Male Fischer Rats Research Articles

Inhalation of formaldehyde does not induce genotoxic effects in broncho-alveolar lavage (BAL) cells of rats

Male Fischer-344 rats were exposed to formaldehyde (FA) by inhalation for 4 weeks (6h/day, 5 days/week). Groups of six rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15ppm. Potential genotoxic effects in the lung were investigated as part of a comprehensive study on local and systemic toxic and genotoxic effects. Broncho-alveolar lavage (BAL) cells were obtained by lung lavage with physiological saline and counted. From one half of the cells, slides for the micronucleus test (MNT) were prepared by cytocentrifugation; with the other half, the comet assay was performed. DNA migration in the comet assay was measured both directly and after irradiation of the cells with 2Gy gamma-radiation. The latter modification of the comet assay was included to increase its sensitivity for the detection of DNA-protein cross-links (DPX). For the comet assay, four slides were analysed from each cell sample, two without and two with irradiation. From each slide, 50 randomly selected cells were measured by image analysis and tail intensity (% tail DNA) and tail moment were evaluated. The frequency of micronucleated BAL cells was determined in acridine orange-stained slides by analysing 2000 cells per animal. FA did not induce any significant effect in any of the genotoxicity tests performed. It can be concluded that inhalation of FA in a 28 days study with FA concentrations up to 15ppm does not lead to genotoxic effects in BAL cells of rats. Because detection of DPX by the comet assay is a very sensitive biomarker of FA exposure of cells, our results suggest that there is no genetically relevant exposure of the lung after FA inhalation. The results of our inhalation study, which was performed under GLP conditions, call into question the biological significance of previously reported genotoxic effects in the lung of rats after FA inhalation.

Abstract B32: Effects of 120 Hz electromagnetic fields on the early hepatocarcinogenesis in F-344 rats

Abstract Background: In recent years the extremely low frequency of electromagnetic fields (ELF-EMF) has been studied with major interest due their possible effects on the human health. Researches about that are contrasting; whereas some authors associate to ELF-EMF exposure with carcinogenesis, studies in experimental models and human being cancer have shown that the ELF-EMF not increases the risk of several cancer types. Previously, a biophysical model has hypothesized that the action mechanism of ELF-EMF on cells is through forced-vibration of the free ions that exist on all plasma membranes and that can move across of them using transmembrane proteins, disordering the electrochemical balance of the plasma membrane and therefore the whole cell function. In this way, cancer development takes place through rapid proliferation and the continuous increase of altered cells modifying the cellular environment including the flow of ionic charges across the cell membrane; considering that hepatocellular carcinoma (HCC) is a common form of cancer and its incidence remains high; in this study we evaluated the effect of 4.5 mT-120 Hz electromagnetic field (EMF) on the development of preneoplastic lesions in experimental hepatocarcinogenesis. Methods: Male Fischer-344 rats were subjected to the modified hepatocyte resistant model and were exposed to uniform and homogenous EMF of 4.5 mT-120 Hz during 50 min per day since seven days before until 25 days after carcinogenesis initiation when animals were sacrificed. The effect of ELF-EMF on early hepatocarcinogenesis, the induction of altered cells death and proliferation, and cell cycle progression was evaluated by histochemical, TUNEL assay, caspasa 3 level, immunohistochemical and western blot analysis. Results: The application of 4.5 mT-120 Hz EMF compared with shamexposed group, decreased over 50% the number and area of γ-glutamyl transpeptidase-positive preneoplastic lesions and the glutathione S-transferase placental expression (P=0.008 and P=0.03, respectively), both markers that identify preneoplastic lesions. Cells TUNEL positive and cleaved caspase 3 levels were unaffected; however, the cell number/mm2 in serial liver tissues of proliferating cell nuclear antigen (PCNA), Ki-67 and cyclin D1 expression decreased significantly (>80%) becoming similar to normal control (P=0.03, P=0.004 and P=0.008, respectively). Finally, western blot analysis revealed a diminution over 50% of PCNA and cyclin D1 expression (P=0.03 and P=0.01, respectively). Conclusion: The application of 4.5 mT-120 Hz EMF affect the early chemically induced carcinogenesis in rat liver through reduction of the proliferation markers such as PCNA and Ki-67 affecting the cyclin D1 expression which participates in the cell cycle continuity, without altering apoptosis process. These results suggest that ELF-EMF could be regulating the altered cellular homeostasis to inhibit the cancer development. Finally, this finding may be the base for clinical applications in the design of strategies to treat HCC. Citation Information: Cancer Res 2009;69(23 Suppl):B32.

