Burst inflation test for measuring biomechanical properties of rat abdominal walls.

PURPOSE: Evaluation of potential grafts to improve upon current strategies for abdominal wall (AW) repair in small animal models typically involves mechanical testing using methods that currently are inadequate to assess physiologically relevant parameters. This study introduces burst inflation testing as a more relevant assessment of the mechanical integrity of the AW compared to traditional tensile testing. METHODS: AWs were excised from 14 healthy adult Fischer 344 rats and tested using either a custom burst inflation device or an Instron tensile testing system. Modulus outcomes from both testing methods were compared. RESULTS: Mechanical analyses of native AW using burst and tensile testing methods resulted in similar average tissue moduli, but with the burst test, there was significantly less variability among specimens. CONCLUSIONS: The burst test had greater repeatability compared to tensile testing and has the ability to test repaired AWs without compromising the integrity of the repair site, making it a useful tool for assessing graft repairs.

Effect of cold on serum substrate and glycogen concentration in young and old Fischer 344 rats.

This investigation evaluated the hypothesis that the age-related decline in cold-induced thermogenesis observed in male (F344) rats is associated with altered substrate concentrations of glucose, lactate, and/or liver and muscle glycogen. Body mass-independent O2 consumption, core temperature, and serum glucose and lactate concentrations were measured at rest and during 4 h of exposure to 5 degrees C in male F344 rats ages 6, 12, and 26 months. At the end of the 4-h cold exposure, liver, soleus, and gastrocnemius tissues were removed, frozen, and analyzed for glycogen concentration and/or citrate synthase activity. Core temperature decreased during cold exposure and was consistently less in the 26-month versus the 6- and 12-month rats. There were no significant differences between the 6- and 12-month-old rats with respect to cold-induced O2 consumption, but measures were significantly lower in the 26-month-old rats. During cold exposure, serum lactate and glucose concentrations increased in the 26-month-old animals compared to those in the 6- and 12-month-old rats, while liver glycogen concentrations decreased in all groups, and gastrocnemius glycogen contents decreased in the 12- and 26-month-old rats. Citrate synthase specific activity (mumol.[min.microgram.protein] -1) did not differ with age. These data suggest that carbohydrate availability (as measured by serum glucose and muscle glycogen) is not a limiting factor in the attenuated cold-exposed thermogenic response of the 26-month-old male F344 rat. However, it appears that the 26-month-old rat may have a diminished capacity to fully oxidize carbohydrate during cold exposure.

Chemical versus dual energy x-ray absorptiometry for detecting age-associated body compositional changes in male rats.

Aging is associated with increases in body mass and fat mass (FM), whereas fat-free mass (FFM) either decreases or remains unchanged. The purpose of this study was to determine whether dual-energy X-ray absorptiometry (DXA) accurately detects age-associated changes in male Fischer 344 x Brown-Norway rats ages 8, 18, and 28 months. Eviscerated animal carcasses were first examined via the Lunar DPX-IQ DXA (small animal software version 1.0; HiRes (0.6 x 1.2 mm) medium mode). Eviscerated carcasses were then weighed, autoclaved, homogenized, and fat isolated from aliquots of homogenate via methanol/chloroform extraction. In both chemical (CHEM) and DXA analysis, carcass mass (CM), FM, and % fat were significantly higher (P < 0.0001) in the 18 and 28 versus 8-month-old rats. CHEM showed greater FFM in the 18 versus 8 months-old rats but not the 28 months-old animals. DXA was unable to detect the age-associated changes in FFM. Regression analysis showed a strong correlation between CHEM and DXA methods for CM (r = 0.98, P < 0.0001) and FM (r = 0.97, P < 0.0001), but less strong for FFM (r = 0.59, P = 0.0002). In conclusion, compared to CHEM, DXA consistently overestimated CM and FM across the age groups by 9% and 77%, respectively, and underestimated FFM by 5%.

Effect of aging and obesity on insulin responsiveness and glut-4 glucose transporter content in skeletal muscle of Fischer 344 x Brown Norway rats.

