PO2 cycling reduces diaphragm fatigue by attenuating ROS formation.

Prolonged muscle exposure to low PO2 conditions may cause oxidative stress resulting in severe muscular injuries. We hypothesize that PO2 cycling preconditioning, which involves brief cycles of diaphragmatic muscle exposure to a low oxygen level (40 Torr) followed by a high oxygen level (550 Torr), can reduce intracellular reactive oxygen species (ROS) as well as attenuate muscle fatigue in mouse diaphragm under low PO2. Accordingly, dihydrofluorescein (a fluorescent probe) was used to monitor muscular ROS production in real time with confocal microscopy during a lower PO2 condition. In the control group with no PO2 cycling, intracellular ROS formation did not appear during the first 15 min of the low PO2 period. However, after 20 min of low PO2, ROS levels increased significantly by ∼30% compared to baseline, and this increase continued until the end of the 30 min low PO2 condition. Conversely, muscles treated with PO2 cycling showed a complete absence of enhanced fluorescence emission throughout the entire low PO2 period. Furthermore, PO2 cycling-treated diaphragm exhibited increased fatigue resistance during prolonged low PO2 period compared to control. Thus, our data suggest that PO2 cycling mitigates diaphragm fatigue during prolonged low PO2. Although the exact mechanism for this protection remains to be elucidated, it is likely that through limiting excessive ROS levels, PO2 cycling initiates ROS-related antioxidant defenses.

The role of exercise training on lipoprotein profiles in adolescent males.

BACKGROUND: Major cardiovascular disorders are being recognized earlier in life. In this study we examined the effects of swimming and soccer training on male adolescent lipid-lipoprotein profiles relative to a maturity matched control group to determine the effects of these exercises on specific cardiovascular risk and anti-risk factors. METHODS: Forty five adolescent males (11.81 ± 1.38 yr) including swimmers (SW), soccer players (SO), and non-athlete, physically active individuals as controls (C), participated in this study. Training groups completed 12-wk exercise programs on three non-consecutive days per week. Plasma low-density lipoprotein (LDL), very low density lipoprotein (VLDL), high density lipoprotein (HDL), apolipoprotein A-I (apoA-I), apolipoprotein B (apoB), total cholesterol (TC), and triglyceride (TG) levels were measured in control, pre-training, during-training, and post-training. RESULTS: In response to the 12-wk training period, the SO group demonstrated a decrease in the mean LDL level compared to the SW and C (SW: 0.15%; SO: -9.51%; C: 19.59%; p < 0.001) groups. There was an increase in both the SW and SO groups vs. the control in mean HDL (SW: 5.66%; SO: 3.07%; C: -7.21%; p < 0.05) and apoA-I (SW: 3.86%; SO: 5.48%; C: -1.01%; p < 0.05). ApoB was considerably lower in the training groups vs. control (SW: -9.52%; SO: -13.87%; C: 21.09%; p < 0.05). ApoA-I/apoB ratio was significantly higher in training groups vs. control (SW: 16.74%; SO: 23.71%; C: -17.35%; p < 0.001). There were no significant differences between groups for other factors. CONCLUSIONS: The favorable alterations in LDL, HDL, apoA-I, and apoB observed in the training groups suggest that both regular swimming or soccer exercise can potentially mitigate cardiovascular risk in adolescent males.

Diagnostic ultrasound imaging of mouse diaphragm function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Molecular characterization of reactive oxygen species in systemic and pulmonary hypertension.

Hypertension, commonly recognized as high blood pressure, is a serious disease that affects millions of people worldwide. Similar to many physiological disorders, hypertension consists of several different cellular signaling pathways that involve various molecular messengers. Recent studies have shown that reactive oxygen species (ROS) play a substantial role in the development of both systemic and pulmonary hypertension, contributing to the pathology of this disease. However, the exact molecular mechanism of ROS in hypertension is not completely understood. In this review, we extensively examine and discuss the most recent experimental findings regarding the role of ROS in both pulmonary and systemic hypertension. Current studies show that excessive ROS not only promote JAK/STAT (janus kinase/signal transducers and activators of transcription)-mediated vascular remodeling in an angiotensin (ANG) II-induced hypertension model but also decrease the nitric oxide bioavailability. Furthermore, it has been shown that ROS generation can be mitigated through the inhibition of upstream ANG II or by blocking key ROS generators, such as nicotinamide adenine dinucleotide phosphate oxidase. Thus, various treatment options have been explored. Yet, as discussed in the current review, the regulation of ROS via novel antioxidant therapies may provide an alternative treatment for hypertension in the future.

Superoxide release from contracting skeletal muscle in pulmonary TNF-α overexpression mice.

