Vitamin B-6 metabolism in the brains of young adult and senescent mice

The steady state levels of pyridoxal-P and pyridoxamine-P, the activities of pyridoxal (pyridoxine) kinase and pyridoxamine-(pyridoxine)-P oxidase, and the metabolism of [³H]pyridoxine were determined in the brains of C57B1/6J mice of selected ages. The steady state concentratioons of the coenzymes and the activities of the enzymes required for pyridoxal-P synthesis did not change significantly as a function of age. The uptake and metabolism of vitamin B-6 by the brain was studied by injecting [³H]pyridoxine in the tail vein of young adult and senescent mice, killing the mice after 15 or 30 min, and separating the B-6 metabolites by ion exchange chromatography. More total radioactivity was accumulated in 15 min in the brains of the senescent mice than the brains of the young mice. The brains from both age groups rapidly synthesized pyridoxal-P from pyridoxine. However, less radioactive pyridoxamine-P and more radioactive pyridoxal were formed in the brains of the senescent mice than in the young mice killed 15 min after injection. These results are similar to those obtained for the metabolim of [³H]-pyridoxine in the liver of these senescent mice. The senescent mice appear to be vitamin B-6 deficient, have decreased brain amino acid transaminase activity, and either increased pyridoxal-P phosphatase activity or decreased protection of brain pyridoxal-P.     

Effect of ageing on the nerve fibre population of rat optic nerve

The age-related changes in the number and density of optic nerve fibres were studied in 3-month-old (young), 12-month-old (adult), 20-month-old (senescent) and 30-month-old (aged) male Wistar rats. Two-micrometer-thick resin-embedded transverse sections of the optic nerve of animals of the different age groups were stained with toluidine blue and examined under a light microscope at low (X5) and high (X500) magnification. The optic nerve cross-sectional area and the number of nerve fibres with diameters shorter or longer than 1 micron were assessed by means of computerized image analysis. Optic nerve cross-sectional area is constant in young and adult rats, but is increased in senescence. The number of optic nerve fibres with a diameter less than 1 micron is decreased by about 18 and 12% in 20-month-old rats versus 3- and 12-month-old rats, respectively. The number of these nerve fibres is decreased by about 43, 39 and 30% in 30-month-old rats versus 3-, 12- and 20-month-old animals, respectively. In contrast no age-dependent changes were observed in nerve fibres with diameters greater than 1 micron. The present data suggest that age-related impairment of nerve cell population described by other authors in rat retina and visual cortex occurs also at the level of the optic nerve.     

Regeneration of beta-adrenergic receptors in senescent rats: a study using an irreversible binding antagonist.

The drug used in this study, bromoacetylalprenololmenthane, has the ability to bind and block irreversibly beta-adrenergic receptors. The drug was bound to membranes prepared from hearts, lungs, and brains of both senescent and young rats with a similar affinity. When this drug was injected into rats in nontoxic doses (up to 70 mg/kg), up to 90% of beta-adrenergic receptors were irreversibly blocked 4 hr after injection, whereas the injection of similar amounts of (+/-)-alprenolol was without effect on receptor number. In senescent animals this blockade lasted considerably longer than in young animals; receptor numbers in hearts and lungs of senescent rats returned to control levels only 1 month after injection. The number of beta-adrenergic receptors in brains of senescent rats was unaffected by this drug. Thus, based upon the long-lasting blockade of beta-adrenergic receptors in therapeutically important organs, it appears that irreversible binding blockers may have potential in the treatments of senescent organisms.     

Increase in substance P levels in the lung of old rats

Substance P levels in the lung of young (3-month-old), adult (12-month-old), senescent (20-month-old) and aged (28-month-old) male Wistar rats were determined by radioimmunoassay. Levels of substance P were constant until 28 months of age when these become significantly higher (approximately more than 2 times, p less than 0.001) than the values found in younger rats. Whether the increase of substance P content in the lung of old rats may reflect changes in the density of substance P containing nerve fibres or in the content of the peptide in single nerve fibres is discussed.     

Change with donor age in the degradation rate of endogenous proteins of mouse hepatocytes in primary culture

The degradation rate of [(3)H]leucine labeled intracellular proteins was compared in hepatocytes derived from young (4-7-month-old) and aged (23-month-old) mice. Average half-lives of long-lived proteins were 40 h and 65 h in the cells from young and old animals, respectively. This observation confirms our earlier results that half-lives of foreign proteins introduced into the cells from old mice are 50-60% longer than those of younger counterparts (Ishigami and Goto, 1988, Mech. Ageing Dev., 46, 125-133; Ishigami and Goto, 1990, Arch. Biochem. Biophys., 277, 189-195).     