This study investigated the metabolic changes with age in the Fischer 344 x Brown Norway rat and its suitability as an animal model of postmaturational insulin resistance. Specifically, we determined whether an age-associated decrease in glucose disposal is associated with diminished whole body insulin responsiveness and/or a decrease in glucose transporter (GLUT-4) protein and mRNA content in medial gastrocnemius muscle of male Fischer 344 x Brown Norway rats of ages 8, 18, and 28 months. Fasting plasma glucose was unchanged with age. There was a significant age effect on visceral adiposity, fasting plasma insulin levels, insulin responsiveness, and GLUT-4 protein content. Insulin responsiveness and GLUT-4 protein were lower in the 18-month-old rats than in the 8-month-old rats. The findings of age-associated increases in visceral adiposity and insulin resistance, and decreases in GLUT-4 in the Fisher 344 x Brown Norway rat, suggest that this rat strain may be an appropriate model for studying the effects of aging on glucose homeostasis.

Effect of age and neurovascular grafting on the mechanical function of medial gastrocnemius muscles of Fischer 344 rats.

Both aging and grafting of whole skeletal muscle are associated with decreased specific force and resistance to fatigue. This study tested the hypothesis that the recovery of mechanical function in nerve-repair skeletal muscle grafts in senescent rats would be impaired compared with recovery in similar grafts in younger animals. Following a 120-day recovery period, the contractile properties of grafted medial gastrocnemius (MGN) muscles from young-mature (6 months), middle-aged (12 months), and senescent (24 months) Fischer 344 rats were measured and compared to age-matched controls. Although there was full recovery of muscle mass, grafting and aging alone both were associated with diminished maximum twitch and tetanic tension, maximum power, and maximum sustained power. In addition, the deleterious effect of grafting on maximum tetanic tension, specific force, and sustained power of MGN muscle was significantly greater in old animals. These findings suggest that aging limits full recovery of the quality of muscle contractions from the nerve-repair grafting procedure, possibly due to an age-related impairment of reinnervation.

Synergist muscle ablation and recovery from nerve-repair grafting: contractile and metabolic function.

After nerve-repair grafting of medial gastrocnemius muscle, there is incomplete recovery of specific force and sustainable power, perhaps due to overcompensation by synergistic muscles. We hypothesized that increased workload due to synergist ablation would enhance graft recovery. Contractile and metabolic properties of control and nerve-repair grafted muscles, with and without synergist ablation, were determined after 120 days recovery. Specific force (N/cm(2)) and normalized power (W/kg) were less in the experimental groups compared with controls. Sustained power (W/kg) in the synergist-ablated nerve-repair grafted muscle was higher than nerve-repair grafted muscle, returning to control values. GLUT-4 protein was higher and glycogen content was diminished in both synergist-ablated groups. In summary, synergist ablation did not enhance the recovery of specific force or normalized power, but sustained power did recover, suggesting that metabolic and not mechanical parameters were responsible for this recovery. The enhanced endurance after synergist ablation was accompanied by increased GLUT-4 protein, suggesting a role for increased uptake of circulating glucose during contraction.

Transient enhancement of GLUT-4 levels in rat epitrochlearis muscle after exercise training.

The purpose of the present study was to examine the effect of detraining on the glucose transport system after short-term swim training (5 days), long-term swim training (5 wk), and treadmill run training (5 wk). Skeletal muscles were isolated from female Wistar rats at 24 or 48 h posttraining. SST produces a 48% increase in GLUT-4 mRNA, a 30% increase in GLUT-4 protein, and a 60% increase in insulin-stimulated glucose transport activity at 24 h posttraining but not at 48 h posttraining. Similar to SST, long-term swim training produces a 60% increase in GLUT-4 mRNA and a 30% increase in GLUT-4 protein content at 24 h posttraining but not at 48 h posttraining. Finally, treadmill run training produces a transient 35% increase in GLUT-4 protein content that is completely reversed at 48 h after the last bout of exercise. These results demonstrate that the increase in GLUT-4 mRNA and GLUT-4 protein occurs during the first week of exercise training and is rapidly lost after training cessation. We believe that the transient enhancement in GLUT-4 protein after exercise training is due to a short GLUT-4 half-life, a process that is primarily regulated by pretranslational mechanisms.

Functional deficits in medial gastrocnemius grafts in rats: relation to muscle metabolism and beta-AR regulation.