The radiation response of cells from 9L gliosarcoma tumours is correlated with [F18]-EF5 uptake

Purpose: Tumour hypoxia affects cancer biology and therapy-resistance in both animals and humans. The purpose of this study was to determine whether EF5 ([2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)-acetamide]) binding and/or radioactive drug uptake correlated with single-dose radiation response in 9L gliosarcoma tumours.Materials and methods: Twenty-two 9L tumours were grown in male Fischer rats. Rats were administered low specific activity 18F-EF5 and their tumours irradiated and assessed for cell survival and hypoxia. Hypoxia assays included EF5 binding measured by antibodies against bound-drug adducts and gamma counts of 18F-EF5 tumour uptake compared with uptake by normal muscle and blood. These assays were compared with cellular radiation response (in vivo to in vitro assay). In six cases, uptake of tumour versus muscle was also assayed using images from a PET (positron emission tomography) camera (PENN G-PET).Results: The intertumoural variation in radiation response of 9L tumour-cells was significantly correlated with uptake of 18F-labelled EF5 (i.e., including both bound and non-bound drug) using either tumour to muscle or tumour to blood gamma count ratios. In the tumours where imaging was performed, there was a significant correlation between the image analysis and gamma count analysis. Intertumoural variation in cellular radiation response of the same 22 tumours was also correlated with mean flow cytometry signal due to EF5 binding.Conclusion: To our knowledge, this is the first animal model/drug combination demonstrating a correlation of radioresponse for tumour-cells from individual tumours with drug metabolism using either immunohistochemical or non-invasive techniques.

Abstract 5262: Progressive Decline in C-Type Natriuretic Peptide With Aging is Associated With Cardiorenal Fibrosis, Myocardial Dysfunction and Proteinuria

Background: C-type natriuretic peptide (CNP) is of endothelial cell origin and represents the most potent anti-fibrotic peptide of the natriuretic peptide family and is important for extracellular matrix homeostasis including mediating bone growth in the young. Cardiorenal aging is associated with altered cardiac and renal structure and function that may contribute to the development of heart and kidney failure, particularly in the elderly. The impact of aging on circulating CNP and associated changes in cardiorenal structure and function are undefined. Hypothesis: We hypothesized that a decrease in endogenous plasma CNP occurs with aging and is associated with an increase in cardiorenal fibrosis and altered left ventricular (LV) and renal structure and function. Methods: Studies were performed in 2, 11 and 20 month old male Fischer rats (n = 8 –10/group). Echo was used to assess LV structure and function. Blood pressure (BP), urinary protein excretion, plasma B-type natriuretic peptide (BNP) and CNP were measured. Kidneys and LV were harvested for gross and histology analysis. Mean±SE, *p<0.05. Results: Aging from 2 to 20 months was associated with progressive reductions in plasma CNP (30±3 to 21±1* to 9±1*pg/ml). Significant and progressive cardiac and renal fibrosis were observed with aging (LV: 9±0.3 to 15±0.7* to 21±1*% and kidney: 1±0.3 to 8±2* to 17±1* %). In contrast to CNP, plasma BNP was modestly increased from the 2 to 20 month old group (21±2 to 26±1*pg/ml). The reduction in CNP and increase in BNP, the latter a biomarker for LV dysfunction, were associated with decreased EF (88±1 to 80±1*%) and increased LVEDd (6.61±0.09 to 7.48±0.09*mm). BP and urinary protein excretion were mildly elevated with aging (91±1 to 101±3* mmHg and 8±0.3 to 20±3* mg/day, respectively). Conclusion: We report for the first time that aging is associated with a progressive decline in circulating CNP and progressive cardiorenal fibrosis, systolic dysfunction and proteinuria. Further studies are warranted to explore the hypothesis that a mechanism of cardiorenal aging with altered cardiorenal structure and function may include a decrease in the bioavailability of CNP.

