Fischer F344 Rats Research Articles

Aging Reduces Cardiac Contractility by Altering Cardiac Regulatory and Contractile Proteins

Aging is associated with an increased incidence of cardiac dysfunction. We hypothesized that aging associated changes in cardiac regulatory and contractile proteins produce contractile dysfunction. We assessed regulatory and contractile protein expression as well as contractility in left ventricular cardiac muscle from Fischer F-344 rats at 6-, 18-, and 24-month of age. Age-related changes in the expression of β-MHC as well as the expression and phosphorylation of troponin I (TnI) and cardiac myosin binding C protein (MyBP-C) were assessed by Western blot. Aging was associated with an increase in the expression of β-MHC and the phosphorylation of both TnI at Ser23/24 and MyBP-C. Mechanical properties were assessed in Triton-X permeabilized anterior papillary muscle from the left ventricle from both 6- and 24-month old rats. In papillary muscle of 24-month old rats, there was a decrease in maximum Ca2+ activated force, Ca2+ sensitivity of force and the rate constant for force recovery after quick release (ktr). Additionally, cardiomyocytes were isolated from the left ventricle at 6-, 18-, and 24-month of age using a modified Langendorff system. Cardiomyocytes were then loaded with fura-2 AM to measure cytosolic calcium ([Ca2+]cyt) responses to electrical field stimulation (0.5 Hz). [Ca2+]cyt and contractile (sarcomere length shortening) responses to stimulation were simultaneously measured using an IonOptix system. In older cardiomyocytes, the amplitude of evoked [Ca2+]cyt responses was reduced while there was no difference in the kinetics of the [Ca2+]cyt transient. The extent of sarcomere length shortening as well as the rates of both shortening and relaxation were reduced. These results demonstrate that aging is associated with changes in the expression and phosphorylation of contractile and regulatory proteins, which produce changes in cardiac contractility. Supported by a Mayo CV Prospective Grant (FVB). This is the full abstract presented at the American Physiology Summit 2024 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.

Comparative genomic analysis of Fischer F344 rat livers exposed for 90 days to 3-methylfuran or its parental compound furan

Furan is a naturally forming compound found in heat-processed foods such as coffee, canned meats, and jarred baby food. It is concurrently found with analogues including 2-methylfuran (2-MF) and 3-methylfuran (3-MF), and toxicity studies demonstrate all are potent liver toxins. Toxicity studies found 3-MF is more toxic than either furan, or 2-MF. The present analysis assesses the transcriptional response in liver samples taken from male Fischer (F344) rats exposed to furan or 3-MF from 0 to 2.0 and 0–1.0 mg/kg bw/day, respectively, for 90 days. Transcriptional analyses found decreased liver function and fatty acid metabolism are common responses to both furan and 3-MF exposure. Furan liver injury promotes a ductular reaction through Hippo and TGFB signalling, which combined with increased immune response results in ameliorating perturbed bile acid homeostasis in treated rats. Failure to activate these pathways in 3-MF exposed rats and decreased p53 activity leads to cholestasis, and increased toxicity. Finally, BMD analysis indicate many of the most sensitive pathways affected by furan and 3-MF exposure relate to metabolism - malate dehydrogenase and glucose metabolism with BMDLs of 0.03 and 0.01 mg/kg bw/day for furan and 3-MF exposure, respectively, which agrees with BMDLs previously reported for apical and microarray data.

The SGLT2i Dapagliflozin Reduces RV Mass Independent of Changes in RV Pressure Induced by Pulmonary Artery Banding.

Sodium glucose linked transporter 2 (SGLT2) inhibition not only reduces morbidity and mortality in patients with diagnosed heart failure but also prevents the development of heart failure hospitalization in those at risk. While studies to date have focused on the role of SGLT2 inhibition in left ventricular failure, whether this drug class is efficacious in the treatment and prevention of right heart failure has not been explored. We hypothesized that SGLT2 inhibition would reduce the structural, functional, and molecular responses to pressure overload of the right ventricle. Thirteen-week-old Fischer F344 rats underwent pulmonary artery banding (PAB) or sham surgery prior to being randomized to receive either the SGLT2 inhibitor: dapagliflozin (0.5mg/kg/day) or vehicle by oral gavage. After 6weeks of treatment, animals underwent transthoracic echocardiography and invasive hemodynamic studies. Animals were then terminated, and their hearts harvested for structural and molecular analyses. PAB induced features consistent with a compensatory response to increased right ventricular (RV) afterload with elevated mass, end systolic pressure, collagen content, and alteration in calcium handling protein expression (all p < 0.05 when compared to sham + vehicle). Dapagliflozin reduced RV mass, including both wet and dry weight as well as normalizing the protein expression of SERCA 2A, phospho-AMPK and LC3I/II ratio expression (all p < 0.05). Dapagliflozin reduces the structural, functional, and molecular manifestations of right ventricular pressure overload. Whether amelioration of these early changes in the RV may ultimately lead to a reduction in RV failure remains to be determined.