This study tested the hypothesis that alterations in the metabolic integrity of grafted muscle contribute to its diminished ability to sustain power. Compared with control muscles, muscles studied 120 days after the grafting procedure had lower specific force and sustained power. The sustained power protocol resulted in a depletion of muscle glycogen in control (83%) and grafted (85%) animals. Grafts had lower pre- and poststimulation glycogen, diminished citrate synthase activity, and greater hexokinase activity. No differences were observed in phosphofructokinase activity, glucose transporter GLUT-4 content, fiber type, beta-adrenergic-receptor (beta-AR) density, or binding affinity. Isoproterenol-stimulated adenylyl cyclase activity was lower in grafted vs. control muscle, suggesting an uncoupling of the beta-AR-effector complex. Thus the diminished ability of the grafted muscle to sustain power may be explained, in part, by a decrease in energy available from glycogen stores and/or a decrease in oxidative capacity.

Effect of photoperiod on body weight and food intake of obese and lean Zucker rats.

Although the rat is usually not considered to be sensitive to photoperiod, under some experimental conditions photoperiod responses are unmasked. In addition, we have observed photoperiod-induced changes in body weight gain in lean and obese Zucker rats. In this experiment, body mass, food intake, body composition, brown adipose tissue (BAT) thermogenic state, and blood concentrations of corticosterone, insulin, and glucose were evaluated under one of two lighting conditions: a short (10 h light: 14 h dark) or a long (14 h light: 10 h dark) photoperiod. Plasma corticosterone and glucose concentrations measured under fasting conditions were unaffected by photoperiod in either genotype. The amount of BAT mitochondrial protein isolated was less in long photoperiod rats. BAT mitochondrial GDP binding was unaffected by photoperiod in the lean rats, but tended to be lower in long photoperiod obese rats than in short photoperiod obese rats. Although, photoperiod had no effect on daily food intake of rats exposed to the short versus long photoperiod, body mass was heaviest in obese rats raised in long photoperiod. Plasma insulin was increased in both lean and obese rats in long photoperiod. In addition, fat storage appeared to shift to internal depots in the lean rats exposed to long photoperiod. Our data demonstrate that photoperiod does have an effect on male Zucker rats with respect to body weight and fat distribution, with the obese rats being more sensitive to changes in photoperiod than the lean rats.

Use of adipose-derived stem cells to fabricate scaffoldless tissue-engineered neural conduits in vitro.

Peripheral nerve injuries resulting from trauma or disease often necessitate surgical intervention. Although the gold standard for such repairs uses nerve autografts, alternatives that do not require invasive harvesting of autologous nerve tissues are currently being designed and evaluated. We previously established the use of scaffoldless engineered neural conduits (ENCs) fabricated from primary cells as one such alternative in sciatic nerve repair in rats [Baltich et al. (2010) In Vitro Cell Dev Biol Anim 46(5):438-444]. The present study establishes protocols for fabricating neural conduits from adipose-derived stem cells (ASCs) differentiated to either a fibroblast or neural lineage and co-cultured into a three-dimensional (3-D) scaffoldless tissue-ENC. Addition of ascorbic acid-2-phosphate and fibroblast growth factor (FGF)-2 to the medium induced and differentiated ASCs to a fibroblast lineage in more than 90% of the cell population, as confirmed by collagen I expression. ASC-differentiated fibroblasts formed monolayers, delaminated, and formed 3-D conduits. Neurospheres were formed by culturing ASCs on non-adherent surfaces in serum-free neurobasal medium with the addition of epidermal growth factor (EGF) and FGF-2. The addition of 10 ng EGF and 10 ng FGF-2 produced larger and more numerous neurospheres than treatments of lower EGF and FGF-2 concentrations. Subsequent differentiation to glial-like cells was confirmed by the expression of S100. ASC-derived fibroblast monolayers and neurospheres were co-cultured to fabricate a 3-D scaffoldless tissue-ENC. Their nerve-like structure and incorporation of glial-like cells, which would associate with regenerating axons, may make these novel, stem cell-derived neural conduits an efficacious technology for repairing critical gaps following peripheral nerve injury.

Effect of aging on rat skeletal muscle beta-AR function in male Fischer 344 x brown Norway rats.