Increased nitric oxide-mediated vasodilation of bone resistance arteries is associated with increased trabecular bone volume after endurance training in rats

Old age-associated osteoporosis is related to diminished bone blood flow and impaired nitric oxide (NO)-mediated vasodilation of the bone vasculature. Endurance exercise training restores the age-associated reduction of vasodilation in numerous vascular beds, as well as improving bone properties. The purpose of this study was to determine whether functional improvements in the bone vasculature are associated with increased bone properties after an endurance training intervention. Young adult (4–6 months) and old (24–26 months) male Fischer-344 rats remained sedentary or were trained (15 m/min walking, 15° incline, 5 days/week, 10–12 weeks). Endothelium-dependent vasodilation of the femoral principal nutrient artery (PNA) was assessed in vitro using acetylcholine (ACh) and inhibitors of NO synthase (NOS) and cyclooxygenase (COX). PNA endothelium-dependent vasodilation was greater after training by 16% in young and by 24% in old animals. The NOS-mediated contribution to endothelium-dependent vasodilation was enhanced by 77% after training in old rats. Distal femur trabecular bone volume (BV/TV, %) was lower with old age in sedentary animals (young: 27±2%, old: 23±1%; P<0.05). Exercise-induced elevations in bone and marrow blood flow and the NOS signaling pathway were associated with greater BV/TV (young trained: 34±2%, old trained: 26±1%; P<0.05) relative to sedentary groups. These data demonstrate that training-induced increases in bone properties are associated with enhanced endothelium-dependent vasodilation through a NOS signaling pathway in the bone vasculature.

Ischemic Preconditioning Augments Survival of Stem Cells via miR-210 Expression by Targeting Caspase-8-associated Protein 2

MicroRNAs (miRs) participate in most cellular functions by posttranscriptional regulation of gene expression albeit with little information regarding their role in ischemic preconditioning (IP) of stem cells. We report that IP of bone marrow-derived mesenchymal stem cells (MSCs) with two cycles of 30-min ischemia/reoxygenation (I/R) supported their survival under subsequent longer exposure to anoxia and following engraftment in the infarcted heart. IP significantly reduced apoptosis in MSCs through activation of Akt (Ser(473)) and ERK1/2 (Thr(202)/Tyr(204)) and nuclear translocation of hypoxia-inducible factor-1alpha (HIF-1alpha). We observed concomitant induction of miR-210 in the preconditioned MSCs ((PC)MSCs). Inhibition of HIF-1alpha or of miR-210 abrogated the cytoprotective effects of preconditioning. Extrapolation of these data to in vivo studies in a rat model of acute myocardial infarction predominantly improved stem cell survival after engraftment with a role for miR-210. Notably, multiple I/R cycles more effectively regulated the miR-210 and hence promoted MSC survival compared with single-cycle hypoxia of an equal duration. Real time PCR array for rat apoptotic genes, computational target gene analyses, and luciferase reporter assay identified FLICE-associated huge protein (FLASH)/caspase-8-associated protein-2 (Casp8ap2) in (PC)MSCs as the target gene of miR-210. Induction of FLASH/CASP8AP2 in miR-210 knocked-down (PC)MSCs resulted in increased cell apoptosis. Taken together, these data demonstrated that cytoprotection afforded by IP was regulated by miR-210 induction via FLASH/Casp8ap2 suppression. These results highlighted that IP by multiple short episodes of I/R is a novel strategy to promote stem cell survival.

Cocaine effects on dopamine and NMDA receptors interactions in the striatum of Fischer rats

Numerous studies have shown that biochemical and behavioral effects of cocaine are mediated by dopamine D1 receptor (D1R) and NMDA R1 receptor (NR1)-mediated transmission. In this study, we investigated the physical interactions between D1R and NR1 in response to acute cocaine administration in a time course of 5–60 min. In the caudate-putamen (CPu) of male Fischer rats, a single cocaine injection (30 mg/kg) reduced D1R–NR1 protein–protein interactions 30 min after treatment. In addition, activation or blockade of the NMDA receptor using NMDA (25 mg/kg) or MK-801 (0.25 mg/kg), respectively, also reduced the D1R–NR1 physical interactions. Acute cocaine administration did not alter total D1R or NR1 protein levels in our time course of study. These results indicate that D1R–NR1 physical interaction rather than total protein levels may regulate the intracellular signaling after acute cocaine administration.

