Effect of advanced age on p-aminohippurate-induced inhibition of renal tubular secretion in male Fischer 344 rats

The effect of aging on glomerular filtration, effective renal plasma flow and on the responsiveness of the renal tubular anion secretory system to inhibition by 4-aminobenzoylglycine (p-aminohippurate, PAH) was examined in young (5-month) and old (22-month) Fischer 344 male rats. Plasma clearance, protein binding and renal extraction of [131I]o-iodohippurate, [125I]iothalamate and HPLC-purified [99mTc]mercaptoacetyltriglycine (MAG3), were used as in vivo probes of renal function. The effect of advanced age, without concomitant PAH, on the disposition of these markers was initially determined in ketamine anesthetized, temperature-maintained male rats, ages 5, 14 and 22 months by means of constant infusion clearance studies. Aging per se decreased (P less than 0.05) the kidney-weight normalized or body weight-normalized GFR and effective renal plasma flow rates. GFR values averaged 1.67, 1.43 and 1.32 ml/min per g kidney for the 5-, 14- and 22-month-old rats, respectively. Kidney- or body weight-normalized clearances of MAG3 and o-iodohippurate showed similar (25-27%) decreases, whereas the absolute values (ml/min) for GFR, o-iodohippurate and MAG3 clearance rates were not altered by aging. The effective filtration fraction, extraction ratio and plasma protein binding were also unchanged by advanced age. Overall, the age-related decreases in renal function were minimal in Fischer-344 rats, compared to other species. Differences in data normalization, species and gender account, in part, for discrepancies observed when comparing results in different studies on the effects of advanced age on renal function. Subsequently, we examined the effect of aging on the renal responsiveness to inhibition of tubular anion secretion using constant rate PAH infusion studies, adjusted for age-related changes in renal function. Aging did not alter PAH-induced inhibition of iodohippurate secretion. Inhibition of MAG3 elimination was more pronounced in the old rats compared to the young controls.     

Age and organ dependent spontaneous generation of nuclear 8-hydroxydeoxyguanosine in male Fischer 344 rats

8-Hydroxydeoxyguanosine (8-OHdG) is a major oxidative DNA adduct playing roles in senescence, carcinogenesis and various disease processes. High-performance liquid chromatography with an electrochemical detection (HPLC-ECD) method has been widely used to assess organ levels of 8-OHdG, and a recently introduced immunohistochemical approach has made it possible to clarify intra-organ localization. In the present study, these methods were employed to reveal age-dependent changes in nuclear 8-OHdG within various tissues of male Fischer 344 rats between 18 fetal days and 104 weeks of age. 8-OHdG was detected in the nuclei of cerebellar small granule and small cortical cells, cerebral nerve cells, and choroid plexus epithelia of the brain and ependymal cells of the spinal cord; parenchymal cells in the anterior lobe of the pituitary and adrenal glands (mainly cortex); bronchial epithelium of the lung; intra-hepatic bile duct, pancreatic duct, glandular gastric and intestinal epithelial cells; renal tubular epithelial cells (mainly medulla); and spermatogonia and spermatocytes of the testis and seminal vesicle epithelia. The nuclear 8-OHdG levels were high (more than two lesions per 10(6) deoxyguanosines) from 7 days to 104 weeks of age in the brain, 3 to 6 weeks in the adrenal gland, 6 to 104 weeks in the lung, and 3 to 52 weeks in the testis. In the other organs, the nuclear 8-OHdG levels remained low throughout. These findings provide a basis for research dealing with oxidative stress by indicating organ-specific and age- but not aging-dependent changes in the localization of spontaneously generated nuclear 8-OHdG in intact rats. The immunohistochemical approach has advantages for assessing variation of 8-OHdG formation at the cellular level not accessible to the HPLC-ECD method.     

Age-related changes in cochlear and brainstem auditory functions in Fischer 344 rats

Auditory function in Fischer 344 (F344) and Long Evans (LE) rats was monitored during their lifespan by evaluating hair cell loss, middle-ear compliance and the recording of otoacoustic emissions and auditory brainstem responses. The results revealed a faster deterioration of hearing function in F344 rats compared with LE rats, resulting in larger hearing threshold shifts, a decrease in the latency and amplitude of click-evoked auditory brainstem responses, diminution of the distortion product otoacoustic emissions and a decrease in middle-ear compliance. However, hair cell loss, observed only at the most basal and apical parts of the organ of Corti, was comparable in older individuals of both rat strains. The results suggest involvement of cochlear (stria vascularis) and extracochlear (middle-ear) pathological changes during ageing. Thus, F344 rats represent a complex mix of conductive hearing loss (with low-frequency threshold shift, declining parameters of the middle-ear admittance and asymmetric otoacoustic emissions) and sensorineural hearing loss (with a decrease in the amplitudes of auditory brainstem response and a high-frequency threshold shift).     

