Female Fischer-344 Research Articles

Abstract P153: The Role of Lipoprotein Lipase Activation in Vascular Aging

Lipoprotein metabolism plays a role in cardiovascular diseases and becomes impaired with aging. Angiopoietin-like proteins (ANGPTLs) regulate lipoprotein lipase (LPL) and endothelial lipase (EL), key hydrolases in triglyceride catabolism. We hypothesized that ANGPTL3 and ANGPTL4 would be elevated in aging and that the response to LPL activation would be impaired in mesenteric resistance arteries (MRAs) from old rats. To assess this, MRAs were isolated from 3 and 16-month-old male and female Fischer344 rats and mounted on wire myographs. Concentration-response curves (CRC) to phenylephrine (PE, 10nM-100μM), Ca 2+ channel agonist Bay-K8644 (10nM-30nM), and acetylcholine (ACh, 0.1nM-100uM) were performed with or without LPL activator, NO-1886 (10uM and 50uM). Protein expression was assessed by Western blot. We show for the first time that circulating ANGPTL3 (young: 1.5±0.8 vs old: 5±0.7) and ANGPTL4 (young: 1.7±0.3 vs old: 2.5±0.5) are increased in aging. In MRAs from young rats, NO-1886 attenuated PE-induced contraction in males (Emax; vehicle: 106±3 vs NO-1886 10uM: 86 ± 5* vs NO-1886 50uM: 69±8*, p<0.05) and females (Emax; vehicle: 104±4 vs NO-1886 10uM: 91±5* vs NO-1886 50uM: 75±8*, p<0.05). While 16-month-old males presented a similar reduction in contraction (Emax; vehicle: 105±6 vs NO-1886 10uM: 88±11* vs NO-1886 50uM: 73±17*, p<0.05), in females only 50uM showed a significant anti-contractile effect in a sex-dependent manner (Emax; vehicle: 100±7 vs NO-1886 10uM: 95±7 vs NO-1886 50uM: 83±22*, p<0.05). CRC to bay-K8644 showed decreased contraction after NO-1886 incubation (Emax; vehicle: 16±8 vs NO-1886 50uM: 5±3) indicating that L-type Ca 2+ channel Ca 2+ influx isn’t involved in NO-1886’s anti-contractile effect. NO-1886 incubation did not change the maximal ACh response in young rat MRAs, but a rightward shift in the curve was seen in both sexes. As expected, MRAs from old males (Emax; young: 97±2 vs old: 86±7) and females (Emax; young: 95±2 vs old: 92±6) had decreased maximal relaxation to ACh, compared to young rats. Interestingly, in old rats NO-1886 induced further impaired relaxation to ACh in males (Emax; vehicle: 86±7 vs NO-1886 10uM: 78±14) and females (Emax; vehicle: 92±6 vs NO-1886 10uM: 77±22). Together, this data suggests (1) ANGPTL3 and ANGPTL4 are increased in old rats, (2) LPL/EL activity diminished with age in females, but not males, and (3) while NO-1886 exerts an anti-contractile effect, it can also induce endothelial dysfunction.

Serine racemase expression profile in the prefrontal cortex and hippocampal subregions during aging in male and female rats.

Aging is associated with a decrease in N-methyl-D-aspartate (NMDA) receptor function, which is critical for maintaining synaptic plasticity, learning, and memory. Activation of the NMDA receptor requires binding of the neurotransmitter glutamate and also the presence of co-agonist D-serine at the glycine site. The enzymatic conversion of L-serine to D-serine is facilitated by the enzyme serine racemase (SR). Subsequently, SR plays a pivotal role in regulating NMDA receptor activity, thereby impacting synaptic plasticity and memory processes in the central nervous system. As such, age-related changes in the expression of SR could contribute to decreased NMDA receptor function. However, age-associated changes in SR expression levels in the medial and lateral prefrontal cortex (mPFC, lPFC), and in the dorsal hippocampal subfields, CA1, CA3, and dentate gyrus (DG), have not been thoroughly elucidated. Therefore, the current studies were designed to determine the SR expression profile, including protein levels and mRNA, for these regions in aged and young male and female Fischer-344 rats. Our results demonstrate a significant reduction in SR expression levels in the mPFC and all hippocampal subfields of aged rats compared to young rats. No sex differences were observed in the expression of SR. These findings suggest that the decrease in SR levels may play a role in the age-associated reduction of NMDA receptor function in brain regions crucial for cognitive function and synaptic plasticity.