Effects of Dose and Route on the Disposition and Kinetics of 1-Butyl-1-methylpyrrolidinium Chloride in Male F-344 Rats

Studies were conducted to characterize the effects of dose and route of administration on the disposition of 1-butyl-1-methylpyrrolidinium (BmPy-Cl) in male Fischer-344 rats. After a single oral administration of [(14)C]BmPy-Cl (50 mg/kg), BmPy-Cl in the blood decreased rapidly after C(max) of 89.1 min with a distribution half-life (t(1/2)(alpha)) of 21 min, an elimination half-life (t(1/2)(beta)) of 5.6 h, and a total body clearance of 7.6 ml/min. After oral administration (50, 5, and 0.5 mg/kg), 50 to 70% of the administered radioactivity was recovered in the feces, with the remainder recovered in the urine. Serial daily oral administrations of [(14)C]BmPy-Cl (50 mg/kg/day for 5 days) did not result in a notable alteration in disposition or elimination. After each administration, 88 to 94% of the dose was eliminated in a 24-h period, with 63 to 76% of dose recovered in the feces. Intravenous administration of [(14)C]BmPy-Cl (5 mg/kg) resulted in biphasic elimination. Oral systemic bioavailability was 43.4%, approximately equal to the dose recovered in urine after oral administration (29-38%). Total dermal absorption of [(14)C]BmPy-Cl (5 mg/kg) was moderate when it was applied in dimethylformamide-water (34 + or - 13%), variable in water (22 + or - 8%), or minimal in ethanol-water (13 + or - 1%) vehicles. Urine was the predominant route of elimination regardless of vehicle. Only parent [(14)C]BmPy-Cl was detected in the urine after all doses and routes of administration. BmPy-Cl was found to be a substrate for (K(t) = 37 microM) and inhibitor of (IC(50/tetraethylammonium) = 0.5 microM) human organic cation transporter 2. In summary, BmPy-Cl is moderately absorbed, extracted by the kidney, and eliminated in the urine as parent compound, independent of dose, number, or route of administration.

Carcinogenic and non-carcinogenic doses of Phenobarbital cause distinct microrna profiles in male Fischer rats after 14 days of dietary exposure

Carcinogenic and non-carcinogenic doses of Phenobarbital cause distinct microrna profiles in male Fischer rats after 14 days of dietary exposure

Regulation of dynamin 2 and G protein‐coupled receptor kinase 2 in rat nucleus accumbens during acute and repeated cocaine administration

Exposure to cocaine causes many neuroadaptations including alterations in several neurotransmitter receptors and transporters. This study investigated potential mechanisms of cocaine-induced receptor and transporter regulation by measuring levels of two proteins involved in receptor and transporter trafficking, dynamin 2 and G protein-coupled receptor kinase 2 (GRK2). Male Fischer rats received three daily injections of cocaine, 15 mg/kg, in a binge-pattern (at 1 h intervals) for 1, 3, or 14 days. Brain regions of interest were collected 30 min after the last injection and proteins measured by Western blot. Acute binge-pattern cocaine administration produced a significant increase in both dynamin 2- and GRK2-immunoreactivity (227% and 358% of control) in the nucleus accumbens and GKR2 (150% of control) in the caudate putamen. Tolerance to this effect occurred, as levels of both proteins returned to baseline after 3 days of cocaine. In contrast, dynamin 2 and GRK2 were significantly decreased in the nucleus accumbens after chronic cocaine. This pattern of regulation was unique to the nucleus accumbens and not seen in the frontal cortex or substantia nigra. Pretreatment with either the dopamine (DA) D1 receptor antagonist SCH 23390 or D2 receptor antagonist eticlopride prior to acute cocaine blocked the upregulation of dynamin 2 and GRK2 in the nucleus accumbens. However, only eticlopride was effective in attenuating the decrease in these proteins following chronic cocaine exposure. These results demonstrate that two proteins involved in receptor and transporter trafficking are selectively regulated in the nucleus accumbens following acute versus chronic cocaine exposure, and dopamine receptor activation is required for this regulation.

