed to evaluate the hemodynamic level, HRV, and APV associating with myocardial inflammation and plasma concentrations of oxide nitric (NO) in SHR and WKY rats with PD.MethodsThree weeks after bilateral ligation of the first mandibular molar, or Sham operation, the rats received catheters into the femoral artery and had their arterial pressure (AP) recorded the following day. Subsequently, plasma, heart, and jaw were collected. The NO was quantified by the chemiluminescence method in plasma, and the myocardial IL-1β concentrations were evaluated by ELISA. In the jaw was evaluated linear alveolar bone loss induced by PD.ResultsThe linear alveolar bone loss in jaws of SHR with PD was higher than in all other groups. AP and heart rate were higher in SHR than in their WKY counterparts. SHR with PD showed lower AP than control SHR. HRV and APV were different between SHR and WKY rats; however, no differences in these parameters were found between the animals with PD and their control counterparts. Plasma NO and myocardial IL-1β concentrations were higher in SHR with PD as compared to control WKY. A significant correlation was found between linear alveolar bone loss and plasma NO and myocardial IL-1β concentrations.ConclusionOur results demonstrated that short-term PD lowered the AP in SHR, which might be due to the higher levels of plasma NO. Even though PD did not affect either HRV or APV, it did induce myocardial inflammation, which can determine cardiovascular dysfunction in long-term PD.

Evaluation of Heart Rate Fragmentation in Spontaneously Hypertensive Rats

Rapid heart rate (HR) variations at respiratory frequency range are well accepted as a marker of cardiac vagal modulation. Nevertheless, a new pattern of ultra-rapid HR variation has been recently studied and named as heart rate fragmentation (HRF). HRF is a type of sinoatrial instability characterized by the presence of numerous inflections points (from one bit to another) in RR interval time series. HRF has been shown to be increased in aging and coronary diseases and seems to be linked to the risk life-threatening cardiovascular events and death. This study aimed to evaluate the HRF in a rat model of spontaneous hypertension. Unanesthetized freely moving spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats at 2 distinct ages, 4 to 6 weeks (young) or 12 to 15 weeks (adult), had their electrocardiogram recorded for 40 minutes. Series of RR intervals were generated and transformed into a sequence of symbols “-1”, “0” or “1” when the difference between successive RR values (transitions) was positive, zero, or negative, respectively. Transitions between symbols “-1” and “1” were labeled as “hard” (H) inflection points while those between “-1” (or “1”) and “0” were labeled as “soft” (S) inflection points. Next, sequences of 4 consecutive symbols (words - W) were distributed according to the number and type of transition they enclose. The total percentage of inflection points (PIP), the percentage of words with zero (W0), one (W1), two (W2) or three (W3) inflection points, as well as percentage of words enclosing only hard (WH), soft (WS) or mixed (WM), i.e., containing both types of inflection points, were quantified. PIP and W3 were lower in the young and adult SHR when compared to the counterparts WKY. In contrast, young and adult SHR showed a higher percentage of W0, W1 and W2 compared to WKY.Regarding the type of transition, the percentage of WH was lower, while the WS was higher, in young and adult SHR when compared to WKY counterparts. The percentage of WM was higher only in young SHR. In conclusion, although evidences indicate that cardiovascular diseases lead to an increase in HRF, we have shown lower HRF in hypertensive rats, with or without the presence of the hypertensive phenotype. However, the mechanisms giving rise to HRF are still unknown. Future studies must be conducted to evaluate whether the HRF pattern observed in young and adult SHR is maintained in elderly animals and to elucidate the mechanisms behind HRF in SHR and normotensive rats.

The antihypertensive agent hydralazine reduced extracellular matrix synthesis and liver fibrosis in nonalcoholic steatohepatitis exacerbated by hypertension.