To explore the change of motor cognitive function in pituitary tumor rats after operation

To study the changes of motor and cognitive function of pituitary tumor rats after the operation. Methods: The experiment was divided into three groups: control group, model group and operation group (30 animals for each group). Female Fischer344 rats were selected. Model group rats were subcutaneously embedded with 10 mg estrogen sustained-release pump to induce a pituitary tumor model, and control group rats were subcutaneously embedded with a normal saline sustained-release pump as control. The operation group was successfully treated by microsurgery after the model was established. The quantitative expressions of PTTG, FGF-2 and VEGF were detected by Western blot. Morris test was used to detect the spatial learning and memory ability of rats. Western blot results showed that compared with the model group, the expression of the operation group was decreased, but still higher than that of the control group (p < 0.05). The water maze test results showed that the incubation period of searching the safe island in the model group was significantly longer than that in the control group on the 8th and 9th day after the injury, and the difference was statistically significant (p < 0.05). The incubation period of searching the safe island on the 8th and 9th day after injury in the operation group was significantly shorter than that in the control group. Through the detection of behavioral-related experimental and protein, the motor memory of rats after pituitary tumor surgery can be improved to some extent.

Adipose stromal vascular fraction reverses mitochondrial dysfunction and hyperfission in aging-induced coronary microvascular disease.

Aging is associated with blunted coronary microvascular vasodilatory function. Previously, systemically administered adipose stromal vascular fraction (SVF) therapy reversed aging-induced attenuation of β1-adrenergic- and flow-mediated dilation dependent on reducing mitochondrial reactive oxygen species. We hypothesized that SVF-mediated recovery of microvascular dilatory function is dependent on recovery of mitochondrial function, specifically by reducing mitochondrial hyperfission. Female Fischer-344 rats were allocated into young control, old control, and old + SVF therapy groups. Pressure myography, immunofluorescent staining, Western blot analysis, and RNA sequencing were performed to determine coronary microvascular mitochondrial dynamics and function. Gene and protein expression of fission-mediator DRP-1 was enhanced with aging but reversed by SVF therapy. SVF facilitated an increase in fusion-mediator MFN-1 gene and protein expression. Mitochondrial morphology was characterized as rod-like and densely networked in young controls, isolated circular and punctate with aging, and less circularity with partially restored mitochondrial branch density with SVF therapy. Decreased mitochondrial membrane potential and ATP bioavailability in aged animals at baseline and during flow-mediated dilation were reversed by SVF and accompanied with enhanced oxygen consumption. Dilation to norepinephrine and flow in young controls were dependent on uninhibited mitochondrial fusion, whereas inhibiting fission did not restore aged microvessel response to norepinephrine or flow. SVF-mediated recovery of β-adrenergic function was dependent on uninhibited mitochondrial fusion, whereas recovery of flow-mediated dilation was dependent on maintained mitochondrial fission. Impaired dilation in aging is mitigated by SVF therapy, which recovers mitochondrial function and fission/fusion balance.NEW & NOTEWORTHY We elucidated the consequences of aging on coronary microvascular mitochondrial health as well as SVF's ability to reverse these effects. Aging shifts gene/protein expression and mitochondrial morphology indicating hyperfission, alongside attenuated mitochondrial membrane potential and ATP bioavailability, all reversed using SVF therapy. Mitochondrial membrane potential and ATP levels correlated with vasodilatory efficiency. Mitochondrial dysfunction is a contributing pathological factor in aging that can be targeted by therapeutic SVF to preserve microvascular dilative function.

Cell therapy rescues aging-induced beta-1 adrenergic receptor and GRK2 dysfunction in the coronary microcirculation.

Our past study showed that coronary arterioles isolated from adipose-derived stromal vascular fraction (SVF)-treated rats showed amelioration of the age-related decrease in vasodilation to beta-adrenergic receptor (β-AR) agonist and improved β-AR-dependent coronary flow and microvascular function in a model of advanced age. We hypothesized that intravenously (i.v.) injected SVF improves coronary microvascular function in aged rats by re-establishing the equilibrium of the negative regulators of the internal adrenergic signaling cascade, G-protein receptor kinase 2 (GRK2) and G-alpha inhibitory (Gαi) proteins, back to youthful levels. Female Fischer-344 rats aged young (3 months, n = 24), old (24 months, n = 26), and old animals that received 1 × 107 green fluorescent protein (GFP+) SVF cells (O + SVF, n = 11) 4 weeks prior to sacrifice were utilized. Overnight urine was collected prior to sacrifice for catecholamine measurements. Cardiac samples were used for western blotting while coronary arterioles were isolated for pressure myography studies, immunofluorescence staining, and RNA sequencing. Coronary microvascular levels of the β1 adrenergic receptor are decreased with advancing age, but this decreased expression was rescued by SVF treatment. Aging led to a decrease in phosphorylated GRK2 in cardiomyocytes vs. young control with restoration of phosphorylation status by SVF. In vessels, there was no change in genetic transcription (RNAseq) or protein expression (immunofluorescence); however, inhibition of GRK2 (paroxetine) led to improved vasodilation to norepinephrine in the old control (OC) and O + SVF, indicating greater GRK2 functional inhibition of β1-AR in aging. SVF works to improve adrenergic-mediated vasodilation by restoring the β1-AR population and mitigating signal cascade inhibitors to improve vasodilation.