Changes in vasopressin and testosterone in the senescent brown-Norway (BN/BiRij) rat

Although earlier reports in the literature suggested degenerative changes in the senescent hypothalamo-neurohypophyseal system (HNS), recent investigation showed hyperactivity of this system in the old Wistar (WAG/Rij) rat. In the brain, changes were found in those sites of extrahypothalamic vasopressin (VP) fiber termination that are testosterone-dependent. In the present study, further evaluation of these changes was performed in male Brown-Norway (BN/BiRij) rats. Immunocytochemical staining of VP binding sites in renal tubuli of the senescent, 33-month-old rats was occasional, and could only slightly be enhanced up to weak staining by in vitro preincubation with the peptide, in contrast to the intense staining observed in young, 3-month-old rats. Although VP plasma levels of young and old rats did not differ significantly and no change in either urine or plasma osmolality was observed, urinary VP concentration and 24-hour urinary VP excretion were significantly increased in senescent rats. The activation of the hypothalamo-neurohypophyseal system (HNS) in the senescent rat seems thus to compensate for a decreased responsiveness to VP in the aged kidney. Testicular weight in the senescent animals declined by 40% and a highly significant decrease of 80-90% was observed in total and free testosterone plasma levels. Consequently, changes in peripheral organs in senescent rats may well underly the changes observed in the brain of these animals.     

Tissue-specific and age-dependent expression of protein arginine methyltransferases (PRMTs) in male rat tissues

Protein arginine methyltransferases (PRMTs) generate asymmetric and symmetric dimethyl-arginines by catalyzing the transfer of methyl groups from S: -adenosyl-L-methionine to arginines in target proteins. Previously, we observed that the expression and activity of PRMTs were significantly down-regulated in replicatively senescent fibroblasts compared to young fibroblasts. In this study, we determined the level of three PRMT family members (PRMT1, PRMT4, and PRMT5) and the arginine methylation status in eight tissues from 6- and 24-month-old rats. We observed tissue-specific down-regulation of individual PRMT members in testis, thymus, kidney, lung, and heart from 24-month-old as compared to 6-month-old rats. Specifically, we observed reduced levels of PRMT1 in thymus and lung, reduced levels of PRMT4 in testis, thymus, and hearts, and reduced levels of PRMT5 in all five tissues. PRMT enzyme activity on histones generally correlated with PRMT expression. Furthermore, we observed a reduction in asymmetric and symmetric dimethylation on proteins in aged thymus and lung, and a reduction in symmetric dimethylation in aged testes relative to the testes harvested from young rats. These results suggest that individual PRMT proteins have tissue-specific functions and are regulated in a tissue-specific and age-dependent manner.     

Identification of histone methylation multiplicities patterns in the brain of senescence-accelerated prone mouse 8

Histone post-translational modifications (PTMs) are involved in diverse biological processes and methylation was regarded as a long-term epigenetic mark. Though aging represented one of the major risk factors for neurodegenerative diseases, no systematic investigations had correlated the patterns of histone PTMs in the brain with aging and the roles of such concerted histone PTMs in brain aging are still unknown. In this study, enzyme digestion, nano-LC, MALDI-TOF/TOF MS analysis and Western blotting were combined to investigate the defined methylation of core histones (H2A, H2B, H3 and H4) in the brain of 12-month-old senescence accelerated mouse prone 8 (SAMP8). The expression of several modified histones in the brain of 3-, and 12-month-old SAMP8 mice as well as that of the age-matched control senescence accelerated-resistant mouse (SAMR1) was compared. In the brain of 12-month-old SAMP8 mice, seven methylation sites (H3K24, H3K27, H3K36, H3K79, H3R128, H4K20 and H2A R89) were detected and most PTMs sites were located on histone H3. Mono-methylated H4K20 decreased significantly in the brain of 12-month-old SAMP8 mice. Methylated H3K27 and H3K36 coexisted in the aged brain with different methylation multiplicities. Di-methylated H3K79 expressed in the neurons of cerebral cortex and hippocampus. This study showed histone methylation patterns in the aged SAMP8 mice brain and provided the experimental evidences for further research on histone PTMs in the aged brain. We hope these results could initiate a platform for the exchange of comprehensive information concerning aging or neurodegenerative disease and help us interpret the change of gene expression and DNA repair ability at epigenetic level.     