PD37-08 PHOSPHATIDYLDIGLYCEROL-BASED THERMOSENSITIVE LIPOSOMES SHOW SUPERIOR THERAPEUTIC EFFICACY AGAINST MUSCLE-INVASIVE BLADDER CANCER IN VIVO

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I (PD37)1 Sep 2021PD37-08 PHOSPHATIDYLDIGLYCEROL-BASED THERMOSENSITIVE LIPOSOMES SHOW SUPERIOR THERAPEUTIC EFFICACY AGAINST MUSCLE-INVASIVE BLADDER CANCER IN VIVO Iris Brummelhuis, Michiel Simons, Lars Lindner, Simone Kort, Sytse de Jong, Martin Hossann, Alfred Witjes, and Egbert Oosterwijk Iris BrummelhuisIris Brummelhuis More articles by this author , Michiel SimonsMichiel Simons More articles by this author , Lars LindnerLars Lindner More articles by this author , Simone KortSimone Kort More articles by this author , Sytse de JongSytse de Jong More articles by this author , Martin HossannMartin Hossann More articles by this author , Alfred WitjesAlfred Witjes More articles by this author , and Egbert OosterwijkEgbert Oosterwijk More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002047.08AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Recommended treatments for muscle-invasive bladder cancer (MIBC) are associated with substantial morbidity. Hyperthermia (HT) triggered drug release from phosphatidyldiglycerol-based thermosensitive liposomes (DPPG2-TSL) might prevent surgical bladder removal and toxicity from systemic chemotherapy. We aimed to assess the efficacy of DPPG2-TSL encapsulated doxorubicin (DOX; DPPG2-TSL-DOX) combined with bladder HT, compared to free systemic and intravesical DOX, in a syngeneic orthotopic rat urothelial carcinoma model. METHODS: A total of 191 female Fischer F344 rats were used. Bladder tumors were initiated by inoculation of AY-27 cells and tumor bearing rats were selected with cystoscopy and semi-randomized over treatment groups. At day 5 and 8, animals were treated with 2 mg/kg DOX in different treatment modalities: intravenous (iv) DPPG2-TSL-DOX with HT, iv free DOX without HT, intravesical DOX without HT, intravesical DOX with HT or no treatment (control group), respectively. Animals were euthanized at day 14 and complete tumor response was assessed by histopathological evaluation. RESULTS: Treatment with iv DPPG2-TSL-DOX with HT resulted in the highest rate of animals with complete tumor response of 70%, which was significantly better than iv free DOX (18%, p=0.02) and no treatment (0%, p=0.001), and non-significantly better than intravesical DOX with (43%, p=0.35) or without HT (50%, p=0.41; Figure 1). All rats receiving intravesical DOX with HT (7/7) and 24% (5/21) of rats treated with DPPG2-TSL-DOX containing the same DOX dose with HT had to be euthanized before day 14 because of >15% body weight loss, while some rats showed dilated ureters (3/7 and 4/21) and urine retention (2/7 and 1/21, respectively).Figure 1. Tumor stage as determined by histopathological evaluation per group. Green colored parts of bars represent T1 tumors, and purple T2. CONCLUSIONS: Treatment with DPPG2-TSL-DOX combined with intravesical HT outperformed systemic and intravesical DOX in a syngeneic orthotopic rat UC model. There might be a role for DPPG2-TSL encapsulating chemotherapeutics in treatment of MIBC in the future. Source of Funding: This study was partially sponsored by Thermosome © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e658-e658 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Iris Brummelhuis More articles by this author Michiel Simons More articles by this author Lars Lindner More articles by this author Simone Kort More articles by this author Sytse de Jong More articles by this author Martin Hossann More articles by this author Alfred Witjes More articles by this author Egbert Oosterwijk More articles by this author Expand All Advertisement Loading ...

DPPG2-based thermosensitive liposomes as drug delivery system for effective muscle-invasive bladder cancer treatment in vivo

Purpose Recommended treatments for muscle-invasive bladder cancer (MIBC) come with considerable morbidity. Hyperthermia (HT) triggered drug release from phosphatidylglycerol-based thermosensitive liposomes (DPPG2-TSL) might prevent surgical bladder removal and toxicity from systemic chemotherapy. We aimed to assess the efficacy of DPPG2-TSL with HT in a syngeneic orthotopic rat urothelial carcinoma model. Methods A total of 191 female Fischer F344 rats were used. Bladder tumors were initiated by inoculation of AY-27 cells and tumor-bearing rats were selected with cystoscopy and semi-randomized over treatment groups. On days 5 and 8, animals were treated with DOX in different treatment modalities: intravenous (iv) DPPG2-TSL-DOX with HT, iv free DOX without HT, intravesical DOX without HT, intravesical DOX with HT or no treatment (control group), respectively. Animals were euthanized on day 14 and complete tumor response was assessed by histopathological evaluation. Results Iv DPPG2-TSL-DOX + HT resulted in a favorable rate of animals with complete tumor response (70%), compared to iv free DOX (18%, p = .02), no treatment (0%, p = .001), and intravesical DOX with (43%, p = .35) or without HT (50%, p = .41). All rats receiving intravesical DOX with HT and 24% of rats treated with DPPG2-TSL-DOX containing the same DOX dose with HT had to be euthanized before day 14 because of substantial bodyweight loss, which was associated with dilated ureters urine retention in a few rats. Conclusion Treatment with DPPG2-TSL-DOX combined with intravesical HT outperformed systemic and intravesical DOX in vivo. There might be a role for DPPG2-TSL encapsulating chemotherapeutics in the treatment of MIBC in the future.

