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 ...

The Ohio State University College of Medicine

Oakland University

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.

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

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.

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

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.

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

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.

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

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.

William J. Roberts

Radiologic Sciences & Respiratory Therapy Division,
School of Health and Rehabilitation Sciences,
Davis Heart and Lung Research Institute,
Radiologic Sciences & Respiratory Therapy Division, School of Health and Rehabilitation Sciences, Davis Heart and Lung Research Institute

The Ohio State University College of Medicine

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function