Chronic obstructive pulmonary disease (COPD) often results in increased levels of tumor necrosis factor-α (TNF-α), a proinflammatory cytokine, which circulates in the blood. However, it is not clear whether pulmonary TNF-α overexpression (a COPD mimic) induces excessive reactive oxygen species (ROS) formation in skeletal muscle and thereby may contribute to the muscle impairment often seen in COPD. We hypothesized that ROS generation in contracting skeletal muscle is elevated when there is TNF-α overproduction in the lung and that this can induce muscle dysfunction. Cytochrome c (cyt c) in the perfusate was used to assay superoxide (O2(·-)) release from isolated contracting soleus muscles from transgenic mice of pulmonary TNF-α overexpression (Tg(+)) and wild-type (WT) mice. Our results showed that Tg(+) muscle released significantly higher levels of O2(·-) than WT during a period of intense contractile activity (in nmol/mg wt; 17.5 ± 2.3 vs. 4.4 ± 1.3, respectively; n = 5; P < 0.05). In addition, the soleus muscle demonstrated a significantly reduced fatigue resistance in Tg(+) mice compared with WT mice. Perfusion of the contracting soleus muscle with superoxide dismutase, which specifically scavenges O2(·-) in the perfusate, resulted in significantly less cyt c reduction, thereby indicating that the type of ROS released from the Tg(+) muscles is O2(·-). Our results demonstrate that pulmonary TNF-α overexpression leads to a greater O2(·-) release from contracting soleus muscle in Tg(+) compared with WT and that the excessive formation of O2(·-) in the contracting muscle of Tg(+) mice leads to earlier fatigue.

Low Po2 conditions induce reactive oxygen species formation during contractions in single skeletal muscle fibers.

Contractions in whole skeletal muscle during hypoxia are known to generate reactive oxygen species (ROS); however, identification of real-time ROS formation within isolated single skeletal muscle fibers has been challenging. Consequently, there is no convincing evidence showing increased ROS production in intact contracting fibers under low Po2 conditions. Therefore, we hypothesized that intracellular ROS generation in single contracting skeletal myofibers increases during low Po2 compared with a value approximating normal resting Po2. Dihydrofluorescein was loaded into single frog (Xenopus) fibers, and fluorescence was used to monitor ROS using confocal microscopy. Myofibers were exposed to two maximal tetanic contractile periods (1 contraction/3 s for 2 min, separated by a 60-min rest period), each consisting of one of the following treatments: high Po2 (30 Torr), low Po2 (3-5 Torr), high Po2 with ebselen (antioxidant), or low Po2 with ebselen. Ebselen (10 µM) was administered before the designated contractile period. ROS formation during low Po2 treatment was greater than during high Po2 treatment, and ebselen decreased ROS generation in both low- and high-Po2 conditions (P < 0.05). ROS accumulated at a faster rate in low vs. high Po2. Force was reduced >30% for each condition except low Po2 with ebselen, which only decreased ~15%. We concluded that single myofibers under low Po2 conditions develop accelerated and more oxidative stress than at Po2 = 30 Torr (normal human resting Po2). Ebselen decreases ROS formation in both low and high Po2, but only mitigates skeletal muscle fatigue during reduced Po2 conditions.

Topical delivery of 5-fluorouracil (5-FU) by 3-alkylcarbonyloxymethyl-5-FU prodrugs.

The 3-alkylcarbonyloxymethyl-5-fluorouracil (3-ACOM-5-FU) prodrugs have been characterized by their solubilities in isopropyl myristate (S(IPM)) and partition coefficients between IPM and pH 4.0 buffer (K(IPM:AQ)). Estimated S(AQ) values have been obtained from S(AQ) = S(IPM)/K(IPM:AQ.) The abilities of the prodrugs to deliver total 5-FU species from IPM suspensions through hairless mouse skin (J(i)) have been evaluated in diffusion cell experiments. All of the prodrugs were much more soluble in IPM than 5-FU, and the propionyloxymethyl (C2) member of the series was almost twice as soluble in pH 4.0 buffer. Except for the acetyloxymethyl (C1) member of the series, the 3-ACOM-5-FU prodrugs exhibited greater S(IPM) and S(AQ) values than the corresponding 1-alkylcarbonyloxymethyl (1-ACOM-5-FU) prodrugs. The 3-ACOM-5-FU prodrugs that exhibited greater S(IPM) and S(AQ) values than the 1-ACOM-5-FU prodrugs also exhibited greater J(i) values, except for the C2 member. The C2 member also gave the largest error in predicting J(i) using the transformed Potts-Guy equation. All of the 3-ACOM-5-FU prodrugs delivered more 5-FU as a percentage of J(i) than the 1-ACOM-5-FU prodrugs but were not more effective as a series at targeting dermal as opposed to transdermal delivery.

Genetic evidence for the correlation of deep dorsal horn Fos protein immunoreactivity with tonic formalin pain behavior.