Toxicity and occupational exposure assessment for hydroprocessed esters and fatty acids (HEFA) alternative jet fuels

ABSTRACTThe U.S. Air Force (USAF) has pursued development of alternative fuels to augment or replace petroleum-based jet fuels. Hydroprocessed esters and fatty acids (HEFA) renewable jet fuel is certified for use in commercial and USAF aircraft. HEFA feedstocks include camelina seed oil (Camelina sativa, HEFA-C); rendered animal fat (tallow, HEFA-T); and mixed fats and oils (HEFA-F). The aim of this study was to examine potential toxic effects associated with HEFA fuels exposures. All 3 HEFA fuels were less dermally irritating to rabbits than petroleum-derived JP-8 currently in use. Inhalation studies using male and female Fischer-344 rats included acute (1 day, with and without an 11-day recovery), 5-, 10- or 90-day durations. Rats were exposed to 0, 200, 700 or 2000 mg/m3 HEFA-F (6 hr/day, 5 days/week). Acute, 5 – and 10-day responses included minor urinalysis effects. Kidney weight increases might be attributed to male rat specific hyaline droplet formation. Nasal cavity changes included olfactory epithelial degeneration at 2000 mg/m3. Alveolar inflammation was observed at ≥700 mg/m3. For the 90-day study using HEFA-C, no significant neurobehavioral effects were detected. Minimal histopathological effects at 2000 mg/m3 included nasal epithelium goblet cell hyperplasia and olfactory epithelium degeneration. A concurrent micronucleus test was negative for evidence of genotoxicity. All HEFA fuels were negative for mutagenicity (Ames test). Sensory irritation (RD50) values were determined to be 9578 mg/m3 for HEFA-C and greater than 10,000 mg/m3 for HEFA-T and HEFA-F in male Swiss-Webster mice. Overall, HEFA jet fuel was less toxic than JP-8. Occupational exposure levels of 200 mg/m3 for vapor and 5 mg/m3 for aerosol are recommended for HEFA-based jet fuels.

Sex- and Age-dependent Differences in Sleep-wake Characteristics of Fisher-344 Rats

Aging is a well-recognized risk factor for sleep disruption. The characteristics of sleep in aging include its disruption by frequent awakenings, a decline in both non-rapid eye movement (nonREM) and REM sleep amounts, and a weaker homeostatic response to sleep loss. Evidence also suggests that sleep in females is more sensitive to changes in the ovarian steroidal milieu. The Fischer-344 rats are commonly used experimental subjects in behavioral and physiological studies, including sleep and aging. Most sleep studies in Fischer-344 rats have used male subjects to avoid interactions between the estrus and sleep-waking cycles. The changes in the sleep-wake organization of female Fischer-344 rats, especially with advancing age, are not well-characterized. We determined sleep-waking features of cycling females across estrus stages. We also compared spontaneous and homeostatic sleep response profiles of young (3-4 months) and old (24-25 months) male and female Fischer-344 rats. The results suggest that: i) sleep-wake architectures across stages of estrus cycle in young females were largely comparable except for a significant suppression of REM sleep at proestrus night and an increase in REM sleep the following day; ii) despite hormonal differences, sleep-wake architecture in male and female rats of corresponding ages were comparable except for the suppression of REM sleep at proestrus night and higher nonREM delta power in recovery sleep; and iii) aging significantly affected sleep-wake amounts, sleep-wake stability, and homeostatic response to sleep loss in both male and female rats and that the adverse effects of aging were largely comparable in both sexes.

Enhanced beta-1 adrenergic receptor responsiveness in coronary arterioles following intravenous stromal vascular fraction therapy in aged rats.

