[Eosinophilic bronchitis: update and review].

Eosinophilic bronchitis (EB) is a common cause of chronic cough, which shares similar airway eosinophilic inflammation with asthma, however, there is no airway hyperresponsiveness and airflow obstruction. The mechanism of the different phenotype between EB and asthma remains unclear. The differences in the location of airway inflammation, the level of inflammatory mediators, the imbalance of important metabolic pathways, and the degree of airway remodeling may result in different pathogenesis between EB and asthma. EB response well to inhaled corticosteroids but recurrence of EB is still high after treatment. The longer duration of treatment with inhaled corticosteroids could decrease the relapse rate. On the prognosis of EB, a long-term follow-up study suggested that EB should be a distinct entity rather than an early stage of asthma or chronic obstructive pulmonary disease.

Synaptic vesicle pools at diaphragm neuromuscular junctions vary with motoneuron soma, not axon terminal, inactivity.

Both spinal hemisection (SH) at C2 and tetrodotoxin (TTX) phrenic nerve blockade result in diaphragm muscle paralysis and inactivity of the phrenic axon terminals. However, phrenic motoneuron somata are inactive with SH but remain active with TTX phrenic nerve blockade. Neuromuscular transmission failure with repeated activation decreases following SH and increases following TTX phrenic nerve blockade, suggesting that matching (or mismatching) of somal and synaptic inactivities of phrenic motoneurons differentially regulates synaptic vesicle pools at diaphragm neuromuscular junctions. At individual type-identified rat diaphragm presynaptic terminals, the size of the releasable pool of synaptic vesicles was analyzed by fluorescence confocal microscopy of N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM4-64) uptake and synaptic vesicle density at active zones was determined using transmission electron microscopy. After 14 days of SH and TTX-induced diaphragm muscle inactivity, neuromuscular junction size was not different at type I or IIa fibers, but increased at type IIx and/or IIb fibers (by 51% in SH and 35% in TTX) compared with control. With SH, synaptic vesicle pool size and density increased at presynaptic terminals innervating type I or IIa fibers (17 and 63%, respectively; P<0.001) and type IIx and/or IIb fibers (41 and 31%, respectively; P<0.001) when compared with controls. Following TTX, synaptic vesicle pool size and density decreased by 64 and 17%, respectively, at presynaptic terminals innervating type I or IIa fibers, and by 50 and 36%, respectively, at type IIx and/or IIb fibers (P<0.001, for all comparisons). Thus, matching motoneuron soma and axon terminal inactivity (SH) increases the size and density of releasable synaptic vesicle pools at adult rat diaphragm neuromuscular junctions. Mismatching motoneuron soma and axon terminal inactivities (TTX) results in converse presynaptic adaptations. Inactivity-induced neuromuscular plasticity reflects specific adaptations in the size and density of synaptic vesicle pools that depend on motoneuron soma rather than axon terminal (or muscle fiber) inactivity.

Exogenous testosterone treatment decreases diaphragm neuromuscular transmission failure in male rats.

The effect of chronic exogenous testosterone (T) treatment on neuromuscular transmission in the diaphragm (Dia) muscle of adult male rats was determined. The contribution of neuromuscular transmission failure (NTF) to Dia fatigue was evaluated by superimposing intermittent direct muscle stimulation on repetitive nerve stimulation of isometric contraction in vitro. T treatment significantly reduced the contribution of NTF to Dia fatigue by approximately 20% (P < 0.001). Fiber type-specific effects on NTF were determined by measuring Dia fiber glycogen levels subsequent to repetitive nerve or muscle stimulation. T treatment had no effect on glycogen depletion in Dia type I and IIa fibers regardless of stimulation route. In the control group, type IIx fibers demonstrated significantly less glycogen depletion after nerve stimulation compared with direct muscle stimulation (P < 0.05), suggesting the presence of NTF. In contrast, T treatment increased glycogen depletion of type IIx fibers during nerve stimulation to levels similar to those after direct muscle stimulation. These data indicate that testosterone treatment substantially improves neuromuscular transmission in the Dia.

Power fatigue of the rat diaphragm muscle.