Blueberry-Enriched Diet Protects Rat Heart from Ischemic Damage

Objectivesto assess the cardioprotective properties of a blueberry enriched diet (BD).BackgroundReactive oxygen species (ROS) play a major role in ischemia-related myocardial injury. The attempts to use synthetic antioxidants to block the detrimental effects of ROS have produced mixed or negative results precipitating the interest in natural products. Blueberries are readily available product with the highest antioxidant capacity among fruits and vegetables.Methods and ResultsFollowing 3-mo of BD or a regular control diet (CD), the threshold for mitochondrial permeability transition (tMPT) was measured in isolated cardiomyocytes obtained from young male Fischer-344 rats. Compared to CD, BD resulted in a 24% increase (p<0.001) of ROS indexed tMPT. The remaining animals were subjected to a permanent ligation of the left descending coronary artery. 24 hrs later resulting myocardial infarction (MI) in rats on BD was 22% less than in CD rats (p<0.01). Significantly less TUNEL(+) cardiomyocytes (2% vs 9%) and 40% less inflammation cells were observed in the myocardial area at risk of BD compared to CD rats (p<0.01). In the subgroup of rats, after coronary ligation the original diet was either continued or switched to the opposite one, and cardiac remodeling and MI expansion were followed by serial echocardiography for 10 weeks. Measurements suggested that continuation of BD or its withdrawal after MI attenuated or accelerated rates of post MI cardiac remodeling and MI expansion.ConclusionA blueberry-enriched diet protected the myocardium from induced ischemic damage and demonstrated the potential to attenuate the development of post MI chronic heart failure.

Orchiectomy upregulates free soluble RANKL in bone marrow of aged rats

The osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL) axis is thought to be involved in the upregulation of bone turnover following sex steroid deficiency. Here, we investigated the effects of orchiectomy (ORX) on bone turnover and free soluble RANKL (sRANKL) in aged rats. Free, bioactive sRANKL is a critical determinant and key mediator for survival and activity of mature osteoclasts. Thirty-three 9-month-old male Fischer-344 rats were either ORX or sham-operated (SHAM). Following in vivo fluorochrome labeling, vehicle (ricinus oil/benzyl benzoate)-treated SHAM and vehicle- or testosterone undecanoate (T, 6 mg/kg s.c. once weekly)-treated ORX rats ( n = 8–9 each) were killed 2 months after surgery. Vehicle-treated ORX rats showed lower seminal vesicle weight, loss of proximal tibial trabecular bone mineral density, and reduced cortical thickness at the tibial shaft as measured by peripheral quantitative computed tomography relative to SHAM controls. Bone loss in vehicle-treated ORX rats was associated with enhanced bone turnover as evidenced by increases in tibial cancellous bone formation rate, osteoclast numbers, urinary excretion of calcium and deoxypyridinoline, and serum osteocalcin. T treatment of ORX rats restored seminal vesicle weight to SHAM control levels, and completely protected against post-ORX bone loss by suppressing bone turnover. Free sRANKL concentrations in bone marrow supernatants harvested from the proximal femur were about 3-fold higher in vehicle-treated ORX relative to SHAM rats, and returned to SHAM control levels in T-treated ORX rats. mRNA abundance of matrix metalloproteinase-14 (MMP-14) in bone marrow was 4-fold higher in vehicle-treated ORX rats relative to SHAM rats. T treatment of ORX rats suppressed MMP-14 mRNA expression to SHAM control levels. We conclude that orchiectomy increases the concentration of free sRANKL in bone marrow of aged rats. In addition, increased shedding of membrane-bound RANKL by MMP-14 may be a pivotal mechanism resulting in augmented free sRANKL concentrations in the bone marrow environment after androgen withdrawal.