Development and characterization of a predictive microCT-based non-union model in Fischer F344 rats.

Non-unions remain a clinical problem and are characterised by the failure to heal after a defined period of time. Current preclinical non-union models apply a wide variety of techniques to diminish intrinsic healing potential deviating from the clinical situation. The aim of this study was to develop and characterise a non-union model in rats using internal plate fixation without the need for additional healing insults, whereby bone healing can be longitudinally assessed using microCT. It was hypothesized that healing/non-unions can be accurately predicted at early time points by microCT. Female, skeletally mature Fischer F344 rats received a 2mm or 1mm femoral osteotomy, stabilized with either a 2mm thick plate or a 1.25mm thick plate. Healing was monitored by microCT over 14weeks and histological analysis at euthanasia. The mechanical environment was characterised using finite element (FE) modelling and biomechanical testing. The majority of animals receiving the 2mm thick plate displayed poor healing responses in both the 2mm and 1mm defect size groups. Bone and cartilage formation were markedly improved using the 1.25mm thick plate. MicroCT could accurately predict bone forming capacity at early time points (3-4weeks). The 2mm thick plating system confers poor healing responses in female Fischer F344 rats, comparable to atrophic non-unions. By reducing plate thickness to increase interfragmentary strain within the defect site healing is improved, leading to borderline healing situations or increased abundance of cartilage tissue present in the defect site with ultimate failure to bridge the defect (hypertrophic non-union). Furthermore, microCT can reliably identify delayed/non-healing animals within 4weeks, thereby allowing their selective targeting for the testing of novel, clinically relevant treatment strategies in different clinical situations aimed at restoring impaired bone healing.

Enhanced MRI-guided radiotherapy with gadolinium-based nanoparticles: preclinical evaluation with an MRI-linac

BackgroundThe AGuIX® (NH TherAguix) nanoparticle has been developed to enhance radiotherapy treatment and provide strong MR contrast. These two properties have previously been investigated separately and progressed to clinical trial following a clinical workflow of separate MR imaging followed some time later by radiotherapy treatment. The recent development of MRI-linacs (combined Magnetic Resonance Imaging–linear accelerator systems enabling MRI-guided radiotherapy) opens up a new workflow where MR confirmation of nanoparticle uptake can be carried out at the time of treatment. A preclinical study was carried out to assess the suitability of a gadolinium-containing nanoparticle AGuIX® (NH TherAguix) for nano-enhanced image-guided radiotherapy on an MRI-linac.MethodsTreatments were carried out on F344 Fischer rats bearing a 9L glioma brain tumour. Animals received either: (A) no treatment; (B) injection of nanoparticles followed by MRI; (C) radiotherapy with MRI; or (D) injection of nanoparticles followed by radiotherapy with MRI. Pre-clinical irradiations were carried out on the 1.0 T, 6 MV in-line Australian MRI-linac. Imaging used a custom head coil specially designed to minimise interference from the radiotherapy beam. Anaesthetised rats were not restrained during treatment but were monitored with a cine-MRI sequence. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis was used to quantify residual gadolinium in the brain in normal and tumour tissue.ResultsA preclinical evaluation of nano-enhanced radiation treatment has been carried out on a 1.0 T MRI-linac, establishing a workflow on these novel systems. Extension of life when combining radiotherapy with nanoparticles was not statistically different from that for rats receiving radiotherapy only. However, there was no detrimental effect for animals receiving nanoparticles and radiation treatment in the magnetic field compared with control branches. Cine-MR imaging was sufficient to carry out monitoring of anaesthetised animals during treatment. AGuIX nanoparticles demonstrated good positive contrast on the MRI-linac system allowing confirmation of tumour extent and nanoparticle uptake at the time of treatment.ConclusionsNovel nano-enhanced radiotherapy with gadolinium-containing nanoparticles is ideally suited for implementation on an MRI-linac, allowing a workflow with time-of-treatment imaging. Live irradiations using this treatment workflow, carried out for the first time at the Australian MRI-linac, confirm the safety and feasibility of performing MRI-guided radiotherapy with AGuIX® nanoparticles. Follow-up studies are needed to demonstrate on an MRI-linac the radiation enhancement effects previously shown with conventional radiotherapy.