Hypertension is an important risk factor for nonalcoholic steatohepatitis. We have previously demonstrated that hypertensive rats fed a high fat and cholesterol (HFC) diet incurred a more severe hepatic inflammatory response and fibrosis. Here we investigated the role of hypertension in NASH by comparing HFC-induced hepatic fibrogenesis between spontaneously hypertensive rats (SHRs) and their normotensive Wistar Kyoto counterpart. Compared to the counterpart, the HFC diet led to stronger aggregation of CD68-positive macrophages in SHRs. HFC feeding also resulted in significantly higher upregulation of the fibrosis-related gene alpha-smooth muscle actin in SHR. The HFC diet induced higher overexpression of serum tissue inhibitor of metalloproteinase-1 (TIMP1) and greater suppression of matrix metalloproteinase-2 (MMP2):TIMP1, MMP8:TIMP1, and MMP9:TIMP1 ratios, as a proxy of the activities of these MMPs in SHR. Administration of the antihypertensive agent hydralazine to SHRs significantly ameliorated HFC-induced liver fibrosis; it suppressed the aggregation of CD68-positive macrophages and the upregulation of platelet-derived growth factor receptor beta, and collagen, type 1, alpha-1 chain. In conclusion, a hypertensive environment exacerbated the hepatic fibrogenetic effects of the HFC diet; while the effects were partially reversed by the antihypertensive agent hydralazine. Our data suggest that antihypertensive drugs hold promise for treating NASH exacerbated by hypertension.

Na+/H+ Exchanger NHE3 is differentially regulated in renal proximal tubules of pre‐hypertensive and hypertensive spontaneously hypertensive rats

The present work aimed to measure NHE3 in vivo transport activity and to define the mechanisms underlying NHE3 regulation in young pre‐hypertensive (5‐week‐old, systolic blood pressure (SBP) = 92 ± 2 mmHg) and adult hypertensive spontaneously hypertensive rats (SHR) (14‐week‐old, SBP = 187 ± 4 mmHg). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo microperfusion. NHE3‐mediated bicarbonate reabsorption was stimulated in 5‐week‐old SHR (44 ± 5%, P<0.01) and reduced in 14‐week‐old SHR (57 ± 4%, P<0.001) compared to Wistar Kyoto (WKY) rats at corresponding age. Stimulated NHE3 transport activity in young pre‐hypertensive SHR was associated with enhanced NHE3 microvillus expression, lower levels of phosphorylation at Ser552 and increased association with the enzyme dipeptidyl peptidase IV (DPPIV). Conversely, adult hypertensive SHR showed decreased NHE3 microvillus membrane content, higher relative ratio of phosphorylated Ser552, and lower amount of the NHE3‐DPPIV complex. No differences on renal cortical NHE3 protein content and mRNA levels were found between SHR and its WKY counterpart. We conclude that NHE3 is differentially regulated before and after development of hypertension in SHR by mechanisms that involve post‐translational modifications, subcellular redistribution and assembly with interacting proteins. Supported by FAPESP and CNPq

Enhanced survival of vascular smooth muscle cells accounts for heightened elastin deposition in arteries of neonatal spontaneously hypertensive rats

Abnormal stiffening and narrowing of arteries are characteristic features of spontaneously hypertensive rats (SHR). In this strain, we have previously demonstrated an increased elastin content and abnormal organization of lamellae in conduit and resistance arteries from neonatal rats that preceded the impending inward remodelling, increased vascular stiffness and development of hypertension. The aim of this study was to assess the mechanism responsible for such excessive and aberrant elastin deposition in SHR vessels during perinatal development. We compared elastin, collagen and fibronectin production (inmunocytochemistry and quantitative assay of metabolically labelled insoluble elastin), DNA content as well as cell proliferation (proliferative cellular nuclear antigen, bromodeoxyuridine incorporation) and death rates (propidium iodide exclusion test, terminal transferase nick and labeling (TUNEL) assay) in cultures of vascular smooth muscle cells (VSMC) derived from neonatal SHR and Wistar-Kyoto (WKY) control rats. Cultures of VSMC derived from neonatal SHR exhibited hypertrophy, produced more elastin, collagen and fibronectin and contained more DNA than equally plated WKY counterparts. Further analysis revealed that the higher net DNA content in SHR-derived cultures was due to increased diploidy, but not to a heightened cell multiplication. The SHR-derived VSMC also exhibited lower rates of cell death and apoptosis, which were associated with increased levels of the anti-apoptotic protein, survivin. We therefore conclude that the peculiar heightened survival of matrix-producing VSMC in neonatal SHR is responsible for accumulation of hard-wearing elastin and other extracellular matrix elements in the growing arteries, thereby contributing to the subsequent development of systemic hypertension.