Inhalation of formaldehyde does not induce systemic genotoxic effects in rats

Male Fischer-344 rats were exposed to formaldehyde (FA) by inhalation for 4 weeks (6 h/day, 5 days/week). Groups of six rats each were exposed to the target concentrations of 0, 0.5, 1, 2, 6, 10 and 15 ppm. Potential systemic genotoxic effects were investigated as part of a comprehensive study on local and systemic toxic and genotoxic effects. For this purpose, peripheral blood samples were obtained by puncturing the retro-orbital venous plexus at the end of the exposure period. Blood sampling was carried out in a randomized sequence and samples were coded by sequence number to ensure blind evaluation. Blood samples were used for the comet assay, the sister chromatid exchange test (SCE test) and the micronucleus test (MNT). DNA migration in the comet assay was measured both directly and after irradiation of the blood samples with 2 Gy gamma-radiation. The latter modification of the comet assay was included to increase its sensitivity for the detection of DNA–protein cross-links (DPX). The following positive control groups were included: one group (six animals) was treated with 50 mg/kg methyl methanesulfonate (MMS) once by gavage 4 h before blood sampling. Another group (six animals) was treated twice orally with 10 mg/kg cyclophosphamide (CP) with an interval of 24 h. The last application of CP was 24 h before blood sampling. For the comet assay, four slides were analysed from each blood sample, two without and two with irradiation. From each slide, 50 randomly selected cells were measured by image analysis, and tail intensity (% tail DNA) and tail moment were evaluated. For the SCE test, blood was cultured for 56 h in the presence of BrdU (10 μg/ml for the last 35 h) and SCE were counted in 30 second-division metaphases per sample. The MNT with peripheral blood was performed according to the instructions for the micronucleus analysis kit MICROFLOW (Litron Laboratories). Approximately 20,000 cells per sample were analysed by flow cytometry and the percentage of reticulocytes with micronuclei (MN) was determined. The positive control substances induced a significant effect in the genotoxicity tests and thus demonstrated the sensitivity of the test systems. FA did not induce any significant effect in any of the genotoxicity tests performed. It can be concluded that inhalation of FA in a 28-day study with FA concentrations up to 15 ppm does not lead to systemic genotoxic effects in the blood of rats.

Effect of glycosidation of isoflavones on their bioavailability and pharmacokinetics in aged male rats

There are limited reports on the bioavailability and pharmacokinetics of isoflavones in elderly humans and aged animals. The present study was conducted to assess the effect of glycosidation of isoflavones on their bioavailability and pharmacokinetics in aged (20 month old) male Fischer-344 (F-344) rats. The F-344 rat, developed by the National Institute on Aging, is an inbred rat model that is commonly used for aging studies and resembles many features of aging humans. Three sources of isoflavones; Novasoy (a commercial supplement), a mixture of synthetic aglycons (daidzein, genistein and glycitein), and a mixture of synthetic glucosides (daidzin, genistin, and glycitin) were tested. Following administration, blood samples were collected at different times (0-48 h post-oral gavage and 0-8 h post-IV dosing). Plasma isoflavones and 7-hydroxy-3-(4'-hydroxyphenyl)-chroman (a metabolite of daidzein) were measured by LC/MS. The extent of absorption was determined by comparing the area under the curve (AUC) of the plasma-concentration time curve after intravenous (IV) administration with that following oral administration. The extent of bioavailability was then calculated as: %bioabailability = (AUC(or)/AUC(IV))x(Dose(IV)/Dose(or))x100. Bioavailabilities for genistein were significantly (p = 0.013) higher for the aglycon (35 +/- 9%) compared with the glucoside forms (11 +/- 3%). In contrast, the bioavailabilities for glycitein were significantly (p = 0.011) higher in Novasoy (27 +/- 13%) and the glucoside form (21 +/- 10%) compared with the aglycon (8 +/- 3%). No significant differences in the bioavailability of daidzein were observed in aged rats dosed with aglycon, glucoside or Novasoy. However, aged rats were able to produce equol as early as 8 h post-dosing. In summary, the source of isoflavones had significant effects on genistein and glycitein bioavailability in aged male rats.

Whole-body synthesis-secretion rates of long-chain n-3 PUFAs from circulating unesterified α-linolenic acid in unanesthetized rats

Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), long-chain n-3 PUFAs important for brain and heart function, can be obtained from dietary fish products or by liver synthesis from alpha-linolenic acid (alpha-LNA). Their daily human dietary requirements are not clear, and their liver synthesis rates in humans and nonhumans are unknown. We estimated whole-body (presumably liver) synthesis rates in unanesthetized rats by infusing [U-(13)C]alpha-LNA intravenously for 2 h and measuring labeled and unlabeled n-3 PUFA in arterial plasma using negative chemical ionization GC-MS. Newly synthesized esterified [(13)C]DHA, [(13)C]EPA, and [(13)C]docosapentaenoic acid (DPA) appeared in arterial plasma after 60 min of infusion, then their concentrations rose in an S-shaped manner. Esterified concentration x plasma volume data were fit with a sigmoidal equation, whose peak first derivatives provided synthesis rates of unlabeled EPA, DPA, and DHA equal to 8.40, 6.27, and 9.84 mumol/day, respectively. The DHA synthesis rate exceeded the published daily rat brain DHA consumption rate by 30-fold, suggesting that liver synthesis from alpha-LNA could maintain brain DHA homeostasis were DHA absent from the diet. This stable isotope infusion method could be used to quantify whole-body DHA synthesis rates in human subjects.