Age-related neuronal loss in the submucosal plexus of the colon of Fischer 344 rats

The submucosal plexus (SMP) of the large intestine plays important roles in secretion and motility, functions that are compromised in the aged. To determine the effects of aging on intrinsic SMP neurons and their extrinsic inputs, whole mounts from the colon of ad libitum fed virgin male Fischer 344 rats ranging in age from 6 to 27 months were processed to visualize neurons and nerve fibers immunoreactive for either tyrosine hydroxylase (TH-IR) or calcitonin gene-related peptide (CGRP-IR). Significant age-related loss of SMP neurons occurred as early as 12 months of age and progressed in a roughly linear manner with age, dropping, for example, by 38% in the distal colon. In aging rats, the colonic SMP routinely contained markedly swollen TH-IR axons and terminals and exhibited a reduction of ganglionic neuropil, whereas CGRP-IR fibers evidenced only modest axonopathies. These findings point to selective deterioration of the SMP and its extrinsic inputs as one possible mechanism for the age-related decline in large intestinal function evidenced in the elderly.     

Activation-induced apoptosis in T cells from young and old Fischer 344 rats

BACKGROUND: Activation-induced apoptosis is believed to limit cell proliferation and eliminate the high number of activated cells during an immune response. METHODS: Activation-induced apoptosis was investigated in splenic T cells isolated from young (6 months) and old (24 months) male Fischer 344 rats. The cells were incubated with anti-CD3, concanavalin A or staphylococcal enterotoxin B (primary stimulus) for 72 h, followed by restimulation with anti-CD3 or concanavalin A (secondary stimulus). Apoptosis was assessed by DNA fragmentation assay and DNA gel electrophoresis. The expression of the apoptotic marker CD95 was analyzed by flow cytometry and the relative levels of CD95 ligand, Bcl-2 and Bax protein were measured by immunoblotting. RESULTS: It was shown that DNA fragmentation was very low in the unstimulated T cells from both young and old rats. However, the level of DNA fragmentation was 45-55% greater in the activated T cells from old rats than in the activated T cells from young rats. The increase in DNA fragmentation was paralleled by an increase in the proportion of cells expressing the CD95 molecule. The proportion of CD95+ cells was approximately 40% higher in T cells from old rats than in T cells from young rats. In addition, it was found that the expression of CD95 ligand and Bax increased and Bcl-2 decreased in the activated T cells from old rats compared to the activated T cells from young rats. CONCLUSION: Our data suggest that the increase in sensitivity of T cells to apoptosis with age may contribute to age-associated immune dysfunction and disorders.     

Effects of age and caloric restriction on cell proliferation in hepatocyte cultures from control and hepatectomized Fischer 344 rats

The effects of age and caloric restriction on cell proliferation,measured as scheduled DNA synthesis (SDS), were evaluated inprimary hepatocyte cultures from control and partially hepatectomized(PH) young to old ad libitum (AL) and caloric-restricted (CR)male Fischer 344 (F344) rats. We reported significant age- orCR-related decreases in SDS in control cultures. PH-inducedcultures exhibited significant increases in SDS compared withtheir control counterparts. Hepatocytes from PH-induced oldCR diet-fed animals exhibited significant increases in SDS comparedwith cultures from control old CR, PH-induced young CR and PH-inducedold AL animals. Alternatively, SDS rates for PH-induced youngCR animals were significantly lower at 48 h and higher at 72h than the rates we reported for cultures from PH-induced youngAL F344 rats. These data suggest that CR decreases and preservesthe proliferative capacity in hepatocytes from young animalsand may permit animals to respond more efficiently with inducedcompensatory cellular replication in old age. ⁴To whom correspondence should be addressed at: National Center for Toxicological Research, Division of Genetic Toxicology, HFT-120, 3900 NCTR Drive, Jefferson, AR 72079, USA     

Spatial reference and working memory across the lifespan of male Fischer 344 rats