Angiotension II receptor 1 blocker modifies the expression of apoptosis‐related proteins and transforming growth factor‐β1 in prostate tissue of spontaneously hypertensive rats

To assess whether angiotensin II (Ang II), important in hypertension and highly expressed in benign prostatic hyperplasia (BPH), is involved in prostate growth, by analysing changes in the histological composition, tissue apoptotic status and level of transforming growth factor-beta1 (TGFbeta1) induced by an Ang II type 1 receptor blocker, losartan, in the prostates of spontaneously hypertensive (SH) rats. We assessed four groups of six rats each: normotensive Wistar-Kyoto counterparts of SH rats; untreated SH rats; SH rats given low-dose losartan (10 mg/kg/day for 10 weeks); and SH given high-dose losartan (30 mg/kg/day for 10 weeks). We evaluated the histological composition and expression of TGFbeta1 and apoptosis-related proteins, i.e. Bax and the 116-kDa poly (adenosine diphosphate-ribose) polymerase (PARP), by Western blotting in the rat prostate ventral lobes. Compared with Wistar-Kyoto rats, untreated SH rats had a significantly increased epithelium component in the prostate (P < 0.01), but with losartan treatment, SH rats showed less of the epithelium component than untreated rats (P < 0.01 for both low- and high-dose losartan). Western-blot analysis showed a significantly increased level of Bax in high-dose losartan-treated rats (P < 0.01). The expression of 116 kDa PARP was also decreased in these rats (P < 0.01), which suggests increased caspase-3 activity. In addition, TGFbeta1 levels were significantly elevated in high-dose losartan-treated rats (P < 0.01). These results show that losartan can induce apoptosis of prostate epithelium and increase the TGFbeta1 expression in SH rats, suggesting that Ang II stimulation might be involved in the pathogenesis of BPH, which might correlate with the regulation of TGFbeta1 expression.

INFLUENCE OF GENDER ON OXIDATIVE STRESS, LIPID PEROXIDATION, PROTEIN DAMAGE AND APOPTOSIS IN HEARTS AND BRAINS FROM SPONTANEOUSLY HYPERTENSIVE RATS

1. Hypertension leads to oxidative stress, lipid and protein damage, apoptosis and impaired cardiac contractile function. However, impact of gender on these hypertension-associated abnormalities has not been elucidated. 2. The present study evaluated the oxidative stress, lipid/protein damage, apoptosis in heart and brain tissues as well as cardiomyocyte contractile function in Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) of both genders. Oxidative stress, lipid peroxidation, protein damage and apoptosis were assessed by glutathione (GSH) : reduced glutathione (GSSG) ratio, malondialdehyde (MDA) levels, protein carbonyl levels and caspase-3 activity, respectively. Cardiomyocyte contractile function was examined including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR90) and maximal velocity of shortening/relengthening (+/-dL/dt). The SHR cardiomyocytes displayed reduced PS and +/-dL/dt compared with gender-matched WKY counterparts. Male but not female SHR cardiomyocytes possessed longer resting cell length, normal TPS and prolonged TR90. All mechanical parameters were comparable between male and female WKY rats with the exception of a higher TR90 in females. Hypertension did not significantly affect the GSH : GSSG ratio in the heart and brain tissues of either gender. Brain from female WKY rats displayed a reduced GSH : GSSG ratio. The MDA levels were unchanged and elevated, respectively, in SHR heart and SHR brain tissues from both genders. Protein carbonyl formation and caspase-3 activity were elevated in male but not female SHR hearts. Nonetheless, brain protein carbonyl level and caspase-3 activity were unaffected by hypertension or gender. 3. In summary, these results suggest that gender affects hypertension-associated oxidative stress, lipid and protein damage, apoptosis in heart and brain tissues and cardiomyocyte contractile function.

Proteomic Analysis of Early Left Ventricular Hypertrophy Secondary to Hypertension

Untreated or poorly controlled arterial hypertension induced development of pathologic left ventricular hypertrophy (LVH), a common finding in hypertensive patients and a strong predictor of cardiovascular morbidity and mortality. The proteomic approach is a powerful technique to analyze a complex mixture of proteins in various settings. An experimental model of hypertension-induced early LVH was performed in spontaneously hypertensive rats, and the cardiac protein pattern compared with the normotensive Wistar Kyoto counterpart was analyzed. Fifteen altered protein spots were shown in the early stage of LVH. Compared with a previous animal model of established and regressed LVH, three protein spots were common in both models. These three altered protein spots corresponded to two unique proteins that were identified as Calsarcin-1 (CS-1) and ubiquinone biosynthesis protein COQ7 homolog. CS-1 is a negative regulator of the calcineurin/NF-AT pathway. Because upregulation in the expression levels of this protein was observed, the activation level of NF-kappaB by oxidative stress as an alternative pathway was investigated. It was found that antihypertensive therapies partially decreased oxidative stress and normalized the activation of NF-kappaB in the kidneys and aorta NF-kappaB activation but just moderately in the heart. This could be due to the interaction of any specific cardiac protein with any component of the NF-kappaB pathway. In this sense, CS-1 could be a good candidate because it is expressed preferentially in heart, to a lesser extent in smooth muscle cells, but not in kidney. Further investigations are necessary to elucidate the exact role of CS-1 and ubiquinone biosynthesis protein COQ7 in the setting of hypertension-induced LVH.