Exercise Training Modulates the Nitric Oxide Synthase Profile in Skeletal Muscle From Old Rats

The effects of exercise training on the nitric oxide synthase (NOS) isoform profile in aging fast-twitch (white gastrocnemius [WG]) and slow-twitch (soleus [SOL]) muscle have not been investigated. Six-month and 27-month male Fischer-344 rats were divided into the following groups: young sedentary (YS), young treadmill exercise trained for 12 weeks, old sedentary (OS), and old exercise trained (OE). Inducible NOS (iNOS) protein expression and activity were significantly higher in OS compared with YS, whereas exercise training significantly reduced iNOS protein and activity levels in the WG. Neuronal NOS protein expression decreased with aging in WG but was upregulated significantly with exercise training in OE for both WG and SOL. Endothelial NOS (eNOS) protein levels were depressed in WG of old rats but were higher in OE than in OS. eNOS was unaffected by aging or exercise in the SOL. Our results indicate that endurance exercise training attenuates age-induced alterations of NOS isoforms with a greater response in fast-twitch compared with slow-twitch muscle.

Anti‐intercellular adhesion molecule‐1 antibody's effect on noise damage

The purpose of this study was to investigate possible preventive effects of anti-intercellular adhesion molecule-1 antibody (anti-ICAM-1 Ab) on noise-induced cochlear damage as assessed by changes in auditory thresholds and cochlear blood flow. A controlled animal study. Pretreated rats with anti-ICAM-1 Ab or saline control, followed with exposure to 72 continuous hours of broad band noise (107 dB SPL), and 24 hours after noise exposure treated again with anti-ICAM-1 Ab or saline. Eighteen healthy male Fischer rats (200-250 g) were used. Sixteen were randomly selected to study noise-induced temporary threshold shifts. The remaining two rats were used to study cochlear blood flow (CBF), using laser Doppler flowmetry and blood pressure measurements. Rats treated with anti-ICAM-1 Ab (1.875 mg/kg, intravenously) showed attenuated temporary threshold shifts (TTS) compared to controls. Both groups showed a partial threshold recovery 72 hours following noise exposure, normal for this noise exposure paradigm. Comparisons of baseline and post-treatment measurements of CBF and mean arterial blood pressure revealed no significant changes. Anti-ICAM-1 Ab animals displayed significantly lower mean auditory threshold shifts at all five test frequencies (P < .05) when compared to control. Blocking the cascade of reactive oxygen species (ROS) generation by using anti-ICAM-Ab protects against noise-induced hearing loss.

Acceleration of spinal fusion using syngeneic and allogeneic adult adipose derived stem cells in a rat model

Posterolateral spinal fusion is the standard treatment for lumbar compression fractures. Adult adipose tissue-derived stem cells (ASCs) promote osteogenesis in vivo and in vitro. The hypothesis tested in this study was that syngeneic and allogeneic ASCs on a biomaterial scaffold composed of tricalcium phosphate and collagen I will accelerate spinal fusion in a rat model. ASCs from male Fischer or ACI rats were loaded onto scaffolds (53,571 cells/mm(3)) and cultured in stromal media for 48 h. Male Fisher rats were assigned to 4 cohorts (n = 14/cohort) after bilateral decortication of the L4 and L5 transverse processes: (1) No treatment; (2) scaffold only; (3) scaffold + syngeneic ASCs; or (4) scaffold + allogeneic ASCs. Half of each cohort was harvested 4 or 8 weeks after surgery. Spinal fusion was evaluated with radiographs, microcomputed tomography, and light microscopy. Callus did not form in spines without scaffolds. There were no significant differences in callus formation among scaffold cohorts 4 weeks after surgery. Callus formation was more mature in both ASC cohorts versus scaffold alone 8 weeks after surgery based on microstructure as well as radiographic and microcomputed tomographic evidence of active bone formation. Inflammatory cell infiltrate was significantly lower in both ASC cohorts (syngeneic = 18.3 +/- 0.85%; allogeneic = 23.5 +/- 2.33%) versus scaffold alone (46.8 +/- 11.8%) 4 weeks after surgery. Results of this study support syngeneic and allogeneic ASC acceleration of posterior lumbar spinal fusion in a rat model.