The formalin test is commonly used as a model of persistent pain. Besides producing pain behavior, hind paw formalin injection induces the expression of the immediate-early gene, c-fos. A current controversy is whether noxious stimulus-induced Fos protein immunoreactivity can be considered a proxy (biomarker) of nociception in the spinal cord. We investigated this issue by exploiting our recent demonstration of genotype-dependent behavioral differences in response to formalin injection among inbred mouse strains. Accordingly, 6 inbred and 2 outbred strains were administered formalin (5% in 25 microL) into the ventral hind paw, monitored for licking behavior, and then sacrificed at 90 minutes after injection for Fos protein immunocytochemistry. Significant strain differences were observed in both licking behavior and Fos counts in superficial and deep laminae. We observed a significant correlation among strains between licking behavior in the late phase (10 to 60 minutes) of the formalin test and Fos expression in laminae V-VI (but not laminae I-II) of the dorsal horn (r = 0.94). These findings reinforce the use of the Fos technique to study the neuronal processing underlying pain but suggest that Fos labeling reliably reflects tonic pain behavior only in neurons located in the neck of the dorsal horn in mice.

Post-sympathectomy neuralgia: hypotheses on peripheral and central neuronal mechanisms.

Post-sympathectomy neuralgia is proposed here to be a complex neuropathic and central deafferentation/reafferentation syndrome dependent on: (a) the transection, during sympathectomy, of paraspinal somatic and visceral afferent axons within the sympathetic trunk; (b) the subsequent cell death of many of the axotomized afferent neurons, resulting in central deafferentation; and (c) the persistent sensitization of spinal nociceptive neurons by painful conditions present prior to sympathectomy. Viscerosomatic convergence, collateral sprouting of afferents, and mechanisms associated with sympathetically maintained pain are all proposed to be important to the development of the syndrome.

Slowly developing placebo responses confound tests of intravenous phentolamine to determine mechanisms underlying idiopathic chronic low back pain.

Phentolamine (30 mg) was administered intravenously to subjects with idiopathic chronic low back pain in a novel placebo-controlled test to determine whether this alpha-adrenergic antagonist would reduce their pain. The effects of infusions on spontaneous pain and stimulus-evoked pains (touch, cold, tapping and deep pressure) were evaluated separately. All subjects gave strong placebo responses (reduced pain) that prevented assessment of specific drug effects. The placebo responses had onset latencies of 15-60 min, developed slowly over the next 15-45 min and persisted for hours or several days. These results not only reinforce the understanding that placebo controls are essential in the evaluation of drugs or other palliative procedures on patients with chronic pain but also indicate that the control paradigms must allow for placebo effects that are slow to develop and very persistent.

Sympathetic activation of cat spinal neurons responsive to noxious stimulation of deep tissues in the low back.

Prior findings from diverse studies have indicated that activity in axons located in the lumbar sympathetic chains contributes to the activation of spinal pain pathways and to low back pain; these studies have utilized sympathetic blocks in patients, electrical stimulation of the chain in conscious humans, and neuroanatomical mapping of afferent fiber projections. In the present study, dorsal horn neurons receiving nociceptor input from lumbar paraspinal tissues were tested for activation by electrical stimulation of the lumbar sympathetic chain in anesthetized cats. Of 83 neurons tested, 70% were responsive to sympathetic trunk stimulation. Excitatory responses, observed in both nociceptive specific and wide-dynamic-range neurons, were differentiable into two classes: non-entrained and entrained responses. Non-entrained responses were attenuated or blocked by systemic administration of the alpha-adrenergic antagonist phentolamine and are thought to result from sympathetic efferent activation of primary afferents in the units' receptive fields. Entrained responses were unaffected by phentolamine and are thought to result from electrical activation of somatic and/or visceral afferent fibers ascending through the sympathetic trunk into the dorsal horn. These findings from nocireceptive neurons serving lumbar paraspinal tissues suggest that low back pain may be exacerbated by activity in both efferent and afferent fibers located in the lumbar sympathetic chain, the efferent actions being mediated indirectly through sympathetic-sensory interactions in somatic and/or visceral tissues.

Characterization of spinal somatosensory neurons having receptive fields in lumbar tissues of cats.