Reproductive age-related changes in the blood brain barrier: Expression of IgG and tight junction proteins

We previously demonstrated that there is a significantly greater transfer of intravenously-injected Evan's blue dye into the forebrain of acyclic (reproductive senescent) females compared to young adult females, indicating that blood brain barrier permeability is compromised in the reproductive senescent forebrain. The present study examined brain IgG expression and microvessel tight junction proteins to assess ovarian age-related changes in microvascular permeability, and further compared young and senescent females with age-matched males to distinguish changes attributable to age and reproductive senescence. Blood brain barrier breakdown are often associated with increased extravasation of plasma proteins and high levels of immunoglobulin G (IgG) in brain. In the present study, IgG expression was dramatically increased in the hippocampus and thalamus, but not the hypothalamus of reproductive senescent females compared to young adult females. In males, IgG expression was increased in all these regions in middle-aged animals (aged-matched to senescent females) as compared to young males (age-matched to the young adult females). Furthermore, the proportion of hippocampal microvessels with perivascular IgG immunoreactivity was significantly greater in reproductive senescent females as compared to young adult females, while middle-aged males and young adult males did not differ. The tight junctions between adjacent microvascular endothelial cells regulated by transmembrane proteins such as claudin-5 and occludin play a critical role in maintaining the blood brain barrier integrity. Increased hippocampal IgG expression in senescent females was paralleled by poor junctional localization of the tight junction protein claudin-5 in hippocampal microvessels. However, there was no difference in hippocampal claudin-5 localization between young adult and middle-aged males, indicating that dysregulation of this junctional protein was associated with ovarian aging. Parallel studies in human brain microvessels also revealed age-dependent disruption in claudin-5 distribution in post-menopausal women compared to pre-menopausal women. Collectively, these data support the hypothesis that constitutive loss of barrier integrity in the forebrain during reproductive senescence may be due, in part, to the selective loss of tight junction proteins in endothelial junctions.     

Age-related defense against infection with intracellular pathogens.

Young adult (6--12 weeks old) and aged (20--24 months old) NMRI mice were infected with various intracellular parasites. The following results were obtained: (1) After a sublethal infection with Listeria monocytogenes, aged mice were found to show a resistance similar to that of young adults. A challenge infection with this pathogen was followed by specific immunity of long duration in both age-groups. (2) On the other hand, young animals were significantly more resistant to Salmonella typhimurium than aged mice. It was concluded that this was due to the LD50 which was 14 times greater for 2-month-old than for 20-month-old mice. Furthermore, during 7 weeks after infection there were more S. typhimurium in the spleens of senescent mice than in those of young adult controls. (3) Aged mice showed highly increased susceptibility to the weakly virulent DX strain of Toxoplasma gondii. Almost all aged animals died whereas the control mice survived. When death of the aged mice was prevented by treatment with sulfadiazine after infection with the DX strain, the aged mice were found to be as well protected against subsequent infection with the strongly virulent BK strains as the young adult mice. These results suggest that the susceptibility of the aged animal to infectious agents may considerably vary from one pathogen to another.     

Altered neuroendocrine status of middle-aged rats prior to the onset of senescent anovulation

The incidence of senescent anovulation (constant estrus) in female rats increases sharply in the age interval 10--14 months. We have compared the neuroendocrine status of 12-month-old rats, which were still cycling, with that of 6-month-old rats in the reproductive prime. Norepinephrine content in the median eminence of the hypothalamus and circulating levels of FSh and androstenedione were significnatly higher in middle-aged rats (12 months old) than in young controls (6 months old). These increases were selective, in that ten other neuroendocrine parameters measured were unchanged. These results indicate that changes occur at multiple levels of the neuroendocrine system during the transitional phase prior to the onset of senescent anovulation.     

Species-specific suppression of histone H1 and H2B production in human/mouse hybrids.