“(−)‐Epicatechin reduces inflammation biomarkers in plasma and ameliorates cardiac fibrosis in a female rat model of pre‐HFpEF”

The consumption of polyphenolic compounds, specifically flavonoids, have been associated with beneficial impacts on cardiovascular health. Flavonoids are found in plants and fruits such as cacao, berries, grapes and tea leaves. Cacao seeds contain high levels of the flavanol (−)‐ epicatechin. (−)‐Epicatechin (Epi) is therefore present in products such as dark chocolate or cocoa powder and has proven to have beneficial effects on the cardiovascular system. In the heart, it can reduce infarct size and apparently, myocardial fibrosis. Thus, we aimed to examine the potential of Epi to reduce myocardial fibrosis and improving passive and active left ventricular function and structure in an aging female rat model of pre‐HFpEF characterized by low estrogen levels, excess weight and hypertension. Female, 18‐month‐old, Fischer F344 rats were allocated into an ovariectomy + 10% fructose (OVF), and ovariectomy + 10% fructose + Epi (OVFE) (n=9–10/group). Epi treatment (1 mg/kg/day) was provided by gavage during a 30‐day period (from 21 to 22 months of age) while control animals received water. Left ventricular (LV) structure/function was assessed monthly by echocardiography. At 22 months of age, animals were anesthetized, and LV function was evaluated using a Millar conductance catheter. Histological measures of chamber morphometry, inflammation and collagen density (fibrosis) were also obtained. With treatment, no major structural and functional modifications were detected by echocardiography or by conductance‐catheter based hemodynamics. Passive left ventricular pressure volume curves were significantly right shifted with Epi while circumferential epicardial strains were reduced. The histological analysis of collagen quantification using Sirius red indicated that Epi treatment significantly reduced overall levels of myocardial fibrosis. Epi also significantly reduced plasma levels of pro‐fibrotic and pro‐inflammatory cytokines. In conclusion, Epi appears capable of reducing cardiac fibrosis in an animal model of pre‐HFpEF which correlate with reductions in cytokine plasma levels. However, reductions in tissue fibrosis did not translate into major modifications in passive or active chamber structure and function. This model may serve to understand the role that estrogen depletion and weight gain may have in the development of HFpEF.Support or Funding InformationFunding was provided by the Department of Defense PR150090 and National Institutes of Health DK98717, AG47326 to F.V. and Consejo Nacional de Ciencia y Tecnologia, Mexico to G. C.

Mechanisms of atrial fibrillation in aged rats with heart failure with preserved ejection fraction

Although ∼20% of the elderly population develops atrial fibrillation (AF), little is known about the mechanisms. Heart failure with preserved ejection fraction (HFpEF), which is associated with AF, is more common in aged women than in men. The purpose of this study was to identify potential mechanisms of AF in an age-related HFpEF model. In aged female Fischer F344 rats (21- to 24-month-old), which are prone to HFpEF, we induced AF by atrial pacing. Young Fischer F344 female rats (3- to 4-month-old) and age-matched Sprague Dawley female rats (27-month-old) served as controls. Phenotyping included echocardiography to assess left ventricular structure/function; invivo electrophysiology and exvivo high-resolution optical mapping to assess AF vulnerability; systemic and atrial inflammatory profiling; atrial histology; and expression of inflammasome signaling proteins. Aged rats developed left ventricular hypertrophy, left atrial enlargement, diastolic dysfunction, and pulmonary congestion, without ejection fraction impairment, thus meeting the criteria for HFpEF. Increased serum inflammatory markers, hypertension, and obesity further characterize aged females. Sinoatrial and atrioventricular node dysfunction was associated with the high inducibility of AF in aged rats. Exvivo electrical activation mapping revealed abnormal β-adrenergic responsiveness and slowed conduction velocity. Atrial inflammasome signaling was enhanced in aged rats, which may contribute to fibrotic remodeling and high AF susceptibility. Together, our data demonstrate that aging-related atrial remodeling and HFpEF are associated with atrial enlargement, fibrosis, conduction abnormalities, and nodal dysfunction, favoring a substrate conducive to AF.

Load-independent effects of empagliflozin contribute to improved cardiac function in experimental heart failure with reduced ejection fraction

Background and aimsSodium–glucose linked cotransporter-2 (SGLT2) inhibitors reduce the likelihood of hospitalization for heart failure and cardiovascular death in both diabetic and non-diabetic individuals with reduced ejection fraction heart failure. Because SGLT2 inhibitors lead to volume contraction with reductions in both preload and afterload, these load-dependent factors are thought to be major contributors to the cardioprotective effects of the drug class. Beyond these effects, we hypothesized that SGLT2 inhibitors may also improve intrinsic cardiac function, independent of loading conditions.MethodsPressure–volume (P–V) relationship analysis was used to elucidate changes in intrinsic cardiac function, independent of alterations in loading conditions in animals with experimental myocardial infarction, a well-established model of HFrEF. Ten-week old, non-diabetic Fischer F344 rats underwent ligation of the left anterior descending (LAD) coronary artery to induce myocardial infarction (MI) of the left ventricle (LV). Following confirmation of infarct size with echocardiography 1-week post MI, animals were randomized to receive vehicle, or the SGLT2 inhibitor, empagliflozin. Cardiac function was assessed by conductance catheterization just prior to termination 6 weeks later.ResultsThe circumferential extent of MI in animals that were subsequently randomized to vehicle or empagliflozin groups was similar. Empagliflozin did not affect fractional shortening (FS) as assessed by echocardiography. In contrast, load-insensitive measures of cardiac function were substantially improved with empagliflozin. Load-independent measures of cardiac contractility, preload recruitable stroke work (PRSW) and end-systolic pressure volume relationship (ESPVR) were higher in rats that had received empagliflozin. Consistent with enhanced cardiac performance in the heart failure setting, systolic blood pressure (SBP) was higher in rats that had received empagliflozin despite its diuretic effects. A trend to improved diastolic function, as evidenced by reduction in left ventricular end-diastolic pressure (LVEDP) was also seen with empagliflozin. MI animals treated with vehicle demonstrated myocyte hypertrophy, interstitial fibrosis and evidence for changes in key calcium handling proteins (all p < 0.05) that were not affected by empagliflozin therapy.ConclusionEmpagliflozin therapy improves cardiac function independent of loading conditions. These findings suggest that its salutary effects are, at least in part, due to actions beyond a direct effect of reduced preload and afterload.