In this study, we tested the hypothesis that, in the male Fischer 344 x Brown Norway (F344xBN) rat, aging would be associated with an increase in sympathetic nervous system activity and a decrease in skeletal muscle beta-adrenergic-receptor (beta-AR) density and function. Radioligand-binding studies using [125I] iodocyanopindolol were done to evaluate beta-AR density (Bmax) and antagonist-binding affinity in gastrocnemius and cardiac muscle from 6-, 18-, and 28-mo-old male F344xBN rats. beta-AR function was measured as adenylyl cyclase (AC) activity stimulated by the beta-AR agonist isoproterenol (Iso, 10(-4) M). Basal arterial plasma norepinephrine (pNE) concentrations were higher in the 28-than in the 6- and 18-mo-old rats. Bmax was greatest and Iso-stimulated AC activity was unchanged in gastrocnemius muscle of the 28-mo-old age group. In contrast, there was an age-associated decrease in Bmax and Iso-stimulated AC activity in cardiac muscle. In conclusion, there was an age-associated increase in pNE concentrations in male F344xBN rats, suggesting an increase in sympathetic nervous system activity. In addition, there was an age-associated increase in skeletal muscle beta-AR density, whereas in skeletal muscle beta-AR-stimulated AC activity remained unchanged with age.

Adrenergic stimulated skeletal muscle glycogenolysis in perfused hindlimbs of young and old male Fischer 344 rats.

Epinephrine (Epi)- and forskolin (FSK)-stimulated glycogenolysis of skeletal muscle was evaluated in perfused hindlimb isolated from male Fischer 344 (F344) rats, ages 6, 12, and 26 mo. Muscle glycogen stores were reduced by sciatic nerve stimulation and replenished by infusing 10 mM glucose, 500 microU insulin, and 5 microCi [14C]glucose via a left carotid artery cannula. Then the hindlimb was perfused with a modified Krebs-Henseleit buffer (pH 7.4). At minute 20 of the perfusion, Epi [0.0 (perfusate), 0.25, 0.50, or 0.75 microM] or 40 microM FSK were infused for 10 min. Radioactivity (14C) in the effluent perfusate was collected every 60 s during a 20-min preinfusion, a 10-min Epi infusion, and a 20-min postinfusion period and was used to determine the rate of muscle glycogen utilization. Total 14C release increased with Epi and 40 microM FSK. However, the pattern of release did not differ significantly with age. In general, the fraction of the perfusate released as 14CO2 increased in the presence of FSK and Epi but did not significantly differ with age. [14C]lactate released in response to Epi increased in the 6-mo-old group, remained unchanged in the 12-mo-old group, and decreased in the 26-mo-old group compared with 0.0 Epi (perfusate) values. It appears that stimulation of skeletal muscle glycogenolysis via adrenergic receptor or postreceptor/adenosine 3',5'-cyclic monophosphate-mediated mechanisms is unaffected by age. However, the utilization of carbohydrate by isolated hindlimb muscle is altered in the aging rat, resulting in a more oxidative metabolism.

Effect of age and high sucrose diet on 2-deoxyglucose uptake in perfused hindlimb.

The purpose of this investigation was to evaluate insulin-stimulated skeletal muscle glucose uptake in male Fischer 344 rats, age 6, 12, and 27 mo, fed either a sucrose (S: 66.6% sucrose, 17.6% protein, 6.4% fat) or sucrose-free (SF: 66.6% starch, 17.6% protein, 6.4% fat) diet for 3 mo. Skeletal muscle glucose uptake (Rg) in perfused hindlimbs was estimated from the uptake and subsequent phosphorylation of radiolabeled 2-deoxyglucose (2DG) in the gastrocnemius (GN), extensor digitorum longus (EDL), and soleus (SOL) muscles. Rat hindlimbs were perfused at a rate of 10 ml.min-1 with a modified Krebs Henseleit buffer containing bovine red blood cells (hematocrit: 40%) and 5.85 mmole.L-1 glucose along with 358 pmoles.L-1 followed by 3580 pmoles.L-1 insulin. There was no effect of diet on plasma glucose levels measured at weeks 1, 7, and 11 of the dietary period. A significant effect of age on estimated glucose uptake in the GN was demonstrated due primarily to greater uptake in the 27-mo compared to the 6-mo-old animals. This significant effect of age was not evident in the EDL or SOL, nor were there significant effects of diet in any muscle. These data suggest that insulin-stimulated glucose uptake in perfused hindlimbs is not attenuated with senescence or with the feeding of a sucrose diet for 3 months.