In pentobarbital anesthetized cats, extracellular unitary recordings were made from neurons in the extreme lateral dorsal horn of spinal segments L4-5. All 118 units reported had receptive fields in deep somatic tissues and/or skin of the lumbar region, hip and/or proximal leg. Neurons were functionally characterized according to their responses to non-noxious and noxious mechanical stimuli and to injections of algogens. Most neurons (92%) were either wide-dynamic range (WDR) or nociceptive specific (NS), and most of these had very large nociceptive receptive fields in the back/hip/leg that included both skin and deep somatic tissues innervated through both the dorsal (back/hip) and ventral (leg/ventral spine) rami. Most (72%) were 'hyperconvergent' in that they were responsive to stimulation of many different somatic tissues including skin, muscles, facet joint capsules, ligaments, and periosteum. Some units were tested and found also to be activated by noxious stimulation of spinal dura and ventral annulus fibrosis and ventral longitudinal ligament. Twelve of 22 neurons tested were found to have ascending axons extending beyond Th10. The nocireceptive neurons (NS and WDR) in the population tested are suitable for processing information about tissue damage in deep somatic tissues in the back, hip and proximal leg. The apparent relative paucity of such neurons and their very large hyperconvergent receptive fields suggest that sensations served by these neurons, such as low back and referred leg pain, would be neither well localized nor attributable to pathology in a specific tissue. These deductions, based on physiological characteristics in cats, are consistent with clinical reports from humans who experience pain as a consequence of spinal or paraspinal injuries.

Identification of afferents contributing to sympathetically evoked activity in wide-dynamic-range neurons.

The purpose of this study was to determine which types of mechanoreceptor afferents contribute to sympathetically evoked activity in wide-dynamic-range (WDR) neurons--the spinal neurons thought to mediate sympathetically maintained pain. The experimental approach was to record and compare activity evoked in single WDR neurons, hair afferents, and slowly adapting type I (SAI) afferents in anesthetized cats. During electrical stimulation of the sympathetic trunk, WDR neurons responded with either an early transient burst of activity, sustained activity, or both. The early transient response was observed only in neurons with piloerection in the receptive field; this response had a similar time course to sympathetically evoked activity in hair afferents with piloerection in the receptive field. The sustained response that occurred in some WDR neurons was independent of piloerection and was similar in time course to the response evoked in SAI afferents by sympathetic stimulation. We conclude that hair and SAI afferents contribute to different components of sympathetically evoked activity in WDR neurons and that both types of afferents are likely to be involved in sympathetically maintained pain in humans.

Spinal recordings suggest that wide-dynamic-range neurons mediate sympathetically maintained pain.

In order to determine which classes of spinal neurons are capable of mediating sympathetically maintained pain, recordings were made from single somatosensory neurons in spinal cords of anesthetized cats. Each neuron was functionally identified with mechanical stimuli, and its responses to electrical stimulation of the sympathetic trunk were recorded. Nearly half (45%) of the wide-dynamic-range (WDR) neurons tested were activated by sympathetic stimulation, but none of the high threshold (nociceptor-specific) neurons and only 17% of the low threshold neurons were activated. Sympathetic activation was most common for WDR neurons that had the following: receptive fields proximal to the toes, low thresholds for mechanical activation, and both rapidly and slowly adapting responses to pressure. The predominant WDR response to sympathetic stimulation was long latency (greater than 1 sec) excitation. Sympathetic activation of WDR neurons was abolished by each of the following procedures: subcutaneous injection of local anesthetic, cooling of the receptive field with ice, and intravenous injection of the alpha-adrenergic blocker, phentolamine. The axons of some sympathetically activated WDR were shown to project to higher centers. These results indicate that WDR neurons are the only spinal nociceptive neurons activated by sympathetic efferent activity in this preparation. Therefore, WDR neurons, rather than high threshold neurons, are most likely to mediate the spinal component of sympathetically maintained pain. These results provide supporting evidence for our previous hypothesis that sympathetically maintained pain is mediated by myelinated mechanoreceptors acting on sensitized WDR neurons. Our results also demonstrate that sympathetic activation of WDR neurons is mediated by an alpha-adrenergic mechanism in the skin.

A hypothesis on the physiological basis for causalgia and related pains.

A hypothesis is presented concerning the neuronal mechanisms which subserve the sympathetically maintained pains such as causalgia and reflex sympathetic dystrophy. The hypothesis rests on two assumptions: that a high rate of firing in spinal wide-dynamic-range (WDR) or multireceptive neurons results in painful sensations; and that nociceptor responses associated with trauma can produce long-term sensitization of WDR neurons. The hypothesis states that chronic sympathetically maintained pains are mediated by activity in low-threshold, myelinated mechanoreceptors, that this afferent activity results from sympathetic efferent actions upon the receptors or upon afferent fibers ending in a neuroma and that these afferent fibers evoke sufficient activity in sensitized spinal WDR neurons to produce a painful sensation. This hypothesis is based on known characteristics of these neuronal populations studied in experimental animals and on the observed sensory disturbances reported in patients successfully treated with sympathetic blocks. This hypothesis does not require nerve injury or dystrophic tissue. It explains both the continuous pain and the allodynia that are common to these syndromes and their abolition by sympathetic block. Specific changes are proposed in the diagnosis and treatment of post-traumatic pains.