Our past study showed that a single tail vein injection of adipose-derived stromal vascular fraction (SVF) into old rats was associated with improved dobutamine-mediated coronary flow reserve. We hypothesize that i.v. injection of SVF improves coronary microvascular function in aged rats via alterations in beta adrenergic microvascular signaling. Female Fischer-344 rats aged young (3 months, n=32) and old (24 months, n=30) were utilized, along with two cell therapies intravenously injected in old rats four weeks prior to sacrifice: 1x107 green fluorescent protein (GFP+) SVF cells (O+SVF, n=21), and 5x106 GFP+ bone-marrow mesenchymal stromal cells (O+BM, n=6), both harvested from young donors. Cardiac ultrasound and pressure-volume measurements were obtained, and coronary arterioles were isolated from each group for microvessel reactivity studies and immunofluorescence staining. Coronary flow reserve decreased with advancing age, but this effect was rescued by the SVF treatment in the O+SVF group. Echocardiography showed an age-related diastolic dysfunction that was improved with SVF to a greater extent than with BM treatment. Coronary arterioles isolated from SVF-treated rats showed amelioration of the age-related decrease in vasodilation to a non-selective β-AR agonist. I.v. injected SVF cells improved β-adrenergic receptor-dependent coronary flow and microvascular function in a model of advanced age.

Abstract 1951: Increased glioma collagen is associated with greater blood flow and peri-tumoral fluid flux, but less infiltration

Abstract Introduction: Composition of the glioma microenvironment (TME) determines tumor growth rate, invasion, metastasis and resistance to treatment. Compared to normal brain, tumor extracellular matrix (ECM) has a higher concentration of structural proteins including collagen, laminin, tenascin, and vinculin. We compared collagen expression in two rat models of glioma, human U251 grown in immune-compromised rats and 9L in syngeneic Fischer-344 rats, representing primary and recurrent glioma features, respectively. Experimental procedures: The U251 and 9L cells were implanted in the right brain hemisphere of female RNU rats (n=10) and Fischer-344 rats (n=7), respectively. Rats were imaged 2 to 3 weeks after implantation by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Tumor size, blood flow, apparent diffusion coefficient (ADC), blood-to-tumor forward volumetric transfer constant (Ktrans), peri-tumoral contrast flux (Flux), hydraulic conductivity (K), and extracellular volume fraction (VD) in the tumor (VD-tumor), tumor rim (VD-rim) and its periphery (VD-peri) along with tumor interstitial fluid pressure (TIFP) were measured. Immediately after imaging, brains were processed for paraffin embedding and histopathology. Brain sections (6 µm) containing the tumor were stained for collagen using Picrosirius red and adjacent brain sections with hematoxylin and eosin (H&amp;E). MRI data were compared by t-tests and significance inferred at p≤0.05. Digital images of Picrosirius red staining were analyzed using ImageJ for collagen expression expressed as % fraction of total tumor area. It was compared to the MRI biomarkers, and to the patterns of tumor cell dispersion into normal brain observed on H&amp;E stained images. Results: Tumor diameters averaged 4 mm for both models at the time of imaging. The U251 tumors showed greater Flux (p=0.03), higher blood flow (p=0.02), smaller VD_tumor (p=0.01) and VD_rim (p=0.02) values than the 9L tumors. TIFP also tended to be higher in the U251 model (p=0.06). Collagen expression in U251 tumors was significantly higher than in 9L tumors (p=0.002). On H&amp;E stained sections, the U251 tumors showed a well delineated tumor margin with very few cells invading the host tissue. In contrast, the 9L tumors showed cancer cells infiltrating singly and in clusters, along white matter tracts as well as in perivascular spaces. Conclusions: Increased collagen content was associated with elevated blood flow, Flux and TIFP, but less tumor cell infiltration in the U251 glioma. Observations of low collagen content in conjunction with increased tumor cell invasion in the 9L model suggest that treatment with collagenase, while increasing drug penetration may also make the tumors more infiltrative. A closer examination of TME to determine the composition of ECM proteins that regulate glioma aggressiveness and modulate its response to treatments is warranted. Note: This abstract was not presented at the meeting. Citation Format: Tavarekere N. Nagaraja, Rasha Elmghirbi, Stephen L. Brown, Ian Y. Lee, Glauber Cabral, Swayamprava Panda, Robert A. Knight, James R. Ewing. Increased glioma collagen is associated with greater blood flow and peri-tumoral fluid flux, but less infiltration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1951.