We hypothesized that decrements in maximum power output (W(max)) of the rat diaphragm (Dia) muscle with repetitive activation are due to a disproportionate reduction in force (force fatigue) compared with a slowing of shortening velocity (velocity fatigue). Segments of midcostal Dia muscle were mounted in vitro (26 degrees C) and stimulated directly at 75 Hz in 400-ms-duration trains repeated each second (duty cycle = 0.4) for 120 s. A novel technique was used to monitor instantaneous reductions in maximum specific force (P(o)) and W(max) during fatigue. During each stimulus train, activation was isometric for the initial 360 ms during which P(o) was measured; the muscle was then allowed to shorten at a constant velocity (30% V(max)) for the final 40 ms, and W(max) was determined. Compared with initial values, after 120 s of repetitive activation, P(o) and W(max) decreased by 75 and 73%, respectively. Maximum shortening velocity was measured in two ways: by extrapolation of the force-velocity relationship (V(max)) and using the slack test [maximum unloaded shortening velocity (V(o))]. After 120 s of repetitive activation, V(max) slowed by 44%, whereas V(o) slowed by 22%. Thus the decrease in W(max) with repetitive activation was dominated by force fatigue, with velocity fatigue playing a secondary role. On the basis of a greater slowing of V(max) vs. V(o), we also conclude that force and power fatigue cannot be attributed simply to the total inactivation of the most fatigable fiber types.

Phrenic motoneuron morphology during rapid diaphragm muscle growth.

In the adult rat, there is a general correspondence between the sizes of motoneurons, motor units, and muscle fibers that has particular functional importance in motor control. During early postnatal development, after the establishment of singular innervation, there is rapid growth of diaphragm muscle (Dia(m)) fibers. In the present study, the association between Dia(m) fiber growth and changes in phrenic motoneuron size (both somal and dendritic) was evaluated from postnatal day 21 (D21) to adulthood. Phrenic motoneurons were retrogradely labeled with fluorescent tetramethylrhodamine dextran (3,000 MW), and motoneuron somal volumes and surface areas were measured using three-dimensional confocal microscopy. In separate animals, phrenic motoneurons retrogradely labeled with choleratoxin B-fragment were visualized using immunocytochemistry, and dendritic arborization was analyzed by camera lucida. Between D21 and adulthood, Dia(m) fiber cross-sectional area increased by approximately 164% overall, with the growth of type II fibers being disproportionate to that of type I fibers. There was also substantial growth of phrenic motoneurons ( approximately 360% increase in total surface area), during this same period, that was primarily attributable to an expansion of dendritic surface area. Comparison of the distribution of phrenic motoneuron surface areas between D21 and adults suggests the establishment of a bimodal distribution that may have functional significance for motor unit recruitment in the adult rat.

Denervation alters myosin heavy chain expression and contractility of developing rat diaphragm muscle.

We hypothesized that unilateral denervation (DNV) of the rat diaphragm muscle (Dia(m)) in neonates at postnatal day 7 (D-7) alters normal transitions of myosin heavy chain (MHC) isoform expression and thereby affects postnatal changes in maximum specific force (P(o)) and maximum unloaded shortening velocity (V(o)). The relative expression of different MHC isoforms was analyzed electrophoretically. With DNV at D-7, expression of MHC(neo) in the Dia(m) persisted, and emergence of MHC(2X) and MHC(2B) was delayed. By D-21 and D-28, relative expression of MHC(2A) and MHC(2B) was reduced in DNV compared with control (CTL) animals. Expression of MHC(neo) also reappeared in adult Dia(m) by 2-3 wk after DNV, and relative expression of MHC(2B) was reduced. At each age, P(o) was reduced and V(o) was slowed by DNV, compared with CTL. In CTL Dia(m), postnatal changes in P(o) and V(o) were associated with an increase in fast MHC isoform expression. In DNV Dia(m), no such association existed. We conclude that, in the Dia(m), DNV induces alterations in both MHC isoform expression and contractile properties, which are not necessarily causally linked.

Regional differences in serotonergic input to canine parasternal intercostal motoneurons.