Loss of mnemonic function is among the earliest and most disconcerting consequences of the aging process. This study was designed to provide a comprehensive profile of spatial mnemonic abilities in male Fischer 344 (F344) rats across the lifespan. Young, middle-aged, and aged F344 rats were trained in spatial reference and working memory versions of the water maze task. There was a progressive age-related decline in spatial reference memory across the lifespan. Reliable individual differences were observed among aged rats, with some aged rats performing as well as young cohorts and others performing outside this range. An age-related delay-dependent decline was observed on a working memory version of the water maze task although no relationship between performance on reference and working memory tasks was present. Notably, middle-aged rats were impaired relative to young on both tasks. Together these data demonstrate that individual differences in spatial reference memory exist among aged F344 rats and provide novel data demonstrating an unrelated decline in working memory across the lifespan, suggesting that age-related mnemonic dysfunction may occur across multiple brain systems.     

Effect of age on the expression of antioxidant enzymes in male Fischer F344 rats

Age-related changes in the activities of superoxide dismutase, catalase, and glutathione peroxidase were determined in brain, heart, hepatocytes, intestinal mucosa, and kidney from male Fischer F344 rats. Superoxide dismutase activity decreased significantly with age in all five tissues studied. The activity of catalase decreased with age in brain, hepatocytes, and kidney while glutathione peroxidase activity decreased significantly with age only in intestinal mucosa and kidney. The relative levels of superoxide dismutase, catalase, and glutathione peroxidase mRNA were measured in brain, hepatocytes, and kidney. An age-related decrease in SOD and catalase mRNA was observed for brain, hepatocytes, and kidney. GPX mRNA levels decreased with age in hepatocytes and kidney but did not change with age in brain. In general, the age-related changes in the activities of SOD, catalase, and GPX were paralleled by a similar change in the relative level of the mRNAs coding for these enzymes.     

Comparison of the dominant lethal effects of acrylonitrile and acrylamide in male Fischer 344 rats

Acrylonitrile (ACN) and acrylamide (AA), structurally similarvinyl monomers, are both animal carcinogens. ACN is weakly mutagenicin bacteria and induces sister-chromatid exchange, unscheduledDNA synthesis and cell transformation in cells in culture. AAinduces chromosomal aberrations in bone marrow, blood and germcells in vivo, and dominant lethal mutations in the germ cellsof male mice and rats. In the current study, the ability ofAA and ACN to induce dominant lethal mutations in the germ cellsof male Fischer 344 rats was compared. Three groups of 50 maleswere gavaged daily for 5 days with ACN (60 mg/kg in normal saline),AA (30 mg/kg in normal saline) or vehicle only; an additionalgroup of 20 males received a single i.p. injection of 0.2 mg/kgtriethylenemelamine (TEM) on the afternoon of day 5. Starting1 day after exposure, each male was bred to one female per weekfor 4 weeks (TEM-exposed group) or 10 weeks (ACN, AA and controlgroups). Mating rates were reduced only during week 1 in theTEM-treated group; pregnancy rates were reduced only duringweek 2 in the AA-exposed group and week 4 in the TEM-treatedgroup. Females were necropsied 13 days after the end of theappropriate mating week and the amount of pre- and post-implantationloss calculated. ACN treatment of male rats induced no increasesin either pre- or post-implantation loss in females in any ofthe 10 weeks post-exposure examined. AA induced significantlyelevated amounts of post-implantation loss for 3 weeks afterexposure and pre-implantation loss for 4 weeks post-exposure;both measures returned to control values for the remaining 6weeks of the study. The positive control TEM caused both indicesto increase during all 4 weeks examined. We conclude that AAis a dominant lethal mutagen in male rat germ cells in vivo,specifically in mature spermatozoa and late-stage spermatids.In contrast, the structurally related chemical ACN has neitherfertility nor dominant lethal effects in the male Fischer 344rat after oral administration, and may not pose a significantmutagenic risk to germ cells.     