Novel Role of Thbs‐1/CD47‐Drp1 signaling following ischemia reperfusion in the aging heart

Ischemia reperfusion (IR) injury leads to activation of dynamin‐related protein (Drp‐1), causing mitochondrial fission and generation of reactive oxygen species (ROS), but the molecular mechanisms that activate Drp‐1 are not known. The purpose of this study was to establish the signaling pathway between Thbs‐1/CD47 and fission protein (Drp‐1) through PGC‐1 following IR in advancing age.MethodsFemale Fischer‐344 rats were divided into 4 groups: Young Control, Young+IR, Old Control and Old+IR. Echocardiography measurements were done at baseline and 72 hours after IR (30 minutes of ischemia,72 hrs of reperfusion). After evaluation of heart function and coronary flow using vevo3100, hearts were explanted and mitochondrial ROS generation was measured using MitoPY1 (O2·−), as well as protein levels of Thbs‐1, PGC‐1, and Drp‐1. In vitro, rat aortic endothelial cells (RAEC) received hypoxic conditions to mimic IR in vivo model. Cells were treated with SiRNA for PGC‐1 or control scrambled SIRNA and then underwent hypoxia challenge to evaluate PGC‐1 effect on Drp‐1. Cell lysate was collected for Western Blot after 36 hours of transfection.ResultsMitochondrial O2·− generation in heart tissue increased in both age groups following IR. Old animals experiencing diastolic dysfunction at the baseline, after IR they exhibited reduced systolic function and exacerbated diastolic dysfunction compared to young controls. IR induces Thbs‐1 and Drp‐1 expression in young and old hearts. SiRNA to PGC‐1 enhanced levels of Drp‐1 in RAECs and increases ROS generation after hypoxia.ConclusionLeft ventricular diastolic dysfunction during IR is potentiated due to increased generation of ROS. These results highlight a novel signaling pathway by which Thbs‐1 regulates mitochondrial fission (Drp‐1) and ROS generation during hypoxia, and presumably, following IR. Inhibiting Thbs‐1 immediately after IR may prevent Drp‐1‐mediated mitochondrial fission and is likely to improve diastolic function of the heart by reducing ROS‐mediated cardiomyocyte damage in the aged population.Support or Funding InformationNIH R01 AG05358 Gheen's FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 4989: Efficacy of erlotinib and/or naproxen when administered by intermittent dosing schedules in the prevention of chemically induced urinary bladder cancers

Abstract Inflammation and epidermal growth factor receptor (EGFR) signaling dysregulation plays an important role in urinary bladder cancer development. We have previously shown that single agent regimens with erlotinib (EGFR inhibitor) or naproxen (nonsteroidal anti-inflammatory drug; NSAID) when given continuously were highly effective in the prevention of OH-BBN-induced urinary bladder cancers in rats. Better tolerated chemoprevention regimens can be obtained by reducing doses and frequency of administration. Low dose combinations may also achieve substantial efficacy with minimal toxicity by targeting complementary pathways. Female Fischer-344 rats were obtained at 28 days of age, placed on Teklad (4% fat) diet, and received OH-BBN (150 mg/gavage) 2x/week for 8 weeks beginning at 56 days of age. In the first study, beginning one week after the final OH-BBN treatment the rats (25/group) received either: Group 1, erlotinib (42 mg/kg BW, 1x/week); Group 2, naproxen (30 mg/kg BW/day, 3 weeks on/ 3 weeks off); Group 3, the combination of erlotinib and naproxen (using the same treatment regimens), and Group 4, vehicle. The rats were palpated for urinary bladder tumors, weighed 1x/week and observed daily for signs of toxicity. At the end of the study (10 months after the initial OH-BBN), the average weights of the urinary bladders (bladder plus tumors combined) were determined. The weights in Groups 1-4 were: 194*, 186*, 136* and 354 mg (*P &amp;lt;0.05, Wilcoxon Rank test). This was accompanied by an increase in tumor latency, a decrease in tumor multiplicity, and a decrease in the number of rats with large palpable tumors (&amp;gt; 200 mg). The second study used the same treatment regimens, but administration of the agents was delayed until three months after the final OH-BBN treatment, at which point microscopic transitional cell carcinomas were present. At the end of the study (11 months after the initial OH-BBN), the weights of the individual bladders in Groups 1-4 were: 584, 234*, 138* and 779 mg (*P&amp;lt; 0.05, Wilcoxon Rank test). Thus, naproxen and particularly the combination were still highly effective. This was accompanied by an increase in tumor latency and a decrease in the number of rats with large palpable tumors (&amp;gt; 200 mg). These studies demonstrate that in protocols meant to reduce the toxicity of agents (weekly erlotinib or intermittent dosing with naproxen), high chemopreventive efficacy was achieved. Of importance, starting erlotinib and/or naproxen intermittent treatments at the time microscopic cancers were present still reduced the size of the urinary bladder cancers without showing observable toxicity. The latter is of particular interest based on the clinical FAP trial showing great efficacy of erlotinib and sulindac. Supported by NCI contract number HHSN261201500036I, Task Order HHSN26100002. Citation Format: Altaf Mohammed, Mark S. Miller, Ronald A. Lubet, Chen Suen, Shizuko Sei, Robert H. Shoemaker, Clinton J. Grubbs. Efficacy of erlotinib and/or naproxen when administered by intermittent dosing schedules in the prevention of chemically induced urinary bladder cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4989.

