Mammalian cells require a continuous and sufficient supply of oxygen to carry out their functions. The oxygen pathway has an overall direction taking O2 from the air to the mitochondria, which is a result of the mitochondrial O2 consumption (VO2) NO various effects on the mitochondria: at low concentrations for short periods NO specifically and irreversibly inhibits cytochrome c and reversibly inhibits cytochrome c oxidase, to decrease VO2. Thus, NO can modulate VO2 of skeletal muscle. The purpose of the present study was to measure VO2 of the rat spinotrapezius muscle under conditions of altered NO. The methods used provide a direct way to measure PO2 in the interstitium (PISFO2) and use it as indicator of local metabolic changes. Intravital microscopy and phosphorescence quenching were used to record PISFO2 in resting muscle for 120 s before, 60 s during, and 420 s after a period of tissue compression that abruptly halted perfusion. Control VO2 measurements were made, followed by those in which the spinotrapezius muscle had been treated by topical application of agents known to alter NO levels (L-NAME, C-PTIO, Sperm/NO). The compression was achieved by rapid inflation of a Saran film air bag, attached to a X20 objective lens, which pressed the muscle against the animal platform. The rapid pressure onset (0-120 mmHg in ISFO2, which started immediately after the airbag inflation, was used to calculate VO2 and was based on the assumption that the amount of blood in the tissue after compression was small. Control VO2 was 5.91 ± 0.2 ml O2 · kg-1 · min-1. Since the presence of RBCs sequestered in capillaries cannot be ruled out during compression, this value can be considered a lower limit for VO2 by resting muscle. Comparison of baseline and treatment measurements of VO2 showed no significant differences between them. This was unexpected based on in vitro studies and may reflect an impaired ability of the agents used to alter NO at the mitochondrial level.

In resting muscle, blood flow is regulated to meet the demand for O2 by the tissue. A modified ischemia (I)/reperfusion(R) investigation was systematically run and PISFO2, PaO2, Q and VO2 were observed. Twenty-nine spinotrapezius muscles from male Sprague-Dawley rats (284±20 grams) were surgically exteriorized for intravital microscopy to test a model relating blood flow, O2 supply and O2 demand. The model can aid in the understanding of the regulation of tissue PO2. The interstitial PO2 (PISFO2) and perivascular PO2 (PaO2) measurements were made using phosphorescence quenching microscopy (PQM). O2 consumption (VO2) values were obtained with a quasi-continuous, flash-synchronized, pressurized airbag to initiate ischemia and sample the rate of O¬2 change (dPO2/dt). Centerline red blood cell velocity was measured with an Optical Doppler Velocimeter and converted to flow using vessel diameter. 5-, 15-, 30-, 60-, 300- and 600-second ischemic durations were used to observe changes in PISFO2, Q, and VO2. A critical point was observed following 30 seconds of (I) where dPISFO2/dt during recovery was the fastest (4.25±0.72 mmHg/s) and was 1.00±0.16 mmHg/s following 600 seconds. Flow recovery, dQ/dt, peaked to 3.88±0.64 (µl•min-1)/s after 60 seconds of (I) but significantly dropped to 2.83±0.55 (µl•min-1)/s following 300 seconds of (I) but increased to 2.92±0.45 (µl•min-1)/s following 600 seconds. This gives evidence to a no-reflow phenomenon occurring in the extended periods of ischemia. A peak in VO¬2 to 309.2±45.0 nl O2/cm3•s with a time course of 160 seconds occurred following 600 seconds of ischemia. As the ischemic duration decreased, the time course and peak VO2 also decreased. VO2 following 300 seconds of (I) was significantly higher than 5-60 seconds of (I) (p <0.05) but was not significantly different from 600 seconds of (I). The information collected during the Q and VO2 studies can be incorporated into a factor, M, that relates VO2, Q and ∆PO2. M calculated for the recovery of 5- through 60-second (I) groups reasonably relates the three variables due to consistency and little variability. However, recovery in 600- and especially 300-second (I) groups showed higher variability in M which requires more consideration.