Loss of N-cadherin and alpha-catenin in the proximal tubules of aging male Fischer 344 rats

Aging is associated with a loss of renal reserve, and increased sensitivity to either xenobiotic or physiologic insult. Given the critical role of the cadherin/catenin complex in establishing and maintaining the integrity and polarity of tubular epithelial cells, it was hypothesized that aging was associated with alterations in renal cadherin/catenin complexes. Histological assessment of aged (24 months) kidneys harvested from male Fischer 344 rats demonstrates mild degeneration of proximal tubules, multifocal chronic lymphocytic infiltration, moderate development of protein casts inside tubules, and tubular dilatation or degeneration. Western blot analysis revealed that N-cadherin protein expression is not constant over 24 months. N-cadherin expression increased from 4 to 9 months, with peak levels at 9 and 13 months. A decrease in expression was seen at 19 months and an almost complete loss of expression was seen at 24 months. In contrast, the expression of E- and Ksp-cadherin was constant over 24 months. A loss of alpha-catenin at was seen at 19 and 24 months in the absence of changes in beta-, gamma-, and p120-catenin. This pattern of N-cadherin expression (increase followed by decrease) was confirmed by real-time PCR analysis, which demonstrated a similar pattern as the Western blot, suggesting that the loss of N-cadherin protein was due to decreased gene expression. The loss of N-cadherin was specific for the kidney, as no changes in N-cadherin expression in the liver, brain, or testes were seen during aging. The conclusion that loss of N-cadherin expression is a critical component of the renal dysfunction associated with aging is supported by the finding that caloric restriction attenuates the loss of N-cadherin, as well as the finding that a significant loss of N-cadherin is seen in the kidneys of ZDF x SHHF rats, a genetic model of end-stage renal disease. Cadherin and catenin expression was further analyzed by immunofluorescence. A significant loss of staining of both N-cadherin and alpha-catenin was seen in the proximal tubules of rats at 24 months. Interestingly, this corresponded with delocalization of the alpha-1 subunit of the Na+K+-ATPase, i.e. aberrant staining on cell-cell borders and some indication of apical staining in proximal tubules. Taken together, these data suggest that aging is associated with decreased expression of N-cadherin and alpha-catenin and is associated with a loss of cell polarity.     

Effects of dietary taurine supplementation or deprivation in aged male Fischer 344 rats

Taurine is a sulfur amino acid that is present in high concentration in mammalian tissues and previously has been reported to decline in a number of tissues with advancing age. The aims of the present study were to examine: (1) the effects of dietary taurine supplementation; (2) the effects of taurine-free diets; (3) the ability of aged rats to conserve urinary taurine; and (4) the consequences of these dietary manipulations on some biochemical parameters. Male F344 rats (n = 30/group) 18 months of age were placed on control diets, diets supplemented with 1.5% taurine in the drinking water, or a taurine-free diet for 10 months. An adult control group (12 months old at the end of the study) on normal diets was included for comparison purposes. Significant (P < 0.05) age-related declines in taurine content were observed in the spleen, kidney, eye, cerebellum and serum. Taurine supplementation corrected these deficits in tissue content in aged rats and in many cases increased taurine content above that of adult controls. Urinary excretion of taurine was significantly (P < 0.05) reduced in aged rats indicating an increased need to conserve taurine. Taurine-deficient diets did not further exacerbate the age-related decline in tissue taurine content, suggesting biosynthetic adaptations to the lack of dietary taurine. Dietary taurine supplementation blunted age-related declines in serum IGF-1 and increases in serum creatinine and blood urinary nitrogen (BUN). These studies suggest that advanced aging results in a taurine-deficient state that can be corrected by dietary supplementation.     

Cardiovascular responses to acute footshock stress in adult and aged Fischer 344 male rats

Tail artery catheters were surgically implanted in Fischer 344 male rats to allow for measurement of mean arterial pressure (MAP, mm Hg) and heart rate (HR, beats/min) in conscious, unrestrained rats. Basal values of MAP and HR were similar for groups of 4, 12 and 24 month old rats. Increments in Map did not differ among rats of the 3 ages following handling and transfer to a shock chamber or immediately or 5 minutes after exposure to inescapable footshock (2.0 mA, 0.6 sec duration, every 6 sec for 1 min). In contrast, there was a significant age-related attenuation of the tachycardia following handling and transfer of rats to the shock chamber and at the end of footshock. These data are consistent with previous findings of a reduced sensitivity of the aged myocardium to stress-induced sympathetic stimulation.     