Activation of GPR30 improves exercise capacity and skeletal muscle strength in senescent female Fischer344 × Brown Norway rats

The molecular mechanisms of muscle weakness and sarcopenia in postmenopausal women are largely unknown. To determine the effect of a new estrogen receptor, GPR30, in the maintenance of exercise capacity and skeletal muscle function in females, the selective GPR30 agonist, G1 (100 μg/kg/day), or vehicle (V, soybean oil) was administered subcutaneously daily (n = 7 per group) to ovariectomized (OVX) 27-month-old Fischer 344 × Brown Norway (F344BN) female rats. Following 8 weeks of treatment, the exercise capacity (treadmill walk time to exhaustion) was reduced in OVX vs. sham rats (5.1 ± 1.4 vs. 11.0 ± 0.9 min, P < 0.05), and chronic G1 restored exercise capacity (12.9 ± 1.2 min; P < 0.05 vs. OVX-V). Similarly, the peak twitch of electrically stimulated soleus muscles was decreased by 22% in OVX vs. sham rats (P < 0.05), and G1 attenuated this decline (P < 0.05). Western blot analysis showed that chronic G1 treatment attenuated OVX-associated decreases in heat shock protein (HSP) 90, HSP70, and HSP27 expressions. In vitro studies using the L6 myoblast cell line demonstrated that G1 increased mRNA levels of HSPs in cultured cells. Collectively, these data demonstrate that the activation of GPR30 mitigates the adverse effects of estrogen loss on exercise capacity and skeletal muscle contractile function in old F344BN rats. The protective effects of GPR30 might be through its upregulation of heat shock proteins in skeletal muscle.

Effect of Age, Estrogen Status, and Late-Life GPER Activation on Cardiac Structure and Function in the Fischer344×Brown Norway Female Rat.

Age-associated changes in cardiac structure and function, together with estrogen loss, contribute to the progression of heart failure with preserved ejection fraction in older women. To investigate the effects of aging and estrogen loss on the development of its precursor, asymptomatic left ventricular diastolic dysfunction, echocardiograms were performed in 10 middle-aged (20 months) and 30 old-aged (30 months) female Fischer344×Brown-Norway rats, 4 and 8 weeks after ovariectomy (OVX) and sham procedures (gonads left intact). The cardioprotective potential of administering chronic G1, the selective agonist to the new G-protein-coupled estrogen receptor (GPER), was further evaluated in old rats (Old-OVX+G1) versus age-matched, vehicle-treated OVX and gonadal intact rats. Advanced age and estrogen loss led to decreases in myocardial relaxation and elevations in filling pressure, in part, due to reductions in phosphorylated phospholamban and increases in cardiac collagen deposition. Eight weeks of G-protein-coupled estrogen receptor activation in Old-OVX+G1 rats reversed the adverse effects of age and estrogen loss on myocardial relaxation through increases in sarcoplasmic reticulum Ca2+ ATPase expression and reductions in interstitial fibrosis. These findings may explain the preponderance of heart failure with preserved ejection fraction in older postmenopausal women and provide a promising, late-life therapeutic target to reverse or halt the progression of left ventricular diastolic dysfunction.

Background Noise Contributes to Organic Solvent Induced Brain Dysfunction.

Occupational exposure to complex blends of organic solvents is believed to alter brain functions among workers. However, work environments that contain organic solvents are also polluted with background noise which raises the issue of whether or not the noise contributed to brain alterations. The purpose of the current study was to determine whether or not repeated exposure to low intensity noise with and without exposure to a complex blend of organic solvents would alter brain activity. Female Fischer344 rats served as subjects in these experiments. Asynchronous volume conductance between the midbrain and cortex was evaluated with a slow vertex recording technique. Subtoxic solvent exposure, by itself, had no statistically significant effects. However, background noise significantly suppressed brain activity and this suppression was exacerbated with solvent exposure. Furthermore, combined exposure produced significantly slow neurotransmission. These abnormal neurophysiologic findings occurred in the absence of hearing loss and detectable damage to sensory cells. The observations from the current experiment raise concern for all occupations where workers are repeatedly exposed to background noise or noise combined with organic solvents. Noise levels and solvent concentrations that are currently considered safe may not actually be safe and existing safety regulations have failed to recognize the neurotoxic potential of combined exposures.