The Role of Tissue Doppler Imaging in the Noninvasive Detection of Chronic Rejection after Heterotopic Cardiac Transplantation in Rats

Chronic rejection is a risk factor for the development of cardiac allograft vasculopathy (CAV) in heart transplant recipients. A useful animal model to study the role of immunosuppressive strategies in the prevention of chronic rejection involves heterotopic abdominal cardiac transplantation in rats. The detection of rejection and concurrent CAV traditionally involves subjective serial palpation of the graft from a scale of 0 to 4, with 4 indicating vigorous beats. Recent advances in murine echocardiography, in particular Tissue Doppler imaging (TDI), may allow for objective in vivo monitoring of chronic rejection in this transplant model. The objective of this study was to compare the diagnostic accuracy of murine echocardiography as compared to the abdominal palpation heart score for the noninvasive detection of chronic cardiac graft rejection. In an animal model of heterotopic cardiac transplantation, 18 male Fischer and Lewis rats were used as donors and recipients, respectively. Abdominal palpation and murine transthoracic echocardiography were performed to assess in vivo function of the transplanted heart. Left ventricular (LV) structure and function and TDI indices, including endocardial velocity (Vendo) and strain rate (SR), were evaluated in the ectopic heart. Graft tissues were processed for histological examination and graded for chronic rejection. Abdominal palpation scores were obtained in all 18 rats; score 1 (n = 5); score 2 (n = 4); score 3 (n = 6); and score 4 (n = 3). The mean LV ejection fraction was significantly (P <0.01) lower in score 3 and 4 grafts as compared to score 1 grafts. There was no correlation between the abdominal palpation score and LV systolic function. There was a significant relationship between decreasing Vendo or SR values and increasing grades of rejection (r = 0.65, P <0.05 and r = 0.75, P < 0.05, respectively). TDI of the transplanted heart in rats is feasible, reproducible, and more sensitive than palpation for the detection of chronic rejection.

Impact of 6-mo caloric restriction on myocardial ischemic tolerance: possible involvement of nitric oxide-dependent increase in nuclear Sirt1

Ischemic tolerance decreases with aging, and the cardioprotective effect of ischemic preconditioning (IPC) is impaired in middle-aged animals. We have demonstrated that short-term caloric restriction (CR) improves myocardial ischemic tolerance in young and old animals via the activation of adiponectin-AMP-activated protein kinase (AMPK)-mediated signaling. However, it is unknown whether prolonged CR confers cardioprotection in a similar manner. Furthermore, little is known regarding the myocardial expression of silent information regulator 1 (Sirt1; which reportedly mediates various aspects of the CR response) with prolonged CR. Thus, 6-mo-old male Fischer-344 rats were randomly divided into ad libitum (AL) and CR groups. Six months later, isolated perfused hearts were subjected to 25 min of global ischemia followed by 120 min of reperfusion with or without IPC. CR improved the recovery of left ventricular function and reduced infarct size after ischemia-reperfusion and restored the IPC effect. Serum adiponectin levels increased, but myocardial levels of total and phosphorylated AMPK did not change with prolonged CR. Total levels of Sirt1 did not change with CR; however, in the nuclear fraction, CR significantly increased Sirt1 and decreased acetyl-histone H3. Eleven rats from each group were given N-nitro-l-arginine methyl ester in their drinking water for 4 wk before death. In these hearts, chronic inhibition of nitric oxide synthase prevented the increase in nuclear Sirt1 content by CR and abrogated CR-induced cardioprotection. These results demonstrate that 1) prolonged CR improves myocardial ischemic tolerance and restores the IPC effect in middle-aged rats and 2) CR-induced cardioprotection is associated with a nitric oxide-dependent increase in nuclear Sirt1 content.