There is a mediolateral gradient in activation of the parasternal intercostal (PI) muscle during inspiration. In the present study, we tested the hypotheses that serotonergic [5-hydroxytryptamine (5-HT)] input from descending central drive and/or intrinsic size-related properties of PI motoneurons leads to the differential activation of PI muscles. In dogs, PI motoneurons innervating the medial and lateral regions of the PI muscles at the T(3)-T(5) interspaces were retrogradely labeled by intramuscular injection of cholera toxin B subunit. After a 10-day survival period, PI motoneurons and 5-HT terminals were visualized by using immunohistochemistry and confocal imaging. There were no differences in motoneuron morphology among motoneurons innervating the medial and lateral regions of the PI muscle. However, the number of 5-HT terminals and the 5-HT terminal density (normalized for surface area) were greater in motoneurons innervating the medial region of the PI muscle compared with the lateral region. These results suggest that differences in distribution of 5-HT input may contribute to regional differences in PI muscle activation during inspiration and that differences in PI motoneuron recruitment do not relate to size.

Effects of genetic selection and voluntary activity on the medial gastrocnemius muscle in house mice.

In a previous study, we found that in house mice both genetic selection (10 generations of artificial selection for high voluntary activity on running wheels) and access to running wheels (7-8 weeks) elicited a modest increase in maximal oxygen consumption. Based on these results, we hypothesized that genetic selection would affect the changes in endurance and oxidative capacity of the medial gastrocnemius (MG) muscle induced by wheel access (training response). Wheel access increased the isotonic endurance of the MG in both genetically selected and random-bred (control) mice. However, this exercise-induced improvement in isotonic endurance of the MG was similar between genetically selected and control mice. Wheel access also increased the succinate dehydrogenase activity of MG muscle fibers in both selected and control lines. However, this exercise-induced increase in succinate dehydrogenase activity was comparable between genetically selected and control animals. Taken together, these results indicate that the modest increase in maximal oxygen consumption associated with genetic selection is not reflected by the training-induced changes in oxidative capacity and endurance of MG muscle fibers.

Inactivity-induced remodeling of neuromuscular junctions in rat diaphragmatic muscle.

We hypothesized that inactivity-induced remodeling of neuromuscular junctions (NMJs) depends on fiber type and the match between muscle fiber and motoneuron (MN) activities. Two inactivity models were studied in rat diaphragmatic muscle: spinal hemisection at C2 (SH), where both diaphragmatic muscle fibers and phrenic MNs were inactive, and tetrodotoxin (TTX) nerve blockade, where only muscle fibers were inactive. After 2 weeks of inactivity, there was increased number of pre- and postsynaptic branches (fragmentation) of NMJs at type IIx/b fibers in both models. In addition, planar NMJ areas at type IIx/b fibers in the SH model were enlarged. In contrast, NMJs at type I and IIa fibers were unaffected in both SH and TTX models. Functionally, neuromuscular transmission in diaphragmatic muscle fibers improved in the SH model, but worsened in the TTX model, compared to controls. These results suggest that NMJ remodeling depends on the level of MN activity. The relative preservation of NMJs at type I and IIa fibers suggests a potential for recovery from diaphragmatic paralysis in the clinical setting, at least for respiratory behaviors.

Corticosteroid effects on diaphragm neuromuscular junctions.

The effects of corticosteroid (CS) treatment (prednisolone continuously administered subcutaneously at a flow rate of 2.5 microl/h, daily dose 5.6 mg/kg, for 3 wk) on neuromuscular junction (NMJ) morphology and neuromuscular transmission in rat diaphragm muscle (Dimus) were compared with weight-matched (Sham) and ad libitum fed control (Ctl) groups. Fibers were classified on the basis of myosin heavy chain (MHC) isoform expression. CS treatment caused significant atrophy of fibers expressing MHC2X (type IIx), either alone or with MHC2B (type IIx/b). Fibers expressing MHCslow (type I) and MHC2A (type IIa) were unaffected by CS. The planar areas of nerve terminals and motor endplates at type IIx/b fibers were smaller in CS-treated Dimus compared with Sham and Ctl. However, CS-induced atrophy of type IIx/b fibers exceeded changes in NMJ morphology. Thus, when normalized for fiber diameter, NMJs were relatively larger in the CS-treated group compared with Ctl. Neuromuscular transmission failure, assessed in vitro by comparing force loss during repetitive (40 Hz) nerve vs. direct muscle stimulation, was less in CS-treated Dimus. These results indicate that alterations in NMJ morphology after CS treatment are dependent on fiber type and may contribute to improved neuromuscular transmission.