Abstract 351: Late-life Activation of GPR30 by G1 Reverses Diastolic Dysfunction in Senescent Fischer344 x Brown Norway Female Rats

Introduction: The aged heart is characterized by impaired lusitropy and increased stiffness, and no proven pharmacologic interventions exist that limit these manifestations of left ventricular diastolic dysfunction (LVDD). G1, a selective agonist to the estrogen receptor GPR30, attenuated the effects of estrogen (E2) loss on LVDD in a hypertensive model. To extend prior studies, we determined the cardioprotective potential of chronic GPR30 activation in the female Fischer344 x Brown Norway (F344BN) rat, a normotensive aging model. Methods and Results: Bilateral oophorectomy (OVX) or sham-operations were performed in middle- (Mid) (18 months) and Old-aged rats (27 months). At 28 months of age, Old rats were randomized to daily G1 (100 μg/kg/day, s.c.) or vehicle (VEH; soybean oil) injections (n=7 rats/group). Following 8 weeks of treatment, left ventricle (LV) mass was greater in Old vs. Mid-aged rats, independent of E2 or G1, while systolic function and LV wall thicknesses were similar among groups. Myocardial relaxation (e’) was reduced (P&lt;0.001) and LV filling pressure (E/e’) was increased (P&lt;0.01) in Old vs. Mid-aged rats, and this was exacerbated by OVX (P&lt;0.01) and reversed by late-life G1 treatment. Systolic blood pressure (SBP) increased by 20% after mid-life OVX, whereas late-life E2 loss had no effect on SBP, irrespective of G1. Cardiac chymase expression was increased in E2 deficient hearts determined by Western blot and G1 modestly limited this E2/age effect. Conclusion: Our data indicate that late-life G1 treatment reverses the detrimental effects of advanced age and E2 deficiency on diastolic function, possibly via interactions with the noncanonical cardiac renin angiotensin system.

Prevention of chemically induced urinary bladder cancers by naproxen: protocols to reduce gastric toxicity in humans do not alter preventive efficacy.

The COX inhibitors (NSAID/Coxibs) are a major focus for the chemoprevention of cancer. The COX-2-specific inhibitors have progressed to clinical trials and have shown preventive efficacy in colon and skin cancers. However, they have significant adverse cardiovascular effects. Certain NSAIDs (e.g., naproxen) have a good cardiac profile, but can cause gastric toxicity. The present study examined protocols to reduce this toxicity of naproxen. Female Fischer-344 rats were treated weekly with the urinary bladder-specific carcinogen hydroxybutyl(butyl)nitrosamine (OH-BBN) for 8 weeks. Rats were dosed daily with NPX (40 mg/kg body weight/day, gavage) or with the proton pump inhibitor omeprazole (4.0 mg/kg body weight/day) either singly or in combination beginning 2 weeks after the final OH-BBN. OH-BBN-treated rats, 96% developed urinary bladder cancers. While omeprazole alone was ineffective (97% cancers), naproxen alone or combined with omeprazole-prevented cancers, yielding 27 and 35% cancers, respectively. In a separate study, OH-BBN -: treated rats were administered naproxen: (A) daily, (B) 1 week daily naproxen/1week vehicle, (C) 3 weeks daily naproxen/3 week vehicle, or (D) daily vehicle beginning 2 weeks after last OH-BBN treatment. In the intermittent dosing study, protocol A, B, C, and D resulted in palpable cancers in 27%, 22%, 19%, and 96% of rats (P < 0.01). Short-term naproxen treatment increased apoptosis, but did not alter proliferation in the urinary bladder cancers. Two different protocols that should decrease the gastric toxicity of NSAIDs in humans did not alter chemopreventive efficacy. This should encourage the use of NSAIDs (e.g., naproxen) in clinical prevention trials.

Aging and Estrogen Status: A Possible Endothelium-Dependent Vascular Coupling Mechanism in Bone Remodeling

Bone loss with aging and menopause may be linked to vascular endothelial dysfunction. The purpose of the study was to determine whether putative modifications in endothelium-dependent vasodilation of the principal nutrient artery (PNA) of the femur are associated with changes in trabecular bone volume (BV/TV) with altered estrogen status in young (6 mon) and old (24 mon) female Fischer-344 rats. Animals were divided into 6 groups: 1) young intact, 2) old intact, 3) young ovariectomized (OVX), 4) old OVX, 5) young OVX plus estrogen replacement (OVX+E2), and 6) old OVX+E2. PNA endothelium-dependent vasodilation was assessed in vitro using acetylcholine. Trabecular bone volume of the distal femoral metaphysis was determined by microCT. In young rats, vasodilation was diminished by OVX and restored with estrogen replacement (intact, 82±7; OVX, 61±9; OVX+E2, 90±4%), which corresponded with similar modifications in BV/TV (intact, 28.7±1.6; OVX, 16.3±0.9; OVX+E2, 25.7±1.4%). In old animals, vasodilation was unaffected by OVX but enhanced with estrogen replacement (intact, 55±8; OVX, 59±7; OVX+E2, 92±4%). Likewise, modifications in BV/TV followed the same pattern (intact, 33.1±1.6; OVX, 34.4±3.7; OVX+E2, 42.4±2.1%). Furthermore, in old animals with low endogenous estrogen (i.e., intact and old OVX), vasodilation was correlated with BV/TV (R2 = 0.630; P<0.001). These data demonstrate parallel effects of estrogen on vascular endothelial function and BV/TV, and provide for a possible coupling mechanism linking endothelium-dependent vasodilation to bone remodeling.