Distinct gene-sex interactions regulate adult rat cardiomyocyte width and length independently.

Wistar-Kyoto (WKY) and WKY-derived hyperactive (WKHA) rats are two genetically-related inbred strains of rats that are both normotensive yet exhibit differences in left ventricular mass (LVM). We had shown previously that cardiomyocytes from male WKHA are wider than that of male WKY, and that there was genetic linkage between LVM and a locus on chromosome 5 (RNO5) in the male progeny of a F2 WKHA/WKY cross. We show here that cardiomyocyte width is linked to the same RNO5 locus in male reciprocal congenic rats derived from WKHA and WKY. Contrary to males, we found no genetic linkage between LVM and the RNO5 locus in female rats. However, ventricular hypertrophy in females might be of a different nature, because cardiomyocytes from female WKHA were shorter than their WKY counterparts (with no difference in width). The RNO5 locus contains that of the natriuretic peptide precursor A (Nppa) gene. In male congenic rats, changes in cardiomyocyte width always correlated with reciprocal changes in the LV concentration of atrial natriuretic factor (ANF, i.e., the peptide product of Nppa). Taken together with other functional data, the small size of the RNO5 locus (approximately 63 cR) increased the likelihood that both cardiomyocyte width and LV ANF concentration could be linked to only one gene (possibly Nppa) in male rats. Moreover, our results support the notion that genes and sex interact to regulate cardiomyocyte width and length independently from one another.

Variations in Dietary Fat and Cholesterol Intakes Modify Antioxidant Status of SHR and WKY Rats

The effects of varying dietary fat saturation [butter (B), beef tallow (BT)] or polyunsaturation [(n-6) soybean oil (SBO), (n-3) menhaden oil (MO)] and cholesterol content (0.05 and 0.5 g/100 g) on systolic blood pressure (SBP), plasma lipids and tissue antioxidant status were investigated in 14-wk-old spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. Varying dietary fat composition for 9 wk had no influence on SBP in either SHR or WKY rats. Rats fed MO diets exhibited smaller (P < 0.05) body weight gains, lower (P < 0.05) feed efficiency ratios and lower (P < 0.05) plasma cholesterol concentrations than those fed the B, BT and SBO diets. Significant (P < 0.05) interactions for animal strain × cholesterol intake and animal strain × fat source were noted for serum cholesterol concentrations. SHR exhibited higher (P < 0.05) RBC and liver catalase (CAT), and heart and liver superoxide dismutase (SOD) activities similar to those of WKY rats. The lower (P <0.01) RBC, heart and liver glutathione peroxidase (GSH-Px) activities observed in SHR coincided with higher (P <0.01) glutathione reductase (GSSG-Red), compared with WKY rats. Dietary cholesterol intake had no effect on RBC, heart and liver total sulfhydryl concentration or GSH-Px activities, but increased (P <0.001) liver GSSG-Red. Feeding MO resulted in lower (P <0.001) RBC and heart GSH-Px activities. In contrast, feeding B and BT resulted in lower GSH-Px in liver. The significant (P < 0.01) animal strain × fat source interaction obtained for liver GSH-Px activity indicated that SHR responded differently to polyunsaturated fatty acid feeding than their WKY counterparts. Diet-induced changes in tissue antioxidant status were tissue specific and did not affect the development of hypertension in SHR.