Longitudinal evaluation of immediate inflammatory responses after intravitreal AAV2 injection in rats by optical coherence tomography

Gene therapy has been proposed as a feasible strategy for RGC survival and optic nerve regeneration. Some preclinical and clinical studies revealed intraocular inflammation after intravitreal injection of adeno-associated virus (AAV) by slit-lamp or indirect ophthalmoscope. Here we evaluate the longitudinal profile of immediate inflammatory responses after AAV2 injection in rat retina and vitreous body by optical coherence tomography (OCT). Adult Fischer F344 rats were intravitreally injected once with saline, AAV2 or zymosan. Retinal thickness and cell infiltration were recorded by OCT longitudinally for 2 months and verified by histological analysis. The transduction rate of single intravitreal AAV2 injection was 21.3 ± 4.9% of whole retina, and the transduction efficiency on RGCs was 91.5 ± 2.5% in the transduced area. Significant increase in cell infiltration was observed from Day 1–3 after AAV2 injection, compared to very few infiltrating cells observed in the saline-injected group. The infiltrating cells ceased at Day 5 after intravitreal injection and remained absent at 2 months. The thicknesses of total and inner retina were increased along Day 1–3 after AAV2 injection, but reverted to normal afterwards. The surviving RGCs in the AAV2-injected groups at Day 14 showed no significant difference compared to saline-injected group. In summary, this study revealed the immediate inflammatory responses and retinal edema after intravitreal AAV2 injection in normal rats, without influencing long-term retinal thickness and RGC survival. OCT can be implemented for the time-lapse in vivo evaluation of inflammatory response after AAV-mediated gene therapy through intravitreal injection.

The impact of short-term machine perfusion on the risk of cancer recurrence after rat liver transplantation with donors after circulatory death.

Hypothermic and normothermic ex vivo liver perfusions promote organ recovery after donation after circulatory death (DCD). We tested whether these perfusions can reduce the risk of hepatocellular carcinoma (HCC) recurrence in a 1h-DCD syngeneic transplantation model, using Fischer F344 rats. DCD grafts were machine perfused for 2h with hypothermic perfusion (HOPE) or normothermic perfusion (NORMO), and transplanted. After reperfusion, we injected HCC cells into the vena porta. On day 28 after transplantation, we assessed tumour volumes by MRI. Control rats included transplantations with Fresh and non-perfused DCD livers. We observed apoptotic-necrotic hepatocyte foci in all DCD grafts, which were more visible than in the Fresh liver grafts. Normothermic perfusion allowed a faster post-transplant recovery, with lower day 1 levels of transaminases compared with the other DCD. Overall, survival was similar in all four groups and all animals developed HCCs. Total tumor volume was lower in the Fresh liver recipients compared to the DCD and DCD+HOPE recipients. Volumes in DCD+NORMO recipients were not significantly different from those in the Fresh group. This experiment confirms that ischemia/reperfusion injury promotes HCC cell engraftment/growth after DCD liver transplantation. Using the present extreme 1h ischemia model, both hypothermic and normothermic perfusions were not effective in reducing this risk.

Immunohistochemical Review of Leydig Cell Lesions in Ochratoxin A-Treated Fischer Rats and Controls.

Ochratoxin A is best known as a potent renal carcinogen in male rats and mice after necessarily protracted ingestion, although valid extrapolation to any human disease has not been verified. The hypothesis that the toxin is a cause of human testicular cancer was proposed a decade ago and has proliferated since, partly through incomplete study of the scientific literature. Archived tumorous rat testes were available from Fischer F344 rats exposed to continuous dietary exposure for half of or the whole life in London in the 2000s. Renal cancer occurred in some of these cases and testicular tumours were observed frequently, as expected, in both treated and untreated animals. Application of clinical immunohistochemistry has for the first time consistently diagnosed the testicular hypertrophy in toxin-treated rats as Leydig cell tumours. Comparison is made with similar analysis of tumorous testes from control (untreated) rats from U.S. National Toxicology Program studies, both of ochratoxin A (1989) and the more recent one on Ginkgo biloba. All have been found to have identical pathology as being of sex cord-stromal origin. Such are rare in humans, most being of germinal cell origin. The absence of experimental evidence of any specific rat testicular cellular pathology attributable to long-term dietary ochratoxin A exposure discredits any experimental animal evidence of testicular tumorigenicity. Thus, no epidemiological connection between ochratoxin A and the incidence of human testicular cancer can be justified scientifically.

Green Tea Extract Ameliorates Ischemia-Induced Retinal Ganglion Cell Degeneration in Rats.