Atrazine and Breast Cancer: A Framework Assessment of the Toxicological and Epidemiological Evidence

The causal relationship between atrazine exposure and the occurrence of breast cancer in women was evaluated using the framework developed by Adami et al. (2011) wherein biological plausibility and epidemiological evidence were combined to conclude that a causal relationship between atrazine exposure and breast cancer is “unlikely”. Carcinogenicity studies in female Sprague-Dawley (SD) but not Fischer-344 rats indicate that high doses of atrazine caused a decreased latency and an increased incidence of combined adenocarcinoma and fibroadenoma mammary tumors. There were no effects of atrazine on any other tumor type in male or female SD or Fischer-344 rats or in three strains of mice. Seven key events that precede tumor expression in female SD rats were identified. Atrazine induces mammary tumors in aging female SD rats by suppressing the luteinizing hormone surge, thereby supporting a state of persistent estrus and prolonged exposure to endogenous estrogen and prolactin. This endocrine mode of action has low biological plausibility for women because women who undergo reproductive senescence have low rather than elevated levels of estrogen and prolactin. Four alternative modes of action (genotoxicity, estrogenicity, upregulation of aromatase gene expression or delayed mammary gland development) were considered and none could account for the tumor response in SD rats. Epidemiological studies provide no support for a causal relationship between atrazine exposure and breast cancer. This conclusion is consistent with International Agency for Research on Cancer’s classification of atrazine as “unclassifiable as to carcinogenicity” and the United States Environmental Protection Agency's classification of atrazine as “not likely to be carcinogenic.”

Neuropeptide Y overflow and metabolism in skeletal muscle arterioles

The purpose of this study was to characterize neuropeptide Y (NPY) overflow and metabolism from isolated skeletal muscle arterioles of female rats. Gastrocnemius first-order arterioles were removed from young (2 months), young adult (6 months) and middle-aged (12 months) F344 female rats. Arterioles were isolated, cannulated and pressurized in a microvessel bath with field stimulation electrodes. NPY overflow from isolated arterioles was assessed at 0 s and 30 s post-field stimulation. Dipeptidyl peptidase IV (DPPIV) activity was quantified via fluorometric assay of whole vessel homogenate. In young adult and middle-aged rats, NPY overflow increased 0 s and 30 s following field stimulation. In young adult rats, DPPIV inhibition resulted in an increase in NPY overflow at 30 s, while middle-aged rats had no increase in NPY overflow with DPPIV inhibition (P <0.05). DPPIV activity was influenced by factors such as age, vessel type, and endothelium (P <0.05). The present data suggest that DPPIV plays a significant role in modulating the actions of NPY in arterioles of young adult females; however, this role appears to diminish with age.

Inhibition of Urinary Bladder Carcinogenesis by Aqueous Extract of Sclerotia of Polyporus umbellatus Fries and Polyporus Polysaccharide

The study aimed to evaluate inhibition effect of sclerotia of Polyporus umbellatus Fries aqueous extract (SPUE) and polyporus polysaccharide (PPS) on bladder cancer, then to measure their effect on mRNA expression of glutathione S-transferase π (GSTPi) and NAD(P)H:quinone oxidoreductase 1 (NQO1) in female Fischer-344 rats model. The model rats were induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) for a period of 8 weeks and saccharin for 12 weeks. SPUE (50 mg/kg, 250 mg/kg, 500 mg/kg) and PPS (28 mg/kg) were orally administrated to the model rats during the whole study. Compared to the control group, a more preventive effect of SPUE and PPS treatment on bladder cancer was discovered, higher mRNA upregulation of GSTpi and NQO1 was seen in the treatment group. Furthermore, the GSTPi and NQO1 mRNA upregulated level in the low-dose group (SPUE 50 mg/kg) was at maximum. In brief, SPUE and PPS are highly effective in inhibiting bladder carcinogenesis in rats, which may be associated with upregulation of GSTPi and NQO1 in the bladder.