Ten human/mouse hybrid cell lines that segregate either human or mouse chromosomes were examined for the expression of human- and mouse-specific histones H1 and H2B. Results of this study indicate that the human and mouse chromosomes in hybrid cells that segregate human chromosomes (M greater than H hybrids) contain only mouse histone H1 and H2B. Chromosomes in hybrid cells that segregate mouse chromosomes (H greater than M hybrids) contain only human H1 and H2B histones. Loss of the ability to produce either human or mouse histones does not seem to be due to the loss of specific human or mouse chromosomes because M greater than H hybrids retaining at least one copy of each human chromosome contain only mouse H1 and H2B and H greater than M hybrids retaining at least one copy of each mouse chromosome contain only human H1 and H2B histones. These results, together with those concerning histone H4 acetylation levels and ratios of variants of histones H3 and H2A that are like those in the dominant parent cell type, indicate that the control mechanisms affecting H1 and H2B expression in H greater than M and in M greater than H hybrid cells affect expression of histones H2A, H3, and H4 genes as well. The present data thus support the hypothesis that none of the histone genes that are active in the recessive parent cell type is expressed in hybrid lines that segregate recessive cell chromosomes.     

Effects of clenbuterol on skeletal muscle mass, body composition, and recovery from surgical stress in senescent rats

Aging decreases skeletal muscle mass and strength, which may be exacerbated by age-related diseases. There is a need for therapeutic agents to prevent or restore loss of skeletal muscle in elderly subjects with muscle wasting disorders. Clenbuterol, a beta 2-adrenergic agonist, dramatically increases skeletal muscle mass in young animals and partially prevents or restores muscle loss in experimental models of muscle wasting. However, the protein anabolic and fat catabolic effects of clenbuterol have not been studied in senescent animals. To determine whether this drug has potential for preventing or repairing muscle loss in elderly subjects, we have examined its effects in young and old rats. Clenbuterol was administered by implanted osmotic minipumps to Fischer-344 rats ages 3, 12, and 23 months, at a dose of 1.5 mg/kg/24 h for 3 weeks. The weights of five hindlimb muscles and carcass protein and fat content were determined. Clenbuterol treatment increased the weight of skeletal muscles 22% to 39% in 3-month-old rats, 19% to 35% in 12-month-old rats, and 22% to 25% in 23-month-old animals. Likewise, clenbuterol increased carcass protein content 19% in 3-month-old rats, 16% in 12-month-old rats, and 24% in 23-month-old animals. Conversely, the drug reduced carcass fat content 36% in 3-month-old rats, 32% in 12-month-old rats, and 38% in 23-month-old rats. Therefore, clenbuterol had similar anabolic and catabolic effects in all age groups. In addition, clenbuterol stimulated recovery of skeletal muscle protein lost following pump implantation in senescent rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes of the H1 and H1(0) histone variants in murine tissues

The relative proportion of the histone H1(0) which is present in chromatin of nondividing and terminally differentiated cells is shown to increase with age. CNBr nonenzymatic cleavage and SDS-polyacrylamide gel electrophoresis of H1(0) extracted from liver chromatin of young and old mice and from age-related hepatocarcinomas showed that H1(0) consists of two variants, one contains methionine the other is methionine-free. The ratio between these two H1(0) variants also changes with age. The relative amounts of specific minor H1 and H1(0) histone fractions which are more loosely bound in chromatin and are extractable with 0.35 M NaCl, together with the HMG nonhistone proteins decrease in ageing mouse tissues. The age-related alteration of the ratio between H1(0) variants probably represents the chromatin repair process, whereas the age-related replacement of H1A and H1B subfractions by H1(0) histones may reflect the continuing process of differentiation.     

Skeletal muscle aging in F344BN F1-hybrid rats: I. Mitochondrial dysfunction contributes to the age-associated reduction in VO2max

Although mitochondrial DNA damage accumulates in aging skeletal muscles, how this relates to the decline in muscle mass-specific skeletal muscle aerobic function is unknown. We used a pump-perfused rat hind-limb model to examine maximal aerobic performance (VO(2max)) in young adult (YA; 8-9-month-old), late middle aged (LMA; 28-30-month-old) and senescent (SEN; 36-month-old) Fischer 344 x Brown Norway F1-hybrid rats at matched rates of convective O(2) delivery (QO(2)). Despite similar muscle QO(2) during a 4-minute contraction bout, muscle mass-specific VO(2max) was reduced in LMA (15%) and SEN (52%) versus YA. In plantaris muscle homogenates, nested polymerase chain reaction revealed an increased frequency of mitochondrial DNA deletions in the older animals. A greater reduction in the flux through electron transport chain complexes I-III than citrate synthase activity in the older animals suggests mitochondrial dysfunction consequent to mitochondrial DNA damage with aging. These results support the hypothesis that a reduced oxidative capacity, due in part to age-related mitochondrial dysfunction, contributes to the decline in aerobic performance in aging skeletal muscles.     