Purpose Oxidative stress induced by reduced blood circulation is a critical pathological damage to retinal ganglion cells (RGCs) in glaucoma. We previously showed that green tea extract (GTE) and its catechin constituents alleviate sodium iodate-induced retinal degeneration in rats. Here, we investigated the therapeutic effect of GTE on ischemia-induced RGC degeneration in rats. Methods RGC degeneration was induced by ischemic reperfusion in adult Fischer F344 rats. Green tea extract (Theaphenon E) was intragastrically administered 4 times within 48 hours after ischemia. RGC survival, pupillary light reflex, expressions of cell apoptosis, oxidative stress, and inflammation-related proteins were studied. Results Ischemic reperfusion significantly induced apoptotic RGCs, RGC loss, and larger constricted pupil area compared to the untreated normal rats. Expressions of activated caspase-3 and caspase-8, Sod2, and inflammation-related proteins as well as p38 phosphorylation were significantly upregulated in the ischemia-injured rats. Compared to the saline-fed ischemic rats, significantly higher number of surviving RGCs, less apoptotic RGCs, and smaller constricted pupil area were observed in the GTE-fed ischemic rats. GTE also reduced the increased protein expressions caused by ischemic injury but enhanced the Jak phosphorylation in the retina. Notably, green tea extract did not affect the survival of RGCs in the uninjured normal rats. Conclusions In summary, GTE offers neuroprotection to RGCs under ischemic challenge, suggesting a potential therapeutic strategy for glaucoma and optic neuropathies.

Rat strain response differences upon exposure to technical or alpha hexabromocyclododecane

Hexabromocyclododecane (HBCD) is a brominated flame retardant which was recommended by a UN expert body under the Stockholm Convention to be eliminated from the global marketplace in 2011; however, due to its ability to persist in the environment, undergo long-range transport and bioaccumulate, it remains a concern for human health. The commercial mix of HBCD (T-HBCD) consists of α-HBCD, β-HBCD and γ-HBCD. Although the γ-HBCD (79%) isomer is the predominant isomer of T-HBCD, the most bioaccumulative isomer detected in mammals is the α-HBCD isomer. This study was undertaken to investigate three rat strains treated with commercial grade (technical) HBCD or HBCD enriched with the α isomer (A-HBCD) and to examine strain- and sex-related differences in response to exposure. Female Sprague Dawley (SD), Wistar (WI) and Fischer F344 (FI) rats were exposed for 28 days to either T-HBCD or A-HBCD in feed, at doses of 0, 250, 1250 and 5000 mg/kg diet. The FI rodent strain was found to be the most sensitive to effects of HBCD based on the greatest number of significantly affected endpoints which indicated that T-HBCD primarily affected liver and thyroid, resulting in multiple health effects. Consequently, male FI were included in the study and exposed to T- and A-HBCD. Histopathological data supports previously reported effects of HBCD on the thyroid and endocrine system although the effects in FI rats are significantly elevated compared to other strains. As with T-HBCD, liver and thyroid were found to be target organs of A-HBCD. Sex differences, specifically in tissue concentration levels, immune response parameters and in number and severity of thyroid and liver lesions, following exposure to either T- or A-HBCD were apparent, with treatment eliciting a greater response in males. Residue analysis revealed that α-HBCD is more bioaccumulative than γ-HBCD in all rodent strains, with levels of HBCD in animals treated with A-HBCD several fold higher for all tissues tested (7–11 fold at the highest dose). Thus, residue data supports the selective uptake (implies there are differences in bioavailability and/or bioaccumulation; is this the case or do certain isomers simply have a longer half-life) of specific isomers, with α-HBCD > γ-HBCD. Taken together, our study highlights the importance of selecting the most appropriate strain and of including both sexes in studies to ensure that sex-related differences in response to test chemical is taken into consideration. Moreover, ours is the first study to show the effects of a sub-acute exposure to a diet containing only HBCD enriched for the α isomer, which better represents the isomer ratios present in the biota due to bioaccumulation.

The Decline of Glutamate‐Stimulated Norepinephrine Release in Aging Rat Brain

Unravelling and understanding the molecular and neurochemical changes of the aging brain is key to early intervention strategies. Glutamate (Glu) neurotransmission, the major excitatory neurotransmitter in the brain, is mechanistically involved in memory, learning performance, and other aspects of cognition. Moreover, as the brain ages, the glutamatergic signaling system mediated through brain N‐methyl‐D‐aspartate (NMDA) receptors becomes hypofunctional, and this decline may be associated with cognitive dysfunction in normal aging and in disease states. We have developed an in vitro assay that measures Glu‐stimulated release of neurotransmitters in rat brain slices. Using this assay, we compared changes in Glu‐stimulated release of [3H]‐norepinephrine (NE) from the cerebral cortex and hippocampus in young (2–3 months old) versus old (21–23 months old) Fischer F344 rats. Our findings demonstrate that that there is a pronounced deficit (approximately 30–40%) of Glu‐stimulated [3H]‐NE release in the aged rat brain in comparison to younger controls. In the same tissues, potassium (K+) stimulated‐NE release is similar in brains from young and old rats, suggesting that the age‐related decline in in Glu‐stimulated NE release is related to a change in NMDA receptors. Notably, the deficit in Glu‐stimulated [3H]‐NE release in both brain regions was completely rescued by the addition of 1 uM d‐amphetamine sulfate to the tissue slices. Overall, these findings reveal an important deficit in NE release in the aging brain, which may have important implications for cognitive dysfunction in aging. Moreover, this deficit can be reversed with amphetamine and possibly with similar drugs. These studies have clear clinical implications for the use of CNS stimulants as a therapeutic intervention for early cognitive impairment.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduction of Muscle-Invasive Tumors by Photodynamic Therapy with Tetrahydroporphyrin-Tetratosylat in an Orthotopic Rat Bladder Cancer Model.