Effect of long-term caloric restriction on brain monoamines in aging male and female Fischer 344 rats

The present study examines the changes in central monoamines and their metabolites in aged male and female rats after long-term caloric restriction. Fischer 344 rats of both sexes (n = 5-10/group) were maintained on one of two dietary regimens: ad libitum NIH 31 diet or 60% by weight of the ad lib. intake (restricted), supplemented with vitamins and minerals. Animals received these diets from the age of 14 weeks until killed at 22.25 months of age. Caudate nucleus (CN), hypothalamus (HYPO), olfactory bulb (OB) and nucleus accumbens (NA) were assayed for content of norepinephrine (NE), dopamine (DA) and its metabolites (dihydroxyphenylacetic acid, DOPAC, and homovanillic acid, HVA) and serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) using HPLC/EC. Relative to the ad lib. group, restricted rats of both sex showed significant decreases in NE content in CN, HYPO and OB. DA and 5-HT content were decreased significantly in the CN and HYPO. No significant changes were found in the levels of DA metabolites in all brain regions studied. While the 5-HIAA level was significantly reduced in the HYPO and NA of the female restricted rats, it was increased several-fold in the OB of the male restricted animals. These preliminary results suggest that long-term caloric restriction alters brain monoamine concentrations, an effect which may in turn modify the normal rate of aging.     

Age-associated changes in hearts of male Fischer 344/Brown Norway F1 rats

Aging is associated with left ventricular hypertrophy, dilatation, and fibrosis of the heart. The Fischer 344/Brown Norway F1 (F344/BNF1) rat is recommended for age-related studies by the National Institutes on Aging because this hybrid rat lives longer and has a lower rate of pathological conditions than inbred rats. However, little is known about age-associated changes in cardiac and aortic function and structure in this model. This study evaluated age-related cardiac changes in male F344/BNF1 rats using ECHO, gross, and microscopic examinations. Rats aged 6-, 30-, and 36-mo were anesthetized and two-dimensional ECHO measurements, two-dimensional guided M-mode, Doppler M-mode, and other recordings from parasternal long- and short-axis views were obtained using a Phillips 5500 ECHO system with a 12 megahertz transducer. Hearts and aortas from sacrificed rats were evaluated grossly and microscopically. The ECHO studies revealed persistent cardiac arrhythmias (chiefly PVCs) in 72% (13/18) of 36-mo rats, 10% (1/10) of 30-mo rats, and none in 6-mo rats (0/16). Gross and microscopic studies showed left ventricular (LV) dilatation, borderline to mild hypertrophy, and areas of fibrosis that were common in 36-mo rats, less evident in 30-mo rats, and absent in 6-mo rats. Aging was associated with mild to moderate decreases of LV diastolic and systolic function. Thus, male F344/BN F1 rats demonstrated progressive age-related (a) decline in cardiac function (diastolic and systolic indices), (b) LV structural changes (chamber dimensions, volumes, and wall thicknesses), and (c) persistent arrhythmias. These changes are consistent with those in humans. The noninvasive ECHO technique offers a means to monitor serial age-related cardiac failure and therapeutic responses in the same rats over designated time intervals.     

Dose-dependent renal tubular toxicity of harman and norharman in male F344 rats.

The renal toxicity of harman and norharman, administered for 2 or 4 weeks at dietary levels of 1,000, 500, or 0 parts per million (ppm), was investigated in 6-week-old male F344/DuCrj rats. Although rats fed 1,000 ppm harman or norharman, but not the 500 ppm level, demonstrated marked body weight retardation from 1 week to termination, no mortalities occurred. Marked elevation of water consumption was evident in rats given harman or norharman at 1,000 ppm, but not at 500 ppm, together with large increases in urine of low specific gravity. Urinary lysosomal enzymes (N-acetyl-beta-D-glucosaminidase, NAG, and lactate dehydrogenase, LDH) and sugar levels were increased, and the brush border enzymes (gamma-glutamyl transpeptidase, GGT, and alkaline phosphatase, ALP) decreased. Furthermore, serum biochemistry revealed clear elevation of parameters indicating renal toxicity in these rats. Histopathologically, rats fed 1,000 ppm harman or norharman, but not 500 ppm, demonstrated focal toxic renal degenerative/necrotic and regenerative lesions in proximal, distal, and collecting tubules. These changes were associated with a clearly increased labeling index (LI) of the nuclei of renal tubular epithelial cells on immunohistochemical staining for 5-bromo-2'-deoxyuridine (BrdU). Chemical specific crystal formation within tubular lumina was evident in rats fed 1,000 ppm, but not 500 ppm, this being considered the cause of the renal tubular lesions. It was concluded that harman and norharman exert renal toxicity at the dietary level of 1,000 ppm, but not 500 ppm, in male F344 rats.     