Plasma and lipoprotein lipid composition and hepatic antioxidant status in spontaneously hypertensive (SHR) and normotensive (WKY) rats

Plasma and lipoprotein lipid composition and endogenous hepatic antioxidant status were investigated in hypertensive, 14-week-old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats fed a standard commercial rat chow. Total plasma calcium and magnesium concentrations were similar between both rat strains; however, systolic blood pressure in SHR was greater than in WKY at 13 weeks of age (197 +/- 12 vs. 132 +/- 14 mmHg; p < or = 0.05), confirming hypertension in SHR. Total plasma cholesterol and triacylglycerol concentrations were lower (p < or = 0.05) in SHR compared with WKY. A lower (p < 0.05) HDL cholesterol level in SHR plasma resulted in a higher LDL to HDL cholesterol ratio compared with WKY counterparts. No significant differences in the relative proportion of HDL apolipoprotein A-I fraction were observed between SHR and WKY. Both SHR VLDL and HDL triacylglycerol fractions were lower (p < 0.05) in SHR than WKY. Analysis of liver antioxidant enzyme activities showed no differences in rat liver superoxide dismutase (SOD), but lower (p < 0.05) liver glutathione peroxidase (GSH-Px) activity in SHR. However, liver glutathione (GSH) levels were similar in SHR and WKY counterparts. A possible compensatory effect to the oxidative status of SHR was suggested by the significant (p < 0.05) increase in both liver catalase (CAT) and glutathione reductase (GSSG-Red) activities. Despite these results, in vitro oxidative challenge studies with H2O2 demonstrated a greater susceptibility of liver to GSH depletion in the SHR, although no parallel change in thiobarbituric acid reactive substances (TBARS) production was observed. The comparatively lower plasma cholesterol observed in hypertensive SHR paralleled specific differences in liver catalase and glutathione redox antioxidant enzyme activities.

Reduced sodium-proton exchange activity in lymphocytes from transgenic rats.

We investigated sodium-proton (Na(+)-H+) exchange activity in transgenic TGR(mRen-2)27 rats, a strain showing fulminant hypertension after the mouse Ren-2d renin gene has been integrated into its genome, in age-matched normotensive Sprague-Dawley (SD) rats, in spontaneously hypertensive rats (SHR) from the Münster strain, and in normotensive Wistar-Kyoto (WKY) rats. From each strain Na(+)-H+ exchange activity was determined in lymphocytes using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) by measuring the recovery rate of cytosolic pH (pHi) after intracellular acidification. Resting pHi was not significantly different in transgenic rats (n = 10) compared with SD rats (n = 10) (7.305 +/- 0.038 versus 7.337 +/- 0.031; mean +/- SEM), but resting pHi was significantly lower in lymphocytes from SHR (n = 12) compared with their normotensive WKY counterparts (n = 12) (7.232 +/- 0.030 versus 7.377 +/- 0.022; P < .01). Na(+)-H+ exchange activity was significantly lower in lymphocytes from transgenic rats compared with SD rats (5.102 +/- 0.561 versus 7.385 +/- 0.491 x 10(-3) dpHi/s; P < .01), whereas Na(+)-H+ exchange was significantly enhanced in lymphocytes from SHR compared with WKY rats (5.564 +/- 0.432 versus 3.921 +/- 0.433 x 10(-3) dpHi/s; P < .05). The apparent half-maximal activation of Na(+)-H+ exchange was not significantly different in the strains tested. The present study indicates that hypertension in transgenic rats is not related to Na(+)-H+ exchange overactivity.

In situ analysis of microvascular pericytes in hypertensive rat brains

We used immunofluorescence microscopy and isoactin-specific antibodies to characterize the pattern and prevalence of pericytes within the brain microcirculation. Blood pressures of normotensive. Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were measured prior to sacrifice and pressure-perfusion fixation. WKY and SHR brains were subdivided into ten major regions prior to ultracryomicrotomy. Sections 0.3–0.5 μm wide were treated with 10–40 μg/ml affinity-purified antibodies to the muscle and non-muscle actin isoforms. These localization studies show that there are four times the number of pericyte-rich capillaries in the SHR motor cortex compared to WKY counterparts (59.9 vs. 15.3%). In contrast, the sensory cortex of both rat strains is deficient in muscle actin staining surrounding the capillaries. The most striking difference in pericyte presence and muscle actin antibody staining between the SHR and WKY was observed in the tegmentum of the brainstem. There is nearly a one-to-one coincidence observed in pericyte and capillary profiles present within thin, frozen sections of the SHR midbrain. SHR pons capillaries were also pericyte-enriched. WKY microvessels of these brainstem regions were pericyte-poor. Transmission electron microscopic analyses of plastic embedded thin sections confirmed the presence of pericytes and their filament-enriched processes encircling the capillaries of the hypertensive brains. These results suggest that pericytes may play important roles in hypertension and cerebrovascular disease processes.