Photodynamic therapy (PDT) is a promising option for minimal-invasive treatment of bladder cancer. Efficacy of PDT in muscle-invasive urothelial cancer is still hampered by low tissue penetration of most photosensitizers due to short excitation wavelength. The novel light reactive agent tetrahydroporphyrin-tetratosylat (THPTS) is excitable at near-infrared (760 nm), allowing tissue penetration of up to 15 mm. Here, we established an orthotopic rat bladder cancer model and examined the effects of THPTS-PDT on tumor growth in vivo, and analyzed molecular mechanisms in vitro We examined pharmacokinetics and subcellular localization, and evoked cell death mode in cultured rat urothelial carcinoma cells (AY-27). We used female F344 Fischer rats for in vivo studies. Ten rats each were used for THPTS-PDT and light-only control. Bladders were evaluated by macroscopy and histology. Temperature-dependent THPTS uptake resulted in endosomal/lysosomal localization. PDT (0-50 μmol/L THPTS; 10 J/cm2) induced early onset of apoptosis leading to dose-dependent cytotoxicity in AY-27 cells. Single-time transurethral THPTS-PDT (100 μmol/L THPTS; 10 J/cm2) in F344 rats led to significant reduction of muscle-invasive tumor number (2/10 vs. 7/10 in controls) and total tumor volume (60% reduction) 2 weeks after PDT, while sparing healthy tissue. Here, we report for the first time effective tumor growth control by PDT in vivo THPTS is a promising new photosensitizer with the advantage of higher therapeutic depth and the potential of high-selective therapy in muscle-invasive urothelial cancer. This approach possibly allows minimal-invasive bladder preserving treatment of bladder cancer without systemic side effects.

Unmasking of oestrogen-dependent changes in left ventricular structure and function in aged female rats: a potential model for pre-heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is seen more frequently in older women; risk factors include age, hypertension and excess weight. No female animal models of early stage remodelling (pre-HFpEF) have examined the effects that the convergence of such factors have on cardiac structure and function. In this study, we demonstrate that ageing can lead to the development of mild chamber remodelling, diffuse fibrosis and loss of diastolic function. The loss of oestrogens further aggravates such changes by leading to a notable drop in cardiac output (while preserving normal ejection fraction) in the presence of diffuse fibrosis that is more predominant in endocardium and is accompanied by papillary fibrosis. Excess weight did not markedly aggravate such findings. This animal model recapitulates many of the features recognized in older, female HFpEF patients and thus, may serve to examine the effects of candidate therapeutic agents. Two-thirds of patients with heart failure with preserved ejection fraction (HFpEF) are older women, and risk factors include hypertension and excess weight/obesity. Pathophysiological factors that drive early disease development (before heart failure ensues) remain obscure and female animal models are lacking. The study evaluated the intersecting roles of ageing, oestrogen depletion and excess weight on altering cardiac structure/function. Female, 18-month-old, Fischer F344 rats were divided into an aged group, aged + ovariectomy (OVX) and aged + ovariectomy + 10% fructose (OVF) in drinking water (n=8-16/group) to induce weight gain. Left ventricular (LV) structure/function was monitored by echocardiography. At 22months of age, animals were anaesthetized and catheter-based haemodynamics evaluated, followed by histological measures of chamber morphometry and collagen density. All aged animals developed hypertension. OVF animals increased body weight. Echocardiography only detected mild chamber remodelling with ageing while intraventricular pressure-volume loop analysis showed significant (P<0.05) decreases vs. ageing in stroke volume (13% OVX and 15% for OVF), stroke work (34% and 52%) and cardiac output (29% and 27%), and increases in relaxation time (10% OVX) with preserved ejection fraction. Histology indicated papillary and interstitial fibrosis with ageing, which was higher in the endocardium of OVX and OVF groups. With ageing, ovariectomy leads to the loss of diastolic and global LV function while preserving ejection fraction. This model recapitulates many cardiovascular features present in HFpEF patients and may help understand the roles that ageing and oestrogen depletion play in early (pre-HFpEF) disease development.