Peripheral arterial occlusive disease (PAOD) is a globally-prevalent cardiovascular disease in which atherosclerotic plaques narrow arterial lumen diameters and restrict blood flow to downstream tissues. The impact of these occlusions can be mitigated by collateral vessels that connect parallel arterial branches and act as natural bypasses to maintain perfusion. In animal models that lack collateral arterioles, capillaries that connect terminal arteriolar segments can arterialize and form functional collaterals following an ischemic event; however, in the early stages of development, vasodilation is impaired. We explored the mechanism of impaired vasodilation in arterialized collateral capillaries (ACCs) and pre-existing collaterals (PECs) by evaluating endothelial-dependent vasodilation and endothelial-independent reactivity at day seven following the ischemic event. We also evaluated functional vasodilation in mature ACCs and PECs at day 21 by applying vasodilation inhibitors during the electrical stimulation of muscle contraction. Arterial occlusion was performed by ligating the cranial-lateral spinotrapezius feed artery in Balb/C mice, a strain that either lacks native arteriolar collaterals or contains a single collateral arteriole (~50% of mice), as opposed to the C57Bl/6 strain, which each contain 10 or more collateral arterioles. At seven days post-surgery, both vasodilation and vasoconstriction were impaired in ACCs when compared to terminal arterioles of similar size in unoperated limbs, but still exhibited significant changes when compared to baseline. The comparable reactivity in both endothelial-dependent and independent vasodilation at day-seven in ACCs indicates that vascular smooth muscle cells are likely responsible for the impairment, as they may still be developing, rearranging, or both, and are not yet fully capable of regulating diameter in immature ACCs. However, by 21 days post-ligation, ACCs regained the capacity to dilate in response to muscle contraction, and utilized similar vasodilation pathways as control vessels. At seven days post-ligation, PECs had impaired endothelialindependent dilation, but successful endothelial-dependent dilation, indicating the use of alternative pathways to dilate. Unlike ACCs, the PECs never completely restored vasodilation capabilities by day 21, which may be due to a variation in smooth muscle phenotype, sensitivity to vasoactive agents, and/or limited growth factor expression. For future work, evaluating collateral formation and vasodilation in a diseased model and investigating molecular variations in the smooth muscle may yield additional knowledge that can improve therapies for patients during ischemic events.

Skeletal muscle dysfunction in chronic obstructive pulmonary disease (COPD) is a condition in which peripheral skeletal muscle undergoes myopathic changes which impair muscle function, limit physical performance, and can lead to significant disability. While the etiology of the dysfunction is unknown, this study was conducted to test the hypothesis that chronic hypoxemia leads to alterations in oxygen transport and muscle function. A primary objective was to validate elastase-induced emphysema in rats as an animal model of skeletal muscle dysfunction in COPD.Arterial blood gases were used to determine the severity of hypoxemia and sodium dodecyl sulfate- polyacrylamide gel electrophoresis was used to determine the proportions of myosin heavy chain isoforms I, IIa, IIx, and IIb. Measures of microvascular oxygenation and blood flow in the spinotrapezius muscle allowed for determination of both convective and diffusive oxygen supply to the muscle, as well as calculation of muscle oxygen consumption at rest and during electrically stimulated three-minute muscle contractions. Muscle performance measures included peak force, force-time integral, and fatigue index. Due to a presumed rat respiratory virus, which likely resulted in the control group being nearly as hypoxemic as the elastase-induced emphysema group, this study was not able to definitively test the hypothesis that chronic hypoxemia leads to both a diminished supply and demand of oxygen in skeletal muscle. Although many of the results of the present study were not statistically significant, they exhibited consistent trends over time and are likely of physiological significance. All measures of muscle performance were lower in the emphysema group. In addition, spinotrapezius muscle oxygen consumption and blood flow were lower in the emphysema group. The addition of supplemental oxygen during isolated, small-muscle mass exercise did increase the force-time integral by ~18% in both groups, suggesting that muscle work in these hypoxemic animals may be limited by oxygen supply. Thus, the data on muscle fiber type, oxygen consumption and muscle performance suggest that elastase-induced emphysema in rats leads to a similar skeletal muscle dysfunction that is observed in humans with COPD, and indicates that it is a valid animal model of skeletal muscle dysfunction in COPD.

There are gaps in the fundamental understanding of the expression of nitric oxide synthases (NOS) in the microvasculature. We examined co-localization of NOS1 (nNOS), NOS2 (iNOS) and NOS3 (eNOS) in the spinotrapezius muscle of young adult male Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SHR) rats according to fiber type using immunohistochemistry and brightfield microscopy. Data regarding fiber distribution, population and morphology data were collected. Alkaline phosphatase staining was used to determine capillary density and average number of capillaries around a fiber. Gel electrophoresis and Western blot techniques were used to compare myosin heavy chain (MHC) protein expression with fiber type population data and to determine NOS1-3 protein expression in whole muscle homogenate. This study should provide a more accurate understanding of differences in NOS expression between these two strains of rats.