Cervical dorsal rhizotomy enhances serotonergic innervation of phrenic motoneurons and serotonin-dependent long-term facilitation of respiratory motor output in rats.

We tested the hypothesis that spinal plasticity elicited by chronic bilateral cervical dorsal rhizotomy (C3-C5; CDR) has functional implications for respiratory motor control. Surgery was performed on rats (CDR or sham-operated) 26 d before phrenic motoneurons were retrogradely labeled with cholera toxin. Rats were killed 2 d later, and their spinal cords were harvested and processed to reveal the cholera toxin-labeled phrenic motoneurons and serotonin-immunoreactive terminals. The number of serotonin-immunoreactive terminals within 5 micrometer of labeled phrenic motoneuron soma and primary dendrites increased 2.1-fold after CDR versus sham-operation. Time-dependent phrenic motor responses to hypoxia were compared among CDR, sham-operated, and control rats. Anesthetized, paralyzed, vagotomized, and artificially ventilated rats were exposed to three, 5 min episodes of isocapnic hypoxia (FiO2 = 0.11), separated by 5 min hyperoxic intervals (FiO2 = 0.5). One hour after hypoxia, a long-lasting, serotonin-dependent enhancement of phrenic motor output (long-term facilitation) was observed in both sham and control rats. After CDR, long-term facilitation was 108 and 163% greater than control and sham responses, respectively. Pretreatment of CDR rats with a 5-HT2 receptor antagonist (ketanserin tartrate, 2 mg/kg, i.v.) before episodic hypoxia prevented long-term facilitation and revealed a modest (-28 +/- 13%; p < 0.05) long-lasting depression of phrenic motor output. The results indicate that CDR: (1) increases serotonergic innervation of the phrenic motor nucleus; and (2) augments serotonin-dependent long-term facilitation of phrenic motor output. These results further suggest a form of plasticity based on changes in the capacity for neuromodulation.

Salbutamol enhances isotonic contractile properties of rat diaphragm muscle.

The effects of the beta2-adrenoceptor agonist salbutamol (Slb) on isometric and isotonic contractile properties of the rat diaphragm muscle (Diamus) were examined. A loading dose of 25 microg/kg Slb was administered intracardially before Diamus excision to ensure adequate diffusion. Studies were then performed with 0.05 microM Slb in the in vitro tissue chamber. cAMP levels were determined by radioimmunoassay. Compared with controls (Ctl), cAMP levels were elevated after Slb treatment. In Slb-treated rats, isometric twitch and maximum tetanic force were increased by approximately 40 and approximately 20%, respectively. Maximum shortening velocity increased by approximately 15% after Slb treatment, and maximum power output increased by approximately 25%. During repeated isotonic activation, the rate of fatigue was faster in the Slb-treated Diamus, but both Slb-treated and Ctl Diamus fatigued to the same maximum power output. Still, endurance time during repetitive isotonic contractions was approximately 10% shorter in the Slb-treated Diamus. These results are consistent with the hypothesis that beta-adrenoceptor stimulation by Slb enhances Diamus contractility and that these effects of Slb are likely mediated, at least in part, by elevated cAMP.

Isotonic contractile and fatigue properties of developing rat diaphragm muscle.

Postnatal transitions in myosin heavy chain (MHC) isoform expression were found to be associated with changes in both isometric and isotonic contractile properties of rat diaphragm muscle (Diam). Expression of MHCneo predominated in neonatal Diam fibers but was usually coexpressed with MHCslow or MHC2A isoforms. Expression of MHCneo disappeared by day 28. Expression of MHC2X and MHC2B emerged at day 14 and increased thereafter. Associated with these MHC transitions in the Diam, maximum isometric tetanic force (Po), maximum shortening velocity, and maximum power output progressively increased during early postnatal development. Maximum power output of the Diam occurred at approximately 40% Po at days 0 and 7 and at approximately 30% Po in older animals. Susceptibility to isometric and isotonic fatigue, defined as a decline in force and power output during repetitive activation, respectively, increased with maturation. Isotonic endurance time, defined as the time for maximum power output to decline to zero, progressively decreased with maturation. In contrast, isometric endurance time, defined as the time for force to decline to 30-40% Po, remained > 300 s until after day 28. We speculate that with the postnatal transition to MHC2X and MHC2B expression energy requirements for contraction increase, especially during isotonic shortening, leading to a greater imbalance between energy supply and demand.