Neurochemical, endocrine and immunological responses to stress in young and old Fischer 344 male rats

Two experiments were performed. In the first, a 20 min conditioned emotional response (CER) paradigm was used to compare the neurochemical, endocrine and immunological responses to stress of 7- and 22-month-old Fischer 344 (F344) male rats. In the second, corticosterone levels 20 min following ether stress, and regional brain type I and II corticosterone receptor densities were examined using 7- and 17.5-month-old F344 male rats. Dopamine (DA) metabolism in old nonstressed rats was significantly reduced in the medial frontal cortex, neostriatum, nucleus accumbens and hypothalamus, but not in the amygdala. The CER procedure, nevertheless, increased medial frontal cortical, nucleus accumbens and amygdaloid DA turnover in both the young and old rats. The young and old nonstressed rats did not evidence differences in norepinephrine (NE) and serotonin (5-HT) concentrations. However, stress resulted in a decrease in medial frontal cortical 5-hydroxyindoleacetic acid (5-HIAA) and hypothalamic 5-HT levels in old but not in young animals. These observations suggest age-related differences in the response of central NE and 5-HT systems to stress. Ether and the CER procedure led to exaggerated corticosterone responses in the old rats (17.5 and 22 month, respectively). Hippocampal type I but not type II corticosterone receptors were decreased by 47% in the 17.5-month-old rats. Thus, age-related changes in hippocampal corticosterone receptor types do not occur in unison, and the exacerbated corticosterone response to stress precedes the reported down-regulation of hippocampal type II corticosterone receptors in aged rats. Age-related changes were not observed in the concentrations of corticosterone receptors in other brain regions, or in the prolactin response to stress. The old rats, however, evidenced a reduction in the availability of the renin substrate, angiotensinogen, and in stress-induced renin secretion. Immune function was impaired in the old nonstressed rats, and further compromised by exposure to the CER procedure. In comparison to the young control rats, the old nonstressed rats showed an increased percentage of splenic large granular lymphocytes, reduced splenic natural killer cytotoxicity, and impaired Con-A-stimulated splenic T lymphocyte proliferation. Reductions in T splenic cell proliferation and natural killer cytotoxicity were observed in the young rats subjected to the CER paradigm, but not to the same extent as in the old rats. These observations indicate that aging male F344 rats evidence major alterations in basal central monoamine, endocrine and immune functions, and an increased sensitivity of these systems to stress.     

Metabolic mass, metabolic rate, caloric restriction, and aging in male Fischer 344 rats

Previous investigators have found the metabolic rate to be the same in calorically-restricted and ad-libitum fed rodents, and hence concluded that the Rate of Living Theory does not help explain the longer lifespan of the calorically-restricted (CR) animal. However, these previous instigators may not have used reliable estimates of metabolic mass in their calculations of metabolic rate. Hence the present study investigated the reliability of ten different estimates of metabolic mass (MM) in 21-month-old male Fischer 344 rats fed three different diets to yield a wide range of body compositions. Two criteria were used to rank each estimate of metabolic mass: strong correlation with daily caloric intake (DCI); and zero Y-intercept on the regression curve of DCI versus the MM. The combined weight of the heart, liver, kidneys and brain (OW) was found to be the best estimate of MM. Statistical analysis of the differences in metabolic rate in the three groups of rats showed that the significance of these differences depended on the estimate of MM used. OW yielded different results than did fat-free mass (FFM), body weight (BW), BW(0.75), and BW(0.67). Therefore, because previous investigators used FFM, BW, BW(0.75), or BW(0.67), rather than a more reliable estimate such as OW, their finding that metabolic rate was not different in the CR and ad-lib groups, and their conclusion that the Rate of Living Theory does not help explain the longer lifespan of the CR animal, are called into question.     

Role of spontaneous interstitial cell testicular tumors on the mitogen reactivity of spleen cells from aged male Fischer-344 rats

Virtually all aged, male, Fischer-344 rats have testicular tumors. The influence of this tumor on lymphocyte reactivity from aged Fischer-344 rats is unknown. In this report we demonstrate that neither the presence of this tumor nor the serum concentration of luteinizing hormone has an effect on the splenic mitogen reactivity of old animals.