Effect of aging on expression of angiogenesis-related factors in mouse skeletal muscle

The molecular mechanisms by which capillary supply is maintained with advancing age remain to be elucidated. To help clarify these mechanisms, we investigated the gene expression levels of angiogenesis-related factors in young (2.5-month-old), adult (6-month-old), and old (22-month-old) mice. To assess the capillary supply, the capillary endothelium in frozen transverse sections was identified by staining for alkaline phosphatase. The mRNA levels for angiogenesis-related factors were analyzed using real-time RT-PCR. The capillary supply to individual muscle fibers, assessed as the number of capillaries around a muscle fiber, did not change with advancing age. Real-time RT-PCR analysis showed that (1) the level of mRNA for VEGF was lower in old animals than young animals; (2) the mRNA levels of Flt-1 and neuropilin-1 are lower in old animals than young animals, while that of KDR/Flk-1 remained unchanged with advancing age; and (3) the levels of mRNA for angiopoietin-1 and -2 remained unchanged, while the mRNA for Tie-2 was lower in old animals than young animals. These findings suggest that capillary supply is maintained irrespective of the down-regulation of several angiogenesis-related factors and that old animals possess the minimum levels of maintenance and reparative abilities needed to preserve the capillary supply.     

Age related increase of brush border enzyme activities along the small intestine.

Intestinal morphology and brush border hydrolase activities were determined along the small intestine of young adult (three months, n = 10), mature (12 months, n = 10), and senescent (29 months, n = 15) rats. The intestinal segments of the senescent rats contained higher mucosal mass and protein content (p less than 0.05) compared with the young and mature animals. A significant reduction of villus height and crypt depth (p less than 0.05) was found in the proximal intestine during aging. A 35% increase in villus height (p less than 0.05) without changes in crypt depth, was observed in the distal ileum in senescent rats. The activities of sucrase and isomaltase were significantly increased during aging in the duodenum and jejunum (p less than 0.05). Lactase and aminopeptidase activities which showed only minor changes between young and mature animals were significantly enhanced in senescent animals (p less than 0.05) with aminopeptidase exhibiting a three-fold increase in activity in the proximal ileum. The results when combined with those of previous studies suggest that in the aged animal, the increased level of intestinal hydrolase activities may be the consequence of prolonged cellular maturation along the villi in the proximal intestine, and of adaptation to increased concentrations of intraluminal substrates in the distal intestine.     

Induction of tyrosine hydroxylase and neuropeptide Y by carbachol: modulation with age

With aging, circulating catecholamines are elevated in both humans and animals. This may be related to the increased basal levels of dopamine beta-hydroxylase (DbetaH) and tyrosine hydroxylase (TH) mRNA levels and TH enzyme activity in the adrenal medulla of senescent compared with younger animals. Cold exposure induces TH and DbetaH mRNA, and the cholinergic pathway is believed to be involved in the cold-stimulated increase in TH expression in the adrenal medulla. However, TH gene expression in the senescent rat is resistant to stimulation by cold exposure, suggesting that the cholinergic pathway may be impaired with age in the adrenal medulla. To investigate this possibility, we administered carbachol (0.5 mg/kg i.p., every 12 hours for 3 consecutive days), a mixed nicotinic-muscarinic agonist, to young (4-month-old) and senescent (24-month-old) male F-344 rats. We examined the induction of TH mRNA, TH immunoreactivity, and TH enzyme activity in the adrenal medulla in young and old rats. In addition DbetaH and NPY mRNA levels were determined in the adrenal medulla with or without carbachol administration. Basal levels of TH mRNA, TH immunoreactivity, and TH activity as well as DbetaH and neuropeptide Y (NPY) mRNA were 1.5- to 4-fold greater in the adrenal medullae of old rats compared with young rats. Carbachol administration increased TH mRNA, TH immunoreactivity, and TH activity as well as DbetaH and NPY mRNA to the same or a greater extent in the senescent compared with the young rats. The present study indicates that the cholinergic induction of TH or DbetaH are not impaired with age, and that senescent rats retain the capacity to respond to carbachol stimulation. The present findings cannot explain why the adrenal medullae from senescent rats are resistant to the cold-induced elevation of TH mRNA and TH activity observed in young rats.     

Age-related changes in renal function, membrane protein metabolism, and Na,K-ATPase activity and abundance in hypokalemic F344 x BNF(1) rats.