Polymorphism at the HLA-DQ locus determines susceptibility to experimental autoimmune myasthenia gravis.

Studies in myasthenia gravis (MG) patients demonstrate that polymorphism at the HLA-DQ locus influences the development of MG. Several studies using the mouse models also demonstrate the influence of class II molecules, especially the H2-A, which is the mouse homologue of HLA-DQ, in experimental autoimmune myasthenia gravis (EAMG). We used transgenic mice expressing two different DQ molecules, DQ8 (DQA1*0301/B1*0302) and DQ6 (DQA1*0103/B1*0601), to evaluate the role of HLA-DQ genes in MG. These mice do not express endogenous mouse class II molecules since they contain the mutant H2-A beta0 gene. The mice were immunized with Torpedo acetylcholine receptor, and EAMG was assessed by clinical evaluation and was confirmed by electrophysiology. Clinical scores for EAMG were highest in HLA-DQ8 transgenic mice, whereas the scores of HLA-DQ6 mice rarely exceeded grade 1. There was no incidence of EAMG in class II-deficient (H2-A beta0) mice. These results demonstrate that polymorphism at the HLA-DQ locus affects the incidence and the severity of EAMG. The manifestation of susceptibility to EAMG in the context of human class II molecules underscores the important roles of these molecules in the initiation and perpetuation of EAMG.

Effects of voluntary activity and genetic selection on aerobic capacity in house mice (Mus domesticus).

An animal model was developed to study effects on components of exercise physiology of both "nature" (10 generations of genetic selection for high voluntary activity on running wheels) and "nurture" (7-8 wk of access or no access to running wheels, beginning at weaning). At the end of the experiment, mice from both wheel-access groups were significantly lighter in body mass than mice from sedentary groups. Within the wheel-access group, a statistically significant, negative relationship existed between activity and final body mass. In measurements of maximum oxygen consumption during forced treadmill exercise (VO2max), mice with wheel access were significantly more cooperative than sedentary mice; however, trial quality was not a significant predictor of individual variation in VO2max. Nested two-way analysis of covariance demonstrated that both genetic selection history and access to wheels had significant positive effects on VO2max. A 12% difference in VO2max existed between wheel-access selected mice, which had the highest mass-corrected VO2max, and sedentary control mice, which had the lowest. The respiratory exchange ratio at VO2max was also significantly lower in the wheel-access group. Our results suggest the existence of a possible genetic correlation between voluntary activity levels (behavior) and aerobic capacity (physiology).

Corticosteroid effects on isotonic contractile properties of rat diaphragm muscle.

The effects of corticosteroids (CS) on diaphragm muscle (Diam) fiber morphology and contractile properties were evaluated in three groups of rats: controls (Ctl), surgical sham and weight-matched controls (Sham), and CS-treated (6 mg . kg-1 . day-1 prednisolone at 2.5 ml/h for 3 wk). In the CS-treated Diam, there was a selective atrophy of type IIx and IIb fibers, compared with a generalized atrophy of all fibers in the Sham group. Maximum isometric force was reduced by 20% in the CS group compared with both Ctl and Sham. Maximum shortening velocity in the CS Diam was slowed by approximately 20% compared with Ctl and Sham. Peak power output of the CS Diam was only 60% of Ctl and 70% of Sham. Endurance to repeated isotonic contractions improved in the CS-treated Diam compared with Ctl. We conclude that the atrophy of type IIx and IIb fibers in the Diam can only partially account for the CS-induced changes in isotonic contractile properties. Other factors such as reduced myofibrillar density or altered cross-bridge cycling kinetics are also likely to contribute to the effects of CS treatment.

Metabolic and phenotypic adaptations of diaphragm muscle fibers with inactivation.