Abstract 274: Selective chemopreventive efficacy of 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp) towards lung cancer

Abstract 1,4-Phenylenebis(methylene)selenocyanate (p-XSC) has been shown to inhibit tobacco carcinogen NNK induced lung tumor development in several animal models. This had placed p-XSC on the National Cancer Institute´s (NCI) list of chemopreventive agents for clinical development, but there were systemic toxicity issues. p-XSC metabolizes through the formation of active bis-selenol (p-XSeH) along with the release of poisonous hydrogen cyanide (HCN). We recently developed p-XS-Asp, with a rationale that it would cleave in vivo to release the active p-XSeH and aspirin, thus making the compound less toxic and possibly more potent than p-XSC. Indeed, we previously presented (AACR Annual Meeting 2014) that p-XS-Asp inhibited NNK-induced lung tumorigenesis in A/J mice more effectively than p-XSC, and was also more tolerable. At doses of 15 ppm and 7.5 ppm Se, p-XS-Asp showed a significantly marked decrease in the percentage of lung cancer incidence in vivo with only 50% and 87% of tumor incidence, as compared to p-XSC (79% and 100%), respectively. NNK-control showed an 100% tumor incidence. Likewise, the tumor multiplicity for p-XS-Asp group was 0.87 and 1.93 tumors/mouse as compared to the NNK-control (11.53) and p-XSC (1.66 and 4.10 tumors/mouse, respectively) at the two doses tested. Notably, blood chemistry and tissue analyses did not show systemic toxicity for the p-XS-Asp fed group. We have now evaluated the underlying mechanisms of lung cancer preventive action of p-XS-Asp and its efficacy for inhibiting azoxymethane (AOM)- and dimethyl hydrazine (DMH)-induced Aberrant Crypt Foci (ACF) in Fischer F344 rats. At a dose of 7.5 ppm Se, p-XS-Asp was able to restore the expression of several genes (MMP9, COX-2, Myc, SphK1 and RELA), that were over-expressed in the NNK group, to control or even lower levels. The AKT1 gene expression was much lower in the lung tissue of p-XS-Asp treated mice at this dose compared to both negative and NNK control groups. Therefore, p-XS-Asp might be exerting its chemopreventive effect on NNK-induce lung tumorigenesis via inhibiting COX-2 mediated PI3K/Akt signaling pathway. Interestingly, contrary to the striking inhibition of lung tumorigenesis, p-XS-Asp failed to significantly inhibit AOM- or DMH-induced formation of ACF in Fischer F344 rats. In both AOM and DMH models, aspirin (positive control) significantly reduced the number of ACF and large ACF per area (cm2) by 37.4% and 33.8%, respectively. On the other hand, both p-XSC and p-XS-Asp showed no significant inhibitory effect on the formation of ACF and large ACF. Taken together, our results have shown p-XS-Asp to selectively prevent the lung, but not colon, tumorigenesis, and thus is a promising candidate for further development as a lung cancer preventive agent. Citation Format: Daniel Plano, Srinivasa Ramisetti, Sang-Yub Kim, Manoj Pandey, Cesar Aliaga, Deepkamal N. Karelia, Arthur Berg, Carmen Sanmartin, Junxuan Lu, Shantu Amin, Arun K. Sharma. Selective chemopreventive efficacy of 1,4-phenylenebis(methylene)seleno-aspirin (p-XS-Asp) towards lung cancer [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 274.

Detrimental Effects of Aging, Ovariectomy and Weight Gain on Left Ventricular Structure and Function: A Potential Preclinical Model of Early Stage HFpEF

The incidence of heart failure with preserved ejection fraction (HFpEF) continues to increase and little is known about its pathophysiology. Two‐thirds of HFpEF patients are senile women and risk factors include hypertension and metabolic syndrome. Currently, no drugs are effective for HFpEF treatment and the development of animal models would assist in novel therapy evaluation. We implemented a rodent female animal model of aging, estrogen depletion and weight gain to evaluate the role of these factors in altering cardiac structure/function. Female, 18 month old, Fischer F344 rats were allocated into an aging (A) group, aging + ovariectomy (O) and aging + ovariectomy and 10% fructose (OF) in drinking water (n=8–13/group). For comparison purposes a young (3 month old, n=8) cohort of female rats was included. Left ventricular (LV) structure/function was monitored monthly by echocardiography. At 22 months of age, animals were anesthetized and LV function evaluated using a Millar conductance catheter. Histological measures of chamber morphometry, inflammation and collagen density (fibrosis) were also obtained. OF animals significantly increased body weight vs. A and O. While echocardiography did not detect major alterations in cardiac endpoints, intraventricular pressure‐volume loop analysis evidenced significant (p&lt;0.05) decreases in stroke volume (19% O and 27% for OF vs. A), stroke work (27 O and 31% OF vs. A), cardiac output (43 O and 44% OF vs. A) and increases in Tau (30–35% in A, O and OF vs. young) and LV end‐diastolic pressure volume relationship slope (i.e. stiffness) with ovariectomy in the presence of preserved ejection fraction for all groups. Histomorphometry did not detect changes in chamber dimensions. However, histological analysis indicated progressively increasing levels of inflammatory infiltration, perivascular and interstitial fibrosis with ovariectomy and with fructose supplementation in particular, in the subendocardium (see figure). In conclusion, in the setting of aging, ovariectomy (which depletes estrogens), further deteriorates myocardial microstructure which may facilitate the loss of diastolic and global functional indices in the apparent presence of preserved ejection fraction. This model may serve to understand the role that estrogen depletion and weight gain may have in the development of HFpEF.Support or Funding InformationDoD 150090 to FVThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.