BACKGROUND: Potassium depletion is a common electrolyte abnormality in elderly humans, usually as a consequence of diuretic use or poor oral intake. Hypokalemia is associated with a number of changes in renal function and an increase in some renal membrane transporters; its growth-promoting effect in young animals is well known. With aging, the renal adaptation to a number of challenges is often diminished. We hypothesized that aging is related to decreases in renal function, renal membrane protein metabolism, as well as Na, K-ATPase protein abundance and activity in both control animals as well as in those with potassium depletion. OBJECTIVE: We examined the effects of dietary-induced hypokalemia in true-aged nonobese rats (30 months old) on renal function, cortical brush border membrane (BBM) and basolateral membrane (BLM) protein metabolism, and Na,K-ATPase protein abundance and activity. We compared the results obtained to those seen in their 4-month-old counterparts similarly treated. Methods: Young (4-month-old) and senescent (30-month-old) male Fisher 344 x Brown-Norway F(1) rats (F344 x BNF(1)) were fed either a normal or potassium-deficient diet for 7 days. At 24 h, the U-(14)C-leucine incorporation was measured for determination of protein metabolism in renal BBM and BLM. Cortical BLM vesicle and microdissected proximal convoluted tubule (PCT) Na, K-ATPase activities were determined along with Western blot analysis of the cortical BLM alpha(1) subunit of Na,K-ATPase. Metabolic and renal function parameters were also examined. RESULTS: Hypokalemia caused hyperbicarbonatemia, hyperglycemia, and azotemia, but only in the senescent animals. The aged control rats had a higher basal level of urine volume, ammonium excretion, and fractional excretion of chloride. By contrast, aging in the F344 x BNF(1) rats was associated with a decrease in plasma aldosterone (by 35%) and phosphate (by 40%) levels as compared with their young controls. Hypokalemia resulted in a significant reduction of plasma aldosterone and a rise in muscle sodium concentration in both age groups; it significantly increased renal BBM and BLM protein concentrations in the young group, while these parameters remained unchanged in the senescent rats. The aged potassium-depleted animals showed a 14% decrease in BBM protein biosynthesis, but there were no changes in the young hypokalemic rats. Both potassium-depleted elderly and young rats had a significant reduction (by 33%) in BLM protein biosynthesis. Hypokalemia significantly increased the Na, K-ATPase activity in both cortical BLM vesicles and in microdissected PCT. The percentage increase in microdissected PCT segments (Na,K-ATPase activity) in elderly potassium-depleted animals was significantly less than that seen in hypokalemic young ones. Aging, per se, was associated with decreased basal microdissected PCT Na,K-ATPase activity in control animals. Hypokalemia had no effect on cortical BLM alpha(1) subunit Na, K-ATPase protein abundance in either age group. CONCLUSIONS: The present study provides the first evidence in nonobese aged rats as to the metabolic parameters, renal function, renal cortical membrane protein metabolism, and transporter Na,K-ATPase activity and abundance during potassium depletion. The aged nonobese F344 x BNF(1) rats responded differently from their young nonobese counterparts following potassium depletion. These differences may contribute substantially to the effects often encountered in elderly humans receiving diuretics or having a poor dietary potassium intake.     

Mechanism of specific force deficit in the senescent rat diaphragm

Aging is associated with a decline in the maximal in vitro specific force in the rat costal diaphragm. The purpose of this study was to determine if this force deficit is associated with a decrease in the concentration of myofibrillar protein in diaphragm fibers of senescent rats. Isometric twitch and tetanic contractile properties were measured on diaphragm strips from young adult (9-month-old; n=12) and senescent (26-month-old; n=13) male specific pathogen free-barrier protected Fischer 344 rats. Maximal tetanic force (P_o) normalized to the cross-sectional area (CSA) of the in vitro diaphragm strips was 16.4% lower in the senescent diaphragms (21.03±0.4 N/cm²) compared to the young adult (25.16±0.5 N/cm²) (p<0.001). Diaphragm water content was significantly higher in the senescent group (75.9% of total wet mass) compared to the young adult (72.1% of total wet mass, p<0.05). Subtracting the contribution of water from the CSA of the diaphragm strips significantly reduced (p<0.05) the senescent specific P_o deficit (from −16.4 to −6.4%). Further, correcting P_o for the contribution of myofibrillar protein to CSA resulted in no age group differences in specific force. These data indicate that the age-related decline in diaphragm in vitro maximal specific P_o can be explained by an age-related increase in the water content of the diaphragm muscle. Future experiments are necessary to determine the mechanism(s) responsible for this observation.