We hypothesized that metabolic adaptations to muscle inactivity are most pronounced when neurotrophic influence is disrupted. In rat diaphragm muscle (Dia(m)), 2 wk of unilateral denervation or tetrodotoxin nerve blockade resulted in a reduction in succinate dehydrogenase (SDH) activity of type I, IIa, and IIx fibers (approximately 50, 70, and 24%, respectively) and a decrease in SDH variability among fibers (approximately 63%). In contrast, inactivity induced by spinal cord hemisection at C2 (ST) resulted in much less change in SDH activity of type I and IIa fibers (approximately 27 and 24%, respectively) and only an approximately 30% reduction in SDH variability among fibers. Actomyosin adenosinetriphosphatase (ATPase) activities of type I, IIx, and IIb fibers in denervated and tetrodotoxin-treated Dia(m) were reduced by approximately 20, 45, and 60%, respectively, and actomyosin ATPase variability among fibers was approximately 60% lower. In contrast, only actomyosin ATPase activity of type IIb fibers was reduced (approximately 20%) in ST Dia(m). These results suggest that disruption of neurotrophic influence has a greater impact on muscle fiber metabolic properties than inactivity per se.

Diaphragm disuse reduces Ca2+ uptake capacity of sarcoplasmic reticulum.

Chronic phrenic tetrodotoxin (TTX) blockade and phrenic denervation (Dnv) of hamster diaphragm result in decreased maximum specific tension, prolonged contraction time, and improved fatigue resistance (W. Z. Zhan and G. C. Sieck, J. Appl. Physiol. 72: 1445-1453, 1992). An underlying increased relative contribution of type I fibers to total muscle mass appears to be consistent with, but does not completely account for, changes in contractile and fatigue properties. The present study was designed to evaluate a potential role for altered cellular Ca2+ metabolism in the adaptive response of the diaphragm to chronic disuse. An analytic method based on simulation and modeling of long-term 45Ca2+ efflux data was used to estimate Ca2+ contents (nmol Ca2+/g wet wt tissue) and exchange fluxes (nmol Ca2+.min-1.g-1) for extracellular and intracellular compartments in the in vitro hamster hemidiaphragm after prolonged disuse. Three groups were compared: control (Con, n = 5), phrenic TTX blockade (TTX, n = 5), and phrenic denervation (Dnv, n = 5). Experimental muscles were loaded with 45Ca2+ for 1 h, and efflux data were collected for 8 h by using a flow-through tissue chamber. Compartmental analysis of efflux data estimated that the Ca2+ contents and Ca2+ exchange fluxes of the largest and slowest intracellular compartment (putative longitudinal reticulum) were reduced by approximately 50% in TTX and Dnv muscle groups compared with Con. In addition, the kinetic model predicted significant decreases in total intracellular Ca2+ and total diaphragm Ca2+ in TTX and Dnv muscles. We conclude that the data support the hypothesis that the capacity of the sarcoplasmic reticulum for Ca2+ sequestration is reduced in chronic diaphragm disuse. The impact of this effect on diaphragm contractile and fatigue properties is discussed.

Hypothyroidism alters diaphragm muscle development.

The impact of hypothyroidism (Hyp) on myosin heavy chain (MHC) isoform expression, maximum specific force (P0), fatigability, and maximum unloaded shortening velocity (V0) was determined in the rat diaphragm muscle (Dia) at 0, 7, 14, 21, and 28 days of age. Hyp was induced by treating pregnant rats with 6-n-propyl-2-thiouracil (0.05% in drinking water) beginning at gestational day 10 and was confirmed by reduced plasma levels of 3,5,3'-triiodothyronine and thyroxine. MHC isoforms were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by densitometry. Isometric P0 and fatigue resistance of the Dia were measured in vitro at 26 degrees C, and V0 was determined at 15 degrees C with the slack test. Compared with control muscles, expression of MHC-slow was higher and expression of adult fast MHC isoforms was lower in Hyp Dia at all ages. The neonatal isoform of MHC continued to be expressed in the Hyp Dia until day 28. At each age, P0 and fatigability were reduced and V0 was slower in the Hyp Dia. We conclude that Hyp-induced alterations in MHC isoform expression do not fully predict the changes in Dia contractile properties.